U.S. patent application number 12/461353 was filed with the patent office on 2009-12-17 for nucleic acid molecules and other molecules associated with the sucrose pathway.
Invention is credited to Nordine Cheikh, Dane K. Fisher, Jingdong Liu.
Application Number | 20090313727 12/461353 |
Document ID | / |
Family ID | 46279431 |
Filed Date | 2009-12-17 |
United States Patent
Application |
20090313727 |
Kind Code |
A1 |
Cheikh; Nordine ; et
al. |
December 17, 2009 |
Nucleic acid molecules and other molecules associated with the
sucrose pathway
Abstract
The present invention is in the field of plant biochemistry.
More specifically the invention relates to nucleic acid sequences
from plant cells, in particular, nucleic acid sequences from maize
and soybean plants associated with the sucrose pathway. The
invention encompasses nucleic acid molecules that encode proteins
and fragments of proteins. In addition, the invention also
encompasses proteins and fragments of proteins so encoded and
antibodies capable of binding these proteins or fragments. The
invention also relates to methods of using the nucleic acid
molecules, proteins and fragments of proteins and antibodies, for
example for genome mapping, gene identification and analysis, plant
breeding, preparation of constructs for use in plant gene
expression and transgenic plants.
Inventors: |
Cheikh; Nordine;
(Manchester, MO) ; Fisher; Dane K.; (O'Fallon,
MO) ; Liu; Jingdong; (Ballwin, MO) |
Correspondence
Address: |
ARNOLD & PORTER LLP
555 TWELFTH STREET, N.W., ATTN: IP DOCKETING
WASHINGTON
DC
20004
US
|
Family ID: |
46279431 |
Appl. No.: |
12/461353 |
Filed: |
August 10, 2009 |
Related U.S. Patent Documents
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Current U.S.
Class: |
800/298 ;
47/58.1R; 536/23.6 |
Current CPC
Class: |
C12N 15/52 20130101;
C12N 15/8245 20130101 |
Class at
Publication: |
800/298 ;
536/23.6; 47/58.1R |
International
Class: |
A01H 5/00 20060101
A01H005/00; C07H 21/04 20060101 C07H021/04; A01G 1/00 20060101
A01G001/00 |
Claims
1-6. (canceled)
7. A transformed plant comprising a nucleic acid molecule which
comprises: (a) an exogenous promoter region which functions in a
plant cell to cause the production of an mRNA molecule; which is
linked to; (b) a structural nucleic acid molecule, wherein said
structural nucleic acid molecule comprises a nucleic acid sequence,
wherein said nucleic acid sequence shares between 100% and 90%
sequence identity with a nucleic acid sequence selected from the
group consisting of SEQ ID NO: 1 through SEQ ID NO: 2814 and the
complement of SEQ ID NO: 1 through SEQ ID NO: 2814, which is linked
to (c) a 3' non-translated sequence that functions in said plant
cell to cause the termination of transcription and the addition of
polyadenylated ribonucleotides to said 3' end of said mRNA
molecule.
8. The transformed plant according to claim 7, wherein said nucleic
acid sequence is the complement of a nucleic acid sequence selected
from the group consisting of SEQ ID NO: 1 through SEQ ID NO:
2814.
9. The transformed plant according to claim 7, wherein said nucleic
acid sequence shares between 100% and 95% sequence identity with a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 1 through SEQ ID NO: 2814 and the complement of SEQ ID NO: 1
through SEQ ID NO: 2814.
10. The transformed plant according to claim 9, wherein said
nucleic acid sequence shares between 100% and 98% sequence identity
with a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 2814 and the complement of SEQ ID
NO: 1 through SEQ ID NO: 2814.
11. The transformed plant according to claim 10, wherein said
nucleic acid sequence shares between 100% and 99% sequence identity
with a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 2814 and the complement of SEQ ID
NO: 1 through SEQ ID NO: 2814.
12. The transformed plant according to claim 11, wherein said
nucleic acid sequence exhibits 100% sequence identity with a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 1 through SEQ ID NO: 2814 and the complement of SEQ ID NO: 1
through SEQ ID NO: 2814.
13. A transformed seed comprising a transformed plant cell
comprising a nucleic acid molecule which comprises: (a) an
exogenous promoter region which functions in said plant cell to
cause the production of an mRNA molecule; which is linked to; (b) a
structural nucleic acid molecule, wherein said structural nucleic
acid molecule comprises a nucleic acid sequence, wherein said
nucleic acid sequence shares between 100% and 90% sequence identity
with a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 2814 and the complement of SEQ ID
NO: 1 through SEQ ID NO: 2814, which is linked to (c) a 3'
non-translated sequence that functions in said plant cell to cause
the termination of transcription and the addition of polyadenylated
ribonucleotides to said 3' end of said mRNA molecule.
14. The transformed seed according to claim 13, wherein said
nucleic acid sequence is the complement of a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 2814.
15. The transformed seed according to claim 13, wherein said
exogenous promoter region functions in a seed cell.
16. The transformed seed according to claim 13, wherein said
nucleic acid sequence shares between 100% and 95% sequence identity
with a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 2814 and the complement of SEQ ID
NO: 1 through SEQ ID NO: 2814.
17. The transformed seed according to claim 16, wherein said
nucleic acid sequence shares between 100% and 98% sequence identity
with a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 2814 and the complement of SEQ ID
NO: 1 through SEQ ID NO: 2814.
18. The transformed seed according to claim 17, wherein said
nucleic acid sequence shares between 100% and 99% sequence identity
with a nucleic acid sequence selected from the group consisting of
SEQ ID NO: 1 through SEQ ID NO: 2814 and the complement of SEQ ID
NO: 1 through SEQ ID NO: 2814.
19. The transformed seed according to claim 18, wherein said
nucleic acid sequence exhibits 100% sequence identity with a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 1 through SEQ ID NO: 2814 and the complement of SEQ ID NO: 1
through SEQ ID NO: 2814.
20. A method of growing a transgenic plant comprising (a) planting
a transformed seed comprising a nucleic acid sequence that shares
between 100% and 90% sequence identity with a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 2814 and the complement of SEQ ID NO: 1 through SEQ ID NO:
2814, and (b) growing a plant from said seed.
21. A substantially purified nucleic acid molecule comprising a
nucleic acid sequence, wherein said nucleic acid sequence shares
between 100% and 90% sequence identity with a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 2814 and the complement of SEQ ID NO: 1 through SEQ ID NO:
2814.
22. The substantially purified nucleic acid molecule of claim 21,
wherein said nucleic acid molecule encodes a soybean protein or
fragment thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 09/237,183 filed Jan. 26, 1999, which claims benefit under 35
U.S.C .sctn. 119(e) of U.S. Provisional Application Nos. 60/067,000
filed Nov. 24, 1997; 60/069,472 filed Dec. 9, 1997; 60/072,888
filed Jan. 27, 1998; 60/074,201 filed Feb. 10, 1998; 60/074,282
filed Feb. 10, 1998; 60/074,280 filed Feb. 10, 1998; 60/074,281
filed Feb. 10, 1998; 60/074,566 filed Feb. 12, 1998; 60/074,567
filed Feb. 12, 1998; 60/074,565 filed Feb. 12, 1998; 60/075,462
filed Feb. 19, 1998; 60/074,789 filed Feb. 19, 1998; 60/075,459
filed Feb. 19, 1998; 60/075,461 filed Feb. 19, 1998; 60/075,464
filed Feb. 19, 1998; 60/075,460 filed Feb. 19, 1998; 60/075,463
filed Feb. 19, 1998; 60/076,912 filed Mar. 6, 1998; 60/077,231
filed Mar. 9, 1998; 60/077,229 filed Mar. 9, 1998; 60/077,230 filed
Mar. 9, 1998; 60/078,368 filed Mar. 18, 1998; 60/080,844 filed Apr.
7, 1998; 60/083,067 filed Apr. 27, 1998; 60/083,386 filed Apr. 29,
1998; 60/083,387 filed Apr. 29, 1998; 60/083,388 filed Apr. 29,
1998; 60/083,389 filed Apr. 29, 1998; 60/083,390 filed Apr. 29,
1998; 60/085,224 filed May 13, 1998; 60/085,223 filed May 13, 1998;
60/085,222 filed May 13, 1998; 60/086,186 filed May 21, 1998;
60/086,187 filed May 21, 1998; 60/086,185 filed May 21, 1998;
60/086,184 filed May 21, 1998; 60/086,183 filed May 21, 1998;
60/086,188 filed May 21, 1998; 60/087,422 filed Jun. 1, 1998;
60/089,524 filed Jun. 16, 1998; 60/089,810 filed Jun. 18, 1998;
60/089,814 filed Jun. 18, 1998; 60/089,793 filed Jun. 18, 1998;
60/090,170 filed Jun. 22, 1998; 60/090,928 filed Jun. 26, 1998;
60/091,035 filed Jun. 29, 1998; 60/091,405 filed Jun. 30, 1998;
60/092,036 filed Jul. 8, 1998; 60/099,667 filed Sep. 9, 1998;
60/099,670 filed Sep. 9, 1998; 60/099,697 filed Sep. 9, 1998;
60/100,674 filed Sep. 16, 1998; 60/100,673 filed Sep. 16, 1998;
60/100,672 filed Sep. 16, 1998; 60/101,131 filed Sep. 21, 1998;
60/101,132 filed Sep. 21, 1998; 60/101,130 filed Sep. 21, 1998;
60/101,508 filed Sep. 22, 1998; 60/101,344 filed Sep. 22, 1998;
60/101,347 filed Sep. 22, 1998; 60/101,343 filed Sep. 22, 1998;
60/101,707 filed Sep. 25, 1998; 60/104,126 filed Oct. 13, 1998;
60/104,128 filed Oct. 13, 1998; 60/104,127 filed Oct. 13, 1998;
60/104,124 filed Oct. 13, 1998; 60/104,123 filed Oct. 13, 1998;
60/109,018 filed Nov. 18, 1998; 60/108,996 filed Nov. 18, 1998;
60/111,981 filed Dec. 11, 1998; and 60/113,224 filed Dec. 22, 1998;
and claims the benefit under 35 U.S.C. .sctn. 120 as a
continuation-in-part application of U.S. application Ser. Nos.
09/119,129 filed Nov. 24, 1998; 09/210,297 filed Dec. 8, 1998; and
09/229,413 filed Jan. 12, 1999, the disclosures of which are herein
incorporated by reference in their entirety.
INCORPORATION OF SEQUENCE LISTING
[0002] The sequence listing is hereby incorporated by reference in
its entirety, (including the file named "SequenceListing.txt" on 3
CD-ROM copies (CRF, Copy 1 and Copy 2), which is 1,352,824 bytes in
size (measured in Windows XP) and was recorded on Aug. 4, 2009,
which is likewise herein incorporated by reference in its
entirety.)
FIELD OF THE INVENTION
[0003] The present invention is in the field of plant biochemistry.
More specifically the invention relates to nucleic acid sequences
from plant cells, in particular, nucleic acid sequences from maize
and soybean plants associated with the sucrose pathway. The
invention encompasses nucleic acid molecules that encode proteins
and fragments of proteins. In addition, the invention also
encompasses proteins and fragments of proteins so encoded and
antibodies capable of binding these proteins or fragments. The
invention also relates to methods of using the nucleic acid
molecules, proteins and fragments of proteins and antibodies, for
example for genome mapping, gene identification and analysis, plant
breeding, preparation of constructs for use in plant gene
expression and transgenic plants.
BACKGROUND OF THE INVENTION
[0004] Carbon fixed during photosynthesis is either retained in the
chloroplast and converted to a storage carbohydrate, for example,
starch, or it is transferred to the cytosol in the form of triose
phosphates and converted to sucrose. The newly synthesized sucrose
in source tissues is a major transported form of reduced carbon in
higher plants and can be either metabolized into other
carbohydrates, stored in the vacuole or exported to other plant
tissues. Plant tissues where sucrose is synthesized, such as
leaves, are often referred to as `source` tissues. Translocated
sucrose is retained in `sink` tissues (such as expanding leaves,
growing seeds, flowers, roots or tubers, and fruit) and may be
assimilated, or further metabolized to sustain cell maintenance or
fuel growth, or be converted to alternative storage compounds
(e.g., starch, fats). The relative type and size of these
carbohydrate pools vary during tissue development, between
different plant species, and within the same species subject to
different environmental conditions. Such differences are reported
to affect the yield and quality of agricultural produce.
[0005] Sucrose synthesis and catabolism are reported to be highly
coordinated and regulated processes that may also be coordinately
regulated with other dedicated metabolic pathways in a particular
plant, plant organ or cell type. Sucrose synthesis is reported to
be coordinately regulated with starch metabolism and photosynthesis
in green `source` plant tissues. Sucrose supply by transport
mechanisms to actively growing `sink` tissues is reported to be
coordinated with plant development. In growing sink tissues, the
supply of carbohydrate is reported to be important to other
metabolic pathways and physiological processes including
respiration, starch biosynthesis, cell wall biogenesis, lipid and
protein biosynthesis. Sucrose synthesis and/or transport is also
reported to play a role in the carbohydrate capacity that is
available to growing fruits and seeds. Sucrose resynthesis during
seed germination is reported to play a role in seedling vigor and
agronomic stand establishment in many plant species during early
plant development.
[0006] In many plant species, enzymes of pathways involved in
sucrose metabolism can play a role in plant physiology and plant
growth and development. Compartmentation and temporal regulation of
genes and enzymes of sucrose metabolic pathways can allow multiple
pathways to utilize sucrose as a common metabolite. Flux through a
particular sucrose metabolic pathway can define the utilization of
sucrose in any tissue or developmental stage. Sucrose and its
metabolite products have been reported to play a role in gene
regulation and expression of the sucrose pathway and other
metabolic pathways in plants.
[0007] Reviews on sucrose metabolism in plants include Avigad, In:
Encyclopedia of Plant Physiology, Vol 13A, Loewus and Tanner, eds.,
Springer Verlag, Heidelberg, 217-347 (1982); Hawker, In:
Biochemistry of Storage Carbohydrates in Green Plants, Dey and
Dixon, eds., Academic Press, London, 1-51 (1985); Huber et al., In:
Carbon Partitioning Within and Between Organisms, Pollock et al.,
eds., Bios Scientific, Oxford, 1-26 (1992); Stitt et al., In:
Biochemistry of Plants, Vol 10, Hatch and Boardman, eds., Academic
Press, New York, 327-407 (1987); Quick and Schaffer, In:
Photoassimilate Distribution In: Plants And Crops, Zamski and
Schaffer, eds., Marcel Dekker Inc., New York, 115-156 (1996), all
of which are herein incorporated by reference in their
entirety.
[0008] The synthesis of sucrose precursors (triose and hexose
phosphates) is derived from either photosynthetic CO.sub.2 fixation
or degradation of previously deposited storage reserves. One
substrate for sucrose synthesis in photosynthetic tissues is three
carbon sugar phosphates. These are exported from the chloroplast
during photosynthesis, predominantly in the form of triose
phosphates. The pool of triose phosphates, dihydroxyacetone
phosphate ("DHAP"), and glyceraldehyde-3-phosphate ("GAP"), is
maintained at equilibrium within the cytoplasm by triose phosphate
isomerase (EC 5.3.1.1). A subsequent reaction involves an aldol
condensation of DHAP and GAP, catalyzed by the enzyme fructose
1,6-bisphosphate aldolase (often called aldolase) (EC 4.1.2.13) to
form fructose 1,6-bisphosphate ("F1,6BP").
Fructose-1,6-bisphosphatase ("FBPase") (EC 3.1.3.11) catalyzes the
cleavage of phosphate from the C1 carbon of
fructose-1,6-bisphosphate to form fructose-6-phosphate ("F6P").
This reaction is essentially irreversible and has been reported to
represent the first committed step within the pathway of sucrose
synthesis. The cytosolic FBPase has been reported to be subject to
allosteric regulation and may serve to coordinate the rate of
sucrose synthesis with that of photosynthesis. Fructose
2,6-bisphosphate ("F2,6BP") is reported to be a regulator of FBPase
(Black et al., In: Regulation of Carbohydrate Partitioning In
Photosynthetic Tissue, Heath and Preiss, eds., Waverly, Baltimore,
109-126 (1985); Stitt et al., In: Biochemistry Of Plants, Vol. 10,
Hatch and Boardman, eds., Academic Press, New York, 327-407 (1987);
both of which are herein incorporated by reference in their
entirety). The concentration of F2,6BP is reported to be controlled
in plants by two enzymes, fructose-2,6-bisphosphatase (F2,6 Bpase)
(EC 3.1.3.46) and fructose-6-phosphate,2-kinase (F6P,2K) (EC
2.7.1.105) (Stitt, Annu. Rev. Plant Physiol. Plant Mol. Biol. 41:
153-181 (1990), the entirety of which is herein incorporated by
reference).
[0009] Glucose-6-phosphate ("G6P") and glucose-1-phosphate ("G1P")
are reported to be maintained in equilibrium with the F6P pool by
the action of phosphoglucoisomerase ("PGI") (EC 5.3.1.9) and
phosphoglucomutase ("PGM") (EC 5.4.2.2), respectively. Uridine
diphosphate glucose ("UDPG") and pyrophosphate ("PPi") are formed
from uridine triphosphate ("UTP") and G1P catalyzed by the enzyme
UDPG-pyrophosphorylase ("UDPGase") (EC 2.7.7.9). This reaction is
reversible and net flux in the direction of sucrose synthesis is
reported to require removal of its products, particularly PPi. A
pyrophosphate-dependent proton pump, vacuolar
H.sup.+-translocating-pyrophosphatase (EC 3.6.1.1), has been
identified within the vacuolar membrane and has been reported to
utilize pyrophosphate to sustain a proton gradient formed between
these two compartments (Rea et al., Trends in Biol. Sci. 17:
348-353 (1992), the entirety of which is herein incorporated by
reference).
[0010] A pyrophosphate-dependent fructose-6-phosphate
phosphotransferase ("PFP") (EC 2.7.1.90) is also present in the
cytoplasm and catalyzes the reversible production of F1,6BP and Pi
from F6P and PPi. One reported function of PFP is to operate in a
futile cycle with the cytosolic FBPase, and function as a
"pseudopyrophosphatase" recycling PPi. Uridine diphosphate glucose
is then combined with F6P to form sucrose-6-phosphate ("S6P"). This
reaction is catalyzed by sucrose phosphate synthase ("SPS") (EC
2.4.1.14). Attachment of UDP to the glucose moiety activates the C1
carbon atom of UDPG, which is necessary for the subsequent
formation of a glycosidic bond in sucrose. In certain organisms,
SPS is capable of using adenine diphosphate glucose ("ADPG"),
instead of UDPG, as a substrate. The use of nucleotide biphosphate
sugars is a feature of metabolic pathways leading to the production
of disaccharides and polysaccharides. SPS is reported to be subject
to allosteric and covalent regulation and, in conjunction with the
cytosolic FBPase, reportedly serves to coordinate the rate of
sucrose synthesis with the rate of photosynthesis. The reported
final reaction in the pathway is catalyzed by sucrose-6-phosphate
phosphatase ("SPPase" or "SPP") (EC 3.1.3.24), which catalyzes the
hydrolysis of S6P to sucrose. It has been reported that SPS and
SPPase may associate to form a multienzyme complex, that the rate
of sucrose-6-phosphate synthesis by SPS is enhanced in the presence
of SPP, and that the rate of sucrose-6-phosphate hydrolysis by SPP
is increased in the presence of SPS (Echeverria et al., Plant
Physiol. 115: 223-227 (1997), herein incorporated by reference in
its entirety).
[0011] I. Sucrose Synthesis
[0012] Reviews describing fructose-1,6-bisphosphatase ("FBPase", EC
3.1.3.11) include those by Hers and Van Shaftingen, Biochem J.
206:1-12 (1982), the entirety of which is herein incorporated by
reference, and Stitt, Annu. Rev. Plant Physiol. Plant Mol. Biol.
41:153-181 (1990). Two isoforms of FBPase are reported to exist in
plants. The first isoform is associated with the plastid and occurs
largely in photosynthetic plastids. The second isoform, located in
the cytoplasm, is reported to be involved in both gluconeogenesis
and sucrose synthesis (Zimmerman et al., J. Biol. Chem. 253:
5952-5956 (1978); Stitt and Heldt, Planta 164: 179-188 (1985), both
of which are hereby incorporated by reference in their entirety).
FBPase catalyzes an irreversible reaction in the direction of F6P
synthesis in vivo and has been reported to represent the first
committed step in the pathway of sucrose synthesis. The properties
of the enzyme are reported to involve the action of several
regulatory metabolites (Stitt et al., In: Biochemistry Of Plants,
Vol. 10, Hatch and Boardman, eds., Academic Press, New York,
327-407 (1987)). The enzyme reportedly has a high affinity for its
substrate F1,6BP, a requirement for Mg.sup.2+, a requirement for a
neutral pH, is weakly inhibited (Km 2-4 .mu.m) by adenosine
monophosphate (AMP), and is strongly inhibited by the regulatory
metabolite F2,6BP (Hers and Van Shaftingen, Biochem J 206: 1-12
(1982); Black et al., In: Regulation of Carbohydrate Partitioning
In Photosynthetic Tissue, Heath and Preiss, eds., Waverly,
Baltimore, 109-126 (1985); Huber, Annu. Rev. Plant Physiol. 37:
233-246 (1986); Stiff et al., In: Biochemistry Of Plants, Vol. 10,
Hatch and Boardman, eds., Academic Press, New York, 327-407 (1987),
all of which are herein incorporated by reference in their
entirety). F2,6BP is also an activator of PFP and reportedly plays
a role in the regulation of gluconeogenetic and respiratory
metabolism.
[0013] The concentration of F2,6BP is reportedly determined in
plants by two enzymes, fructose-2,6-bisphosphatase ("F2,6BPase")
(EC 3.1.3.46) and fructose-6-phosphate,2-kinase ("F6P,2K") (EC
2.7.1.105). A review of these enzymes is provided by Stitt, Annu.
Rev. Plant Physiol. Plant Mol. Biol. 41: 153-181 (1990). Regulation
of the activity of the F1,6FBPase and the rate of sucrose synthesis
is reported to be, at least in part, brought about by changes in
the concentration of F2,6BP.
[0014] Sucrose phosphate synthase (SPS (EC 2.4.1.14)) catalyzes a
reaction that is displaced from equilibrium in vivo in the
direction of S6P synthesis and is reported as an essentially
irreversible reaction in vivo (Stitt et al., In: Biochemistry Of
Plants, Vol. 10, Hatch and Boardman, eds., Academic Press, New
York, 327-407 (1987); Lunn and Rees, Biochem. J. 267: 739-743
(1990), the entirety of which is herein incorporated by reference;
U.S. Pat. No. 5,665,892, the entirety of which is herein
incorporated by reference). SPS has been purified from spinach and
Zea mays, and the amino acid and cDNA sequences have been published
(Worrel et al., Plant Cell 3:1121-1130 (1991); Klein et al., Planta
190: 498-510 (1993); Sonnewald et al., Planta 189: 174-181 (1993),
all of which are herein incorporated by reference in their
entirety). The enzyme has a subunit molecular weight of 117 kDa
from spinach (Klein et al., Planta 190: 498-510 (1993); Sonnewald
et al., Planta 189: 174-181 (1993), both of which are herein
incorporated by reference) and pea (Lunn and Rees, Phytochem. 29:
1057-1063 (1990), the entirety of which is herein incorporated by
reference) and 135 kDa from Zea mays (Worrel et al., Plant Cell
3:1121-1130 (1991)). The native enzyme reportedly exists as a
tetramer (Walker and Huber, Plant Physiol. 89: 518-524 (1988); Lunn
and Rees, Phytochem. 29: 1057-1063 (1990); Worrel et al., Plant
Cell 3:1121-1130 (1991), although dimeric molecular weights have
been reported (Klein et al., Planta 190: 498-510 (1993), the
entirety of which is herein incorporated by reference). Activity
has been observed for SPS at both dimeric and tetrameric molecular
weights (Sonnewald et al., Planta 189:174-181 (1993), the entirety
of which is herein incorporated by reference).
[0015] SPS is located in the cytosol, has a neutral pH optimum, and
has been detected in all plant tissues which undertake active
sucrose synthesis. SPS is also reported to undertake active sucrose
synthesis. An increase in abundance of the enzyme is has been
reported during the development of leaves, germination of seeds and
ripening of fruit. The enzyme has been reported to be subject to
regulation by metabolites and is activated by G6P and is inhibited
by Pi. Pi and GP6 are reported to act competitively at an
allosteric site of the enzyme. In the presence of high Pi
concentrations, the enzyme is phosphorylated which reduces activity
of the enzyme. It has also been reported that light-induced
photosynthesis increases the activity of SPS in crude extracts
(Sicher and Kremer, Plant Physiol. 79: 910-912 (1984), Sicher and
Kremer, Plant Physiol. 79: 695-698 (1985); Pollock and Housley,
Ann. Bot. 55: 593-596 (1985), all of which are herein incorporated
by reference in their entirety). In addition, it has been reported
that compounds altering the phosphate status of the leaf can
simulate the effects of light. Feeding leaves mannose, which
sequesters phosphate by its conversion to the non-metabolized
mannose-6-P, has been reported to cause activation of SPS (Stitt et
al., Planta 174: 217-230 (1988), the entirety of which is herein
incorporated by reference).
[0016] The phosphorylation and dephosphorylation of SPS is
catalyzed by SPS-phosphatase and SPS-kinase, respectively (Huber et
al., Plant Physiol. 99: 1275-1278 (1992). Hydrolysis of sucrose-6-P
to sucrose is catalyzed by sucrose-6-phosphatase (SPPase or SPP)
(EC 3.1.3.24). The activity of both SPS and SPP is reported to be
affected by a multienzyme complex between SPS and SPP (Echeverria
et al., Plant Physiol. 115: 223-227 (1997)).
[0017] Regulatory properties of SPS and FBPase are reported to
coordinate the rate of sucrose synthesis with that of
photosynthesis (Stitt, In: Plant Physiology, Biochemistry and
Molecular Biology, Dennis and Turpin, eds., Singapore, London,
319-340 (1990), the entirety of which is herein incorporated by
reference). When photosynthesis produces triose phosphate in excess
of the rate of sucrose synthesis, a feed-forward activation of
sucrose synthesis occurs. Triose phosphate crosses the chloroplast
membrane in exchange for cytosolic Pi. Under these conditions,
F6P,2-kinase activity is reduced and the inhibition of F2,6Bpase is
decreased.
[0018] As cytosolic F2,6BP falls, F2,6BPase activity increases, and
F6P levels increase. Hexose phosphate levels are reported to
increase due to PGM and PGI, and with low Pi, activate SPS and
F1,6BPase. Reduction in rate of photosynthesis must result in a
deactivation of sucrose synthesis, which occurs through decreased
cytosolic triose-P, increased Pi and ultimately increased F2,6BP
concentration and reduced SPS activity (Stitt, Phil. Trans. R Soc.
Lond. B 342: 225-233 (1993); Huber et al., Plant Physiol. 99:
1275-1278 (1992); Neuhaus et al., Planta 181: 583-592 (1990), both
of which are herein incorporated by reference).
[0019] II. Metabolic Pathways of Sucrose Catabolism
[0020] Sucrose can initially be cleaved by invertases (EC 3.2.1.26)
or by sucrose synthases (EC 2.4.1.13). Invertases, which are
classified as acid or alkaline in pH preference (Karuppiah et al.,
Plant Physiol. 91: 993-998 (1989); Fahrendorf and Beck, Planta 180:
237-244 (1990); Iwatsubo et al., Biosc. Biotech. Biochem. 56:
1959-1962 (1992); Unger et al., Plant Physiol. 104: 1351-1357
(1994); Avigad, In: Encyclopedia of Plant Physiology, Vol 13A,
Loewus and Tanner, eds., Springer Verlag, Heidelberg, 217-347
(1982), all of which are herein incorporated by reference in their
entirety), irreversibly cleave sucrose into glucose and fructose,
both of which is usually phosphorylated for further metabolism. The
invertase pathway usually is associated with rapidly growing sink
tissues such as expanding leaves, expanding internodes, flower
petals, and early fruit development (Avigad, In: Encyclopedia of
Plant Physiology, Vol 13A, Loewus and Tanner, eds., Springer
Verlag, Heidelberg, 217-347 (1982); Huber, Plant Physiol.
91:656-662 (1989); Morris and Arthur, Phytochem. 23: 2163-2167
(1984); Hawker et al., Phytochem. 15: 1441-1443 (1976); Schaffer et
al., Plant Physiol. 69: 151-155 (1987), all of which are herein
incorporated by reference in their entirety).
[0021] Sucrose synthase carries out the kinetically reversible
transglycosylation of sucrose and UDP into fructose and UDPG,
requiring only the phosphorylation of fructose for additional
metabolism. Polysaccharide biosynthesis in sink tissues may utilize
a sucrose synthase mediated sucrose catabolism (Avigad, In:
Encyclopedia of Plant Physiology, Vol 13A, Loewus and Tanner, eds.,
Springer Verlag, Heidelberg, 217-347 (1982); Doehlert et al., Plant
Physiol. 86: 1013-1019 (1988); Dale and Housley Plant Physiol. 82:
7-10 (1986), all of which are herein incorporated by reference).
Respiring tissues reportedly utilize either sucrose synthase or
invertase metabolic pathways (Echeverria and Humphreys, Phytochem.
23: 2173-2178 (1984); Uritani and Asahi, In: The Biochemistry of
Plants Vol. 2, Davies, ed., Academic Press, New York, 463-487
(1980), all of which are herein incorporated by reference in their
entirety). Tissues that are undergoing respiration, starch
biosynthesis, amino acid and fatty acid synthesis, rapid expansion
or growth, and other cellular metabolism, can utilize several
sucrose metabolic pathways which may be temporally or
compartmentally regulated (Doehlert et al., Plant Physiol. 86:
1013-1019 (1988); Doehlert, Plant Physiol. 78: 560-567 (1990);
Doehlert and Choury, In: Recent Advances in Phloem Transport and
Assimilate Compartmentation, Bonnemain et al., eds., Ouest
editions, Nantes, France, 187-195 (1991); Delmer and Stone, In: The
Biochemistry of Plants, Vol. 14, Preiss, ed., Academic Press, San
Diego, 373-420 (1988); Maas et al., EMBO J. 9: 3447-3452 (1990),
all of which are herein incorporated by reference in their
entirety).
[0022] Hexose kinases are a class of enzymes responsible for the
phosphorylation of hexoses, and are classified into two groups.
Hexokinase (EC 2.7.1.1) can phosphorylate either glucose or
fructose, with different isoforms often unique to different tissues
or plant species. Different isoforms can have affinities for
different hexoses (Turner and Copeland, Plant Physiol. 68:
1123-1127 (1981), the entirety of which is herein incorporated by
reference; Copeland and Turner, In: The Biochemistry of Plants,
Vol. 11, Stumpf and Conn, eds., Academic Press, New York, 107-128
(1987), the entirety of which is herein incorporated by reference).
Hexokinases include fructokinases (EC 2.7.1.11), which typically
have specific affinities for fructose (Doehlert, Plant Physiol. 89:
1042-1048 (1989); Renz and Stitt Planta 190: 166-175 (1993), both
of which are herein incorporated by reference). Fructokinases can
also be specific in their affinity for nucleotides. The extent to
which a fructokinase utilizes UTP may play a physiological role in
how efficiently UDP can be recycled for sucrose synthase activity
in a particular tissue (Huber and Akazawa, Plant Physiol. 81:
1008-1013 (1986); Xu et al., Plant Physiol. 90: 635-642 (1989),
both of which are herein incorporated by reference). UDP levels for
the sucrose synthase reaction may be maintained, even in the case
of an ATP-specific fructokinase, by the enzyme NDP-kinase (EC
2.7.4.6).
[0023] NDP-kinase has been reported in several plant tissues
(Kirkland and Turner, J. Biochem. 72: 716-720 (1959); Bryce and
Nelson, Plant Physiol. 63: 312-317 (1979); Dancer et al., Plant
Physiol. 92: 637-641 (1990); Yano et al., Plant Molec. Biol. 23:
1087-1090 (1993), all of which are herein incorporated by reference
in their entirety). Fructokinase can be substrate inhibited by
fructose. In addition, sucrose synthase can be inhibited by
fructose (Doehlert, Plant Sci. 52: 153-157 (1987); Morell and
Copeland, Plant Physiol. 78: 140-154 (1985), Ross and Davies, Plant
Physiol. 100: 1008-1013 (1992), all of which are herein
incorporated by reference in their entirety). Whereas plant tissues
where sucrose is catabolized by sucrose synthase predominantly
contain fructokinases (Xu et al., Plant Physiol. 90: 635-642
(1989); Kursanov et al., Soviet Plant Physiol. 37: 507-515 (1990);
Ross et al., Plant Physiol. 90: 748-756 (1994)), plant tissues
where sucrose is catabolized by invertase often contain hexokinases
(Nakamura et al., Plant Physiol. 81: 215-220 (1991)). Tissues which
have both invertase and sucrose synthase activity may contain both
hexose kinases (Nakamura et al., Plant Physiol. 81: 215-220 (1991),
the entirety of which is herein incorporated by reference). F6P
resulting from hexose kinase activity can be further metabolized in
glycolysis or used in resynthesis of sucrose by SPS. G6P resulting
from hexose kinase activity can enter the pentose phosphate
pathway, via G6P dehydrogenase (EC 1.1.1.49), or be converted to
F6P by phosphoglucoisomerase ("PGI") (EC 5.3.1.9) or GIP by
phosphoglucomutase ("PGM") (EC 5.4.2.2) (Rees, In: Encyclopedia of
Plant Physiology Vol 18, Douce and Day, eds., Springer Verlag,
Berlin, 391-417 (1985); Copeland and Turner, In: The Biochemistry
of Plants Vol. 11, Stumpf and Conn, eds., Academic Press, New York,
107-128 (1987); Foster and Smith, Planta 180: 237-244 (1993), all
of which are herein incorporated by reference in their
entirety).
[0024] PGI and PGM are reported to be ubiquitous and reversible
with commitments of G6P to either F6P or G1P resulting from fluxes
in metabolites further along each pathway, i.e., depending on the
cell needs for glycolysis (F6P) or starch biosynthesis (G1P)
(Edwards and Rees, Phytochem. 25: 2033-2039 (1986); Kursanov et
al., Soviet Plant Physiol. 37: 507-515 (1990); Tobias et al., Plant
Physiol. 99: 140-145 (1992), all of which are herein incorporated
by reference in their entirety). UDPG formed by sucrose synthase
may be utilized directly for cellulose or callose biosynthesis via
UDP-glucose dehydrogenase (EC 1.1.1.2) (Robertson et al.,
Phytochem. 39: 21-28 (1995), the entirety of which is herein
incorporated by reference), can be used for sucrose synthesis by
SPS or sucrose synthase, or for glycolysis or starch metabolism
dependent on further metabolism by UDP-glucose pyrophosphorylase
(EC 2.7.7.9). UDP-glucose phosphorylase has been reported to be a
largely reversible enzyme (Kleczkowski, Phytochem. 37: 1507-1515
(1994), the entirety of which is herein incorporated by reference).
Flux through UDP-glucose pyrophosphorylase is reported to be
influenced by metabolite levels and utilization of reaction
products further along in the pathways (Doehlert et al., Plant
Physiol. 86: 1013-1019 (1988); Huber and Akazawa, Plant Physiol.
81: 1008-1013 (1986); Zrenner et al., Planta 190: 247-252 (1993),
all of which are herein incorporated by reference in their
entirety). The reversibility of PGI, PGM and UDPGPPase has been
reported to provide for metabolic variability and networking in
metabolism, independent of which initial enzyme cleaved
sucrose.
[0025] The fate of F6P reportedly plays a role in carbohydrate
metabolism. NTP-phosphofructokinase (PFK) (EC 2.7.1.11) (Copeland
and Turner, In: The Biochemistry of Plants Vol. 11, Stumpf and
Conn, eds., Academic Press, New York, 107-128 (1987); Dennis and
Greyson, Plant Physiol. 69: 395-404 (1987); Rees, In: The
Biochemistry of Plants Vol. 14, Preiss, ed., Academic Press, San
Diego, 1-33 (1988), all of which are herein incorporated by
reference in their entirety) is reported to irreversibly convert
F6P to F16BP and is associated with glycolysis. The reverse
reaction of F16BP to F6P, associated with gluconeogenesis, is
essentially irreversible, and is catalyzed by FBPase (EC 3.1.3.11)
(Black et al., Plant Physiol. 69: 387-394 (1987). Both reactions
may be carried out in a reversible manner by a PPi-dependent
fructose-6-phosphate phosphotransferase or PPi-phosphofructokinase
(PFP; EC 2.7.1.90) (Black et al., Plant Physiol. 69: 387-394
(1987).
[0026] PPi-dependent fructose-6-phosphate phosphotransferase or
PPi-phosphofructokinase is reported to play a role in the
generation of biosynthetic intermediates (Dennis and Greyson, Plant
Physiol. 69: 395-404 (1987); Tobias et al., Plant Physiol. 99:
146-152 (1992), the entirety of which is herein incorporated by
reference) in addition to the cycling of PPi for UDPGPPase and
ultimately UDP for sucrose synthase (Huber and Akazawa, Plant
Physiol. 81: 1008-1013 (1986); Black et al., Plant Physiol. 69:
387-394 (1987); Rees, In: The Biochemistry of Plants Vol. 14,
Preiss, ed., Academic Press, San Diego, 1-33 (1988), all of which
are herein incorporated by reference in their entirety).
[0027] II. Expressed Sequence Tag Nucleic Acid Molecules
[0028] Expressed sequence tags, or ESTs are randomly sequenced
members of a cDNA library (or complementary DNA) (McCombie et al.,
Nature Genetics 1:124-130 (1992); Kurata et al., Nature Genetics
8:365-372 (1994); Okubo et al., Nature Genetics 2:173-179 (1992),
all of which references are incorporated herein in their entirety).
The randomly selected clones comprise insets that can represent a
copy of up to the full length of a mRNA transcript.
[0029] Using conventional methodologies, cDNA libraries can be
constructed from the mRNA (messenger RNA) of a given tissue or
organism using poly dT primers and reverse transcriptase
(Efstratiadis et al., Cell 7:279-3680 (1976), the entirety of which
is herein incorporated by reference; Higuchi et al., Proc. Natl.
Acad. Sci. (U.S.A.) 73:3146-3150 (1976), the entirety of which is
herein incorporated by reference; Maniatis et al., Cell 8:163-182
(1976) the entirety of which is herein incorporated by reference;
Land et al., Nucleic Acids Res. 9:2251-2266 (1981), the entirety of
which is herein incorporated by reference; Okayama et al., Mol.
Cell. Biol. 2:161-170 (1982), the entirety of which is herein
incorporated by reference; Gubler et al., Gene 25:263-269 (1983),
the entirety of which is herein incorporated by reference).
[0030] Several methods may be employed to obtain full-length cDNA
constructs. For example, terminal transferase can be used to add
homopolymeric tails of dC residues to the free 3' hydroxyl groups
(Land et al., Nucleic Acids Res. 9:2251-2266 (1981), the entirety
of which is herein incorporated by reference). This tail can then
be hybridized by a poly dG oligo which can act as a primer for the
synthesis of full length second strand cDNA. Okayama and Berg, Mol.
Cell. Biol. 2:161-170 (1982), the entirety of which is herein
incorporated by reference, report a method for obtaining full
length cDNA constructs. This method has been simplified by using
synthetic primer-adapters that have both homopolymeric tails for
priming the synthesis of the first and second strands and
restriction sites for cloning into plasmids (Coleclough et al.,
Gene 34:305-314 (1985), the entirety of which is herein
incorporated by reference) and bacteriophage vectors (Krawinkel et
al., Nucleic Acids Res. 14:1913 (1986), the entirety of which is
herein incorporated by reference; Han et al., Nucleic Acids Res.
15:6304 (1987), the entirety of which is herein incorporated by
reference).
[0031] These strategies have been coupled with additional
strategies for isolating rare mRNA populations. For example, a
typical mammalian cell contains between 10,000 and 30,000 different
mRNA sequences (Davidson, Gene Activity in Early Development, 2nd
ed., Academic Press, New York (1976), the entirety of which is
herein incorporated by reference). The number of clones required to
achieve a given probability that a low-abundance mRNA will be
present in a cDNA library is N=(ln(1-P))/(ln(1-1/n)) where N is the
number of clones required, P is the probability desired and 1/n is
the fractional proportion of the total mRNA that is represented by
a single rare mRNA (Sambrook et al., Molecular Cloning: A
Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press
(1989), the entirety of which is herein incorporated by
reference).
[0032] A method to enrich preparations of mRNA for sequences of
interest is to fractionate by size. One such method is to
fractionate by electrophoresis through an agarose gel (Pennica et
al., Nature 301:214-221 (1983), the entirety of which is herein
incorporated by reference). Another such method employs sucrose
gradient centrifugation in the presence of an agent, such as
methylmercuric hydroxide, that denatures secondary structure in RNA
(Schweinfest et al., Proc. Natl. Acad. Sci. (U.S.A.) 79:4997-5000
(1982), the entirety of which is herein incorporated by
reference).
[0033] A frequently adopted method is to construct equalized or
normalized cDNA libraries (Ko, Nucleic Acids Res. 18:5705-5711
(1990), the entirety of which is herein incorporated by reference;
Patanjali et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1943-1947
(1991), the entirety of which is herein incorporated by reference).
Typically, the cDNA population is normalized by subtractive
hybridization (Schmid et al., J. Neurochem. 48:307-312 (1987), the
entirety of which is herein incorporated by reference; Fargnoli et
al., Anal. Biochem. 187:364-373 (1990), the entirety of which is
herein incorporated by reference; Travis et al., Proc. Natl. Acad.
Sci. (U.S.A.) 85:1696-1700 (1988), the entirety of which is herein
incorporated by reference; Kato, Eur. J. Neurosci. 2:704-711
(1990); and Schweinfest et al., Genet. Anal. Tech. Appl. 7:64-70
(1990), the entirety of which is herein incorporated by reference).
Subtraction represents another method for reducing the population
of certain sequences in the cDNA library (Swaroop et al., Nucleic
Acids Res. 19:1954 (1991), the entirety of which is herein
incorporated by reference).
[0034] ESTs can be sequenced by a number of methods. Two basic
methods may be used for DNA sequencing, the chain termination
method of Sanger et al., Proc. Natl. Acad. Sci. (U.S.A.)
74:5463-5467 (1977), the entirety of which is herein incorporated
by reference and the chemical degradation method of Maxam and
Gilbert, Proc. Nat. Acad. Sci. (U.S.A.) 74:560-564 (1977), the
entirety of which is herein incorporated by reference. Automation
and advances in technology such as the replacement of radioisotopes
with fluorescence-based sequencing have reduced the effort required
to sequence DNA (Craxton, Methods 2:20-26 (1991), the entirety of
which is herein incorporated by reference; Ju et al., Proc. Natl.
Acad. Sci. (U.S.A) 92:4347-4351 (1995), the entirety of which is
herein incorporated by reference; Tabor and Richardson, Proc. Natl.
Acad. Sci. (U.S.A.) 92:6339-6343 (1995), the entirety of which is
herein incorporated by reference). Automated sequencers are
available from, for example, Pharmacia Biotech, Inc., Piscataway,
N.J. (Pharmacia ALF), LI-COR, Inc., Lincoln, Nebr. (LI-COR 4,000)
and Millipore, Bedford, Mass. (Millipore BaseStation).
[0035] In addition, advances in capillary gel electrophoresis have
also reduced the effort required to sequence DNA and such advances
provide a rapid high resolution approach for sequencing DNA samples
(Swerdlow and Gesteland, Nucleic Acids Res. 18:1415-1419 (1990);
Smith, Nature 349:812-813 (1991); Luckey et al., Methods Enzymol.
218:154-172 (1993); Lu et al., J Chromatog. A. 680:497-501 (1994);
Carson et al., Anal. Chem. 65:3219-3226 (1993); Huang et al., Anal.
Chem. 64:2149-2154 (1992); Kheterpal et al., Electrophoresis
17:1852-1859 (1996); Quesada and Zhang, Electrophoresis
17:1841-1851 (1996); Baba, Yakugaku Zasshi 117:265-281 (1997), all
of which are herein incorporated by reference in their
entirety).
[0036] ESTs longer than 150 nucleotides have been found to be
useful for similarity searches and mapping (Adams et al., Science
252:1651-1656 (1991), herein incorporated by reference). ESTs,
which can represent copies of up to the full length transcript, may
be partially or completely sequenced. Between 150-450 nucleotides
of sequence information is usually generated as this is the length
of sequence information that is routinely and reliably produced
using single run sequence data. Typically, only single run sequence
data is obtained from the cDNA library (Adams et al., Science
252:1651-1656 (1991). Automated single run sequencing typically
results in an approximately 2-3% error or base ambiguity rate
(Boguski et al., Nature Genetics 4:332-333 (1993), the entirety of
which is herein incorporated by reference).
[0037] EST databases have been constructed or partially constructed
from, for example, C. elegans (McCombrie et al., Nature Genetics
1:124-131 (1992)), human liver cell line HepG2 (Okubo et al.,
Nature Genetics 2:173-179 (1992)), human brain RNA (Adams et al.,
Science 252:1651-1656 (1991); Adams et al., Nature 355:632-635
(1992)), Arabidopsis, (Newman et al., Plant Physiol. 106:1241-1255
(1994)); and rice (Kurata et al., Nature Genetics 8:365-372
(1994)).
[0038] III. Sequence Comparisons
[0039] A characteristic feature of a DNA sequence is that it can be
compared with other DNA sequences. Sequence comparisons can be
undertaken by determining the similarity of the test or query
sequence with sequences in publicly available or proprietary
databases ("similarity analysis") or by searching for certain
motifs ("intrinsic sequence analysis") (e.g. cis elements)
(Coulson, Trends in Biotechnology 12:76-80 (1994), the entirety of
which is herein incorporated by reference); Birren et al., Genome
Analysis 1: Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. 543-559 (1997), the entirety of which is herein
incorporated by reference).
[0040] Similarity analysis includes database search and alignment.
Examples of public databases include the DNA Database of Japan
(DDBJ) (available on the worldwide web at ddbj.nig.acjp); Genebank
(available on the worldwide web at the ncbi website at:
/Web/Search/Index.html); and the European Molecular Biology
Laboratory Nucleic Acid Sequence Database (EMBL) (available on the
worldwide web at ebi.ac.uldebi_docs/emb1_db/embl-db.html). Other
appropriate databases include dbEST (available on the worldwide web
at the ncbi website at: /dbEST/index.html), SwisProt (available on
the worldwide web at
ebi.ac.ulc/ebi_docs/swisprot_db/swisshome.html), PIR (available on
the worldwide web at nbrt.georgetown.edu/pir), and The Institute
for Genome Research (available on the worldwide web at
tigr.org/tdb/tdb.html).
[0041] A number of different search algorithms have been developed,
one example of which are the suite of programs referred to as BLAST
programs. There are five implementations of BLAST, three designed
for nucleotide sequences queries (BLASTN, BLASTX and TBLASTX) and
two designed for protein sequence queries (BLASTP and TBLASTN)
(Coulson, Trends in Biotechnology 12:76-80 (1994); Birren et al.,
Genome Analysis 1, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y. 543-559 (1997)).
[0042] BLASTN takes a nucleotide sequence (the query sequence) and
its reverse complement and searches them against a nucleotide
sequence database. BLASTN was designed for speed, not maximum
sensitivity and may not find distantly related coding sequences.
BLASTX takes a nucleotide sequence, translates it in three forward
reading frames and three reverse complement reading frames and then
compares the six translations against a protein sequence database.
BLASTX is useful for sensitive analysis of preliminary
(single-pass) sequence data and is tolerant of sequencing errors
(Gish and States, Nature Genetics 3:266-272 (1993), the entirety of
which is herein incorporated by reference). BLASTN and BLASTX may
be used in concert for analyzing EST data (Coulson, Trends in
Biotechnology 12:76-80 (1994); Birren et al., Genome Analysis
1:543-559 (1997)).
[0043] Given a coding nucleotide sequence and the protein it
encodes, it is often preferable to use the protein as the query
sequence to search a database because of the greatly increased
sensitivity to detect more subtle relationships. This is due to the
larger alphabet of proteins (20 amino acids) compared with the
alphabet of nucleic acid sequences (4 bases), where it is far
easier to obtain a match by chance. In addition, with nucleotide
alignments, only a match (positive score) or a mismatch (negative
score) is obtained, but with proteins, the presence of conservative
amino acid substitutions can be taken into account. Here, a
mismatch may yield a positive score if the non-identical residue
has physical/chemical properties similar to the one it replaced.
Various scoring matrices are used to supply the substitution scores
of all possible amino acid pairs. A general purpose scoring system
is the BLOSUM62 matrix (Henikoff and Henikoff, Proteins 17:49-61
(1993), the entirety of which is herein incorporated by reference),
which is currently the default choice for BLAST programs. BLOSUM62
is tailored for alignments of moderately diverged sequences and
thus may not yield the best results under all conditions. Altschul,
J. Mol. Biol. 36:290-300 (1993), the entirety of which is herein
incorporated by reference, describes a combination of three
matrices to cover all contingencies. This may improve sensitivity,
but at the expense of slower searches. In practice, a single
BLOSUM62 matrix is often used but others (PAM40 and PAM250) may be
attempted when additional analysis is necessary. Low PAM matrices
are directed at detecting very strong but localized sequence
similarities, whereas high PAM matrices are directed at detecting
long but weak alignments between very distantly related
sequences.
[0044] Homologues in other organisms are available that can be used
for comparative sequence analysis. Multiple alignments are
performed to study similarities and differences in a group of
related sequences. CLUSTAL W is a multiple sequence alignment
package that performs progressive multiple sequence alignments
based on the method of Feng and Doolittle, J. Mol. Evol. 25:351-360
(1987), the entirety of which is herein incorporated by reference.
Each pair of sequences is aligned and the distance between each
pair is calculated; from this distance matrix, a guide tree is
calculated and all of the sequences are progressively aligned based
on this tree. A feature of the program is its sensitivity to the
effect of gaps on the alignment; gap penalties are varied to
encourage the insertion of gaps in probable loop regions instead of
in the middle of structured regions. Users can specify gap
penalties, choose between a number of scoring matrices, or supply
their own scoring matrix for both pairwise alignments and multiple
alignments. CLUSTAL W for UNIX and VMS systems is available by
anonymous ftp at: ftp,e13.i,ae,ak ebi.ac.uk. Another program is
MACAW (Schuler et al., Proteins Struct. Func. Genet. 9:180-190
(1991), the entirety of which is herein incorporated by reference,
for which both Macintosh and Microsoft Windows versions are
available. MACAW uses a graphical interface, provides a choice of
several alignment algorithms and is available by anonymous ftp at
the ncbi website at: ncbi.nlm.nih.gov nlm.nih.gov
(directory/pub/macaw).
[0045] Sequence motifs are derived from multiple alignments and can
be used to examine individual sequences or an entire database for
subtle patterns. With motifs, it is sometimes possible to detect
distant relationships that may not be demonstrable based on
comparisons of primary sequences alone. Currently, the largest
collection of sequence motifs in the world is PROSITE (Bairoch and
Bucher, Nucleic Acid Research 22:3583-3589 (1994), the entirety of
which is herein incorporated by reference). PROSITE may be accessed
via either the ExPASy server on the World Wide Web or anonymous ftp
site. Many commercial sequence analysis packages also provide
search programs that use PROSITE data.
[0046] A resource for searching protein motifs is the BLOCKS E-mail
server developed by Henikoff, Trends Biochem Sci. 18:267-268
(1993), the entirety of which is herein incorporated by reference;
Henikoff and Henikoff, Nucleic Acid Research 19:6565-6572 (1991),
the entirety of which is herein incorporated by reference; Henikoff
and Henikoff, Proteins 17:49-61 (1993). BLOCKS searches a protein
or nucleotide sequence against a database of protein motifs or
"blocks." Blocks are defined as short, ungapped multiple alignments
that represent highly conserved protein patterns. The blocks
themselves are derived from entries in PROSITE as well as other
sources. Either a protein query or a nucleotide query can be
submitted to the BLOCKS server; if a nucleotide sequence is
submitted, the sequence is translated in all six reading frames and
motifs are sought for these conceptual translations. Once the
search is completed, the server will return a ranked list of
significant matches, along with an alignment of the query sequence
to the matched BLOCKS entries.
[0047] Conserved protein domains can be represented by
two-dimensional matrices, which measure either the frequency or
probability of the occurrences of each amino acid residue and
deletions or insertions in each position of the domain. This type
of model, when used to search against protein databases, is
sensitive and usually yields more accurate results than simple
motif searches. Two popular implementations of this approach are
profile searches such as GCG program ProfileSearch and Hidden
Markov Models (HMMs) (Krough et al., J. Mol. Biol. 235:1501-1531,
(1994); Eddy, Current Opinion in Structural Biology 6:361-365,
(1996), both of which are herein incorporated by reference in their
entirety). In both cases, a large number of common protein domains
have been converted into profiles, as present in the PROSITE
library, or HHM models, as in the Pfam protein domain library
(Sonnhammer et al., Proteins 28:405-420 (1997), the entirety of
which is herein incorporated by reference). Pfam contains more than
500 HMM models for enzymes, transcription factors, signal
transduction molecules and structural proteins. Protein databases
can be queried with these profiles or HMM models, which will
identify proteins containing the domain of interest. For example,
HMMSW or HMMFS, two programs in a public domain package called
HMMER (Sonnhammer et al., Proteins 28:405-420 (1997)) can be
used.
[0048] PROSITE and BLOCKS represent collected families of protein
motifs. Thus, searching these databases entails submitting a single
sequence to determine whether or not that sequence is similar to
the members of an established family. Programs working in the
opposite direction compare a collection of sequences with
individual entries in the protein databases. An example of such a
program is the Motif Search Tool, or MoST (Tatusov et al., Proc.
Natl. Acad. Sci. (U.S.A.) 91:12091-12095 (1994), the entirety of
which is herein incorporated by reference). On the basis of an
aligned set of input sequences, a weight matrix is calculated by
using one of four methods (selected by the user). A weight matrix
is simply a representation, position by position of how likely a
particular amino acid will appear. The calculated weight matrix is
then used to search the databases. To increase sensitivity, newly
found sequences are added to the original data set, the weight
matrix is recalculated and the search is performed again. This
procedure continues until no new sequences are found.
SUMMARY OF THE INVENTION
[0049] The present invention provides a substantially purified
nucleic acid molecule that encodes a maize or a soybean enzyme or
fragment thereof, wherein the maize or the soybean enzyme is
selected from the group consisting of: (a) triose phosphate
isomerase; (b) fructose 1,6-bisphosphate aldolase; (c) fructose
1,6-bisphosphate; (d) fructose 6-phosphate 2-kinase; (e)
phosphoglucoisomerase; (f) vacuolar H.sup.+
translocating-pyrophosphatase; (g) pyrophosphate-dependent
fructose-6-phosphate phosphotransferase; (h) invertase; (i) sucrose
synthase; (j) hexokinase; (k) fructokinase; (l) NDP-kinase; (m)
glucose-6-phosphate 1-dehydrogenase; (n) phosphoglucomutase and (o)
UDP-glucose pyrophosphorylase.
[0050] The present invention also provides a substantially purified
nucleic acid molecule that encodes a plant sucrose pathway enzyme
or fragment thereof, wherein the nucleic acid molecule is selected
from the group consisting of a nucleic acid molecule that encodes a
maize or a soybean triose phosphate isomerase enzyme or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
fructose 1,6-bisphosphate aldolase enzyme or fragment thereof, a
nucleic acid molecule that encodes a maize or a soybean fructose
1,6-bisphosphate enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean fructose 6-phosphate
2-kinase enzyme or fragment thereof, a nucleic acid molecule that
encodes a maize or a soybean phosphoglucoisomerase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean vacuolar H.sup.+ translocating-pyrophosphatase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean invertase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean sucrose synthase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean hexokinase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean fructokinase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean NDP-kinase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean glucose-6-phosphate 1-dehydrogenase enzyme or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
phosphoglucomutase enzyme or fragment thereof and a nucleic acid
molecule that encodes a maize or a soybean UDP-glucose
pyrophosphorylase enzyme or fragment thereof.
[0051] The present invention also provides a substantially purified
maize or soybean enzyme or fragment thereof, wherein the maize or
soybean enzyme is selected from the group consisting of (a) triose
phosphate isomerase; (b) fructose 1,6-bisphosphate aldolase; (c)
fructose 1,6-bisphosphate; (d) fructose 6-phosphate 2-kinase; (e)
phosphoglucoisomerase; (f) vacuolar H+
translocating-pyrophosphatase; (g) pyrophosphate-dependent
fructose-6-phosphate phosphotransferase; (h) invertase; (i) sucrose
synthase; (j) hexokinase; (k) fructokinase; (l) NDP-kinase; (m)
glucose-6-phosphate 1-dehydrogenase; (n) phosphoglucomutase and (o)
UDP-glucose pyrophosphorylase.
[0052] The present invention also provides a substantially purified
maize or soybean sucrose pathway protein or fragment thereof
encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 1 through SEQ ID NO:
2814.
[0053] The present invention also provides a substantially purified
maize or soybean triose phosphate isomerase enzyme or fragment
thereof encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 1 through SEQ ID NO:
206 and SEQ ID NO: 1538 through SEQ ID NO: 1707.
[0054] The present invention also provides a substantially purified
maize or soybean triose phosphate isomerase enzyme or fragment
thereof encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1 through SEQ ID NO: 206 and SEQ ID NO:
1538 through SEQ ID NO: 1707.
[0055] The present invention also provides a substantially purified
maize or soybean fructose 1,6-bisphosphate aldolase enzyme or
fragment thereof encoded by a first nucleic acid molecule which
specifically hybridizes to a second nucleic acid molecule, the
second nucleic acid molecule having a nucleic acid sequence
selected from the group consisting of a complement of SEQ ID NO:
207 through SEQ ID NO: 232 and SEQ ID NO: 1708 through SEQ ID NO:
2113.
[0056] The present invention also provides a substantially purified
maize or soybean fructose 1,6-bisphosphate aldolase enzyme or
fragment thereof encoded by a nucleic acid sequence selected from
the group consisting SEQ ID NO: 207 through SEQ ID NO: 232 and SEQ
ID NO: 1708 through SEQ ID NO: 2113.
[0057] The present invention also provides a substantially purified
maize or soybean fructose 1,6-bisphosphate enzyme or fragment
thereof encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 233 through SEQ ID
NO: 258 and SEQ ID NO: 2114 through SEQ ID NO: 2162.
[0058] The present invention also provides a substantially purified
maize or soybean fructose 1,6-bisphosphate e enzyme or fragment
thereof encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 233 through SEQ ID NO: 258 and SEQ ID NO:
2114 through SEQ ID NO: 2162.
[0059] The present invention also provides a substantially purified
maize or soybean fructose 6-phosphate 2-kinase enzyme or fragment
thereof encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 259 through SEQ ID
NO: 275 and SEQ ID NO: 2163 through SEQ ID NO: 2166.
[0060] The present invention also provides a substantially purified
maize or soybean fructose 6-phosphate 2-kinase enzyme or fragment
thereof encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 259 through SEQ ID NO: 275 and SEQ ID NO:
2163 through SEQ ID NO: 2166.
[0061] The present invention also provides a substantially purified
maize or soybean phosphoglucoisomerase enzyme or fragment thereof
encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 276 through SEQ ID
NO: 340 and SEQ ID NO: 2167 through SEQ ID NO: 2182.
[0062] The present invention also provides a substantially purified
maize or soybean phosphoglucoisomerase enzyme or fragment thereof
encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 276 through SEQ ID NO: 340 and SEQ ID NO:
2167 through SEQ ID NO: 2182.
[0063] The present invention also provides a substantially purified
maize or soybean vacuolar H.sup.+ translocating-pyrophosphatase
enzyme or fragment thereof encoded by a first nucleic acid molecule
which specifically hybridizes to a second nucleic acid molecule,
the second nucleic acid molecule having a nucleic acid sequence
selected from the group consisting of a complement of SEQ ID NO:
341 through SEQ ID NO: 497 and SEQ ID NO: 2183 through SEQ ID NO:
2241.
[0064] The present invention also provides a substantially purified
maize or soybean vacuolar H.sup.+ translocating-pyrophosphatase
enzyme or fragment thereof encoded by a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 341 through SEQ ID
NO: 497 and SEQ ID NO: 2183 through SEQ ID NO: 2241.
[0065] The present invention also provides a substantially purified
maize or soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or fragment thereof encoded by a first
nucleic acid molecule which specifically hybridizes to a second
nucleic acid molecule, the second nucleic acid molecule having a
nucleic acid sequence selected from the group consisting of a
complement of SEQ ID NO: 498 through SEQ ID NO: 507 and SEQ ID NO:
2442.
[0066] The present invention also provides a substantially purified
maize or soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or fragment thereof encoded by a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 498
through SEQ ID NO: 507 and SEQ ID NO: 2442.
[0067] The present invention also provides a substantially purified
maize or soybean invertase enzyme or fragment thereof encoded by a
first nucleic acid molecule which specifically hybridizes to a
second nucleic acid molecule, the second nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of a complement of SEQ ID NO: 508 through SEQ ID NO: 510 and SEQ ID
NO: 2243 through SEQ ID NO: 2254.
[0068] The present invention also provides a substantially purified
maize or soybean invertase enzyme or fragment thereof encoded by a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 508 through SEQ ID NO: 510 and SEQ ID NO: 2243 through SEQ ID
NO: 2254.
[0069] The present invention also provides a substantially purified
maize or soybean sucrose synthase enzyme or fragment thereof
encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 511 through SEQ ID
NO: 1086 and SEQ ID NO: 2255 through SEQ ID NO: 2590.
[0070] The present invention also provides a substantially purified
maize or soybean sucrose synthase enzyme or fragment thereof
encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 511 through SEQ ID NO: 1086 and SEQ ID NO:
2255 through SEQ ID NO: 2590.
[0071] The present invention also provides a substantially purified
maize or soybean hexokinase enzyme or fragment thereof encoded by a
first nucleic acid molecule which specifically hybridizes to a
second nucleic acid molecule, the second nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of a complement of SEQ ID NO: 1087 through SEQ ID NO: 1135 and SEQ
ID NO: 2591 through SEQ ID NO: 2634.
[0072] The present invention also provides a substantially purified
maize or soybean hexokinase enzyme or fragment thereof encoded by a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 1087 through SEQ ID NO: 1135 and SEQ ID NO: 2591 through SEQ ID
NO: 2634.
[0073] The present invention also provides a substantially purified
maize or soybean fructokinase enzyme or fragment thereof encoded by
a first nucleic acid molecule which specifically hybridizes to a
second nucleic acid molecule, the second nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of a complement of SEQ ID NO: 1136 through SEQ ID NO: 1215 and SEQ
ID NO: 2635 through SEQ ID NO: 2678.
[0074] The present invention also provides a substantially purified
maize or soybean fructokinase enzyme or fragment thereof encoded by
a nucleic acid sequence selected from the group consisting of SEQ
ID NO: 1136 through SEQ ID NO: 1215 and SEQ ID NO: 2635 through SEQ
ID NO: 2678.
[0075] The present invention also provides a substantially purified
maize or soybean NDP-kinase enzyme or fragment thereof encoded by a
first nucleic acid molecule which specifically hybridizes to a
second nucleic acid molecule, the second nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of a complement of SEQ ID NO: 1216 through SEQ ID NO: 1251 and SEQ
ID NO: 2679 through SEQ ID NO: 2681.
[0076] The present invention also provides a substantially purified
maize or soybean NDP-kinase e enzyme or fragment thereof encoded by
a nucleic acid sequence selected from the group consisting of SEQ
ID NO: 1216 through SEQ ID NO: 1251 and SEQ ID NO: 2679 through SEQ
ID NO: 2681.
[0077] The present invention also provides a substantially purified
maize or soybean glucose-6-phosphate 1-dehydrogenase enzyme or
fragment thereof encoded by a first nucleic acid molecule which
specifically hybridizes to a second nucleic acid molecule, the
second nucleic acid molecule having a nucleic acid sequence
selected from the group consisting of a complement of SEQ ID NO:
1252 through SEQ ID NO: 1254 and SEQ ID NO: 2682 through SEQ ID NO:
2689.
[0078] The present invention also provides a substantially purified
maize or soybean glucose-6-phosphate 1-dehydrogenase enzyme or
fragment thereof encoded by a nucleic acid sequence selected from
the group consisting of SEQ ID NO: 1252 through SEQ ID NO: 1254 and
SEQ ID NO: 2682 through SEQ ID NO: 2689.
[0079] The present invention also provides a substantially purified
maize or soybean phosphoglucomutase enzyme or fragment thereof
encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 1255 through SEQ ID
NO: 1360 and SEQ ID NO: 2690 through SEQ ID NO: 2740.
[0080] The present invention also provides a substantially purified
maize or soybean phosphoglucomutase enzyme or fragment thereof
encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1255 through SEQ ID NO: 1360 and SEQ ID
NO: 2690 through SEQ ID NO: 2740.
[0081] The present invention also provides a substantially purified
maize or soybean UDP-glucose pyrophosphorylase enzyme or fragment
thereof encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 1361 through SEQ ID
NO: 1537 and SEQ ID NO: 2741 through SEQ ID NO: 2814.
[0082] The present invention also provides a substantially purified
maize or soybean UDP-glucose pyrophosphorylase enzyme or fragment
thereof encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1361 through SEQ ID NO: 1537 and SEQ ID
NO: 2741 through SEQ ID NO: 2814.
[0083] The present invention also provides a purified antibody or
fragment thereof which is capable of specifically binding to a
maize or soybean enzyme or fragment thereof, wherein the maize or
soybean enzyme or fragment thereof is encoded by a nucleic acid
molecule comprising a nucleic acid sequence selected from the group
consisting of consisting of SEQ ID NO: 1 through SEQ ID NO:
2814.
[0084] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean triose
phosphate isomerase enzyme or fragment thereof encoded by a first
nucleic acid molecule which specifically hybridizes to a second
nucleic acid molecule, the second nucleic acid molecule having a
nucleic acid sequence selected from the group consisting of a
complement of SEQ ID NO: 1 through SEQ ID NO: 206 and SEQ ID NO:
1538 through SEQ ID NO: 1707 and a maize or soybean triose
phosphate isomerase enzyme or fragment thereof encoded by a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 1
through SEQ ID NO: 206 and SEQ ID NO: 1538 through SEQ ID NO:
1707.
[0085] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean fructose
1,6-bisphosphate aldolase enzyme or fragment thereof encoded by a
first nucleic acid molecule which specifically hybridizes to a
second nucleic acid molecule, the second nucleic acid molecule
having a nucleic acid sequence selected from the group consisting
of a complement of SEQ ID NO: 207 through SEQ ID NO: 232 and SEQ ID
NO: 1708 through SEQ ID NO: 2113 and a maize or soybean fructose
1,6-bisphosphate aldolase enzyme or fragment thereof encoded by a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 207 through SEQ ID NO: 232 and SEQ ID NO: 1708 through SEQ ID
NO: 2113.
[0086] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean fructose
1,6-bisphosphate enzyme or fragment thereof encoded by a first
nucleic acid molecule which specifically hybridizes to a second
nucleic acid molecule, the second nucleic acid molecule having a
nucleic acid sequence selected from the group consisting of a
complement of SEQ ID NO: 233 through SEQ ID NO: 258 and SEQ ID NO:
2114 through SEQ ID NO: 2162 and a maize or soybean fructose
1,6-bisphosphate enzyme or fragment thereof encoded by a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 233
through SEQ ID NO: 258 and SEQ ID NO: 2114 through SEQ ID NO:
2162.
[0087] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean fructose
6-phosphate 2-kinase enzyme or fragment thereof encoded by a first
nucleic acid molecule which specifically hybridizes to a second
nucleic acid molecule, the second nucleic acid molecule having a
nucleic acid sequence selected from the group consisting of a
complement of SEQ ID SEQ ID NO: 259 through SEQ ID NO: 275 and SEQ
ID NO: 2163 through SEQ ID NO: 2166 and a maize or soybean fructose
6-phosphate 2-kinase enzyme or fragment thereof encoded by a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 259 through SEQ ID NO: 275 and SEQ ID NO: 2163 through SEQ ID
NO: 2166.
[0088] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean
phosphoglucoisomerase enzyme or fragment thereof encoded by a first
nucleic acid molecule which specifically hybridizes to a second
nucleic acid molecule, the second nucleic acid molecule having a
nucleic acid sequence selected from the group consisting of a
complement of SEQ ID NO: 276 through SEQ ID NO: 340 and SEQ ID NO:
2167 through SEQ ID NO: 2182 and a maize or soybean
phosphoglucoisomerase enzyme or fragment thereof encoded by a
nucleic acid sequence selected from the group consisting of SEQ ID
NO: 276 through SEQ ID NO: 340 and SEQ ID NO: 2167 through SEQ ID
NO: 2182.
[0089] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean vacuolar
H.sup.+ translocating-pyrophosphatase enzyme or fragment thereof
encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 341 through SEQ ID
NO: 497 and SEQ ID NO: 2183 through SEQ ID NO: 2241 and a maize or
soybean vacuolar H.sup.+ translocating-pyrophosphatase enzyme or
fragment thereof encoded by a nucleic acid sequence selected from
the group consisting of SEQ ID NO: 341 through SEQ ID NO: 497 and
SEQ ID NO: 2183 through SEQ ID NO: 2241.
[0090] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean
pyrophosphate-dependent fructose-6-phosphate phosphotransferase
enzyme or fragment thereof encoded by a first nucleic acid molecule
which specifically hybridizes to a second nucleic acid molecule,
the second nucleic acid molecule having a nucleic acid sequence
selected from the group consisting of a complement of SEQ ID NO:
498 through SEQ ID NO: 507 and SEQ ID NO: 2442 and a maize or
soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or fragment thereof encoded by a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 498
through SEQ ID NO: 507 and SEQ ID NO: 2442.
[0091] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean invertase
enzyme or fragment thereof encoded by a first nucleic acid molecule
which specifically hybridizes to a second nucleic acid molecule,
the second nucleic acid molecule having a nucleic acid sequence
selected from the group consisting of a complement of SEQ ID NO:
508 through SEQ ID NO: 510 and SEQ ID NO: 2243 through SEQ ID NO:
2254 and a maize or soybean invertase enzyme or fragment thereof
encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 508 through SEQ ID NO: 510 and SEQ ID NO:
2243 through SEQ ID NO: 2254.
[0092] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean sucrose
synthase enzyme or fragment thereof encoded by a first nucleic acid
molecule which specifically hybridizes to a second nucleic acid
molecule, the second nucleic acid molecule having a nucleic acid
sequence selected from the group consisting of a complement of SEQ
ID NO: 511 through SEQ ID NO: 1086 and SEQ ID NO: 2255 through SEQ
ID NO: 2590 and a maize or soybean sucrose synthase enzyme or
fragment thereof encoded by a nucleic acid sequence selected from
the group consisting of SEQ ID NO: 511 through SEQ ID NO: 1086 and
SEQ ID NO: 2255 through SEQ ID NO: 2590.
[0093] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean hexokinase
enzyme or fragment thereof encoded by a first nucleic acid molecule
which specifically hybridizes to a second nucleic acid molecule,
the second nucleic acid molecule having a nucleic acid sequence
selected from the group consisting of a complement of SEQ ID NO:
1087 through SEQ ID NO: 1135 and SEQ ID NO: 2591 through SEQ ID NO:
2634 and a maize or soybean hexokinase enzyme or fragment thereof
encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1087 through SEQ ID NO: 1135 and SEQ ID
NO: 2591 through SEQ ID NO: 2634.
[0094] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean
fructokinase enzyme or fragment thereof encoded by a first nucleic
acid molecule which specifically hybridizes to a second nucleic
acid molecule, the second nucleic acid molecule having a nucleic
acid sequence selected from the group consisting of a complement of
SEQ ID NO: 1136 through SEQ ID NO: 1215 and SEQ ID NO: 2635 through
SEQ ID NO: 2678 and a maize or soybean fructokinase enzyme or
fragment thereof encoded by a nucleic acid sequence selected from
the group consisting of SEQ ID NO: 1136 through SEQ ID NO: 1215 and
SEQ ID NO: 2635 through SEQ ID NO: 2678.
[0095] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean NDP-kinase
enzyme or fragment thereof encoded by a first nucleic acid molecule
which specifically hybridizes to a second nucleic acid molecule,
the second nucleic acid molecule having a nucleic acid sequence
selected from the group consisting of a complement of SEQ ID NO:
1216 through SEQ ID NO: 1251 and SEQ ID NO: 2679 through SEQ ID NO:
2681 and a maize or soybean NDP-kinase enzyme or fragment thereof
encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1216 through SEQ ID NO: 1251 and SEQ ID
NO: 2679 through SEQ ID NO: 2681.
[0096] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean
glucose-6-phosphate 1-dehydrogenase enzyme or fragment thereof
encoded by a first nucleic acid molecule which specifically
hybridizes to a second nucleic acid molecule, the second nucleic
acid molecule having a nucleic acid sequence selected from the
group consisting of a complement of SEQ ID NO: 1252 through SEQ ID
NO: 1254 and SEQ ID NO: 2682 through SEQ ID NO: 2689 and a maize or
soybean glucose-6-phosphate 1-dehydrogenase enzyme or fragment
thereof encoded by a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1252 through SEQ ID NO: 1254 and SEQ ID
NO: 2682 through SEQ ID NO: 2689.
[0097] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean
phosphoglucomutase enzyme or fragment thereof encoded by a first
nucleic acid molecule which specifically hybridizes to a second
nucleic acid molecule, the second nucleic acid molecule having a
nucleic acid sequence selected from the group consisting of a
complement of SEQ ID NO: 1255 through SEQ ID NO: 1360 and SEQ ID
NO: 2690 through SEQ ID NO: 2740 and a maize or soybean
phosphoglucomutase enzyme or fragment thereof encoded by a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 1255
through SEQ ID NO: 1360 and SEQ ID NO: 2690 through SEQ ID NO:
2740.
[0098] The present invention also provides a substantially purified
antibody or fragment thereof, the antibody or fragment thereof
capable of specifically binding to a maize or a soybean UDP-glucose
pyrophosphorylase enzyme or fragment thereof encoded by a first
nucleic acid molecule which specifically hybridizes to a second
nucleic acid molecule, the second nucleic acid molecule having a
nucleic acid sequence selected from the group consisting of a
complement of SEQ ID NO: 1361 through SEQ ID NO: 1537 and SEQ ID
NO: 2741 through SEQ ID NO: 2814 and a maize or soybean UDP-glucose
pyrophosphorylase enzyme or fragment thereof encoded by a nucleic
acid sequence selected from the group consisting of SEQ ID NO: 1361
through SEQ ID NO: 1537 and SEQ ID NO: 2741 through SEQ ID NO:
2814.
[0099] The present invention also provides a transformed plant
having a nucleic acid molecule which comprises: (A) an exogenous
promoter region which functions in a plant cell to cause the
production of a mRNA molecule; (B) a structural nucleic acid
molecule comprising a nucleic acid sequence selected from the group
consisting of (a) a nucleic acid sequence which encodes for triose
phosphate isomerase or fragment thereof; (b) a nucleic acid
sequence which encodes for fructose 1,6-bisphosphate aldolase or
fragment thereof; (c) a nucleic acid sequence which encodes for
fructose 1,6-bisphosphate or fragment thereof; (d) a nucleic acid
sequence which encodes for fructose 6-phosphate 2-kinase or
fragment thereof; (e) a nucleic acid sequence which encodes for
phosphoglucoisomerase or fragment thereof; (f) a nucleic acid
sequence which encodes for vacuolar H.sup.+
translocating-pyrophosphatase or fragment thereof; (g) a nucleic
acid sequence which encodes for pyrophosphate-dependent
fructose-6-phosphate phosphotransferase or fragment thereof; (h) a
nucleic acid sequence which encodes for invertase or fragment
thereof; (i) a nucleic acid sequence which encodes for sucrose
synthase or fragment thereof; (j) a nucleic acid sequence which
encodes for hexokinase or fragment thereof; (k) a nucleic acid
sequence which encodes for fructokinase or fragment thereof; (l) a
nucleic acid sequence which encodes for NDP-kinase or fragment
thereof; (m) a nucleic acid sequence which encodes for
glucose-6-phosphate 1-dehydrogenase or fragment thereof; (n) a
nucleic acid sequence which encodes for phosphoglucomutase or
fragment thereof (o) a nucleic acid sequence which encodes for
UDP-glucose pyrophosphorylase or fragment thereof and (p) a nucleic
acid sequence which is complementary to any of the nucleic acid
sequences of (a) through (o); and (C) a 3' non-translated sequence
that functions in the plant cell to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of the mRNA molecule.
[0100] The present invention also provides a transformed plant
having a nucleic acid molecule which comprises: (A) an exogenous
promoter region which functions in a plant cell to cause the
production of a mRNA molecule; which is linked to (B) a structural
nucleic acid molecule, wherein the structural nucleic acid molecule
encodes a plant sucrose pathway enzyme or fragment thereof, the
structural nucleic acid molecule comprising a nucleic acid sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 2814 or fragment thereof; which is linked to (C) a 3'
non-translated sequence that functions in the plant cell to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA molecule.
[0101] The present invention also provides a transformed plant
having a nucleic acid molecule which comprises: (A) an exogenous
promoter region which functions in a plant cell to cause the
production of a mRNA molecule; which is linked to (B) a structural
nucleic acid molecule, wherein the structural nucleic acid molecule
is selected from the group consisting of a nucleic acid molecule
that encodes a maize or a soybean triose phosphate isomerase enzyme
or fragment thereof, a nucleic acid molecule that encodes a maize
or a soybean fructose 1,6-bisphosphate aldolase enzyme or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
fructose 1,6-bisphosphate enzyme or fragment thereof, a nucleic
acid molecule that encodes a maize or a soybean fructose
6-phosphate 2-kinase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean phosphoglucoisomerase
enzyme or fragment thereof, a nucleic acid molecule that encodes a
maize or a soybean vacuolar H.sup.+ translocating-pyrophosphatase
enzyme or fragment thereof, a nucleic acid molecule that encodes a
maize or a soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean invertase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean sucrose synthase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean hexokinase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean fructokinase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean NDP-kinase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean glucose-6-phosphate 1-dehydrogenase enzyme or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
phosphoglucomutase enzyme or fragment thereof and a nucleic acid
molecule that encodes a maize or a soybean UDP-glucose
pyrophosphorylase enzyme or fragment thereof; which is linked to
(C) a 3' non-translated sequence that functions in the plant cell
to cause termination of transcription and addition of
polyadenylated ribonucleotides to a 3' end of the mRNA
molecule.
[0102] The present invention also provides a transformed plant
having a nucleic acid molecule which comprises: (A) an exogenous
promoter region which functions in a plant cell to cause the
production of a mRNA molecule; which is linked to (B) a transcribed
nucleic acid molecule with a transcribed strand and a
non-transcribed strand, wherein the transcribed strand is
complementary to a nucleic acid molecule comprising a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 2814 or fragment thereof; which is linked to (C) a 3'
non-translated sequence that functions in plant cells to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of the mRNA molecule.
[0103] The present invention also provides a transformed plant
having a nucleic acid molecule which comprises: (A) an exogenous
promoter region which functions in a plant cell to cause the
production of a mRNA molecule; which is linked to: (B) a
transcribed nucleic acid molecule with a transcribed strand and a
non-transcribed strand, wherein a transcribed mRNA of the
transcribed strand is complementary to an endogenous mRNA molecule
having a nucleic acid sequence selected from the group consisting
of an endogenous mRNA molecule that encodes a maize or a soybean
triose phosphate isomerase enzyme or fragment thereof, an
endogenous mRNA molecule that encodes a maize or a soybean fructose
1,6-bisphosphate aldolase enzyme or fragment thereof, an endogenous
mRNA molecule that encodes a maize or a soybean fructose
1,6-bisphosphate enzyme or fragment thereof, an endogenous mRNA
molecule that encodes a maize or a soybean fructose 6-phosphate
2-kinase enzyme or fragment thereof, an endogenous mRNA molecule
that encodes a maize or a soybean phosphoglucoisomerase enzyme or
fragment thereof, an endogenous mRNA molecule that encodes a maize
or a soybean vacuolar H.sup.+ translocating-pyrophosphatase enzyme
or fragment thereof, an endogenous mRNA molecule that encodes a
maize or a soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or fragment thereof, an endogenous mRNA
molecule that encodes a maize or a soybean invertase enzyme or
fragment thereof, an endogenous mRNA molecule that encodes a maize
or a soybean sucrose synthase enzyme or fragment thereof, an
endogenous mRNA molecule that encodes a maize or a soybean
hexokinase enzyme or fragment thereof, an endogenous mRNA molecule
that encodes a maize or a soybean fructokinase enzyme or fragment
thereof, an endogenous mRNA molecule that encodes a maize or a
soybean NDP-kinase enzyme or fragment thereof, an endogenous mRNA
molecule that encodes a maize or a soybean glucose-6-phosphate
1-dehydrogenase enzyme or fragment thereof, an endogenous mRNA
molecule that encodes a maize or a soybean phosphoglucomutase
enzyme or fragment thereof and an endogenous mRNA molecule that
encodes a maize or a soybean UDP-glucose pyrophosphorylase enzyme
or fragment thereof; which is linked to (C) a 3' non-translated
sequence that functions in the plant cell to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of the mRNA molecule.
[0104] The present invention also provides a method for determining
a level or pattern in a plant cell of an enzyme in a plant
metabolic pathway comprising: (A) incubating, under conditions
permitting nucleic acid hybridization, a marker nucleic acid
molecule, the marker nucleic acid molecule selected from the group
of marker nucleic acid molecules which specifically hybridize to a
nucleic acid molecule having the nucleic acid sequence of SEQ ID
NO: 1 through SEQ ID NO: 2814 or compliments thereof, with a
complementary nucleic acid molecule obtained from the plant cell or
plant tissue, wherein nucleic acid hybridization between the marker
nucleic acid molecule and the complementary nucleic acid molecule
obtained from the plant cell or plant tissue permits the detection
of an mRNA for the enzyme; (B) permitting hybridization between the
marker nucleic acid molecule and the complementary nucleic acid
molecule obtained from the plant cell or plant tissue; and (C)
detecting the level or pattern of the complementary nucleic acid,
wherein the detection of the complementary nucleic acid is
predictive of the level or pattern of the enzyme in the plant
metabolic pathway.
[0105] The present invention also provides a method for determining
a level or pattern of a plant sucrose pathway enzyme in a plant
cell or plant tissue comprising: (A) incubating, under conditions
permitting nucleic acid hybridization, a marker nucleic acid
molecule, the marker nucleic acid molecule having a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 2814 or complements thereof or fragment of either, with
a complementary nucleic acid molecule obtained from the plant cell
or plant tissue, wherein nucleic acid hybridization between the
marker nucleic acid molecule and the complementary nucleic acid
molecule obtained from the plant cell or plant tissue permits the
detection of the plant sucrose pathway enzyme; (B) permitting
hybridization between the marker nucleic acid molecule and the
complementary nucleic acid molecule obtained from the plant cell or
plant tissue; and (C) detecting the level or pattern of the
complementary nucleic acid, wherein the detection of the
complementary nucleic acid is predictive of the level or pattern of
the plant sucrose pathway enzyme.
[0106] The present invention also provides a method for determining
a level or pattern of a plant sucrose pathway enzyme in a plant
cell or plant tissue comprising: (A) incubating, under conditions
permitting nucleic acid hybridization, a marker nucleic acid
molecule, the marker nucleic acid molecule comprising a nucleic
acid molecule that encodes a maize or a soybean triose phosphate
isomerase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean fructose
1,6-bisphosphate aldolase enzyme or complement thereof or fragment
of either, a nucleic acid molecule that encodes a maize or a
soybean fructose 1,6-bisphosphate enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean fructose 6-phosphate 2-kinase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean phosphoglucoisomerase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean vacuolar H.sup.+ translocating-pyrophosphatase
enzyme or complement thereof or fragment of either, a nucleic acid
molecule that encodes a maize or a soybean pyrophosphate-dependent
fructose-6-phosphate phosphotransferase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean invertase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean sucrose synthase enzyme or complement thereof or fragment
of either, a nucleic acid molecule that encodes a maize or a
soybean hexokinase enzyme or complement thereof or fragment of
either, a nucleic acid molecule that encodes a maize or a soybean
fructokinase enzyme or complement thereof or fragment of either f,
a nucleic acid molecule that encodes a maize or a soybean
NDP-kinase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean
glucose-6-phosphate 1-dehydrogenase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean phosphoglucomutase enzyme or complement thereof or
fragment of either and a nucleic acid molecule that encodes a maize
or a soybean UDP-glucose pyrophosphorylase enzyme or complement
thereof or fragment of either, with a complementary nucleic acid
molecule obtained from the plant cell or plant tissue, wherein
nucleic acid hybridization between the marker nucleic acid molecule
and the complementary nucleic acid molecule obtained from the plant
cell or plant tissue permits the detection of the plant sucrose
pathway enzyme; (B) permitting hybridization between the marker
nucleic acid molecule and the complementary nucleic acid molecule
obtained from the plant cell or plant tissue; and (C) detecting the
level or pattern of the complementary nucleic acid, wherein the
detection of the complementary nucleic acid is predictive of the
level or pattern of the plant sucrose pathway enzyme.
[0107] The present invention also provides a method for determining
a level or pattern of a plant sucrose pathway enzyme in a plant
cell or plant tissue under evaluation which comprises assaying the
concentration of a molecule, whose concentration is dependent upon
the expression of a gene, the gene specifically hybridizes to a
nucleic acid molecule having a nucleic acid sequence selected from
the group consisting of SEQ ID NO: 1 through SEQ ID NO: 2814 or
complements thereof, in comparison to the concentration of that
molecule present in a reference plant cell or a reference plant
tissue with a known level or pattern of the plant sucrose pathway
enzyme, wherein the assayed concentration of the molecule is
compared to the assayed concentration of the molecule in the
reference plant cell or reference plant tissue with the known level
or pattern of the plant sucrose pathway enzyme.
[0108] The present invention also provides a method for determining
a level or pattern of a plant sucrose pathway enzyme in a plant
cell or plant tissue under evaluation which comprises assaying the
concentration of a molecule, whose concentration is dependent upon
the expression of a gene, the gene specifically hybridizes to a
nucleic acid molecule selected from the group consisting of a
nucleic acid molecule that encodes a maize or a soybean triose
phosphate isomerase enzyme or complement thereof, a nucleic acid
molecule that encodes a maize or a soybean fructose
1,6-bisphosphate aldolase enzyme or complement thereof, a nucleic
acid molecule that encodes a maize or a soybean fructose
1,6-bisphosphate enzyme or complement thereof, a nucleic acid
molecule that encodes a maize or a soybean fructose 6-phosphate
2-kinase enzyme or complement thereof, a nucleic acid molecule that
encodes a maize or a soybean phosphoglucoisomerase enzyme or
complement thereof, a nucleic acid molecule that encodes a maize or
a soybean vacuolar H.sup.+ translocating-pyrophosphatase enzyme or
complement thereof, a nucleic acid molecule that encodes a maize or
a soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or complement thereof, a nucleic acid
molecule that encodes a maize or a soybean invertase enzyme or
complement thereof, a nucleic acid molecule that encodes a maize or
a soybean sucrose synthase enzyme or complement thereof, a nucleic
acid molecule that encodes a maize or a soybean hexokinase enzyme
or complement thereof, a nucleic acid molecule that encodes a maize
or a soybean fructokinase enzyme or complement thereof, a nucleic
acid molecule that encodes a maize or a soybean NDP-kinase enzyme
or complement thereof, a nucleic acid molecule that encodes a maize
or a soybean glucose-6-phosphate 1-dehydrogenase enzyme or
complement thereof, a nucleic acid molecule that encodes a maize or
a soybean phosphoglucomutase enzyme or complement thereof and a
nucleic acid molecule that encodes a maize or a soybean UDP-glucose
pyrophosphorylase enzyme or complement thereof, in comparison to
the concentration of that molecule present in a reference plant
cell or a reference plant tissue with a known level or pattern of
the plant sucrose pathway enzyme, wherein the assayed concentration
of the molecule is compared to the assayed concentration of the
molecule in the reference plant cell or the reference plant tissue
with the known level or pattern of the plant sucrose pathway
enzyme.
[0109] The present invention provides a method of determining a
mutation in a plant whose presence is predictive of a mutation
affecting a level or pattern of a protein comprising the steps: (A)
incubating, under conditions permitting nucleic acid hybridization,
a marker nucleic acid, the marker nucleic acid selected from the
group of marker nucleic acid molecules which specifically hybridize
to a nucleic acid molecule having a nucleic acid sequence selected
from the group of SEQ ID NO: 1 through SEQ ID NO: 2814 or
complements thereof and a complementary nucleic acid molecule
obtained from the plant, wherein nucleic acid hybridization between
the marker nucleic acid molecule and the complementary nucleic acid
molecule obtained from the plant permits the detection of a
polymorphism whose presence is predictive of a mutation affecting
the level or pattern of the plant sucrose pathway enzyme in the
plant; (B) permitting hybridization between the marker nucleic acid
molecule and the complementary nucleic acid molecule obtained from
the plant; and (C) detecting the presence of the polymorphism,
wherein the detection of the polymorphism is predictive of the
mutation.
[0110] The present invention also provides a method for determining
a mutation in a plant whose presence is predictive of a mutation
affecting the level or pattern of a plant sucrose pathway enzyme
comprising the steps: (A) incubating, under conditions permitting
nucleic acid hybridization, a marker nucleic acid molecule, the
marker nucleic acid molecule comprising a nucleic acid molecule
that is linked to a gene, the gene specifically hybridizes to a
nucleic acid molecule having a nucleic acid sequence selected from
the group consisting of SEQ ID NO: 1 through SEQ ID NO: 2814 or
complements thereof and a complementary nucleic acid molecule
obtained from the plant, wherein nucleic acid hybridization between
the marker nucleic acid molecule and the complementary nucleic acid
molecule obtained from the plant permits the detection of a
polymorphism whose presence is predictive of a mutation affecting
the level or pattern of the plant sucrose pathway enzyme in the
plant; (B) permitting hybridization between the marker nucleic acid
molecule and the complementary nucleic acid molecule obtained from
the plant; and (C) detecting the presence of the polymorphism,
wherein the detection of the polymorphism is predictive of the
mutation.
[0111] The present invention also provides a method for determining
a mutation in a plant whose presence is predictive of a mutation
affecting the level or pattern of a plant sucrose pathway enzyme
comprising the steps: (A) incubating, under conditions permitting
nucleic acid hybridization, a marker nucleic acid molecule, the
marker nucleic acid molecule comprising a nucleic acid molecule
that is linked to a gene, the gene specifically hybridizes to a
nucleic acid molecule selected from the group consisting of a
nucleic acid molecule that encodes a maize or a soybean triose
phosphate isomerase enzyme or complement thereof, a nucleic acid
molecule that encodes a maize or a soybean fructose
1,6-bisphosphate aldolase enzyme or complement thereof, a nucleic
acid molecule that encodes a maize or a soybean fructose
1,6-bisphosphate enzyme or complement thereof, a nucleic acid
molecule that encodes a maize or a soybean fructose 6-phosphate
2-kinase enzyme or complement thereof, a nucleic acid molecule that
encodes a maize or a soybean phosphoglucoisomerase enzyme or
complement thereof, a nucleic acid molecule that encodes a maize or
a soybean vacuolar H.sup.+ translocating-pyrophosphatase enzyme or
complement thereof, a nucleic acid molecule that encodes a maize or
a soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or complement thereof, a nucleic acid
molecule that encodes a maize or a soybean invertase enzyme or
complement thereof, a nucleic acid molecule that encodes a maize or
a soybean sucrose synthase enzyme or complement thereof, a nucleic
acid molecule that encodes a maize or a soybean hexokinase enzyme
or complement thereof, a nucleic acid molecule that encodes a maize
or a soybean fructokinase enzyme or complement thereof, a nucleic
acid molecule that encodes a maize or a soybean NDP-kinase enzyme
or complement thereof, a nucleic acid molecule that encodes a maize
or a soybean glucose-6-phosphate 1-dehydrogenase enzyme or
complement thereof, a nucleic acid molecule that encodes a maize or
a soybean phosphoglucomutase enzyme or complement thereof and a
nucleic acid molecule that encodes a maize or a soybean UDP-glucose
pyrophosphorylase enzyme or complement thereof and a complementary
nucleic acid molecule obtained from the plant, wherein nucleic acid
hybridization between the marker nucleic acid molecule and the
complementary nucleic acid molecule obtained from the plant permits
the detection of a polymorphism whose presence is predictive of a
mutation affecting the level or pattern of the plant sucrose
pathway enzyme in the plant; (B) permitting hybridization between
the marker nucleic acid molecule and the complementary nucleic acid
molecule obtained from the plant; and (C) detecting the presence of
the polymorphism, wherein the detection of the polymorphism is
predictive of the mutation.
[0112] The present invention also provides a method of producing a
plant containing an overexpressed protein comprising: (A)
transforming the plant with a functional nucleic acid molecule,
wherein the functional nucleic acid molecule comprises a promoter
region, wherein the promoter region is linked to a structural
region, wherein the structural region has a nucleic acid sequence
selected from group consisting of SEQ ID NO: 1 through SEQ ID NO:
2814 wherein the structural region is linked to a 3' non-translated
sequence that functions in the plant to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of a mRNA molecule; and wherein the functional nucleic acid
molecule results in overexpression of the protein; and (B) growing
the transformed plant.
[0113] The present invention also provides a method of producing a
plant containing an overexpressed plant sucrose enzyme comprising:
(A) transforming the plant with a functional nucleic acid molecule,
wherein the functional nucleic acid molecule comprises a promoter
region, wherein the promoter region is linked to a structural
region, wherein the structural region comprises a nucleic acid
molecule having a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1 through SEQ ID NO: 2814 or fragment
thereof; wherein the structural region is linked to a 3'
non-translated sequence that functions in the plant to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of a mRNA molecule; and wherein the
functional nucleic acid molecule results in overexpression of the
plant sucrose pathway enzyme; and (B) growing the transformed
plant.
[0114] The present invention also provides a method of producing a
plant containing an overexpressed plant sucrose pathway enzyme
comprising: (A) transforming the plant with a functional nucleic
acid molecule, wherein the functional nucleic acid molecule
comprises a promoter region, wherein the promoter region is linked
to a structural region, wherein the structural region comprises a
nucleic acid molecule selected from the group consisting of a
nucleic acid molecule that encodes a maize or a soybean triose
phosphate isomerase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean fructose
1,6-bisphosphate aldolase enzyme or fragment thereof, a nucleic
acid molecule that encodes a maize or a soybean fructose
1,6-bisphosphate enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean fructose 6-phosphate
2-kinase enzyme or fragment thereof, a nucleic acid molecule that
encodes a maize or a soybean phosphoglucoisomerase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean vacuolar H.sup.+ translocating-pyrophosphatase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean invertase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean sucrose synthase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean hexokinase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean fructokinase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean NDP-kinase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean glucose-6-phosphate 1-dehydrogenase enzyme or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
phosphoglucomutase enzyme or fragment thereof and a nucleic acid
molecule that encodes a maize or a soybean UDP-glucose
pyrophosphorylase enzyme or fragment thereof, wherein the
structural region is linked to a 3' non-translated sequence that
functions in the plant to cause termination of transcription and
addition of polyadenylated ribonucleotides to a 3' end of a mRNA
molecule; and wherein the functional nucleic acid molecule results
in overexpression of the plant sucrose pathway enzyme protein; and
(B) growing the transformed plant.
[0115] The present invention also provides a method of producing a
plant containing reduced levels of a plant sucrose pathway enzyme
comprising: (A) transforming the plant with a functional nucleic
acid molecule, wherein the functional nucleic acid molecule
comprises a promoter region, wherein the promoter region is linked
to a structural region, wherein the structural region comprises a
nucleic acid molecule having a nucleic acid sequence selected from
the group consisting of SEQ ID NO: 1 through SEQ ID NO: 2814;
wherein the structural region is linked to a 3' non-translated
sequence that functions in the plant to cause termination of
transcription and addition of polyadenylated ribonucleotides to a
3' end of a mRNA molecule; and wherein the functional nucleic acid
molecule results in co-suppression of the plant sucrose pathway
enzyme protein; and (B) growing the transformed plant.
[0116] The present invention also provides a method of producing a
plant containing reduced levels of a plant sucrose pathway enzyme
comprising: (A) transforming the plant with a functional nucleic
acid molecule, wherein the functional nucleic acid molecule
comprises a promoter region, wherein the promoter region is linked
to a structural region, wherein the structural region comprises a
nucleic acid molecule having a nucleic acid sequence selected from
the group consisting of a nucleic acid molecule that encodes a
maize or a soybean triose phosphate isomerase enzyme or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
fructose 1,6-bisphosphate aldolase enzyme or fragment thereof, a
nucleic acid molecule that encodes a maize or a soybean fructose
1,6-bisphosphate enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean fructose 6-phosphate
2-kinase enzyme or fragment thereof, a nucleic acid molecule that
encodes a maize or a soybean phosphoglucoisomerase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean vacuolar H.sup.+ translocating-pyrophosphatase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean invertase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean sucrose synthase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean hexokinase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean fructokinase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean NDP-kinase enzyme or
fragment thereof, a nucleic acid molecule that encodes a maize or a
soybean glucose-6-phosphate 1-dehydrogenase enzyme or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
phosphoglucomutase enzyme or fragment thereof and a nucleic acid
molecule that encodes a maize or a soybean UDP-glucose
pyrophosphorylase enzyme or fragment thereof, wherein the
structural region is linked to a 3' non-translated sequence that
functions in the plant to cause termination of transcription and
addition of polyadenylated ribonucleotides to a 3' end of a mRNA
molecule; and wherein the functional nucleic acid molecule results
in co-suppression of the plant sucrose pathway enzyme; and (B)
growing the transformed plant.
[0117] The present invention also provides a method for reducing
expression of a plant sucrose pathway enzyme in a plant comprising:
(A) transforming the plant with a nucleic acid molecule, the
nucleic acid molecule having an exogenous promoter region which
functions in a plant cell to cause the production of a mRNA
molecule, wherein the exogenous promoter region is linked to a
transcribed nucleic acid molecule having a transcribed strand and a
non-transcribed strand, wherein the transcribed strand is
complementary to a nucleic acid molecule having a nucleic acid
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 2814 or complements thereof or fragments of either and
the transcribed strand is complementary to an endogenous mRNA
molecule; and wherein the transcribed nucleic acid molecule is
linked to a 3' non-translated sequence that functions in the plant
cell to cause termination of transcription and addition of
polyadenylated ribonucleotides to a 3' end of a mRNA molecule; and
(B) growing the transformed plant.
[0118] The present invention also provides a method for reducing
expression of a plant sucrose pathway enzyme in a plant comprising:
(A) transforming the plant with a nucleic acid molecule, the
nucleic acid molecule having an exogenous promoter region which
functions in a plant cell to cause the production of a mRNA
molecule, wherein the exogenous promoter region is linked to a
transcribed nucleic acid molecule having a transcribed strand and a
non-transcribed strand, wherein a transcribed mRNA of the
transcribed strand is complementary to a nucleic acid molecule
selected from the group consisting of an endogenous mRNA molecule
that encodes a maize or a soybean triose phosphate isomerase enzyme
or fragment thereof, an endogenous mRNA molecule that encodes a
maize or a soybean fructose 1,6-bisphosphate aldolase enzyme or
fragment thereof, an endogenous mRNA molecule that encodes a maize
or a soybean fructose 1,6-bisphosphate enzyme or fragment thereof,
an endogenous mRNA molecule that encodes a maize or a soybean
fructose 6-phosphate 2-kinase enzyme or fragment thereof, an
endogenous mRNA molecule that encodes a maize or a soybean
phosphoglucoisomerase enzyme or fragment thereof, an endogenous
mRNA molecule that encodes a maize or a soybean vacuolar H.sup.+
translocating-pyrophosphatase enzyme or fragment thereof, an
endogenous mRNA molecule that encodes a maize or a soybean
pyrophosphate-dependent fructose-6-phosphate phosphotransferase
enzyme or fragment thereof, an endogenous mRNA molecule that
encodes a maize or a soybean invertase enzyme or fragment thereof,
an endogenous mRNA molecule that encodes a maize or a soybean
sucrose synthase enzyme or fragment thereof, an endogenous mRNA
molecule that encodes a maize or a soybean hexokinase enzyme or
fragment thereof, an endogenous mRNA molecule that encodes a maize
or a soybean fructokinase enzyme or fragment thereof, an endogenous
mRNA molecule that encodes a maize or a soybean NDP-kinase enzyme
or fragment thereof, an endogenous mRNA molecule that encodes a
maize or a soybean glucose-6-phosphate 1-dehydrogenase enzyme or
fragment thereof, an endogenous mRNA molecule that encodes a maize
or a soybean phosphoglucomutase enzyme or fragment thereof and an
endogenous mRNA molecule that encodes a maize or a soybean
UDP-glucose pyrophosphorylase enzyme or fragment thereof, and
wherein the transcribed nucleic acid molecule is linked to a 3'
non-translated sequence that functions in the plant cell to cause
termination of transcription and addition of polyadenylated
ribonucleotides to a 3' end of a mRNA molecule; and (B) growing the
transformed plant.
[0119] The present invention also provides a method of determining
an association between a polymorphism and a plant trait comprising:
(A) hybridizing a nucleic acid molecule specific for the
polymorphism to genetic material of a plant, wherein the nucleic
acid molecule has a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1 through SEQ ID NO: 2814 or complements
thereof or fragment of either; and (B) calculating the degree of
association between the polymorphism and the plant trait.
[0120] The present invention also provides a method of determining
an association between a polymorphism and a plant trait comprising:
(A) hybridizing a nucleic acid molecule specific for the
polymorphism to genetic material of a plant, wherein the nucleic
acid molecule is selected from the group consisting of a nucleic
acid molecule that encodes a maize or a soybean triose phosphate
isomerase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean fructose
1,6-bisphosphate aldolase enzyme or complement thereof or fragment
of either, a nucleic acid molecule that encodes a maize or a
soybean fructose 1,6-bisphosphate enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean fructose 6-phosphate 2-kinase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean phosphoglucoisomerase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean vacuolar H.sup.+ translocating-pyrophosphatase
enzyme or complement thereof or fragment of either, a nucleic acid
molecule that encodes a maize or a soybean pyrophosphate-dependent
fructose-6-phosphate phosphotransferase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean invertase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean sucrose synthase enzyme or complement thereof or fragment
of either, a nucleic acid molecule that encodes a maize or a
soybean hexokinase enzyme or complement thereof or fragment of
either, a nucleic acid molecule that encodes a maize or a soybean
fructokinase enzyme or complement thereof or fragment of either f,
a nucleic acid molecule that encodes a maize or a soybean
NDP-kinase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean
glucose-6-phosphate 1-dehydrogenase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean phosphoglucomutase enzyme or complement thereof or
fragment of either and a nucleic acid molecule that encodes a maize
or a soybean UDP-glucose pyrophosphorylase enzyme or complement
thereof or fragment of either and (B) calculating the degree of
association between the polymorphism and the plant trait.
[0121] The present invention also provides a method of isolating a
nucleic acid that encodes a plant sucrose pathway enzyme or
fragment thereof comprising: (A) incubating under conditions
permitting nucleic acid hybridization, a first nucleic acid
molecule comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NO: 1 through SEQ ID NO: 2814 or complements
thereof or fragment of either with a complementary second nucleic
acid molecule obtained from a plant cell or plant tissue; (B)
permitting hybridization between the first nucleic acid molecule
and the second nucleic acid molecule obtained from the plant cell
or plant tissue; and (C) isolating the second nucleic acid
molecule.
[0122] The present invention also provides a method of isolating a
nucleic acid molecule that encodes a plant sucrose pathway enzyme
or fragment thereof comprising: (A) incubating under conditions
permitting nucleic acid hybridization, a first nucleic acid
molecule selected from the group consisting of a nucleic acid
molecule that encodes a maize or a soybean triose phosphate
isomerase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean fructose
1,6-bisphosphate aldolase enzyme or complement thereof or fragment
of either, a nucleic acid molecule that encodes a maize or a
soybean fructose 1,6-bisphosphate enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean fructose 6-phosphate 2-kinase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean phosphoglucoisomerase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean vacuolar H.sup.+ translocating-pyrophosphatase
enzyme or complement thereof or fragment of either, a nucleic acid
molecule that encodes a maize or a soybean pyrophosphate-dependent
fructose-6-phosphate phosphotransferase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean invertase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean sucrose synthase enzyme or complement thereof or fragment
of either, a nucleic acid molecule that encodes a maize or a
soybean hexokinase enzyme or complement thereof or fragment of
either, a nucleic acid molecule that encodes a maize or a soybean
fructokinase enzyme or complement thereof or fragment of either f,
a nucleic acid molecule that encodes a maize or a soybean
NDP-kinase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean
glucose-6-phosphate 1-dehydrogenase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean phosphoglucomutase enzyme or complement thereof or
fragment of either and a nucleic acid molecule that encodes a maize
or a soybean UDP-glucose pyrophosphorylase enzyme or complement
thereof or fragment of either, with a complementary second nucleic
acid molecule obtained from a plant cell or plant tissue; (B)
permitting hybridization between the plant sucrose pathway nucleic
acid molecule and the complementary nucleic acid molecule obtained
from the plant cell or plant tissue; and (C) isolating the second
nucleic acid molecule.
DETAILED DESCRIPTION OF THE INVENTION
Definitions and Agents of the Present Invention
Definitions
[0123] As used herein, a sucrose pathway enzyme is any enzyme that
is associated with the synthesis or degradation of sucrose.
[0124] As used herein, a sucrose synthesis enzyme is any enzyme
that is associated with the synthesis of sucrose.
[0125] As used herein, a sucrose degradation enzyme is any enzyme
that is associated with the degradation of sucrose.
[0126] As used herein, triose phosphate isomerase is any enzyme
that maintains at equilibrium the pool of triose phosphates,
dihydroxyacetone phosphate ("DHAP"), and glyceraldehyde-3-phosphate
("GAP") within the cytoplasm.
[0127] As used herein, fructose 1,6-bisphosphate aldolase is any
enzyme that catalyzes an aldol condensation of DHAP and GAP to form
fructose 1,6-bisphosphate ("F1,6BP").
[0128] As used herein, fructose-1,6-bisphosphatase ("FBPase") is
any enzyme that catalyzes the cleavage of phosphate from the C1
carbon of fructose-1,6-bisphosphate to form fructose-6-phosphate
("F6P").
[0129] As used herein, fructose 6-phosphate 2-kinase is any enzyme
that controls the concentration of fructose 2,6-bisphosphate.
[0130] As used herein, phosphoglucoisomerase is any enzyme that
maintains glucose-6-phosphate ("G6P") and glucose-1-phosphate
("GIP") in equilibrium with the F6P pool.
[0131] As used herein, vacuolar H.sup.+
translocating-pyrophosphatase is any enzyme that utilizes
pyrophosphate to sustain a proton gradient formed within the
vacuolar membrane.
[0132] As used herein, pyrophosphate-dependent fructose-6-phosphate
phosphotransferase is any enzyme that catalyzes the reversible
production of F1,6BP and Pi from F6P and PPi.
[0133] As used herein, invertase is any enzyme that irreversibly
cleaves sucrose into glucose and fructose.
[0134] As used herein, sucrose synthase is any enzyme that carries
out the kinetically reversible transglycosylation of sucrose and
UDP into fructose and UDPG.
[0135] As used herein, hexokinase is any enzyme that can
phosphorylate either glucose or fructose.
[0136] As used herein, fructokinase is any enzyme that typically
has a specific affinity for fructose.
[0137] As used herein, NDP-kinase is any enzyme that can maintain
UDP levels for sucrose synthase reactions, even in the case of an
ATP-specific fructokinase.
[0138] As used herein, glucose-6-phosphate 1-dehydrogenase is any
enzyme that allows G6P resulting from hexose kinase activity to
enter the pentose phosphate pathway.
[0139] As used herein, UDP-glucose dehydrogenase is any enzyme that
allows UDPG formed by sucrose synthase to be utilized directly for
cellulose or callose biosynthesis.
[0140] As used herein, phosphoglucomutase is any enzyme that is
ubiquitous and reversible with commitments of G6P to either F6P or
GIP resulting from fluxes in metabolites further along each
pathway.
Agents
[0141] (a) Nucleic Acid Molecules
[0142] Agents of the present invention include plant nucleic acid
molecules and more preferably include maize and soybean nucleic
acid molecules and more preferably include nucleic acid molecules
of the maize genotypes B73 (Illinois Foundation Seeds, Champaign,
Illinois U.S.A.), B73.times.Mol7 (Illinois Foundation Seeds,
Champaign, Illinois U.S.A.), DK604 (Dekalb Genetics, Dekalb,
Illinois U.S.A.), H99 (Illinois Foundation Seeds, Champaign,
Illinois U.S.A.), RX601 (Asgrow Seed Company, Des Moines, Iowa),
Mol7 (Illinois Foundation Seeds, Champaign, Illinois U.S.A.), and
soybean types Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa),
C1944 (United States Department of Agriculture (USDA) Soybean
Germplasm Collection, Urbana, Ill. U.S.A.), Cristalina (USDA
Soybean Germplasm Collection, Urbana, Ill. U.S.A.), FT108 (Monsoy,
Brazil), Hartwig (USDA Soybean Germplasm Collection, Urbana, Ill.
U.S.A.), BW211S Null (Tohoku University, Morioka, Japan), PI507354
(USDA Soybean Germplasm Collection, Urbana, Ill. U.S.A.), Asgrow
A4922 (Asgrow Seed Company, Des Moines, Iowa U.S.A.), P1227687
(USDA Soybean Germplasm Collection, Urbana, Ill. U.S.A.), P1229358
(USDA Soybean Germplasm Collection, Urbana, Ill. U.S.A.) and Asgrow
A3237 (Asgrow Seed Company, Des Moines, Iowa U.S.A.).
[0143] A subset of the nucleic acid molecules of the present
invention includes nucleic acid molecules that are marker
molecules. Another subset of the nucleic acid molecules of the
present invention include nucleic acid molecules that encode a
protein or fragment thereof. Another subset of the nucleic acid
molecules of the present invention are EST molecules.
[0144] Fragment nucleic acid molecules may encode significant
portion(s) of, or indeed most of, these nucleic acid molecules.
Alternatively, the fragments may comprise smaller oligonucleotides
(having from about 15 to about 250 nucleotide residues and more
preferably, about 15 to about 30 nucleotide residues).
[0145] As used herein, an agent, be it a naturally occurring
molecule or otherwise may be "substantially purified," if desired,
such that one or more molecules that is or may be present in a
naturally occurring preparation containing that molecule will have
been removed or will be present at a lower concentration than that
at which it would normally be found.
[0146] The agents of the present invention will preferably be
"biologically active" with respect to either a structural
attribute, such as the capacity of a nucleic acid to hybridize to
another nucleic acid molecule, or the ability of a protein to be
bound by an antibody (or to compete with another molecule for such
binding). Alternatively, such an attribute may be catalytic and
thus involve the capacity of the agent to mediate a chemical
reaction or response.
[0147] The agents of the present invention may also be recombinant.
As used herein, the term recombinant means any agent (e.g. DNA,
peptide etc.), that is, or results, however indirect, from human
manipulation of a nucleic acid molecule.
[0148] It is understood that the agents of the present invention
may be labeled with reagents that facilitate detection of the agent
(e.g. fluorescent labels, Prober et al., Science 238:336-340
(1987); Albarella et al., EP 144914; chemical labels, Sheldon et
al., U.S. Pat. No. 4,582,789; Albarella et al., U.S. Pat. No.
4,563,417; modified bases, Miyoshi et al., EP 119448, all of which
are hereby incorporated by reference in their entirety).
[0149] It is further understood, that the present invention
provides recombinant bacterial, mammalian, microbial, insect,
fungal and plant cells and viral constructs comprising the agents
of the present invention (See, for example, Uses of the Agents of
the Invention, Section (a) Plant Constructs and Plant
Transformants; Section (b) Fungal Constructs and Fungal
Transformants; Section (c) Mammalian Constructs and Transformed
Mammalian Cells; Section (d) Insect Constructs and Transformed
Insect Cells; and Section (e) Bacterial Constructs and Transformed
Bacterial Cells).
[0150] Nucleic acid molecules or fragments thereof of the present
invention are capable of specifically hybridizing to other nucleic
acid molecules under certain circumstances. As used herein, two
nucleic acid molecules are said to be capable of specifically
hybridizing to one another if the two molecules are capable of
forming an anti-parallel, double-stranded nucleic acid structure. A
nucleic acid molecule is said to be the "complement" of another
nucleic acid molecule if they exhibit complete complementarity. As
used herein, molecules are said to exhibit "complete
complementarity" when every nucleotide of one of the molecules is
complementary to a nucleotide of the other. Two molecules are said
to be "minimally complementary" if they can hybridize to one
another with sufficient stability to permit them to remain annealed
to one another under at least conventional "low-stringency"
conditions. Similarly, the molecules are said to be "complementary"
if they can hybridize to one another with sufficient stability to
permit them to remain annealed to one another under conventional
"high-stringency" conditions. Conventional stringency conditions
are described by Sambrook et al., Molecular Cloning, A Laboratory
Manual, 2nd Ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
(1989) and by Haymes et al., Nucleic Acid Hybridization, A
Practical Approach, IRL Press, Washington, D.C. (1985), the
entirety of which is herein incorporated by reference. Departures
from complete complementarity are therefore permissible, as long as
such departures do not completely preclude the capacity of the
molecules to form a double-stranded structure. Thus, in order for a
nucleic acid molecule to serve as a primer or probe it need only be
sufficiently complementary in sequence to be able to form a stable
double-stranded structure under the particular solvent and salt
concentrations employed.
[0151] Appropriate stringency conditions which promote DNA
hybridization, for example, 6.0.times. sodium chloride/sodium
citrate (SSC) at about 45.degree. C., followed by a wash of
2.0.times.SSC at 50.degree. C., are known to those skilled in the
art or can be found in Current Protocols in Molecular Biology, John
Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the salt
concentration in the wash step can be selected from a low
stringency of about 2.0.times.SSC at 50.degree. C. to a high
stringency of about 0.2.times.SSC at 50.degree. C. In addition, the
temperature in the wash step can be increased from low stringency
conditions at room temperature, about 22.degree. C., to high
stringency conditions at about 65.degree. C. Both temperature and
salt may be varied, or either the temperature or the salt
concentration may be held constant while the other variable is
changed.
[0152] In a preferred embodiment, a nucleic acid of the present
invention will specifically hybridize to one or more of the nucleic
acid molecules set forth in SEQ ID NO: 1 through SEQ ID NO: 2814 or
complements thereof under moderately stringent conditions, for
example at about 2.0.times.SSC and about 65.degree. C.
[0153] In a particularly preferred embodiment, a nucleic acid of
the present invention will include those nucleic acid molecules
that specifically hybridize to one or more of the nucleic acid
molecules set forth in SEQ ID NO: 1 through SEQ ID NO: 2814 or
complements thereof under high stringency conditions such as
0.2.times.SSC and about 65.degree. C.
[0154] In one aspect of the present invention, the nucleic acid
molecules of the present invention have one or more of the nucleic
acid sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 2814 or
complements thereof. In another aspect of the present invention,
one or more of the nucleic acid molecules of the present invention
share between 100% and 90% sequence identity with one or more of
the nucleic acid sequences set forth in SEQ ID NO: 1 through SEQ ID
NO: 2814 or complements thereof. In a further aspect of the present
invention, one or more of the nucleic acid molecules of the present
invention share between 100% and 95% sequence identity with one or
more of the nucleic acid sequences set forth in SEQ ID NO: 1
through SEQ ID NO: 2814 or complements thereof. In a more preferred
aspect of the present invention, one or more of the nucleic acid
molecules of the present invention share between 100% and 98%
sequence identity with one or more of the nucleic acid sequences
set forth in SEQ ID NO: 1 through SEQ ID NO: 2814 or complements
thereof. In an even more preferred aspect of the present invention,
one or more of the nucleic acid molecules of the present invention
share between 100% and 99% sequence identity with one or more of
the sequences set forth in SEQ ID NO: 1 through SEQ ID NO: 2814 or
complements thereof.
[0155] In a further more preferred aspect of the present invention,
one or more of the nucleic acid molecules of the present invention
exhibit 100% sequence identity with a nucleic acid molecule present
within MONN01, SATMON001 through SATMON031, SATMON033, SATMON034,
SATMON.about.001, SATMONN01, SATMONN04 through SATMONN006, CMz029
through CMz031, CMz033, CMz035 through CMz037, CMz039 through
CMz042, CMz044 through CMz045, CMz047 through CMz050, SOYMON001
through SOYMON038, Soy51 through Soy56, Soy58 through Soy62, Soy65
through Soy66, Soy 68 through Soy73 and Soy76 through Soy77, Lib9,
Lib22 through Lib25, Lib35, Lib80 through Lib81, Lib 144, Lib146,
Lib147, Lib190, Lib3032 through Lib3036 and Lib3099 (Monsanto
Company, St. Louis, Mo. U.S.A.).
[0156] (i) Nucleic Acid Molecules Encoding Proteins or Fragments
Thereof
[0157] Nucleic acid molecules of the present invention can comprise
sequences that encode a sucrose pathway protein or fragment
thereof. Such proteins or fragments thereof include homologues of
known proteins in other organisms.
[0158] In a preferred embodiment of the present invention, a maize
or a soybean protein or fragment thereof of the present invention
is a homologue of another plant protein. In another preferred
embodiment of the present invention, a maize or a soybean protein
or fragment thereof of the present invention is a homologue of a
fungal protein. In another preferred embodiment of the present
invention, a maize or a soybean protein of the present invention is
a homologue of mammalian protein. In another preferred embodiment
of the present invention, a maize or a soybean protein or fragment
thereof of the present invention is a homologue of a bacterial
protein. In another preferred embodiment of the present invention,
a soybean protein or fragment thereof of the present invention is a
homologue of a maize protein. In another preferred embodiment of
the present invention, a maize protein homologue or fragment
thereof of the present invention is a homologue of a soybean
protein.
[0159] In a preferred embodiment of the present invention, the
nucleic molecule of the present invention encodes a maize or a
soybean protein or fragment thereof where a maize or a soybean
protein exhibits a BLAST probability score of greater than 1E-12,
preferably a BLAST probability score of between about 1E-30 and
about 1E-12, even more preferably a BLAST probability score of
greater than 1E-30 with its homologue.
[0160] In another preferred embodiment of the present invention,
the nucleic acid molecule encoding a maize or a soybean protein or
fragment thereof exhibits a % identity with its homologue of
between about 25% and about 40%, more preferably of between about
40 and about 70%, even more preferably of between about 70% and
about 90% and even more preferably between about 90% and 99%. In
another preferred embodiment, of the present invention, a maize or
a soybean protein or fragments thereof exhibits a % identity with
its homologue of 100%.
[0161] In a preferred embodiment of the present invention, the
nucleic molecule of the present invention encodes a maize or a
soybean protein or fragment thereof where a maize or a soybean
protein exhibits a BLAST score of greater than 120, preferably a
BLAST score of between about 1450 and about 120, even more
preferably a BLAST score of greater than 1450 with its
homologue.
[0162] Nucleic acid molecules of the present invention also include
non-maize, non-soybean homologues. Preferred non-maize and soybean
homologues are selected from the group consisting of alfalfa,
Arabidopsis, barley, Brassica, broccoli, cabbage, citrus, cotton,
garlic, oat, oilseed rape, onion, canola, flax, an ornamental
plant, pea, peanut, pepper, potato, rice, rye, sorghum, strawberry,
sugarcane, sugarbeet, tomato, wheat, poplar, pine, fir, eucalyptus,
apple, lettuce, lentils, grape, banana, tea, turf grasses,
sunflower, oil palm and Phaseolus.
[0163] In a preferred embodiment, nucleic acid molecules having SEQ
ID NO: 1 through SEQ ID NO: 2814 or complements and fragments of
either can be utilized to obtain such homologues.
[0164] The degeneracy of the genetic code, which allows different
nucleic acid sequences to code for the same protein or peptide, is
known in the literature. (U.S. Pat. No. 4,757,006, the entirety of
which is herein incorporated by reference).
[0165] In an aspect of the present invention, one or more of the
nucleic acid molecules of the present invention differ in nucleic
acid sequence from those encoding a maize or a soybean protein or
fragment thereof in SEQ ID NO: 1 through SEQ ID NO: 2814 due to the
degeneracy in the genetic code in that they encode the same protein
but differ in nucleic acid sequence.
[0166] In another further aspect of the present invention, one or
more of the nucleic acid molecules of the present invention differ
in nucleic acid sequence from those encoding a maize or a soybean
protein or fragment thereof in SEQ ID NO: 1 through SEQ ID NO: 2814
due to fact that the different nucleic acid sequence encodes a
protein having one or more conservative amino acid residue.
Examples of conservative substitutions are set forth in Table 1. It
is understood that codons capable of coding for such conservative
substitutions are known in the art.
TABLE-US-00001 TABLE 1 Original Residue Conservative Substitutions
Ala Ser Arg Lys Asn Gln; His Asp Glu Cys Ser; Ala Gln Asn Glu Asp
Gly Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu
Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe
Val Ile; Leu
[0167] In a further aspect of the present invention, one or more of
the nucleic acid molecules of the present invention differ in
nucleic acid sequence from those encoding a maize or a soybean
protein or fragment thereof set forth in SEQ ID NO: 1 through SEQ
ID NO: 2814 or fragment thereof due to the fact that one or more
codons encoding an amino acid has been substituted for a codon that
encodes a nonessential substitution of the amino acid originally
encoded.
[0168] Agents of the present invention include nucleic acid
molecules that encode a maize or a soybean sucrose pathway protein
or fragment thereof and particularly substantially purified nucleic
acid molecules selected from the group consisting of a nucleic acid
molecule that encodes a maize or a soybean triose phosphate
isomerase protein or fragment thereof, a nucleic acid molecule that
encodes a maize or a soybean fructose 1,6-bisphosphate aldolase
protein or fragment thereof, a nucleic acid molecule that encodes a
maize or a soybean fructose 1,6-bisphosphate protein or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
fructose 6-phosphate 2-kinase protein or fragment thereof, a
nucleic acid molecule that encodes a maize or a soybean
phosphoglucoisomerase protein or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean vacuolar H.sup.+
translocating-pyrophosphatase protein or fragment thereof, a
nucleic acid molecule that encodes a maize or a soybean
pyrophosphate-dependent fructose-6-phosphate phosphotransferase
protein or fragment thereof, a nucleic acid molecule that encodes a
maize or a soybean invertase protein or fragment thereof, a nucleic
acid molecule that encodes a maize or a soybean sucrose synthase
protein or fragment thereof, a nucleic acid molecule that encodes a
maize or a soybean hexokinase protein or fragment thereof, a
nucleic acid molecule that encodes a maize or a soybean
fructokinase protein or fragment thereof, a nucleic acid molecule
that encodes a maize or a soybean NDP-kinase protein or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
glucose-6-phosphate 1-dehydrogenase protein or fragment thereof, a
nucleic acid molecule that encodes a maize or a soybean
phosphoglucomutase protein or fragment thereof and a nucleic acid
molecule that encodes a maize or a soybean UDP-glucose
pyrophosphorylase protein or fragment thereof.
[0169] Non-limiting examples of such nucleic acid molecules of the
present invention are nucleic acid molecules comprising: SEQ ID NO:
1 through SEQ ID NO: 2814 or fragment thereof that encode for a
sucrose pathway protein or fragment thereof, SEQ ID NO: 1 through
SEQ ID NO: 206 and SEQ ID NO: 1538 through SEQ ID NO: 1707 or
fragment thereof that encode for a triose phosphate isomerase
protein or fragment thereof, SEQ ID NO: 207 through SEQ ID NO: 232
and SEQ ID NO: 1708 through SEQ ID NO: 2113 or fragment thereof
that encode for a fructose 1,6-bisphosphate aldolase protein or
fragment thereof, SEQ ID NO: 233 through SEQ ID NO: 258 and SEQ ID
NO: 2114 through SEQ ID NO: 2162 or fragment thereof that encode
for a fructose 1,6-bisphosphate protein or fragment thereof, SEQ ID
NO: 259 through SEQ ID NO: 275 and SEQ ID NO: 2163 through SEQ ID
NO: 2166 or fragment thereof that encode for a fructose 6-phosphate
2-kinase protein or fragment thereof, SEQ ID NO: 276 through SEQ ID
NO: 340 and SEQ ID NO: 2167 through SEQ ID NO: 2182 or fragment
thereof that encode for a phosphoglucoisomerase protein or fragment
thereof, SEQ ID NO: 341 through SEQ ID NO: 497 and SEQ ID NO: 2183
through SEQ ID NO: 2241 or fragment thereof that encode for a
vacuolar H.sup.+ translocating-pyrophosphatase protein or fragment
thereof, SEQ ID NO: 498 through SEQ ID NO: 507 and SEQ ID NO: 2442
or fragment thereof that encode for a pyrophosphate-dependent
fructose-6-phosphate phosphotransferase protein or fragment
thereof, SEQ ID NO: 508 through SEQ ID NO: 510 and SEQ ID NO: 2243
through SEQ ID NO: 2254 or fragment thereof that encode for an
invertase protein or fragment thereof, SEQ ID NO: 511 through SEQ
ID NO: 1086 and SEQ ID NO: 2255 through SEQ ID NO: 2590 or fragment
thereof that encode for a sucrose synthase protein or fragment
thereof, SEQ ID NO: 1087 through SEQ ID NO: 1135 and SEQ ID NO:
2591 through SEQ ID NO: 2634 or fragment thereof that encode for a
hexokinase protein or fragment thereof, SEQ ID NO: 1136 through SEQ
ID NO: 1215 and SEQ ID NO: 2635 through SEQ ID NO: 2678 or fragment
thereof that encode for a fructokinase protein or fragment thereof,
SEQ ID NO: 1216 through SEQ ID NO: 1251 and SEQ ID NO: 2679 through
SEQ ID NO: 2681 or fragment thereof that encode for a NDP-kinase
protein or fragment thereof, SEQ ID NO: 1252 through SEQ ID NO:
1254 and SEQ ID NO: 2682 through SEQ ID NO: 2689 or fragment
thereof that encode for a glucose-6-phosphate 1-dehydrogenase
protein or fragment thereof, SEQ ID NO: 1255 through SEQ ID NO:
1360 and SEQ ID NO: 2690 through SEQ ID NO: 2740 or fragment
thereof that encode for a phosphoglucomutase protein or fragment
thereof and SEQ ID NO: 1361 through SEQ ID NO: 1537 and SEQ ID NO:
2741 through SEQ ID NO: 2814 or fragment thereof that encode for an
UDP-glucose pyrophosphorylase protein or fragment thereof.
[0170] A nucleic acid molecule of the present invention can also
encode a homologue of a maize or a soybean triose phosphate
isomerase or fragment thereof, a maize or a soybean fructose
1,6-bisphosphate aldolase or fragment thereof, a maize or a soybean
fructose 1,6-bisphosphate or fragment thereof, a maize or a soybean
fructose 6-phosphate 2-kinase or fragment thereof, a maize or a
soybean phosphoglucoisomerase or fragment thereof, a maize or a
soybean vacuolar H.sup.+ translocating-pyrophosphatase or fragment
thereof, a maize or a soybean pyrophosphate-dependent
fructose-6-phosphate phosphotransferase or fragment thereof, a
maize or a soybean invertase or fragment thereof, a maize or a
soybean sucrose synthase or fragment thereof, a maize or a soybean
hexokinase or fragment thereof, a maize or a soybean fructokinase
or fragment thereof, a maize or a soybean NDP-kinase or fragment
thereof, a maize or a soybean glucose-6-phosphate 1-dehydrogenase
or fragment thereof, a maize or a soybean phosphoglucomutase or
fragment thereof and a maize or a soybean UDP-glucose
pyrophosphorylase or fragment thereof. As used herein a homologue
protein molecule or fragment thereof is a counterpart protein
molecule or fragment thereof in a second species (e.g., maize
triose phosphate isomerase protein is a homologue of soybean triose
phosphate isomerase protein).
[0171] (ii) Nucleic Acid Molecule Markers and Probes
[0172] One aspect of the present invention concerns markers that
include nucleic acid molecules SEQ ID NO: 1 through SEQ ID NO: 2814
or complements thereof or fragments of either that can act as
markers or other nucleic acid molecules of the present invention
that can act as markers. Genetic markers of the present invention
include "dominant" or "codominant" markers "Codominant markers"
reveal the presence of two or more alleles (two per diploid
individual) at a locus. "Dominant markers" reveal the presence of
only a single allele per locus. The presence of the dominant marker
phenotype (e.g., a band of DNA) is an indication that one allele is
present in either the homozygous or heterozygous condition. The
absence of the dominant marker phenotype (e.g. absence of a DNA
band) is merely evidence that "some other" undefined allele is
present. In the case of populations where individuals are
predominantly homozygous and loci are predominately dimorphic,
dominant and codominant markers can be equally valuable. As
populations become more heterozygous and multi-allelic, codominant
markers often become more informative of the genotype than dominant
markers. Marker molecules can be, for example, capable of detecting
polymorphisms such as single nucleotide polymorphisms (SNPs).
[0173] SNPs are single base changes in genomic DNA sequence. They
occur at greater frequency and are spaced with a greater uniformly
throughout a genome than other reported forms of polymorphism. The
greater frequency and uniformity of SNPs means that there is
greater probability that such a polymorphism will be found near or
in a genetic locus of interest than would be the case for other
polymorphisms. SNPs are located in protein-coding regions and
noncoding regions of a genome. Some of these SNPs may result in
defective or variant protein expression (e.g., as a result of
mutations or defective splicing). Analysis (genotyping) of
characterized SNPs can require only a plus/minus assay rather than
a lengthy measurement, permitting easier automation.
[0174] SNPs can be characterized using any of a variety of methods.
Such methods include the direct or indirect sequencing of the site,
the use of restriction enzymes (Botstein et al., Am. J. Hum. Genet.
32:314-331 (1980), the entirety of which is herein incorporated
reference; Konieczny and Ausubel, Plant J. 4:403-410 (1993), the
entirety of which is herein incorporated by reference), enzymatic
and chemical mismatch assays (Myers et al., Nature 313:495-498
(1985), the entirety of which is herein incorporated by reference),
allele-specific PCR (Newton et al., Nucl. Acids Res. 17:2503-2516
(1989), the entirety of which is herein incorporated by reference;
Wu et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:2757-2760 (1989), the
entirety of which is herein incorporated by reference), ligase
chain reaction (Barany, Proc. Natl. Acad. Sci. (U.S.A.) 88:189-193
(1991), the entirety of which is herein incorporated by reference),
single-strand conformation polymorphism analysis (Labrune et al.,
Am. J. Hum. Genet. 48: 1115-1120 (1991), the entirety of which is
herein incorporated by reference), primer-directed nucleotide
incorporation assays (Kuppuswami et al., Proc. Natl. Acad. Sci. USA
88:1143-1147 (1991), the entirety of which is herein incorporated
by reference), dideoxy fingerprinting (Sarkar et al., Genomics
13:441-443 (1992), the entirety of which is herein incorporated by
reference), solid-phase ELISA-based oligonucleotide ligation assays
(Nikiforov et al., Nucl. Acids Res. 22:4167-4175 (1994), the
entirety of which is herein incorporated by reference),
oligonucleotide fluorescence-quenching assays (Livak et al., PCR
Methods Appl. 4:357-362 (1995), the entirety of which is herein
incorporated by reference), 5'-nuclease allele-specific
hybridization TaqMan assay (Livak et al., Nature Genet. 9:341-342
(1995), the entirety of which is herein incorporated by reference),
template-directed dye-terminator incorporation (TDI) assay (Chen
and Kwok, Nucl. Acids Res. 25:347-353 (1997), the entirety of which
is herein incorporated by reference), allele-specific molecular
beacon assay (Tyagi et al., Nature Biotech. 16: 49-53 (1998), the
entirety of which is herein incorporated by reference), PinPoint
assay (Haff and Smirnov, Genome Res. 7: 378-388 (1997), the
entirety of which is herein incorporated by reference) and dCAPS
analysis (Neff et al., Plant J 14:387-392 (1998), the entirety of
which is herein incorporated by reference).
[0175] Additional markers, such as AFLP markers, RFLP markers and
RAPD markers, can be utilized (Walton, Seed World 22-29 (July,
1993), the entirety of which is herein incorporated by reference;
Burow and Blake, Molecular Dissection of Complex Traits, 13-29,
Paterson (ed.), CRC Press, New York (1988), the entirety of which
is herein incorporated by reference). DNA markers can be developed
from nucleic acid molecules using restriction endonucleases, the
PCR and/or DNA sequence information. RFLP markers result from
single base changes or insertions/deletions. These codominant
markers are highly abundant in plant genomes, have a medium level
of polymorphism and are developed by a combination of restriction
endonuclease digestion and Southern blotting hybridization. CAPS
are similarly developed from restriction nuclease digestion but
only of specific PCR products. These markers are also codominant,
have a medium level of polymorphism and are highly abundant in the
genome. The CAPS result from single base changes and
insertions/deletions.
[0176] Another marker type, RAPDs, are developed from DNA
amplification with random primers and result from single base
changes and insertions/deletions in plant genomes. They are
dominant markers with a medium level of polymorphisms and are
highly abundant. AFLP markers require using the PCR on a subset of
restriction fragments from extended adapter primers. These markers
are both dominant and codominant are highly abundant in genomes and
exhibit a medium level of polymorphism.
[0177] SSRs require DNA sequence information. These codominant
markers result from repeat length changes, are highly polymorphic
and do not exhibit as high a degree of abundance in the genome as
CAPS, AFLPs and RAPDs SNPs also require DNA sequence information.
These codominant markers result from single base substitutions.
They are highly abundant and exhibit a medium of polymorphism
(Rafalski et al., In: Nonmammalian Genomic Analysis, Birren and Lai
(ed.), Academic Press, San Diego, Calif., pp. 75-134 (1996), the
entirety of which is herein incorporated by reference). It is
understood that a nucleic acid molecule of the present invention
may be used as a marker.
[0178] A PCR probe is a nucleic acid molecule capable of initiating
a polymerase activity while in a double-stranded structure to with
another nucleic acid. Various methods for determining the structure
of PCR probes and PCR techniques exist in the art. Computer
generated searches using programs such as Primer3
(www-genome.wi.mit.edu/cgi-bin/primer/primer3.cgi), STSPipeline
(www-genome.wi.mit.edu/cgi-bin/www-STS_Pipeline), or GeneUp (Pesole
et al., BioTechniques 25:112-123 (1998) the entirety of which is
herein incorporated by reference), for example, can be used to
identify potential PCR primers.
[0179] It is understood that a fragment of one or more of the
nucleic acid molecules of the present invention may be a probe and
specifically a PCR probe.
[0180] (b) Protein and Peptide Molecules
[0181] A class of agents comprises one or more of the protein or
fragments thereof or peptide molecules encoded by SEQ ID NO: 1
through SEQ ID NO: 2814 or one or more of the protein or fragment
thereof and peptide molecules encoded by other nucleic acid agents
of the present invention. As used herein, the term "protein
molecule" or "peptide molecule" includes any molecule that
comprises five or more amino acids. It is well known in the art
that proteins may undergo modification, including
post-translational modifications, such as, but not limited to,
disulfide bond formation, glycosylation, phosphorylation, or
oligomerization. Thus, as used herein, the term "protein molecule"
or "peptide molecule" includes any protein molecule that is
modified by any biological or non-biological process. The terms
"amino acid" and "amino acids" refer to all naturally occurring
L-amino acids. This definition is meant to include norleucine,
ornithine, homocysteine and homoserine.
[0182] Non-limiting examples of the protein or fragment thereof of
the present invention include a maize or a soybean sucrose pathway
protein or fragment thereof; a maize or a soybean triose phosphate
isomerase or fragment thereof, a maize or a soybean fructose
1,6-bisphosphate aldolase or fragment thereof, a maize or a soybean
fructose 1,6-bisphosphate or fragment thereof, a maize or a soybean
fructose 6-phosphate 2-kinase or fragment thereof, a maize or a
soybean phosphoglucoisomerase or fragment thereof, a maize or a
soybean vacuolar H.sup.+ translocating-pyrophosphatase or fragment
thereof, a maize or a soybean pyrophosphate-dependent
fructose-6-phosphate phosphotransferase or fragment thereof, a
maize or a soybean invertase or fragment thereof, a maize or a
soybean sucrose synthase or fragment thereof, a maize or a soybean
hexokinase or fragment thereof, a maize or a soybean fructokinase
or fragment thereof, a maize or a soybean NDP-kinase or fragment
thereof, a maize or a soybean glucose-6-phosphate 1-dehydrogenase
or fragment thereof, a maize or a soybean phosphoglucomutase or
fragment thereof and a maize or a soybean UDP-glucose
pyrophosphorylase or fragment thereof.
[0183] Non-limiting examples of the protein or fragment molecules
of the present invention are a sucrose pathway protein or fragment
thereof encoded by: SEQ ID NO: 1 through SEQ ID NO: 2814 or
fragment thereof that encode for a sucrose pathway protein or
fragment thereof, SEQ ID NO: 1 through SEQ ID NO: 206 and SEQ ID
NO: 1538 through SEQ ID NO: 1707 or fragment thereof that encode
for a triose phosphate isomerase protein or fragment thereof, SEQ
ID NO: 207 through SEQ ID NO: 232 and SEQ ID NO: 1708 through SEQ
ID NO: 2113 or fragment thereof that encode for a fructose
1,6-bisphosphate aldolase protein or fragment thereof, SEQ ID NO:
233 through SEQ ID NO: 258 and SEQ ID NO: 2114 through SEQ ID NO:
2162 or fragment thereof that encode for a fructose
1,6-bisphosphate protein or fragment thereof, SEQ ID NO: 259
through SEQ ID NO: 275 and SEQ ID NO: 2163 through SEQ ID NO: 2166
or fragment thereof that encode for a fructose 6-phosphate 2-kinase
protein or fragment thereof, SEQ ID NO: 276 through SEQ ID NO: 340
and SEQ ID NO: 2167 through SEQ ID NO: 2182 or fragment thereof
that encode for a phosphoglucoisomerase protein or fragment
thereof, SEQ ID NO: 341 through SEQ ID NO: 497 and SEQ ID NO: 2183
through SEQ ID NO: 2241 or fragment thereof that encode for a
vacuolar H.sup.+ translocating-pyrophosphatase protein or fragment
thereof, SEQ ID NO: 498 through SEQ ID NO: 507 and SEQ ID NO: 2442
or fragment thereof that encode for a pyrophosphate-dependent
fructose-6-phosphate phosphotransferase protein or fragment
thereof, SEQ ID NO: 508 through SEQ ID NO: 510 and SEQ ID NO: 2243
through SEQ ID NO: 2254 or fragment thereof that encode for an
invertase protein or fragment thereof, SEQ ID NO: 511 through SEQ
ID NO: 1086 and SEQ ID NO: 2255 through SEQ ID NO: 2590 or fragment
thereof that encode for a sucrose synthase protein or fragment
thereof, SEQ ID NO: 1087 through SEQ ID NO: 1135 and SEQ ID NO:
2591 through SEQ ID NO: 2634 or fragment thereof that encode for a
hexokinase protein or fragment thereof, SEQ ID NO: 1136 through SEQ
ID NO: 1215 and SEQ ID NO: 2635 through SEQ ID NO: 2678 or fragment
thereof that encode for a fructokinase protein or fragment thereof,
SEQ ID NO: 1216 through SEQ ID NO: 1251 and SEQ ID NO: 2679 through
SEQ ID NO: 2681 or fragment thereof that encode for a NDP-kinase
protein or fragment thereof, SEQ ID NO: 1252 through SEQ ID NO:
1254 and SEQ ID NO: 2682 through SEQ ID NO: 2689 or fragment
thereof that encode for a glucose-6-phosphate 1-dehydrogenase
protein or fragment thereof, SEQ ID NO: 1255 through SEQ ID NO:
1360 and SEQ ID NO: 2690 through SEQ ID NO: 2740 or fragment
thereof that encode for a phosphoglucomutase protein or fragment
thereof and SEQ ID NO: 1361 through SEQ ID NO: 1537 and SEQ IID NO:
2741 through SEQ ID NO: 2814 or fragment thereof that encode for an
UDP-glucose pyrophosphorylase protein or fragment thereof.
[0184] One or more of the protein or fragment of peptide molecules
may be produced via chemical synthesis, or more preferably, by
expressing in a suitable bacterial or eucaryotic host. Suitable
methods for expression are described by Sambrook et al., (In:
Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. (1989)), or similar texts.
For example, the protein may be expressed in, for example, Uses of
the Agents of the Invention, Section (a) Plant Constructs and Plant
Transformants; Section (b) Fungal Constructs and Fungal
Transformants; Section (c) Mammalian Constructs and Transformed
Mammalian Cells; Section (d) Insect Constructs and Transformed
Insect Cells; and Section (e) Bacterial Constructs and Transformed
Bacterial Cells.
[0185] A "protein fragment" is a peptide or polypeptide molecule
whose amino acid sequence comprises a subset of the amino acid
sequence of that protein. A protein or fragment thereof that
comprises one or more additional peptide regions not derived from
that protein is a "fusion" protein. Such molecules may be
derivatized to contain carbohydrate or other moieties (such as
keyhole limpet hemocyanin, etc.). Fusion protein or peptide
molecules of the present invention are preferably produced via
recombinant means.
[0186] Another class of agents comprise protein or peptide
molecules or fragments or fusions thereof encoded by SEQ ID NO: 1
through SEQ ID NO: 2814 or complements thereof in which
conservative, non-essential or non-relevant amino acid residues
have been added, replaced or deleted. Computerized means for
designing modifications in protein structure are known in the art
(Dahiyat and Mayo, Science 278:82-87 (1997), the entirety of which
is herein incorporated by reference).
[0187] The protein molecules of the present invention include plant
homologue proteins. An example of such a homologue is a homologue
protein of a non-maize or non-soybean plant species, that include
but not limited to alfalfa, Arabidopsis, barley, Brassica,
broccoli, cabbage, citrus, cotton, garlic, oat, oilseed rape,
onion, canola, flax, an ornamental plant, pea, peanut, pepper,
potato, rice, rye, sorghum, strawberry, sugarcane, sugarbeet,
tomato, wheat, poplar, pine, fir, eucalyptus, apple, lettuce,
lentils, grape, banana, tea, turf grasses, sunflower, oil palm,
Phaseolus etc. Particularly preferred non-maize or non-soybean for
use for the isolation of homologs would include, Arabidopsis,
barley, cotton, oat, oilseed rape, rice, canola, ornamentals,
sugarcane, sugarbeet, tomato, potato, wheat and turf grasses. Such
a homologue can be obtained by any of a variety of methods. Most
preferably, as indicated above, one or more of the disclosed
sequences (SEQ ID NO: 1 through SEQ ID NO: 2814 or complements
thereof) will be used to define a pair of primers that may be used
to isolate the homologue-encoding nucleic acid molecules from any
desired species. Such molecules can be expressed to yield
homologues by recombinant means.
[0188] (c) Antibodies
[0189] One aspect of the present invention concerns antibodies,
single-chain antigen binding molecules, or other proteins that
specifically bind to one or more of the protein or peptide
molecules of the present invention and their homologues, fusions or
fragments. Such antibodies may be used to quantitatively or
qualitatively detect the protein or peptide molecules of the
present invention. As used herein, an antibody or peptide is said
to "specifically bind" to a protein or peptide molecule of the
present invention if such binding is not competitively inhibited by
the presence of non-related molecules.
[0190] Nucleic acid molecules that encode all or part of the
protein of the present invention can be expressed, via recombinant
means, to yield protein or peptides that can in turn be used to
elicit antibodies that are capable of binding the expressed protein
or peptide. Such antibodies may be used in immunoassays for that
protein. Such protein-encoding molecules, or their fragments may be
a "fusion" molecule (i.e., a part of a larger nucleic acid
molecule) such that, upon expression, a fusion protein is produced.
It is understood that any of the nucleic acid molecules of the
present invention may be expressed, via recombinant means, to yield
proteins or peptides encoded by these nucleic acid molecules.
[0191] The antibodies that specifically bind proteins and protein
fragments of the present invention may be polyclonal or monoclonal
and may comprise intact immunoglobulins, or antigen binding
portions of immunoglobulins fragments (such as (F(ab'),
F(ab').sub.2), or single-chain immunoglobulins producible, for
example, via recombinant means. It is understood that practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of antibodies (see, for example, Harlow
and Lane, In: Antibodies: A Laboratory Manual, Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. (1988), the entirety of which is
herein incorporated by reference).
[0192] Murine monoclonal antibodies are particularly preferred.
BALB/c mice are preferred for this purpose, however, equivalent
strains may also be used. The animals are preferably immunized with
approximately 25 .mu.g of purified protein (or fragment thereof)
that has been emulsified in a suitable adjuvant (such as TiterMax
adjuvant (Vaxcel, Norcross, Ga.)). Immunization is preferably
conducted at two intramuscular sites, one intraperitoneal site and
one subcutaneous site at the base of the tail. An additional i.v.
injection of approximately 25 .mu.g of antigen is preferably given
in normal saline three weeks later. After approximately 11 days
following the second injection, the mice may be bled and the blood
screened for the presence of anti-protein or peptide antibodies.
Preferably, a direct binding Enzyme-Linked Immunoassay (ELISA) is
employed for this purpose.
[0193] More preferably, the mouse having the highest antibody titer
is given a third i.v. injection of approximately 25 .mu.g of the
same protein or fragment. The splenic leukocytes from this animal
may be recovered 3 days later and then permitted to fuse, most
preferably, using polyethylene glycol, with cells of a suitable
myeloma cell line (such as, for example, the P3X63Ag8.653 myeloma
cell line). Hybridoma cells are selected by culturing the cells
under "HAT" (hypoxanthine-aminopterin-thymine) selection for about
one week. The resulting clones may then be screened for their
capacity to produce monoclonal antibodies ("mAbs"), preferably by
direct ELISA.
[0194] In one embodiment, anti-protein or peptide monoclonal
antibodies are isolated using a fusion of a protein or peptide of
the present invention, or conjugate of a protein or peptide of the
present invention, as immunogens. Thus, for example, a group of
mice can be immunized using a fusion protein emulsified in Freund's
complete adjuvant (e.g. approximately 50 .mu.g of antigen per
immunization). At three week intervals, an identical amount of
antigen is emulsified in Freund's incomplete adjuvant and used to
immunize the animals. Ten days following the third immunization,
serum samples are taken and evaluated for the presence of antibody.
If antibody titers are too low, a fourth booster can be employed.
Polysera capable of binding the protein or peptide can also be
obtained using this method.
[0195] In a preferred procedure for obtaining monoclonal
antibodies, the spleens of the above-described immunized mice are
removed, disrupted and immune splenocytes are isolated over a
ficoll gradient. The isolated splenocytes are fused, using
polyethylene glycol with BALB/c-derived HGPRT (hypoxanthine guanine
phosphoribosyl transferase) deficient P3.times.63xAg8.653
plasmacytoma cells. The fused cells are plated into 96 well
microtiter plates and screened for hybridoma fusion cells by their
capacity to grow in culture medium supplemented with hypothanthine,
aminopterin and thymidine for approximately 2-3 weeks.
[0196] Hybridoma cells that arise from such incubation are
preferably screened for their capacity to produce an immunoglobulin
that binds to a protein of interest. An indirect ELISA may be used
for this purpose. In brief, the supernatants of hybridomas are
incubated in microtiter wells that contain immobilized protein.
After washing, the titer of bound immunoglobulin can be determined
using, for example, a goat anti-mouse antibody conjugated to
horseradish peroxidase. After additional washing, the amount of
immobilized enzyme is determined (for example through the use of a
chromogenic substrate). Such screening is performed as quickly as
possible after the identification of the hybridoma in order to
ensure that a desired clone is not overgrown by non-secreting
neighbor cells. Desirably, the fusion plates are screened several
times since the rates of hybridoma growth vary. In a preferred
sub-embodiment, a different antigenic form may be used to screen
the hybridoma. Thus, for example, the splenocytes may be immunized
with one immunogen, but the resulting hybridomas can be screened
using a different immunogen. It is understood that any of the
protein or peptide molecules of the present invention may be used
to raise antibodies.
[0197] As discussed below, such antibody molecules or their
fragments may be used for diagnostic purposes. Where the antibodies
are intended for diagnostic purposes, it may be desirable to
derivatize them, for example with a ligand group (such as biotin)
or a detectable marker group (such as a fluorescent group, a
radioisotope or an enzyme).
[0198] The ability to produce antibodies that bind the protein or
peptide molecules of the present invention permits the
identification of mimetic compounds of those molecules. A "mimetic
compound" is a compound that is not that compound, or a fragment of
that compound, but which nonetheless exhibits an ability to
specifically bind to antibodies directed against that compound.
[0199] It is understood that any of the agents of the present
invention can be substantially purified and/or be biologically
active and/or recombinant.
Uses of the Agents of the Invention
[0200] Nucleic acid molecules and fragments thereof of the present
invention may be employed to obtain other nucleic acid molecules
from the same species (e.g., ESTs or fragment thereof from maize
may be utilized to obtain other nucleic acid molecules from maize).
Such nucleic acid molecules include the nucleic acid molecules that
encode the complete coding sequence of a protein and promoters and
flanking sequences of such molecules. In addition, such nucleic
acid molecules include nucleic acid molecules that encode for other
isozymes or gene family members. Such molecules can be readily
obtained by using the above-described nucleic acid molecules or
fragments thereof to screen cDNA or genomic libraries obtained from
maize or soybean. Methods for forming such libraries are well known
in the art.
[0201] Nucleic acid molecules and fragments thereof of the present
invention may also be employed to obtain nucleic acid homologues.
Such homologues include the nucleic acid molecule of other plants
or other organisms (e.g., alfalfa, Arabidopsis, barley, Brassica,
broccoli, cabbage, citrus, cotton, garlic, oat, oilseed rape,
onion, canola, flax, an ornamental plant, pea, peanut, pepper,
potato, rice, rye, sorghum, strawberry, sugarcane, sugarbeet,
tomato, wheat, poplar, pine, fir; eucalyptus, apple, lettuce,
lentils, grape, banana, tea, turf grasses, sunflower, oil palm,
Phaseolus, etc.) including the nucleic acid molecules that encode,
in whole or in part, protein homologues of other plant species or
other organisms, sequences of genetic elements such as promoters
and transcriptional regulatory elements. Such molecules can be
readily obtained by using the above-described nucleic acid
molecules or fragments thereof to screen cDNA or genomic libraries
obtained from such plant species. Methods for forming such
libraries are well known in the art. Such homologue molecules may
differ in their nucleotide sequences from those found in one or
more of SEQ ID NO: 1 through SEQ ID NO: 2814 or complements thereof
because complete complementarity is not needed for stable
hybridization. The nucleic acid molecules of the present invention
therefore also include molecules that, although capable of
specifically hybridizing with the nucleic acid molecules, may lack
"complete complementarity."
[0202] Any of a variety of methods may be used to obtain one or
more of the above-described nucleic acid molecules (Zamechik et
al., Proc. Natl. Acad. Sci. (U.S.A) 83:4143-4146 (1986), the
entirety of which is herein incorporated by reference; Goodchild et
al., Proc. Natl. Acad. Sci. (U.S.A.) 85:5507-5511 (1988), the
entirety of which is herein incorporated by reference; Wickstrom et
al., Proc. Natl. Acad. Sci. (U.S.A.) 85:1028-1032 (1988), the
entirety of which is herein incorporated by reference; Holt et al.,
Molec. Cell. Biol. 8:963-973 (1988), the entirety of which is
herein incorporated by reference; Gerwirtz et al., Science
242:1303-1306 (1988), the entirety of which is herein incorporated
by reference; Anfossi et al., Proc. Natl. Acad. Sci. (U.S.A.)
86:3379-3383 (1989), the entirety of which is herein incorporated
by reference; Becker et al., EMBO J. 8:3685-3691 (1989); the
entirety of which is herein incorporated by reference). Automated
nucleic acid synthesizers may be employed for this purpose. In lieu
of such synthesis, the disclosed nucleic acid molecules may be used
to define a pair of primers that can be used with the polymerase
chain reaction (Mullis et al., Cold Spring Harbor Symp. Quant.
Biol. 51:263-273 (1.986); Erlich et al., European Patent 50,424;
European Patent 84,796; European Patent 258,017; European Patent
237,362; Mullis, European Patent 201,184; Mullis et al., U.S. Pat.
No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki et al.,
U.S. Pat. No. 4,683,194, all of which are herein incorporated by
reference in their entirety) to amplify and obtain any desired
nucleic acid molecule or fragment.
[0203] Promoter sequence(s) and other genetic elements, including
but not limited to transcriptional regulatory flanking sequences,
associated with one or more of the disclosed nucleic acid sequences
can also be obtained using the disclosed nucleic acid sequence
provided herein. In one embodiment, such sequences are obtained by
incubating EST nucleic acid molecules or preferably fragments
thereof with members of genomic libraries (e.g. maize and soybean)
and recovering clones that hybridize to the EST nucleic acid
molecule or fragment thereof. In a second embodiment, methods of
"chromosome walking," or inverse PCR may be used to obtain such
sequences (Frohman et al., Proc. Natl. Acad. Sci. (U.S.A.)
85:8998-9002 (1988); Ohara et al., Proc. Natl. Acad. Sci. (U.S.A)
86:5673-5677 (1989); Pang et al., Biotechniques 22:1046-1048
(1977); Huang et al., Methods Mol. Biol. 69:89-96 (1997); Huang et
al., Method Mol. Biol. 67:287-294 (1997); Benkel et al., Genet.
Anal. 13:123-127 (1996); Hartl et al., Methods Mol. Biol.
58:293-301 (1996), all of which are herein incorporated by
reference in their entirety).
[0204] The nucleic acid molecules of the present invention may be
used to isolate promoters of cell enhanced, cell specific, tissue
enhanced, tissue specific, developmentally or environmentally
regulated expression profiles. Isolation and functional analysis of
the 5' flanking promoter sequences of these genes from genomic
libraries, for example, using genomic screening methods and PCR
techniques would result in the isolation of useful promoters and
transcriptional regulatory elements. These methods are known to
those of skill in the art and have been described (See, for
example, Birren et al., Genome Analysis: Analyzing DNA, 1, (1997),
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., the
entirety of which is herein incorporated by reference). Promoters
obtained utilizing the nucleic acid molecules of the present
invention could also be modified to affect their control
characteristics. Examples of such modifications would include but
are not limited to enhanced sequences as reported in Uses of the
Agents of the Invention, Section (a) Plant Constructs and Plant
Transformants. Such genetic elements could be used to enhance gene
expression of new and existing traits for crop improvements.
[0205] In one sub-aspect, such an analysis is conducted by
determining the presence and/or identity of polymorphism(s) by one
or more of the nucleic acid molecules of the present invention and
more preferably one or more of the EST nucleic acid molecule or
fragment thereof which are associated with a phenotype, or a
predisposition to that phenotype.
[0206] Any of a variety of molecules can be used to identify such
polymorphism(s). In one embodiment, one or more of the EST nucleic
acid molecules (or a sub-fragment thereof) may be employed as a
marker nucleic acid molecule to identify such polymorphism(s).
Alternatively, such polymorphisms can be detected through the use
of a marker nucleic acid molecule or a marker protein that is
genetically linked to (i.e., a polynucleotide that co-segregates
with) such polymorphism(s).
[0207] In an alternative embodiment, such polymorphisms can be
detected through the use of a marker nucleic acid molecule that is
physically linked to such polymorphism(s). For this purpose, marker
nucleic acid molecules comprising a nucleotide sequence of a
polynucleotide located within lmb of the polymorphism(s) and more
preferably within 100 kb of the polymorphism(s) and most preferably
within 1 Okb of the polymorphism(s) can be employed.
[0208] The genomes of animals and plants naturally undergo
spontaneous mutation in the course of their continuing evolution
(Gusella, Ann. Rev. Biochem. 55:831-854 (1986)). A "polymorphism"
is a variation or difference in the sequence of the gene or its
flanking regions that arises in some of the members of a species.
The variant sequence and the "original" sequence co-exist in the
species' population. In some instances, such co-existence is in
stable or quasi-stable equilibrium.
[0209] A polymorphism is thus said to be "allelic," in that, due to
the existence of the polymorphism, some members of a species may
have the original sequence (i.e., the original "allele") whereas
other members may have the variant sequence (i.e., the variant
"allele"). In the simplest case, only one variant sequence may
exist and the polymorphism is thus said to be di-allelic. In other
cases, the species' population may contain multiple alleles and the
polymorphism is termed tri-allelic, etc. A single gene may have
multiple different unrelated polymorphisms. For example, it may
have a di-allelic polymorphism at one site and a multi-allelic
polymorphism at another site.
[0210] The variation that defines the polymorphism may range from a
single nucleotide variation to the insertion or deletion of
extended regions within a gene. In some cases, the DNA sequence
variations are in regions of the genome that are characterized by
short tandem repeats (STRs) that include tandem di- or
tri-nucleotide repeated motifs of nucleotides. Polymorphisms
characterized by such tandem repeats are referred to as "variable
number tandem repeat" ("VNTR") polymorphisms. VNTRs have been used
in identity analysis (Weber, U.S. Pat. No. 5,075,217; Armour et
al., FEBS Lett. 307:113-115 (1992); Jones et al., Eur. J. Haematol.
39:144-147 (1987); Horn et al., PCT Patent Application WO91/14003;
Jeffreys, European Patent Application 370,719; Jeffreys, U.S. Pat.
No. 5,175,082; Jeffreys et al., Amer. J. Hum. Genet. 39:11-24
(1986); Jeffreys et al., Nature 316:76-79 (1985); Gray et al.,
Proc. R. Acad. Soc. Lond. 243:241-253 (1991); Moore et al.,
Genomics 10:654-660 (1991); Jeffreys et al., Anim. Genet. 18:1-15
(1987); Hillel et al., Anim. Genet. 20:145-155 (1989); Hillel et
al., Genet. 124:783-789 (1990), all of which are herein
incorporated by reference in their entirety).
[0211] The detection of polymorphic sites in a sample of DNA may be
facilitated through the use of nucleic acid amplification methods.
Such methods specifically increase the concentration of
polynucleotides that span the polymorphic site, or include that
site and sequences located either distal or proximal to it. Such
amplified molecules can be readily detected by gel electrophoresis
or other means.
[0212] The most preferred method of achieving such amplification
employs the polymerase chain reaction ("PCR") (Mullis et al., Cold
Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich et al.,
European Patent Appln. 50,424; European Patent Appln. 84,796;
European Patent Application 258,017; European Patent Appln.
237,362; Mullis, European Patent Appln. 201,184; Mullis et al.,
U.S. Pat. No. 4,683,202; Erlich, U.S. Pat. No. 4,582,788; and Saiki
et al., U.S. Pat. No. 4,683,194), using primer pairs that are
capable of hybridizing to the proximal sequences that define a
polymorphism in its double-stranded form.
[0213] In lieu of PCR, alternative methods, such as the "Ligase
Chain Reaction" ("LCR") may be used (Barany, Proc. Natl. Acad. Sci.
(U.S.A) 88:189-193 (1991), the entirety of which is herein
incorporated by reference). LCR uses two pairs of oligonucleotide
probes to exponentially amplify a specific target. The sequences of
each pair of oligonucleotides is selected to permit the pair to
hybridize to abutting sequences of the same strand of the target.
Such hybridization forms a substrate for a template-dependent
ligase. As with PCR, the resulting products thus serve as a
template in subsequent cycles and an exponential amplification of
the desired sequence is obtained.
[0214] LCR can be performed with oligonucleotides having the
proximal and distal sequences of the same strand of a polymorphic
site. In one embodiment, either oligonucleotide will be designed to
include the actual polymorphic site of the polymorphism. In such an
embodiment, the reaction conditions are selected such that the
oligonucleotides can be ligated together only if the target
molecule either contains or lacks the specific nucleotide that is
complementary to the polymorphic site present on the
oligonucleotide. Alternatively, the oligonucleotides may be
selected such that they do not include the polymorphic site (see,
Segev, PCT Application WO 90/01069, the entirety of which is herein
incorporated by reference).
[0215] The "Oligonucleotide Ligation Assay" ("OLA") may
alternatively be employed (Landegren et al., Science 241:1077-1080
(1988), the entirety of which is herein incorporated by reference).
The OLA protocol uses two oligonucleotides which are designed to be
capable of hybridizing to abutting sequences of a single strand of
a target. OLA, like LCR, is particularly suited for the detection
of point mutations. Unlike LCR, however, OLA results in "linear"
rather than exponential amplification of the target sequence.
[0216] Nickerson et al., have described a nucleic acid detection
assay that combines attributes of PCR and OLA (Nickerson et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927 (1990), the entirety
of which is herein incorporated by reference). In this method, PCR
is used to achieve the exponential amplification of target DNA,
which is then detected using OLA. In addition to requiring multiple
and separate, processing steps, one problem associated with such
combinations is that they inherit all of the problems associated
with PCR and OLA.
[0217] Schemes based on ligation of two (or more) oligonucleotides
in the presence of nucleic acid having the sequence of the
resulting "di-oligonucleotide", thereby amplifying the
di-oligonucleotide, are also known (Wu et al., Genomics 4:560-569
(1989), the entirety of which is herein incorporated by reference)
and may be readily adapted to the purposes of the present
invention.
[0218] Other known nucleic acid amplification procedures, such as
allele-specific oligomers, branched DNA technology,
transcription-based amplification systems, or isothermal
amplification methods may also be used to amplify and analyze such
polymorphisms (Malek et al., U.S. Pat. No. 5,130,238; Davey et al.,
European Patent Application 329,822; Schuster et al., U.S. Pat. No.
5,169,766; Miller et al., PCT Patent Application WO 89/06700; Kwoh
et al., Proc. Natl. Acad. Sci. (U.S.A) 86:1173-1177 (1989);
Gingeras et al., PCT Patent Application WO 88/10315; Walker et al.,
Proc. Natl. Acad. Sci. (U.S.A) 89:392-396 (1992), all of which are
herein incorporated by reference in their entirety).
[0219] The identification of a polymorphism can be determined in a
variety of ways. By correlating the presence or absence of it in a
plant with the presence or absence of a phenotype, it is possible
to predict the phenotype of that plant. If a polymorphism creates
or destroys a restriction endonuclease cleavage site, or if it
results in the loss or insertion of DNA (e.g., a VNTR
polymorphism), it will alter the size or profile of the DNA
fragments that are generated by digestion with that restriction
endonuclease. As such, individuals that possess a variant sequence
can be distinguished from those having the original sequence by
restriction fragment analysis. Polymorphisms that can be identified
in this manner are termed "restriction fragment length
polymorphisms" ("RFLPs"). RFLPs have been widely used in human and
plant genetic analyses (Glassberg, UK Patent Application 2135774;
Skolnick et al., Cytogen. Cell Genet. 32:58-67 (1982); Botstein et
al., Ann. J. Hum. Genet. 32:314-331 (1980); Fischer et al., (PCT
Application WO90/13668); Uhlen, PCT Application WO90/11369).
[0220] Polymorphisms can also be identified by Single Strand
Conformation Polymorphism (SSCP) analysis. SSCP is a method capable
of identifying most sequence variations in a single strand of DNA,
typically between 150 and 250 nucleotides in length (Elles, Methods
in Molecular Medicine Molecular Diagnosis of Genetic Diseases,
Humana Press (1996), the entirety of which is herein incorporated
by reference); Orita et al., Genomics 5:874-879 (1989), the
entirety of which is herein incorporated by reference). Under
denaturing conditions a single strand of DNA will adopt a
conformation that is uniquely dependent on its sequence
conformation. This conformation usually will be different, even if
only a single base is changed. Most conformations have been
reported to alter the physical configuration or size sufficiently
to be detectable by electrophoresis. A number of protocols have
been described for SSCP including, but not limited to, Lee et al.,
Anal. Biochem. 205:289-293 (1992), the entirety of which is herein
incorporated by reference; Suzuki et al., Anal. Biochem. 192:82-84
(1991), the entirety of which is herein incorporated by reference;
Lo et al., Nucleic Acids Research 20:1005-1009 (1992), the entirety
of which is herein incorporated by reference; Sarkar et al.,
Genomics 13:441-443 (1992), the entirety of which is herein
incorporated by reference. It is understood that one or more of the
nucleic acids of the present invention, may be utilized as markers
or probes to detect polymorphisms by SSCP analysis.
[0221] Polymorphisms may also be found using a DNA fingerprinting
technique called amplified fragment length polymorphism (AFLP),
which is based on the selective PCR amplification of restriction
fragments from a total digest of genomic DNA to profile that DNA
(Vos et al., Nucleic Acids Res. 23:4407-4414 (1995), the entirety
of which is herein incorporated by reference). This method allows
for the specific co-amplification of high numbers of restriction
fragments, which can be visualized by PCR without knowledge of the
nucleic acid sequence.
[0222] AFLP employs basically three steps. Initially, a sample of
genomic DNA is cut with restriction enzymes and oligonucleotide
adapters are ligated to the restriction fragments of the DNA. The
restriction fragments are then amplified using PCR by using the
adapter and restriction sequence as target sites for primer
annealing. The selective amplification is achieved by the use of
primers that extend into the restriction fragments, amplifying only
those fragments in which the primer extensions match the nucleotide
flanking the restriction sites. These amplified fragments are then
visualized on a denaturing polyacrylamide gel.
[0223] AFLP analysis has been performed on Salix (Beismann et al.,
Mol. Ecol. 6:989-993 (1997), the entirety of which is herein
incorporated by reference), Acinetobacter (Janssen et al., Int. J.
Syst. Bacteriol. 47:1179-1187 (1997), the entirety of which is
herein incorporated by reference), Aeromonas popoffi (Huys et al.,
Int. J. Syst. Bacteriol. 47:1165-1171 (1997), the entirety of which
is herein incorporated by reference), rice (McCouch et al., Plant
Mol. Biol. 35:89-99 (1997), the entirety of which is herein
incorporated by reference; Nandi et al., Mol. Gen. Genet. 255:1-8
(1997), the entirety of which is herein incorporated by reference;
Cho et al., Genome 39:373-378 (1996), the entirety of which is
herein incorporated by reference), barley (Hordeum vulgare)(Simons
et al., Genomics 44:61-70 (1997), the entirety of which is herein
incorporated by reference; Waugh et al., Mol. Gen. Genet.
255:311-321 (1997), the entirety of which is herein incorporated by
reference; Qi et al., Mol. Gen. Genet. 254:330-336 (1997), the
entirety of which is herein incorporated by reference; Becker et
al., Mol. Gen. Genet. 249:65-73 (1995), the entirety of which is
herein incorporated by reference), potato (Van der Voort et al.,
Mol. Gen. Genet. 255:438-447 (1997), the entirety of which is
herein incorporated by reference; Meksem et al., Mol. Gen. Genet.
249:74-81 (1995), the entirety of which is herein incorporated by
reference), Phytophthora infestans (Van der Lee et al., Fungal
Genet. Biol. 21:278-291 (1997), the entirety of which is herein
incorporated by reference), Bacillus anthracis (Keim et al., J.
Bacteriol. 179:818-824 (1997), the entirety of which is herein
incorporated by reference), Astragalus cremnophylax (Travis et al.,
Mol. EcoL 5:735-745 (1996), the entirety of which is herein
incorporated by reference), Arabidopsis (Cnops et al., Mol. Gen.
Genet. 253:32-41 (1996), the entirety of which is herein
incorporated by reference), Escherichia coli (Lin et al., Nucleic
Acids Res. 24:3649-3650 (1996), the entirety of which is herein
incorporated by reference), Aeromonas (Huys et al., Int. J. Syst.
Bacteriol. 46:572-580 (1996), the entirety of which is herein
incorporated by reference), nematode (Folkertsma et al., Mol.
Plant. Microbe Interact. 9:47-54 (1996), the entirety of which is
herein incorporated by reference), tomato (Thomas et al., Plant J
8:785-794 (1995), the entirety of which is herein incorporated by
reference) and human (Latorra et al., PCR Methods Appl. 3:351-358
(1994), the entirety of which is herein incorporated by reference).
AFLP analysis has also been used for fingerprinting mRNA (Money et
al., Nucleic Acids Res. 24:2616-2617 (1996), the entirety of which
is herein incorporated by reference; Bachem et al., Plant J.
9:745-753 (1996), the entirety of which is herein incorporated by
reference). It is understood that one or more of the nucleic acids
of the present invention, may be utilized as markers or probes to
detect polymorphisms by AFLP analysis or for fingerprinting
RNA.
[0224] Polymorphisms may also be found using random amplified
polymorphic DNA (RAPD) (Williams et al., Nucl. Acids Res.
18:6531-6535 (1990), the entirety of which is herein incorporated
by reference) and cleaveable amplified polymorphic sequences (CAPS)
(Lyamichev et al., Science 260:778-783 (1993), the entirety of
which is herein incorporated by reference). It is understood that
one or more of the nucleic acid molecules of the present invention,
may be utilized as markers or probes to detect polymorphisms by
RAPD or CAPS analysis.
[0225] Through genetic mapping, a fine scale linkage map can be
developed using DNA markers and, then, a genomic DNA library of
large-sized fragments can be screened with molecular markers linked
to the desired trait. Molecular markers are advantageous for
agronomic traits that are otherwise difficult to tag, such as
resistance to pathogens, insects and nematodes, tolerance to
abiotic stress, quality parameters and quantitative traits such as
high yield potential.
[0226] The essential requirements for marker-assisted selection in
a plant breeding program are: (1) the marker(s) should co-segregate
or be closely linked with the desired trait; (2) an efficient means
of screening large populations for the molecular marker(s) should
be available; and (3) the screening technique should have high
reproducibility across laboratories and preferably be economical to
use and be user-friendly.
[0227] The genetic linkage of marker molecules can be established
by a gene mapping model such as, without limitation, the flanking
marker model reported by Lander and Botstein, Genetics 121:185-199
(1989) and the interval mapping, based on maximum likelihood
methods described by Lander and Botstein, Genetics 121:185-199
(1989) and implemented in the software package MAPMAKER/QTL
(Lincoln and Lander, Mapping Genes Controlling Quantitative Traits
Using MAPMAKER/QTL, Whitehead Institute for Biomedical Research,
Massachusetts, (1990). Additional software includes Qgene, Version
2.23 (1996), Department of Plant Breeding and Biometry, 266 Emerson
Hall, Cornell University, Ithaca, N.Y., the manual of which is
herein incorporated by reference in its entirety). Use of Qgene
software is a particularly preferred approach.
[0228] A maximum likelihood estimate (MLE) for the presence of a
marker is calculated, together with an MLE assuming no QTL effect,
to avoid false positives. A log.sub.10 of an odds ratio (LOD) is
then calculated as: LOD=log.sub.10 (MLE for the presence of a
QTL/MLE given no linked QTL).
[0229] The LOD score essentially indicates how much more likely the
data are to have arisen assuming the presence of a QTL than in its
absence. The LOD threshold value for avoiding a false positive with
a given confidence, say 95%, depends on the number of markers and
the length of the genome. Graphs indicating LOD thresholds are set
forth in Lander and Botstein, Genetics 121:185-199 (1989) the
entirety of which is herein incorporated by reference and further
described by Ar s and Moreno-Gonzalez, Plant Breeding, Hayward et
al., (eds.) Chapman & Hall, London, pp. 314-331 (1993), the
entirety of which is herein incorporated by reference.
[0230] Additional models can be used. Many modifications and
alternative approaches to interval mapping have been reported,
including the use non-parametric methods (Kruglyak and Lander,
Genetics 139:1421-1428 (1995), the entirety of which is herein
incorporated by reference). Multiple regression methods or models
can be also be used, in which the trait is regressed on a large
number of markers (Jansen, Biometrics in Plant Breeding, van Oijen
and Jansen (eds.), Proceedings of the Ninth Meeting of the Eucarpia
Section Biometrics in Plant Breeding, The Netherlands, pp. 116-124
(1994); Weber and Wricke, Advances in Plant Breeding, Blackwell,
Berlin, 16 (1994), both of which is herein incorporated by
reference in their entirety). Procedures combining interval mapping
with regression analysis, whereby the phenotype is regressed onto a
single putative QTL at a given marker interval and at the same time
onto a number of markers that serve as `cofactors,` have been
reported by Jansen and Stam, Genetics 136:1447-1455 (1994), the
entirety of which is herein incorporated by reference and Zeng,
Genetics 136:1457-1468 (1994) the entirety of which is herein
incorporated by reference. Generally, the use of cofactors reduces
the bias and sampling error of the estimated QTL positions (Utz and
Melchinger, Biometrics in Plant Breeding, van Oijen and Jansen
(eds.) Proceedings of the Ninth Meeting of the Eucarpia Section
Biometrics in Plant Breeding, The Netherlands, pp. 195-204 (1994),
the entirety of which is herein incorporated by reference, thereby
improving the precision and efficiency of QTL mapping (Zeng,
Genetics 136:1457-1468 (1994)). These models can be extended to
multi-environment experiments to analyze genotype-environment
interactions (Jansen et al., Theo. Appl. Genet. 91:33-37 (1995),
the entirety of which is herein incorporated by reference).
[0231] Selection of an appropriate mapping populations is important
to map construction. The choice of appropriate mapping population
depends on the type of marker systems employed (Tanksley et al.,
Molecular mapping plant chromosomes. Chromosome structure
andfunction: Impact of new concepts, Gustafson and Appels (eds.),
Plenum Press, New York, pp 157-173 (1988), the entirety of which is
herein incorporated by reference). Consideration must be given to
the source of parents (adapted vs. exotic) used in the mapping
population. Chromosome pairing and recombination rates can be
severely disturbed (suppressed) in wide crosses
(adapted.times.exotic) and generally yield greatly reduced linkage
distances. Wide crosses will usually provide segregating
populations with a relatively large array of polymorphisms when
compared to progeny in a narrow cross (adapted.times.adapted).
[0232] An F.sub.2 population is the first generation of selfing
after the hybrid seed is produced. Usually a single F.sub.1 plant
is selfed to generate a population segregating for all the genes in
Mendelian (1:2:1) fashion. Maximum genetic information is obtained
from a completely classified F.sub.2 population using a codominant
marker system (Mather, Measurement of Linkage in Heredity, Methuen
and Co., (1938), the entirety of which is herein incorporated by
reference). In the case of dominant markers, progeny tests (e.g.
F.sub.3, BCF.sub.2) are required to identify the heterozygotes,
thus making it equivalent to a completely classified F.sub.2
population. However, this procedure is often prohibitive because of
the cost and time involved in progeny testing. Progeny testing of
F.sub.2 individuals is often used in map construction where
phenotypes do not consistently reflect genotype (e.g. disease
resistance) or where trait expression is controlled by a QTL.
Segregation data from progeny test populations (e.g. F.sub.3 or
BCF.sub.2) can be used in map construction. Marker-assisted
selection can then be applied to cross progeny based on
marker-trait map associations (F.sub.2, F.sub.3), where linkage
groups have not been completely disassociated by recombination
events (i.e., maximum disequillibrium).
[0233] Recombinant inbred lines (RIL) (genetically related lines;
usually >F.sub.5, developed from continuously selfing F.sub.2
lines towards homozygosity) can be used as a mapping population.
Information obtained from dominant markers can be maximized by
using RIL because all loci are homozygous or nearly so. Under
conditions of tight linkage (i.e., about <10% recombination),
dominant and co-dominant markers evaluated in RIL populations
provide more information per individual than either marker type in
backcross populations (Reiter et al., Proc. Natl. Acad. Sci.
(U.S.A.) 89:1477-1481 (1992), the entirety of which is herein
incorporated by reference). However, as the distance between
markers becomes larger (i.e., loci become more independent), the
information in RIL populations decreases dramatically when compared
to codominant markers.
[0234] Backcross populations (e.g., generated from a cross between
a successful variety (recurrent parent) and another variety (donor
parent) carrying a trait not present in the former) can be utilized
as a mapping population. A series of backcrosses to the recurrent
parent can be made to recover most of its desirable traits. Thus a
population is created consisting of individuals nearly like the
recurrent parent but each individual carries varying amounts or
mosaic of genomic regions from the donor parent. Backcross
populations can be useful for mapping dominant markers if all loci
in the recurrent parent are homozygous and the donor and recurrent
parent have contrasting polymorphic marker alleles (Reiter et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 89:1477-1481 (1992)). Information
obtained from backcross populations using either codominant or
dominant markers is less than that obtained from F.sub.2
populations because one, rather than two, recombinant gametes are
sampled per plant. Backcross populations, however, are more
informative (at low marker saturation) when compared to RILs as the
distance between linked loci increases in RIL populations (i.e.
about 15% recombination). Increased recombination can be beneficial
for resolution of tight linkages, but may be undesirable in the
construction of maps with low marker saturation.
[0235] Near-isogenic lines (NIL) created by many backcrosses to
produce an array of individuals that are nearly identical in
genetic composition except for the trait or genomic region under
interrogation can be used as a mapping population. In mapping with
NILs, only a portion of the polymorphic loci are expected to map to
a selected region.
[0236] Bulk segregant analysis (BSA) is a method developed for the
rapid identification of linkage between markers and traits of
interest (Michelmore et al., Proc. Natl. Acad. Sci. (U.S.A.)
88:9828-9832 (1991), the entirety of which is herein incorporated
by reference). In BSA, two bulked DNA samples are drawn from a
segregating population originating from a single cross. These bulks
contain individuals that are identical for a particular trait
(resistant or susceptible to particular disease) or genomic region
but arbitrary at unlinked regions (i.e. heterozygous). Regions
unlinked to the target region will not differ between the bulked
samples of many individuals in BSA.
[0237] It is understood that one or more of the nucleic acid
molecules of the present invention may be used as molecular
markers. It is also understood that one or more of the protein
molecules of the present invention may be used as molecular
markers.
[0238] In accordance with this aspect of the present invention, a
sample nucleic acid is obtained from plants cells or tissues. Any
source of nucleic acid may be used. Preferably, the nucleic acid is
genomic DNA. The nucleic acid is subjected to restriction
endonuclease digestion. For example, one or more nucleic acid
molecule or fragment thereof of the present invention can be used
as a probe in accordance with the above-described polymorphic
methods. The polymorphism obtained in this approach can then be
cloned to identify the mutation at the coding region which alters
the protein's structure or regulatory region of the gene which
affects its expression level.
[0239] In an aspect of the present invention, one or more of the
nucleic molecules of the present invention are used to determine
the level (i.e., the concentration of mRNA in a sample, etc.) in a
plant (preferably maize or soybean) or pattern (i.e., the kinetics
of expression, rate of decomposition, stability profile, etc.) of
the expression of a protein encoded in part or whole by one or more
of the nucleic acid molecule of the present invention
(collectively, the "Expression Response" of a cell or tissue). As
used herein, the Expression Response manifested by a cell or tissue
is said to be "altered" if it differs from the Expression Response
of cells or tissues of plants not exhibiting the phenotype. To
determine whether a Expression Response is altered, the Expression
Response manifested by the cell or tissue of the plant exhibiting
the phenotype is compared with that of a similar cell or tissue
sample of a plant not exhibiting the phenotype. As will be
appreciated, it is not necessary to re-determine the Expression
Response of the cell or tissue sample of plants not exhibiting the
phenotype each time such a comparison is made; rather, the
Expression Response of a particular plant may be compared with
previously obtained values of normal plants. As used herein, the
phenotype of the organism is any of one or more characteristics of
an organism (e.g. disease resistance, pest tolerance, environmental
tolerance such as tolerance to abiotic stress, male sterility,
quality improvement or yield etc.). A change in genotype or
phenotype may be transient or permanent. Also as used herein, a
tissue sample is any sample that comprises more than one cell. In a
preferred aspect, a tissue sample comprises cells that share a
common characteristic (e.g. derived from root, seed, flower, leaf,
stem or pollen etc.).
[0240] In one aspect of the present invention, an evaluation can be
conducted to determine whether a particular mRNA molecule is
present. One or more of the nucleic acid molecules of the present
invention, preferably one or more of the EST nucleic acid molecules
or fragments thereof of the present invention are utilized to
detect the presence or quantity of the mRNA species. Such molecules
are then incubated with cell or tissue extracts of a plant under
conditions sufficient to permit nucleic acid hybridization. The
detection of double-stranded probe-mRNA hybrid molecules is
indicative of the presence of the mRNA; the amount of such hybrid
formed is proportional to the amount of mRNA. Thus, such probes may
be used to ascertain the level and extent of the mRNA production in
a plant's cells or tissues. Such nucleic acid hybridization may be
conducted under quantitative conditions (thereby providing a
numerical value of the amount of the mRNA present). Alternatively,
the assay may be conducted as a qualitative assay that indicates
either that the mRNA is present, or that its level exceeds a user
set, predefined value.
[0241] A principle of in situ hybridization is that a labeled,
single-stranded nucleic acid probe will hybridize to a
complementary strand of cellular DNA or RNA and, under the
appropriate conditions, these molecules will form a stable hybrid.
When nucleic acid hybridization is combined with histological
techniques, specific DNA or RNA sequences can be identified within
a single cell. An advantage of in situ hybridization over more
conventional techniques for the detection of nucleic acids is that
it allows an investigator to determine the precise spatial
population (Angerer et al., Dev. Biol. 101:477-484 (1984), the
entirety of which is herein incorporated by reference; Angerer et
al., Dev. Biol. 112:157-166 (1985), the entirety of which is herein
incorporated by reference; Dixon et al., EMBO J. 10:1317-1324
(1991), the entirety of which is herein incorporated by reference).
In situ hybridization may be used to measure the steady-state level
of RNA accumulation. It is a sensitive technique and RNA sequences
present in as few as 5-10 copies per cell can be detected (Hardin
et al., J. Mol. Biol. 202:417-431 (1989), the entirety of which is
herein incorporated by reference). A number of protocols have been
devised for in situ hybridization, each with tissue preparation,
hybridization and washing conditions (Meyerowitz, Plant Mol. Biol.
Rep. 5:242-250 (1987), the entirety of which is herein incorporated
by reference; Cox and Goldberg, In: Plant Molecular Biology: A
Practical Approach, Shaw (ed.), pp 1-35, IRL Press, Oxford (1988),
the entirety of which is herein incorporated by reference; Raikhel
et al., In situ RNA hybridization in plant tissues, In: Plant
Molecular Biology Manual, vol. B9: 1-32, Kluwer Academic Publisher,
Dordrecht, Belgium (1989), the entirety of which is herein
incorporated by reference).
[0242] In situ hybridization also allows for the localization of
proteins within a tissue or cell (Wilkinson, In Situ Hybridization,
Oxford University Press, Oxford (1992), the entirety of which is
herein incorporated by reference; Langdale, In Situ Hybridization
In: The Maize Handbook, Freeling and Walbot (eds.), pp 165-179,
Springer-Verlag, New York (1994), the entirety of which is herein
incorporated by reference). It is understood that one or more of
the molecules of the present invention, preferably one or more of
the EST nucleic acid molecules or fragments thereof of the present
invention or one or more of the antibodies of the present invention
may be utilized to detect the level or pattern of a sucrose pathway
protein or mRNA thereof by in situ hybridization.
[0243] Fluorescent in situ hybridization allows the localization of
a particular DNA sequence along a chromosome which is useful, among
other uses, for gene mapping, following chromosomes in hybrid lines
or detecting chromosomes with translocations, transversions or
deletions. In situ hybridization has been used to identify
chromosomes in several plant species (Griffor et al., Plant Mol.
Biol. 17:101-109 (1991), the entirety of which is herein
incorporated by reference; Gustafson et al., Proc. Natl. Acad. Sci.
(U.S.A) 87:1899-1902 (1990), herein incorporated by reference;
Mukai and Gill, Genome 34:448-452 (1991), the entirety of which is
herein incorporated by reference; Schwarzacher and Heslop-Harrison,
Genome 34:317-323 (1991); Wang et al., Jpn. J. Genet. 66:313-316
(1991), the entirety of which is herein incorporated by reference;
Parra and Windle, Nature Genetics 5:17-21 (1993), the entirety of
which is herein incorporated by reference). It is understood that
the nucleic acid molecules of the present invention may be used as
probes or markers to localize sequences along a chromosome.
[0244] Another method to localize the expression of a molecule is
tissue printing. Tissue printing provides a way to screen, at the
same time on the same membrane many tissue sections from different
plants or different developmental stages. Tissue-printing
procedures utilize films designed to immobilize proteins and
nucleic acids. In essence, a freshly cut section of a tissue is
pressed gently onto nitrocellulose paper, nylon membrane or
polyvinylidene difluoride membrane. Such membranes are commercially
available (e.g. Millipore, Bedford, Mass. U.S.A.). The contents of
the cut cell transfer onto the membrane and the contents and are
immobilized to the membrane. The immobilized contents form a latent
print that can be visualized with appropriate probes. When a plant
tissue print is made on nitrocellulose paper, the cell walls leave
a physical print that makes the anatomy visible without further
treatment (Varner and Taylor, Plant Physiol. 91:31-33 (1989), the
entirety of which is herein incorporated by reference).
[0245] Tissue printing on substrate films is described by Daoust,
Exp. Cell Res. 12:203-211 (1957), the entirety of which is herein
incorporated by reference, who detected amylase, protease,
ribonuclease and deoxyribonuclease in animal tissues using starch,
gelatin and agar films. These techniques can be applied to plant
tissues (Yomo and Taylor, Planta 112:35-43 (1973); the entirety of
which is herein incorporated by reference; Harris and Chrispeels,
Plant Physiol. 56:292-299 (1975), the entirety of which is herein
incorporated by reference). Advances in membrane technology have
increased the range of applications of Daoust's tissue-printing
techniques allowing (Cassab and Varner, J. Cell. Biol.
105:2581-2588 (1987), the entirety of which is herein incorporated
by reference) the histochemical localization of various plant
enzymes and deoxyribonuclease on nitrocellulose paper and nylon
(Spruce et al., Phytochemistry 26:2901-2903 (1987), the entirety of
which is herein incorporated by reference; Barres et al., Neuron
5:527-544 (1990), the entirety of which is herein incorporated by
reference; Reid and Pont-Lezica, Tissue Printing: Tools for the
Study of Anatomy, Histochemistry and Gene Expression, Academic
Press, New York, N.Y. (1992), the entirety of which is herein
incorporated by reference; Reid et al., Plant Physiol. 93:160-165
(1990), the entirety of which is herein incorporated by reference;
Ye et al., Plant J. 1: 175-183 (1991), the entirety of which is
herein incorporated by reference).
[0246] It is understood that one or more of the molecules of the
present invention, preferably one or more of the EST nucleic acid
molecules or fragments thereof of the present invention or one or
more of the antibodies of the present invention may be utilized to
detect the presence or quantity of a sucrose pathway protein by
tissue printing.
[0247] Further it is also understood that any of the nucleic acid
molecules of the present invention may be used as marker nucleic
acids and or probes in connection with methods that require probes
or marker nucleic acids. As used herein, a probe is an agent that
is utilized to determine an attribute or feature (e.g. presence or
absence, location, correlation, etc.) of a molecule, cell, tissue
or plant. As used herein, a marker nucleic acid is a nucleic acid
molecule that is utilized to determine an attribute or feature
(e.g. presence or absence, location, correlation, etc.) or a
molecule, cell, tissue or plant.
[0248] A microarray-based method for high-throughput monitoring of
plant gene expression may be utilized to measure gene-specific
hybridization targets. This `chip`-based approach involves using
microarrays of nucleic acid molecules as gene-specific
hybridization targets to quantitatively measure expression of the
corresponding plant genes (Schena et al., Science 270:467-470
(1995), the entirety of which is herein incorporated by reference;
Shalon, Ph.D. Thesis, Stanford University (1996), the entirety of
which is herein incorporated by reference). Every nucleotide in a
large sequence can be queried at the same time. Hybridization can
be used to efficiently analyze nucleotide sequences.
[0249] Several microarray methods have been described. One method
compares the sequences to be analyzed by hybridization to a set of
oligonucleotides representing all possible subsequences (Bains and
Smith, J. Theor. Biol. 135:303-307 (1989), the entirety of which is
herein incorporated by reference). A second method hybridizes the
sample to an array of oligonucleotide or cDNA molecules. An array
consisting of oligonucleotides complementary to subsequences of a
target sequence can be used to determine the identity of a target
sequence, measure its amount and detect differences between the
target and a reference sequence. Nucleic acid molecules microarrays
may also be screened with protein molecules or fragments thereof to
determine nucleic acid molecules that specifically bind protein
molecules or fragments thereof.
[0250] The microarray approach may be used with polypeptide targets
(U.S. Pat. No. 5,445,934; U.S. Pat. No. 5,143,854; U.S. Pat. No.
5,079,600; U.S. Pat. No. 4,923,901, all of which are herein
incorporated by reference in their entirety). Essentially,
polypeptides are synthesized on a substrate (microarray) and these
polypeptides can be screened with either protein molecules or
fragments thereof or nucleic acid molecules in order to screen for
either protein molecules or fragments thereof or nucleic acid
molecules that specifically bind the target polypeptides. (Fodor et
al., Science 251:767-773 (1991), the entirety of which is herein
incorporated by reference). It is understood that one or more of
the nucleic acid molecules or protein or fragments thereof of the
present invention may be utilized in a microarray based method.
[0251] In a preferred embodiment of the present invention
microarrays may be prepared that comprise nucleic acid molecules
where such nucleic acid molecules encode at least one, preferably
at least two, more preferably at least three or preferably at least
four, preferably at least five, preferably at least six, preferably
at least seven, preferably at least eight, preferably at least
nine, preferably at least ten, preferably at least eleven,
preferably at least twelve, preferably at least thirteen,
preferably at least fourteen preferably at least fifteen sucrose
pathway enzymes. In a preferred embodiment the nucleic acid
molecules are selected from the group consisting of a nucleic acid
molecule that encodes a maize or a soybean triose phosphate
isomerase enzyme or fragment thereof, a nucleic acid molecule that
encodes a maize or a soybean fructose 1,6-bisphosphate aldolase
enzyme or fragment thereof, a nucleic acid molecule that encodes a
maize or a soybean fructose 1,6-bisphosphate enzyme or fragment
thereof, a nucleic acid molecule that encodes a maize or a soybean
fructose 6-phosphate 2-kinase enzyme or fragment thereof, a nucleic
acid molecule that encodes a maize or a soybean
phosphoglucoisomerase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean vacuolar H.sup.+
translocating-pyrophosphatase enzyme or fragment thereof, a nucleic
acid molecule that encodes a maize or a soybean
pyrophosphate-dependent fructose-6-phosphate phosphotransferase
enzyme or fragment thereof, a nucleic acid molecule that encodes a
maize or a soybean invertase enzyme or fragment thereof, a nucleic
acid molecule that encodes a maize or a soybean sucrose synthase
enzyme or fragment thereof, a nucleic acid molecule that encodes a
maize or a soybean hexokinase enzyme or fragment thereof, a nucleic
acid molecule that encodes a maize or a soybean fructokinase enzyme
or fragment thereof, a nucleic acid molecule that encodes a maize
or a soybean NDP-kinase enzyme or fragment thereof, a nucleic acid
molecule that encodes a maize or a soybean glucose-6-phosphate
1-dehydrogenase enzyme or fragment thereof, a nucleic acid molecule
that encodes a maize or a soybean phosphoglucomutase enzyme or
fragment thereof and a nucleic acid molecule that encodes a maize
or a soybean UDP-glucose pyrophosphorylase enzyme or fragment
thereof.
[0252] Site directed mutagenesis may be utilized to modify nucleic
acid sequences, particularly as it is a technique that allows one
or more of the amino acids encoded by a nucleic acid molecule to be
altered (e.g. a threonine to be replaced by a methionine). Three
basic methods for site directed mutagenesis are often employed.
These are cassette mutagenesis (Wells et al., Gene 34:315-323
(1985), the entirety of which is herein incorporated by reference),
primer extension (Gilliam et al., Gene 12:129-137 (1980), the
entirety of which is herein incorporated by reference; Zoller and
Smith, Methods Enzymol. 100:468-500 (1983), the entirety of which
is herein incorporated by reference; Dalbadie-McFarland et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 79:6409-6413 (1982), the entirety
of which is herein incorporated by reference) and methods based
upon PCR (Scharf et al., Science 233:1076-1078 (1986), the entirety
of which is herein incorporated by reference; Higuchi et al.,
Nucleic Acids Res. 16:7351-7367 (1988), the entirety of which is
herein incorporated by reference). Site directed mutagenesis
approaches are also described in European Patent 0 385 962, the
entirety of which is herein incorporated by reference; European
Patent 0 359 472, the entirety of which is herein incorporated by
reference; and PCT Patent Application WO 93/07278, the entirety of
which is herein incorporated by reference.
[0253] Site directed mutagenesis strategies have been applied to
plants for both in vitro as well as in vivo site directed
mutagenesis (Lanz et al., J. Biol. Chem. 266:9971-9976 (1991), the
entirety of which is herein incorporated by reference; Kovgan and
Zhdanov, Biotekhnologiya 5:148-154; No. 207160n, Chemical Abstracts
110:225 (1989), the entirety of which is herein incorporated by
reference; Ge et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:4037-4041
(1989), the entirety of which is herein incorporated by reference;
Zhu et al., J. Biol. Chem. 271:18494-18498 (1996), the entirety of
which is herein incorporated by reference; Chu et al., Biochemistry
33:6150-6157 (1994), the entirety of which is herein incorporated
by reference; Small et al., EMBO J. 11:1291-1296 (1992), the
entirety of which is herein incorporated by reference; Cho et al.,
Mol. Biotechnol. 8:13-16 (1997), the entirety of which is herein
incorporated by reference; Kita et al., J. Biol. Chem.
271:26529-26535 (1996), the entirety of which is herein
incorporated by reference, Jin et al., Mol. Microbiol. 7:555-562
(1993), the entirety of which is herein incorporated by reference;
Hatfield and Vierstra, J. Biol. Chem. 267:14799-14803 (1992), the
entirety of which is herein incorporated by reference; Zhao et al.,
Biochemistry 31:5093-5099 (1992), the entirety of which is herein
incorporated by reference).
[0254] Any of the nucleic acid molecules of the present invention
may either be modified by site directed mutagenesis or used as, for
example, nucleic acid molecules that are used to target other
nucleic acid molecules for modification. It is understood that
mutants with more than one altered nucleotide can be constructed
using techniques that practitioners are familiar with such as
isolating restriction fragments and ligating such fragments into an
expression vector (see, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Press (1989)).
[0255] Sequence-specific DNA-binding proteins play a role in the
regulation of transcription. The isolation of recombinant cDNAs
encoding these proteins facilitates the biochemical analysis of
their structural and functional properties. Genes encoding such
DNA-binding proteins have been isolated using classical genetics
(Vollbrecht et al., Nature 350: 241-243 (1991), the entirety of
which is herein incorporated by reference) and molecular
biochemical approaches, including the screening of recombinant cDNA
libraries with antibodies (Landschulz et al., Genes Dev. 2:786-800
(1988), the entirety of which is herein incorporated by reference)
or DNA probes (Bodner et al., Cell 55:505-518 (1988), the entirety
of which is herein incorporated by reference). In addition, an in
situ screening procedure has been used and has facilitated the
isolation of sequence-specific DNA-binding proteins from various
plant species (Gilmartin et al., Plant Cell 4:839-849 (1992), the
entirety of which is herein incorporated by reference; Schindler et
al., EMBO J. 11: 1261-1273 (1992), the entirety of which is herein
incorporated by reference). An in situ screening protocol does not
require the purification of the protein of interest (Vinson et al.,
Genes Dev. 2:801-806 (1988), the entirety of which is herein
incorporated by reference; Singh et al., Cell 52:415-423 (1988),
the entirety of which is herein incorporated by reference).
[0256] Two steps may be employed to characterize DNA-protein
interactions. The first is to identify promoter fragments that
interact with DNA-binding proteins, to titrate binding activity, to
determine the specificity of binding and to determine whether a
given DNA-binding activity can interact with related DNA sequences
(Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd
edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y. (1989)). Electrophoretic mobility-shift assay is a widely used
assay. The assay provides a rapid and sensitive method for
detecting DNA-binding proteins based on the observation that the
mobility of a DNA fragment through a nondenaturing, low-ionic
strength polyacrylamide gel is retarded upon association with a
DNA-binding protein (Fried and Crother, Nucleic Acids Res.
9:6505-6525 (1981), the entirety of which is herein incorporated by
reference). When one or more specific binding activities have been
identified, the exact sequence of the DNA bound by the protein may
be determined. Several procedures for characterizing
protein/DNA-binding sites are used, including methylation and
ethylation interference assays (Maxam and Gilbert, Methods Enzymol.
65:499-560 (1980), the entirety of which is herein incorporated by
reference; Wissman and Hillen, Methods Enzymol. 208:365-379 (1991),
the entirety of which is herein incorporated by reference),
footprinting techniques employing DNase I (Galas and Schmitz,
Nucleic Acids Res. 5:3157-3170 (1978), the entirety of which is
herein incorporated by reference), 1,10-phenanthroline-copper ion
methods (Sigman et al., Methods Enzymol. 208:414-433 (1991), the
entirety of which is herein incorporated by reference) and hydroxyl
radicals methods (Dixon et al., Methods Enzymol. 208:414-433
(1991), the entirety of which is herein incorporated by reference).
It is understood that one or more of the nucleic acid molecules of
the present invention may be utilized to identify a protein or
fragment thereof that specifically binds to a nucleic acid molecule
of the present invention. It is also understood that one or more of
the protein molecules or fragments thereof of the present invention
may be utilized to identify a nucleic acid molecule that
specifically binds to it.
[0257] A two-hybrid system is based on the fact that many cellular
functions are carried out by proteins, such as transcription
factors, that interact (physically) with one another. Two-hybrid
systems have been used to probe the function of new proteins (Chien
et al., Proc. Natl. Acad. Sci. (U.S.A) 88:9578-9582 (1991) the
entirety of which is herein incorporated by reference; Durfee et
al., Genes Dev. 7:555-569 (1993) the entirety of which is herein
incorporated by reference; Choi et al., Cell 78:499-512 (1994), the
entirety of which is herein incorporated by reference; Kranz et
al., Genes Dev. 8:313-327 (1994), the entirety of which is herein
incorporated by reference).
[0258] Interaction mating techniques have facilitated a number of
two-hybrid studies of protein-protein interaction. Interaction
mating has been used to examine interactions between small sets of
tens of proteins (Finley and Brent, Proc. Natl. Acad. Sci. (U.S.A.)
91:12098-12984 (1994), the entirety of which is herein incorporated
by reference), larger sets of hundreds of proteins (Bendixen et
al., Nucl. Acids Res. 22:1778-1779 (1994), the entirety of which is
herein incorporated by reference) and to comprehensively map
proteins encoded by a small genome (Bartel et al., Nature Genetics
12:72-77 (1996), the entirety of which is herein incorporated by
reference). This technique utilizes proteins fused to the
DNA-binding domain and proteins fused to the activation domain.
They are expressed in two different haploid yeast strains of
opposite mating type and the strains are mated to determine if the
two proteins interact. Mating occurs when haploid yeast strains
come into contact and result in the fusion of the two haploids into
a diploid yeast strain. An interaction can be determined by the
activation of a two-hybrid reporter gene in the diploid strain. An
advantage of this technique is that it reduces the number of yeast
transformations needed to test individual interactions. It is
understood that the protein-protein interactions of protein or
fragments thereof of the present invention may be investigated
using the two-hybrid system and that any of the nucleic acid
molecules of the present invention that encode such proteins or
fragments thereof may be used to transform yeast in the two-hybrid
system.
[0259] (a) Plant Constructs and Plant Transformants
[0260] One or more of the nucleic acid molecules of the present
invention may be used in plant transformation or transfection.
Exogenous genetic material may be transferred into a plant cell and
the plant cell regenerated into a whole, fertile or sterile plant.
Exogenous genetic material is any genetic material, whether
naturally occurring or otherwise, from any source that is capable
of being inserted into any organism. Such genetic material may be
transferred into either monocotyledons and dicotyledons including,
but not limited to maize (pp 63-69), soybean (pp 50-60),
Arabidopsis (p 45), phaseolus (pp 47-49), peanut (pp 49-50),
alfalfa (p 60), wheat (pp 69-71), rice (pp 72-79), oat (pp 80-81),
sorghum (p 83), rye (p 84), tritordeum (p 84), millet (p85), fescue
(p 85), perennial ryegrass (p 86), sugarcane (p87), cranberry
(p101), papaya (pp 101-102), banana (p 103), banana (p 103),
muskmelon (p 104), apple (p 104), cucumber (p 105), dendrobium (p
109), gladiolus (p110), chrysanthemum (p 110), liliacea (p 111),
cotton (pp113-114), eucalyptus (p 115), sunflower (p 118), canola
(p 118), turfgrass (p 121), sugarbeet (p 122), coffee (p 122) and
dioscorea (p 122), (Christou, In: Particle Bombardment for Genetic
Engineering of Plants, Biotechnology Intelligence Unit. Academic
Press, San Diego, Calif. (1996), the entirety of which is herein
incorporated by reference).
[0261] Transfer of a nucleic acid that encodes for a protein can
result in overexpression of that protein in a transformed cell or
transgenic plant. One or more of the proteins or fragments thereof
encoded by nucleic acid molecules of the present invention may be
overexpressed in a transformed cell or transformed plant.
Particularly, any of the sucrose pathway proteins or fragments
thereof may be overexpressed in a transformed cell or transgenic
plant. Such overexpression may be the result of transient or stable
transfer of the exogenous genetic material.
[0262] Exogenous genetic material may be transferred into a plant
cell and the plant cell by the use of a DNA vector or construct
designed for such a purpose. Design of such a vector is generally
within the skill of the art (See, Plant Molecular Biology: A
Laboratory Manual, Clark (ed.), Springier, New York (1997), the
entirety of which is herein incorporated by reference).
[0263] A construct or vector may include a plant promoter to
express the protein or protein fragment of choice. A number of
promoters which are active in plant cells have been described in
the literature. These include the nopaline synthase (NOS) promoter
(Ebert et al., Proc. Natl. Acad. Sci. (U.S.A.) 84:5745-5749 (1987),
the entirety of which is herein incorporated by reference), the
octopine synthase (OCS) promoter (which are carried on
tumor-inducing plasmids of Agrobacterium tumefaciens), the
caulimovirus promoters such as the cauliflower mosaic virus (CaMV)
19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324 (1987), the
entirety of which is herein incorporated by reference) and the CAMV
35S promoter (Odell et al., Nature 313:810-812 (1985), the entirety
of which is herein incorporated by reference), the figwort mosaic
virus 35S-promoter, the light-inducible promoter from the small
subunit of ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the
Adh promoter (Walker et al., Proc. Natl. Acad. Sci. (U.S.A.)
84:6624-6628 (1987), the entirety of which is herein incorporated
by reference), the sucrose synthase promoter (Yang et al., Proc.
Natl. Acad. Sci. (U.S.A) 87:4144-4148 (1990), the entirety of which
is herein incorporated by reference), the R gene complex promoter
(Chandler et al., The Plant Cell 1:1175-1183 (1989), the entirety
of which is herein incorporated by reference) and the chlorophyll
a/b binding protein gene promoter, etc. These promoters have been
used to create DNA constructs which have been expressed in plants;
see, e.g., PCT publication WO 84/02913, herein incorporated by
reference in its entirety.
[0264] Promoters which are known or are found to cause
transcription of DNA in plant cells can be used in the present
invention. Such promoters may be obtained from a variety of sources
such as plants and plant viruses. It is preferred that the
particular promoter selected should be capable of causing
sufficient expression to result in the production of an effective
amount of the sucrose pathway protein to cause the desired
phenotype. In addition to promoters that are known to cause
transcription of DNA in plant cells, other promoters may be
identified for use in the current invention by screening a plant
cDNA library for genes which are selectively or preferably
expressed in the target tissues or cells.
[0265] For the purpose of expression in source tissues of the
plant, such as the leaf, seed, root or stem, it is preferred that
the promoters utilized in the present invention have relatively
high expression in these specific tissues. For this purpose, one
may choose from a number of promoters for genes with tissue- or
cell-specific or -enhanced expression. Examples of such promoters
reported in the literature include the chloroplast glutamine
synthetase GS2 promoter from pea (Edwards et al., Proc. Natl. Acad.
Sci. (U.S.A.) 87:3459-3463 (1990), herein incorporated by reference
in its entirety), the chloroplast fructose-1,6-biphosphatase
(FBPase) promoter from wheat (Lloyd et al., Mol. Gen. Genet.
225:209-216 (1991), herein incorporated by reference in its
entirety), the nuclear photosynthetic ST-LS1 promoter from potato
(Stockhaus et al., EMBO J. 8:2445-2451 (1989), herein incorporated
by reference in its entirety), the serine/threonine kinase (PAL)
promoter and the glucoamylase (CHS) promoter from Arabidopsis
thaliana. Also reported to be active in photosynthetically active
tissues are the ribulose-1,5-bisphosphate carboxylase (RbcS)
promoter from eastern larch (Larix laricina), the promoter for the
cab gene, cab6, from pine (Yamamoto et al., Plant Cell Physiol;
35:773-778 (1994), herein incorporated by reference in its
entirety), the promoter for the Cab-1 gene from wheat (Fejes et
al., Plant Mol. Biol. 15:921-932 (1990), herein incorporated by
reference in its entirety), the promoter for the CAB-1 gene from
spinach (Lubberstedt et al., Plant Physiol. 104:997-1006 (1994),
herein incorporated by reference in its entirety), the promoter for
the cab1R gene from rice (Luan et al., Plant Cell. 4:971-981
(1992), the entirety of which is herein incorporated by reference),
the pyruvate, orthophosphate dikinase (PPDK) promoter from maize
(Matsuoka et al., Proc. Natl. Acad. Sci. (U.S.A.) 90: 9586-9590
(1993), herein incorporated by reference in its entirety), the
promoter for the tobacco Lhcb1*2 gene (Cerdan et al., Plant Mol.
Biol. 33:245-255 (1997), herein incorporated by reference in its
entirety), the Arabidopsis thaliana SUC2 sucrose-H+ symporter
promoter (Truernit et al., Planta. 196:564-570 (1995), herein
incorporated by reference in its entirety) and the promoter for the
thylakoid membrane proteins from spinach (psaD, psaF, psaE, PC,
FNR, atpC, atpD, cab, rbcS). Other promoters for the chlorophyll
a/b-binding proteins may also be utilized in the present invention,
such as the promoters for LhcB gene and PsbP gene from white
mustard (Sinapis alba; Kretsch et al., Plant Mol. Biol. 28:219-229
(1995), the entirety of which is herein incorporated by
reference).
[0266] For the purpose of expression in sink tissues of the plant,
such as the tuber of the potato plant, the fruit of tomato, or the
seed of maize, wheat, rice and barley, it is preferred that the
promoters utilized in the present invention have relatively high
expression in these specific tissues. A number of promoters for
genes with tuber-specific or -enhanced expression are known,
including the class I patatin promoter (Bevan et al., EMBO J.
8:1899-1906 (1986); Jefferson et al., Plant Mol. Biol. 14:995-1006
(1990), both of which are herein incorporated by reference in its
entirety), the promoter for the potato tuber ADPGPP genes, both the
large and small subunits, the sucrose synthase promoter (Salanoubat
and Belliard, Gene. 60:47-56 (1987), Salanoubat and Belliard, Gene.
84:181-185 (1989), both of which are incorporated by reference in
their entirety), the promoter for the major tuber proteins
including the 22 kd protein complexes and proteinase inhibitors
(Hannapel, Plant Physiol. 101:703-704 (1993), herein incorporated
by reference in its entirety), the promoter for the granule bound
starch synthase gene (GBSS) (Visser et al., Plant Mol. Biol.
17:691-699 (1991), herein incorporated by reference in its
entirety) and other class I and II patatins promoters
(Koster-Topfer et al., Mol Gen Genet. 219:390-396 (1989); Mignery
et al., Gene. 62:27-44 (1988), both of which are herein
incorporated by reference in their entirety).
[0267] Other promoters can also be used to express a sucrose
pathway protein or fragment thereof in specific tissues, such as
seeds or fruits. The promoter for .beta.-conglycinin (Chen et al.
Dev. Genet. 10: 112-122 (1989), herein incorporated by reference in
its entirety) or other seed-specific promoters such as the napin
and phaseolin promoters, can be used. The zeins are a group of
storage proteins found in maize endosperm. Genomic clones for zein
genes have been isolated (Pedersen et al., Cell 29:1015-1026
(1982), herein incorporated by reference in its entirety) and the
promoters from these clones, including the 15 kD, 16 kD, 19 kD, 22
kD, 27 kD and .gamma. genes, could also be used. Other promoters
known to function, for example, in maize include the promoters for
the following genes: waxy, Brittle, Shrunken 2, Branching enzymes I
and II, starch synthases, debranching enzymes, oleosins, glutelins
and sucrose synthases. A particularly preferred promoter for maize
endosperm expression is the promoter for the glutelin gene from
rice, more particularly the Osgt-1 promoter (Zheng et al., Mol.
Cell. Biol. 13:5829-5842 (1993), herein incorporated by reference
in its entirety). Examples of promoters suitable for expression in
wheat include those promoters for the ADPglucose pyrosynthase
(ADPGPP) subunits, the granule bound and other starch synthase, the
branching and debranching enzymes, the embryogenesis-abundant
proteins, the gliadins and the glutenins. Examples of such
promoters in rice include those promoters for the ADPGPP subunits,
the granule bound and other starch synthase, the branching enzymes,
the debranching enzymes, sucrose synthases and the glutelins. A
particularly preferred promoter is the promoter for rice glutelin,
Osgt-1. Examples of such promoters for barley include those for the
ADPGPP subunits, the granule bound and other starch synthase, the
branching enzymes, the debranching enzymes, sucrose synthases, the
hordeins, the embryo globulins and the aleurone specific
proteins.
[0268] Root specific promoters may also be used. An example of such
a promoter is the promoter for the acid chitinase gene (Samac et
al., Plant Mol. Biol. 25:587-596 (1994), the entirety of which is
herein incorporated by reference). Expression in root tissue could
also be accomplished by utilizing the root specific subdomains of
the CaMV35S promoter that have been identified (Lam et al., Proc.
Natl. Acad. Sci. (U.S.) 86:7890-7894 (1989), herein incorporated by
reference in its entirety). Other root cell specific promoters
include those reported by Conkling et al. (Conkling et al., Plant
Physiol. 93:1203-1211 (1990), the entirety of which is herein
incorporated by reference).
[0269] Additional promoters that may be utilized are described, for
example, in U.S. Pat. Nos. 5,378,619; 5,391,725; 5,428,147;
5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435;
and 4,633,436, all of which are herein incorporated in their
entirety. In addition, a tissue specific enhancer may be used
(Fromm et al., The Plant Cell 1:977-984 (1989), the entirety of
which is herein incorporated by reference).
[0270] Constructs or vectors may also include with the coding
region of interest a nucleic acid sequence that acts, in whole or
in part, to terminate transcription of that region. For example,
such sequences have been isolated including the Tr7 3' sequence and
the NOS 3' sequence (Ingelbrecht et al., The Plant Cell 1:671-680
(1989), the entirety of which is herein incorporated by reference;
Bevan et al., Nucleic Acids Res. 11:369-385 (1983), the entirety of
which is herein incorporated by reference), or the like.
[0271] A vector or construct may also include regulatory elements.
Examples of such include the Adh intron 1 (Callis et al., Genes and
Develop. 1:1183-1200 (1987), the entirety of which is herein
incorporated by reference), the sucrose synthase intron (Vasil et
al., Plant Physiol. 91:1575-1579 (1989), the entirety of which is
herein incorporated by reference) and the TMV omega element (Gallie
et al., The Plant Cell 1:301-311 (1989), the entirety of which is
herein incorporated by reference). These and other regulatory
elements may be included when appropriate.
[0272] A vector or construct may also include a selectable marker.
Selectable markers may also be used to select for plants or plant
cells that contain the exogenous genetic material. Examples of such
include, but are not limited to, a neo gene (Potrykus et al., Mol.
Gen. Genet. 199:183-188 (1985), the entirety of which is herein
incorporated by reference) which codes for kanamycin resistance and
can be selected for using kanamycin, G418, etc.; a bar gene which
codes for bialaphos resistance; a mutant EPSP synthase gene
(Hinchee et al., Bio/Technology 6:915-922 (1988), the entirety of
which is herein incorporated by reference) which encodes glyphosate
resistance; a nitrilase gene which confers resistance to bromoxynil
(Stalker et al., J. Biol. Chem. 263:6310-6314 (1988), the entirety
of which is herein incorporated by reference); a mutant
acetolactate synthase gene (ALS) which confers imidazolinone or
sulphonylurea resistance (European Patent Application 154,204 (Sep.
11, 1985), the entirety of which is herein incorporated by
reference); and a methotrexate resistant DHFR gene (Thillet et al.,
J. Biol. Chem. 263:12500-12508 (1988), the entirety of which is
herein incorporated by reference).
[0273] A vector or construct may also include a transit peptide.
Incorporation of a suitable chloroplast transit peptide may also be
employed (European Patent Application Publication Number 0218571,
the entirety of which is herein incorporated by reference).
Translational enhancers may also be incorporated as part of the
vector DNA. DNA constructs could contain one or more 5'
non-translated leader sequences which may serve to enhance
expression of the gene products from the resulting mRNA
transcripts. Such sequences may be derived from the promoter
selected to express the gene or can be specifically modified to
increase translation of the mRNA. Such regions may also be obtained
from viral RNAs, from suitable eukaryotic genes, or from a
synthetic gene sequence. For a review of optimizing expression of
transgenes, see Koziel et al., Plant Mol. Biol. 32:393-405 (1996),
the entirety of which is herein incorporated by reference.
[0274] A vector or construct may also include a screenable marker.
Screenable markers may be used to monitor expression. Exemplary
screenable markers include a .beta.-glucuronidase or uidA gene
(GUS) which encodes an enzyme for which various chromogenic
substrates are known (Jefferson, Plant Mol. Biol, Rep. 5:387-405
(1987), the entirety of which is herein incorporated by reference;
Jefferson et al., EMBO J. 6:3901-3907 (1987), the entirety of which
is herein incorporated by reference); an R-locus gene, which
encodes a product that regulates the production of anthocyanin
pigments (red color) in plant tissues (Dellaporta et al., Stadler
Symposium 11:263-282 (1988), the entirety of which is herein
incorporated by reference); a .alpha.-lactamase gene (Sutcliffe et
al., Proc. Natl. Acad. Sci. (U.S.) 75:3737-3741 (1978), the
entirety of which is herein incorporated by reference), a gene
which encodes an enzyme for which various chromogenic substrates
are known (e.g., PADAC, a chromogenic cephalosporin); a luciferase
gene (Ow et al., Science 234:856-859 (1986), the entirety of which
is herein incorporated by reference); a xylE gene (Zukowsky et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 80:1101-1105 (1983), the entirety
of which is herein incorporated by reference) which encodes a
catechol diozygenase that can convert chromogenic catechols; an
.alpha.-amylase gene (Ikatu et al., Bio/Technol. 8:241-242 (1990),
the entirety of which is herein incorporated by reference); a
tyrosinase gene (Katz et al., J. Gen. Microbiol. 129:2703-2714
(1983), the entirety of which is herein incorporated by reference)
which encodes an enzyme capable of oxidizing tyrosine to DOPA and
dopaquinone which in turn condenses to melanin; an
.alpha.-galactosidase, which will turn a chromogenic
.alpha.-galactose substrate.
[0275] Included within the terms "selectable or screenable marker
genes" are also genes which encode a secretable marker whose
secretion can be detected as a means of identifying or selecting
for transformed cells. Examples include markers which encode a
secretable antigen that can be identified by antibody interaction,
or even secretable enzymes which can be detected catalytically.
Secretable proteins fall into a number of classes, including small,
diffusible proteins which are detectable, (e.g., by ELISA), small
active enzymes which are detectable in extracellular solution
(e.g., .alpha.-amylase, .beta.-lactamase, phosphinothricin
transferase), or proteins which are inserted or trapped in the cell
wall (such as proteins which include a leader sequence such as that
found in the expression unit of extension or tobacco PR-S). Other
possible selectable and/or screenable marker genes will be apparent
to those of skill in the art.
[0276] There are many methods for introducing transforming nucleic
acid molecules into plant cells. Suitable methods are believed to
include virtually any method by which nucleic acid molecules may be
introduced into a cell, such as by Agrobacterium infection or
direct delivery of nucleic acid molecules such as, for example, by
PEG-mediated transformation, by electroporation or by acceleration
of DNA coated particles, etc (Potrykus, Ann. Rev. Plant Physiol.
Plant Mol. Biol. 42:205-225 (1991), the entirety of which is herein
incorporated by reference; Vasil, Plant Mol. Biol. 25:925-937
(1994), the entirety of which is herein incorporated by reference).
For example, electroporation has been used to transform maize
protoplasts (Fromm et al., Nature 312:791-793 (1986), the entirety
of which is herein incorporated by reference).
[0277] Other vector systems suitable for introducing transforming
DNA into a host plant cell include but are not limited to binary
artificial chromosome (BIBAC) vectors (Hamilton et al., Gene
200:107-116 (1997), the entirety of which is herein incorporated by
reference); and transfection with RNA viral vectors (Della-Cioppa
et al., Ann. N.Y. Acad. Sci. (1996), 792 (Engineering Plants for
Commercial Products and Applications), 57-61, the entirety of which
is herein incorporated by reference). Additional vector systems
also include plant selectable YAC vectors such as those described
in Mullen et al., Molecular Breeding 4:449-457 (1988), the entirety
of which is herein incorporated by reference).
[0278] Technology for introduction of DNA into cells is well known
to those of skill in the art. Four general methods for delivering a
gene into cells have been described: (1) chemical methods (Graham
and van der Eb, Virology 54:536-539 (1973), the entirety of which
is herein incorporated by reference); (2) physical methods such as
microinjection (Capecchi, Cell 22:479-488 (1980), the entirety of
which is herein incorporated by reference), electroporation (Wong
and Neumann, Biochem. Biophys. Res. Commun. 107:584-587 (1982);
Fromm et al., Proc. Natl. Acad. Sci. (U.S.A) 82:5824-5828 (1985);
U.S. Pat. No. 5,384,253, all of which are herein incorporated in
their entirety); and the gene gun (Johnston and Tang, Methods Cell
Biol. 43:353-365 (1994), the entirety of which is herein
incorporated by reference); (3) viral vectors (Clapp, Clin.
Perinatol. 20:155-168 (1993); Lu et al., J. Exp. Med. 178:2089-2096
(1993); Eglitis and Anderson, Biotechniques 6:608-614 (1988), all
of which are herein incorporated in their entirety); and (4)
receptor-mediated mechanisms (Curiel et al., Hum. Gen. Ther.
3:147-154 (1992), Wagner et al., Proc. Natl. Acad. Sci. (U.S.A)
89:6099-6103 (1992), both of which are incorporated by reference in
their entirety).
[0279] Acceleration methods that may be used include, for example,
microprojectile bombardment and the like. One example of a method
for delivering transforming nucleic acid molecules to plant cells
is microprojectile bombardment. This method has been reviewed by
Yang and Christou (eds.), Particle Bombardment Technology for Gene
Transfer, Oxford Press, Oxford, England (1994), the entirety of
which is herein incorporated by reference). Non-biological
particles (microprojectiles) that may be coated with nucleic acids
and delivered into cells by a propelling force. Exemplary particles
include those comprised of tungsten, gold, platinum and the
like.
[0280] A particular advantage of microprojectile bombardment, in
addition to it being an effective means of reproducibly
transforming monocots, is that neither the isolation of protoplasts
(Cristou et al., Plant Physiol. 87:671-674 (1988), the entirety of
which is herein incorporated by reference) nor the susceptibility
of Agrobacterium infection are required. An illustrative embodiment
of a method for delivering DNA into maize cells by acceleration is
a biolistics .alpha.-particle delivery system, which can be used to
propel particles coated with DNA through a screen, such as a
stainless steel or Nytex screen, onto a filter surface covered with
corn cells cultured in suspension. Gordon-Kamm et al., describes
the basic procedure for coating tungsten particles with DNA
(Gordon-Kamm et al., Plant Cell 2:603-618 (1990), the entirety of
which is herein incorporated by reference). The screen disperses
the tungsten nucleic acid particles so that they are not delivered
to the recipient cells in large aggregates. A particle delivery
system suitable for use with the present invention is the helium
acceleration PDS-1000/He gun is available from Bio-Rad Laboratories
(Bio-Rad, Hercules, Calif.) (Sanford et al., Technique 3:3-16
(1991), the entirety of which is herein incorporated by
reference).
[0281] For the bombardment, cells in suspension may be concentrated
on filters. Filters containing the cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the gun and the cells to be bombarded.
[0282] Alternatively, immature embryos or other target cells may be
arranged on solid culture medium. The cells to be bombarded are
positioned at an appropriate distance below the microprojectile
stopping plate. If desired, one or more screens are also positioned
between the acceleration device and the cells to be bombarded.
Through the use of techniques set forth herein one may obtain up to
1000 or more foci of cells transiently expressing a marker gene.
The number of cells in a focus which express the exogenous gene
product 48 hours post-bombardment often range from one to ten and
average one to three.
[0283] In bombardment transformation, one may optimize the
pre-bombardment culturing conditions and the bombardment parameters
to yield the maximum numbers of stable transformants. Both the
physical and biological parameters for bombardment are important in
this technology. Physical factors are those that involve
manipulating the DNA/microprojectile precipitate or those that
affect the flight and velocity of either the macro- or
microprojectiles. Biological factors include all steps involved in
manipulation of cells before and immediately after bombardment, the
osmotic adjustment of target cells to help alleviate the trauma
associated with bombardment and also the nature of the transforming
DNA, such as linearized DNA or intact supercoiled plasmids. It is
believed that pre-bombardment manipulations are especially
important for successful transformation of immature embryos.
[0284] In another alternative embodiment, plastids can be stably
transformed. Methods disclosed for plastid transformation in higher
plants include the particle gun delivery of DNA containing a
selectable marker and targeting of the DNA to the plastid genome
through homologous recombination (Svab et al., Proc. Natl. Acad.
Sci. (U.S.A) 87:8526-8530 (1990); Svab and Maliga, Proc. Natl.
Acad. Sci. (U.S.A.) 90:913-917 (1993); Staub and Maliga, EMBO J.
12:601-606 (1993); U.S. Pat. Nos. 5,451,513 and 5,545,818, all of
which are herein incorporated by reference in their entirety).
[0285] Accordingly, it is contemplated that one may wish to adjust
various aspects of the bombardment parameters in small scale
studies to fully optimize the conditions. One may particularly wish
to adjust physical parameters such as gap distance, flight
distance, tissue distance and helium pressure. One may also
minimize the trauma reduction factors by modifying conditions which
influence the physiological state of the recipient cells and which
may therefore influence transformation and integration
efficiencies. For example, the osmotic state, tissue hydration and
the subculture stage or cell cycle of the recipient cells may be
adjusted for optimum transformation. The execution of other routine
adjustments will be known to those of skill in the art in light of
the present disclosure.
[0286] Agrobacterium-mediated transfer is a widely applicable
system for introducing genes into plant cells because the DNA can
be introduced into whole plant tissues, thereby bypassing the need
for regeneration of an intact plant from a protoplast. The use of
Agrobacterium-mediated plant integrating vectors to introduce DNA
into plant cells is well known in the art. See, for example the
methods described by Fraley et al., Bio/Technology 3:629-635 (1985)
and Rogers et al., Methods Enzymol. 153:253-277 (1987), both of
which are herein incorporated by reference in their entirety.
Further, the integration of the Ti-DNA is a relatively precise
process resulting in few rearrangements. The region of DNA to be
transferred is defined by the border sequences and intervening DNA
is usually inserted into the plant genome as described (Spielmann
et al., Mol. Gen. Genet. 205:34 (1986), the entirety of which is
herein incorporated by reference).
[0287] Modern Agrobacterium transformation vectors are capable of
replication in E. coli as well as Agrobacterium, allowing for
convenient manipulations as described (Klee et al., In: Plant DNA
Infectious Agents, Hohn and Schell (eds.), Springer-Verlag, New
York, pp. 179-203 (1985), the entirety of which is herein
incorporated by reference. Moreover, technological advances in
vectors for Agrobacterium-mediated gene transfer have improved the
arrangement of genes and restriction sites in the vectors to
facilitate construction of vectors capable of expressing various
polypeptide coding genes. The vectors described have convenient
multi-linker regions flanked by a promoter and a polyadenylation
site for direct expression of inserted polypeptide coding genes and
are suitable for present purposes (Rogers et al., Methods Enzymol.
153:253-277 (1987)). In addition, Agrobacterium containing both
armed and disarmed Ti genes can be used for the transformations. In
those plant strains where Agrobacterium-mediated transformation is
efficient, it is the method of choice because of the facile and
defined nature of the gene transfer.
[0288] A transgenic plant formed using Agrobacterium transformation
methods typically contains a single gene on one chromosome. Such
transgenic plants can be referred to as being heterozygous for the
added gene. More preferred is a transgenic plant that is homozygous
for the added structural gene; i.e., a transgenic plant that
contains two added genes, one gene at the same locus on each
chromosome of a chromosome pair. A homozygous transgenic plant can
be obtained by sexually mating (selfing) an independent segregant
transgenic plant that contains a single added gene, germinating
some of the seed produced and analyzing the resulting plants
produced for the gene of interest.
[0289] It is also to be understood that two different transgenic
plants can also be mated to produce offspring that contain two
independently segregating added, exogenous genes. Selfing of
appropriate progeny can produce plants that are homozygous for both
added, exogenous genes that encode a polypeptide of interest.
Back-crossing to a parental plant and out-crossing with a
non-transgenic plant are also contemplated, as is vegetative
propagation.
[0290] Transformation of plant protoplasts can be achieved using
methods based on calcium phosphate precipitation, polyethylene
glycol treatment, electroporation and combinations of these
treatments (See, for example, Potrykus et al., Mol. Gen. Genet.
205:193-200 (1986); Lorz et al., Mol. Gen. Genet. 199:178 (1985);
Fromm et al., Nature 319:791 (1986); Uchimiya et al., Mol. Gen.
Genet. 204:204 (1986); Marcotte et al., Nature 335:454-457 (1988),
all of which are herein incorporated by reference in their
entirety).
[0291] Application of these systems to different plant strains
depends upon the ability to regenerate that particular plant strain
from protoplasts. Illustrative methods for the regeneration of
cereals from protoplasts are described (Fujimura et al., Plant
Tissue Culture Letters 2:74 (1985); Toriyama et al., Theor Appl.
Genet. 205:34 (1986); Yamada et al., Plant Cell Rep. 4:85 (1986);
Abdullah et al., Biotechnology 4:1087 (1986), all of which are
herein incorporated by reference in their entirety).
[0292] To transform plant strains that cannot be successfully
regenerated from protoplasts, other ways to introduce DNA into
intact cells or tissues can be utilized. For example, regeneration
of cereals from immature embryos or explants can be effected as
described (Vasil, Biotechnology 6:397 (1988), the entirety of which
is herein incorporated by reference). In addition, "particle gun"
or high-velocity microprojectile technology can be utilized (Vasil
et al., Bio/Technology 10:667 (1992), the entirety of which is
herein incorporated by reference).
[0293] Using the latter technology, DNA is carried through the cell
wall and into the cytoplasm on the surface of small metal particles
as described (Klein et al., Nature 328:70 (1987); Klein et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 85:8502-8505 (1988); McCabe et al.,
Bio/Technology 6:923 (1988), all of which are herein incorporated
by reference in their entirety). The metal particles penetrate
through several layers of cells and thus allow the transformation
of cells within tissue explants.
[0294] Other methods of cell transformation can also be used and
include but are not limited to introduction of DNA into plants by
direct DNA transfer into pollen (Zhou et al., Methods Enzymol.
101:433 (1983); Hess et al., Intern Rev. Cytol. 107:367 (1987); Luo
et al., Plant Mol. Biol. Reporter 6:165 (1988), all of which are
herein incorporated by reference in their entirety), by direct
injection of DNA into reproductive organs of a plant (Pena et al.,
Nature 325:274 (1987), the entirety of which is herein incorporated
by reference), or by direct injection of DNA into the cells of
immature embryos followed by the rehydration of desiccated embryos
(Neuhaus et al., Theor. Appl. Genet. 75:30 (1987), the entirety of
which is herein incorporated by reference).
[0295] The regeneration, development and cultivation of plants from
single plant protoplast transformants or from various transformed
explants is well known in the art (Weissbach and Weissbach, In:
Methods for Plant Molecular Biology, Academic Press, San Diego,
Calif., (1988), the entirety of which is herein incorporated by
reference). This regeneration and growth process typically includes
the steps of selection of transformed cells, culturing those
individualized cells through the usual stages of embryonic
development through the rooted plantlet stage. Transgenic embryos
and seeds are similarly regenerated. The resulting transgenic
rooted shoots are thereafter planted in an appropriate plant growth
medium such as soil.
[0296] The development or regeneration of plants containing the
foreign, exogenous gene that encodes a protein of interest is well
known in the art. Preferably, the regenerated plants are
self-pollinated to provide homozygous transgenic plants. Otherwise,
pollen obtained from the regenerated plants is crossed to
seed-grown plants of agronomically important lines. Conversely,
pollen from plants of these important lines is used to pollinate
regenerated plants. A transgenic plant of the present invention
containing a desired polypeptide is cultivated using methods well
known to one skilled in the art.
[0297] There are a variety of methods for the regeneration of
plants from plant tissue. The particular method of regeneration
will depend on the starting plant tissue and the particular plant
species to be regenerated.
[0298] Methods for transforming dicots, primarily by use of
Agrobacterium tumefaciens and obtaining transgenic plants have been
published for cotton (U.S. Pat. No. 5,004,863; U.S. Pat. No.
5,159,135; U.S. Pat. No. 5,518,908, all of which are herein
incorporated by reference in their entirety); soybean (U.S. Pat.
No. 5,569,834; U.S. Pat. No. 5,416,011; McCabe et. al.,
Biotechnology 6:923 (1988); Christou et al., Plant Physiol.
87:671-674 (1988); all of which are herein incorporated by
reference in their entirety); Brassica (U.S. Pat. No. 5,463,174,
the entirety of which is herein incorporated by reference); peanut
(Cheng et al., Plant Cell Rep. 15:653-657 (1996), McKently et al.,
Plant Cell Rep. 14:699-703 (1995), all of which are herein
incorporated by reference in their entirety); papaya; and pea
(Grant et al., Plant Cell Rep. 15:254-258 (1995), the entirety of
which is herein incorporated by reference).
[0299] Transformation of monocotyledons using electroporation,
particle bombardment and Agrobacterium have also been reported.
Transformation and plant regeneration have been achieved in
asparagus (Bytebier et al., Proc. Natl. Acad. Sci. (USA) 84:5354
(1987), the entirety of which is herein incorporated by reference);
barley (Wan and Lemaux, Plant Physiol 104:37 (1994), the entirety
of which is herein incorporated by reference); maize (Rhodes et
al., Science 240:204 (1988); Gordon-Kamm et al., Plant Cell
2:603-618 (1990); Fromm et al., Bio/Technology 8:833 (1990); Koziel
et al., Bio/Technology 11:194 (1993); Armstrong et al., Crop
Science 35:550-557 (1995); all of which are herein incorporated by
reference in their entirety); oat (Somers et al., Bio/Technology
10:1589 (1992), the entirety of which is herein incorporated by
reference); orchard grass (Horn et al., Plant Cell Rep. 7:469
(1988), the entirety of which is herein incorporated by reference);
rice (Toriyama et al., Theor Appl. Genet. 205:34 (1986); Part et
al., Plant Mol. Biol. 32:1135-1148 (1996); Abedinia et al., Aust.
J. Plant Physiol. 24:133-141 (1997); Zhang and Wu, Theor. Appl.
Genet. 76:835 (1988); Zhang et al., Plant Cell Rep. 7:379 (1988);
Battraw and Hall, Plant Sci. 86:191-202 (1992); Christou et al.,
Bio/Technology 9:957 (1991), all of which are herein incorporated
by reference in their entirety); rye (De la Pena et al., Nature
325:274 (1987), the entirety of which is herein incorporated by
reference); sugarcane (Bower and Birch, Plant J 2:409 (1992), the
entirety of which is herein incorporated by reference); tall fescue
(Wang et al., Bio/Technology 10:691 (1992), the entirety of which
is herein incorporated by reference) and wheat (Vasil et al.,
Bio/Technology 10:667 (1992), the entirety of which is herein
incorporated by reference; U.S. Pat. No. 5,631,152, the entirety of
which is herein incorporated by reference.)
[0300] Assays for gene expression based on the transient expression
of cloned nucleic acid constructs have been developed by
introducing the nucleic acid molecules into plant cells by
polyethylene glycol treatment, electroporation, or particle
bombardment (Marcotte et al., Nature 335:454-457 (1988), the
entirety of which is herein incorporated by reference; Marcotte et
al., Plant Cell 1:523-532 (1989), the entirety of which is herein
incorporated by reference; McCarty et al., Cell 66:895-905 (1991),
the entirety of which is herein incorporated by reference; Hattori
et al., Genes Dev. 6:609-618 (1992), the entirety of which is
herein incorporated by reference; Goff et al., EMBO J. 9:2517-2522
(1990), the entirety of which is herein incorporated by reference).
Transient expression systems may be used to functionally dissect
gene constructs (see generally, Mailga et al., Methods in Plant
Molecular Biology, Cold Spring Harbor Press (1995)).
[0301] Any of the nucleic acid molecules of the present invention
may be introduced into a plant cell in a permanent or transient
manner in combination with other genetic elements such as vectors,
promoters, enhancers etc. Further, any of the nucleic acid
molecules of the present invention may be introduced into a plant
cell in a manner that allows for overexpression of the protein or
fragment thereof encoded by the nucleic acid molecule.
[0302] Cosuppression is the reduction in expression levels, usually
at the level of RNA, of a particular endogenous gene or gene family
by the expression of a homologous sense construct that is capable
of transcribing mRNA of the same strandedness as the transcript of
the endogenous gene (Napoli et al., Plant Cell 2:279-289 (1990),
the entirety of which is herein incorporated by reference; van der
Krol et al., Plant Cell 2:291-299 (1990), the entirety of which is
herein incorporated by reference). Cosuppression may result from
stable transformation with a single copy nucleic acid molecule that
is homologous to a nucleic acid sequence found with the cell
(Prolls and Meyer, Plant J. 2:465-475 (1992), the entirety of which
is herein incorporated by reference) or with multiple copies of a
nucleic acid molecule that is homologous to a nucleic acid sequence
found with the cell (Mittlesten et al., Mol. Gen. Genet.
244:325-330 (1994), the entirety of which is herein incorporated by
reference). Genes, even though different, linked to homologous
promoters may result in the cosuppression of the linked genes
(Vaucheret, C.R. Acad. Sci. III 316:1471-1483 (1993), the entirety
of which is herein incorporated by reference).
[0303] This technique has, for example, been applied to generate
white flowers from red petunia and tomatoes that do not ripen on
the vine. Up to 50% of petunia transformants that contained a sense
copy of the glucoamylase (CHS) gene produced white flowers or
floral sectors; this was as a result of the post-transcriptional
loss of mRNA encoding CHS (Flavell, Proc. Natl. Acad. Sci. (U.S.A)
91:3490-3496 (1994), the entirety of which is herein incorporated
by reference); van Blokland et al., Plant J. 6:861-877 (1994), the
entirety of which is herein incorporated by reference).
Cosuppression may require the coordinate transcription of the
transgene and the endogenous gene and can be reset by a
developmental control mechanism (Jorgensen, Trends Biotechnol.
8:340-344 (1990), the entirety of which is herein incorporated by
reference; Meins and Kunz, In: Gene Inactivation and Homologous
Recombination in Plants, Paszkowski (ed.), pp. 335-348, Kluwer
Academic, Netherlands (1994), the entirety of which is herein
incorporated by reference).
[0304] It is understood that one or more of the nucleic acids of
the present invention may be introduced into a plant cell and
transcribed using an appropriate promoter with such transcription
resulting in the cosuppression of an endogenous sucrose pathway
protein.
[0305] Antisense approaches are a way of preventing or reducing
gene function by targeting the genetic material (Mol et al., FEBS
Lett. 268:427-430 (1990), the entirety of which is herein
incorporated by reference). The objective of the antisense approach
is to use a sequence complementary to the target gene to block its
expression and create a mutant cell line or organism in which the
level of a single chosen protein is selectively reduced or
abolished. Antisense techniques have several advantages over other
`reverse genetic` approaches. The site of inactivation and its
developmental effect can be manipulated by the choice of promoter
for antisense genes or by the timing of external application or
microinjection. Antisense can manipulate its specificity by
selecting either unique regions of the target gene or regions where
it shares homology to other related genes (Hiatt et al., In:
Genetic Engineering, Setlow (ed.), Vol. 11, New York: Plenum 49-63
(1989), the entirety of which is herein incorporated by
reference).
[0306] The principle of regulation by antisense RNA is that RNA
that is complementary to the target mRNA is introduced into cells,
resulting in specific RNA:RNA duplexes being formed by base pairing
between the antisense substrate and the target mRNA (Green et al.,
Annu. Rev. Biochem. 55:569-597 (1986), the entirety of which is
herein incorporated by reference). Under one embodiment, the
process involves the introduction and expression of an antisense
gene sequence. Such a sequence is one in which part or all of the
normal gene sequences are placed under a promoter in inverted
orientation so that the `wrong` or complementary strand is
transcribed into a noncoding antisense RNA that hybridizes with the
target mRNA and interferes with its expression (Takayama and
Inouye, Crit. Rev. Biochem. Mol. Biol. 25:155-184 (1990), the
entirety of which is herein incorporated by reference). An
antisense vector is constructed by standard procedures and
introduced into cells by transformation, transfection,
electroporation, microinjection, infection, etc. The type of
transformation and choice of vector will determine whether
expression is transient or stable. The promoter used for the
antisense gene may influence the level, timing, tissue,
specificity, or inducibility of the antisense inhibition.
[0307] It is understood that the activity of a sucrose pathway
protein in a plant cell may be reduced or depressed by growing a
transformed plant cell containing a nucleic acid molecule whose
non-transcribed strand encodes a sucrose pathway protein or
fragment thereof.
[0308] Antibodies have been expressed in plants (Hiatt et al.,
Nature 342:76-78 (1989), the entirety of which is herein
incorporated by reference; Conrad and Fielder, Plant Mol. Biol.
26:1023-1030 (1994), the entirety of which is herein incorporated
by reference). Cytoplamsic expression of a scFv (single-chain Fv
antibodies) has been reported to delay infection by artichoke
mottled crinkle virus. Transgenic plants that express antibodies
directed against endogenous proteins may exhibit a physiological
effect (Philips et al., EMBO J. 16:4489-4496 (1997), the entirety
of which is herein incorporated by reference; Marion-Poll, Trends
in Plant Science 2:447-448 (1997), the entirety of which is herein
incorporated by reference). For example, expressed anti-abscisic
antibodies have been reported to result in a general perturbation
of seed development (Philips et al., EMBO J. 16: 4489-4496
(1997)).
[0309] Antibodies that are catalytic may also be expressed in
plants (abzymes). The principle behind abzymes is that since
antibodies may be raised against many molecules, this recognition
ability can be directed toward generating antibodies that bind
transition states to force a chemical reaction forward (Persidas,
Nature Biotechnology 15:1313-1315 (1997), the entirety of which is
herein incorporated by reference; Baca et al., Ann. Rev. Biophys.
Biomol. Struct. 26:461-493 (1997), the entirety of which is herein
incorporated by reference). The catalytic abilities of abzymes may
be enhanced by site directed mutagenesis. Examples of abzymes are,
for example, set forth in U.S. Pat. No. 5,658,753; U.S. Pat. No.
5,632,990; U.S. Pat. No. 5,631,137; U.S. Pat. No. 5,602,015; U.S.
Pat. No. 5,559,538; U.S. Pat. No. 5,576,174; U.S. Pat. No.
5,500,358; U.S. Pat. No. 5,318,897; U.S. Pat. No. 5,298,409; U.S.
Pat. No. 5,258,289 and U.S. Pat. No. 5,194,585, all of which are
herein incorporated in their entirety.
[0310] It is understood that any of the antibodies of the present
invention may be expressed in plants and that such expression can
result in a physiological effect. It is also understood that any of
the expressed antibodies may be catalytic.
[0311] (b) Fungal Constructs and Fungal Transformants
[0312] The present invention also relates to a fungal recombinant
vector comprising exogenous genetic material. The present invention
also relates to a fungal cell comprising a fungal recombinant
vector. The present invention also relates to methods for obtaining
a recombinant fungal host cell comprising introducing into a fungal
host cell exogenous genetic material.
[0313] Exogenous genetic material may be transferred into a fungal
cell. In a preferred embodiment the exogenous genetic material
includes a nucleic acid molecule of the present invention having a
sequence selected from the group consisting of SEQ ID NO: 1 through
SEQ ID NO: 2814 or complements thereof or fragments of either or
other nucleic acid molecule of the present invention. The fungal
recombinant vector may be any vector which can be conveniently
subjected to recombinant DNA procedures. The choice of a vector
will typically depend on the compatibility of the vector with the
fungal host cell into which the vector is to be introduced. The
vector may be a linear or a closed circular plasmid. The vector
system may be a single vector or plasmid or two or more vectors or
plasmids which together contain the total DNA to be introduced into
the genome of the fungal host.
[0314] The fungal vector may be an autonomously replicating vector,
i.e., a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the fungal cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. For integration, the vector may rely on the
nucleic acid sequence of the vector for stable integration of the
vector into the genome by homologous or nonhomologous
recombination. Alternatively, the vector may contain additional
nucleic acid sequences for directing integration by homologous
recombination into the genome of the fungal host. The additional
nucleic acid sequences enable the vector to be integrated into the
host cell genome at a precise location(s) in the chromosome(s). To
increase the likelihood of integration at a precise location, there
should be preferably two nucleic acid sequences which individually
contain a sufficient number of nucleic acids, preferably 400 bp to
1500 bp, more preferably 800 bp to 1000 bp, which are highly
homologous with the corresponding target sequence to enhance the
probability of homologous recombination. These nucleic acid
sequences may be any sequence that is homologous with a target
sequence in the genome of the fungal host cell and, furthermore,
may be non-encoding or encoding sequences.
[0315] For autonomous replication, the vector may further comprise
an origin of replication enabling the vector to replicate
autonomously in the host cell in question. Examples of origin of
replications for use in a yeast host cell are the 2 micron origin
of replication and the combination of CEN3 and ARS1. Any origin of
replication may be used which is compatible with the fungal host
cell of choice.
[0316] The fungal vectors of the present invention preferably
contain one or more selectable markers which permit easy selection
of transformed cells. A selectable marker is a gene the product of
which provides, for example biocide or viral resistance, resistance
to heavy metals, prototrophy to auxotrophs and the like. The
selectable marker may be selected from the group including, but not
limited to, amdS (acetamidase), argB (ornithine
carbamoyltransferase), bar (phosphinothricin acetyltransferase),
hygB (hygromycin phosphotransferase), niaD (nitrate reductase),
pyrG (orotidine-5'-phosphate decarboxylase) and sC (sulfate
adenyltransferase) and trpC (anthranilate synthase). Preferred for
use in an Aspergillus cell are the amdS and pyrG markers of
Aspergillus nidulans or Aspergillus oryzae and the bar marker of
Streptomyces hygroscopicus. Furthermore, selection may be
accomplished by co-transformation, e.g., as described in WO
91/17243, the entirety of which is herein incorporated by
reference. A nucleic acid sequence of the present invention may be
operably linked to a suitable promoter sequence. The promoter
sequence is a nucleic acid sequence which is recognized by the
fungal host cell for expression of the nucleic acid sequence. The
promoter sequence contains transcription and translation control
sequences which mediate the expression of the protein or fragment
thereof.
[0317] A promoter may be any nucleic acid sequence which shows
transcriptional activity in the fungal host cell of choice and may
be obtained from genes encoding polypeptides either homologous or
heterologous to the host cell. Examples of suitable promoters for
directing the transcription of a nucleic acid construct of the
invention in a filamentous fungal host are promoters obtained from
the genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor
miehei aspartic proteinase, Aspergillus niger neutral
alpha-amylase, Aspergillus niger acid stable alpha-amylase,
Aspergillus niger or Aspergillus awamori glucoamylase (glaA),
Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease,
Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans
acetamidase and hybrids thereof. In a yeast host, a useful promoter
is the Saccharomyces cerevisiae enolase (eno-1) promoter.
Particularly preferred promoters are the TAKA amylase, NA2-tpi (a
hybrid of the promoters from the genes encoding Aspergillus niger
neutral alpha-amylase and Aspergillus oryzae triose phosphate
isomerase) and glaA promoters.
[0318] A protein or fragment thereof encoding nucleic acid molecule
of the present invention may also be operably linked to a
terminator sequence at its 3' terminus. The terminator sequence may
be native to the nucleic acid sequence encoding the protein or
fragment thereof or may be obtained from foreign sources. Any
terminator which is functional in the fungal host cell of choice
may be used in the present invention, but particularly preferred
terminators are obtained from the genes encoding Aspergillus oryzae
TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans
anthranilate synthase, Aspergillus niger alpha-glucosidase and
Saccharomyces cerevisiae enolase.
[0319] A protein or fragment thereof encoding nucleic acid molecule
of the present invention may also be operably linked to a suitable
leader sequence. A leader sequence is a nontranslated region of a
mRNA which is important for translation by the fungal host. The
leader sequence is operably linked to the 5' terminus of the
nucleic acid sequence encoding the protein or fragment thereof. The
leader sequence may be native to the nucleic acid sequence encoding
the protein or fragment thereof or may be obtained from foreign
sources. Any leader sequence which is functional in the fungal host
cell of choice may be used in the present invention, but
particularly preferred leaders are obtained from the genes encoding
Aspergillus oryzae TAKA amylase and Aspergillus oryzae triose
phosphate isomerase.
[0320] A polyadenylation sequence may also be operably linked to
the 3' terminus of the nucleic acid sequence of the present
invention. The polyadenylation sequence is a sequence which when
transcribed is recognized by the fungal host to add polyadenosine
residues to transcribed mRNA. The polyadenylation sequence may be
native to the nucleic acid sequence encoding the protein or
fragment thereof or may be obtained from foreign sources. Any
polyadenylation sequence which is functional in the fungal host of
choice may be used in the present invention, but particularly
preferred polyadenylation sequences are obtained from the genes
encoding Aspergillus oryzae TAKA amylase, Aspergillus niger
glucoamylase, Aspergillus nidulans anthranilate synthase and
Aspergillus niger alpha-glucosidase.
[0321] To avoid the necessity of disrupting the cell to obtain the
protein or fragment thereof and to minimize the amount of possible
degradation of the expressed protein or fragment thereof within the
cell, it is preferred that expression of the protein or fragment
thereof gives rise to a product secreted outside the cell. To this
end, a protein or fragment thereof of the present invention may be
linked to a signal peptide linked to the amino terminus of the
protein or fragment thereof. A signal peptide is an amino acid
sequence which permits the secretion of the protein or fragment
thereof from the fungal host into the culture medium. The signal
peptide may be native to the protein or fragment thereof of the
invention or may be obtained from foreign sources. The 5' end of
the coding sequence of the nucleic acid sequence of the present
invention may inherently contain a signal peptide coding region
naturally linked in translation reading frame with the segment of
the coding region which encodes the secreted protein or fragment
thereof. Alternatively, the 5' end of the coding sequence may
contain a signal peptide coding region which is foreign to that
portion of the coding sequence which encodes the secreted protein
or fragment thereof. The foreign signal peptide may be required
where the coding sequence does not normally contain a signal
peptide coding region. Alternatively, the foreign signal peptide
may simply replace the natural signal peptide to obtain enhanced
secretion of the desired protein or fragment thereof. The foreign
signal peptide coding region may be obtained from a glucoamylase or
an amylase gene from an Aspergillus species, a lipase or proteinase
gene from Rhizomucor miehei, the gene for the alpha-factor from
Saccharomyces cerevisiae, or the calf preprochymosin gene. An
effective signal peptide for fungal host cells is the Aspergillus
oryzae TAKA amylase signal, Aspergillus niger neutral amylase
signal, the Rhizomucor miehei aspartic proteinase signal, the
Humicola lanuginosus cellulase signal, or the Rhizomucor miehei
lipase signal. However, any signal peptide capable of permitting
secretion of the protein or fragment thereof in a fungal host of
choice may be used in the present invention.
[0322] A protein or fragment thereof encoding nucleic acid molecule
of the present invention may also be linked to a propeptide coding
region. A propeptide is an amino acid sequence found at the amino
terminus of aproprotein or proenzyme. Cleavage of the propeptide
from the proprotein yields a mature biochemically active protein.
The resulting polypeptide is known as a propolypeptide or proenzyme
(or a zymogen in some cases). Propolypeptides are generally
inactive and can be converted to mature active polypeptides by
catalytic or autocatalytic cleavage of the propeptide from the
propolypeptide or proenzyme. The propeptide coding region may be
native to the protein or fragment thereof or may be obtained from
foreign sources. The foreign propeptide coding region may be
obtained from the Saccharomyces cerevisiae alpha-factor gene or
Myceliophthora thermophila laccase gene (WO 95/33836, the entirety
of which is herein incorporated by reference).
[0323] The procedures used to ligate the elements described above
to construct the recombinant expression vector of the present
invention are well known to one skilled in the art (see, for
example, Sambrook et al., Molecular Cloning, A Laboratory Manual,
2nd ed., Cold Spring Harbor, N.Y., (1989)).
[0324] The present invention also relates to recombinant fungal
host cells produced by the methods of the present invention which
are advantageously used with the recombinant vector of the present
invention. The cell is preferably transformed with a vector
comprising a nucleic acid sequence of the invention followed by
integration of the vector into the host chromosome. The choice of
fungal host cells will to a large extent depend upon the gene
encoding the protein or fragment thereof and its source. The
fuingal host cell may, for example, be a yeast cell or a
filamentous fungal cell.
[0325] "Yeast" as used herein includes Ascosporogenous yeast
(Endomycetales), Basidiosporogenous yeast and yeast belonging to
the Fungi Imperfecti (Blastomycetes). The Ascosporogenous yeasts
are divided into the families Spermophthoraceae and
Saccharomycetaceae. The latter is comprised of four subfamilies,
Schizosaccharomycoideae (for example, genus Schizosaccharomyces),
Nadsonioideae, Lipomycoideae and Saccharomycoideae (for example,
genera Pichia, Kluyveromyces and Saccharomyces). The
Basidiosporogenous yeasts include the genera Leucosporidim,
Rhodosporidium, Sporidiobolus, Filobasidium and Filobasidiella.
Yeast belonging to the Fungi Imperfecti are divided into two
families, Sporobolomycetaceae (for example, genera Sorobolomyces
and Bullera) and Cryptococcaceae (for example, genus Candida).
Since the classification of yeast may change in the future, for the
purposes of this invention, yeast shall be defined as described in
Biology and Activities of Yeast (Skinner et al., Soc. App.
Bacteriol. Symposium Series No. 9, (1980), the entirety of which is
herein incorporated by reference). The biology of yeast and
manipulation of yeast genetics are well known in the art (see, for
example, Biochemistry and Genetics of Yeast, Bacil et al. (ed.),
2nd edition, 1987; The Yeasts, Rose and Harrison (eds.), 2nd ed.,
(1987); and The Molecular Biology of the Yeast Saccharomyces,
Strathern et al. (eds.), (1981), all of which are herein
incorporated by reference in their entirety).
[0326] "Fungi" as used herein includes the phyla Ascomycota,
Basidiomycota, Chytridiomycota and Zygomycota (as defined by
Hawksworth et al., In: Ainsworth and Bisby's Dictionary of The
Fungi, 8th edition, 1995, CAB International, University Press,
Cambridge, UK; the entirety of which is herein incorporated by
reference) as well as the Oomycota (as cited in Hawksworth et al.,
In: Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,
1995, CAB International, University Press, Cambridge, UK) and all
mitosporic fungi (Hawksworth et al., In: Ainsworth and Bisby's
Dictionary of The Fungi, 8th edition, 1995, CAB International,
University Press, Cambridge, UK). Representative groups of
Ascomycota include, for example, Neurospora, Eupenicillium
(=Penicillium), Emericella (=Aspergillus), Eurotiun (=Aspergillus)
and the true yeasts listed above. Examples of Basidiomycota include
mushrooms, rusts and smuts. Representative groups of
Chytridiomycota include, for example, Allomyces, Blastocladiella,
Coelomomyces and aquatic fungi. Representative groups of Oomycota
include, for example, Saprolegniomycetous aquatic fungi (water
molds) such as Achlya. Examples of mitosporic fungi include
Aspergillus, Penicilliun, Candida and Alternaria. Representative
groups of Zygomycota include, for example, Rhizopus and Mucor.
[0327] "Filamentous fungi" include all filamentous forms of the
subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,
In: Ainsworth and Bisby's Dictionary of The Fungi, 8th edition,
1995, CAB International, University Press, Cambridge, UK). The
filamentous fungi are characterized by a vegetative mycelium
composed of chitin, cellulose, glucan, chitosan, mannan and other
complex polysaccharides. Vegetative growth is by hyphal elongation
and carbon catabolism is obligately aerobic. In contrast,
vegetative growth by yeasts such as Saccharomyces cerevisiae is by
budding of a unicellular thallus and carbon catabolism may be
fermentative.
[0328] In one embodiment, the fungal host cell is a yeast cell. In
a preferred embodiment, the yeast host cell is a cell of the
species of Candida, Kluyveromyces, Saccharomyces,
Schizosaccharomyces, Pichia and Yarrowia. In a preferred
embodiment, the yeast host cell is a Saccharomyces cerevisiae cell,
a Saccharomyces carlsbergensis, Saccharomyces diastaticus cell, a
Saccharomyces douglasii cell, a Saccharomyces kluyveri cell, a
Saccharomyces norbensis cell, or a Saccharomyces oviformis cell. In
another preferred embodiment, the yeast host cell is a
Kluyveromyces lactis cell. In another preferred embodiment, the
yeast host cell is a Yarrowia lipolytica cell.
[0329] In another embodiment, the fungal host cell is a filamentous
fungal cell. In a preferred embodiment, the filamentous fungal host
cell is a cell of the species of, but not limited to, Acremonium,
Aspergillus, Fusarium, Humicola, Myceliophthora, Mucor, Neurospora,
Penicillium, Thielavia, Tolypocladium and Trichoderma. In a
preferred embodiment, the filamentous fungal host cell is an
Aspergillus cell. In another preferred embodiment, the filamentous
fungal host cell is an Acremonium cell. In another preferred
embodiment, the filamentous fungal host cell is a Fusarium cell. In
another preferred embodiment, the filamentous fungal host cell is a
Humicola cell. In another preferred embodiment, the filamentous
fungal host cell is a Myceliophthora cell. In another even
preferred embodiment, the filamentous fungal host cell is a Mucor
cell. In another preferred embodiment, the filamentous fungal host
cell is a Neurospora cell. In another preferred embodiment, the
filamentous fungal host cell is a Penicillium cell. In another
preferred embodiment, the filamentous fungal host cell is a
Thielavia cell. In another preferred embodiment, the filamentous
fungal host cell is a Tolypocladiun cell. In another preferred
embodiment, the filamentous fungal host cell is a Trichoderma cell.
In a preferred embodiment, the filamentous fungal host cell is an
Aspergillus oryzae cell, an Aspergillus niger cell, an Aspergillus
foetidus cell, or an Aspergillus japonicus cell. In another
preferred embodiment, the filamentous fungal host cell is a
Fusarium oxysporum cell or a Fusarium graminearum cell. In another
preferred embodiment, the filamentous fungal host cell is a
Humicola insolens cell or a Humicola lanuginosus cell. In another
preferred embodiment, the filamentous fungal host cell is a
Myceliophthora thermophila cell. In a most preferred embodiment,
the filamentous fungal host cell is a Mucor miehei cell. In a most
preferred embodiment, the filamentous fungal host cell is a
Neurospora crassa cell. In a most preferred embodiment, the
filamentous fungal host cell is a Penicillium purpurogenum cell. In
another most preferred embodiment, the filamentous fungal host cell
is a Thielavia terrestris cell. In another most preferred
embodiment, the Trichoderma cell is a Trichoderma reesei cell, a
Trichoderma viride cell, a Trichoderma longibrachiatum cell, a
Trichoderma harzianum cell, or a Trichoderma koningii cell. In a
preferred embodiment, the fungal host cell is selected from an A.
nidulans cell, an A. niger cell, an A. oryzae cell and an A. sojae
cell. In a further preferred embodiment, the fungal host cell is an
A. nidulans cell.
[0330] The recombinant fungal host cells of the present invention
may further comprise one or more sequences which encode one or more
factors that are advantageous in the expression of the protein or
fragment thereof, for example, an activator (e.g., a trans-acting
factor), a chaperone and a processing protease. The nucleic acids
encoding one or more of these factors are preferably not operably
linked to the nucleic acid encoding the protein or fragment
thereof. An activator is a protein which activates transcription of
a nucleic acid sequence encoding a polypeptide (Kudla et al., EMBO
9:1355-1364 (1990); Jarai and Buxton, Current Genetics 26:2238-244
(1994); Verdier, Yeast 6:271-297 (1990), all of which are herein
incorporated by reference in their entirety). The nucleic acid
sequence encoding an activator may be obtained from the genes
encoding Saccharomyces cerevisiae heme activator protein 1 (hap1),
Saccharomyces cerevisiae galactose metabolizing protein 4 (gal4)
and Aspergillus nidulans ammonia regulation protein (areA). For
further examples, see Verdier, Yeast 6:271-297 (1990); MacKenzie et
al., Journal of Gen. Microbiol. 139:2295-2307 (1993), both of which
are herein incorporated by reference in their entirety). A
chaperone is a protein which assists another protein in folding
properly (Hartl et al., TIBS 19:20-25 (1994); Bergeron et al., TIBS
19:124-128 (1994); Demolder et al., J Biotechnology 32:179-189
(1994); Craig, Science 260:1902-1903 (1993); Gething and Sambrook,
Nature 355:33-45 (1992); Puig and Gilbert, J. Biol. Chem.
269:7764-7771 (1994); Wang and Tsou, FASEB Journal 7:1515-11157
(1993); Robinson et al., Bio/Technology 1:381-384 (1994), all of
which are herein incorporated by reference in their entirety). The
nucleic acid sequence encoding a chaperone may be obtained from the
genes encoding Aspergillus oryzae protein disulphide isomerase,
Saccharomyces cerevisiae calnexin, Saccharomyces cerevisiae
BiP/GRP78 and Saccharomyces cerevisiae Hsp70. For further examples,
see Gething and Sambrook, Nature 355:33-45 (1992); Hartl et al.,
TIBS 19:20-25 (1994). A processing protease is a protease that
cleaves a propeptide to generate a mature biochemically active
polypeptide (Enderlin and Ogrydziak, Yeast 10:67-79 (1994); Fuller
et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:1434-1438 (1989); Julius
et al., Cell 37:1075-1089 (1984); Julius et al., Cell 32:839-852
(1983), all of which are incorporated by reference in their
entirety). The nucleic acid sequence encoding a processing protease
may be obtained from the genes encoding Aspergillus niger Kex2,
Saccharomyces cerevisiae dipeptidylaminopeptidase, Saccharomyces
cerevisiae Kex2 and Yarrowia lipolytica dibasic processing
endoprotease (xpr6). Any factor that is functional in the fungal
host cell of choice may be used in the present invention.
[0331] Fungal cells may be transformed by a process involving
protoplast formation, transformation of the protoplasts and
regeneration of the cell wall in a manner known per se. Suitable
procedures for transformation of Aspergillus host cells are
described in EP 238 023 and Yelton et al., Proc. Natl. Acad. Sci.
(U.S.A.) 81:1470-1474 (1984), both of which are herein incorporated
by reference in their entirety. A suitable method of transforming
Fusarium species is described by Malardier et al., Gene 78:147-156
(1989), the entirety of which is herein incorporated by reference.
Yeast may be transformed using the procedures described by Becker
and Guarente, In: Abelson and Simon, (eds.), Guide to Yeast
Genetics and Molecular Biology, Methods Enzymol. Volume 194, pp
182-187, Academic Press, Inc., New York; Ito et al., J Bacteriology
153:163 (1983); Hinnen et al., Proc. Natl. Acad. Sci. (U.S.A.)
75:1920 (1978), all of which are herein incorporated by reference
in their entirety.
[0332] The present invention also relates to methods of producing
the protein or fragment thereof comprising culturing the
recombinant fungal host cells under conditions conducive for
expression of the protein or fragment thereof. The fungal cells of
the present invention are cultivated in a nutrient medium suitable
for production of the protein or fragment thereof using methods
known in the art. For example, the cell may be cultivated by shake
flask cultivation, small-scale or large-scale fermentation
(including continuous, batch, fed-batch, or solid state
fermentations) in laboratory or industrial fermentors performed in
a suitable medium and under conditions allowing the protein or
fragment thereof to be expressed and/or isolated. The cultivation
takes place in a suitable nutrient medium comprising carbon and
nitrogen sources and inorganic salts, using procedures known in the
art (see, e.g., Bennett and LaSure (eds.), More Gene Manipulations
in Fungi, Academic Press, CA, (1991), the entirety of which is
herein incorporated by reference). Suitable media are available
from commercial suppliers or may be prepared according to published
compositions (e.g., in catalogues of the American Type Culture
Collection, Manassas, Va.). If the protein or fragment thereof is
secreted into the nutrient medium, a protein or fragment thereof
can be recovered directly from the medium. If the protein or
fragment thereof is not secreted, it is recovered from cell
lysates.
[0333] The expressed protein or fragment thereof may be detected
using methods known in the art that are specific for the particular
protein or fragment. These detection methods may include the use of
specific antibodies, formation of an enzyme product, or
disappearance of an enzyme substrate. For example, if the protein
or fragment thereof has enzymatic activity, an enzyme assay may be
used. Alternatively, if polyclonal or monoclonal antibodies
specific to the protein or fragment thereof are available,
immunoassays may be employed using the antibodies to the protein or
fragment thereof. The techniques of enzyme assay and immunoassay
are well known to those skilled in the art.
[0334] The resulting protein or fragment thereof may be recovered
by methods known in the arts. For example, the protein or fragment
thereof may be recovered from the nutrient medium by conventional
procedures including, but not limited to, centrifugation,
filtration, extraction, spray-drying, evaporation, or
precipitation. The recovered protein or fragment thereof may then
be further purified by a variety of chromatographic procedures,
e.g., ion exchange chromatography, gel filtration chromatography,
affinity chromatography, or the like.
[0335] (c) Mammalian Constructs and Transformed Mammalian Cells
[0336] The present invention also relates to methods for obtaining
a recombinant mammalian host cell, comprising introducing into a
mammalian host cell exogenous genetic material. The present
invention also relates to a mammalian cell comprising a mammalian
recombinant vector. The present invention also relates to methods
for obtaining a recombinant mammalian host cell, comprising
introducing into a mammalian cell exogenous genetic material. In a
preferred embodiment the exogenous genetic material includes a
nucleic acid molecule of the present invention having a sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 2814 or complements thereof or fragments of either or other
nucleic acid molecule of the present invention.
[0337] Mammalian cell lines available as hosts for expression are
known in the art and include many immortalized cell lines available
from the American Type Culture Collection (ATCC, Manassas, Va.),
such as HeLa cells, Chinese hamster ovary (CHO) cells, baby hamster
kidney (BHK) cells and a number of other cell lines. Suitable
promoters for mammalian cells are also known in the art and include
viral promoters such as that from Simian Virus 40 (SV40) (Fiers et
al., Nature 273:113 (1978), the entirety of which is herein
incorporated by reference), Rous sarcoma virus (RSV), adenovirus
(ADV) and bovine papilloma virus (BPV). Mammalian cells may also
require terminator sequences and poly-A addition sequences.
Enhancer sequences which increase expression may also be included
and sequences which promote amplification of the gene may also be
desirable (for example methotrexate resistance genes).
[0338] Vectors suitable for replication in mammalian cells may
include viral replicons, or sequences which insure integration of
the appropriate sequences encoding HCV epitopes into the host
genome. For example, another vector used to express foreign DNA is
vaccinia virus. In this case, for example, a nucleic acid molecule
encoding a protein or fragment thereof is inserted into the
vaccinia genome. Techniques for the insertion of foreign DNA into
the vaccinia virus genome are known in the art and may utilize, for
example, homologous recombination. Such heterologous DNA is
generally inserted into a gene which is non-essential to the virus,
for example, the thymidine kinase gene (tk), which also provides a
selectable marker. Plasmid vectors that greatly facilitate the
construction of recombinant viruses have been described (see, for
example, Mackett et al, J. Virol. 49:857 (1984); Chakrabarti et
al., Mol. Cell. Biol. 5:3403 (1985); Moss, In: Gene Transfer
Vectors For Mammalian Cells (Miller and Calos, eds., Cold Spring
Harbor Laboratory, N.Y., p. 10, (1987); all of which are herein
incorporated by reference in their entirety). Expression of the HCV
polypeptide then occurs in cells or animals which are infected with
the live recombinant vaccinia virus.
[0339] The sequence to be integrated into the mammalian sequence
may be introduced into the primary host by any convenient means,
which includes calcium precipitated DNA, spheroplast fusion,
transformation, electroporation, biolistics, lipofection,
microinjection, or other convenient means. Where an amplifiable
gene is being employed, the amplifiable gene may serve as the
selection marker for selecting hosts into which the amplifiable
gene has been introduced. Alternatively, one may include with the
amplifiable gene another marker, such as a drug resistance marker,
e.g. neomycin resistance (G418 in mammalian cells), hygromycin in
resistance etc., or an auxotrophy marker (HIS3, TRP1, LEU2, URA3,
ADE2, LYS2, etc.) for use in yeast cells.
[0340] Depending upon the nature of the modification and associated
targeting construct, various techniques may be employed for
identifying targeted integration. Conveniently, the DNA may be
digested with one or more restriction enzymes and the fragments
probed with an appropriate DNA fragment which will identify the
properly sized restriction fragment associated with
integration.
[0341] One may use different promoter sequences, enhancer
sequences, or other sequence which will allow for enhanced levels
of expression in the expression host. Thus, one may combine an
enhancer from one source, a promoter region from another source, a
5'-noncoding region upstream from the initiation sucrose from the
same or different source as the other sequences and the like. One
may provide for an intron in the non-coding region with appropriate
splice sites or for an alternative 3'-untranslated sequence or
polyadenylation site. Depending upon the particular purpose of the
modification, any of these sequences may be introduced, as
desired.
[0342] Where selection is intended, the sequence to be integrated
will have with it a marker gene, which allows for selection. The
marker gene may conveniently be downstream from the target gene and
may include resistance to a cytotoxic agent, e.g. antibiotics,
heavy metals, or the like, resistance or susceptibility to HAT,
gancyclovir, etc., complementation to an auxotrophic host,
particularly by using an auxotrophic yeast as the host for the
subject manipulations, or the like. The marker gene may also be on
a separate DNA molecule, particularly with primary mammalian cells.
Alternatively, one may screen the various transformants, due to the
high efficiency of recombination in yeast, by using hybridization
analysis, PCR, sequencing, or the like.
[0343] For homologous recombination, constructs can be prepared
where the amplifiable gene will be flanked, normally on both sides
with DNA homologous with the DNA of the target region. Depending
upon the nature of the integrating DNA and the purpose of the
integration, the homologous DNA will generally be within 100 kb,
usually 50 kb, preferably about 25 kb, of the transcribed region of
the target gene, more preferably within 2 kb of the target gene.
Where modeling of the gene is intended, homology will usually be
present proximal to the site of the mutation. The homologous DNA
may include the 5'-upstream region outside of the transcriptional
regulatory region or comprising any enhancer sequences,
transcriptional initiation sequences, adjacent sequences, or the
like. The homologous region may include a portion of the coding
region, where the coding region may be comprised only of an open
reading frame or combination of exons and introns. The homologous
region may comprise all or a portion of an intron, where all or a
portion of one or more exons may also be present. Alternatively,
the homologous region may comprise the 3'-region, so as to comprise
all or a portion of the transcriptional termination region, or the
region 3' of this region. The homologous regions may extend over
all or a portion of the target gene or be outside the target gene
comprising all or a portion of the transcriptional regulatory
regions and/or the structural gene.
[0344] The integrating constructs may be prepared in accordance
with conventional ways, where sequences may be synthesized,
isolated from natural sources, manipulated, cloned, ligated,
subjected to in vitro mutagenesis, primer repair, or the like. At
various stages, the joined sequences may be cloned and analyzed by
restriction analysis, sequencing, or the like. Usually during the
preparation of a construct where various fragments are joined, the
fragments, intermediate constructs and constructs will be carried
on a cloning vector comprising a replication system functional in a
prokaryotic host, e.g., E. Coli and a marker for selection, e.g.,
biocide resistance, complementation to an auxotrophic host, etc.
Other functional sequences may also be present, such as
polylinkers, for ease of introduction and excision of the construct
or portions thereof, or the like. A large number of cloning vectors
are available such as pBR322, the pUC series, etc. These constructs
may then be used for integration into the primary mammalian
host.
[0345] In the case of the primary mammalian host, a replicating
vector may be used. Usually, such vector will have a viral
replication system, such as SV40, bovine papilloma virus,
adenovirus, or the like. The linear DNA sequence vector may also
have a selectable marker for identifying transfected cells.
Selectable markers include the neo gene, allowing for selection
with G418, the herpes tk gene for selection with HAT medium, the
gpt gene with mycophenolic acid, complementation of an auxotrophic
host, etc.
[0346] The vector may or may not be capable of stable maintenance
in the host. Where the vector is capable of stable maintenance, the
cells will be screened for homologous integration of the vector
into the genome of the host, where various techniques for curing
the cells may be employed. Where the vector is not capable of
stable maintenance, for example, where a temperature sensitive
replication system is employed, one may change the temperature from
the permissive temperature to the non-permissive temperature, so
that the cells may be cured of the vector. In this case, only those
cells having integration of the construct comprising the
amplifiable gene and, when present, the selectable marker, will be
able to survive selection.
[0347] Where a selectable marker is present, one may select for the
presence of the targeting construct by means of the selectable
marker. Where the selectable marker is not present, one may select
for the presence of the construct by the amplifiable gene. For the
neo gene or the herpes tk gene, one could employ a medium for
growth of the transformants of about 0.1-1 mg/ml of G418 or may use
HAT medium, respectively. Where DHFR is the amplifiable gene, the
selective medium may include from about 0.01-0.5 .mu.M of
methotrexate or be deficient in glycine-hypoxanthine-thymidine and
have dialysed serum (GHT media).
[0348] The DNA can be introduced into the expression host by a
variety of techniques that include calcium phosphate/DNA
co-precipitates, microinjection of DNA into the nucleus,
electroporation, yeast protoplast fusion with intact cells,
transfection, polycations, e.g., polybrene, polyornithine, etc., or
the like. The DNA may be single or double stranded DNA, linear or
circular. The various techniques for transforming mammalian cells
are well known (see Keown et al., Methods Enzymol. (1989); Keown et
al., Methods Enzymol. 185:527-537 (1990); Mansour et al., Nature
336:348-352, (1988); all of which are herein incorporated by
reference in their entirety).
[0349] (d) Insect Constructs and Transformed Insect Cells
[0350] The present invention also relates to an insect recombinant
vectors comprising exogenous genetic material. The present
invention also relates to an insect cell comprising an insect
recombinant vector. The present invention also relates to methods
for obtaining a recombinant insect host cell, comprising
introducing into an insect cell exogenous genetic material. In a
preferred embodiment the exogenous genetic material includes a
nucleic acid molecule of the present invention having a sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 2814 or complements thereof or fragments of either or other
nucleic acid molecule of the present invention.
[0351] The insect recombinant vector may be any vector which can be
conveniently subjected to recombinant DNA procedures and can bring
about the expression of the nucleic acid sequence. The choice of a
vector will typically depend on the compatibility of the vector
with the insect host cell into which the vector is to be
introduced. The vector may be a linear or a closed circular
plasmid. The vector system may be a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the insect host. In addition, the
insect vector may be an expression vector. Nucleic acid molecules
can be suitably inserted into a replication vector for expression
in the insect cell under a suitable promoter for insect cells. Many
vectors are available for this purpose and selection of the
appropriate vector will depend mainly on the size of the nucleic
acid molecule to be inserted into the vector and the particular
host cell to be transformed with the vector. Each vector contains
various components depending on its function (amplification of DNA
or expression of DNA) and the particular host cell with which it is
compatible. The vector components for insect cell transformation
generally include, but are not limited to, one or more of the
following: a signal sequence, origin of replication, one or more
marker genes and an inducible promoter.
[0352] The insect vector may be an autonomously replicating vector,
i.e., a vector which exists as an extrachromosomal entity, the
replication of which is independent of chromosomal replication,
e.g., a plasmid, an extrachromosomal element, a minichromosome, or
an artificial chromosome. The vector may contain any means for
assuring self-replication. Alternatively, the vector may be one
which, when introduced into the insect cell, is integrated into the
genome and replicated together with the chromosome(s) into which it
has been integrated. For integration, the vector may rely on the
nucleic acid sequence of the vector for stable integration of the
vector into the genome by homologous or nonhomologous
recombination. Alternatively, the vector may contain additional
nucleic acid sequences for directing integration by homologous
recombination into the genome of the insect host. The additional
nucleic acid sequences enable the vector to be integrated into the
host cell genome at a precise location(s) in the chromosome(s). To
increase the likelihood of integration at a precise location, there
should be preferably two nucleic acid sequences which individually
contain a sufficient number of nucleic acids, preferably 400 bp to
1500 bp, more preferably 800 bp to 1000 bp, which are highly
homologous with the corresponding target sequence to enhance the
probability of homologous recombination. These nucleic acid
sequences may be any sequence that is homologous with a target
sequence in the genome of the insect host cell and, furthermore,
may be non-encoding or encoding sequences.
[0353] Baculovirus expression vectors (BEVs) have become important
tools for the expression of foreign genes, both for basic research
and for the production of proteins with direct clinical
applications in human and veterinary medicine (Doerfler, Curr. Top.
Microbiol. Immunol. 131:51-68 (1968); Luckow and Summers,
Bio/Technology 6:47-55 (1988a); Miller, Annual Review of Microbiol.
42:177-199 (1988); Summers, Curr. Comm. Molecular Biology, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1988); all of which
are herein incorporated by reference in their entirety). BEVs are
recombinant insect viruses in which the coding sequence for a
chosen foreign gene has been inserted behind a baculovirus promoter
in place of the viral gene, e.g., polyhedrin (Smith and Summers,
U.S. Pat. No. 4,745,051, the entirety of which is incorporated
herein by reference).
[0354] The use of baculovirus vectors relies upon the host cells
being derived from Lepidopteran insects such as Spodoptera
frugiperda or Trichoplusia ni. The preferred Spodoptera frugiperda
cell line is the cell line Sf9. The Spodoptera frugiperda Sf9 cell
line was obtained from American Type Culture Collection (Manassas,
Va.) and is assigned accession number ATCC CRL 1711 (Summers and
Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell
Culture Procedures, Texas Ag. Exper. Station Bulletin No. 1555
(1988), the entirety of which is herein incorporated by reference).
Other insect cell systems, such as the silkworm B. mori may also be
used.
[0355] The proteins expressed by the BEVs are, therefore,
synthesized, modified and transported in host cells derived from
Lepidopteran insects. Most of the genes that have been inserted and
produced in the baculovirus expression vector system have been
derived from vertebrate species. Other baculovirus genes in
addition to the polyhedrin promoter may be employed to advantage in
a baculovirus expression system. These include immediate-early
(alpha), delayed-early (.beta.), late (.gamma.), or very late
(delta), according to the phase of the viral infection during which
they are expressed. The expression of these genes occurs
sequentially, probably as the result of a "cascade" mechanism of
transcriptional regulation. (Guarino and Summers, J. Virol.
57:563-571 (1986); Guarino and Summers, J. Virol. 61:2091-2099
(1987); Guarino and Summers, Virol. 162:444-451 (1988); all of
which are herein incorporated by reference in their entirety).
[0356] Insect recombinant vectors are useful as intermediates for
the infection or transformation of insect cell systems. For
example, an insect recombinant vector containing a nucleic acid
molecule encoding a baculovirus transcriptional promoter followed
downstream by an insect signal DNA sequence is capable of directing
the secretion of the desired biologically active protein from the
insect cell. The vector may utilize a baculovirus transcriptional
promoter region derived from any of the over 500 baculoviruses
generally infecting insects, such as for example the Orders
Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera,
including for example but not limited to the viral DNAs of
Autographa californica MNPV, Bombyx mori NPV, Trichoplusia ni MNPV,
Rachiplusia ou MNPV or Galleria mellonella MNPV, wherein said
baculovirus transcriptional promoter is a baculovirus
immediate-early gene IE1 or IEN promoter; an immediate-early gene
in combination with a baculovirus delayed-early gene promoter
region selected from the group consisting of 39K and a HindIII-k
fragment delayed-early gene; or a baculovirus late gene promoter.
The immediate-early or delayed-early promoters can be enhanced with
transcriptional enhancer elements. The insect signal DNA sequence
may code for a signal peptide of a Lepidopteran adipokinetic
hormone precursor or a signal peptide of the Manduca sexta
adipokinetic hormone precursor (Summers, U.S. Pat. No. 5,155,037;
the entirety of which is herein incorporated by reference). Other
insect signal DNA sequences include a signal peptide of the
Orthoptera Schistocerca gregaria locust adipokinetic hormone
precurser and the Drosophila melanogaster cuticle genes CP1, CP2,
CP3 or CP4 or for an insect signal peptide having substantially a
similar chemical composition and function (Summers, U.S. Pat. No.
5,155,037).
[0357] Insect cells are distinctly different from animal cells.
Insects have a unique life cycle and have distinct cellular
properties such as the lack of intracellular plasminogen activators
in which are present in vertebrate cells. Another difference is the
high expression levels of protein products ranging from 1 to
greater than 500 mg/liter and the ease at which cDNA can be cloned
into cells (Frasier, In Vitro Cell. Dev. Biol. 25:225 (1989);
Summers and Smith, In: A Manual of Methods for Baculovirus Vectors
and Insect Cell Culture Procedures; Texas Ag. Exper. Station
Bulletin No. 1555 (1988), both of which are incorporated by
reference in their entirety).
[0358] Recombinant protein expression in insect cells is achieved
by viral infection or stable transformation. For viral infection,
the desired gene is cloned into baculovirus at the site of the
wild-type polyhedron gene (Webb and Summers, Technique 2:173
(1990); Bishop and Posse, Adv. Gene Technol. 1:55 (1990); both of
which are incorporated by reference in their entirety). The
polyhedron gene is a component of a protein coat in occlusions
which encapsulate virus particles. Deletion or insertion in the
polyhedron gene results the failure to form occlusion bodies.
Occlusion negative viruses are morphologically different from
occlusion positive viruses and enable one skilled in the art to
identify and purify recombinant viruses.
[0359] The vectors of present invention preferably contain one or
more selectable markers which permit easy selection of transformed
cells. A selectable marker is a gene the product of which provides,
for example biocide or viral resistance, resistance to heavy
metals, prototrophy to auxotrophs and the like. Selection may be
accomplished by co-transformation, e.g., as described in WO
91/17243, a nucleic acid sequence of the present invention may be
operably linked to a suitable promoter sequence. The promoter
sequence is a nucleic acid sequence which is recognized by the
insect host cell for expression of the nucleic acid sequence. The
promoter sequence contains transcription and translation control
sequences which mediate the expression of the protein or fragment
thereof. The promoter may be any nucleic acid sequence which shows
transcriptional activity in the insect host cell of choice and may
be obtained from genes encoding polypeptides either homologous or
heterologous to the host cell.
[0360] For example, a nucleic acid molecule encoding a protein or
fragment thereof may also be operably linked to a suitable leader
sequence. A leader sequence is a nontranslated region of a mRNA
which is important for translation by the fungal host. The leader
sequence is operably linked to the 5' terminus of the nucleic acid
sequence encoding the protein or fragment thereof. The leader
sequence may be native to the nucleic acid sequence encoding the
protein or fragment thereof or may be obtained from foreign
sources. Any leader sequence which is functional in the insect host
cell of choice may be used in the present invention.
[0361] A polyadenylation sequence may also be operably linked to
the 3' terminus of the nucleic acid sequence of the present
invention. The polyadenylation sequence is a sequence which when
transcribed is recognized by the insect host to add polyadenosine
residues to transcribed mRNA. The polyadenylation sequence may be
native to the nucleic acid sequence encoding the protein or
fragment thereof or may be obtained from foreign sources. Any
polyadenylation sequence which is functional in the fungal host of
choice may be used in the present invention.
[0362] To avoid the necessity of disrupting the cell to obtain the
protein or fragment thereof and to minimize the amount of possible
degradation of the expressed polypeptide within the cell, it is
preferred that expression of the polypeptide gene gives rise to a
product secreted outside the cell. To this end, the protein or
fragment thereof of the present invention may be linked to a signal
peptide linked to the amino terminus of the protein or fragment
thereof. A signal peptide is an amino acid sequence which permits
the secretion of the protein or fragment thereof from the insect
host into the culture medium. The signal peptide may be native to
the protein or fragment thereof of the invention or may be obtained
from foreign sources. The 5' end of the coding sequence of the
nucleic acid sequence of the present invention may inherently
contain a signal peptide coding region naturally linked in
translation reading frame with the segment of the coding region
which encodes the secreted protein or fragment thereof.
[0363] At present, a mode of achieving secretion of a foreign gene
product in insect cells is by way of the foreign gene's native
signal peptide. Because the foreign genes are usually from
non-insect organisms, their signal sequences may be poorly
recognized by insect cells and hence, levels of expression may be
suboptimal. However, the efficiency of expression of foreign gene
products seems to depend primarily on the characteristics of the
foreign protein. On average, nuclear localized or non-structural
proteins are most highly expressed, secreted proteins are
intermediate and integral membrane proteins are the least
expressed. One factor generally affecting the efficiency of the
production of foreign gene products in a heterologous host system
is the presence of native signal sequences (also termed
presequences, targeting signals, or leader sequences) associated
with the foreign gene. The signal sequence is generally coded by a
DNA sequence immediately following (5' to 3') the translation start
site of the desired foreign gene.
[0364] The expression dependence on the type of signal sequence
associated with a gene product can be represented by the following
example: If a foreign gene is inserted at a site downstream from
the translational start site of the baculovirus polyhedrin gene so
as to produce a fusion protein (containing the N-terminus of the
polyhedrin structural gene), the fused gene is highly expressed.
But less expression is achieved when a foreign gene is inserted in
a baculovirus expression vector immediately following the
transcriptional start site and totally replacing the polyhedrin
structural gene.
[0365] Insertions into the region -50 to -1 significantly alter
(reduce) steady state transcription which, in turn, reduces
translation of the foreign gene product. Use of the pVL941 vector
optimizes transcription of foreign genes to the level of the
polyhedrin gene transcription. Even though the transcription of a
foreign gene may be optimal, optimal translation may vary because
of several factors involving processing: signal peptide
recognition, mRNA and ribosome binding, glycosylation, disulfide
bond formation, sugar processing, oligomerization, for example.
[0366] The properties of the insect signal peptide are expected to
be more optimal for the efficiency of the translation process in
insect cells than those from vertebrate proteins. This phenomenon
can generally be explained by the fact that proteins secreted from
cells are synthesized as precursor molecules containing hydrophobic
N-terminal signal peptides. The signal peptides direct transport of
the select protein to its target membrane and are then cleaved by a
peptidase on the membrane, such as the endoplasmic reticulum, when
the protein passes through it.
[0367] Another exemplary insect signal sequence is the sequence
encoding for Drosophila cuticle proteins such as CP1, CP2, CP3 or
CP4 (Summers, U.S. Pat. No. 5,278,050; the entirety of which is
herein incorporated by reference). Most of a 9 kb region of the
Drosophila genome containing genes for the cuticle proteins has
been sequenced. Four of the five cuticle genes contains a signal
peptide coding sequence interrupted by a short intervening sequence
(about 60 base pairs) at a conserved site. Conserved sequences
occur in the 5' mRNA untranslated region, in the adjacent 35 base
pairs of upstream flanking sequence and at -200 base pairs from the
mRNA start position in each of the cuticle genes.
[0368] Standard methods of insect cell culture, cotransfection and
preparation of plasmids are set forth in Summers and Smith (Summers
and Smith, A Manual of Methods for Baculovirus Vectors and Insect
Cell Culture Procedures, Texas Agricultural Experiment Station
Bulletin No. 1555, Texas A&M University (1987)). Procedures for
the cultivation of viruses and cells are described in Volkman and
Summers, J. Virol 19:820-832 (1975) and Volkman et al., J. Virol
19:820-832 (1976); both of which are herein incorporated by
reference in their entirety.
[0369] (e) Bacterial Constructs and Transformed Bacterial Cells
[0370] The present invention also relates to a bacterial
recombinant vector comprising exogenous genetic material. The
present invention also relates to a bacteria cell comprising a
bacterial recombinant vector. The present invention also relates to
methods for obtaining a recombinant bacteria host cell, comprising
introducing into a bacterial host cell exogenous genetic material.
In a preferred embodiment the exogenous genetic material includes a
nucleic acid molecule of the present invention having a sequence
selected from the group consisting of SEQ ID NO: 1 through SEQ ID
NO: 2814 or complements thereof or fragments of either or other
nucleic acid molecule of the present invention.
[0371] The bacterial recombinant vector may be any vector which can
be conveniently subjected to recombinant DNA procedures. The choice
of a vector will typically depend on the compatibility of the
vector with the bacterial host cell into which the vector is to be
introduced. The vector may be a linear or a closed circular
plasmid. The vector system may be a single vector or plasmid or two
or more vectors or plasmids which together contain the total DNA to
be introduced into the genome of the bacterial host. In addition,
the bacterial vector may be an expression vector. Nucleic acid
molecules encoding protein homologues or fragments thereof can, for
example, be suitably inserted into a replicable vector for
expression in the bacterium under the control of a suitable
promoter for bacteria. Many vectors are available for this purpose
and selection of the appropriate vector will depend mainly on the
size of the nucleic acid to be inserted into the vector and the
particular host cell to be transformed with the vector. Each vector
contains various components depending on its function
(amplification of DNA or expression of DNA) and the particular host
cell with which it is compatible. The vector components for
bacterial transformation generally include, but are not limited to,
one or more of the following: a signal sequence, an origin of
replication, one or more marker genes and an inducible
promoter.
[0372] In general, plasmid vectors containing replicon and control
sequences that are derived from species compatible with the host
cell are used in connection with bacterial hosts. The vector
ordinarily carries a replication site, as well as marking sequences
that are capable of providing phenotypic selection in transformed
cells. For example, E. coli is typically transformed using pBR322,
a plasmid derived from an E. coli species (see, e.g., Bolivar et
al., Gene 2:95 (1977); the entirety of which is herein incorporated
by reference). pBR322 contains genes for ampicillin and
tetracycline resistance and thus provides easy means for
identifying transformed cells. The pBR322 plasmid, or other
microbial plasmid or phage, also generally contains, or is modified
to contain, promoters that can be used by the microbial organism
for expression of the selectable marker genes.
[0373] Nucleic acid molecules encoding protein or fragments thereof
may be expressed not only directly, but also as a fusion with
another polypeptide, preferably a signal sequence or other
polypeptide having a specific cleavage site at the N-terminus of
the mature polypeptide. In general, the signal sequence may be a
component of the vector, or it may be a part of the polypeptide DNA
that is inserted into the vector. The heterologous signal sequence
selected should be one that is recognized and processed (i.e.,
cleaved by a signal peptidase) by the host cell. For bacterial host
cells that do not recognize and process the native polypeptide
signal sequence, the signal sequence is substituted by a bacterial
signal sequence selected, for example, from the group consisting of
the alkaline phosphatase, penicillinase, lpp, or heat-stable
enterotoxin II leaders.
[0374] Both expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomal DNA and includes origins of replication or autonomously
replicating sequences. Such sequences are well known for a variety
of bacteria. The origin of replication from the plasmid pBR322 is
suitable for most Gram-negative bacteria.
[0375] Expression and cloning vectors also generally contain a
selection gene, also termed a selectable marker. This gene encodes
a protein necessary for the survival or growth of transformed host
cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will not
survive in the culture medium. Typical selection genes encode
proteins that (a) confer resistance to antibiotics or other toxins,
e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)
complement auxotrophic deficiencies, or (c) supply critical
nutrients not available from complex media, e.g., the gene encoding
D-alanine racemase for Bacilli. One example of a selection scheme
utilizes a drug to arrest growth of a host cell. Those cells that
are successfully transformed with a heterologous protein homologue
or fragment thereof produce a protein conferring drug resistance
and thus survive the selection regimen.
[0376] The expression vector for producing a protein or fragment
thereof can also contains an inducible promoter that is recognized
by the host bacterial organism and is operably linked to the
nucleic acid encoding, for example, the nucleic acid molecule
encoding the protein homologue or fragment thereof of interest.
Inducible promoters suitable for use with bacterial hosts include
the .beta.-lactamase and lactose promoter systems (Chang et al.,
Nature 275:615 (1978); Goeddel et al., Nature 281:544 (1979); both
of which are herein incorporated by reference in their entirety),
the arabinose promoter system (Guzman et al., J. Bacteriol.
174:7716-7728 (1992); the entirety of which is herein incorporated
by reference), alkaline phosphatase, a tryptophan (trp) promoter
system (Goeddel, Nucleic Acids Res. 8:4057 (1980); EP 36,776; both
of which are herein incorporated by reference in their entirety)
and hybrid promoters such as the tac promoter (deBoer et al., Proc.
Natl. Acad. Sci. (USA) 80:21-25 (1983); the entirety of which is
herein incorporated by reference). However, other known bacterial
inducible promoters are suitable (Siebenlist et al., Cell 20:269
(1980); the entirety of which is herein incorporated by
reference).
[0377] Promoters for use in bacterial systems also generally
contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA
encoding the polypeptide of interest. The promoter can be removed
from the bacterial source DNA by restriction enzyme digestion and
inserted into the vector containing the desired DNA.
[0378] Construction of suitable vectors containing one or more of
the above-listed components employs standard ligation techniques.
Isolated plasmids or DNA fragments are cleaved, tailored and
re-ligated in the form desired to generate the plasmids required.
Examples of available bacterial expression vectors include, but are
not limited to, the multifunctional E. coli cloning and expression
vectors such as Bluescript.TM. (Stratagene, La Jolla, Calif.), in
which, for example, encoding an A. nidulans protein homologue or
fragment thereof homologue, may be ligated into the vector in frame
with sequences for the amino-terminal Met and the subsequent 7
residues of .beta.-galactosidase so that a hybrid protein is
produced; pIN vectors (Van Heeke and Schuster, J. Biol. Chem.
264:5503-5509 (1989), the entirety of which is herein incorporated
by reference); and the like. pGEX vectors (Promega, Madison Wis.
U.S.A.) may also be used to express foreign polypeptides as fusion
proteins with glutathione S-transferase (GST). In general, such
fusion proteins are soluble and can easily be purified from lysed
cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. Proteins made in such
systems are designed to include heparin, thrombin or factor XA
protease cleavage sites so that the cloned polypeptide of interest
can be released from the GST moiety at will.
[0379] Suitable host bacteria for a bacterial vector include
archaebacteria and eubacteria, especially eubacteria and most
preferably Enterobacteriaceae. Examples of useful bacteria include
Escherichia, Enterobacter, Azotobacter, Erwinia, Bacillus,
Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella,
Rhizobia, Vitreoscilla and Paracoccus. Suitable E. coli hosts
include E. coli W3110 (American Type Culture Collection (ATCC)
27,325, Manassas, Va. U.S.A.), E. coli 294 (ATCC 31,446), E. coli B
and E. coli X1776 (ATCC 31,537). These examples are illustrative
rather than limiting. Mutant cells of any of the above-mentioned
bacteria may also be employed. It is, of course, necessary to
select the appropriate bacteria taking into consideration
replicability of the replicon in the cells of a bacterium. For
example, E. coli, Serratia, or Salmonella species can be suitably
used as the host when well known plasmids such as pBR322, pBR325,
pACYC177, or pKN410 are used to supply the replicon. E. coli strain
W3110 is a preferred host or parent host because it is a common
host strain for recombinant DNA product fermentations. Preferably,
the host cell should secrete minimal amounts of proteolytic
enzymes.
[0380] Host cells are transfected and preferably transformed with
the above-described vectors and cultured in conventional nutrient
media modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0381] Numerous methods of transfection are known to the ordinarily
skilled artisan, for example, calcium phosphate and
electroporation. Depending on the host cell used, transformation is
done using standard techniques appropriate to such cells. The
calcium treatment employing calcium chloride, as described in
section 1.82 of Sambrook et al., Molecular Cloning: A Laboratory
Manual, New York: Cold Spring Harbor Laboratory Press, (1989), is
generally used for bacterial cells that contain substantial
cell-wall barriers. Another method for transformation employs
polyethylene glycol/DMSO, as described in Chung and Miller (Chung
and Miller, Nucleic Acids Res. 16:3580 (1988); the entirety of
which is herein incorporated by reference). Yet another method is
the use of the technique termed electroporation.
[0382] Bacterial cells used to produce the polypeptide of interest
for purposes of this invention are cultured in suitable media in
which the promoters for the nucleic acid encoding the heterologous
polypeptide can be artificially induced as described generally,
e.g., in Sambrook et al., Molecular Cloning: A Laboratory Manual,
New York: Cold Spring Harbor Laboratory Press, (1989). Examples of
suitable media are given in U.S. Pat. Nos. 5,304,472 and 5,342,763;
both of which are incorporated by reference in their entirety.
[0383] In addition to the above discussed procedures, practitioners
are familiar with the standard resource materials which describe
specific conditions and procedures for the construction,
manipulation and isolation of macromolecules (e.g., DNA molecules,
plasmids, etc.), generation of recombinant organisms and the
screening and isolating of clones, (see for example, Sambrook et
al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor
Press (1989); Mailga et al., Methods in Plant Molecular Biology,
Cold Spring Harbor Press (1995), the entirety of which is herein
incorporated by reference; Birren et al., Genome Analysis:
Analyzing DNA, 1, Cold Spring Harbor, N.Y., the entirety of which
is herein incorporated by reference).
[0384] (f) Computer Readable Media
[0385] The nucleotide sequence provided in SEQ ID NO: 1 through SEQ
ID NO: 2814 or fragment thereof, or complement thereof, or a
nucleotide sequence at least 90% identical, preferably 95%,
identical even more preferably 99% or 100% identical to the
sequence provided in SEQ ID NO: 1 through SEQ ID NO: 2814 or
fragment thereof, or complement thereof, can be "provided" in a
variety of mediums to facilitate use. Such a medium can also
provide a subset thereof in a form that allows a skilled artisan to
examine the sequences.
[0386] A preferred subset of nucleotide sequences are those nucleic
acid sequences that encodes a maize or a soybean triose phosphate
isomerase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean fructose
1,6-bisphosphate aldolase enzyme or complement thereof or fragment
of either, a nucleic acid molecule that encodes a maize or a
soybean fructose 1,6-bisphosphate enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean fructose 6-phosphate 2-kinase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean phosphoglucoisomerase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean vacuolar H.sup.+ translocating-pyrophosphatase
enzyme or complement thereof or fragment of either, a nucleic acid
molecule that encodes a maize or a soybean pyrophosphate-dependent
fructose-6-phosphate phosphotransferase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean invertase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean sucrose synthase enzyme or complement thereof or fragment
of either, a nucleic acid molecule that encodes a maize or a
soybean hexokinase enzyme or complement thereof or fragment of
either, a nucleic acid molecule that encodes a maize or a soybean
fructokinase enzyme or complement thereof or fragment of either f,
a nucleic acid molecule that encodes a maize or a soybean
NDP-kinase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean
glucose-6-phosphate 1-dehydrogenase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean phosphoglucomutase enzyme or complement thereof or
fragment of either and a nucleic acid molecule that encodes a maize
or a soybean UDP-glucose pyrophosphorylase enzyme or complement
thereof or fragment of either.
[0387] A further preferred subset of nucleic acid sequences is
where the subset of sequences is two proteins or fragments thereof,
more preferably three proteins or fragments thereof and even more
preferable four proteins or fragments thereof, these nucleic acid
sequences are selected from the group that comprises a maize or a
soybean triose phosphate isomerase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean fructose 1,6-bisphosphate aldolase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean fructose 1,6-bisphosphate enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean fructose 6-phosphate 2-kinase enzyme or
complement thereof or fragment of either, a nucleic acid molecule
that encodes a maize or a soybean phosphoglucoisomerase enzyme or
complement thereof or fragment of either, a nucleic acid molecule
that encodes a maize or a soybean vacuolar H.sup.+
translocating-pyrophosphatase enzyme or complement thereof or
fragment of either, a nucleic acid molecule that encodes a maize or
a soybean pyrophosphate-dependent fructose-6-phosphate
phosphotransferase enzyme or complement thereof or fragment of
either, a nucleic acid molecule that encodes a maize or a soybean
invertase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean sucrose
synthase enzyme or complement thereof or fragment of either, a
nucleic acid molecule that encodes a maize or a soybean hexokinase
enzyme or complement thereof or fragment of either, a nucleic acid
molecule that encodes a maize or a soybean fructokinase enzyme or
complement thereof or fragment of either f, a nucleic acid molecule
that encodes a maize or a soybean NDP-kinase enzyme or complement
thereof or fragment of either, a nucleic acid molecule that encodes
a maize or a soybean glucose-6-phosphate 1-dehydrogenase enzyme or
complement thereof or fragment of either, a nucleic acid molecule
that encodes a maize or a soybean phosphoglucomutase enzyme or
complement thereof or fragment of either and a nucleic acid
molecule that encodes a maize or a soybean UDP-glucose
pyrophosphorylase enzyme or complement thereof or fragment of
either.
[0388] In one application of this embodiment, a nucleotide sequence
of the present invention can be recorded on computer readable
media. As used herein, "computer readable media" refers to any
medium that can be read and accessed directly by a computer. Such
media include, but are not limited to: magnetic storage media, such
as floppy discs, hard disc, storage medium and magnetic tape:
optical storage media such as CD-ROM; electrical storage media such
as RAM and ROM; and hybrids of these categories such as
magnetic/optical storage media. A skilled artisan can readily
appreciate how any of the presently known computer readable mediums
can be used to create a manufacture comprising computer readable
medium having recorded thereon a nucleotide sequence of the present
invention.
[0389] As used herein, "recorded" refers to a process for storing
information on computer readable medium. A skilled artisan can
readily adopt any of the presently known methods for recording
information on computer readable medium to generate media
comprising the nucleotide sequence information of the present
invention. A variety of data storage structures are available to a
skilled artisan for creating a computer readable medium having
recorded thereon a nucleotide sequence of the present invention.
The choice of the data storage structure will generally be based on
the means chosen to access the stored information. In addition, a
variety of data processor programs and formats can be used to store
the nucleotide sequence information of the present invention on
computer readable medium. The sequence information can be
represented in a word processing text file, formatted in
commercially-available software such as WordPerfect and Microsoft
Word, or represented in the form of an ASCII file, stored in a
database application, such as DB2, Sybase, Oracle, or the like. A
skilled artisan can readily adapt any number of data processor
structuring formats (e.g. text file or database) in order to obtain
computer readable medium having recorded thereon the nucleotide
sequence information of the present invention.
[0390] By providing one or more of nucleotide sequences of the
present invention, a skilled artisan can routinely access the
sequence information for a variety of purposes. Computer software
is publicly available which allows a skilled artisan to access
sequence information provided in a computer readable medium. The
examples which follow demonstrate how software which implements the
BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990), the
entirety of which is herein incorporated by reference) and BLAZE
(Brutlag et al., Comp. Chem. 17:203-207 (1993), the entirety of
which is herein incorporated by reference) search algorithms on a
Sybase system can be used to identify open reading frames (ORFs)
within the genome that contain homology to ORFs or proteins from
other organisms. Such ORFs are protein-encoding fragments within
the sequences of the present invention and are useful in producing
commercially important proteins such as enzymes used in amino acid
biosynthesis, metabolism, transcription, translation, RNA
processing, nucleic acid and a protein degradation, protein
modification and DNA replication, restriction, modification,
recombination and repair.
[0391] The present invention further provides systems, particularly
computer-based systems, which contain the sequence information
described herein. Such systems are designed to identify
commercially important fragments of the nucleic acid molecule of
the present invention. As used herein, "a computer-based system"
refers to the hardware means, software means and data storage means
used to analyze the nucleotide sequence information of the present
invention. The minimum hardware means of the computer-based systems
of the present invention comprises a central processing unit (CPU),
input means, output means and data storage means. A skilled artisan
can readily appreciate that any one of the currently available
computer-based system are suitable for use in the present
invention.
[0392] As indicated above, the computer-based systems of the
present invention comprise a data storage means having stored
therein a nucleotide sequence of the present invention and the
necessary hardware means and software means for supporting and
implementing a search means. As used herein, "data storage means"
refers to memory that can store nucleotide sequence information of
the present invention, or a memory access means which can access
manufactures having recorded thereon the nucleotide sequence
information of the present invention. As used herein, "search
means" refers to one or more programs which are implemented on the
computer-based system to compare a target sequence or target
structural motif with the sequence information stored within the
data storage means. Search means are used to identify fragments or
regions of the sequence of the present invention that match a
particular target sequence or target motif. A variety of known
algorithms are disclosed publicly and a variety of commercially
available software for conducting search means are available can be
used in the computer-based systems of the present invention.
Examples of such software include, but are not limited to,
MacPattern (EMBL), BLASTIN and BLASTIX (NCBIA). One of the
available algorithms or implementing software packages for
conducting homology searches can be adapted for use in the present
computer-based systems.
[0393] The most preferred sequence length of a target sequence is
from about 10 to 100 amino acids or from about 30 to 300 nucleotide
residues. However, it is well recognized that during searches for
commercially important fragments of the nucleic acid molecules of
the present invention, such as sequence fragments involved in gene
expression and protein processing, may be of shorter length.
[0394] As used herein, "a target structural motif," or "target
motif," refers to any rationally selected sequence or combination
of sequences in which the sequences the sequence(s) are chosen
based on a three-dimensional configuration which is formed upon the
folding of the target motif. There are a variety of target motifs
known in the art. Protein target motifs include, but are not
limited to, enzymatic active sites and signal sequences. Nucleic
acid target motifs include, but are not limited to, promoter
sequences, cis elements, hairpin structures and inducible
expression elements (protein binding sequences).
[0395] Thus, the present invention further provides an input means
for receiving a target sequence, a data storage means for storing
the target sequences of the present invention sequence identified
using a search means as described above and an output means for
outputting the identified homologous sequences. A variety of
structural formats for the input and output means can be used to
input and output information in the computer-based systems of the
present invention. A preferred format for an output means ranks
fragments of the sequence of the present invention by varying
degrees of homology to the target sequence or target motif. Such
presentation provides a skilled artisan with a ranking of sequences
which contain various amounts of the target sequence or target
motif and identifies the degree of homology contained in the
identified fragment.
[0396] A variety of comparing means can be used to compare a target
sequence or target motif with the data storage means to identify
sequence fragments sequence of the present invention. For example,
implementing software which implement the BLAST and BLAZE
algorithms (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) can
be used to identify open frames within the nucleic acid molecules
of the present invention. A skilled artisan can readily recognize
that any one of the publicly available homology search programs can
be used as the search means for the computer-based systems of the
present invention.
[0397] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration and are not
intended to be limiting of the present invention, unless
specified.
Example 1
[0398] The MONN01 cDNA library is a normalized library generated
from maize (DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.) total
leaf tissue at the V6 plant development stage. Seeds are planted at
a depth of approximately 3 cm into 2-3 inch peat pots containing
Metro 200 growing medium. After 2-3 weeks growth they are
transplanted into 10 inch pots containing the same growing medium.
Plants are watered daily before transplantation and three times a
week after transplantation. Peters 15-16-17 fertilizer is applied
three times per week after transplanting at a strength of 150 ppm
N. Two to three times during the lifetime of the plant, from
transplanting to flowering, a total of 900 mg Fe is added to each
pot. Maize plants are grown in the greenhouse in 15 hr day/9 hr
night cycles. The daytime temperature is approximately 80.degree.
F. and the nighttime temperature is approximately 70.degree. F.
Supplemental lighting is provided by 1000 W sodium vapor lamps.
Tissue is collected when the maize plant is at the 6-leaf
development stage. The older, more juvenile leaves, which are in a
basal position, as well as the younger, more adult leaves, which
are more apical are cut at the base of the leaves. The leaves are
then pooled and immediately transferred to liquid nitrogen
containers in which the pooled leaves are crushed. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
[0399] The SATMON001 cDNA library is generated from maize (B73,
Illinois Foundation Seeds, Champaign, Illinois U.S.A.) immature
tassels at the V6 plant development stage. Seeds are planted at a
depth of approximately 3 cm into 2-3 inch peat pots containing
Metro 200 growing medium. After 2-3 weeks growth they are
transplanted into 10 inch pots containing the same growing medium.
Plants are watered daily before transplantation and three times a
week after transplantation. Peters 15-16-17 fertilizer is applied
three times per week after transplanting at a strength of 150 ppm
N. Two to three times during the lifetime of the plant, from
transplanting to flowering, a total of 900 mg Fe is added to each
pot. Maize plants are grown in a greenhouse in 15 hr day/9 hr night
cycles. The daytime temperature is approximately 80.degree. F. and
the nighttime temperature is approximately 70.degree. F.
Supplemental lighting is provided by 1000 W sodium vapor lamps.
Tissue from the maize plant is collected at the V6 stage. At that
stage the tassel is an immature tassel of about 2-3 cm in length.
The tassels are removed and frozen in liquid nitrogen. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0400] The SATMON003 library is generated from maize (B73.times.Mol
7, Illinois Foundation Seeds, Champaign, Illinois U.S.A.) roots at
the V6 developmental stage. Seeds are planted at a depth of
approximately 3 cm in coil into 2-3 inch peat pots containing Metro
200 growing medium. After 2-3 weeks growth, the seedlings are
transplanted into 10 inch pots containing the Metro 200 growing
medium. Plants are watered daily before transplantation and
approximately 3 times a week after transplantation. Peters 15-16-17
fertilizer is applied approximately three times per week after
transplanting at a concentration of 150 ppm N. Two to three times
during the life time of the plant from transplanting to flowering a
total of approximately 900 mg Fe is added to each pot. Maize plants
are grown in the green house in approximately 15 hr day/9 hr night
cycles. The daytime temperature is approximately 80.degree. F. and
the nighttime temperature is approximately 70.degree. F.
Supplemental lighting is provided by 1000 W sodium vapor lamps.
Tissue is collected when the maize plant is at the 6 leaf
development stage. The root system is cut from maize plant and
washed with water to free it from the soil. The tissue is then
immediately frozen in liquid nitrogen. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0401] The SATMON004 cDNA library is generated from maize
(B73.times.Mol7, Illinois Foundation Seeds, Champaign, Illinois
U.S.A.) total leaf tissue at the V6 plant development stage. Seeds
are planted at a depth of approximately 3 cm into 2-3 inch peat
pots containing Metro 200 growing medium. After 2-3 weeks growth
they are transplanted into 10 inch pots containing the same growing
medium. Plants are watered daily before transplantation and three
times a week after transplantation. Peters 15-16-17 fertilizer is
applied three times per week after transplanting at a strength of
150 ppm N. Two to three times during the lifetime of the plant,
from transplanting to flowering, a total of 900 mg Fe is added to
each pot. Maize plants are grown in the greenhouse in 15 hr day/9
hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the
6-leaf development stage. The older, more juvenile leaves, which
are in a basal position, as well as the younger, more adult leaves,
which are more apical are cut at the base of the leaves. The leaves
are then pooled and immediately transferred to liquid nitrogen
containers in which the pooled leaves are crushed. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
[0402] The SATMON005 cDNA library is generated from maize
(B73.times.Mol7, Illinois Foundation Seeds, Champaign Illinois,
U.S.A.) root tissue at the V6 development stage. Seeds are planted
at a depth of approximately 3 cm into 2-3 inch peat pots containing
Metro 200 growing medium. After 2-3 weeks growth they are
transplanted into 10 inch pots containing the same growing medium.
Plants are watered daily before transplantation and three times a
week after transplantation. Peters 15-16-17 fertilizer is applied
three times per week after transplanting at a strength of 150 ppm
N. Two to three times during the lifetime of the plant, from
transplanting to flowering, a total of 900 mg Fe is added to each
pot. Maize plants are grown in the green house in 15 hr day/9 hr
night cycles. The daytime temperature is approximately 80.degree.
F. and the nighttime temperature is approximately 70.degree. F.
Supplemental lighting is provided by 1000 W sodium vapor lamps.
Tissue is collected when the maize plant is at the 6-leaf
development stage. The root system is cut from the mature maize
plant and washed with water to free it from the soil. The tissue is
immediately frozen in liquid nitrogen and the harvested tissue is
then stored at -80.degree. C. until RNA preparation.
[0403] The SATMON006 cDNA library is generated from maize
(B73.times.Mol7, Illinois Foundation Seeds, Champaign Illinois,
U.S.A.) total leaf tissue at the V6 plant development stage. Seeds
are planted at a depth of approximately 3 cm into 2-3 inch peat
pots containing Metro 200 growing medium. After 2-3 weeks growth
they are transplanted into 10 inch pots containing the same growing
medium. Plants are watered daily before transplantation and three
times a week after transplantation. Peters 15-16-17 fertilizer is
applied three times per week after transplanting at a strength of
150 ppm N. Two to three times during the lifetime of the plant,
from transplanting to flowering, a total of 900 mg Fe is added to
each pot. Maize plants are grown in the greenhouse in 15 hr day/9
hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the
6-leaf development stage. The older more juvenile leaves, which are
in a basal position, as well as the younger more adult leaves,
which are more apical are cut at the base of the leaves. The leaves
are then pooled and immediately transferred to liquid nitrogen
containers in which the pooled leaves are crushed. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
[0404] The SATMON007 cDNA library is generated from the primary
root tissue of 5 day old maize (DK604, Dekalb Genetics, Dekalb,
Illinois U.S.A.) seedlings. Seeds are planted on a moist filter
paper on a covered tray that is kept in the dark until germination
(one day). After germination, the trays, along with the moist
paper, are moved to a greenhouse where the maize plants are grown
in the greenhouse in 15 hr day/9 hr night cycles for approximately
5 days. The daytime temperature is approximately 80.degree. F. and
the nighttime temperature is approximately 70.degree. F.
Supplemental lighting is provided by 1000 W sodium vapor lamps. The
primary root tissue is collected when the seedlings are 5 days old.
At this stage, the primary root (radicle) is pushed through the
coleorhiza which itself is pushed through the seed coat. The
primary root, which is about 2-3 cm long, is cut and immediately
frozen in liquid nitrogen and then stored at -80.degree. C. until
RNA preparation.
[0405] The SATMON008 cDNA library is generated from the primary
shoot (coleoptile 2-3 cm) of maize (DK604, Dekalb Genetics, Dekalb,
Illinois U.S.A.) seedlings which are approximately 5 days old.
Seeds are planted on a moist filter paper on a covered tray that is
kept in the dark until germination (one day). Then the trays
containing the seeds are moved to a greenhouse at 15 hr daytime/9
hr nighttime cycles and grown until they are 5 days post
germination. The daytime temperature is approximately 80.degree. F.
and the nighttime temperature is approximately 70.degree. F. Tissue
is collected when the seedlings are 5 days old. At this stage, the
primary shoot (coleoptile) is pushed through the seed coat and is
about 2-3 cm long. The coleoptile is dissected away from the rest
of the seedling, immediately frozen in liquid nitrogen and then
stored at -80.degree. C. until RNA preparation.
[0406] The SATMON009 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) leaves at the 8 leaf
stage (V8 plant development stage). Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Maize plants
are grown in the green house in 15 hr day/9 hr night cycles. The
daytime temperature is 80.degree. F. and the nighttime temperature
is 70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the
8-leaf development stage. The older more juvenile leaves, which are
in a basal position, as well as the younger more adult leaves,
which are more apical, are cut at the base of the leaves. The
leaves are then pooled and then immediately transferred to liquid
nitrogen containers in which the pooled leaves are crushed. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0407] The SATMON010 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) root tissue at the V8
plant development stage. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Maize plants
are grown in the green house in 15 hr day/9 hr night cycles. The
daytime temperature is 80.degree. F. and the nighttime temperature
is 70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the V8
development stage. The root system is cut from this mature maize
plant and washed with water to free it from the soil. The tissue is
immediately frozen in liquid nitrogen. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0408] The SATMON011 cDNA library is generated from undeveloped
maize (DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.) leaf at the
V6 plant development stage. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Maize plants
are grown in the green house in 15 hr day/9 hr night cycles. The
daytime temperature is approximately 80.degree. F. and the
nighttime temperature is approximately 70.degree. F. Supplemental
lighting is provided by 1000 W sodium vapor lamps. Tissue is
collected when the maize plant is at the 6-leaf development stage.
The second youngest leaf which is at the base of the apical leaf of
V6 stage maize plant is cut at the base and immediately transferred
to liquid nitrogen containers in which the leaf is crushed. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0409] The SATMON012 cDNA library is generated from 2 day post
germination maize (DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.)
seedlings. Seeds are planted on a moist filter paper on a covered
tray that is kept in the dark until germination (one day). Then the
trays containing the seeds are moved to the greenhouse and grown at
15 hr daytime/9 hr nighttime cycles until 2 days post germination.
The daytime temperature is approximately 80.degree. F. and the
nighttime temperature is approximately 70.degree. F. Tissue is
collected when the seedlings are 2 days old. At the two day stage,
the coleorhiza is pushed through the seed coat and the primary root
(the radicle) is pierced the coleorhiza but is barely visible.
Also, at this two day stage, the coleoptile is just emerging from
the seed coat. The 2 days post germination seedlings are then
immersed in liquid nitrogen and crushed. The harvested tissue is
stored at -80.degree. C. until preparation of total RNA.
[0410] The SATMON013 cDNA library is generated from apical maize
(DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.) meristem founder
at the V4 plant development stage. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Maize plants
are grown in the greenhouse in 15 hr day/9 hr night cycles. The
daytime temperature is approximately 80.degree. F. and the
nighttime temperature is approximately 70.degree. F. Supplemental
lighting is provided by 1000 W sodium vapor lamps. Prior to tissue
collection, the plant is at the 4 leaf stage. The lead at the apex
of the V4 stage maize plant is referred to as the meristem founder.
This apical meristem founder is cut, immediately frozen in liquid
nitrogen and crushed. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0411] The SATMON014 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) endosperm fourteen days
after pollination. Seeds are planted at a depth of approximately 3
cm into 2-3 inch peat pots containing Metro 200 growing medium.
After 2-3 weeks growth they are transplanted into 10 inch pots
containing the same growing medium. Plants are watered daily before
transplantation and three times a week after transplantation.
Peters 15-16-17 fertilizer is applied three times per week after
transplanting at a strength of 150 ppm N. Two to three times during
the lifetime of the plant, from transplanting to flowering, a total
of 900 mg Fe is added to each pot. Maize plants are grown in the
greenhouse in 15 hr day/9 hr night cycles. The daytime temperature
is approximately 80.degree. F. and the nighttime temperature is
approximately 70.degree. F. Supplemental lighting is provided by
1000 W sodium vapor lamps. After the V10 stage, the maize plant ear
shoots are ready for fertilization. At this stage, the ear shoots
are enclosed in a paper bag before silk emergence to withhold the
pollen. The ear shoots are pollinated and 14 days after
pollination, the ears are pulled out and then the kernels are
plucked out of the ears. Each kernel is then dissected into the
embryo and the endosperm and the aleurone layer is removed. After
dissection, the endosperms are immediately frozen in liquid
nitrogen and then stored at -80.degree. C. until RNA
preparation.
[0412] The SATMON016 library is a maize (DK604, Dekalb Genetics,
Dekalb, Illinois U.S.A.) sheath library collected at the V8
developmental stage. Seeds are planted in a depth of approximately
3 cm in solid into 2-3 inch pots containing Metro growing medium.
After 2-3 weeks growth, they are transplanted into 10'' pots
containing the same. Plants are watered daily before
transplantation and approximately the times a week after
transplantation. Peters 15-16-17 fertilizer is applied
approximately three times per week after transplanting, at a
strength of 150 ppm N. Two to three times during the life time of
the plant from transplanting to flowering, a total of approximately
900 mg Fe is added to each pot. Maize plants are grown in the green
house in 15 hr day/9 hr night cycles. The daytime temperature is
approximately 80.degree. F. and the nighttime temperature is
approximately 70.degree. F. Supplemental lighting is provided by
1000 W sodium vapor lamps. When the maize plants are at the V8
stage the 5.sup.th and 6.sup.th leaves from the bottom exhibit
fully developed leaf blades. At the base of these leaves, the
ligule is differentiated and the leaf blade is joined to the
sheath. The sheath is dissected away from the base of the leaf then
the sheath is frozen in liquid nitrogen and crushed. The tissue is
then stored at -80.degree. C. until RNA preparation.
[0413] The SATMON017 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) embryo seventeen days
after pollination. Seeds are planted at a depth of approximately 3
cm into 2-3 inch peat pots containing Metro 200 growing medium.
After 2-3 weeks growth the seeds are transplanted into 10 inch pots
containing the same growing medium. Plants are watered daily before
transplantation and three times a week after transplantation.
Peters 15-16-17 fertilizer is applied three times per week after
transplanting at a strength of 150 ppm N. Two to three times during
the lifetime of the plant, from transplanting to flowering, a total
of 900 mg Fe is added to each pot. Maize plants are grown in the
green house in 15 hr day/9 hr night cycles. The daytime temperature
is approximately 80.degree. F. and the nighttime temperature is
approximately 70.degree. F. Supplemental lighting is provided by
1000 W sodium vapor lamps. After the VI 0 stage, the ear shoots of
maize plant, which are ready for fertilization, are enclosed in a
paper bag before silk emergence to withhold the pollen. The ear
shoots are fertilized and 21 days after pollination, the ears are
pulled out and the kernels are plucked out of the ears. Each kernel
is then dissected into the embryo and the endosperm and the
aleurone layer is removed. After dissection, the embryos are
immediately frozen in liquid nitrogen and then stored at
-80.degree. C. until RNA preparation.
[0414] The SATMON019 (Lib3054) cDNA library is generated from maize
(DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.) culm (stem) at
the V8 developmental stage. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Maize plants
are grown in the green house in 15 hr day/9 hr night cycles. The
daytime temperature is approximately 80.degree. F. and the
nighttime temperature is approximately 70.degree. F. Supplemental
lighting is provided by 1000 W sodium vapor lamps. When the maize
plant is at the V8 stage, the 5th and 6th leaves from the bottom
have fully developed leaf blades. The region between the nodes of
the 5th and the sixth leaves from the bottom is the region of the
stem that is collected. The leaves are pulled out and the sheath is
also torn away from the stem. This stem tissue is completely free
of any leaf and sheath tissue. The stem tissue is then frozen in
liquid nitrogen and stored at -80.degree. C. until RNA
preparation.
[0415] The SATMON020 cDNA library is from a maize (DK604, Dekalb
Genetics, Dekalb, Illinois U.S.A.) Hill Type 1'-Initiated Callus.
Petri plates containing approximately 25 ml of Type II initiation
media are prepared. This medium contains N6 salts and vitamins, 3%
sucrose, 2.3 g/liter proline 0.1 g/liter enzymatic casein
hydrolysate, 2 mg/liter 2,4- dichloro phenoxy-acetic acid (2,4, D),
15.3 mg/liter AgNO.sub.3 and 0.8% bacto agar and is adjusted to pH
6.0 before autoclaving. At 9-11 days after pollination, an ear with
immature embryos measuring approximately 1-2 mm in length is
chosen. The husks and silks are removed and then the ear is broken
into halves and placed in an autoclaved solution of Clorox/TWEEN 20
sterilizing solution. Then the ear is rinsed with deionized water.
Then each embryo is extracted from the kernel. Intact embryos are
placed in contact with the medium, scutellar side up). Multiple
embryos are plated on each plate and the plates are incubated in
the dark at 25.degree. C. Type II calluses are friable, can be
subcultured with a spatula, frequently regenerate via somatic
embryogenesis and are relatively undifferentiated. As seen in the
microscope, the Tape II calluses show color ranging from
translucent to light yellow and heterogeneity on with respect to
embryoid structure as well as stage of embryoid development. Once
Type II callus are formed, the calluses is transferred to type II
callus maintenance medium without AgNO.sub.3. Every 7-10 days, the
callus is subcultured. About 4 weeks after embryo isolation the
callus is removed from the plates and then frozen in liquid
nitrogen. The harvested tissue is stored at -80.degree. C. until
RNA preparation.
[0416] The SATMON021 cDNA library is generated from the immature
maize (DK604, Dekalb Genetics, Dekalb Illinois, U.S.A.) tassel at
the V8 plant development stage. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Maize plants
are grown in the green house in 15 hr day/9 hr night cycles. The
daytime temperature is approximately 80.degree. F. and the
nighttime temperature is approximately 70.degree. F. Supplemental
lighting is provided by 1000 W sodium vapor lamps. As the maize
plant enters the V8 stage, tassels which are 15-20 cm in length are
collected and frozen in liquid nitrogen. The harvested tissue is
stored at -80.degree. C. until RNA preparation.
[0417] The SATMON022 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) ear (growing silks) at
the V8 plant development stage. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Zea mays
plants are grown in the greenhouse in 15 hr day/9 hr night cycles.
The daytime temperature is approximately 80.degree. F. and the
nighttime temperature is approximately 70.degree. F. Supplemental
lighting is provided by 1000 W sodium vapor lamps. Tissue is
collected when the plant is in the V8 stage. At this stage, some
immature ear shoots are visible. The immature ear shoots
(approximately 1 cm in length) are pulled out, frozen in liquid
nitrogen and then stored at -80.degree. C. until RNA
preparation.
[0418] The SATMON23 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) ear (growing silk) at the
V8 development stage. Seeds are planted at a depth of approximately
3 cm into 2-3 inch peat pots containing Metro 200 growing medium.
After 2-3 weeks growth they are transplanted into 10 inch pots
containing the same growing medium. Plants are watered daily before
transplantation and three times a week after transplantation.
Peters 15-16-17 fertilizer is applied three times per week after
transplanting at a strength of 150 ppm N. Two to three times during
the lifetime of the plant, from transplanting to flowering, a total
of 900 mg Fe is added to each pot. Maize plants are grown in the
greenhouse in 15 hr day/9 hr night cycles. The daytime temperature
is approximately 80.degree. F. and the nighttime temperature is
approximately 70.degree. F. When the tissue is harvested at the V8
stage, the length of the ear that is harvested is about 10-15 cm
and the silks are just exposed (approximately 1 inch). The ear
along with the silks is frozen in liquid nitrogen and then the
tissue is stored at -80.degree. C. until RNA preparation.
[0419] The SATMON024 cDNA library is generated from the immature
maize (DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.) tassel at
the V9 development stage. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Maize plants
are grown in the green house in 15 hr day/9 hr night cycles. The
daytime temperature is approximately 80.degree. F. and the
nighttime temperature is approximately 70.degree. F. As a maize
plant enters the V9 stage, the tassel is rapidly developing and a
37 cm tassel along with the glume, anthers and pollen is collected
and frozen in liquid nitrogen. The harvested tissue is stored at
-80.degree. C. until RNA preparation.
[0420] The SATMON025 cDNA library is from maize (DK604, Dekalb
Genetics, Dekalb, Illinois U.S.A.) Hill Type II-Regenerated Callus.
Type II callus is grown in initiation media as described for
SATMON020 and then the embryoids on the surface of the Type II
callus are allowed to mature and germinate. The 1-2 gm fresh weight
of the soft friable type callus containing numerous embryoids are
transferred to 100.times.15 mm petri plates containing 25 ml of
regeneration media. Regeneration media consists of Murashige and
Skoog (MS) basal salts, modified White's vitamins (0.2 g/liter
glycine and 0.5 g/liter myo-inositoland 0.8% bacto agar (6SMSOD)).
The plates are then placed in the dark after covering with
parafilm. After 1 week, the plates are moved to a lighted growth
chamber with 16 hr light and 8 hr dark photoperiod. Three weeks
after plating the Type II callus to 6SMSOD, the callus exhibit
shoot formation. The callus and the shoots are transferred to fresh
6SMSOD plates for another 2 weeks. The callus and the shoots are
then transferred to petri plates with reduced sucrose (3SMSOD).
Upon distinct formation of a root and shoot, the newly developed
green plants are then removed out with a spatula and frozen in
liquid nitrogen containers. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0421] The SATMON026 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) juvenile/adult shift
leaves at the V8 plant development stage. Seeds are planted at a
depth of approximately 3 cm into 2-3 inch peat pots containing
Metro 200 growing medium. After 2-3 weeks growth they are
transplanted into 10 inch pots containing the same growing medium.
Plants are watered daily before transplantation and three times a
week after transplantation. Peters 15-16-17 fertilizer is applied
three times per week after transplanting at a strength of 150 ppm
N. Two to three times during the lifetime of the plant, from
transplanting to flowering, a total of 900 mg Fe is added to each
pot. Maize plants are grown in the green house in 15 hr day/9 hr
night cycles. The daytime temperature is approximately 80.degree.
F. and the nighttime temperature is approximately 70.degree. F.
Supplemental lighting is provided by 1000 W sodium vapor lamps.
Tissue is collected when the maize plants are at the 8-leaf
development stage. Leaves are founded sequentially around the
meristem over weeks of time and the older, more juvenile leaves
arise earlier and in a more basal position than the younger, more
adult leaves, which are in a more apical position. In a V8 plant,
some leaves which are in the middle portion of the plant exhibit
characteristics of both juvenile as well as adult leaves. They
exhibit a yellowing color but also exhibit, in part, a green color.
These leaves are termed juvenile/adult shift leaves. The
juvenile/adult shift leaves (the 4th, 5th leaves from the bottom)
are cut at the base, pooled and transferred to liquid nitrogen in
which they are then crushed. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0422] The SATMON027 cDNA library is generated from 6 day maize
(DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.) leaves. Seeds are
planted at a depth of approximately 3 cm into 2-3 inch peat pots
containing Metro 200 growing medium. After 2-3 weeks growth they
are transplanted into 10 inch pots containing the Metro 200 growing
medium. Plants are watered daily before transplantation and three
times a week after transplantation. Peters 15-16-17 fertilizer is
applied three times per week after transplanting at a strength of
150 ppm N. Two to three times during the lifetime of the plant,
from transplanting to flowering, a total of 900 mg Fe is added to
each pot. Zea mays plants are grown in the greenhouse in 15 hr
day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Prior to tissue collection, when the plant is at the
8-leaf stage, water is held back for six days. The older, more
juvenile leaves, which are in a basal position, as well as the
younger, more adult leaves, which are more apical, are all cut at
the base of the leaves. All the leaves exhibit significant wilting.
The leaves are then pooled and immediately transferred to liquid
nitrogen containers in which the pooled leaves are then crushed.
The harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0423] The SATMON028 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) roots at the V8
developmental stage that are subject to six days water stress.
Seeds are planted at a depth of approximately 3 cm into 2-3 inch
peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the Metro
200 growing medium. Plants are watered daily before transplantation
and three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in the greenhouse in 15
hr day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Prior to tissue collection, when the plant is at the
8-leaf stage, water is held back for six days. The root system is
cut, shaken and washed to remove soil. Root tissue is then pooled
and immediately transferred to liquid nitrogen containers in which
the pooled leaves are then crushed. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0424] The SATMON029 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) seedlings at the
etiolated stage. Seeds are planted on a moist filter paper on a
covered tray that is kept in the dark for 4 days at approximately
70.degree. F. Tissue is collected when the seedlings are 4 days
old. By 4 days, the primary root has penetrated the coleorhiza and
is about 4-5 cm and the secondary lateral roots have also made
their appearance. The coleoptile has also pushed through the seed
coat and is about 4-5 cm long. The seedlings are frozen in liquid
nitrogen and crushed. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0425] The SATMON030 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) root tissue at the V4
plant development stage. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth, they are transplanted into
10 inch pots containing the same. Plants are watered daily before
transplantation and approximately 3 times a week after
transplantation. Peters 15-16-17 fertilizer is applied
approximately three times per week after transplanting, at a
strength of 150 ppm N. Two to three times during the life time of
the plant, from transplanting to flowering, a total of
approximately 900 mg Fe is added to each pot. Maize plants are
grown in the green house in 15 hr day/9 hr night cycles. The
daytime temperature is approximately 80.degree. F. and the
nighttime temperature is approximately 70.degree. F. Supplemental
lighting is provided by 1000 sodium vapor lamps. Tissue is
collected when the maize plant is at the 4 leaf development stage.
The root system is cut from the mature maize plant and washed with
water to free it from the soil. The tissue is then immediately
frozen in liquid nitrogen. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0426] The SATMON031 cDNA library is generated from the maize
(DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.) leaf tissue at
the V4 plant development stage. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Maize plants
are grown in the green house in 15 hr day/9 hr night cycles. The
daytime temperature is 80.degree. F. and the nighttime temperature
is 70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the
4-leaf development stage. The third leaf from the bottom is cut at
the base and immediately frozen in liquid nitrogen and crushed. The
tissue is immediately frozen in liquid nitrogen. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
[0427] The SATMON033 cDNA library is generated from maize (DK604,
Dekalb Genetics, Dekalb, Illinois U.S.A.) embryo tissue 13 days
after pollination. Seeds are planted at a depth of approximately 3
cm into 2-3 inch peat pots containing Metro 200 growing medium.
After 2-3 weeks growth they are transplanted into 10 inch pots
containing the same growing medium. Plants are watered daily before
transplantation and three times a week after transplantation.
Peters 15-16-17 fertilizer is applied three times per week after
transplanting at a strength of 150 ppm N. Two to three times during
the lifetime of the plant, from transplanting to flowering, a total
of 900 mg Fe is added to each pot. Maize plants are grown in the
greenhouse in 15 hr day/9 hr night cycles. The daytime temperature
is approximately 80.degree. F. and the nighttime temperature is
approximately 70.degree. F. Supplemental lighting is provided by
1000 W sodium vapor lamps. After the VI 0 stage, the ear shoots of
the maize plant, which are ready for fertilization, are enclosed in
a paper bag before silk emergent to withhold the pollen. The ear
shoots are pollinated and 13 days after pollination, the ears are
pulled out and then the kernels are plucked cut of the ears. Each
kernel is then dissected into the embryo and the endosperm and the
aleurone layer is removed. After dissection, the embryos are
immediately frozen in liquid nitrogen and then stored at
-80.degree. C. until RNA preparation.
[0428] The SATMON034 cDNA library is generated from cold stressed
maize (DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.) seedlings.
Seeds are planted on a moist filter paper on a covered tray that is
kept on at 10.degree. C. for 7 days. After 7 days, the temperature
is shifted to 15.degree. C. for one day until germination of the
seed. Tissue is collected once the seedlings are 1 day old. At this
point, the coleorhiza has just pushed out of the seed coat and the
primary root is just making its appearance. The coleoptile has not
yet pushed completely through the seed coat and is also just making
its appearance. These 1 day old cold stressed seedlings are frozen
in liquid nitrogen and crushed. The harvested tissue is then stored
at -80.degree. C. until RNA preparation.
[0429] The SATMON.about.001 (Lib36, Lib83, Lib84) cDNA library is
generated from maize leaves at the V8 plant development stage.
Seeds are planted at a depth of approximately 3 cm into 2-3 inch
peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in a greenhouse in 15 hr
day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue from the maize plant is collected at the V8
stage. The older more juvenile leaves in a basal position was well
as the younger more adult leaves which are more apical are all cut
at the base, pooled and frozen in liquid nitrogen. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
[0430] The SATMONN01 cDNA library is generated from maize (B73,
Illinois Foundation Seeds, Champaign, Illinois U.S.A.) normalized
immature tassels at the V6 plant development stage normalized
tissue. Seeds are planted at a depth of approximately 3 cm into 2-3
inch peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in a greenhouse in 15 hr
day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue from the maize plant is collected at the V6
stage. At that stage the tassel is an immature tassel of about 2-3
cm in length. The tassels are removed and frozen in liquid
nitrogen. The harvested tissue is then stored at -80.degree. C.
until RNA preparation. Single stranded and double stranded DNA
representing approximately 1.times.10.sup.6 colony forming units
are isolated using standard protocols. RNA, complementary to the
single stranded DNA, is synthesized using the double stranded DNA
as a template. Biotinylated dATP is incorporated into the RNA
during the synthesis reaction. The single stranded DNA is mixed
with the biotinylated RNA in a 1:10 molar ratio) and allowed to
hybridize. DNA-RNA hybrids are captured on Dynabeads M280
streptavidin (Dynabeads, Dynal Corporation, Lake Success, New York
U.S.A.). The dynabeads with captured hybrids are collected with a
magnet. The non-hybridized single stranded molecules remaining
after hybrid capture are converted to double stranded form and
represent the primary normalized library.
[0431] The SATMONN04 cDNA library is generated from maize
(B73.times.Mol7, Illinois Foundation Seeds, Champaign, Illinois
U.S.A.) normalized total leaf tissue at the V6 plant development
stage. Seeds are planted at a depth of approximately 3 cm into 2-3
inch peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in the greenhouse in 15
hr day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the
6-leaf development stage. The older, more juvenile leaves, which
are in a basal position, as well as the younger, more adult leaves,
which are more apical are cut at the base of the leaves. The leaves
are then pooled and immediately transferred to liquid nitrogen
containers in which the pooled leaves are crushed. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
Single stranded and double stranded DNA representing approximately
1.times.10.sup.6 colony forming units are isolated using standard
protocols. RNA, complementary to the single stranded DNA, is
synthesized using the double stranded DNA as a template.
Biotinylated DATP is incorporated into the RNA during the synthesis
reaction. The single stranded DNA is mixed with the biotinylated
RNA in a 1:10 molar ratio) and allowed to hybridize. DNA-RNA
hybrids are captured on Dynabeads M280 streptavidin (Dynabeads,
Dynal Corporation, Lake Success, New York U.S.A.). The dynabeads
with captured hybrids are collected with a magnet. The
non-hybridized single stranded molecules remaining after hybrid
capture are converted to double stranded form and represent the
primary normalized library.
[0432] The SATMONN05 cDNA library is generated from maize
(B73.times.Mol7, Illinois Foundation Seeds, Champaign Illinois,
U.S.A.) normalized root tissue at the V6 development stage. Seeds
are planted at a depth of approximately 3 cm into 2-3 inch peat
pots containing Metro 200 growing medium. After 2-3 weeks growth
they are transplanted into 10 inch pots containing the same growing
medium. Plants are watered daily before transplantation and three
times a week after transplantation. Peters 15-16-17 fertilizer is
applied three times per week after transplanting at a strength of
150 ppm N. Two to three times during the lifetime of the plant,
from transplanting to flowering, a total of 900 mg Fe is added to
each pot. Maize plants are grown in the green house in 15 hr day/9
hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the
6-leaf development stage. The root system is cut from the mature
maize plant and washed with water to free it from the soil. The
tissue is immediately frozen in liquid nitrogen and the harvested
tissue is then stored at -80.degree. C. until RNA preparation. The
single stranded and double stranded DNA representing approximately
1.times.1.sup.6 colony forming units are isolated using standard
protocols. RNA, complementary to the single stranded DNA, is
synthesized using the double stranded DNA as a template.
Biotinylated dATP is incorporated into the RNA during the synthesis
reaction. The single stranded DNA is mixed with the biotinylated
RNA in a 1:10 molar ratio) and allowed to hybridize. DNA-RNA
hybrids are captured on Dynabeads M280 streptavidin (Dynabeads,
Dynal Corporation, Lake Success, New York U.S.A.). The dynabeads
with captured hybrids are collected with a magnet. The
non-hybridized single stranded molecules remaining after hybrid
capture are converted to double stranded form and represent the
primary normalized library.
[0433] The SATMONN06 cDNA library is generated from maize
(B73.times.Mol7, Illinois Foundation Seeds, Champaign Illinois,
U.S.A.) normalized total leaf tissue at the V6 plant development
stage. Seeds are planted at a depth of approximately 3 cm into 2-3
inch peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in the greenhouse in 15
hr day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the
6-leaf development stage. The older more juvenile leaves, which are
in a basal position, as well as the younger more adult leaves,
which are more apical are cut at the base of the leaves. The leaves
are then pooled and immediately transferred to liquid nitrogen
containers in which the pooled leaves are crushed. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
Single stranded and double stranded DNA representing approximately
1.times.10.sup.6 colony forming units are isolated using standard
protocols. RNA, complementary to the single stranded DNA, is
synthesized using the double stranded DNA as a template.
Biotinylated dATP is incorporated into the RNA during the synthesis
reaction. The single stranded DNA is mixed with the biotinylated
RNA in a 1:10 molar ratio) and allowed to hybridize. DNA-RNA
hybrids are captured on Dynabeads M280 streptavidin (Dynabeads,
Dynal Corporation, Lake Success, New York U.S.A.). The dynabeads
with captured hybrids are collected with a magnet. The
non-hybridized single stranded molecules remaining after hybrid
capture are converted to double stranded form and represent the
primary normalized library.
[0434] The CMZ029 (SATMON036) cDNA library is generated from maize
(DK604, Dekalb Genetics, Dekalb, Illinois U.S.A.) endosperm 22 days
after pollination. Seeds are planted at a depth of approximately 3
cm into 2-3 inch peat pots containing Metro 200 growing medium.
After 2-3 weeks growth they are transplanted into 10 inch pots
containing the same growing medium. Plants are watered daily before
transplantation and three times a week after transplantation.
Peters 15-16-17 fertilizer is applied three times per week after
transplanting at a strength of 150 ppm N. Two to three times during
the lifetime of the plant, from transplanting to flowering, a total
of 900 mg Fe is added to each pot. Maize plants are grown in the
green house in 15 hr day/9 hr night cycles. The daytime temperature
is approximately 80.degree. F. and the nighttime temperature is
approximately 70.degree. F. Supplemental lighting is provided by
1000 W sodium vapor lamps. After the V10 stage, the ear shoots of
the maize plant, which are ready for fertilization, are enclosed in
a paper bag before silk emergent to withhold the pollen. The ear
shoots are pollinated and 22 days after pollination, the ears are
pulled out and then the kernels are plucked out of the ears. Each
kernel is then dissected into the embryo and the endosperm and the
alurone layer is removed. After dissection, the endosperms are
immediately frozen in liquid nitrogen and then stored at
-80.degree. C. until RNA preparation.
[0435] The CMz030 (Lib143) cDNA library is generated from maize
seedling tissue two days post germination. Seeds are planted on a
moist filter paper on a covered try that is keep in the dark until
germination. The trays are then moved to the bench top at 15 hr
daytime/9 hr nighttime cycles for 2 days post-germination. The day
time temperature is 80.degree. F. and the nighttime temperature is
70.degree. F. Tissue is collected when the seedlings are 2 days
old. At this stage, the colehrhiza has pushed through the seed coat
and the primary root (the radicle) is just piercing the colehrhiza
and is barely visible. The seedlings are placed at 42.degree. C.
for 1 hour. Following the heat shock treatment, the seedlings are
immersed in liquid nitrogen and crushed. The harvested tissue is
stored at -80.degree. until RNA preparation.
[0436] The CMz031 (Lib148) cDNA library is generated from maize
pollen tissue at the V10+ plant development stage. Seeds are
planted at a depth of approximately 3 cm into 2-3 inch peat pots
containing Metro 200 growing medium. After 2-3 weeks growth they
are transplanted into 10 inch pots containing the same growing
medium. Plants are watered daily before transplantation and three
times a week after transplantation. Peters 15-16-17 fertilizer is
applied three times per week after transplanting at a strength of
150 ppm N. Two to three times during the lifetime of the plant,
from transplanting to flowering, a total of 900 mg Fe is added to
each pot. Maize plants are grown in the greenhouse in 15 hr day/9
hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected from V10+ stage plants. The ear
shoots, which are ready for fertilization, are enclosed in a paper
bag to withhold pollen. Twenty-one days after pollination, prior to
removing the ears, the paper bag is shaken to collect the mature
pollen. The mature pollen is immediately frozen in liquid nitrogen
containers and the pollen is crushed. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0437] The CMz033 (Lib189) cDNA library is generated from maize
pooled leaf tissue. Samples are harvested from open pollinated
plants. Tissue is collected from maize leaves at the anthesis
stage. The leaves are collect from 10-12 plants and frozen in
liquid nitrogen. The harvested tissue is then stored at -80.degree.
C. until RNA preparation.
[0438] The CMz034 (Lib3060) cDNA library is generated from maize
mature tissue at 40 days post pollination plant development stage.
Seeds are planted at a depth of approximately 3 cm into 2-3 inch
peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in the greenhouse in 15
hr day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected from leaves located two leaves
below the ear leaf. This sample represents those genes expressed
during onset and early stages of leaf senescence. The leaves are
pooled and immediately transferred to liquid nitrogen. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0439] The CMz035 (Lib3061) cDNA library is generated from maize
endosperm tissue at the V10+ plant development stage. Seeds are
planted at a depth of approximately 3 cm into 2-3 inch peat pots
containing Metro 200 growing medium. After 2-3 weeks growth they
are transplanted into 10 inch pots containing the same growing
medium. Plants are watered daily before transplantation and three
times a week after transplantation. Peters 15-16-17 fertilizer is
applied three times per week after transplanting at a strength of
150 ppm N. Two to three times during the lifetime of the plant,
from transplanting to flowering, a total of 900 mg Fe is added to
each pot. Maize plants are grown in the greenhouse in 15 hr day/9
hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected from V10+ stage plants. The ear
shoots, which are ready for fertilization, are enclosed in a paper
bag prior to silk emergence to withhold pollen. Thirty-two days
after pollination, the ears are pulled out and the kernels are
removed from the cob. Each kernel is dissected into the embryo and
the endosperm and the aleurone layer is removed. After dissection,
the endosperms are immediately transferred to liquid nitrogen. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0440] The CMz036 (Lib3062) cDNA library is generated from maize
husk tissue at the 8 week old plant development stage. Seeds are
planted at a depth of approximately 3 cm into 2-3 inch peat pots
containing Metro 200 growing medium. After 2-3 weeks growth they
are transplanted into 10 inch pots containing the same growing
medium. Plants are watered daily before transplantation and three
times a week after transplantation. Peters 15-16-17 fertilizer is
applied three times per week after transplanting at a strength of
150 ppm N. Two to three times during the lifetime of the plant,
from transplanting to flowering, a total of 900 mg Fe is added to
each pot. Maize plants are grown in the greenhouse in 15 hr day/9
hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected from 8 week old plants. The husk
is separated from the ear and immediately transferred to liquid
nitrogen containers. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0441] The CMz037 (Lib3059) cDNA library is generated from maize
pooled kernal at 12-15 days after pollination plant development
stage. Sample were collected from field grown material. Whole
kernals from hand pollinated (control pollination) are harvested as
whole ears and immediately frozen on dry ice. Kernels from 10-12
ears were pooled and ground together in liquid nitrogen. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0442] The CMz039 (Lib3066) cDNA library is generated from maize
immature anther tissue at the 7 week old immature tassel stage.
Seeds are planted at a depth of approximately 3 cm into 2-3 inch
peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in the greenhouse in 15
hr day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the 7
week old immature tassel stage. At this stage, prior to anthesis,
the immature anthers are green and enclosed in the staminate
spikelet. The developing anthers are dissected away from the 7 week
old immature tassel and immediately frozen in liquid nitrogen. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0443] The CMz040 (Lib3067) cDNA library is generated from maize
kernel tissue at the V10+ plant development stage. Seeds are
planted at a depth of approximately 3 cm into 2-3 inch peat pots
containing Metro 200 growing medium. After 2-3 weeks growth they
are transplanted into 10 inch pots containing the same growing
medium. Plants are watered daily before transplantation and three
times a week after transplantation. Peters 15-16-17 fertilizer is
applied three times per week after transplanting at a strength of
150 ppm N. Two to three times during the lifetime of the plant,
from transplanting to flowering, a total of 900 mg Fe is added to
each pot. Maize plants are grown in the greenhouse in 15 hr day/9
hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected from V10+ stage plants. The ear
shoots, which are ready for fertilization, are enclosed in a paper
bag before silk emergence to withhold pollen. Five to eight days
after controlled pollination. The ears are pulled and the kernels
removed. The kernels are immediately frozen in liquid nitrogen. The
harvested kernels tissue is then stored at -80.degree. C. until RNA
preparation. This sample represents gene expressed in early kernel
development, during periods of cell division, amyloplast biogenesis
and early carbon flow across the material to filial tissue.
[0444] The CMz041 (Lib3068) cDNA library is generated from maize
pollen germinating silk tissue at the V10+ plant development stage.
Seeds are planted at a depth of approximately 3 cm into 2-3 inch
peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in the greenhouse in 15
hr day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected from V10+ stage plants when the
ear shoots are ready for fertilization at the silk emergence stage.
The emerging silks are pollinated with an excess of pollen under
controlled pollination conditions in the green house. Eighteen
hours after pollination the silks are removed from the ears and
immediately frozen in liquid nitrogen containers. This sample
represents genes expressed in both pollen and silk tissue early in
pollination. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0445] The CMz042 (Lib3069) cDNA library is generated from maize
ear tissue excessively pollinated at the V10+ plant development
stage. Seeds are planted at a depth of approximately 3 cm into 2-3
inch peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in the greenhouse in 15
hr day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected from V10+ stage plants and the ear
shoots which are ready for fertilization are at the silk emergence
stage. The immature ears are pollinated with an excess of pollen
under controlled pollination conditions. Eighteen hours
post-pollination, the ears are removed and immediately transferred
to liquid nitrogen containers. The harvested tissue is then stored
at -80.degree. C. until RNA preparation.
[0446] The CMz044 (Lib3075) cDNA library is generated from maize
microspore tissue at the V10+ plant development stage. Seeds are
planted at a depth of approximately 3 cm into 2-3 inch peat pots
containing Metro 200 growing medium. After 2-3 weeks growth they
are transplanted into 10 inch pots containing the same growing
medium. Plants are watered daily before transplantation and three
times a week after transplantation. Peters 15-16-17 fertilizer is
applied three times per week after transplanting at a strength of
150 ppm N. Two to three times during the lifetime of the plant,
from transplanting to flowering, a total of 900 mg Fe is added to
each pot. Maize plants are grown in the greenhouse in 15 hr day/9
hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected from immature anthers from 7 week
old tassels. The immature anthers are first dissected from the 7
week old tassel with a scalpel on a glass slide covered with water.
The microspores (immature pollen) are released into the water and
are recovered by centrifugation. The microspore suspension is
immediately frozen in liquid nitrogen. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0447] The CMz045 (Lib3076) cDNA library is generated from maize
immature ear megaspore tissue. Seeds are planted at a depth of
approximately 3 cm into 2-3 inch peat pots containing Metro 200
growing medium. After 2-3 weeks growth they are transplanted into
10 inch pots containing the same growing medium. Plants are watered
daily before transplantation and three times a week after
transplantation. Peters 15-16-17 fertilizer is applied three times
per week after transplanting at a strength of 150 ppm N. Two to
three times during the lifetime of the plant, from transplanting to
flowering, a total of 900 mg Fe is added to each pot. Maize plants
are grown in the greenhouse in 15 hr day/9 hr night cycles. The
daytime temperature is approximately 80.degree. F. and the
nighttime temperature is approximately 70.degree. F. Supplemental
lighting is provided by 1000 W sodium vapor lamps. Tissue is
collected from immature ear (megaspore) obtained from 7 week old
plants. The immature ears are harvested from the 7 week old plants
and are approximately 2.5 to 3 cm in length. The kernels are
removed from the cob immediately frozen in liquid nitrogen. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0448] The CMz047 (Lib3078) cDNA library is generated from maize
CO.sub.2 treated high-exposure shoot tissue at the V10+ plant
development stage. RX601 maize seeds are sterilized for minute with
a 10% clorox solution. The seeds are rolled in germination paper,
and germinated in 0.5 mM calcium sulfate solution for two days are
30.degree. C. The seedlings are planted at a depth of approximately
3 cm into 2-3 inch peat pots containing Metro 200 growing medium at
a rate of 2-3 seedlings per pot. Twenty pots are placed into a high
CO.sub.2 environment (approximately 1000 ppm CO.sub.2). Twenty
plants were grown under ambient greenhouse CO.sub.2 (approximately
450 ppm CO.sub.2). Plants are watered daily before transplantation
and three times a week after transplantation. Peters 20-20-20
fertilizer is also lightly applied. Maize plants are grown in the
greenhouse in 15 hr day/9 hr night cycles. The daytime temperature
is approximately 80.degree. F. and the nighttime temperature is
approximately 70.degree. F. Supplemental lighting is provided by
1000 W sodium vapor lamps. At ten days post planting, the shoots
from both atmosphere are frozen in liquid nitrogen and lightly
ground. The roots are washed in deionized water to remove the
support media and the tissue is immediately transferred to liquid
nitrogen containers. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0449] The CMz048 (Lib3079) cDNA library is generated from maize
basal endosperm transfer layer tissue at the V10+ plant development
stage. Seeds are planted at a depth of approximately 3 cm into 2-3
inch peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in the greenhouse in 15
hr day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected from V10+maize plants. The ear
shoots, which are ready for fertilization, are enclosed in a paper
bag prior to silk emergence, to withhold the pollen. Kernels are
harvested at 12 days post-pollination and placed on wet ice for
dissection. The kernels are cross sectioned laterally, dissecting
just above the pedicel region, including 1-2 mm of the lower
endosperm and the basal endosperm transfer region. The pedicel and
lower endosperm region containing the basal endosperm transfer
layer is pooled and immediately frozen in liquid nitrogen. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0450] The CMz049(Lib3088) cDNA library is generated from maize
immature anther tissue at the 7 week old immature tassel stage.
Seeds are planted at a depth of approximately 3 cm into 2-3 inch
peat pots containing Metro 200 growing medium. After 2-3 weeks
growth they are transplanted into 10 inch pots containing the same
growing medium. Plants are watered daily before transplantation and
three times a week after transplantation. Peters 15-16-17
fertilizer is applied three times per week after transplanting at a
strength of 150 ppm N. Two to three times during the lifetime of
the plant, from transplanting to flowering, a total of 900 mg Fe is
added to each pot. Maize plants are grown in the greenhouse in 15
hr day/9 hr night cycles. The daytime temperature is approximately
80.degree. F. and the nighttime temperature is approximately
70.degree. F. Supplemental lighting is provided by 1000 W sodium
vapor lamps. Tissue is collected when the maize plant is at the 7
week old immature tassel stage. At this stage, prior to anthesis,
the immature anthers are green and enclosed in the staminate
spikelet. The developing anthers are dissected away from the 7 week
old immature tassel and immediately transferred to liquid nitrogen
container. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0451] The CMz050 (Lib3114) cDNA library is generated from maize
silk tissue at the V10+ plant development stage. Seeds are planted
at a depth of approximately 3 cm into 2-3 inch peat pots containing
Metro 200 growing medium. After 2-3 weeks growth they are
transplanted into 10 inch pots containing the same growing medium.
Plants are watered daily before transplantation and three times a
week after transplantation. Peters 15-16-17 fertilizer is applied
three times per week after transplanting at a strength of 150 ppm
N. Two to three times during the lifetime of the plant, from
transplanting to flowering, a total of 900 mg Fe is added to each
pot. Maize plants are grown in the greenhouse in 15 hr day/9 hr
night cycles. The daytime temperature is approximately 80.degree.
F. and the nighttime temperature is approximately 70.degree. F.
Supplemental lighting is provided by 1000 W sodium vapor lamps.
Tissue is collected when the maize plant is beyond the 10-leaf
development stage and the ear shoots are approximately 15-20 cm in
length. The ears are pulled and silks are separated from the ears
and immediately transferred to liquid nitrogen containers. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0452] The SOYMON001 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
total leaf tissue at the V4 plant development stage. Leaf tissue
from 38, field grown V4 stage plants is harvested from the 4.sup.th
node. Leaf tissue is removed from the plants and immediately frozen
in dry-ice. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0453] The SOYMON002 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
root tissue at the V4 plant development stage. Root tissue from 76,
field grown V4 stage plants is harvested. The root systems is cut
from the soybean plant and washed with water to free it from the
soil and immediately frozen in dry-ice. The harvested tissue is
then stored at -80.degree. C. until RNA preparation.
[0454] The SOYMON003 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
seedling hypocotyl axis tissue harvested 2 day post-imbibition.
Seeds are planted at a depth of approximately 2 cm into 2-3 inch
peat pots containing Metromix 350 medium. Trays are placed in an
environmental chamber and grown at 12 hr daytime/12 hr nighttime
cycles. The daytime temperature is approximately 29.degree. C. and
the nighttime temperature approximately 24.degree. C. Soil is
checked and watered daily to maintain even moisture conditions.
Tissue is collected 2 days after the start of imbibition. The 2
days after imbibition samples are separated into 3 collections
after removal of any adhering seed coat. At the 2 day stage, the
hypocotyl axis is emerging from the soil. A few seedlings have
cracked the soil surface and exhibited slight greening of the
exposed cotyledons. The seedlings are washed in water to remove
soil, hypocotyl axis harvested and immediately frozen in liquid
nitrogen. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0455] The SOYMON004 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
seedling cotyledon tissue harvested 2 day post-imbibition. Seeds
are planted at a depth of approximately 2 cm into 2-3 inch peat
pots containing Metromix 350 medium. Trays are placed in an
environmental chamber and grown at 12 hr daytime/12 hr nighttime
cycles. The daytime temperature is approximately 29.degree. C. and
the nighttime temperature approximately 24.degree. C. Soil is
checked and watered daily to maintain even moisture conditions.
Tissue is collected 2 days after the start of imbibition. The 2
days after imbibition samples are separated into 3 collections
after removal of any adhering seed coat. At the 2 day stage, the
hypocotyl axis is emerging from the soil. A few seedlings have
cracked the soil surface and exhibited slight greening of the
exposed cotyledons. The seedlings are washed in water to remove
soil, hypocotyl axis harvested and immediately frozen in liquid
nitrogen. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0456] The SOYMON005 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
seedling hypocotyl axis tissue harvested 6 hour post-imbibition.
Seeds are planted at a depth of approximately 2 cm into 2-3 inch
peat pots containing Metromix 350 medium. Trays are placed in an
environmental chamber and grown at 12 hr daytime/12 hr nighttime
cycles. The daytime temperature is approximately 29.degree. C. and
the nighttime temperature approximately 24.degree. C. Soil is
checked and watered daily to maintain even moisture conditions.
Tissue is collected 6 hours after the start of imbibition. The 6
hours after imbibition samples are separated into 3 collections
after removal of any adhering seed coat. The 6 hours after
imbibition sample is collected over the course of approximately 2
hours starting at 6 hours post imbibition. At the 6 hours after
imbibition stage, not all cotyledons have become fully hydrated and
germination, or radicle protrusion, has not occurred. The seedlings
are washed in water to remove soil, hypocotyl axis harvested and
immediately frozen in liquid nitrogen. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0457] The SOYMON006 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
seedling cotyledons tissue harvest 6 hour post-imbibition. Seeds
are planted at a depth of approximately 2 cm into 2-3 inch peat
pots containing Metromix 350 medium. Trays are placed in an
environmental chamber and grown at 12 hr daytime/12 hr nightime
cycles. The daytime temperature is approximately 29.degree. C. and
the nighttime temperature approximately 24.degree. C. Soil is
checked and watered daily to maintain even moisture conditions.
Tissue is collected 6 hours after imbibition. The 6 hours after
imbibition samples are separated into 3 collections after removal
of any adhering seed coat. The 6 hours after imbibition sample is
collected over the course of approximately 2 hours starting at 6
hours post-imbibition. At the 6 hours after imbibition, not all
cotyledons have become fully hydrated and germination or radicle
protrusion, have not occurred. The seedlings are washed in water to
remove soil, cotyledon harvested and immediately frozen in liquid
nitrogen. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0458] The SOYMON007 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
seed tissue harvested 25 and 35 days post-flowering. Seed pods from
field grown plants are harvested 25 and 35 days after flowering and
the seeds extracted from the pods. Approximately 4.4 g and 19.3 g
of seeds are harvested from the respective seed pods and
immediately frozen in dry ice. The harvested tissue is then stored
at -80.degree. C. until RNA preparation.
[0459] The SOYMON008 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
leaf tissue harvested from 25 and 35 days post-flowering plants.
Total leaf tissue is harvested from field grown plants.
Approximately 19 g and 29 g of leaves are harvested from the fourth
node of the plant 25 and 35 days post-flowering and immediately
frozen in dry ice. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0460] The SOYMON009 cDNA library is generated from soybean
cutlivar C1944 (USDA Soybean Germplasm Collection, Urbana, Ill.
U.S.A.) pod and seed tissue harvested 15 days post-flowering. Pods
from field grown plants are harvested 15 days post-flowering.
Approximately 3 g of pod tissue is harvested and immediately frozen
in dry-ice. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0461] The SOYMON010 cDNA library is generated from soybean
cultivar C1944 (USDA Soybean Germplasm Collection, Urbana, Ill.
U.S.A.) seed tissue harvested 40 days post-flowering. Pods from
field grown plants are harvested 40 days post-flowering. Pods and
seeds are separated, approximately 19 g of seed tissue is harvested
and immediately frozen in dry-ice. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0462] The SOYMON011 cDNA library is generated from soybean
cultivars Cristalina (USDA Soybean Germplasm Collection, Urbana,
Ill. U.S.A.) and FT108 (Monsoy, Brazil) (tropical germ plasma) leaf
tissue. Leaves are harvested from plants grown in an environmental
chamber under 12 hr daytime/12 hr nighttime cycles. The daytime
temperature is approximately 29.degree. C. and the nighttime
temperature approximately 24.degree. C. Soil is checked and watered
daily to maintain even moisture conditions. Approximately 30 g of
leaves are harvested from the 4.sup.th node of each of the
Cristalina and FT108 cultivars and immediately frozen in dry ice.
The harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0463] The SOYMON012 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
leaf tissue. Leaves from field grown plants are harvested from the
fourth node 15 days post-flowering. Approximately 12 g of leaves
are harvested and immediately frozen in dry ice. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
[0464] The SOYMON013 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
root and nodule tissue. Approximately, 28 g of root tissue from
field grown plants is harvested 15 days post-flowering. The root
system is cut from the soybean plant, washed with water to free it
from the soil and immediately frozen in dry-ice. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
[0465] The SOYMON014 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
seed tissue harvested 25 and 35 days after flowering. Seed pods
from field grown plants are harvested 15 days after flowering and
the seeds extracted from the pods. Approximately 5 g of seeds are
harvested from the respective seed pods and immediately frozen in
dry ice. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0466] The SOYMON015 cDNA is generated from soybean cultivar Asgrow
3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.) seed tissue
harvested 45 and 55 days post-flowering. Seed pods from field grown
plants are harvested 45 and 55 days after flowering and the seeds
extracted from the pods. Approximately 19 g and 31 g of seeds are
harvested from the respective seed pods and immediately frozen in
dry ice. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0467] The SOYMON016 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
root tissue. Approximately, 61 g and 38 g of root tissue from field
grown plants is harvested 25 and 35 days post-flowering is
harvested. The root system is cut from the soybean plant and washed
with water to free it from the soil. The tissue is placed in 14 ml
polystyrene tubes and immediately frozen in dry-ice. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
[0468] The SOYMON017 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
root tissue. Approximately 28 g of root tissue from field grown
plants is harvested 45 and 55 days post-flowering. The root system
is cut from the soybean plant, washed with water to free it from
the soil and immediately frozen in dry-ice. The harvested tissue is
then stored at -80.degree. C. until RNA preparation.
[0469] The SOYMON018 cDNA is generated from soybean cultivar Asgrow
3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.) leaf tissue
harvested 45 and 55 days post-flowering. Leaves from field grown
plants are harvested 45 and 55 days after flowering from the fourth
node. Approximately 27 g and 33 g of seeds are harvested from the
respective seed pods and immediately frozen in dry ice. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0470] The SOYMON019 cDNA library is generated from soybean
cultivars Cristalina (USDA Soybean Gemplasm Collection, Urbana,
Ill. U.S.A.) and FT108 (Monsoy, Brazil) (tropical germ plasma) root
tissue. Roots are harvested from plants grown in an environmental
chamber under 12 hr daytime/12 hr nighttime cycles. The daytime
temperature is approximately 29.degree. C. and the nighttime
temperature approximately 24.degree. C. Soil is checked and watered
daily to maintain even moisture conditions. Approximately 50 g and
56 g of roots are harvested from each of the Cristalina and FT108
cultivars and immediately frozen in dry ice. The harvested tissue
is then stored at -80.degree. C. until RNA preparation.
[0471] The SOYMON020 cDNA is generated from soybean cultivar Asgrow
3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.) seed tissue
harvested 65 and 75 days post-flowering. Seed pods from field grown
plants are harvested 45 and 55 days after flowering and the seeds
extracted from the pods. Approximately 14 g and 31 g of seeds are
harvested from the respective seed pods and immediately frozen in
dry ice. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0472] The SOYMON021 cDNA library is generated from Soybean Cyst
Nematode-resistant soybean cultivar Hartwig (USDA Soybean Germplasm
Collection, Urbana, Ill. U.S.A.) root tissue. Plants are grown in
tissue culture at room temperature. At approximately 6 weeks
post-germination, the plants are exposed to sterilized Soybean Cyst
Nematode eggs. Infection is then allowed to progress for 10 days.
After the 10 day infection process, the tissue is harvested. Agar
from the culture medium and nematodes are removed and the root
tissue is immediately frozen in dry ice. The harvested tissue is
then stored at -80.degree. C. until RNA preparation.
[0473] The SOYMON022 (Lib3030) cDNA library is generated from
soybean cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa
U.S.A.) partially opened flower tissue. Partially to fully opened
flower tissue is harvested from plants grown in an environmental
chamber under 12 hr daytime/12 hr nighttime cycles. The daytime
temperature is approximately 29.degree. C. and the nighttime
temperature approximately 24.degree. C. Soil is checked and watered
daily to maintain even moisture conditions. A total of 3 g of
flower tissue is harvested and immediately frozen in dry ice. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0474] The SOYMON023 cDNA library is generated from soybean
genotype BW211S Null (Tohoku University, Morioka, Japan) seed
tissue harvested 15 and 40 days post-flowering. Seed pods from
field grown plants are harvested 15 and 40 days post-flowering and
the seeds extracted from the pods. Approximately 0.7 g and 14.2 g
of seeds are harvested from the respective seed pods and
immediately frozen in dry ice. The harvested tissue is then stored
at -80.degree. C. until RNA preparation.
[0475] The SOYMON024 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
internode-2 tissue harvested 18 days post-imbibition. Seeds are
planted at a depth of approximately 2 cm into 2-3 inch peat pots
containing Metromix 350 medium. The plants are grown in a
greenhouse for 18 days after the start of imbibition at ambient
temperature. Soil is checked and watered daily to maintain even
moisture conditions. Stem tissue is harvested 18 days after the
start of imbibition. The samples are divided into hypocotyl and
internodes 1 through 5. The fifth internode contains some leaf bud
material. Approximately 3 g of each sample is harvested and
immediately frozen in dry ice. The harvested tissue is then stored
at -80.degree. C. until RNA preparation.
[0476] The SOYMON025 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
leaf tissue harvested 65 days post-flowering. Leaves are harvested
from the fourth node of field grown plants 65 days post-flowering.
Approximately 18.4 g of leaf tissue is harvested and immediately
frozen in dry ice. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0477] SOYMON026 cDNA library is generated from soybean cultivar
Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.) root
tissue harvested 65 and 75 days post-flowering. Approximately 27 g
and 40 g of root tissue from field grown plants is harvested 65 and
75 days post-flowering. The root system is cut from the soybean
plant, washed with water to free it from the soil and immediately
frozen in dry-ice. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0478] The SOYMON027 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
seed tissue harvested 25 days post-flowering. Seed pods from field
grown plants are harvested 25 days post-flowering and the seeds
extracted from the pods. Approximately 17 g of seeds are harvested
from the seed pods and immediately frozen in dry ice. The harvested
tissue is then stored at -80.degree. C. until RNA preparation.
[0479] The SOYMON028 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
drought-stressed root tissue. The plants are grown in an
environmental chamber under 12 hr daytime/12 hr nighttime cycles.
The daytime temperature is approximately 29.degree. C. and the
nighttime temperature 24.degree. C. Soil is checked and watered
daily to maintain even moisture conditions. At the R3 stage of
development, water is withheld from half of the plant collection
(drought stressed population). After 3 days, half of the plants
from the drought stressed condition and half of the plants from the
control population are harvested. After another 3 days (6 days post
drought induction) the remaining plants are harvested. A total of
27 g and 40 g of root tissue is harvested and immediately frozen in
dry ice. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0480] The SOYMON029 cDNA library is generated from Soybean Cyst
Nematode-resistant soybean cultivar PI07354 (USDA Soybean Germplasm
Collection, Urbana, Ill. U.S.A.) root tissue. Late fall to early
winter greenhouse grown plants are exposed to Soybean Cyst Nematode
eggs. At 10 days post-infection, the plants are uprooted, rinsed
briefly and the roots frozen in liquid nitrogen. Approximately 20
grams of root tissue is harvested from the infected plants. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0481] The SOYMON030 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
flower bud tissue. Seeds are planted at a depth of approximately 2
cm into 2-3 inch peat pots containing Metromix 350 medium and the
plants are grown in an environmental chamber under 12 hr daytime/12
hr nighttime cycles. The daytime temperature is approximately
29.degree. C. and the nighttime temperature approximately
24.degree. C. Soil is checked and watered daily to maintain even
moisture conditions. Flower buds are removed from the plant at the
pedicel. A total of 100 mg of flower buds are harvested and
immediately frozen in liquid nitrogen. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0482] The SOYMON031 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
carpel and stamen tissue. Seeds are planted at a depth of
approximately 2 cm into 2-3 inch peat pots containing Metromix 350
medium and the plants are grown in an environmental chamber under
12 hr daytime/12 hr nighttime cycles. The daytime temperature is
approximately 29.degree. C. and the nighttime temperature
approximately 24.degree. C. Soil is checked and watered daily to
maintain even moisture conditions. Flower buds are removed from the
plant at the pedicel. Flowers are dissected to separate petals,
sepals and reproductive structures (carpels and stamens). A total
of 300 mg of carpel and stamen tissue are harvested and immediately
frozen in liquid nitrogen. The harvested tissue is then stored at
-80.degree. C. until RNA preparation.
[0483] The SOYMON032 cDNA library is prepared from the Asgrow
cultivar A4922 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
rehydrated dry soybean seed meristem tissue. Surface sterilized
seeds are germinated in liquid media for 24 hours. The seed axis is
then excised from the barely germinating seed, placed on tissue
culture media and incubated overnight at 20.degree. C. in the dark.
The supportive tissue is removed from the explant prior to harvest.
Approximately 570 mg of tissue is harvested and frozen in liquid
nitrogen. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0484] The SOYMON033 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
heat-shocked seedling tissue without cotyledons. Seeds are imbibed
and germinated in vermiculite for 2 days under constant
illumination. After 48 hours, the seedlings are transferred to an
incubator set at 40.degree. C. under constant illumination. After
30, 60 and 180 minutes seedlings are harvested and dissected. A
portion of the seedling consisting of the root, hypocotyl and
apical hook is frozen in liquid nitrogen and stored at -80.degree.
C. The seedlings after 2 days of imbibition are beginning to emerge
from the vermiculite surface. The apical hooks are dark green in
appearance. Total RNA and poly A.sup.+ RNA is prepared from equal
amounts of pooled tissue.
[0485] The SOYMON034 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
cold-shocked seedling tissue without cotyledons. Seeds are imbibed
and germinated in vermiculite for 2 days under constant
illumination. After 48 hours, the seedlings are transferred to a
cold room set at 5.degree. C. under constant illumination. After
30, 60 and 180 minutes seedlings are harvested and dissected. A
portion of the seedling consisting of the root, hypocotyl and
apical hook is frozen in liquid nitrogen and stored at -80.degree.
C. The seedlings after 2 days of imbibition are beginning to emerge
from the vermiculite surface. The apical hooks are dark green in
appearance.
[0486] The SOYMON035 cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
seed coat tissue. Seeds are planted at a depth of approximately 2
cm into 2-3 inch peat pots containing Metromix 350 medium and the
plants are grown in an environmental chamber under 12 hr daytime/12
hr nighttime cycles. The daytime temperature is approximately
29.degree. C. and the nighttime temperature 24.degree. C. Soil is
checked and watered daily to maintain even moisture conditions.
Seeds are harvested from mid to nearly full maturation (seed coats
are not yellowing). The entire embryo proper is removed from the
seed coat sample and the seed coat tissue are harvested and
immediately frozen in liquid nitrogen. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0487] The SOYMON036 cDNA library is generated from soybean
cultivars PI171451, PI227687 and PI229358 (USDA Soybean Germplasm
Collection, Urbana, Ill. U.S.A.) insect challenged leaves. Plants
from each of the three cultivars are grown in screenhouse
conditions. The screenhouse is divided in half and one half of the
screenhouse is infested with soybean looper and the other half
infested with velvetbean caterpillar. A single leaf is taken from
each of the representative plants at 3 different time points, 11
days after infestation, 2 weeks after infestation and 5 weeks after
infestation and immediately frozen in liquid nitrogen. The
harvested tissue is then stored at -80.degree. C. until RNA
preparation. Total RNA and poly A+RNA is isolated from pooled
tissue consisting of equal quantities of all 18 samples (3
genotypes.times.3 sample times.times.2 insect genotypes).
[0488] The SOYMON037 cDNA library is generated from soybean
cultivar A3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
etiolated axis and radical tissue. Seeds are planted in moist
vermiculite, wrapped and kept at room temperature in complete
darkness until harvest. Etiolated axis and hypocotyl tissue is
harvested at 2, 3 and 4 days post-planting. A total of 1 gram of
each tissue type is harvested at 2, 3 and 4 days after planting and
immediately frozen in liquid nitrogen. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0489] The SOYMON038 cDNA library is generated from soybean variety
Asgrow A3237 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
rehydrated dry seeds. Explants are prepared for transformation
after germination of surface-sterilized seeds on solid tissue
media. After 6 days, at 28.degree. C. and 18 hours of light per
day, the germinated seeds are cold shocked at 4.degree. C. for 24
hours. Meristemic tissue and part of the hypocotyl is remove and
cotyledon excised. The prepared explant is then wounded for
Agrobacterium infection. The 2 grams of harvested tissue is frozen
in liquid nitrogen and stored at -80.degree. C. until RNA
preparation.
[0490] The Soy51 (LIB3027) cDNA library is prepared from equal
amounts tissue harvested from SOYMON007, SOYMON015 and SOYMON020
prepared tissue. Single stranded and double stranded DNA
representing approximately 1.times.10.sup.6 colony forming units
are isolated using standard protocols. RNA, complementary to the
single stranded DNA, is synthesized using the double stranded DNA
as a template. Biotinylated dATP is incorporated into the RNA
during the synthesis reaction. The single stranded DNA is mixed
with the biotinylated RNA in a 1:10 molar ratio) and allowed to
hybridize. DNA-RNA hybrids are captured on Dynabeads M280
streptavidin (Dynabeads, Dynal Corporation, Lake Success, New York
U.S.A.). The dynabeads with captured hybrids are collected with a
magnet. The non-hybridized single stranded molecules remaining
after hybrid capture are converted to double stranded form and
represent the primary normalized library.
[0491] The Soy52 (LIB3028) cDNA library is generated from
normalized flower DNA. Single stranded DNA representing
approximately 1.times.10.sup.6 colony forming units of SOYMON022
harvested tissue is used as the starting material for
normalization. RNA, complementary to the single stranded DNA, is
synthesized using the double stranded DNA as a template.
Biotinylated dATP is incorporated into the RNA during the synthesis
reaction. The single stranded DNA is mixed with the biotinylated
RNA in a 1:10 molar ratio) and allowed to hybridize. DNA-RNA
hybrids are captured on Dynabeads M280 streptavidin (Dynabeads,
Dynal Corporation, Lake Success, New York U.S.A.). The dynabeads
with captured hybrids are collected with a magnet. The
non-hybridized single stranded molecules remaining after hybrid
capture are converted to double stranded form and represent the
primary normalized library.
[0492] The Soy53 (LIB3039) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
seedling shoot apical meristem tissue. Seeds are planted at a depth
of approximately 2 cm into 2-3 inch peat pots containing Metromix
350 medium and the plants are grown in an environmental chamber
under 12 hr daytime/12 hr nighttime cycles. The daytime temperature
is approximately 29.degree. C. and the nighttime temperature
24.degree. C. Soil is checked and watered daily to maintain even
moisture conditions. Apical tissue is harvested from seedling shoot
meristem tissue, 7-8 days after the start of imbibition. The apex
of each seedling is dissected to include the fifth node to the
apical meristem. The fifth node corresponds to the third trifoliate
leaf in the very early stages of development. Stipules completely
envelop the leaf primordia at this time. A total of 200 mg of
apical tissue is harvested and immediately frozen in liquid
nitrogen. The harvested tissue is then stored at -80.degree. C.
until RNA preparation.
[0493] The Soy54 (LIB3040) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
heart to torpedo stage embryo tissue. Seeds are planted at a depth
of approximately 2 cm into 2-3 inch peat pots containing Metromix
350 medium and the plants are grown in an environmental chamber
under 12 hr daytime/12 hr nighttime cycles. The daytime temperature
is approximately 29.degree. C. and the nighttime temperature
24.degree. C. Soil is checked and watered daily to maintain even
moisture conditions. Seeds are collected and embryos removed from
surrounding endosperm and maternal tissues. Embryos from globular
to young torpedo stages (by corresponding analogy to Arabidopsis)
are collected with a bias towards the middle of this spectrum.
Embryos which are beginning to show asymmetric development of
cotyledons are considered the upper developmental boundary for the
collection and are excluded. A total of 12 mg embryo tissue is
frozen in liquid nitrogen. The harvested tissue is stored at
-80.degree. C. until RNA preparation.
[0494] Soy55 (LIB3049) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
young seed tissue. Seeds are planted at a depth of approximately 2
cm into 2-3 inch peat pots containing Metromix 350 medium and the
plants are grown in an environmental chamber under 12 hr daytime/12
hr nighttime cycles. The daytime temperature is approximately
29.degree. C. and the nighttime temperature 24.degree. C. Soil is
checked and watered daily to maintain even moisture conditions.
Seeds are collected from very young pods (5 to 15 days after
flowering). A total of 100 mg of seeds are harvested and frozen in
liquid nitrogen. The harvested tissue is stored at -80.degree. C.
until RNA preparation.
[0495] Soy56 (LIB3029) cDNA library is prepared from equal amounts
tissue harvested from SOYMON007, SOYMON015 and SOYMON020 prepared
tissue. Single stranded and double stranded DNA representing
approximately 1.times.10.sup.6 colony forming units are isolated
using standard protocols. RNA, complementary to the single stranded
DNA, is synthesized using the double stranded DNA as a template.
Biotinylated dATP is incorporated into the RNA during the synthesis
reaction. The single stranded DNA is mixed with the biotinylated
RNA in a 1:10 molar ratio and allowed to hybridize. DNA-RNA hybrids
are captured on Dynabeads M280 streptavidin (Dynabeads, Dynal
Corporation, Lake Success, New York U.S.A.). The dynabeads with
captured hybrids are collected with a magnet. The non-hybridized
single stranded molecules remaining after hybrid capture are not
converted to double stranded form and represent a non-normalized
seed pool for comparison to Soy51 cDNA libraries.
[0496] The Soy58 (LIB3050) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
drought stressed root tissue subtracted from control root tissue.
Seeds are planted at a depth of approximately 2 cm into 2-3 inch
peat pots containing Metromix 350 medium and the plants are grown
in an environmental chamber under 12 hr daytime/12 hr nighttime
cycles. The daytime temperature is approximately 29.degree. C. and
the nighttime temperature 24.degree. C. Soil is checked and watered
daily to maintain even moisture conditions. At the R3 stage of the
plant drought is induced by withholding water. After 3 and 6 days
root tissue from both drought stressed and control (watered
regularly) plants are collected and frozen in dry-ice. The
harvested tissue is stored at -80.degree. C. until RNA preparation.
For subtraction, target cDNA is made from the drought stressed
tissue total RNA using the SMART cDNA synthesis system from
Clonetech (Clonetech Laboratories, Palo Alto, Calif. U.S.A.).
Driver first strand cDNA is covalently linked to Dynabeads
following a protocol similar to that described in the Dynal
literature (Dynabeads, Dynal Corporation, Lake Success, New York
U.S.A.). The target cDNA is then heat denatured and the second
strand trapped using Dynabeads oligo-dT. The target second strand
cDNA is then hybridized to the driver cDNA in 400 .mu.l
2.times.SSPE for two rounds of hybridization at 65.degree. C. and
20 hours. After each hybridization, the hybridization solution is
removed from the system and the hybridized target cDNA removed from
the driver by heat denaturation in water. After hybridization, the
remaining cDNA is trapped with Dynabeads oligo-dT. The trapped cDNA
is then amplified as in previous PCR based libraries and the
resulting cDNA ligated into the pSPORT vector (Invitrogen, Carlsbad
Calif. U.S.A.).
[0497] The Soy59 (LIB3051) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
endosperm tissue. Seeds are germinated on paper towels under
laboratory ambient light conditions. At 8, 10 and 14 hours after
imbibition, the seed coats are harvested. The endosperm consists of
a very thin layer of tissue affixed to the inside of the seed coat.
The seed coat and endosperm are frozen immediately after harvest in
liquid nitrogen. The harvested tissue is stored at -80.degree. C.
until RNA preparation.
[0498] The Soy60 (LIB3072) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
drought stressed seed plus pod subtracted from control seed plus
pod tissue. Seeds are planted at a depth of approximately 2 cm into
2-3 inch peat pots containing Metromix 350 medium and the plants
are grown in an environmental chamber under 12 hr daytime/12 hr
nighttime cycles. The daytime temperature is approximately
26.degree. C. and the nighttime temperature 21.degree. C. and 70%
relative humidity. Soil is checked and watered daily to maintain
even moisture conditions. At the R3 stage of the plant drought is
induced by withholding water. After 3 and 6 days seeds and pods
from both drought stressed and control (watered regularly) plants
are collected from the fifth and sixth node and frozen in dry-ice.
The harvested tissue is stored at -80.degree. C. until RNA
preparation. For subtraction, target cDNA is made from the drought
stressed tissue total RNA using the SMART cDNA synthesis system
from Clonetech (Clonetech Laboratories, Palo Alto, Calif. U.S.A.).
Driver first strand cDNA is covalently linked to Dynabeads
following a protocol similar to that described in the Dynal
literature (Dynabeads, Dynal Corporation, Lake Success, New York
U.S.A.). The target cDNA is then heat denatured and the second
strand trapped using Dynabeads oligo-dT. The target second strand
cDNA is then hybridized to the driver cDNA in 400 .mu.l
2.times.SSPE for two rounds of hybridization at 65.degree. C. and
20 hours. After each hybridization, the hybridization solution is
removed from the system and the hybridized target cDNA removed from
the driver by heat denaturation in water. After hybridization, the
remaining cDNA is trapped with Dynabeads oligo-dT. The trapped cDNA
is then amplified as in previous PCR based libraries and the
resulting cDNA ligated into the pSPORT vector (Invitrogen, Carlsbad
Calif. U.S.A.).
[0499] The Soy61 (LIB3073) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
jasmonic acid treated seedling subtracted from control tissue.
Seeds are planted at a depth of approximately 2 cm into 2-3 inch
peat pots containing Metromix 350 medium and the plants are grown
in a greenhouse. The daytime temperature is approximately
29.4.degree. C. and the nighttime temperature 20.degree. C. Soil is
checked and watered daily to maintain even moisture conditions. At
9 days post planting, the plantlets are sprayed with either control
buffer of 0.1% Tween-20 or jasmonic acid (Sigma J-2500, Sigma, St.
Louis, Mo. U.S.A.) at 1 mg/ml in 0.1% Tween-20. Plants are sprayed
until runoff and the soil and the stem is socked with the spraying
solution. At 18 hours post application of jasmonic acid, the
soybean plantlets appear growth retarded. After 18 hours, 24 hours
and 48 hours post treatment, the cotyledons are removed and the
remaining leaf and stem tissue above the soil is harvested and
frozen in liquid nitrogen. The harvested tissue is stored at
-80.degree. C. until RNA preparation. To make RNA, the three sample
timepoints were combined and ground. For subtraction, target cDNA
is made from the jasmonic acid treated tissue total RNA using the
SMART cDNA synthesis system from Clonetech (Clonetech Laboratories,
Palo Alto, Calif. U.S.A.). Driver first strand cDNA is covalently
linked to Dynabeads following a protocol similar to that described
in the Dynal literature (Dynabeads, Dynal Corporation, Lake
Success, New York U.S.A.). The target cDNA is then heat denatured
and the second strand trapped using Dynabeads oligo-dT. The target
second strand cDNA is then hybridized to the driver cDNA in 400
.mu.l 2.times.SSPE for two rounds of hybridization at 65.degree. C.
and 20 hours. After each hybridization, the hybridization solution
is removed from the system and the hybridized target cDNA removed
from the driver by heat denaturation in water. After hybridization,
the remaining cDNA is trapped with Dynabeads oligo-dT. The trapped
cDNA is then amplified as in previous PCR based libraries and the
resulting cDNA ligated into the pSPORT vector (Invitrogen, Carlsbad
Calif. U.S.A.). For this library's construction, the eighth
fraction of the cDNA size fractionation step was used for
ligation.
[0500] The Soy62 (LIB3074) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
jasmonic acid treated seedling subtracted from control tissue.
Seeds are planted at a depth of approximately 2 cm into 2-3 inch
peat pots containing Metromix 350 medium and the plants are grown
in a greenhouse. The daytime temperature is approximately
29.4.degree. C. and the nighttime temperature 20.degree. C. Soil is
checked and watered daily to maintain even moisture conditions. At
9 days post planting, the plantlets are sprayed with either control
buffer of 0.1% Tween-20 or jasmonic acid (Sigma J-2500, Sigma, St.
Louis, Mo. U.S.A.) at 1 mg/ml in 0.1% Tween-20. Plants are sprayed
until runoff and the soil and the stem is socked with the spraying
solution. At 18 hours post application of jasmonic acid, the
soybean plantlets appear growth retarded. After 18 hours, 24 hours
and 48 hours post treatment, the cotyledons are removed and the
remaining leaf and stem tissue above the soil is harvested and
frozen in liquid nitrogen. The harvested tissue is stored at
-80.degree. C. until RNA preparation. To make RNA, the three sample
timepoints were combined and ground. For subtraction, target cDNA
is made from the j asmonic acid treated tissue total RNA using the
SMART cDNA synthesis system from Clonetech (Clonetech Laboratories,
Palo Alto, Calif. U.S.A.). Driver first strand cDNA is covalently
linked to Dynabeads following a protocol similar to that described
in the Dynal literature (Dynabeads, Dynal Corporation, Lake
Success, New York U.S.A.). The target cDNA is then heat denatured
and the second strand trapped using Dynabeads oligo-dT. The target
second strand cDNA is then hybridized to the driver cDNA in 400
.mu.l 2.times.SSPE for two rounds of hybridization at 65.degree. C.
and 20 hours. After each hybridization, the hybridization solution
is removed from the system and the hybridized target cDNA removed
from the driver by heat denaturation in water. After hybridization,
the remaining cDNA is trapped with Dynabeads oligo-dT. The trapped
cDNA is then amplified as in previous PCR based libraries and the
resulting cDNA ligated into the pSPORT vector (Invitrogen, Carlsbad
Calif. U.S.A.). For this library's construction, the ninth fraction
of the cDNA size fractionation step was used for ligation.
[0501] The Soy65 (LIB133107) 07cDNA library is generated from
soybean cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa
U.S.A.) drought-stressed abscission zone tissue. Seeds are planted
at a depth of approximately 2 cm into 2-3 inch peat pots containing
Metromix 350 medium and the plants are grown in an environmental
chamber under 12 hr daytime/12 hr nighttime cycles. The daytime
temperature is approximately 29.degree. C. and the nighttime
temperature 24.degree. C. Soil is checked and watered daily to
maintain even moisture conditions. Plants are irrigated with
15-16-17 Peter's Mix. At the R3 stage of development, drought is
imposed by withholding water. At 3, 4, 5 and 6 days, tissue is
harvested and wilting is not obvious until the fourth day.
Abscission layers from reproductive organs are harvested by cutting
less than one millimeter proximal and distal to the layer and
immediately frozen in liquid nitrogen. The harvested tissue is
stored at -80.degree. C. until RNA preparation.
[0502] The Soy66 (LIB3109) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
non-drought stressed abscission zone tissue. Seeds are planted at a
depth of approximately 2 cm into 2-3 inch peat pots containing
Metromix 350 medium and the plants are grown in an environmental
chamber under 12 hr daytime/12 hr nighttime cycles. The daytime
temperature is approximately 29.degree. C. and the nighttime
temperature approximately 24.degree. C. Soil is checked and watered
daily to maintain even moisture conditions. Plants are irrigated
with 15-16-17 Peter's Mix. At 3, 4, 5 and 6 days, control
abscission layer tissue is harvested. Abscission layers from
reproductive organs are harvested by cutting less than one
millimeter proximal and distal to the layer and immediately frozen
in liquid nitrogen. The harvested tissue is stored at -80.degree.
C. until RNA preparation.
[0503] Soy67 (LIB3065) cDNA library is prepared from equal amounts
tissue harvested from SOYMON007, SOYMON015 and SOYMON020 prepared
tissue. Single stranded and double stranded DNA representing
approximately 1.times.10.sup.6 colony forming units are isolated
using standard protocols. RNA, complementary to the single stranded
DNA, is synthesized using the double stranded DNA as a template.
Biotinylated dATP is incorporated into the RNA during the synthesis
reaction. The single stranded DNA is mixed with the biotinylated
RNA in a 1:10 molar ratio) and allowed to hybridize. DNA-RNA
hybrids are captured on Dynabeads M280 streptavidin (Dynabeads,
Dynal Corporation, Lake Success, New York U.S.A.). The dynabeads
with captured hybrids are collected with a magnet. Captured hybrids
are eluted with water.
[0504] Soy68 (LIB3052) cDNA library is prepared from equal amounts
tissue harvested from SOYMON007, SOYMON015 and SOYMON020 prepared
tissue. Single stranded and double stranded DNA representing
approximately 1.times.10.sup.6 colony forming units are isolated
using standard protocols. RNA, complementary to the single stranded
DNA, is synthesized using the double stranded DNA as a template.
Biotinylated dATP is incorporated into the RNA during the synthesis
reaction. The single stranded DNA is mixed with the biotinylated
RNA in a 1:10 molar ratio) and allowed to hybridize. DNA-RNA
hybrids are captured on Dynabeads M280 streptavidin (Dynabeads,
Dynal Corporation, Lake Success, New York U.S.A.). The dynabeads
with captured hybrids are collected with a magnet. Captured hybrids
are eluted with water.
[0505] Soy69 (LIB3053) cDNA library is generated from soybean
cultivars Cristalina (USDA Soybean Germplasm Collection, Urbana,
Ill. U.S.A.) and FT108 (Monsoy, Brazil) (tropical germ plasma)
normalized leaf tissue. Leaves are harvested from plants grown in
an environmental chamber under 12 hr daytime/12 hr nighttime
cycles. The daytime temperature is approximately 29.degree. C. and
the nighttime temperature approximately 24.degree. C. Soil is
checked and watered daily to maintain even moisture conditions.
Approximately 30 g of leaves are harvested from the 4.sup.th node
of each of the Cristalina and FT108 cultivars and immediately
frozen in dry ice. The harvested tissue is then stored at
-80.degree. C. until RNA preparation. Single stranded and double
stranded DNA representing approximately 1.times.10.sup.6 colony
forming units are isolated using standard protocols. RNA,
complementary to the single stranded DNA, is synthesized using the
double stranded DNA as a template. Biotinylated dATP is
incorporated into the RNA during the synthesis reaction. The single
stranded DNA is mixed with the biotinylated RNA in a 1:10 molar
ratio) and allowed to hybridize. DNA-RNA hybrids are captured on
Dynabeads M280 streptavidin (Dynabeads, Dynal Corporation, Lake
Success, New York U.S.A.). The dynabeads with captured hybrids are
collected with a magnet. The non-hybridized single stranded
molecules remaining after hybrid capture are converted to double
stranded form and represent the primary normalized library.
[0506] Soy70 (LIB3055) cDNA library is generated from soybean
cultivars Cristalina (USDA Soybean Germplasm Collection, Urbana,
Ill. U.S.A.) and FT108 (Monsoy, Brazil) (tropical germ plasma) leaf
tissue. Leaves are harvested from plants grown in an environmental
chamber under 12 hr daytime/12 hr nighttime cycles. The daytime
temperature is approximately 29.degree. C. and the nighttime
temperature approximately 24.degree. C. Soil is checked and watered
daily to maintain even moisture conditions. Approximately 30 g of
leaves are harvested from the 4.sup.th node of each of the
Cristalina and FT108 cultivars and immediately frozen in dry ice.
The harvested tissue is then stored at -80.degree. C. until RNA
preparation.
[0507] Soy71 (LIB3056) cDNA library is generated from soybean
cultivars Cristalina and FT108 (tropical germ plasma) root tissue.
Roots are harvested from plants grown in an environmental chamber
under 12 hr daytime/12 hr nighttime cycles. The daytime temperature
is approximately 29.degree. C. and the nighttime temperature
approximately 24.degree. C. Soil is checked and watered daily to
maintain even moisture conditions. Approximately 50 g and 56 g of
roots are harvested from each of the Cristalina and FT108 cultivars
and immediately frozen in dry ice. The harvested tissue is then
stored at -80.degree. C. until RNA preparation.
[0508] Soy72 (LIB3093) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
drought stressed leaf control tissue. Seeds are planted at a depth
of approximately 2 cm into 2-3 inch peat pots containing Metromix
350 medium and the plants are grown in an environmental chamber
under 12 hr daytime/12 hr nighttime cycles. The daytime temperature
is approximately 26.degree. C. and the nighttime temperature
21.degree. C. and 70% relative humidity. Soil is checked and
watered daily to maintain even moisture conditions. At the R3 stage
of the plant drought is induced by withholding water. After 3 and 6
days seeds and pods from both drought stressed and control (watered
regularly) plants are collected from the fifth and sixth node and
frozen in dry-ice. The harvested tissue is stored at -80.degree. C.
until RNA preparation. For subtraction, target cDNA is made from
the drought stressed tissue total RNA using the SMART cDNA
synthesis system from Clonetech (Clonetech Laboratories, Palo Alto,
Calif. U.S.A.). Driver first strand cDNA is covalently linked to
Dynabeads following a protocol similar to that described in the
Dynal literature (Dynabeads, Dynal Corporation, Lake Success, New
York U.S.A.). The target cDNA is then heat denatured and the second
strand trapped using Dynabeads oligo-dT. The target second strand
cDNA is then hybridized to the driver cDNA in 400 .mu.l
2.times.SSPE for two rounds of hybridization at 65.degree. C. and
20 hours. After each hybridization, the hybridization solution is
removed from the system and the hybridized target cDNA removed from
the driver by heat denaturation in water. After hybridization, the
remaining cDNA is trapped with Dynabeads oligo-dT. The trapped cDNA
is then amplified as in previous PCR based libraries and the
resulting cDNA ligated into the pSPORT vector (Invitrogen, Carlsbad
Calif. U.S.A.).
[0509] Soy73 (LIB3093) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
drought stressed leaf subtracted from control tissue. Seeds are
planted at a depth of approximately 2 cm into 2-3 inch peat pots
containing Metromix 350 medium and the plants are grown in an
environmental chamber under 12 hr daytime/12 hr nighttime cycles.
The daytime temperature is approximately 26.degree. C. and the
nighttime temperature 21.degree. C. and 70% relative humidity. Soil
is checked and watered daily to maintain even moisture conditions.
At the R3 stage of the plant drought is induced by withholding
water. After 3 and 6 days seeds and pods from both drought stressed
and control (watered regularly) plants are collected from the fifth
and sixth node and frozen in dry-ice. The harvested tissue is
stored at -80.degree. C. until RNA preparation. For subtraction,
target cDNA is made from the drought stressed tissue total RNA
using the SMART cDNA synthesis system from Clonetech (Clonetech
Laboratories, Palo Alto, Calif. U.S.A.). Driver first strand cDNA
is covalently linked to Dynabeads following a protocol similar to
that described in the Dynal literature (Dynabeads, Dynal
Corporation, Lake Success, New York U.S.A.). The target cDNA is
then heat denatured and the second strand trapped using Dynabeads
oligo-dT. The target second strand cDNA is then hybridized to the
driver cDNA in 400 .mu.l 2.times.SSPE for two rounds of
hybridization at 65.degree. C. and 20 hours. After each
hybridization, the hybridization solution is removed from the
system and the hybridized target cDNA removed from the driver by
heat denaturation in water. After hybridization, the remaining cDNA
is trapped with Dynabeads oligo-dT. The trapped cDNA is then
amplified as in previous PCR based libraries and the resulting cDNA
ligated into the pSPORT vector (Invitrogen, Carlsbad Calif.
U.S.A.).
[0510] The Soy76 (Lib3106) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
jasmonic acid and arachidonic treated seedling subtracted from
control tissue. Seeds are planted at a depth of approximately 2 cm
into 2-3 inch peat pots containing Metromix 350 medium and the
plants are grown in a greenhouse. The daytime temperature is
approximately 29.4.degree. C. and the nighttime temperature
20.degree. C. Soil is checked and watered daily to maintain even
moisture conditions. At 9 days post planting, the plantlets are
sprayed with either control buffer of 0.1% Tween-20 or jasmonic
acid (Sigma J-2500, Sigma, St. Louis, Mo. U.S.A.) at 1 mg/ml in
0.1% Tween-20. Plants are sprayed until runoff and the soil and the
stem is socked with the spraying solution. At 18 hours post
application of jasmonic acid, the soybean plantlets appear growth
retarded. Arachidonic treated seedlings are sprayed with 1 m/ml
arachidonic acid in 0.1% Tween-20. After 18 hours, 24 hours and 48
hours post treatment, the cotyledons are removed and the remaining
leaf and stem tissue above the soil is harvested and frozen in
liquid nitrogen. The harvested tissue is stored at -80.degree. C.
until RNA preparation. To make RNA, the three sample timepoints
were combined and ground. The RNA from the arachidonic treated
seedlings is isolated separately. For subtraction, target cDNA is
made from the jasmonic acid treated tissue total RNA using the
SMART cDNA synthesis system from Clonetech (Clonetech Laboratories,
Palo Alto, Calif. U.S.A.). Driver first strand cDNA is covalently
linked to Dynabeads following a protocol similar to that described
in the Dynal literature (Dynabeads, Dynal Corporation, Lake
Success, New York U.S.A.). The target cDNA is then heat denatured
and the second strand trapped using Dynabeads oligo-dT. The target
second strand cDNA is then hybridized to the driver cDNA in 400
.mu.l 2.times.SSPE for two rounds of hybridization at 65.degree. C.
and 20 hours. After each hybridization, the hybridization solution
is removed from the system and the hybridized target cDNA removed
from the driver by heat denaturation in water. After hybridization,
the remaining cDNA is trapped with Dynabeads oligo-dT. The trapped
cDNA is then amplified as in previous PCR based libraries and the
resulting cDNA ligated into the pSPORT vector (Invitrogen, Carlsbad
Calif. U.S.A.). Fraction 10 of the size fractionated cDNA is
ligated into the pSPORT vector (Invitrogen, Carlsbad Calif. U.S.A.)
in order to capture some of the smaller transcripts characteristic
of antifungal proteins.
[0511] Soy77 (LIB3108) cDNA library is generated from soybean
cultivar Asgrow 3244 (Asgrow Seed Company, Des Moines, Iowa U.S.A.)
jasmonic acid control tissue. Seeds are planted at a depth of
approximately 2 cm into 2-3 inch peat pots containing Metromix 350
medium and the plants are grown in a greenhouse. The daytime
temperature is approximately 29.4.degree. C. and the nighttime
temperature 20.degree. C. Soil is checked and watered daily to
maintain even moisture conditions. At 9 days post planting, the
plantlets are sprayed with either control buffer of 0.1% Tween-20
or jasmonic acid (Sigma J-2500, Sigma, St. Louis, Mo. U.S.A.) at 1
mg/ml in 0.1% Tween-20. Plants are sprayed until runoff and the
soil and the stem is socked with the spraying solution. At 18 hours
post application of jasmonic acid, the soybean plantlets appear
growth retarded. Arachidonic treated seedlings are sprayed with 1
m/ml arachidonic acid in 0.1% Tween-20. After 18 hours, 24 hours
and 48 hours post treatment, the cotyledons are removed and the
remaining leaf and stem tissue above the soil is harvested and
frozen in liquid nitrogen. The harvested tissue is stored at
-80.degree. C. until RNA preparation. To make RNA, the three sample
timepoints were combined and ground. The RNA from the arachidonic
treated seedlings is isolated separately. For subtraction, target
cDNA is made from the jasmonic acid treated tissue total RNA using
the SMART cDNA synthesis system from Clonetech (Clonetech
Laboratories, Palo Alto, Calif. U.S.A.). Driver first strand cDNA
is covalently linked to Dynabeads following a protocol similar to
that described in the Dynal literature (Dynabeads, Dynal
Corporation, Lake Success, New York U.S.A.). The target cDNA is
then heat denatured and the second strand trapped using Dynabeads
oligo-dT. The target second strand cDNA is then hybridized to the
driver cDNA in 400 .mu.l 2.times.SSPE for two rounds of
hybridization at 65.degree. C. and 20 hours. After each
hybridization, the hybridization solution is removed from the
system and the hybridized target cDNA removed from the driver by
heat denaturation in water. After hybridization, the remaining cDNA
is trapped with Dynabeads oligo-dT. The trapped cDNA is then
amplified as in previous PCR based libraries and the resulting cDNA
ligated into the pSPORT vector (Invitrogen, Carlsbad Calif.
U.S.A.). Fraction 10 of the size fractionated cDNA is ligated into
the pSPORT vector in order to capture some of the smaller
transcripts characteristic of antifungal proteins.
[0512] The Lib9 cDNA library is prepared from Arabidopsis thaliana,
Columbia ecotype, leaf tissue. Wild type Arabidopsis thaliana seeds
are planted in commonly used planting pots and grown in an
environmental chamber. Leaf blades were cut with sharp scissors at
seven weeks after planting. The tissue was immediately frozen in
liquid nitrogen. The harvested tissue is stored at -80.degree. C.
until RNA extraction. PolyA mRNA is purified from the total RNA
preparation using Dynabeads.RTM. Oligo(dT).sub.25 (Dynal Inc., Lake
Success, N.Y.), or equivalent methods. This library was normalized
using a PCR-based protocol.
[0513] The Lib22 cDNA library is prepared from Arabidopsis thaliana
Columbia ecotype, root tissue. Wild type Arabidopsis thaliana seeds
are planted in commonly used planting pots and grown in an
environmental chamber. After 5-6 weeks the plants are in the
reproductive growth phase. Stems are bolting from the base of the
plants. After 7 weeks, more stems, floral buds appear, and a few
flowers are starting to open. The 7-week old plants are rinsed
intensively by tope water remove dirt from the roots, and blotted
by paper towel. The tissues are immediately frozen in liquid
nitrogen. The harvested tissue is stored at -80.degree. C. until
RNA preparation.
[0514] The Lib23 cDNA library is prepared from Arabidopsis
thaliana, Columbia ecotype, stem tissue. Wild type Arabidopsis
thaliana seeds are planted in commonly used planting pots and grown
in an environmental chamber. Stems were collected seven to eight
weeks after planting by cutting the stems from the base and cutting
the top of the plant to remove the floral tissue. The tissue was
immediately frozen in liquid nitrogen and stored at -80.degree. C.
until total RNA extraction. PolyA mRNA is purified from the total
RNA preparation using Dynabeads.RTM. Oligo(dT).sub.25 (Dynal Inc.,
Lake Success, N.Y.), or equivalent methods. This library was
normalized using a PCR-based protocol.
[0515] The Lib24 cDNA library is prepared from Arabidopsis
thaliana, Columbia ecotype, flower bud tissue. Wild type
Arabidopsis thaliana seeds are planted in commonly used planting
pots and grown in an environmental chamber. Flower buds are green
and unopened and harvested about seven weeks after planting. The
tissue is immediately frozen in liquid nitrogen. The harvested
tissue is stored at -80.degree. C. until total RNA extraction.
PolyA mRNA is purified from the total RNA preparation using
Dynabeads.RTM. Oligo(dT).sub.25 (Dynal Inc., Lake Success, N.Y.),
or equivalent methods. This library was normalized using a
PCR-based protocol.
[0516] The Lib25 cDNA library is prepared from Arabidopsis
thaliana, Columbia ecotype, open flower tissue. Wild type
Arabidopsis thaliana seeds are planted in commonly used planting
pots and grown in an environmental chamber. Flowers are completely
opened with all parts of floral structure observable, but no
siliques are appearing. The tissue was immediately frozen in liquid
nitrogen and stored at -80.degree. C. until total RNA extraction.
PolyA mRNA is purified from the total RNA preparation using
Dynabeads.RTM. Oligo(dT).sub.25 (Dynal Inc., Lake Success, N.Y.),
or equivalent methods. This library was normalized using a
PCR-based protocol.
[0517] The Lib35 cDNA library of the present invention, was
prepared from Arabidopsis thaliana Columbia ecotype leaf tissue.
Wild type Arabidopsis thaliana seeds are planted in commonly used
planting pots and grown in an environmental chamber. After 5-6
weeks the plants are in the reproductive growth phase. Stems are
bolting from the base of the plants. After 7 weeks, more stems and
floral buds appeared and a few flowers were starting to open. Leaf
blades were collected by cutting with sharp scissors. The tissues
were immediately frozen in liquid nitrogen and stored at
-80.degree. C. until use. PolyA mRNA is purified from the total RNA
preparation using Dynabeads.RTM. Oligo(dT).sub.25 (Dynal Inc., Lake
Success, N.Y.), or equivalent methods. This library was normalized
using a PCR-based protocol.
[0518] The Lib146 cDNA library is prepared from Arabidopsis
thaliana, Columbia ecotype, immature seed tissue. Wild type
Arabidopsis thaliana seeds are planted in commonly used planting
pots and grown in an environmental chamber. At approximately 7-8
weeks of age, the seeds are harvested. The seeds ranged in maturity
from the smallest seeds that could be dissected from silques to
just before starting to turn yellow in color. The tissue is
immediately frozen in liquid nitrogen. The harvested tissue is
stored at -80.degree. C. until RNA extraction. PolyA mRNA is
purified from the total RNA preparation using Dynabeads.RTM.
Oligo(dT).sub.25 (Dynal Inc., Lake Success, N.Y.), or equivalent
methods. This library is normalized using a PCR-based protocol.
[0519] The Lib3032 (Lib80) cDNA libraries are generated from
Brassica napus seeds harvested 30 days after pollination. The cDNA
libraries are constructed using the SuperScript Plasmid system for
cDNA synthesis and plasmid cloning (Life Technologies,
Gaithersburg, Md. U.S.A.) according to the manufacturers protocol
with the following modification: 40 micrograms of total RNA is used
as the starting material for cDNA synthesis, and first strand cDNA
synthesis is carried out at 45.degree. C.
[0520] The Lib3034 (Lib82) cDNA libraries are generated from
Brassica napus seeds harvested 15 and 18 days after pollination.
The cDNA libraries are constructed using the SuperScript Plasmid
system for cDNA synthesis and plasmid cloning (Life Technologies,
Gaithersburg, Md. U.S.A.) according to the manufacturers protocol
with the following modification: 40 micrograms of total RNA is used
as the starting material for cDNA synthesis, and first strand cDNA
synthesis was carried out at 45.degree. C.
[0521] The Lib3099 cDNA library is generated by a subtraction
procedure. The library contains cDNAs whose abundance is enriched
in the Brassica napus 15 and 18 day after pollination seed tissues
when compared to Brassica leaf tissues. The cDNA synthesis is
performed on Brassica leaf RNA and Brassica RNA isolated from seeds
harvested 15 and 18 days after pollination using a Smart PCR cDNA
synthesis kit according to the manufacturers protocol (Clontech,
Palo Alto, Calif. U.S.A.). The subtracted cDNA is generated using
the Clontech PCR-Select subtraction kit according to the
manufacturers protocol (Clontech, Palo Alto, Calif. U.S.A.). The
subtracted cDNA was cloned into plasmid vector pCR2.1 according to
the manufacturers protocol (Invitrogen, Carlsbad, Calif.
U.S.A.).
[0522] The Lib3033 (Lib81) cDNA libraries are generated from the
Schizochytrium species cells. The Schizochytrium species cells are
grown in liquid media until saturation. The culture is centrifuged
to pellet the cells, the medium is decanted off, and pellet
immediately frozen in liquid nitrogen. Wax esters are produced
under such dark, anaerobic, rich-medium conditions. High wax
production by the cultures is verified by microscopy (fluorescein
staining of wax bodies) and by lipid extraction/TLC/GC. The
harvested cells are stored at -80.degree. C. until RNA preparation.
RNA is prepared from the frozen Euglena cell pellet as follows. The
pellet is pulverized to a powder in liquid nitrogen using a mortar
and pestle. The powder is transferred to tubes containing 6 ml each
of lysis buffer (100 mM Tris, pH 8, 0.6 M NaCl, 10 mM EDTA, and 4%
(w/v) SDS) and buffered phenol, vortexed, and disrupted with a
Polytron. The mixture is centrifuged 20 min at 10,000.times.g in
Corex glass tubes to separate the phases. 5 ml of the upper phase
is removed, vortexed with 5 ml fresh phenol, and centrifuged. The
upper phase is removed and the RNA is precipitated overnight at
4.degree. C. by adding 1.5 volumes of 4 M LiCl. The RNA is further
purified on Rneasy columns according to the manufacturers protocol
(Qiagen, Valencia, Calif. U.S.A.). The cDNA library is constructed
using the SuperScript Plasmid system for cDNA synthesis and plasmid
cloning (Life Technologies, Gaithersburg, Md. U.S.A.) according to
the manufacturers protocol with the following modification: 40
micrograms of total RNA was used as the starting material for cDNA
synthesis, and first strand cDNA synthesis was carried out at
45.degree. C.
[0523] The Lib47 cDNA library is generated from Euglena gracilus
strain 753 (ATTC No. 30285, ATCC Manasas, Va. U.S.A.) grown in
liquid culture. A liquid culture is innoculated with 1/10 volume of
a previously grown saturated culture, and the new culture for 4
days under near-anaerobic conditions (near-anaerobic cultures are
not agitated, just gently swirled once a day) in the dark in
2.times. Beef (10 g/l bacto peptone, 4 g/l yeast extract, 2 g/l
beef extract, 6 g/l glucose). The culture is then centrifuged to
pellet the cells, the medium is decanted off, and pellet
immediately frozen in liquid nitrogen. Wax esters are produced
under such dark, anaerobic, rich-medium conditions. High wax
production by the cultures is verified by microscopy (fluorescein
staining of wax bodies) and by lipid extraction/TLC/GC. The
harvested cells are stored at -80.degree. C. until RNA preparation.
RNA is prepared from the frozen Euglena cell pellet as follows. The
pellet is pulverized to a powder in liquid nitrogen using a mortar
and pestle. The powder is transferred to tubes containing 6 ml each
of lysis buffer (100 mM Tris, pH 8, 0.6 M NaCl, 10 mM EDTA, and 4%
(w/v) SDS) and buffered phenol, vortexed, and disrupted with a
Polytron. The mixture is centrifuged 20 min at 10,000.times.g in
Corex glass tubes to separate the phases. 5 ml of the upper phase
is removed, vortexed with 5 ml fresh phenol, and centrifuged. The
upper phase is removed and the RNA is precipitated overnight at
4.degree. C. by adding 1.5 volumes of 4 M LiCl. The RNA is further
purified on Rneasy columns according to the manufacturers protocol
(Qiagen, Valencia, Calif. U.S.A.). The cDNA library is constructed
using the SuperScript Plasmid system for cDNA synthesis and plasmid
cloning (Life Technologies, Gaithersburg, Md. U.S.A.) according to
the manufacturers protocol with the following modification: 40
micrograms of total RNA was used as the starting material for cDNA
synthesis, and first strand cDNA synthesis was carried out at
45.degree. C.
[0524] The Lib44 cDNA library is generated from Phaeodactylum
tricornatum grown in modified Jones medium for 3 days. The cells
were harvested by centrifugation and the resulting pellet frozen
immediately in liquid nitrogen. The harvested cells are stored at
-80.degree. C. until RNA preparation. RNA is prepared from the
frozen Phaeodactylum cell pellet as follows. The pellet is
pulverized to a powder in liquid nitrogen using a mortar and
pestle. The powder is transferred to tubes containing 6 ml each of
lysis buffer (100 mM Tris, pH 8, 0.6 M NaCl, 10 mM EDTA, and 4%
(w/v) SDS) and buffered phenol, vortexed, and disrupted with a
Polytron. The mixture is centrifuged 20 min at 10,000.times.g in
Corex glass tubes to separate the phases. 5 ml of the upper phase
is removed, vortexed with 5 ml fresh phenol, and centrifuged. The
upper phase is removed and the RNA is precipitated overnight at
4.degree. C. by adding 1.5 volumes of 4 M LiCl. The RNA is further
purified on Rneasy columns according to the manufacturers protocol
(Qiagen, Valencia, Calif. U.S.A.). The cDNA library is constructed
using the SuperScript Plasmid system for cDNA synthesis and plasmid
cloning (Life Technologies, Gaithersburg, Md. U.S.A.) according to
the manufacturers protocol with the following modification: 40
micrograms of total RNA was used as the starting material for cDNA
synthesis, and first strand cDNA synthesis was carried out at 45
degrees centigrade.
[0525] The LIB3036 genomic library is generated from Mycobacterium
neoaurum US52 (ATCC No. 23072, ATCC, Manasas, Va. U.S.A.) cells.
Mycobacterium neoaurum US52 is a gram-positive Actinomycete
bacterium. Mycobacterium neoaurum US52 is genetically related to
Mycobacterium tuberculosis, but there is no reason to believe that
it is a primary pathogen. It normally is saprophytic, i.e. it lives
in soil and gets nutrients from decaying matter. Genomic DNA
obtained from Mycobacterium neoaurum US52 is digested for various
times with the restriction enzyme Sau3A. The DNA fractions are
size-separated on an agarose gel, and the first fraction wherein
most of the partially-digested fragments are about 10 kB is used to
isolated fragments in the range of 2-3 kB. For LE33036, the 2-3 kB
DNA is cloned into vector pRY401 (Invitrogen, Carlsbad, Calif.
U.S.A.). The vector pZERO-2 (Invitrogen, Carlsbad, Calif. U.S.A.).
is used for the construction of LIB3104.
[0526] The stored RNA is purified using Trizol reagent from Life
Technologies (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), essentially as recommended by the manufacturer. Poly A+
RNA (mRNA) is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, New York U.S.A.).
[0527] Construction of plant cDNA libraries is well-known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0528] Normalized libraries are made using essentially the Soares
procedure (Soares et al., Proc. Natl. Acad. Sci. (U.S.A.)
91:9228-9232 (1994), the entirety of which is herein incorporated
by reference). This approach is designed to reduce the initial
10,000-fold variation in individual cDNA frequencies to achieve
abundances within one order of magnitude while maintaining the
overall sequence complexity of the library. In the normalization
process, the prevalence of high-abundance cDNA clones decreases
dramatically, clones with mid-level abundance are relatively
unaffected and clones for rare transcripts are effectively
increased in abundance.
Example 2
[0529] The cDNA libraries are plated on LB agar containing the
appropriate antibiotics for selection and incubated at 37.degree.
for a sufficient time to allow the growth of individual colonies.
Single colonies are individually placed in each well of a 96-well
microtiter plates containing LB liquid including the selective
antibiotics. The plates are incubated overnight at approximately
37.degree. C. with gentle shaking to promote growth of the
cultures. The plasmid DNA is isolated from each clone using Qiaprep
plasmid isolation kits, using the conditions recommended by the
manufacturer (Qiagen Inc., Santa Clara, Calif. U.S.A.).
[0530] Template plasmid DNA clones are used for subsequent
sequencing. For sequencing, the ABI PRISM dRhodamine Terminator
Cycle Sequencing Ready Reaction Kit with AmpliTaq.RTM. DNA
Polymerase, FS, is used (PE Applied Biosystems, Foster City, Calif.
U.S.A.).
Example 3
[0531] Nucleic acid sequences that encode for the following
proteins: triose phosphate isomerase, fructose 1,6-bisphosphate
aldolase, fructose 1,6-bisphosphate, fructose 6-phosphate 2-kinase,
phosphoglucoisomerase, vacuolar H.sup.+
translocating-pyrophosphatase, pyrophosphate-dependent
fructose-6-phosphate phosphotransferase, invertase, sucrose
synthase, hexokinase, fructokinase, NDP-kinase, glucose-6-phosphate
1-dehydrogenase, phosphoglucomutase and UDP-glucose
pyrophosphorylase are identified from the Monsanto EST PhytoSeq
database using TBLASTN (default values) (TBLASTN compares a protein
query against the six reading frames of a nucleic acid sequence).
Matches found with BLAST P values equal or less than 0.001
(probability) or BLAST Score of equal or greater than 90 are
classified as hits. If the program used to determine the hit is
HMMSW then the score refers to HMMSW score.
[0532] In addition, the GenBank database is searched with BLASTN
and BLASTX (default values) using ESTs as queries. EST that pass
the hit probability threshold of 10e.sup.-8 for the following
enzymes are combined with the hits generated by using TBLASTN
(described above) and classified by enzyme (see Table A below).
[0533] A cluster refers to a set of overlapping clones in the
PhytoSeq database. Such an overlapping relationship among clones is
designated as a "cluster" when BLAST scores from pairwise sequence
comparisons of the member clones meets a predetermined minimum
value or product score of 50 or more (Product Score=(BLAST
SCORE.times.Percentage Identity)/(5.times.minimum [length (Seq1),
length (Seq2)]))
[0534] Since clusters are formed on the basis of single-linkage
relationships, it is possible for two non-overlapping clones to be
members of the same cluster if, for instance, they both overlap a
third clone with at least the predetermined minimum BLAST score
(stringency). A cluster ID is arbitrarily assigned to all of those
clones which belong to the same cluster at a given stringency and a
particular clone will belong to only one cluster at a given
stringency. If a cluster contains only a single clone (a
"singleton"), then the cluster ID number will be negative, with an
absolute value equal to the clone ID number of its single member.
Clones grouped in a cluster in most cases represent a contiguous
sequence.
TABLE-US-00002 TABLE A* Seq No. Cluster ID Clone ID Library NCBI gi
Method Score P-value % Ident MAIZE TRIOSE PHOSPHATE ISOMERASE 1
-700019675 700019675H1 SATMON001 g546735 BLASTX 134 1e-11 78 2
-700073894 700073894H1 SATMON007 g609261 BLASTN 257 1e-10 84 3
-700167260 700167260H1 SATMON013 g609261 BLASTN 644 1e-44 79 4
-700380595 700380595H1 SATMON021 g609261 BLASTN 1121 1e-84 87 5
-700449667 700449667H1 SATMON028 g217973 BLASTN 204 1e-18 93 6
-700449720 700449720H2 SATMON028 g217973 BLASTN 216 1e-18 88 7
-700570661 700570661H1 SATMON030 g168647 BLASTX 131 1e-11 88 8
-700616770 700616770H1 SATMON033 g407525 BLASTX 149 1e-13 83 9
-701170944 701170944H1 SATMONN05 g217921 BLASTX 188 1e-20 53 10
11337 700337974H1 SATMON020 g256119 BLASTN 535 1e-61 78 11 11337
700027829H1 SATMON003 g256119 BLASTN 726 1e-51 80 12 126
700050046H1 SATMON003 g1785947 BLASTN 440 1e-26 92 13 282
700077320H1 SATMON007 g217973 BLASTN 666 1e-108 97 14 282
700104541H1 SATMON010 g217973 BLASTN 631 1e-106 97 15 282
700047476H1 SATMON003 g217973 BLASTN 648 1e-105 97 16 282
700211559H1 SATMON016 g217973 BLASTN 525 1e-104 97 17 282
700073553H1 SATMON007 g217973 BLASTN 981 1e-103 98 18 282
700613011H1 SATMON033 g217973 BLASTN 552 1e-102 98 19 282
700352119H1 SATMON023 g217973 BLASTN 666 1e-101 97 20 282
700088148H1 SATMON011 g217973 BLASTN 666 1e-100 98 21 282
700351626H1 SATMON023 g217973 BLASTN 401 1e-99 98 22 282
700240096H1 SATMON010 g217973 BLASTN 666 1e-98 97 23 282
700083660H1 SATMON011 g217973 BLASTN 666 1e-97 99 24 282
700208721H1 SATMON016 g217973 BLASTN 497 1e-96 98 25 282
700203144H1 SATMON003 g217973 BLASTN 511 1e-96 96 26 282
700430425H1 SATMONN01 g217973 BLASTN 666 1e-96 98 27 282
700206091H1 SATMON003 g217973 BLASTN 497 1e-94 97 28 282
700077017H1 SATMON007 g217973 BLASTN 614 1e-93 93 29 282
700618792H1 SATMON034 g217973 BLASTN 546 1e-92 96 30 282
700572532H1 SATMON030 g407524 BLASTN 1212 1e-92 84 31 282
700106512H1 SATMON010 g217973 BLASTN 632 1e-91 97 32 282
700195031H1 SATMON014 g217973 BLASTN 471 1e-90 97 33 282
700168131H1 SATMON013 g217973 BLASTN 497 1e-89 98 34 282
700197039H1 SATMON014 g217973 BLASTN 546 1e-89 98 35 282
700572688H1 SATMON030 g169820 BLASTN 1114 1e-89 85 36 282
700021313H1 SATMON001 g217973 BLASTN 913 1e-87 97 37 282
700452417H1 SATMON028 g217973 BLASTN 425 1e-86 95 38 282
700346119H1 SATMON021 g217973 BLASTN 444 1e-86 96 39 282
700082359H1 SATMON011 g217973 BLASTN 542 1e-86 93 40 282
700240042H1 SATMON010 g217973 BLASTN 596 1e-86 97 41 282
700030064H1 SATMON003 g217973 BLASTN 587 1e-85 94 42 282
700615185H1 SATMON033 g217973 BLASTN 430 1e-84 98 43 282
700196125H1 SATMON014 g217973 BLASTN 581 1e-84 100 44 282
700243429H1 SATMON010 g217973 BLASTN 632 1e-84 97 45 282
700474112H1 SATMON025 g217973 BLASTN 570 1e-83 98 46 282
700572282H1 SATMON030 g407524 BLASTN 838 1e-83 82 47 282
700622238H1 SATMON034 g169820 BLASTN 917 1e-80 86 48 282
700095609H1 SATMON008 g169820 BLASTN 1067 1e-80 82 49 282
700218886H1 SATMON011 g217973 BLASTN 551 1e-79 93 50 282
700018688H1 SATMON001 g217973 BLASTN 1066 1e-79 99 51 282
700049775H1 SATMON003 g217973 BLASTN 362 1e-78 91 52 282
700575972H1 SATMON030 g169820 BLASTN 894 1e-78 79 53 282
700215519H1 SATMON016 g217973 BLASTN 497 1e-76 97 54 282
700161120H1 SATMON012 g217973 BLASTN 622 1e-76 98 55 282
700581760H1 SATMON031 g217973 BLASTN 533 1e-75 90 56 282
700104672H1 SATMON010 g169820 BLASTN 1012 1e-75 83 57 282
700346053H1 SATMON021 g169820 BLASTN 1012 1e-75 83 58 282
701166592H1 SATMONN04 g217973 BLASTN 661 1e-74 95 59 282
700968667H1 SATMONN04 g217973 BLASTN 497 1e-73 92 60 282
700205627H1 SATMON003 g217973 BLASTN 666 1e-73 99 61 282
700029005H1 SATMON003 g169820 BLASTN 979 1e-72 85 62 282
700476479H1 SATMON025 g169820 BLASTN 554 1e-71 84 63 282
700050148H1 SATMON003 g169820 BLASTN 608 1e-70 83 64 282
700259846H1 SATMON017 g217973 BLASTN 283 1e-69 94 65 282
700344093H1 SATMON021 g169820 BLASTN 934 1e-69 83 66 282
700082327H1 SATMON011 g169820 BLASTN 943 1e-69 85 67 282
700020156H1 SATMON001 g217973 BLASTN 420 1e-68 99 68 282
700577714H1 SATMON031 g169820 BLASTN 928 1e-68 85 69 282
700104904H1 SATMON010 g169820 BLASTN 913 1e-67 84 70 282
700104685H1 SATMON010 g169820 BLASTN 897 1e-66 84 71 282
700053463H1 SATMON009 g169820 BLASTN 907 1e-66 85 72 282
700171639H1 SATMON013 g217973 BLASTN 401 1e-65 98 73 282
700574233H1 SATMON030 g169820 BLASTN 651 1e-65 83 74 282
700262653H1 SATMON017 g169820 BLASTN 877 1e-64 84 75 282
700456738H1 SATMON029 g169820 BLASTN 877 1e-64 84 76 282
700611806H1 SATMON022 g169820 BLASTN 877 1e-64 83 77 282
700381177H1 SATMON023 g169820 BLASTN 884 1e-64 84 78 282
700103347H1 SATMON010 g169820 BLASTN 861 1e-63 84 79 282
700103605H1 SATMON010 g169820 BLASTN 868 1e-63 84 80 282
700578536H1 SATMON031 g169820 BLASTN 856 1e-62 84 81 282
700258606H1 SATMON017 g169820 BLASTN 807 1e-61 83 82 282
700335703H1 SATMON019 g217973 BLASTN 376 1e-60 90 83 282
700351044H1 SATMON023 g169820 BLASTN 471 1e-59 83 84 282
700346364H1 SATMON021 g169820 BLASTN 813 1e-59 85 85 282
700619037H1 SATMON034 g169820 BLASTN 814 1e-59 84 86 282
700465160H1 SATMON025 g169820 BLASTN 751 1e-57 84 87 282
700235687H1 SATMON010 g169820 BLASTN 791 1e-57 82 88 282
700105645H1 SATMON010 g169820 BLASTN 793 1e-57 83 89 282
700082237H1 SATMON011 g169820 BLASTN 793 1e-57 84 90 282
700261906H1 SATMON017 g169820 BLASTN 796 1e-57 83 91 282
700456154H1 SATMON029 g169820 BLASTN 799 1e-57 84 92 282
700047696H1 SATMON003 g169820 BLASTN 561 1e-56 83 93 282
700449905H1 SATMON028 g169820 BLASTN 788 1e-56 84 94 282
700336106H1 SATMON019 g217973 BLASTN 325 1e-55 92 95 282
700381867H1 SATMON023 g2529386 BLASTN 422 1e-55 97 96 282
700051335H1 SATMON003 g169820 BLASTN 608 1e-55 83 97 282
700050988H1 SATMON003 g169820 BLASTN 768 1e-55 86 98 282
700029471H1 SATMON003 g169820 BLASTN 772 1e-55 84 99 282
700106806H1 SATMON010 g169820 BLASTN 773 1e-55 84 100 282
700071749H1 SATMON007 g217973 BLASTN 362 1e-54 85 101 282
700207607H1 SATMON016 g217973 BLASTN 362 1e-54 85 102 282
700573465H2 SATMON030 g169820 BLASTN 753 1e-54 86 103 282
700220908H1 SATMON011 g169820 BLASTN 758 1e-54 84 104 282
700467719H1 SATMON025 g169820 BLASTN 761 1e-54 85 105 282
700456018H1 SATMON029 g169820 BLASTN 764 1e-54 81 106 282
700453767H1 SATMON029 g217973 BLASTN 296 1e-52 94 107 282
700026118H1 SATMON003 g217973 BLASTN 341 1e-52 93 108 282
700026760H1 SATMON003 g217973 BLASTN 421 1e-52 99 109 282
700029525H1 SATMON003 g169820 BLASTN 738 1e-52 85 110 282
700457972H1 SATMON029 g169820 BLASTN 723 1e-51 85 111 282
700455866H1 SATMON029 g169820 BLASTN 726 1e-51 84 112 282
700165290H1 SATMON013 g169820 BLASTN 726 1e-51 84 113 282
700351190H1 SATMON023 g169820 BLASTN 672 1e-50 81 114 282
700154095H1 SATMON007 g169820 BLASTN 696 1e-49 84 115 282
700450438H1 SATMON028 g217973 BLASTN 430 1e-48 99 116 282
700044892H1 SATMON004 g169820 BLASTN 683 1e-48 85 117 282
700185095H1 SATMON014 g169820 BLASTN 673 1e-47 84 118 282
700575506H1 SATMON030 g169820 BLASTN 680 1e-47 83 119 282
700161966H1 SATMON012 g217973 BLASTN 335 1e-46 98 120 282
700343401H1 SATMON021 g169820 BLASTN 426 1e-45 77 121 282
700152354H1 SATMON007 g169820 BLASTN 653 1e-45 84 122 282
701164924H1 SATMONN04 g169820 BLASTN 397 1e-44 84 123 282
700346896H1 SATMON021 g169820 BLASTN 496 1e-42 84 124 282
700210157H1 SATMON016 g169820 BLASTN 617 1e-42 84 125 282
700383103H1 SATMON024 g169820 BLASTN 531 1e-41 84 126 282
701158829H1 SATMONN04 g407524 BLASTN 549 1e-40 80 127 282
700619883H1 SATMON034 g217973 BLASTN 325 1e-38 99 128 282
700168219H1 SATMON013 g169820 BLASTN 540 1e-36 83 129 282
700155210H1 SATMON007 g169820 BLASTN 545 1e-36 83 130 282
700334861H1 SATMON019 g169820 BLASTN 484 1e-31 82 131 282
700355663H1 SATMON024 g217973 BLASTN 213 1e-30 88 132 282
700074764H1 SATMON007 g546734 BLASTN 387 1e-27 84 133 282
700621934H1 SATMON034 g217973 BLASTN 430 1e-26 100 134 282
700802084H1 SATMON036 g217973 BLASTN 270 1e-24 98 135 3039
700620444H1 SATMON034 g1785947 BLASTN 473 1e-56 75 136 3039
700356205H1 SATMON024 g1785947 BLASTN 332 1e-32 72 137 3039
700215549H1 SATMON016 g414549 BLASTN 443 1e-26 72 138 3039
700620318H1 SATMON034 g556171 BLASTX 214 1e-25 79 139 3039
700028742H1 SATMON003 g556171 BLASTX 156 1e-20 86 140 3039
700150060H1 SATMON007 g556171 BLASTX 181 1e-17 89 141 3039
700448477H1 SATMON027 g556171 BLASTX 137 1e-12 85 142 3039
700336489H1 SATMON019 g556171 BLASTX 126 1e-10 81 143 3414
700099709H1 SATMON009 g609261 BLASTN 600 1e-49 84 144 3414
700075837H1 SATMON007 g609261 BLASTN 494 1e-41 84 145 3414
700045678H1 SATMON004 g609261 BLASTN 340 1e-29 73 146 3414
700097852H1 SATMON009 g609261 BLASTN 436 1e-27 84 147 3414
700053342H1 SATMON009 g609261 BLASTN 346 1e-25 73 148 3414
700041954H1 SATMON004 g609261 BLASTN 340 1e-24 82 149 3414
700217471H1 SATMON016 g609261 BLASTN 265 1e-21 71 150 3414
700264437H1 SATMON017 g609261 BLASTN 231 1e-17 69 151 3414
700218371H1 SATMON016 g609261 BLASTN 156 1e-10 68 152 5593
700381686H1 SATMON023 g609261 BLASTN 534 1e-44 89 153 5593
700356082H1 SATMON024 g609261 BLASTN 246 1e-24 90 154 5593
700622077H1 SATMON034 g609261 BLASTN 292 1e-20 86 155 5593
700470822H1 SATMON025 g609262 BLASTX 134 1e-11 79 156 6525
700083139H1 SATMON011 g256119 BLASTN 880 1e-64 76 157 6525
700205474H1 SATMON003 g169820 BLASTN 849 1e-62 77 158 6991
700336856H1 SATMON019 g609261 BLASTN 1131 1e-85 85 159 6991
700042717H1 SATMON004 g609261 BLASTN 1028 1e-76 85 160 6991
700379491H1 SATMON020 g609261 BLASTN 995 1e-74 81 161 6991
700156635H1 SATMON012 g609261 BLASTN 877 1e-64 84 162 6991
700046340H1 SATMON004 g609261 BLASTN 852 1e-62 84 163 6991
700081869H1 SATMON011 g609261 BLASTN 266 1e-14 80 164 6991
700426102H1 SATMONN01 g806312 BLASTX 134 1e-13 89 165 7384
700613626H1 SATMON033 g609261 BLASTN 920 1e-87 85 166 7384
700101506H1 SATMON009 g609261 BLASTN 1124 1e-84 85 167 7384
700206445H1 SATMON003 g609261 BLASTN 987 1e-73 79 168 7384
700220160H1 SATMON011 g609261 BLASTN 878 1e-64 85 169 -L1431527
LIB143-004- LIB143 g217973 BLASTN 290 1e-13 93 Q1-E1-C5 170
-L30613868 LIB3061-017- LIB3061 g217973 BLASTN 182 1e-13 70
Q1-K1-C9 171 -L30623620 LIB3062-034- LIB3062 g609261 BLASTN 599
1e-39 74 Q1-K1-A8 172 -L361705 LIB36-021- LIB36 g609261 BLASTN 266
1e-14 80 Q1-E1-E7 173 23992 LIB3062-056- LIB3062 g1200507 BLASTX
285 1e-64 61 Q1-K1-F9 174 282 LIB3067-047- LIB3067 g217973 BLASTN
1076 1e-164 96 Q1-K1-H2 175 282 LIB3067-055- LIB3067 g217973 BLASTN
1076 1e-133 93 Q1-K1-G8 176 282 LIB3067-059- LIB3067 g169820 BLASTN
1401 1e-115 84 Q1-K1-D10 177 282 LIB3067-027- LIB3067 g407524
BLASTN 995 1e-113 83 Q1-K1-B10 178 282 LIB189-032- LIB189 g217973
BLASTN 629 1e-111 93 Q1-E1-H2 179 282 LIB3059-023- LIB3059 g407524
BLASTN 1436 1e-111 83 Q1-K1-A7 180 282 LIB3069-016- LIB3069 g169820
BLASTN 1301 1e-107 81 Q1-K1-D9 181 282 LIB143-006- LIB143 g169820
BLASTN 1373 1e-105 84 Q1-E1-A8 182 282 LIB3068-054- LIB3068 g169820
BLASTN 1327 1e-102 82 Q1-K1-C11 183 282 LIB3067-034- LIB3067
g407524 BLASTN 1321 1e-101 83 Q1-K1-B7 184 282 LIB143-031- LIB143
g169820 BLASTN 1311 1e-100 84 Q1-E1-E5 185 282 LIB3069-055- LIB3069
g169820 BLASTN 1046 1e-97 75 Q1-K1-H12 186 282 LIB3061-027- LIB3061
g169820 BLASTN 936 1e-96 83 Q1-K1-A8 187 282 LIB3078-008- LIB3078
g169820 BLASTN 1210 1e-92 82 Q1-K1-E5 188 282 LIB3066-027- LIB3066
g407524 BLASTN 1196 1e-91 82 Q1-K1-E1 189 282 LIB3067-032- LIB3067
g169820 BLASTN 1122 1e-84 84 Q1-K1-E5 190 282 LIB3078-029- LIB3078
g169820 BLASTN 827 1e-83 82 Q1-K1-F7 191 282 LIB3061-006- LIB3061
g169820 BLASTN 1091 1e-82 78 Q1-K1-B7 192 282 LIB143-048- LIB143
g169820 BLASTN 644 1e-74 75 Q1-E1-F8 193 282 LIB3078-033- LIB3078
g169820 BLASTN 584 1e-73 79 Q1-K1-B10 194 282 LIB3069-046- LIB3069
g169820 BLASTN 819 1e-59 79 Q1-K1-C4 195 282 LIB3061-049- LIB3061
g169820 BLASTN 587 1e-47 80 Q1-K1-H2 196 282 LIB143-029- LIB143
g169820 BLASTN 679 1e-47 84 Q1-E1-G4 197 282 LIB84-027- LIB84
g169820 BLASTN 613 1e-46 78 Q1-E1-E5 198 282 LIB3062-001- LIB3062
g169820 BLASTN 507 1e-33 80 Q1-K2-F7 199 282 LIB3066-014- LIB3066
g169820 BLASTN 385 1e-25 76 Q1-K1-H11 200 29645 LIB3069-014-
LIB3069 g168647 BLASTX 131 1e-27 34 Q1-K1-C11 201 29645
LIB3069-013- LIB3069 g168647 BLASTX 124 1e-24 33 Q1-K1-C11 202 3039
LIB3062-045- LIB3062 g1785947 BLASTN 1119 1e-84 72 Q1-K1-F6 203
5593 LIB3067-045- LIB3067 g609261 BLASTN 702 1e-58 75 Q1-K1-E5 204
6991 LIB3059-026- LIB3059 g609261 BLASTN 1493 1e-115 84 Q1-K1-G9
205 6991 LIB3078-049- LIB3078 g609261 BLASTN 747 1e-55 83 Q1-K1-E4
206 7384 LIB3062-034- LIB3062 g609261 BLASTN 1351 1e-107 85
Q1-K1-A4
MAIZE FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE 207 -700026544 700026544H1
SATMON003 g22144 BLASTN 215 1e-30 88 208 -700073329 700073329H1
SATMON007 g22144 BLASTN 590 1e-89 95 209 -700151987 700151987H1
SATMON007 g22144 BLASTN 212 1e-8 78 210 -700206575 700206575H1
SATMON003 g22144 BLASTN 1009 1e-109 96 211 -700333727 700333727H1
SATMON019 g1217893 BLASTX 154 1e-16 61 212 -700429795 700429795H1
SATMONN01 g1619605 BLASTX 102 1e-16 77 213 -700804137 700804137H1
SATMON036 g22144 BLASTN 742 1e-52 92 214 1182 700449930H1 SATMON028
g22632 BLASTN 856 1e-62 79 215 1182 701185559H1 SATMONN06 g22632
BLASTN 793 1e-57 79 216 1182 700203130H1 SATMON003 g22632 BLASTN
799 1e-57 78 217 1182 700083459H1 SATMON011 g22632 BLASTN 800 1e-57
76 218 1182 700465449H1 SATMON025 g22632 BLASTN 405 1e-50 76 219
1182 701165344H1 SATMONN04 g22632 BLASTN 326 1e-29 78 220 1182
700427538H1 SATMONN01 g438275 BLASTX 96 1e-9 88 221 38 700224356H1
SATMON011 g22144 BLASTN 1290 1e-98 96 222 38 700048169H1 SATMON003
g22144 BLASTN 528 1e-72 98 223 38 700616610H1 SATMON033 g22144
BLASTN 278 1e-31 91 224 38 700355765H1 SATMON024 g20204 BLASTX 141
1e-12 96 225 6547 700194431H1 SATMON014 g2636513 BLASTX 181 1e-17
47 226 6547 700469777H1 SATMON025 g2636513 BLASTX 174 1e-16 48 227
8494 700425929H1 SATMONN01 g927507 BLASTX 67 1e-11 89 228
-L30603643 LIB3060-046- LIB3060 g169037 BLASTX 155 1e-44 66
Q1-K1-G7 229 1182 LIB3079-006- LIB3079 g22632 BLASTN 598 1e-39 65
Q1-K1-H8 230 28633 LIB3062-015- LIB3062 g1208898 BLASTX 116 1e-24
45 Q1-K1-G12 231 38 LIB3061-025- LIB3061 g22144 BLASTN 895 1e-133
94 Q1-K1-C9 232 38 LIB3059-020- LIB3059 g22144 BLASTN 745 1e-53 98
Q1-K1-H3 MAIZE FRUCTOSE-1,6-BISPHOSPHATASE 233 -700262935
700262935H1 SATMON017 g3041775 BLASTX 184 1e-18 94 234 -700432173
700432173H1 SATMONN01 g1790679 BLASTX 123 1e-16 56 235 -700455709
700455709H1 SATMON029 g3041776 BLASTN 597 1e-40 85 236 -700573083
700573083H1 SATMON030 g3041775 BLASTX 69 1e-10 64 237 12846
700101851H1 SATMON009 g3041776 BLASTN 1312 1e-100 91 238 12846
700101541H1 SATMON009 g3041776 BLASTN 1252 1e-95 90 239 12846
700581510H1 SATMON031 g3041776 BLASTN 872 1e-82 90 240 15627
700046054H1 SATMON004 g21736 BLASTN 1213 1e-92 91 241 15627
700421605H1 SATMONN01 g3041776 BLASTN 664 1e-77 90 242 15627
700445495H1 SATMON027 g21736 BLASTN 1004 1e-74 84 243 15627
700042188H1 SATMON004 g3041776 BLASTN 875 1e-64 88 244 16870
700100752H1 SATMON009 g3041776 BLASTN 257 1e-33 75 245 16870
700044805H1 SATMON004 g3041776 BLASTN 194 1e-14 76 246 16870
700099217H1 SATMON009 g21736 BLASTN 246 1e-9 59 247 5480
700442189H1 SATMON026 g3041774 BLASTN 536 1e-54 93 248 8243
700264654H1 SATMON017 g3041774 BLASTN 942 1e-69 84 249 8243
700479624H1 SATMON034 g3041774 BLASTN 902 1e-66 82 250 8243
700448974H1 SATMON028 g3041774 BLASTN 876 1e-64 84 251 -L1485381
LIB148-057- LIB148 g440591 BLASTX 80 1e-30 63 Q1-E1-E6 252
-L30662839 LIB3066-035- LIB3066 g3041774 BLASTN 215 1e-15 77
Q1-K1-F11 253 -L362913 LIB36-013- LIB36 g3041776 BLASTN 937 1e-69
88 Q1-E1-D10 254 -L832444 LIB83-005- LIB83 g3041776 BLASTN 575
1e-37 93 Q1-E1-D2 255 12846 LIB83-008- LIB83 g3041776 BLASTN 1610
1e-135 92 Q1-E1-A8 256 12846 LIB3078-003- LIB3078 g3041776 BLASTN
873 1e-98 93 Q1-K1-C7 257 16870 LIB3060-052- LIB3060 g21736 BLASTN
377 1e-66 70 Q1-K1-D11 258 26002 LIB83-008- LIB83 g3041776 BLASTN
378 1e-20 86 Q1-E1-B10 MAIZE FRUCTOSE-6-PHOSPHATE,2-KINASE 259
-700093724 700093724H1 SATMON008 g3170230 BLASTX 123 1e-21 53 260
-700099547 700099547H1 SATMON009 g3309582 BLASTN 630 1e-43 80 261
-700100682 700100682H1 SATMON009 g3170230 BLASTX 269 1e-39 65 262
-700173085 700173085H1 SATMON013 g2286154 BLASTN 1165 1e-88 100 263
-700217623 700217623H1 SATMON016 g3170229 BLASTN 593 1e-40 73 264
-700219340 700219340H1 SATMON011 g3170230 BLASTX 190 1e-20 56 265
-700265353 700265353H1 SATMON017 g2286154 BLASTN 1268 1e-107 98 266
-700379777 700379777H1 SATMON021 g3309582 BLASTN 905 1e-66 76 267
-700620963 700620963H1 SATMON034 g2286154 BLASTN 376 1e-52 85 268
-701159590 701159590H1 SATMONN04 g3309582 BLASTN 682 1e-48 73 269
20094 700209789H1 SATMON016 g2286154 BLASTN 1093 1e-96 92 270 20094
700550375H1 SATMON022 g3309582 BLASTN 780 1e-58 81 271 29193
700021150H1 SATMON001 g2286154 BLASTN 466 1e-75 92 272 -L30593297
LIB3059-029- LIB3059 g2286154 BLASTN 401 1e-22 70 Q1-K1-B3 273
-L30614892 LIB3061-021- LIB3061 g2286154 BLASTN 469 1e-38 79
Q1-K1-G9 274 -L30623700 LIB3062-031- LIB3062 g3170229 BLASTN 230
1e-10 70 Q1-K1-E8 275 29193 LIB83-007- LIB83 g2286154 BLASTN 595
1e-113 90 Q1-E1-C11 MAIZE PHOSPHOGLUCOISOMERASE 276 -700086021
700086021H1 SATMON011 g1100771 BLASTX 225 1e-28 51 277 -700169489
700169489H1 SATMON013 g1100771 BLASTX 152 1e-13 59 278 -700222638
700222638H1 SATMON011 g1100771 BLASTX 256 1e-28 60 279 -700445574
700445574H1 SATMON027 g1100771 BLASTX 143 1e-12 54 280 -700475232
700475232H1 SATMON025 g596022 BLASTN 845 1e-61 90 281 -700612774
700612774H1 SATMON033 g596022 BLASTN 1574 1e-122 95 282 14393
700222547H1 SATMON011 g1100771 BLASTX 239 1e-25 60 283 14393
700220357H1 SATMON011 g1100771 BLASTX 218 1e-23 68 284 14393
700050317H1 SATMON003 g1100771 BLASTX 120 1e-22 63 285 14393
700163544H1 SATMON013 g1100771 BLASTX 214 1e-22 62 286 15724
700207164H1 SATMON017 g1100771 BLASTX 135 1e-17 67 287 15724
700552402H1 SATMON022 g1100771 BLASTX 135 1e-11 60 288 15724
700086085H1 SATMON011 g1100771 BLASTX 137 1e-11 45 289 20643
700577051H1 SATMON031 g1100771 BLASTX 241 1e-26 66 290 20643
700201592H1 SATMON003 g1100771 BLASTX 113 1e-19 45 291 20643
700576644H1 SATMON030 g1100771 BLASTX 113 1e-17 43 292 2351
700208928H1 SATMON016 g1100771 BLASTX 274 1e-43 73 293 2351
700240758H1 SATMON010 g1100771 BLASTX 283 1e-43 79 294 2351
700352502H1 SATMON023 g1100771 BLASTX 197 1e-36 70 295 2351
700581930H1 SATMON031 g1100771 BLASTX 164 1e-34 72 296 2351
700028642H1 SATMON003 g1100771 BLASTX 294 1e-33 65 297 2351
700106092H1 SATMON010 g1100771 BLASTX 294 1e-33 62 298 2351
700082102H1 SATMON011 g1100771 BLASTX 300 1e-33 62 299 2351
700083446H1 SATMON011 g1100771 BLASTX 274 1e-30 65 300 2351
700580585H1 SATMON031 g1100771 BLASTX 163 1e-29 69 301 2351
700550608H1 SATMON022 g1100771 BLASTX 265 1e-29 61 302 2351
700106079H1 SATMON010 g1100771 BLASTX 261 1e-28 54 303 2351
700244248H1 SATMON010 g1100771 BLASTX 238 1e-25 67 304 2351
700152233H1 SATMON007 g1100771 BLASTX 167 1e-22 72 305 2351
700455043H1 SATMON029 g1100771 BLASTX 168 1e-21 68 306 2351
700615809H1 SATMON033 g1100771 BLASTX 207 1e-21 66 307 2351
701165320H1 SATMONN04 g1100771 BLASTX 122 1e-14 63 308 32930
700042996H1 SATMON004 g596022 BLASTN 476 1e-95 98 309 4222
700222539H1 SATMON011 g596022 BLASTN 1160 1e-87 100 310 4222
700104023H1 SATMON010 g596022 BLASTN 1060 1e-84 100 311 4222
700101580H1 SATMON009 g596022 BLASTN 871 1e-74 99 312 4222
700473395H1 SATMON025 g596022 BLASTN 368 1e-46 95 313 4222
700800179H1 SATMON036 g596022 BLASTN 240 1e-11 100 314 8858
700221523H1 SATMON011 g1100771 BLASTX 278 1e-31 59 315 895
700100965H1 SATMON009 g596022 BLASTN 1611 1e-125 99 316 895
700620985H1 SATMON034 g596022 BLASTN 1418 1e-114 98 317 895
700082062H1 SATMON011 g596022 BLASTN 1365 1e-110 97 318 895
700573782H1 SATMON030 g596022 BLASTN 920 1e-107 98 319 895
700236138H1 SATMON010 g596022 BLASTN 1395 1e-107 100 320 895
700086336H1 SATMON011 g596022 BLASTN 1370 1e-105 100 321 895
700801467H1 SATMON036 g596022 BLASTN 1249 1e-99 95 322 895
700801458H1 SATMON036 g596022 BLASTN 1245 1e-98 100 323 895
700475024H1 SATMON025 g596022 BLASTN 1162 1e-97 93 324 895
700243164H1 SATMON010 g596022 BLASTN 1105 1e-96 100 325 895
700804665H1 SATMON036 g596022 BLASTN 1266 1e-96 99 326 895
700021931H1 SATMON001 g596022 BLASTN 1126 1e-84 99 327 895
700805540H1 SATMON036 g596022 BLASTN 776 1e-55 99 328 895
700172576H1 SATMON013 g596022 BLASTN 571 1e-38 98 329 895
700105116H1 SATMON010 g596022 BLASTN 558 1e-37 99 330 895
700472931H1 SATMON025 g596022 BLASTN 379 1e-31 97 331 20643
LIB3069-009- LIB3069 g1100771 BLASTX 215 1e-44 50 Q1-K1-B3 332 2351
LIB3079-007- LIB3079 g1100771 BLASTX 304 1e-77 72 Q1-K1-C11 333
32930 LIB189-001- LIB189 g596022 BLASTN 794 1e-115 95 Q1-E1-E4 334
4222 LIB3079-001- LIB3079 g596022 BLASTN 1132 1e-101 89 Q1-K1-H7
335 895 LIB148-049- LIB148 g596022 BLASTN 2194 1e-178 97 Q1-E1-D6
336 895 LIB3066-052- LIB3066 g596022 BLASTN 2178 1e-172 97 Q1-K1-G8
337 895 LIB148-016- LIB148 g596022 BLASTN 1567 1e-161 99 Q1-E1-G5
338 895 LIB143-032- LIB143 g596022 BLASTN 1914 1e-155 99 Q1-E1-E10
339 895 LIB3061-013- LIB3061 g596022 BLASTN 1738 1e-136 88 Q1-K1-F7
340 895 LIB143-047- LIB143 g596022 BLASTN 1490 1e-119 88 Q1-E1-D4
MAIZE VACUOLAR H+-TRANSLOCATING-PYROPHOSPHATASE 341 -700163331
700163331H1 SATMON013 2g534915 BLASTN 751 1e-53 77 342 -700171438
700171438H1 SATMON013 g2258073 BLASTN 256 1e-10 76 343 -700202576
700202576H1 SATMON003 g2668746 BLASTX 214 1e-23 84 344 -700206487
700206487H1 SATMON003 g2570501 BLASTX 174 1e-17 86 345 -700217292
700217292H1 SATMON016 g2668746 BLASTX 214 1e-23 100 346 -700240889
700240889H1 SATMON010 g2570500 BLASTN 639 1e-47 84 347 -700347658
700347658H1 SATMON023 g2668746 BLASTX 215 1e-23 95 348 -700454151
700454151H1 SATMON029 g2668745 BLASTN 172 1e-10 90 349 -700454532
700454532H1 SATMON029 g2668745 BLASTN 259 1e-38 93 350 -700552133
700552133H1 SATMON022 g457744 BLASTX 176 1e-19 68 351 -700611864
700611864H1 SATMON022 g2668745 BLASTN 203 1e-9 84 352 107
700622451H1 SATMON034 g2668745 BLASTN 1645 1e-129 100 353 107
700571235H1 SATMON030 g2668745 BLASTN 1406 1e-125 98 354 107
700266126H1 SATMON017 g2668745 BLASTN 1145 1e-121 100 355 107
700621607H1 SATMON034 g2668745 BLASTN 1375 1e-121 99 356 107
700345080H1 SATMON021 g2668745 BLASTN 1195 1e-117 100 357 107
700624257H1 SATMON034 g2668745 BLASTN 825 1e-115 100 358 107
700030359H1 SATMON003 g2668745 BLASTN 1470 1e-114 100 359 107
700214462H1 SATMON016 g2668745 BLASTN 1223 1e-110 98 360 107
700356050H1 SATMON024 g2668745 BLASTN 1430 1e-110 100 361 107
701181128H1 SATMONN06 g2668745 BLASTN 1368 1e-105 98 362 107
700349795H1 SATMON023 g2668745 BLASTN 1370 1e-105 95 363 107
700473278H1 SATMON025 g2668745 BLASTN 1355 1e-104 100 364 107
700157057H1 SATMON012 g2668745 BLASTN 1345 1e-103 100 365 107
700622505H1 SATMON034 g2668745 BLASTN 762 1e-100 96 366 107
700219661H1 SATMON011 g2668745 BLASTN 942 1e-98 99 367 107
700619032H1 SATMON034 g2668745 BLASTN 989 1e-98 96 368 107
700620065H1 SATMON034 g2668745 BLASTN 1069 1e-98 94 369 107
700569179H1 SATMON030 g2668745 BLASTN 1233 1e-97 98 370 107
700156773H1 SATMON012 g2668745 BLASTN 1276 1e-97 99 371 107
700207120H1 SATMON017 g2668745 BLASTN 740 1e-96 99 372 107
700030407H1 SATMON003 g2668745 BLASTN 480 1e-95 98 373 107
700457309H1 SATMON029 g2668745 BLASTN 979 1e-95 99 374 107
700195681H1 SATMON014 g2668745 BLASTN 1246 1e-95 99 375 107
700444838H1 SATMON027 g2668745 BLASTN 1249 1e-95 96 376 107
700581619H1 SATMON031 g2668745 BLASTN 943 1e-94 96 377 107
700351021H1 SATMON023 g2668745 BLASTN 853 1e-91 92 378 107
700205723H1 SATMON003 g2668745 BLASTN 1138 1e-91 95 379 107
700159712H1 SATMON012 g2668745 BLASTN 1199 1e-91 94 380 107
700158937H1 SATMON012 g2668745 BLASTN 1132 1e-90 96 381 107
700336255H1 SATMON019 g2668745 BLASTN 489 1e-85 94 382 107
700422922H1 SATMONN01 g2668745 BLASTN 642 1e-84 95 383 107
700347429H1 SATMON023 g2668745 BLASTN 891 1e-83 92 384 107
700350695H1 SATMON023 g2668745 BLASTN 960 1e-83 91 385 107
700212988H1 SATMON016 g2668745 BLASTN 988 1e-82 96 386 107
700345278H1 SATMON021 g2668745 BLASTN 989 1e-82 95 387 107
700264475H1 SATMON017 g2668745 BLASTN 1089 1e-82 99 388 107
700211923H1 SATMON016 g2668745 BLASTN 991 1e-81 94 389 107
700620974H1 SATMON034 g2668745 BLASTN 907 1e-80 92 390 107
700156401H1 SATMON012 g2668745 BLASTN 1058 1e-79 90 391 107
700172547H1 SATMON013 g2668745 BLASTN 1042 1e-78 96 392 107
700552384H1 SATMON022 g2668745 BLASTN 916 1e-76 96 393 107
700219926H1 SATMON011 g2668745 BLASTN 1005 1e-75 100 394 107
700357492H1 SATMON024 g2668745 BLASTN 610 1e-74 99 395 107
700343365H1 SATMON021 g2668745 BLASTN 891 1e-74 94 396 107
700018618H1 SATMON001 g2668745 BLASTN 1001 1e-74 93 397 107
700570755H1 SATMON030 g2668745 BLASTN 845 1e-71 93 398 107
700194777H1 SATMON014 g2668745 BLASTN 940 1e-69 100 399 107
700453790H1 SATMON029 g2668745 BLASTN 925 1e-68 92 400 107
700197306H1 SATMON014 g2668745 BLASTN 928 1e-68 85 401 107
700355750H1 SATMON024 g2668745 BLASTN 393 1e-66 93 402 107
700172940H1 SATMON013 g2668745 BLASTN 902 1e-66 97 403 107
700102133H1 SATMON010 g2668745 BLASTN 850 1e-62 100 404 107
700350332H1 SATMON023 g2668745 BLASTN 539 1e-57 97 405 107
700450285H1 SATMON028 g2668745 BLASTN 750 1e-53 100 406 107
700165003H1 SATMON013 g2668745 BLASTN 548 1e-52 83 407 107
700016136H1 SATMON001 g2668745 BLASTN 527 1e-50 85 408 107
700171557H1 SATMON013 g2668745 BLASTN 714 1e-50 95 409 107
700238156H1 SATMON010 g2668745 BLASTN 715 1e-50 96 410 107
700425175H1 SATMONN01 g2668745 BLASTN 698 1e-49 94 411 107
700354402H1 SATMON024 g2668745 BLASTN 616 1e-48 91 412 107
700159204H1 SATMON012 g2668745 BLASTN 617 1e-42 94 413 107
700623602H1 SATMON034 g2668745 BLASTN 460 1e-38 100 414 107
700612844H1 SATMON033 g2668745 BLASTN 421 1e-36 84 415 107
700621062H2 SATMON034 g2668745 BLASTN 285 1e-25 89 416 107
700335685H1 SATMON019 g2668745 BLASTN 339 1e-25 91 417 13843
700334949H1 SATMON019 g2570500 BLASTN 680 1e-55 83 418 13843
700346817H1 SATMON021 g2570500 BLASTN 705 1e-54 83 419 13843
700103380H1 SATMON010 g2570500 BLASTN 710 1e-54 83 420 13843
700348280H1 SATMON023 g2570500 BLASTN 669 1e-51 83 421 13843
700453203H1 SATMON028 g2570500 BLASTN 659 1e-50 82 422 13843
700381101H1 SATMON023 g2570500 BLASTN 621 1e-47 82 423 13843
700347617H1 SATMON023 g2570500 BLASTN 592 1e-44 85 424 13843
700043259H1 SATMON004 g2570500 BLASTN 530 1e-39 84 425 13843
701184447H1 SATMONN06 g2570500 BLASTN 481 1e-35 78
426 21076 700241354H1 SATMON010 g166634 BLASTX 201 1e-20 58 427
24066 700423113H1 SATMONN01 g457744 BLASTX 124 1e-23 54 428 24266
700577157H1 SATMON031 g2570500 BLASTN 1001 1e-74 89 429 2531
700099364H1 SATMON009 g2570500 BLASTN 669 1e-51 86 430 2531
700336387H1 SATMON019 g2570500 BLASTN 389 1e-47 85 431 2531
700217095H1 SATMON016 g2570500 BLASTN 451 1e-33 86 432 2531
700155869H1 SATMON007 g2570500 BLASTN 385 1e-27 89 433 2531
700575534H1 SATMON030 g2570500 BLASTN 365 1e-26 88 434 2531
700163562H1 SATMON013 g2570501 BLASTX 145 1e-24 94 435 32364
700204306H1 SATMON003 g2668745 BLASTN 471 1e-28 74 436 32856
700166756H1 SATMON013 g534915 BLASTN 744 1e-53 76 437 32856
700042535H1 SATMON004 g534915 BLASTN 644 1e-44 73 438 3384
700237775H1 SATMON010 g2258073 BLASTN 911 1e-67 81 439 3384
700342456H1 SATMON021 g2258073 BLASTN 648 1e-64 78 440 3384
700073654H1 SATMON007 g2668745 BLASTN 860 1e-63 78 441 3384
700577805H1 SATMON031 g2258073 BLASTN 840 1e-61 78 442 3384
700028881H1 SATMON003 g534915 BLASTN 835 1e-60 78 443 3384
700215076H1 SATMON016 g534915 BLASTN 824 1e-59 78 444 3384
700017479H1 SATMON001 g534915 BLASTN 766 1e-55 80 445 3384
700204495H1 SATMON003 g534915 BLASTN 373 1e-51 81 446 3384
700206347H1 SATMON003 g2706449 BLASTN 685 1e-48 80 447 3384
700351040H1 SATMON023 g2706449 BLASTN 436 1e-45 78 448 3384
700345264H1 SATMON021 g2706449 BLASTN 616 1e-42 82 449 3384
700196795H1 SATMON014 g2570500 BLASTN 579 1e-39 80 450 3384
700019241H1 SATMON001 g2706449 BLASTN 583 1e-39 78 451 3384
700018612H1 SATMON001 g2668745 BLASTN 518 1e-34 76 452 3384
700102142H1 SATMON010 g2668745 BLASTN 539 1e-34 78 453 3384
700348430H1 SATMON023 g534915 BLASTN 489 1e-30 78 454 3384
700337745H1 SATMON020 g2706449 BLASTN 471 1e-28 79 455 3384
700439515H1 SATMON026 g534915 BLASTN 437 1e-27 75 456 3384
700074977H1 SATMON007 g534915 BLASTN 434 1e-25 76 457 3384
700615213H1 SATMON033 g2570501 BLASTX 125 1e-21 93 458 3384
700074109H1 SATMON007 g2668746 BLASTX 197 1e-20 72 459 3384
700549517H1 SATMON022 g2668746 BLASTX 172 1e-17 75 460 3384
700030347H1 SATMON003 g2668746 BLASTX 171 1e-16 77 461 3384
700221176H1 SATMON011 g2668746 BLASTX 171 1e-16 77 462 3384
700433360H1 SATMONN01 g2668746 BLASTX 95 1e-13 74 463 5000
700026151H1 SATMON003 g2903 BLASTX 261 1e-28 54 464 5000
700347165H1 SATMON021 g2624379 BLASTX 223 1e-24 51 465 5000
700430341H1 SATMONN01 g2903 BLASTX 185 1e-18 56 466 5000
700457781H1 SATMON029 g2903 BLASTX 133 1e-16 49 467 5861
700104993H1 SATMON010 g2258073 BLASTN 456 1e-27 73 468 5861
700203452H1 SATMON003 g2258073 BLASTN 428 1e-26 72 469 -L1431590
LIB143-006- LIB143 g16347 BLASTN 286 1e-13 61 Q1-E1-C9 470
-L1433414 LIB143-026- LIB143 g2258073 BLASTN 480 1e-29 70 Q1-E1-C3
471 -L1482832 LIB148-009- LIB148 g2258073 BLASTN 1086 1e-81 78
Q1-E1-D8 472 -L30674379 LIB3067-042- LIB3067 g2668745 BLASTN 305
1e-21 68 Q1-K1-H8 473 -L30675678 LIB3067-034- LIB3067 g2706449
BLASTN 286 1e-12 73 Q1-K1-E3 474 107 LIB3059-036- LIB3059 g2668745
BLASTN 1965 1e-166 100 Q1-K1-B10 475 107 LIB3061-035- LIB3061
g2668745 BLASTN 948 1e-138 93 Q1-K1-C9 476 107 LIB3061-032- LIB3061
g2668745 BLASTN 1685 1e-138 96 Q1-K1-A12 477 107 LIB3062-044-
LIB3062 g2668745 BLASTN 1492 1e-134 95 Q1-K1-F8 478 107
LIB3068-025- LIB3068 g2668745 BLASTN 1687 1e-132 96 Q1-K1-E5 479
107 LIB3067-022- LIB3067 g2668745 BLASTN 1581 1e-128 91 Q1-K1-D11
480 107 LIB3067-016- LIB3067 g2668745 BLASTN 1305 1e-126 97
Q1-K1-G4 481 107 LIB3067-029- LIB3067 g2668745 BLASTN 1560 1e-125
90 Q1-K1-C6 482 107 LIB189-031- LIB189 g2668745 BLASTN 897 1e-81 85
Q1-E1-D3 483 24066 LIB3069-047- LIB3069 g166634 BLASTX 173 1e-45 55
Q1-K1-C4 484 24266 LIB3069-006- LIB3069 g2570500 BLASTN 717 1e-57
83 Q1-K1-F4 485 293 LIB3068-043- LIB3068 g633598 BLASTN 552 1e-34
78 Q1-K1-A2 486 32364 LIB3066-001- LIB3066 g2668745 BLASTN 612
1e-40 73 Q1-K1-B7 487 32856 LIB189-028- LIB189 g534915 BLASTN 986
1e-73 73 Q1-E1-C4 488 3384 LIB143-026- LIB143 g534915 BLASTN 1284
1e-98 78 Q1-E1-C1 489 3384 LIB3068-013- LIB3068 g534915 BLASTN 1074
1e-80 78 Q1-K1-H2 490 3384 LIB3062-033- LIB3062 g2668745 BLASTN
1009 1e-75 76 Q1-K1-D2 491 3384 LIB83-002- LIB83 g2706449 BLASTN
820 1e-59 78 Q1-E1-D2 492 3384 LIB3062-057- LIB3062 g2668745 BLASTN
801 1e-58 73 Q1-K1-B7 493 3384 LIB3062-001- LIB3062 g16347 BLASTN
802 1e-57 77 Q1-K2-H5 494 3384 LIB189-022- LIB189 g2668745 BLASTN
646 1e-43 75 Q1-E1-D5 495 3384 LIB189-012- LIB189 g2570501 BLASTX
138 1e-32 72 Q1-E1-F4 496 5000 LIB36-015- LIB36 g2624379 BLASTX 236
1e-41 51 Q1-E1-D6 497 5000 LIB83-016- LIB83 g4198 BLASTN 534 1e-33
61 Q1-E1-H7 MAIZE PYROPHOSPHATE-DEPENDENT FRUCTOSE-6-PHOSPHATE
PHOSPHOTRANSFERASE 498 -700208959 700208959H1 SATMON016 g169538
BLASTX 107 1e-19 50 499 -700237606 700237606H1 SATMON010 g169538
BLASTX 114 1e-11 62 500 3456 700083478H1 SATMON011 g169538 BLASTX
121 1e-39 88 501 3652 700242182H1 SATMON010 g169538 BLASTX 155
1e-13 82 502 4965 700475352H1 SATMON025 g169538 BLASTX 123 1e-9 69
503 4965 700550752H1 SATMON022 g169538 BLASTX 123 1e-9 69 504 5359
700347441H1 SATMON023 g169538 BLASTX 139 1e-11 70 505 -L30594734
LIB3059-018- LIB3059 g169538 BLASTX 145 1e-49 83 Q1-K1-H3 506
-L30622375 LIB3062-009- LIB3062 g169538 BLASTX 157 1e-30 65
Q1-K1-B3 507 32156 LIB189-021- LIB189 g169538 BLASTX 123 1e-25 78
Q1-E1-G8 MAIZE INVERTASES 508 -700240132 700240132H1 SATMON010
g397631 BLASTX 134 1e-11 74 509 1923 700574932H1 SATMON030 g393390
BLASTX 152 1e-14 65 510 4355 700379641H1 SATMON021 g1177601 BLASTX
175 1e-19 85 MAIZE SUCROSE SYNTHASE 511 -700151470 700151470H1
SATMON007 g1196837 BLASTX 197 1e-27 64 512 -700214035 700214035H1
SATMON016 g22485 BLASTN 523 1e-34 79 513 -700262270 700262270H1
SATMON017 g2570066 BLASTN 866 1e-63 76 514 -700334686 700334686H1
SATMON019 g1100216 BLASTN 424 1e-31 88 515 -700381593 700381593H1
SATMON023 g22485 BLASTN 219 1e-13 97 516 -700404808 700404808H1
SATMON026 g2570066 BLASTN 859 1e-70 82 517 -700456905 700456905H1
SATMON029 g22485 BLASTN 528 1e-64 90 518 -700571529 700571529H1
SATMON030 g19106 BLASTX 139 1e-24 56 519 -700576567 700576567H1
SATMON030 g22485 BLASTN 285 1e-14 92 520 -700800659 700800659H1
SATMON036 g22485 BLASTN 558 1e-37 97 521 -700802941 700802941H1
SATMON036 g22485 BLASTN 316 1e-29 97 522 -701181030 701181030H1
SATMONN06 g2606080 BLASTN 669 1e-46 72 523 13723 700203023H1
SATMON003 g2570066 BLASTN 820 1e-68 84 524 13723 700215119H1
SATMON016 g2570066 BLASTN 680 1e-47 86 525 13723 700473266H1
SATMON025 g2570066 BLASTN 537 1e-35 85 526 15661 700440404H1
SATMON026 g2570066 BLASTN 364 1e-36 74 527 15661 700168252H1
SATMON013 g16525 BLASTN 433 1e-27 80 528 20925 700551647H1
SATMON022 g2570066 BLASTN 307 1e-35 73 529 20925 700257052H1
SATMON017 g2570067 BLASTX 118 1e-9 64 530 20934 700217752H1
SATMON016 g514945 BLASTN 1397 1e-107 98 531 20934 700332156H1
SATMON019 g514945 BLASTN 589 1e-97 95 532 30444 700257522H1
SATMON017 g1100216 BLASTN 760 1e-54 95 533 32909 700264718H1
SATMON017 g2570066 BLASTN 702 1e-57 76 534 405 700091402H1
SATMON011 g514945 BLASTN 1830 1e-143 100 535 405 700572549H1
SATMON030 g514945 BLASTN 1658 1e-129 99 536 405 700203058H1
SATMON003 g22485 BLASTN 1360 1e-127 100 537 405 700091753H1
SATMON011 g514945 BLASTN 1245 1e-126 99 538 405 700090929H1
SATMON011 g514945 BLASTN 1620 1e-126 100 539 405 700091711H1
SATMON011 g514945 BLASTN 1621 1e-126 99 540 405 700084254H1
SATMON011 g514945 BLASTN 1600 1e-124 100 541 405 700082305H1
SATMON011 g514945 BLASTN 1601 1e-124 99 542 405 700048236H1
SATMON003 g22485 BLASTN 1583 1e-123 99 543 405 700086713H1
SATMON011 g514945 BLASTN 1584 1e-123 99 544 405 700049353H1
SATMON003 g514945 BLASTN 1586 1e-123 99 545 405 700082766H1
SATMON011 g22485 BLASTN 1589 1e-123 98 546 405 700086055H1
SATMON011 g514945 BLASTN 1590 1e-123 100 547 405 700215105H1
SATMON016 g514945 BLASTN 1590 1e-123 100 548 405 700104149H1
SATMON010 g22485 BLASTN 1594 1e-123 98 549 405 700101601H1
SATMON009 g514945 BLASTN 1270 1e-122 100 550 405 700206869H1
SATMON003 g22485 BLASTN 1574 1e-122 97 551 405 700088163H1
SATMON011 g22485 BLASTN 1581 1e-122 99 552 405 700089166H1
SATMON011 g514945 BLASTN 1565 1e-121 100 553 405 700266251H1
SATMON017 g514945 BLASTN 1570 1e-121 100 554 405 700332710H1
SATMON019 g514945 BLASTN 1570 1e-121 100 555 405 700571106H1
SATMON030 g514945 BLASTN 1227 1e-120 98 556 405 700081893H1
SATMON011 g514945 BLASTN 1550 1e-120 98 557 405 700074739H1
SATMON007 g514945 BLASTN 1550 1e-120 100 558 405 700095163H1
SATMON008 g514945 BLASTN 1555 1e-120 100 559 405 700612766H1
SATMON033 g514945 BLASTN 883 1e-119 96 560 405 700267271H1
SATMON017 g514945 BLASTN 1535 1e-119 100 561 405 700083175H1
SATMON011 g514945 BLASTN 1535 1e-119 100 562 405 700088993H1
SATMON011 g22485 BLASTN 1545 1e-119 98 563 405 700094087H1
SATMON008 g22485 BLASTN 1526 1e-118 99 564 405 700086708H1
SATMON011 g514945 BLASTN 1529 1e-118 97 565 405 700090671H1
SATMON011 g514945 BLASTN 1530 1e-118 100 566 405 700209809H1
SATMON016 g22485 BLASTN 1532 1e-118 99 567 405 700084625H1
SATMON011 g514945 BLASTN 1533 1e-118 99 568 405 700089718H1
SATMON011 g514945 BLASTN 1120 1e-117 100 569 405 700213014H1
SATMON016 g514945 BLASTN 1405 1e-117 100 570 405 700086555H1
SATMON011 g514945 BLASTN 1514 1e-117 98 571 405 700475892H1
SATMON025 g514945 BLASTN 1516 1e-117 99 572 405 700047374H1
SATMON003 g22485 BLASTN 1516 1e-117 99 573 405 700090018H1
SATMON011 g514945 BLASTN 1519 1e-117 99 574 405 700076107H1
SATMON007 g514945 BLASTN 1520 1e-117 93 575 405 700213105H1
SATMON016 g514945 BLASTN 972 1e-116 99 576 405 700103806H1
SATMON010 g514945 BLASTN 1503 1e-116 99 577 405 700090748H1
SATMON011 g514945 BLASTN 1505 1e-116 100 578 405 700052006H1
SATMON003 g514945 BLASTN 1506 1e-116 99 579 405 700614963H1
SATMON033 g514945 BLASTN 957 1e-115 93 580 405 700337255H1
SATMON020 g22485 BLASTN 995 1e-115 97 581 405 700102778H1 SATMON010
g22485 BLASTN 1493 1e-115 99 582 405 700405466H1 SATMON029 g22485
BLASTN 1493 1e-115 99 583 405 700209634H1 SATMON016 g514945 BLASTN
1495 1e-115 100 584 405 700220467H1 SATMON011 g514945 BLASTN 1495
1e-115 100 585 405 700266637H1 SATMON017 g514945 BLASTN 1480 1e-114
100 586 405 700267579H1 SATMON017 g514945 BLASTN 1484 1e-114 99 587
405 700088475H1 SATMON011 g514945 BLASTN 1465 1e-113 100 588 405
700332618H1 SATMON019 g514945 BLASTN 1466 1e-113 99 589 405
700211347H1 SATMON016 g514945 BLASTN 1470 1e-113 100 590 405
700477206H1 SATMON025 g514945 BLASTN 1471 1e-113 99 591 405
700336768H1 SATMON019 g514945 BLASTN 1473 1e-113 99 592 405
700105305H1 SATMON010 g22485 BLASTN 1473 1e-113 99 593 405
700087114H1 SATMON011 g514945 BLASTN 1473 1e-113 99 594 405
700105366H1 SATMON010 g22485 BLASTN 1474 1e-113 98 595 405
700104831H1 SATMON010 g22485 BLASTN 825 1e-112 98 596 405
700620134H1 SATMON034 g22485 BLASTN 1179 1e-112 92 597 405
700211934H1 SATMON016 g22485 BLASTN 1215 1e-112 98 598 405
700096103H1 SATMON008 g514945 BLASTN 1391 1e-112 99 599 405
700264979H1 SATMON017 g514945 BLASTN 1454 1e-112 98 600 405
700053864H1 SATMON011 g514945 BLASTN 1455 1e-112 100 601 405
700211782H1 SATMON016 g514945 BLASTN 1460 1e-112 100 602 405
700102063H1 SATMON010 g22485 BLASTN 1461 1e-112 99 603 405
700207024H1 SATMON003 g514945 BLASTN 825 1e-111 100 604 405
700207970H1 SATMON016 g514945 BLASTN 1186 1e-111 98 605 405
700336624H1 SATMON019 g514945 BLASTN 1440 1e-111 100 606 405
700104357H1 SATMON010 g514945 BLASTN 1448 1e-111 98 607 405
700222053H1 SATMON011 g514945 BLASTN 1449 1e-111 99 608 405
700350806H1 SATMON023 g514945 BLASTN 660 1e-110 99 609 405
700091159H1 SATMON011 g514945 BLASTN 870 1e-110 100 610 405
700081810H1 SATMON011 g514945 BLASTN 926 1e-110 99 611 405
700102954H1 SATMON010 g514945 BLASTN 926 1e-110 97 612 405
700085307H1 SATMON011 g514945 BLASTN 1035 1e-110 100 613 405
700094295H1 SATMON008 g22485 BLASTN 1137 1e-110 96 614 405
700089176H1 SATMON011 g514945 BLASTN 1393 1e-110 97 615 405
700093643H1 SATMON008 g514945 BLASTN 1427 1e-110 95 616 405
700082421H1 SATMON011 g514945 BLASTN 1430 1e-110 98 617 405
700211788H1 SATMON016 g514945 BLASTN 1431 1e-110 99 618 405
700026724H1 SATMON003 g514945 BLASTN 1433 1e-110 97 619 405
700085275H1 SATMON011 g514945 BLASTN 1435 1e-110 100 620 405
700472161H1 SATMON025 g514945 BLASTN 755 1e-109 99 621 405
700084926H1 SATMON011 g514945 BLASTN 825 1e-109 100 622 405
700084592H1 SATMON011 g514945 BLASTN 920 1e-109 100 623 405
700053811H1 SATMON011 g514945 BLASTN 1296 1e-109 96 624 405
700216963H1 SATMON016 g514945 BLASTN 1415 1e-109 100 625 405
700085273H1 SATMON011 g22485 BLASTN 1416 1e-109 98 626 405
700082127H1 SATMON011 g514945 BLASTN 1420 1e-109 100 627 405
700085731H1 SATMON011 g514945 BLASTN 1425 1e-109 100 628 405
700088595H1 SATMON011 g22485 BLASTN 1426 1e-109 99 629 405
700470903H1 SATMON025 g514945 BLASTN 1426 1e-109 99 630 405
700265288H1 SATMON017 g514945 BLASTN 1375 1e-108 100 631 405
700072245H1 SATMON007 g514945 BLASTN 1404 1e-108 99 632 405
700347692H1 SATMON023 g514945 BLASTN 1405 1e-108 98 633 405
700214447H1 SATMON016 g514945 BLASTN 1406 1e-108 99 634 405
700476252H1 SATMON025 g514945 BLASTN 1407 1e-108 99 635 405
700336746H1 SATMON019 g514945 BLASTN 1409 1e-108 99 636 405
700053833H1 SATMON011 g514945 BLASTN 1410 1e-108 100 637 405
700094342H1 SATMON008 g514945 BLASTN 1410 1e-108 100 638 405
700202813H1 SATMON003 g514945 BLASTN 1032 1e-107 97 639 405
700050589H1 SATMON003 g514945 BLASTN 1035 1e-107 100 640 405
700050011H1 SATMON003 g514945 BLASTN 1078 1e-107 99 641 405
700215426H1 SATMON016 g514945 BLASTN 1189 1e-107 96
642 405 700472461H1 SATMON025 g514945 BLASTN 1392 1e-107 99 643 405
700336684H1 SATMON019 g22485 BLASTN 1393 1e-107 98 644 405
700449826H2 SATMON028 g514945 BLASTN 1395 1e-107 100 645 405
700216443H1 SATMON016 g514945 BLASTN 1396 1e-107 99 646 405
700240793H1 SATMON010 g514945 BLASTN 1399 1e-107 98 647 405
700215985H1 SATMON016 g514945 BLASTN 1400 1e-107 100 648 405
700336740H1 SATMON019 g514945 BLASTN 915 1e-106 99 649 405
700047958H1 SATMON003 g514945 BLASTN 987 1e-106 96 650 405
700085447H1 SATMON011 g514945 BLASTN 1030 1e-106 100 651 405
700084978H1 SATMON011 g514945 BLASTN 1121 1e-106 91 652 405
700800439H1 SATMON036 g22485 BLASTN 1379 1e-106 99 653 405
700219631H1 SATMON011 g514945 BLASTN 1380 1e-106 100 654 405
700220740H1 SATMON011 g514945 BLASTN 1380 1e-106 100 655 405
700243367H1 SATMON010 g514945 BLASTN 1381 1e-106 99 656 405
700220363H1 SATMON011 g514945 BLASTN 1387 1e-106 99 657 405
700215869H1 SATMON016 g514945 BLASTN 1390 1e-106 100 658 405
700216519H1 SATMON016 g514945 BLASTN 1131 1e-105 97 659 405
700052206H1 SATMON003 g514945 BLASTN 1264 1e-105 96 660 405
700094975H1 SATMON008 g514945 BLASTN 1368 1e-105 99 661 405
700220837H1 SATMON011 g514945 BLASTN 1369 1e-105 98 662 405
700221108H1 SATMON011 g514945 BLASTN 1370 1e-105 98 663 405
700222850H1 SATMON011 g514945 BLASTN 1370 1e-105 100 664 405
700214429H1 SATMON016 g514945 BLASTN 1373 1e-105 99 665 405
700473857H1 SATMON025 g514945 BLASTN 1375 1e-105 98 666 405
700213762H1 SATMON016 g514945 BLASTN 1378 1e-105 99 667 405
700405254H1 SATMON028 g22485 BLASTN 1242 1e-104 99 668 405
700029978H1 SATMON003 g22485 BLASTN 1324 1e-104 97 669 405
700238315H1 SATMON010 g514945 BLASTN 1355 1e-104 100 670 405
700241686H1 SATMON010 g514945 BLASTN 1358 1e-104 99 671 405
700237721H1 SATMON010 g22485 BLASTN 1360 1e-104 100 672 405
700217344H1 SATMON016 g514945 BLASTN 1360 1e-104 100 673 405
700030048H1 SATMON003 g514945 BLASTN 1363 1e-104 99 674 405
700211866H1 SATMON016 g514945 BLASTN 1363 1e-104 99 675 405
700214860H1 SATMON016 g514945 BLASTN 1365 1e-104 100 676 405
700085490H1 SATMON011 g514945 BLASTN 900 1e-103 98 677 405
700048568H1 SATMON003 g514945 BLASTN 980 1e-103 100 678 405
700381034H1 SATMON023 g22485 BLASTN 1269 1e-103 98 679 405
700220930H1 SATMON011 g514945 BLASTN 1347 1e-103 99 680 405
700030261H1 SATMON003 g514945 BLASTN 1353 1e-103 98 681 405
700081835H1 SATMON011 g22485 BLASTN 797 1e-102 98 682 405
700205270H1 SATMON003 g514945 BLASTN 1024 1e-102 94 683 405
700093612H1 SATMON008 g514945 BLASTN 1065 1e-102 99 684 405
700333392H1 SATMON019 g514945 BLASTN 1108 1e-102 97 685 405
700575385H1 SATMON030 g514945 BLASTN 1171 1e-102 96 686 405
700241061H1 SATMON010 g514945 BLASTN 1174 1e-102 99 687 405
700239916H1 SATMON010 g514945 BLASTN 1255 1e-102 100 688 405
700090248H1 SATMON011 g514945 BLASTN 1334 1e-102 98 689 405
700222923H1 SATMON011 g514945 BLASTN 1334 1e-102 98 690 405
700216993H1 SATMON016 g514945 BLASTN 1335 1e-102 100 691 405
700215984H1 SATMON016 g514945 BLASTN 1340 1e-102 100 692 405
700213182H1 SATMON016 g514945 BLASTN 1340 1e-102 98 693 405
700219845H1 SATMON011 g514945 BLASTN 1340 1e-102 100 694 405
700237762H1 SATMON010 g514945 BLASTN 1340 1e-102 100 695 405
700551043H1 SATMON022 g514945 BLASTN 1342 1e-102 99 696 405
700219254H1 SATMON011 g514945 BLASTN 1252 1e-101 99 697 405
700210348H1 SATMON016 g514945 BLASTN 1320 1e-101 97 698 405
700215089H1 SATMON016 g514945 BLASTN 1320 1e-101 100 699 405
700217251H1 SATMON016 g514945 BLASTN 1320 1e-101 100 700 405
700219240H1 SATMON011 g514945 BLASTN 1320 1e-101 100 701 405
700082094H1 SATMON011 g514945 BLASTN 1321 1e-101 99 702 405
700219385H1 SATMON011 g514945 BLASTN 1322 1e-101 99 703 405
700220052H1 SATMON011 g514945 BLASTN 1325 1e-101 100 704 405
700210366H1 SATMON016 g514945 BLASTN 1329 1e-101 93 705 405
700083089H1 SATMON011 g514945 BLASTN 1330 1e-101 98 706 405
700340286H1 SATMON020 g22485 BLASTN 677 1e-100 98 707 405
700221062H1 SATMON011 g514945 BLASTN 845 1e-100 98 708 405
700382272H1 SATMON024 g22485 BLASTN 958 1e-100 96 709 405
700209310H1 SATMON016 g514945 BLASTN 1187 1e-100 97 710 405
700052340H1 SATMON003 g514945 BLASTN 1188 1e-100 94 711 405
700467851H1 SATMON025 g22485 BLASTN 1245 1e-100 97 712 405
700088014H1 SATMON011 g514945 BLASTN 1270 1e-100 98 713 405
700214596H1 SATMON016 g514945 BLASTN 1295 1e-100 100 714 405
700157215H1 SATMON012 g22485 BLASTN 1310 1e-100 98 715 405
700223892H1 SATMON011 g514945 BLASTN 1310 1e-100 100 716 405
700218981H1 SATMON011 g514945 BLASTN 1310 1e-100 100 717 405
700081945H1 SATMON011 g514945 BLASTN 1311 1e-100 96 718 405
700217817H1 SATMON016 g514945 BLASTN 1315 1e-100 100 719 405
700469042H1 SATMON025 g514945 BLASTN 561 1e-99 98 720 405
700474709H1 SATMON025 g514945 BLASTN 801 1e-99 99 721 405
700201736H1 SATMON003 g514945 BLASTN 1168 1e-99 98 722 405
700223516H1 SATMON011 g514945 BLASTN 1201 1e-99 99 723 405
700453941H1 SATMON029 g22485 BLASTN 1295 1e-99 95 724 405
700212970H1 SATMON016 g514945 BLASTN 1297 1e-99 99 725 405
700215662H1 SATMON016 g22485 BLASTN 1297 1e-99 99 726 405
700802209H1 SATMON036 g22485 BLASTN 1300 1e-99 98 727 405
700343716H1 SATMON021 g514945 BLASTN 1300 1e-99 100 728 405
700223322H1 SATMON011 g514945 BLASTN 1300 1e-99 100 729 405
700217238H1 SATMON016 g514945 BLASTN 1300 1e-99 100 730 405
700195066H1 SATMON014 g22485 BLASTN 1300 1e-99 98 731 405
700072395H1 SATMON007 g514945 BLASTN 1301 1e-99 95 732 405
700212752H1 SATMON016 g22485 BLASTN 1305 1e-99 98 733 405
700222204H1 SATMON011 g514945 BLASTN 1305 1e-99 100 734 405
700550572H1 SATMON022 g22485 BLASTN 713 1e-98 97 735 405
700213879H1 SATMON016 g514945 BLASTN 866 1e-98 99 736 405
700551585H1 SATMON022 g514945 BLASTN 916 1e-98 99 737 405
700195025H1 SATMON014 g22485 BLASTN 1283 1e-98 98 738 405
700800710H1 SATMON036 g22485 BLASTN 1283 1e-98 98 739 405
700222985H1 SATMON011 g22485 BLASTN 1283 1e-98 98 740 405
700798823H1 SATMON036 g22485 BLASTN 1284 1e-98 98 741 405
700104391H1 SATMON010 g22485 BLASTN 1289 1e-98 98 742 405
700466592H1 SATMON025 g22485 BLASTN 1289 1e-98 95 743 405
700027037H1 SATMON003 g514945 BLASTN 919 1e-97 91 744 405
700214371H1 SATMON016 g514945 BLASTN 1033 1e-97 95 745 405
700799077H1 SATMON036 g22485 BLASTN 1091 1e-97 99 746 405
700467028H1 SATMON025 g514945 BLASTN 1103 1e-97 98 747 405
700219393H1 SATMON011 g514945 BLASTN 1250 1e-97 99 748 405
700197602H1 SATMON014 g22485 BLASTN 1273 1e-97 97 749 405
700801226H1 SATMON036 g22485 BLASTN 1276 1e-97 99 750 405
700216371H1 SATMON016 g514945 BLASTN 1278 1e-97 98 751 405
700805695H1 SATMON036 g22485 BLASTN 1280 1e-97 98 752 405
700334076H1 SATMON019 g514945 BLASTN 503 1e-96 98 753 405
700082647H1 SATMON011 g514945 BLASTN 735 1e-96 100 754 405
700458687H1 SATMON029 g22485 BLASTN 751 1e-96 95 755 405
700220750H1 SATMON011 g514945 BLASTN 1187 1e-96 96 756 405
700194931H1 SATMON014 g22485 BLASTN 1262 1e-96 99 757 405
700800522H1 SATMON036 g22485 BLASTN 1265 1e-96 98 758 405
700244185H1 SATMON010 g514945 BLASTN 1265 1e-96 100 759 405
700240785H1 SATMON010 g514945 BLASTN 1268 1e-96 98 760 405
700551959H1 SATMON022 g514945 BLASTN 1270 1e-96 100 761 405
700085057H1 SATMON011 g514945 BLASTN 682 1e-95 97 762 405
700332020H1 SATMON019 g514945 BLASTN 713 1e-95 97 763 405
700208841H1 SATMON016 g514945 BLASTN 822 1e-95 95 764 405
700193023H1 SATMON014 g22485 BLASTN 1248 1e-95 98 765 405
700153902H1 SATMON007 g514945 BLASTN 1250 1e-95 100 766 405
700196173H1 SATMON014 g22485 BLASTN 1252 1e-95 99 767 405
700804187H1 SATMON036 g22485 BLASTN 1253 1e-95 98 768 405
700339656H1 SATMON020 g22485 BLASTN 1257 1e-95 99 769 405
700238537H1 SATMON010 g22485 BLASTN 1258 1e-95 99 770 405
700551221H1 SATMON022 g514945 BLASTN 1204 1e-94 99 771 405
700801807H1 SATMON036 g22485 BLASTN 1235 1e-94 100 772 405
700085096H1 SATMON011 g514945 BLASTN 1235 1e-94 100 773 405
700020516H1 SATMON001 g514945 BLASTN 1236 1e-94 98 774 405
700193482H1 SATMON014 g22485 BLASTN 1236 1e-94 99 775 405
700217793H1 SATMON016 g514945 BLASTN 1237 1e-94 98 776 405
700088752H1 SATMON011 g514945 BLASTN 1240 1e-94 100 777 405
700087940H1 SATMON011 g514945 BLASTN 1241 1e-94 99 778 405
700089541H1 SATMON011 g533251 BLASTN 1067 1e-93 91 779 405
700346461H1 SATMON021 g514945 BLASTN 1190 1e-93 99 780 405
700156258H1 SATMON007 g514945 BLASTN 1225 1e-93 100 781 405
700195532H1 SATMON014 g22485 BLASTN 1226 1e-93 99 782 405
700196548H1 SATMON014 g22485 BLASTN 1227 1e-93 99 783 405
700213282H1 SATMON016 g514945 BLASTN 1227 1e-93 96 784 405
700194939H1 SATMON014 g22485 BLASTN 1228 1e-93 98 785 405
700340787H1 SATMON020 g22485 BLASTN 697 1e-92 94 786 405
700105064H1 SATMON010 g22485 BLASTN 706 1e-92 98 787 405
700805132H1 SATMON036 g22485 BLASTN 1046 1e-92 99 788 405
700803414H1 SATMON036 g22485 BLASTN 1211 1e-92 99 789 405
700214594H1 SATMON016 g514945 BLASTN 1212 1e-92 98 790 405
700215080H1 SATMON016 g514945 BLASTN 1213 1e-92 92 791 405
700224219H1 SATMON011 g514945 BLASTN 1213 1e-92 99 792 405
700048190H1 SATMON003 g514945 BLASTN 1218 1e-92 92 793 405
700223284H1 SATMON011 g514945 BLASTN 1220 1e-92 100 794 405
700152413H1 SATMON007 g514945 BLASTN 1220 1e-92 100 795 405
700218003H1 SATMON016 g514945 BLASTN 1221 1e-92 96 796 405
700160155H1 SATMON012 g22485 BLASTN 1222 1e-92 99 797 405
700087943H1 SATMON011 g22485 BLASTN 1222 1e-92 99 798 405
700474049H1 SATMON025 g514945 BLASTN 632 1e-91 97 799 405
700216994H1 SATMON016 g514945 BLASTN 1043 1e-91 99 800 405
700346892H1 SATMON021 g514945 BLASTN 1210 1e-91 96 801 405
700142782H1 SATMON013 g514945 BLASTN 1190 1e-90 100 802 405
700244157H1 SATMON010 g514945 BLASTN 1197 1e-90 97 803 405
700469243H1 SATMON025 g22485 BLASTN 701 1e-89 98 804 405
700618387H1 SATMON033 g514945 BLASTN 853 1e-89 93 805 405
700211154H1 SATMON016 g533251 BLASTN 917 1e-89 90 806 405
700081933H1 SATMON011 g533251 BLASTN 955 1e-89 91 807 405
700235229H1 SATMON010 g514945 BLASTN 955 1e-89 97 808 405
700209241H1 SATMON016 g514945 BLASTN 1076 1e-89 98 809 405
700265841H1 SATMON017 g514945 BLASTN 1097 1e-89 95 810 405
700193804H1 SATMON014 g22485 BLASTN 1180 1e-89 98 811 405
700167742H1 SATMON013 g22485 BLASTN 1180 1e-89 100 812 405
700163256H1 SATMON013 g514945 BLASTN 1182 1e-89 97 813 405
700184973H1 SATMON014 g22485 BLASTN 1182 1e-89 99 814 405
700171785H1 SATMON013 g22485 BLASTN 1184 1e-89 97 815 405
700216915H1 SATMON016 g514945 BLASTN 1185 1e-89 100 816 405
700806685H1 SATMON036 g22485 BLASTN 1186 1e-89 99 817 405
700803846H1 SATMON036 g22485 BLASTN 892 1e-88 95 818 405
700218514H1 SATMON011 g533251 BLASTN 907 1e-88 91 819 405
700574674H1 SATMON030 g22485 BLASTN 957 1e-88 84 820 405
700196082H1 SATMON014 g22485 BLASTN 1054 1e-88 94 821 405
700241637H1 SATMON010 g22485 BLASTN 1081 1e-88 98 822 405
700801876H1 SATMON036 g22485 BLASTN 1163 1e-88 98 823 405
700213443H1 SATMON016 g514945 BLASTN 1171 1e-88 99 824 405
700465181H1 SATMON025 g22485 BLASTN 797 1e-87 92 825 405
700798732H1 SATMON036 g22485 BLASTN 1159 1e-87 96 826 405
700153473H1 SATMON007 g514945 BLASTN 1160 1e-87 100 827 405
700165496H1 SATMON013 g514945 BLASTN 1160 1e-87 97 828 405
700264250H1 SATMON017 g514945 BLASTN 643 1e-86 100 829 405
700335490H1 SATMON019 g514945 BLASTN 671 1e-86 97 830 405
700575891H1 SATMON030 g22485 BLASTN 780 1e-86 92 831 405
700222931H1 SATMON011 g514945 BLASTN 1117 1e-86 91 832 405
700163588H1 SATMON013 g514945 BLASTN 1140 1e-86 100 833 405
700161111H1 SATMON012 g22485 BLASTN 1142 1e-86 99 834 405
700016023H1 SATMON001 g514945 BLASTN 1147 1e-86 99 835 405
700209043H1 SATMON016 g514945 BLASTN 659 1e-85 98 836 405
700333556H1 SATMON019 g514945 BLASTN 783 1e-85 89 837 405
700570471H1 SATMON030 g22485 BLASTN 831 1e-85 89 838 405
700171040H1 SATMON013 g22485 BLASTN 1127 1e-85 99 839 405
700196146H1 SATMON014 g22485 BLASTN 1128 1e-85 96 840 405
700021837H1 SATMON001 g514945 BLASTN 1130 1e-85 98 841 405
700169062H1 SATMON013 g514945 BLASTN 1130 1e-85 100 842 405
700218263H1 SATMON016 g514945 BLASTN 1135 1e-85 100 843 405
700091281H1 SATMON011 g514945 BLASTN 1135 1e-85 100 844 405
700221221H1 SATMON011 g22485 BLASTN 735 1e-84 98 845 405
700239933H1 SATMON010 g22485 BLASTN 779 1e-84 97 846 405
700193089H1 SATMON014 g22485 BLASTN 979 1e-84 97 847 405
700216767H1 SATMON016 g514945 BLASTN 1005 1e-84 98 848 405
700085786H1 SATMON011 g514945 BLASTN 1117 1e-84 99 849 405
700163868H1 SATMON013 g22485 BLASTN 1120 1e-84 100 850 405
700167028H1 SATMON013 g514945 BLASTN 1120 1e-84 100 851 405
700170416H1 SATMON013 g514945 BLASTN 1120 1e-84 100 852 405
700266595H1 SATMON017 g514945 BLASTN 1120 1e-84 100 853 405
700377630H1 SATMON019 g514945 BLASTN 649 1e-83 95 854 405
700207830H1 SATMON016 g514945 BLASTN 877 1e-83 97 855 405
700241726H1 SATMON010 g514945 BLASTN 1104 1e-83 97 856 405
700806447H1 SATMON036 g22485 BLASTN 1106 1e-83 93 857 405
700018166H1 SATMON001 g514945 BLASTN 1108 1e-83 98 858 405
700083463H1 SATMON011 g514945 BLASTN 633 1e-82 92 859 405
700548890H1 SATMON022 g22485 BLASTN 727 1e-82 93 860 405
700218553H1 SATMON011 g22485 BLASTN 979 1e-82 95 861 405
700016408H1 SATMON001 g514945 BLASTN 1026 1e-82 97 862 405
700569427H2 SATMON030 g514945 BLASTN 1095 1e-82 97 863 405
700172546H1 SATMON013 g514945 BLASTN 1100 1e-82 100 864 405
700804387H1 SATMON036 g22485 BLASTN 668 1e-81 97 865 405
700155788H1 SATMON007 g514945 BLASTN 840 1e-81 100 866 405
700807167H1 SATMON036 g22485 BLASTN 1024 1e-81 97 867 405
700472356H1 SATMON025 g22485 BLASTN 1080 1e-81 98 868 405
700193535H1 SATMON014 g22485 BLASTN 1082 1e-81 99 869 405
700171177H1 SATMON013 g22485 BLASTN 1086 1e-81 98 870 405
700799867H1 SATMON036 g22485 BLASTN 1087 1e-81 96 871 405
700263716H1 SATMON017 g514945 BLASTN 1089 1e-81 92 872 405
700476045H1 SATMON025 g22485 BLASTN 608 1e-80 88 873 405
700803344H1 SATMON036 g22485 BLASTN 834 1e-80 97 874 405
700168924H1 SATMON013 g514945 BLASTN 860 1e-80 99 875 405
700218569H1 SATMON011 g22485 BLASTN 900 1e-80 98 876 405
700088574H1 SATMON011 g514945 BLASTN 900 1e-80 86 877 405
700471932H1 SATMON025 g530978 BLASTN 1064 1e-80 83 878 405
700020011H1 SATMON001 g22485 BLASTN 1067 1e-80 99 879 405
700167511H1 SATMON013 g22485 BLASTN 1070 1e-80 100 880 405
700219249H1 SATMON011 g514945 BLASTN 1070 1e-80 100 881 405
700804846H1 SATMON036 g22485 BLASTN 1075 1e-80 90 882 405
700150388H1 SATMON007 g22485 BLASTN 1075 1e-80 100 883 405
700807395H1 SATMON036 g22485 BLASTN 571 1e-79 90 884 405
700090864H1 SATMON011 g514945 BLASTN 630 1e-79 100 885 405
700217812H1 SATMON016 g514945 BLASTN 646 1e-79 91 886 405
700203618H1 SATMON003 g22485 BLASTN 913 1e-79 96 887 405
700203302H1 SATMON003 g514945 BLASTN 1030 1e-79 100 888 405
700163192H1 SATMON013 g22485 BLASTN 1056 1e-79 97 889 405
700805065H1 SATMON036 g22485 BLASTN 1066 1e-79 95 890 405
700086763H1 SATMON011 g514945 BLASTN 901 1e-78 98 891 405
700240070H1 SATMON010 g533251 BLASTN 923 1e-78 90 892 405
700018847H1 SATMON001 g22485 BLASTN 1045 1e-78 98
893 405 700803420H1 SATMON036 g22485 BLASTN 1048 1e-78 96 894 405
700799936H1 SATMON036 g22485 BLASTN 1050 1e-78 96 895 405
700207637H1 SATMON016 g514945 BLASTN 828 1e-77 98 896 405
700807034H1 SATMON036 g22485 BLASTN 808 1e-76 91 897 405
700198035H1 SATMON016 g514945 BLASTN 1025 1e-76 100 898 405
700169076H1 SATMON013 g514945 BLASTN 1028 1e-76 99 899 405
700020564H1 SATMON001 g514945 BLASTN 1030 1e-76 98 900 405
700799968H1 SATMON036 g22485 BLASTN 701 1e-75 99 901 405
700378056H1 SATMON019 g22485 BLASTN 802 1e-75 97 902 405
700219691H1 SATMON011 g514945 BLASTN 1018 1e-75 99 903 405
700168945H1 SATMON013 g22485 BLASTN 848 1e-74 94 904 405
700242730H1 SATMON010 g514945 BLASTN 1006 1e-74 99 905 405
700210096H1 SATMON016 g514945 BLASTN 756 1e-73 93 906 405
700333941H1 SATMON019 g514945 BLASTN 923 1e-73 99 907 405
700576645H1 SATMON030 g22485 BLASTN 991 1e-73 99 908 405
700333494H1 SATMON019 g514945 BLASTN 601 1e-72 91 909 405
700023296H1 SATMON003 g514945 BLASTN 726 1e-72 95 910 405
700802508H1 SATMON036 g22485 BLASTN 811 1e-72 94 911 405
700223382H1 SATMON011 g22485 BLASTN 865 1e-72 98 912 405
700215535H1 SATMON016 g514945 BLASTN 942 1e-72 96 913 405
700017549H1 SATMON001 g514945 BLASTN 973 1e-72 97 914 405
700799113H1 SATMON036 g22485 BLASTN 787 1e-70 99 915 405
700168696H1 SATMON013 g514945 BLASTN 946 1e-69 89 916 405
700088269H1 SATMON011 g514945 BLASTN 946 1e-69 93 917 405
700194522H1 SATMON014 g22485 BLASTN 875 1e-68 97 918 405
700203476H1 SATMON003 g22485 BLASTN 923 1e-68 86 919 405
700549205H1 SATMON022 g22485 BLASTN 300 1e-66 89 920 405
700196217H1 SATMON014 g22485 BLASTN 907 1e-66 96 921 405
700163647H1 SATMON013 g22485 BLASTN 888 1e-65 98 922 405
700804485H1 SATMON036 g22485 BLASTN 896 1e-65 99 923 405
700193074H1 SATMON014 g22485 BLASTN 605 1e-63 96 924 405
700203370H1 SATMON003 g514945 BLASTN 857 1e-62 98 925 405
700201575H1 SATMON003 g514945 BLASTN 335 1e-60 87 926 405
700378020H1 SATMON019 g514945 BLASTN 833 1e-60 97 927 405
700242865H1 SATMON010 g514945 BLASTN 823 1e-59 91 928 405
700344036H1 SATMON021 g514945 BLASTN 825 1e-59 100 929 405
700215849H1 SATMON016 g514945 BLASTN 805 1e-58 100 930 405
700443538H1 SATMON027 g22485 BLASTN 814 1e-58 98 931 405
700804448H1 SATMON036 g22485 BLASTN 791 1e-57 99 932 405
700155008H1 SATMON007 g22485 BLASTN 802 1e-57 98 933 405
700201244H1 SATMON003 g22485 BLASTN 530 1e-56 97 934 405
700616378H1 SATMON033 g22485 BLASTN 682 1e-56 97 935 405
700333357H1 SATMON019 g22485 BLASTN 780 1e-56 80 936 405
700222360H1 SATMON011 g514945 BLASTN 777 1e-55 92 937 405
700214724H1 SATMON016 g514945 BLASTN 763 1e-54 98 938 405
700571283H1 SATMON030 g514945 BLASTN 736 1e-52 99 939 405
700020194H1 SATMON001 g22485 BLASTN 415 1e-51 99 940 405
700620551H1 SATMON034 g22485 BLASTN 473 1e-51 95 941 405
700446320H1 SATMON027 g22485 BLASTN 475 1e-50 87 942 405
700241357H1 SATMON010 g22485 BLASTN 701 1e-49 99 943 405
700617094H1 SATMON033 g22485 BLASTN 673 1e-47 97 944 405
700206691H1 SATMON003 g514945 BLASTN 680 1e-47 90 945 405
700091580H1 SATMON011 g514945 BLASTN 680 1e-47 100 946 405
700574515H1 SATMON030 g514945 BLASTN 369 1e-46 74 947 405
700155148H1 SATMON007 g514945 BLASTN 397 1e-45 97 948 405
700612388H1 SATMON033 g514945 BLASTN 625 1e-43 100 949 405
700474681H1 SATMON025 g22485 BLASTN 379 1e-41 91 950 405
700800401H1 SATMON036 g22485 BLASTN 395 1e-40 90 951 405
700155657H1 SATMON007 g514945 BLASTN 591 1e-40 95 952 405
700076002H1 SATMON007 g514945 BLASTN 575 1e-39 100 953 405
700802090H1 SATMON036 g22485 BLASTN 577 1e-39 98 954 405
700170104H1 SATMON013 g22485 BLASTN 565 1e-38 100 955 405
701183763H1 SATMONN06 g514945 BLASTN 569 1e-38 90 956 405
700084688H1 SATMON011 g514945 BLASTN 380 1e-36 98 957 405
700473655H1 SATMON025 g22485 BLASTN 530 1e-35 100 958 405
700615166H1 SATMON033 g514945 BLASTN 531 1e-35 94 959 405
700085562H1 SATMON011 g533251 BLASTN 532 1e-35 98 960 405
700153049H1 SATMON007 g514945 BLASTN 537 1e-35 94 961 405
700090656H1 SATMON011 g514945 BLASTN 489 1e-34 98 962 405
700802054H1 SATMON036 g22485 BLASTN 345 1e-31 99 963 405
700802284H1 SATMON036 g22485 BLASTN 488 1e-31 97 964 405
700802312H1 SATMON036 g22485 BLASTN 270 1e-30 100 965 405
700153683H1 SATMON007 g22485 BLASTN 461 1e-29 98 966 405
700028453H1 SATMON003 g22485 BLASTN 321 1e-27 99 967 405
700089391H1 SATMON011 g514945 BLASTN 404 1e-24 96 968 405
700381969H1 SATMON023 g22485 BLASTN 385 1e-23 94 969 405
700800135H1 SATMON036 g22485 BLASTN 180 1e-21 100 970 405
700088173H1 SATMON011 g514945 BLASTN 347 1e-20 95 971 405
700202170H1 SATMON003 g19108 BLASTX 133 1e-11 96 972 537
700209929H1 SATMON016 g22485 BLASTN 1478 1e-114 99 973 537
700096948H1 SATMON008 g22485 BLASTN 911 1e-113 99 974 537
700476287H1 SATMON025 g22485 BLASTN 1403 1e-108 98 975 537
700803088H1 SATMON036 g22485 BLASTN 1336 1e-107 96 976 537
700799436H1 SATMON036 g22485 BLASTN 1361 1e-104 99 977 537
700224822H1 SATMON011 g22485 BLASTN 1300 1e-103 96 978 537
700241134H1 SATMON010 g22485 BLASTN 1302 1e-99 99 979 537
700803625H1 SATMON036 g22485 BLASTN 1292 1e-98 99 980 537
700802549H1 SATMON036 g22485 BLASTN 1232 1e-93 99 981 537
700477992H1 SATMON025 g22485 BLASTN 943 1e-92 97 982 537
700150953H1 SATMON007 g22485 BLASTN 1152 1e-87 99 983 537
700205638H1 SATMON003 g22485 BLASTN 1086 1e-81 99 984 537
700803732H1 SATMON036 g22487 BLASTN 379 1e-79 97 985 537
700165461H1 SATMON013 g22485 BLASTN 1064 1e-79 98 986 537
700807069H1 SATMON036 g22485 BLASTN 957 1e-77 96 987 537
700800902H1 SATMON036 g22485 BLASTN 762 1e-54 86 988 537
700466671H1 SATMON025 g22485 BLASTN 520 1e-44 95 989 537
700799118H1 SATMON036 g22485 BLASTN 626 1e-43 99 990 537
700802273H1 SATMON036 g22485 BLASTN 616 1e-42 99 991 537
700804848H1 SATMON036 g22485 BLASTN 306 1e-33 98 992 8549
700103190H1 SATMON010 g1100216 BLASTN 615 1e-92 98 993 8549
700075574H1 SATMON007 g1100216 BLASTN 701 1e-92 100 994 8549
700218547H1 SATMON011 g514945 BLASTN 1208 1e-91 99 995 8549
700213873H1 SATMON016 g1100216 BLASTN 673 1e-90 95 996 8549
700221147H1 SATMON011 g1100216 BLASTN 646 1e-89 98 997 8549
700207093H1 SATMON003 g1100216 BLASTN 701 1e-87 100 998 8549
700210112H1 SATMON016 g1100216 BLASTN 615 1e-84 98 999 8549
700096984H1 SATMON008 g514945 BLASTN 1111 1e-83 99 1000 8549
700221070H1 SATMON011 g1100216 BLASTN 645 1e-82 96 1001 8549
700332046H1 SATMON019 g1100216 BLASTN 601 1e-76 89 1002 8549
700150377H1 SATMON007 g1100216 BLASTN 621 1e-74 100 1003 8549
700084780H1 SATMON011 g514945 BLASTN 585 1e-39 100 1004 8549
700153082H1 SATMON007 g1100216 BLASTN 495 1e-36 89 1005 8549
700261144H1 SATMON017 g1100216 BLASTN 339 1e-35 87 1006 8549
700264112H1 SATMON017 g1100216 BLASTN 428 1e-34 91 1007 8549
700473660H1 SATMON025 g1100216 BLASTN 415 1e-28 100 1008 8549
700473628H1 SATMON025 g514945 BLASTN 329 1e-26 88 1009 8549
700351060H1 SATMON023 g1100216 BLASTN 291 1e-22 91 1010 -L30595280
LIB3059-039- LIB3059 g22485 BLASTN 473 1e-30 79 Q1-K1-A5 1011
-L30612133 LIB3061-024- LIB3061 g22485 BLASTN 849 1e-61 80 Q1-K1-H5
1012 -L30616296 LIB3061-043- LIB3061 g22485 BLASTN 479 1e-98 82
Q1-K1-A10 1013 -L30623037 LIB3062-030- LIB3062 g514945 BLASTN 684
1e-48 78 Q1-K1-F12 1014 -L30625289 LIB3062-021- LIB3062 g514945
BLASTN 1180 1e-111 79 Q1-K1-C2 1015 -L30663565 LIB3066-053- LIB3066
g530978 BLASTN 568 1e-36 76 Q1-K1-D6 1016 -L30784420 LIB3078-039-
LIB3078 g514945 BLASTN 484 1e-40 81 Q1-K1-A4 1017 30444
LIB3069-052- LIB3069 g1100216 BLASTN 558 1e-77 89 Q1-K1-F8 1018
32909 LIB143-057- LIB143 g2570066 BLASTN 902 1e-69 74 Q1-E1-F6 1019
405 LIB3062-021- LIB3062 g514945 BLASTN 2368 1e-188 99 Q1-K1-C5
1020 405 LIB3078-024- LIB3078 g514945 BLASTN 2356 1e-187 98
Q1-K1-C5 1021 405 LIB3059-028- LIB3059 g22485 BLASTN 2163 1e-171 98
Q1-K1-D5 1022 405 LIB3059-015- LIB3059 g22485 BLASTN 2167 1e-171 98
Q1-K1-E7 1023 405 LIB3059-044- LIB3059 g514945 BLASTN 2170 1e-171
98 Q1-K1-E7 1024 405 LIB3061-029- LIB3061 g22485 BLASTN 2055 1e-170
98 Q1-K1-G11 1025 405 LIB3059-011- LIB3059 g22485 BLASTN 2137
1e-169 98 Q1-K1-F5 1026 405 LIB3062-009- LIB3062 g514945 BLASTN
2122 1e-167 98 Q1-K1-D1 1027 405 LIB3061-011- LIB3061 g22485 BLASTN
2091 1e-165 98 Q1-K1-D9 1028 405 LIB3067-040- LIB3067 g514945
BLASTN 1916 1e-164 99 Q1-K1-E8 1029 405 LIB3062-041- LIB3062
g514945 BLASTN 2082 1e-164 97 Q1-K1-D4 1030 405 LIB3062-022-
LIB3062 g514945 BLASTN 2084 1e-164 99 Q1-K1-C9 1031 405
LIB3062-033- LIB3062 g514945 BLASTN 1854 1e-161 95 Q1-K1-C7 1032
405 LIB3062-002- LIB3062 g514945 BLASTN 1854 1e-161 97 Q1-K2-F9
1033 405 LIB3059-010- LIB3059 g22485 BLASTN 2018 1e-159 99 Q1-K1-C9
1034 405 LIB3059-013- LIB3059 g22485 BLASTN 2022 1e-159 98
Q1-K1-B10 1035 405 LIB3061-020- LIB3061 g22485 BLASTN 1771 1e-158
97 Q1-K1-F2 1036 405 LIB3061-022- LIB3061 g22485 BLASTN 1909 1e-158
98 Q1-K1-C2 1037 405 LIB3062-023- LIB3062 g22485 BLASTN 1508 1e-157
96 Q1-K1-D10 1038 405 LIB3061-008- LIB3061 g22485 BLASTN 1983
1e-156 97 Q1-K1-H11 1039 405 LIB3059-024- LIB3059 g22485 BLASTN
1051 1e-154 99 Q1-K1-H4 1040 405 LIB3062-048- LIB3062 g22485 BLASTN
1187 1e-154 94 Q1-K1-G5 1041 405 LIB3061-025- LIB3061 g22485 BLASTN
1803 1e-154 95 Q1-K1-B1 1042 405 LIB3061-028- LIB3061 g22485 BLASTN
1963 1e-154 97 Q1-K1-C4 1043 405 LIB3078-057- LIB3078 g514945
BLASTN 1412 1e-153 92 Q1-K1-D9 1044 405 LIB3061-021- LIB3061 g22485
BLASTN 1465 1e-153 96 Q1-K1-A8 1045 405 LIB3061-025- LIB3061 g22485
BLASTN 1524 1e-153 96 Q1-K1-B5 1046 405 LIB3061-008- LIB3061 g22485
BLASTN 1879 1e-153 94 Q1-K1-C7 1047 405 LIB3078-039- LIB3078
g514945 BLASTN 1853 1e-151 96 Q1-K1-A8 1048 405 LIB3061-049-
LIB3061 g22485 BLASTN 1801 1e-150 98 Q1-K1-E5 1049 405 LIB3062-001-
LIB3062 g514945 BLASTN 1916 1e-150 94 Q1-K2-G2 1050 405
LIB3061-021- LIB3061 g22485 BLASTN 1918 1e-150 92 Q1-K1-G6 1051 405
LIB3061-039- LIB3061 g22485 BLASTN 1361 1e-149 96 Q1-K1-D2 1052 405
LIB3061-051- LIB3061 g22485 BLASTN 1768 1e-148 98 Q1-K1-G8 1053 405
LIB3061-015- LIB3061 g22485 BLASTN 1667 1e-146 93 Q1-K1-A12 1054
405 LIB3059-040- LIB3059 g22485 BLASTN 1835 1e-146 97 Q1-K1-H11
1055 405 LIB3061-002- LIB3061 g22485 BLASTN 1845 1e-144 89 Q1-K2-G5
1056 405 LIB3062-002- LIB3062 g22485 BLASTN 1672 1e-142 99
Q1-K2-G12 1057 405 LIB3059-048- LIB3059 g22485 BLASTN 1822 1e-142
99 Q1-K1-H5 1058 405 LIB3078-040- LIB3078 g514945 BLASTN 1801
1e-141 97 Q1-K1-F8 1059 405 LIB3078-001- LIB3078 g22485 BLASTN 1246
1e-139 95 Q1-K1-C7 1060 405 LIB3061-024- LIB3061 g22485 BLASTN 1376
1e-139 94 Q1-K1-A12 1061 405 LIB3061-026- LIB3061 g22485 BLASTN
1643 1e-138 93 Q1-K1-D3 1062 405 LIB3061-056- LIB3061 g22485 BLASTN
1763 1e-138 92 Q1-K1-D8 1063 405 LIB3069-041- LIB3069 g514945
BLASTN 1758 1e-137 97 Q1-K1-G12 1064 405 LIB3059-025- LIB3059
g22485 BLASTN 1532 1e-132 94 Q1-K1-E5 1065 405 LIB3061-014- LIB3061
g22485 BLASTN 1294 1e-130 88 Q1-K1-D4 1066 405 LIB3061-005- LIB3061
g22485 BLASTN 1540 1e-130 97 Q1-K1-C9 1067 405 LIB3061-016- LIB3061
g22485 BLASTN 1251 1e-129 85 Q1-K1-G2 1068 405 LIB3069-029- LIB3069
g514945 BLASTN 1657 1e-129 88 Q1-K1-B2 1069 405 LIB3078-012-
LIB3078 g514945 BLASTN 857 1e-128 86 Q1-K1-F7 1070 405 LIB3078-016-
LIB3078 g514945 BLASTN 1335 1e-128 87 Q1-K1-D7 1071 405
LIB3062-049- LIB3062 g514945 BLASTN 1609 1e-128 88 Q1-K1-A8 1072
405 LIB143-006- LIB143 g514945 BLASTN 1614 1e-125 96 Q1-E1-G12 1073
405 LIB3059-024- LIB3059 g22485 BLASTN 1529 1e-123 83 Q1-K1-E5 1074
405 LIB3069-008- LIB3069 g514945 BLASTN 1036 1e-115 94 Q1-K1-C1
1075 405 LIB3059-018- LIB3059 g514945 BLASTN 910 1e-103 93
Q1-K1-F11 1076 405 LIB3078-001- LIB3078 g514945 BLASTN 952 1e-98 90
Q1-K1-E8
1077 405 LIB3059-017- LIB3059 g22485 BLASTN 1170 1e-88 92 Q1-K1-G4
1078 405 LIB3067-045- LIB3067 g533251 BLASTN 917 1e-87 87 Q1-K1-E9
1079 405 LIB3062-015- LIB3062 g514945 BLASTN 1066 1e-86 96 Q1-K1-C1
1080 405 LIB3059-039- LIB3059 g22485 BLASTN 856 1e-82 92 Q1-K1-A3
1081 405 LIB3062-024- LIB3062 g514945 BLASTN 548 1e-79 88 Q1-K1-C3
1082 405 LIB3059-029- LIB3059 g22485 BLASTN 925 1e-74 94 Q1-K1-F1
1083 405 LIB3059-006- LIB3059 g22485 BLASTN 530 1e-50 83 Q1-K1-F4
1084 405 LIB3067-017- LIB3067 g533251 BLASTN 425 1e-26 100 Q1-K1-C3
1085 405 LIB3061-028- LIB3061 g19106 BLASTX 118 1e-25 100 Q1-K1-A9
1086 537 LIB3066-009- LIB3066 g22485 BLASTN 1369 1e-122 96 Q1-K1-B9
MAIZE HEXOKINASE 1087 -700018381 700018381H1 SATMON001 g1899025
BLASTX 166 1e-16 48 1088 -700051079 700051079H1 SATMON003 g1899025
BLASTX 84 1e-11 50 1089 -700101579 700101579H1 SATMON009 g881521
BLASTX 217 1e-23 66 1090 -700105594 700105594H1 SATMON010 g3087888
BLASTX 181 1e-17 57 1091 -700106018 700106018H1 SATMON010 g3087888
BLASTX 195 1e-19 64 1092 -700157233 700157233H1 SATMON012 g3087888
BLASTX 198 1e-20 58 1093 -700202992 700202992H1 SATMON003 g3087888
BLASTX 89 1e-9 58 1094 -700224204 700224204H1 SATMON011 g1899024
BLASTN 520 1e-34 70 1095 -700241273 700241273H1 SATMON010 g3087888
BLASTX 184 1e-18 58 1096 -700352183 700352183H1 SATMON023 g1899024
BLASTN 481 1e-31 70 1097 -700573814 700573814H1 SATMON030 g1899024
BLASTN 535 1e-34 67 1098 -700612458 700612458H1 SATMON033 g619928
BLASTX 229 1e-26 61 1099 -701168774 701168774H1 SATMONN05 g619927
BLASTN 252 1e-10 62 1100 1195 700457430H1 SATMON029 g3087888 BLASTX
122 1e-19 53 1101 13262 700102942H1 SATMON010 g3087888 BLASTX 113
1e-18 53 1102 1378 700456148H1 SATMON029 g1899025 BLASTX 267 1e-29
59 1103 1378 700455837H1 SATMON029 g1899025 BLASTX 166 1e-21 60
1104 17305 700460742H1 SATMON031 g619928 BLASTX 131 1e-15 57 1105
17305 700614972H1 SATMON033 g1899025 BLASTX 100 1e-8 53 1106 1842
700089135H1 SATMON011 g619928 BLASTX 405 1e-49 70 1107 1842
700430234H1 SATMONN01 g619927 BLASTN 461 1e-28 72 1108 1842
700166122H1 SATMON013 g619928 BLASTX 183 1e-18 84 1109 24376
700053677H1 SATMON010 g1899024 BLASTN 642 1e-44 70 1110 24376
700152328H1 SATMON007 g619927 BLASTN 555 1e-37 69 1111 24376
700623451H1 SATMON034 g619928 BLASTX 197 1e-32 72 1112 28388
700089065H1 SATMON011 g619928 BLASTX 186 1e-30 61 1113 3345
700072110H1 SATMON007 g619928 BLASTX 125 1e-24 66 1114 3345
700472061H1 SATMON025 g619928 BLASTX 112 1e-20 55 1115 3345
701173753H1 SATMONN05 g619928 BLASTX 135 1e-16 54 1116 3345
700202130H1 SATMON003 g619928 BLASTX 113 1e-11 68 1117 5073
700582054H1 SATMON031 g619928 BLASTX 247 1e-29 66 1118 5073
700053432H1 SATMON009 g619928 BLASTX 233 1e-25 60 1119 6731
700099009H1 SATMON009 g619927 BLASTN 736 1e-52 72 1120 6731
700089738H1 SATMON011 g1899024 BLASTN 700 1e-49 70 1121 6731
700171542H1 SATMON013 g619927 BLASTN 530 1e-35 74 1122 7565
700356773H1 SATMON024 g1899025 BLASTX 177 1e-17 62 1123 9695
700212172H1 SATMON016 g1899024 BLASTN 832 1e-60 74 1124 9695
700212124H1 SATMON016 g1899024 BLASTN 835 1e-60 75 1125 9695
700094278H1 SATMON008 g1899024 BLASTN 819 1e-59 74 1126 -L30621307
LIB3062-001- LIB3062 g1899025 BLASTX 95 1e-32 53 Q1-K2-G11 1127
-L30782665 LIB3078-007- LIB3078 g3087888 BLASTX 130 1e-39 47
Q1-K1-E9 1128 24376 LIB3069-041- LIB3069 g1899024 BLASTN 608 1e-61
70 Q1-K1-E7 1129 28244 LIB3061-004- LIB3061 g687676 BLASTN 499
1e-30 65 Q1-K1-F9 1130 28388 LIB3066-030- LIB3066 g619928 BLASTX
299 1e-63 64 Q1-K1-G10 1131 3364 LIB3078-051- LIB3078 g687676
BLASTN 619 1e-41 67 Q1-K1-B3 1132 3364 LIB3078-053- LIB3078 g687676
BLASTN 627 1e-41 69 Q1-K1-C9 1133 3364 LIB84-015- LIB84 g687676
BLASTN 554 1e-35 69 Q1-E1-F7 1134 6731 LIB3061-028- LIB3061
g1899024 BLASTN 831 1e-60 70 Q1-K1-C1 1135 9695 LIB143-065- LIB143
g1899024 BLASTN 1096 1e-82 73 Q1-E1-C10 MAIZE FRUCTOKINASE 1136
-700106058 700106058H1 SATMON010 g1052972 BLASTN 220 1e-9 68 1137
-700151135 700151135H1 SATMON007 g297014 BLASTN 351 1e-18 75 1138
-700169310 700169310H1 SATMON013 g1052972 BLASTN 273 1e-12 59 1139
-700210226 700210226H1 SATMON016 g1052973 BLASTX 188 1e-24 68 1140
-700257901 700257901H1 SATMON017 g297015 BLASTX 200 1e-20 72 1141
-700621274 700621274H1 SATMON034 g1052973 BLASTX 141 1e-24 64 1142
11678 700105513H1 SATMON010 g1052972 BLASTN 580 1e-39 64 1143 11678
700170725H1 SATMON013 g1052972 BLASTN 478 1e-31 66 1144 2526
700159958H1 SATMON012 g1052973 BLASTX 152 1e-14 64 1145 2754
700102678H1 SATMON010 g1052972 BLASTN 707 1e-50 69 1146 2754
700102312H1 SATMON010 g1052972 BLASTN 701 1e-49 69 1147 2754
700205695H1 SATMON003 g1915973 BLASTN 633 1e-43 69 1148 2754
700221511H1 SATMON011 g1915973 BLASTN 587 1e-40 69 1149 2754
700469079H1 SATMON025 g1052972 BLASTN 584 1e-39 72 1150 2754
701173520H1 SATMONN05 g1915973 BLASTN 342 1e-36 70 1151 2754
700267332H1 SATMON017 g1052972 BLASTN 541 1e-35 64 1152 2754
701164907H1 SATMONN04 g1052973 BLASTX 280 1e-33 57 1153 2754
700450050H2 SATMON028 g1052973 BLASTX 160 1e-31 60 1154 2754
701182860H1 SATMONN06 g297015 BLASTX 188 1e-27 65 1155 2754
700467520H1 SATMON025 g1915974 BLASTX 242 1e-26 60 1156 2754
700159848H1 SATMON012 g1052973 BLASTX 197 1e-24 63 1157 3287
700088103H1 SATMON011 g2102693 BLASTX 239 1e-43 74 1158 3287
700210913H1 SATMON016 g2102693 BLASTX 250 1e-35 77 1159 3287
700167609H1 SATMON013 g1052973 BLASTX 300 1e-35 68 1160 3287
700085916H1 SATMON011 g1052972 BLASTN 553 1e-35 64 1161 3287
700262715H1 SATMON017 g1915974 BLASTX 201 1e-33 71 1162 3287
700170179H1 SATMON013 g1052973 BLASTX 289 1e-33 67 1163 3287
700615671H1 SATMON033 g1052972 BLASTN 515 1e-32 63 1164 3287
700223640H1 SATMON011 g1052973 BLASTX 219 1e-31 67 1165 3287
700215234H1 SATMON016 g1052973 BLASTX 190 1e-30 67 1166 3287
700203946H1 SATMON003 g1052973 BLASTX 198 1e-30 60 1167 3287
700028411H1 SATMON003 g2102693 BLASTX 110 1e-29 57 1168 3287
700224307H1 SATMON011 g1052973 BLASTX 159 1e-29 87 1169 3287
700072013H1 SATMON007 g1052973 BLASTX 191 1e-29 65 1170 3287
700215669H1 SATMON016 g1052973 BLASTX 260 1e-29 57 1171 3287
700353954H1 SATMON024 g1052973 BLASTX 260 1e-29 61 1172 3287
700342211H1 SATMON021 g1052973 BLASTX 137 1e-28 67 1173 3287
700085462H1 SATMON011 g297014 BLASTN 466 1e-28 62 1174 3287
700220972H1 SATMON011 g1052973 BLASTX 109 1e-27 83 1175 3287
700451141H1 SATMON028 g1052973 BLASTX 245 1e-27 63 1176 3287
700087484H1 SATMON011 g1052972 BLASTN 440 1e-26 64 1177 3287
700343411H1 SATMON021 g1052973 BLASTX 163 1e-25 67 1178 3287
700217263H1 SATMON016 g1915973 BLASTN 393 1e-25 68 1179 3287
700030665H1 SATMON003 g1052973 BLASTX 176 1e-24 71 1180 3287
700343380H1 SATMON021 g1052973 BLASTX 228 1e-24 57 1181 3287
701159743H2 SATMONN04 g1052973 BLASTX 183 1e-23 55 1182 3287
700221543H1 SATMON011 g1052973 BLASTX 217 1e-23 50 1183 3287
700333946H1 SATMON019 g1052973 BLASTX 178 1e-22 66 1184 3287
700091730H1 SATMON011 g1052973 BLASTX 171 1e-21 64 1185 3287
700570521H1 SATMON030 g1915974 BLASTX 98 1e-18 58 1186 3287
700048604H1 SATMON003 g1052973 BLASTX 88 1e-15 54 1187 3287
700208681H1 SATMON016 g1052973 BLASTX 129 1e-15 55 1188 3287
700028328H1 SATMON003 g1052973 BLASTX 162 1e-15 66 1189 3287
700220530H1 SATMON011 g1052973 BLASTX 141 1e-14 88 1190 3287
700243726H1 SATMON010 g1052973 BLASTX 153 1e-14 68 1191 3287
700142502H1 SATMON012 g1052973 BLASTX 157 1e-14 47 1192 3287
700336537H1 SATMON019 g1052973 BLASTX 141 1e-12 50 1193 3287
700205308H1 SATMON003 g1052973 BLASTX 133 1e-11 75 1194 5966
700084171H1 SATMON011 g1052972 BLASTN 448 1e-26 66 1195 5966
700084951H1 SATMON011 g2102693 BLASTX 214 1e-22 73 1196 5966
700089353H1 SATMON011 g2102691 BLASTX 195 1e-20 72 1197 5966
700220723H1 SATMON011 g1915974 BLASTX 198 1e-20 73 1198 5966
700084412H1 SATMON011 g2102693 BLASTX 179 1e-19 76 1199 5966
700085628H1 SATMON011 g2102691 BLASTX 180 1e-18 72 1200 5966
700027982H1 SATMON003 g2102691 BLASTX 178 1e-17 72 1201 5966
700106884H1 SATMON010 g1915974 BLASTX 148 1e-13 75 1202 5966
700053135H1 SATMON008 g1915974 BLASTX 131 1e-11 73 1203 5966
700027988H1 SATMON003 g1915974 BLASTX 134 1e-11 65 1204 5966
700207083H1 SATMON003 g1915974 BLASTX 100 1e-10 46 1205 5966
700158574H1 SATMON012 g1915974 BLASTX 120 1e-9 50 1206 2754
LIB3061-030- LIB3061 g1052972 BLASTN 882 1e-64 67 Q1-K1-G12 1207
2754 LIB3061-030- LIB3061 g1052972 BLASTN 751 1e-52 68 Q1-K1-G11
1208 3287 LIB3067-040- LIB3067 g1052972 BLASTN 657 1e-44 64
Q1-K1-H10 1209 3287 LIB84-024- LIB84 g1052972 BLASTN 638 1e-42 64
Q1-E1-H7 1210 3287 LIB3069-045- LIB3069 g1052972 BLASTN 592 1e-38
61 Q1-K1-F6 1211 3287 LIB3061-014- LIB3061 g1052973 BLASTX 175
1e-36 41 Q1-K1-A3 1212 3287 LIB3062-019- LIB3062 g1052973 BLASTX
154 1e-30 68 Q1-K1-H11 1213 3287 LIB3067-054- LIB3067 g1052972
BLASTN 495 1e-30 61 Q1-K1-C9 1214 3287 LIB3067-022- LIB3067
g1052973 BLASTX 141 1e-27 68 Q1-K1-H4 1215 3287 LIB3069-045-
LIB3069 g1052972 BLASTN 439 1e-25 57 Q1-K1-F2 MAIZE NDP-KINASE 1216
-700575072 700575072H1 SATMON030 g303849 BLASTX 74 1e-13 89 1217
-701170773 701170773H1 SATMONN05 g1777930 BLASTX 132 1e-30 71 1218
2462 700050003H1 SATMON003 g218233 BLASTN 656 1e-58 83 1219 2462
700204789H1 SATMON003 g218233 BLASTN 780 1e-58 87 1220 2462
700049819H1 SATMON003 g218233 BLASTN 786 1e-58 86 1221 2462
700204211H1 SATMON003 g218233 BLASTN 786 1e-58 86 1222 2462
700205742H1 SATMON003 g218233 BLASTN 763 1e-57 86 1223 2462
700207611H1 SATMON016 g218233 BLASTN 764 1e-57 87 1224 2462
700072505H1 SATMON007 g218233 BLASTN 740 1e-55 86 1225 2462
700236468H1 SATMON010 g218233 BLASTN 710 1e-52 86 1226 2462
701181270H1 SATMONN06 g218233 BLASTN 445 1e-51 86 1227 2462
700573201H1 SATMON030 g218233 BLASTN 691 1e-51 81 1228 2462
700452623H1 SATMON028 g218233 BLASTN 694 1e-51 85 1229 2462
700351523H1 SATMON023 g218233 BLASTN 679 1e-50 86 1230 2462
700042795H1 SATMON004 g218233 BLASTN 630 1e-45 86 1231 2462
700445979H1 SATMON027 g218233 BLASTN 595 1e-43 86 1232 2462
700201855H1 SATMON003 g218233 BLASTN 604 1e-43 87 1233 2462
700573101H1 SATMON030 g218233 BLASTN 594 1e-42 78 1234 2462
700049543H1 SATMON003 g218233 BLASTN 577 1e-41 79 1235 2462
700432359H1 SATMONN01 g218233 BLASTN 561 1e-40 81 1236 2462
701182021H1 SATMONN06 g218233 BLASTN 561 1e-40 85 1237 2462
701182019H1 SATMONN06 g218233 BLASTN 566 1e-40 86 1238 2462
700150928H1 SATMON007 g218233 BLASTN 569 1e-40 85 1239 2462
700202824H1 SATMON003 g218233 BLASTN 336 1e-39 86 1240 2462
700451056H1 SATMON028 g218233 BLASTN 553 1e-39 85 1241 2462
700449958H1 SATMON028 g218233 BLASTN 544 1e-38 86 1242 2462
700347592H1 SATMON023 g218233 BLASTN 403 1e-34 78 1243 2462
700573195H1 SATMON030 g218233 BLASTN 200 1e-22 84 1244 2462
700582836H1 SATMON031 g303849 BLASTX 157 1e-15 83 1245 2462
700029459H1 SATMON003 g303849 BLASTX 134 1e-11 84 1246 27065
700583429H1 SATMON031 g1064895 BLASTX 72 1e-13 54 1247 -L1482546
LIB148-007- LIB148 g218233 BLASTN 359 1e-19 75 Q1-E1-E6 1248 2462
LIB3067-039- LIB3067 g218233 BLASTN 711 1e-52 82 Q1-K1-B10 1249
2462 LIB3078-001- LIB3078 g218233 BLASTN 488 1e-49 85 Q1-K1-F3 1250
2462 LIB3067-029- LIB3067 g1236951 BLASTX 166 1e-31 96 Q1-K1-C3
1251 25174 LIB189-022- LIB189 g758643 BLASTN 440 1e-25 76 Q1-E1-E9
MAIZE GLUCOSE-6-PHOSPHATE 1-DEHYDROGENASE 1252 -700047645
700047645H1 SATMON003 g471345 BLASTX 193 1e-21 58 1253 -700210379
700210379H1 SATMON016 g1480344 BLASTX 103 1e-10 85 1254 9135
700203121H1 SATMON003 g1166405 BLASTX 108 1e-10 78 MAIZE
PHOSPHOGLUCOMUTASE 1255 -700045655 700045655H1 SATMON004 g534982
BLASTX 144 1e-12 65 1256 -700053330 700053330H1 SATMON009 g3294467
BLASTX 211 1e-23 71 1257 -700102193 700102193H1 SATMON010 g534982
BLASTX 145 1e-14 53 1258 -700166982 700166982H1 SATMON013 g2795876
BLASTX 168 1e-16 52 1259 -700169540 700169540H1 SATMON013 g534982
BLASTX 180 1e-17 61 1260 -700210088 700210088H1 SATMON016 g534982
BLASTX 328 1e-38 55 1261 -700573194 700573194H1 SATMON030 g534982
BLASTX 192 1e-21 54 1262 -700616588 700616588H1 SATMON033 g3294468
BLASTN 593 1e-48 95 1263 119 700574655H1 SATMON030 g3294466 BLASTN
1705 1e-133 98 1264 119 700574672H1 SATMON030 g3294466 BLASTN 820
1e-121 100 1265 119 700100992H1 SATMON009 g3294466 BLASTN 1545
1e-119 99 1266 119 700615409H1 SATMON033 g3294466 BLASTN 1050
1e-118 100 1267 119 700210693H1 SATMON016 g3294468 BLASTN 1515
1e-117 100 1268 119 700381526H1 SATMON023 g3294468 BLASTN 1490
1e-115 100 1269 119 700026372H1 SATMON003 g3294466 BLASTN 1463
1e-113 99 1270 119 700201578H1 SATMON003 g3294468 BLASTN 677 1e-112
96 1271 119 700101083H1 SATMON009 g3294468 BLASTN 1430 1e-110 100
1272 119 700217101H1 SATMON016 g3294468 BLASTN 1420 1e-109 100 1273
119 700222466H1 SATMON011 g3294466 BLASTN 957 1e-106 97 1274 119
700072492H1 SATMON007 g3294466 BLASTN 1381 1e-106 99 1275 119
700043724H1 SATMON004 g3294468 BLASTN 1390 1e-106 100 1276 119
700346762H1 SATMON021 g3294468 BLASTN 1333 1e-102 94 1277 119
700347741H1 SATMON023 g3294468 BLASTN 1339 1e-102 97 1278 119
700550792H1 SATMON022 g3294466 BLASTN 731 1e-101 99 1279 119
700380144H1 SATMON021 g3294466 BLASTN 1216 1e-98 97 1280 119
700241526H1 SATMON010 g3294466 BLASTN 1285 1e-98 100 1281 119
700380456H1 SATMON021 g3294468 BLASTN 650 1e-97 99 1282 119
700238734H1 SATMON010 g3294466 BLASTN 974 1e-97 97 1283 119
700083634H1 SATMON011 g3294468 BLASTN 1265 1e-96 100 1284 119
700383086H1 SATMON024 g3294466 BLASTN 961 1e-94 96 1285 119
700169630H1 SATMON013 g3294466 BLASTN 1245 1e-94 100 1286 119
701177766H1 SATMONN05 g3294466 BLASTN 1187 1e-93 97 1287 119
700142461H1 SATMON012 g3294466 BLASTN 1231 1e-93 99
1288 119 700044235H1 SATMON004 g3294466 BLASTN 1175 1e-89 100 1289
119 700216921H1 SATMON016 g3294466 BLASTN 1165 1e-88 100 1290 119
700333779H1 SATMON019 g3294466 BLASTN 996 1e-87 96 1291 119
700021881H1 SATMON001 g3294468 BLASTN 1120 1e-84 100 1292 119
700049194H1 SATMON003 g3294468 BLASTN 940 1e-82 98 1293 119
700164477H1 SATMON013 g3294466 BLASTN 1091 1e-82 99 1294 119
700169514H1 SATMON013 g3294468 BLASTN 865 1e-80 100 1295 119
700050896H1 SATMON003 g3294466 BLASTN 591 1e-76 94 1296 119
700172394H1 SATMON013 g3294466 BLASTN 1024 1e-76 99 1297 119
700211437H1 SATMON016 g3294466 BLASTN 994 1e-73 99 1298 119
700084535H1 SATMON011 g3294468 BLASTN 973 1e-72 99 1299 119
700203439H1 SATMON003 g3294466 BLASTN 765 1e-71 100 1300 119
700257833H1 SATMON017 g3294468 BLASTN 611 1e-69 94 1301 119
700621831H1 SATMON034 g3294466 BLASTN 412 1e-52 90 1302 119
700354511H1 SATMON024 g3294468 BLASTN 703 1e-52 91 1303 119
700203525H1 SATMON003 g3294468 BLASTN 708 1e-50 99 1304 119
700020476H1 SATMON001 g3294468 BLASTN 658 1e-45 99 1305 119
700050562H1 SATMON003 g3294466 BLASTN 544 1e-42 88 1306 119
700613868H1 SATMON033 g3294466 BLASTN 615 1e-42 100 1307 119
700574982H1 SATMON030 g3294466 BLASTN 473 1e-35 97 1308 119
700049512H1 SATMON003 g3294466 BLASTN 268 1e-29 95 1309 119
700260372H2 SATMON017 g3294466 BLASTN 226 1e-10 89 1310 16726
700082801H1 SATMON011 g2829893 BLASTX 278 1e-30 55 1311 16726
700212054H1 SATMON016 g2829893 BLASTX 220 1e-23 53 1312 19462
700097450H1 SATMON009 g1814400 BLASTN 323 1e-29 64 1313 19462
700441165H1 SATMON026 g1408296 BLASTX 239 1e-25 61 1314 24348
700379424H1 SATMON020 g3294466 BLASTN 707 1e-50 98 1315 2587
700089556H1 SATMON011 g2829893 BLASTX 117 1e-8 67 1316 3016
700204345H1 SATMON003 g3294468 BLASTN 1784 1e-139 98 1317 3016
700098713H1 SATMON009 g3294468 BLASTN 1516 1e-117 99 1318 3016
700084751H1 SATMON011 g3294466 BLASTN 1475 1e-114 100 1319 3016
700351326H1 SATMON023 g3294468 BLASTN 1460 1e-112 100 1320 3016
700097161H1 SATMON009 g3294466 BLASTN 1308 1e-109 98 1321 3016
700266423H1 SATMON017 g3294468 BLASTN 1065 1e-108 96 1322 3016
700349605H1 SATMON023 g3294466 BLASTN 1335 1e-107 100 1323 3016
700350209H1 SATMON023 g3294468 BLASTN 1188 1e-106 97 1324 3016
700265291H1 SATMON017 g3294468 BLASTN 873 1e-100 98 1325 3016
700457572H1 SATMON029 g3294466 BLASTN 1288 1e-98 98 1326 3016
700334810H1 SATMON019 g3294468 BLASTN 863 1e-97 99 1327 3016
700194444H1 SATMON014 g3294466 BLASTN 1265 1e-96 100 1328 3016
700457426H1 SATMON029 g3294466 BLASTN 1236 1e-94 98 1329 3016
700210958H1 SATMON016 g3294466 BLASTN 1148 1e-92 98 1330 3016
700075135H1 SATMON007 g3294468 BLASTN 1219 1e-92 97 1331 3016
700152065H1 SATMON007 g3294466 BLASTN 1135 1e-90 99 1332 3016
700219672H1 SATMON011 g3294468 BLASTN 823 1e-89 99 1333 3016
700170425H1 SATMON013 g3294466 BLASTN 1110 1e-83 100 1334 3016
700153495H1 SATMON007 g3294468 BLASTN 640 1e-82 100 1335 3016
700348567H1 SATMON023 g3294468 BLASTN 557 1e-81 87 1336 3016
700803158H1 SATMON036 g3294468 BLASTN 630 1e-60 85 1337 3016
700264923H1 SATMON017 g3294468 BLASTN 340 1e-50 98 1338 3016
700615715H1 SATMON033 g3294466 BLASTN 567 1e-48 96 1339 3016
700027830H1 SATMON003 g3294468 BLASTN 632 1e-43 95 1340 3016
700350539H1 SATMON023 g3294466 BLASTN 333 1e-41 96 1341 4562
700044891H1 SATMON004 g3294466 BLASTN 650 1e-45 74 1342 4562
700215538H1 SATMON016 g3294466 BLASTN 555 1e-37 67 1343 9894
700220429H1 SATMON011 g3294468 BLASTN 1302 1e-99 99 1344 9894
700236461H1 SATMON010 g3294466 BLASTN 1054 1e-90 97 1345 -L30594453
LIB3059-042- LIB3059 g1814401 BLASTX 290 1e-49 58 Q1-K1-B5 1346
-L30605287 LIB3060-049- LIB3060 g534982 BLASTX 172 1e-34 77
Q1-K1-B7 1347 119 LIB3059-019- LIB3059 g1881692 BLASTN 2094 1e-165
98 Q1-K1-H1 1348 119 LIB3059-031- LIB3059 g1881692 BLASTN 1926
1e-151 96 Q1-K1-H10 1349 119 LIB3069-012- LIB3069 g1881692 BLASTN
1188 1e-146 90 Q1-K1-F2 1350 119 LIB36-019- LIB36 g1881692 BLASTN
1783 1e-139 90 Q1-E1-A7 1351 119 LIB3078-023- LIB3078 g1881692
BLASTN 860 1e-124 87 Q1-K1-C3 1352 119 LIB3067-058- LIB3067
g1881692 BLASTN 991 1e-114 99 Q1-K1-G1 1353 119 LIB3062-048-
LIB3062 g1881692 BLASTN 1181 1e-103 97 Q1-K1-B7 1354 119
LIB3069-023- LIB3069 g1881692 BLASTN 1176 1e-87 84 Q1-K1-G4 1355
119 LIB3069-025- LIB3069 g1881692 BLASTN 611 1e-65 91 Q1-K1-B6 1356
24348 LIB3066-043- LIB3066 g1881692 BLASTN 560 1e-37 100 Q1-K1-F11
1357 24348 LIB3067-048- LIB3067 g1881692 BLASTN 543 1e-36 99
Q1-K1-F3 1358 3016 LIB143-002- LIB143 g2829893 BLASTX 224 1e-51 72
Q1-E1-C12 1359 3016 LIB189-034- LIB189 g2829893 BLASTX 216 1e-48 68
Q1-E1-A11 1360 3016 LIB3069-043- LIB3069 g1814401 BLASTX 98 1e-32
64 Q1-K1-D5 MAIZE UDP-GLUCOSE PYROPHOSPHORYLASE 1361 -700197315
700197315H1 SATMON014 g1388021 BLASTX 122 1e-9 70 1362 -700203530
700203530H1 SATMON003 g1212995 BLASTN 568 1e-38 78 1363 -700267284
700267284H1 SATMON017 g1212996 BLASTX 150 1e-13 87 1364 -700336683
700336683H1 SATMON019 g1752677 BLASTX 150 1e-27 82 1365 -700342324
700342324H1 SATMON021 g3107931 BLASTX 95 1e-14 80 1366 -700354856
700354856H1 SATMON024 g1388021 BLASTX 121 1e-22 75 1367 -700613858
700613858H1 SATMON033 g1212995 BLASTN 776 1e-59 88 1368 14982
700028996H1 SATMON003 g1212995 BLASTN 560 1e-37 76 1369 14982
700155115H1 SATMON007 g1212995 BLASTN 399 1e-31 81 1370 14982
700356747H1 SATMON024 g1388021 BLASTX 166 1e-15 76 1371 19537
700573761H1 SATMON030 g1212995 BLASTN 954 1e-70 79 1372 19537
700208049H1 SATMON016 g1212995 BLASTN 901 1e-66 78 1373 19537
700086382H1 SATMON011 g1212995 BLASTN 885 1e-64 77 1374 69
700091881H1 SATMON011 g1212995 BLASTN 844 1e-105 89 1375 69
700624406H1 SATMON034 g1212995 BLASTN 816 1e-97 88 1376 69
700211464H1 SATMON016 g1212995 BLASTN 1251 1e-95 88 1377 69
700099836H1 SATMON009 g1212995 BLASTN 1239 1e-94 88 1378 69
700084756H1 SATMON011 g1212995 BLASTN 1240 1e-94 90 1379 69
700076136H1 SATMON007 g1212995 BLASTN 1243 1e-94 89 1380 69
700073071H1 SATMON007 g1212995 BLASTN 1163 1e-88 86 1381 69
700614228H1 SATMON033 g1212995 BLASTN 1013 1e-87 84 1382 69
700379926H1 SATMON021 g1212995 BLASTN 1138 1e-86 88 1383 69
700089172H1 SATMON011 g1212995 BLASTN 1141 1e-86 88 1384 69
700265063H1 SATMON017 g1212995 BLASTN 1147 1e-86 86 1385 69
700085964H1 SATMON011 g1212995 BLASTN 1135 1e-85 85 1386 69
700282281H2 SATMON023 g1212995 BLASTN 1136 1e-85 86 1387 69
700429855H1 SATMONN01 g1212995 BLASTN 1114 1e-84 89 1388 69
700347453H1 SATMON023 g1212995 BLASTN 1117 1e-84 87 1389 69
700265087H1 SATMON017 g1212995 BLASTN 1120 1e-84 87 1390 69
700092705H1 SATMON008 g1212995 BLASTN 1122 1e-84 87 1391 69
700212686H1 SATMON016 g1212995 BLASTN 1123 1e-84 91 1392 69
700623332H1 SATMON034 g1212995 BLASTN 800 1e-83 86 1393 69
700041787H1 SATMON004 g1212995 BLASTN 1091 1e-82 91 1394 69
700219031H1 SATMON011 g1212995 BLASTN 1093 1e-82 89 1395 69
700218632H1 SATMON011 g1212995 BLASTN 1086 1e-81 90 1396 69
700211962H1 SATMON016 g1212995 BLASTN 1086 1e-81 86 1397 69
700220729H1 SATMON011 g1212995 BLASTN 916 1e-80 84 1398 69
700197025H1 SATMON014 g1212995 BLASTN 1063 1e-79 89 1399 69
700086546H1 SATMON011 g1212995 BLASTN 1049 1e-78 86 1400 69
700217064H1 SATMON016 g1212995 BLASTN 1051 1e-78 88 1401 69
700799128H1 SATMON036 g1212995 BLASTN 618 1e-77 88 1402 69
700265488H1 SATMON017 g1212995 BLASTN 1030 1e-77 84 1403 69
700043842H1 SATMON004 g1212995 BLASTN 1035 1e-77 87 1404 69
700236833H1 SATMON010 g1212995 BLASTN 1035 1e-77 87 1405 69
700219083H1 SATMON011 g1212995 BLASTN 1036 1e-77 88 1406 69
700042338H1 SATMON004 g1212995 BLASTN 1037 1e-77 87 1407 69
700352484H1 SATMON023 g1212995 BLASTN 1038 1e-77 85 1408 69
700083771H1 SATMON011 g1212995 BLASTN 613 1e-76 91 1409 69
700473855H1 SATMON025 g1212995 BLASTN 755 1e-76 85 1410 69
700353922H1 SATMON024 g1212995 BLASTN 1024 1e-76 85 1411 69
700023267H1 SATMON003 g1212995 BLASTN 1007 1e-75 89 1412 69
700157596H1 SATMON012 g1212995 BLASTN 1008 1e-75 87 1413 69
700218718H1 SATMON011 g1212995 BLASTN 1012 1e-75 86 1414 69
700162316H1 SATMON012 g1212995 BLASTN 626 1e-74 80 1415 69
700046475H1 SATMON004 g1212995 BLASTN 1003 1e-74 85 1416 69
700466010H1 SATMON025 g1212995 BLASTN 558 1e-73 82 1417 69
700571392H1 SATMON030 g1212995 BLASTN 985 1e-73 85 1418 69
700165241H1 SATMON013 g1212995 BLASTN 987 1e-73 85 1419 69
700457410H1 SATMON029 g1212995 BLASTN 988 1e-73 87 1420 69
700194672H1 SATMON014 g1212995 BLASTN 963 1e-71 86 1421 69
700089746H1 SATMON011 g1212995 BLASTN 964 1e-71 83 1422 69
700801620H1 SATMON036 g1212995 BLASTN 536 1e-70 91 1423 69
700264785H1 SATMON017 g1212995 BLASTN 952 1e-70 84 1424 69
700244093H1 SATMON010 g1212995 BLASTN 954 1e-70 85 1425 69
700043787H1 SATMON004 g1212995 BLASTN 957 1e-70 85 1426 69
700267269H1 SATMON017 g1212995 BLASTN 867 1e-69 85 1427 69
700167985H1 SATMON013 g1212995 BLASTN 940 1e-69 89 1428 69
700799042H1 SATMON036 g1212995 BLASTN 812 1e-68 89 1429 69
700163824H1 SATMON013 g1212995 BLASTN 888 1e-65 86 1430 69
700098307H1 SATMON009 g1212995 BLASTN 461 1e-63 81 1431 69
700805267H1 SATMON036 g1212995 BLASTN 734 1e-63 88 1432 69
700204843H1 SATMON003 g1212995 BLASTN 854 1e-62 88 1433 69
700206721H1 SATMON003 g1212995 BLASTN 461 1e-61 81 1434 69
700018559H1 SATMON001 g1212995 BLASTN 847 1e-61 85 1435 69
700026241H1 SATMON003 g1212995 BLASTN 847 1e-61 87 1436 69
700099987H1 SATMON009 g1212995 BLASTN 461 1e-60 81 1437 69
700475628H1 SATMON025 g1212995 BLASTN 750 1e-59 80 1438 69
700016675H1 SATMON001 g1212995 BLASTN 814 1e-59 86 1439 69
700150144H1 SATMON007 g1212995 BLASTN 822 1e-59 86 1440 69
700267260H1 SATMON017 g1212995 BLASTN 461 1e-58 80 1441 69
700261336H1 SATMON017 g1212995 BLASTN 564 1e-58 82 1442 69
700618652H1 SATMON033 g1212995 BLASTN 730 1e-58 78 1443 69
700469914H1 SATMON025 g1212995 BLASTN 735 1e-58 89 1444 69
700048027H1 SATMON003 g1212995 BLASTN 807 1e-58 83 1445 69
700165703H1 SATMON013 g1212995 BLASTN 796 1e-57 85 1446 69
700265403H1 SATMON017 g1212995 BLASTN 797 1e-57 78 1447 69
700099428H1 SATMON009 g1212995 BLASTN 474 1e-56 88 1448 69
700243212H1 SATMON010 g1212995 BLASTN 779 1e-56 84 1449 69
700092996H1 SATMON008 g1212995 BLASTN 789 1e-56 84 1450 69
700803035H1 SATMON036 g1212995 BLASTN 436 1e-54 80 1451 69
700235803H1 SATMON010 g1212995 BLASTN 688 1e-54 79 1452 69
700172581H1 SATMON013 g1212995 BLASTN 754 1e-54 79 1453 69
700214715H1 SATMON016 g1212995 BLASTN 762 1e-54 86 1454 69
700223082H1 SATMON011 g1212995 BLASTN 764 1e-54 84 1455 69
700093483H1 SATMON008 g1212995 BLASTN 357 1e-51 88 1456 69
700261920H1 SATMON017 g1212995 BLASTN 363 1e-51 82 1457 69
700221718H1 SATMON011 g1212995 BLASTN 363 1e-51 83 1458 69
700453106H1 SATMON028 g1212995 BLASTN 670 1e-51 82 1459 69
700210506H1 SATMON016 g1212995 BLASTN 461 1e-50 85 1460 69
700212333H1 SATMON016 g1212995 BLASTN 443 1e-49 83 1461 69
700072654H1 SATMON007 g1212995 BLASTN 443 1e-49 79 1462 69
700218282H1 SATMON016 g1212995 BLASTN 452 1e-49 85 1463 69
700263725H1 SATMON017 g1212995 BLASTN 662 1e-49 80 1464 69
700343083H1 SATMON021 g1212995 BLASTN 388 1e-48 80 1465 69
700219739H1 SATMON011 g1212995 BLASTN 443 1e-48 81 1466 69
700620336H1 SATMON034 g1212995 BLASTN 621 1e-48 88 1467 69
700264630H1 SATMON017 g1212995 BLASTN 377 1e-47 80 1468 69
700439242H1 SATMON026 g1212995 BLASTN 648 1e-47 83 1469 69
700259658H1 SATMON017 g1212995 BLASTN 511 1e-45 79 1470 69
700263521H1 SATMON017 g1212995 BLASTN 461 1e-44 79 1471 69
700261387H1 SATMON017 g1212995 BLASTN 461 1e-44 80 1472 69
700439277H1 SATMON026 g1212995 BLASTN 461 1e-43 84 1473 69
700452839H1 SATMON028 g1212995 BLASTN 544 1e-43 77 1474 69
700220236H1 SATMON011 g1212995 BLASTN 448 1e-40 84 1475 69
700472602H1 SATMON025 g1212995 BLASTN 254 1e-38 81 1476 69
700266424H1 SATMON017 g1212995 BLASTN 499 1e-37 80 1477 69
700449187H1 SATMON028 g1212995 BLASTN 540 1e-36 81 1478 69
700202731H1 SATMON003 g1212995 BLASTN 543 1e-36 79 1479 69
700156144H2 SATMON007 g1212995 BLASTN 441 1e-35 76 1480 69
700442679H1 SATMON026 g1212995 BLASTN 533 1e-35 80 1481 69
700449879H2 SATMON028 g1212995 BLASTN 535 1e-35 81 1482 69
700266832H1 SATMON017 g1212995 BLASTN 346 1e-34 77 1483 69
700332389H1 SATMON019 g1212995 BLASTN 382 1e-34 84 1484 69
700804202H1 SATMON036 g1212995 BLASTN 436 1e-34 76 1485 69
700151037H1 SATMON007 g1212995 BLASTN 443 1e-34 79 1486 69
700802810H1 SATMON036 g1212995 BLASTN 525 1e-34 85 1487 69
700455879H1 SATMON029 g1212995 BLASTN 448 1e-32 72 1488 69
700427769H1 SATMONN01 g1212995 BLASTN 481 1e-31 81 1489 69
700464626H1 SATMON025 g1212995 BLASTN 388 1e-30 76 1490 69
700439228H1 SATMON026 g1212995 BLASTN 470 1e-30 77 1491 69
700256847H1 SATMON017 g1212995 BLASTN 264 1e-29 85 1492 69
700204881H1 SATMON003 g1212995 BLASTN 430 1e-29 81 1493 69
700076032H1 SATMON007 g1212995 BLASTN 218 1e-26 72 1494 69
700426342H1 SATMONN01 g1212995 BLASTN 443 1e-26 79 1495 69
700209062H1 SATMON016 g1212995 BLASTN 279 1e-24 80 1496 69
700076988H1 SATMON007 g1212995 BLASTN 337 1e-24 83 1497 69
700349778H1 SATMON023 g1212995 BLASTN 406 1e-24 81 1498 69
700261886H1 SATMON017 g1212995 BLASTN 287 1e-15 80 1499 69
700426642H1 SATMONN01 g1388021 BLASTX 161 1e-14 76 1500 69
700155195H1 SATMON007 g1212995 BLASTN 155 1e-10 81 1501 69
700211992H1 SATMON016 g1212996 BLASTX 118 1e-9 85 1502 -L1485255
LIB148-053- LIB148 g1212995 BLASTN 691 1e-48 80 Q1-E1-E12 1503
-L30663959 LIB3066-015- LIB3066 g218000 BLASTN 251 1e-9 65
Q1-K1-F12 1504 19537 LIB3066-025- LIB3066 g1212995 BLASTN 1001
1e-74 79 Q1-K1-E5 1505 69 LIB3059-023- LIB3059 g1212995 BLASTN 1301
1e-133 89 Q1-K1-C8 1506 69 LIB3078-022- LIB3078 g1212995 BLASTN
1656 1e-129 86 Q1-K1-C1 1507 69 LIB3059-037- LIB3059 g1212995
BLASTN 1646 1e-128 86 Q1-K1-H5 1508 69 LIB3061-030- LIB3061
g1212995 BLASTN 1493 1e-124 86 Q1-K1-A12 1509 69 LIB3061-023-
LIB3061 g1212995 BLASTN 1598 1e-124 86 Q1-K1-A1 1510 69
LIB3079-001- LIB3079 g1212995 BLASTN 1600 1e-124 83 Q1-K1-D12 1511
69 LIB189-028- LIB189 g1212995 BLASTN 1583 1e-123 87 Q1-E1-E3
1512 69 LIB3067-017- LIB3067 g1212995 BLASTN 1364 1e-120 88
Q1-K1-D9 1513 69 LIB3068-007- LIB3068 g1212995 BLASTN 1501 1e-116
85 Q1-K1-F9 1514 69 LIB3069-025- LIB3069 g1212995 BLASTN 1487
1e-115 85 Q1-K1-E9 1515 69 LIB3069-026- LIB3069 g1212995 BLASTN
1453 1e-112 85 Q1-K1-E11 1516 69 LIB3066-006- LIB3066 g1212995
BLASTN 1077 1e-107 83 Q1-K1-G12 1517 69 LIB3067-027- LIB3067
g1212995 BLASTN 1401 1e-107 86 Q1-K1-D12 1518 69 LIB189-010- LIB189
g1212995 BLASTN 1368 1e-105 85 Q1-E1-H10 1519 69 LIB3066-015-
LIB3066 g1212995 BLASTN 1289 1e-104 82 Q1-K1-G12 1520 69
LIB3061-016- LIB3061 g1212995 BLASTN 1180 1e-102 84 Q1-K1-G11 1521
69 LIB3059-032- LIB3059 g1212995 BLASTN 1334 1e-102 87 Q1-K1-G11
1522 69 LIB3067-059- LIB3067 g1212995 BLASTN 1090 1e-100 85
Q1-K1-G12 1523 69 LIB3061-049- LIB3061 g1212995 BLASTN 1223 1e-98
79 Q1-K1-C8 1524 69 LIB3062-044- LIB3062 g1212995 BLASTN 1259 1e-96
83 Q1-K1-F2 1525 69 LIB3061-010- LIB3061 g1212995 BLASTN 1180 1e-95
84 Q1-K1-F5 1526 69 LIB3067-018- LIB3067 g1212995 BLASTN 1127 1e-89
82 Q1-K1-A12 1527 69 LIB3067-030- LIB3067 g1212995 BLASTN 1171
1e-88 83 Q1-K1-F4 1528 69 LIB3062-021- LIB3062 g1212995 BLASTN 1138
1e-86 87 Q1-K1-F10 1529 69 LIB3061-034- LIB3061 g1212995 BLASTN
1148 1e-86 85 Q1-K1-D8 1530 69 LIB3066-049- LIB3066 g1212995 BLASTN
1134 1e-85 83 Q1-K1-C1 1531 69 LIB3078-002- LIB3078 g1212995 BLASTN
859 1e-77 86 Q1-K1-F5 1532 69 LIB84-011- LIB84 g1212995 BLASTN 1020
1e-76 83 Q1-E1-G9 1533 69 LIB3067-043- LIB3067 g1212995 BLASTN 574
1e-59 77 Q1-K1-D2 1534 69 LIB189-003- LIB189 g1212995 BLASTN 247
1e-40 77 Q1-E1-G5 1535 69 LIB3062-008- LIB3062 g1212995 BLASTN 576
1e-37 63 Q1-K1-E6 1536 69 LIB189-016- LIB189 g1212996 BLASTX 156
1e-30 78 Q1-E1-H7 1537 69 LIB3067-007- LIB3067 g1212996 BLASTX 145
1e-28 82 Q1-K1-G4 SOYBEAN TRIOSE PHOSPHATE ISOMERASE 1538
-700743237 700743237H1 SOYMON012 g407525 BLASTX 173 1e-17 91 1539
-700977730 700977730H1 SOYMON009 g602589 BLASTN 373 1e-20 71 1540
-701056176 701056176H1 SOYMON032 g806311 BLASTN 752 1e-53 74 1541
-701110172 701110172H1 SOYMON036 g806311 BLASTN 801 1e-57 78 1542
10244 700995141H1 SOYMON011 g806311 BLASTN 470 1e-30 87 1543 10244
701124548H1 SOYMON037 g806311 BLASTN 490 1e-30 88 1544 10244
700739771H1 SOYMON012 g806311 BLASTN 329 1e-16 77 1545 10244
700999820H1 SOYMON018 g806312 BLASTX 147 1e-13 84 1546 10244
701119858H1 SOYMON037 g806312 BLASTX 118 1e-9 72 1547 10535
700988684H1 SOYMON009 g806311 BLASTN 905 1e-66 79 1548 10535
700902425H1 SOYMON027 g806311 BLASTN 872 1e-63 80 1549 1357
701069004H1 SOYMON034 g806311 BLASTN 832 1e-60 81 1550 1357
701151554H1 SOYMON031 g806311 BLASTN 568 1e-38 82 1551 1357
700659936H1 SOYMON004 g806311 BLASTN 545 1e-36 79 1552 16
700680927H1 SOYMON008 g256119 BLASTN 1020 1e-81 78 1553 16
700656871H1 SOYMON004 g256119 BLASTN 903 1e-66 81 1554 16
701124364H1 SOYMON037 g256119 BLASTN 872 1e-64 80 1555 16
701134707H2 SOYMON038 g256119 BLASTN 874 1e-64 81 1556 16
700673750H1 SOYMON007 g256119 BLASTN 781 1e-60 81 1557 16
701123269H1 SOYMON037 g602589 BLASTN 819 1e-59 78 1558 16
701004846H1 SOYMON019 g256119 BLASTN 801 1e-58 80 1559 16
700993362H1 SOYMON011 g256119 BLASTN 808 1e-58 80 1560 16
701005445H1 SOYMON019 g256119 BLASTN 630 1e-56 78 1561 16
701134327H1 SOYMON038 g602589 BLASTN 782 1e-56 79 1562 16
701148169H1 SOYMON031 g602589 BLASTN 574 1e-51 76 1563 16
701153410H1 SOYMON031 g602589 BLASTN 451 1e-50 80 1564 16
700830168H1 SOYMON019 g256119 BLASTN 705 1e-50 77 1565 16
701120627H1 SOYMON037 g602589 BLASTN 715 1e-50 78 1566 16
700975358H1 SOYMON009 g602589 BLASTN 628 1e-49 77 1567 16
700755979H1 SOYMON014 g602589 BLASTN 697 1e-49 79 1568 16
701131374H1 SOYMON038 g602589 BLASTN 703 1e-49 79 1569 16
700994166H1 SOYMON011 g602589 BLASTN 513 1e-47 77 1570 16
701138038H1 SOYMON038 g602589 BLASTN 672 1e-47 77 1571 16
700974248H1 SOYMON005 g602589 BLASTN 658 1e-46 77 1572 16
700655832H1 SOYMON004 g602589 BLASTN 664 1e-46 78 1573 16
700758320H1 SOYMON015 g602589 BLASTN 409 1e-45 80 1574 16
701064709H1 SOYMON034 g602589 BLASTN 477 1e-45 78 1575 16
701138504H1 SOYMON038 g602589 BLASTN 591 1e-45 76 1576 16
700980284H1 SOYMON009 g602589 BLASTN 652 1e-45 79 1577 16
701133585H2 SOYMON038 g602589 BLASTN 634 1e-44 78 1578 16
700674706H1 SOYMON007 g602589 BLASTN 634 1e-44 78 1579 16
700964927H1 SOYMON022 g602589 BLASTN 639 1e-44 78 1580 16
700830923H1 SOYMON019 g602589 BLASTN 626 1e-43 76 1581 16
700662845H1 SOYMON005 g602589 BLASTN 617 1e-42 76 1582 16
701133824H1 SOYMON038 g602589 BLASTN 619 1e-42 78 1583 16
700848913H1 SOYMON021 g602589 BLASTN 603 1e-41 77 1584 16
701005984H1 SOYMON019 g602589 BLASTN 604 1e-41 78 1585 16
701140769H1 SOYMON038 g602589 BLASTN 605 1e-41 76 1586 16
700753357H1 SOYMON014 g602589 BLASTN 328 1e-40 78 1587 16
701056336H1 SOYMON032 g602589 BLASTN 344 1e-40 77 1588 16
700895411H1 SOYMON027 g602589 BLASTN 593 1e-40 78 1589 16
701060188H1 SOYMON033 g602589 BLASTN 277 1e-39 80 1590 16
700739461H1 SOYMON012 g602589 BLASTN 573 1e-39 77 1591 16
700941104H1 SOYMON024 g602589 BLASTN 579 1e-39 79 1592 16
700732960H1 SOYMON010 g602589 BLASTN 581 1e-39 78 1593 16
700686476H1 SOYMON008 g602589 BLASTN 583 1e-39 79 1594 16
701054231H1 SOYMON032 g602589 BLASTN 583 1e-39 77 1595 16
700671690H1 SOYMON006 g602589 BLASTN 566 1e-38 77 1596 16
700941174H1 SOYMON024 g602589 BLASTN 569 1e-38 78 1597 16
701125091H1 SOYMON037 g256119 BLASTN 358 1e-37 74 1598 16
700989827H1 SOYMON011 g602589 BLASTN 555 1e-37 78 1599 16
700835006H1 SOYMON019 g602589 BLASTN 555 1e-37 75 1600 16
700834847H1 SOYMON019 g602589 BLASTN 559 1e-37 78 1601 16
700953411H1 SOYMON022 g602589 BLASTN 314 1e-36 80 1602 16
700869222H1 SOYMON016 g602589 BLASTN 541 1e-36 78 1603 16
700850633H1 SOYMON023 g602589 BLASTN 544 1e-36 78 1604 16
700890283H1 SOYMON024 g602589 BLASTN 310 1e-35 80 1605 16
700727079H1 SOYMON009 g414549 BLASTN 358 1e-35 73 1606 16
700892544H1 SOYMON024 g602589 BLASTN 486 1e-35 78 1607 16
700869230H1 SOYMON016 g602589 BLASTN 528 1e-35 78 1608 16
700993034H1 SOYMON011 g602589 BLASTN 518 1e-34 75 1609 16
700975553H1 SOYMON009 g414549 BLASTN 524 1e-34 79 1610 16
700651326H1 SOYMON003 g602589 BLASTN 356 1e-33 80 1611 16
701215308H1 SOYMON035 g414549 BLASTN 450 1e-33 75 1612 16
700654480H1 SOYMON004 g414549 BLASTN 511 1e-33 80 1613 16
701045128H1 SOYMON032 g414549 BLASTN 512 1e-33 78 1614 16
701060759H1 SOYMON033 g414549 BLASTN 513 1e-33 80 1615 16
700741652H1 SOYMON012 g602589 BLASTN 493 1e-32 79 1616 16
700675469H1 SOYMON007 g602589 BLASTN 494 1e-32 78 1617 16
700657787H1 SOYMON004 g414549 BLASTN 495 1e-32 79 1618 16
701009957H2 SOYMON019 g414549 BLASTN 495 1e-32 80 1619 16
700983693H1 SOYMON009 g414549 BLASTN 495 1e-32 80 1620 16
701156784H1 SOYMON031 g602589 BLASTN 495 1e-32 78 1621 16
700893935H1 SOYMON024 g602589 BLASTN 481 1e-31 79 1622 16
701144619H1 SOYMON031 g414549 BLASTN 485 1e-31 78 1623 16
701148851H1 SOYMON031 g602589 BLASTN 487 1e-31 79 1624 16
701058218H1 SOYMON033 g602589 BLASTN 495 1e-31 78 1625 16
700975165H1 SOYMON009 g414549 BLASTN 466 1e-30 80 1626 16
701100165H1 SOYMON028 g602589 BLASTN 485 1e-30 79 1627 16
701150241H1 SOYMON031 g602589 BLASTN 455 1e-29 79 1628 16
701098308H1 SOYMON028 g414549 BLASTN 460 1e-29 79 1629 16
701150440H1 SOYMON031 g602589 BLASTN 462 1e-29 78 1630 16
700685125H1 SOYMON008 g414549 BLASTN 471 1e-29 81 1631 16
701061565H1 SOYMON033 g414549 BLASTN 471 1e-29 81 1632 16
700991418H1 SOYMON011 g602589 BLASTN 394 1e-28 68 1633 16
701156156H1 SOYMON031 g602589 BLASTN 456 1e-28 78 1634 16
701007231H2 SOYMON019 g602589 BLASTN 461 1e-28 79 1635 16
700829667H1 SOYMON019 g414549 BLASTN 333 1e-27 73 1636 16
701156033H1 SOYMON031 g602589 BLASTN 432 1e-27 78 1637 16
701014293H1 SOYMON019 g414549 BLASTN 446 1e-27 77 1638 16
701152138H1 SOYMON031 g414549 BLASTN 450 1e-27 81 1639 16
700945665H1 SOYMON024 g414549 BLASTN 450 1e-27 81 1640 16
701001407H1 SOYMON018 g169820 BLASTN 219 1e-26 72 1641 16
700983185H1 SOYMON009 g414549 BLASTN 435 1e-26 72 1642 16
700752364H1 SOYMON014 g414549 BLASTN 441 1e-26 76 1643 16
700992409H1 SOYMON011 g414549 BLASTN 427 1e-25 75 1644 16
701109396H1 SOYMON036 g414549 BLASTN 420 1e-24 76 1645 16
701151402H1 SOYMON031 g556171 BLASTX 151 1e-23 85 1646 16
701149617H1 SOYMON031 g556171 BLASTX 158 1e-23 86 1647 16
700747310H1 SOYMON013 g414549 BLASTN 406 1e-23 73 1648 16
701139569H1 SOYMON038 g556171 BLASTX 191 1e-22 84 1649 16
701213275H1 SOYMON035 g602589 BLASTN 255 1e-22 80 1650 16
701157185H1 SOYMON031 g556171 BLASTX 197 1e-20 90 1651 16
700655520H1 SOYMON004 g556171 BLASTX 166 1e-19 86 1652 16
701010779H1 SOYMON019 g556171 BLASTX 173 1e-19 64 1653 16
701044104H1 SOYMON032 g556171 BLASTX 188 1e-19 89 1654 16
700867605H1 SOYMON016 g556171 BLASTX 160 1e-17 70 1655 16
701058593H1 SOYMON033 g168647 BLASTX 169 1e-16 94 1656 16
701070286H1 SOYMON034 g168647 BLASTX 164 1e-15 91 1657 16
700877219H1 SOYMON018 g168647 BLASTX 154 1e-14 93 1658 16
700876790H1 SOYMON018 g168647 BLASTX 154 1e-14 93 1659 16
700877212H1 SOYMON018 g168647 BLASTX 154 1e-14 93 1660 16
700760847H1 SOYMON015 g556171 BLASTX 138 1e-13 86 1661 16
700893711H1 SOYMON024 g168647 BLASTX 140 1e-13 82 1662 16
700557532H1 SOYMON001 g256120 BLASTX 115 1e-12 88 1663 16
700793802H1 SOYMON017 g556171 BLASTX 138 1e-12 93 1664 16
700659725H1 SOYMON004 g556171 BLASTX 144 1e-12 47 1665 16
701044545H1 SOYMON032 g556171 BLASTX 144 1e-12 92 1666 16
701037485H1 SOYMON029 g556171 BLASTX 135 1e-11 96 1667 16
700683524H1 SOYMON008 g168647 BLASTX 136 1e-11 90 1668 16
700876711H1 SOYMON018 g168647 BLASTX 109 1e-10 85 1669 16
701155437H1 SOYMON031 g556171 BLASTX 130 1e-10 92 1670 28599
700997892H1 SOYMON018 g806311 BLASTN 834 1e-60 78 1671 31
701053174H1 SOYMON032 g806311 BLASTN 572 1e-37 73 1672 31
700754467H1 SOYMON014 g806312 BLASTX 145 1e-21 66 1673 31
701107430H1 SOYMON036 g806312 BLASTX 199 1e-20 63 1674 31
700985855H1 SOYMON009 g806312 BLASTX 145 1e-18 64 1675 31
701038167H1 SOYMON029 g806312 BLASTX 179 1e-17 61 1676 31
700670393H1 SOYMON006 g806312 BLASTX 167 1e-16 78 1677 31
700559280H1 SOYMON001 g609262 BLASTX 164 1e-15 69 1678 31
700793048H1 SOYMON017 g806312 BLASTX 97 1e-12 60 1679 31
700993683H1 SOYMON011 g806312 BLASTX 103 1e-11 60 1680 31
700663233H1 SOYMON005 g806312 BLASTX 130 1e-11 56 1681 31
700908079H1 SOYMON022 g806312 BLASTX 103 1e-10 60 1682 31
701043447H1 SOYMON029 g609262 BLASTX 126 1e-10 84 1683 31
700740188H1 SOYMON012 g806312 BLASTX 103 1e-8 60 1684 7466
700742922H1 SOYMON012 g806311 BLASTN 435 1e-27 76 1685 7466
700606255H1 SOYMON008 g806312 BLASTX 117 1e-17 80 1686 16
LIB3053-005- LIB3053 g602589 BLASTN 1000 1e-74 77 Q1-N1-F9 1687 16
LIB3039-035- LIB3039 g602589 BLASTN 979 1e-72 78 Q1-E1-C5 1688 16
LIB3039-031- LIB3039 g256119 BLASTN 911 1e-71 80 Q1-E1-A8 1689 16
LIB3030-003- LIB3030 g602589 BLASTN 949 1e-70 78 Q1-B1-C9 1690 16
LIB3039-023- LIB3039 g602589 BLASTN 913 1e-67 78 Q1-E1-H12 1691 16
LIB3039-047- LIB3039 g602589 BLASTN 566 1e-65 75 Q1-E1-D8 1692 16
LIB3039-052- LIB3039 g602589 BLASTN 890 1e-65 77 Q1-E1-D6 1693 16
LIB3039-051- LIB3039 g602589 BLASTN 855 1e-62 78 Q1-E1-A1 1694 16
LIB3049-009- LIB3049 g602589 BLASTN 783 1e-56 78 Q1-E1-G5 1695 16
LIB3039-009- LIB3039 g602589 BLASTN 805 1e-56 78 Q1-E1-C1 1696 16
LIB3055-006- LIB3055 g256119 BLASTN 481 1e-54 78 Q1-N1-H3 1697 16
LIB3055-013- LIB3055 g256119 BLASTN 769 1e-54 79 Q1-N1-C3 1698 16
LIB3049-034- LIB3049 g602589 BLASTN 626 1e-51 76 Q1-E1-A2 1699 16
LIB3049-022- LIB3049 g602589 BLASTN 519 1e-43 78 Q1-E1-F9 1700 16
LIB3049-030- LIB3049 g602589 BLASTN 572 1e-38 77 Q1-E1-C7 1701 16
LIB3040-035- LIB3040 g556171 BLASTX 175 1e-33 82 Q1-E1-C5 1702 16
LIB3040-005- LIB3040 g169820 BLASTN 324 1e-33 76 Q1-E1-H8 1703 16
LIB3028-025- LIB3028 g602589 BLASTN 464 1e-33 78 Q1-B1-D1 1704 16
LIB3039-022- LIB3039 g602589 BLASTN 357 1e-32 73 Q1-E1-D5 1705 16
LIB3052-001- LIB3052 G414549 BLASTN 327 1e-29 73 Q1-B1-C5 1706
28599 LIB3039-047- LIB3039 G806311 BLASTN 1183 1e-94 81 Q1-E1-D9
1707 28599 LIB3039-048- LIB3039 G806311 BLASTN 1007 1e-92 81
Q1-E1-D12 SOYBEAN FRUCTOSE 1,6-BISPHOSPHATE ALDOLASE 1708
-700565253 700565253H1 SOYMON002 G3021337 BLASTN 352 1e-39 76 1709
-700865276 700865276H1 SOYMON016 G3021337 BLASTN 629 1e-43 76 1710
-700873022 700873022H1 SOYMON018 G3696 BLASTX 211 1e-26 70 1711
-700943855 700943855H1 SOYMON024 G20204 BLASTX 202 1e-20 86 1712
-700974965 700974965H1 SOYMON005 g3021337 BLASTN 259 1e-10 84
1713 -701039850 701039850H1 SOYMON029 g22632 BLASTN 408 1e-23 76
1714 -701206840 701206840H1 SOYMON035 g3021338 BLASTX 151 1e-13 83
1715 11792 700654881H1 SOYMON004 g20204 BLASTX 150 1e-13 76 1716
11792 700746016H1 SOYMON013 g3021337 BLASTN 284 1e-12 67 1717 12314
701037190H1 SOYMON029 g3021337 BLASTN 634 1e-44 78 1718 12314
701042664H1 SOYMON029 g3021338 BLASTX 197 1e-20 66 1719 16
700651596H1 SOYMON003 g3021337 BLASTN 1101 1e-83 86 1720 16
700750439H1 SOYMON013 g3021337 BLASTN 1078 1e-81 86 1721 16
700649475H1 SOYMON003 g3021337 BLASTN 1082 1e-81 84 1722 16
700652995H1 SOYMON003 g3021337 BLASTN 1084 1e-81 82 1723 16
700981967H1 SOYMON009 g3021337 BLASTN 1071 1e-80 85 1724 16
700863243H1 SOYMON023 g3021337 BLASTN 1044 1e-78 86 1725 16
700558625H1 SOYMON001 g3021337 BLASTN 1041 1e-77 84 1726 16
700564806H1 SOYMON002 g3021337 BLASTN 1021 1e-76 80 1727 16
700746368H1 SOYMON013 g3021337 BLASTN 897 1e-75 86 1728 16
700960290H1 SOYMON022 g3021337 BLASTN 1009 1e-75 87 1729 16
701055132H1 SOYMON032 g3021337 BLASTN 1011 1e-75 86 1730 16
701056109H1 SOYMON032 g3021337 BLASTN 1012 1e-75 84 1731 16
701119884H1 SOYMON037 g3021337 BLASTN 1014 1e-75 87 1732 16
700898149H1 SOYMON027 g3021337 BLASTN 1015 1e-75 86 1733 16
700661436H1 SOYMON005 g3021337 BLASTN 596 1e-74 83 1734 16
701042223H1 SOYMON029 g3021337 BLASTN 997 1e-74 84 1735 16
700676004H1 SOYMON007 g3021337 BLASTN 984 1e-73 85 1736 16
700747718H1 SOYMON013 g3021337 BLASTN 988 1e-73 87 1737 16
700751133H1 SOYMON014 g3021337 BLASTN 989 1e-73 86 1738 16
701215247H1 SOYMON035 g3021337 BLASTN 989 1e-73 84 1739 16
700652484H1 SOYMON003 g3021337 BLASTN 910 1e-72 85 1740 16
700869785H1 SOYMON016 g3021337 BLASTN 970 1e-72 87 1741 16
700981960H1 SOYMON009 g3021337 BLASTN 970 1e-72 87 1742 16
700969335H1 SOYMON005 g3021337 BLASTN 972 1e-72 82 1743 16
700854174H1 SOYMON023 g3021337 BLASTN 965 1e-71 84 1744 16
700761638H1 SOYMON015 g3021337 BLASTN 966 1e-71 86 1745 16
700984860H1 SOYMON009 g3021337 BLASTN 967 1e-71 84 1746 16
701005716H1 SOYMON019 g3021337 BLASTN 967 1e-71 83 1747 16
700941053H1 SOYMON024 g3021337 BLASTN 968 1e-71 86 1748 16
700561358H1 SOYMON002 g3021337 BLASTN 968 1e-71 82 1749 16
700564906H1 SOYMON002 g3021337 BLASTN 562 1e-70 82 1750 16
700833951H1 SOYMON019 g3021337 BLASTN 954 1e-70 88 1751 16
701117626H1 SOYMON037 g3021337 BLASTN 957 1e-70 85 1752 16
700729103H1 SOYMON009 g3021337 BLASTN 535 1e-69 86 1753 16
700670615H1 SOYMON006 g3021337 BLASTN 936 1e-69 83 1754 16
701053635H1 SOYMON032 g3021337 BLASTN 941 1e-69 84 1755 16
700982280H1 SOYMON009 g3021337 BLASTN 923 1e-68 82 1756 16
701119874H1 SOYMON037 g3021337 BLASTN 925 1e-68 88 1757 16
700758937H1 SOYMON015 g3021337 BLASTN 926 1e-68 87 1758 16
701214027H1 SOYMON035 g3021337 BLASTN 928 1e-68 82 1759 16
700972858H1 SOYMON005 g3021337 BLASTN 929 1e-68 84 1760 16
701099780H1 SOYMON028 g3021337 BLASTN 930 1e-68 85 1761 16
700829560H1 SOYMON019 g3021337 BLASTN 932 1e-68 85 1762 16
700971973H1 SOYMON005 g3021337 BLASTN 576 1e-67 85 1763 16
701142336H1 SOYMON038 g3021337 BLASTN 750 1e-67 81 1764 16
701132605H1 SOYMON038 g3021337 BLASTN 759 1e-67 85 1765 16
700969222H1 SOYMON005 g3021337 BLASTN 913 1e-67 84 1766 16
700670956H1 SOYMON006 g3021337 BLASTN 920 1e-67 84 1767 16
700895725H1 SOYMON027 g3021337 BLASTN 921 1e-67 84 1768 16
701013771H1 SOYMON019 g3021337 BLASTN 921 1e-67 81 1769 16
701055481H1 SOYMON032 g3021337 BLASTN 654 1e-66 80 1770 16
700753940H1 SOYMON014 g3021337 BLASTN 899 1e-66 84 1771 16
700974141H1 SOYMON005 g3021337 BLASTN 900 1e-66 81 1772 16
700562408H1 SOYMON002 g3021337 BLASTN 902 1e-66 82 1773 16
700685292H1 SOYMON008 g3021337 BLASTN 903 1e-66 83 1774 16
700985157H1 SOYMON009 g3021337 BLASTN 907 1e-66 82 1775 16
701038194H1 SOYMON029 g3021337 BLASTN 907 1e-66 82 1776 16
700986633H1 SOYMON009 g3021337 BLASTN 908 1e-66 83 1777 16
700564282H1 SOYMON002 g3021337 BLASTN 517 1e-65 83 1778 16
700733754H1 SOYMON010 g3021337 BLASTN 680 1e-65 84 1779 16
700988179H1 SOYMON009 g3021337 BLASTN 887 1e-65 82 1780 16
700555591H1 SOYMON001 g3021337 BLASTN 887 1e-65 82 1781 16
701206717H1 SOYMON035 g3021337 BLASTN 888 1e-65 81 1782 16
700968494H1 SOYMON036 g3021337 BLASTN 889 1e-65 86 1783 16
700906271H1 SOYMON022 g3021337 BLASTN 894 1e-65 82 1784 16
700677674H1 SOYMON007 g3021337 BLASTN 894 1e-65 83 1785 16
700970391H1 SOYMON005 g3021337 BLASTN 896 1e-65 83 1786 16
700753641H1 SOYMON014 g3021337 BLASTN 897 1e-65 82 1787 16
700646593H1 SOYMON014 g3021337 BLASTN 468 1e-64 80 1788 16
700565615H1 SOYMON002 g3021337 BLASTN 667 1e-64 80 1789 16
700746523H1 SOYMON013 g3021337 BLASTN 744 1e-64 83 1790 16
700899019H1 SOYMON027 g3021337 BLASTN 875 1e-64 83 1791 16
701127167H1 SOYMON037 g3021337 BLASTN 876 1e-64 84 1792 16
701131053H1 SOYMON038 g3021337 BLASTN 879 1e-64 84 1793 16
700670980H1 SOYMON006 g3021337 BLASTN 881 1e-64 83 1794 16
701055811H1 SOYMON032 g3021337 BLASTN 881 1e-64 85 1795 16
700900103H1 SOYMON027 g3021337 BLASTN 882 1e-64 83 1796 16
700975609H1 SOYMON009 g3021337 BLASTN 882 1e-64 84 1797 16
701102865H1 SOYMON028 g3021337 BLASTN 883 1e-64 85 1798 16
701145255H1 SOYMON031 g3021337 BLASTN 509 1e-63 80 1799 16
701210875H1 SOYMON035 g3021337 BLASTN 616 1e-63 84 1800 16
700646664H1 SOYMON014 g3021337 BLASTN 862 1e-63 85 1801 16
700897337H1 SOYMON027 g3021337 BLASTN 865 1e-63 86 1802 16
700736783H1 SOYMON010 g3021337 BLASTN 867 1e-63 83 1803 16
701059586H1 SOYMON033 g3021337 BLASTN 869 1e-63 81 1804 16
701127063H1 SOYMON037 g3021337 BLASTN 412 1e-62 84 1805 16
700556614H1 SOYMON001 g3021337 BLASTN 475 1e-62 86 1806 16
700672681H1 SOYMON006 g3021337 BLASTN 818 1e-62 82 1807 16
700727057H1 SOYMON009 g3021337 BLASTN 850 1e-62 82 1808 16
701042141H1 SOYMON029 g3021337 BLASTN 851 1e-62 83 1809 16
700561860H1 SOYMON002 g3021337 BLASTN 854 1e-62 81 1810 16
700677460H1 SOYMON007 g3021337 BLASTN 855 1e-62 83 1811 16
700971671H1 SOYMON005 g3021337 BLASTN 856 1e-62 81 1812 16
700749578H1 SOYMON013 g3021337 BLASTN 856 1e-62 81 1813 16
700672288H1 SOYMON006 g3021337 BLASTN 860 1e-62 81 1814 16
701068481H1 SOYMON034 g3021337 BLASTN 861 1e-62 81 1815 16
700729913H1 SOYMON009 g3021337 BLASTN 661 1e-61 79 1816 16
700739449H1 SOYMON012 g3021337 BLASTN 724 1e-61 85 1817 16
700830902H1 SOYMON019 g3021337 BLASTN 814 1e-61 83 1818 16
700895304H1 SOYMON027 g3021337 BLASTN 840 1e-61 82 1819 16
700605676H2 SOYMON005 g3021337 BLASTN 842 1e-61 84 1820 16
700677453H1 SOYMON007 g3021337 BLASTN 843 1e-61 83 1821 16
700983108H1 SOYMON009 g3021337 BLASTN 843 1e-61 81 1822 16
700889170H1 SOYMON024 g3021337 BLASTN 845 1e-61 86 1823 16
701004956H1 SOYMON019 g3021337 BLASTN 849 1e-61 82 1824 16
700958213H1 SOYMON022 g3021337 BLASTN 849 1e-61 82 1825 16
701129305H1 SOYMON037 g3021337 BLASTN 659 1e-60 85 1826 16
701014446H1 SOYMON019 g3021337 BLASTN 669 1e-60 85 1827 16
700832047H1 SOYMON019 g3021337 BLASTN 738 1e-60 83 1828 16
700669966H1 SOYMON006 g3021337 BLASTN 829 1e-60 82 1829 16
700758028H1 SOYMON015 g3021337 BLASTN 829 1e-60 81 1830 16
700659491H1 SOYMON004 g3021337 BLASTN 829 1e-60 83 1831 16
701003560H1 SOYMON019 g3021337 BLASTN 829 1e-60 82 1832 16
701060964H1 SOYMON033 g3021337 BLASTN 833 1e-60 81 1833 16
700548284H1 SOYMON002 g3021337 BLASTN 834 1e-60 82 1834 16
700894957H1 SOYMON024 g3021337 BLASTN 837 1e-60 81 1835 16
700646551H1 SOYMON014 g3021337 BLASTN 479 1e-59 83 1836 16
700967633H1 SOYMON032 g3021337 BLASTN 530 1e-59 81 1837 16
700754430H1 SOYMON014 g3021337 BLASTN 654 1e-59 85 1838 16
700865919H1 SOYMON016 g3021337 BLASTN 814 1e-59 81 1839 16
700980426H1 SOYMON009 g3021337 BLASTN 815 1e-59 80 1840 16
701048203H1 SOYMON032 g3021337 BLASTN 816 1e-59 81 1841 16
700846414H1 SOYMON021 g3021337 BLASTN 819 1e-59 81 1842 16
700851608H1 SOYMON023 g3021337 BLASTN 822 1e-59 81 1843 16
700970160H1 SOYMON005 g3021337 BLASTN 822 1e-59 82 1844 16
700834462H1 SOYMON019 g3021337 BLASTN 823 1e-59 81 1845 16
701206312H1 SOYMON035 g3021337 BLASTN 823 1e-59 85 1846 16
700562478H1 SOYMON002 g3021337 BLASTN 487 1e-58 84 1847 16
700788114H1 SOYMON011 g3021337 BLASTN 751 1e-58 83 1848 16
700753792H1 SOYMON014 g3021337 BLASTN 804 1e-58 84 1849 16
700837427H1 SOYMON020 g3021337 BLASTN 805 1e-58 86 1850 16
700753668H1 SOYMON014 g3021337 BLASTN 806 1e-58 85 1851 16
700667315H1 SOYMON006 g3021337 BLASTN 809 1e-58 81 1852 16
700808315H1 SOYMON024 g3021337 BLASTN 558 1e-57 80 1853 16
700670207H1 SOYMON006 g3021337 BLASTN 791 1e-57 87 1854 16
700849886H1 SOYMON021 g3021337 BLASTN 791 1e-57 83 1855 16
700839033H1 SOYMON020 g3021337 BLASTN 791 1e-57 81 1856 16
700751117H1 SOYMON014 g3021337 BLASTN 799 1e-57 86 1857 16
700851803H1 SOYMON023 g3021337 BLASTN 799 1e-57 86 1858 16
700669164H1 SOYMON006 g3021337 BLASTN 800 1e-57 80 1859 16
700548285H1 SOYMON002 g3021337 BLASTN 801 1e-57 85 1860 16
701065620H1 SOYMON034 g3021337 BLASTN 426 1e-56 82 1861 16
700727996H1 SOYMON009 g3021337 BLASTN 468 1e-56 79 1862 16
700869176H1 SOYMON016 g3021337 BLASTN 786 1e-56 85 1863 16
700973141H1 SOYMON005 g3021337 BLASTN 440 1e-55 79 1864 16
700969555H1 SOYMON005 g3021337 BLASTN 448 1e-55 81 1865 16
700866138H1 SOYMON016 g3021337 BLASTN 641 1e-55 86 1866 16
700904813H1 SOYMON022 g3021337 BLASTN 699 1e-55 85 1867 16
700894146H1 SOYMON024 g3021337 BLASTN 773 1e-55 86 1868 16
700669945H1 SOYMON006 g3021337 BLASTN 773 1e-55 86 1869 16
701060489H1 SOYMON033 g3021337 BLASTN 664 1e-54 85 1870 16
701125675H1 SOYMON037 g3021337 BLASTN 721 1e-54 85 1871 16
700754750H1 SOYMON014 g3021337 BLASTN 722 1e-54 86 1872 16
701142770H1 SOYMON038 g3021337 BLASTN 755 1e-54 88 1873 16
700731095H1 SOYMON009 g3021337 BLASTN 755 1e-54 87 1874 16
700667966H1 SOYMON006 g3021337 BLASTN 756 1e-54 84 1875 16
700673606H1 SOYMON007 g3021337 BLASTN 760 1e-54 83 1876 16
700605289H2 SOYMON003 g3021337 BLASTN 763 1e-54 84 1877 16
700965253H1 SOYMON022 g3021337 BLASTN 763 1e-54 86 1878 16
700732985H1 SOYMON010 g3021337 BLASTN 765 1e-54 87 1879 16
700986523H1 SOYMON009 g3021337 BLASTN 474 1e-53 85 1880 16
701100040H2 SOYMON028 g3021337 BLASTN 602 1e-53 85 1881 16
700895328H1 SOYMON027 g3021337 BLASTN 742 1e-53 83 1882 16
701141083H1 SOYMON038 g3021337 BLASTN 751 1e-53 85 1883 16
700829878H1 SOYMON019 g3021337 BLASTN 417 1e-52 86 1884 16
700671825H1 SOYMON006 g3021337 BLASTN 431 1e-52 79 1885 16
700755240H1 SOYMON014 g3021337 BLASTN 731 1e-52 88 1886 16
701011659H1 SOYMON019 g3021337 BLASTN 734 1e-52 86 1887 16
701011547H1 SOYMON019 g3021337 BLASTN 381 1e-51 84 1888 16
700835614H1 SOYMON019 g3021337 BLASTN 437 1e-51 80 1889 16
700671849H1 SOYMON006 g3021337 BLASTN 471 1e-51 87 1890 16
700734822H1 SOYMON010 g3021337 BLASTN 486 1e-51 79 1891 16
700830223H1 SOYMON019 g3021337 BLASTN 622 1e-51 84 1892 16
700659970H1 SOYMON004 g3021337 BLASTN 722 1e-51 82 1893 16
701101779H1 SOYMON028 g3021337 BLASTN 728 1e-51 86 1894 16
700852553H1 SOYMON023 g3021337 BLASTN 490 1e-50 88 1895 16
700853857H1 SOYMON023 g3021337 BLASTN 711 1e-50 88 1896 16
700980358H1 SOYMON009 g3021337 BLASTN 712 1e-50 85 1897 16
700672182H1 SOYMON006 g3021337 BLASTN 714 1e-50 89 1898 16
700748455H1 SOYMON013 g3021337 BLASTN 396 1e-49 85 1899 16
700657257H1 SOYMON004 g3021337 BLASTN 694 1e-49 75 1900 16
700729301H1 SOYMON009 g3021337 BLASTN 702 1e-49 80 1901 16
700726175H1 SOYMON009 g3021337 BLASTN 704 1e-49 80 1902 16
700966844H1 SOYMON028 g3021337 BLASTN 414 1e-47 81 1903 16
700960965H1 SOYMON022 g3021337 BLASTN 452 1e-47 85 1904 16
700678326H1 SOYMON007 g3021337 BLASTN 480 1e-47 83 1905 16
700751042H1 SOYMON014 g3021337 BLASTN 675 1e-47 87 1906 16
700830863H1 SOYMON019 g3021337 BLASTN 343 1e-46 84 1907 16
701213640H1 SOYMON035 g3021337 BLASTN 667 1e-46 87 1908 16
700870215H1 SOYMON016 g3021337 BLASTN 667 1e-46 80 1909 16
700658278H1 SOYMON004 g3021337 BLASTN 425 1e-44 87 1910 16
700942532H1 SOYMON024 g3021337 BLASTN 583 1e-44 83 1911 16
700986276H1 SOYMON009 g3021337 BLASTN 630 1e-43 81 1912 16
700870216H1 SOYMON016 g3021337 BLASTN 457 1e-42 82 1913 16
700899828H1 SOYMON027 g3021337 BLASTN 464 1e-42 83 1914 16
700678816H1 SOYMON007 g3021337 BLASTN 618 1e-42 86 1915 16
700666809H1 SOYMON005 g3021337 BLASTN 621 1e-42 82 1916 16
701098073H1 SOYMON028 g3021337 BLASTN 285 1e-41 83 1917 16
700669492H1 SOYMON006 g3021337 BLASTN 504 1e-39 83 1918 16
700975340H1 SOYMON009 g3021337 BLASTN 574 1e-39 81 1919 16
700753528H1 SOYMON014 g3021337 BLASTN 576 1e-39 81 1920 16
700665923H1 SOYMON005 g3021337 BLASTN 373 1e-35 84 1921 16
701038320H1 SOYMON029 g3021337 BLASTN 518 1e-34 84 1922 16
700755605H1 SOYMON014 g3021337 BLASTN 431 1e-33 81 1923 16
700890349H1 SOYMON024 g3021337 BLASTN 511 1e-33 88 1924 16
700669817H1 SOYMON006 g3021337 BLASTN 363 1e-31 87 1925 16
701097640H1 SOYMON028 g3021337 BLASTN 476 1e-30 67 1926 16
700562959H1 SOYMON002 g3021337 BLASTN 482 1e-30 81 1927 16
700852454H1 SOYMON023 g3021337 BLASTN 446 1e-28 77 1928 16
701121443H1 SOYMON037 g3021337 BLASTN 418 1e-24 84 1929 16
701118247H1 SOYMON037 g3021337 BLASTN 280 1e-18 85 1930 16
700665401H1 SOYMON005 g927505 BLASTX 172 1e-16 94 1931 16
700750038H1 SOYMON013 g3021338 BLASTX 162 1e-15 84 1932 16
700665414H1 SOYMON005 g3021337 BLASTN 273 1e-13 88 1933 16
700889072H1 SOYMON024 g3021338 BLASTX 136 1e-11 83 1934 16
700727964H1 SOYMON009 g927505 BLASTX 137 1e-11 86 1935 16
700680648H1 SOYMON008 g3021337 BLASTN 226 1e-10 73 1936 16
701044547H1 SOYMON032 g927505 BLASTX 91 1e-9 76 1937 16 700649174H1
SOYMON003 g3021338 BLASTX 126 1e-9 83 1938 16531 701120682H1
SOYMON037 g3021337 BLASTN 716 1e-50 77 1939 1701 700993909H1
SOYMON011 g22633 BLASTX 112 1e-31 78 1940 1701 700955490H1
SOYMON022 g22633 BLASTX 176 1e-25 70 1941 1701 700682081H1
SOYMON008 g22633 BLASTX 147 1e-20 68 1942 1701 700988843H1
SOYMON011 g22633 BLASTX 90 1e-14 67 1943 1701 700740531H1 SOYMON012
g22633 BLASTX 92 1e-12 64 1944 1701 700790059H2 SOYMON011 g22633
BLASTX 92 1e-12 67 1945 1701 700872670H1 SOYMON018 g169037 BLASTX
144 1e-12 90 1946 1701 700990591H1 SOYMON011 g22632 BLASTN 199
1e-11 68 1947 1701 700743120H1 SOYMON012 g22633 BLASTX 92 1e-9 68
1948 1701 700994931H1 SOYMON011 g22633 BLASTX 92 1e-8 64 1949 1938
700738074H1 SOYMON012 g927507 BLASTX 134 1e-11 90 1950 239
701126904H1 SOYMON037 g169037 BLASTX 231 1e-24 81 1951 239
700668532H1 SOYMON006 g169037 BLASTX 202 1e-20 83 1952 239
700943660H1 SOYMON024 g169037 BLASTX 180 1e-17 84 1953 239
701009915H2 SOYMON019 g169037 BLASTX 180 1e-17 84 1954 239
701100047H2 SOYMON028 g169037 BLASTX 160 1e-15 84 1955 239
700794458H1 SOYMON017 g22633 BLASTX 131 1e-10 58 1956 239
700738441H1 SOYMON012 g169037 BLASTX 118 1e-8 78 1957 3425
700984050H1 SOYMON009 g3021337 BLASTN 874 1e-64 80 1958 3425
701014509H1 SOYMON019 g3021337 BLASTN 520 1e-60 80 1959 3425
701138819H1 SOYMON038 g3021337 BLASTN 815 1e-59 80 1960 3425
700977309H1 SOYMON009 g3021337 BLASTN 809 1e-58 80 1961 3425
700984876H1 SOYMON009 g3021337 BLASTN 813 1e-58 80 1962 3425
701046151H1 SOYMON032 g3021337 BLASTN 730 1e-52 80 1963 3425
700889668H1 SOYMON024 g3021337 BLASTN 737 1e-52 81
1964 3425 700976571H1 SOYMON009 g3021337 BLASTN 737 1e-52 81 1965
3425 701045371H1 SOYMON032 g3021337 BLASTN 716 1e-50 79 1966 3425
700548283H1 SOYMON002 g3021337 BLASTN 700 1e-49 81 1967 3425
701103461H1 SOYMON028 g3021337 BLASTN 705 1e-49 81 1968 3425
700898446H1 SOYMON027 g3021337 BLASTN 686 1e-48 83 1969 3425
701006432H1 SOYMON019 g3021337 BLASTN 688 1e-48 83 1970 3425
701041476H1 SOYMON029 g3021337 BLASTN 693 1e-48 81 1971 3425
700568335H1 SOYMON002 g3021337 BLASTN 678 1e-47 82 1972 3425
701046312H1 SOYMON032 g3021337 BLASTN 650 1e-45 85 1973 3425
701050171H1 SOYMON032 g3021337 BLASTN 650 1e-45 85 1974 3425
700685063H1 SOYMON008 g3021337 BLASTN 643 1e-44 83 1975 3425
701010250H2 SOYMON019 g3021337 BLASTN 542 1e-36 86 1976 3425
700665454H1 SOYMON005 g3021337 BLASTN 520 1e-34 80 1977 3425
701043888H1 SOYMON032 g3021337 BLASTN 495 1e-32 85 1978 3425
700726806H1 SOYMON009 g3021337 BLASTN 213 1e-23 76 1979 491
700997879H1 SOYMON018 g22632 BLASTN 789 1e-56 77 1980 491
700646208H1 SOYMON012 g22632 BLASTN 733 1e-52 76 1981 491
700559796H1 SOYMON001 g22632 BLASTN 715 1e-50 76 1982 491
700789784H1 SOYMON011 g22632 BLASTN 664 1e-46 76 1983 491
700683122H1 SOYMON008 g22632 BLASTN 485 1e-41 86 1984 491
701105914H1 SOYMON036 g22632 BLASTN 504 1e-41 73 1985 491
700558789H1 SOYMON001 g22632 BLASTN 607 1e-41 74 1986 491
700873051H1 SOYMON018 g22632 BLASTN 608 1e-41 75 1987 491
700684010H1 SOYMON008 g22632 BLASTN 597 1e-40 75 1988 491
700786096H2 SOYMON011 g22632 BLASTN 576 1e-39 75 1989 491
700731865H1 SOYMON010 g22632 BLASTN 582 1e-39 75 1990 491
701108111H1 SOYMON036 g22632 BLASTN 467 1e-38 75 1991 491
700740887H1 SOYMON012 g22632 BLASTN 567 1e-38 74 1992 491
700559579H1 SOYMON001 g22632 BLASTN 572 1e-38 75 1993 491
700996104H1 SOYMON018 g22632 BLASTN 476 1e-37 76 1994 491
700682145H1 SOYMON008 g22632 BLASTN 542 1e-36 74 1995 491
700737263H1 SOYMON010 g22632 BLASTN 526 1e-35 74 1996 491
700547963H1 SOYMON001 g22632 BLASTN 527 1e-35 73 1997 491
700686296H1 SOYMON008 g22632 BLASTN 527 1e-35 73 1998 491
700646072H1 SOYMON011 g22632 BLASTN 537 1e-35 74 1999 491
701106662H1 SOYMON036 g22632 BLASTN 514 1e-34 74 2000 491
700684335H1 SOYMON008 g22632 BLASTN 516 1e-34 74 2001 491
701000609H1 SOYMON018 g22632 BLASTN 520 1e-34 74 2002 491
700685658H1 SOYMON008 g22632 BLASTN 520 1e-34 74 2003 491
700875532H1 SOYMON018 g22632 BLASTN 521 1e-34 73 2004 491
700730264H1 SOYMON009 g22632 BLASTN 502 1e-33 74 2005 491
700872948H1 SOYMON018 g22632 BLASTN 502 1e-33 74 2006 491
700685813H1 SOYMON008 g22632 BLASTN 502 1e-33 74 2007 491
701104554H1 SOYMON036 g22632 BLASTN 503 1e-33 74 2008 491
700960601H1 SOYMON022 g22632 BLASTN 503 1e-33 74 2009 491
700876633H1 SOYMON018 g22632 BLASTN 503 1e-33 74 2010 491
700739662H1 SOYMON012 g22632 BLASTN 504 1e-33 72 2011 491
700685904H1 SOYMON008 g22632 BLASTN 505 1e-33 72 2012 491
700995183H1 SOYMON011 g22632 BLASTN 513 1e-33 73 2013 491
700901996H1 SOYMON027 g22632 BLASTN 513 1e-33 74 2014 491
700727070H1 SOYMON009 g22632 BLASTN 490 1e-32 72 2015 491
700685790H1 SOYMON008 g22632 BLASTN 492 1e-32 74 2016 491
700998652H1 SOYMON018 g22632 BLASTN 494 1e-32 72 2017 491
700740465H1 SOYMON012 g22632 BLASTN 482 1e-31 74 2018 491
700682621H2 SOYMON008 g22632 BLASTN 484 1e-31 74 2019 491
700874316H1 SOYMON018 g22632 BLASTN 466 1e-30 73 2020 491
700686477H1 SOYMON008 g22632 BLASTN 473 1e-30 73 2021 491
700739979H1 SOYMON012 g22632 BLASTN 476 1e-30 74 2022 491
700739416H1 SOYMON012 g22632 BLASTN 476 1e-30 74 2023 491
700685976H1 SOYMON008 g22632 BLASTN 476 1e-30 74 2024 491
700739629H1 SOYMON012 g22632 BLASTN 486 1e-30 70 2025 491
700989163H1 SOYMON011 g22632 BLASTN 468 1e-29 72 2026 491
701000555H1 SOYMON018 g22632 BLASTN 477 1e-29 72 2027 491
700872702H1 SOYMON018 g22632 BLASTN 436 1e-28 72 2028 491
701000781H1 SOYMON018 g22632 BLASTN 460 1e-28 73 2029 491
700682760H1 SOYMON008 g22632 BLASTN 463 1e-28 72 2030 491
700740390H1 SOYMON012 g22632 BLASTN 440 1e-27 73 2031 491
700685346H1 SOYMON008 g22632 BLASTN 451 1e-27 72 2032 491
700557272H1 SOYMON001 g22632 BLASTN 250 1e-26 78 2033 491
700953343H1 SOYMON022 g22632 BLASTN 349 1e-26 74 2034 491
700741960H1 SOYMON012 g22632 BLASTN 430 1e-26 73 2035 491
700680247H2 SOYMON008 g22632 BLASTN 425 1e-25 67 2036 491
700680002H2 SOYMON008 g22632 BLASTN 241 1e-24 72 2037 491
700684827H1 SOYMON008 g22632 BLASTN 379 1e-24 74 2038 491
700956353H1 SOYMON022 g22632 BLASTN 410 1e-24 72 2039 491
700787513H1 SOYMON011 g22632 BLASTN 235 1e-22 72 2040 491
700725070H1 SOYMON009 g22632 BLASTN 241 1e-22 71 2041 491
700741111H1 SOYMON012 g22632 BLASTN 304 1e-22 73 2042 491
700985308H1 SOYMON009 g22632 BLASTN 241 1e-21 80 2043 491
700738230H1 SOYMON012 g22632 BLASTN 241 1e-21 72 2044 491
700991396H1 SOYMON011 g22632 BLASTN 350 1e-21 72 2045 491
700741276H1 SOYMON012 g22632 BLASTN 379 1e-21 71 2046 491
700740223H1 SOYMON012 g22632 BLASTN 241 1e-20 72 2047 491
700738808H1 SOYMON012 g22632 BLASTN 241 1e-20 72 2048 491
700997995H1 SOYMON018 g22632 BLASTN 241 1e-19 81 2049 491
700875139H1 SOYMON018 g22632 BLASTN 241 1e-19 71 2050 491
700989713H1 SOYMON011 g22632 BLASTN 241 1e-19 73 2051 491
700958366H1 SOYMON022 g22632 BLASTN 241 1e-18 71 2052 491
700683887H1 SOYMON008 g22632 BLASTN 344 1e-18 70 2053 491
700740788H1 SOYMON012 g22632 BLASTN 339 1e-17 70 2054 491
700743058H1 SOYMON012 g22632 BLASTN 205 1e-16 81 2055 491
700996423H1 SOYMON018 g22632 BLASTN 234 1e-16 80 2056 491
700686075H1 SOYMON008 g22632 BLASTN 241 1e-16 71 2057 491
700738811H1 SOYMON012 g22632 BLASTN 193 1e-15 72 2058 491
700998312H1 SOYMON018 g22632 BLASTN 234 1e-15 73 2059 491
700681825H1 SOYMON008 g22632 BLASTN 241 1e-15 81 2060 491
701109105H1 SOYMON036 g22632 BLASTN 290 1e-14 69 2061 491
701203741H2 SOYMON035 g22632 BLASTN 230 1e-13 78 2062 491
700740785H1 SOYMON012 g22632 BLASTN 287 1e-13 68 2063 491
700738486H1 SOYMON012 g22632 BLASTN 295 1e-13 64 2064 491
700739078H1 SOYMON012 g22632 BLASTN 178 1e-12 73 2065 491
701002287H1 SOYMON018 g22632 BLASTN 255 1e-12 74 2066 491
700742470H1 SOYMON012 g22632 BLASTN 278 1e-12 69 2067 491
700743421H1 SOYMON012 g22632 BLASTN 261 1e-11 71 2068 491
700744039H1 SOYMON012 g22632 BLASTN 265 1e-11 69 2069 491
700789444H2 SOYMON011 g22632 BLASTN 158 1e-10 87 2070 491
700741074H1 SOYMON012 g22632 BLASTN 178 1e-10 77 2071 491
700998877H1 SOYMON018 g22632 BLASTN 235 1e-10 72 2072 491
700740005H1 SOYMON012 g22633 BLASTX 75 1e-9 64 2073 491 700872703H1
SOYMON018 g169037 BLASTX 116 1e-9 83 2074 491 700743301H1 SOYMON012
g22632 BLASTN 241 1e-9 76 2075 491 700875039H1 SOYMON018 g22632
BLASTN 241 1e-9 72 2076 491 700742515H1 SOYMON012 g22632 BLASTN 241
1e-9 76 2077 491 700990557H1 SOYMON011 g22632 BLASTN 241 1e-9 76
2078 491 700743995H1 SOYMON012 g22632 BLASTN 241 1e-9 76 2079 491
700743495H1 SOYMON012 g22632 BLASTN 241 1e-9 76 2080 491
701001909H1 SOYMON018 g22632 BLASTN 241 1e-9 76 2081 491
701001445H1 SOYMON018 g169037 BLASTX 115 1e-8 92 2082 491
700554881H1 SOYMON001 g169037 BLASTX 116 1e-8 94 2083 491
700954194H1 SOYMON022 g169037 BLASTX 116 1e-8 94 2084 491
700996869H1 SOYMON018 g22632 BLASTN 230 1e-8 76 2085 491
700897820H1 SOYMON027 g22632 BLASTN 234 1e-8 74 2086 491
700742574H1 SOYMON012 g22632 BLASTN 234 1e-8 74 2087 491
700684738H1 SOYMON008 g22632 BLASTN 235 1e-8 75 2088 7368
700739343H1 SOYMON012 g927507 BLASTX 164 1e-15 88 2089 -GM32379
LIB3051-015- LIB3051 g3021337 BLASTN 260 1e-28 77 Q1-E1-B12 2090
-GM8265 LIB3039-048- LIB3039 g3021337 BLASTN 481 1e-29 65 Q1-E1-F11
2091 16 LIB3027-010- LIB3027 g3021337 BLASTN 1393 1e-107 82
Q1-B1-B7 2092 16 LIB3039-049- LIB3039 g3021337 BLASTN 1297 1e-99 83
Q1-E1-B8 2093 16 LIB3051-061- LIB3051 g3021337 BLASTN 1303 1e-99 84
Q1-K1-E11 2094 16 LIB3056-009- LIB3056 g3021337 BLASTN 1126 1e-96
84 Q1-N1-A10 2095 16 LIB3051-025- LIB3051 g3021337 BLASTN 1262
1e-96 83 Q1-K1-E11 2096 16 LIB3056-014- LIB3056 g3021337 BLASTN
1077 1e-94 81 Q1-N1-E1 2097 16 LIB3055-005- LIB3055 g3021337 BLASTN
1227 1e-93 84 Q1-N1-A8 2098 16 LIB3040-045- LIB3040 g3021337 BLASTN
1211 1e-92 83 Q1-E1-A4 2099 16 LIB3028-010- LIB3028 g3021337 BLASTN
1215 1e-92 83 Q1-B1-G9 2100 16 LIB3056-010- LIB3056 g3021337 BLASTN
1217 1e-92 84 Q1-N1-G8 2101 16 LIB3039-029- LIB3039 g3021337 BLASTN
1128 1e-85 85 Q1-E1-A6 2102 16 LIB3051-014- LIB3051 g3021337 BLASTN
716 1e-80 83 Q1-E1-D2 2103 16 LIB3030-010- LIB3030 g3021337 BLASTN
1052 1e-78 83 Q1-B1-D7 2104 16 LIB3051-094- LIB3051 g3021337 BLASTN
778 1e-74 83 Q1-K1-A9 2105 16 LIB3028-030- LIB3028 g3021337 BLASTN
953 1e-70 85 Q1-B1-C9 2106 16 LIB3052-004- LIB3052 g3021337 BLASTN
868 1e-63 82 Q1-N1-D8 2107 16 LIB3065-014- LIB3065 g3021337 BLASTN
540 1e-61 79 Q1-N1-A3 2108 16 LIB3050-019- LIB3050 g168420 BLASTX
223 1e-40 63 Q1-K1-H1 2109 16 LIB3051-062- LIB3051 g3021337 BLASTN
541 1e-38 79 Q1-K1-B5 2110 3425 LIB3051-067- LIB3051 g3021337
BLASTN 1082 1e-81 78 Q1-K1-E7 2111 3425 LIB3050-006- LIB3050
g3021337 BLASTN 752 1e-57 75 Q1-E1-G7 2112 491 LIB3028-011- LIB3028
g22632 BLASTN 911 1e-67 75 Q1-B1-B9 2113 491 LIB3028-011- LIB3028
g22632 BLASTN 886 1e-65 77 Q1-B1-F2 SOYBEAN
FRUCTOSE-1,6-BISPHOSPHATASE 2114 -700685384 700685384H1 SOYMON008
g21244 BLASTN 597 1e-49 80 2115 -700737915 700737915H1 SOYMON012
g515746 BLASTN 1316 1e-100 97 2116 -700741457 700741457H1 SOYMON012
g3041774 BLASTN 692 1e-58 80 2117 -700874831 700874831H1 SOYMON018
g515746 BLASTN 1295 1e-99 100 2118 -700996155 700996155H1 SOYMON018
g3041774 BLASTN 651 1e-45 83 2119 -700996632 700996632H1 SOYMON018
g515746 BLASTN 507 1e-51 90 2120 -700998027 700998027H1 SOYMON018
g515746 BLASTN 636 1e-65 94 2121 -701209548 701209548H1 SOYMON035
g3041774 BLASTN 642 1e-44 83 2122 10129 700870828H1 SOYMON018
g21244 BLASTN 827 1e-60 79 2123 10129 700741669H1 SOYMON012 g21244
BLASTN 657 1e-53 80 2124 10348 700555754H1 SOYMON001 g21244 BLASTN
466 1e-29 77 2125 10348 700991527H1 SOYMON011 g440591 BLASTX 169
1e-16 88 2126 13716 700898719H1 SOYMON027 g515746 BLASTN 1186 1e-90
97 2127 13716 700993540H1 SOYMON011 g515746 BLASTN 1179 1e-89 98
2128 13716 700909657H1 SOYMON022 g515746 BLASTN 568 1e-57 86 2129
1894 700555054H1 SOYMON001 g515746 BLASTN 1320 1e-101 100 2130 1894
700685264H1 SOYMON008 g515746 BLASTN 1323 1e-101 99 2131 1894
700558854H1 SOYMON001 g515746 BLASTN 695 1e-98 100 2132 1894
700554755H1 SOYMON001 g515746 BLASTN 767 1e-98 99 2133 1894
701000504H1 SOYMON018 g515746 BLASTN 626 1e-95 98 2134 1894
700738115H1 SOYMON012 g515746 BLASTN 1230 1e-93 100 2135 1894
700992933H1 SOYMON011 g515746 BLASTN 1074 1e-91 98 2136 1894
701107444H1 SOYMON036 g515746 BLASTN 1201 1e-91 99 2137 1894
700852823H1 SOYMON023 g515746 BLASTN 1041 1e-90 98 2138 1894
700733478H1 SOYMON010 g515746 BLASTN 1150 1e-90 97 2139 1894
701105185H1 SOYMON036 g515746 BLASTN 641 1e-87 89 2140 1894
700737830H1 SOYMON012 g515746 BLASTN 1060 1e-87 100 2141 1894
700685110H1 SOYMON008 g515746 BLASTN 597 1e-86 90 2142 1894
700968307H1 SOYMON036 g515746 BLASTN 1113 1e-84 97 2143 1894
700653014H1 SOYMON003 g515746 BLASTN 587 1e-82 90 2144 1894
700555504H1 SOYMON001 g515746 BLASTN 626 1e-81 88 2145 1894
700751540H1 SOYMON014 g515746 BLASTN 585 1e-77 91 2146 1894
700901976H1 SOYMON027 g515746 BLASTN 505 1e-73 87 2147 1894
700986496H1 SOYMON009 g515746 BLASTN 559 1e-73 90 2148 1894
700751580H1 SOYMON014 g515746 BLASTN 569 1e-72 89 2149 1894
700751532H1 SOYMON014 g515746 BLASTN 571 1e-72 90 2150 1894
700990937H1 SOYMON011 g515746 BLASTN 544 1e-71 88 2151 1894
700740789H1 SOYMON012 g515746 BLASTN 630 1e-69 100 2152 1894
700743994H1 SOYMON012 g515746 BLASTN 945 1e-69 100 2153 1894
700754374H1 SOYMON014 g515746 BLASTN 460 1e-62 91 2154 1894
701001295H1 SOYMON018 g515746 BLASTN 541 1e-62 97 2155 1894
701155952H1 SOYMON031 g515746 BLASTN 568 1e-51 83 2156 1894
700872212H1 SOYMON018 g515746 BLASTN 670 1e-47 100 2157 1894
700682196H1 SOYMON008 g515746 BLASTN 609 1e-41 98 2158 1894
700738779H1 SOYMON012 g515746 BLASTN 252 1e-16 82 2159 26568
700844816H1 SOYMON021 g21244 BLASTN 649 1e-45 78 2160 27512
701128049H1 SOYMON037 g440591 BLASTX 185 1e-18 87 2161 7128
700649846H1 SOYMON003 g440591 BLASTX 125 1e-15 81 2162 10348
LIB3030-010- LIB3030 g21244 BLASTN 476 1e-28 76 Q1-B1-C7
FRUCTOSE-6-PHOSPHATE,2-KINASE 2163 -700730441 700730441H1 SOYMON009
g3309583 BLASTX 179 1e-17 82 2164 -700953509 700953509H1 SOYMON022
g3170229 BLASTN 674 1e-47 75 2165 -700955121 700955121H1 SOYMON022
g3309582 BLASTN 303 1e-14 68 2166 -GM28972 LIB3050-012- LIB3050
g3170229 BLASTN 1073 1e-80 80 Q1-E1-E9 SOYBEAN
PHOSPHOGLUCOISOMERASE 2167 -700568558 700568558H1 SOYMON002
g1369950 BLASTX 165 1e-15 80 2168 -700845275 700845275H1 SOYMON021
g1100771 BLASTX 124 1e-10 53 2169 -700960755 700960755H1 SOYMON022
g1100771 BLASTX 153 1e-14 52 2170 18663 700838363H1 SOYMON020
g1100771 BLASTX 215 1e-22 63 2171 18663 700838355H1 SOYMON020
g1100771 BLASTX 155 1e-14 81 2172 19355 700897450H1 SOYMON027
g1100771 BLASTX 273 1e-31 74 2173 19355 700744258H1 SOYMON013
g1100771 BLASTX 207 1e-29 69 2174 19355 701153832H1 SOYMON031
g1100771 BLASTX 226 1e-23 58 2175 20088 700856114H1 SOYMON023
g1100771 BLASTX 176 1e-33 75 2176 20088 700670380H1 SOYMON006
g1100771 BLASTX 207 1e-33 71 2177 20088 700788785H2 SOYMON011
g1100771 BLASTX 120 1e-32 74 2178 20088 700847659H1 SOYMON021
g1100771 BLASTX 192 1e-31 84 2179 20088 701136417H1 SOYMON038
g1100771 BLASTX 169 1e-27 66 2180 31255 701207622H1 SOYMON035
g1100771 BLASTX 168 1e-29 61 2181 20088 LIB3051-014- LIB3051
g1100771 BLASTX 400 1e-68 73 Q1-E1-G3 2182 31255 LIB3056-008-
LIB3056 g1100771 BLASTX 188 1e-52 62 Q1-N1-G8
SOYBEAN VACUOLAR H+-TRANSLOCATING-PYROPHOSPHATASE 2183 -700660662
700660662H1 SOYMON004 g16347 BLASTN 540 1e-36 79 2184 -700793860
700793860H1 SOYMON017 g2706449 BLASTN 808 1e-58 78 2185 -700837007
700837007H1 SOYMON020 g16347 BLASTN 776 1e-55 78 2186 -700890647
700890647H1 SOYMON024 g790474 BLASTN 826 1e-60 81 2187 -700942978
700942978H1 SOYMON024 g790478 BLASTN 605 1e-63 82 2188 -700944280
700944280H1 SOYMON024 g790479 BLASTX 119 1e-10 76 2189 -700974544
700974544H1 SOYMON005 g1103711 BLASTN 854 1e-62 83 2190 -700984449
700984449H1 SOYMON009 g1103711 BLASTN 287 1e-12 71 2191 -700989248
700989248H1 SOYMON011 g534915 BLASTN 276 1e-14 67 2192 -701102931
701102931H1 SOYMON028 g2706449 BLASTN 438 1e-46 76 2193 -701106870
701106870H1 SOYMON036 g790478 BLASTN 623 1e-47 75 2194 -701122796
701122796H1 SOYMON037 g2258074 BLASTX 71 1e-15 73 2195 -701132123
701132123H1 SOYMON038 g790478 BLASTN 627 1e-43 81 2196 -701136557
701136557H1 SOYMON038 g16347 BLASTN 376 1e-33 77 2197 14021
700973215H1 SOYMON005 g2668745 BLASTN 435 1e-39 80 2198 14021
701109310H1 SOYMON036 g2668745 BLASTN 281 1e-25 83 2199 16
700891764H1 SOYMON024 g790479 BLASTX 172 1e-16 68 2200 19232
701061126H1 SOYMON033 g790474 BLASTN 935 1e-69 81 2201 19232
700962864H1 SOYMON022 g790474 BLASTN 874 1e-64 82 2202 20872
700754883H1 SOYMON014 g790478 BLASTN 824 1e-59 81 2203 20872
700971147H1 SOYMON005 g1103711 BLASTN 564 1e-54 79 2204 2813
700797861H1 SOYMON017 g16347 BLASTN 731 1e-52 79 2205 2813
700944850H1 SOYMON024 g2570500 BLASTN 738 1e-52 82 2206 2813
701056207H1 SOYMON032 g2570500 BLASTN 556 1e-46 80 2207 2813
700605115H2 SOYMON003 g2570500 BLASTN 478 1e-42 80 2208 2813
700897063H1 SOYMON027 g2570500 BLASTN 596 1e-40 80 2209 2813
700561829H1 SOYMON002 g2570500 BLASTN 570 1e-38 80 2210 2813
701204883H1 SOYMON035 g2668745 BLASTN 545 1e-36 77 2211 2813
700754984H1 SOYMON014 g2570500 BLASTN 527 1e-35 75 2212 2813
700854552H1 SOYMON023 g2570500 BLASTN 536 1e-35 79 2213 2813
700873337H1 SOYMON018 g2570500 BLASTN 505 1e-33 75 2214 2813
700873349H1 SOYMON018 g2570500 BLASTN 506 1e-33 75 2215 2813
700952403H1 SOYMON022 g2668745 BLASTN 499 1e-32 76 2216 2813
700846561H1 SOYMON021 g2570500 BLASTN 488 1e-31 75 2217 2813
700953987H1 SOYMON022 g2570500 BLASTN 461 1e-29 75 2218 2813
700568667H1 SOYMON002 g2570500 BLASTN 296 1e-24 79 2219 2813
700895231H1 SOYMON024 g2258074 BLASTX 207 1e-22 80 2220 2813
701101791H1 SOYMON028 g2668746 BLASTX 147 1e-13 77 2221 8040
701121224H1 SOYMON037 g534915 BLASTN 298 1e-14 77 2222 8040
700743066H1 SOYMON012 g2668746 BLASTX 140 1e-12 80 2223 8531
701005139H1 SOYMON019 g2258073 BLASTN 871 1e-63 79 2224 8531
701008308H1 SOYMON019 g534915 BLASTN 789 1e-57 76 2225 8531
700559054H1 SOYMON001 g2570500 BLASTN 790 1e-57 77 2226 8531
700942540H1 SOYMON024 g2706449 BLASTN 755 1e-54 80 2227 8531
700790983H1 SOYMON011 g2258073 BLASTN 431 1e-52 77 2228 8531
701007949H1 SOYMON019 g2570500 BLASTN 404 1e-41 70 2229 8531
701123827H1 SOYMON037 g534915 BLASTN 436 1e-26 75 2230 8531
701013616H1 SOYMON019 g534915 BLASTN 431 1e-25 78 2231 8531
700565624H1 SOYMON002 g2570501 BLASTX 169 1e-16 85 2232 8531
701121092H1 SOYMON037 g2570501 BLASTX 110 1e-15 60 2233 16
LIB3040-003- LIB3040 g633598 BLASTN 523 1e-51 74 Q1-E1-F6 2234 16
LIB3051-114- LIB3051 g790478 BLASTN 457 1e-48 79 Q1-K1-G5 2235 16
LIB3039-020- LIB3039 g790478 BLASTN 338 1e-30 74 Q1-E1-A2 2236 2813
LIB3028-026- LIB3028 g2570500 BLASTN 1029 1e-77 80 Q1-B1-B7 2237
8040 LIB3049-045- LIB3049 g2706449 BLASTN 752 1e-52 72 Q1-E1-C3
2238 8040 LIB3049-005- LIB3049 g2570501 BLASTX 154 1e-32 61
Q1-E1-A7 2239 8531 LIB3050-013- LIB3050 g2570500 BLASTN 748 1e-53
72 Q1-E1-G8 2240 8531 LIB3073-025- LIB3073 g534915 BLASTN 711 1e-49
78 Q1-K1-D6 2241 8531 LIB3050-012- LIB3050 g2258074 BLASTX 93 1e-31
74 Q1-E1-D1 SOYBEAN PYROPHOSPHATE-DEPENDENT FRUCTOSE-6-PHOSPHATE
PHOSPHOTRANSFERASE 2242 7899 701008645H1 SOYMON019 g169538 BLASTX
160 1e-15 83 INVERTASES 2243 -700653543 700653543H1 SOYMON003
g1160487 BLASTN 541 1e-55 84 2244 -700992760 700992760H1 SOYMON011
g550319 BLASTX 117 1e-12 49 2245 -701005703 701005703H1 SOYMON019
g861157 BLASTX 213 1e-22 46 2246 -701047324 701047324H1 SOYMON032
g1160487 BLASTN 647 1e-45 81 2247 -701130328 701130328H1 SOYMON037
g167551 BLASTX 215 1e-22 61 2248 20460 700658149H1 SOYMON004
g861157 BLASTX 198 1e-20 72 2249 20460 701041452H1 SOYMON029
g402740 BLASTX 105 1e-13 76 2250 -GM31611 LIB3051-002- LIB3051
g1160487 BLASTN 1033 1e-77 77 Q1-E1-B9 2251 -GM34282 LIB3O51-025-
LIB3051 g1160487 BLASTN 1069 1e-80 79 Q1-K1-C4 2252 -GM34976
LIB3051-031- LIB3051 g1160487 BLASTN 769 1e-66 80 Q1-K1-A9 2253
31949 LIB3051-093- LIB3051 g1160487 BLASTN 948 1e-92 77 Q1-K1-B1
2254 31949 LIB3051-054- LIB3051 g1160487 BLASTN 903 1e-90 82
Q1-K2-D11 SOYBEAN SUCROSE SYNTHASE 2255 -700565776 700565776H1
SOYMON002 g3169544 BLASTX 89 1e-8 64 2256 -700606005 700606005H2
SOYMON007 g2570066 BLASTN 1069 1e-80 89 2257 -700664186 700664186H1
SOYMON005 g2606080 BLASTN 426 1e-62 91 2258 -700668119 700668119H1
SOYMON006 g2570066 BLASTN 279 1e-14 83 2259 -700668348 700668348H1
SOYMON006 g2570066 BLASTN 693 1e-48 88 2260 -700671225 700671225H1
SOYMON006 g16525 BLASTN 617 1e-42 72 2261 -700673918 700673918H1
SOYMON007 g218332 BLASTN 152 1e-9 92 2262 -700726266 700726266H1
SOYMON009 g2606080 BLASTN 237 1e-21 79 2263 -700747171 700747171H1
SOYMON013 g2606080 BLASTN 735 1e-52 89 2264 -700747359 700747359H1
SOYMON013 g218332 BLASTN 447 1e-28 78 2265 -700787443 700787443H2
SOYMON011 g22485 BLASTN 1171 1e-95 98 2266 -700796035 700796035H1
SOYMON017 g2570066 BLASTN 1039 1e-77 90 2267 -700832792 700832792H1
SOYMON019 g2606080 BLASTN 444 1e-31 88 2268 -700836673 700836673H1
SOYMON020 g2570066 BLASTN 843 1e-61 85 2269 -700841855 700841855H1
SOYMON020 g2570066 BLASTN 425 1e-35 84 2270 -700851758 700851758H1
SOYMON023 g2570066 BLASTN 211 1e-15 91 2271 -700851991 700851991H1
SOYMON023 g2570066 BLASTN 768 1e-55 81 2272 -700852943 700852943H1
SOYMON023 g2606080 BLASTN 250 1e-13 85 2273 -700853396 700853396H1
SOYMON023 g2570067 BLASTX 145 1e-13 65 2274 -700872206 700872206H1
SOYMON018 g1488570 BLASTX 235 1e-25 64 2275 -700876641 700876641H1
SOYMON018 g2606080 BLASTN 410 1e-53 88 2276 -700890526 700890526H1
SOYMON024 g2606080 BLASTN 652 1e-60 83 2277 -700893784 700893784H1
SOYMON024 g3169543 BLASTN 217 1e-11 82 2278 -700909222 700909222H1
SOYMON022 g2570066 BLASTN 440 1e-44 72 2279 -700944438 700944438H1
SOYMON024 g3169543 BLASTN 669 1e-46 73 2280 -700945733 700945733H1
SOYMON024 g1488569 BLASTN 504 1e-33 66 2281 -700969926 700969926H1
SOYMON005 g2570066 BLASTN 674 1e-47 72 2282 -701001986 701001986H1
SOYMON018 g1146237 BLASTX 106 1e-9 45 2283 -701005687 701005687H1
SOYMON019 g2606080 BLASTN 591 1e-40 85 2284 -701012195 701012195H1
SOYMON019 g2606080 BLASTN 418 1e-46 77 2285 -701046403 701046403H1
SOYMON032 g2606080 BLASTN 574 1e-38 76 2286 -701058966 701058966H1
SOYMON033 g218332 BLASTN 529 1e-56 84 2287 -701150574 701150574H1
SOYMON031 g1041247 BLASTX 155 1e-14 74 2288 -701205210 701205210H1
SOYMON035 g218332 BLASTN 981 1e-72 85 2289 10445 700605276H2
SOYMON003 g2606080 BLASTN 860 1e-65 84 2290 10445 700832417H1
SOYMON019 g2606080 BLASTN 876 1e-64 82 2291 10445 700833214H1
SOYMON019 g2606080 BLASTN 740 1e-58 83 2292 10445 700832409H1
SOYMON019 g2606080 BLASTN 800 1e-57 84 2293 10445 701007169H1
SOYMON019 g2606080 BLASTN 691 1e-55 81 2294 10445 701005913H1
SOYMON019 g2606080 BLASTN 680 1e-52 83 2295 10445 701204549H2
SOYMON035 g2606080 BLASTN 732 1e-52 83 2296 10445 701208347H1
SOYMON035 g2606080 BLASTN 656 1e-49 83 2297 10445 700958980H1
SOYMON022 g2606080 BLASTN 670 1e-49 83 2298 10445 700988126H1
SOYMON009 g2606080 BLASTN 324 1e-47 78 2299 10445 700830464H1
SOYMON019 g2606080 BLASTN 347 1e-47 79 2300 10445 700763911H1
SOYMON019 g3169543 BLASTN 517 1e-47 75 2301 10445 700891996H1
SOYMON024 g2606080 BLASTN 667 1e-46 88 2302 10445 700725104H1
SOYMON009 g2606080 BLASTN 577 1e-45 81 2303 10445 701124001H1
SOYMON037 g2606080 BLASTN 648 1e-45 86 2304 10445 700833919H1
SOYMON019 g2606080 BLASTN 496 1e-41 79 2305 10445 701006692H1
SOYMON019 g2606080 BLASTN 536 1e-41 86 2306 10445 700905349H1
SOYMON022 g2606080 BLASTN 585 1e-39 75 2307 10445 701204596H2
SOYMON035 g2606080 BLASTN 521 1e-38 79 2308 10445 700958885H1
SOYMON022 g2606080 BLASTN 351 1e-36 81 2309 10445 701208390H1
SOYMON035 g2606080 BLASTN 259 1e-29 86 2310 10445 701003131H1
SOYMON019 g2606080 BLASTN 442 1e-26 76 2311 10445 701207712H1
SOYMON035 g2606080 BLASTN 260 1e-17 78 2312 10445 701215107H1
SOYMON035 g2606080 BLASTN 260 1e-14 88 2313 10445 700852649H1
SOYMON023 g2606080 BLASTN 254 1e-13 74 2314 11259 701063407H1
SOYMON033 g2570066 BLASTN 1100 1e-82 87 2315 11259 700674761H1
SOYMON007 g2570066 BLASTN 739 1e-71 86 2316 11259 700839148H1
SOYMON020 g2570066 BLASTN 919 1e-67 87 2317 11259 700674815H1
SOYMON007 g2570066 BLASTN 904 1e-66 87 2318 12890 701103318H1
SOYMON028 g2570066 BLASTN 1005 1e-74 86 2319 12890 700855911H1
SOYMON023 g2570066 BLASTN 569 1e-69 86 2320 12890 700850874H1
SOYMON023 g2570066 BLASTN 937 1e-69 90 2321 12890 700837552H1
SOYMON020 g2570066 BLASTN 888 1e-65 89 2322 14264 700677058H1
SOYMON007 g2606080 BLASTN 578 1e-39 99 2323 14264 700679301H1
SOYMON007 g2606080 BLASTN 325 1e-18 90 2324 14740 701214452H1
SOYMON035 g2570066 BLASTN 1072 1e-80 89 2325 14740 701044972H1
SOYMON032 g2570066 BLASTN 537 1e-43 87 2326 14740 701040560H1
SOYMON029 g2570066 BLASTN 302 1e-24 75 2327 14740 700793901H1
SOYMON017 g2570066 BLASTN 231 1e-14 84 2328 15394 701136903H1
SOYMON038 g2606080 BLASTN 936 1e-69 81 2329 15394 701004431H1
SOYMON019 g218332 BLASTN 942 1e-69 80 2330 15394 701006153H1
SOYMON019 g218332 BLASTN 920 1e-67 83 2331 15394 701138281H1
SOYMON038 g218332 BLASTN 485 1e-40 82 2332 15394 701209319H1
SOYMON035 g3169543 BLASTN 508 1e-33 81 2333 16344 700746372H1
SOYMON013 g2606080 BLASTN 471 1e-65 85 2334 16344 700945706H1
SOYMON024 g2606080 BLASTN 635 1e-65 84 2335 17781 700960671H1
SOYMON022 g2570066 BLASTN 966 1e-71 88 2336 17781 700838540H1
SOYMON020 g2570066 BLASTN 532 1e-62 83 2337 20151 700847184H1
SOYMON021 g2570066 BLASTN 762 1e-72 90 2338 20151 700831558H1
SOYMON019 g2570066 BLASTN 980 1e-72 89 2339 22196 701046171H1
SOYMON032 g2606080 BLASTN 1321 1e-101 99 2340 22196 701207390H1
SOYMON035 g2606080 BLASTN 1258 1e-95 98 2341 25275 701013025H1
SOYMON019 g2606080 BLASTN 1353 1e-103 98 2342 25275 700561738H1
SOYMON002 g2606080 BLASTN 953 1e-84 91 2343 25380 700667735H1
SOYMON006 g2570066 BLASTN 959 1e-71 87 2344 25380 701047629H1
SOYMON032 g2570066 BLASTN 774 1e-55 89 2345 26818 701047072H1
SOYMON032 g2606080 BLASTN 830 1e-60 87 2346 26818 700737511H1
SOYMON010 g3169543 BLASTN 607 1e-57 83 2347 31182 701098655H1
SOYMON028 g2570066 BLASTN 951 1e-70 85 2348 318 701052316H1
SOYMON032 g2606080 BLASTN 1555 1e-120 100 2349 318 701053115H1
SOYMON032 g2606080 BLASTN 1281 1e-111 96 2350 318 700983049H1
SOYMON009 g2606080 BLASTN 1438 1e-110 96 2351 318 701058416H1
SOYMON033 g2606080 BLASTN 1385 1e-106 100 2352 318 701013289H1
SOYMON019 g2606080 BLASTN 1374 1e-105 99 2353 318 701002784H2
SOYMON019 g2606080 BLASTN 1365 1e-104 100 2354 318 700868516H1
SOYMON016 g2606080 BLASTN 1195 1e-103 100 2355 318 700978851H1
SOYMON009 g2606080 BLASTN 1325 1e-101 98 2356 318 701204954H1
SOYMON035 g2606080 BLASTN 770 1e-100 100 2357 318 700889102H1
SOYMON024 g2606080 BLASTN 1048 1e-100 99 2358 318 701053120H1
SOYMON032 g218332 BLASTN 1109 1e-100 90 2359 318 700731734H1
SOYMON010 g2606080 BLASTN 1308 1e-100 97 2360 318 700972625H1
SOYMON005 g2606080 BLASTN 1120 1e-98 99 2361 318 701006566H1
SOYMON019 g2606080 BLASTN 983 1e-97 99 2362 318 700952789H1
SOYMON022 g2606080 BLASTN 1276 1e-97 97 2363 318 701141518H1
SOYMON038 g2606080 BLASTN 716 1e-96 99 2364 318 700653475H1
SOYMON003 g3169543 BLASTN 1262 1e-96 87 2365 318 700650832H1
SOYMON003 g2606080 BLASTN 643 1e-95 97 2366 318 700678981H1
SOYMON007 g2606080 BLASTN 1142 1e-95 96 2367 318 700890311H1
SOYMON024 g2606080 BLASTN 1200 1e-95 100 2368 318 700892212H1
SOYMON024 g2606080 BLASTN 1250 1e-95 97 2369 318 700943424H1
SOYMON024 g2606080 BLASTN 1251 1e-95 99 2370 318 700833982H1
SOYMON019 g2606080 BLASTN 1255 1e-95 100 2371 318 700834361H1
SOYMON019 g2606080 BLASTN 981 1e-94 99 2372 318 700746379H1
SOYMON013 g2606080 BLASTN 1108 1e-94 96 2373 318 700889648H1
SOYMON024 g2606080 BLASTN 1238 1e-94 99 2374 318 701054868H1
SOYMON032 g2606080 BLASTN 1243 1e-94 95 2375 318 700959914H1
SOYMON022 g2606080 BLASTN 1226 1e-93 96 2376 318 701011518H1
SOYMON019 g2606080 BLASTN 705 1e-92 99 2377 318 700734053H1
SOYMON010 g2606080 BLASTN 765 1e-92 100 2378 318 701005295H1
SOYMON019 g2606080 BLASTN 962 1e-92 93 2379 318 700945690H1
SOYMON024 g2606080 BLASTN 1054 1e-92 99 2380 318 701118196H1
SOYMON037 g2606080 BLASTN 1100 1e-92 95 2381 318 700673512H1
SOYMON007 g2606080 BLASTN 1211 1e-92 97 2382 318 700852712H1
SOYMON023 g2606080 BLASTN 1215 1e-92 98 2383 318 701004755H1
SOYMON019 g2606080 BLASTN 1221 1e-92 99 2384 318 700677915H1
SOYMON007 g2606080 BLASTN 685 1e-91 99 2385 318 700977846H1
SOYMON009 g2606080 BLASTN 731 1e-91 99 2386 318 700831789H1
SOYMON019 g2606080 BLASTN 1204 1e-91 97 2387 318 700754901H1
SOYMON014 g2606080 BLASTN 1205 1e-91 100 2388 318 700666594H1
SOYMON005 g2606080 BLASTN 1210 1e-91 100 2389 318 700750890H1
SOYMON014 g2606080 BLASTN 1188 1e-90 99 2390 318 700890229H1
SOYMON024 g2606080 BLASTN 1195 1e-90 100 2391 318 700732660H1
SOYMON010 g2606080 BLASTN 1154 1e-89 95 2392 318 700764730H1
SOYMON023 g2606080 BLASTN 1181 1e-89 99 2393 318 701050015H1
SOYMON032 g218332 BLASTN 1185 1e-89 89 2394 318 700870180H1
SOYMON016 g2606080 BLASTN 710 1e-88 100 2395 318 701204236H2
SOYMON035 g2606080 BLASTN 904 1e-88 98 2396 318 700645782H1
SOYMON010 g2606080 BLASTN 633 1e-87 95 2397 318 700831711H1
SOYMON019 g2606080 BLASTN 1025 1e-87 96 2398 318 701056026H1
SOYMON032 g2606080 BLASTN 1158 1e-87 96 2399 318 700678853H1
SOYMON007 g2606080 BLASTN 1161 1e-87 97 2400 318 700852424H1
SOYMON023 g2606080 BLASTN 913 1e-86 95 2401 318 701049116H1
SOYMON032 g3169543 BLASTN 1146 1e-86 89 2402 318 700977788H1
SOYMON009 g2606080 BLASTN 642 1e-85 94 2403 318 700833546H1
SOYMON019 g2606080 BLASTN 1134 1e-85 94 2404 318 701004915H1
SOYMON019 g2606080 BLASTN 591 1e-84 96 2405 318 700730093H1
SOYMON009 g2606080 BLASTN 755 1e-84 96 2406 318 701119060H1
SOYMON037 g2606080 BLASTN 824 1e-84 97 2407 318 700963024H1
SOYMON022 g2606080 BLASTN 1116 1e-84 90 2408 318 700563532H1
SOYMON002 g22037 BLASTN 1116 1e-84 87 2409 318 700755891H1
SOYMON014 g2606080 BLASTN 1117 1e-84 94 2410 318 700850605H1
SOYMON023 g2606080 BLASTN 1118 1e-84 94 2411 318 700888245H1
SOYMON024 g2606080 BLASTN 643 1e-83 98 2412 318 701037091H1
SOYMON029 g2606080 BLASTN 821 1e-83 95 2413 318 700673790H1
SOYMON007 g2606080 BLASTN 1104 1e-83 95 2414 318 700845518H1
SOYMON021 g2606080 BLASTN 673 1e-82 91
2415 318 700854591H1 SOYMON023 g2606080 BLASTN 606 1e-81 95 2416
318 700907167H1 SOYMON022 g2606080 BLASTN 920 1e-81 96 2417 318
700978575H1 SOYMON009 g218332 BLASTN 971 1e-81 91 2418 318
700853484H1 SOYMON023 g2606080 BLASTN 1079 1e-81 92 2419 318
701124012H1 SOYMON037 g218332 BLASTN 1083 1e-81 89 2420 318
700835387H1 SOYMON019 g2606080 BLASTN 1087 1e-81 96 2421 318
700749133H1 SOYMON013 g2606080 BLASTN 571 1e-80 98 2422 318
700727185H1 SOYMON009 g2606080 BLASTN 730 1e-80 98 2423 318
700869024H1 SOYMON016 g2606080 BLASTN 807 1e-79 96 2424 318
701013537H1 SOYMON019 g2606080 BLASTN 929 1e-79 87 2425 318
701010402H1 SOYMON019 g218332 BLASTN 1055 1e-79 85 2426 318
701107955H1 SOYMON036 g2606080 BLASTN 1058 1e-79 87 2427 318
700731653H1 SOYMON010 g2606080 BLASTN 578 1e-78 94 2428 318
700888950H1 SOYMON024 g218332 BLASTN 765 1e-78 88 2429 318
700894112H1 SOYMON024 g2606080 BLASTN 842 1e-78 98 2430 318
701005565H1 SOYMON019 g2606080 BLASTN 1024 1e-78 92 2431 318
700548286H1 SOYMON002 g2606080 BLASTN 1045 1e-78 88 2432 318
700975854H1 SOYMON009 g22037 BLASTN 1053 1e-78 86 2433 318
700944525H1 SOYMON024 g218332 BLASTN 1054 1e-78 89 2434 318
701061312H1 SOYMON033 g2606080 BLASTN 773 1e-77 87 2435 318
700831277H1 SOYMON019 g2606080 BLASTN 947 1e-77 97 2436 318
700788482H1 SOYMON011 g2606080 BLASTN 1038 1e-77 89 2437 318
701055686H1 SOYMON032 g2606080 BLASTN 1039 1e-77 90 2438 318
701054768H1 SOYMON032 g2606080 BLASTN 786 1e-76 88 2439 318
700854891H1 SOYMON023 g2606080 BLASTN 1030 1e-76 93 2440 318
701215276H1 SOYMON035 g2606080 BLASTN 1030 1e-76 90 2441 318
700944860H1 SOYMON024 g2606080 BLASTN 887 1e-75 96 2442 318
701010957H1 SOYMON019 g2606080 BLASTN 1011 1e-75 87 2443 318
701007175H1 SOYMON019 g2606080 BLASTN 1013 1e-75 90 2444 318
700725567H1 SOYMON009 g2606080 BLASTN 1013 1e-75 93 2445 318
700904972H1 SOYMON022 g22037 BLASTN 1015 1e-75 89 2446 318
700747391H1 SOYMON013 g2606080 BLASTN 1017 1e-75 87 2447 318
700747523H1 SOYMON013 g22037 BLASTN 836 1e-74 86 2448 318
700561819H1 SOYMON002 g218332 BLASTN 999 1e-74 82 2449 318
700835961H1 SOYMON019 g218332 BLASTN 1006 1e-74 87 2450 318
700562318H1 SOYMON002 g2606080 BLASTN 986 1e-73 84 2451 318
700745092H1 SOYMON013 g2606080 BLASTN 987 1e-73 88 2452 318
700832618H1 SOYMON019 g2606080 BLASTN 975 1e-72 87 2453 318
700891092H1 SOYMON024 g2606080 BLASTN 982 1e-72 88 2454 318
701119264H1 SOYMON037 g2606080 BLASTN 690 1e-71 89 2455 318
700894436H1 SOYMON024 g2606080 BLASTN 901 1e-71 91 2456 318
700894532H1 SOYMON024 g22037 BLASTN 959 1e-71 89 2457 318
700891712H1 SOYMON024 g22037 BLASTN 960 1e-71 89 2458 318
700895985H1 SOYMON027 g2606080 BLASTN 964 1e-71 89 2459 318
701203243H1 SOYMON035 g2606080 BLASTN 969 1e-71 88 2460 318
700985945H1 SOYMON009 g218332 BLASTN 713 1e-70 90 2461 318
700984768H1 SOYMON009 g2606080 BLASTN 781 1e-69 84 2462 318
700675710H1 SOYMON007 g2606080 BLASTN 784 1e-69 91 2463 318
700829561H1 SOYMON019 g218332 BLASTN 935 1e-69 87 2464 318
700964918H1 SOYMON022 g22037 BLASTN 942 1e-69 83 2465 318
701046747H1 SOYMON032 g2606080 BLASTN 422 1e-68 84 2466 318
700745512H1 SOYMON013 g3169543 BLASTN 457 1e-68 85 2467 318
700666671H1 SOYMON005 g218332 BLASTN 506 1e-68 87 2468 318
700889555H1 SOYMON024 g3169543 BLASTN 930 1e-68 86 2469 318
701147844H1 SOYMON031 g3169543 BLASTN 932 1e-68 86 2470 318
701206247H1 SOYMON035 g3169543 BLASTN 934 1e-68 82 2471 318
701103801H1 SOYMON036 g218332 BLASTN 723 1e-67 88 2472 318
700943746H1 SOYMON024 g218332 BLASTN 913 1e-67 86 2473 318
700745956H1 SOYMON013 g22037 BLASTN 921 1e-67 83 2474 318
700893512H1 SOYMON024 g218332 BLASTN 835 1e-66 90 2475 318
700897675H1 SOYMON027 g22037 BLASTN 899 1e-66 83 2476 318
700565777H1 SOYMON002 g2606080 BLASTN 510 1e-65 89 2477 318
700749851H1 SOYMON013 g2606080 BLASTN 887 1e-65 89 2478 318
700746286H1 SOYMON013 g2606080 BLASTN 876 1e-64 82 2479 318
700869142H1 SOYMON016 g2606080 BLASTN 885 1e-64 100 2480 318
700892442H1 SOYMON024 g2606080 BLASTN 872 1e-63 84 2481 318
700964153H1 SOYMON022 g22037 BLASTN 873 1e-63 83 2482 318
700898176H1 SOYMON027 g3169543 BLASTN 873 1e-63 84 2483 318
701056245H1 SOYMON032 g218332 BLASTN 543 1e-61 84 2484 318
700835360H1 SOYMON019 g218332 BLASTN 839 1e-61 88 2485 318
700749067H1 SOYMON013 g3169543 BLASTN 473 1e-60 86 2486 318
701008962H1 SOYMON019 g3169543 BLASTN 614 1e-60 90 2487 318
700980315H1 SOYMON009 g3169543 BLASTN 655 1e-60 84 2488 318
701202680H1 SOYMON035 g2606080 BLASTN 678 1e-60 89 2489 318
701202364H1 SOYMON035 g2606080 BLASTN 711 1e-60 85 2490 318
701037195H1 SOYMON029 g218332 BLASTN 439 1e-59 86 2491 318
701011681H1 SOYMON019 g3169543 BLASTN 459 1e-59 83 2492 318
700976368H1 SOYMON009 g218332 BLASTN 363 1e-58 85 2493 318
700829847H1 SOYMON019 g218332 BLASTN 384 1e-58 86 2494 318
700561920H1 SOYMON002 g2606080 BLASTN 809 1e-58 88 2495 318
701004573H1 SOYMON019 g2606080 BLASTN 813 1e-58 77 2496 318
701049462H1 SOYMON032 g3169543 BLASTN 450 1e-57 82 2497 318
700866272H1 SOYMON016 g3169543 BLASTN 421 1e-54 77 2498 318
700892632H1 SOYMON024 g2606080 BLASTN 453 1e-54 84 2499 318
701215184H1 SOYMON035 g218332 BLASTN 464 1e-54 88 2500 318
700831177H1 SOYMON019 g2606080 BLASTN 759 1e-54 85 2501 318
700835115H1 SOYMON019 g2606080 BLASTN 762 1e-54 81 2502 318
701015056H1 SOYMON019 g3169543 BLASTN 447 1e-53 81 2503 318
700675496H1 SOYMON007 g2606080 BLASTN 465 1e-53 95 2504 318
701052767H1 SOYMON032 g2606080 BLASTN 753 1e-53 88 2505 318
700833078H1 SOYMON019 g3169543 BLASTN 414 1e-52 84 2506 318
700869165H1 SOYMON016 g3169543 BLASTN 534 1e-51 84 2507 318
700831532H1 SOYMON019 g2606080 BLASTN 655 1e-51 100 2508 318
701010104H2 SOYMON019 g2606080 BLASTN 698 1e-51 85 2509 318
700890513H1 SOYMON024 g22037 BLASTN 575 1e-50 88 2510 318
700890952H1 SOYMON024 g2606080 BLASTN 709 1e-50 75 2511 318
700567301H1 SOYMON002 g22037 BLASTN 716 1e-50 82 2512 318
700945284H1 SOYMON024 g3169543 BLASTN 701 1e-49 75 2513 318
701206626H1 SOYMON035 g3169543 BLASTN 702 1e-49 81 2514 318
700748456H1 SOYMON013 g2606080 BLASTN 384 1e-48 77 2515 318
700981883H1 SOYMON009 g2606080 BLASTN 419 1e-48 85 2516 318
700942575H1 SOYMON024 g22037 BLASTN 340 1e-46 82 2517 318
700945125H1 SOYMON024 g2606080 BLASTN 405 1e-46 81 2518 318
700830469H1 SOYMON019 g3169543 BLASTN 636 1e-44 83 2519 318
700991669H1 SOYMON011 g218332 BLASTN 630 1e-43 83 2520 318
700866064H1 SOYMON016 g3169543 BLASTN 453 1e-41 84 2521 318
700866806H1 SOYMON016 g218332 BLASTN 607 1e-41 96 2522 318
700893154H1 SOYMON024 g2606080 BLASTN 539 1e-38 87 2523 318
700893118H1 SOYMON024 g2606080 BLASTN 539 1e-38 87 2524 318
701142963H2 SOYMON038 g218332 BLASTN 569 1e-38 90 2525 318
700945968H1 SOYMON024 g218332 BLASTN 572 1e-38 86 2526 318
700945788H1 SOYMON024 g2606080 BLASTN 514 1e-36 90 2527 318
700563455H1 SOYMON002 g2606080 BLASTN 496 1e-32 83 2528 318
700888936H1 SOYMON024 g3169543 BLASTN 498 1e-32 86 2529 318
701039594H1 SOYMON029 g22037 BLASTN 254 1e-28 84 2530 318
701015024H1 SOYMON019 g218333 BLASTX 65 1e-14 66 2531 318
700893166H1 SOYMON024 g22037 BLASTN 232 1e-8 85 2532 4258
700646449H1 SOYMON013 g22037 BLASTN 584 1e-39 70 2533 4258
700952838H1 SOYMON022 g20373 BLASTN 557 1e-37 70 2534 4413
700902256H1 SOYMON027 g2606080 BLASTN 1215 1e-99 97 2535 4413
700900032H1 SOYMON027 g2606080 BLASTN 720 1e-95 98 2536 4413
701006182H1 SOYMON019 g2606080 BLASTN 1179 1e-89 99 2537 4413
700831710H1 SOYMON019 g2606080 BLASTN 1070 1e-80 97 2538 4413
701008850H1 SOYMON019 g2606080 BLASTN 999 1e-74 99 2539 4413
701015432H1 SOYMON019 g2606080 BLASTN 813 1e-68 95 2540 4413
700987094H1 SOYMON009 g2606080 BLASTN 928 1e-68 84 2541 4413
700736179H1 SOYMON010 g2606080 BLASTN 753 1e-63 96 2542 4413
700890230H1 SOYMON024 g2606080 BLASTN 798 1e-57 95 2543 4413
701015314H1 SOYMON019 g2606080 BLASTN 639 1e-49 97 2544 4413
701052019H1 SOYMON032 g2606080 BLASTN 448 1e-37 95 2545 4748
701209527H1 SOYMON035 g2606080 BLASTN 1207 1e-91 93 2546 4748
700561984H1 SOYMON002 g2606080 BLASTN 542 1e-81 94 2547 4748
700895166H1 SOYMON024 g2606080 BLASTN 1004 1e-74 98 2548 4748
700843735H1 SOYMON021 g2606080 BLASTN 227 1e-20 93 2549 869
700650545H1 SOYMON003 g2606080 BLASTN 804 1e-107 94 2550 869
701205255H1 SOYMON035 g2606080 BLASTN 1135 1e-101 98 2551 869
700562091H1 SOYMON002 g2606080 BLASTN 1311 1e-100 92 2552 869
701213906H1 SOYMON035 g2606080 BLASTN 1300 1e-99 100 2553 869
700567712H1 SOYMON002 g2606080 BLASTN 634 1e-95 97 2554 869
701010943H1 SOYMON019 g2606080 BLASTN 1236 1e-94 99 2555 869
701006976H1 SOYMON019 g2606080 BLASTN 601 1e-93 98 2556 869
700752409H1 SOYMON014 g2606080 BLASTN 1080 1e-92 100 2557 869
701204769H1 SOYMON035 g2606080 BLASTN 795 1e-90 100 2558 869
701042737H1 SOYMON029 g2606080 BLASTN 1058 1e-90 99 2559 869
700832091H1 SOYMON019 g2606080 BLASTN 1116 1e-88 99 2560 869
701049161H1 SOYMON032 g2606080 BLASTN 1053 1e-86 96 2561 869
700906541H1 SOYMON022 g2606080 BLASTN 1087 1e-86 96 2562 869
701008182H1 SOYMON019 g2606080 BLASTN 1111 1e-86 92 2563 869
700831609H1 SOYMON019 g2606080 BLASTN 611 1e-84 92 2564 869
700834954H1 SOYMON019 g2606080 BLASTN 835 1e-84 100 2565 869
701037284H1 SOYMON029 g2606080 BLASTN 858 1e-83 94 2566 869
700561458H1 SOYMON002 g2606080 BLASTN 1019 1e-83 93 2567 869
701208357H1 SOYMON035 g2606080 BLASTN 1113 1e-83 99 2568 869
700747138H1 SOYMON013 g2606080 BLASTN 985 1e-80 93 2569 869
701014835H1 SOYMON019 g2606080 BLASTN 891 1e-78 89 2570 869
700956359H1 SOYMON022 g2606080 BLASTN 1052 1e-78 96 2571 869
701012740H1 SOYMON019 g2606080 BLASTN 643 1e-77 93 2572 869
701042523H1 SOYMON029 g2606080 BLASTN 667 1e-74 95 2573 869
701205775H1 SOYMON035 g2606080 BLASTN 745 1e-74 100 2574 869
701049184H1 SOYMON032 g2606080 BLASTN 600 1e-72 95 2575 869
700889179H1 SOYMON024 g2606080 BLASTN 942 1e-69 92 2576 869
700963920H1 SOYMON022 g2606080 BLASTN 718 1e-66 90 2577 869
700737476H1 SOYMON010 g2606080 BLASTN 548 1e-44 97 2578 869
701044544H1 SOYMON032 g2606080 BLASTN 462 1e-43 96 2579 869
700737636H1 SOYMON010 g2606080 BLASTN 426 1e-34 95 2580 9398
700837013H1 SOYMON020 g2570066 BLASTN 1025 1e-76 88 2581 9398
700891526H1 SOYMON024 g2570066 BLASTN 868 1e-63 87 2582 14740
LIB3051-038- LIB3051 g2570066 BLASTN 1331 1e-102 86 Q1-K1-E10 2583
31182 LIB3051-015- LIB3051 g2570066 BLASTN 1540 1e-119 88 Q1-E1-F1
2584 318 LIB3050-024- LIB3050 g2606080 BLASTN 1736 1e-135 95
Q1-K1-H5 2585 318 LIB3050-012- LIB3050 g2606080 BLASTN 1564 1e-125
98 Q1-E1-F10 2586 318 LIB3056-013- LIB3056 g3169543 BLASTN 1617
1e-125 86 Q1-N1-H11 2587 318 LIB3028-026- LIB3028 g3169543 BLASTN
1393 1e-107 84 Q1-B1-F6 2588 318 LIB3049-031- LIB3049 g3169543
BLASTN 1290 1e-98 90 Q1-E1-B6 2589 33428 LIB3051-085- LIB3051
g2570066 BLASTN 679 1e-53 86 Q1-K1-D11 2590 869 LIB3056-014-
LIB3056 g2606080 BLASTN 1503 1e-132 96 Q1-N1-G8 SOYBEAN HEXOKINASE
2591 -700560085 700560085H1 SOYMON001 g1899024 BLASTN 456 1e-27 67
2592 -700752579 700752579H1 SOYMON014 g836808 BLASTX 113 1e-8 54
2593 -700753182 700753182H1 SOYMON014 g619928 BLASTX 234 1e-25 63
2594 -700838622 700838622H1 SOYMON020 g619927 BLASTN 767 1e-55 78
2595 -700840271 700840271H1 SOYMON020 g619927 BLASTN 525 1e-34 67
2596 -700844132 700844132H1 SOYMON021 g619927 BLASTN 474 1e-51 77
2597 -700898308 700898308H1 SOYMON027 g619927 BLASTN 464 1e-29 72
2598 -700904279 700904279H1 SOYMON022 g881521 BLASTX 129 1e-10 67
2599 -700904320 700904320H1 SOYMON022 g1899024 BLASTN 612 1e-42 71
2600 -700946357 700946357H1 SOYMON024 g619928 BLASTX 112 1e-18 69
2601 -700998007 700998007H1 SOYMON018 g1899024 BLASTN 367 1e-20 71
2602 -701097096 701097096H1 SOYMON028 g619927 BLASTN 488 1e-30 73
2603 -701102877 701102877H1 SOYMON028 g619927 BLASTN 551 1e-37 70
2604 -701103285 701103285H1 SOYMON028 g619928 BLASTX 179 1e-17 77
2605 -701105838 701105838H1 SOYMON036 g619928 BLASTX 274 1e-30 63
2606 -701138291 701138291H1 SOYMON038 g619927 BLASTN 819 1e-59 79
2607 12404 701065794H1 SOYMON034 g3087888 BLASTX 84 1e-11 44 2608
12404 701131030H1 SOYMON038 g1899025 BLASTX 120 1e-9 45 2609 12693
700846513H1 SOYMON021 g619927 BLASTN 459 1e-28 70 2610 12693
700656744H1 SOYMON004 g619927 BLASTN 251 1e-10 57 2611 12917
700906858H1 SOYMON022 g3087888 BLASTX 183 1e-32 80 2612 12917
700830011H1 SOYMON019 g619927 BLASTN 495 1e-32 70 2613 12917
701068501H1 SOYMON034 g619927 BLASTN 475 1e-29 72 2614 12917
701153981H1 SOYMON031 g3087887 BLASTN 440 1e-26 69 2615 222
700663332H1 SOYMON005 g619927 BLASTN 724 1e-51 76 2616 222
701142003H1 SOYMON038 g881520 BLASTN 542 1e-39 72 2617 222
700657213H1 SOYMON004 g881520 BLASTN 524 1e-34 73 2618 222
700833679H1 SOYMON019 g1899024 BLASTN 453 1e-28 80 2619 222
700556060H1 SOYMON001 g619927 BLASTN 463 1e-28 82 2620 23610
700984359H1 SOYMON009 g1899024 BLASTN 611 1e-42 73 2621 23610
701003284H1 SOYMON019 g1899024 BLASTN 577 1e-39 75 2622 25188
700760643H1 SOYMON015 g619927 BLASTN 701 1e-49 73 2623 25188
701056127H1 SOYMON032 g1899024 BLASTN 649 1e-45 70 2624 27316
701054167H1 SOYMON032 g3087888 BLASTX 177 1e-17 47 2625 27316
701054157H1 SOYMON032 g3087888 BLASTX 177 1e-17 47 2626 488
700682650H2 SOYMON008 g687676 BLASTN 730 1e-52 77 2627 488
700849894H1 SOYMON021 g687676 BLASTN 582 1e-39 76 2628 -GM32703
LIB3051-008- LIB3051 g1899024 BLASTN 981 1e-76 77 Q1-E1-C12 2629
-GM9523 LIB3049-003- LIB3049 g619928 BLASTX 203 1e-37 64 Q1-E1-A6
2630 12693 LIB3051-106- LIB3051 g619927 BLASTN 459 1e-38 71
Q1-K1-A9 2631 488 LIB3040-006- LIB3040 g687676 BLASTN 622 1e-41 76
Q1-E1-A12 2632 488 LIB3053-008- LIB3053 g687676 BLASTN 597 1e-39 75
Q1-N1-C6 2633 488 LIB3055-008- LIB3055 g687676 BLASTN 559 1e-36 75
Q1-N1-F5 2634 488 LIB3053-010- LIB3053 g687676 BLASTN 514 1e-32 75
Q1-N1-D8 SOYBEAN FRUCTOKINASE 2635 -700834049 700834049H1 SOYMON019
g1915974 BLASTX 112 1e-10 97 2636 -700905716 700905716H1 SOYMON022
g1915973 BLASTN 774 1e-55 77 2637 -700978126 700978126H1 SOYMON009
g1915973 BLASTN 565 1e-38 77 2638 -700983171 700983171H1 SOYMON009
g1915974 BLASTX 96 1e-9 93 2639 -701069652 701069652H1 SOYMON034
g297014 BLASTN 447 1e-27 73 2640 -701118004 701118004H2 SOYMON037
g2102690 BLASTN 440 1e-26 73 2641 -701209270 701209270H1 SOYMON035
g1052972 BLASTN 648 1e-45 79 2642 1174 700832430H1 SOYMON019
g1915973 BLASTN 638 1e-44 81 2643 1174 701101576H1 SOYMON028
g1915973 BLASTN 592 1e-40 79 2644 1174 700754333H1 SOYMON014
g1915973 BLASTN 323 1e-37 80 2645 1174 701004323H1 SOYMON019
g297014 BLASTN 560 1e-37 80 2646 1174 700988192H1 SOYMON009
g1915973 BLASTN 508 1e-33 78 2647 1174 700646337H1 SOYMON013
g1915974 BLASTX 153 1e-30 79
2648 1174 701039647H1 SOYMON029 g1915973 BLASTN 275 1e-12 80 2649
16472 701155250H1 SOYMON031 g1915973 BLASTN 642 1e-50 78 2650 16472
700953304H1 SOYMON022 g1915973 BLASTN 690 1e-48 79 2651 16472
700725996H1 SOYMON009 g1915973 BLASTN 362 1e-28 73 2652 17936
700965277H1 SOYMON022 g2102690 BLASTN 375 1e-42 77 2653 17936
700746240H1 SOYMON013 g2102690 BLASTN 606 1e-41 74 2654 22120
701215393H1 SOYMON035 g2102691 BLASTX 133 1e-11 86 2655 22586
701009695H1 SOYMON019 g2102690 BLASTN 696 1e-49 76 2656 22586
700900731H1 SOYMON027 g2102690 BLASTN 422 1e-26 76 2657 23551
701053585H1 SOYMON032 g2102691 BLASTX 120 1e-9 92 2658 28587
701156878H1 SOYMON031 g2102690 BLASTN 448 1e-33 72 2659 3876
700942858H1 SOYMON024 g297014 BLASTN 705 1e-49 74 2660 3876
701063105H1 SOYMON033 g1052972 BLASTN 679 1e-47 73 2661 3876
700844831H1 SOYMON021 g1915973 BLASTN 466 1e-37 72 2662 5530
700733713H1 SOYMON010 g1915974 BLASTX 156 1e-26 81 2663 5530
701057239H1 SOYMON033 g1915974 BLASTX 176 1e-17 92 2664 5530
700985231H1 SOYMON009 g297014 BLASTN 222 1e-16 79 2665 5805
701010614H1 SOYMON019 g1915973 BLASTN 958 1e-71 80 2666 5805
701003106H1 SOYMON019 g1915973 BLASTN 679 1e-64 81 2667 5805
700748895H1 SOYMON013 g1915973 BLASTN 475 1e-55 83 2668 5805
700892801H1 SOYMON024 g1915973 BLASTN 639 1e-55 80 2669 5805
700891914H1 SOYMON024 g1915973 BLASTN 639 1e-55 81 2670 5805
700962529H1 SOYMON022 g1915973 BLASTN 622 1e-54 82 2671 5805
700869294H1 SOYMON016 g1915973 BLASTN 760 1e-54 80 2672 5805
700986530H1 SOYMON009 g1915973 BLASTN 761 1e-54 80 2673 5805
700661115H1 SOYMON005 g1915973 BLASTN 682 1e-48 78 2674 5805
701041987H1 SOYMON029 g297014 BLASTN 475 1e-45 83 2675 5805
701006803H1 SOYMON019 g1915973 BLASTN 607 1e-41 80 2676 28587
LIB3028-008- LIB3028 g2102690 BLASTN 900 1e-66 68 Q1-B1-H3 2677
5530 LIB3055-004- LIB3055 g297014 BLASTN 606 1e-39 76 Q1-N1-H3 2678
5805 LIB3065-006- LIB3065 g1915973 BLASTN 954 1e-81 79 Q1-N1-F11
SOYBEAN NDP-KINASE 2679 33331 701108520H1 SOYMON036 g758643 BLASTN
473 1e-31 75 2680 23595 LIB3050-018- LIB3050 g758643 BLASTN 295
1e-13 76 Q1-E1-C4 2681 33331 LIB3040-037- LIB3040 g758643 BLASTN
413 1e-47 79 Q1-E1-D6 SOYBEAN GLUCOSE-6-PHOSPHATE 1-DEHYDROGENASE
2682 -700869140 700869140H1 SOYMON016 g2829880 BLASTX 164 1e-15 44
2683 -701065174 701065174H1 SOYMON034 g603219 BLASTX 86 1e-9 76
2684 -701130434 701130434H1 SOYMON037 g1197385 BLASTX 189 1e-19 55
2685 -701149522 701149522H1 SOYMON031 g603219 BLASTX 99 1e-8 71
2686 26484 701003905H1 SOYMON019 g1197385 BLASTX 138 1e-15 81 2687
9136 701038169H1 SOYMON029 g603219 BLASTX 139 1e-21 73 2688 9136
700903571H1 SOYMON022 g603219 BLASTX 144 1e-20 81 2689 9136
701045122H1 SOYMON032 g603219 BLASTX 100 1e-13 79 SOYBEAN
PHOSPHOGLUCOMUTASE 2690 -700554424 700554424H1 SOYMON001 g534982
BLASTX 133 1e-25 60 2691 -700556670 700556670H1 SOYMON001 g3294468
BLASTN 355 1e-43 74 2692 -700563871 700563871H1 SOYMON002 g2795876
BLASTX 101 1e-16 75 2693 -700565101 700565101H1 SOYMON002 g3294466
BLASTN 588 1e-40 68 2694 -700566749 700566749H1 SOYMON002 g1814400
BLASTN 475 1e-41 73 2695 -700681382 700681382H2 SOYMON008 g3294467
BLASTX 98 1e-11 48 2696 -700763827 700763827H1 SOYMON018 g3192042
BLASTX 257 1e-29 60 2697 -700865583 700865583H1 SOYMON016 g3192042
BLASTX 134 1e-17 57 2698 -700891379 700891379H1 SOYMON024 g534982
BLASTX 167 1e-15 53 2699 -700942816 700942816H1 SOYMON024 g3294466
BLASTN 636 1e-44 74 2700 -701004954 701004954H1 SOYMON019 g1814400
BLASTN 790 1e-56 78 2701 -701011364 701011364H1 SOYMON019 g534982
BLASTX 284 1e-32 67 2702 -701057063 701057063H2 SOYMON033 g1814401
BLASTX 121 1e-9 60 2703 -701119491 701119491H1 SOYMON037 g1814400
BLASTN 762 1e-54 76 2704 -701149254 701149254H1 SOYMON031 g534982
BLASTX 147 1e-19 52 2705 10032 700988921H1 SOYMON011 g1814400
BLASTN 908 1e-66 80 2706 10032 701136003H1 SOYMON038 g1814400
BLASTN 842 1e-61 78 2707 10032 700953253H1 SOYMON022 g1814400
BLASTN 808 1e-58 77 2708 10032 701103083H1 SOYMON028 g1814400
BLASTN 813 1e-58 78 2709 10131 701104852H1 SOYMON036 g3294466
BLASTN 302 1e-27 74 2710 10131 700970420H1 SOYMON005 g2829893
BLASTX 240 1e-26 56 2711 1180 701125681H1 SOYMON037 g2829893 BLASTX
163 1e-15 82 2712 1180 700559947H1 SOYMON001 g2829893 BLASTX 163
1e-15 82 2713 1180 700556009H1 SOYMON001 g2829893 BLASTX 102 1e-14
87 2714 13262 701006086H2 SOYMON019 g3294466 BLASTN 734 1e-52 75
2715 13262 701137937H1 SOYMON038 g3294466 BLASTN 491 1e-32 71 2716
13262 701004207H1 SOYMON019 g3294466 BLASTN 271 1e-30 75 2717 13262
700904551H1 SOYMON022 g3294466 BLASTN 473 1e-30 75 2718 13262
701014357H1 SOYMON019 g1814401 BLASTX 210 1e-21 83 2719 13262
701146638H1 SOYMON031 g1814401 BLASTX 111 1e-20 80 2720 13262
700833416H1 SOYMON019 g1814400 BLASTN 374 1e-20 74 2721 13262
701148967H1 SOYMON031 g1814401 BLASTX 194 1e-19 82 2722 13262
701156042H1 SOYMON031 g1814401 BLASTX 182 1e-18 67 2723 13262
700943365H1 SOYMON024 g1814401 BLASTX 168 1e-16 76 2724 13262
701105762H1 SOYMON036 g1814401 BLASTX 165 1e-15 83 2725 13262
701038338H1 SOYMON029 g1814400 BLASTN 186 1e-13 77 2726 13262
700645989H1 SOYMON011 g1814401 BLASTX 133 1e-11 78 2727 13262
700868941H1 SOYMON016 g1814400 BLASTN 181 1e-9 80 2728 19312
701121150H1 SOYMON037 g3294468 BLASTN 501 1e-61 79 2729 19312
700742959H1 SOYMON012 g3294468 BLASTN 440 1e-45 83 2730 19312
701135418H1 SOYMON038 g3294468 BLASTN 459 1e-42 79 2731 19312
700979514H2 SOYMON009 g1814400 BLASTN 612 1e-42 78 2732 19883
701133631H2 SOYMON038 g1814400 BLASTN 758 1e-54 75 2733 19883
700970758H1 SOYMON005 g1814400 BLASTN 717 1e-50 77 2734 19883
701153416H1 SOYMON031 g1814400 BLASTN 691 1e-48 76 2735 26278
701214005H1 SOYMON035 g534982 BLASTX 118 1e-8 47 2736 -GM1647
LIB3028-009- LIB3028 g534982 BLASTX 192 1e-42 57 Q1-B1-F3 2737
-GM17162 LIB3055-012- LIB3055 g1814400 BLASTN 491 1e-29 62 Q1-N1-B3
2738 13262 LIB3028-003- LIB3028 g1814400 BLASTN 1069 1e-80 76
Q1-B1-B11 2739 13262 LIB3054-009- LIB3054 g1814400 BLASTN 612 1e-40
73 Q1-N1-A12 2740 13262 LIB3054-009- LIB3054 g1814401 BLASTX 200
1e-36 73 Q1-N1-A5 SOYBEAN UDP-GLUCOSE PYROPHOSPHORYLASE 2741
-700665357 700665357H1 SOYMON005 g1388021 BLASTX 183 1e-18 69 2742
-700674325 700674325H1 SOYMON007 g218000 BLASTN 645 1e-44 72 2743
-700835903 700835903H1 SOYMON019 g1388021 BLASTX 135 1e-11 68 2744
-700841466 700841466H1 SOYMON020 g1388021 BLASTX 115 1e-14 56 2745
-700846570 700846570H1 SOYMON021 g3107930 BLASTN 486 1e-31 70 2746
-700888547 700888547H1 SOYMON024 g3107930 BLASTN 582 1e-39 81 2747
-700973436 700973436H1 SOYMON005 g1212996 BLASTX 132 1e-15 51 2748
-700985779 700985779H1 SOYMON009 g3107930 BLASTN 958 1e-71 83 2749
-700992994 700992994H1 SOYMON011 g1388021 BLASTX 103 1e-10 64 2750
-701061122 701061122H1 SOYMON033 g1388021 BLASTX 129 1e-19 73 2751
-701063465 701063465H1 SOYMON033 g3107930 BLASTN 426 1e-62 82 2752
-701118256 701118256H1 SOYMON037 g3107930 BLASTN 378 1e-31 83 2753
11810 700952705H1 SOYMON022 g3107930 BLASTN 613 1e-54 80 2754 11810
701060568H1 SOYMON033 g3107930 BLASTN 652 1e-45 81 2755 11810
701002783H2 SOYMON019 g3107930 BLASTN 458 1e-43 80 2756 11810
701202674H1 SOYMON035 g218000 BLASTN 331 1e-33 73 2757 11810
700871590H1 SOYMON018 g218000 BLASTN 326 1e-27 76 2758 11810
700747279H1 SOYMON013 g1388021 BLASTX 154 1e-21 75 2759 11810
701014424H1 SOYMON019 g1388021 BLASTX 131 1e-20 84 2760 11810
701039454H1 SOYMON029 g1388021 BLASTX 157 1e-16 80 2761 11810
701054271H1 SOYMON032 g1388021 BLASTX 154 1e-15 71 2762 11810
700955092H1 SOYMON022 g1388021 BLASTX 154 1e-14 71 2763 11810
701107189H1 SOYMON036 g1388021 BLASTX 155 1e-14 72 2764 11810
701107930H1 SOYMON036 g218000 BLASTN 308 1e-14 75 2765 11810
700904384H1 SOYMON022 g1388021 BLASTX 149 1e-13 72 2766 11810
700729516H1 SOYMON009 g1388021 BLASTX 155 1e-13 72 2767 11810
701009325H1 SOYMON019 g1388021 BLASTX 143 1e-12 75 2768 11821
701060627H1 SOYMON033 g218000 BLASTN 253 1e-26 74 2769 11821
701004671H1 SOYMON019 g21599 BLASTX 166 1e-22 77 2770 11821
700964889H1 SOYMON022 g1388021 BLASTX 167 1e-16 67 2771 13178
700562308H1 SOYMON002 g3107930 BLASTN 1198 1e-91 87 2772 13178
701049018H1 SOYMON032 g3107930 BLASTN 1101 1e-82 88 2773 13178
701126215H1 SOYMON037 g3107930 BLASTN 1072 1e-80 88 2774 13178
701211745H1 SOYMON035 g3107930 BLASTN 1038 1e-77 87 2775 13178
700850417H1 SOYMON023 g3107930 BLASTN 1022 1e-76 87 2776 13178
700665292H1 SOYMON005 g3107930 BLASTN 980 1e-72 88 2777 13178
700994009H1 SOYMON011 g3107930 BLASTN 958 1e-71 86 2778 13178
700895203H1 SOYMON024 g3107930 BLASTN 864 1e-68 86 2779 13178
701151725H1 SOYMON031 g3107930 BLASTN 800 1e-66 87 2780 13178
700988803H1 SOYMON011 g3107930 BLASTN 896 1e-65 80 2781 13178
700646581H1 SOYMON014 g3107930 BLASTN 483 1e-62 81 2782 13178
701153726H1 SOYMON031 g3107930 BLASTN 832 1e-60 87 2783 13178
701152333H1 SOYMON031 g3107930 BLASTN 674 1e-56 79 2784 13178
700756960H1 SOYMON015 g3107930 BLASTN 787 1e-56 86 2785 13178
700556901H1 SOYMON001 g218000 BLASTN 772 1e-55 84 2786 13178
701063605H1 SOYMON033 g3107930 BLASTN 566 1e-51 89 2787 13178
701212385H1 SOYMON035 g3107930 BLASTN 390 1e-23 78 2788 13178
700889518H1 SOYMON024 g3107931 BLASTX 131 1e-10 64 2789 17057
700740176H1 SOYMON012 g3107930 BLASTN 798 1e-57 81 2790 17057
700905747H1 SOYMON022 g3107930 BLASTN 511 1e-33 81 2791 1955
701059208H1 SOYMON033 g3107930 BLASTN 1034 1e-77 85 2792 1955
700984109H1 SOYMON009 g3107930 BLASTN 970 1e-72 84 2793 1955
701209482H1 SOYMON035 g3107930 BLASTN 931 1e-68 84 2794 1955
700554847H1 SOYMON001 g3107930 BLASTN 493 1e-66 83 2795 1955
701150363H1 SOYMON031 g3107930 BLASTN 898 1e-66 84 2796 1955
700986014H1 SOYMON009 g3107930 BLASTN 907 1e-66 84 2797 1955
700564270H1 SOYMON002 g3107930 BLASTN 501 1e-65 84 2798 1955
700844253H1 SOYMON021 g3107930 BLASTN 875 1e-64 84 2799 1955
701140892H1 SOYMON038 g3107930 BLASTN 879 1e-64 83 2800 1955
700685893H1 SOYMON008 g3107930 BLASTN 832 1e-60 81 2801 1955
700789732H1 SOYMON011 g3107930 BLASTN 554 1e-57 84 2802 1955
700902418H1 SOYMON027 g3107930 BLASTN 731 1e-52 83 2803 1955
701128306H1 SOYMON037 g3107930 BLASTN 466 1e-46 82 2804 1955
701057973H1 SOYMON033 g3107930 BLASTN 422 1e-43 78 2805 21035
700946288H1 SOYMON024 g3107931 BLASTX 175 1e-17 72 2806 21035
701043539H1 SOYMON029 g3107931 BLASTX 147 1e-13 71 2807 30564
701063642H1 SOYMON033 g3107931 BLASTX 181 1e-25 75 2808 -GM18453
LIB3065-001- LIB3065 g1212996 BLASTX 68 1e-29 57 Q1-N1-H4 2809
-GM32502 LIB3051-013- LIB3051 g3107931 BLASTX 227 1e-47 51 Q1-E1-A6
2810 11810 LIB3030-010- LIB3030 g21598 BLASTN 1115 1e-84 76
Q1-B1-H12 2811 13178 LIB3056-014- LIB3056 g3107930 BLASTN 1145
1e-99 84 Q1-N1-G7 2812 1955 LIB3056-012- LIB3056 g3107930 BLASTN
856 1e-62 80 Q1-N1-D4 2813 30564 LIB3050-003- LIB3050 g3107930
BLASTN 1078 1e-81 74 Q1-E1-D8 2814 30564 LIB3050-010- LIB3050
g3107930 BLASTN 1050 1e-78 75 Q1-E1-D6 *Table Headings Cluster ID A
cluster ID is arbitrarily assigned to all of those clones which
belong to the same cluster at a given stringency and a particular
clone will belong to only one cluster at a given stringency. If a
cluster contains only a single clone (a "singleton"), then the
cluster ID number will be negative, with an absolute value equal to
the clone ID number of its single member. The cluster ID entries in
the table refer to the cluster with which the particular clone in
each row is associated. Clone ID The clone ID number refers to the
particular clone in the PhytoSeq database. Each clone ID entry in
the table refers to the clone whose sequence is used for (1) the
sequence comparison whose scores are presented and/or (2)
assignment to the particular cluster which is presented. Note that
a clone may be included in this table even if its sequence
comparison scores fail to meet the minimum standards for
similarity. In such a case, the clone is included due solely to its
association with a particular cluster for which sequences of one or
more other member clones possess the required level of similarity.
Library The library ID refers to the particular cDNA library from
which a given clone is obtained. Each cDNA library is associated
with the particular tissue(s), line(s) and developmental stage(s)
from which it is isolated. NCBI gi Each sequence in the GenBank
public database is arbitrarily assigned a unique NCBI gi (National
Center for Biotechnology Information GenBank Identifier) number. In
this table, the NCBI gi number which is associated (in the same
row) with a given clone refers to the particular GenBank sequence
which is used in the sequence comparison. This entry is omitted
when a clone is included solely due to its association with a
particular cluster. Method The entry in the "Method" column of the
table refers to the type of BLAST search that is used for the
sequence comparison. "CLUSTER" is entered when the sequence
comparison scores for a given clone fail to meet the minimum values
required for significant similarity. In such cases, the clone is
listed in the table solely as a result of its association with a
given cluster for which sequences of one or more other member
clones possess the required level of similarity. Score Each entry
in the "Score" column of the table refers to the BLAST score that
is generated by sequence comparison of the designated clone with
the designated GenBank sequence using the designated BLAST method.
This entry is omitted when a clone is included solely due to its
association with a particular cluster. If the program used to
determine the hit is HMMSW then the score refers to HMMSW score.
P-Value The entries in the P-Value column refer to the probability
that such matches occur by chance. % Ident The entries in the "%
Ident" column of the table refer to the percentage of identically
matched nucleotides (or residues) that exist along the length of
that portion of the sequences which is aligned by the BLAST
comparison to generate the statistical scores presented. This entry
is omitted when a clone is included solely due to its association
with a particular cluster.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090313727A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20090313727A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References