U.S. patent application number 12/640674 was filed with the patent office on 2010-06-24 for method for speeding up plant growth and improving yield by introducing phosphatases in transgenic plant.
Invention is credited to Boon Leong Lim.
Application Number | 20100159065 12/640674 |
Document ID | / |
Family ID | 42266496 |
Filed Date | 2010-06-24 |
United States Patent
Application |
20100159065 |
Kind Code |
A1 |
Lim; Boon Leong |
June 24, 2010 |
METHOD FOR SPEEDING UP PLANT GROWTH AND IMPROVING YIELD BY
INTRODUCING PHOSPHATASES IN TRANSGENIC PLANT
Abstract
Transgenic plants having increased growth rate, increased sugar
content, and increase yield are disclosed, and methods for making
the same. The transgenic plants have a gene coding for a
phosphatase having a C-terminal motif under control of a
heterologous promoter incorporated into the genomic DNA of the
plant.
Inventors: |
Lim; Boon Leong; (Hong Kong,
HK) |
Correspondence
Address: |
TUROCY & WATSON, LLP
127 Public Square, 57th Floor, Key Tower
CLEVELAND
OH
44114
US
|
Family ID: |
42266496 |
Appl. No.: |
12/640674 |
Filed: |
December 17, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61138918 |
Dec 18, 2008 |
|
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Current U.S.
Class: |
426/2 ;
435/320.1; 435/419; 435/468; 800/290; 800/298 |
Current CPC
Class: |
C12N 15/8261 20130101;
Y02A 40/146 20180101; C12N 9/16 20130101; C12N 15/8245
20130101 |
Class at
Publication: |
426/2 ; 800/290;
800/298; 435/320.1; 435/419; 435/468 |
International
Class: |
A23K 1/18 20060101
A23K001/18; A01H 1/00 20060101 A01H001/00; A01H 5/00 20060101
A01H005/00; C12N 15/82 20060101 C12N015/82; C12N 5/10 20060101
C12N005/10 |
Claims
1. A method to make a transgenic plant having increased rate of
plant growth and elevate plant yields comprising: introducing a
gene coding for a phosphatase into a plant, the phosphatase gene
encodes for a polypeptide comprising a C-terminal motif having the
sequence of SEQ ID NO: 66 or a homologue thereof, and one or more
sequences selected from the group consisting of SEQ ID NOS:
48-53.
2. The method of claim 1, wherein the phosphatase gene is a purple
acid phosphatase gene comprising a nucleotide sequence encoding a
polypeptide comprising an amino acid sequence of one or more
selected from SEQ ID NOS: 2, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33,
35, and 47, and homologues thereof.
3. The method of claim 1, wherein the phosphatase gene is a purple
acid phosphatase gene comprising a nucleotide sequence selected
from SEQ ID NOS: 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, and
46 and homologues thereof.
4. The method of claim 2, wherein said nucleotide sequence
comprises one or more selected from the group consisting of SEQ ID
NOs 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 46 and
homologues thereof.
5. The method of claim 2, wherein said nucleotide sequence
comprises a nucleotide sequence encoding a polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO: 2 and conservatively substituted variants thereof.
6. The method of claim 2, wherein said nucleotide sequence
comprises a nucleotide sequence encoding a polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 54 and 55 and conservatively substituted variants thereof.
7. The method of claim 6, wherein said nucleotide sequence
comprises a nucleotide sequence encoding a polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 50 and 51.
8. The method of claim 2, wherein said nucleotide sequence
comprises a nucleotide sequence encoding a polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 57-60 and conservatively substituted variants thereof.
9. The method of claim 8, wherein said nucleotide sequence
comprises a nucleotide sequence encoding a polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 50 and 51.
10. The method of claim 2, wherein said nucleotide sequence
comprises a nucleotide sequence encoding a polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOS: 61-64.
11. The method of claim 1, wherein introducing the phosphatase gene
up-regulates the enzymatic activity of sucrose phosphate synthase
in the transgenic plant relative to the wild-type plant.
12. The method of claim 1, wherein introducing the phosphatase gene
up-regulates the sucrose and/or glucose level in the transgenic
plant relative to the wild-type plant.
13. The method of claim 1, wherein introducing the phosphatase gene
increases the growth rate of the transgenic plant relative to the
wild-type plants.
14. The method of claim 1, wherein introducing the phosphatase gene
results in a higher crop yield of the transgenic plant relative to
the wild-type plants.
15. The method of claim 1, wherein the plant is a species selected
from one of the group consisting of the following genera:
Asparagus, Bromus, Hemerocalli, Hordeum, Loliu, Panicum,
Pennisetum, Saccharum, Sorghum, Trigonell, Triticum, Zea,
Antirrhinum, Arabidopsis, Arachis, Atropa, Brassica, Browallia,
Capsicum, Carthamus, Cichorium, Citrus, Chrysanthemum, Cucumis,
Datura, Daucus, Digitalis, Fragaria, Geranium, Glycine, Helianthus,
Hyscyamus, Ipomoea, Latuca, Linum, Lotus, Solanum lycopersicon,
Majorana, Malva, Gossypium, Manihot, Medicago, Nemesia, Nicotiana,
Onobrychis, Pelargonium, Petunia, Ranunculus, Raphanus,
Salpiglossis, Senecio, Sinapis, Solanum, Trifolium, Vigna, and
Vitis.
16. The method of claim 1, wherein the plant is a species selected
from the family Brassica.
17. A transformed plant, comprising: a plant comprising at least
one additional gene coding for a phosphatase relative to a
corresponding wild-type plant, the phosphatase gene encodes for a
polypeptide comprising a C-terminal motif having the sequence of
SEQ ID NO: 66 or a homologue thereof, and one or more sequences
selected from the group consisting of SEQ ID NOS: 48-53.
18. The transformed plant of claim 17, wherein the phosphatase gene
is a purple acid phosphatase gene comprising a nucleotide sequence
encoding a polypeptide comprising an amino acid sequence of one or
more selected from SEQ ID NOS: 2, 6, 8, 19, 21, 23, 25, 27, 29, 31,
33, 35, and 47, and homologues thereof.
19. The transgenic plant of claim 17, wherein the phosphatase gene
is a purple acid phosphatase gene comprising a nucleotide sequence
selected from SEQ ID NOS: 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32,
34, and 46 and homologues thereof.
20. The transgenic plant of claim 17, wherein the phosphatase gene
is a purple acid phosphatase gene comprising a nucleotide sequence
selected from SEQ ID NO: 1 or a homologue thereof.
21. The transformed plant of claim 17, wherein the phosphatase gene
is a purple acid phosphatase gene comprising a nucleotide sequence
encoding a polypeptide comprising an amino acid sequence of one or
more selected from SEQ ID NOS: 2 or a homologue thereof.
22. The transformed plant of claim 17, wherein said plant is of a
monocotyledonous species.
23. The transformed plant of claim 17, wherein said plant is of a
dicotyledonous species.
24. The transformed plant of claim 17, wherein the plant is a
species selected from one of the group consisting of the following
genera: Asparagus, Bromus, Hemerocalli, Hordeum, Loliu, Panicum,
Pennisetum, Saccharum, Sorghum, Trigonell, Triticum, Zea,
Antirrhinum, Arabidopsis, Arachis, Atropa, Brassica, Browallia,
Capsicum, Carthamus, Cichorium, Citrus, Chrysanthemum, Cucumis,
Datura, Daucus, Digitalis, Fragaria, Geranium, Glycine, Helianthus,
Hyscyamus, Ipomoea, Latuca, Linum, Lotus, Solanum lycopersicon,
Majorana, Malva, Gossypium, Manihot, Medicago, Nemesia, Nicotiana,
Onobrychis, Pelargonium, Petunia, Ranunculus, Raphanus,
Salpiglossis, Senecio, Sinapis, Solanum, Trifolium, Vigna, and
Vitis.
25. The transformed plant of claim 17, wherein the plant is a
species selected from the family Brassica.
26. A vector comprising a plasmid comprising a phosphatase gene
comprising a nucleic acid sequence selected from the group
consisting of SEQ ID NOS: 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32,
34, and 46 and homologues thereof, wherein the plasmid is capable
of transforming a bacterial cell.
27. The vector of claim 26, wherein the bacterial cell is
Agrobacterium tumefaciens.
28. The vector of claim 26, wherein said phosphatase gene encodes
an amino acid sequence comprising one or more selected from the
group consisting of SEQ ID NOS: 2, 6, 8, 19, 21, 23, 25, 27, 29,
31, 33, 35, and 47 and homologues thereof, except that all or an
N-terminal portion of amino acid residues 1 to 50 of these amino
acid sequences are replaced by a plant signal peptide such that
said polypeptide is sorted to various organelles or compartments of
plant cells upon expression of the phosphatase gene in a
transformed host plant cell.
29. The vector of claim 26, wherein the phosphatase gene comprises
a nucleic acid sequence selected from SEQ ID NO: 1 or a homologue
thereof.
30. A host cell comprising the vector of claim 26.
31. The host cell of claim 30, wherein the phosphatase gene
comprising the vector is operably linked to a heterologous
promoter.
32. The host cell of claim 31, wherein the heterologous promoter is
a plant promoter.
33. The host cell of claim 31, wherein the heterologous promoter is
a promoter derived from cauliflower mosaic virus.
34. The host cell of claim 30, wherein the host cell is a plant
species.
35. A method for preparing a cell or progeny thereof capable of
expressing a purple acid phosphatase in a host cell, comprising:
transforming a bacterial cell with the vector of claim 26 and
transforming the host cell by transfer of DNA from the bacterial
cell to the host cell.
36. The method of claim 35, wherein the bacterial cell is
Agrobacterium tumefaciens and the host cell is a plant cell.
37. The method of claim 36, wherein the host cell is from the
family Brassica.
38. An expression cassette comprising a phosphatase gene comprising
a nucleotide sequence encoding an enzyme having phosphatase
activity, wherein said nucleotide sequence hybridizes to a
nucleotide sequences selected from the group consisting of SEQ ID
NOS: 1, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, and 46, under
stringent condition and is operably linked to regulatory nucleotide
sequences such that said regulatory nucleotide sequences cause
expression of the nucleotide sequence in plant cells.
39. The expression cassette of claim 38, wherein said nucleotide
sequence hybridizes to the nucleotide sequence of SEQ ID NO: 1.
40. The Expression cassette of claim 38, wherein said regulator
nucleotide sequence is a cauliflower mosaic virus promoter.
41. A method of feeding animals, comprising: providing a feed
comprising matter derived from the transformed plant of claim 17 to
an animal for consumption by the animal.
Description
RELATED APPLICATIONS
[0001] This application claims priority to provisional application
Ser. No. 61/138,918, filed on Dec. 18, 2008, which is incorporated
herein by reference.
1. TECHNICAL FIELD
[0002] The present disclosure provides methods that speeds up plant
growth and elevates plant yields by introducing phosphatases with a
C-terminal motif into plants. The present disclosure relates to
phosphatases with a C-terminal motif, and their respectively
encoded protein products, as well as fragments, derivatives,
homologues, and variants thereof. Methods for introducing these
genes into plants to (1) speed up the growth rate of plants, (2) to
increase the sugar contents of plants, and (3) to increase of yield
of plants, are provided.
2. BACKGROUND
[0003] Purple acid phosphatases (PAPs) catalyze the hydrolysis of a
wide range of activated phosphoric acid mono- and di-esters and
anhydrides (Klabunde et al., 1996). The PAP proteins are
characterized by seven conserved amino acid residues (shown in bold
face) in the five conserved motifs XDXX, XDXXY, GNH(D/E), XXXH,
XHXH, which are involved in the coordination of the dimetal nuclear
center (Fe.sup.3+-Me.sup.2+) in the active site (Li et al., 2002),
where Me is a transition metal and Me.sup.2+ is mostly found to be
Fe.sup.2+ in mammalian, and Zn.sup.2+, or Mn.sup.2+ in plants
(Klabunde and Krebs, 1997; Schenk et al., 1999).
[0004] Purple acid phosphatases are distinguished from the other
phosphatases by their characteristic purple color, which is caused
by a charge transfer transition at 560 nm from a metal-coordinating
tyrosine to the metal ligand Fe.sup.3+ (Klabunde and Krebs, 1997;
Schenk et al., 2000). Different from the other acid phosphatases,
PAPs are insensitive to inhibition by tartrate, so they are also
known as tartrate-resistant acid phosphatases (TRAPs).
[0005] The biochemical properties of some plant PAPs have been
characterized, firstly in red kidney bean, and later in soybean
suspension cell, soybean seedlings, rice culture cells, spinach
leaves, sweet potato tubers, tomato, yellow lupin seeds, medicago
and Arabidopsis, etc. (Schenk et al., 1999). Plant PAPs are
generally considered to mediate phosphorus acquisition and
redistribution based on their ability to hydrolyze phosphate
compounds (Cashikar et al., 1997; Bozzo et al., 2004; Lung et al.,
2008). Regulation of some plant PAPs transcripts by external
phosphate level in medium or soil, strongly suggest their involving
in phosphate acquisition. For example, the transcription level of
Medicago MtPAP1 in roots was increased under P stress, implicating
a role in P acquisition or internal mobilization (Xiao et al.,
2005; Xiao et al., 2006). Some plant PAPs could be secreted from
root cells to extracellular environment, then hydrolyze various
phosphate esters. Lung et al. purified a secreted PAP phosphatase
from tobacco, which could hydrolyze broad substrates and help to
alleviate P starvation (Lung et al., 2008). Certain plant PAPs can
also hydrolyze phytate, a major storage compound of phosphorus in
plants. Hegeman and Grabau (2001) purified a novel PAPs (GmPhy)
from the cotyledon of the germinating soybean seedlings. GmPhy was
introduced into soybean tissue culture and was assayed to show
phosphatase activity. Most recently, AtPAP15 and 23 in Arabidopsis
sharing high sequence homology (73-52%) with this soybean PAP, were
found to exhibit phytase activity (Zhu et al., 2005; Zhang et al.,
2008).
[0006] Besides involvement in P acquisition, plant PAPs may perform
some other physiological roles. For example, the PAPs AtACP5
(AtPAP17), SAP1, and SAP2 (del Pozo et al., 1999; Bozzo et al.,
2002) display not only phosphatase but also peroxidase activity,
suggesting their involvement in the removal of reactive oxygen
compounds in plant organs. A pollen-specific PAP from Ester lily
was suggested to function as an iron carrier in mature pollen (Kim
and Gynheung, 1996). Other studies indicate that plant PAPs may
also be involved in NaCl stress adaption or cell regeneration
(Kaida, 2003; Liao et al., 2003).
[0007] In the Arabidopsis genome, twenty-nine potential PAP genes
were identified based on sequence comparison. Twenty-four of these
putative enzymes contain seven conserved amino-acids residues
involved in metal binding. One (AtPAP13) lacked four of these seven
residues, and the other four (AtPAP14, 16, 28 and 29) lacked either
the first, the second, or both motifs of the five conserved motifs.
Twenty-eight are actively transcribed in Arabidopsis (Zhu et al.,
2005).
[0008] To date, relatively little is known about AtPAPs biochemical
properties and physiological roles, though several members have
been characterized (del Pozo et al., 1999). AtPAP17 (AtACP5) was
first known to be induced by phosphorus starvation. The
transcription of AtPAP17 was also responsive to ABA, salt stress
(NaCl), oxidative stress (H.sub.2O.sub.2) and leaves senescence,
according to GUS activity assay. No alteration in the expression of
AtPAP17 was observed during the nitrogen or potassium starvation,
and paraquat or salicylic acid. Like the other type5 acid
phosphatases, AtPAP17 displayed peroxidation activity, which may be
involved in the metabolism of reactive oxygen species in stressed
or senescent parts of plants.
[0009] Besides AtPAP17, several AtPAPs were found to be involved in
phosphorus metabolism in Arabidopsis. Root secretion of AtPAP12 was
induced by P stress, and its regulation was mainly at
transcriptional level (Patel et al., 1998; Coello, 2002/11).
AtPAP4, as well as AtPAP10, AtPAP11 and AtPAP12 were involved in
phosphorus starvation response since their transcription levels
increased during phosphate deprivation (Li et al., 2002; Wu et al.,
2003). In contrast, AtPAP20, 21 and 22 were irrespective to P
starvation and expressed constitutively in Pi sufficient or
deficient condition. Fluorescent signals were detected in the
cytoplasm via the baculovirus expression system, indicating that
they may function in the cytoplasm (Li and Wang, 2003).
[0010] AtPAP26 was purified and characterized from Pi-starved
Arabidopsis suspension cell culture (Veljanovski et al., 2006). It
exists as a homodimer with 55 kDa glycosylated protein, showing
wide substrate specificity with the highest activity against
phosphoenolpyruvate (PEP) and polypeptide phosphate. AtPAP26 also
displayed alkaline peroxidase activity with the probable roles in
the metabolism of reactive oxygen species. Proteomic study
suggested that it may be localized in vacuole, and involved in
recycling Pi from intracellular P metabolites (Shimaoka et al.,
2004).
[0011] PAPs can act on a wide range of substrates, but not all of
them exhibit phytase activity. An enzyme assay involving the
GST-AtPAP23 fusion protein revealed that AtPAP23 exhibits phytase
activity. A GUS study showed that AtPAP23 is exclusively expressed
in the flower of the Arabidopsis, and may play certain roles in
flower development (Zhu et al., 2005). In a recent report, a
recombinant AtPAP15 expressed and partial purified in E. coli and
yeast was also found to exhibit phytase activity (Zhang et al.,
2008). It was proposed that AtPAP15 may be involved in ascorbic
acid biosynthesis with the end product myo-inositol of phytate
hydrolysis as the precursor of ascorbic acid synthesis.
[0012] As stated above, most of the functions of characterized
plant PAPs are related to phosphorus metabolism. None of the
functionally or biochemically characterized plant PAPs carry
transmembrane motif, and none of them were shown to be associated
with membrane. Furthermore, to date, no AtPAPs or any plant PAPs,
have been showed to affect sugar signalling and carbon metabolism
in plant.
[0013] The first report of transgenic expression of plant PAP in
plant was reported in 2005 (Xiao et al., 2005). The PAP-phosphatase
gene from Medicago (MtPHY1) was expressed in transgenic
Arabidopsis, resulting in increased capacity of P acquisition from
phytate in agar culture (Xiao et al., 2005). Nonetheless, the
growth performance of the plants was not reported to be different
under normal growth.
3. SUMMARY
[0014] The present disclosure provides a method that speeds up
plant growth and elevates plant yields by introducing phosphatases
with a C-terminal motif into plants. Phosphatases with a C-terminal
motif, and their respectively encoded protein products, as well as
fragments, derivatives, homologues, and variants thereof are
disclosed. Methods for introducing this class of genes into plants
to speed up the growth rate of plants, to increase the sugar
contents of plants, and to increase of yield of plants, are
provided. Without wishing to be bound by any particular theory, the
C-terminal motif is believed to function as a transmembrane
structural element (transmembrane motif).
[0015] As stated above in the Background section, most of the
functions of characterized plant PAPs are related to phosphorus
metabolism. None of the functionally or biochemically characterized
plant PAPs carry transmembrane motif, and none of them were shown
to be associated with membrane. Furthermore, to date, no AtPAPs or
any plant PAPs, have been showed to affect sugar signalling and
carbon metabolism in plant.
[0016] The first report of transgenic expression of plant PAP in
plant was reported in 2005 (Xiao et al., 2005). The PAP-phosphatase
gene from Medicago (MtPHY1) was expressed in transgenic
Arabidopsis, resulting in increased capacity of P acquisition from
phytate in agar culture. Nonetheless, the growth performance of the
plants was not reported to be different under normal growth.
[0017] We also produced transgenic tobacco and Arabidopsis that
overexpressed AtPAP15, a PAP with phosphatase activity, which does
not carry any C-terminal motif equivalent to that of AtPAP2;
phosphatase activity was secreted into extracellular growth medium.
Significant secretion of phosphatase activity was observed in the
transgenic plants and the transgenic plants showed larger biomass
than the control plants in agar and soil supplemented with
exogenous phytate. Higher P content was also obtained in
overexpressed transgenic lines in phytate treatment. However, the
growth of transgenic plants overexpressing AtPAP15 did not show any
difference in growth phenotypes when it was compared with the
wild-type, under treatments of K--P or No--P, or in soil.
[0018] Here, we have developed a technology to speed up plant
growth and improve seed yield by overexpressing a phosphatase with
a C-terminal motif in plants. An example is the use of a purple
acid phosphatase (PAP). This disclosure is the first report to show
that overexpressing a phosphatase with a C-terminal motif in
transgenic plant is able to speed up the growth of the plants, to
increase the sugar contents of plants, and to increase the yield of
plants, by altering the carbon metabolism of the plants.
[0019] The present advances are based, in part, on the
characterization of a group of purple acid phosphatases (SEQ ID
NOS: 1-8 and 18-47) from plants and the observations that
overexpression of a purple acid phosphatase (AtPAP2, SEQ ID NO:1)
of this group in plants resulted in rapid plant growth, higher
sugar content, and higher yield. Accordingly, nucleotide sequences
of a group of purple acid phosphatase genes (SEQ ID NOs:1, 3, 5, 7,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46),
which share a C-terminal motif/domain, from plants and amino acid
sequences of their encoded proteins (SEQ ID NOS:2, 4, 6, 8, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47), as well as
fragments, derivatives, homologues, and variants thereof, as
defined herein, are disclosed. Furthermore, nucleic acid molecules
encoding the polypeptides of interest and include cDNA, genomic
DNA, and RNA, are disclosed.
[0020] As used herein, italicizing the name of a gene shall
indicate the gene, in contrast to its encoded protein or
polypeptide product which is indicated by the name of the gene in
the absence of any italicizing. For example, "Gene" shall mean the
Gene gene, whereas "Gene" shall indicate the protein or polypeptide
product of the Gene gene.
[0021] In one embodiment, isolated nucleic acid molecules hybridize
under stringent conditions, as defined herein, to nucleic acids
having the sequence of SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26,
28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or homologues thereof,
wherein the nucleic acid molecules encode proteins or polypeptides
which exhibit at least one structural and/or functional feature of
the polypeptides of the invention.
[0022] Another embodiment includes, nucleic acid molecules, which
are suitable for use as primers or hybridization probes for the
detection of nucleic acids encoding one of the disclosed
phosphatase polypeptides or other sequences.
[0023] Yet another embodiment includes vectors, e.g., recombinant
expression vectors, comprising a nucleic acid molecule of the
invention. Furthermore, host cells containing such a vector or
engineered to contain and/or express a nucleic acid molecule of the
invention and host cells containing a nucleotide sequence of the
invention operably linked to a heterologous promoter are
disclosed.
[0024] A further embodiment includes methods for preparing a
polypeptide of the invention by a recombinant DNA technology in
which the host cells containing a recombinant expression vector
encoding a polypeptide of the invention or a nucleotide sequence
encoding a polypeptide of the invention operably linked to a
heterologous promoter, are cultured, and the polypeptide of the
invention are produced.
[0025] In still further another embodiment, a transgenic plant
contains a nucleic acid molecule which encodes an isolated
polypeptides or proteins comprising the five conserved motifs of
purple acid phosphatases, including XDXX, XDXXY, GNH(D/E), XXXH,
XHXH, and linked to a C-terminal motif.
[0026] Embodiments further provide antibodies that
immunospecifically bind a polypeptide of the invention. Such
antibodies include, but are not limited to, antibodies from various
animals, humanized, chimeric, polyclonal, monoclonal, bi-specific,
multi-specific, single chain antibodies, Fab fragments,
F(ab').sub.2 fragments, disulfide-linked Fvs, fragments containing
either a VL or VH domain or even a complementary determining region
(CDR), that immunospecifically binds to a polypeptide of the
invention.
[0027] In an additional embodiment, method for detecting the
presence, activity or expression of a polypeptide of the invention
or similar polypeptide in a biological material, such as cells,
culture media, and so forth are provided. The increased or
decreased activity or expression of the polypeptide in a sample
relative to a control sample can be determined by contacting the
biological material with an agent that can detect directly or
indirectly the presence, activity or expression of the polypeptide
of the invention. In a particular embodiment, such an agent is an
antibody or a fragment thereof which immunospecifically binds to a
one of the disclosed polypeptides.
[0028] In a still another embodiment, a fusion protein comprising a
bioactive molecule and one or more domains of a disclosed
polypeptide or fragment thereof is provided. In particular, fusion
proteins comprising a bioactive molecule recombinantly fused or
chemically conjugated (including both covalent and non-covalent
conjugations) to one or more domains of a disclosed polypeptide or
fragments thereof.
[0029] We also produced transgenic tobacco and Arabidopsis that
overexpressed AtPAP15, a PAP with phosphatase activity, which does
not carry any C-terminal motif and was found to be secreted into
extracellular growth medium. Significant secretion of phosphatase
activity was observed in the transgenic plants and the transgenic
plants showed larger biomass than the control plants in agar and
soil supplemented with exogenous phytate. Higher P content was also
obtained in overexpressed transgenic lines in phytate treatment.
However, the growth of transgenic plants overexpressing AtPAP15 did
not show any difference in growth phenotypes when it was compared
with the wild-type, under treatments of K--P or No--P, or in
soil.
[0030] In conclusion, this disclosure is the first report to show
that overexpressing a phosphatase with a C-terminal motif in
transgenic plant is able to speed up the growth of the plants, to
increase the sugar contents of plants, and to increase the yield of
plants, by altering the carbon metabolism of the plants.
3.1 DEFINITIONS
[0031] The term "acidic" or "acid pH" as used herein refers to a pH
value of less than about 6.0.
[0032] The term "homologue" as used herein refers to a polypeptide
that possesses a similar or identical function to polypeptides
encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45 and 47, and/or a fragment of these
polypeptides, that do not have an identical amino acid sequence of
these polypeptides and/or a fragment of these polypeptides. A
polypeptide that has a similar amino acid sequence included in the
definition of the term "homologue" includes a polypeptide that
satisfied at least one of the following: (i) polypeptide having an
amino acid sequence that is one or more of at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70%, at least about 80%, at least about 90%, and at least
about 98% identical. (ii) a polypeptide encoded by a nucleotide
sequence that is one or more of at least about 30%, at least about
40%, at least about 50%, at least about 60%, at least about 70%, at
least about 80%, at least about 90%, and at least about 98%
identical and/or conservatively substituted to one or more of the
nucleotide sequences encoding the polypeptides of SEQ ID NOS: 2, 4,
6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and
47, and/or a fragment of the these polypeptides; (iii) a
polypeptide encoded by a nucleotide sequence that hybridizes under
stringent conditions as defined herein to one or more of nucleotide
sequences SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44 and 6; (iv) a polypeptide having an amino
acid sequence that is one or more of at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, and at least about 98%
identical and/or conservatively substituted; (v) a nucleic acid
sequence encoding an amino acid sequence that is one or more of at
least about 30%, at least about 40%, at least about 50%, at least
about 60%, at least about 70%, at least about 80%, at least about
90%, and at least about 98% identical and/or conservatively
substituted; (vi) a fragment of any of the polypeptides or nucleic
acid sequences described in (i) through (v) having one of at least
20 amino acid residues, at least 25 amino acid residues, at least
40 amino acid residues, at least 80 amino acid residues, at least
90 amino acid residues, at least 100 amino acid residues, at least
125 amino acid residues, at least 150 amino acid residues, at least
175 amino acid residues, at least 200 amino acid residues, at least
225 amino acid residues, at least 250 amino acid residues, at least
275 amino acid residues, at least 300 amino acid residues, at least
325 amino acid residues, at least 350 amino acid residues, or at
least 375 amino acid residues; (vii) a polypeptide with similar
structure and function or a nucleotide sequence encoding a
polypeptide with similar structure and function, exhibiting the
antigenicity, immunogenicity, catalytic activity, and other readily
assayable activities, to polypeptides encoded by SEQ ID NOS: 2, 4,
6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and
47, and/or a fragment of these polypeptides, refers to a
polypeptide that has a similar secondary, tertiary, or quaternary
structure of these polypeptides, or a fragment of these
polypeptides. The structure of a polypeptide can be determined by
methods known to those skilled in the art, including but not
limited to, X-ray crystallography, nuclear magnetic resonance, and
crystallographic electron microscopy. The term "homologue" is used
herein to describe a sequence that has sequence homology. A
sequence having sequence homology can be made using standard
molecular biology techniques including site-directed mutagenesis
including insertion or deletion of sequences. The term "homologue"
is not limited to homologous genes or proteins originating from
different species and expressly includes artificial modification to
the sequences disclosed herein.
[0033] The term "conservatively substituted variant" refers to a
polypeptide or a nucleic acid sequence encoding a homologue
polypeptide in which one or more amino acid residues or codons have
been modified by conservative substitution with an amino acid
residue or a codon coding for an amino acid residue of similar
chemical-type, as described below.
[0034] The term "an antibody or an antibody fragment which
immunospecifically binds to polypeptides encoded by SEQ ID NOS: 2,
4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and
47," as used herein refers to an antibody or a fragment thereof
that immunospecifically binds to polypeptides encoded by SEQ ID
NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45 and 47, or a fragment of these polypeptide and does not
non-specifically bind to other polypeptides. An antibody or a
fragment thereof that immunospecifically binds to polypeptides
encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45 and 47, or a fragment of these polypeptide,
may cross-react with other antigens. Preferably, an antibody or a
fragment thereof that immunospecifically binds to polypeptides
encoded by SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33,
35, 37, 39, 41, 43, 45 and 47, or a fragment of these polypeptides,
does not cross-react with other antigens. An antibody or a fragment
thereof that immunospecifically binds to polypeptides encoded by
SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39,
41, 43, 45 and 47, or a fragment of these polypeptide, can be
identified by, for example, immunoassays or other techniques known
to those skilled in the art. An antibody or an antibody fragment
which immunospecifically binds polypeptides encoded by SEQ ID NOS:
2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45
and 47, may be interchangeably referred to as "anti-PAP
antibody".
[0035] The term "derivative" as used herein refers to a given
peptide or protein that is otherwise modified, e.g., by covalent
attachment of any type of molecule, preferably having bioactivity,
to the peptide or protein, including the incorporation of
non-naturally occurring amino acids. The resulting bioactivity
retains one or more biological activities of the peptide
protein.
[0036] The term "fragment" as used herein refers to a fragment of a
nucleic acid molecule containing one of at least about 25, at least
about 30, at least about 35, at least about 40, at least about 45,
at least about 100, at least about 150, at least about 200, at
least about 250, at least about 300, at least about 350, at least
about 400, at least about 450, at least about 500, at least about
550, at least about 600, at least about 650, at least about 700, at
least about 750, at least about 800, at least about 850, at least
about 900, at least about 950, at least about 1000, at least about
1050, at least about 1100, at least about 1150, at least about
1200, at least about 1250, at least about 1300, at least about
1350, from about 500 to about 2000, from about 1000 to about 2000
from about 200 to about 500, from about 500 to about 1000, form
about 1000 to about 1500, and from about 1500 to about 2000 nucleic
acid bases in length of the relevant nucleic acid molecule and
having at least one functional feature of the nucleic acid molecule
(or the encoded protein has one functional feature of the protein
encoded by the nucleic acid molecule); or a fragment of a protein
or a polypeptide containing one or more of at least about 5, at
least about 10, at least about 15, at least about 20, at least
about 25, at least about 30, at least about 35, at least about 40,
at least about 45, at least about 50, at least about 55, at least
about 60, at least about 65, at least about 70, at least about 75,
at least about 80, at least about 90, at least about 100, at least
about 120, at least about 140, at least about 160, at least about
180, at least about 200, at least about 220, at least about 240, at
least about 260, at least about 280, at least about 300, at least
about 320, at least about 340, at least about 360, from about 250
to about 660, from about 350 to about 660, form about 450 to about
660, and form about 550 to about 660 amino acid residues in length
of the relevant protein or polypeptide and having at least one
functional feature of the protein or polypeptide, such functional
features include ability to bind a Fe.sup.3+-Me.sup.2+ dimetal
nuclear center and form a C-terminal motif.
[0037] An "isolated" nucleic acid molecule is one which is
separated from other nucleic acid molecules which are present in
the natural source of the nucleic acid molecule. Moreover, an
"isolated" nucleic acid molecule, such as a cDNA molecule, can be
substantially free of other cellular materials, or culture medium
when produced by recombinant techniques, or substantially free of
chemical precursors or other chemicals when chemically synthesized,
but excludes nucleic acid molecules present in recombinant DNA
libraries. In a preferred embodiment, nucleic acid molecules
encoding the disclosed polypeptides/proteins are isolated or
purified.
[0038] The term "operably linked" as used herein refers to when
transcription under the control of the "operably linked" promoter
produces a functional messenger RNA, translation of which results
in the production of the polypeptide encoded by the DNA operably
linked to the promoter.
[0039] The term "under stringent condition" refers to hybridization
and washing conditions under which nucleotide sequences having
homology to each other remain hybridized to each other. Such
hybridization conditions are described in, for example but not
limited to, Current Protocols in Molecular Biology, John Wiley
& Sons, N.Y. (1989), 6.3.1-6.3.6.; Basic Methods in Molecular
Biology, Elsevier Science Publishing Co., Inc., N.Y. (1986), pp.
75-78, and 84-87; and Molecular Cloning, Cold Spring Harbor
Laboratory, N.Y. (1982), pp. 387-389, and are well known to those
skilled in the art. A preferred, non-limiting example of stringent
hybridization conditions is hybridization in 6.times. sodium
chloride/sodium citrate (SSC), 0.5% SDS at about 68.degree. C.
followed by one or more washes in 2.times.SSC, 0.5% SDS at room
temperature. Another preferred, non-limiting example of stringent
hybridization conditions is hybridization in 6.times.SSC at about
45.degree. C. followed by one or more washes in 0.2.times.SSC, 0.1%
SDS at about 50-65.degree. C.
[0040] The term "variant" as used herein refers either to a
naturally occurring allelic variation of a given peptide or a
recombinantly prepared variation of a given peptide or protein in
which one or more amino acid residues have been modified by amino
acid substitution, addition, or deletion.
[0041] The term "aligned" as used herein refers to a homology
alignment between two or more sequences using a standard algorithm
such as BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
[0042] The term "predicted to form a transmembrane motif by TMHMM
analysis" or "predicted to form a C-terminal motif by TMHMM
analysis" (http://www.cbs.dtu.dk/services/TMHMM/) herein refers to
a probability that is equal to or greater than about 0.5.
BRIEF DESCRIPTION OF THE FIGURES
[0043] The following figures illustrate the embodiments and are not
meant to limit the scope of the invention encompassed by the
claims.
[0044] FIG. 1 shows the phylogenetic tree of PAP-like sequences in
the Arabidopsis genome. Twenty-nine PAPs were aligned using
ClustalX and the phylogenetic tree was created by the
neighbor-joining algorithm of the MEGA4 program. The accession
numbers of the PAP-like, transmembrane-like C-terminal motif
containing, polypeptide from Zea mays (ZmPAP2) and Oryza sativa
(OsPAP2) were ACG47621 and BAC15853.1, respectively.
[0045] FIG. 2A is the amino acid alignment of the C-terminal
transmembrane-like motifs in AtPAP2 with other PAP sequences.
[0046] FIG. 2B is the amino acid alignment of AtPAP2 with other PAP
sequences, showing the full length of each sequence. These
sequences include homologous sequences from B. napus (BnPAP2), G.
max (GmPAP2) and Z. may (ZmPAP2). The five conserved motifs (XDXX,
XDXXY, GNH(D/E), XXXH, XHXH) are boxed. Residues in shades have low
or no homology. Hydrophobic motifs at the C-termini of these
polypeptides are underlined by a bar (614.sup.th-636.sup.th amino
acid), which is absent from the sequence of AtPAP15. As shown,
AtPAP15 does not have a C-terminal region corresponding to the
other PAP sequences.
[0047] FIG. 3 shows that a unique hydrophobic motif is present at
the C-termini of AtPAP2 and ZmPAP2 by TMHMM analysis. This
transmembrane-like C-terminal motif is absent from AtPAP15.
[0048] FIG. 4 shows the characteristics of the T-DNA lines. The
T-DNA line (Salk.sub.--013567) was obtained from TAIR. The AtPAP2
genomic sequence carries two exons and the T-DNA was inserted in
exon 2 and causes a disruption of the AtPAP2 mRNA (a). Three PCR
primers (A, B and C) were designed for the differentiation of the
wild-type (WT) and the T-DNA line (atpap2-8) and they were used for
PCR screening of genomic DNA extracted from WT and the T-DNA line
(b). Total RNA was extracted from 10-day-old seedlings grown on MS
with 2% sucrose using the TRIzol RNA isolation method and were used
for RT-PCR (c). 50 .mu.g of seedlings proteins were loaded for
Western blotting studies, using the anti-AtPAP2 specific antiserum
(Section 6.3).
[0049] FIG. 5 is the schematic diagram of the expression vector
pBV-AtPAP2. CaMV 35S: 35S promoter of the cauliflower mosaic virus;
NOS: polyadenylation signal of nopaline synthase gene; aadA:
bacterial streptomycin/spectinomycin resistance gene encoding
aminoglycoside-3''-adenyltransferase; pNOS:BAR: bialaphos
resistance gene under the control of the nopaline synthase
promoter; bom: basis of mobility from pBR322; ColE1: replication
origin from pBR322; pVS1-REP: replication origin from pVS1;
pVS1-STA: STA region from pVS1 plasmid; LB: left border T-DNA
repeat; RB: right border T-DNA repeat. (Hajdukiewicz et al.,
1994).
[0050] FIG. 6A shows the results of the Western blot analysis of
the overexpression lines (OE), wild-type (WT), T-DNA and the
complementation lines (CP) of AtPAP2 and FIG. 6B shows the results
of the Western blot analysis of the overexpression lines (C-15) and
wild-type (WT) of AtPAP15.
[0051] FIG. 7 shows the expression analysis of AtPAP2. The mRNA
expression profile was analysed by the Spot History program of NASC
(a). The protein expression profiles of 30 day old, soil-grown
plant (b), seedlings germinated on MS agar (c) and 2 week old
plants transferred to Pi-sufficient/Pi-deficient MS agar for 3 days
(d), were analyzed by Western blotting using the anti-PAP2
antiserum.
[0052] FIG. 8 shows the growth performance of the wild-type, T-DNA
and overexpression lines in soil. Seeds were germinated in MS agar
with 2% sucrose for 10 days. Seedlings with 2 small visible rosette
leaves (.about.1 mm) were transferred to soil and grown under 16
h/8 h light/dark cycles.
[0053] FIG. 9 shows the levels of sucrose and glucose in the
rosette leaves of 21-day-old, soil grown seedlings.
[0054] FIG. 10 shows the recovery of various lines after prolonged
darkness treatment. Seeds were germinated in MS agar with 2%
sucrose for 10 days. Seedlings with 2 small visible rosette leaves
(.about.1 mm) were transferred to soil and grown for 12 days under
16 h/8 h light/dark cycles. The lights of the growth chamber were
then switched off for 12 days and the plants were allowed to
recover under 16 h/8 h light/dark cycles for 1 week. n=9-12 per
line.
[0055] FIG. 11. Detection of AtPAP2 protein in subcellular
fractions by Western blotting. Mito.: Mitochondria; Chlorop.:
Chloroplasts.
[0056] FIG. 12. shows a schematic representation of two vector
constructs incorporating the AtPAP2 gene.
[0057] FIG. 13. shows Western blot analysis results for
overexpression of AtPAP2 proteins missing the C-terminal motif.
DETAILED DESCRIPTION
5.1 Method of Speeding Up Plant Growth and Improving Crop Yield
[0058] The present disclosure provides a method that speeds up
plant growth and elevates plant yields by introducing phosphatases
with a C-terminal motif into plants. In an embodiment, the present
disclosure relates to a class of genes of purple acid phosphatases,
and their respectively encoded protein products, as well as
fragments, derivatives, homologues, and variants thereof. Methods
for introducing this class of genes into plants to speed up the
growth rate of plants, to increase the sugar contents of plants,
and to increase of yield of plants, are provided.
[0059] A group of purple acid phosphatases (PAPs) which carry seven
conserved amino acid residues (shown in bold face) in the five
conserved motifs XDXX (example GDXG (SEQ ID NO: 48)), XDXXY (SEQ ID
NO: 49), GNH(D/E) (SEQ ID NOS: 50-51), XXXH (example ZXGH (SEQ ID
NO: 52)), XHXH (SEQ ID NO: 53), where X is any amino acid and Z is
any amino acid selected from L, I, V, F, and M, and a
transmembrane-like motif at their C-termini were identified in the
genomes of a number of plants (FIGS. 1, 2A, and 2B). The presence
of the C-terminal transmembrane-like motif enables the localization
of this group of PAPs to the membrane fraction (FIGS. 3 and 11).
This property makes this group of PAPs differ from the other
previously characterized PAPs because all previously characterized
PAPs did not carry any C-terminal motif (FIGS. 2A, 2B, and 3). By
using the protein sequence of a representative gene of this group,
AtPAP2, to blast the NCBI database and various EST databases, a
number of genomic or cDNA sequences were identified to encodes
polypeptides that carry the five conserved motifs XDXX, XDXXY,
GNH(D/E), XXXH, XHXH of PAPs and a transmembrane motif at their
C-termini (FIG. 2B).
[0060] The introduction of a representative gene of this group of
phosphatases, AtPAP2, into the genome of Arabidopsis by transgenic
technology produced transgenic Arabidopsis that grew faster than
the wild-type plants (FIG. 8), and the yield of seeds were elevated
by approximately 40% (Table 3). However, transgenic plant that
expressed AtPAP15 did not show these phenotypes. The sugar
contents, including glucose and sucrose, in the leaf of the
transgenic lines, were also found to be higher than that of the
wild-types (FIG. 9).
[0061] Thus, this disclosure provides a method that speeds up plant
growth and elevates plant yields by introducing phosphatases into
plants. In an embodiment, a group of genes of purple acid
phosphatases, and their respectively encoded protein products, as
well as fragments, derivatives, homologues, and variants thereof
are described.
5.2 Homologues, Derivatives, and Variants of Phosphatases
[0062] In addition to the nucleic acid molecules (SEQ ID NOS: 1, 3,
5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and
46) and polypeptides (SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27,
29, 31, 33, 35, 37, 39, 41, 43, 45 and 47) described in claims
9-16, the nucleic acid molecules and polypeptides also encompass
those nucleic acid molecules and polypeptides having a common
biological activity, similar or identical structural domain and/or
having sufficient nucleotide sequence or amino acid identity
(homologues) to those of the nucleic acid molecules and
polypeptides described above.
[0063] Such common biological activities of the polypeptides
include antigenicity, immunogenicity, catalytic activity especially
phosphatase activity, ability to bind a Fe.sup.3+-Me.sup.2+ dimetal
nuclear center, fold into or form a transmembrane-like C-terminal
motif and other activities readily assayable by the skilled
artisan.
[0064] A polypeptide that has a similar amino acid sequence
(homologue) refers to a polypeptide that satisfied at least one of
the following: (i) a polypeptide having an amino acid sequence that
is one of at least about 30%, at least about 40%, at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, and at least about 95%, and at least about 98%
identical and/or conservatively substituted to the amino acid
sequence of a AtPAP2 (SEQ ID NO: 2) and/or other PAPs with a
transmembrane-like C-terminal motif including SEQ ID NOS: 4, 6, 8,
19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and/or 47, a
fragment of AtPAP2, and having at least one biological feature of
the described polypeptides; (ii) a polypeptide encoded by a
nucleotide sequence that is one of at least about 30%, at least
about 40%, at least about 40%, at least about 50%, at least about
60%, at least about 70%, at least about 80%, at least about 90%, at
least about 95%, and at least about 98% identical to the nucleotide
sequence encoding AtPAP2 (SEQ ID NO: 1) and/or other PAPs with a
transmembrane-like C-terminal motif including SEQ ID NOS: 3, 5, 7,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and/or 46, a
fragment of AtPAP2 and having at least one structural and/or
biological feature of AtPAP2; (iii) a polypeptide encoded by a
nucleotide sequence that hybridizes under stringent conditions as
defined herein to a nucleotide sequence encoding AtPAP2 (SEQ ID NO:
1) and/or other PAPs with a motif including SEQ ID NOS: 3, 5, 7,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and/or 46, a
fragment of AtPAP2 and having at least one structural and/or
biological feature of AtPAP2. A polypeptide with similar structure
to AtPAP2, or a fragment of AtPAP2, refers to a polypeptide that
has a similar secondary, tertiary, or quaternary structure of
AtPAP2, a fragment of AtPAP2 and has at least one functional
feature of a AtPAP2, including one or more of ability to bind a
Fe.sup.3+-Me.sup.2+ dimetal nuclear center and fold into or form a
transmembrane-like C-terminal motif. The structure of a polypeptide
can be determined by methods known to those skilled in the art,
including but not limited to, X-ray crystallography, nuclear
magnetic resonance, and crystallographic electron microscopy.
[0065] Those having skill in the art will readily recognized that
mutations, deletions or insertions can be made in any of the
sequences disclosed herein, including SEQ ID NOS: 1-8 and 18-47,
without affecting function. Sequences useful in practicing the
embodiments include sequences having homology to SEQ ID NOS: 1-8
and 18-47 and being a protein, polypeptide, or polynucleotide
coding for such protein or peptide having functionality to bind a
dimetal nuclear center (Fe.sup.3+-Me.sup.2+) and being a protein,
polypeptide, or polynucleotide coding for such protein or peptide
having a C-terminal motif. That is, those skilled in the art will
recognize that many mutations can be made to any of SEQ ID NOS: 1-8
and 18-47 without affecting the catalytic functionality nor
interrupting the transmembrane-like C-terminal motif. Such modified
sequences that maintain catalytic activity and a transmembrane-like
C-terminal motif are defined as homologues to SEQ ID NOS: 1-8 and
18-47 and are including within the scope of useful sequences.
[0066] In one embodiment, such homologues can have about 30% or
more identity to the sequences disclosed herein. In another
embodiment, such homologues can have about 40% or more identity to
the sequences disclosed herein. In yet another embodiment, such
homologues can have about 50% or more identity to the sequences
disclosed herein. In sill yet another embodiment, such homologues
can have about 60% or more identity to the sequences disclosed
herein. In even sill yet another embodiment, such homologues can
have about 70% or more identity to the sequences disclosed herein.
In a further embodiment, such homologues can have about 80% or more
identity to the sequences disclosed herein. In yet a still further
embodiment, homologues can have about 90% or more identity to the
sequences disclosed herein. In a still further embodiment,
homologues can have about 98% or more identity to the sequences
disclosed herein.
[0067] Those having skill in the art will recognize that mutations
can be made to proteins and peptides and/or to polynucleotides
coding for protein and peptides or complementary thereto that
substitute amino acid residue for other amino acids residues having
similar chemical properties (conservative substitutions) and that
such mutations are less likely to cause structural changes that
affect functionality including catalytic activity and/or the
function of a transmembrane-like C-terminal motif. Conservatively
substituting amino acids are substituting an amino acid residue
belong to any of the following 11 chemical groups with another
amino acid from the same chemical group: (1) acidic (negatively
charged) amino acids such as aspartic acid and glutamic acid; (2)
basic (positively charged) amino acids such as arginine, histidine,
and lysine; (3) neutral polar amino acids such as glycine, serine,
threonine, cysteine, tyrosine, asparagine, and glutamine; (4)
neutral nonpolar (hydrophobic) amino acids such as alanine,
leucine, isoleucine, valine, proline, phenylalanine, tryptophan,
and methionine; (5) amino acids having aliphatic side chains such
as glycine, alanine, valine, leucine, and isoleucine; (6) amino
acids having aliphatic-hydroxyl side chains such as serine and
threonine; (7) amino acids having amide-containing side chains such
as asparagine and glutamine; (8) amino acids having aromatic side
chains such as phenylalanine, tyrosine, and tryptophan; (9) amino
acids having basic side chains such as lysine, arginine, and
histidine; (10) amino acids having sulfur-containing side chains
such as cysteine and methionine; (11); amino acids having similar
geometry and hydrogen bonding patterns such as aspartic acid,
asparagine, glutamic acid and glutamine.
[0068] In one embodiment, homologues can have about 30% or more
identity and/or conservative substitutions to the sequences
disclosed herein. In another embodiment, homologues can have about
40% or more identity and/or conservative substitutions to the
sequences disclosed herein. In yet another embodiment, homologues
can have about 50% or more identity and/or conservative
substitutions to the sequences disclosed herein. In still yet
another embodiment, homologues can have about 60% or more identity
and/or conservative substitutions to the sequences disclosed
herein. In a further embodiment, homologues can have about 70% or
more identity and/or conservative substitutions to the sequences
disclosed herein. In a still further embodiment, homologues can
have about 80% or more identity and/or conservative substitutions
to the sequences disclosed herein. In still another embodiment,
homologues can have about 90% or more identity and/or conservative
substitutions to the sequences disclosed herein. In still another
further embodiment, homologues can have about 98% or more identity
and/or conservative substitutions to the sequences disclosed
herein.
[0069] Embodiments further provide isolated nucleic acid molecules
which comprise or consist of one or more of at least about 25, at
least about 30, at least about 35, at least about 40, at least
about 45, at least about 100, at least about 150, at least about
200, at least about 250, at least about 300, at least about 350, at
least about 400, at least about 450, at least about 500, at least
about 550, at least about 600, at least about 650, at least about
700, at least about 750, at least about 800, at least about 850, at
least about 900, at least about 950, at least about 1000, at least
about 1050, at least about 1100, at least about 1150, at least
about 1200, at least about 1250, at least about 1300, at least
about 1350, from about 500 to about 2000, from about 1000 to about
2000, from about 200 to about 500, from about 500 to about 1000,
form about 1000 to about 1500, and from about 1500 to about 2000
nucleotides of the nucleotide sequences of SEQ ID NOS: 1, 3, 5, 7,
18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 and 46, or a
complement thereof encoding a protein or polypeptide having one or
more activity of the amino acid sequences of their encoded proteins
(SEQ ID NOS: 2, 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45 and 47). The activity includes one or more of
antigenicity, immunogenicity, catalytic activity (e.g., phosphatase
activity), ability to bind a Fe.sup.3+-Me.sup.2+ dimetal nuclear
center, fold into or form a transmembrane-like C-terminal motif,
and other activities readily assayable.
[0070] Embodiments provide isolated polypeptides or proteins
consisting of an amino acid sequence that contains one of about 5,
at least about 10, at least about 15, at least about 20, at least
about 25, at least about 30, at least about 35, at least about 40,
at least about 45, at least about 50, at least about 55, at least
about 60, at least about 65, at least about 70, at least about 75,
at least about 80, at least about 90, at least about 100, at least
about 120, at least about 140, at least about 160, at least about
180, at least about 200, at least about 220, at least about 240, at
least about 260, at least about 280, at least about 300, at least
about 320, at least about 340, at least about 360, from about 250
to about 660, from about 350 to about 660, form about 450 to about
660, and form about 550 to about 660 amino acid bases in length of
the relevant protein or polypeptide and having at least one
functional feature of the protein or polypeptide, such functional
features including ability to bind a Fe.sup.3+-Me.sup.2+ dimetal
nuclear center and form a transmembrane-like C-terminal motif.
[0071] Additional embodiments are any of the phosphatases and
homologues thereof with the identity and/or conservative
substitutions to SEQ ID NOS: 1-8 and 18-47 described above that
additionally consist of a protein, polypeptide, or polynucleotide
encoding a protein having the five conserved motifs in purple acid
phosphatases, including XDXX, XDXXY, GNH(D/E), XXXH, XHXH, where X
is any amino acid. In one embodiment, the described phosphatases
and homologues thereof consist of a protein, polypeptide, or
polynucleotide encoding one of the sequences YHVCIGNHEYDF (SEQ ID
NO: 54) and YHVCIGNHEYDW (SEQ ID NO: 55). In one embodiment, the
described phosphatases and homologues thereof consist of a protein,
polypeptide, or polynucleotide encoding a sequence of amino acid
residues having one of the sequences YHVCIGNFIEYD(W/F) (SEQ ID NO:
54) and YHVCIGNHEYN(W/F) (SEQ ID NO: 55) or a protein, polypeptide,
or polynucleotide encoding a homologue to one of the foregoing
sequences with only conservative substitutions, as described above,
to those sequences. In yet another embodiment, the described
phosphatases and homologues thereof consist of a protein,
polypeptide, or polynucleotide encoding a sequence of amino acid
residues having one of the sequences GNHE (SEQ ID NO: 51) and GNHD
(SEQ ID NO: 50). In still yet another embodiment, the described
phosphatases and homologues thereof consist of a protein,
polypeptide, or polynucleotide encoding a sequence of amino acid
residues having one of the sequences GNHE (SEQ ID NO: 51) and GNHD
(SEQ ID NO: 50) or a protein, polypeptide, or polynucleotide
encoding a protein having a homologous sequence to one of the
foregoing sequences SEQ 1N NOS: 50-51 with only conservative
substitutions.
[0072] Additional embodiments are any of the phosphatases and
homologues thereof with the identity and/or conservative
substitutions to SEQ ID NOS: 1-8 and 18-47 described above that
additionally consist of a protein, polypeptide, or polynucleotide
encoding a sequence having at least about 70% or more identity
and/or conservative substitutions to amino acid residues 302-315 of
SEQ ID NO: 2 when such sequence is aligned with SEQ ID NO: 2. In
another embodiment, the described phosphatases and homologues
thereof consist of a protein, polypeptide, or polynucleotide
encoding a protein having about 80% or more identity and/or
conservative substitutions to amino acid residues 302-315 of SEQ ID
NO: 2 when such sequence is aligned with SEQ ID NO: 2. In another
embodiment, the described phosphatases and homologues consist of a
protein, polypeptide, or polynucleotide encoding a sequence of
amino acid residues having at least about 70% or more identity to
the sequence HIGDISYARGYSW (SEQ ID NO: 56). In another embodiment,
the described phosphatases and homologues thereof consist of a
protein, polypeptide, or polynucleotide encoding a sequence of
amino acid residues having the sequence HIGDISYARGYSW (SEQ ID NO:
56) or a protein, polypeptide, or polynucleotide encoding a protein
having a homologous sequence to the foregoing sequences with only
conservative substitutions, as described above, to those
sequences.
[0073] In another embodiment, the described phosphatases and
homologues thereof consist of a protein, polypeptide, or
polynucleotide encoding a sequence of amino acid residues having at
least about 70% or more identity to the sequences
KEKLTVSFVGNHDGEVHD (SEQ ID NO: 57), KERLTLSYVGNHDGEVHD (SEQ ID NO:
58), REKLTLTYVGNHDGQVHD (SEQ ID NO: 59), and KEKLTLTYIGNHDGQVHD
(SEQ ID NO: 60). In still yet another embodiment, the described
phosphatases and homologues thereof consist of a protein,
polypeptide, or polynucleotide encoding a sequence of amino acid
residues having one or more of the sequences KEKLTVSFVGNHDGEVHD
(SEQ ID NO: 57), KERLTLSYVGNHDGEVHD (SEQ ID NO: 58),
REKLTLTYVGNHDGQVHD (SEQ ID NO: 59), and KEKLTLTYIGNHDGQVHD (SEQ ID
NO: 60) or a protein, polypeptide, or polynucleotide encoding a
protein having a homologous sequence to one of the foregoing
sequences with only conservative substitutions, as described above,
to those sequences.
[0074] In a further embodiment, the described phosphatases and
homologues thereof consist of a protein, polypeptide, or
polynucleotide encoding a sequence of amino acid residues having
the sequence (F/Y)(V/I)GNHDGXXH (SEQ ID NOS: 61-64), where the
first residue of the sequence can be F or Y and the second residue
of the sequence can be V or I. In a still further embodiment, the
described phosphatases and homologues thereof consist of a protein,
polypeptide, or polynucleotide encoding a sequence of amino acid
residues having the sequence (F/Y)(V/I)GNHDGXXH (SEQ ID NOS:
61-64), where the first residue of the sequence can be F or Y and
the second residue of the sequence can be V or I, or a protein,
polypeptide, or polynucleotide encoding a protein having a
homologous sequence to the foregoing sequence with only
conservative substitutions, as described above, to the foregoing
sequence. In a yet still further embodiment, the described
phosphatases and homologues thereof consist of a protein,
polypeptide, or polynucleotide encoding a sequence of amino acid
residues having the sequence (F/Y)(V/I)GNHDGXXH (SEQ ID NOS:
61-64), where the first residue of the sequence can be F or Y and
the second residue of the sequence can be V or I, or a protein,
polypeptide, or polynucleotide encoding a protein having a
homologous sequence having at least about 70% identity and/or
conservative substitution, as described above, to the foregoing
sequence.
[0075] In one embodiment, the described phosphatases and homologues
thereof consist of a protein, polypeptide, or polynucleotide
encoding a sequence of amino acid residues having at least about
60% or more identity and/or conservative substitutions to amino
acid residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65) and/or having
at least about 60% or more identity and/or conservative
substitutions to the sequence of 23 amino acid residues of SEQ ID
NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33 and 47 aligned with
residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65), and where amino
acid residues aligned with amino acid residues 614-636 of SEQ ID
NO: 2 are predicted to form a transmembrane-like C-terminal motif
by TMHMM analysis (http://www.cbs.dtu.dk/services/TMHMM/). In one
embodiment, the described phosphatases and homologues thereof
consist of a protein, polypeptide, or polynucleotide encoding a
sequence of amino acid residues having at least about 70% or more
identity and/or conservative substitutions to amino acid residues
614-636 of SEQ ID NO: 2 (SEQ ID NO: 65) and/or having at least
about 60% or more identity and/or conservative substitutions to the
sequence of 23 amino acid residues of SEQ ID NOS: 4, 6, 8, 19, 21,
23, 25, 27, 29, 31, 33 and 47 aligned with residues 614-636 of SEQ
ID NO: 2 (SEQ ID NO: 65), and where amino acid residues aligned
with amino acid residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65)
are predicted to form a transmembrane-like C-terminal motif by
TMHMM analysis (http://www.cbs.dtu.dk/services/TMHMM/). In one
embodiment, the described phosphatases and homologues thereof
consist of a protein, polypeptide, or polynucleotide encoding a
sequence of amino acid residues having at least about 80% or more
identity and/or conservative substitutions to amino acid residues
614-636 of SEQ ID NO: 2 and/or having at least about 60% or more
identity and/or conservative substitutions to the sequence of 23
amino acid residues of SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29,
31, 33 and 47 aligned with residues 614-636 of SEQ ID NO: 2 (SEQ ID
NO: 65), and where amino acid residues aligned with amino acid
residues 614-636 of SEQ ID NO: 2 (SEQ ID NO: 65) are predicted to
form a transmembrane-like C-terminal motif by TMHMM analysis
(http://www.cbs.dtu.dk/services/TMHMM/). In one embodiment, the
described phosphatases and homologues thereof consist of a protein,
polypeptide, or polynucleotide encoding a sequence of amino acid
residues having at least about 90% or more identity and/or
conservative substitutions to amino acid residues 614-636 of SEQ ID
NO: 2 and/or having at least about 90% or more identity and/or
conservative substitutions to the sequence of 23 amino acid
residues of SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31, 33 and
47 aligned with residues 614-636 of SEQ ID NO: 2, and where amino
acid residues aligned with amino acid residues 614-636 of SEQ ID
NO: 2 are predicted to form a transmembrane-like C-terminal motif
by TMHMM analysis (http://www.cbs.dtu.dk/services/TMHMM/).
[0076] In one embodiment, the described phosphatases or phosphatase
genes consist of a protein, polypeptide, or polynucleotide encoding
the sequence (L/M/V)-(L/M/V)-Z-(G/A)-(V/A/L)-Z-Z-G-(F/Y)-X-Z-G (SEQ
ID NO: 66), where Z is any of the hydrophobic residues L, I, V, F,
and M. In another embodiment, the described phosphatase or
phosphatase genes consist of a protein, polypeptide, or
polynucleotide encoding the sequence
(L/M/V)-(L/M/V)-Z-(G/A)-(V/A/L)-Z-Z-G-(F/Y)-X-Z-G (SEQ ID NO: 66),
or a protein, polypeptide, or polynucleotide encoding a sequence
having at least 70% identity and/or conservative substitution to
the foregoing sequence.
[0077] Embodiments also encompass derivatives of the disclosed
polypeptides. For example, but not by way of limitation,
derivatives may include peptides or proteins that have been
modified, e.g., by glycosylation, acetylation, pegylation,
phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a
cellular ligand or other protein, etc. Any of numerous chemical
modifications may be carried out by known techniques, including,
but not limited to, specific chemical cleavage, acetylation,
formylation, etc. Additionally, the derivative may contain one or
more non-classical amino acids.
[0078] In another aspect, an isolated nucleic acid molecule encodes
a variant of a polypeptide in which the amino acid sequences have
been modified by genetic engineering so that biological activities
of the polypeptides are either enhanced or reduced, or the local
structures thereof are changed without significantly altering the
biological activities. In one aspect, these variants can act as
either agonists or as antagonists. An agonist can retain
substantially the same or a portion of the biological activities of
the polypeptides and an antagonist can inhibit one or more of the
activities of the polypeptides. Such modifications include amino
acid substitution, deletion, and/or insertion. Amino acid
modifications can be made by any method known in the art and
various methods are available to and routine for those skilled in
the art.
[0079] For example, mutagenesis may be performed in accordance with
any of the techniques known in the art including, but not limited
to, synthesizing an oligonucleotide having one or more
modifications within the sequence of a given polypeptide to be
modified. Site-specific mutagenesis can be conducted using specific
oligonucleotide sequences which encode the nucleotide sequence
containing the desired mutations in addition to a sufficient number
of adjacent nucleotides in the polypeptide. Such oligonucleotides
can serve as primers which can form a stable duplex on both sides
of the deletion junction being traversed. Typically, a primer of
about 15 to about 75 nucleotides or more in length is preferred,
with about 10 to about 25 or more residues on both sides of the
junction of the sequence being altered. A number of such primers
introducing a variety of different mutations at one or more
positions can be used to generate a library of mutants.
[0080] The technique of site-specific mutagenesis is well known in
the art, as described in various publications (e.g., Kunkel et al.,
Methods Enzymol., 154:367-82, 1987, which is hereby incorporated by
reference in its entirety). In general, site-directed mutagenesis
is performed by first obtaining a single-stranded vector or melting
apart of two strands of a double stranded vector which includes
within its sequence a DNA sequence which encodes the desired
peptide. An oligonucleotide primer bearing the desired mutated
sequence is prepared, generally synthetically. This primer is then
annealed with the single-stranded vector, and subjected to DNA
polymerizing enzymes such as T7 DNA polymerase, in order to
complete the synthesis of the mutation-bearing strand. Thus, a
heteroduplex is formed wherein one strand encodes the original
non-mutated sequence and the second strand bears the desired
mutation. This heteroduplex vector is then used to transform or
transfect appropriate cells, such as E. coli cells, and clones are
selected which include recombinant vectors bearing the mutated
sequence arrangement. As will be appreciated, the technique
typically employs a phage vector which exists in both a single
stranded and double stranded form. Typical vectors useful in
site-directed mutagenesis include vectors such as the M13 phage.
These phages are readily commercially available and their use is
generally well known to those skilled in the art. Double stranded
plasmids are also routinely employed in site directed mutagenesis
which eliminates the step of transferring the gene of interest from
a plasmid to a phage.
[0081] Alternatively, the use of PCR with commercially available
thermostable enzymes such as Taq DNA polymerase may be used to
incorporate a mutagenic oligonucleotide primer into an amplified
DNA fragment that can then be cloned into an appropriate cloning or
expression vector. See, e.g., Tomic et al., Nucleic Acids Res.,
18(6):1656, 1987, and Upender et al., Biotechniques, 18(1):29-30,
32, 1995, for PCR-mediated mutagenesis procedures, which are hereby
incorporated in their entireties. PCR employing a thermostable
ligase in addition to a thermostable polymerase may also be used to
incorporate a phosphorylated mutagenic oligonucleotide into an
amplified DNA fragment that may then be cloned into an appropriate
cloning or expression vector (see e.g., Michael, Biotechniques,
16(3):410-2, 1994, which is hereby incorporated by reference in its
entirety).
[0082] Other methods known to those skilled in art of producing
sequence variants of a given polypeptide or a fragment thereof can
be used. For example, recombinant vectors encoding the amino acid
sequence of the polypeptide or a fragment thereof may be treated
with mutagenic agents, such as hydroxylamine, to obtain sequence
variants.
[0083] Optionally, the amino acid residues to be modified are
surface exposed residues. Additionally, in making amino acid
substitutions, preferably the amino acid residue to be substituted
is a conservative amino acid substitution, for example, a polar
residue is substituted with a polar residue, a hydrophilic residue
with a hydrophilic residue, hydrophobic residue with a hydrophobic
residue, a positively charged residue with a positively charged
residue, or a negatively charged residue with a negatively charged
residue. Moreover, the amino acid residue that can be modified is
not highly or completely conserved across strains or species and/or
is critical to maintain the biological activities of the
protein.
[0084] Accordingly, included in the scope of the disclosure are
nucleic acid molecules encoding a polypeptide of the invention that
contains amino acid modifications that are not critical to its
biological activity.
5.3 Fusion Proteins
[0085] The present disclosure further encompasses fusion proteins
in which the polypeptides or fragments thereof, are recombinantly
fused or chemically conjugated (e.g., covalent and non-covalent
conjugations) to heterologous polypeptides (i.e., an unrelated
polypeptide or portion thereof, preferably at least 10, at least
20, at least 30, at least 40, at least 50, at least 60, at least
70, at least 80, at least 90 or at least 100 amino acids of the
polypeptide) to generate fusion proteins. The fusion can be direct,
but may occur through linker sequences.
[0086] In one aspect, the fusion protein comprises a polypeptide
which is fused to a heterologous signal sequence at its N-terminus.
For example, the signal sequence naturally found in the polypeptide
can be replaced by a signal sequence which is derived from a
heterologous origin. Various signal sequences are commercially
available.
[0087] In another embodiment, a polypeptide can be fused to tag
sequences, e.g., a hexa-histidine peptide, among others, many of
which are commercially available. As described in Gentz et al.,
1989, Proc. Natl. Acad. Sci. USA, 86:821-824, for instance,
hexa-histidine provides for convenient purification of the fusion
protein. Other examples of peptide tags are the hemagglutinin "HA"
tag, which corresponds to an epitope derived from the influenza
hemagglutinin protein (Wilson et al., 1984, Cell, 37:767) and the
"flag" tag (Knappik et al., 1994, Biotechniques, 17(4):754-761).
These tags are especially useful for purification of recombinantly
produced polypeptides.
[0088] Fusion proteins can be produced by standard recombinant DNA
techniques or by protein synthetic techniques, e.g., by use of a
DNA synthesizer. For example, a nucleic acid molecule encoding a
fusion protein can be synthesized by conventional techniques
including automated DNA synthesizers. Alternatively, PCR
amplification of gene fragments can be carried out using anchor
primers which give rise to complementary overhangs between two
consecutive gene fragments which can subsequently be annealed and
reamplified to generate a chimeric gene sequence (see, e.g.,
Current Protocols in Molecular Biology, Ausubel et al., eds., John
Wiley & Sons, 1992).
[0089] The nucleotide sequence coding for a fusion protein can be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted protein-coding sequence.
[0090] In a specific embodiment, the expression of a fusion protein
is regulated by an inducible promoter.
5.4 Preparation of Transgenic Plants
[0091] Carbon flow is a key process in plant biology and high
energy carbon molecules (e.g. glucose) were harvested by plant
through photosynthesis. The carbon molecules were then converted
into more complicated carbohydrate molecules such as starch,
cellulose, etc. Cellulose is the major component of cell wall and
starch is the major storage form of glucose in plant cells and
plant seeds. Therefore, the efficiency and/or the equilibrium of
the carbon flow process become a limiting factor for plant growth
and crop yield.
[0092] The present disclosure is based upon the discovery that
overexpression of a membrane-bound phosphatase can enhance the
growth performance of plants by altering its carbon metabolism, as
indicated by, for example, a faster growth rate, a higher sugar
contents, and a higher seed yield.
[0093] In an embodiment, the present disclosure provides a
transgenic plant containing a nucleic acid molecule that encodes
and expresses a phosphatase having a C-terminal transmembrane-like
domain. The transgenic plants disclosed herein have faster growth
rate, and higher seed yield to comparable unengineered plants i.e.
same species (strain). In a specific embodiment, such a phosphatase
is from a plant species having a phosphatase activity and a
C-terminal motif. In another embodiment, a transgenic plant
disclosed herein comprises a nucleic acid molecule encoding
phosphatase and expresses AtPAP2 (SEQ ID NO: 2) and/or other PAPs
with a C-terminal motif including one or more of SEQ ID NOS: 4, 6,
8, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and 47.
In another embodiment, the phosphatase is expressed on cellular
membrane, for example, the ER or the Golgi apparatus. Such a
membrane expression of a phosphatase in plants can be achieved by
fusing onto the C-terminus with a nucleotide sequence encoding a
C-terminal motif peptide which can efficiently attach the
phosphatase upon translation thereof from the cells of a given
plant. Accordingly, in another embodiment, a transgenic plant
comprises a nucleic acid molecule encoding phosphatase and
expresses AtPAP2 (SEQ ID NO: 2) and/or other PAPs with a C-terminal
motif including SEQ ID NOS: 4, 6, 8, 19, 21, 23, 25, 27, 29, 31,
33, 35, 37, 39, 41, 43, 45 and 47, except that all or a portion,
particularly an N-terminal portion, of amino acid residues 1 to 80,
preferably all or a portion of amino acid residues 1 to 30, of SEQ
ID NO: 2 or all or a portion, particularly an N-terminal portion,
of amino acid residues 1 to 80, preferably all or a portion of
amino acid residues 1 to 30, of SEQ ID NOS: 4, 6, 8, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 or 47, are replaced by a
heterologous plant signal peptide by genetic engineering. In such a
transgenic plant, the phosphatases are directed to various
organelles/compartments of the cells. In another embodiment, a
transgenic plant comprises a nucleic acid molecule encoding
phosphatase and expresses homologues, derivatives, and/or fragments
thereof having at least one functional feature and/or structural
feature of a phosphatase polypeptide. In all embodiments where all
or a portion of the N-terminal portion of SEQ ID NOS: 4, 6, 8, 19,
21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45 and/or 47 are
replaced, the embodiments include homologues to such sequences, as
described above, having at least one functional feature and/or
structural feature of a phosphatase polypeptide. In yet another
embodiment, a transgenic plant comprises a nucleic acid molecule
that hybridizes under stringent conditions, as defined herein, to a
nucleic acid molecule having the sequence of SEQ ID NOS: 1, 3, 5,
7, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46, or
a complement thereof, and encodes a protein or polypeptide that
exhibits at least one structural and/or functional feature of the
disclosed phosphatase polypeptides. Specifically, the production of
transgenic plant that overexpressed a membrane-bound phosphatase,
which contributes to improving plant physiology, such as plant
growth rate and characteristics, for example, in seed yield, is
provided.
[0094] Accordingly, also provided are chimeric gene constructs for
genetic modification of plants to increase their growth rate and
improve the yield. The chimeric gene constructs comprise a sequence
that encodes substantially solely for a phosphatase enzyme that
carry a C-terminal transmembrane-like motif. Such a phosphatase
enzyme can be derived from the purple acid phosphatase family. In a
specific embodiment, the chimeric gene constructs comprise a
nucleic acid having the sequence of SEQ ID NOS: 1, 3, 5, 7, 18, 20,
22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44 or 46. In another
embodiment, the chimeric gene constructs comprise a nucleic acid
molecule that encodes a homologue or fragment thereof having at
least one functional feature and/or structural feature of a
phosphatase polypeptide. In another specific embodiment, the
chimeric gene constructs comprise a sequence that hybridizes under
stringent conditions, as defined herein, to a nucleic acid having
the sequence of SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30,
32, 34, 36, 38, 40, 42, 44 or 46, or a complement thereof, wherein
the sequence encodes a protein or a polypeptide that exhibits at
least one structural and/or functional feature of the phosphatase
polypeptides. Furthermore, the phosphatases encoded by the nucleic
acid molecules contained in the chimeric gene constructs can be any
other phosphatases that have similar structural characteristics,
such as having a C-terminal transmembrane-like motif, to those of
the phosphatases described herein. Such phosphatase include, but
not limited to, the following polypeptides: Purple acid
phosphatases from Zea mays (Accession No: ACG47621); and Oryza
sativa (Accession No: BAC15853.1).
[0095] The phosphatase-coding sequence is operatively linked to
upstream and downstream regulatory components, preferably
heterologous to the phosphatase sequence; for example CMV 35S
promoter, which acts to cause expression of the gene (production of
the enzyme) in plant cells (see Section 6.2). When a construct
containing a gene for a phosphatase according to this disclosure,
is introduced into plant cells by a conventional transformation
method, such as microparticle bombardment, Agrobacterium infection,
or microinjection, the gene is expressed in the cells under the
control of the regulatory sequences. The expressed phosphatase
successfully interacts with the biosynthetic machinery that is
naturally present in the plant cells to alter the carbon
metabolism. By altering the carbon metabolism, the method described
herein also favors the growth rate of the plant, resulting in
faster growth rate and higher yield. Thus, the time required for
the maturation of the plant and the time required for flowering is
shortened. Also provided are methods for increasing growth rate and
yield of plants, comprising the step of inserting into such plant
cells or the cells of such whole plants a chimeric gene
construct.
[0096] In specific embodiments, Arabidopsis (see Section 6) was
adopted as the model system. An overexpression construct the gene
coding for phosphatase were introduced into Arabidopsis.
[0097] In an embodiment, the phosphatase from Arabidopsis is used.
The results obtained with this disclosure indicate that the growth
rate and the seed yield of transgenic Arabidopsis were enhanced by
overexpressing this gene (see Section 6.5 and FIG. 8 and Table
3).
[0098] While any plant species can be modified using the expression
cassette and methods described herein, preferably included without
limitation are species from the following genera with
representative species in parentheses:
[0099] Monocots: genera Asparagus (asparagus), Bromus (cheatgrass),
Hemerocallis (daylily), Hordeum (barley), Lolium (ryegrass), Oryza
(rice), Panicum (Switchgrass), Pennisetum (fountaingrass),
Saccharum (Sugar cane), Sorghum, Trigonella (fenu grass), Triticum
(wheat), Zea (corn); and
[0100] Dicots: genera Antirrhinum (flower sp.), Arabidopsis
(thaliana), Arachis (peanut), Atropa (deadly nightshade), Brassica
(rapeseed), Browallia, Capsicum (pepper), Carthamus (safflower),
Cichorium (chicory), Citrus (orange, lemon), Chrysanthemum, Cucumis
(cucumber), Datura (thorn apple), Daucus (carrot), Digitalis
(foxglove), Fragaria (strawberry), Geranium (flower sp.), Glycine
(soybean), Helianthus (sunflower), Hyscyamus, Ipomoea (morning
glory), Latuca (lettuce), Linum (linseed), Lotus (flower sp.),
Lycopersicon (tomato), Majorana, Malva (cotton), Manihot, Medicago
(alfalfa), Nemesia, Nicotiana (tobacco), Onobrychis, Pelargonium
(citrosa), Petunia (flower sp.), Ranunculus (flower sp.), Raphanus
(radishes), Salpiglossis, Senecio (flower sp.), Sinapis (albae
semen), Solanum (potato), Trifolium (clovers), Vigna (mungbean,
faba bean), Vitis (grape).
[0101] Genetic engineering of plants can be achieved in several
ways. The most common method is Agrobacterium-mediated
transformation. In this method, A. tumefaciens, which in nature
infects plants by inserting tumor causing genes into a plant's
genome, is altered. Selected genes are engineered into the T-DNA of
the bacterial Ti (tumor-inducing) plasmid of A. tumefaciens in
laboratory conditions so that they become integrated into the plant
chromosomes when the T-DNA is transferred to the plant by the
bacteria's own internal transfer mechanisms. The only essential
parts of the T-DNA are its two small (25 base pair) border repeats,
at least one of which is needed for plant transformation. The
bacterial genes encoding for plant hormones that promote tumor
growth are excised from the T-DNA and replaced with a sequence of
DNA that typically contains: a selectable marker (e.g. an
antibiotic-resistance gene; usually kanamycin resistance), a
restriction site--a site with a specific sequence of nucleotides
where a restriction enzyme will cut the DNA, and the desired genes
to be incorporated into the plant (B. Tinland, 1996. The
integration of T-DNA into plant genomes. Trends in Plant Science 1,
178-184; D. Grierson (ed.) 1991. Plant Genetic Engineering.
Blackie, Glasgow). Agrobacterium can be added to plant protoplasts
(plant cells with cell walls removed) in culture, that are then
allowed to regenerate cell walls at which point non-transformed
plants are killed with antibiotics for which the transformed plants
have been given resistance genes. Plantlets are then regenerated
from the surviving transformed cells using standard plant tissue
culture techniques. In an alternative technique, sterile disks or
fragments of vegetative portions of plants are place in liquid
culture medium with Agrobacterium, then hormones are used to induce
rooting thereby regenerate plantlets which are grown on selection
media. A third technique for delivering genes is possible for some
plants such as Arabidopsis where the Agrobacterium or even "naked"
DNA can be infused through the seed coat to cause transformation
(Clough S J and Bent A F, 1998. Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana.
Plant J 16:735-43).
[0102] The biolistic method for genetic engineering of plants was
developed more recently and is becoming more widely employed. In
this method, very small particles (microprojectiles) of tungsten or
gold coated with biologically active DNA are propelled at
high-velocities into plant cells using an electrostatic pulse, air
pressure, or gunpowder percussion. As the particles pass through
the cell, the DNA dissolves and can then integrate into the genome
of that cell and its progeny. It has been demonstrated this method
can produce stable transformants (Christou, P., et al., 1988.
Stable transformation of soybean callus by DNA-coated gold
particles, Plant Physiology 87:671-674). The method can be
practiced on whole plants and is particularly effective on
meristematic tissue. It is also capable of delivering DNA either to
the nucleus or into mitochondria (Johnston, S. A., et al., 1988.
Mitochondrial transformation in yeast by bombardment with
microprojectiles (Science 240, 1538-41) and chloroplasts (Svab, Z.,
et al., 1990, Stable transformation of plastids in higher plants,
Proc Natl Acad. Sci. USA 87, 8526-8530).
[0103] The electroporation method of plant genetic engineering has
met with less success. In this technique, protoplasts in culture
take up pure DNA when treated with certain membrane-active agents
or with electroporation, a rapid pulse of high-voltage direct
current. Once the DNA has entered the protoplast it can be
integrated into the cells genome. Standard tissue culture
techniques are then used to regenerate transgenic plants.
[0104] The microinjection method of plant genetic engineering is
perhaps the most difficult. In this method, DNA is microinjected
into target plant cells using very thin glass needles in a method
similar to that used with animals. The technique is laborious,
ineffective, and impractical for generating large numbers of
transgenic plants.
[0105] The method chosen for genetically engineering plants is most
often dependent on the targeted plant species and which methods
have been proven effective therein.
5.5 Preparation of Antibodies
[0106] Antibodies which specifically recognize one of the described
phosphatase polypeptides or fragments thereof can be used for
detecting, screening, and isolating the polypeptide of the
invention or fragments thereof, or similar sequences that might
encode similar enzymes from the other organisms. For example, in
one specific embodiment, an antibody which immunospecifically binds
AtPAP2 or fragments thereof can be used for various in vitro
detection assays, including enzyme-linked immunosorbent assays
(ELISA), radioimmunoassays, Western blot, etc., for the detection
of the polypeptide of the invention or fragments, derivatives,
homologues, or variants thereof, or similar molecules having the
similar enzymatic activities as the phosphatase polypeptides, in
samples, for example, a biological material, including plant cells,
plants, food, drinks, or any materials derived from plants.
[0107] Antibodies specific for the described phosphatase
polypeptides can be generated by any suitable method known in the
art. Polyclonal antibodies to an antigen-of-interest can be
produced by various procedures well known in the art. For example,
an antigen derived from the phosphatase polypeptide can be
administered to various host animals including, but not limited to,
rabbits, mice, rats, etc., to induce the production of antisera
containing polyclonal antibodies specific for the antigen. Various
adjuvants may be used to increase the immunological response,
depending on the host species, and include but are not limited to,
Freund's (complete and incomplete) adjuvant, mineral gels such as
aluminum hydroxide, surface active substances such as lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanins, dinitrophenol, and potentially useful adjuvants
for humans such as BCG (Bacille Calmette-Guerin) and
Corynebacterium parvum. Such adjuvants are also well known in the
art.
[0108] Monoclonal antibodies can be prepared using a wide variety
of techniques known in the art including the use of hybridoma,
recombinant, and phage display technologies, or a combination
thereof. For example, monoclonal antibodies can be produced using
hybridoma techniques including those known in the art and taught,
for example, in Harlow et al., Antibodies: A Laboratory Manual,
(Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et
al., in: Monoclonal Antibodies and T-Cell Hybridomas, pp. 563-681
(Elsevier, N.Y., 1981) (both of which are incorporated by reference
in their entireties). The term "monoclonal antibody" as used herein
is not limited to antibodies produced through hybridoma technology.
The term "monoclonal antibody" refers to an antibody that is
derived from a single clone, including any eukaryotic, prokaryotic,
or phage clone, and not the method by which it is produced.
[0109] Methods for producing and screening for specific antibodies
using hybridoma technology are routine and well known in the art.
In a non-limiting example, mice can be immunized with an antigen of
interest or a cell expressing such an antigen. Once an immune
response is detected, e.g., antibodies specific for the antigen are
detected in the mouse serum, the mouse spleen is harvested and
splenocytes isolated. The splenocytes are then fused by well known
techniques to any suitable myeloma cells. Hybridomas are selected
and cloned by limiting dilution. The hybridoma clones are then
assayed by methods known in the art for cells that secrete
antibodies capable of binding the antigen. Ascites fluid, which
generally contains high levels of antibodies, can be generated by
inoculating mice intraperitoneally with positive hybridoma
clones.
[0110] Antibody fragments which recognize specific epitopes may be
generated by known techniques. For example, Fab and F(ab').sub.2
fragments may be produced by proteolytic cleavage of immunoglobulin
molecules, using enzymes such as papain (to produce Fab fragments)
or pepsin (to produce F(ab').sub.2 fragments). F(ab').sub.2
fragments contain the complete light chain, and the variable
region, the CH1 region and the hinge region of the heavy chain.
[0111] The antibodies or fragments thereof can be also produced by
any method known in the art for the synthesis of antibodies, in
particular, by chemical synthesis or preferably, by recombinant
expression techniques.
[0112] The nucleotide sequence encoding an antibody may be obtained
from any information available to those skilled in the art (i.e.,
from Genbank, the literature, or by routine cloning). If a clone
containing a nucleic acid encoding a particular antibody or an
epitope-binding fragment thereof is not available, but the sequence
of the antibody molecule or epitope-binding fragment thereof is
known, a nucleic acid encoding the immunoglobulin may be chemically
synthesized or obtained from a suitable source (e.g., an antibody
cDNA library, or a cDNA library generated from, or nucleic acid,
preferably poly A+ RNA, isolated from any tissue or cells
expressing the antibody, such as hybridoma cells selected to
express an antibody) by PCR amplification using synthetic primers
hybridizable to the 3' and 5' ends of the sequence or by cloning
using an oligonucleotide probe specific for the particular gene
sequence to identify, e.g., a cDNA clone from a cDNA library that
encodes the antibody. Amplified nucleic acids generated by PCR may
then be cloned into replicable cloning vectors using any method
well known in the art.
[0113] Once the nucleotide sequence of the antibody is determined,
the nucleotide sequence of the antibody may be manipulated using
methods well known in the art for the manipulation of nucleotide
sequences, e.g., recombinant DNA techniques, site directed
mutagenesis, PCR, etc. (see, for example, the techniques described
in Sambrook et al., supra; and Ausubel et al., eds., 1998, Current
Protocols in Molecular Biology, John Wiley & Sons, NY, which
are both incorporated by reference herein in their entireties), to
generate antibodies having a different amino acid sequence by, for
example, introducing amino acid substitutions, deletions, and/or
insertions into the epitope-binding domain regions of the
antibodies or any portion of antibodies which may enhance or reduce
biological activities of the antibodies.
[0114] Recombinant expression of an antibody requires construction
of an expression vector containing a nucleotide sequence that
encodes the antibody. Once a nucleotide sequence encoding an
antibody molecule or a heavy or light chain of an antibody, or
portion thereof has been obtained, the vector for the production of
the antibody molecule may be produced by recombinant DNA technology
using techniques well known in the art as discussed in the previous
sections. Methods which are well known to those skilled in the art
can be used to construct expression vectors containing antibody
coding sequences and appropriate transcriptional and translational
control signals. These methods include, for example, in vitro
recombinant DNA techniques, synthetic techniques, and in vivo
genetic recombination. The nucleotide sequence encoding the
heavy-chain variable region, light-chain variable region, both the
heavy-chain and light-chain variable regions, an epitope-binding
fragment of the heavy- and/or light-chain variable region, or one
or more complementarity determining regions (CDRs) of an antibody
may be cloned into such a vector for expression. Thus-prepared
expression vector can be then introduced into appropriate host
cells for the expression of the antibody. Accordingly, embodiments
include host cells containing a polynucleotide encoding an antibody
specific for the disclosed phosphatase polypeptides or fragments
thereof.
[0115] The host cell can be co-transfected with two expression
vectors, the first vector encoding a heavy chain derived
polypeptide and the second vector encoding a light chain derived
polypeptide. The two vectors may contain identical selectable
markers which enable equal expression of heavy and light chain
polypeptides or different selectable markers to ensure maintenance
of both plasmids. Alternatively, a single vector may be used which
encodes, and is capable of expressing, both heavy and light chain
polypeptides. In such situations, the light chain should be placed
before the heavy chain to avoid an excess of toxic free heavy chain
(Proudfoot, 1986, Nature, 322:52; and Kohler, 1980, Proc. Natl.
Acad. Sci. USA, 77:2197). The coding sequences for the heavy and
light chains may comprise cDNA or genomic DNA.
[0116] In another embodiment, antibodies can also be generated
using various phage display methods known in the art. In phage
display methods, functional antibody domains are displayed on the
surface of phage particles which carry the polynucleotide sequences
encoding them. In a particular embodiment, such phage can be
utilized to display antigen binding domains, such as Fab and Fv or
disulfide-bond stabilized Fvs, expressed from a repertoire or
combinatorial antibody library (e.g., human or murine). Phage
expressing an antigen binding domain that binds the antigen of
interest can be selected or identified with antigen, e.g., using
labeled antigen or antigen bound or captured to a solid surface or
bead. Phages used in these methods are typically filamentous phage,
including fd and M13. The antigen binding domains are expressed as
a recombinantly fused protein to either the phage gene III or gene
VIII protein. Examples of phage display methods that can be used to
make the immunoglobulins, or fragments thereof, include those
disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50;
Ames et al., 1995, J. Immunol. Methods 184:177-186; Kettleborough
et al., 1994, Eur. J. Immunol., 24:952-958; Persic et al., 1997,
Gene, 187:9-18; Burton et al., 1994, Advances in Immunology
57:191-280; PCT application No. PCT/GB91/01134; PCT publications WO
90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO
95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;
5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;
5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and
5,969,108; each of which is incorporated herein by reference in its
entirety.
[0117] As described in the above documents, after phage selection,
the antibody coding regions from the phage can be isolated and used
to generate whole antibodies, including human antibodies, or any
other desired fragments, and expressed in any desired host,
including mammalian cells, insect cells, plant cells, yeast, and
bacteria, e.g., as described in detail below. For example,
techniques to recombinantly produce Fab, Fab' and F(ab').sub.2
fragments can also be employed using methods known in the art such
as those disclosed in PCT publication WO 92/22324; Mullinax et al.,
1992, BioTechniques 12(6):864-869; and Sawai et al., 1995, AJRI
34:26-34; and Better et al., Science, 240:1041-1043, 1988 (each of
which is incorporated by reference in its entirety). Examples of
techniques which can be used to produce single-chain Fvs and
antibodies include those described in U.S. Pat. Nos. 4,946,778 and
5,258,498; Huston et al., 1991, Methods in Enzymology 203:46-88;
Shu et al., 1993, PNAS 90:7995-7999; and Skerra et al., 1988,
Science 240:1038-1040.
[0118] Once an antibody molecule has been produced by any methods
described above, it may then be purified by any method known in the
art for purification of an immunoglobulin molecule, for example, by
chromatography (e.g., ion exchange, affinity, particularly by
affinity for the specific antigen after Protein A or Protein G
purification, and sizing column chromatography), centrifugation,
differential solubility, or by any other standard techniques for
the purification of proteins. Further, the antibodies or fragments
thereof may be fused to heterologous polypeptide sequences
described herein or otherwise known in the art to facilitate
purification.
[0119] Antibodies fused or conjugated to heterologous polypeptides
may be used in in vitro immunoassays and in purification methods
(e.g., affinity chromatography) well known in the art. See e.g.,
PCT publication Number WO 93/21232; EP 439,095; Naramura et al.,
1994, Immunol. Lett. 39:91-99; U.S. Pat. No. 5,474,981; Gillies et
al., 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol.
146:2446-2452, which are incorporated herein by reference in their
entireties.
[0120] Antibodies may also be attached to solid supports, which are
particularly useful for immunoassays or purification of the
described polypeptides or fragments, derivatives, homologues, or
variants thereof, or similar molecules having the similar enzymatic
activities as the polypeptide of the invention. Such solid supports
include, but are not limited to, glass, cellulose, polyacrylamide,
nylon, polystyrene, polyvinyl chloride or polypropylene.
5.6 Detection Assays
[0121] An exemplary method for detecting the presence or absence of
an over-expressed phosphatase polypeptide or an inserted
phosphatase-encoding nucleic acid in a biological sample involves
obtaining a biological sample from various sources and contacting
the sample with a compound or an agent capable of detecting a
polypeptide or nucleic acid (e.g., mRNA, genomic DNA) such that the
presence of a heterologous polypeptide or nucleic acid is detected
in the sample. An exemplary agent for detecting mRNA or genomic DNA
encoding an inserted phosphatase polypeptide is a labeled nucleic
acid probe capable of hybridizing to mRNA or genomic DNA encoding
any of the described phosphatase polypeptides. The nucleic acid
probe can be, for example, a full-length cDNA, such as the nucleic
acid of SEQ ID NOS: 1, 3, 5, 7, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44 or 46, or a portion thereof, such as an
oligonucleotide of at least one of at least about 15, at least
about 20, at least about 25, at least about 30, at least about 50,
at least about 100, at least about 250, at least about 500, or more
nucleotides in length and sufficient to specifically hybridize
under stringent conditions to a mRNA or genomic DNA encoding a
polypeptide of the invention.
[0122] An exemplary agent for detecting an over-expressed
phosphatase polypeptide is an antibody capable of binding to a
phosphatase polypeptide product of an inserted phosphatase gene,
preferably an antibody with a detectable label. Antibodies can be
polyclonal, or more preferably, monoclonal. An intact antibody, or
a fragment thereof. (e.g., Fab or F(ab').sub.2) can be used. See
also the detailed descriptions about antibodies in Section 5.5.
[0123] The term "labeled", with regard to the probe or antibody, is
intended to encompass direct labeling of the probe or antibody by
coupling (i.e., physically linking) a detectable substance to the
probe or antibody, as well as indirect labeling of the probe or
antibody by reactivity with another reagent that is directly
labeled. Examples of indirect labeling include detection of a
primary antibody using a fluorescently labeled secondary antibody
and end-labeling of a DNA probe with biotin such that it can be
detected with fluorescently labeled streptavidin. The detection
method can be used to detect mRNA, protein, or genomic DNA in a
sample in vitro as well as in vivo. For example, in vitro
techniques for detection of mRNA include Northern hybridizations
and in situ hybridizations. In vitro techniques for detection of a
heterologous polypeptide include enzyme linked immunosorbent assays
(ELISAs), Western blots, immunoprecipitations and
immunofluorescence. In vitro techniques for detection of genomic
DNA include Southern hybridizations. Furthermore, in vivo
techniques for detection of a heterologous polypeptide include
introducing into a subject organism a labeled antibody directed
against the polypeptide. For example, the antibody can be labeled
with a radioactive marker whose presence and location in the
subject organism can be detected by standard imaging techniques,
including autoradiography.
[0124] In a specific embodiment, the methods further involve
obtaining a control sample from a control subject, contacting the
control sample with a compound or agent capable of detecting an
over-expressed polypeptide product or the mRNA transcription
product or genomic DNA encoding an inserted phospatase gene, such
that the presence of the polypeptide or mRNA or genomic DNA
encoding the phosphatase polypeptide is detected in the sample, and
comparing the presence of the phosphatase polypeptide or mRNA or
genomic DNA encoding the polypeptide in the control sample with the
presence of the polypeptide or mRNA or genomic DNA encoding
endogenous phosphatase polypeptides in the test sample.
[0125] Embodiments also encompasse kits for detecting the presence
of a heterologous polypeptide or nucleic acid in a test sample.
[0126] The kit, for example, can comprise a labeled compound or
agent capable of detecting the polypeptide or mRNA encoding the
polypeptide in a test sample and means for determining the amount
of the polypeptide or mRNA in the sample (e.g., an antibody which
binds the polypeptide or an oligonucleotide probe which binds to
DNA or mRNA encoding the polypeptide). Kits can also optionally
include instructions for use.
[0127] For antibody-based kits, the kit can comprise, for example:
(1) a first antibody (e.g., attached to a solid support) which
binds to a phosphatase polypeptide; and, optionally, (2) a second,
different antibody which binds to either the polypeptide or the
first antibody and is conjugated to a detectable agent.
[0128] For oligonucleotide-based kits, the kit can comprise, for
example: (1) an oligonucleotide, e.g., a detectably labeled
oligonucleotide, which hybridizes to a nucleic acid sequence
encoding an inserted phosphatase polypeptide or (2) a pair of
primers useful for amplifying a nucleic acid molecule encoding an
inserted phosphatase polypeptide. The kit can also comprise, e.g.,
a buffering agent, a preservative, or a protein stabilizing agent.
The kit can also comprise components necessary for detecting the
detectable agent (e.g., an enzyme or a substrate). The kit can also
contain a control sample or a series of control samples which can
be assayed and compared to the test sample contained. Each
component of the kit is usually enclosed within an individual
container and all of the various containers are within a single
package along with instructions for use.
5.7 Commercial Application of Transgenic Plants
[0129] The transgenic plants generated can have many useful
applications, including food, feed, biomass, biofuels (starch,
cellulose, seed lipids) and wood pulp. The enhanced growth rate of
the transgenic plants may provide additional carbon dioxide
fixation per hectare of land per year and thus generate carbon
credits.
6. EXAMPLES
[0130] The following examples illustrate the cloning of AtPAP2, its
overexpression in transgenic Arabidopsis, and the characterization
of the transgenic plants. These examples should not be construed as
limiting. The following examples illustrate some embodiments.
Unless otherwise indicated in the following examples and elsewhere
in the specification and claims, all parts and percentages are by
weight, all temperatures are in degrees Centigrade, and pressure is
at or near atmospheric pressure.
6.1 Sequence Alignment and Phylogenetic Analysis
[0131] PAP2 locus and its genomic organization, including its
intron/exon boundaries, were identified in the Arabidopsis Col-0
ecotype (http://www.arabidopsis.org). Sequence alignment and
phylogenetic tree were conducted using MEGA4 (Kumar et al., 2004)
and ClustalW program
(http://www.ebi.ac.uk/Tools/clustalw2/index.html). Amino acid
sequence comparisons were performed using CLC Sequence Viewer 5.1.1
(www.cicbio.com).
[0132] Twenty nine PAP-like sequences were identified from the
Arabidopsis genome and a phylogenetic tree was produced by
neighbor-joining algorithm (FIG. 1). The gene locus of AtPAP2
(At1g13900) composes of two exons and the coding region is 1971 bp
in length (SEQ ID NO: 1), which is predicted to encode a
polypeptide of .about.73.7-KD. Among the twenty nine PAP-like
protein sequences, only AtPAP2 and AtPAP9 carry a unique
hydrophobic motif at their C-termini by TMHMM analysis
(http://www.cbs.dtu.dk/services/TMHMM-2.0/) (FIG. 3). AtPAP2 was
found to share 72% sequence identity in amino acid sequence with
AtPAP9. Two sequences from Zea mays (Accession No: ACG47621) and
Oryza sativa (Accession No: BAC15853.1) were found to share 58% and
57% a.a. identity with AtPAP2, respectively. Their sequences were
aligned in FIG. 2.
[0133] AtPAP2-like sequences from other plant species that carry a
hydrophobic motif at their C-termini were retrieved by tblastn
program from Plant GDB database (http://www.plantgdb.org/) and NCBI
database (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the amino
acid sequence of AtPAP2 as the search sequence. cDNA and protein
sequences that share high homology with that of AtPAP2 were
identified in Zea mays (SEQ ID NOs: 7 and 8), Brassica rapa (SEQ ID
NOs: 18 and 19), Hordeum vulgare (SEQ ID NOs: 20 and 21), Medicago
truncatula (SEQ ID NOs: 22 and 23), Physcomitrella patens (SEQ ID
NOs: 24 and 25), Populus trichocarpa (SEQ ID NOs: 26 and 27),
Saccharum officinarum (SEQ ID NOs: 28 and 29), Solanum tuberosum
(SEQ ID NOs: 30 and 31), Vitis vinifera (SEQ ID NOs: 32 and 33),
Oryza sativa (SEQ ID NOs: 34 and 35), Gossypium hirsutum (SEQ ID
NOs: 36 and 37) Panicum virgatum (SEQ ID NOs: 38 and 39), Solanum
lycopersicum (SEQ ID NOs: 40 and 41), Sorghum bicolor (SEQ ID NOs:
42 and 43) and Triticum aestivum (SEQ ID NOs: 44 and 45).
[0134] The cDNA sequences of AtPAP-like sequences were amplified
from a local Glycine max variety (SEQ ID NO: 5) and the Brassica
napus cultivar Westar (SEQ ID NO: 46) by RT-PCR using primers
designed from corresponding EST sequences, which were retrieved
from the Plant GDB database (http://www.plantgdb.org/).
6.2 Screening of T-DNA Line and Production of Overexpression Lines
and Complementation Lines in Arabidopsis
[0135] T-DNA insertion lines of PAP2 gene (Arabidopsis genomic
locus name: Salk.sub.--013567), in the Col ecotype were obtained
from Arabidopsis Biological Resources Center (Alonso et al., 2003).
Homologous T-DNA lines were identified by genomic PCR screening
from SIGnAl database (http://signal.salk.edu/cgi-bin/tdnaexpress)
by using the primers (LBa-1,5'-TGGTTCACGTAGTGGGCCATCG-3', SEQ ID
NO: 9) and PAP2 specific forward primer (P2LP,
5'-TTGAAGTTTAACATGCCTGGG-3, SEQ ID NO: 10) and reverse primer
(P2RP, 5'-TCCAATGCTCGA TTGATTAGC-3', SEQ ID NO: 11). The PCR
product was sequenced and the T-DNA insertion site was confirmed.
To exclude the possibility that another T-DNA locus interferes with
the PAP2 mutant site, homologous pap2 mutant lines were backcrossed
to the wild-type to dilute the potential T-DNA sites. The produced
heterozygous pap2 mutants were grown on the MS plates containing 50
mg/ml Kanamycin. The ratio of the resistant to sensitive plants was
about 3:1. These results demonstrated a single insertion locus site
of the T-DNA line (pap2-8) lines.
[0136] The inability of the T-DNA line to express full length
AtPAP2 mRNA was confirmed by RT-PCR. Total RNA was extracted from
10-day-old seedlings grown on MS with 2% (w/v) sucrose using the
TRIzol RNA isolation method (Invitrogen) with DNase I treatment.
cDNAs were generated using Superscript III reverse transcriptase
(Invitrogen, Carlsbad, Calif., USA) using an oligo dT primer. Two
gene-specific primers, P2YF (5'-GGCCGTCGACATGATCGTTAATT TCTCTTTC-3'
SEQ ID NO: 12) and P2NR (5'-CCGGACTAGTTCATGTCTCCTCGTTCTTGAC-3' SEQ
ID NO: 13), were used to amplify a 1971 bp coding region of AtPAP2.
For each sample, 1 .mu.g of cDNA was amplified for 30 cycles, with
an annealing temperature of 50.degree. C. and using elongation
factor (EF) primers, EF-1 (5'-GTTTCACATCAACATTGTGGTCA TTGG-3, SEQ
ID NO: 14) and EF-2 (5'-GAGTACTTGGGGGTAGTGGCATCC-3, SEQ ID NO: 15)
(Axelos et al., 1989) for control experiment.
[0137] The inability of the T-DNA line to express protein was
confirmed by Western blotting analysis (FIG. 4). Antiserum specific
to AtPAP2 was raised in rabbit as described in Section 6.3.
[0138] To create transgenic AtPAP2 overexpressing lines or
expressing this gene in the knockout mutants, the full length
coding region of the AtPAP2 cDNA was amplified by PCR using primers
P2YF (SEQ ID NO: 12) and P2NR (SEQ ID NO: 13). A SalI site and a
SpeI site were engineered into P2YF and P2NR, respectively. The
resulting product (1976 bp) was inserted into the XhoI/Spe I sites
of a binary vector, immediately downstream to the cauliflower
mosaic virus (CaMV) 35S promoter (FIG. 5).
[0139] The vector was introduced into Agrobacterium tumefaciens
strain GV3101 and then transformed by the floral dip method (Clough
and Bent, 1998), into wild-type Col-0 to generate
PAP2-overexpressing lines or into homologous pap2 plants (T-DNA
lines) to generate complementation lines. Through 2 generations of
selection on MS agar plate with 50 mg/l Basta (Riedel-deHaen),
homologous 35S:PAP2 transgenic lines were obtained. The resistant
plants were transferred to soil to grow to maturity, and their
transgenic status was further confirmed by PCR and immunoblot
analyses. As shown in FIG. 6A, AtPAP2 protein was overexpressed in
OE lines but was absence from the T-DNA line. The homozygous T3
seeds of the transgenic plants were used for further analysis.
[0140] To create transgenic AtPAP15 overexpression lines, the cDNA
of AtPAP15 was also amplified by RT-PCR and then subcloned into a
plant binary vector which bared a kanamycin-resistant gene and a
cauliflower mosaic virus 35S promoter (CaMV). This expression
construct named was then mobilized into Agrobacterium tumefaciens
strain EHA105 by freeze-thaw transformation (Hofgen and Willmitzer,
1988) and transformed into Arabidopsis. Transgenic status was
further confirmed by PCR and immunoblot analyses using an
anti-AtPAP15 antiserum. As shown in FIG. 6B, AtPAP15 protein was
overexpressed in OE lines. The homozygous T3 seeds of the
transgenic plants were used for further analysis.
6.3 Production of PAP2 Polycolonal Antiserum and Western Blots
Analysis
[0141] A fragment of AtPAP2 cDNA corresponding to the N terminal
120 amino acids (from 21 to 141) was amplified using forward primer
P2AF (5'-GGTTGAGCTCGATTCTAAAGCGACCATTTC-3', SEQ ID NO: 16) and
reverse primer P2AR (5'-TTTTGGTACCTCAGGATCCGAA AGTCAGC-3', SEQ ID
NO: 17). The PCR product was cleaved by Sad and KpnI and cloned
into the pRsetA vector (Invitrogen) so that the coding sequence of
the first 120 a.a. of AtPAP2 was fused to a His-tag sequence. The
resulting plasmid was transformed into Escherichia coli strain BL21
(DE3). The BL21 cells were induced at 30.degree. C. by 0.1 mM
isopropylthio-.beta.-D-galactoside for 4 h and resuspended in 100
mM NaCl and 50 mM Tris-HCl, pH 7.5, 2 mM phenylmethylsulphonyl
fluoride (PMSF). The lysates were sonicated 5 times for 30 s each.
The overexpressed His-AtPAP2 fusion proteins in inclusion bodies
were centrifuged at 5000.times.g for 15 mM, and the pellets were
solubilized in 150 mM NaCl, 8 M urea, and 20 mM Tris-HCl, pH 7.5.
The fusion proteins were purified on a HisTrap FF (GE Healthcare)
column and were used for standard immunization protocols in
rabbits.
6.4 Expression Analysis of AtPAP2 mRNA and its Protein Levels
[0142] The mRNA expression level of AtPAP2 was analyzed by the Spot
History program
(http://affymetrix.arabidopsis.info/narrays/spothistory.pl) that
presented the expression levels of a given gene in thousands of
microarray (Affymetrix ATH1 microarray) database. Spot history
analysis indicated that the expression of AtPAP2 was constitutive
but is relative low in most experimental circumstances (FIG. 7a).
To determine AtPAP2 expression levels, different tissues of
wild-type A. thaliana (Col-0) were collected.
[0143] The expression level of proteins were also studied by
western blotting, using the anti-AtPAP2 antiserum generated from
Section 6.3. Total plant soluble protein was extracted from
wild-type A. thaliana, T-DNA line, AtPAP2-overexpress lines in
grinding buffer (Tris-HCl 50 mM, pH7.4 containing 150 mM NaCl, 1 mM
EDTA, 0.2 mM PMSF) on ice. Protein extracts were centrifuged at
16000.times.g and supernatants were collected for Bradford protein
concentration determination assay. Equal amount of protein samples
(50-90 .mu.g/lane in different experiments) were loaded and
separated in 12% (w/v) SDS-PAGE. The separated proteins were
transferred to Hybond C-Extra membranes (Amersham Biosciences) (400
mA, 1 h). Membranes were blocked with 5% (w/v) non-fat milk in TTBS
washing buffer (pH 7.6) for 2 hours and probed with specific
anti-AtPAP2 antiserum for 3 hours or overnight at an 1:1000
dilution at 4.degree. C. After rinsing the membrane with three
changes of TTBS washing buffer (20 mM Tris-HCl, pH7.6, 136 mM NaCl,
0.1% Tween20) in half an hour, HRP-labeled secondary antibody,
diluted 1:10,000 in TTBS washing buffer was added. After 2 hours,
the membrane was washed thrice before the bands were visualized by
Enhanced Chemiluminescence method (Amersham Biosciences). As shown
in FIG. 7b, AtPAP2 protein was expressed in all tissues tested
(Leaf, Flower, Stem, Root, Silique) at equal levels. The protein
expression level of AtPAP2 during germination was very stable too
(FIG. 7c) and was independent of phosphorus status (FIG. 7d).
[0144] 6.5 Growth Phenotypes of WT, T-DNA Line and OE Lines
[0145] Arabidopsis seeds were soaked in water at 4.degree. C. for 3
days. The seeds were surface sterilized and sown on Murashige and
Skoog (MS) medium supplemented with 2% (w/v) sucrose for 10 days.
Seedlings with 2 rosette leaves of the same size were transferred
to soil under Long Day (16 h light at 22.degree. C./8 h dark at
18.degree. C.) or Short Day (8 h light at 22.degree. C./16 h dark
at 18.degree. C.) conditions in a plant growth chamber. Flowering
time was started to be measured by scoring the number of rosette
leaves and cauline leaves when the primary inflorescence
florescence reached 1 cm above the rosette leaves. Ten to 20 plants
were scored for each line (Liu et al., 2008; Wu et al., 2008).
[0146] The inflorescences of OE lines of AtPAP2 emerged earlier
(5-6 days for Long Day, 14-16 days for Short Day) than that of the
WT and T-DNA lines (Table 1). Under Long Day conditions, the number
of rosette leaves of the OE lines were less (5-6 leaves) than the
WT during the emergence of inflorescence (Table 1 and FIG. 8). At
day 28 (Long Day), the OE lines of AtPAP2 had more cauline leaves
and inflorescences than the WT and T-DNA lines, but had less
rosette leaves (Table 2.). This phenotype observation was repeated
at least four times and the results of one of the experiments were
shown here.
TABLE-US-00001 TABLE 1 AtPAP2 OE lines flowered at an earlier
developmental stage. Long Day (16 h/8 h) Short Day (8 h/16 h) Lines
AEI SD NRL SD AEI SD NRL SD Col-0 26.9 1.2 13.0 0.8 41.0 4.7 18.0
3.0 T-DNA 25.7 0.7 11.6 1.1 40.7 4.9 15.0 3.0 OE7 20.0* 1.1 6.4*
0.5 25.6* 1.3 5.3* 0.5 OE21 20.8* 0.6 6.5* 0.7 26.0* 1.1 5.4* 0.5
AEI: Average date of emergence of inflorescence NRL: No. of rosette
leaves at the first appearance of inflorescence *Statistically (p
< 0.001) different from the wild-type (n = 15).
TABLE-US-00002 TABLE 2 Phenotypes of AtPAP2 OE lines at Day 28
(Long Day). No. of No. of No. of Lines Rosette Leaf SD Cauline Leaf
SD Inflorescence SD Col-0 14.5 1.2 1.6 0.5 1.0 0.0 T-DNA 16.7 1.7
1.9 0.6 1.0 0.0 OE7 9.9* 1.0 6.0* 1.2 3.6* 0.7 OE21 10.2* 1.8 7.2*
1.6 3.7* 1.1 *Statistically (p < 0.001) different from the
wild-type (n = 15).
[0147] At maturity (Long Day), the number of siliques and the total
weight of seeds harvested from each line were recorded. Two
separate experimental trials are shown in Tables 3A and 3B. Our
results showed that overexpression of AtPAP2 resulted in increase
number of siliques per plant and the seed yield per plant. Compared
to that of the wild-type, the seed yield of the two overexpression
lines shown in Table 3A increased 38-40%. Compared to that of the
wild-type, the seed yield of the two overexpression lines shown in
Table 3B increased 54-58%.
TABLE-US-00003 TABLE 3A OE lines produced more siliques and seeds
(Trial 1). Weight of seeds Lines No. of siliques/plant SD (g)/plant
SD N Col-0 327.4 53.3 0.188 0.047 5 T-DNA 236.6* 60.2 0.121* 0.040
7 OE7 453.2** 62.1 0.264** 0.039 5 OE21 498.2.sup.# 52.5 0.260**
0.049 7 Statistically (p < 0.02*, p < 0.01**, p <
0.001.sup.#) different from the wild-type.
TABLE-US-00004 TABLE 3B OE lines produced more siliques and seeds
(Trial 2). Weight of seeds Lines No. of siliques/plant SD (g)/plant
SD N Col-0 396.4 89.5 0.225 0.058 13 T-DNA 386.3 70.4 0.240 0.049
12 OE7 610.9* 76.6 0.351* 0.050 7 OE21 624.9* 94.7 0.355* 0.066 11
Statistically (p < 0.0001*) different from the wild-type.
[0148] However, the OE lines of AtPAP15 grew normally and were not
different from the wild-types. Therefore, the enhanced growth
performance was due to the overexpression of AtPAP2, which bears a
transmembrane-like motif at its C-terminus (FIGS. 2 and 3).
6.6 Growth Phenotypes of Truncated AtPAP2 Constructs
[0149] An alternate vector construct employing the sequence for
AtPAP2 was also constructed using analogous techniques to those
described above. As shown in FIG. 12, a construct equivalent to the
OE lines of AtPAP2 missing the C-terminal motif (residues 614-636
of SEQ ID NO: 2) was constructed (P2C lines). Transgenic plants
were generated using substantially identical techniques to those
described above. Western blot analysis was used to confirm the
over-expression of the AtPAP2 fragment proteins in transformed
plant lines. Performance of Western blot analysis was identical to
that reported above. As show in FIG. 13, the P2C lines were
strongly overexpressed. The growth phenotype of the P2C lines
appeared to be indifferent from the wild-type, which is indicative
of the importance of the C-terminal domain of AtPAP2 in developing
an increased growth phenotype.
6.7 MS/MS Analysis of Sucrose and Glucose Levels in Leaf
[0150] Rosette leaves of plants of various developmental stages
were harvested at the end of the light period of 21-day-old plants.
Soluble sugars were extracted from Arabidopsis using
chloroform/methanol method (Lunn et al., 2006; Antonio et al.,
2007; Luo et al., 2007). 100 mg plant tissues were ground to a fine
powder in liquid nitrogen and mixed and vortexed with 250 .mu.l
ice-cold chloroform:methanol (3:7, v/v). Soluble metabolites were
then extracted at -20.degree. C. overnight. 200 .mu.l water was
added to the mixture with repeated shaking. The extracts were
centrifuged at 16000.times.g for 10 min and the supernatant was
collected. The pellet was re-extracted by 204.1 water and the
supernatant was collected by centrifugation as described above. The
combined supernatant was evaporated to dryness using a SpeedVac and
the pellet was re-dissolved in 200 .mu.l water. Finally, debris was
removed by centrifugation at 16000.times.g for 30 min.
[0151] 20 .mu.l filtered samples were analyzed by an API-3000
triple-quadrupole mass spectrometer (Applied Biosystems) via an
electrospray ionization source. The parameters, optimized by 0-40
.mu.g/ml glucose and sucrose standards, were as following: curtain
gas (CUR) 25, nebulizer gas (GS1) 50, auxiliary gas (GS2) 30,
ionspray voltage -4.5 k V, temperature 400.degree. C., declustering
potential (DP) -106 V, entrance potential (EP) -8.5 V, collision
cell entrance potential (CEP) -46.7 V, collision energy (CE) 20 V.
The peaks were identified by comparison with glucose and sucrose
standards and the amount of sugars were quantified by standard
curves of these sugars. The Analyst 1.3.1 software (Applied
Biosystems) was used for data acquisition, peak integration, and
calculation. The amount of sucrose and glucose at the end of day in
the shoots of 21-day-old soil grown plants were shown in FIG. 9. It
was found that the levels of both sugars were significantly higher
than that in WT.
6.8 Recovery of Plants after Prolonged Darkness Treatment
[0152] Seeds of wild-type, T-DNA, OE7 and OE21 lines were
germinated in MS (2% sucrose) medium for 10 days. Seedlings with 2
small visible rosette leaves (1 mm) of the same size were
transferred to soil for another 12 days in normal growth conditions
(LDs, 16 h/light (22.degree. C.)/8 h darkness (18.degree. C.)). The
light source of the growth chamber was then switched off for 12
days. Then the plants were allowed to recover under the 16 h/light
(22.degree. C.)/8 h darkness (18.degree. C.) cycle for 10 days. The
plants that stayed green and that continued to emerge inflorescence
were recorded in Table 4.
TABLE-US-00005 TABLE 4 Surviving rate and flowering ratio after
prolonged darkness treatment. Flowering after Recovery (leaf
recovery greening) Wild-type 8/12 11/12 T-DNA 5/12 8/12 OE7 9/9 9/9
OE21 12/12 12/12
[0153] Extended darkness could induce carbohydrate starvation
(Thompson et al., 2005). Our data showed that the OE lines
exhibited 100% recovery rate under prolonged (12 days) darkness
treatment, which was higher than that of the WT and the T-DNA line
(FIG. 10). This could be attributed to a higher endogenous sugar
levels (FIG. 9) in the OE lines.
6.9 Phenotypes of Plants Under NaCl and ABA Treatments
[0154] 5-day-old seedlings grown on MS agar were transferred to MS
agar with NaCl (50 mM, 100 mM, 150 mM), ABA (0.1 uM, 0.2 uM. 0.5
uM, 1 uM, 2 uM) or sorbitol (300 mM, 400 mM, 500 mM).
Alternatively, seeds were directly germinated on the treatment
media. Wild-type, T-DNA and OE lines did not show remarkable
phenotypic differences under the above conditions.
6.10 Subcellular Fractionation
[0155] Rosette leaves of three-week-old wild-type (Col-0)
Arabidopsis were harvested and stored at -80.degree. C. freezer
until use. Tissues (4-5 g) were ground to fine powder in liquid
nitrogen using a mortar with a pestle. The powder was transferred
into 10 ml grinding buffer (0.3 M sucrose, 40 mM Tris-HCl (pH 7.8),
5 mM MgCl.sub.2, 1 mM PMSF) and swelled on ice for 5 min.
Homogenization was performed for two 30-second pulses at high-speed
setting. The homogenate was filtered through two layers of
Miracloth (Tetko, Elmsford, N.Y., USA). Subsequently, the
homogenate was separated by centrifugation at 350 g for 10 min at
4.degree. C. The pellet (crude nuclear) was further layered onto 1
ml of 2.3 M sucrose, 50 mM Tris-HCl (pH 8.8), 5 mM MgCl.sub.2 in an
Eppendorf tube for centrifugation at 15,000 g 10 min at 4.degree.
C., to obtain the nuclear fraction in the derived pellet.
Supernatants from the first low-speed centrifugation (350 g) were
centrifuged at 12,000.times.g for 20 min at 4.degree. C. The pellet
contained large particles including mitochondria, chloroplasts and
peroxisomes. The supernatant was further centrifuged at
100,000.times.g for 1 h at 4.degree. C. to yield the soluble
cytosol fraction in the resulting supernatant. The pellet
representing the membrane fraction was resuspended in 0.1 ml
grinding buffer. Protein concentration in the extract was
determined following the method of Bradford (Bradford, 1976) using
the Bio-Rad Protein Assay Kit I.
[0156] To isolate cell wall, leaf tissues were homogenized in
grinding buffer (62.5 mM Tris-HCl, pH 7.5, 5 mM DTT, 1% (v/v)
bovine serum albumin, 2 mM phenylmethylsulfonyl fluoride, 2
.mu.g/ml leupeptin, 2 .mu.g/ml E-64, 2 .mu.g/ml pepstatin A) using
a Polytron (full speed, 3.times.10 s). The homogenate was
centrifuged at 1,000.times.g for 3 min. The pellet was washed with
ice-cold grinding buffer (without 1% BSA) 10 times. Finally the
(cell wall) pellet was washed by resuspending in 500 mM CaCl.sub.2,
20 mM NaCl, 62.5 mM Tris-HCl, pH 7.5, and spinning at
10,000.times.g for 15 min (He et al., 1996).
[0157] The subcellular fractions were run in a SDS-PAGE gel and
were probed with anti-AtPAP2 antiserum. AtPAP2 was detected in
membrane and soluble protein fractions but not in nucleus,
mitochondria nor chloroplasts (FIG. 11).
[0158] In summary, Arabidopsis plants transformed with the AtPAP2
gene have the following phenotypes when they were compared with the
wild-type: (1) Faster growth rate (Tables 1 and 2); (2) Higher
sucrose content (FIG. 9); (3) Higher glucose content (FIG. 9); and
(4) Higher crop yield (Table 3).
[0159] Those skilled in the art will recognize, or be able to
ascertain many equivalents to the specific embodiments described
herein using no more than routine experimentation. Such equivalents
are intended to be encompassed by the following claims.
[0160] With respect to any figure or numerical range for a given
characteristic, a figure or a parameter from one range may be
combined with another figure or a parameter from a different range
for the same characteristic to generate a numerical range.
[0161] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference.
[0162] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
patent application.
[0163] While the embodiments have been explained in relation to
certain embodiments, it is to be understood that various
modifications thereof will become apparent to those skilled in the
art upon reading the specification. Therefore, it is to be
understood that the embodiments disclosed herein are intended to
cover such modifications as fall within the scope of the appended
claims. Features of two or more of any of the above embodiments can
be combined to form additional embodiments.
[0164] Other than in the operating examples, or where otherwise
indicated, all numbers, values and/or expressions referring to
quantities of ingredients, reaction conditions, etc., used in the
specification and claims are to be understood as modified in all
instances by the term "about."
6.11 Assays of Enzymes Involved in Sucrose Metabolism
[0165] Sucrose phosphate synthesis (SPS), sucrose synthesis (SuSy),
cytosolic invertase and cell wall invertase activities in the shoot
of 20-day-old plants were determined. Samples were collected 8 h
after the light and dark period (Long Day). SPS activity was
measured under both optimal (Vmax) and limiting (V limit) assay
conditions (Park et al., 2008). SuSy, cytosolic invertase and
insoluble cell wall invertase activities were also determined
(Doehlert, 1987). The assays were repeated three times and the SPS
(Vmax and V limit) activities of both independent lines were
significantly higher than that of the wild-type and T-DNA lines in
all three repeated experiments. The data of a representative
experiment is shown in table 5. In contrast to SPS, SuSy, cytosolic
invertase and cell wall invertase activities were not different
among the lines.
TABLE-US-00006 TABLE 5 Enzyme assays Plant line WT T-DNA OE7 OE21
Sucrose phosphate synthase (.mu.M sucrose/.mu.g enzyme
extracts/hour) Vmax (Day) 108.8 .+-. 18.1 116.1 .+-. 11.9 157.9
.+-. 21.7** 159.5 .+-. 19.0** Vlimit (Day) 63.6 .+-. 6.4 69.5 .+-.
2.5 90.8 .+-. 18.5** 79.8 .+-. 13.7* Vmax (Night) 118.3 .+-. 11.4
104.9 .+-. 14.4 150.9 .+-. 19.4** 136.5 .+-. 15.1* Vlimit (Night)
74.9 .+-. 6.3 56.7 .+-. 3.3 93.4 .+-. 3.6** 97.3 .+-. 10.9**
Sucrose synthase (.mu.M glucose/.mu.g enzyme extracts/hour) Day
249.2 .+-. 4.6 247.0 .+-. 24.0 248.6 .+-. 4.5 255.2 .+-. 5.4 Night
249.4 .+-. 7.2 252.7 .+-. 8.8 250.8 .+-. 8.7 258.8 .+-. 5.5
Cytosolic invertase (.mu.M glucose/.mu.g enzyme extracts/hour) Day
(Acid) 14.3 .+-. 2.3 12.1 .+-. 5.4 18.3 .+-. 9.1 18.0 .+-. 0.8
Night (Acid) 14.0 .+-. 6.8 26.1 .+-. 10.9 17.6 .+-. 4.3 13.8 .+-.
0.8 Day (Alkaline) 169.7 .+-. 9.8 161.2 .+-. 32.3 160.9 .+-. 27.9
178.8 .+-. 16.9 Night (Alkaline) 130.0 .+-. 8.2 105.1 .+-. 12.6
136.1 .+-. 13.6 136.6 .+-. 1.4 Cell wall invertase (.mu.M
sucrose/.mu.g enzyme extracts/hour) Day (Acid) 22.3 .+-. 3.9 18.0
.+-. 4.9 23.5 .+-. 6.3 24.0 .+-. 6.6 Night (Acid) 21.6 .+-. 10.1
17.0 .+-. 4.3 28.3 .+-. 4.2 22.3 .+-. 3.3 Day (Alkaline) 121.7 .+-.
2.8 127.1 .+-. 2.2 101.8 .+-. 6.4 105.5 .+-. 2.0 Night (Alkaline)
138.9 .+-. 6.3 151.5 .+-. 8.7 123.4 .+-. 5.6 135.4 .+-. 14.7 (**P
< 0.01; *P < 0.05)
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Sequence CWU 1
1
6611971DNAArabidopsis thaliana 1atgatcgtta atttctcttt cttcctcctt
ctcttcgtct ccgtcttcgt ttcctctgct 60gattctaaag cgaccatttc aatttcccct
aatgctctca atcgatctgg cgattccgtt 120gtgatacaat ggtccggtgt
cgattctccg tcagatctcg attggttagg actctactcg 180ccgccggagt
ctcctaatga tcactttatt ggttacaaat tcctcaatga atcgtccact
240tggaaagatg gtttcggttc gatttctctt cctttaacca atctccgatc
aaattacaca 300ttccggatct tccgttggag cgaatccgag attgatccga
aacataagga tcatgatcag 360aatcctttac caggaactaa acatcttcta
gctgaatcgg agcagctgac tttcggatcc 420ggtgttggta tgccggagca
gatccatttg tcgttcacaa atatggttaa cacgatgcgt 480gttatgtttg
tagctggaga tggtgaagaa cgttttgtta gatacggtga atcgaaggat
540ttgttaggta attccgcggc ggcgcgtggg atgaggtacg agagagagca
catgtgtgat 600tcgccggcga attccactat tggttggaga gatcctggtt
ggatttttga taccgtcatg 660aagaatttga atgatggcgt tagatactat
tatcaggttg ggagtgattc taagggatgg 720agtgagatcc atagctacat
tgctcgagat gtgactgcag aagaaaccgt agctttcatg 780tttggagata
tgggttgtgc tacaccatac acgacattta tccgcacaca agatgagagc
840atatctacag tgaagtggat cctccgtgac attgaagctc ttggtgataa
gccagctatg 900atttcacaca ttggagatat aagttatgct cgtggttact
cgtgggtatg ggatgagttc 960tttgctcagg ttgagcctat tgcctcgaca
gttccttacc atgtttgcat tggtaaccat 1020gagtatgatt tctctactca
gccgtggaaa cctgattggg cagcttctat ttatggaaac 1080gatggtggtg
gcgaatgtgg tgtgccgtat agcttgaagt ttaacatgcc tgggaattct
1140tcagagtcta caggaatgaa agctcctccg acaaggaatt tatattattc
ttatgatatg 1200ggaacggtcc atttcgttta tatctccaca gagacgaatt
ttcttaaagg aggtagtcaa 1260tatgaattca taaagcgaga tctagagtct
gtagacagga agaaaacacc gtttgttgtt 1320gtgcaaggac atagaccaat
gtacactacg agcaacgagg ttagagacac tatgattcga 1380caaaagatgg
ttgagcatct agaacctttg tttgtgaaaa acaatgtcac acttgctcta
1440tggggacatg ttcatagata cgaaaggttt tgtcccataa gcaacaacac
ttgcggcaca 1500cagtggcaag gaaatccggt tcatcttgtg atcggtatgg
ctggtcaaga ttggcaaccg 1560atttggcagc ctagaccaaa ccatccagat
cttcctatat tccctcagcc tgaacaatca 1620atgtatcgta caggtgagtt
tggttacact cgtttagttg caaacaaaga aaagctcact 1680gtttcttttg
tgggtaatca cgatggcgaa gttcatgata ctgttgagat gttagcatct
1740ggggtagtaa tcagtgggag caaagagagt actaaaatcc caaatctgaa
aaccgttcct 1800gcttctgcta cacttatggg aaaatcagaa tctaatgctt
tgtggtatgc caaaggagca 1860ggcttgatgg ttgtgggtgt gcttttaggg
ttcattatcg ggttttttac ccggggaaag 1920aaatcttcgt ctggaaaccg
ttggatccca gtcaagaacg aggagacata a 19712656PRTArabidopsis thaliana
2Met Ile Val Asn Phe Ser Phe Phe Leu Leu Leu Phe Val Ser Val Phe1 5
10 15Val Ser Ser Ala Asp Ser Lys Ala Thr Ile Ser Ile Ser Pro Asn
Ala 20 25 30Leu Asn Arg Ser Gly Asp Ser Val Val Ile Gln Trp Ser Gly
Val Asp 35 40 45Ser Pro Ser Asp Leu Asp Trp Leu Gly Leu Tyr Ser Pro
Pro Glu Ser 50 55 60Pro Asn Asp His Phe Ile Gly Tyr Lys Phe Leu Asn
Glu Ser Ser Thr65 70 75 80Trp Lys Asp Gly Phe Gly Ser Ile Ser Leu
Pro Leu Thr Asn Leu Arg 85 90 95Ser Asn Tyr Thr Phe Arg Ile Phe Arg
Trp Ser Glu Ser Glu Ile Asp 100 105 110Pro Lys His Lys Asp His Asp
Gln Asn Pro Leu Pro Gly Thr Lys His 115 120 125Leu Leu Ala Glu Ser
Glu Gln Leu Thr Phe Gly Ser Gly Val Gly Met 130 135 140Pro Glu Gln
Ile His Leu Ser Phe Thr Asn Met Val Asn Thr Met Arg145 150 155
160Val Met Phe Val Ala Gly Asp Gly Glu Glu Arg Phe Val Arg Tyr Gly
165 170 175Glu Ser Lys Asp Leu Leu Gly Asn Ser Ala Ala Ala Arg Gly
Met Arg 180 185 190Tyr Glu Arg Glu His Met Cys Asp Ser Pro Ala Asn
Ser Thr Ile Gly 195 200 205Trp Arg Asp Pro Gly Trp Ile Phe Asp Thr
Val Met Lys Asn Leu Asn 210 215 220Asp Gly Val Arg Tyr Tyr Tyr Gln
Val Gly Ser Asp Ser Lys Gly Trp225 230 235 240Ser Glu Ile His Ser
Tyr Ile Ala Arg Asp Val Thr Ala Glu Glu Thr 245 250 255Val Ala Phe
Met Phe Gly Asp Met Gly Cys Ala Thr Pro Tyr Thr Thr 260 265 270Phe
Ile Arg Thr Gln Asp Glu Ser Ile Ser Thr Val Lys Trp Ile Leu 275 280
285Arg Asp Ile Glu Ala Leu Gly Asp Lys Pro Ala Met Ile Ser His Ile
290 295 300Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Val Trp Asp
Glu Phe305 310 315 320Phe Ala Gln Val Glu Pro Ile Ala Ser Thr Val
Pro Tyr His Val Cys 325 330 335Ile Gly Asn His Glu Tyr Asp Phe Ser
Thr Gln Pro Trp Lys Pro Asp 340 345 350Trp Ala Ala Ser Ile Tyr Gly
Asn Asp Gly Gly Gly Glu Cys Gly Val 355 360 365Pro Tyr Ser Leu Lys
Phe Asn Met Pro Gly Asn Ser Ser Glu Ser Thr 370 375 380Gly Met Lys
Ala Pro Pro Thr Arg Asn Leu Tyr Tyr Ser Tyr Asp Met385 390 395
400Gly Thr Val His Phe Val Tyr Ile Ser Thr Glu Thr Asn Phe Leu Lys
405 410 415Gly Gly Ser Gln Tyr Glu Phe Ile Lys Arg Asp Leu Glu Ser
Val Asp 420 425 430Arg Lys Lys Thr Pro Phe Val Val Val Gln Gly His
Arg Pro Met Tyr 435 440 445Thr Thr Ser Asn Glu Val Arg Asp Thr Met
Ile Arg Gln Lys Met Val 450 455 460Glu His Leu Glu Pro Leu Phe Val
Lys Asn Asn Val Thr Leu Ala Leu465 470 475 480Trp Gly His Val His
Arg Tyr Glu Arg Phe Cys Pro Ile Ser Asn Asn 485 490 495Thr Cys Gly
Thr Gln Trp Gln Gly Asn Pro Val His Leu Val Ile Gly 500 505 510Met
Ala Gly Gln Asp Trp Gln Pro Ile Trp Gln Pro Arg Pro Asn His 515 520
525Pro Asp Leu Pro Ile Phe Pro Gln Pro Glu Gln Ser Met Tyr Arg Thr
530 535 540Gly Glu Phe Gly Tyr Thr Arg Leu Val Ala Asn Lys Glu Lys
Leu Thr545 550 555 560Val Ser Phe Val Gly Asn His Asp Gly Glu Val
His Asp Thr Val Glu 565 570 575Met Leu Ala Ser Gly Val Val Ile Ser
Gly Ser Lys Glu Ser Thr Lys 580 585 590Ile Pro Asn Leu Lys Thr Val
Pro Ala Ser Ala Thr Leu Met Gly Lys 595 600 605Ser Glu Ser Asn Ala
Leu Trp Tyr Ala Lys Gly Ala Gly Leu Met Val 610 615 620Val Gly Val
Leu Leu Gly Phe Ile Ile Gly Phe Phe Thr Arg Gly Lys625 630 635
640Lys Ser Ser Ser Gly Asn Arg Trp Ile Pro Val Lys Asn Glu Glu Thr
645 650 65531956DNAArabidopsis thaliana 3atgatcgccg ccgtttacac
tctcttcttc ttcttcctct taatctcctc tgtttattcc 60aaagccacga tttcaatctc
tccccaaacc ttaaaccgat ccggcgacat agtcgtgatc 120aaatggtccg
gcgtcgaatc accgtccgat ctcgactggt taggaatcta ctcgccaccg
180gactctcctc acgatcactt catcggctac aaattcctct ccgattcacc
cacgtggcaa 240tccgggtcgg gttcgatttc acttccctta accaatctcc
gatcaaatta cactttccgg 300atctttcatt ggacccaatc cgaaataaac
ccgaaacacc aagaccatga tcacaatcct 360ttacccggaa ctcgtcatct
cttaaccgaa tcaaaccagt taaatttccg gttcgctgtt 420aaccgaccgg
agcagattca tttaagttac acagataaca tcaacgagat gagagtagtg
480tttgtaaccg gagatggaga agaacgagaa gctcgctacg gtgaggttaa
ggacaagctc 540gataacatag cggtggcgcg tggagttagg tacgagatag
aacatatgtg tcacgcgccg 600gcgaattcta cagtcggatg gagagatcca
ggttggacat tcgatgccgt gatgaagaat 660ctaaaacaag ggattaggta
ttattatcag gttgggagtg atttaaaagg atggagtgag 720attcatagct
ttgtttctcg aaatgagggt tcagaagaaa cattagcttt catgtttggt
780gatatgggat gttatacacc ttatacaaca tttatccgtg gagaagaaga
aagtttatca 840actgtgaaat ggattttaag agacattgaa gctttaggtg
atgataagcc tgtgattgta 900tcacatatcg gagatataag ttatgcacga
ggttactcgt ggatttggga tgagttcttt 960actcagattg agcctattgc
ttctaaagtg ccttaccatg tatgtattgg taaccatgag 1020tatgattggc
ctaaccagcc ttggaaacca gattgggctg cttatgttta tggtaaagat
1080agtggtggtg aatgtggtgt accgtatagt gttaagttca acatgcctgg
taattcaacg 1140gaagctactg gtatggttaa gggacctcaa agtcggaacc
tttactattc ttatgatatg 1200ggttctgttc atttcgttta tatttcgaca
gagactgatt ttcttaaagg tgggaagcag 1260tatagttttt tgaagagtga
tttggagtct gttaatagga gtaagacacc gtttgttgtt 1320gtccaagggc
atagacctat gtacactacg agtaggaaga tcagagacgc tgctataaga
1380gagaagatga tcgagcattt ggaaccgttg ttagtgaaga acaatgtgac
ggttgcttta 1440tggggacatg tacatagata tgaaaggttt tgtgcgatta
gtaacaatac ttgtggtgaa 1500cgttggcaag gaaatccagt tcatcttgtg
attggtatgg ctggaaaaga ctcacaaccg 1560atgtgggaac cgagagctaa
tcacgaggat gtcccgatct ttcctcagcc tgctaactca 1620atgtaccgtg
gaggcgagtt tgggtacatt cgtttggttg ctaataagga aagacttact
1680ctttcttatg tgggaaacca tgacggagaa gttcatgatg ttgttgagat
tttggcttct 1740ggggaagtta ttagcggtag tgatgatggt actaaagact
caaactttgg atcagaatct 1800gactttgcag tcttgtggta cattgaagga
gcaagtgtga tggttgtggg agtgattttt 1860gggtactttg tcggttttct
tagtcgtaaa aagaaagaat ctggagttgg atcatctaat 1920cgtagttgga
tccaagtgaa aaacgaggag acatga 19564651PRTArabidopsis thaliana 4Met
Ile Ala Ala Val Tyr Thr Leu Phe Phe Phe Phe Leu Leu Ile Ser1 5 10
15Ser Val Tyr Ser Lys Ala Thr Ile Ser Ile Ser Pro Gln Thr Leu Asn
20 25 30Arg Ser Gly Asp Ile Val Val Ile Lys Trp Ser Gly Val Glu Ser
Pro 35 40 45Ser Asp Leu Asp Trp Leu Gly Ile Tyr Ser Pro Pro Asp Ser
Pro His 50 55 60Asp His Phe Ile Gly Tyr Lys Phe Leu Ser Asp Ser Pro
Thr Trp Gln65 70 75 80Ser Gly Ser Gly Ser Ile Ser Leu Pro Leu Thr
Asn Leu Arg Ser Asn 85 90 95Tyr Thr Phe Arg Ile Phe His Trp Thr Gln
Ser Glu Ile Asn Pro Lys 100 105 110His Gln Asp His Asp His Asn Pro
Leu Pro Gly Thr Arg His Leu Leu 115 120 125Thr Glu Ser Asn Gln Leu
Asn Phe Arg Phe Ala Val Asn Arg Pro Glu 130 135 140Gln Ile His Leu
Ser Tyr Thr Asp Asn Ile Asn Glu Met Arg Val Val145 150 155 160Phe
Val Thr Gly Asp Gly Glu Glu Arg Glu Ala Arg Tyr Gly Glu Val 165 170
175Lys Asp Lys Leu Asp Asn Ile Ala Val Ala Arg Gly Val Arg Tyr Glu
180 185 190Ile Glu His Met Cys His Ala Pro Ala Asn Ser Thr Val Gly
Trp Arg 195 200 205Asp Pro Gly Trp Thr Phe Asp Ala Val Met Lys Asn
Leu Lys Gln Gly 210 215 220Ile Arg Tyr Tyr Tyr Gln Val Gly Ser Asp
Leu Lys Gly Trp Ser Glu225 230 235 240Ile His Ser Phe Val Ser Arg
Asn Glu Gly Ser Glu Glu Thr Leu Ala 245 250 255Phe Met Phe Gly Asp
Met Gly Cys Tyr Thr Pro Tyr Thr Thr Phe Ile 260 265 270Arg Gly Glu
Glu Glu Ser Leu Ser Thr Val Lys Trp Ile Leu Arg Asp 275 280 285Ile
Glu Ala Leu Gly Asp Asp Lys Pro Val Ile Val Ser His Ile Gly 290 295
300Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Ile Trp Asp Glu Phe
Phe305 310 315 320Thr Gln Ile Glu Pro Ile Ala Ser Lys Val Pro Tyr
His Val Cys Ile 325 330 335Gly Asn His Glu Tyr Asp Trp Pro Asn Gln
Pro Trp Lys Pro Asp Trp 340 345 350Ala Ala Tyr Val Tyr Gly Lys Asp
Ser Gly Gly Glu Cys Gly Val Pro 355 360 365Tyr Ser Val Lys Phe Asn
Met Pro Gly Asn Ser Thr Glu Ala Thr Gly 370 375 380Met Val Lys Gly
Pro Gln Ser Arg Asn Leu Tyr Tyr Ser Tyr Asp Met385 390 395 400Gly
Ser Val His Phe Val Tyr Ile Ser Thr Glu Thr Asp Phe Leu Lys 405 410
415Gly Gly Lys Gln Tyr Ser Phe Leu Lys Ser Asp Leu Glu Ser Val Asn
420 425 430Arg Ser Lys Thr Pro Phe Val Val Val Gln Gly His Arg Pro
Met Tyr 435 440 445Thr Thr Ser Arg Lys Ile Arg Asp Ala Ala Ile Arg
Glu Lys Met Ile 450 455 460Glu His Leu Glu Pro Leu Leu Val Lys Asn
Asn Val Thr Val Ala Leu465 470 475 480Trp Gly His Val His Arg Tyr
Glu Arg Phe Cys Ala Ile Ser Asn Asn 485 490 495Thr Cys Gly Glu Arg
Trp Gln Gly Asn Pro Val His Leu Val Ile Gly 500 505 510Met Ala Gly
Lys Asp Ser Gln Pro Met Trp Glu Pro Arg Ala Asn His 515 520 525Glu
Asp Val Pro Ile Phe Pro Gln Pro Ala Asn Ser Met Tyr Arg Gly 530 535
540Gly Glu Phe Gly Tyr Ile Arg Leu Val Ala Asn Lys Glu Arg Leu
Thr545 550 555 560Leu Ser Tyr Val Gly Asn His Asp Gly Glu Val His
Asp Val Val Glu 565 570 575Ile Leu Ala Ser Gly Glu Val Ile Ser Gly
Ser Asp Asp Gly Thr Lys 580 585 590Asp Ser Asn Phe Gly Ser Glu Ser
Asp Phe Ala Val Leu Trp Tyr Ile 595 600 605Glu Gly Ala Ser Val Met
Val Val Gly Val Ile Phe Gly Tyr Phe Val 610 615 620Gly Phe Leu Ser
Arg Lys Lys Lys Glu Ser Gly Val Gly Ser Ser Asn625 630 635 640Arg
Ser Trp Ile Gln Val Lys Asn Glu Glu Thr 645 65051989DNAGlycine max
5atgattcccg atctacccct ccccttcctc ttctccttat tcatcatctt cttccacctc
60gctgaatcca aaccctccct caccgccacg ccaaccaccc tcccagcctc cggcgccacc
120gtcaatctcc gctggtccgg catcccttcc ccctccgacc tcgacttcct
cgccatctat 180tcccctccga cctccccgca cgacaacttc atcggatacc
tcttcctctc gcagtccgcc 240acgtggcgca ccggctccgg caacctctcc
ctccccctcg tcgacctccg ctccaactac 300tccttccgca tcttcagctg
gacccgcgcc gagatcaacc ccaagcgcca ggaccacgat 360cacaaccctc
tcccggtcac acgccacctc ctcgcgtttt cggaggaggt ctccttcgcc
420cctcaccgtg ggccccaaca gatccacctg gcgttcgttg gggcccacgg
caaggaggag 480gatatgcgcg tgatgtacat cgcgcgcgat ccgagagaaa
cctacgtaag gtacggggag 540agggaggata agcttgatgg gattgcggtc
gcacgtgtgg agaggtacga gagggagcac 600atgtgcgacg cgcctgccaa
cacgagtgtt gggtggaggg accctgggtt tatcaacgac 660gccgttctca
taggtttgaa gaagggacag aggtattatt acaaggttgg aaatgataac
720ggaggttgga gtgcaactca aagttttgtg tcgaggaata gtgattcaga
cgaaacaata 780gctttcctgt ttggtgacat gggaacagct gtaccataca
atacgtttct gcgaacgcag 840gatgaaagca tatcaaccat gaagtggatc
ctccgtgatg ttgaagctct aggcgacaag 900ccagcctttg tgtcgcacat
tggagacatt agttatgcaa gaggttattc ctggttgtgg 960gaccattttt
ttgcccagat tgaacctgtt gcctcccaag tggcatacca tgtttgcatt
1020ggcaatcatg agtatgactg gcctttgcag ccatggaaac ctgattgggc
cagttatgga 1080aaagatgggg gtggtgagtg tggtgtgcct tacagtttaa
ggtttaacat gcccggaaac 1140tcttcagaac tcactggaaa tgctgcagcc
ccaccaacta ggaatcttta ttactcattt 1200gatatgggag cagtacactt
tgtgtatatt tccacggaga ccaattttgt tcctgggagc 1260aaacagtacg
acttcttgaa gcatgatttg gaatcggtta acaggagcaa gactcctttt
1320gtggtggtgc aagggcacag gcccatgtac actaccagcc atgaaaatag
ggatgctgct 1380ttaagaggaa agatgcttga gcacttggaa cctctgttgg
tgaataacaa tgtgacactt 1440gccctttggg gtcatgttca tagatacgag
agattttgtc cactgaataa cttcacttgt 1500ggtgttaatg cgggtcacaa
tgcaggggac aaaaaaggat atactgttca cattgtgatc 1560ggcatggcag
ggcaagactg gcaacctgtc tgggaaccaa ggccagacca tcccgatgat
1620ccaatctttc cacagccaaa atggtctctg taccgcggag gcgagtttgg
gtacacaaga 1680ctcgtcgcta caaagcagaa gctcgtgctt tcttatgtgg
gaaaccatga cggtgaggtg 1740catgatcagt tggaaattct ggcatctggg
gaagttgtca gtggtgacgg aggctgtagt 1800attgctgatg ctaattctaa
agctggaaat gtgattgtgg aatccacatt gtcttggtat 1860gtcaagggag
gaagtgtgct gctgcttggt gcattcatgg gttacgtttt tggttacgtt
1920acaagtgcaa ggaagaagtc tgaggtgcca gagagcaatt ggactccggt
gaagactgag 1980gaaacttga 19896662PRTGlycine max 6Met Ile Pro Asp
Leu Pro Leu Pro Phe Leu Phe Ser Leu Phe Ile Ile1 5 10 15Phe Phe His
Leu Ala Glu Ser Lys Pro Ser Leu Thr Ala Thr Pro Thr 20 25 30Thr Leu
Pro Ala Ser Gly Ala Thr Val Asn Leu Arg Trp Ser Gly Ile 35 40 45Pro
Ser Pro Ser Asp Leu Asp Phe Leu Ala Ile Tyr Ser Pro Pro Thr 50 55
60Ser Pro His Asp Asn Phe Ile Gly Tyr Leu Phe Leu Ser Gln Ser Ala65
70 75 80Thr Trp Arg Thr Gly Ser Gly Asn Leu Ser Leu Pro Leu Val Asp
Leu 85 90 95Arg Ser Asn Tyr Ser Phe Arg Ile Phe Ser Trp Thr Arg Ala
Glu Ile 100 105 110Asn Pro Lys Arg Gln Asp His Asp His Asn Pro Leu
Pro Val Thr Arg 115 120 125His Leu Leu Ala Phe Ser Glu Glu Val Ser
Phe Ala Pro His Arg Gly 130 135 140Pro Gln Gln Ile His Leu Ala Phe
Val Gly Ala His Gly Lys Glu Glu145 150 155
160Asp Met Arg Val Met Tyr Ile Ala Arg Asp Pro Arg Glu Thr Tyr Val
165 170 175Arg Tyr Gly Glu Arg Glu Asp Lys Leu Asp Gly Ile Ala Val
Ala Arg 180 185 190Val Glu Arg Tyr Glu Arg Glu His Met Cys Asp Ala
Pro Ala Asn Thr 195 200 205Ser Val Gly Trp Arg Asp Pro Gly Phe Ile
Asn Asp Ala Val Leu Ile 210 215 220Gly Leu Lys Lys Gly Gln Arg Tyr
Tyr Tyr Lys Val Gly Asn Asp Asn225 230 235 240Gly Gly Trp Ser Ala
Thr Gln Ser Phe Val Ser Arg Asn Ser Asp Ser 245 250 255Asp Glu Thr
Ile Ala Phe Leu Phe Gly Asp Met Gly Thr Ala Val Pro 260 265 270Tyr
Asn Thr Phe Leu Arg Thr Gln Asp Glu Ser Ile Ser Thr Met Lys 275 280
285Trp Ile Leu Arg Asp Val Glu Ala Leu Gly Asp Lys Pro Ala Phe Val
290 295 300Ser His Ile Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp
Leu Trp305 310 315 320Asp His Phe Phe Ala Gln Ile Glu Pro Val Ala
Ser Gln Val Ala Tyr 325 330 335His Val Cys Ile Gly Asn His Glu Tyr
Asp Trp Pro Leu Gln Pro Trp 340 345 350Lys Pro Asp Trp Ala Ser Tyr
Gly Lys Asp Gly Gly Gly Glu Cys Gly 355 360 365Val Pro Tyr Ser Leu
Arg Phe Asn Met Pro Gly Asn Ser Ser Glu Leu 370 375 380Thr Gly Asn
Ala Ala Ala Pro Pro Thr Arg Asn Leu Tyr Tyr Ser Phe385 390 395
400Asp Met Gly Ala Val His Phe Val Tyr Ile Ser Thr Glu Thr Asn Phe
405 410 415Val Pro Gly Ser Lys Gln Tyr Asp Phe Leu Lys His Asp Leu
Glu Ser 420 425 430Val Asn Arg Ser Lys Thr Pro Phe Val Val Val Gln
Gly His Arg Pro 435 440 445Met Tyr Thr Thr Ser His Glu Asn Arg Asp
Ala Ala Leu Arg Gly Lys 450 455 460Met Leu Glu His Leu Glu Pro Leu
Leu Val Asn Asn Asn Val Thr Leu465 470 475 480Ala Leu Trp Gly His
Val His Arg Tyr Glu Arg Phe Cys Pro Leu Asn 485 490 495Asn Phe Thr
Cys Gly Val Asn Ala Gly His Asn Ala Gly Asp Lys Lys 500 505 510Gly
Tyr Thr Val His Ile Val Ile Gly Met Ala Gly Gln Asp Trp Gln 515 520
525Pro Val Trp Glu Pro Arg Pro Asp His Pro Asp Asp Pro Ile Phe Pro
530 535 540Gln Pro Lys Trp Ser Leu Tyr Arg Gly Gly Glu Phe Gly Tyr
Thr Arg545 550 555 560Leu Val Ala Thr Lys Gln Lys Leu Val Leu Ser
Tyr Val Gly Asn His 565 570 575Asp Gly Glu Val His Asp Gln Leu Glu
Ile Leu Ala Ser Gly Glu Val 580 585 590Val Ser Gly Asp Gly Gly Cys
Ser Ile Ala Asp Ala Asn Ser Lys Ala 595 600 605Gly Asn Val Ile Val
Glu Ser Thr Leu Ser Trp Tyr Val Lys Gly Gly 610 615 620Ser Val Leu
Leu Leu Gly Ala Phe Met Gly Tyr Val Phe Gly Tyr Val625 630 635
640Thr Ser Ala Arg Lys Lys Ser Glu Val Pro Glu Ser Asn Trp Thr Pro
645 650 655Val Lys Thr Glu Glu Thr 66071965DNAZea mays 7atgtaccccg
aaaaccccca cctccgcttc ctcctcttcc tcgccgtcgc ggcagttgcc 60gccggcgggg
ctgcggcgaa caccaccctc accgcgtccc tctccggcaa ccagatcaag
120atcatctggt ccggactccc ggccccggac ggcctcgact acgttgccat
ctactcgccg 180ccgtcctccc tcgaccgcga cttcctcggc tatctcttcc
tcaacggctc cgcctcctgg 240cgcggcggct ccggggagct ctccctcccg
ctcctcccga cgctccgcgc gccctaccag 300ttccgtctct ttcgctggcc
cgccaaggag tactcctacc accacgtcga ccacgaccag 360aacccgctcc
cccacggcaa gcaccgcgtc gccgtctccg ccgacgtctc cgtcggcgac
420cccgcccgcc ccgagcagct gcacctcgcg tttgcggatg aggtcgacga
gatgcgggtc 480ctgttcgtgt gcggcgaccg cggggagagg gtcgtcaggt
acgggctgca gaaggaggac 540gacaaggagt ggaaggaggt gggcacggat
gtgagcacgt acgagcagag gcacatgtgc 600gattggccgg ccaacagcag
cgtcgcctgg agggatccgg gattcgtctt cgacggcctc 660atgaagggat
tggagcccgg aaggaggtac ttttacaagg ttggtagtga cacaggagga
720tggagtgaga tatacagctt catttcacgt gacagtgaag ccagtgagac
caatgctttt 780ctatttggtg acatgggaac ttatgtgcct tataacacct
acattcgcac acaatctgag 840agcttgtcca ctgtaaagtg gatccttcgt
gatattgaag cccttggaga taaacccgcc 900tttatttcac acattgggga
catcagctat gctagaggtt attcttgggt ctggtatcat 960ttcttcagcc
agatcgagcc tattgctgcc aatactccat accatgtctg tataggaaat
1020catgagtatg attggccatc acaaccctgg aaaccatggt gggctacata
tggaacggac 1080ggtggaggcg aatgtggaat accttatagt gtcaggttca
gaatgccagg caattctatt 1140ctacctacag gtaatggtgg cccagacacc
aggaatcttt attactcctt cgactcaggc 1200gtggtgcatt tcgtctacat
gtcgaccgaa acaaattttg ttcagggaag tgagcagcac 1260aacttcttga
aagcggacct tgagaaggtg aaccgaagta gaacaccctt tgttgttttt
1320cagggccacc gccccatgta cacctcgagc gatgaaacca gggacgcggc
tttgaaacag 1380cagatgctcc agaatctgga accgctgctg gtgacataca
atgtgaccct cgcgctatgg 1440ggacatgtcc acaggtacga gaggttctgc
ccgatgcaga attcccaatg tgtcaacact 1500tcatcaagct tccagtactc
tggcgctcct gtgcatcttg tgatcgggat gggcggccaa 1560gactggcaac
ctgtatggca accgaggcct gatcacccag acgtccctat ctttcctcag
1620cctgagcgtt ccatgtaccg cggtggcgag tttggatacg ccagacttgt
ggcaacaagg 1680gagaagctaa cattgactta tgtggggaac catgatgggc
aggtccatga tatggtggag 1740atattttctg gcctggtatc ccccagtaac
agtagtgttg ctgaggcggt ggatggaacc 1800aaacttggca caggagtcag
caccgtgcgg aaaatttctc cgctgtactt ggaaattgga 1860ggcagtgtga
tgtttgcgct gctcctggga ttttcctttg ggatacttgt caggagaaag
1920aaagaagctg cacagtggac tcaagtaaag aatgaggaat cgtag
19658654PRTZea mays 8Met Tyr Pro Glu Asn Pro His Leu Arg Phe Leu
Leu Phe Leu Ala Val1 5 10 15Ala Ala Val Ala Ala Gly Gly Ala Ala Ala
Asn Thr Thr Leu Thr Ala 20 25 30Ser Leu Ser Gly Asn Gln Ile Lys Ile
Ile Trp Ser Gly Leu Pro Ala 35 40 45Pro Asp Gly Leu Asp Tyr Val Ala
Ile Tyr Ser Pro Pro Ser Ser Leu 50 55 60Asp Arg Asp Phe Leu Gly Tyr
Leu Phe Leu Asn Gly Ser Ala Ser Trp65 70 75 80Arg Gly Gly Ser Gly
Glu Leu Ser Leu Pro Leu Leu Pro Thr Leu Arg 85 90 95Ala Pro Tyr Gln
Phe Arg Leu Phe Arg Trp Pro Ala Lys Glu Tyr Ser 100 105 110Tyr His
His Val Asp His Asp Gln Asn Pro Leu Pro His Gly Lys His 115 120
125Arg Val Ala Val Ser Ala Asp Val Ser Val Gly Asp Pro Ala Arg Pro
130 135 140Glu Gln Leu His Leu Ala Phe Ala Asp Glu Val Asp Glu Met
Arg Val145 150 155 160Leu Phe Val Cys Gly Asp Arg Gly Glu Arg Val
Val Arg Tyr Gly Leu 165 170 175Gln Lys Glu Asp Asp Lys Glu Trp Lys
Glu Val Gly Thr Asp Val Ser 180 185 190Thr Tyr Glu Gln Arg His Met
Cys Asp Trp Pro Ala Asn Ser Ser Val 195 200 205Ala Trp Arg Asp Pro
Gly Phe Val Phe Asp Gly Leu Met Lys Gly Leu 210 215 220Glu Pro Gly
Arg Arg Tyr Phe Tyr Lys Val Gly Ser Asp Thr Gly Gly225 230 235
240Trp Ser Glu Ile Tyr Ser Phe Ile Ser Arg Asp Ser Glu Ala Ser Glu
245 250 255Thr Asn Ala Phe Leu Phe Gly Asp Met Gly Thr Tyr Val Pro
Tyr Asn 260 265 270Thr Tyr Ile Arg Thr Gln Ser Glu Ser Leu Ser Thr
Val Lys Trp Ile 275 280 285Leu Arg Asp Ile Glu Ala Leu Gly Asp Lys
Pro Ala Phe Ile Ser His 290 295 300Ile Gly Asp Ile Ser Tyr Ala Arg
Gly Tyr Ser Trp Val Trp Tyr His305 310 315 320Phe Phe Ser Gln Ile
Glu Pro Ile Ala Ala Asn Thr Pro Tyr His Val 325 330 335Cys Ile Gly
Asn His Glu Tyr Asp Trp Pro Ser Gln Pro Trp Lys Pro 340 345 350Trp
Trp Ala Thr Tyr Gly Thr Asp Gly Gly Gly Glu Cys Gly Ile Pro 355 360
365Tyr Ser Val Arg Phe Arg Met Pro Gly Asn Ser Ile Leu Pro Thr Gly
370 375 380Asn Gly Gly Pro Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp
Ser Gly385 390 395 400Val Val His Phe Val Tyr Met Ser Thr Glu Thr
Asn Phe Val Gln Gly 405 410 415Ser Glu Gln His Asn Phe Leu Lys Ala
Asp Leu Glu Lys Val Asn Arg 420 425 430Ser Arg Thr Pro Phe Val Val
Phe Gln Gly His Arg Pro Met Tyr Thr 435 440 445Ser Ser Asp Glu Thr
Arg Asp Ala Ala Leu Lys Gln Gln Met Leu Gln 450 455 460Asn Leu Glu
Pro Leu Leu Val Thr Tyr Asn Val Thr Leu Ala Leu Trp465 470 475
480Gly His Val His Arg Tyr Glu Arg Phe Cys Pro Met Gln Asn Ser Gln
485 490 495Cys Val Asn Thr Ser Ser Ser Phe Gln Tyr Ser Gly Ala Pro
Val His 500 505 510Leu Val Ile Gly Met Gly Gly Gln Asp Trp Gln Pro
Val Trp Gln Pro 515 520 525Arg Pro Asp His Pro Asp Val Pro Ile Phe
Pro Gln Pro Glu Arg Ser 530 535 540Met Tyr Arg Gly Gly Glu Phe Gly
Tyr Ala Arg Leu Val Ala Thr Arg545 550 555 560Glu Lys Leu Thr Leu
Thr Tyr Val Gly Asn His Asp Gly Gln Val His 565 570 575Asp Met Val
Glu Ile Phe Ser Gly Leu Val Ser Pro Ser Asn Ser Ser 580 585 590Val
Ala Glu Ala Val Asp Gly Thr Lys Leu Gly Thr Gly Val Ser Thr 595 600
605Val Arg Lys Ile Ser Pro Leu Tyr Leu Glu Ile Gly Gly Ser Val Met
610 615 620Phe Ala Leu Leu Leu Gly Phe Ser Phe Gly Ile Leu Val Arg
Arg Lys625 630 635 640Lys Glu Ala Ala Gln Trp Thr Gln Val Lys Asn
Glu Glu Ser 645 650922DNAArtificial SequenceSynthetic primer
9tggttcacgt agtgggccat cg 221021DNAArtificial SequenceSynthetic
primer 10ttgaagttta acatgcctgg g 211121DNAArtificial
sequenceSynthetic primer 11tccaatgctc gattgattag c
211231DNAArtificial SequenceSynthetic primer 12ggccgtcgac
atgatcgtta atttctcttt c 311331DNAArtificial SequenceSynthetic
primer 13ccggactagt tcatgtctcc tcgttcttga c 311427DNAArtificial
SequenceSynthetic primer 14gtttcacatc aacattgtgg tcattgg
271524DNAArtificial SequenceSynthetic primer 15gagtacttgg
gggtagtggc atcc 241630DNAArtificial SequenceSynthetic primer
16ggttgagctc gattctaaag cgaccatttc 301729DNAArtificial
SequenceSynthetic primer 17ttttggtacc tcaggatccg aaagtcagc
29181959DNABrassica rapa 18atgatcgtcg agttctctac cttcatcctc
ttcctctccg tcttcgtctc ctcagctaac 60gccaaagcaa ccttatccat ctcccccaaa
actctaagcc gatccggcga ttccatcctc 120atcaaatggt ccaacctcga
ctctccctcc gatctcgact ggctaggcat ctactccccc 180ccagcctctc
cccacgacca cttcatcggc tacaagttcc tcaacgcctc ccccacgtgg
240caatccggct ccggcgcgat ctccctcccc ctcaccaacc tccgatcgaa
ctacacgttc 300cgtatcttcc gatggacgca gtccgagatc aatccgaagc
acaaggacca cgaccagaat 360cccttaccgg gaacgaagca ccttctggcg
gaatcggagc aggtggggtt cggatccgcc 420ggcgtgggga ggccggagca
gatccatttg gcgttcgagg ataaggttaa caggatgcag 480gtcacgttcg
tagctgggga tggggaagaa aggttcgtga ggtacggaga ggcggaggat
540gcgttggcga actccgcggc ggcgcgcggg attaggtacg agagggagca
tatgtgtaat 600gctccggcta attccaccgt gggatggaga gatcccgggt
ggatttttca taccgttatg 660aagaatttga acggtggcgt taggtattat
tatcaggttg ggagtgattc aaagggatgg 720agtgagatcc acagcttcat
cgctcgagat atctactcag aagaaaccat agctttcatg 780ttcggagaca
tgggttgcgc tacaccttac aataccttta tccggacgca ggacgagagt
840atgtccacag tgaagtggat actccgcgac atcgaagctc ttggtgacaa
gccggctctc 900gtttcgcaca ttggagatat aagctacgct cgtggttact
cctgggtgtg ggatgagttc 960tttgctcaga tcgagcctat tgcctcgaga
gttccttacc acgtctgcat tggtaaccac 1020gagtatgact tccctactca
gccgtggaaa cctgattggg gaacttacgg taatgacggt 1080gggggagagt
gcggtgtgcc gtatagtctc aagttcaaca tgcctggaaa ctcgtcggaa
1140ccaacgggaa cgaaagctcc tcctacaagg aatttgtatt actcttacga
catggggtcg 1200gttcatttcc tttacatctc caccgagacg aactttctca
aaggagggag gcaatacgag 1260tttataaagc gagatcttga gtctgtgaac
agggagaaaa caccgtttgt tgtcgtgcaa 1320ggacacagac cgatgtacac
cacgagcaac gaggtgagag acgcgatgat taggcaaaag 1380atggtggagc
atttggagcc gctgtttgtg gagaacaacg tgacgcttgc tctgtgggga
1440catgttcata gatacgagag gttttgtccg ataagcaaca acacgtgtgg
gaaacagtgg 1500agaggaagcc cggttcatct tgtgatcggt atgggtggtc
aagactggca accgatttgg 1560cagccgagac cgaaccatcc gggtcttcct
atattccctc agcctgaaca gtcgatgtac 1620aggacgggtg agtttgggta
cactcgtttg gttgcgaaca aagagaagct cactgtttcg 1680tttgtgggta
accatgatgg agaagttcat gatagtgttg agatgtttgc gtctggggaa
1740gtaatcagtg ggaggaaaga ggaaactatt aagaccgttc ctgtatctgc
aacacttgtg 1800gggaaacctg agtctgatgt cttatggtat gttaaaggag
caggcttgtt ggttattggt 1860gtgcttttag ggttcattat agggtttttt
acaaggggga agaaaggatc ttcttcatct 1920gataaccgtt ggatcccagt
caagaacgag gagacatga 195919652PRTBrassica rapa 19Met Ile Val Glu
Phe Ser Thr Phe Ile Leu Phe Leu Ser Val Phe Val1 5 10 15Ser Ser Ala
Asn Ala Lys Ala Thr Leu Ser Ile Ser Pro Lys Thr Leu 20 25 30Ser Arg
Ser Gly Asp Ser Ile Leu Ile Lys Trp Ser Asn Leu Asp Ser 35 40 45Pro
Ser Asp Leu Asp Trp Leu Gly Ile Tyr Ser Pro Pro Ala Ser Pro 50 55
60His Asp His Phe Ile Gly Tyr Lys Phe Leu Asn Ala Ser Pro Thr Trp65
70 75 80Gln Ser Gly Ser Gly Ala Ile Ser Leu Pro Leu Thr Asn Leu Arg
Ser 85 90 95Asn Tyr Thr Phe Arg Ile Phe Arg Trp Thr Gln Ser Glu Ile
Asn Pro 100 105 110Lys His Lys Asp His Asp Gln Asn Pro Leu Pro Gly
Thr Lys His Leu 115 120 125Leu Ala Glu Ser Glu Gln Val Gly Phe Gly
Ser Ala Gly Val Gly Arg 130 135 140Pro Glu Gln Ile His Leu Ala Phe
Glu Asp Lys Val Asn Arg Met Gln145 150 155 160Val Thr Phe Val Ala
Gly Asp Gly Glu Glu Arg Phe Val Arg Tyr Gly 165 170 175Glu Ala Glu
Asp Ala Leu Ala Asn Ser Ala Ala Ala Arg Gly Ile Arg 180 185 190Tyr
Glu Arg Glu His Met Cys Asn Ala Pro Ala Asn Ser Thr Val Gly 195 200
205Trp Arg Asp Pro Gly Trp Ile Phe His Thr Val Met Lys Asn Leu Asn
210 215 220Gly Gly Val Arg Tyr Tyr Tyr Gln Val Gly Ser Asp Ser Lys
Gly Trp225 230 235 240Ser Glu Ile His Ser Phe Ile Ala Arg Asp Ile
Tyr Ser Glu Glu Thr 245 250 255Ile Ala Phe Met Phe Gly Asp Met Gly
Cys Ala Thr Pro Tyr Asn Thr 260 265 270Phe Ile Arg Thr Gln Asp Glu
Ser Met Ser Thr Val Lys Trp Ile Leu 275 280 285Arg Asp Ile Glu Ala
Leu Gly Asp Lys Pro Ala Leu Val Ser His Ile 290 295 300Gly Asp Ile
Ser Tyr Ala Arg Gly Tyr Ser Trp Val Trp Asp Glu Phe305 310 315
320Phe Ala Gln Ile Glu Pro Ile Ala Ser Arg Val Pro Tyr His Val Cys
325 330 335Ile Gly Asn His Glu Tyr Asp Phe Pro Thr Gln Pro Trp Lys
Pro Asp 340 345 350Trp Gly Thr Tyr Gly Asn Asp Gly Gly Gly Glu Cys
Gly Val Pro Tyr 355 360 365Ser Leu Lys Phe Asn Met Pro Gly Asn Ser
Ser Glu Pro Thr Gly Thr 370 375 380Lys Ala Pro Pro Thr Arg Asn Leu
Tyr Tyr Ser Tyr Asp Met Gly Ser385 390 395 400Val His Phe Leu Tyr
Ile Ser Thr Glu Thr Asn Phe Leu Lys Gly Gly 405 410 415Arg Gln Tyr
Glu Phe Ile Lys Arg Asp Leu Glu Ser Val Asn Arg Glu 420 425 430Lys
Thr Pro Phe Val Val Val Gln Gly His Arg Pro Met Tyr Thr Thr 435 440
445Ser Asn Glu Val Arg Asp Ala Met Ile Arg Gln Lys Met Val Glu His
450 455 460Leu Glu Pro Leu Phe Val Glu Asn Asn Val Thr Leu Ala Leu
Trp Gly465 470 475 480His Val His Arg Tyr Glu Arg Phe Cys Pro Ile
Ser Asn Asn Thr Cys 485 490 495Gly Lys Gln Trp Arg Gly Ser Pro Val
His Leu Val Ile Gly Met Gly 500
505 510Gly Gln Asp Trp Gln Pro Ile Trp Gln Pro Arg Pro Asn His Pro
Gly 515 520 525Leu Pro Ile Phe Pro Gln Pro Glu Gln Ser Met Tyr Arg
Thr Gly Glu 530 535 540Phe Gly Tyr Thr Arg Leu Val Ala Asn Lys Glu
Lys Leu Thr Val Ser545 550 555 560Phe Val Gly Asn His Asp Gly Glu
Val His Asp Ser Val Glu Met Phe 565 570 575Ala Ser Gly Glu Val Ile
Ser Gly Arg Lys Glu Glu Thr Ile Lys Thr 580 585 590Val Pro Val Ser
Ala Thr Leu Val Gly Lys Pro Glu Ser Asp Val Leu 595 600 605Trp Tyr
Val Lys Gly Ala Gly Leu Leu Val Ile Gly Val Leu Leu Gly 610 615
620Phe Ile Ile Gly Phe Phe Thr Arg Gly Lys Lys Gly Ser Ser Ser
Ser625 630 635 640Asp Asn Arg Trp Ile Pro Val Lys Asn Glu Glu Thr
645 650201965DNAHordeum vulgare 20atgcacccca aaaccccgcc tctcctcctc
gtcctcctct tcttcgccgc cggcgaggcc 60gcggggacca ccctcacggc caccccggcg
aagctcaccc aatccgacca agaaatcacg 120atccggtggt ccgacctccc
gtccccggat ggcctcgacc acgtggcgat ctactccccg 180ccgtcctcca
gcgaccgcga cttcctaggc tacatcttcc tcaatggctc cgcctcctgg
240cgcagcggcc gcggagagct caccctccca cggctcccca acctgcgggc
gccctaccag 300ttccgcctct tccgctggcc cgccagagag tactcctacc
accacgtcga ccacgacggc 360aacccgctcc cccacggcca ccaccgcgtc
gccctatccg gcgaggtcgc tttcgcgggc 420tcggccgcgc ggcccgagca
ggtgcacctc gcgttcgccg atagggccga cgagatgcgg 480gtgatgttcg
tgtgcgcgga cgccggcaag agggccgtga ggtacgggct tgagaaggag
540gaggagaagg gctggacgga agtgggcacg gaggtgagga cgtacgagca
gaagcacatg 600tgcgacacgc cggcgaacga caccgtaggg tggagggatc
cgggcttcgt cttcgatggc 660ctcatgaatg ggttggagcc cggaaggagg
tacttttaca aggtcggtag tgacctggga 720ggatggagcg agacatacag
ctttatttca cgtgacagtg aggccaatga gaccattgct 780tttctcttcg
gtgatatggg cacttatgta ccatacaaca cctacatccg cacacaagat
840gagagcttgt caacggtgaa gtggatcctc cgtgatattg aagcccttgg
agataagcct 900gcatttattt cgcacattgg ggacatcagt tatgccagag
gctatgcttg ggtgtgggat 960catttcttca gccagattga gcctattgca
gccaatactc cataccatgt ctgcatagga 1020aatcatgagt atgattggcc
ttcacaacct tggaaacctt catggtctac atatgggaag 1080gatggtggag
gtgaatgtgg aataccatac agtgtcaagt tcagaatgcc tggggattct
1140gttctaccta ctggcaatgg agctccggac acacggaatc tctactactc
ttttgattca 1200ggcgtcgtgc attttgtgta catgtcgact gaaactaatt
tcgttcaggg cagcgaccaa 1260cacaatttcc taaaagctga tctggagaag
gtgaaccgaa gcagaacccc atttgttgtg 1320tttcagggcc accggcccat
gtatacctcg agcaacgaag ccagggattc tgccatgaga 1380cagcagatgg
tccagcatct tgaaccgctc ttggtgatat acaatgtgac gcttgccctg
1440tggggacatg tccataggta tgagaggttc tgccccatga agaattcaca
gtgtctgaac 1500acatcatcaa gcttcgtata ccctggtgcc cctgttcatg
ttgtgatcgg gatggctgga 1560caagattggc aaccgatctg gcaaccaagg
cgtgatcatc caaatgttcc catctttcca 1620cagcctggga tctccatgta
ccgtggtggt gagttcgggt acacaaagct cgcagctaac 1680agggagaagc
taacgctgat gtacgttggg aaccacgatg gacaagtcca tgacatggtg
1740gaaatattct ctggacaaac atctactgaa gctagcgcta ctgaggcggt
caatcaaaca 1800aagctcagct cgggagccag cgccaagctg aagatttccc
caatatactt ggaaattgga 1860ggtagtgtga tgtttgccct aatgcttggt
tttgccttgg gatttctcct caggaagaag 1920agagaagctg cacaatggac
tccggtcaag aacgaggaat cctaa 196521654PRTHordeum vulgare 21Met His
Pro Lys Thr Pro Pro Leu Leu Leu Val Leu Leu Phe Phe Ala1 5 10 15Ala
Gly Glu Ala Ala Gly Thr Thr Leu Thr Ala Thr Pro Ala Lys Leu 20 25
30Thr Gln Ser Asp Gln Glu Ile Thr Ile Arg Trp Ser Asp Leu Pro Ser
35 40 45Pro Asp Gly Leu Asp His Val Ala Ile Tyr Ser Pro Pro Ser Ser
Ser 50 55 60Asp Arg Asp Phe Leu Gly Tyr Ile Phe Leu Asn Gly Ser Ala
Ser Trp65 70 75 80Arg Ser Gly Arg Gly Glu Leu Thr Leu Pro Arg Leu
Pro Asn Leu Arg 85 90 95Ala Pro Tyr Gln Phe Arg Leu Phe Arg Trp Pro
Ala Arg Glu Tyr Ser 100 105 110Tyr His His Val Asp His Asp Gly Asn
Pro Leu Pro His Gly His His 115 120 125Arg Val Ala Leu Ser Gly Glu
Val Ala Phe Ala Gly Ser Ala Ala Arg 130 135 140Pro Glu Gln Val His
Leu Ala Phe Ala Asp Arg Ala Asp Glu Met Arg145 150 155 160Val Met
Phe Val Cys Ala Asp Ala Gly Lys Arg Ala Val Arg Tyr Gly 165 170
175Leu Glu Lys Glu Glu Glu Lys Gly Trp Thr Glu Val Gly Thr Glu Val
180 185 190Arg Thr Tyr Glu Gln Lys His Met Cys Asp Thr Pro Ala Asn
Asp Thr 195 200 205Val Gly Trp Arg Asp Pro Gly Phe Val Phe Asp Gly
Leu Met Asn Gly 210 215 220Leu Glu Pro Gly Arg Arg Tyr Phe Tyr Lys
Val Gly Ser Asp Leu Gly225 230 235 240Gly Trp Ser Glu Thr Tyr Ser
Phe Ile Ser Arg Asp Ser Glu Ala Asn 245 250 255Glu Thr Ile Ala Phe
Leu Phe Gly Asp Met Gly Thr Tyr Val Pro Tyr 260 265 270Asn Thr Tyr
Ile Arg Thr Gln Asp Glu Ser Leu Ser Thr Val Lys Trp 275 280 285Ile
Leu Arg Asp Ile Glu Ala Leu Gly Asp Lys Pro Ala Phe Ile Ser 290 295
300His Ile Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ala Trp Val Trp
Asp305 310 315 320His Phe Phe Ser Gln Ile Glu Pro Ile Ala Ala Asn
Thr Pro Tyr His 325 330 335Val Cys Ile Gly Asn His Glu Tyr Asp Trp
Pro Ser Gln Pro Trp Lys 340 345 350Pro Ser Trp Ser Thr Tyr Gly Lys
Asp Gly Gly Gly Glu Cys Gly Ile 355 360 365Pro Tyr Ser Val Lys Phe
Arg Met Pro Gly Asp Ser Val Leu Pro Thr 370 375 380Gly Asn Gly Ala
Pro Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser385 390 395 400Gly
Val Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Val Gln 405 410
415Gly Ser Asp Gln His Asn Phe Leu Lys Ala Asp Leu Glu Lys Val Asn
420 425 430Arg Ser Arg Thr Pro Phe Val Val Phe Gln Gly His Arg Pro
Met Tyr 435 440 445Thr Ser Ser Asn Glu Ala Arg Asp Ser Ala Met Arg
Gln Gln Met Val 450 455 460Gln His Leu Glu Pro Leu Leu Val Ile Tyr
Asn Val Thr Leu Ala Leu465 470 475 480Trp Gly His Val His Arg Tyr
Glu Arg Phe Cys Pro Met Lys Asn Ser 485 490 495Gln Cys Leu Asn Thr
Ser Ser Ser Phe Val Tyr Pro Gly Ala Pro Val 500 505 510His Val Val
Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Ile Trp Gln 515 520 525Pro
Arg Arg Asp His Pro Asn Val Pro Ile Phe Pro Gln Pro Gly Ile 530 535
540Ser Met Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Lys Leu Ala Ala
Asn545 550 555 560Arg Glu Lys Leu Thr Leu Met Tyr Val Gly Asn His
Asp Gly Gln Val 565 570 575His Asp Met Val Glu Ile Phe Ser Gly Gln
Thr Ser Thr Glu Ala Ser 580 585 590Ala Thr Glu Ala Val Asn Gln Thr
Lys Leu Ser Ser Gly Ala Ser Ala 595 600 605Lys Leu Lys Ile Ser Pro
Ile Tyr Leu Glu Ile Gly Gly Ser Val Met 610 615 620Phe Ala Leu Met
Leu Gly Phe Ala Leu Gly Phe Leu Leu Arg Lys Lys625 630 635 640Arg
Glu Ala Ala Gln Trp Thr Pro Val Lys Asn Glu Glu Ser 645
650221980DNAMedicago truncatula 22atgatccctc acacaatact cttcacctta
ctcttatcct caaccttcac ctctaccctc 60gcccaatcaa aacccaccct aacagtaacc
ccaaccaccc tcacaaaatc tggcgacacc 120gtcaccctcc ggtggtccgg
tatccaatcc ccctccgatc tcgacttcct cgcaatctac 180tccccaccta
cctccgccca caaaaactac atcggctacc tcttcctctc caaatccccc
240acctggcaat ccggctccgg caacctctct cttcctctca tcaacctccg
ttccaactac 300tccttccgta tcttccactg gtcccaatcc gaaatcaacc
ctaaacgtca agatcacgat 360cataatccct taccacaaac gcatcacctt
cttgctttct ctgatgaagt ttcttttcca 420tcccttcgac cggagcagat
tcatcttgct tttgcagatg aagaagatgc tatgagggtg 480atgtatgtga
cgggggttcc gaagaagacg tatgtgagat atggagaaag agaggatatg
540atggatagat tggttgttgc gaatgtgaag agatatgaga gagagcatat
gtgtgatgct 600cctgctaatc agagtgttgg ttggagggat cctggtttta
ttcatgatgc tttgattact 660ggtttggaca aaggaagaag atattactac
caggttggaa atgataatgg aggttggagt 720gcaacccata gctttgtgtc
gaggaatagt gattcaaatg aaacaatagc tttccttttc 780ggtgacatgg
gaacatttac agcatacaat acgtatttgc gtacacaaga tgagagcata
840tcaaccatga agtggatcct gcgtgatgtt gaagctctag gaaacaagcc
cgcctttata 900tcacacattg gagacacaag ttatgcaaga ggttatgcgt
ggttgtggga tcattttttc 960gcacagattg aacctgttgc aaccaaagtg
gcataccatg tatgcattgg caatcacgag 1020tataactggc ctttacagcc
gtggaaacct gattgggcta attatagaac agatggaggt 1080ggtgaatgtg
gtgtacccta cagtttaagg ttcaacatgc caggaaactc ttcagaaccc
1140actggaactg tagctccagc cactaggaat ctttattact catttgatat
gggagcagta 1200cattttgttt atatttccac agagaccaat ttccttcctg
ggagcaatca gtataacttc 1260ttaaagcgtg atttggaatc agttgacagg
aacaagactc cttttgtagt agtccaaggg 1320caccgaccca tgtacacaac
aagcaatgaa tttagggatg ctgcgttaag aggaaagatg 1380gttgagcacc
tggaacctct attggtgaat aaccatgtaa cccttgccct ttggggtcat
1440gttcataggt acgagagatt ttgtccacta aacaacttta cttgtggaaa
tggtgtgggt 1500cggagagcag gggaaaaagg tcataccatt catcttgtga
tcggcatggc agggcaagac 1560tggcaaccca tgtggcgacc aagaccggat
catcccgatg tcccaatcta tccacaacca 1620aaacgatctt tgtaccgcgg
gggtgagttc ggatacatta gattgatggc tacaaagcag 1680aatctcgtga
tttcttatgt tggtaatcat gatggcgagg tgcatgacac attggagatt
1740ctggaatctg gagaagttgt tagtggtggt ggtggtaacg ataatgttaa
tggcggtatt 1800ggtagtgcta aacctgaagg tcagattaaa gaatccacgt
tgtcgtggta tgtccaggga 1860ggaagtgtac tagtgcttgg ggcctttatg
ggctacattc ttggtttcgt ttcacatgct 1920aggaagaagc agcccgagtc
caggagtggt tttagccccg tgaagactga ggaaacatga 198023659PRTMedicago
truncatula 23Met Ile Pro His Thr Ile Leu Phe Thr Leu Leu Leu Ser
Ser Thr Phe1 5 10 15Thr Ser Thr Leu Ala Gln Ser Lys Pro Thr Leu Thr
Val Thr Pro Thr 20 25 30Thr Leu Thr Lys Ser Gly Asp Thr Val Thr Leu
Arg Trp Ser Gly Ile 35 40 45Gln Ser Pro Ser Asp Leu Asp Phe Leu Ala
Ile Tyr Ser Pro Pro Thr 50 55 60Ser Ala His Lys Asn Tyr Ile Gly Tyr
Leu Phe Leu Ser Lys Ser Pro65 70 75 80Thr Trp Gln Ser Gly Ser Gly
Asn Leu Ser Leu Pro Leu Ile Asn Leu 85 90 95Arg Ser Asn Tyr Ser Phe
Arg Ile Phe His Trp Ser Gln Ser Glu Ile 100 105 110Asn Pro Lys Arg
Gln Asp His Asp His Asn Pro Leu Pro Gln Thr His 115 120 125His Leu
Leu Ala Phe Ser Asp Glu Val Ser Phe Pro Ser Leu Arg Pro 130 135
140Glu Gln Ile His Leu Ala Phe Ala Asp Glu Glu Asp Ala Met Arg
Val145 150 155 160Met Tyr Val Thr Gly Val Pro Lys Lys Thr Tyr Val
Arg Tyr Gly Glu 165 170 175Arg Glu Asp Met Met Asp Arg Leu Val Val
Ala Asn Val Lys Arg Tyr 180 185 190Glu Arg Glu His Met Cys Asp Ala
Pro Ala Asn Gln Ser Val Gly Trp 195 200 205Arg Asp Pro Gly Phe Ile
His Asp Ala Leu Ile Thr Gly Leu Asp Lys 210 215 220Gly Arg Arg Tyr
Tyr Tyr Gln Val Gly Asn Asp Asn Gly Gly Trp Ser225 230 235 240Ala
Thr His Ser Phe Val Ser Arg Asn Ser Asp Ser Asn Glu Thr Ile 245 250
255Ala Phe Leu Phe Gly Asp Met Gly Thr Phe Thr Ala Tyr Asn Thr Tyr
260 265 270Leu Arg Thr Gln Asp Glu Ser Ile Ser Thr Met Lys Trp Ile
Leu Arg 275 280 285Asp Val Glu Ala Leu Gly Asn Lys Pro Ala Phe Ile
Ser His Ile Gly 290 295 300Asp Thr Ser Tyr Ala Arg Gly Tyr Ala Trp
Leu Trp Asp His Phe Phe305 310 315 320Ala Gln Ile Glu Pro Val Ala
Thr Lys Val Ala Tyr His Val Cys Ile 325 330 335Gly Asn His Glu Tyr
Asn Trp Pro Leu Gln Pro Trp Lys Pro Asp Trp 340 345 350Ala Asn Tyr
Arg Thr Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr Ser 355 360 365Leu
Arg Phe Asn Met Pro Gly Asn Ser Ser Glu Pro Thr Gly Thr Val 370 375
380Ala Pro Ala Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Met Gly Ala
Val385 390 395 400His Phe Val Tyr Ile Ser Thr Glu Thr Asn Phe Leu
Pro Gly Ser Asn 405 410 415Gln Tyr Asn Phe Leu Lys Arg Asp Leu Glu
Ser Val Asp Arg Asn Lys 420 425 430Thr Pro Phe Val Val Val Gln Gly
His Arg Pro Met Tyr Thr Thr Ser 435 440 445Asn Glu Phe Arg Asp Ala
Ala Leu Arg Gly Lys Met Val Glu His Leu 450 455 460Glu Pro Leu Leu
Val Asn Asn His Val Thr Leu Ala Leu Trp Gly His465 470 475 480Val
His Arg Tyr Glu Arg Phe Cys Pro Leu Asn Asn Phe Thr Cys Gly 485 490
495Asn Gly Val Gly Arg Arg Ala Gly Glu Lys Gly His Thr Ile His Leu
500 505 510Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Met Trp Arg
Pro Arg 515 520 525Pro Asp His Pro Asp Val Pro Ile Tyr Pro Gln Pro
Lys Arg Ser Leu 530 535 540Tyr Arg Gly Gly Glu Phe Gly Tyr Ile Arg
Leu Met Ala Thr Lys Gln545 550 555 560Asn Leu Val Ile Ser Tyr Val
Gly Asn His Asp Gly Glu Val His Asp 565 570 575Thr Leu Glu Ile Leu
Glu Ser Gly Glu Val Val Ser Gly Gly Gly Gly 580 585 590Asn Asp Asn
Val Asn Gly Gly Ile Gly Ser Ala Lys Pro Glu Gly Gln 595 600 605Ile
Lys Glu Ser Thr Leu Ser Trp Tyr Val Gln Gly Gly Ser Val Leu 610 615
620Val Leu Gly Ala Phe Met Gly Tyr Ile Leu Gly Phe Val Ser His
Ala625 630 635 640Arg Lys Lys Gln Pro Glu Ser Arg Ser Gly Phe Ser
Pro Val Lys Thr 645 650 655Glu Glu Thr 242007DNAPhyscomitrella
patens 24atgggatcgc aagtattcca ttttcttctg gtgttctttg ggtactttct
gcatggagct 60tcatcagaat ctgtgatttt ggacgcgaga cctacaatat tacaacattc
aggagaaaat 120atcactcttg cttggaaggg tgtgaattta ccgacgaaat
acgattggct gggtatatat 180acgcctccta cttctcctga cgaccagcat
atcgggtaca tacttctctc ttcctgttca 240acatggacaa caggcgcctg
ctccttgcag atccccttgg tcaacatgcg tgctccttac 300agtttccgaa
ttttcagagg cgtgttcgta aatgtatctg caagtacaaa tgtgactgga
360tcaaacaatg gggctacaac gatatcattg gatcgggagg gtaatcctct
accagatgtc 420acgaaacggt tagctgcaag cccagttgtt caattctcca
attacaacga gccaacacaa 480attcatctag ctctttcctc ggacgagact
gctgttaggg ttatgtttgt cactagggat 540cctctgagaa gccaagtaag
attcggggaa gatggagatg aactgggcaa cacagttgat 600gctacatcag
tcacatactc tcaaattgat atgtgcgatg aacctgcaag ttcttatggg
660tggagatctc cgggatacat acataatgtt gtgatggggg ggctgaatcc
tgggagtcgc 720tatttctatc gggtaggaag caatgtagga ggatggagct
cgacctatag cttcatcgct 780ccacatcctc gtgctgatga aacaaatgct
ctcatattcg gtgacatggg tacttcgatt 840ccttattcaa cgtatcaata
cacgcagagc gagagcaaga ataccgtgaa gtggctcaca 900cgggacctag
aacaaatagg tgacaaacct agcttcgtag cgcacattgg tgacataagc
960tatgctcgtg gtttatcttg gctctgggac aacttcttca cccaaatcga
gcccgtagct 1020gcaagatcac catatcatgt ttgcatggga aaccacgaat
atgattggcc tgggcaacct 1080ttcaagccag actggtcacc ataccaaaca
gatggaggcg gagaatgtgg cgtgccatat 1140agcttacgct tcatcatgcc
gggaaactcc tccttaccca ctggaactac ctccccagcc 1200accaaaaacc
tctattattc cattgatgtt ggggttgtgc atttcctctt ctattctacc
1260gaaaccgatt tccaggtagg ctccccccag tacactttta tagccaacga
cttgagaaca 1320gttgacagga acaagacgcc ctttgtggta tttttgggcc
atcggccgct ctatacaacc 1380gattaccgag ccttgttaga cacgatgaca
cagaaattag ttcaaacttt tgagcctttg 1440ttgatagata ccaatgtcac
tgtagccttt tgtggccatg tccataagta cgagcgaatg 1500tgccccttga
aaaattacac ctgtattgaa ccatctaagg caaacggtga gcttccaatt
1560catatggtgg tgggaatggg aggtgctgat caccaaccca ttgatgaccc
tctccccagt 1620caaagtcagc ctatctttcc tcagcccagc tggtcagtat
ttcgaacatt tgaatgggga 1680tatatcaggc tacatgcaac gagacatctc
atgacgattt catatgttgg taaccacgat 1740gggaaggtgc atgatgttgt
cgaaattcca gttctggatg atatcaagtc tggagcatat 1800gttgagtcga
gggagtcttt ttttgacact gccagcggag tgcaaatacc ttgtggcagg
1860tctgagaata ttgtagcatt cctgtttgtt ttagcgttgg gttgtggatg
cggggcggct 1920gctactcttt ttttcatgcg gaggcagcag aggaagcaga
tttggcagcc tgtcaaccgt 1980gaggaagcta gttcttctca attataa
200725668PRTPhyscomitrella patens 25Met Gly Ser Gln Val Phe His Phe
Leu Leu Val Phe Phe Gly Tyr Phe1
5 10 15Leu His Gly Ala Ser Ser Glu Ser Val Ile Leu Asp Ala Arg Pro
Thr 20 25 30Ile Leu Gln His Ser Gly Glu Asn Ile Thr Leu Ala Trp Lys
Gly Val 35 40 45Asn Leu Pro Thr Lys Tyr Asp Trp Leu Gly Ile Tyr Thr
Pro Pro Thr 50 55 60Ser Pro Asp Asp Gln His Ile Gly Tyr Ile Leu Leu
Ser Ser Cys Ser65 70 75 80Thr Trp Thr Thr Gly Ala Cys Ser Leu Gln
Ile Pro Leu Val Asn Met 85 90 95Arg Ala Pro Tyr Ser Phe Arg Ile Phe
Arg Gly Val Phe Val Asn Val 100 105 110Ser Ala Ser Thr Asn Val Thr
Gly Ser Asn Asn Gly Ala Thr Thr Ile 115 120 125Ser Leu Asp Arg Glu
Gly Asn Pro Leu Pro Asp Val Thr Lys Arg Leu 130 135 140Ala Ala Ser
Pro Val Val Gln Phe Ser Asn Tyr Asn Glu Pro Thr Gln145 150 155
160Ile His Leu Ala Leu Ser Ser Asp Glu Thr Ala Val Arg Val Met Phe
165 170 175Val Thr Arg Asp Pro Leu Arg Ser Gln Val Arg Phe Gly Glu
Asp Gly 180 185 190Asp Glu Leu Gly Asn Thr Val Asp Ala Thr Ser Val
Thr Tyr Ser Gln 195 200 205Ile Asp Met Cys Asp Glu Pro Ala Ser Ser
Tyr Gly Trp Arg Ser Pro 210 215 220Gly Tyr Ile His Asn Val Val Met
Gly Gly Leu Asn Pro Gly Ser Arg225 230 235 240Tyr Phe Tyr Arg Val
Gly Ser Asn Val Gly Gly Trp Ser Ser Thr Tyr 245 250 255Ser Phe Ile
Ala Pro His Pro Arg Ala Asp Glu Thr Asn Ala Leu Ile 260 265 270Phe
Gly Asp Met Gly Thr Ser Ile Pro Tyr Ser Thr Tyr Gln Tyr Thr 275 280
285Gln Ser Glu Ser Lys Asn Thr Val Lys Trp Leu Thr Arg Asp Leu Glu
290 295 300Gln Ile Gly Asp Lys Pro Ser Phe Val Ala His Ile Gly Asp
Ile Ser305 310 315 320Tyr Ala Arg Gly Leu Ser Trp Leu Trp Asp Asn
Phe Phe Thr Gln Ile 325 330 335Glu Pro Val Ala Ala Arg Ser Pro Tyr
His Val Cys Met Gly Asn His 340 345 350Glu Tyr Asp Trp Pro Gly Gln
Pro Phe Lys Pro Asp Trp Ser Pro Tyr 355 360 365Gln Thr Asp Gly Gly
Gly Glu Cys Gly Val Pro Tyr Ser Leu Arg Phe 370 375 380Ile Met Pro
Gly Asn Ser Ser Leu Pro Thr Gly Thr Thr Ser Pro Ala385 390 395
400Thr Lys Asn Leu Tyr Tyr Ser Ile Asp Val Gly Val Val His Phe Leu
405 410 415Phe Tyr Ser Thr Glu Thr Asp Phe Gln Val Gly Ser Pro Gln
Tyr Thr 420 425 430Phe Ile Ala Asn Asp Leu Arg Thr Val Asp Arg Asn
Lys Thr Pro Phe 435 440 445Val Val Phe Leu Gly His Arg Pro Leu Tyr
Thr Thr Asp Tyr Arg Ala 450 455 460Leu Leu Asp Thr Met Thr Gln Lys
Leu Val Gln Thr Phe Glu Pro Leu465 470 475 480Leu Ile Asp Thr Asn
Val Thr Val Ala Phe Cys Gly His Val His Lys 485 490 495Tyr Glu Arg
Met Cys Pro Leu Lys Asn Tyr Thr Cys Ile Glu Pro Ser 500 505 510Lys
Ala Asn Gly Glu Leu Pro Ile His Met Val Val Gly Met Gly Gly 515 520
525Ala Asp His Gln Pro Ile Asp Asp Pro Leu Pro Ser Gln Ser Gln Pro
530 535 540Ile Phe Pro Gln Pro Ser Trp Ser Val Phe Arg Thr Phe Glu
Trp Gly545 550 555 560Tyr Ile Arg Leu His Ala Thr Arg His Leu Met
Thr Ile Ser Tyr Val 565 570 575Gly Asn His Asp Gly Lys Val His Asp
Val Val Glu Ile Pro Val Leu 580 585 590Asp Asp Ile Lys Ser Gly Ala
Tyr Val Glu Ser Arg Glu Ser Phe Phe 595 600 605Asp Thr Ala Ser Gly
Val Gln Ile Pro Cys Gly Arg Ser Glu Asn Ile 610 615 620Val Ala Phe
Leu Phe Val Leu Ala Leu Gly Cys Gly Cys Gly Ala Ala625 630 635
640Ala Thr Leu Phe Phe Met Arg Arg Gln Gln Arg Lys Gln Ile Trp Gln
645 650 655Pro Val Asn Arg Glu Glu Ala Ser Ser Ser Gln Leu 660
665261944DNAPopulus trichocarpa 26atgaagctcc ctatcttcct cctcctcctc
ctcctctccc tcatcactca aacttccctc 60tccaaagtca ccatctccgt gactccaaca
accctccaga aatccggtga cacagtaacc 120atttcctggt ccaacgttga
ttcaccttcc aaactcgact ggctcgggct ctattcacct 180cctgactcac
ctcacgacca cttcattggc tacaagttcc tttcttcctc tccttcatgg
240caatccgggt cgggttccat ttccttgccc atcaccaacc tccgctccaa
ttactctttc 300cggatcttcc actggaccga atccgaaatc aaccccaaac
gccatgacca tgatcacaac 360cccctccctg ggacggccca ttttctggcg
gagtcggatg ttgtcgggtt cgagtcgggt 420catgggccag agcagatcca
tttggcatat acggatgatg aggacgagat gagggtgatg 480tttgtggtgg
gtgatggaga ggagaggggt gtgaagtggg gagagaggga cggggagtgg
540agtcacgtga gtggggcacg tgtggtgagg tatgaaaggg aggatatgtg
tgatgctccg 600gcaaatggga gtattgggtg gagagatccg ggttggatcc
atgatggggt aatgaaggat 660ttgaagaaag gtgttaggta ttattatcag
gttggaagcg actctaaggg ttggagcaca 720actaggagct ttgtctctcg
gaatggagac tcggatgaaa caatagcctt cctgtttggg 780gacatgggaa
cttcaacacc atatgctacc tttatccgta cacaagatga aagcatatca
840accatgaagt ggatcctccg agacatagaa gctattggtg acaagcatgc
ttttgtttct 900catataggag atatcagcta tgcaagaggg tactcatggt
tgtgggacca tttttttacc 960caagtggaac ctgttgcttc caaagtgcca
taccatgtgt gcattggtaa tcatgagtac 1020gattggccct tacagccctg
gaaaccagat tgggccaatg cagtttacgg aactgatggt 1080ggtggtgaat
gtggggttcc ttacagcctt aaatttaaca tgccagggaa ctcttcagac
1140tcaactggga cccgtgctcc tgcaacccga aacctttact actcttttga
cacaggggct 1200gtacattttg tgtacatatc aactgagacc aattttgttg
ctgggagcag ccaatataac 1260tttataaagc aagatctgga atcagttgac
cggagcaaga ctccttttgt ggtagtccaa 1320gggcacagac caatgtatac
tactagcaat gaaaacaggg atgccccaat gaggaacaaa 1380atgcttgagc
acttggaacc tttgtttacg aaatacaatg ttacccttgc actgtggggt
1440catgtgcata gatacgaaag gttttgtcca gtgaataact tcatctgcgg
aagcacttgg 1500aagggatttc cagtccatgc tgtgattggc atggcaggac
aagactggca gcccatctgg 1560gagccaagat cagaccaccc aaatgatcca
atttttccac agccagccag gtctatgttc 1620cgtggggggg agttcgggta
caccaaattg gttgccacaa aggagaagct aacacttact 1680tatgtaggta
accatgatgg aaagatgcac gatatggttg agtttttggc atctggagaa
1740gttctcagtg gtgatgacag cattagtgtg gatgctggag ccaggattgg
ggtggttgat 1800tctacgttct catggtatgt caagggggca agtgttcttg
tccttggggc ttttgtgggc 1860tatactcttg gctacgcatc ccattccagg
aagcaaaatg gtaacaaggc cagctggact 1920cctgtgaaaa gtgaggatat atga
194427647PRTPopulus trichocarpa 27Met Lys Leu Pro Ile Phe Leu Leu
Leu Leu Leu Leu Ser Leu Ile Thr1 5 10 15Gln Thr Ser Leu Ser Lys Val
Thr Ile Ser Val Thr Pro Thr Thr Leu 20 25 30Gln Lys Ser Gly Asp Thr
Val Thr Ile Ser Trp Ser Asn Val Asp Ser 35 40 45Pro Ser Lys Leu Asp
Trp Leu Gly Leu Tyr Ser Pro Pro Asp Ser Pro 50 55 60His Asp His Phe
Ile Gly Tyr Lys Phe Leu Ser Ser Ser Pro Ser Trp65 70 75 80Gln Ser
Gly Ser Gly Ser Ile Ser Leu Pro Ile Thr Asn Leu Arg Ser 85 90 95Asn
Tyr Ser Phe Arg Ile Phe His Trp Thr Glu Ser Glu Ile Asn Pro 100 105
110Lys Arg His Asp His Asp His Asn Pro Leu Pro Gly Thr Ala His Phe
115 120 125Leu Ala Glu Ser Asp Val Val Gly Phe Glu Ser Gly His Gly
Pro Glu 130 135 140Gln Ile His Leu Ala Tyr Thr Asp Asp Glu Asp Glu
Met Arg Val Met145 150 155 160Phe Val Val Gly Asp Gly Glu Glu Arg
Gly Val Lys Trp Gly Glu Arg 165 170 175Asp Gly Glu Trp Ser His Val
Ser Gly Ala Arg Val Val Arg Tyr Glu 180 185 190Arg Glu Asp Met Cys
Asp Ala Pro Ala Asn Gly Ser Ile Gly Trp Arg 195 200 205Asp Pro Gly
Trp Ile His Asp Gly Val Met Lys Asp Leu Lys Lys Gly 210 215 220Val
Arg Tyr Tyr Tyr Gln Val Gly Ser Asp Ser Lys Gly Trp Ser Thr225 230
235 240Thr Arg Ser Phe Val Ser Arg Asn Gly Asp Ser Asp Glu Thr Ile
Ala 245 250 255Phe Leu Phe Gly Asp Met Gly Thr Ser Thr Pro Tyr Ala
Thr Phe Ile 260 265 270Arg Thr Gln Asp Glu Ser Ile Ser Thr Met Lys
Trp Ile Leu Arg Asp 275 280 285Ile Glu Ala Ile Gly Asp Lys His Ala
Phe Val Ser His Ile Gly Asp 290 295 300Ile Ser Tyr Ala Arg Gly Tyr
Ser Trp Leu Trp Asp His Phe Phe Thr305 310 315 320Gln Val Glu Pro
Val Ala Ser Lys Val Pro Tyr His Val Cys Ile Gly 325 330 335Asn His
Glu Tyr Asp Trp Pro Leu Gln Pro Trp Lys Pro Asp Trp Ala 340 345
350Asn Ala Val Tyr Gly Thr Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr
355 360 365Ser Leu Lys Phe Asn Met Pro Gly Asn Ser Ser Asp Ser Thr
Gly Thr 370 375 380Arg Ala Pro Ala Thr Arg Asn Leu Tyr Tyr Ser Phe
Asp Thr Gly Ala385 390 395 400Val His Phe Val Tyr Ile Ser Thr Glu
Thr Asn Phe Val Ala Gly Ser 405 410 415Ser Gln Tyr Asn Phe Ile Lys
Gln Asp Leu Glu Ser Val Asp Arg Ser 420 425 430Lys Thr Pro Phe Val
Val Val Gln Gly His Arg Pro Met Tyr Thr Thr 435 440 445Ser Asn Glu
Asn Arg Asp Ala Pro Met Arg Asn Lys Met Leu Glu His 450 455 460Leu
Glu Pro Leu Phe Thr Lys Tyr Asn Val Thr Leu Ala Leu Trp Gly465 470
475 480His Val His Arg Tyr Glu Arg Phe Cys Pro Val Asn Asn Phe Ile
Cys 485 490 495Gly Ser Thr Trp Lys Gly Phe Pro Val His Ala Val Ile
Gly Met Ala 500 505 510Gly Gln Asp Trp Gln Pro Ile Trp Glu Pro Arg
Ser Asp His Pro Asn 515 520 525Asp Pro Ile Phe Pro Gln Pro Ala Arg
Ser Met Phe Arg Gly Gly Glu 530 535 540Phe Gly Tyr Thr Lys Leu Val
Ala Thr Lys Glu Lys Leu Thr Leu Thr545 550 555 560Tyr Val Gly Asn
His Asp Gly Lys Met His Asp Met Val Glu Phe Leu 565 570 575Ala Ser
Gly Glu Val Leu Ser Gly Asp Asp Ser Ile Ser Val Asp Ala 580 585
590Gly Ala Arg Ile Gly Val Val Asp Ser Thr Phe Ser Trp Tyr Val Lys
595 600 605Gly Ala Ser Val Leu Val Leu Gly Ala Phe Val Gly Tyr Thr
Leu Gly 610 615 620Tyr Ala Ser His Ser Arg Lys Gln Asn Gly Asn Lys
Ala Ser Trp Thr625 630 635 640Pro Val Lys Ser Glu Asp Ile
645281962DNASaccharum officinarum 28atgtcccccg aaaaccccca
cctccgcttc ctcctcttcc tcgccgtcgc ggccgtcgcc 60gccggcgggg ctgcggcggg
caccaccctc accgcgaccc tctccagcga ccagatcaag 120atccgctgga
caggcctccc ggccccggac ggcctcgact acgtcggcat ctactcgccg
180ccgtcctccc gcgaccgcga cttcctcggc tacctcttcc tcaacggctc
cgcctcctgg 240cgcggcggct caggggagct ctccctcccg cgcctcccga
ccctgcgcgc gccctaccag 300ttccgcctct tccgctggcc cgccaacgag
tactcctacc accacatcga ccatgaccgg 360aacccgctcc cccacggcaa
gcaccgcgtc gccgtctccg ccgacgtctc cgtcggcgac 420cccgcgcgcc
ccgagcaggt gcacctcgcg ttcgcggatg ggatcgacga gatgcgggtc
480ctgttcgtgt gcggcgaccg cgggaagagg gtcgtcaggt acgggctgca
aaaggaagac 540gagaaggagt ggaaggaggt gggcacggat gtgagcacgt
acaagcaaaa gcacatgtgc 600gattggccgc cgaacagcag cgtcgcctgg
agggatcccg gattcgtctt cgacgggctc 660atgaagggat tggagcctgg
aaggaggtac ttttacaagg ttggtagtga cactggagga 720tggagtgaga
tatacagctt tatttcacgt gacagtgaag ccaatgagac caacacattt
780ctgtttggtg acatgggaac ttatgtgcct tatcacacct acattcgcac
acaagatgag 840agcttgtcca ctgtaaagtg gatccttcgt gatattgaag
cccttgggga taaacccgcc 900tttatttcac acattgggga catcagctat
gctagaggtt attcttgggt atgggatcat 960ttcttcagtc agattgagcc
aattgctgcc aataccccat accatgtctg tataggaaat 1020catgagtatg
attggccatc tcaaccttgg aaaccatggt gggctacata tggaaaggat
1080ggtggaggcg aatgtggaat accgtatagc gtcaagttca gaatgcctgg
caattctatt 1140ctaccaactg gtaatggtgg cccagacacc aggaatcttt
attactcctt tgactcaggt 1200gtggtgcatt tcgtctacat gtcaaccgaa
acaaattttg ttcagggcag tgatcagtac 1260aacttcttga aagcggacct
tgagaaggtg aaccgaagta gaacaccatt tgttgttttt 1320cagggccacc
gccccatgta cacctcaagt gatgaaacca gggacgcggc cttgagacag
1380cagatgctcc agcatttgga accgctgctg gtgacataca gtgtgaccct
tgcactatgg 1440ggacatgtcc acaggtacga gaggttctgc ccgatgaaga
acttccaatg tgtcaacact 1500tcatcaagct tccaatactc tggtgctcct
gtgcatcttg tgattgggat gggcggggca 1560gactgggcaa ccatatggca
accgaggcct gatcacccag acgtccccat ctttccacag 1620cctgagaggt
ccatgtaccg tggcggtgag tttggataca caaggcttgc agcaacaagg
1680gagaagctaa cattaaccta tgtggggaac catgatgggc aagtccatga
tataatggag 1740atattttccg gcctggtatc tagtaacagt agtgttgctg
aggtggtgga tgatactaaa 1800catggcacag gagtcagcac cgtgcgaaaa
atatctccgt tgtacttgga aatcggaggc 1860agtgtattgt ttgcactgct
tctgggattt tcctttggat ttcttatcag gagaaagaaa 1920gaagctgcac
agtggactcc agtaaagaac gaggaatcgt aa 196229653PRTSaccharum
officinarum 29Met Ser Pro Glu Asn Pro His Leu Arg Phe Leu Leu Phe
Leu Ala Val1 5 10 15Ala Ala Val Ala Ala Gly Gly Ala Ala Ala Gly Thr
Thr Leu Thr Ala 20 25 30Thr Leu Ser Ser Asp Gln Ile Lys Ile Arg Trp
Thr Gly Leu Pro Ala 35 40 45Pro Asp Gly Leu Asp Tyr Val Gly Ile Tyr
Ser Pro Pro Ser Ser Arg 50 55 60Asp Arg Asp Phe Leu Gly Tyr Leu Phe
Leu Asn Gly Ser Ala Ser Trp65 70 75 80Arg Gly Gly Ser Gly Glu Leu
Ser Leu Pro Arg Leu Pro Thr Leu Arg 85 90 95Ala Pro Tyr Gln Phe Arg
Leu Phe Arg Trp Pro Ala Asn Glu Tyr Ser 100 105 110Tyr His His Ile
Asp His Asp Arg Asn Pro Leu Pro His Gly Lys His 115 120 125Arg Val
Ala Val Ser Ala Asp Val Ser Val Gly Asp Pro Ala Arg Pro 130 135
140Glu Gln Val His Leu Ala Phe Ala Asp Gly Ile Asp Glu Met Arg
Val145 150 155 160Leu Phe Val Cys Gly Asp Arg Gly Lys Arg Val Val
Arg Tyr Gly Leu 165 170 175Gln Lys Glu Asp Glu Lys Glu Trp Lys Glu
Val Gly Thr Asp Val Ser 180 185 190Thr Tyr Lys Gln Lys His Met Cys
Asp Trp Pro Pro Asn Ser Ser Val 195 200 205Ala Trp Arg Asp Pro Gly
Phe Val Phe Asp Gly Leu Met Lys Gly Leu 210 215 220Glu Pro Gly Arg
Arg Tyr Phe Tyr Lys Val Gly Ser Asp Thr Gly Gly225 230 235 240Trp
Ser Glu Ile Tyr Ser Phe Ile Ser Arg Asp Ser Glu Ala Asn Glu 245 250
255Thr Asn Thr Phe Leu Phe Gly Asp Met Gly Thr Tyr Val Pro Tyr His
260 265 270Thr Tyr Ile Arg Thr Gln Asp Glu Ser Leu Ser Thr Val Lys
Trp Ile 275 280 285Leu Arg Asp Ile Glu Ala Leu Gly Asp Lys Pro Ala
Phe Ile Ser His 290 295 300Ile Gly Asp Ile Ser Tyr Ala Arg Gly Tyr
Ser Trp Val Trp Asp His305 310 315 320Phe Phe Ser Gln Ile Glu Pro
Ile Ala Ala Asn Thr Pro Tyr His Val 325 330 335Cys Ile Gly Asn His
Glu Tyr Asp Trp Pro Ser Gln Pro Trp Lys Pro 340 345 350Trp Trp Ala
Thr Tyr Gly Lys Asp Gly Gly Gly Glu Cys Gly Ile Pro 355 360 365Tyr
Ser Val Lys Phe Arg Met Pro Gly Asn Ser Ile Leu Pro Thr Gly 370 375
380Asn Gly Gly Pro Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser
Gly385 390 395 400Val Val His Phe Val Tyr Met Ser Thr Glu Thr Asn
Phe Val Gln Gly 405 410 415Ser Asp Gln Tyr Asn Phe Leu Lys Ala Asp
Leu Glu Lys Val Asn Arg 420 425 430Ser Arg Thr Pro Phe Val Val Phe
Gln Gly His Arg Pro Met Tyr Thr 435 440 445Ser Ser Asp Glu Thr Arg
Asp Ala Ala Leu Arg Gln Gln Met Leu Gln 450 455 460His Leu Glu Pro
Leu Leu Val Thr Tyr Ser Val Thr Leu Ala Leu Trp465 470 475 480Gly
His Val His Arg Tyr Glu Arg Phe Cys Pro Met Lys Asn Phe Gln 485 490
495Cys Val Asn Thr Ser Ser Ser Phe Gln Tyr Ser Gly
Ala Pro Val His 500 505 510Leu Val Ile Gly Met Gly Gly Ala Asp Trp
Ala Thr Ile Trp Gln Pro 515 520 525Arg Pro Asp His Pro Asp Val Pro
Ile Phe Pro Gln Pro Glu Arg Ser 530 535 540Met Tyr Arg Gly Gly Glu
Phe Gly Tyr Thr Arg Leu Ala Ala Thr Arg545 550 555 560Glu Lys Leu
Thr Leu Thr Tyr Val Gly Asn His Asp Gly Gln Val His 565 570 575Asp
Ile Met Glu Ile Phe Ser Gly Leu Val Ser Ser Asn Ser Ser Val 580 585
590Ala Glu Val Val Asp Asp Thr Lys His Gly Thr Gly Val Ser Thr Val
595 600 605Arg Lys Ile Ser Pro Leu Tyr Leu Glu Ile Gly Gly Ser Val
Leu Phe 610 615 620Ala Leu Leu Leu Gly Phe Ser Phe Gly Phe Leu Ile
Arg Arg Lys Lys625 630 635 640Glu Ala Ala Gln Trp Thr Pro Val Lys
Asn Glu Glu Ser 645 650301950DNASolanum tuberosum 30atgatgatcc
cttttgtcac cttcacctta ctcatcttct tcaacttgat ttcatcatct 60tcttcttcac
aaatctccat ttctgtaacc ccaaaaacct tatcaaaatc tggtgatttt
120gttacaatca aatggactgg tatcccctca ccttctaaac tcgatttctt
aggaatttac 180tctccaccca gttcactcca cgacaatttc attggctata
ttttcctatc ttcaacaccc 240gaatgggaat ctgggtcggg ttcaatttcc
atccctttag tcaatcttcg atctgggtat 300cagtttcgga tattcagatg
gacggaatcg gagattgtac cggatctagt ggatcatgac 360cacaatccat
tgccgcagac gaagcatatt cttgcggtgt cggaggaggt tgggtttgtt
420tcgggtcggg gacccgaaca ggttcatttg gctttaacgg gttttgaaga
tgagatgcgg 480gttatgtttg ttacgcctga cgggaaagag agttatgtga
gatatgggtt gacccggggt 540agattgggtc gggttgtgaa aactcgggtt
gtgaggtatg agaaggaaga tttatgtgat 600gcaccagcta atagtagtat
tggatggaga gatcctgggt atatacatga tggtgttatg 660cttaatttga
aaaagggaaa gaagtattat tatcaggttg gcagtgattc agggggctgg
720agcaccattt acagctttgt gtcacagaat agagactcag gtgaaacatt
tgctttcttg 780tttggagaca tggggactgc taccccatac ttgacatttc
ttcgtacaca ggacgaaagt 840aaatcaacga ttaagtggat tagccgtgat
attgaagctc ttggtaataa gcctgccctt 900atctcacata ttggagatat
cagctacgct agaggatact cttggttgtg ggacaacttt 960tttactcagg
tagaacctgt tgcatccaga gttccatacc atgtatgcat cggaaaccat
1020gaatatgatt ggccacttca accttggaag cctgattggt caagctacgg
gaaagatggg 1080ggaggtgaat gtggtgtacc ctacaggtca tacttccata
tgccaagaaa ctcttcagtg 1140ccgactggaa tgcatgctcc tgcaactcgg
aatctttatt actcatttga ttctgggccc 1200gttcactttg tctatatgtc
aactgaaacc aatttccttc caggtagtaa ccagtatgac 1260tttttaaagc
atgacttgga atcagttgat cgagtaaaaa ctccttttgt tgtctttcaa
1320gggcacagac caatgtacag ttcaagtagc ggagcaaaag atatatcttt
gaggaagaga 1380atgatggagt atttggaacc tcttcttgtg aagaacaatg
tgaatcttgt attgtggggg 1440catgttcata ggtatgagag gttttgccct
ttgaataact tcacctgtgg aagcttggcc 1500ttgaatggga aggagcaaaa
ggctttccca gttcaaattg tgattgggat ggcaggacag 1560gactggcagc
ctatctgggc accaagagaa gaccacccta cggatcctat tttcccacag
1620cctctgcaat ctctgtaccg tgggagtgaa tttggctacg tgaggctgca
tgccacaaag 1680aaaaagctta cactttctta tgtaggaaac catgacggag
aggtgcatga taaggtggag 1740ttcctagctt caggactact tctcagtgct
ggtatccgtg atggtcctgc agatgcagta 1800cacatggagt ctaagttctc
atggtatgta aaggttggaa gtgtgctaat gcttggagct 1860tttatgggtt
acatagttgg attcttatct catgctcgga aaaattctgc tgataaagga
1920tggagaccta taaaaactga ggaaatatga 195031649PRTSolanum tuberosum
31Met Met Ile Pro Phe Val Thr Phe Thr Leu Leu Ile Phe Phe Asn Leu1
5 10 15Ile Ser Ser Ser Ser Ser Ser Gln Ile Ser Ile Ser Val Thr Pro
Lys 20 25 30Thr Leu Ser Lys Ser Gly Asp Phe Val Thr Ile Lys Trp Thr
Gly Ile 35 40 45Pro Ser Pro Ser Lys Leu Asp Phe Leu Gly Ile Tyr Ser
Pro Pro Ser 50 55 60Ser Leu His Asp Asn Phe Ile Gly Tyr Ile Phe Leu
Ser Ser Thr Pro65 70 75 80Glu Trp Glu Ser Gly Ser Gly Ser Ile Ser
Ile Pro Leu Val Asn Leu 85 90 95Arg Ser Gly Tyr Gln Phe Arg Ile Phe
Arg Trp Thr Glu Ser Glu Ile 100 105 110Val Pro Asp Leu Val Asp His
Asp His Asn Pro Leu Pro Gln Thr Lys 115 120 125His Ile Leu Ala Val
Ser Glu Glu Val Gly Phe Val Ser Gly Arg Gly 130 135 140Pro Glu Gln
Val His Leu Ala Leu Thr Gly Phe Glu Asp Glu Met Arg145 150 155
160Val Met Phe Val Thr Pro Asp Gly Lys Glu Ser Tyr Val Arg Tyr Gly
165 170 175Leu Thr Arg Gly Arg Leu Gly Arg Val Val Lys Thr Arg Val
Val Arg 180 185 190Tyr Glu Lys Glu Asp Leu Cys Asp Ala Pro Ala Asn
Ser Ser Ile Gly 195 200 205Trp Arg Asp Pro Gly Tyr Ile His Asp Gly
Val Met Leu Asn Leu Lys 210 215 220Lys Gly Lys Lys Tyr Tyr Tyr Gln
Val Gly Ser Asp Ser Gly Gly Trp225 230 235 240Ser Thr Ile Tyr Ser
Phe Val Ser Gln Asn Arg Asp Ser Gly Glu Thr 245 250 255Phe Ala Phe
Leu Phe Gly Asp Met Gly Thr Ala Thr Pro Tyr Leu Thr 260 265 270Phe
Leu Arg Thr Gln Asp Glu Ser Lys Ser Thr Ile Lys Trp Ile Ser 275 280
285Arg Asp Ile Glu Ala Leu Gly Asn Lys Pro Ala Leu Ile Ser His Ile
290 295 300Gly Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Leu Trp Asp
Asn Phe305 310 315 320Phe Thr Gln Val Glu Pro Val Ala Ser Arg Val
Pro Tyr His Val Cys 325 330 335Ile Gly Asn His Glu Tyr Asp Trp Pro
Leu Gln Pro Trp Lys Pro Asp 340 345 350Trp Ser Ser Tyr Gly Lys Asp
Gly Gly Gly Glu Cys Gly Val Pro Tyr 355 360 365Arg Ser Tyr Phe His
Met Pro Arg Asn Ser Ser Val Pro Thr Gly Met 370 375 380His Ala Pro
Ala Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser Gly Pro385 390 395
400Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Leu Pro Gly Ser
405 410 415Asn Gln Tyr Asp Phe Leu Lys His Asp Leu Glu Ser Val Asp
Arg Val 420 425 430Lys Thr Pro Phe Val Val Phe Gln Gly His Arg Pro
Met Tyr Ser Ser 435 440 445Ser Ser Gly Ala Lys Asp Ile Ser Leu Arg
Lys Arg Met Met Glu Tyr 450 455 460Leu Glu Pro Leu Leu Val Lys Asn
Asn Val Asn Leu Val Leu Trp Gly465 470 475 480His Val His Arg Tyr
Glu Arg Phe Cys Pro Leu Asn Asn Phe Thr Cys 485 490 495Gly Ser Leu
Ala Leu Asn Gly Lys Glu Gln Lys Ala Phe Pro Val Gln 500 505 510Ile
Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Ile Trp Ala Pro 515 520
525Arg Glu Asp His Pro Thr Asp Pro Ile Phe Pro Gln Pro Leu Gln Ser
530 535 540Leu Tyr Arg Gly Ser Glu Phe Gly Tyr Val Arg Leu His Ala
Thr Lys545 550 555 560Lys Lys Leu Thr Leu Ser Tyr Val Gly Asn His
Asp Gly Glu Val His 565 570 575Asp Lys Val Glu Phe Leu Ala Ser Gly
Leu Leu Leu Ser Ala Gly Ile 580 585 590Arg Asp Gly Pro Ala Asp Ala
Val His Met Glu Ser Lys Phe Ser Trp 595 600 605Tyr Val Lys Val Gly
Ser Val Leu Met Leu Gly Ala Phe Met Gly Tyr 610 615 620Ile Val Gly
Phe Leu Ser His Ala Arg Lys Asn Ser Ala Asp Lys Gly625 630 635
640Trp Arg Pro Ile Lys Thr Glu Glu Ile 645321959DNAVitis vinifera
32atgtttccaa ttttatcttt ctgcctcttc ttcgtcctcg ctcctcctct cctcgcatct
60tcttctccag tctccataac cctaaccgcc aaaatcttag ccaaatcggg cgacccgatc
120cgaatcaaat ggtcggggat cgactccccg tccgacctcg actggctcgg
catctactcg 180ccgccgtcct ccgcccacga caacttcatt ggctatgttt
ttctgtcgtc atgtcccaca 240tgggaatctg gatcgggttc gatcagctta
cccctggtta atctccgtgc taactactct 300tttcggatat tccggtggtc
ccggtccgag gtcgacccga cccggatgga ccacgaccac 360aatcccttgc
cggggacaac gcatctggtg gcggagtccg gggaggtggg gttcgggggc
420ggcggggggc cggagcagat ccatttggcg tacacggata gggaggatga
gatgcgggtg 480atgttcgtga cgggggacgc gggcgtgagg actgtgaggt
atggcttgag cagggacgcg 540atgcacaggg tggtgacggc ggcggtgggg
agatatgaga gggaggacat gtgtgactcg 600ccagcgaatg agagtgttgg
gtggagagat ccgggtttta ttcaagatgc ggtgatgagg 660aatttgaaga
aagggaagag atattattat aaggttggaa gtgattcagg aggttggagc
720gcaattcaca actttatgtc acgggatatg gactctgaaa aaacaatagc
ttttctattt 780ggtgacatgg ggacagcaac accatactca acctttcttc
gtacacaaga ggaaagcaag 840tcaaccgtta aatggatcct ccgtgacatt
gaggctcttg atgacaaccc tgccttcatc 900tcgcatattg gagatattag
ctatgctaga ggttattcat ggttgtggga caattttttc 960actcaggttg
aacctatcgc ctccagactc ccataccatg tgtgtattgg taatcatgaa
1020tatgattggc cattgcagcc ttggaaacct gattggtcct ccacagttta
tggaacagat 1080ggtggcggtg aatgtggagt gccctacagc cttaagttca
aaatgcctgg aaactcttca 1140gaactaactg gaacccgtgc cccagccact
cgaaacctct tctactcatt tgatacgaag 1200gcagtgcatt ttgtgtacat
atcaactgag accaatttcc ttccagggag cagccaatat 1260gactttataa
agcaggattt ggagtcagtt gatcggaaaa aaaccccttt tgtggttgtc
1320caagggcaca gaccaatgta cacaacaagc aatgaactta gagatgcccc
agtgagggag 1380aggatgctca agtatttgga acctcttttt gtgaagaaca
atgtgaccct tgcactctgg 1440ggtcatgtcc acagatatga gaggttttgc
ccaataaata acttcacttg tggaaacatg 1500ggattgaatg gggaatacct
ggggggattg cctgttcata tcgtgattgg gatggcaggg 1560caagactggc
agcccacatg ggaaccaaga ccagaccacc cgaaggaccc tgtctaccca
1620caacctaaat ggtcattgta ccgtgggggt gagtttgggt acactaggtt
ggttgccacc 1680aaagagaagc taactctttc ttatgtagga aaccatgatg
gtgaggtgca tgatactgtt 1740gagattctgg catctggaca agttctcagt
ggtgttggag aggatgatgc tcaacccaga 1800gttgaggtgg cagagtacac
attttcatgg tatgttaagg gggcaagtat cttggtgctg 1860ggggctttta
tgggctatgt tattgggttc gtatcacatg ccaggagaga agctgccttg
1920agaaagaact ggactccagt gaagatcgaa gatagctga 195933652PRTVitis
vinifera 33Met Phe Pro Ile Leu Ser Phe Cys Leu Phe Phe Val Leu Ala
Pro Pro1 5 10 15Leu Leu Ala Ser Ser Ser Pro Val Ser Ile Thr Leu Thr
Ala Lys Ile 20 25 30Leu Ala Lys Ser Gly Asp Pro Ile Arg Ile Lys Trp
Ser Gly Ile Asp 35 40 45Ser Pro Ser Asp Leu Asp Trp Leu Gly Ile Tyr
Ser Pro Pro Ser Ser 50 55 60Ala His Asp Asn Phe Ile Gly Tyr Val Phe
Leu Ser Ser Cys Pro Thr65 70 75 80Trp Glu Ser Gly Ser Gly Ser Ile
Ser Leu Pro Leu Val Asn Leu Arg 85 90 95Ala Asn Tyr Ser Phe Arg Ile
Phe Arg Trp Ser Arg Ser Glu Val Asp 100 105 110Pro Thr Arg Met Asp
His Asp His Asn Pro Leu Pro Gly Thr Thr His 115 120 125Leu Val Ala
Glu Ser Gly Glu Val Gly Phe Gly Gly Gly Gly Gly Pro 130 135 140Glu
Gln Ile His Leu Ala Tyr Thr Asp Arg Glu Asp Glu Met Arg Val145 150
155 160Met Phe Val Thr Gly Asp Ala Gly Val Arg Thr Val Arg Tyr Gly
Leu 165 170 175Ser Arg Asp Ala Met His Arg Val Val Thr Ala Ala Val
Gly Arg Tyr 180 185 190Glu Arg Glu Asp Met Cys Asp Ser Pro Ala Asn
Glu Ser Val Gly Trp 195 200 205Arg Asp Pro Gly Phe Ile Gln Asp Ala
Val Met Arg Asn Leu Lys Lys 210 215 220Gly Lys Arg Tyr Tyr Tyr Lys
Val Gly Ser Asp Ser Gly Gly Trp Ser225 230 235 240Ala Ile His Asn
Phe Met Ser Arg Asp Met Asp Ser Glu Lys Thr Ile 245 250 255Ala Phe
Leu Phe Gly Asp Met Gly Thr Ala Thr Pro Tyr Ser Thr Phe 260 265
270Leu Arg Thr Gln Glu Glu Ser Lys Ser Thr Val Lys Trp Ile Leu Arg
275 280 285Asp Ile Glu Ala Leu Asp Asp Asn Pro Ala Phe Ile Ser His
Ile Gly 290 295 300Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp Leu Trp
Asp Asn Phe Phe305 310 315 320Thr Gln Val Glu Pro Ile Ala Ser Arg
Leu Pro Tyr His Val Cys Ile 325 330 335Gly Asn His Glu Tyr Asp Trp
Pro Leu Gln Pro Trp Lys Pro Asp Trp 340 345 350Ser Ser Thr Val Tyr
Gly Thr Asp Gly Gly Gly Glu Cys Gly Val Pro 355 360 365Tyr Ser Leu
Lys Phe Lys Met Pro Gly Asn Ser Ser Glu Leu Thr Gly 370 375 380Thr
Arg Ala Pro Ala Thr Arg Asn Leu Phe Tyr Ser Phe Asp Thr Lys385 390
395 400Ala Val His Phe Val Tyr Ile Ser Thr Glu Thr Asn Phe Leu Pro
Gly 405 410 415Ser Ser Gln Tyr Asp Phe Ile Lys Gln Asp Leu Glu Ser
Val Asp Arg 420 425 430Lys Lys Thr Pro Phe Val Val Val Gln Gly His
Arg Pro Met Tyr Thr 435 440 445Thr Ser Asn Glu Leu Arg Asp Ala Pro
Val Arg Glu Arg Met Leu Lys 450 455 460Tyr Leu Glu Pro Leu Phe Val
Lys Asn Asn Val Thr Leu Ala Leu Trp465 470 475 480Gly His Val His
Arg Tyr Glu Arg Phe Cys Pro Ile Asn Asn Phe Thr 485 490 495Cys Gly
Asn Met Gly Leu Asn Gly Glu Tyr Leu Gly Gly Leu Pro Val 500 505
510His Ile Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Thr Trp Glu
515 520 525Pro Arg Pro Asp His Pro Lys Asp Pro Val Tyr Pro Gln Pro
Lys Trp 530 535 540Ser Leu Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Arg
Leu Val Ala Thr545 550 555 560Lys Glu Lys Leu Thr Leu Ser Tyr Val
Gly Asn His Asp Gly Glu Val 565 570 575His Asp Thr Val Glu Ile Leu
Ala Ser Gly Gln Val Leu Ser Gly Val 580 585 590Gly Glu Asp Asp Ala
Gln Pro Arg Val Glu Val Ala Glu Tyr Thr Phe 595 600 605Ser Trp Tyr
Val Lys Gly Ala Ser Ile Leu Val Leu Gly Ala Phe Met 610 615 620Gly
Tyr Val Ile Gly Phe Val Ser His Ala Arg Arg Glu Ala Ala Leu625 630
635 640Arg Lys Asn Trp Thr Pro Val Lys Ile Glu Asp Ser 645
650341962DNAOryza sativa 34atgcttctct tcctcctctt cctcctcgcc
gccggcgagg ccgcggcggc ggcggcggcc 60accacgctca ccgcgacgcc ggcgaagctc
acccagtccg accgcgagat cacgatccgg 120tggtcgggcc tcccggaccc
ggacggcctc gactacgtcg gcatctactc gccgccgacc 180tcctccgacc
gcgacttcct cggctacctc ttcctcaacg gctcggccac ctggcgcacg
240ggcaccggcg agctcaccct cccgcgcctc cccaacctgc gcgcgcccta
ccagttccgc 300ctcttccgct ggcccgcgag ggagtactcc taccaccaca
tcgaccacga cgggaacccg 360ctcccccacg gccgccaccg cgtcgccgcc
tccggtgagg tcgccttcga ctccccctcc 420cgccccgacc aggtgcacct
ctcgttcgcc gacggggtcg acgagatgcg ggtcatgttc 480gtctgcggcg
acggcgggag gagggtggtg aggtacgggc cggcgaagga ggagggggag
540ggctggaagg aggtggccgc ggaggtgagg acgtacgagc agaagcacat
gtgcgactcg 600ccggcgaact cctccgtcgg gtggagggat ccagggttcg
tcttcgatgg actcatgaag 660ggattggagc ccgggaggag gtacttctac
aaggttggta gcaactcttc aggatggagc 720gatacgtaca gcttcatttc
acgtgacaac gaagccaatg aaactattgc atttctcttt 780ggtgacatgg
gcacttatat accatataac acctatgtcc gcacgcaaga tgaaagcttg
840tcgactgtaa agtggatact tcgtgatatt caagcccttg gagataagcc
tgcatttatt 900tcacacattg gggacatcag ctatgctaga ggttatgctt
gggtatggga tcacttcttc 960aaccagattg agcctattgc tgccaatacc
ccataccatg tctgcatagg aaatcatgaa 1020tatgattggc cattgcaacc
ttggaaacct tggtgggcaa ctggtatata tggaacagat 1080ggtggaggtg
aatgtggcat accttacagc gtaaagttca gaatgcctgg caattctttt
1140gtgcctactg gcaatggagc tcccgacacc cgaaatcttt actactcctt
cgattcaggg 1200gttgtgcatt ttgtttacat gtcaactgag actaattttg
ttcagggcag tgaccaatac 1260aacttcataa aagctgacct agagaaggtc
aaccgaagta gaactccttt cattgtgttt 1320cagggccacc ggccaatgta
tacatcaagc aatgaagcta gggattttgc tcatagacag 1380cagatgctcc
agaacctgga accactcttg gtaacataca aagtgaccct tgcactctgg
1440ggacatgtcc acaggtacga gaggttctgc cccatgaaaa acttccaatg
tgtcaacatg 1500tcatcaagct tcgtataccc tggtgcccct gttcatcttg
tgatcgggat gggtggtcaa 1560gattatcaac cattctggca gccaaggaag
gatcaccctg atgtacctgt ctatccgcag 1620cctgagaggt ctatgtaccg
tggtggggag tttggataca caaaacttgt agctacaaag 1680gagaagttga
cactaacgta catcggcaac catgatgggc aagtccatga tatggtggag
1740atattctctg ggcaagtatc taataacaat ggtgttcctg aggtgatcga
tgatacaaag 1800ctcagcacag gggtcagcac caaactgaaa atccctctgt
tctccttgga aattgtaggc 1860agcgtgatgt ttgcactggt tctgggtttc
tctcttggat ttctgatcag aaggaagaaa 1920gaagctgcac agtggacccc
agtgaagaac gaggagacct aa 196235653PRTOryza sativa 35Met Leu Leu Phe
Leu Leu Phe Leu Leu Ala Ala Gly Glu Ala Ala Ala1 5 10 15Ala Ala Ala
Ala Thr Thr Leu Thr Ala Thr Pro Ala Lys Leu Thr Gln 20 25
30Ser Asp Arg Glu Ile Thr Ile Arg Trp Ser Gly Leu Pro Asp Pro Asp
35 40 45Gly Leu Asp Tyr Val Gly Ile Tyr Ser Pro Pro Thr Ser Ser Asp
Arg 50 55 60Asp Phe Leu Gly Tyr Leu Phe Leu Asn Gly Ser Ala Thr Trp
Arg Thr65 70 75 80Gly Thr Gly Glu Leu Thr Leu Pro Arg Leu Pro Asn
Leu Arg Ala Pro 85 90 95Tyr Gln Phe Arg Leu Phe Arg Trp Pro Ala Arg
Glu Tyr Ser Tyr His 100 105 110His Ile Asp His Asp Gly Asn Pro Leu
Pro His Gly Arg His Arg Val 115 120 125Ala Ala Ser Gly Glu Val Ala
Phe Asp Ser Pro Ser Arg Pro Asp Gln 130 135 140Val His Leu Ser Phe
Ala Asp Gly Val Asp Glu Met Arg Val Met Phe145 150 155 160Val Cys
Gly Asp Gly Gly Arg Arg Val Val Arg Tyr Gly Pro Ala Lys 165 170
175Glu Glu Gly Glu Gly Trp Lys Glu Val Ala Ala Glu Val Arg Thr Tyr
180 185 190Glu Gln Lys His Met Cys Asp Ser Pro Ala Asn Ser Ser Val
Gly Trp 195 200 205Arg Asp Pro Gly Phe Val Phe Asp Gly Leu Met Lys
Gly Leu Glu Pro 210 215 220Gly Arg Arg Tyr Phe Tyr Lys Val Gly Ser
Asn Ser Ser Gly Trp Ser225 230 235 240Asp Thr Tyr Ser Phe Ile Ser
Arg Asp Asn Glu Ala Asn Glu Thr Ile 245 250 255Ala Phe Leu Phe Gly
Asp Met Gly Thr Tyr Ile Pro Tyr Asn Thr Tyr 260 265 270Val Arg Thr
Gln Asp Glu Ser Leu Ser Thr Val Lys Trp Ile Leu Arg 275 280 285Asp
Ile Gln Ala Leu Gly Asp Lys Pro Ala Phe Ile Ser His Ile Gly 290 295
300Asp Ile Ser Tyr Ala Arg Gly Tyr Ala Trp Val Trp Asp His Phe
Phe305 310 315 320Asn Gln Ile Glu Pro Ile Ala Ala Asn Thr Pro Tyr
His Val Cys Ile 325 330 335Gly Asn His Glu Tyr Asp Trp Pro Leu Gln
Pro Trp Lys Pro Trp Trp 340 345 350Ala Thr Gly Ile Tyr Gly Thr Asp
Gly Gly Gly Glu Cys Gly Ile Pro 355 360 365Tyr Ser Val Lys Phe Arg
Met Pro Gly Asn Ser Phe Val Pro Thr Gly 370 375 380Asn Gly Ala Pro
Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser Gly385 390 395 400Val
Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Val Gln Gly 405 410
415Ser Asp Gln Tyr Asn Phe Ile Lys Ala Asp Leu Glu Lys Val Asn Arg
420 425 430Ser Arg Thr Pro Phe Ile Val Phe Gln Gly His Arg Pro Met
Tyr Thr 435 440 445Ser Ser Asn Glu Ala Arg Asp Phe Ala His Arg Gln
Gln Met Leu Gln 450 455 460Asn Leu Glu Pro Leu Leu Val Thr Tyr Lys
Val Thr Leu Ala Leu Trp465 470 475 480Gly His Val His Arg Tyr Glu
Arg Phe Cys Pro Met Lys Asn Phe Gln 485 490 495Cys Val Asn Met Ser
Ser Ser Phe Val Tyr Pro Gly Ala Pro Val His 500 505 510Leu Val Ile
Gly Met Gly Gly Gln Asp Tyr Gln Pro Phe Trp Gln Pro 515 520 525Arg
Lys Asp His Pro Asp Val Pro Val Tyr Pro Gln Pro Glu Arg Ser 530 535
540Met Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Lys Leu Val Ala Thr
Lys545 550 555 560Glu Lys Leu Thr Leu Thr Tyr Ile Gly Asn His Asp
Gly Gln Val His 565 570 575Asp Met Val Glu Ile Phe Ser Gly Gln Val
Ser Asn Asn Asn Gly Val 580 585 590Pro Glu Val Ile Asp Asp Thr Lys
Leu Ser Thr Gly Val Ser Thr Lys 595 600 605Leu Lys Ile Pro Leu Phe
Ser Leu Glu Ile Val Gly Ser Val Met Phe 610 615 620Ala Leu Val Leu
Gly Phe Ser Leu Gly Phe Leu Ile Arg Arg Lys Lys625 630 635 640Glu
Ala Ala Gln Trp Thr Pro Val Lys Asn Glu Glu Thr 645
65036738DNAGossypium hirsutum 36gcacgagcgg gaagcagcca atatgacttt
ctgaagcatg atctagagtc ggttgatcgg 60atgaagaccc cttttgttgt agttcaaggg
catagaccaa tgtacactac aagtttcgaa 120agtagggacg ccccattgag
agagaaaatg cttgagcatt tggaaccttt atttgtgaaa 180aacaatgtga
accttgcatt atggggccat gttcatcggt acgagaggtt ttgtccattg
240aagaacttca catgtggaag catggggcag aaggggaagg attgggaggc
atttccagtt 300catgttgtga ttgggatggc aggacaagac tggcaaccaa
catgggaacc tcgaccagac 360catccaacga tcccgtctac ccacaacccc
aagaggtctt tgtaccgcac aggcgagttt 420gggtacacta gattaattgc
tacaaaagag aaacttacac tatcgttcgt aggaaaccat 480gacggggagg
tgcatgacat ggttgagatt ttggcatctg ggcaagttct caatggtggt
540gatgataaca atggtaaagt cggagcagtc cataaggttg atgatgtgac
acggtactca 600ttttcacact atgtctgggg tggtagtgtc ttggtgcttg
gtggttttgt tggctatgtt 660ctgggtttcg tttcacatgc taggagacaa
attgcaacag aaagaggctg gacttccttg 720aaaaccgagg agcaatga
73837245PRTGossypium hirsutum 37Ala Arg Ala Gly Ser Ser Gln Tyr Asp
Phe Leu Lys His Asp Leu Glu1 5 10 15Ser Val Asp Arg Met Lys Thr Pro
Phe Val Val Val Gln Gly His Arg 20 25 30Pro Met Tyr Thr Thr Ser Phe
Glu Ser Arg Asp Ala Pro Leu Arg Glu 35 40 45Lys Met Leu Glu His Leu
Glu Pro Leu Phe Val Lys Asn Asn Val Asn 50 55 60Leu Ala Leu Trp Gly
His Val His Arg Tyr Glu Arg Phe Cys Pro Leu65 70 75 80Lys Asn Phe
Thr Cys Gly Ser Met Gly Gln Lys Gly Lys Asp Trp Glu 85 90 95Ala Phe
Pro Val His Val Val Ile Gly Met Ala Gly Gln Asp Trp Gln 100 105
110Pro Thr Trp Glu Pro Arg Pro Asp His Pro Thr Ile Pro Ser Thr His
115 120 125Asn Pro Lys Arg Ser Leu Tyr Arg Thr Gly Glu Phe Gly Tyr
Thr Arg 130 135 140Leu Ile Ala Thr Lys Glu Lys Leu Thr Leu Ser Phe
Val Gly Asn His145 150 155 160Asp Gly Glu Val His Asp Met Val Glu
Ile Leu Ala Ser Gly Gln Val 165 170 175Leu Asn Gly Gly Asp Asp Asn
Asn Gly Lys Val Gly Ala Val His Lys 180 185 190Val Asp Asp Val Thr
Arg Tyr Ser Phe Ser His Tyr Val Trp Gly Gly 195 200 205Ser Val Leu
Val Leu Gly Gly Phe Val Gly Tyr Val Leu Gly Phe Val 210 215 220Ser
His Ala Arg Arg Gln Ile Ala Thr Glu Arg Gly Trp Thr Ser Leu225 230
235 240Lys Thr Glu Glu Gln 24538930DNAPanicum virgatum 38tggccatcac
aaccttggaa accatggtgg gctacatatg gaaaggacgg tgggggtgaa 60tgtggaatac
catacagtgt caagttcaga atgcctggca attcagttct acctactggt
120aatggtggtc cagacaccag gaatctttat tactcctttg attcaggtgt
ggtgcatttc 180gtgtacatgt caactgaaac taattttctt cagggcagtg
accagtacaa cttcttaaaa 240gcggaccttg agaaggtgaa ccgaactaga
acaccattcg ttgtttttca gggccaccgt 300cccatgtaca cctcaagtga
tgaaaccagg gatgctgctt tgaaacagca gatgctccag 360aatttggaac
cactgctggt gacatacaat gtgacccttg cactctgggg acatgtccac
420aggtatgaga ggttctgccc catgaagaac ttccaatgtg ttaacacttc
gtcaagcttc 480caataccctg gcgcccctgt gcatcttgtg atcgggatgg
gtggtcaaga ctggcaacct 540atatggcaac caaggcctga tcaccctgat
gttcccatct ttccgcagcc tgagaggtct 600atgtaccgtg gtggtgtgtt
tggatacaca agacttgtag ctacaaggga gaagctaaca 660ctaacgtatg
tggggaacca tgatgggcaa gtccatgata tggtggagat attttctggc
720caagtatcca gcaacagcag tgttgctgag gctgttgatg gtgcaaaact
cagcacagga 780gtcagcaccg tgcgaaaaat gcctcctttg tacttggaaa
tcggaggcag tgtgatgttt 840gcactactgc tggggtttgg ttttggattt
cttgtcagga gaaagaaaga agctgcacaa 900tgggctccgg taaagaacga
ggaatcttaa 93039309PRTPanicum virgatum 39Trp Pro Ser Gln Pro Trp
Lys Pro Trp Trp Ala Thr Tyr Gly Lys Asp1 5 10 15Gly Gly Gly Glu Cys
Gly Ile Pro Tyr Ser Val Lys Phe Arg Met Pro 20 25 30Gly Asn Ser Val
Leu Pro Thr Gly Asn Gly Gly Pro Asp Thr Arg Asn 35 40 45Leu Tyr Tyr
Ser Phe Asp Ser Gly Val Val His Phe Val Tyr Met Ser 50 55 60Thr Glu
Thr Asn Phe Leu Gln Gly Ser Asp Gln Tyr Asn Phe Leu Lys65 70 75
80Ala Asp Leu Glu Lys Val Asn Arg Thr Arg Thr Pro Phe Val Val Phe
85 90 95Gln Gly His Arg Pro Met Tyr Thr Ser Ser Asp Glu Thr Arg Asp
Ala 100 105 110Ala Leu Lys Gln Gln Met Leu Gln Asn Leu Glu Pro Leu
Leu Val Thr 115 120 125Tyr Asn Val Thr Leu Ala Leu Trp Gly His Val
His Arg Tyr Glu Arg 130 135 140Phe Cys Pro Met Lys Asn Phe Gln Cys
Val Asn Thr Ser Ser Ser Phe145 150 155 160Gln Tyr Pro Gly Ala Pro
Val His Leu Val Ile Gly Met Gly Gly Gln 165 170 175Asp Trp Gln Pro
Ile Trp Gln Pro Arg Pro Asp His Pro Asp Val Pro 180 185 190Ile Phe
Pro Gln Pro Glu Arg Ser Met Tyr Arg Gly Gly Val Phe Gly 195 200
205Tyr Thr Arg Leu Val Ala Thr Arg Glu Lys Leu Thr Leu Thr Tyr Val
210 215 220Gly Asn His Asp Gly Gln Val His Asp Met Val Glu Ile Phe
Ser Gly225 230 235 240Gln Val Ser Ser Asn Ser Ser Val Ala Glu Ala
Val Asp Gly Ala Lys 245 250 255Leu Ser Thr Gly Val Ser Thr Val Arg
Lys Met Pro Pro Leu Tyr Leu 260 265 270Glu Ile Gly Gly Ser Val Met
Phe Ala Leu Leu Leu Gly Phe Gly Phe 275 280 285Gly Phe Leu Val Arg
Arg Lys Lys Glu Ala Ala Gln Trp Ala Pro Val 290 295 300Lys Asn Glu
Glu Ser305401179DNASolanum lycopersicum 40gctttcttgt ttggagacat
ggggactgct acgccatact tgacatttct tcgtacacag 60gaagaaagta aatcaacgat
taagtggata agccgtgata ttgaagctct tggtaataag 120cctgccctta
tctcacatat tggagatatc agctacgcca gaggatactc ttggttgtgg
180gacaactttt ttactcaggt ggaacctgtt gcatccagag ttccatacca
tgtatgcatc 240ggaaaccatg aatatgattg gccacttcaa ccttggaagc
ctgattggtc aagctacggg 300aaagatgggg gaggtgaatg tggtgtaccc
tacagtcata agttccatat gccaggaaac 360tcttcagtgc cgactggaat
gcatgctcct gcaactcgga atctttatta ctcatttgat 420tctgggcccg
ttcactttgt ctatatgtca actgaaacaa atttcctgcc aggtagtaac
480cagtatgact ttttaaagca tgacttggaa tcagttgatc gagtaaaaac
tccttttgtc 540gtctttcaag ggcacagacc aatgtacagt tcaagtagcg
gaacaaaaga tatatctttg 600aggaagagaa tggttgagta tttggaacct
cttcttgtga agaacaatgt gaatcttgta 660ttgtgggggc atgttcatag
gtatgagagg ttttgccctt tgaataactt cacctgtgga 720agcttggcct
tgaacgggaa ggagcaaaag gctttccctg ttcaaattgt gatcgggatg
780gcaggacagg actggcagcc tatctgggca ccaagagaag accaccctac
ggatcctatt 840ttcccacagc ctctgcaatc tctgtaccgt gggagtgaat
ttggatacat gaggctgcat 900gccacaaagg aaaagcttac actttcttat
gtaggaaacc atgacggaga ggtgcatgat 960aaggtggagt tcctagcttc
aggacaactt ctcaatgctg gtatccgtga tggtcctgca 1020gatacagtac
acatggagtc taacttctca tggtatgtaa aggttggaag tgtgctaatg
1080cttggagctt tgatgggtta catagttgga ttcatatctc atgctcggaa
aaattctgct 1140gataatggtt ggaggcctat aaaaactgag gtaatatga
117941392PRTSolanum lycopersicum 41Ala Phe Leu Phe Gly Asp Met Gly
Thr Ala Thr Pro Tyr Leu Thr Phe1 5 10 15Leu Arg Thr Gln Glu Glu Ser
Lys Ser Thr Ile Lys Trp Ile Ser Arg 20 25 30Asp Ile Glu Ala Leu Gly
Asn Lys Pro Ala Leu Ile Ser His Ile Gly 35 40 45Asp Ile Ser Tyr Ala
Arg Gly Tyr Ser Trp Leu Trp Asp Asn Phe Phe 50 55 60Thr Gln Val Glu
Pro Val Ala Ser Arg Val Pro Tyr His Val Cys Ile65 70 75 80Gly Asn
His Glu Tyr Asp Trp Pro Leu Gln Pro Trp Lys Pro Asp Trp 85 90 95Ser
Ser Tyr Gly Lys Asp Gly Gly Gly Glu Cys Gly Val Pro Tyr Ser 100 105
110His Lys Phe His Met Pro Gly Asn Ser Ser Val Pro Thr Gly Met His
115 120 125Ala Pro Ala Thr Arg Asn Leu Tyr Tyr Ser Phe Asp Ser Gly
Pro Val 130 135 140His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Leu
Pro Gly Ser Asn145 150 155 160Gln Tyr Asp Phe Leu Lys His Asp Leu
Glu Ser Val Asp Arg Val Lys 165 170 175Thr Pro Phe Val Val Phe Gln
Gly His Arg Pro Met Tyr Ser Ser Ser 180 185 190Ser Gly Thr Lys Asp
Ile Ser Leu Arg Lys Arg Met Val Glu Tyr Leu 195 200 205Glu Pro Leu
Leu Val Lys Asn Asn Val Asn Leu Val Leu Trp Gly His 210 215 220Val
His Arg Tyr Glu Arg Phe Cys Pro Leu Asn Asn Phe Thr Cys Gly225 230
235 240Ser Leu Ala Leu Asn Gly Lys Glu Gln Lys Ala Phe Pro Val Gln
Ile 245 250 255Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Ile Trp
Ala Pro Arg 260 265 270Glu Asp His Pro Thr Asp Pro Ile Phe Pro Gln
Pro Leu Gln Ser Leu 275 280 285Tyr Arg Gly Ser Glu Phe Gly Tyr Met
Arg Leu His Ala Thr Lys Glu 290 295 300Lys Leu Thr Leu Ser Tyr Val
Gly Asn His Asp Gly Glu Val His Asp305 310 315 320Lys Val Glu Phe
Leu Ala Ser Gly Gln Leu Leu Asn Ala Gly Ile Arg 325 330 335Asp Gly
Pro Ala Asp Thr Val His Met Glu Ser Asn Phe Ser Trp Tyr 340 345
350Val Lys Val Gly Ser Val Leu Met Leu Gly Ala Leu Met Gly Tyr Ile
355 360 365Val Gly Phe Ile Ser His Ala Arg Lys Asn Ser Ala Asp Asn
Gly Trp 370 375 380Arg Pro Ile Lys Thr Glu Val Ile385
39042406DNASorghum bicolor 42accatgacca gaacccatat ggcaaccgag
gcccgatcac ccagatgtcc ccatctttcc 60acagcctgag aggtccatgt accgtggcgg
tgagtttgga tacacaaggc ttgtagcaac 120aagggagaag ctaacattaa
cctatgtggg gaaccatgat gggcaagtcc atggtatggt 180ggagatattt
tctggcctgg tatccagtaa cagtagtgtt gctgtggcag tgcatgacac
240caaacttggc acagaagtca gcaccgtgcg aaaaatatct ccattgtact
tggaaatcgg 300aggcagtgta ttgtttgcac tgctcctggg attttccttt
ggatttctta tcaggagaaa 360ggaagaagct gcacagtgga ctccagtaaa
gaacgaggaa tcataa 40643130PRTSorghum bicolor 43Pro Ile Trp Gln Pro
Arg Pro Asp His Pro Asp Val Pro Ile Phe Pro1 5 10 15Gln Pro Glu Arg
Ser Met Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Arg 20 25 30Leu Val Ala
Thr Arg Glu Lys Leu Thr Leu Thr Tyr Val Gly Asn His 35 40 45Asp Gly
Gln Val His Gly Met Val Glu Ile Phe Ser Gly Leu Val Ser 50 55 60Ser
Asn Ser Ser Val Ala Val Ala Val His Asp Thr Lys Leu Gly Thr65 70 75
80Glu Val Ser Thr Val Arg Lys Ile Ser Pro Leu Tyr Leu Glu Ile Gly
85 90 95Gly Ser Val Leu Phe Ala Leu Leu Leu Gly Phe Ser Phe Gly Phe
Leu 100 105 110Ile Arg Arg Lys Glu Glu Ala Ala Gln Trp Thr Pro Val
Lys Asn Glu 115 120 125Glu Ser 13044960DNATriticum aestivum
44catgtctgca taggaaatca tgagtatgat tggccttcac aaccttggaa accttcgtgg
60tctacatatg gaaaggatgg tggaggtgaa tgcggaatac catacagtgt caagttcagg
120atgcctggga attctgttct acctactggc aatggagctc cggacacacg
gaatctctat 180tactcttttg attcaggtgt tgtgcatttt gtgtacatgt
cgactgaaac taatttcgtt 240cagggcagcg accaacacaa tttcctaaaa
gctgacctag agaaggtgaa ccgaagtaga 300accccatttg ttgtgtttca
gggccaccgg cccatgtata cctcgagcaa cgaagccagg 360gattttgcca
tgagacagca gatgatccag catcttgaac tgctcttggt gatgtacaat
420gtgacccttg ccctgtgggg acatgtccat aggtatgaga ggttctgccc
catgaagaat 480tcacagtgtc tgaacacatc atcaagcttc atataccctg
gtgcccctgt tcatgttgtg 540atcgggatgg ccggacaaga ctggcaaccg
atctggcaac caaggcgtga tcatccagat 600gttcccatct ttccacagcc
tgggatctcc atgtaccgtg gtggtgagtt cgggtacaca 660aaactggtag
ctaccaggga gaagctaacg ctgatgtacg tcgggaacca tgacggacaa
720gtccatgaca tggtggagat attctctgga caaacatcta ctgaagctag
tgctaccgag 780gcggtcaatc aaacaaagct cggctcggga accagcgcca
agctgaagat ttccccatta 840tacttggaaa ttggaggtag tgtgatgttg
gcattgctgc ttggttttgc cttgggattt 900ctcctcagga agaagagaga
agcggcacaa tggactccgg tgaagaacga ggaatcctaa 96045319PRTTriticum
aestivum 45His Val Cys Ile Gly Asn His Glu Tyr Asp Trp Pro Ser Gln
Pro Trp1 5 10 15Lys Pro Ser Trp Ser Thr Tyr Gly Lys Asp Gly Gly Gly
Glu Cys Gly 20 25 30Ile Pro
Tyr Ser Val Lys Phe Arg Met Pro Gly Asn Ser Val Leu Pro 35 40 45Thr
Gly Asn Gly Ala Pro Asp Thr Arg Asn Leu Tyr Tyr Ser Phe Asp 50 55
60Ser Gly Val Val His Phe Val Tyr Met Ser Thr Glu Thr Asn Phe Val65
70 75 80Gln Gly Ser Asp Gln His Asn Phe Leu Lys Ala Asp Leu Glu Lys
Val 85 90 95Asn Arg Ser Arg Thr Pro Phe Val Val Phe Gln Gly His Arg
Pro Met 100 105 110Tyr Thr Ser Ser Asn Glu Ala Arg Asp Phe Ala Met
Arg Gln Gln Met 115 120 125Ile Gln His Leu Glu Leu Leu Leu Val Met
Tyr Asn Val Thr Leu Ala 130 135 140Leu Trp Gly His Val His Arg Tyr
Glu Arg Phe Cys Pro Met Lys Asn145 150 155 160Ser Gln Cys Leu Asn
Thr Ser Ser Ser Phe Ile Tyr Pro Gly Ala Pro 165 170 175Val His Val
Val Ile Gly Met Ala Gly Gln Asp Trp Gln Pro Ile Trp 180 185 190Gln
Pro Arg Arg Asp His Pro Asp Val Pro Ile Phe Pro Gln Pro Gly 195 200
205Ile Ser Met Tyr Arg Gly Gly Glu Phe Gly Tyr Thr Lys Leu Val Ala
210 215 220Thr Arg Glu Lys Leu Thr Leu Met Tyr Val Gly Asn His Asp
Gly Gln225 230 235 240Val His Asp Met Val Glu Ile Phe Ser Gly Gln
Thr Ser Thr Glu Ala 245 250 255Ser Ala Thr Glu Ala Val Asn Gln Thr
Lys Leu Gly Ser Gly Thr Ser 260 265 270Ala Lys Leu Lys Ile Ser Pro
Leu Tyr Leu Glu Ile Gly Gly Ser Val 275 280 285Met Leu Ala Leu Leu
Leu Gly Phe Ala Leu Gly Phe Leu Leu Arg Lys 290 295 300Lys Arg Glu
Ala Ala Gln Trp Thr Pro Val Lys Asn Glu Glu Ser305 310
315461959DNABrassica napus 46atgatcgtcg acttctctac cttcatcctc
ttcatctccg tcttcatttc ctcagctaac 60gccaaagcaa ccttatccat ctcccccaaa
actctaagcc gatccggcga ttccatcctc 120atcaaatggt ccaacgtcga
ctctccctcc gatctcgact ggctaggcat ctactccccc 180ccagactctc
cccacgacca cttcatcggc tacaaattcc tcaacgtctc ccccacgtgg
240caatccggct ccggcgcgat ctccctcccc ctcaccaacc tccgatcgaa
ctacacgttc 300cgtatcttcc gatggacgca gtccgagatc aatccgaagc
acaaggacca cgaccagaat 360cccttaccgg gaacgaagca ccttctggcg
gaatcggagc aggtggggtt cggatccgcc 420ggcgtgggga ggccggagca
gatccatttg gcgttcgagg ataaggttaa caggatgcgg 480gtcacgttcg
tagctgggga tggggaagaa aggttcgtga ggtacggaga ggggaaggac
540gcgttggcga actccgcggc ggcgcgcggg attaggtacg agagggagca
tatgtgtaat 600gctccggcta attccaccgt gggatggaga gatcccgggt
ggatttttca taccgttatg 660aagaatttga acggtggcgt taggtattat
tatcaggttg ggagtgattc aaagggatgg 720agtgagatcc acagctttat
cgctcgagat atctactcag aagaaaccat agctttcatg 780ttcggagaca
tgggttgcgc tacaccttac aataccttta tccggacgca ggacgagagt
840atctcaacag ttaagtggat actccgcgac atcgaagctc ttggtgacaa
gccagctctt 900gtttcgcaca ttggtgatat aagctacgct cgtggttact
cgtgggtgtg ggatgagttc 960ttcgctcaga tcgagcctat tgcctcgaga
gttccttacc acgtctgcat tggtaaccac 1020gagtatgact tccctactca
gccgtggaaa cctgattggg gaacttacgg taatgacggt 1080gggggagagt
gcggtgtgcc gtatagtctc aagttcaaca tgcctggaaa ctcgtcggaa
1140ccaacgggaa cgaaagctcc tcctacaagg aatttgtatt actcttacga
catggggtcg 1200gttcatttcc tttacatctc caccgagacg aactttctca
aaggagggag gcaatacgag 1260tttataaagc gagatcttga gtctgtgaac
agggagaaaa caccgtttgt tgtcgtgcaa 1320ggacacagac cgatgtacac
cacgagcaac gaggtgagag acgcgatgat taggcaaaag 1380atggtggagc
atttggagcc gctgtttgtg gagaacaacg tgacgcttgc tctgtgggga
1440catgttcata gatacgagag gttttgtccg ataagcaaca acacgtgtgg
gaaacagtgg 1500agaggaagcc cggttcatct tgtgatcggt atgggcggtc
aagactggca accgatttgg 1560cagccgagac cgaaccatcc gggtcttcct
atattccctc agcctgaaca gtcgatgtac 1620aggacgggtg agtttgggta
cactcgtttg gttgcgaaca aagagaagct tactgtttcg 1680tttgtgggta
accatgatgg agaagttcat gatagtgttg agatcttagc gtctggggaa
1740gtaatcagtg ggaggaaaga ggaaactatt aagaccgttc ctgtatctgc
aacacttgtg 1800gggaaacctg agtctgatgt cttatggtat gttaaaggag
caggcttgtt ggttatgggt 1860gtgcttttag ggttccttat agggtttttt
acaaggggga agaaaggatc ttcttcatct 1920gataaccgtt ggatcccagt
caagaacgag gagacatga 195947652PRTBrassica napus 47Met Ile Val Asp
Phe Ser Thr Phe Ile Leu Phe Ile Ser Val Phe Ile1 5 10 15Ser Ser Ala
Asn Ala Lys Ala Thr Leu Ser Ile Ser Pro Lys Thr Leu 20 25 30Ser Arg
Ser Gly Asp Ser Ile Leu Ile Lys Trp Ser Asn Val Asp Ser 35 40 45Pro
Ser Asp Leu Asp Trp Leu Gly Ile Tyr Ser Pro Pro Asp Ser Pro 50 55
60His Asp His Phe Ile Gly Tyr Lys Phe Leu Asn Val Ser Pro Thr Trp65
70 75 80Gln Ser Gly Ser Gly Ala Ile Ser Leu Pro Leu Thr Asn Leu Arg
Ser 85 90 95Asn Tyr Thr Phe Arg Ile Phe Arg Trp Thr Gln Ser Glu Ile
Asn Pro 100 105 110Lys His Lys Asp His Asp Gln Asn Pro Leu Pro Gly
Thr Lys His Leu 115 120 125Leu Ala Glu Ser Glu Gln Val Gly Phe Gly
Ser Ala Gly Val Gly Arg 130 135 140Pro Glu Gln Ile His Leu Ala Phe
Glu Asp Lys Val Asn Arg Met Arg145 150 155 160Val Thr Phe Val Ala
Gly Asp Gly Glu Glu Arg Phe Val Arg Tyr Gly 165 170 175Glu Gly Lys
Asp Ala Leu Ala Asn Ser Ala Ala Ala Arg Gly Ile Arg 180 185 190Tyr
Glu Arg Glu His Met Cys Asn Ala Pro Ala Asn Ser Thr Val Gly 195 200
205Trp Arg Asp Pro Gly Trp Ile Phe His Thr Val Met Lys Asn Leu Asn
210 215 220Gly Gly Val Arg Tyr Tyr Tyr Gln Val Gly Ser Asp Ser Lys
Gly Trp225 230 235 240Ser Glu Ile His Ser Phe Ile Ala Arg Asp Ile
Tyr Ser Glu Glu Thr 245 250 255Ile Ala Phe Met Phe Gly Asp Met Gly
Cys Ala Thr Pro Tyr Asn Thr 260 265 270Phe Ile Arg Thr Gln Asp Glu
Ser Ile Ser Thr Val Lys Trp Ile Leu 275 280 285Arg Asp Ile Glu Ala
Leu Gly Asp Lys Pro Ala Leu Val Ser His Ile 290 295 300Gly Asp Ile
Ser Tyr Ala Arg Gly Tyr Ser Trp Val Trp Asp Glu Phe305 310 315
320Phe Ala Gln Ile Glu Pro Ile Ala Ser Arg Val Pro Tyr His Val Cys
325 330 335Ile Gly Asn His Glu Tyr Asp Phe Pro Thr Gln Pro Trp Lys
Pro Asp 340 345 350Trp Gly Thr Tyr Gly Asn Asp Gly Gly Gly Glu Cys
Gly Val Pro Tyr 355 360 365Ser Leu Lys Phe Asn Met Pro Gly Asn Ser
Ser Glu Pro Thr Gly Thr 370 375 380Lys Ala Pro Pro Thr Arg Asn Leu
Tyr Tyr Ser Tyr Asp Met Gly Ser385 390 395 400Val His Phe Leu Tyr
Ile Ser Thr Glu Thr Asn Phe Leu Lys Gly Gly 405 410 415Arg Gln Tyr
Glu Phe Ile Lys Arg Asp Leu Glu Ser Val Asn Arg Glu 420 425 430Lys
Thr Pro Phe Val Val Val Gln Gly His Arg Pro Met Tyr Thr Thr 435 440
445Ser Asn Glu Val Arg Asp Ala Met Ile Arg Gln Lys Met Val Glu His
450 455 460Leu Glu Pro Leu Phe Val Glu Asn Asn Val Thr Leu Ala Leu
Trp Gly465 470 475 480His Val His Arg Tyr Glu Arg Phe Cys Pro Ile
Ser Asn Asn Thr Cys 485 490 495Gly Lys Gln Trp Arg Gly Ser Pro Val
His Leu Val Ile Gly Met Gly 500 505 510Gly Gln Asp Trp Gln Pro Ile
Trp Gln Pro Arg Pro Asn His Pro Gly 515 520 525Leu Pro Ile Phe Pro
Gln Pro Glu Gln Ser Met Tyr Arg Thr Gly Glu 530 535 540Phe Gly Tyr
Thr Arg Leu Val Ala Asn Lys Glu Lys Leu Thr Val Ser545 550 555
560Phe Val Gly Asn His Asp Gly Glu Val His Asp Ser Val Glu Ile Leu
565 570 575Ala Ser Gly Glu Val Ile Ser Gly Arg Lys Glu Glu Thr Ile
Lys Thr 580 585 590Val Pro Val Ser Ala Thr Leu Val Gly Lys Pro Glu
Ser Asp Val Leu 595 600 605Trp Tyr Val Lys Gly Ala Gly Leu Leu Val
Met Gly Val Leu Leu Gly 610 615 620Phe Leu Ile Gly Phe Phe Thr Arg
Gly Lys Lys Gly Ser Ser Ser Ser625 630 635 640Asp Asn Arg Trp Ile
Pro Val Lys Asn Glu Glu Thr 645 650484PRTArabidopsis
thalianaMISC_FEATURE(3)..(4)Xaa is any amino acid 48Gly Asp Xaa
Gly1495PRTArtificial SequenceSequence from plant purple acid
phosphatases found in plant species such as Arabidopsis thaliana
49Xaa Asp Xaa Xaa Tyr1 5504PRTArabidopsis thaliana 50Gly Asn His
Asp1514PRTArabidopsis thaliana 51Gly Asn His Glu1524PRTArtificial
SequenceSequence from plant purple acid phosphatases found in plant
species such as Arabidopsis thaliana 52Xaa Xaa Gly
His1534PRTArtificial SequenceSequence from plant purple acid
phosphatases found in plant species such as Arabidopsis thaliana
53Xaa His Xaa His15412PRTArabidopsis
thalianaMISC_FEATURE(12)..(12)Xaa is Phe or Trp 54Tyr His Val Cys
Ile Gly Asn His Glu Tyr Asp Xaa1 5 105512PRTArtificial
SequenceSequence from plant purple acid phosphatases found in plant
species such as Arabidopsis thaliana 55Tyr His Val Cys Ile Gly Asn
His Glu Tyr Asn Xaa1 5 105613PRTArabidopsis thaliana 56His Ile Gly
Asp Ile Ser Tyr Ala Arg Gly Tyr Ser Trp1 5 105718PRTArabidopsis
thaliana 57Lys Glu Lys Leu Thr Val Ser Phe Val Gly Asn His Asp Gly
Glu Val1 5 10 15His Asp5818PRTArtificial SequenceSequence derived
from plant purple acid phosphatases found in plant species such as
Arabidopsis thaliana 58Lys Glu Arg Leu Thr Leu Ser Tyr Val Gly Asn
His Asp Gly Glu Val1 5 10 15His Asp5918PRTZea mays 59Arg Glu Lys
Leu Thr Leu Thr Tyr Val Gly Asn His Asp Gly Gln Val1 5 10 15His
Asp6018PRTArtificial SequenceSequence derived from plant purple
acid phosphatases found in plant species such as Arabidopsis
thaliana 60Lys Glu Lys Leu Thr Leu Thr Tyr Ile Gly Asn His Asp Gly
Gln Val1 5 10 15His Asp6110PRTArabidopsis
thalianaMISC_FEATURE(8)..(9)Xaa is any amino acid 61Phe Val Gly Asn
His Asp Gly Xaa Xaa His1 5 106210PRTArtificial SequenceSequence
derived from plant purple acid phosphatases found in plant species
such as Arabidopsis thaliana 62Phe Ile Gly Asn His Asp Gly Xaa Xaa
His1 5 106310PRTZea maysMISC_FEATURE(8)..(9)Xaa is any amino acid
63Tyr Val Gly Asn His Asp Gly Xaa Xaa His1 5 106410PRTArtificial
SequenceSequence derived from plant purple acid phosphatases found
in plant species such as Arabidopsis thaliana 64Tyr Ile Gly Asn His
Asp Gly Xaa Xaa His1 5 106523PRTArabidopsis thaliana 65Leu Trp Tyr
Ala Lys Gly Ala Gly Leu Met Val Val Gly Val Leu Leu1 5 10 15Gly Phe
Ile Ile Gly Phe Phe 206612PRTArtificial SequenceSequence derived
from plant purple acid phosphatases found in plant species such as
Arabidopsis thaliana 66Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa
Gly1 5 10
* * * * *
References