U.S. patent application number 12/607089 was filed with the patent office on 2010-05-06 for manipulation of glutamine synthetases (gs) to improve nitrogen use efficiency and grain yield in higher plants.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL INC.. Invention is credited to Kanwarpal S. Dhugga, Rajeev Gupta.
Application Number | 20100115662 12/607089 |
Document ID | / |
Family ID | 42133133 |
Filed Date | 2010-05-06 |
United States Patent
Application |
20100115662 |
Kind Code |
A1 |
Gupta; Rajeev ; et
al. |
May 6, 2010 |
MANIPULATION OF GLUTAMINE SYNTHETASES (GS) TO IMPROVE NITROGEN USE
EFFICIENCY AND GRAIN YIELD IN HIGHER PLANTS
Abstract
The present invention provides polynucleotides and related
polypeptides of the protein GS. The invention provides genomic
sequence for the GS gene. GS is responsible for controlling
nitrogen utilization efficiency in plants. Glutamine synthase
sequences are provided for improving grain yield and plant growth.
The invention further provides recombinant expression cassettes,
host cells and transgenic plants.
Inventors: |
Gupta; Rajeev; (Johnston,
IA) ; Dhugga; Kanwarpal S.; (Johnston, IA) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL, INC.
7250 N.W. 62ND AVENUE, P.O. BOX 552
JOHNSTON
IA
50131-0552
US
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL
INC.
Johnston
IA
|
Family ID: |
42133133 |
Appl. No.: |
12/607089 |
Filed: |
October 28, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61109651 |
Oct 30, 2008 |
|
|
|
Current U.S.
Class: |
800/278 ;
435/320.1; 435/419; 536/23.1; 800/298; 800/312; 800/314; 800/320;
800/320.1; 800/320.2; 800/320.3 |
Current CPC
Class: |
A01H 1/00 20130101; C12N
9/93 20130101; C12N 15/8271 20130101; C12N 15/63 20130101; C12N
15/8261 20130101; Y02A 40/146 20180101 |
Class at
Publication: |
800/278 ;
536/23.1; 435/320.1; 435/419; 800/298; 800/320.1; 800/312; 800/320;
800/320.3; 800/314; 800/320.2 |
International
Class: |
A01H 1/00 20060101
A01H001/00; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63; C12N 5/10 20060101 C12N005/10; A01H 5/00 20060101
A01H005/00 |
Claims
1. An isolated polynucleotide selected from the group consisting
of: a. a polynucleotide having at least 80% sequence identity, as
determined by the GAP algorithm under default parameters, to the
full length sequence of a polynucleotide selected from the group
consisting of SEQ ID NOS: 43, 45, 47, 49, 51 and 53; wherein the
polynucleotide encodes a polypeptide that functions as a modifier
of GS; b. a polynucleotide encoding a polypeptide selected from the
group consisting of SEQ ID NO: 44, 46, 48, 50, 52 and 54; c. a
polynucleotide selected from the group consisting of SEQ ID NOS:
43, 45, 47, 49, 51 and 53; and d. A polynucleotide which is
complementary to the polynucleotide of (a), (b) or (c).
2. A recombinant expression cassette, comprising the polynucleotide
of claim 1, wherein the polynucleotide is operably linked, in sense
orientation, to a promoter.
3. A host cell comprising the expression cassette of claim 2.
4. A transgenic plant comprising the recombinant expression
cassette of claim 2.
5. The transgenic plant of claim 4, wherein said plant is a
monocot.
6. The transgenic plant of claim 4, wherein said plant is a
dicot.
7. The transgenic plant of claim 4, wherein said plant is selected
from the group consisting of: maize, soybean, sunflower, sorghum,
canola, wheat, alfalfa, cotton, rice, barley, millets, peanut,
switchgrass, myscanthus, triticale and cocoa.
8. A transgenic seed from the transgenic plant of claim 4.
9. A method of modulating the GS in a plant, comprising: a.
introducing into a plant cell a recombinant expression cassette
comprising the polynucleotide of claim 1 operably linked to a
promoter; b. culturing the plant cell under plant cell growing
conditions; and c. regenerating a plant form said plant cell;
wherein the GS in said plant is modulated.
10. The method of claim 9, wherein the plant is selected from the
group consisting of: maize, soybean, alfalfa, barley, canola,
cocoa, cotton, millets, myscanthus, peanut, rice, rye, sorghum,
sugar cane, switchgrass, triticale and wheat.
11. A method of decreasing the GS enzyme polypeptide activity in a
plant cell, comprising: a. providing a nucleotide sequence
comprising at least 15 consecutive nucleotides of the complement of
SEQ ID NO: 43, 45, 47, 49, 51 and 53; b. providing a plant cell
comprising an mRNA having the sequence set forth in SEQ ID NO: 43,
45, 47, 49, 51 and 53; and c. introducing the nucleotide sequence
of step (a) into the plant cell, wherein the nucleotide sequence
inhibits expression of the mRNA in the plant cell.
12. The method of claim 9, wherein said plant cell is from a
monocot.
13. The method of claim 12, wherein said monocot is maize, soybean,
alfalfa, barley, canola, cocoa, cotton, millets, myscanthus,
peanut, rice, rye, sorghum, sugar cane, switchgrass, triticale or
wheat
14. The method of claim 9, wherein said plant cell is from a
dicot.
15. The transgenic plant of claim 4, wherein the GS enzyme activity
in said plant is increased.
16. The transgenic plant of claim 15, wherein the plant has
increased seedling vigor.
17. The transgenic plant of claim 15, wherein the plant has
enhanced silk emergence.
18. The transgenic plant of claim 15, wherein the plant has
enhanced nitrogen assimilation in roots.
19. The transgenic plant of claim 15, wherein the plant has
increased seed number per plant.
20. The transgenic plant of claim 15, wherein the plant has
increased seed size and mass.
21. The transgenic plant of claim 15, wherein the plant has seed
with increased embryo size.
22. The transgenic plant of claim 15, wherein the plant has
increased nitrogen assimilation in the leaf.
23. The transgenic plant of claim 15, wherein the plant has
increased ear size.
24. The transgenic plant of claim 15, wherein the plant has
enhanced nitrogen utilization efficiency.
25. The transgenic plant of claim 15, wherein the plant has
increased nitrogen remobilization during senescence.
26. The transgenic plant of claim 15, wherein the plant has
increased nitrogen remobilization during grain development.
Description
CROSS REFERENCE
[0001] This utility application claims the benefit U.S. Provisional
Patent Application Ser. No. 61/109,651, filed Oct. 30, 2008, which
is incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to the field of molecular
biology.
BACKGROUND OF THE INVENTION
[0003] Nitrogen (N) is the most abundant inorganic nutrient taken
up from the soil by plants for growth and development. Maize roots
absorb most of the N from the soil in the form of nitrate, the
majority of which is transported to the leaf for reduction and
assimilation. Nitrate is reduced to nitrite by nitrate reductase
(NR) in the cytosol and then nitrite is transported into
chloroplast where it is reduced by nitrite reductase (NiR) to
ammonium. Ammonium is assimilated into glutamine by the glutamine
synthase-glutamate synthase system (Crawford and Glass, (1998)
Trends in Plant Science 3:389-395). Also, it has long been known
that significant amounts of N are lost from the plant aerial parts
by volatilization (Glyan'ko, et al., (1980) Agrokhimiya 8:19-26;
Hooker, et al., (1980) Agronomy Journal 72(5):789-792; Silva, et
al., (1981) Crop Science 21(6):913-916; Stutte, et al., (1981) Crop
Science 21(4):596-600; Foster, et al., (1986) Annals of Botany
57(3):305-307; Parton, et al., (1988) Agronomy Journal
80(3):419-425; Kamiji, et al., (1989) Japanese Journal of Crop
Science 58(1):140-142; Morgan, et al., (1989) Crop Science
29(3):726-731; O'Deen, (1989) Agronomy Journal 81(6):980-985;
Guindo, et al., (1994) Arkansas Farm Research 43(1):12-13;
Heckathorn, et al., (1995) Oecologia 101(3):361-365; Cabezas, et
al., (1997) Revista Brasileira de Ciencia do Solo 21(3):481-487).
Experimental evidence supports the loss of N through ammonium and
not through N oxides (Hooker, et al., 1980). Treatment with
chemicals that inhibit glutamine or glutamate synthase activities
led to increased loss of ammonium through volatilization (Foster,
et al., 1986). Loss of N is not only limited to C-3 species as C-4
plants have also been reported to lose N through volatilization
(Heckathorn, et al., 1995).
[0004] Several independent lines of evidence indicate that
glutamine synthetase (GS) is involved in yield formation and its
expression levels affect nitrogen use efficiency (NUE) in maize. GS
carries out two main functions in plant cells: (1) assimilate
ammonium resulting from nitrate reduction into organic form during
the biosynthetic phase and (2) assimilate ammonium generated by
photorespiration, deaminases and glutamate dehydrogenase, for
example, during seed germination and leaf senescence when proteins
are remobilized as N source or used as source of energy. The
cytosolic GS is referred to as GS1 and the plastidial form as GS2.
In a recent report (Martin, et al., (2006) The Plant Cell
18(11):3252-74), a reverse genetics strategy was used to show that
GS indeed is a limiting factor for grain number and grain weight,
both components of grain yield in maize. Earlier QTL mapping
experiments also implicated GS isozymes in the determination of
yield and NUE (Gallais and Hirel, (2004) J Exp Bot.
55(396):295-306). In other experiments, two GS genes located on
chromosome 1, including one expressed in the root, show significant
(p=10.sup.-4) association with biomass at 1 and 5 mM applied N
(data not shown). During leaf senescence, remobilization of N takes
place from source (leaf) to sink (developing grain) tissues.
Proteins are broken down into amino-acids, which are then
transported through phloem to the sink tissue. Grain protein
accounts for .about.60-70% of the total plant N at maturity in
maize, which means 30-40% N still remains in the stover. The
current invention involves efforts to over-express the cytosolic
isoforms of GS under the control of different promoters in maize to
improve NUE and thus grain yield.
SUMMARY OF THE INVENTION
[0005] The present invention provides polynucleotides, related
polypeptides and all conservatively modified variants of the
present GS sequences. The invention provides sequences for the GS
genes. 6 Arabidopsis, 6 maize, 4 rice, 3 sorghum and 8 soybean GS
genes were identified. Table 1 lists these genes and their sequence
ID numbers.
TABLE-US-00001 TABLE 1 SEQUENCE ID NUMBER IDENTITY SEQ ID NO: 1
AT1G48470 Polynucleotide SEQ ID NO: 2 AT1G48470 Polypeptide SEQ ID
NO: 3 AT1G66200 Polynucleotide SEQ ID NO: 4 AT1G66200 Polypeptide
SEQ ID NO: 5 AT3G17820 Polynucleotide SEQ ID NO: 6 AT3G17820
Polypeptide SEQ ID NO: 7 AT5G16570 Polynucleotide SEQ ID NO: 8
AT5G16570 Polypeptide SEQ ID NO: 9 AT5G35630 Polynucleotide SEQ ID
NO: 10 AT5G35630 Polypeptide SEQ ID NO: 11 AT5G37600 Polynucleotide
SEQ ID NO: 12 AT5G37600 Polypeptide SEQ ID NO: 13 Gm0005x00111
Polynucleotide SEQ ID NO: 14 Gm0005x00111 Polypeptide SEQ ID NO: 15
Gm0015x00387 Polynucleotide SEQ ID NO: 16 Gm0015x00387 Polypeptide
SEQ ID NO: 17 Gm0030x00147 Polynucleotide SEQ ID NO: 18
Gm0030x00147 Polypeptide SEQ ID NO: 19 Gm0040x00114 Polynucleotide
SEQ ID NO: 20 Gm0040x00114 Polypeptide SEQ ID NO: 21 Gm0081x00134
Polynucleotide SEQ ID NO: 22 Gm0081x00134 Polypeptide SEQ ID NO: 23
Gm0136x00208 Polynucleotide SEQ ID NO: 24 Gm0136x00208 Polypeptide
SEQ ID NO: 25 Gm0232x00015 Polynucleotide SEQ ID NO: 26
Gm0232x00015 Polypeptide SEQ ID NO: 27 Gm0271x00039 Polynucleotide
SEQ ID NO: 28 Gm0271x00039 Polypeptide SEQ ID NO: 29 Os02g50240
Polynucleotide SEQ ID NO: 30 Os02g50240 Polypeptide SEQ ID NO: 31
Os03g12290 Polynucleotide SEQ ID NO: 32 Os03g12290 Polypeptide SEQ
ID NO: 33 Os03g50490 Polynucleotide SEQ ID NO: 34 Os03g50490
Polypeptide SEQ ID NO: 35 Os04g56400 Polynucleotide SEQ ID NO: 36
Os04g56400 Polypeptide SEQ ID NO: 37 Sb01g143820 Polynucleotide SEQ
ID NO: 38 Sb01g143820 Polypeptide SEQ ID NO: 39 Sb04g133790
Polynucleotide SEQ ID NO: 40 Sb04g133790 Polypeptide SEQ ID NO: 41
Sb06g147820 Polynucleotide SEQ ID NO: 42 Sb06g147820 Polypeptide
SEQ ID NO: 43 ZmGS1-1 Polynucleotide SEQ ID NO: 44 ZmGS1-1
Polypeptide SEQ ID NO: 45 ZmGS1-2 Polynucleotide SEQ ID NO: 46
ZmGS1-2 Polypeptide SEQ ID NO: 47 ZmGS1-3 Polynucleotide SEQ ID NO:
48 ZmGS1-3 Polypeptide SEQ ID NO: 49 ZmGS1-4 Polynucleotide SEQ ID
NO: 50 ZmGS1-4 Polypeptide SEQ ID NO: 51 ZmGS1-5 Polynucleotide SEQ
ID NO: 52 ZmGS1-5 Polypeptide SEQ ID NO: 53 ZmGS2-Polynucleotide
SEQ ID NO: 54 ZmGS2-Polypeptide
[0006] Therefore, in one aspect, the present invention relates to
an isolated nucleic acid comprising an isolated polynucleotide
sequence encoding GS protein. One embodiment of the invention is an
isolated polynucleotide comprising a nucleotide sequence selected
from the group consisting of: (a) the nucleotide sequence
comprising SEQ ID NO: 43, 45, 47, 49, 51, 53; (b) the nucleotide
sequence encoding an amino acid sequence comprising SEQ ID NO: 44,
46, 48, 50, 52 and 54 and (c) the nucleotide sequence comprising at
least 70% sequence identity to SEQ ID NO: 43, 45, 47, 49, 51, 53,
wherein said polynucleotide encodes a polypeptide having GS enzyme
activity.
[0007] Compositions of the invention include an isolated
polypeptide comprising an amino acid sequence selected from the
group consisting of: (a) the amino acid sequence comprising SEQ ID
NO: 44, 46, 48, 50, 52 and 54 and (b) the amino acid sequence
comprising at least 70% sequence identity to SEQ ID NO: 44, 46, 48,
50, 52 and 54, wherein said polypeptide has GS enzyme activity.
[0008] In another aspect, the present invention relates to a
recombinant expression cassette comprising a nucleic acid as
described. Additionally, the present invention relates to a vector
containing the recombinant expression cassette. Further, the vector
containing the recombinant expression cassette can facilitate the
transcription and translation of the nucleic acid in a host cell.
The present invention also relates to the host cells able to
express the polynucleotide of the present invention. A number of
host cells could be used, such as but not limited to, microbial,
mammalian, plant or insect.
[0009] In yet another embodiment, the present invention is directed
to a transgenic plant or plant cells, containing the nucleic acids
of the present invention. Preferred plants containing the
polynucleotides of the present invention include but are not
limited to maize, soybean, sunflower, sorghum, canola, wheat,
alfalfa, cotton, rice, barley, tomato, switchgrass, myscanthus,
triticale and millet. In another embodiment, the transgenic plant
is a maize plant or plant cells. Another embodiment is the
transgenic seeds from the transgenic plant. Another embodiment of
the invention includes plants comprising a GS polypeptide of the
invention operably linked to a promoter that drives expression in
the plant. The plants of the invention can have altered GS as
compared to a control plant. In some plants, the GS is altered in a
vegetative tissue, a reproductive tissue, or a vegetative tissue
and a reproductive tissue. Plants of the invention can have at
least one of the following phenotypes including but not limited to:
increased leaf size, increased ear size, increased seed size,
increased endosperm size, alterations in the relative size of
embryos and endosperms leading to changes in the relative levels of
protein, oil and/or starch in the seeds, absence of tassels,
absence of functional pollen bearing tassels or increased plant
size.
[0010] Another embodiment of the invention would be plants that
have been genetically modified at a genomic locus, wherein the
genomic locus encodes a GS polypeptide of the invention.
[0011] Methods for increasing the activity of a GS polypeptide in a
plant are provided. The method can comprise introducing into the
plant a GS polynucleotide of the invention. Providing the
polypeptide can decrease the number of cells in plant tissue,
modulating the tissue growth and size.
[0012] Methods for reducing or eliminating the level of a GS
polypeptide in the plant are provided. The level or activity of the
polypeptide could also be reduced or eliminated in specific
tissues, causing increased GS in said tissues. Reducing the level
and/or activity of the GS polypeptide increases the number of cells
produced in the associated tissue.
[0013] Compositions further include plants and seed having a DNA
construct comprising a nucleotide sequence of interest operably
linked to a promoter of the current invention.
[0014] In specific embodiments, the DNA construct is stably
integrated into the genome of the plant. The method comprises
introducing into a plant a nucleotide sequence of interest operably
linked to a promoter of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1: Sequence alignment of GS proteins from Arabidopsis,
soybean, rice, sorghum and maize. The polypeptide alignment of all
27 sequences is shown in FIG. 1. Several regions of very high
homology were identified by this alignment. All these polypeptides
from different species (except SEQ ID NO: 20) show a sequence
identity in the range of 70-95% among different members. Due to
several insertions, SEQ ID NO: 20 shows an identity in the range of
53-74% with different GS polypeptides from different species. SEQ
ID NOS: 10, 18, 28, 36, 42 and 54 belong to the GS2 group
(chloroplast-localized) as in all the polypeptide a clear
chloroplast targeting peptide was identified.
[0016] FIG. 2: Phylogenetic tree of GS proteins from Arabidopsis,
rice, soybean, sorghum and maize. Analysis of all the 27
polypeptides are shown in FIG. 2. ZMGS1-1/1-5, ZMGS1-3/1-4, ZMGS1-2
and ZMGS2 along with members from other species were clustered in
four different clades. There is a soybean-specific Glade with SEQ
ID NOS: 14, 22, 24 and 26.
[0017] FIG. 3: Expression analyses of GS genes from maize were
conducted on a MPSS database consisting of more than 300 different
tissue libraries. GS1-1 and GS2 were expressed predominantly in
roots and leaves, respectively (FIG. 3A). GS1-2 expresses more or
less in all the tissues with a slightly higher expression in pollen
(FIG. 3A). GS1-3 and 1-4 were expressed at very low levels in most
of the tissues examined whereas GS1-5 expresses at about 100 ppm in
roots (FIG. 3A). GS1-1 showed 15-20.times. higher expression in
root-cortex as compared to other isoforms (FIG. 3B). Among all the
isoforms, only GS1-2 and 1-5 show the expression in the range of
.about.150-700 PPM in pedicel (FIG. 3C).
[0018] FIG. 4: GS activity in leaves of T0 events of ETX. GS enzyme
activity was determined in the leaves of field-grown T0 inbred
(ETX) events transformed with PHP32005, 32006, 32007, 32008, 38267,
28268 and 38269. The results from the individual events (FIG. 4A,
4C) and average of all the events (FIG. 4B, 4D) in each construct
are summarized. In case of ZM-GS1-3 over-expression PHPs, the
highest activity (on an average 12.times. higher) was observed in
PHP32008 (ZmPEPC1 PRO:ZmGS1-3) followed by PHP32007 (ZmUBI
PRO:ZmGS1-3) where the activity was slightly higher than the
controls in PHP32005 (pZmSSU PRO:ZmGS1-3). In case of PHP32006
(ZmRM2 PRO:ZmGS1-3) leaf samples, the activity was comparable to
control as expected as RM2 is a root-preferred promoter. In case of
PHP32006, the roots of T1 events showed significantly higher GS
activity as compared to non-transgenic sibs. For ZM-GS1-4, the
highest GS activity was observed in PHP38269 (pZM-PEPC::ZM-GS1-4)
followed by PHP38267 (pZM-UBI::ZM-GS1-4). In case of PHP32268
(ZmRM2 PRO:ZmGS1-4) leaf samples the activity was comparable to
control as expected as RM2 is a root-preferred promoter. The
average activities of all the events in each construct are
summarized in FIGS. 4B and 4D.
[0019] FIG. 5: GS activity in roots and leaves of T1 events of FAST
corn all five isoforms ZM-GS1 were also over-expressed in FAST
(Functional Analyses System Traits) (see, U.S. patent application
Ser. No. 10/367,417, filed Feb. 13, 2003) corn system under the
control of a root-preferred (RM2) or constitutive promoters (UBI).
Transgenic seeds segregating 1:1 hemizygous and wildtype were
separated using ELISA and planted in 4 inch square plastic pots
filled with Turface MVP.RTM. and thinned to 1 plant per pot after
emergence. Three weeks after germination and growth under normal N
condition, the leaves and roots were harvested for GS enzyme
activity analyses. The GS activities in individual events and the
average of all the events within a PHP are shown in FIGS. 5A, 5C
and FIG. 5B, 5D, respectively. In case of transgenic events where
various GS1 isoforms were driven by a root preferred promoter
(RM2), significantly higher GS activities were observed in roots as
compare to null controls (FIG. 5A, 5B). In case of a constitutive
promoter (UBI) driven GS1 isoforms events, a higher GS activity was
observed as compared to null controls (FIG. 5C, 5D).
[0020] FIG. 6: Improved specific growth rate in T0 events of FAST
corn. Five isoforms of ZM-GS1 were over-expressed in FAST corn
system under the control of a root-preferred (RM2) or constitutive
promoters (UBI). On an average, 10 independent transgenic events
were generated from each construct. (See, U.S. patent application
Ser. No. 10/367,417, filed Feb. 13, 2003). In all the T0 events,
measurements recorded included but were not limited to specific
growth rate, maximum total area, days to shed, seed number, ear
length and yield estimates. The data from specific growth rate
(SGR, measured from 14-28 days after germination) from this
experiment are shown in FIG. 6. Most of the events from each of the
6 constructs (out of total 10) tested showed significantly better
specific growth rate as compare to controls (0.00) (FIG. 6A).
PHP32772 (RM2 PRO:ZmGS1-4) performed best with a P value
>10.sup.-6 followed by PHP32779 (RM2 PRO:ZmGS1-3) with a P value
>10.sup.-5 (FIG. 6A). Other 4 constructs also show better SGR
with a P value ranging from 10.sup.-2 to 10.sup.-4) (FIG. 6A). Most
of the events in each construct performed significantly better than
control (FIG. 6B). More than 80% and 70% events exceeded the
performance of control in PHP32779 and 32772, respectively (FIG.
6B).
[0021] FIG. 7: Improved agronomic traits in T0 FAST events of
PHP32743. Over-expression of ZM-GS1-5 under the control of a
root-specific promoter resulted in improvement of several agronomic
traits in T0 phenomics measurements. The results from average of
nine events for several of these variables are summarized in FIG.
7. Multiple transgenic events from PHP32743 showed .about.50%
increase in ear length, .about.25% increase in seed number and
yield estimates and .about.18% increase in maximum total area over
the control.
[0022] FIG. 8: Improved growth and N concentrations in PHP32006
(pZMRM2:ZmGS1-3) and PHP 32007 (pUBI:ZMGS1-3) in low N conditions.
Testcross seeds of PHP32006 (FIG. 8a) and 32007 (FIG. 8b) were
assayed in green house under low N conditions. The data for root
dry weight, shoot dry weight, total dry weight and total N were
collected. Four out of six and 3 out of 5 events were significantly
better (denotes with asterisk in FIGS. 8a and 8b) than null control
in all the parameters measured in PHP32006 (FIG. 8a) and 32007
(FIG. 8b), respectively.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Unless
mentioned otherwise, the techniques employed or contemplated herein
are standard methodologies well known to one of ordinary skill in
the art. The materials, methods and examples are illustrative only
and not limiting. The following is presented by way of illustration
and is not intended to limit the scope of the invention.
[0024] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0025] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
[0026] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of botany,
microbiology, tissue culture, molecular biology, chemistry,
biochemistry and recombinant DNA technology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Langenheim and Thimann, BOTANY: PLANT
BIOLOGY AND ITS RELATION TO HUMAN AFFAIRS, John Wiley (1982); CELL
CULTURE AND SOMATIC CELL GENETICS OF PLANTS, vol. 1, Vasil, ed.
(1984); Stanier, et al., THE MICROBIAL WORLD, 5.sup.th ed.,
Prentice-Hall (1986); Dhringra and Sinclair, BASIC PLANT PATHOLOGY
METHODS, CRC Press (1985); Maniatis, et al., MOLECULAR CLONING: A
LABORATORY MANUAL (1982); DNA CLONING, vols. I and II, Glover, ed.
(1985); OLIGONUCLEOTIDE SYNTHESIS, Gait, ed. (1984); NUCLEIC ACID
HYBRIDIZATION, Hames and Higgins, eds. (1984) and the series
METHODS IN ENZYMOLOGY, Colowick and Kaplan, eds, Academic Press,
Inc., San Diego, Calif.
[0027] Units, prefixes and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxy orientation,
respectively. Numeric ranges are inclusive of the numbers defining
the range. Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes. The terms defined below are more
fully defined by reference to the specification as a whole.
[0028] In describing the present invention, the following terms
will be employed and are intended to be defined as indicated
below.
[0029] By "microbe" is meant any microorganism (including both
eukaryotic and prokaryotic microorganisms), such as fungi, yeast,
bacteria, actinomycetes, algae and protozoa, as well as other
unicellular structures.
[0030] By "amplified" is meant the construction of multiple copies
of a nucleic acid sequence or multiple copies complementary to the
nucleic acid sequence using at least one of the nucleic acid
sequences as a template. Amplification systems include the
polymerase chain reaction (PCR) system, ligase chain reaction (LCR)
system, nucleic acid sequence based amplification (NASBA, Cangene,
Mississauga, Ontario), O-Beta Replicase systems,
transcription-based amplification system (TAS), and strand
displacement amplification (SDA). See, e.g., DIAGNOSTIC MOLECULAR
MICROBIOLOGY: PRINCIPLES AND APPLICATIONS, Persing, et al., eds.,
American Society for Microbiology, Washington, D.C. (1993). The
product of amplification is termed an amplicon.
[0031] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refer to
those nucleic acids that encode identical or conservatively
modified variants of the amino acid sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations" and represent one species
of conservatively modified variation. Every nucleic acid sequence
herein that encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of ordinary skill will
recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine; one exception is
Micrococcus rubens, for which GTG is the methionine codon
(Ishizuka, et al., (1993) J. Gen. Microbiol. 139:425-32) can be
modified to yield a functionally identical molecule. Accordingly,
each silent variation of a nucleic acid, which encodes a
polypeptide of the present invention, is implicit in each described
polypeptide sequence and incorporated herein by reference.
[0032] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" when
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Thus, any number of amino acid
residues selected from the group of integers consisting of from 1
to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7 or 10
alterations can be made. Conservatively modified variants typically
provide similar biological activity as the unmodified polypeptide
sequence from which they are derived. For example, substrate
specificity, enzyme activity, or ligand/receptor binding is
generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, preferably
60-90% of the native protein for it's native substrate.
Conservative substitution tables providing functionally similar
amino acids are well known in the art.
[0033] The following six groups each contain amino acids that are
conservative substitutions for one another:
[0034] 1) Alanine (A), Serine (S), Threonine (T);
[0035] 2) Aspartic acid (D), Glutamic acid (E);
[0036] 3) Asparagine (N), Glutamine (Q);
[0037] 4) Arginine (R), Lysine (K);
[0038] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0039] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton, PROTEINS, W.H. Freeman and Co. (1984).
[0040] As used herein, "consisting essentially of" means the
inclusion of additional sequences to an object polynucleotide where
the additional sequences do not selectively hybridize, under
stringent hybridization conditions, to the same cDNA as the
polynucleotide and where the hybridization conditions include a
wash step in 0.1.times.SSC and 0.1% sodium dodecyl sulfate at
65.degree. C.
[0041] By "encoding" or "encoded," with respect to a specified
nucleic acid, is meant comprising the information for translation
into the specified protein. A nucleic acid encoding a protein may
comprise non-translated sequences (e.g., introns) within translated
regions of the nucleic acid, or may lack such intervening
non-translated sequences (e.g., as in cDNA). The information by
which a protein is encoded is specified by the use of codons.
Typically, the amino acid sequence is encoded by the nucleic acid
using the "universal" genetic code. However, variants of the
universal code, such as is present in some plant, animal and fungal
mitochondria, the bacterium Mycoplasma capricolum (Yamao, et al.,
(1985) Proc. Natl. Acad. Sci. USA 82:2306-9) or the ciliate
Macronucleus, may be used when the nucleic acid is expressed using
these organisms.
[0042] When the nucleic acid is prepared or altered synthetically,
advantage can be taken of known codon preferences of the intended
host where the nucleic acid is to be expressed. For example,
although nucleic acid sequences of the present invention may be
expressed in both monocotyledonous and dicotyledonous plant
species, sequences can be modified to account for the specific
codon preferences and GC content preferences of monocotyledonous
plants or dicotyledonous plants as these preferences have been
shown to differ (Murray, et al., (1989) Nucleic Acids Res.
17:477-98 and herein incorporated by reference). Thus, the maize
preferred codon for a particular amino acid might be derived from
known gene sequences from maize. Maize codon usage for 28 genes
from maize plants is listed in Table 4 of Murray, et al.,
supra.
[0043] As used herein, "heterologous" in reference to a nucleic
acid is a nucleic acid that originates from a foreign species, or,
if from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a
heterologous structural gene is from a species different from that
from which the structural gene was derived or, if from the same
species, one or both are substantially modified from their original
form. A heterologous protein may originate from a foreign species
or, if from the same species, is substantially modified from its
original form by deliberate human intervention.
[0044] By "host cell" is meant a cell, which comprises a
heterologous nucleic acid sequence of the invention, which contains
a vector and supports the replication and/or expression of the
expression vector. Host cells may be prokaryotic cells such as E.
coli, or eukaryotic cells such as yeast, insect, plant, amphibian
or mammalian cells. Preferably, host cells are monocotyledonous or
dicotyledonous plant cells, including but not limited to maize,
sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola,
barley, millet, switchgrass, myscanthus, triticale and tomato. A
particularly preferred monocotyledonous host cell is a maize host
cell.
[0045] The term "hybridization complex" includes reference to a
duplex nucleic acid structure formed by two single-stranded nucleic
acid sequences selectively hybridized with each other.
[0046] The term "introduced" in the context of inserting a nucleic
acid into a cell, means "transfection" or "transformation" or
"transduction" and includes reference to the incorporation of a
nucleic acid into a eukaryotic or prokaryotic cell where the
nucleic acid may be incorporated into the genome of the cell (e.g.,
chromosome, plasmid, plastid or mitochondrial DNA), converted into
an autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0047] The terms "isolated" refers to material, such as a nucleic
acid or a protein, which is substantially or essentially free from
components which normally accompany or interact with it as found in
its naturally occurring environment. The isolated material
optionally comprises material not found with the material in its
natural environment. Nucleic acids, which are "isolated", as
defined herein, are also referred to as "heterologous" nucleic
acids. Unless otherwise stated, the term "GS nucleic acid" means a
nucleic acid comprising a polynucleotide ("GS polynucleotide")
encoding a full length or partial length GS polypeptide.
[0048] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues having the essential nature of natural nucleotides
in that they hybridize to single-stranded nucleic acids in a manner
similar to naturally occurring nucleotides (e.g., peptide nucleic
acids).
[0049] By "nucleic acid library" is meant a collection of isolated
DNA or RNA molecules, which comprise and substantially represent
the entire transcribed fraction of a genome of a specified
organism. Construction of exemplary nucleic acid libraries, such as
genomic and cDNA libraries, is taught in standard molecular biology
references such as Berger and Kimmel, GUIDE TO MOLECULAR CLONING
TECHNIQUES, from the series METHODS IN ENZYMOLOGY, vol. 152,
Academic Press, Inc., San Diego, Calif. (1987); Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2.sup.nd ed., vols. 1-3
(1989) and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, et al.,
eds, Current Protocols, a joint venture between Greene Publishing
Associates, Inc. and John Wiley & Sons, Inc. (1994
Supplement).
[0050] As used herein "operably linked" includes reference to a
functional linkage between a first sequence, such as a promoter and
a second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA corresponding to the second
sequence. Generally, operably linked means that the nucleic acid
sequences being linked are contiguous and, where necessary to join
two protein coding regions, contiguous and in the same reading
frame.
[0051] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and
plant cells and progeny of same. Plant cell, as used herein
includes, without limitation, seeds, suspension cultures, embryos,
meristematic regions, callus tissue, leaves, roots, shoots,
gametophytes, sporophytes, pollen and microspores. The class of
plants, which can be used in the methods of the invention, is
generally as broad as the class of higher plants amenable to
transformation techniques, including both monocotyledonous and
dicotyledonous plants including species from the genera: Cucurbita,
Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis,
Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot,
Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum,
Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,
Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus,
Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium,
Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis,
Browallia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena,
Hordeum, Secale, Allium and Triticum. A particularly preferred
plant is Zea mays.
[0052] As used herein, "yield" may include reference to bushels per
acre of a grain crop at harvest, as adjusted for grain moisture
(15% typically for maize, for example). Grain moisture is measured
in the grain at harvest. The adjusted test weight of grain is
determined to be the weight in pounds per bushel, adjusted for
grain moisture level at harvest.
[0053] As used herein, "polynucleotide" includes reference to a
deoxyribopolynucleotide, ribopolynucleotide or analogs thereof that
have the essential nature of a natural ribonucleotide in that they
hybridize, under stringent hybridization conditions, to
substantially the same nucleotide sequence as naturally occurring
nucleotides and/or allow translation into the same amino acid(s) as
the naturally occurring nucleotide(s). A polynucleotide can be
full-length or a subsequence of a native or heterologous structural
or regulatory gene. Unless otherwise indicated, the term includes
reference to the specified sequence as well as the complementary
sequence thereof. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons are "polynucleotides" as that term
is intended herein. Moreover, DNAs or RNAs comprising unusual
bases, such as inosine, or modified bases, such as tritylated
bases, to name just two examples, are polynucleotides as the term
is used herein. It will be appreciated that a great variety of
modifications have been made to DNA and RNA that serve many useful
purposes known to those of skill in the art. The term
polynucleotide as it is employed herein embraces such chemically,
enzymatically or metabolically modified forms of polynucleotides,
as well as the chemical forms of DNA and RNA characteristic of
viruses and cells, including inter alia, simple and complex
cells.
[0054] The terms "polypeptide," "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0055] As used herein "promoter" includes reference to a region of
DNA upstream from the start of transcription and involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription. A "plant promoter" is a promoter capable of
initiating transcription in plant cells. Exemplary plant promoters
include, but are not limited to, those that are obtained from
plants, plant viruses and bacteria which comprise genes expressed
in plant cells such Agrobacterium or Rhizobium. Examples are
promoters that preferentially initiate transcription in certain
tissues, such as leaves, roots, seeds, fibres, xylem vessels,
tracheids or sclerenchyma. Such promoters are referred to as
"tissue preferred." A "cell type" specific promoter primarily
drives expression in certain cell types in one or more organs, for
example, vascular cells in roots or leaves. An "inducible" or
"regulatable" promoter is a promoter, which is under environmental
control. Examples of environmental conditions that may effect
transcription by inducible promoters include anaerobic conditions
or the presence of light. Another type of promoter is a
developmentally regulated promoter, for example, a promoter that
drives expression during pollen development. Tissue preferred, cell
type specific, developmentally regulated and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter, which is active under most
environmental conditions.
[0056] The term "GS polypeptide" refers to one or more amino acid
sequences. The term is also inclusive of fragments, variants,
homologs, alleles or precursors (e.g., preproproteins or
proproteins) thereof. A "GS protein" comprises a GS polypeptide.
Unless otherwise stated, the term "GS nucleic acid" means a nucleic
acid comprising a polynucleotide ("GS polynucleotide") encoding a
GS polypeptide.
[0057] As used herein "recombinant" includes reference to a cell or
vector, that has been modified by the introduction of a
heterologous nucleic acid or that the cell is derived from a cell
so modified. Thus, for example, recombinant cells express genes
that are not found in identical form within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all as a result of deliberate human intervention; or may have
reduced or eliminated expression of a native gene. The term
"recombinant" as used herein does not encompass the alteration of
the cell or vector by naturally occurring events (e.g., spontaneous
mutation, natural transformation/transduction/transposition) such
as those occurring without deliberate human intervention.
[0058] As used herein, a "recombinant expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically,
with a series of specified nucleic acid elements, which permit
transcription of a particular nucleic acid in a target cell. The
recombinant expression cassette can be incorporated into a plasmid,
chromosome, mitochondrial DNA, plastid DNA, virus or nucleic acid
fragment. Typically, the recombinant expression cassette portion of
an expression vector includes, among other sequences, a nucleic
acid to be transcribed, and a promoter.
[0059] The term "residue" or "amino acid residue" or "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide, or peptide (collectively
"protein"). The amino acid may be a naturally occurring amino acid
and, unless otherwise limited, may encompass known analogs of
natural amino acids that can function in a similar manner as
naturally occurring amino acids.
[0060] The term "selectively hybridizes" includes reference to
hybridization, under stringent hybridization conditions, of a
nucleic acid sequence to a specified nucleic acid target sequence
to a detectably greater degree (e.g., at least 2-fold over
background) than its hybridization to non-target nucleic acid
sequences and to the substantial exclusion of non-target nucleic
acids. Selectively hybridizing sequences typically have about at
least 40% sequence identity, preferably 60-90% sequence identity
and most preferably 100% sequence identity (i.e., complementary)
with each other.
[0061] The terms "stringent conditions" or "stringent hybridization
conditions" include reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than other sequences (e.g., at least 2-fold over
background).
[0062] Stringent conditions are sequence-dependent and will be
different in different circumstances. By controlling the stringency
of the hybridization and/or washing conditions, target sequences
can be identified which can be up to 100% complementary to the
probe (homologous probing). Alternatively, stringency conditions
can be adjusted to allow some mismatching in sequences so that
lower degrees of similarity are detected (heterologous probing).
Optimally, the probe is approximately 500 nucleotides in length,
but can vary greatly in length from less than 500 nucleotides to
equal to the entire length of the target sequence.
[0063] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide or Denhardt's. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C. and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at
37.degree. C. and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.
and a wash in 0.1.times.SSC at 60 to 65.degree. C. Specificity is
typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash
solution. For DNA-DNA hybrids, the T.sub.m can be approximated from
the equation of Meinkoth and Wahl, (1984) Anal. Biochem.,
138:267-84: T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61
(% form)-500/L; where M is the molarity of monovalent cations, % GC
is the percentage of guanosine and cytosine nucleotides in the DNA,
% form is the percentage of formamide in the hybridization solution
and L is the length of the hybrid in base pairs. The T.sub.m is the
temperature (under defined ionic strength and pH) at which 50% of a
complementary target sequence hybridizes to a perfectly matched
probe. T.sub.m is reduced by about 1.degree. C. for each 1% of
mismatching; thus, T.sub.m, hybridization and/or wash conditions
can be adjusted to hybridize to sequences of the desired identity.
For example, if sequences with .gtoreq.90% identity are sought, the
T.sub.m can be decreased 10.degree. C. Generally, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence and its
complement at a defined ionic strength and pH. However, severely
stringent conditions can utilize a hybridization and/or wash at 1,
2, 3 or 4.degree. C. lower than the thermal melting point
(T.sub.m); moderately stringent conditions can utilize a
hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C. lower than
the thermal melting point (T.sub.m); low stringency conditions can
utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or
20.degree. C. lower than the thermal melting point (T.sub.m). Using
the equation, hybridization and wash compositions, and desired
T.sub.m, those of ordinary skill will understand that variations in
the stringency of hybridization and/or wash solutions are
inherently described. If the desired degree of mismatching results
in a T.sub.m of less than 45.degree. C. (aqueous solution) or
32.degree. C. (formamide solution) it is preferred to increase the
SSC concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR
BIOLOGY--HYBRIDIZATION WITH NUCLEIC ACID PROBES, part I, chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," Elsevier, N.Y. (1993); and CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, chapter 2, Ausubel, et al., eds,
Greene Publishing and Wiley-Interscience, New York (1995). Unless
otherwise stated, in the present application high stringency is
defined as hybridization in 4.times.SSC, 5.times.Denhardt's (5 g
Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500
ml of water), 0.1 mg/ml boiled salmon sperm DNA and 25 mM Na
phosphate at 65.degree. C., and a wash in 0.1.times.SSC, 0.1% SDS
at 65.degree. C.
[0064] As used herein, "transgenic plant" includes reference to a
plant, which comprises within its genome a heterologous
polynucleotide. Generally, the heterologous polynucleotide is
stably integrated within the genome such that the polynucleotide is
passed on to successive generations. The heterologous
polynucleotide may be integrated into the genome alone or as part
of a recombinant expression cassette. "Transgenic" is used herein
to include any cell, cell line, callus, tissue, plant part or
plant, the genotype of which has been altered by the presence of
heterologous nucleic acid including those transgenics initially so
altered as well as those created by sexual crosses or asexual
propagation from the initial transgenic. The term "transgenic" as
used herein does not encompass the alteration of the genome
(chromosomal or extra-chromosomal) by conventional plant breeding
methods or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition or spontaneous mutation.
[0065] As used herein, "vector" includes reference to a nucleic
acid used in transfection of a host cell and into which can be
inserted a polynucleotide. Vectors are often replicons. Expression
vectors permit transcription of a nucleic acid inserted
therein.
[0066] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides
or polypeptides: (a) "reference sequence," (b) "comparison window,"
(c) "sequence identity," (d) "percentage of sequence identity" and
(e) "substantial identity."
[0067] As used herein, "reference sequence" is a defined sequence
used as a basis for sequence comparison. A reference sequence may
be a subset or the entirety of a specified sequence; for example,
as a segment of a full-length cDNA or gene sequence or the complete
cDNA or gene sequence.
[0068] As used herein, "comparison window" means includes reference
to a contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence may be compared to a reference
sequence and wherein the portion of the polynucleotide sequence in
the comparison window may comprise additions or deletions (i.e.,
gaps) compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Generally, the comparison window is at least 20 contiguous
nucleotides in length, and optionally can be 30, 40, 50, 100 or
longer. Those of skill in the art understand that to avoid a high
similarity to a reference sequence due to inclusion of gaps in the
polynucleotide sequence a gap penalty is typically introduced and
is subtracted from the number of matches.
[0069] Methods of alignment of nucleotide and amino acid sequences
for comparison are well known in the art. The local homology
algorithm (BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math
2:482, may conduct optimal alignment of sequences for comparison;
by the homology alignment algorithm (GAP) of Needleman and Wunsch,
(1970) J. Mol. Biol. 48:443-53; by the search for similarity method
(Tfasta and Fasta) of Pearson and Lipman, (1988) Proc. Natl. Acad.
Sci. USA 85:2444; by computerized implementations of these
algorithms, including, but not limited to: CLUSTAL in the PC/Gene
program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT,
BLAST, FASTA and TFASTA in the Wisconsin Genetics Software
Package.RTM., Version 8 (available from Genetics Computer Group
(GCG.RTM. programs (Accelrys, Inc., San Diego, Calif.)). The
CLUSTAL program is well described by Higgins and Sharp, (1988) Gene
73:237-44; Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et
al., (1988) Nucleic Acids Res.
[0070] 16:10881-90; Huang, et al., (1992) Computer Applications in
the Biosciences 8:155-65, and Pearson, et al., (1994) Meth. Mol.
Biol. 24:307-31. The preferred program to use for optimal global
alignment of multiple sequences is PileUp (Feng and Doolittle,
(1987) J. Mol. Evol., 25:351-60 which is similar to the method
described by Higgins and Sharp, (1989) CABIOS 5:151-53 and hereby
incorporated by reference). The BLAST family of programs which can
be used for database similarity searches includes: BLASTN for
nucleotide query sequences against nucleotide database sequences;
BLASTX for nucleotide query sequences against protein database
sequences; BLASTP for protein query sequences against protein
database sequences; TBLASTN for protein query sequences against
nucleotide database sequences; and TBLASTX for nucleotide query
sequences against nucleotide database sequences. See, CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, Chapter 19, Ausubel, et al., eds.,
Greene Publishing and Wiley-Interscience, New York (1995).
[0071] GAP uses the algorithm of Needleman and Wunsch, supra, to
find the alignment of two complete sequences that maximizes the
number of matches and minimizes the number of gaps. GAP considers
all possible alignments and gap positions and creates the alignment
with the largest number of matched bases and the fewest gaps. It
allows for the provision of a gap creation penalty and a gap
extension penalty in units of matched bases. GAP must make a profit
of gap creation penalty number of matches for each gap it inserts.
If a gap extension penalty greater than zero is chosen, GAP must,
in addition, make a profit for each gap inserted of the length of
the gap times the gap extension penalty. Default gap creation
penalty values and gap extension penalty values in Version 10 of
the Wisconsin Genetics Software Package.RTM. are 8 and 2,
respectively. The gap creation and gap extension penalties can be
expressed as an integer selected from the group of integers
consisting of from 0 to 100. Thus, for example, the gap creation
and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 15, 20, 30, 40, 50 or greater.
[0072] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar.
[0073] Symbols that are across from gaps are ignored. A similarity
is scored when the scoring matrix value for a pair of symbols is
greater than or equal to 0.50, the similarity threshold. The
scoring matrix used in Version 10 of the Wisconsin Genetics
Software Package.RTM. is BLOSUM62 (see, Henikoff and Henikoff,
(1989) Proc. Natl. Acad. Sci. USA 89:10915). Unless otherwise
stated, sequence identity/similarity values provided herein refer
to the value obtained using the BLAST 2.0 suite of programs using
default parameters (Altschul, et al., (1997) Nucleic Acids Res.
25:3389-402).
[0074] As those of ordinary skill in the art will understand, BLAST
searches assume that proteins can be modeled as random sequences.
However, many real proteins comprise regions of nonrandom
sequences, which may be homopolymeric tracts, short-period repeats
or regions enriched in one or more amino acids. Such low-complexity
regions may be aligned between unrelated proteins even though other
regions of the protein are entirely dissimilar. A number of
low-complexity filter programs can be employed to reduce such
low-complexity alignments. For example, the SEG (Wooten and
Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie and
States, (1993) Comput. Chem. 17:191-201) low-complexity filters can
be employed alone or in combination.
[0075] As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences includes
reference to the residues in the two sequences, which are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences, which differ by such conservative substitutions, are
said to have "sequence similarity" or "similarity." Means for
making this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., according to the algorithm of
Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17,
e.g., as implemented in the program PC/GENE (Intelligenetics,
Mountain View, Calif., USA).
[0076] As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0077] The term "substantial identity" of polynucleotide sequences
means that a polynucleotide comprises a sequence that has between
50-100% sequence identity, preferably at least 50% sequence
identity, preferably at least 60% sequence identity, preferably at
least 70%, more preferably at least 80%, more preferably at least
90% and most preferably at least 95%, compared to a reference
sequence using one of the alignment programs described using
standard parameters. One of skill will recognize that these values
can be appropriately adjusted to determine corresponding identity
of proteins encoded by two nucleotide sequences by taking into
account codon degeneracy, amino acid similarity, reading frame
positioning and the like. Substantial identity of amino acid
sequences for these purposes normally means sequence identity of
between 55-100%, preferably at least 55%, preferably at least 60%,
more preferably at least 70%, 80%, 90% and most preferably at least
95%.
[0078] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. The degeneracy of the genetic code
allows for many amino acids substitutions that lead to variety in
the nucleotide sequence that code for the same amino acid, hence it
is possible that the DNA sequence could code for the same
polypeptide but not hybridize to each other under stringent
conditions. This may occur, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code. One indication that two nucleic acid sequences are
substantially identical is that the polypeptide, which the first
nucleic acid encodes, is immunologically cross reactive with the
polypeptide encoded by the second nucleic acid.
[0079] The terms "substantial identity" in the context of a peptide
indicates that a peptide comprises a sequence with between 55-100%
sequence identity to a reference sequence preferably at least 55%
sequence identity, preferably 60% preferably 70%, more preferably
80%, most preferably at least 90% or 95% sequence identity to the
reference sequence over a specified comparison window. Preferably,
optimal alignment is conducted using the homology alignment
algorithm of Needleman and Wunsch, supra. An indication that two
peptide sequences are substantially identical is that one peptide
is immunologically reactive with antibodies raised against the
second peptide. Thus, a peptide is substantially identical to a
second peptide, for example, where the two peptides differ only by
a conservative substitution. In addition, a peptide can be
substantially identical to a second peptide when they differ by a
non-conservative change if the epitope that the antibody recognizes
is substantially identical. Peptides, which are "substantially
similar" share sequences as, noted above except that residue
positions, which are not identical, may differ by conservative
amino acid changes.
[0080] The invention discloses GS polynucleotides and polypeptides.
The novel nucleotides and proteins of the invention have an
expression pattern which indicates that they regulate ammonium
transport and thus play an important role in plant development. The
polynucleotides are expressed in various plant tissues. The
polynucleotides and polypeptides thus provide an opportunity to
manipulate plant development to alter seed and vegetative tissue
development, timing or composition. This may be used to create aa
plant with altered N composition in souce and sink.
Nucleic Acids
[0081] The present invention provides, inter alia, isolated nucleic
acids of RNA, DNA and analogs and/or chimeras thereof, comprising a
GS polynucleotide.
[0082] The present invention also includes polynucleotides
optimized for expression in different organisms. For example, for
expression of the polynucleotide in a maize plant, the sequence can
be altered to account for specific codon preferences and to alter
GC content as according to Murray, et al., supra. Maize codon usage
for 28 genes from maize plants is listed in Table 4 of Murray et
al., supra.
[0083] The GS nucleic acids of the present invention comprise
isolated GS polynucleotides which are inclusive of: [0084] (a) a
polynucleotide encoding a GS polypeptide and conservatively
modified and polymorphic variants thereof; [0085] (b) a
polynucleotide having at least 70% sequence identity with
polynucleotides of (a) or (b); [0086] (c) complementary sequences
of polynucleotides of (a) or (b).
Construction of Nucleic Acids
[0087] The isolated nucleic acids of the present invention can be
made using (a) standard recombinant methods, (b) synthetic
techniques, or combinations thereof. In some embodiments, the
polynucleotides of the present invention will be cloned, amplified
or otherwise constructed from a fungus or bacteria.
[0088] The nucleic acids may conveniently comprise sequences in
addition to a polynucleotide of the present invention. For example,
a multi-cloning site comprising one or more endonuclease
restriction sites may be inserted into the nucleic acid to aid in
isolation of the polynucleotide. Also, translatable sequences may
be inserted to aid in the isolation of the translated
polynucleotide of the present invention. For example, a
hexa-histidine marker sequence provides a convenient means to
purify the proteins of the present invention. The nucleic acid of
the present invention--excluding the polynucleotide sequence--is
optionally a vector, adapter or linker for cloning and/or
expression of a polynucleotide of the present invention. Additional
sequences may be added to such cloning and/or expression sequences
to optimize their function in cloning and/or expression, to aid in
isolation of the polynucleotide or to improve the introduction of
the polynucleotide into a cell. Typically, the length of a nucleic
acid of the present invention less the length of its polynucleotide
of the present invention is less than 20 kilobase pairs, often less
than 15 kb, and frequently less than 10 kb. Use of cloning vectors,
expression vectors, adapters and linkers is well known in the art.
Exemplary nucleic acids include such vectors as: M13, lambda ZAP
Express, lambda ZAP II, lambda gt10, lambda gt11, pBK-CMV, pBK-RSV,
pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4,
pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/-, pSG5, pBK,
pCR-Script, pET, pSPUTK, p3'SS, pGEM, pSK+/-, pGEX, pSPORTI and II,
pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pOG44,
pOG45, pFRTf.beta.GAL, pNEO.beta.GAL, pRS403, pRS404, pRS405,
pRS406, pRS413, pRS414, pRS415, pRS416, lambda MOSSlox and lambda
MOSElox. Optional vectors for the present invention, include but
are not limited to, lambda ZAP II and pGEX. For a description of
various nucleic acids see, e.g., Stratagene Cloning Systems,
Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life
Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).
Synthetic Methods for Constructing Nucleic Acids
[0089] The isolated nucleic acids of the present invention can also
be prepared by direct chemical synthesis by methods such as the
phosphotriester method of Narang, et al., (1979) Meth. Enzymol.
68:90-9; the phosphodiester method of Brown, et al., (1979) Meth.
Enzymol. 68:109-51; the diethylphosphoramidite method of Beaucage,
et al., (1981) Tetra. Letts. 22(20):1859-62; the solid phase
phosphoramidite triester method described by Beaucage, et al.,
supra, e.g., using an automated synthesizer, e.g., as described in
Needham-VanDevanter, et al., (1984) Nucleic Acids Res. 12:6159-68
and the solid support method of U.S. Pat. No. 4,458,066. Chemical
synthesis generally produces a single stranded oligonucleotide.
This may be converted into double stranded DNA by hybridization
with a complementary sequence or by polymerization with a DNA
polymerase using the single strand as a template. One of skill will
recognize that while chemical synthesis of DNA is limited to
sequences of about 100 bases, longer sequences may be obtained by
the ligation of shorter sequences.
UTRs and Codon Preference
[0090] In general, translational efficiency has been found to be
regulated by specific sequence elements in the 5' non-coding or
untranslated region (5' UTR) of the RNA. Positive sequence motifs
include translational initiation consensus sequences (Kozak, (1987)
Nucleic Acids Res. 15:8125) and the 5<G> 7 methyl GpppG RNA
cap structure (Drummond, et al., (1985) Nucleic Acids Res.
13:7375). Negative elements include stable intramolecular 5' UTR
stem-loop structures (Muesing, et al., (1987) Cell 48:691) and AUG
sequences or short open reading frames preceded by an appropriate
AUG in the 5' UTR (Kozak, supra, Rao, et al., (1988) Mol. and Cell.
Biol. 8:284). Accordingly, the present invention provides 5' and/or
3' UTR regions for modulation of translation of heterologous coding
sequences.
[0091] Further, the polypeptide-encoding segments of the
polynucleotides of the present invention can be modified to alter
codon usage. Altered codon usage can be employed to alter
translational efficiency and/or to optimize the coding sequence for
expression in a desired host or to optimize the codon usage in a
heterologous sequence for expression in maize. Codon usage in the
coding regions of the polynucleotides of the present invention can
be analyzed statistically using commercially available software
packages such as "Codon Preference" available from the University
of Wisconsin Genetics Computer Group. See, Devereaux, et al.,
(1984) Nucleic Acids Res. 12:387-395 or MacVector 4.1 (Eastman
Kodak Co., New Haven, Conn.). Thus, the present invention provides
a codon usage frequency characteristic of the coding region of at
least one of the polynucleotides of the present invention. The
number of polynucleotides (3 nucleotides per amino acid) that can
be used to determine a codon usage frequency can be any integer
from 3 to the number of polynucleotides of the present invention as
provided herein. Optionally, the polynucleotides will be
full-length sequences. An exemplary number of sequences for
statistical analysis can be at least 1, 5, 10, 20, 50 or 100.
Sequence Shuffling
[0092] The present invention provides methods for sequence
shuffling using polynucleotides of the present invention, and
compositions resulting therefrom. Sequence shuffling is described
in PCT Publication Number 96/19256. See also, Zhang, et al., (1997)
Proc. Natl. Acad. Sci. USA 94:4504-9 and Zhao, et al., (1998)
Nature Biotech 16:258-61. Generally, sequence shuffling provides a
means for generating libraries of polynucleotides having a desired
characteristic, which can be selected or screened for. Libraries of
recombinant polynucleotides are generated from a population of
related sequence polynucleotides, which comprise sequence regions,
which have substantial sequence identity and can be homologously
recombined in vitro or in vivo. The population of
sequence-recombined polynucleotides comprises a subpopulation of
polynucleotides which possess desired or advantageous
characteristics and which can be selected by a suitable selection
or screening method. The characteristics can be any property or
attribute capable of being selected for or detected in a screening
system, and may include properties of: an encoded protein, a
transcriptional element, a sequence controlling transcription, RNA
processing, RNA stability, chromatin conformation, translation or
other expression property of a gene or transgene, a replicative
element, a protein-binding element, or the like, such as any
feature which confers a selectable or detectable property. In some
embodiments, the selected characteristic will be an altered K.sub.m
and/or K.sub.cat over the wild-type protein as provided herein. In
other embodiments, a protein or polynucleotide generated from
sequence shuffling will have a ligand binding affinity greater than
the non-shuffled wild-type polynucleotide. In yet other
embodiments, a protein or polynucleotide generated from sequence
shuffling will have an altered pH optimum as compared to the
non-shuffled wild-type polynucleotide. The increase in such
properties can be at least 110%, 120%, 130%, 140% or greater than
150% of the wild-type value.
Recombinant Expression Cassettes
[0093] The present invention further provides recombinant
expression cassettes comprising a nucleic acid of the present
invention. A nucleic acid sequence coding for the desired
polynucleotide of the present invention, for example a cDNA or a
genomic sequence encoding a polypeptide long enough to code for an
active protein of the present invention, can be used to construct a
recombinant expression cassette which can be introduced into the
desired host cell. A recombinant expression cassette will typically
comprise a polynucleotide of the present invention operably linked
to transcriptional initiation regulatory sequences which will
direct the transcription of the polynucleotide in the intended host
cell, such as tissues of a transformed plant.
[0094] For example, plant expression vectors may include (1) a
cloned plant gene under the transcriptional control of 5' and 3'
regulatory sequences and (2) a dominant selectable marker. Such
plant expression vectors may also contain, if desired, a promoter
regulatory region (e.g., one conferring inducible or constitutive,
environmentally- or developmentally-regulated or cell- or
tissue-specific/selective expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site and/or a polyadenylation signal.
[0095] A plant promoter fragment can be employed which will direct
expression of a polynucleotide of the present invention in all
tissues of a regenerated plant. Such promoters are referred to
herein as "constitutive" promoters and are active under most
environmental conditions and states of development or cell
differentiation. Examples of constitutive promoters include the 1'-
or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the
Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S.
Pat. No. 5,683,439), the Nos promoter, the rubisco promoter, the
GRP1-8 promoter, the 35S promoter from cauliflower mosaic virus
(CaMV), as described in Odell, et al., (1985) Nature 313:810-2;
rice actin (McElroy, et al., (1990) Plant Cell 163-171); ubiquitin
(Christensen, et al., (1992) Plant Mol. Biol. 12:619-632 and
Christensen, et al., (1992) Plant Mol. Biol. 18:675-89); pEMU
(Last, et al., (1991) Theor. Appl. Genet. 81:581-8); MAS (Velten,
et al., (1984) EMBO J. 3:2723-30) and maize H3 histone (Lepetit, et
al., (1992) Mol. Gen. Genet. 231:276-85 and Atanassvoa, et al.,
(1992) Plant Journal 2(3):291-300); ALS promoter, as described in
PCT Application Number WO 96/30530 and other transcription
initiation regions from various plant genes known to those of
skill. For the present invention ubiquitin is the preferred
promoter for expression in monocot plants.
[0096] Alternatively, the plant promoter can direct expression of a
polynucleotide of the present invention in a specific tissue or may
be otherwise under more precise environmental or developmental
control. Such promoters are referred to here as "inducible"
promoters. Environmental conditions that may effect transcription
by inducible promoters include pathogen attack, anaerobic
conditions or the presence of light. Examples of inducible
promoters are the Adh1 promoter, which is inducible by hypoxia or
cold stress, the Hsp70 promoter, which is inducible by heat stress
and the PPDK promoter, which is inducible by light.
[0097] Examples of promoters under developmental control include
promoters that initiate transcription only, or preferentially, in
certain tissues, such as leaves, roots, fruit, seeds or flowers.
The operation of a promoter may also vary depending on its location
in the genome. Thus, an inducible promoter may become fully or
partially constitutive in certain locations.
[0098] If polypeptide expression is desired, it is generally
desirable to include a polyadenylation region at the 3'-end of a
polynucleotide coding region. The polyadenylation region can be
derived from a variety of plant genes or from T-DNA. The 3' end
sequence to be added can be derived from, for example, the nopaline
synthase or octopine synthase genes or alternatively from another
plant gene or less preferably from any other eukaryotic gene.
Examples of such regulatory elements include, but are not limited
to, 3' termination and/or polyadenylation regions such as those of
the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan,
et al., (1983) Nucleic Acids Res. 12:369-85); the potato proteinase
inhibitor II (PINII) gene (Keil, et al., (1986) Nucleic Acids Res.
14:5641-50 and An, et al., (1989) Plant Cell 1:115-22) and the CaMV
19S gene (Mogen, et al., (1990) Plant Cell 2:1261-72).
[0099] An intron sequence can be added to the 5' untranslated
region or the coding sequence of the partial coding sequence to
increase the amount of the mature message that accumulates in the
cytosol. Inclusion of a spliceable intron in the transcription unit
in both plant and animal expression constructs has been shown to
increase gene expression at both the mRNA and protein levels up to
1000-fold (Buchman and Berg, (1988) Mol. Cell Biol. 8:4395-4405;
Callis, et al., (1987) Genes Dev. 1:1183-200). Such intron
enhancement of gene expression is typically greatest when placed
near the 5' end of the transcription unit. Use of maize introns
Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the
art. See generally, THE MAIZE HANDBOOK, Chapter 116, Freeling and
Walbot, eds., Springer, N.Y. (1994).
[0100] Plant signal sequences, including, but not limited to,
signal-peptide encoding DNA/RNA sequences which target proteins to
the extracellular matrix of the plant cell (Dratewka-Kos, et al.,
(1989) J. Biol. Chem. 264:4896-900), such as the Nicotiana
plumbaginifolia extension gene (DeLoose, et al., (1991) Gene
99:95-100); signal peptides which target proteins to the vacuole,
such as the sweet potato sporamin gene (Matsuka, et al., (1991)
Proc. Natl. Acad. Sci. USA 88:834) and the barley lectin gene
(Wilkins, et al., (1990) Plant Cell, 2:301-13); signal peptides
which cause proteins to be secreted, such as that of PRIb (Lind, et
al., (1992) Plant Mol. Biol. 18:47-53) or the barley alpha amylase
(BAA) (Rahmatullah, et al., (1989) Plant Mol. Biol. 12:119, and
hereby incorporated by reference) or signal peptides which target
proteins to the plastids such as that of rapeseed enoyl-Acp
reductase (Verwaert, et al., (1994) Plant Mol. Biol. 26:189-202)
are useful in the invention. The barley alpha amylase signal
sequence fused to the GS polynucleotide is the preferred construct
for expression in maize for the present invention.
[0101] The vector comprising the sequences from a polynucleotide of
the present invention will typically comprise a marker gene, which
confers a selectable phenotype on plant cells. Usually, the
selectable marker gene will encode antibiotic resistance, with
suitable genes including genes coding for resistance to the
antibiotic spectinomycin (e.g., the aada gene), the streptomycin
phosphotransferase (SPT) gene coding for streptomycin resistance,
the neomycin phosphotransferase (NPTII) gene encoding kanamycin or
geneticin resistance, the hygromycin phosphotransferase (HPT) gene
coding for hygromycin resistance, genes coding for resistance to
herbicides which act to inhibit the action of acetolactate synthase
(ALS), in particular the sulfonylurea-type herbicides (e.g., the
acetolactate synthase (ALS) gene containing mutations leading to
such resistance in particular the S4 and/or Hra mutations), genes
coding for resistance to herbicides which act to inhibit action of
glutamine synthase, such as phosphinothricin or basta (e.g., the
bar gene), or other such genes known in the art. The bar gene
encodes resistance to the herbicide basta and the ALS gene encodes
resistance to the herbicide chlorsulfuron. Typical vectors useful
for expression of genes in higher plants are well known in the art
and include vectors derived from the tumor-inducing (Ti) plasmid of
Agrobacterium tumefaciens described by Rogers, et al., (1987) Meth.
Enzymol. 153:253-77. These vectors are plant integrating vectors in
that on transformation, the vectors integrate a portion of vector
DNA into the genome of the host plant. Exemplary A. tumefaciens
vectors useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et
al., (1987) Gene 61:1-11 and Berger, et al., (1989) Proc. Natl.
Acad. Sci. USA, 86:8402-6. Another useful vector herein is plasmid
pBI101.2 that is available from CLONTECH Laboratories, Inc. (Palo
Alto, Calif.).
Expression of Proteins in Host Cells
[0102] Using the nucleic acids of the present invention, one may
express a protein of the present invention in a recombinantly
engineered cell such as bacteria, yeast, insect, mammalian or
preferably plant cells. The cells produce the protein in a
non-natural condition (e.g., in quantity, composition, location
and/or time), because they have been genetically altered through
human intervention to do so.
[0103] It is expected that those of skill in the art are
knowledgeable in the numerous expression systems available for
expression of a nucleic acid encoding a protein of the present
invention. No attempt to describe in detail the various methods
known for the expression of proteins in prokaryotes or eukaryotes
will be made.
[0104] In brief summary, the expression of isolated nucleic acids
encoding a protein of the present invention will typically be
achieved by operably linking, for example, the DNA or cDNA to a
promoter (which is either constitutive or inducible), followed by
incorporation into an expression vector. The vectors can be
suitable for replication and integration in either prokaryotes or
eukaryotes. Typical expression vectors contain transcription and
translation terminators, initiation sequences and promoters useful
for regulation of the expression of the DNA encoding a protein of
the present invention. To obtain high level expression of a cloned
gene, it is desirable to construct expression vectors which
contain, at the minimum, a strong promoter, such as ubiquitin, to
direct transcription, a ribosome binding site for translational
initiation and a transcription/translation terminator. Constitutive
promoters are classified as providing for a range of constitutive
expression. Thus, some are weak constitutive promoters and others
are strong constitutive promoters. Generally, by "weak promoter" is
intended a promoter that drives expression of a coding sequence at
a low level. By "low level" is intended at levels of about 1/10,000
transcripts to about 1/100,000 transcripts to about 1/500,000
transcripts. Conversely, a "strong promoter" drives expression of a
coding sequence at a "high level," or about 1/10 transcripts to
about 1/100 transcripts to about 1/1,000 transcripts.
[0105] One of skill would recognize that modifications could be
made to a protein of the present invention without diminishing its
biological activity. Some modifications may be made to facilitate
the cloning, expression or incorporation of the targeting molecule
into a fusion protein. Such modifications are well known to those
of skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site or additional
amino acids (e.g., poly His) placed on either terminus to create
conveniently located restriction sites or termination codons or
purification sequences.
Expression in Prokaryotes
[0106] Prokaryotic cells may be used as hosts for expression.
Prokaryotes most frequently are represented by various strains of
E. coli; however, other microbial strains may also be used.
Commonly used prokaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding site
sequences, include such commonly used promoters as the beta
lactamase (penicillinase) and lactose (lac) promoter systems
(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp)
promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057)
and the lambda derived P L promoter and N-gene ribosome binding
site (Shimatake, et al., (1981) Nature 292:128). The inclusion of
selection markers in DNA vectors transfected in E. coli is also
useful. Examples of such markers include genes specifying
resistance to ampicillin, tetracycline or chloramphenicol.
[0107] The vector is selected to allow introduction of the gene of
interest into the appropriate host cell. Bacterial vectors are
typically of plasmid or phage origin. Appropriate bacterial cells
are infected with phage vector particles or transfected with naked
phage vector DNA. If a plasmid vector is used, the bacterial cells
are transfected with the plasmid vector DNA. Expression systems for
expressing a protein of the present invention are available using
Bacillus sp. and Salmonella (Palva, et al., (1983) Gene 22:229-35;
Mosbach, et al., (1983) Nature 302:543-5). The pGEX-4T-1 plasmid
vector from Pharmacia is the preferred E. coli expression vector
for the present invention.
Expression in Eukaryotes
[0108] A variety of eukaryotic expression systems such as yeast,
insect cell lines, plant and mammalian cells, are known to those of
skill in the art. As explained briefly below, the present invention
can be expressed in these eukaryotic systems. In some embodiments,
transformed/transfected plant cells, as discussed infra, are
employed as expression systems for production of the proteins of
the instant invention.
[0109] Synthesis of heterologous proteins in yeast is well known.
Sherman, et al., METHODS IN YEAST GENETICS, Cold Spring Harbor
Laboratory (1982) is a well recognized work describing the various
methods available to produce the protein in yeast.
[0110] Two widely utilized yeasts for production of eukaryotic
proteins are Saccharomyces cerevisiae and Pichia pastoris. Vectors,
strains and protocols for expression in Saccharomyces and Pichia
are known in the art and available from commercial suppliers (e.g.,
Invitrogen). Suitable vectors usually have expression control
sequences, such as promoters, including 3-phosphoglycerate kinase
or alcohol oxidase and an origin of replication, termination
sequences and the like as desired.
[0111] A protein of the present invention, once expressed, can be
isolated from yeast by lysing the cells and applying standard
protein isolation techniques to the lysates or the pellets. The
monitoring of the purification process can be accomplished by using
Western blot techniques or radioimmunoassay of other standard
immunoassay techniques.
[0112] The sequences encoding proteins of the present invention can
also be ligated to various expression vectors for use in
transfecting cell cultures of, for instance, mammalian, insect or
plant origin. Mammalian cell systems often will be in the form of
monolayers of cells although mammalian cell suspensions may also be
used. A number of suitable host cell lines capable of expressing
intact proteins have been developed in the art, and include the
HEK293, BHK21 and CHO cell lines. Expression vectors for these
cells can include expression control sequences, such as an origin
of replication, a promoter (e.g., the CMV promoter, a HSV tk
promoter or pgk (phosphoglycerate kinase) promoter), an enhancer
(Queen, et al., (1986) Immunol. Rev. 89:49) and necessary
processing information sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly
A addition site) and transcriptional terminator sequences. Other
animal cells useful for production of proteins of the present
invention are available, for instance, from the American Type
Culture Collection Catalogue of Cell Lines and Hybridomas (7.sup.th
ed., 1992).
[0113] Appropriate vectors for expressing proteins of the present
invention in insect cells are usually derived from the SF9
baculovirus. Suitable insect cell lines include mosquito larvae,
silkworm, armyworm, moth and Drosophila cell lines such as a
Schneider cell line (see, e.g., Schneider, (1987) J. Embryol. Exp.
Morphol. 27:353-65).
[0114] As with yeast, when higher animal or plant host cells are
employed, polyadenylation or transcription terminator sequences are
typically incorporated into the vector. An example of a terminator
sequence is the polyadenylation sequence from the bovine growth
hormone gene. Sequences for accurate splicing of the transcript may
also be included. An example of a splicing sequence is the VP1
intron from SV40 (Sprague, et al., (1983) J. Virol. 45:773-81).
Additionally, gene sequences to control replication in the host
cell may be incorporated into the vector such as those found in
bovine papilloma virus type-vectors (Saveria-Campo, "Bovine
Papilloma Virus DNA a Eukaryotic Cloning Vector," in DNA CLONING: A
PRACTICAL APPROACH, vol. II, Glover, ed., IRL Press, Arlington,
Va., pp. 213-38 (1985)).
[0115] In addition, the gene for GS placed in the appropriate plant
expression vector can be used to transform plant cells. The
polypeptide can then be isolated from plant callus or the
transformed cells can be used to regenerate transgenic plants. Such
transgenic plants can be harvested, and the appropriate tissues
(seed or leaves, for example) can be subjected to large scale
protein extraction and purification techniques.
Plant Transformation Methods
[0116] Numerous methods for introducing foreign genes into plants
are known and can be used to insert a GS polynucleotide into a
plant host, including biological and physical plant transformation
protocols. See, e.g., Miki et al., "Procedure for Introducing
Foreign DNA into Plants," in METHODS IN PLANT MOLECULAR BIOLOGY AND
BIOTECHNOLOGY, Glick and Thompson, eds., CRC Press, Inc., Boca
Raton, pp. 67-88 (1993). The methods chosen vary with the host
plant, and include chemical transfection methods such as calcium
phosphate, microorganism-mediated gene transfer such as
Agrobacterium (Horsch, et al., (1985) Science 227:1229-31),
electroporation, micro-injection and biolistic bombardment.
[0117] Expression cassettes and vectors and in vitro culture
methods for plant cell or tissue transformation and regeneration of
plants are known and available. See, e.g., Gruber, et al., "Vectors
for Plant Transformation," in METHODS IN PLANT MOLECULAR BIOLOGY
AND BIOTECHNOLOGY, supra, pp. 89-119.
[0118] The isolated polynucleotides or polypeptides may be
introduced into the plant by one or more techniques typically used
for direct delivery into cells. Such protocols may vary depending
on the type of organism, cell, plant or plant cell, i.e. monocot or
dicot, targeted for gene modification. Suitable methods of
transforming plant cells include microinjection (Crossway, et al.,
(1986) Biotechniques 4:320-334 and U.S. Pat. No. 6,300,543),
electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA
83:5602-5606, direct gene transfer (Paszkowski, et al., (1984) EMBO
J. 3:2717-2722) and ballistic particle acceleration (see, for
example, Sanford, et al., U.S. Pat. No. 4,945,050; WO 91/10725 and
McCabe, et al., (1988) Biotechnology 6:923-926). Also see, Tomes,
et al., Direct DNA Transfer into Intact Plant Cells Via
Microprojectile Bombardment. pp. 197-213 in Plant Cell, Tissue and
Organ Culture, Fundamental Methods. eds. Gamborg and Phillips,
Springer-Verlag Berlin Heidelberg N.Y., 1995; U.S. Pat. No.
5,736,369 (meristem); Weissinger, et al., (1988) Ann. Rev. Genet.
22:421-477; Sanford, et al., (1987) Particulate Science and
Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol.
87:671-674 (soybean); Datta, et al., (1990) Biotechnology 8:736-740
(rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA
85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563
(maize); WO 91/10725 (maize); Klein, et al., (1988) Plant Physiol.
91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839
and Gordon-Kamm, et al., (1990) Plant Cell 2:603-618 (maize);
Hooydaas-Van Slogteren and Hooykaas, (1984) Nature (London)
311:763-764; Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA
84:5345-5349 (Liliaceae); De Wet, et al., (1985) In The
Experimental Manipulation of Ovule Tissues, ed. Chapman, et al.,
pp. 197-209 Longman, N.Y. (pollen); Kaeppler, et al., (1990) Plant
Cell Reports 9:415-418 and Kaeppler, et al., (1992) Theor. Appl.
Genet. 84:560-566 (whisker-mediated transformation); U.S. Pat. No.
5,693,512 (sonication); D'Halluin, et al., (1992) Plant Cell
4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell
Reports 12:250-255 and Christou and Ford (1995) Annals of Botany
75:407-413 (rice); Osjoda, et al., (1996) Nature Biotech.
14:745-750; Agrobacterium mediated maize transformation (U.S. Pat.
No. 5,981,840); silicon carbide whisker methods (Frame, et al.,
(1994) Plant J. 6:941-948); laser methods (Guo, et al., (1995)
Physiologia Plantarum 93:19-24); sonication methods (Bao, et al.,
(1997) Ultrasound in Medicine & Biology 23:953-959; Finer and
Finer, (2000) Lett Appl Microbiol. 30:406-10; Amoah, et al., (2001)
J Exp Bot 52:1135-42); polyethylene glycol methods (Krens, et al.,
(1982) Nature 296:72-77); protoplasts of monocot and dicot cells
can be transformed using electroporation (Fromm, et al., (1985)
Proc. Natl. Acad. Sci. USA 82:5824-5828) and microinjection
(Crossway, et al., (1986) Mol. Gen. Genet. 202:179-185), all of
which are herein incorporated by reference.
Agrobacterium-Mediated Transformation
[0119] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium. A. tumefaciens and A.
rhizogenes are plant pathogenic soil bacteria, which genetically
transform plant cells. The Ti and Ri plasmids of A. tumefaciens and
A. rhizogenes, respectively, carry genes responsible for genetic
transformation of plants. See, e.g., Kado, (1991) Crit. Rev. Plant
Sci. 10:1. Descriptions of the Agrobacterium vector systems and
methods for Agrobacterium-mediated gene transfer are provided in
Gruber, et al., supra; Miki, et al., supra and Moloney, et al.,
(1989) Plant Cell Reports 8:238.
[0120] Similarly, the gene can be inserted into the T-DNA region of
a Ti or Ri plasmid derived from A. tumefaciens or A. rhizogenes,
respectively. Thus, expression cassettes can be constructed as
above, using these plasmids. Many control sequences are known which
when coupled to a heterologous coding sequence and transformed into
a host organism show fidelity in gene expression with respect to
tissue/organ specificity of the original coding sequence. See,
e.g., Benfey and Chua, (1989) Science 244:174-81. Particularly
suitable control sequences for use in these plasmids are promoters
for constitutive leaf-specific expression of the gene in the
various target plants. Other useful control sequences include a
promoter and terminator from the nopaline synthase gene (NOS). The
NOS promoter and terminator are present in the plasmid pARC2,
available from the American Type Culture Collection and designated
ATCC 67238. If such a system is used, the virulence (vir) gene from
either the Ti or Ri plasmid must also be present, either along with
the T-DNA portion, or via a binary system where the vir gene is
present on a separate vector. Such systems, vectors for use
therein, and methods of transforming plant cells are described in
U.S. Pat. No. 4,658,082; U.S. patent application Ser. No. 913,914,
filed Oct. 1, 1986, as referenced in U.S. Pat. No. 5,262,306,
issued Nov. 16, 1993 and Simpson, et al., (1986) Plant Mol. Biol.
6:403-15 (also referenced in the '306 patent), all incorporated by
reference in their entirety.
[0121] Once constructed, these plasmids can be placed into A.
rhizogenes or A. tumefaciens and these vectors used to transform
cells of plant species, which are ordinarily susceptible to
Fusarium or Alternaria infection. Several other transgenic plants
are also contemplated by the present invention including but not
limited to soybean, corn, sorghum, alfalfa, rice, clover, cabbage,
banana, coffee, celery, tobacco, cowpea, cotton, melon,
switchgrass, myscanthus, triticale and pepper. The selection of
either A. tumefaciens or A. rhizogenes will depend on the plant
being transformed thereby. In general A. tumefaciens is the
preferred organism for transformation. Most dicotyledonous plants,
some gymnosperms, and a few monocotyledonous plants (e.g., certain
members of the Liliales and Arales) are susceptible to infection
with A. tumefaciens. A. rhizogenes also has a wide host range,
embracing most dicots and some gymnosperms, which includes members
of the Leguminosae, Compositae and Chenopodiaceae. Monocot plants
can now be transformed with some success. EP Patent Application
Number 604 662 A1 discloses a method for transforming monocots
using Agrobacterium. EP Patent Application Number 672 752 A1
discloses a method for transforming monocots with Agrobacterium
using the scutellum of immature embryos. Ishida, et al., discuss a
method for transforming maize by exposing immature embryos to A.
tumefaciens (Nature Biotechnology 14:745-50 (1996)).
[0122] Once transformed, these cells can be used to regenerate
transgenic plants. For example, whole plants can be infected with
these vectors by wounding the plant and then introducing the vector
into the wound site. Any part of the plant can be wounded,
including leaves, stems and roots. Alternatively, plant tissue, in
the form of an explant, such as cotyledonary tissue or leaf disks,
can be inoculated with these vectors, and cultured under
conditions, which promote plant regeneration. Roots or shoots
transformed by inoculation of plant tissue with A. rhizogenes or A.
tumefaciens, containing the gene coding for the fumonisin
degradation enzyme, can be used as a source of plant tissue to
regenerate fumonisin-resistant transgenic plants, either via
somatic embryogenesis or organogenesis. Examples of such methods
for regenerating plant tissue are disclosed in Shahin, (1985)
Theor. Appl. Genet. 69:235-40; U.S. Pat. No. 4,658,082; Simpson, et
al., supra and U.S. patent application Ser. Nos. 913,913 and
913,914, both filed Oct. 1, 1986, as referenced in U.S. Pat. No.
5,262,306, issued Nov. 16, 1993, the entire disclosures therein
incorporated herein by reference.
Direct Gene Transfer
[0123] Despite the fact that the host range for
Agrobacterium-mediated transformation is broad, some major cereal
crop species and gymnosperms have generally been recalcitrant to
this mode of gene transfer, even though some success has recently
been achieved in rice (Hiei, et al., (1994) The Plant Journal
6:271-82). Several methods of plant transformation, collectively
referred to as direct gene transfer, have been developed as an
alternative to Agrobacterium-mediated transformation.
[0124] A generally applicable method of plant transformation is
microprojectile-mediated transformation, where DNA is carried on
the surface of microprojectiles measuring about 1 to 4 .mu.m. The
expression vector is introduced into plant tissues with a biolistic
device that accelerates the microprojectiles to speeds of 300 to
600 m/s which is sufficient to penetrate the plant cell walls and
membranes (Sanford, et al., (1987) Part. Sci. Technol. 5:27;
Sanford, (1988) Trends Biotech 6:299; Sanford, (1990) Physiol.
Plant 79:206 and Klein, et al., (1992) Biotechnology 10:268).
[0125] Another method for physical delivery of DNA to plants is
sonication of target cells as described in Zang, et al., (1991)
BioTechnology 9:996. Alternatively, liposome or spheroplast fusions
have been used to introduce expression vectors into plants. See,
e.g., Deshayes, et al., (1985) EMBO J. 4:2731 and Christou, et al.,
(1987) Proc. Natl. Acad. Sci. USA 84:3962. Direct uptake of DNA
into protoplasts using CaCl.sub.2 precipitation, polyvinyl alcohol
or poly-L-ornithine has also been reported. See, e.g., Hain, et
al., (1985) Mol. Gen. Genet. 199:161 and Draper, et al., (1982)
Plant Cell Physiol. 23:451. Electroporation of protoplasts and
whole cells and tissues has also been described. See, e.g., Donn,
et al., (1990) in Abstracts of the VIIth Int'l. Congress on Plant
Cell and Tissue Culture IAPTC, A2-38, p. 53; D'Halluin, et al.,
(1992) Plant Cell 4:1495-505 and Spencer, et al., (1994) Plant Mol.
Biol. 24:51-61.
Increasing the Activity and/or Level of a GS Polypeptide
[0126] Methods are provided to increase the activity and/or level
of the GS polypeptide of the invention. An increase in the level
and/or activity of the GS polypeptide of the invention can be
achieved by providing to the plant a GS polypeptide. The GS
polypeptide can be provided by introducing the amino acid sequence
encoding the GS polypeptide into the plant, introducing into the
plant a nucleotide sequence encoding a GS polypeptide or
alternatively by modifying a genomic locus encoding the GS
polypeptide of the invention.
[0127] As discussed elsewhere herein, many methods are known the
art for providing a polypeptide to a plant including, but not
limited to, direct introduction of the polypeptide into the plant,
introducing into the plant (transiently or stably) a polynucleotide
construct encoding a polypeptide having GS enzyme activity. It is
also recognized that the methods of the invention may employ a
polynucleotide that is not capable of directing, in the transformed
plant, the expression of a protein or an RNA. Thus, the level
and/or activity of a GS polypeptide may be increased by altering
the gene encoding the GS polypeptide or its promoter. See, e.g.,
Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al., PCT/US93/03868.
Therefore mutagenized plants that carry mutations in GS genes,
where the mutations increase expression of the GS gene or increase
the GS enzyme activity of the encoded GS polypeptide are
provided.
Reducing the Activity and/or Level of a GS Polypeptide
[0128] Methods are provided to reduce or eliminate the activity of
a GS polypeptide of the invention by transforming a plant cell with
an expression cassette that expresses a polynucleotide that
inhibits the expression of the GS polypeptide. The polynucleotide
may inhibit the expression of the GS polypeptide directly, by
preventing transcription or translation of the GS messenger RNA, or
indirectly, by encoding a polypeptide that inhibits the
transcription or translation of a GS gene encoding a GS
polypeptide. Methods for inhibiting or eliminating the expression
of a gene in a plant are well known in the art, and any such method
may be used in the present invention to inhibit the expression of a
GS polypeptide.
[0129] In accordance with the present invention, the expression of
a GS polypeptide is inhibited if the protein level of the GS
polypeptide is less than 70% of the protein level of the same GS
polypeptide in a plant that has not been genetically modified or
mutagenized to inhibit the expression of that GS polypeptide. In
particular embodiments of the invention, the protein level of the
GS polypeptide in a modified plant according to the invention is
less than 60%, less than 50%, less than 40%, less than 30%, less
than 20%, less than 10%, less than 5% or less than 2% of the
protein level of the same GS polypeptide in a plant that is not a
mutant or that has not been genetically modified to inhibit the
expression of that GS polypeptide. The expression level of the GS
polypeptide may be measured directly, for example, by assaying for
the level of GS polypeptide expressed in the plant cell or plant,
or indirectly, for example, by measuring the GS enzyme activity of
the GS polypeptide in the plant cell or plant or by measuring the
GS in the plant. Methods for performing such assays are described
elsewhere herein.
[0130] In other embodiments of the invention, the activity of the
GS polypeptides is reduced or eliminated by transforming a plant
cell with an expression cassette comprising a polynucleotide
encoding a polypeptide that inhibits the activity of a GS
polypeptide. The GS enzyme activity of a GS polypeptide is
inhibited according to the present invention if the GS enzyme
activity of the GS polypeptide is less than 70% of the GS enzyme
activity of the same GS polypeptide in a plant that has not been
modified to inhibit the GS enzyme activity of that GS polypeptide.
In particular embodiments of the invention, the GS enzyme activity
of the GS polypeptide in a modified plant according to the
invention is less than 60%, less than 50%, less than 40%, less than
30%, less than 20%, less than 10% or less than 5% of the GS enzyme
activity of the same GS polypeptide in a plant that that has not
been modified to inhibit the expression of that GS polypeptide. The
GS enzyme activity of a GS polypeptide is "eliminated" according to
the invention when it is not detectable by the assay methods
described elsewhere herein. Methods of determining the GS enzyme
activity of a GS polypeptide are described elsewhere herein.
[0131] In other embodiments, the activity of a GS polypeptide may
be reduced or eliminated by disrupting the gene encoding the GS
polypeptide. The invention encompasses mutagenized plants that
carry mutations in GS genes, where the mutations reduce expression
of the GS gene or inhibit the GS enzyme activity of the encoded GS
polypeptide.
[0132] Thus, many methods may be used to reduce or eliminate the
activity of a GS polypeptide. In addition, more than one method may
be used to reduce the activity of a single GS polypeptide.
Non-limiting examples of methods of reducing or eliminating the
expression of GS polypeptides are given below.
[0133] 1. Polynucleotide-Based Methods:
[0134] In some embodiments of the present invention, a plant is
transformed with an expression cassette that is capable of
expressing a polynucleotide that inhibits the expression of a GS
polypeptide of the invention. The term "expression" as used herein
refers to the biosynthesis of a gene product, including the
transcription and/or translation of said gene product. For example,
for the purposes of the present invention, an expression cassette
capable of expressing a polynucleotide that inhibits the expression
of at least one GS polypeptide is an expression cassette capable of
producing an RNA molecule that inhibits the transcription and/or
translation of at least one GS polypeptide of the invention. The
"expression" or "production" of a protein or polypeptide from a DNA
molecule refers to the transcription and translation of the coding
sequence to produce the protein or polypeptide, while the
"expression" or "production" of a protein or polypeptide from an
RNA molecule refers to the translation of the RNA coding sequence
to produce the protein or polypeptide.
[0135] Examples of polynucleotides that inhibit the expression of a
GS polypeptide are given below.
[0136] i. Sense Suppression/Cosuppression
[0137] In some embodiments of the invention, inhibition of the
expression of a GS polypeptide may be obtained by sense suppression
or cosuppression. For cosuppression, an expression cassette is
designed to express an RNA molecule corresponding to all or part of
a messenger RNA encoding a GS polypeptide in the "sense"
orientation. Over expression of the RNA molecule can result in
reduced expression of the native gene. Accordingly, multiple plant
lines transformed with the cosuppression expression cassette are
screened to identify those that show the greatest inhibition of GS
polypeptide expression.
[0138] The polynucleotide used for cosuppression may correspond to
all or part of the sequence encoding the GS polypeptide, all or
part of the 5' and/or 3' untranslated region of a GS polypeptide
transcript, or all or part of both the coding sequence and the
untranslated regions of a transcript encoding a GS polypeptide. In
some embodiments where the polynucleotide comprises all or part of
the coding region for the GS polypeptide, the expression cassette
is designed to eliminate the start codon of the polynucleotide so
that no protein product will be translated.
[0139] Cosuppression may be used to inhibit the expression of plant
genes to produce plants having undetectable protein levels for the
proteins encoded by these genes. See, for example, Broin, et al.,
(2002) Plant Cell 14:1417-1432. Cosuppression may also be used to
inhibit the expression of multiple proteins in the same plant. See,
for example, U.S. Pat. No. 5,942,657. Methods for using
cosuppression to inhibit the expression of endogenous genes in
plants are described in Flavell, et al., (1994) Proc. Natl. Acad.
Sci. USA 91:3490-3496; Jorgensen, et al., (1996) Plant Mol. Biol.
31:957-973; Johansen and Carrington, (2001) Plant Physiol.
126:930-938; Broin, et al., (2002) Plant Cell 14:1417-1432;
Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Yu, et
al., (2003) Phytochemistry 63:753-763; and U.S. Pat. Nos.
5,034,323, 5,283,184 and 5,942,657, each of which is herein
incorporated by reference. The efficiency of cosuppression may be
increased by including a poly-dT region in the expression cassette
at a position 3' to the sense sequence and 5' of the
polyadenylation signal. See, US Patent Application Publication
Number 2002/0048814, herein incorporated by reference. Typically,
such a nucleotide sequence has substantial sequence identity to the
sequence of the transcript of the endogenous gene, optimally
greater than about 65% sequence identity, more optimally greater
than about 85% sequence identity, most optimally greater than about
95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323,
herein incorporated by reference.
[0140] ii. Sense Suppression
[0141] In some embodiments of the invention, inhibition of the
expression of the GS polypeptide may be obtained by sense
suppression. For sense suppression, the expression cassette is
designed to express an RNA molecule complementary to all or part of
a messenger RNA encoding the GS polypeptide. Over expression of the
sense RNA molecule can result in reduced expression of the native
gene. Accordingly, multiple plant lines transformed with the sense
suppression expression cassette are screened to identify those that
show the greatest inhibition of GS polypeptide expression. The
polynucleotide for use in sense suppression may correspond to all
or part of the complement of the sequence encoding the GS
polypeptide, all or part of the complement of the 5' and/or 3'
untranslated region of the GS transcript or all or part of the
complement of both the coding sequence and the untranslated regions
of a transcript encoding the GS polypeptide. In addition, the sense
polynucleotide may be fully complementary (i.e., 100% identical to
the complement of the target sequence) or partially complementary
(i.e., less than 100% identical to the complement of the target
sequence) to the target sequence. Sense suppression may be used to
inhibit the expression of multiple proteins in the same plant. See,
for example, U.S. Pat. No. 5,942,657. Furthermore, portions of the
sense nucleotides may be used to disrupt the expression of the
target gene. Generally, sequences of at least 50 nucleotides, 100
nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or greater
may be used. Methods for using sense suppression to inhibit the
expression of endogenous genes in plants are described, for
example, in Liu, et al., (2002) Plant Physiol. 129:1732-1743 and
U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein
incorporated by reference. Efficiency of sense suppression may be
increased by including a poly-dT region in the expression cassette
at a position 3' to the sense sequence and 5' of the
polyadenylation signal. See, US Patent Application Publication
Number 2002/0048814, herein incorporated by reference.
[0142] iii. Double-Stranded RNA Interference
[0143] In some embodiments of the invention, inhibition of the
expression of a GS polypeptide may be obtained by double-stranded
RNA (dsRNA) interference. For dsRNA interference, a sense RNA
molecule like that described above for cosuppression and a
anti-sense RNA molecule that is fully or partially complementary to
the sense RNA molecule are expressed in the same cell, resulting in
inhibition of the expression of the corresponding endogenous
messenger RNA.
[0144] Expression of the sense and sense molecules can be
accomplished by designing the expression cassette to comprise both
a sense sequence and a sense sequence. Alternatively, separate
expression cassettes may be used for the sense and sense sequences.
Multiple plant lines transformed with the dsRNA interference
expression cassette or expression cassettes are then screened to
identify plant lines that show the greatest inhibition of GS
polypeptide expression. Methods for using dsRNA interference to
inhibit the expression of endogenous plant genes are described in
Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA
95:13959-13964, Liu, et al., (2002) Plant Physiol. 129:1732-1743
and WO 99/49029, WO 99/53050, WO 99/61631 and WO 00/49035, each of
which is herein incorporated by reference.
[0145] iv. Hairpin RNA Interference and Intron-Containing Hairpin
RNA Interference
[0146] In some embodiments of the invention, inhibition of the
expression of a GS polypeptide may be obtained by hairpin RNA
(hpRNA) interference or intron-containing hairpin RNA (ihpRNA)
interference. These methods are highly efficient at inhibiting the
expression of endogenous genes. See, Waterhouse and Helliwell,
(2003) Nat. Rev. Genet. 4:29-38 and the references cited
therein.
[0147] For hpRNA interference, the expression cassette is designed
to express an RNA molecule that hybridizes with itself to form a
hairpin structure that comprises a single-stranded loop region and
a base-paired stem. The base-paired stem region comprises a sense
sequence corresponding to all or part of the endogenous messenger
RNA encoding the gene whose expression is to be inhibited and a
sense sequence that is fully or partially complementary to the
sense sequence. Alternatively, the base-paired stem region may
correspond to a portion of a promoter sequence controlling
expression of the gene to be inhibited. Thus, the base-paired stem
region of the molecule generally determines the specificity of the
RNA interference. hpRNA molecules are highly efficient at
inhibiting the expression of endogenous genes and the RNA
interference they induce is inherited by subsequent generations of
plants. See, for example, Chuang and Meyerowitz, (2000) Proc. Natl.
Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant
Physiol. 129:1723-1731 and Waterhouse and Helliwell, (2003) Nat.
Rev. Genet. 4:29-38. Methods for using hpRNA interference to
inhibit or silence the expression of genes are described, for
example, in Chuang and Meyerowitz, (2000) Proc. Natl. Acad. Sci.
USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol.
129:1723-1731; Waterhouse and Helliwell, (2003) Nat. Rev. Genet.
4:29-38; Pandolfini, et al., BMC Biotechnology 3:7 and US Patent
Application Publication Number 2003/0175965, each of which is
herein incorporated by reference. A transient assay for the
efficiency of hpRNA constructs to silence gene expression in vivo
has been described by Panstruga, et al., (2003) Mol. Biol. Rep.
30:135-140, herein incorporated by reference.
[0148] For ihpRNA, the interfering molecules have the same general
structure as for hpRNA, but the RNA molecule additionally comprises
an intron that is capable of being spliced in the cell in which the
ihpRNA is expressed. The use of an intron minimizes the size of the
loop in the hairpin RNA molecule following splicing, and this
increases the efficiency of interference. See, for example, Smith,
et al., (2000) Nature 407:319-320. In fact, Smith, et al., show
100% suppression of endogenous gene expression using
ihpRNA-mediated interference. Methods for using ihpRNA interference
to inhibit the expression of endogenous plant genes are described,
for example, in Smith, et al., (2000) Nature 407:319-320; Wesley,
et al., (2001) Plant J. 27:581-590; Wang and Waterhouse, (2001)
Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell, (2003)
Nat. Rev. Genet. 4:29-38; Helliwell and Waterhouse, (2003) Methods
30:289-295 and US Patent Application Publication Number
2003/0180945, each of which is herein incorporated by
reference.
[0149] The expression cassette for hpRNA interference may also be
designed such that the sense sequence and the sense sequence do not
correspond to an endogenous RNA. In this embodiment, the sense and
sense sequence flank a loop sequence that comprises a nucleotide
sequence corresponding to all or part of the endogenous messenger
RNA of the target gene. Thus, it is the loop region that determines
the specificity of the RNA interference. See, for example, WO
02/00904, Mette, et al., (2000) EMBO J 19:5194-5201; Matzke, et
al., (2001) Curr. Opin. Genet. Devel. 11:221-227; Scheid, et al.,
(2002) Proc. Natl. Acad. Sci., USA 99:13659-13662; Aufsaftz, et
al., (2002) Proc. Nat'l. Acad. Sci. 99(4):16499-16506; Sijen, et
al., Curr. Biol. (2001) 11:436-440), herein incorporated by
reference.
[0150] v. Amplicon-Mediated Interference
[0151] Amplicon expression cassettes comprise a plant virus-derived
sequence that contains all or part of the target gene but generally
not all of the genes of the native virus. The viral sequences
present in the transcription product of the expression cassette
allow the transcription product to direct its own replication. The
transcripts produced by the amplicon may be either sense or sense
relative to the target sequence (i.e., the messenger RNA for the GS
polypeptide). Methods of using amplicons to inhibit the expression
of endogenous plant genes are described, for example, in Angell and
Baulcombe, (1997) EMBO J. 16:3675-3684, Angell and Baulcombe,
(1999) Plant J. 20:357-362 and U.S. Pat. No. 6,646,805, each of
which is herein incorporated by reference.
[0152] vi. Ribozymes
[0153] In some embodiments, the polynucleotide expressed by the
expression cassette of the invention is catalytic RNA or has
ribozyme activity specific for the messenger RNA of the GS
polypeptide. Thus, the polynucleotide causes the degradation of the
endogenous messenger RNA, resulting in reduced expression of the GS
polypeptide. This method is described, for example, in U.S. Pat.
No. 4,987,071, herein incorporated by reference.
[0154] vii. Small Interfering RNA or Micro RNA
[0155] In some embodiments of the invention, inhibition of the
expression of a GS polypeptide may be obtained by RNA interference
by expression of a gene encoding a micro RNA (miRNA). miRNAs are
regulatory agents consisting of about 22 ribonucleotides. miRNA are
highly efficient at inhibiting the expression of endogenous genes.
See, for example, Javier, et al., (2003) Nature 425:257-263, herein
incorporated by reference.
[0156] For miRNA interference, the expression cassette is designed
to express an RNA molecule that is modeled on an endogenous miRNA
gene. The miRNA gene encodes an RNA that forms a hairpin structure
containing a 22-nucleotide sequence that is complementary to
another endogenous gene (target sequence). For suppression of GS
expression, the 22-nucleotide sequence is selected from a GS
transcript sequence and contains 22 nucleotides of said GS sequence
in sense orientation and 21 nucleotides of a corresponding sense
sequence that is complementary to the sense sequence. miRNA
molecules are highly efficient at inhibiting the expression of
endogenous genes and the RNA interference they induce is inherited
by subsequent generations of plants.
[0157] 2. Polypeptide-Based Inhibition of Gene Expression
[0158] In one embodiment, the polynucleotide encodes a GS protein
that binds to a gene encoding a GS polypeptide, resulting in
reduced expression of the gene. In particular embodiments, the GS
protein binds to a regulatory region of a GS gene. In other
embodiments, the GS protein binds to a messenger RNA encoding a GS
polypeptide and prevents its translation. Methods of selecting
sites for targeting by GS proteins have been described, for
example, in U.S. Pat. No. 6,453,242, and methods for using GS
proteins to inhibit the expression of genes in plants are
described, for example, in US Patent Application Publication Number
2003/0037355, each of which is herein incorporated by
reference.
[0159] 3. Polypeptide-Based Inhibition of Protein Activity
[0160] In some embodiments of the invention, the polynucleotide
encodes an antibody that binds to at least one GS polypeptide and
reduces the GS enzyme activity of the GS polypeptide. In another
embodiment, the binding of the antibody results in increased
turnover of the antibody-GS complex by cellular quality control
mechanisms. The expression of antibodies in plant cells and the
inhibition of molecular pathways by expression and binding of
antibodies to proteins in plant cells are well known in the art.
See, for example, Conrad and Sonnewald, (2003) Nature Biotech.
21:35-36, incorporated herein by reference.
[0161] 4. Gene Disruption
[0162] In some embodiments of the present invention, the activity
of a GS polypeptide is reduced or eliminated by disrupting the gene
encoding the GS polypeptide. The gene encoding the GS polypeptide
may be disrupted by any method known in the art. For example, in
one embodiment, the gene is disrupted by transposon tagging. In
another embodiment, the gene is disrupted by mutagenizing plants
using random or targeted mutagenesis, and selecting for plants that
have reduced GS enzyme activity.
[0163] i. Transposon Tagging
[0164] In one embodiment of the invention, transposon tagging is
used to reduce or eliminate the GS activity of one or more GS
polypeptide. Transposon tagging comprises inserting a transposon
within an endogenous GS gene to reduce or eliminate expression of
the GS polypeptide. "GS gene" is intended to mean the gene that
encodes a GS polypeptide according to the invention.
[0165] In this embodiment, the expression of one or more GS
polypeptide is reduced or eliminated by inserting a transposon
within a regulatory region or coding region of the gene encoding
the GS polypeptide. A transposon that is within an exon, intron, 5'
or 3' untranslated sequence, a promoter or any other regulatory
sequence of a GS gene may be used to reduce or eliminate the
expression and/or activity of the encoded GS polypeptide.
[0166] Methods for the transposon tagging of specific genes in
plants are well known in the art. See, for example, Maes, et al.,
(1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti, (1999) FEMS
Microbiol. Lett. 179:53-59; Meissner, et al., (2000) Plant J.
22:265-274; Phogat, et al., (2000) J. Biosci. 25:57-63; Walbot
(2000) Curr. Opin. Plant Biol. 2:103-107; Gai, et al., (2000)
Nucleic Acids Res. 28:94-96; Fitzmaurice, et al., (1999) Genetics
153:1919-1928). In addition, the TUSC process for selecting Mu
insertions in selected genes has been described in Bensen, et al.,
(1995) Plant Cell 7:75-84; Mena, et al., (1996) Science
274:1537-1540 and U.S. Pat. No. 5,962,764, each of which is herein
incorporated by reference.
[0167] ii. Mutant Plants with Reduced Activity
[0168] Additional methods for decreasing or eliminating the
expression of endogenous genes in plants are also known in the art
and can be similarly applied to the instant invention. These
methods include other forms of mutagenesis, such as ethyl
methanesulfonate-induced mutagenesis, deletion mutagenesis and fast
neutron deletion mutagenesis used in a reverse genetics sense (with
PCR) to identify plant lines in which the endogenous gene has been
deleted. For examples of these methods see, Ohshima, et al., (1998)
Virology 243:472-481; Okubara, et al., (1994) Genetics 137:867-874
and Quesada, et al., (2000) Genetics 154:421-436, each of which is
herein incorporated by reference. In addition, a fast and
automatable method for screening for chemically induced mutations,
TILLING (Targeting Induced Local Lesions In Genomes), using
denaturing HPLC or selective endonuclease digestion of selected PCR
products is also applicable to the instant invention. See,
McCallum, et al., (2000) Nat. Biotechnol. 18:455-457, herein
incorporated by reference.
[0169] Mutations that impact gene expression or that interfere with
the function (GS enzyme activity) of the encoded protein are well
known in the art. Insertional mutations in gene exons usually
result in null-mutants. Mutations in conserved residues are
particularly effective in inhibiting the GS enzyme activity of the
encoded protein.
[0170] Conserved residues of plant GS polypeptides suitable for
mutagenesis with the goal to eliminate GS enzyme activity have been
described. Such mutants can be isolated according to well-known
procedures, and mutations in different GS loci can be stacked by
genetic crossing. See, for example, Gruis, et al., (2002) Plant
Cell 14:2863-2882.
[0171] In another embodiment of this invention, dominant mutants
can be used to trigger RNA silencing due to gene inversion and
recombination of a duplicated gene locus. See, for example, Kusaba,
et al., (2003) Plant Cell 15:1455-1467.
[0172] The invention encompasses additional methods for reducing or
eliminating the activity of one or more GS polypeptide. Examples of
other methods for altering or mutating a genomic nucleotide
sequence in a plant are known in the art and include, but are not
limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors,
RNA:DNA repair vectors, mixed-duplex oligonucleotides,
self-complementary RNA:DNA oligonucleotides and recombinogenic
oligonucleobases. Such vectors and methods of use are known in the
art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181;
5,756,325; 5,760,012; 5,795,972 and 5,871,984, each of which are
herein incorporated by reference. See also, WO 98/49350, WO
99/07865, WO 99/25821 and Beetham, et al., (1999) Proc. Natl. Acad.
Sci. USA 96:8774-8778, each of which is herein incorporated by
reference.
[0173] iii. Modulating GS Enzyme Activity
[0174] In specific methods, the level and/or activity of a GS
regulator in a plant is decreased by increasing the level or
activity of the GS polypeptide in the plant. Methods for increasing
the level and/or activity of GS polypeptides in a plant are
discussed elsewhere herein. Briefly, such methods comprise
providing a GS polypeptide of the invention to a plant and thereby
increasing the level and/or activity of the GS polypeptide. In
other embodiments, a GS nucleotide sequence encoding a GS
polypeptide can be provided by introducing into the plant a
polynucleotide comprising a GS nucleotide sequence of the
invention, expressing the GS sequence, increasing the activity of
the GS polypeptide and thereby decreasing the ammonium uptake or
transport in the plant or plant part. In other embodiments, the GS
nucleotide construct introduced into the plant is stably
incorporated into the genome of the plant.
[0175] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate the level/activity of a GS
enzyme in the plant. Exemplary promoters for this embodiment have
been disclosed elsewhere herein.
[0176] Accordingly, the present invention further provides plants
having a modified number of cells when compared to the number of
cells of a control plant tissue. In one embodiment, the plant of
the invention has an increased level/activity of the GS polypeptide
of the invention and thus has an increased Ammonium transport in
the plant tissue. In other embodiments, the plant of the invention
has a reduced or eliminated level of the GS polypeptide of the
invention and thus has an increased NUE in the plant tissue. In
other embodiments, such plants have stably incorporated into their
genome a nucleic acid molecule comprising a GS nucleotide sequence
of the invention operably linked to a promoter that drives
expression in the plant cell.
[0177] iv. Modulating Root Development
[0178] Methods for modulating root development in a plant are
provided. By "modulating root development" is intended any
alteration in the development of the plant root when compared to a
control plant. Such alterations in root development include, but
are not limited to, alterations in the growth rate of the primary
root, the fresh root weight, the extent of lateral and adventitious
root formation, the vasculature system, meristem development or
radial expansion.
[0179] Methods for modulating root development in a plant are
provided. The methods comprise modulating the level and/or activity
of the GS polypeptide in the plant. In one method, a GS sequence of
the invention is provided to the plant. In another method, the GS
nucleotide sequence is provided by introducing into the plant a
polynucleotide comprising a GS nucleotide sequence of the
invention, expressing the GS sequence and thereby modifying root
development. In still other methods, the GS nucleotide construct
introduced into the plant is stably incorporated into the genome of
the plant.
[0180] In other methods, root development is modulated by altering
the level or activity of the GS polypeptide in the plant. A
decrease in GS activity can result in at least one or more of the
following alterations to root development, including, but not
limited to, larger root meristems, increased in root growth,
enhanced radial expansion, an enhanced vasculature system,
increased root branching, more adventitious roots and/or an
increase in fresh root weight when compared to a control plant.
[0181] As used herein, "root growth" encompasses all aspects of
growth of the different parts that make up the root system at
different stages of its development in both monocotyledonous and
dicotyledonous plants. It is to be understood that enhanced root
growth can result from enhanced growth of one or more of its parts
including the primary root, lateral roots, adventitious roots,
etc.
[0182] Methods of measuring such developmental alterations in the
root system are known in the art. See, for example, US Patent
Application Publication Number 2003/0074698 and Werner, et al.,
(2001) PNAS 18:10487-10492, both of which are herein incorporated
by reference.
[0183] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate root development in the
plant. Exemplary promoters for this embodiment include constitutive
promoters and root-preferred promoters. Exemplary root-preferred
promoters have been disclosed elsewhere herein.
[0184] Stimulating root growth and increasing root mass by
decreasing the activity and/or level of the GS polypeptide also
finds use in improving the standability of a plant. The term
"resistance to lodging" or "standability" refers to the ability of
a plant to fix itself to the soil. For plants with an erect or
semi-erect growth habit, this term also refers to the ability to
maintain an upright position under adverse (environmental)
conditions. This trait relates to the size, depth and morphology of
the root system. In addition, stimulating root growth and
increasing root mass by decreasing the level and/or activity of the
GS polypeptide also finds use in promoting in vitro propagation of
explants.
[0185] Furthermore, higher root biomass production due to a
decreased level and/or activity of GS activity has a direct effect
on the yield and an indirect effect of production of compounds
produced by root cells or transgenic root cells or cell cultures of
said transgenic root cells. One example of an interesting compound
produced in root cultures is shikonin, the yield of which can be
advantageously enhanced by said methods.
[0186] Accordingly, the present invention further provides plants
having modulated root development when compared to the root
development of a control plant. In some embodiments, the plant of
the invention has an increased level/activity of the GS polypeptide
of the invention and has enhanced root growth and/or root biomass.
In other embodiments, such plants have stably incorporated into
their genome a nucleic acid molecule comprising a GS nucleotide
sequence of the invention operably linked to a promoter that drives
expression in the plant cell.
[0187] v. Modulating Shoot and Leaf Development
[0188] Methods are also provided for modulating shoot and leaf
development in a plant. By "modulating shoot and/or leaf
development" is intended any alteration in the development of the
plant shoot and/or leaf. Such alterations in shoot and/or leaf
development include, but are not limited to, alterations in shoot
meristem development, in leaf number, leaf size, leaf and stem
vasculature, internode length and leaf senescence. As used herein,
"leaf development" and "shoot development" encompasses all aspects
of growth of the different parts that make up the leaf system and
the shoot system, respectively, at different stages of their
development, both in monocotyledonous and dicotyledonous plants.
Methods for measuring such developmental alterations in the shoot
and leaf system are known in the art. See, for example, Werner, et
al., (2001) PNAS 98:10487-10492 and US Patent Application
Publication Number 2003/0074698, each of which is herein
incorporated by reference.
[0189] The method for modulating shoot and/or leaf development in a
plant comprises modulating the activity and/or level of a GS
polypeptide of the invention. In one embodiment, a GS sequence of
the invention is provided. In other embodiments, the GS nucleotide
sequence can be provided by introducing into the plant a
polynucleotide comprising a GS nucleotide sequence of the
invention, expressing the GS sequence and thereby modifying shoot
and/or leaf development. In other embodiments, the GS nucleotide
construct introduced into the plant is stably incorporated into the
genome of the plant.
[0190] In specific embodiments, shoot or leaf development is
modulated by increasing the level and/or activity of the GS
polypeptide in the plant. An increase in GS activity can result in
at least one or more of the following alterations in shoot and/or
leaf development, including, but not limited to, leaf number, leaf
surface, vasculature, internode length and leaf senescence, when
compared to a control plant.
[0191] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate shoot and leaf development
of the plant. Exemplary promoters for this embodiment include
constitutive promoters, shoot-preferred promoters, shoot
meristem-preferred promoters and leaf-preferred promoters.
Exemplary promoters have been disclosed elsewhere herein.
[0192] As discussed above, modulation GS activity in the plant
modulates both root and shoot growth. Thus, the present invention
further provides methods for altering the root/shoot ratio. Shoot
or leaf development can further be modulated by decreasing the
level and/or activity of the GS polypeptide in the plant.
[0193] Accordingly, the present invention further provides plants
having modulated shoot and/or leaf development when compared to a
control plant. In some embodiments, the plant of the invention has
an increased level/activity of the GS polypeptide of the invention.
In other embodiments, the plant of the invention has a decreased
level/activity of the GS polypeptide of the invention.
[0194] vi Modulating Reproductive Tissue Development
[0195] Methods for modulating reproductive tissue development are
provided. In one embodiment, methods are provided to modulate
floral development in a plant. By "modulating floral development"
is intended any alteration in a structure of a plant's reproductive
tissue as compared to a control plant in which the activity or
level of the GS polypeptide has not been modulated. "Modulating
floral development" further includes any alteration in the timing
of the development of a plant's reproductive tissue (i.e., a
delayed or a accelerated timing of floral development) when
compared to a control plant in which the activity or level of the
GS polypeptide has not been modulated. Macroscopic alterations may
include changes in size, shape, number or location of reproductive
organs, the developmental time period that these structures form or
the ability to maintain or proceed through the flowering process in
times of environmental stress. Microscopic alterations may include
changes to the types or shapes of cells that make up the
reproductive organs.
[0196] The method for modulating floral development in a plant
comprises modulating GS activity in a plant. In one method, a GS
sequence of the invention is provided. A GS nucleotide sequence can
be provided by introducing into the plant a polynucleotide
comprising a GS nucleotide sequence of the invention, expressing
the GS sequence, and thereby modifying floral development. In other
embodiments, the GS nucleotide construct introduced into the plant
is stably incorporated into the genome of the plant.
[0197] In specific methods, floral development is modulated by
increasing the level or activity of the GS polypeptide in the
plant. An increase in GS activity can result in at least one or
more of the following alterations in floral development, including,
but not limited to, retarded flowering, reduced number of flowers,
partial male sterility and reduced seed set, when compared to a
control plant. Inducing delayed flowering or inhibiting flowering
can be used to enhance yield in forage crops such as alfalfa.
Methods for measuring such developmental alterations in floral
development are known in the art. See, for example, Mouradov, et
al., (2002) The Plant Cell S111-S130, herein incorporated by
reference.
[0198] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate floral development of the
plant. Exemplary promoters for this embodiment include constitutive
promoters, inducible promoters, shoot-preferred promoters and
inflorescence-preferred promoters.
[0199] In other methods, floral development is modulated by
decreasing the level and/or activity of the GS sequence of the
invention. Such methods can comprise introducing a GS nucleotide
sequence into the plant and decreasing the activity of the GS
polypeptide.
[0200] In other methods, the GS nucleotide construct introduced
into the plant is stably incorporated into the genome of the plant.
Decreasing expression of the GS sequence of the invention can
modulate floral development during periods of stress. Such methods
are described elsewhere herein. Accordingly, the present invention
further provides plants having modulated floral development when
compared to the floral development of a control plant. Compositions
include plants having a decreased level/activity of the GS
polypeptide of the invention and having an altered floral
development. Compositions also include plants having a decreased
level/activity of the GS polypeptide of the invention wherein the
plant maintains or proceeds through the flowering process in times
of stress. Methods are also provided for the use of the GS
sequences of the invention to increase nitrogen use efficiency. The
method comprises decreasing or increasing the activity of the GS
sequences in a plant or plant part, such as the roots, shoot,
epidermal cells, etc.
[0201] As discussed above, one of skill will recognize the
appropriate promoter to use to manipulate the expression of GS.
Exemplary promoters of this embodiment include constitutive
promoters, inducible promoters, and root or shoot or leaf preferred
promoters.
[0202] vii. Method of Use for Gs Promoter Polynucleotides
[0203] The polynucleotides comprising the GS promoters disclosed in
the present invention, as well as variants and fragments thereof,
are useful in the genetic manipulation of any host cell, preferably
plant cell, when assembled with a DNA construct such that the
promoter sequence is operably linked to a nucleotide sequence
comprising a polynucleotide of interest. In this manner, the GS
promoter polynucleotides of the invention are provided in
expression cassettes along with a polynucleotide sequence of
interest for expression in the host cell of interest. GS promoter
sequences of the invention are expressed in a variety of tissues
and thus the promoter sequences can find use in regulating the
temporal and/or the spatial expression of polynucleotides of
interest.
[0204] Synthetic hybrid promoter regions are known in the art. Such
regions comprise upstream promoter elements of one polynucleotide
operably linked to the promoter element of another polynucleotide.
In an embodiment of the invention, heterologous sequence expression
is controlled by a synthetic hybrid promoter comprising the GS
promoter sequences of the invention, or a variant or fragment
thereof, operably linked to upstream promoter element(s) from a
heterologous promoter. Upstream promoter elements that are involved
in the plant defense system have been identified and may be used to
generate a synthetic promoter. See, for example, Rushton, et al.,
(1998) Curr. Opin. Plant Biol. 1:311-315. Alternatively, a
synthetic GS promoter sequence may comprise duplications of the
upstream promoter elements found within the GS promoter
sequences.
[0205] It is recognized that the promoter sequence of the invention
may be used with its native GS coding sequences. A DNA construct
comprising the GS promoter operably linked with its native GS gene
may be used to transform any plant of interest to bring about a
desired phenotypic change, such as, modulating root, shoot, leaf,
floral and embryo development, stress tolerance and any other
phenotype described elsewhere herein.
[0206] The promoter nucleotide sequences and methods disclosed
herein are useful in regulating expression of any heterologous
nucleotide sequence in a host plant in order to vary the phenotype
of a plant. Various changes in phenotype are of interest including
modifying the fatty acid composition in a plant, altering the amino
acid content of a plant, altering a plant's pathogen defense
mechanism and the like. These results can be achieved by providing
expression of heterologous products or increased expression of
endogenous products in plants. Alternatively, the results can be
achieved by providing for a reduction of expression of one or more
endogenous products, particularly enzymes or cofactors in the
plant. These changes result in a change in phenotype of the
transformed plant.
[0207] Genes of interest are reflective of the commercial markets
and interests of those involved in the development of the crop.
Crops and markets of interest change, and as developing nations
open up world markets, new crops and technologies will emerge also.
In addition, as our understanding of agronomic traits and
characteristics such as yield and heterosis increase, the choice of
genes for transformation will change accordingly. General
categories of genes of interest include, for example, those genes
involved in information, such as GSs, those involved in
communication, such as kinases, and those involved in housekeeping,
such as heat shock proteins. More specific categories of
transgenes, for example, include genes encoding important traits
for agronomics, insect resistance, disease resistance, herbicide
resistance, sterility, grain characteristics and commercial
products. Genes of interest include, generally, those involved in
oil, starch, carbohydrate or nutrient metabolism as well as those
affecting kernel size, sucrose loading and the like.
[0208] In certain embodiments the nucleic acid sequences of the
present invention can be used in combination ("stacked") with other
polynucleotide sequences of interest in order to create plants with
a desired phenotype. The combinations generated can include
multiple copies of any one or more of the polynucleotides of
interest. The polynucleotides of the present invention may be
stacked with any gene or combination of genes to produce plants
with a variety of desired trait combinations, including but not
limited to traits desirable for animal feed such as high oil genes
(e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g.,
hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802 and
5,703,409); barley high lysine (Williamson, et al., (1987) Eur. J.
Biochem. 165:99-106 and WO 98/20122) and high methionine proteins
(Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et
al., (1988) Gene 71:359 and Musumura, et al., (1989) Plant Mol.
Biol. 12: 123)); increased digestibility (e.g., modified storage
proteins (U.S. patent application Ser. No. 10/053,410, filed Nov.
7, 2001) and thioredoxins (U.S. patent application Ser. No.
10/005,429, filed Dec. 3, 2001)), the disclosures of which are
herein incorporated by reference. The polynucleotides of the
present invention can also be stacked with traits desirable for
insect, disease or herbicide resistance (e.g., Bacillus
thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450;
5,737,514; 5723,756; 5,593,881; Geiser, et al., (1986) Gene
48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol.
24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931);
avirulence and disease resistance genes (Jones, et al., (1994)
Science 266:789; Martin, et al., (1993) Science 262:1432;
Mindrinos, et al., (1994) Cell 78:1089); acetolactate synthase
(ALS) mutants that lead to herbicide resistance such as the S4
and/or Hra mutations; inhibitors of glutamine synthase such as
phosphinothricin or basta (e.g., bar gene) and glyphosate
resistance (EPSPS gene)) and traits desirable for processing or
process products such as high oil (e.g., U.S. Pat. No. 6,232,529);
modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.
5,952,544; WO 94/11516)); modified starches (e.g., ADPG
pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE) and starch debranching enzymes (SDBE)) and
polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;
beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA reductase (Schubert, et al., (1988) J. Bacteriol.
170:5837-5847) facilitate expression of polyhydroxyalkanoates
(PHAs)), the disclosures of which are herein incorporated by
reference. One could also combine the polynucleotides of the
present invention with polynucleotides affecting agronomic traits
such as male sterility (e.g., see, U.S. Pat. No. 5,583,210), stalk
strength, flowering time or transformation technology traits such
as cell cycle regulation or gene targeting (e.g., WO 99/61619; WO
00/17364; WO 99/25821), the disclosures of which are herein
incorporated by reference.
[0209] In one embodiment, sequences of interest improve plant
growth and/or crop yields. For example, sequences of interest
include agronomically important genes that result in improved
primary or lateral root systems. Such genes include, but are not
limited to, nutrient/water transporters and growth induces.
Examples of such genes, include but are not limited to, maize
plasma membrane H.sup.+-ATPase (MHA2) (Frias, et al., (1996) Plant
Cell 8:1533-44); AKT1, a component of the potassium uptake
apparatus in Arabidopsis, (Spalding, et al., (1999) J Gen Physiol
113:909-18); RML genes which activate cell division cycle in the
root apical cells (Cheng, et al., (1995) Plant Physiol 108:881);
maize glutamine synthetase genes (Sukanya, et al., (1994) Plant Mol
Biol 26:1935-46) and hemoglobin (Duff, et al., (1997) J. Biol.
Chem. 27:16749-16752, Arredondo-Peter, et al., (1997) Plant
Physiol. 115:1259-1266; Arredondo-Peter, et al., (1997) Plant
Physiol 114:493-500 and references sited therein). The sequence of
interest may also be useful in expressing sense nucleotide
sequences of genes that that negatively affects root
development.
[0210] Additional, agronomically important traits such as oil,
starch, and protein content can be genetically altered in addition
to using traditional breeding methods. Modifications include
increasing content of oleic acid, saturated and unsaturated oils,
increasing levels of lysine and sulfur, providing essential amino
acids, and also modification of starch. Hordothionin protein
modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801,
5,885,802 and 5,990,389, herein incorporated by reference. Another
example is lysine and/or sulfur rich seed protein encoded by the
soybean 2S albumin described in U.S. Pat. No. 5,850,016 and the
chymotrypsin inhibitor from barley, described in Williamson, et
al., (1987) Eur. J. Biochem. 165:99-106, the disclosures of which
are herein incorporated by reference.
[0211] Derivatives of the coding sequences can be made by
site-directed mutagenesis to increase the level of preselected
amino acids in the encoded polypeptide. For example, the gene
encoding the barley high lysine polypeptide (BHL) is derived from
barley chymotrypsin inhibitor, U.S. patent application Ser. No.
08/740,682, filed Nov. 1, 1996 and WO 98/20133, the disclosures of
which are herein incorporated by reference. Other proteins include
methionine-rich plant proteins such as from sunflower seed (Lilley,
et al., (1989) Proceedings of the World Congress on Vegetable
Protein Utilization in Human Foods and Animal Feedstuffs, ed.
Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.
497-502; herein incorporated by reference); corn (Pedersen, et al.,
(1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene
71:359; both of which are herein incorporated by reference) and
rice (Musumura, et al., (1989) Plant Mol. Biol. 12:123, herein
incorporated by reference). Other agronomically important genes
encode latex, Floury 2, growth factors, seed storage factors and
transcription factors.
[0212] Insect resistance genes may encode resistance to pests that
have great yield drag such as rootworm, cutworm, European Corn
Borer and the like. Such genes include, for example, Bacillus
thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892;
5,747,450; 5,736,514; 5,723,756; 5,593,881 and Geiser, et al.,
(1986) Gene 48:109), and the like.
[0213] Genes encoding disease resistance traits include
detoxification genes, such as against fumonosin (U.S. Pat. No.
5,792,931); avirulence (avr) and disease resistance (R) genes
(Jones, et al., (1994) Science 266:789; Martin, et al., (1993)
Science 262:1432 and Mindrinos, et al., (1994) Cell 78:1089), and
the like.
[0214] Herbicide resistance traits may include genes coding for
resistance to herbicides that act to inhibit the action of
acetolactate synthase (ALS), in particular the sulfonylurea-type
herbicides (e.g., the acetolactate synthase (ALS) gene containing
mutations leading to such resistance, in particular the S4 and/or
Hra mutations), genes coding for resistance to herbicides that act
to inhibit action of glutamine synthase, such as phosphinothricin
or basta (e.g., the bar gene) or other such genes known in the art.
The bar gene encodes resistance to the herbicide basta, the nptII
gene encodes resistance to the antibiotics kanamycin and geneticin
and the ALS-gene mutants encode resistance to the herbicide
chlorsulfuron.
[0215] Sterility genes can also be encoded in an expression
cassette and provide an alternative to physical detasseling.
Examples of genes used in such ways include male tissue-preferred
genes and genes with male sterility phenotypes such as QM,
described in U.S. Pat. No. 5,583,210. Other genes include kinases
and those encoding compounds toxic to either male or female
gametophytic development.
[0216] The quality of grain is reflected in traits such as levels
and types of oils, saturated and unsaturated, quality and quantity
of essential amino acids, and levels of cellulose. In corn,
modified hordothionin proteins are described in U.S. Pat. Nos.
5,703,049, 5,885,801, 5,885,802 and 5,990,389.
[0217] Commercial traits can also be encoded on a gene or genes
that could increase for example, starch for ethanol production, or
provide expression of proteins. Another important commercial use of
transformed plants is the production of polymers and bioplastics
such as described in U.S. Pat. No. 5,602,321. Genes such as
.beta.-Ketothiolase, PHBase (polyhydroxybutyrate synthase) and
acetoacetyl-CoA reductase (see, Schubert, et al., (1988) J.
Bacteriol. 170:5837-5847) facilitate expression of
polyhydroxyalkanoates (PHAs).
[0218] Exogenous products include plant enzymes and products as
well as those from other sources including procaryotes and other
eukaryotes. Such products include enzymes, cofactors, hormones and
the like. The level of proteins, particularly modified proteins
having improved amino acid distribution to improve the nutrient
value of the plant, can be increased. This is achieved by the
expression of such proteins having enhanced amino acid content.
[0219] This invention can be better understood by reference to the
following non-limiting examples. It will be appreciated by those
skilled in the art that other embodiments of the invention may be
practiced without departing from the spirit and the scope of the
invention as herein disclosed and claimed.
EXAMPLES
Example 1
Identification and Phylogenetic Analyses of GS Sequences from
Arabidopsis, Soybean, Rice, Sorghum and Maize
[0220] A routine for identifying all members of a given species'
glutamine synthetase (GS) gene family was employed. First, a
diverse set of the known available members of the gene family as
protein sequences was prepared from public and proprietary sources.
Then, as in the example of maize, these protein query sequences
were searched using a BLAST algorithm against a combination of
proprietary and public genomic or transcript, nucleotide sequence
datasets and a non-redundant set of candidate GS or `hits` was
identified. These sequences were combined with any existing maize
gene family sequences, and then curated and edited to arrive at a
new working set of unique maize GS gene or transcript sequences and
their translations. This search for gene family members was
repeated. If new sequences were recovered that were unique (not
same-gene matches), the process was repeated until completion, that
is until no new and distinct nucleotide sequences were found. In
this way it was determined that the maize GS gene family consisted
of 6 members. Eight and 3 distinct soybean and sorghum sequences
were found, respectively. Without the complete genome sequences of
maize or soybean available, researchers were less certain of the
exact gene family size, than they were for Arabidopsis (6 members)
and rice (4 members). The availability of complete genome sequences
for Arabidopsis and rice simplified the search, aided also by
availability of fairly mature gene models and annotations for these
species. All the Sequence IDs along with the annotation identity
were cataloged in Table 1. The polypeptide alignment of all 27
sequences is shown in FIG. 1. Several regions of very high homology
were identified by this alignment. All these polypeptides from
different species (except SEQ ID NO: 20) show a sequence identity
in the range of 70-95% among different members. Due to several
insertions, SEQ ID NO: 20 show an identity in the range of 53-74%
with different GS polypeptides from different species. SEQ ID NOS:
10, 18, 28, 36, 42 and 54 belong to the GS2 group
(chloroplast-localized) as in all the polypeptides a clear
chloroplast targeting peptide was identified. Phylogenetic analyses
of all 27 polypeptides are shown in FIG. 2. Clearly, ZMGS1-1/1-5,
ZMGS1-3/1-4, ZMGS1-2 and ZMGS2 along with members from other
species were clustered in four different clades. There seems a
soybean specific Glade with SEQ ID NOS: 14, 22, 24 and 26.
Example 2
MPSS Expression Analyses of Different Gs Isoforms from Maize
[0221] Massively Parallel Signature Sequencing (MPSS) expression
analyses were performed for expression of GS isoforms from a maize
database consisting of more than 300 tissue libraries. The results
from these analyses are summarized in FIG. 3. GS1-1 and GS2 were
expressed predominantly in roots and leaves, respectively (FIG. 3,
top panel). GS1-2 expresses more or less in all the tissues with a
slightly higher expression in the pollen (FIG. 3, top panel). GS1-3
and 1-4 are expressed at very low levels in most of the tissues
examined whereas GS1-5 expresses at .about.100 ppm (parts per
million) in the roots (FIG. 3, top panel). GS1-1 showed 15-20-fold
higher level expression in the root-cortex as compared to other
isoforms (FIG. 3, middle panel). Among all the isoforms, only GS1-2
and 1-5 are expressed in the pedicel (FIG. 3, bottom panel)
Example 3
Transformation and Regeneration of Transgenic Plants by
Agrobacterium-Mediated Transformation
[0222] Several vectors were transformed in maize (FAST/GS3xGF or
ETX inbred) by Agrobacterium mediated transformation. The
description of these vectors is provided in Table 2.
TABLE-US-00002 TABLE 2 ZmGS Promoter Target PHP Isoform Promoter
Specificity Genotype 32754 GS1-1 Ubiquitin Constitutive FAST
(GS3xGF) 32794 GS1-1 RM2 Roots FAST (GS3xGF) 32781 GS1-2 Ubiquitin
Constitutive FAST (GS3xGF) 32786 GS1-2 RM2 Roots FAST (GS3xGF)
32760 GS1-3 Ubiquitin Constitutive FAST (GS3xGF) 32779 GS1-3 RM2
Roots FAST (GS3xGF) 32753 GS1-4 Ubiquitin Constitutive FAST
(GS3xGF) 32772 GS1-4 RM2 Roots FAST (GS3xGF) 32755 GS1-5 Ubiquitin
Constitutive FAST (GS3xGF) 32743 GS1-5 RM2 Roots FAST (GS3xGF)
32007 GS1-3 Ubiquitin Constitutive Inbred (ETX) 32006 GS1-3 RM2
Roots Inbred (ETX) 32005 GS1-3 SSU leaf (bundlesheath) Inbred (ETX)
32008 GS1-3 PEPC leaf (mesophyl) Inbred (ETX) 38267 GS1-4 Ubiquitin
Constitutive Inbred (ETX) 38268 GS1-4 RM2 Roots Inbred (ETX) 38269
GS1-4 PEPC leaf (mesophyl) Inbred (ETX) 38930 GS1-5 Ubiquitin
Constitutive Inbred (ETX) 38931 GS1-5 RM2 Roots Inbred (ETX) 38932
GS1-5 PEPC leaf (mesophyl) Inbred (ETX)
[0223] For Agrobacterium-mediated transformation of maize with a
sense sequence of the GS sequence of the present invention,
preferably the method of Zhao is employed (U.S. Pat. No. 5,981,840
and PCT Patent Publication WO98/32326, the contents of which are
hereby incorporated by reference). Briefly, immature embryos are
isolated from maize and the embryos contacted with a suspension of
Agrobacterium, where the bacteria are capable of transferring the
sense GS sequences to at least one cell of at least one of the
immature embryos (step 1: the infection step). In this step the
immature embryos are preferably immersed in an Agrobacterium
suspension for the initiation of inoculation. The embryos are
co-cultured for a time with the Agrobacterium (step 2: the
co-cultivation step). Preferably the immature embryos are cultured
on solid medium following the infection step. Following this
co-cultivation period an optional "resting" step is contemplated.
In this resting step, the embryos are incubated in the presence of
at least one antibiotic known to inhibit the growth of
Agrobacterium without the addition of a selective agent for plant
transformants (step 3: resting step). Preferably the immature
embryos are cultured on solid medium with antibiotic, but without a
selecting agent, for elimination of Agrobacterium and for a resting
phase for the infected cells. Next, inoculated embryos are cultured
on medium containing a selective agent and growing transformed
callus is recovered (step 4: the selection step). Preferably, the
immature embryos are cultured on solid medium with a selective
agent resulting in the selective growth of transformed cells. The
callus is then regenerated into plants (step 5: the regeneration
step), and preferably calli grown on selective medium are cultured
on solid medium to regenerate the plants. Plants are monitored and
scored for a modulation in tissue development.
[0224] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing the GS sequence operably linked
to constitutive or tissue specific promoter (Vilardell, et al.,
(1990) Plant Mol Biol 14:423-432) and the selectable marker gene
PAT, which confers resistance to the herbicide Bialaphos.
Alternatively, the selectable marker gene is provided on a separate
plasmid. Transformation is performed as follows. Media recipes
follow below.
[0225] Preparation of Target Tissue:
[0226] The ears are husked and surface sterilized in 30%
Clorox.RTM. bleach plus 0.5% Micro detergent for 20 minutes, and
rinsed two times with sterile water. The immature embryos are
excised and placed embryo axis side down (scutellum side up), 25
embryos per plate, on 560Y medium for 4 hours and then aligned
within the 2.5-cm target zone in preparation for bombardment.
[0227] Preparation of DNA:
[0228] A plasmid vector comprising the GS sequence operably linked
to an ubiquitin promoter is made. This plasmid DNA plus plasmid DNA
containing a PAT selectable marker is precipitated onto 1.1 .mu.m
(average diameter) tungsten pellets using a CaCl.sub.2
precipitation procedure as follows:
[0229] 100 .mu.l prepared tungsten particles in water
[0230] 10 .mu.l (1 .mu.g) DNA in Tris EDTA buffer (1 .mu.g total
DNA)
[0231] 100 .mu.l 2.5 M CaCl.sub.2
[0232] 10 .mu.l 0.1 M spermidine
[0233] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 ml 100%
ethanol and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.l spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
[0234] Particle Gun Treatment:
[0235] The sample plates are bombarded at level #4 in particle gun
#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
[0236] Subsequent Treatment:
[0237] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288J medium to initiate plant
regeneration. Following somatic embryo maturation (2-4 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5'' pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
for increased drought tolerance. Assays to measure improved drought
tolerance are routine in the art and include, for example,
increased kernel-earring capacity yields under drought conditions
when compared to control maize plants under identical environmental
conditions. Alternatively, the transformed plants can be monitored
for a modulation in meristem development (i.e., a decrease in
spikelet formation on the ear). See, for example, Bruce, et al.,
(2002) Journal of Experimental Botany 53:1-13.
[0238] Bombardment and Culture Media:
[0239] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts
(SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose,
1.0 mg/l 2,4-D and 2.88 g/l L-proline (brought to volume with
D-1H.sub.2O following adjustment to pH 5.8 with KOH); 2.0 g/l
Gelrite.RTM. (added after bringing to volume with D-1H.sub.2O); and
8.5 mg/l silver nitrate (added after sterilizing the medium and
cooling to room temperature). Selection medium (560R) comprises 4.0
g/l N6 basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose,
and 2.0 mg/l 2,4-D (brought to volume with D-1H.sub.2O following
adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite.RTM. (added after
bringing to volume with D-1H.sub.2O) and 0.85 mg/l silver nitrate
and 3.0 mg/l bialaphos (both added after sterilizing the medium and
cooling to room temperature).
[0240] Plant regeneration medium (288J) comprises 4.3 g/l MS salts
(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL and
0.40 g/l glycine brought to volume with polished D-I H.sub.2O)
(Murashige and Skoog, (1962) Physiol. Plant. 15:473), 100 mg/l
myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose and 1.0 ml/l of 0.1
mM abscisic acid (brought to volume with polished D-I H.sub.2O
after adjusting to pH 5.6); 3.0 g/l Gelrite.RTM. (added after
bringing to volume with D-I H.sub.2O) and 1.0 mg/l indoleacetic
acid and 3.0 mg/l bialaphos (added after sterilizing the medium and
cooling to 60.degree. C.). Hormone-free medium (272V) comprises 4.3
g/l MS salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution
(0.100 g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l
pyridoxine HCL, and 0.40 g/l glycine brought to volume with
polished D-I H.sub.2O), 0.1 g/1 myo-inositol and 40.0 g/l sucrose
(brought to volume with polished D-I H.sub.2O after adjusting pH to
5.6) and 6 g/l Bacto.TM.-agar (added after bringing to volume with
polished D-I H.sub.2O), sterilized and cooled to 60.degree. C.
Example 4
Soybean Embryo Transformation
[0241] Soybean embryos are bombarded with a plasmid containing a
sense GS sequences operably linked to an ubiquitin promoter as
follows. To induce somatic embryos, cotyledons, 3-5 mm in length
dissected from surface-sterilized, immature seeds of the soybean
cultivar A2872, are cultured in the light or dark at 26.degree. C.
on an appropriate agar medium for six to ten weeks. Somatic embryos
producing secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos that multiplied as early, globular-staged embryos,
the suspensions are maintained as described below.
[0242] Soybean embryogenic suspension cultures can be maintained in
35 ml liquid media on a rotary shaker, 150 rpm, at 26.degree. C.
with florescent lights on a 16:8 hour day/night schedule. Cultures
are subcultured every two weeks by inoculating approximately 35 mg
of tissue into 35 ml of liquid medium.
[0243] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein, et
al., (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0244] A selectable marker gene that can be used to facilitate
soybean transformation is a transgene composed of the 35S promoter
from Cauliflower Mosaic Virus (Odell, et al., (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz, et al., (1983) Gene 25:179-188) and
the 3' region of the nopaline synthase gene from the T-DNA of the
Ti plasmid of Agrobacterium tumefaciens. The expression cassette
comprising a sense GS sequence operably linked to the ubiquitin
promoter can be isolated as a restriction fragment. This fragment
can then be inserted into a unique restriction site of the vector
carrying the marker gene.
[0245] To 50 .mu.l of a 60 mg/ml 1 .mu.m gold particle suspension
is added (in order): 5 .mu.l DNA (1 .mu.g/.mu.l), 20 .mu.l
spermidine (0.1 M), and 50 .mu.l CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.l 70% ethanol and
resuspended in 40 .mu.l of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
microliters of the DNA-coated gold particles are then loaded on
each macro carrier disk.
[0246] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi,
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0247] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media and eleven to twelve days
post-bombardment with fresh media containing 50 mg/ml hygromycin.
This selective media can be refreshed weekly. Seven to eight weeks
post-bombardment, green, transformed tissue may be observed growing
from untransformed, necrotic embryogenic clusters. Isolated green
tissue is removed and inoculated into individual flasks to generate
new, clonally propagated, transformed embryogenic suspension
cultures. Each new line may be treated as an independent
transformation event. These suspensions can then be subcultured and
maintained as clusters of immature embryos or regenerated into
whole plants by maturation and germination of individual somatic
embryos.
Example 5
Sunflower Meristem Tissue Transformation
[0248] Sunflower meristem tissues are transformed with an
expression cassette containing a sense GS sequences operably linked
to a ubiquitin promoter as follows (see also, EP Patent Number 0
486233, herein incorporated by reference and Malone-Schoneberg, et
al., (1994) Plant Science 103:199-207). Mature sunflower seed
(Helianthus annuus L.) are dehulled using a single wheat-head
thresher. Seeds are surface sterilized for 30 minutes in a 20%
Clorox.RTM. bleach solution with the addition of two drops of
Tween.RTM. 20 per 50 ml of solution. The seeds are rinsed twice
with sterile distilled water.
[0249] Split embryonic axis explants are prepared by a modification
of procedures described by Schrammeijer, et al., (Schrammeijer, et
al., (1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in
distilled water for 60 minutes following the surface sterilization
procedure. The cotyledons of each seed are then broken off,
producing a clean fracture at the plane of the embryonic axis.
Following excision of the root tip, the explants are bisected
longitudinally between the primordial leaves. The two halves are
placed, cut surface up, on GBA medium consisting of Murashige and
Skoog mineral elements (Murashige, et al., (1962) Physiol. Plant.,
15:473-497), Shepard's vitamin additions (Shepard, (1980) in
Emergent Techniques for the Genetic Improvement of Crops
(University of Minnesota Press, St. Paul, Minn.), 40 mg/l adenine
sulfate, 30 g/l sucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25
mg/l indole-3-acetic acid (IAA), 0.1 mg/l gibberellic acid
(GA.sub.3), pH 5.6 and 8 g/l Phytagar.
[0250] The explants are subjected to microprojectile bombardment
prior to Agrobacterium treatment (Bidney, et al., (1992) Plant Mol.
Biol. 18:301-313). Thirty to forty explants are placed in a circle
at the center of a 60.times.20 mm plate for this treatment.
Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are
resuspended in 25 ml of sterile TE buffer (10 mM Tris HCl, 1 mM
EDTA, pH 8.0) and 1.5 ml aliquots are used per bombardment. Each
plate is bombarded twice through a 150 mm nytex screen placed 2 cm
above the samples in a PDS1000.RTM. particle acceleration
device.
[0251] Disarmed Agrobacterium tumefaciens strain EHA105 is used in
all transformation experiments. A binary plasmid vector comprising
the expression cassette that contains the GS gene operably linked
to the ubiquitin promoter is introduced into Agrobacterium strain
EHA105 via freeze-thawing as described by Holsters, et al., (1978)
Mol. Gen. Genet. 163:181-187. This plasmid further comprises a
kanamycin selectable marker gene (i.e, nptII). Bacteria for plant
transformation experiments are grown overnight (28.degree. C. and
100 RPM continuous agitation) in liquid YEP medium (10 gm/l yeast
extract, 10 gm/l Bacto.RTM.peptone and 5 gm/l NaCl, pH 7.0) with
the appropriate antibiotics required for bacterial strain and
binary plasmid maintenance. The suspension is used when it reaches
an OD.sub.600 of about 0.4 to 0.8. The Agrobacterium cells are
pelleted and resuspended at a final OD.sub.600 of 0.5 in an
inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l
NH.sub.4Cl and 0.3 gm/l MgSO.sub.4.
[0252] Freshly bombarded explants are placed in an Agrobacterium
suspension, mixed, and left undisturbed for 30 minutes. The
explants are then transferred to GBA medium and co-cultivated, cut
surface down, at 26.degree. C. and 18-hour days. After three days
of co-cultivation, the explants are transferred to 374B (GBA medium
lacking growth regulators and a reduced sucrose level of 1%)
supplemented with 250 mg/l cefotaxime and 50 mg/l kanamycin
sulfate. The explants are cultured for two to five weeks on
selection and then transferred to fresh 374B medium lacking
kanamycin for one to two weeks of continued development. Explants
with differentiating, antibiotic-resistant areas of growth that
have not produced shoots suitable for excision are transferred to
GBA medium containing 250 mg/l cefotaxime for a second 3-day
phytohormone treatment. Leaf samples from green,
kanamycin-resistant shoots are assayed for the presence of NPTII by
ELISA and for the presence of transgene expression by assaying for
a modulation in meristem development (i.e., an alteration of size
and appearance of shoot and floral meristems).
[0253] NPTII-positive shoots are grafted to Pioneer.RTM. hybrid
6440 in vitro-grown sunflower seedling rootstock. Surface
sterilized seeds are germinated in 48-0 medium (half-strength
Murashige and Skoog salts, 0.5% sucrose, 0.3% Gelrite.RTM., pH 5.6)
and grown under conditions described for explant culture. The upper
portion of the seedling is removed, a 1 cm vertical slice is made
in the hypocotyl, and the transformed shoot inserted into the cut.
The entire area is wrapped with Parafilm.RTM. to secure the shoot.
Grafted plants can be transferred to soil following one week of in
vitro culture. Grafts in soil are maintained under high humidity
conditions followed by a slow acclimatization to the greenhouse
environment. Transformed sectors of T0 plants (parental generation)
maturing in the greenhouse are identified by NPTII ELISA and/or by
GS activity analysis of leaf extracts while transgenic seeds
harvested from NPTII-positive T0 plants are identified by GS
activity analysis of small portions of dry seed cotyledon.
[0254] An alternative sunflower transformation protocol allows the
recovery of transgenic progeny without the use of chemical
selection pressure. Seeds are dehulled and surface-sterilized for
20 minutes in a 20% Clorox.RTM. bleach solution with the addition
of two to three drops of Tween.RTM. 20 per 100 ml of solution, then
rinsed three times with distilled water. Sterilized seeds are
imbibed in the dark at 26.degree. C. for 20 hours on filter paper
moistened with water. The cotyledons and root radical are removed,
and the meristem explants are cultured on 374E (GBA medium
consisting of MS salts, Shepard vitamins, 40 mg/l adenine sulfate,
3% sucrose, 0.5 mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA and 0.8%
Phytagar at pH 5.6) for 24 hours under the dark. The primary leaves
are removed to expose the apical meristem, around 40 explants are
placed with the apical dome facing upward in a 2 cm circle in the
center of 374M (GBA medium with 1.2% Phytagar) and then cultured on
the medium for 24 hours in the dark.
[0255] Approximately 18.8 mg of 1.8 .mu.m tungsten particles are
resuspended in 150 .mu.l absolute ethanol. After sonication, 8
.mu.l of it is dropped on the center of the surface of
macrocarrier. Each plate is bombarded twice with 650 psi rupture
discs in the first shelf at 26 mm of Hg helium gun vacuum.
[0256] The plasmid of interest is introduced into Agrobacterium
tumefaciens strain EHA105 via freeze thawing as described
previously. The pellet of overnight-grown bacteria at 28.degree. C.
in a liquid YEP medium (10 g/l yeast extract, 10 g/l
Bacto.RTM.peptone and 5 g/l NaCl, pH 7.0) in the presence of 50
.mu.g/l kanamycin is resuspended in an inoculation medium (12.5 mM
2-mM 2-(N-morpholino) ethanesulfonic acid, MES, 1 g/l NH.sub.4Cl
and 0.3 g/l MgSO.sub.4 at pH 5.7) to reach a final concentration of
4.0 at OD.sub.600. Particle-bombarded explants are transferred to
GBA medium (374E) and a droplet of bacteria suspension is placed
directly onto the top of the meristem. The explants are
co-cultivated on the medium for 4 days, after which the explants
are transferred to 374C medium (GBA with 1% sucrose and no BAP,
IAA, GA3 and supplemented with 250 .mu.g/ml cefotaxime). The
plantlets are cultured on the medium for about two weeks under
16-hour day and 26.degree. C. incubation conditions.
[0257] Explants (around 2 cm long) from two weeks of culture in
374C medium are screened for a modulation in meristem development
(i.e., an alteration of size and appearance of shoot and floral
meristems). After positive (i.e., a decrease in GS expression)
explants are identified, those shoots that fail to exhibit a
decrease in GS activity are discarded and every positive explant is
subdivided into nodal explants. One nodal explant contains at least
one potential node. The nodal segments are cultured on GBA medium
for three to four days to promote the formation of auxiliary buds
from each node. Then they are transferred to 374C medium and
allowed to develop for an additional four weeks. Developing buds
are separated and cultured for an additional four weeks on 374C
medium. Pooled leaf samples from each newly recovered shoot are
screened again by the appropriate protein activity assay. At this
time, the positive shoots recovered from a single node will
generally have been enriched in the transgenic sector detected in
the initial assay prior to nodal culture.
[0258] Recovered shoots positive for a decreased GS expression are
grafted to Pioneer hybrid 6440 in vitro-grown sunflower seedling
rootstock. The rootstocks are prepared in the following manner.
Seeds are dehulled and surface-sterilized for 20 minutes in a 20%
Clorox.RTM. bleach solution with the addition of two to three drops
of Tween.RTM. 20 per 100 ml of solution and are rinsed three times
with distilled water. The sterilized seeds are germinated on the
filter moistened with water for three days, then they are
transferred into 48 medium (half-strength MS salt, 0.5% sucrose,
0.3% Gelrite.RTM. pH 5.0) and grown at 26.degree. C. under the dark
for three days, then incubated at 16-hour-day culture conditions.
The upper portion of selected seedling is removed, a vertical slice
is made in each hypocotyl, and a transformed shoot is inserted into
a V-cut. The cut area is wrapped with Parafilm.RTM.. After one week
of culture on the medium, grafted plants are transferred to soil.
In the first two weeks, they are maintained under high humidity
conditions to acclimatize to a greenhouse environment.
Example 6
Molecular Analyses for Transgene Expression
[0259] All the transgenic T0 and T1 events were characterized at
molecular level by genomic and RT-PCR using transgene specific PCR
primers. The single-copy and transgene expressing events were
advanced for further experiments. Actin expression was used as an
internal control in all the PCR reactions. In most cases transgene
expression was as expected from the promoter specificity used for
driving the transgene.
Example 7
Glutamine Synthase (GS) Enzyme Activity in Transgenic Plants
[0260] Glutamine synthase activity was indirectly measured by the
transferase assay shown below.
##STR00001##
.gamma.-glutamylhydroxamate (.gamma.-GHA) thus produced is measured
with acidified FeCl.sub.3, which yields a brown color that absorbs
maximally at 540 nm wavelength.
[0261] GS enzyme activity was determined in the leaves of
field-grown T0 transgenic events transformed with PHP32005, 32006,
32007, 32008, 38267, 28268 and 38269 in an inbred, ETX. The results
from the individual events (FIG. 4A, 4C) and average of all the
events (FIG. 4B, 4D) for each construct are summarized. In case of
ZM-GS1-3 over-expression PHPs, the highest activity (on an average
12.times. higher) was observed in PHP32008 (ZmPEPC1 PRO:ZmGS1-3)
followed by PHP32007 (ZmUBI PRO:ZmGS1-3) where the activity was
slightly higher than the controls in PHP32005 (pZmSSU PRO:ZmGS1-3).
In case of PHP32006 (ZmRM2 PRO:ZmGS1-3) leaf samples the activity
was comparable to control as expected because RM2 is a
root-preferred promoter. The roots of these events, however, showed
significantly higher GS activity as compare to non-transgenic sibs.
In the case of ZM-GS1-4 over-expression PHPs the highest GS
activity was observed in PHP38269 (pZM-PEPC::ZM-GS1-4) followed by
PHP38267 (pZM-UBI:2M-GS1-4). In the case of PHP32268 (ZmRM2
PRO:ZmGS1-4) leaf samples the activity was comparable to control,
as is expected because RM2 promoter is a root-preferred promoter.
The average activities of all the events in each construct are
summarized in FIGS. 4B and 4D.
[0262] As described in Table 2, all five isoforms ZM-GS1 were also
over-expressed in FAST corn system under the control of a
root-preferred (RM2) or constitutive promoters (UBI). T1 seeds of
all these transgenic events along with non-transgenic segregating
seeds were grown in Turface. Three weeks after germination, the
leaves and roots were harvested for GS enzyme activity analyses.
The results from these experiments are summarized in FIG. 5. For
the transgenic events where various GS1 isoforms were driven by a
root-preferred promoter (RM2), significantly higher GS activities
were observed in roots as compare to null controls (FIG. 5A, 5B).
In the constitutive promoter (UBI) driven GS1 isoforms events, GS
activity was increased as compared to null controls (FIG. 5C,
5D).
Example 8
Improved Specific Growth Rate (SGR) in T0 FAST Events
[0263] As described in Table 2, all five isoforms ZM-GS1 were also
over-expressed in FAST corn system under the control of a
root-preferred (RM2) or constitutive promoters (UBI). On an
average, 10 independent transgenic events were generated from each
construct. In all the T0 events, measurements recorded included but
were not limited to specific growth rate, max total area, days to
shed, seed number, ear length and yield estimates. The data from
specific growth rate (SGR, measured from 14-28 days after
germination) from this experiment are shown in FIG. 6. Most of the
events from each of the 6 constructs (out of total 10) tested
showed significantly better specific growth rate as compare to
controls (0.00) (FIG. 6, upper panel). PHP32772 (RM2 PRO:ZmGS1-4)
performed best with a P value >10.sup.-6 followed by PHP32779
(RM2 PRO:ZmGS1-3) with a P value 10.sup.-5 (FIG. 6a). Other 4
constructs also show better SGR with a P value ranging from
10.sup.-2 to 10.sup.-4 (FIG. 6A). Most of the events in each
construct performed significantly better than control (FIG. 6B).
For example, more than 80% and 70% events exceeded the performance
of control in PHP32779 and 32779, respectively (FIG. 6B).
Example 9
Improved Agronomic Traits in T.sub.0 FAST Events of PHP32743
ZM-RM2-PRO:ZM-GS1-5)
[0264] Over-expression of ZM-GS1-5 under the control of a
root-specific promoter resulted in improvement of several agronomic
traits in T0 phenomics measurements. The results from average of
nine events for several of these variables are summarized in FIG.
7. Multiple transgenic events from PHP32743 showed .about.50%
increase in ear length, .about.25% increase in seed number and
yield estimates and .about.18% increase in maximum total area over
the control.
Example 10
Improved Growth and N Concentrations in PHP32006 (pZMRM2:ZmGS1-3)
and PHP 32007 (pUBI:ZMGS1-3) in Low N Conditions
[0265] To test the effect of increased GS activity on plant
performance, that is, alteration in growth rate, N concentration in
the plant and total N accumulated, the plants were grown in a
semi-hydroponics system similar to that described by Tollenaar and
Migus (Tollenaar and Migus, (1984) Can J. Plant Sci. 64:465-485).
Transgenic seeds from testcrosses segregating 1:1
hemizygous:wildtype for pRM2:ZMGS1-3 and pUBI:ZMGS1-3 were
separated using a seed marker and planted, two seeds in each 4 inch
square plastic pot filled with Turface MVP.RTM. and thinned to 1
plant per pot after emergence. These were watered four times a day
with 400 ml of nutrient solution (1 mM KNO.sub.3, 2 mM MgSO.sub.4,
1 mM CaCl.sub.2, 0.5 mM KH.sub.2PO.sub.4, 3 mM KCl, 83 ppm
Sprint330, 3 .mu.M H.sub.3BO.sub.4, 1 .mu.M MnCl.sub.2, 1 .mu.M
ZnSO.sub.4, 0.1 .mu.M CuSO.sub.4, 0.1 .mu.M NaMoO.sub.4 and
sufficient H.sub.2SO.sub.4 to attain a pH of 5.5). Nineteen days
after planting, seedlings were removed from the pot, the rooting
material washed from the roots, the roots and shoots separated and
the plant parts dried at 70.degree. C. for 70 hr. Root, shoot and
total dry weights were determined, the dried plants ground to a
fine powder and approximately 35 mg tissue used to determine total
reduced N by micro-Kjeldahl method (Yasuhura and Nokihara, (2001) J
Agric Food Chem 49:4581-4583). Data were analyzed as described
(Loussaert, (1992) Agron J. 84:256-259) and transgenic mean
parameters compared to the corresponding null mean parameters.
There were 9 replicates of each treatment combination. The data for
root dry weight, shoot dry weight, total dry weight and total N
were collected and summarized in FIG. 8. Four out of six and 3 out
of 5 events significantly outperformed (denotes with asterisk in
FIGS. 8a and 8b) the null control for all the parameters measured
in PHP32006 (FIG. 8a) and 32007 (FIG. 8b), respectively.
Example 12
Variants of GS Sequences
[0266] A. Variant Nucleotide Sequences of Gs that do not Alter the
Encoded Amino Acid Sequence
[0267] The GS nucleotide sequences are used to generate variant
nucleotide sequences having the nucleotide sequence of the open
reading frame with about 70%, 75%, 80%, 85%, 90% and 95% nucleotide
sequence identity when compared to the starting unaltered ORF
nucleotide sequence of the corresponding SEQ ID NO. These
functional variants are generated using a standard codon table.
While the nucleotide sequence of the variants are altered, the
amino acid sequence encoded by the open reading frames do not
change.
[0268] B. Variant Amino Acid Sequences of GS Polypeptides
[0269] Variant amino acid sequences of the GS polypeptides are
generated. In this example, one amino acid is altered.
Specifically, the open reading frames are reviewed to determine the
appropriate amino acid alteration. The selection of the amino acid
to change is made by consulting the protein alignment (with the
other orthologs and other gene family members from various
species). An amino acid is selected that is deemed not to be under
high selection pressure (not highly conserved) and which is rather
easily substituted by an amino acid with similar chemical
characteristics (i.e., similar functional side-chain). Using the
protein alignment set forth in FIG. 1, an appropriate amino acid
can be changed. Once the targeted amino acid is identified, the
procedure outlined in the following section C is followed. Variants
having about 70%, 75%, 80%, 85%, 90% and 95% nucleic acid sequence
identity are generated using this method.
[0270] C. Additional Variant Amino Acid Sequences of GS
Polypeptides
[0271] In this example, artificial protein sequences are created
having 80%, 85%, 90% and 95% identity relative to the reference
protein sequence. This latter effort requires identifying conserved
and variable regions from the alignment set forth in FIG. 1 and
then the judicious application of an amino acid substitutions
table. These parts will be discussed in more detail below.
[0272] Largely, the determination of which amino acid sequences are
altered is made based on the conserved regions among GS protein or
among the other GS polypeptides. Based on the sequence alignment,
the various regions of the GS polypeptide that can likely be
altered are represented in lower case letters, while the conserved
regions are represented by capital letters. It is recognized that
conservative substitutions can be made in the conserved regions
below without altering function. In addition, one of skill will
understand that functional variants of the GS sequence of the
invention can have minor non-conserved amino acid alterations in
the conserved domain.
[0273] Artificial protein sequences are then created that are
different from the original in the intervals of 80-85%, 85-90%,
90-95% and 95-100% identity. Midpoints of these intervals are
targeted, with liberal latitude of plus or minus 1%, for example.
The amino acids substitutions will be effected by a custom Perl
script. The substitution table is provided below in Table 3.
TABLE-US-00003 TABLE 3 Substitution Table Strongly Similar and Rank
of Optimal Order to Amino Acid Substitution Change Comment I L, V 1
50:50 substitution L I, V 2 50:50 substitution V I, L 3 50:50
substitution A G 4 G A 5 D E 6 E D 7 W Y 8 Y W 9 S T 10 T S 11 K R
12 R K 13 N Q 14 Q N 15 F Y 16 M L 17 First methionine cannot
change H Na No good substitutes C Na No good substitutes P Na No
good substitutes
[0274] First, any conserved amino acids in the protein that should
not be changed is identified and "marked off" for insulation from
the substitution. The start methionine will of course be added to
this list automatically. Next, the changes are made.
[0275] H, C and P are not changed in any circumstance. The changes
will occur with isoleucine first, sweeping N-terminal to
C-terminal. Then leucine, and so on down the list until the desired
target it reached. Interim number substitutions can be made so as
not to cause reversal of changes. The list is ordered 1-17, so
start with as many isoleucine changes as needed before leucine, and
so on down to methionine. Clearly many amino acids will in this
manner not need to be changed. L, I and V will involve a 50:50
substitution of the two alternate optimal substitutions.
[0276] The variant amino acid sequences are written as output. Perl
script is used to calculate the percent identities. Using this
procedure, variants of the GS polypeptides are generating having
about 80%, 85%, 90% and 95% amino acid identity to the starting
unaltered ORF nucleotide sequence of SEQ ID NO: 43, 45, 47, 49, 51
and 53.
[0277] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated by reference.
[0278] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the invention.
Sequence CWU 1
1
5411307DNAArabidopsis thaliana 1ataaaaatag tgagagtgac tgagatcaat
agaaccaatc tcagaatcat cttctttctc 60tttcggaaca aaaatgacgt ctcctctctc
agatctccta aaccttgatc tatcagacac 120caagaaaatc atcgctgaat
acatatggat cggtggctct ggaatggata ttagaagcaa 180agccaggaca
ttaccaggac cagtaagtaa tccaacaaag cttccaaaat ggaactacga
240tggatctagc accgatcaag ctgccggaga tgatagtgaa gtcattcttt
atcctcaggc 300aatatttaag gacccattca ggaaggggaa caacattctg
gtgatgtgtg atgcttacag 360accggccgga gatccaattc cgaccaacaa
taggcacaag gccgtaaaaa tcttcgatca 420tcccaatgtg aaggctgaag
agccttggtt tgggatagag caagaataca cattacttaa 480aaaagatgtg
aagtggccac taggttggcc tcttggtggc tttcctggtc ctcagggacc
540gtactattgt gcagtaggtg cagacaaagc ttttggtcgt gacattgtcg
atgctcacta 600taaagcttgt ctatactccg gtttgagtat tggtggtgcc
aatggtgaag tcatgcctgg 660acaatgggag tttcaaatca gtcctactgt
tggtattggt gcaggtgatc aattatgggt 720tgctcgttac attcttgaga
ggattactga gatatgcggt gtgattgtct cattcgatcc 780aaaaccaatc
cagggtgatt ggaatggagc agccgctcat acgaacttca gtacaaaatc
840gatgaggaaa gatggaggac tggatttgat taaggaagca ataaagaagc
ttgaagtgaa 900acacaaacaa cacattgctg cttatggtga aggcaacgag
aggcgtctca ctgggaagca 960tgaaactgca gacatcaaca ctttctcttg
gggagtggcg gatcgtggag catcggtgag 1020agtaggaaga gatacggaga
aagaaggtaa agggtatttt gaagatcgaa ggccttcgtc 1080taatatggat
ccttacctag ttacctccat gattgctgaa accaccatcc tctaagcttt
1140agacttttct tcgttttggt tctttgtatg ttcttcgaat ttcggtttga
tatggtttaa 1200tttcgcattt agacttttct ttcaaataag ttacgaaatg
ttatgtgatt tctattgttt 1260gatccggtta cggttcactt ttaagccaaa
aaatctaccg ttatgac 13072353PRTArabidopsis thaliana 2Met Thr Ser Pro
Leu Ser Asp Leu Leu Asn Leu Asp Leu Ser Asp Thr1 5 10 15Lys Lys Ile
Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp 20 25 30Ile Arg
Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Ser Asn Pro Thr 35 40 45Lys
Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Asp Gln Ala Ala 50 55
60Gly Asp Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp65
70 75 80Pro Phe Arg Lys Gly Asn Asn Ile Leu Val Met Cys Asp Ala Tyr
Arg 85 90 95Pro Ala Gly Asp Pro Ile Pro Thr Asn Asn Arg His Lys Ala
Val Lys 100 105 110Ile Phe Asp His Pro Asn Val Lys Ala Glu Glu Pro
Trp Phe Gly Ile 115 120 125Glu Gln Glu Tyr Thr Leu Leu Lys Lys Asp
Val Lys Trp Pro Leu Gly 130 135 140Trp Pro Leu Gly Gly Phe Pro Gly
Pro Gln Gly Pro Tyr Tyr Cys Ala145 150 155 160Val Gly Ala Asp Lys
Ala Phe Gly Arg Asp Ile Val Asp Ala His Tyr 165 170 175Lys Ala Cys
Leu Tyr Ser Gly Leu Ser Ile Gly Gly Ala Asn Gly Glu 180 185 190Val
Met Pro Gly Gln Trp Glu Phe Gln Ile Ser Pro Thr Val Gly Ile 195 200
205Gly Ala Gly Asp Gln Leu Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile
210 215 220Thr Glu Ile Cys Gly Val Ile Val Ser Phe Asp Pro Lys Pro
Ile Gln225 230 235 240Gly Asp Trp Asn Gly Ala Ala Ala His Thr Asn
Phe Ser Thr Lys Ser 245 250 255Met Arg Lys Asp Gly Gly Leu Asp Leu
Ile Lys Glu Ala Ile Lys Lys 260 265 270Leu Glu Val Lys His Lys Gln
His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr
Gly Lys His Glu Thr Ala Asp Ile Asn Thr Phe 290 295 300Ser Trp Gly
Val Ala Asp Arg Gly Ala Ser Val Arg Val Gly Arg Asp305 310 315
320Thr Glu Lys Glu Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ser Ser
325 330 335Asn Met Asp Pro Tyr Leu Val Thr Ser Met Ile Ala Glu Thr
Thr Ile 340 345 350Leu 31499DNAArabidopsis thaliana 3gtactaccac
aaccacgaac tctaaagcat catctcatta acaaaaataa aacacacaat 60ctcaagattt
tctacttctt attacaaaga ttcaatcttc ttgtttcttc ttgcaaccat
120gagtcttctt gcagatcttg ttaaccttga catctcagac aacagtgaaa
agatcatcgc 180tgaatacata tgggttggtg gttctggtat ggacatgaga
agcaaagcca ggactctccc 240tggacctgtg accgatccat caaaacttcc
aaagtggaac tatgatggtt caagcactgg 300tcaagctcct ggtcaagaca
gtgaagtgat cttataccct caagcaattt tcaaagatcc 360attccgtaga
ggcaacaaca tccttgttat gtgtgatgct tacactccag cgggagagcc
420aatccctact aacaagcgac atgctgcggc tgagatcttt gctaaccctg
atgttattgc 480tgaagtgcca tggtatggaa tcgaacaaga atacactttg
ttgcagaagg atgtgaactg 540gcctcttgga tggcccattg gtggcttccc
tggccctcag ggaccatact actgcagtat 600tggagctgac aaatcttttg
gaagagacat tgttgatgct cactacaaag cctctttgta 660tgctggaatc
aacatcagtg ggatcaatgg agaagtcatg ccgggacaat gggagttcca
720agtcggccca tcggtcggta tctcagctgc tgatgaaata tggatcgctc
gttacatttt 780ggagaggatc acagagattg ctggtgtggt tgtatctttt
gacccaaaac ctattcctgg 840tgactggaat ggagctggtg ctcacaccaa
ttacagtact aaatcaatga gggaagaagg 900aggatacgag ataatcaaga
aggcgatcga gaagcttggc ttgagacaca aggaacacat 960ttccgcttac
ggtgaaggaa acgagcgtcg tctcacggga caccatgaaa ctgctgacat
1020caacactttc ctttggggtg ttgcgaaccg tggtgcatcg atccgagtag
gacgtgacac 1080cgagaaagaa gggaagggat actttgagga taggaggcca
gcttcaaaca tggaccctta 1140cgttgttact tccatgattg cagagactac
actcctctgg aacccttgaa aggatgatcc 1200gtaactcttg aagttgcttc
tgattgggtt ttttggaagt tccaagcttg tcttttctct 1260acagtgtgta
ttaagcaatt gtaccggttg acactgccgg agtttgtgat ttggggcctt
1320tctttctttt tcttcttttt ataatctttt gggttctgtg gttagagcaa
attcggtttg 1380ctctgtttgt ttgaccttta ttgaaacctt tggtattggt
actaataata caatctgaaa 1440aggcctcttc atgtttcaat gttagagact
aattaaagat ctcttttatt tttcatttt 14994356PRTArabidopsis thaliana
4Met Ser Leu Leu Ala Asp Leu Val Asn Leu Asp Ile Ser Asp Asn Ser1 5
10 15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Met
Asp 20 25 30Met Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Thr Asp
Pro Ser 35 40 45Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly
Gln Ala Pro 50 55 60Gly Gln Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala
Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu Val
Met Cys Asp Ala Tyr Thr 85 90 95Pro Ala Gly Glu Pro Ile Pro Thr Asn
Lys Arg His Ala Ala Ala Glu 100 105 110Ile Phe Ala Asn Pro Asp Val
Ile Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr
Leu Leu Gln Lys Asp Val Asn Trp Pro Leu Gly 130 135 140Trp Pro Ile
Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ser145 150 155
160Ile Gly Ala Asp Lys Ser Phe Gly Arg Asp Ile Val Asp Ala His Tyr
165 170 175Lys Ala Ser Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn
Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro
Ser Val Gly Ile 195 200 205Ser Ala Ala Asp Glu Ile Trp Ile Ala Arg
Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Val Val Val
Ser Phe Asp Pro Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn Gly
Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Glu
Glu Gly Gly Tyr Glu Ile Ile Lys Lys Ala Ile Glu Lys 260 265 270Leu
Gly Leu Arg His Lys Glu His Ile Ser Ala Tyr Gly Glu Gly Asn 275 280
285Glu Arg Arg Leu Thr Gly His His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300Leu Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val Gly
Arg Asp305 310 315 320Thr Glu Lys Glu Gly Lys Gly Tyr Phe Glu Asp
Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Ser
Met Ile Ala Glu Thr Thr Leu 340 345 350Leu Trp Asn Pro
35551341DNAArabidopsis thaliana 5ctctataaac acacactctc aggagagaag
ttgtattgat cgtcttctct ttccctaaac 60acactgatta ttttctctcc gacgccgcca
tgtctctgct ctcagatctc gttaacctca 120acctcaccga tgccaccggg
aaaatcatcg ccgaatacat atggatcggt ggatctggaa 180tggatatcag
aagcaaagcc aggacactac caggaccagt gactgatcca tcaaagcttc
240ccaagtggaa ctacgacgga tccagcaccg gtcaggctgc tggagaagac
agtgaagtca 300ttctataccc tcaggcaata ttcaaggatc ccttcaggaa
aggcaacaac atcctggtga 360tgtgtgatgc ttacacacca gctggtgatc
ctattccaac caacaagagg cacaacgctg 420ctaagatctt cagccacccc
gacgttgcca aggaggagcc ttggtatggg attgagcaag 480aatacacttt
gatgcaaaag gatgtgaact ggccaattgg ttggcctgtt ggtggctacc
540ctggccctca gggaccttac tactgtggtg tgggagctga caaagccatt
ggtcgtgaca 600ttgtggatgc tcactacaag gcctgtcttt acgccggtat
tggtatttct ggtatcaatg 660gagaagtcat gccaggccag tgggagttcc
aagtcggccc tgttgagggt attagttctg 720gtgatcaagt ctgggttgct
cgataccttc tcgagaggat cactgagatc tctggtgtaa 780ttgtcagctt
cgacccgaaa ccagtcccgg gtgactggaa tggagctgga gctcactgca
840actacagcac taagacaatg agaaacgatg gaggattaga agtgatcaag
aaagcgatag 900ggaagcttca gctgaaacac aaagaacaca ttgctgctta
cggtgaagga aacgagcgtc 960gtctcactgg aaagcacgaa accgcagaca
tcaacacatt ctcttgggga gtcgcgaacc 1020gtggagcgtc agtgagagtg
ggacgtgaca cagagaagga aggtaaaggg tacttcgaag 1080acagaaggcc
agcttctaac atggatcctt acgttgtcac ctccatgatc gctgagacga
1140ccatactcgg ttgatgacac atttcatgat ttgatttctc tccaatttgg
tttttttttt 1200ttcccttttg attgcacttt tcgataataa aaaaataatt
cttattatgg gcgtattgtt 1260gtgacatttt gtgttttgtt tcgaataatt
aaataagcgc ttcttaaggt gaaaataaat 1320aataattagt gatttttaat c
13416354PRTArabidopsis thaliana 6Met Ser Leu Leu Ser Asp Leu Val
Asn Leu Asn Leu Thr Asp Ala Thr1 5 10 15Gly Lys Ile Ile Ala Glu Tyr
Ile Trp Ile Gly Gly Ser Gly Met Asp 20 25 30Ile Arg Ser Lys Ala Arg
Thr Leu Pro Gly Pro Val Thr Asp Pro Ser 35 40 45Lys Leu Pro Lys Trp
Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Ala 50 55 60Gly Glu Asp Ser
Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp65 70 75 80Pro Phe
Arg Lys Gly Asn Asn Ile Leu Val Met Cys Asp Ala Tyr Thr 85 90 95Pro
Ala Gly Asp Pro Ile Pro Thr Asn Lys Arg His Asn Ala Ala Lys 100 105
110Ile Phe Ser His Pro Asp Val Ala Lys Glu Glu Pro Trp Tyr Gly Ile
115 120 125Glu Gln Glu Tyr Thr Leu Met Gln Lys Asp Val Asn Trp Pro
Ile Gly 130 135 140Trp Pro Val Gly Gly Tyr Pro Gly Pro Gln Gly Pro
Tyr Tyr Cys Gly145 150 155 160Val Gly Ala Asp Lys Ala Ile Gly Arg
Asp Ile Val Asp Ala His Tyr 165 170 175Lys Ala Cys Leu Tyr Ala Gly
Ile Gly Ile Ser Gly Ile Asn Gly Glu 180 185 190Val Met Pro Gly Gln
Trp Glu Phe Gln Val Gly Pro Val Glu Gly Ile 195 200 205Ser Ser Gly
Asp Gln Val Trp Val Ala Arg Tyr Leu Leu Glu Arg Ile 210 215 220Thr
Glu Ile Ser Gly Val Ile Val Ser Phe Asp Pro Lys Pro Val Pro225 230
235 240Gly Asp Trp Asn Gly Ala Gly Ala His Cys Asn Tyr Ser Thr Lys
Thr 245 250 255Met Arg Asn Asp Gly Gly Leu Glu Val Ile Lys Lys Ala
Ile Gly Lys 260 265 270Leu Gln Leu Lys His Lys Glu His Ile Ala Ala
Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr Gly Lys His Glu
Thr Ala Asp Ile Asn Thr Phe 290 295 300Ser Trp Gly Val Ala Asn Arg
Gly Ala Ser Val Arg Val Gly Arg Asp305 310 315 320Thr Glu Lys Glu
Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Met
Asp Pro Tyr Val Val Thr Ser Met Ile Ala Glu Thr Thr Ile 340 345
350Leu Gly71269DNAArabidopsis thaliana 7accaaaaaaa aaaaggttta
ttattctttg agattcctaa gatatgtctt cacttgcaga 60tttaatcaat ctcgatctct
ccgattccac tgaccagatc atcgccgagt acatatggat 120tggtggatcg
ggcttggata tgagaagcaa agcaaggact ttgcctggac cagtgacgga
180tccatcgcag ttaccgaaat ggaactacga cggttcaagc accggccaag
ctccgggcga 240tgacagtgaa gtcatcatct accctcaagc tatcttcaaa
gaccccttca gaagaggcaa 300caacatcctt gtgatgtgtg acgcatatac
accggcagga gagccgattc cgacgaacaa 360aaggcatgcg gcggctaaga
tctttgaaga ccctagtgtt gtcgccgaag aaacatggta 420cggaattgaa
caagagtata ccttgttgca aaaggatatt aagtggccgg taggttggcc
480ggtcggcggt ttcccaggtc ctcagggacc gtactactgt ggagttggag
cagacaaagc 540ctttggaaga gacatcgttg attctcatta caaagcttgt
ctttacgccg gaatcaatgt 600cagtgggact aacggcgaag ttatgcctgg
ccagtgggag ttccaagtcg gtcccaccgt 660tggaatcgct gccgccgatc
aggtctgggt tgctcgttac attcttgaga ggatcacaga 720attggctgga
gttgttctgt ctctagaccc taaaccaatt ccgggagatt ggaatggtgc
780aggggcacac acaaattaca gtacgaagtc gatgagagaa gatggagggt
acgaggtgat 840aaagaaagca atagagaagc ttggattgcg tcacaaggaa
cacattgctg cttatggtga 900aggcaacgag cgtcgtctca ccggaaaaca
tgaaaccgcc gatatcaaca ctttcttatg 960gggtgtggca aaccgtgggg
catcgattag ggttgggcgt gacactgagc aggctggaaa 1020aggatacttt
gaagatcgta ggccagcttc gaacatggat ccttacactg tgacctccat
1080gattgctgaa tccacaatcc tttggaaacc atgaaagaag aaaccttgag
cctcaaggaa 1140tctctataat atcagttcat gttcattctt ctatggtctc
tttctcattc tgaaacagtt 1200ctcatgtgtt ctttgtttat tatgtttgat
ttgaagtctt caatttgttt ctgagaacga 1260tagttcctc
12698356PRTArabidopsis thaliana 8Met Ser Ser Leu Ala Asp Leu Ile
Asn Leu Asp Leu Ser Asp Ser Thr1 5 10 15Asp Gln Ile Ile Ala Glu Tyr
Ile Trp Ile Gly Gly Ser Gly Leu Asp 20 25 30Met Arg Ser Lys Ala Arg
Thr Leu Pro Gly Pro Val Thr Asp Pro Ser 35 40 45Gln Leu Pro Lys Trp
Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro 50 55 60Gly Asp Asp Ser
Glu Val Ile Ile Tyr Pro Gln Ala Ile Phe Lys Asp65 70 75 80Pro Phe
Arg Arg Gly Asn Asn Ile Leu Val Met Cys Asp Ala Tyr Thr 85 90 95Pro
Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg His Ala Ala Ala Lys 100 105
110Ile Phe Glu Asp Pro Ser Val Val Ala Glu Glu Thr Trp Tyr Gly Ile
115 120 125Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile Lys Trp Pro
Val Gly 130 135 140Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro
Tyr Tyr Cys Gly145 150 155 160Val Gly Ala Asp Lys Ala Phe Gly Arg
Asp Ile Val Asp Ser His Tyr 165 170 175Lys Ala Cys Leu Tyr Ala Gly
Ile Asn Val Ser Gly Thr Asn Gly Glu 180 185 190Val Met Pro Gly Gln
Trp Glu Phe Gln Val Gly Pro Thr Val Gly Ile 195 200 205Ala Ala Ala
Asp Gln Val Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile 210 215 220Thr
Glu Leu Ala Gly Val Val Leu Ser Leu Asp Pro Lys Pro Ile Pro225 230
235 240Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys
Ser 245 250 255Met Arg Glu Asp Gly Gly Tyr Glu Val Ile Lys Lys Ala
Ile Glu Lys 260 265 270Leu Gly Leu Arg His Lys Glu His Ile Ala Ala
Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr Gly Lys His Glu
Thr Ala Asp Ile Asn Thr Phe 290 295 300Leu Trp Gly Val Ala Asn Arg
Gly Ala Ser Ile Arg Val Gly Arg Asp305 310 315 320Thr Glu Gln Ala
Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Met
Asp Pro Tyr Thr Val Thr Ser Met Ile Ala Glu Ser Thr Ile 340 345
350Leu Trp Lys Pro 35591604DNAArabidopsis thaliana 9cttcttaatt
gtttcctctt gtgttttgtt aacttttttt ctagcattct tgatctgttg 60ttcttgtcac
ttgttttgtt ttctgggatc atcaatccaa tggctcagat cttagcagct
120tctccaacat gtcagatgag agtgcctaaa cactcatcag tcattgcatc
atcatccaag 180ttatggagct ctgttgtgtt gaaacagaag aagcagagca
acaacaaagt cagaggcttt 240agagttcttg ctctccaatc tgataacagt
actgtcaata gagttgagac tcttctcaat 300ttagacacca aaccttactc
tgacaggatc attgctgaat acatttggat cggaggatct 360ggaattgacc
ttagaagcaa gtcaaggact atcgaaaagc cggtggagga tccttctgag
420ctacctaagt ggaactatga tggttcgagt accggtcaag cacctggtga
agatagtgaa 480gtgattctat acccgcaagc tatcttcaga gatcctttcc
gtggaggcaa taacatcttg 540gttatctgtg atacttggac accagctggt
gagccaattc caacaaacaa acgtgctaaa 600gctgctgaga tcttcagtaa
caagaaggtc tctggcgagg ttccatggtt cggcattgaa
660caagagtaca ctttacttca gcaaaacgtc aaatggcctt taggttggcc
tgttggagcg 720ttccctggtc ctcagggtcc ttactactgt ggagttggag
ctgacaagat ttgggggcgt 780gacatttcag atgctcatta caaagcttgt
ttatatgctg gaattaacat tagtggtact 840aatggtgaag ttatgcctgg
acagtgggag ttccaagttg gcccgagcgt aggaattgat 900gcaggtgatc
atgtttggtg tgctagatac cttcttgaga gaatcacaga acaagctggt
960gttgtcctaa cacttgatcc caaaccgata gagggtgact ggaacggtgc
tggttgccac 1020accaattaca gtaccaagag catgagagag gaaggaggat
ttgaagtgat caagaaggct 1080atcttgaacc tctcgcttcg ccacaaggag
cacatcagtg cctacggtga aggaaacgag 1140agaaggttga ccggaaagca
cgagacagct agtattgacc agttctcatg gggcgtggct 1200aaccgtggat
gctctattcg tgtgggacgt gacaccgagg cgaaaggaaa aggttactta
1260gaagatcgcc gtccagcatc taacatggac ccatacattg tgacctcact
tttggcagag 1320accacactcc tgtgggagcc aactcttgag gctgaagccc
ttgcagctca aaagctttct 1380ttgaatgttt aaaattagtc gaaactttca
tgaatctgat gaacacacgt gtctatgtgg 1440tctctcaagt tgtttaaaca
ttcggattaa gacattgttt gttgtctttt catttgcatt 1500tttaaaactc
agaattgtat ggacaatgtt catcctttta tattggttct tttgactgtt
1560agagcatgtc caatggttga atttaagctg gttcttaact gttg
160410430PRTArabidopsis thaliana 10Met Ala Gln Ile Leu Ala Ala Ser
Pro Thr Cys Gln Met Arg Val Pro1 5 10 15Lys His Ser Ser Val Ile Ala
Ser Ser Ser Lys Leu Trp Ser Ser Val 20 25 30Val Leu Lys Gln Lys Lys
Gln Ser Asn Asn Lys Val Arg Gly Phe Arg 35 40 45Val Leu Ala Leu Gln
Ser Asp Asn Ser Thr Val Asn Arg Val Glu Thr 50 55 60Leu Leu Asn Leu
Asp Thr Lys Pro Tyr Ser Asp Arg Ile Ile Ala Glu65 70 75 80Tyr Ile
Trp Ile Gly Gly Ser Gly Ile Asp Leu Arg Ser Lys Ser Arg 85 90 95Thr
Ile Glu Lys Pro Val Glu Asp Pro Ser Glu Leu Pro Lys Trp Asn 100 105
110Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro Gly Glu Asp Ser Glu Val
115 120 125Ile Leu Tyr Pro Gln Ala Ile Phe Arg Asp Pro Phe Arg Gly
Gly Asn 130 135 140Asn Ile Leu Val Ile Cys Asp Thr Trp Thr Pro Ala
Gly Glu Pro Ile145 150 155 160Pro Thr Asn Lys Arg Ala Lys Ala Ala
Glu Ile Phe Ser Asn Lys Lys 165 170 175Val Ser Gly Glu Val Pro Trp
Phe Gly Ile Glu Gln Glu Tyr Thr Leu 180 185 190Leu Gln Gln Asn Val
Lys Trp Pro Leu Gly Trp Pro Val Gly Ala Phe 195 200 205Pro Gly Pro
Gln Gly Pro Tyr Tyr Cys Gly Val Gly Ala Asp Lys Ile 210 215 220Trp
Gly Arg Asp Ile Ser Asp Ala His Tyr Lys Ala Cys Leu Tyr Ala225 230
235 240Gly Ile Asn Ile Ser Gly Thr Asn Gly Glu Val Met Pro Gly Gln
Trp 245 250 255Glu Phe Gln Val Gly Pro Ser Val Gly Ile Asp Ala Gly
Asp His Val 260 265 270Trp Cys Ala Arg Tyr Leu Leu Glu Arg Ile Thr
Glu Gln Ala Gly Val 275 280 285Val Leu Thr Leu Asp Pro Lys Pro Ile
Glu Gly Asp Trp Asn Gly Ala 290 295 300Gly Cys His Thr Asn Tyr Ser
Thr Lys Ser Met Arg Glu Glu Gly Gly305 310 315 320Phe Glu Val Ile
Lys Lys Ala Ile Leu Asn Leu Ser Leu Arg His Lys 325 330 335Glu His
Ile Ser Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu Thr Gly 340 345
350Lys His Glu Thr Ala Ser Ile Asp Gln Phe Ser Trp Gly Val Ala Asn
355 360 365Arg Gly Cys Ser Ile Arg Val Gly Arg Asp Thr Glu Ala Lys
Gly Lys 370 375 380Gly Tyr Leu Glu Asp Arg Arg Pro Ala Ser Asn Met
Asp Pro Tyr Ile385 390 395 400Val Thr Ser Leu Leu Ala Glu Thr Thr
Leu Leu Trp Glu Pro Thr Leu 405 410 415Glu Ala Glu Ala Leu Ala Ala
Gln Lys Leu Ser Leu Asn Val 420 425 430111494DNAArabidopsis
thaliana 11tgtggagagc caaaaagtct ccaaagtctt cacgtcaccc tcttcctcaa
tctctgcacc 60cacccctcct ccttctataa gtactactct tcatatctct ctctaccaaa
atatcaaaac 120acgagacaga tttgattcca tttttattac tgttactatc
atccaaaccc ttggtatttg 180tagccatgag tcttgtttca gatctcatca
accttaacct ctcagactcc actgacaaaa 240tcattgctga atacatatgg
gttggtggtt ctggaatgga catgagaagc aaagccagga 300ctctacctgg
accagtgact gacccttcgc agctaccaaa gtggaactat gatggttcaa
360gcacaggcca agctcctggt gaagacagtg aagtcatctt ataccctcaa
gccatattca 420aggatccttt ccgtagagga aacaacattc ttgtcatgtg
cgatgcgtac actcccgcgg 480gtgaaccaat cccgactaac aaaagacacg
ctgcggctaa ggtctttagc aaccctgatg 540ttgcagctga agtgccatgg
tatggtattg agcaagaata cactttactc cagaaagatg 600tgaagtggcc
tgttggttgg cctattggtg gttatcccgg ccctcaggga ccgtactatt
660gcggtattgg agcagacaaa tcttttggca gagatgttgt tgattctcac
tacaaggcct 720gcttatacgc tgggatcaac attagtggca tcaatggaga
agtcatgccg ggtcagtggg 780agttccaggt cggtccagct gttggtatct
cggctgctga tgaaatttgg gtcgctcgtt 840acattttgga gaggatcaca
gagattgctg gtgtagtggt atcttttgac ccgaaaccga 900ttcccggtga
ctggaacggt gctggtgctc actgcaacta cagtaccaag tcaatgaggg
960aagaaggcgg ttacgagatc atcaagaaag caatcgataa attgggactg
agacacaaag 1020aacacattgc tgcttacggt gaaggcaatg agcgtcgtct
cacaggacac cacgagactg 1080ctgacatcaa cactttcctt tggggtgttg
cgaaccgtgg agcatcgatc cgagtaggac 1140gtgatacgga gaaagaaggg
aaaggatact ttgaggacag gaggccagct tcgaacatgg 1200atccttacat
tgtcacttcc atgattgcag agactacaat cctctggaat ccttgatgat
1260catcagatca agaaaaaatc ttgaatgtca ctcaaatttg tgtttcttgc
aagattcaaa 1320gtttgtgttc tctatcaagc aatgtcttag gataagtcaa
agatttgctc tgcttattct 1380gctttttatt tacttcacat cctattgaaa
acatttctgt gtattattta tgaataaaca 1440ttatcttaaa agggctgatt
tatttactaa tgcatgcatt caccacttaa gatc 149412356PRTArabidopsis
thaliana 12Met Ser Leu Val Ser Asp Leu Ile Asn Leu Asn Leu Ser Asp
Ser Thr1 5 10 15Asp Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser
Gly Met Asp 20 25 30Met Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val
Thr Asp Pro Ser 35 40 45Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser
Thr Gly Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro
Gln Ala Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile
Leu Val Met Cys Asp Ala Tyr Thr 85 90 95Pro Ala Gly Glu Pro Ile Pro
Thr Asn Lys Arg His Ala Ala Ala Lys 100 105 110Val Phe Ser Asn Pro
Asp Val Ala Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu
Tyr Thr Leu Leu Gln Lys Asp Val Lys Trp Pro Val Gly 130 135 140Trp
Pro Ile Gly Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150
155 160Ile Gly Ala Asp Lys Ser Phe Gly Arg Asp Val Val Asp Ser His
Tyr 165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile
Asn Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly
Pro Ala Val Gly Ile 195 200 205Ser Ala Ala Asp Glu Ile Trp Val Ala
Arg Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Val Val
Val Ser Phe Asp Pro Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn
Gly Ala Gly Ala His Cys Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg
Glu Glu Gly Gly Tyr Glu Ile Ile Lys Lys Ala Ile Asp Lys 260 265
270Leu Gly Leu Arg His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn
275 280 285Glu Arg Arg Leu Thr Gly His His Glu Thr Ala Asp Ile Asn
Thr Phe 290 295 300Leu Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg
Val Gly Arg Asp305 310 315 320Thr Glu Lys Glu Gly Lys Gly Tyr Phe
Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Ile Val
Thr Ser Met Ile Ala Glu Thr Thr Ile 340 345 350Leu Trp Asn Pro
355131364DNAGlycine max 13accctatcaa agaaagctac ctagagcttg
cacctattgg tatcttctac aatatcctct 60catagtgctc ttcttcttct tcattttcat
tatcaagatg tctttgcttt cggatctcat 120caacctcaat ctctcagaat
ccacagaaaa gatcgttgct gagtacatat gggttggtgg 180atctggtatg
gacctcagaa gcaaagccag gactcttcct gggccagtga gtgaccctgc
240aaagcttcca aagtggaact acgatggctc tagcacagac caagctccag
gggatgacag 300tgaagtcatc ctatacccac aagctatttt caaggacccc
tttaggagag gcaacaatat 360tcttgtgatt tgtgatgttt acacccccgc
tggtgagcca cttccaacca acaagaggta 420tgatgctgcc aaaattttca
gccaccctga cgttgctgct gaggaaccat ggtatggtat 480tgagcaagaa
tataccttgt tgcagaaaga tgtaaattgg ccacttgggt ggccacttgg
540tgggtttcct ggaccacagg gcccatacta ctgtggaact ggtgctgata
aagcatatgg 600ccgtgatatt gtagatgcac attacaaagc ttgtatttat
gctggcatca atattagtgg 660catcaatgga gaggttatgc ctggtcagtg
ggaatttcaa gttggtcctt ctgttggtat 720atctgctgga gatgaggtgt
gggcagctcg gtacattttg gagaggatta cagagatggc 780cggagtaatt
gtttcatttg atcccaagcc tattccggga gattggaatg gagctggagc
840tcactcaaac tacagcacca agtccatgag agatgagggt ggttatgagg
tgattaagaa 900ggccattgaa aagcttggat tgaggcacaa ggagcacatt
gcagcatatg gagaaggcaa 960cgagagacgt ctcactggaa gacatgaaac
tgcagacatc aacaccttct cttggggtgt 1020ggcaaaccgt ggaagctcca
ttagagttgg aagagacaca gagaaaaatg gcaaaggtta 1080ctttgaggac
agaaggcctg cttctaatat ggatccatat gtagtcacct ccatgatcgc
1140agagactacc atcctctgga aaccatgaaa aacagtcata tagtctctag
atttggacca 1200ctaaaaattg tgttcaatag tcatttgatc taaaaattta
tatttgcaag gtgatgttta 1260gttaggaatt tctaagtggt ctttttgagc
ctccatgtgc catgtctatg gttgagaata 1320atttcgtcat taataacaag
aatttcccat acactgttcc gtgc 136414356PRTGlycine max 14Met Ser Leu
Leu Ser Asp Leu Ile Asn Leu Asn Leu Ser Glu Ser Thr1 5 10 15Glu Lys
Ile Val Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Met Asp 20 25 30Leu
Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Ser Asp Pro Ala 35 40
45Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Asp Gln Ala Pro
50 55 60Gly Asp Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys
Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu Val Ile Cys Asp
Val Tyr Thr 85 90 95Pro Ala Gly Glu Pro Leu Pro Thr Asn Lys Arg Tyr
Asp Ala Ala Lys 100 105 110Ile Phe Ser His Pro Asp Val Ala Ala Glu
Glu Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr Leu Leu Gln
Lys Asp Val Asn Trp Pro Leu Gly 130 135 140Trp Pro Leu Gly Gly Phe
Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155 160Thr Gly Ala
Asp Lys Ala Tyr Gly Arg Asp Ile Val Asp Ala His Tyr 165 170 175Lys
Ala Cys Ile Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu 180 185
190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile
195 200 205Ser Ala Gly Asp Glu Val Trp Ala Ala Arg Tyr Ile Leu Glu
Arg Ile 210 215 220Thr Glu Met Ala Gly Val Ile Val Ser Phe Asp Pro
Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn Gly Ala Gly Ala His
Ser Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Asp Glu Gly Gly Tyr
Glu Val Ile Lys Lys Ala Ile Glu Lys 260 265 270Leu Gly Leu Arg His
Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg
Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe 290 295 300Ser
Trp Gly Val Ala Asn Arg Gly Ser Ser Ile Arg Val Gly Arg Asp305 310
315 320Thr Glu Lys Asn Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala
Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Ser Met Ile Ala Glu
Thr Thr Ile 340 345 350Leu Trp Lys Pro 355151369DNAGlycine max
15aggaagagaa agaaatttgt ttctctctaa agagtctccg ctgaactttt tggtttcttg
60aagatgtcgt tactctccga tcttatcaac cttaacctct ccgacatcac cgataaggtg
120atcgccgagt acatatgggt tggtggatct ggcatggata tgaggagcaa
agcaaggact 180ctctcgggac tggttaatga cccttccaag cttcccaagt
ggaactatga tggttccagc 240actggtcaag ctcctggaca agatagtgaa
gtgatcttat atccacaagc aatttttcgg 300gatccattca ggaggggtaa
caatatcctg gttatgtgtg atgcttacac tcctgctggg 360gaacccattc
ctaccaacaa gagaaataaa gctgcaaaga tattcagtaa tccggatgtt
420gctgctgaag aaccctggta tggtcttgag caggaatata cattattgca
gaaagatgtc 480caatggcctc ttggatggcc tcttggtggg tttcctgggc
cccagggacc atactattgt 540ggaactggtg ctaacaaggc ttttgggcgt
gatattgttg actcacatta caaagcatgt 600atttatgcgg gaattaacat
aagtggaatc aatggagaag tgatgcccgg tcagtgggaa 660ttccaagttg
gtccatcggt tggcatctct gctgctgacg agttgtgggt tgctcgttac
720attttggaga ggatcaccga gattgctgga gtggtgcttt cctttgaccc
taaaccaatt 780cagggtgatt ggaatggtgc tggtgctcac acaaattaca
gtaccaagtt gatgagaaac 840gatggtggct atgaaatcat caaaaaagca
attgctaagt tggaaaagag gcacaaagag 900cacattgctg cttacggaga
aggcaatgaa cgtcgtttga ccggacgaca cgagacggct 960gacatgaaca
cctttttatg gggtgttgca aaccgtggtg cttctattag ggtagggaga
1020gacactgaaa aggcagggaa gggatacttt gaagatagga ggcctgcctc
taacatggac 1080ccttatgtgg tcacttccat gattgctgag acaactattc
tttggaaacc ataagcaacg 1140tcaaaacaat cacatggtgc cttccgcata
gcattgttgt ttagatggtc aatttgtttt 1200tctatgtttt tgtgtgcatt
ctagttgtga ctacctcgcc tgttgttagg tattgtttgt 1260tggtggtact
catgattacc aagcgaggaa ttgttgtttc attttcttaa tgtacgtttt
1320aagtgttcca ataatgtgta atggccctca agtattgtta tttgctgcg
136916356PRTGlycine max 16Met Ser Leu Leu Ser Asp Leu Ile Asn Leu
Asn Leu Ser Asp Ile Thr1 5 10 15Asp Lys Val Ile Ala Glu Tyr Ile Trp
Val Gly Gly Ser Gly Met Asp 20 25 30Met Arg Ser Lys Ala Arg Thr Leu
Ser Gly Leu Val Asn Asp Pro Ser 35 40 45Lys Leu Pro Lys Trp Asn Tyr
Asp Gly Ser Ser Thr Gly Gln Ala Pro 50 55 60Gly Gln Asp Ser Glu Val
Ile Leu Tyr Pro Gln Ala Ile Phe Arg Asp65 70 75 80Pro Phe Arg Arg
Gly Asn Asn Ile Leu Val Met Cys Asp Ala Tyr Thr 85 90 95Pro Ala Gly
Glu Pro Ile Pro Thr Asn Lys Arg Asn Lys Ala Ala Lys 100 105 110Ile
Phe Ser Asn Pro Asp Val Ala Ala Glu Glu Pro Trp Tyr Gly Leu 115 120
125Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Val Gln Trp Pro Leu Gly
130 135 140Trp Pro Leu Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr
Cys Gly145 150 155 160Thr Gly Ala Asn Lys Ala Phe Gly Arg Asp Ile
Val Asp Ser His Tyr 165 170 175Lys Ala Cys Ile Tyr Ala Gly Ile Asn
Ile Ser Gly Ile Asn Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu
Phe Gln Val Gly Pro Ser Val Gly Ile 195 200 205Ser Ala Ala Asp Glu
Leu Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile
Ala Gly Val Val Leu Ser Phe Asp Pro Lys Pro Ile Gln225 230 235
240Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Leu
245 250 255Met Arg Asn Asp Gly Gly Tyr Glu Ile Ile Lys Lys Ala Ile
Ala Lys 260 265 270Leu Glu Lys Arg His Lys Glu His Ile Ala Ala Tyr
Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr Gly Arg His Glu Thr
Ala Asp Met Asn Thr Phe 290 295 300Leu Trp Gly Val Ala Asn Arg Gly
Ala Ser Ile Arg Val Gly Arg Asp305 310 315 320Thr Glu Lys Ala Gly
Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Met Asp
Pro Tyr Val Val Thr Ser Met Ile Ala Glu Thr Thr Ile 340 345 350Leu
Trp Lys Pro 355171579DNAGlycine max 17cacttcccac tgtgtctcag
ggtctgtgac acacacagac tcacttcaag ttcccagctt 60ttgccatttt tcccactgtt
tattgaacat ggcacagatt ttggctccct ctacgcaatg 120gcagatgaga
atctcaaaat cctctcccaa tgcaactccc attacatcaa acatgtggag
180ttctttattg tggaaacaaa ataagaaagt ttcacctacc agttctgcta
aatttagagt 240gctggcaatt aagtctgaca atagcaccat caacaggctc
gagggtctac ttaatttgga 300tatcactcca ttcactgaca agataattgc
tgagtacatt tggattgggg ggacaggaat 360tgatgtgcgc agtaaatcaa
gaacaatatc aaagcctgtt gaagatccct ctgagctccc 420taaatggaac
tatgatggat ctagcactgg acaggcacct ggtgatgata gtgaagtaat
480cctatatcct caagcaattt tcaaagatcc tttccgtggc ggtaacaata
ttttggtcat
540ttgcgattct tacaccccac aaggtgagcc tatccctaca aacaagagac
acagagctgc 600tgaaattttc agtaacccaa aggtccaagc tgaagttcca
tggtatggaa tagaacaaga 660gtacacctta cttcaaacaa atgtgaaatg
gccattagga tggccggttg gtggctatcc 720cggtcctcag ggtccttatt
attgcagtgc tggggcagat aagtcatttg gacgtgacat 780atctgatgct
cattacaagg cttgcttata tgctggaatt aacatcagtg gcaccaatgg
840ggaggttatg cctgggcagt gggagtacca agttggtcct agtgtaggta
ttgaggctgg 900tgatcatatc tgggcttcaa ggtacatcct cgagagaatt
actgagcaag ctggtgttgt 960gctctctctt gatccaaaac caatagaggg
tgactggaat ggagcaggat gccacaccaa 1020ttacagtaca aagagcatga
gggaagatgg aggctttgag gtaataaaga aggcaatttt 1080gaatctatcg
ctacgccaca aggatcacat cagtgcatat ggagaaggaa atgagagaag
1140gttgacagga aagcatgaga cagcaagcat taacacattt tcttggggag
tggctaaccg 1200tggttgctca atccgtgtgg gaagagacac agagaagaat
ggcaaaggtt acttggaaga 1260caggcgaccg gcttcaaaca tggatccata
tgttgtgaca tcattacttg cagagactac 1320actattgtgg gagccaactc
tggaggctga agctcttgca gctcagaagt tagcattgaa 1380ggtctaaacc
tattgaatga tggcattctg gatgcaaaat cactttcctt ttagattatc
1440tatatgtatt ctaatgatct tgtttggact aaagaggttg ccatgcccag
ttattggtta 1500tcatatgaaa tgcacattgt atatcagaag tttggttggt
actatttgct tcaggacaaa 1560ttttctttga tgcttggtt 157918432PRTGlycine
max 18Met Ala Gln Ile Leu Ala Pro Ser Thr Gln Trp Gln Met Arg Ile
Ser1 5 10 15Lys Ser Ser Pro Asn Ala Thr Pro Ile Thr Ser Asn Met Trp
Ser Ser 20 25 30Leu Leu Trp Lys Gln Asn Lys Lys Val Ser Pro Thr Ser
Ser Ala Lys 35 40 45Phe Arg Val Leu Ala Ile Lys Ser Asp Asn Ser Thr
Ile Asn Arg Leu 50 55 60Glu Gly Leu Leu Asn Leu Asp Ile Thr Pro Phe
Thr Asp Lys Ile Ile65 70 75 80Ala Glu Tyr Ile Trp Ile Gly Gly Thr
Gly Ile Asp Val Arg Ser Lys 85 90 95Ser Arg Thr Ile Ser Lys Pro Val
Glu Asp Pro Ser Glu Leu Pro Lys 100 105 110Trp Asn Tyr Asp Gly Ser
Ser Thr Gly Gln Ala Pro Gly Asp Asp Ser 115 120 125Glu Val Ile Leu
Tyr Pro Gln Ala Ile Phe Lys Asp Pro Phe Arg Gly 130 135 140Gly Asn
Asn Ile Leu Val Ile Cys Asp Ser Tyr Thr Pro Gln Gly Glu145 150 155
160Pro Ile Pro Thr Asn Lys Arg His Arg Ala Ala Glu Ile Phe Ser Asn
165 170 175Pro Lys Val Gln Ala Glu Val Pro Trp Tyr Gly Ile Glu Gln
Glu Tyr 180 185 190Thr Leu Leu Gln Thr Asn Val Lys Trp Pro Leu Gly
Trp Pro Val Gly 195 200 205Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr
Cys Ser Ala Gly Ala Asp 210 215 220Lys Ser Phe Gly Arg Asp Ile Ser
Asp Ala His Tyr Lys Ala Cys Leu225 230 235 240Tyr Ala Gly Ile Asn
Ile Ser Gly Thr Asn Gly Glu Val Met Pro Gly 245 250 255Gln Trp Glu
Tyr Gln Val Gly Pro Ser Val Gly Ile Glu Ala Gly Asp 260 265 270His
Ile Trp Ala Ser Arg Tyr Ile Leu Glu Arg Ile Thr Glu Gln Ala 275 280
285Gly Val Val Leu Ser Leu Asp Pro Lys Pro Ile Glu Gly Asp Trp Asn
290 295 300Gly Ala Gly Cys His Thr Asn Tyr Ser Thr Lys Ser Met Arg
Glu Asp305 310 315 320Gly Gly Phe Glu Val Ile Lys Lys Ala Ile Leu
Asn Leu Ser Leu Arg 325 330 335His Lys Asp His Ile Ser Ala Tyr Gly
Glu Gly Asn Glu Arg Arg Leu 340 345 350Thr Gly Lys His Glu Thr Ala
Ser Ile Asn Thr Phe Ser Trp Gly Val 355 360 365Ala Asn Arg Gly Cys
Ser Ile Arg Val Gly Arg Asp Thr Glu Lys Asn 370 375 380Gly Lys Gly
Tyr Leu Glu Asp Arg Arg Pro Ala Ser Asn Met Asp Pro385 390 395
400Tyr Val Val Thr Ser Leu Leu Ala Glu Thr Thr Leu Leu Trp Glu Pro
405 410 415Thr Leu Glu Ala Glu Ala Leu Ala Ala Gln Lys Leu Ala Leu
Lys Val 420 425 430191425DNAGlycine max 19atgtcgttgc tctccgatct
tatcaacctt aacctctccg acatcaccga taaaacactc 60tcaggaccgg ttaaagaccc
ttcgaagctt cccaagtgga actatgatgg ttccagcact 120ggtcaagctc
ctgggcaaga tagtgaagtg atcttatatc cacaagcaat tttcaaggat
180ccattcagga ggggtagcaa tatcctggtt atgtgtgatg cttacactcc
tgctggggaa 240cccattccta caaacaagag aaataatgct gcaaagatat
tcggccatcc tgatgttgct 300gctgaagaac cctgttactg catgattttc
aaacagggac catattattg tggtactggt 360gctaacaagg ctttcgggcg
tgatattgtt gactcacatt acaaagcatg tatttatgcg 420ggcattaaca
tcagtggaat caatggagaa gtgatgcctg gtcagaggat caccgagatt
480gcaggagtgg tgctttcctt tgaccctaaa ccaattcagt ccatgagaaa
cgatggtggc 540tatgaagtca tcaaaaaagc aattgctaag ttggaaaaga
gacacaagga gcacattgca 600gcttacggag aaggcaacga acgtcgtttg
actggacgac acgagacagc tgacatgaac 660acctttgtat ggagttgcca
atggtggtgt tgggtggagg ttgggcacaa tggcgtttgg 720tgggtggcta
cagaagtagt tttatctgtt tgggtgtttt tggaggtgca agaagtaaaa
780agggtgatgc aggaagtaaa agacggtaaa catagttttg atttcttgaa
gatgtcgtta 840ctctccgatc taatcaacat taacctctcc gacaccacca
agaagggtcc atactattgt 900ggtattggtg ctaacaaggc ttttggacgt
gacattgttg actctcattt caaagcctgt 960ctttatgcag acatcaacat
tactggaatt aatgcagaag tgatgcctgg tcagtgggaa 1020ttccgtgttg
gtccatcgct ggcatctctg cgtgtgacga cttgtgggtt gctcgctaca
1080ttttggaggt tgttagcaca cgactactct catcatcaaa ttctttcttt
tgtaaatatt 1140gcagcgaata tctctgtttg tgctaatatc tctgtttgtg
ctaatatctc tgtggtggtg 1200ctttcctttt atcctcaacc gattaagggt
gattggaatt gtgctagtgc tcacacgaat 1260tacagtacca agtcgatgag
aaatgatggt ggctatgaag tcattagaaa agcaactgcc 1320aagttggaaa
aaaggcataa ggagcacatt gctgcttatg gagaaggcaa tgaacgtcgt
1380ttgacaggtc aacatgagac agctgatatt aacaccttca taagg
142520475PRTGlycine max 20Met Ser Leu Leu Ser Asp Leu Ile Asn Leu
Asn Leu Ser Asp Ile Thr1 5 10 15Asp Lys Thr Leu Ser Gly Pro Val Lys
Asp Pro Ser Lys Leu Pro Lys 20 25 30Trp Asn Tyr Asp Gly Ser Ser Thr
Gly Gln Ala Pro Gly Gln Asp Ser 35 40 45Glu Val Ile Leu Tyr Pro Gln
Ala Ile Phe Lys Asp Pro Phe Arg Arg 50 55 60Gly Ser Asn Ile Leu Val
Met Cys Asp Ala Tyr Thr Pro Ala Gly Glu65 70 75 80Pro Ile Pro Thr
Asn Lys Arg Asn Asn Ala Ala Lys Ile Phe Gly His 85 90 95Pro Asp Val
Ala Ala Glu Glu Pro Cys Tyr Cys Met Ile Phe Lys Gln 100 105 110Gly
Pro Tyr Tyr Cys Gly Thr Gly Ala Asn Lys Ala Phe Gly Arg Asp 115 120
125Ile Val Asp Ser His Tyr Lys Ala Cys Ile Tyr Ala Gly Ile Asn Ile
130 135 140Ser Gly Ile Asn Gly Glu Val Met Pro Gly Gln Arg Ile Thr
Glu Ile145 150 155 160Ala Gly Val Val Leu Ser Phe Asp Pro Lys Pro
Ile Gln Ser Met Arg 165 170 175Asn Asp Gly Gly Tyr Glu Val Ile Lys
Lys Ala Ile Ala Lys Leu Glu 180 185 190Lys Arg His Lys Glu His Ile
Ala Ala Tyr Gly Glu Gly Asn Glu Arg 195 200 205Arg Leu Thr Gly Arg
His Glu Thr Ala Asp Met Asn Thr Phe Val Trp 210 215 220Ser Cys Gln
Trp Trp Cys Trp Val Glu Val Gly His Asn Gly Val Trp225 230 235
240Trp Val Ala Thr Glu Val Val Leu Ser Val Trp Val Phe Leu Glu Val
245 250 255Gln Glu Val Lys Arg Val Met Gln Glu Val Lys Asp Gly Lys
His Ser 260 265 270Phe Asp Phe Leu Lys Met Ser Leu Leu Ser Asp Leu
Ile Asn Ile Asn 275 280 285Leu Ser Asp Thr Thr Lys Lys Gly Pro Tyr
Tyr Cys Gly Ile Gly Ala 290 295 300Asn Lys Ala Phe Gly Arg Asp Ile
Val Asp Ser His Phe Lys Ala Cys305 310 315 320Leu Tyr Ala Asp Ile
Asn Ile Thr Gly Ile Asn Ala Glu Val Met Pro 325 330 335Gly Gln Trp
Glu Phe Arg Val Gly Pro Ser Leu Ala Ser Leu Arg Val 340 345 350Thr
Thr Cys Gly Leu Leu Ala Thr Phe Trp Arg Leu Leu Ala His Asp 355 360
365Tyr Ser His His Gln Ile Leu Ser Phe Val Asn Ile Ala Ala Asn Ile
370 375 380Ser Val Cys Ala Asn Ile Ser Val Cys Ala Asn Ile Ser Val
Val Val385 390 395 400Leu Ser Phe Tyr Pro Gln Pro Ile Lys Gly Asp
Trp Asn Cys Ala Ser 405 410 415Ala His Thr Asn Tyr Ser Thr Lys Ser
Met Arg Asn Asp Gly Gly Tyr 420 425 430Glu Val Ile Arg Lys Ala Thr
Ala Lys Leu Glu Lys Arg His Lys Glu 435 440 445His Ile Ala Ala Tyr
Gly Glu Gly Asn Glu Arg Arg Leu Thr Gly Gln 450 455 460His Glu Thr
Ala Asp Ile Asn Thr Phe Ile Arg465 470 475211454DNAGlycine max
21acatcttctt ttacgtattg aatctcagaa ttctctaaaa gagatctttt tctgctcttt
60gaagaaagaa gggtctttgc ttgattttgg agatgtctct gctctcagat ctcatcaacc
120ttaacctctc cgataccacc gagaaggtga tcgcagagta catatggatc
ggtggatcag 180gaatggacct gaggagcaaa gcaaggactc tcccaggacc
agttagcgac ccttcagagc 240ttcccaagtg gaactatgat ggttccagca
caggtcaagc tcctggtgaa gacagtgaag 300tgattttata cccacaagcc
attttcaggg atccattcag aaggggtaac aatatcttgg 360ttatctgtga
tgcctacact cctgctggag aacctattcc cactaacaag aggcacgctg
420ctgccaaggt tttcagccat cctgatgttg ttgctgaagt gccatggtac
ggtattgaac 480aagaatacac cttgttgcag aaagatatcc aatggcctct
tgggtggcct gttggtggtt 540tccctggacc tcagggtcca tactactgtg
gtgttggcgc tgacaaggct tttggccgtg 600acattgttga cgcacactac
aaagcctgta tttatgctgg catcaacatc agtggaatta 660atggagaagt
gatgcccggt cagtgggaat tccaagttgg accttcagtt ggaatctcag
720ctggtgatga gatttgggca gctcgttaca tcttggagag gatcactgag
attgctggtg 780tggtggtttc ctttgacccc aagccaatta agggtgattg
gaatggtgct ggtgctcaca 840caaactacag caccaagtcc atgagagaag
atggtggcta tgaagtgatc aaagcagcaa 900ttgacaagtt ggggaagaag
cacaaggagc acattgctgc ttatggagaa ggcaacgaac 960gtcgtttgac
aggacgccac gaaaccgctg acatcaacac cttcttatgg ggagttgcaa
1020accgtggagc ttctgttagg gttgggagag acacagagaa agcagggaag
ggatattttg 1080aggacagaag gccagcttcc aacatggacc catacgtggt
tacttccatg attgcagaca 1140caaccattct gtggaagcca tgagcaaaac
ctgcatgttt tctccctttg gatggaaagg 1200aacagttatg cttttcttag
taggatttgg tctctctctc tttttacctt ttgattggta 1260ctatggttgg
tgccttgttg gttggtgcaa ctaactggca agggttgttc attgttttct
1320tctattcctt tccctcgttt tccgattgtt acaatgacaa taatttaatg
gttattatca 1380gtcttgaaca aagaaatgct gattgtgaag tataataata
atatatgaaa ttgtcatgtt 1440cattggagta ggaa 145422356PRTGlycine max
22Met Ser Leu Leu Ser Asp Leu Ile Asn Leu Asn Leu Ser Asp Thr Thr1
5 10 15Glu Lys Val Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met
Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Ser Asp
Pro Ser 35 40 45Glu Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly
Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala
Ile Phe Arg Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu Val
Ile Cys Asp Ala Tyr Thr 85 90 95Pro Ala Gly Glu Pro Ile Pro Thr Asn
Lys Arg His Ala Ala Ala Lys 100 105 110Val Phe Ser His Pro Asp Val
Val Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr
Leu Leu Gln Lys Asp Ile Gln Trp Pro Leu Gly 130 135 140Trp Pro Val
Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155
160Val Gly Ala Asp Lys Ala Phe Gly Arg Asp Ile Val Asp Ala His Tyr
165 170 175Lys Ala Cys Ile Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn
Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro
Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Ile Trp Ala Ala Arg
Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Val Val Val
Ser Phe Asp Pro Lys Pro Ile Lys225 230 235 240Gly Asp Trp Asn Gly
Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Glu
Asp Gly Gly Tyr Glu Val Ile Lys Ala Ala Ile Asp Lys 260 265 270Leu
Gly Lys Lys His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280
285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300Leu Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly
Arg Asp305 310 315 320Thr Glu Lys Ala Gly Lys Gly Tyr Phe Glu Asp
Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Ser
Met Ile Ala Asp Thr Thr Ile 340 345 350Leu Trp Lys Pro
355231446DNAGlycine max 23aagattctaa gagagatttt gctgctcttt
gaagaagggt gtttgcttga ttttggagat 60gtcgctgctc tcagatctca tcaaccttaa
cctctcagac actactgaga aggtgatcgc 120agagtacata tggatcggtg
gatcaggaat ggacctgagg agcaaagcaa ggactctccc 180aggaccagtt
agcgaccctt caaagcttcc caagtggaac tatgatggtt ccagcacagg
240ccaagctcct ggagaagaca gtgaagtgat tatataccca caagccattt
tcagggatcc 300attcagaagg ggcaacaata tcttggttat ctgtgatact
tacactccag ctggagaacc 360cattcccact aacaagaggc acgatgctgc
caaggttttc agccatcctg atgttgttgc 420tgaagagaca tggtatggta
ttgagcagga atacaccttg ttgcagaaag atatccaatg 480gcctcttggg
tggcctgttg gtggtttccc tggaccacag ggtccatact actgtggtgt
540tggcgctgac aaggcttttg gccgtgacat tgttgacgca cattacaaag
cctgtcttta 600tgctggcatc aacatcagtg gaattaatgg agaagtgatg
cccggtcagt gggaattcca 660agttggacct tcagttggaa tctcagctgg
tgacgaggtg tgggcagctc gttacatctt 720ggagaggatc actgagattg
ctggtgtggt ggtttccttt gatcccaagc caattcaggg 780tgattggaat
ggtgctggtg ctcacacaaa ctacagcact aagtccatga gaaatgatgg
840tggctatgaa gtgatcaaaa ccgccattga gaagttgggg aagagacaca
aggagcacat 900tgctgcttat ggagaaggca acgagcgtcg tttgacaggg
cgccacgaaa ccgctgacat 960caacaccttc ttatggggag ttgcaaaccg
tggagcttca gttagggttg ggagggacac 1020agagaaagca gggaagggat
attttgagga cagaaggcca gcttctaaca tggacccata 1080tgtggttact
tccatgattg cagacacaac cattctgtgg aagccatgag caaaacttgc
1140atgttgtctc cctttggatg gaacaaggaa caaggaacaa ggaacaagga
acagttatgc 1200ttttctaagt agggtttggt cctttttatt ttttaccttt
tgatttttct aggatttcga 1260tttgtggcta ctttggttgg tgcaaccaac
tgccaagggt tgttcattgt tttctattcc 1320tttccctcgt tttccgattg
ttacaataat aataatgtaa tatggttatt ttcagtctca 1380aacaaaagta
atgctgattg tgaagtataa taatatatga aattgtcatg tccattggag 1440ttggga
144624356PRTGlycine max 24Met Ser Leu Leu Ser Asp Leu Ile Asn Leu
Asn Leu Ser Asp Thr Thr1 5 10 15Glu Lys Val Ile Ala Glu Tyr Ile Trp
Ile Gly Gly Ser Gly Met Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Leu
Pro Gly Pro Val Ser Asp Pro Ser 35 40 45Lys Leu Pro Lys Trp Asn Tyr
Asp Gly Ser Ser Thr Gly Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val
Ile Ile Tyr Pro Gln Ala Ile Phe Arg Asp65 70 75 80Pro Phe Arg Arg
Gly Asn Asn Ile Leu Val Ile Cys Asp Thr Tyr Thr 85 90 95Pro Ala Gly
Glu Pro Ile Pro Thr Asn Lys Arg His Asp Ala Ala Lys 100 105 110Val
Phe Ser His Pro Asp Val Val Ala Glu Glu Thr Trp Tyr Gly Ile 115 120
125Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile Gln Trp Pro Leu Gly
130 135 140Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr
Cys Gly145 150 155 160Val Gly Ala Asp Lys Ala Phe Gly Arg Asp Ile
Val Asp Ala His Tyr 165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn
Ile Ser Gly Ile Asn Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu
Phe Gln Val Gly Pro Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu
Val Trp Ala Ala Arg Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile
Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile Gln225 230 235
240Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser
245 250 255Met Arg Asn Asp Gly Gly Tyr Glu Val Ile Lys Thr Ala Ile
Glu Lys 260 265 270Leu Gly Lys Arg His Lys Glu His Ile Ala Ala Tyr
Gly Glu Gly Asn 275
280 285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr
Phe 290 295 300Leu Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val
Gly Arg Asp305 310 315 320Thr Glu Lys Ala Gly Lys Gly Tyr Phe Glu
Asp Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr
Ser Met Ile Ala Asp Thr Thr Ile 340 345 350Leu Trp Lys Pro
355251525DNAGlycine max 25gaaaaccata gattatcgct atcttatatc
ttattccggg tctcaagatt caactgtgag 60gaagaaaaag gtctccaaac acatagaagc
acgtgtatgt atgtattgtg caagtcatag 120tattcaacgt taacaccact
gactcactta taaaaggcgc tttaacacca aacggagtct 180ctctatcaaa
aaaagctacc tagagcttgc acctattggt atcttctaca atatcctaaa
240gtgtttttct tcttcttcat caccatgtct ttactttcag acctcatcaa
cctcaatctc 300tcagaatcca cagaaaagat cattgctgag tacatatggg
ttggtggatc tggtatggac 360ctcagaagca aagccaggac tcttcctggg
ccagtgagtg accctgcaaa acttccgaag 420tggaactatg atgggtctag
cacagatcaa gctccagggg atgacagtga agtcattcta 480tacccacaag
ctattttcaa ggaccccttt aggagaggaa acaatattct tgtcatttgt
540gatgtgtaca ccccagctgg tgagccactt ccaaccaaca agaggtatgg
tgctgccaaa 600attttcagtc accctgatgt tgctgctgag gaaccatggt
atggtattga gcaagagtat 660accttattgc agaaagatgt aaattggcca
cttgggtggc cccttggtgg gtttcctgga 720ccacagggcc catactactg
tggaattggt gctgataaag cctatggccg tgatattgta 780gatgcacatt
acaaagcttg tatttatgct ggcattaaca ttagtggcat caatggagag
840gttatgcctg gccagtggga atttcaagtt ggtccttctg ttggtatctc
tgctggagat 900gaggtgtggg ctgctcgcta cattttggag aggattacag
agatagctgg agcaattgtt 960tcatttgatc ccaagcctat tccgggagat
tggaatggag ctggagctca ctcaaactac 1020agcaccaagt ccatgagaga
agagggtggt tatgaggtga tcaagaaggc cattgaaaag 1080cttggattga
ggcacaagga gcacatcgca gcatatggag aaggcaacga gagacgtctc
1140acgggaagac atgaaactgc agacatcaac accttctctt ggggtgtggc
aaaccgtgga 1200agctccatta gagttggaag agacacagag aaaaatggca
aaggttactt cgaggacaga 1260aggcctgctt ctaatatgga tccctatgta
gtcacctcca tgatcgcaga gactaccatc 1320ctctggaaac catgaaaaca
cagtcatatg tctctagatt tggaccactt aaaattgtgt 1380gttcaatagt
catttgatct aaaattttat atttgcaagg tgttgtttag ttaggaattg
1440ccaagtggtc ttttgagcct ccatgtacca tgtgtatggt agagaataat
ctcttcatta 1500ataacaagaa ttgcttcttg atttc 152526356PRTGlycine max
26Met Ser Leu Leu Ser Asp Leu Ile Asn Leu Asn Leu Ser Glu Ser Thr1
5 10 15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Met
Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Ser Asp
Pro Ala 35 40 45Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Asp
Gln Ala Pro 50 55 60Gly Asp Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala
Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu Val
Ile Cys Asp Val Tyr Thr 85 90 95Pro Ala Gly Glu Pro Leu Pro Thr Asn
Lys Arg Tyr Gly Ala Ala Lys 100 105 110Ile Phe Ser His Pro Asp Val
Ala Ala Glu Glu Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr
Leu Leu Gln Lys Asp Val Asn Trp Pro Leu Gly 130 135 140Trp Pro Leu
Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155
160Ile Gly Ala Asp Lys Ala Tyr Gly Arg Asp Ile Val Asp Ala His Tyr
165 170 175Lys Ala Cys Ile Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn
Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro
Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Val Trp Ala Ala Arg
Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Ala Ile Val
Ser Phe Asp Pro Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn Gly
Ala Gly Ala His Ser Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Glu
Glu Gly Gly Tyr Glu Val Ile Lys Lys Ala Ile Glu Lys 260 265 270Leu
Gly Leu Arg His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280
285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300Ser Trp Gly Val Ala Asn Arg Gly Ser Ser Ile Arg Val Gly
Arg Asp305 310 315 320Thr Glu Lys Asn Gly Lys Gly Tyr Phe Glu Asp
Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Ser
Met Ile Ala Glu Thr Thr Ile 340 345 350Leu Trp Lys Pro
355271692DNAGlycine max 27aaaccaattt catccactcg taacgtaccc
ctatcggttt tagaaaagcc aacaaagttt 60gtgtccacca acctctattt tacacgagtc
tctcatattc tgatactata gctacactta 120ccactgtgtc tcagagggtc
tgtgacacac agactcactt ccaagttcca agctttggcc 180atttattccc
actgtttatt gaacatggca cagattttgg ctccctctac gcaatggcag
240atgagaatct caaaatcctc tcccaatgca agtcccatta catcaaacat
gtggagttct 300ttattgtgga aacaaaataa gaaagtttca cccacaagtt
ctgctaaatt tagagtgatg 360gcaattaagt ctgacaatag catcatcaac
aggctagagg gtctacttaa tttggatatc 420actccattca cggacaagat
aattgctgag tacatttgga ttggggggac aggaattgat 480gtgcgcagta
aatcaagaac aatatcaaag cctgttgaac atccctctga gctccctaaa
540tggaactatg atggatctag cactggacag gcaccgggtg atgatagtga
agtaatccta 600tatcctcaag caattttcaa agatcctttc cgtggtggta
acaatatttt ggtcatttgc 660gattcttaca ccccacaagg tgagcctatc
cctacaaaca agagacacag agctgctgaa 720attttcagta acccaaaggt
ccaagcagaa gttccatggt atggaataga acaagagtac 780accttacttc
aaacaaatgt gaaatggcca ttaggttggc ccgttggtgg ctatcctggt
840cctcagggtc cttattattg cagcgctggg gcagacaagt catttggacg
tgacatatct 900gatgctcatt acaaggcttg cttatatgct ggaattaaca
tcagtggtac caatggggag 960gttatgcctg ggcagtggga gtaccaagtt
ggtcctagtg taggtattga ggctggtgat 1020catatctggg cttcaaggta
catcctcgag agaattaccg agcaagctgg tgttgtgctc 1080tctcttgatc
caaaaccaat agagggtgac tggaatggag caggatgcca caccaattac
1140agtacaaaga gcatgaggga agatggaggc tttgaggtaa taaagaaggc
aatattgaat 1200ctatcgcttc gccacaaaga tcacatcagt gcatatggag
aaggaaatga gagaaggttg 1260actggaaagc atgagacagc aagcatcaac
acattttctt ggggagtggc taaccgtggt 1320tgctcaatcc gtgtgggaag
agacactgag aagaatggca aaggttactt ggaagatagg 1380cgaccggctt
caaacatgga tccatatgtt gtgacatcat tacttgcaga gactacacta
1440ttgtgggagc caactctgga ggctgaagct cttgcagctc agaagttagc
attgaaggtc 1500taaacctatt gattgatgag gagctggaaa atactttcac
tttcctttta gattatctat 1560attataatga tcttgtttgg actaaagagg
ttgccatgcc cagttattgg ttgtcatatg 1620aaatgcatat tgtatatcag
aagtttggtt ggtactattt gcttcaggac aaatttgcat 1680tgatgcttgg tt
169228432PRTGlycine max 28Met Ala Gln Ile Leu Ala Pro Ser Thr Gln
Trp Gln Met Arg Ile Ser1 5 10 15Lys Ser Ser Pro Asn Ala Ser Pro Ile
Thr Ser Asn Met Trp Ser Ser 20 25 30Leu Leu Trp Lys Gln Asn Lys Lys
Val Ser Pro Thr Ser Ser Ala Lys 35 40 45Phe Arg Val Met Ala Ile Lys
Ser Asp Asn Ser Ile Ile Asn Arg Leu 50 55 60Glu Gly Leu Leu Asn Leu
Asp Ile Thr Pro Phe Thr Asp Lys Ile Ile65 70 75 80Ala Glu Tyr Ile
Trp Ile Gly Gly Thr Gly Ile Asp Val Arg Ser Lys 85 90 95Ser Arg Thr
Ile Ser Lys Pro Val Glu His Pro Ser Glu Leu Pro Lys 100 105 110Trp
Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro Gly Asp Asp Ser 115 120
125Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp Pro Phe Arg Gly
130 135 140Gly Asn Asn Ile Leu Val Ile Cys Asp Ser Tyr Thr Pro Gln
Gly Glu145 150 155 160Pro Ile Pro Thr Asn Lys Arg His Arg Ala Ala
Glu Ile Phe Ser Asn 165 170 175Pro Lys Val Gln Ala Glu Val Pro Trp
Tyr Gly Ile Glu Gln Glu Tyr 180 185 190Thr Leu Leu Gln Thr Asn Val
Lys Trp Pro Leu Gly Trp Pro Val Gly 195 200 205Gly Tyr Pro Gly Pro
Gln Gly Pro Tyr Tyr Cys Ser Ala Gly Ala Asp 210 215 220Lys Ser Phe
Gly Arg Asp Ile Ser Asp Ala His Tyr Lys Ala Cys Leu225 230 235
240Tyr Ala Gly Ile Asn Ile Ser Gly Thr Asn Gly Glu Val Met Pro Gly
245 250 255Gln Trp Glu Tyr Gln Val Gly Pro Ser Val Gly Ile Glu Ala
Gly Asp 260 265 270His Ile Trp Ala Ser Arg Tyr Ile Leu Glu Arg Ile
Thr Glu Gln Ala 275 280 285Gly Val Val Leu Ser Leu Asp Pro Lys Pro
Ile Glu Gly Asp Trp Asn 290 295 300Gly Ala Gly Cys His Thr Asn Tyr
Ser Thr Lys Ser Met Arg Glu Asp305 310 315 320Gly Gly Phe Glu Val
Ile Lys Lys Ala Ile Leu Asn Leu Ser Leu Arg 325 330 335His Lys Asp
His Ile Ser Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu 340 345 350Thr
Gly Lys His Glu Thr Ala Ser Ile Asn Thr Phe Ser Trp Gly Val 355 360
365Ala Asn Arg Gly Cys Ser Ile Arg Val Gly Arg Asp Thr Glu Lys Asn
370 375 380Gly Lys Gly Tyr Leu Glu Asp Arg Arg Pro Ala Ser Asn Met
Asp Pro385 390 395 400Tyr Val Val Thr Ser Leu Leu Ala Glu Thr Thr
Leu Leu Trp Glu Pro 405 410 415Thr Leu Glu Ala Glu Ala Leu Ala Ala
Gln Lys Leu Ala Leu Lys Val 420 425 430291556DNAOryza sativa
29accaccctcc ttgttacagc tgtgccgcct cttgcttcct cctcctcatc gtccgccatg
60gcttctctca ccgatctcgt caacctcaac ctctccgaca ccacggagaa gatcatcgcc
120gagtacatat ggatcggtgg atctggcatg gatctcagga gcaaggctag
gactctctcc 180ggccctgtga ctgatcccag caagctgccc aagtggaact
acgatggctc cagcaccggc 240caggcccccg gcgaggacag tgaggtcatc
ctgtacccac aggctatctt caaggaccca 300ttcaggaagg gaaacaacat
ccttgtcatg tgcgattgct acacgccagc cggagaaccg 360atccccacca
acaagaggca caatgctgcc aagatcttca gctcccctga ggttgcttct
420gaggagccct ggtacggtat tgagcaagag tacaccctcc tccagaagga
catcaactgg 480ccccttggct ggcctgttgg tggcttccct ggtcctcagg
gtccttacta ctgtggtatc 540ggtgctgaca agtcttttgg gcgtgatatt
gttgactccc actacaaggc ttgcctctat 600gccggcatca acatcagtgg
aatcaacggc gaggtcatgc caggacagtg ggagttccaa 660gttggcccgt
ctgtcggcat ttctgccggt gatcaggtgt gggttgctcg ctacattctt
720gagaggatca ccgagatcgc cggagtcgtc gtctcatttg accccaagcc
catcccggga 780gactggaacg gtgctggtgc tcacaccaac tacagcacca
agtcgatgag gaacgatggt 840ggctacgaga tcatcaagtc cgccattgag
aagctcaagc tcaggcacaa ggagcacatc 900tccgcctacg gcgagggcaa
cgagcgccgg ctcaccggca ggcacgagac cgccgacatc 960aacaccttca
gctggggagt tgccaaccgc ggcgcctcgg tccgcgtcgg ccgggagacg
1020gagcagaacg gcaagggcta cttcgaggat cgccggccgg cgtccaacat
ggacccttac 1080atcgtcacct ccatgatcgc cgagaccacc atcatctgga
agccctgaag cggcttcttg 1140acgccacgac atcctcgtca tcgtcctccc
cagctcgccg tgtcgctccg gttgctccat 1200tgatcggacg atctggtgaa
ttgcatttgt gctgggagaa gtaaaaaaaa aaggaaagag 1260aaaaaaaaga
aaatcacgcc aaaaaaaatt ctcattccat ttcgatttgg ttgcatgcta
1320ccactactac tacattgctc atctgccatt tagattagct cctttttctt
cgtcttttgg 1380gtgagtgcgt ttgggtgctc ttgtgtaatc ctccaataat
ggccgtacct acggtacttg 1440tcccatcctg tggatcatcg tcctcctttc
cacatgtggt tttatcatca ttgttattag 1500tgatcacctt tatataaagt
tcttgctggg cttccaatag ccgtggcttt tgcgtt 155630356PRTOryza sativa
30Met Ala Ser Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Asp Thr Thr1
5 10 15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met
Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Leu Ser Gly Pro Val Thr Asp
Pro Ser 35 40 45Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly
Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala
Ile Phe Lys Asp65 70 75 80Pro Phe Arg Lys Gly Asn Asn Ile Leu Val
Met Cys Asp Cys Tyr Thr 85 90 95Pro Ala Gly Glu Pro Ile Pro Thr Asn
Lys Arg His Asn Ala Ala Lys 100 105 110Ile Phe Ser Ser Pro Glu Val
Ala Ser Glu Glu Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr
Leu Leu Gln Lys Asp Ile Asn Trp Pro Leu Gly 130 135 140Trp Pro Val
Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155
160Ile Gly Ala Asp Lys Ser Phe Gly Arg Asp Ile Val Asp Ser His Tyr
165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn
Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro
Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Gln Val Trp Val Ala Arg
Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Val Val Val
Ser Phe Asp Pro Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn Gly
Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Asn
Asp Gly Gly Tyr Glu Ile Ile Lys Ser Ala Ile Glu Lys 260 265 270Leu
Lys Leu Arg His Lys Glu His Ile Ser Ala Tyr Gly Glu Gly Asn 275 280
285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300Ser Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly
Arg Glu305 310 315 320Thr Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp
Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Ile Val Thr Ser
Met Ile Ala Glu Thr Thr Ile 340 345 350Ile Trp Lys Pro
355311169DNAOryza sativa 31ttctacacct cattttccgc ttgcatcttg
ctcattcaga tctcttctgc tttgagcaat 60ggccaacctc accgacctcg ttaacctcaa
cctcagcgac tgcagcgaca agatcatcgc 120cgagtacatc tgggttggag
gatcgggcat agacctcagg agcaaagcga ggactgtgaa 180aggccccatc
accgatgtga gccagctgcc gaagtggaac tacgacggct ccagcaccgg
240gcaggctccc ggcgaggaca gcgaagtgat cctctaccct caagccattt
tcaaggaccc 300gttcaggagg ggcgacaaca tccttgtgat gtgcgactgc
tacacgccac aaggtgagcc 360aatccccact aacaagaggc acagtgccgc
caagatcttc agccaccctg atgttgttgc 420tgaggtgcca tggtacggta
ttgagcagga gtacacactc cttcaaaagg atgtgaactg 480gccccttggc
tggccagttg gtggcttccc tggcccacag ggaccatact actgcgctgc
540cggtgccgaa aaggcgttcg gccgcgacat cgtggacgcc cactacaagg
cctgcatcta 600cgccgggatc aacatcagtg gcatcaacgg ggaagtcatg
cccggccagt gggagttcca 660agttggcccg tcagttggca tcgccgctgc
tgaccaagtg tgggttgccc gctacatcct 720cgagagggtc acagaggtgg
ccggagtcgt gctctccctt gacccgaagc cgatcccggg 780tgactggaat
ggcgctggtg cccacaccaa cttcagcacc aagtcgatga gggagccggg
840aggctacgag gtgatcaaga aggcgatcga caagctcgcg ctgaggcaca
aggagcacat 900cgccgcctac ggcgagggca acgagcgccg cctcaccggc
cgccacgaga ccgccgacat 960caacaccttc aaatggggcg tggcgaaccg
cggcgcgtcc atccgcgtgg ggcgcgacac 1020ggagaaggag ggcaaggggt
acttcgagga caggaggccg gcgtccaaca tggacccata 1080cgtcgtcacc
ggcatgatcg ccgagaccac gctgctgtgg aagcagaact aagccgtccg
1140gcgggcctct cccgtgcatt tctgcgccc 116932357PRTOryza sativa 32Met
Ala Asn Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Asp Cys Ser1 5 10
15Asp Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Ile Asp
20 25 30Leu Arg Ser Lys Ala Arg Thr Val Lys Gly Pro Ile Thr Asp Val
Ser 35 40 45Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln
Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile
Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asp Asn Ile Leu Val Met
Cys Asp Cys Tyr Thr 85 90 95Pro Gln Gly Glu Pro Ile Pro Thr Asn Lys
Arg His Ser Ala Ala Lys 100 105 110Ile Phe Ser His Pro Asp Val Val
Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr Leu
Leu Gln Lys Asp Val Asn Trp Pro Leu Gly 130 135 140Trp Pro Val Gly
Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala145 150 155 160Ala
Gly Ala Glu Lys Ala Phe Gly Arg Asp Ile Val Asp Ala His Tyr 165 170
175Lys Ala Cys Ile Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu
180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val
Gly Ile 195 200 205Ala Ala Ala Asp Gln Val Trp Val Ala Arg Tyr Ile
Leu Glu Arg Val 210 215 220Thr Glu Val Ala Gly Val Val Leu Ser Leu
Asp Pro Lys Pro Ile Pro225
230 235 240Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Phe Ser Thr
Lys Ser 245 250 255Met Arg Glu Pro Gly Gly Tyr Glu Val Ile Lys Lys
Ala Ile Asp Lys 260 265 270Leu Ala Leu Arg His Lys Glu His Ile Ala
Ala Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr Gly Arg His
Glu Thr Ala Asp Ile Asn Thr Phe 290 295 300Lys Trp Gly Val Ala Asn
Arg Gly Ala Ser Ile Arg Val Gly Arg Asp305 310 315 320Thr Glu Lys
Glu Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn
Met Asp Pro Tyr Val Val Thr Gly Met Ile Ala Glu Thr Thr Leu 340 345
350Leu Trp Lys Gln Asn 355331495DNAOryza sativa 33attgatagcc
tgtgcgtctc caagaagagg cttgccgctg ccgccattgg agccctctcg 60tttctgctcg
agctctgcat ttcttcagta ggaggaggag gaggaagagt tggagtcgcc
120atgtcgtcgt ccctgctcac tgacctcgtt aacctcgacc tgtcggagag
cacggacaag 180gtcatcgccg agtacatatg ggttggtggt actgggatgg
atgtgaggag caaagccaga 240acgttgtctg gacctgttga tgacccaagc
aagcttccaa agtggaactt tgatggctcc 300agcaccggtc aggctaccgg
tgacgacagt gaagtcatcc tccaccctca agccatcttc 360agagacccat
tcaggaaggg gaagaacatc ctggtcatgt gtgactgtta tgcgccgaat
420ggcgagccga ttccgacgaa caaccggtac aatgcagcaa ggatcttcag
tcatcctgat 480gtcaaggctg aagagccatg gtatgggatt gagcaggagt
acacccttct tcagaagcac 540atcaactggc ctcttggctg gccactaggt
ggctatccag gccctcaggg tccgtactac 600tgtgcggcgg gagccgataa
atcgtacggg cgcgacatcg ttgatgccca ctacaaggcc 660tgcctgtttg
ccggcatcaa catcagcggg atcaacgcag aagtcatgcc ggggcagtgg
720gagttccaga ttggccctgt cgttggcgtc tccgcagggg atcatgtctg
ggtggcacgc 780tacattcttg agaggatcac tgagattgct ggcgtcgtcg
tgtccttcga ccccaagccc 840attccgggag actggaatgg cgccggtgct
cacaccaact acagcaccaa gtcgatgagg 900agcaatggcg gctacgaggt
gatcaagaaa gcgatcaaga agcttggcat gcgccaccgt 960gagcacatcg
ccgcctacgg cgacggcaac gagcgccgcc tcaccggccg ccacgagacc
1020gccgacatca acaacttcgt ctggggcgta gcgaaccgcg gcgcgtcggt
gcgtgtcggc 1080cgggacaccg agaaggacgg caaaggttac ttcgaggaca
ggaggccggc gtccaacatg 1140gacccgtacc tggtgaccgc catgatcgcc
gagaccacca tcctctggga gcccagccac 1200ggccacggcc acggccaatc
caacggcaag tgaggaggag tcgcctcgcc cgggttgatg 1260aactgctttc
tcgcgttctg ggtttcatgg aaatctgtgt gtgtgtgttc tctgacgctg
1320gtgctgttag aaacttccaa taattcagaa ataactgcga tgtgctctca
aatttctcat 1380gaggccatca cctgcagcat ctcatgaaat agatctattg
caatgacaat accaatggca 1440acgcaaaatt ttatggtacc tccagatacc
atctactctc ctcaataatg acaat 149534370PRTOryza sativa 34Met Ser Ser
Ser Leu Leu Thr Asp Leu Val Asn Leu Asp Leu Ser Glu1 5 10 15Ser Thr
Asp Lys Val Ile Ala Glu Tyr Ile Trp Val Gly Gly Thr Gly 20 25 30Met
Asp Val Arg Ser Lys Ala Arg Thr Leu Ser Gly Pro Val Asp Asp 35 40
45Pro Ser Lys Leu Pro Lys Trp Asn Phe Asp Gly Ser Ser Thr Gly Gln
50 55 60Ala Thr Gly Asp Asp Ser Glu Val Ile Leu His Pro Gln Ala Ile
Phe65 70 75 80Arg Asp Pro Phe Arg Lys Gly Lys Asn Ile Leu Val Met
Cys Asp Cys 85 90 95Tyr Ala Pro Asn Gly Glu Pro Ile Pro Thr Asn Asn
Arg Tyr Asn Ala 100 105 110Ala Arg Ile Phe Ser His Pro Asp Val Lys
Ala Glu Glu Pro Trp Tyr 115 120 125Gly Ile Glu Gln Glu Tyr Thr Leu
Leu Gln Lys His Ile Asn Trp Pro 130 135 140Leu Gly Trp Pro Leu Gly
Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr145 150 155 160Cys Ala Ala
Gly Ala Asp Lys Ser Tyr Gly Arg Asp Ile Val Asp Ala 165 170 175His
Tyr Lys Ala Cys Leu Phe Ala Gly Ile Asn Ile Ser Gly Ile Asn 180 185
190Ala Glu Val Met Pro Gly Gln Trp Glu Phe Gln Ile Gly Pro Val Val
195 200 205Gly Val Ser Ala Gly Asp His Val Trp Val Ala Arg Tyr Ile
Leu Glu 210 215 220Arg Ile Thr Glu Ile Ala Gly Val Val Val Ser Phe
Asp Pro Lys Pro225 230 235 240Ile Pro Gly Asp Trp Asn Gly Ala Gly
Ala His Thr Asn Tyr Ser Thr 245 250 255Lys Ser Met Arg Ser Asn Gly
Gly Tyr Glu Val Ile Lys Lys Ala Ile 260 265 270Lys Lys Leu Gly Met
Arg His Arg Glu His Ile Ala Ala Tyr Gly Asp 275 280 285Gly Asn Glu
Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn 290 295 300Asn
Phe Val Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly305 310
315 320Arg Asp Thr Glu Lys Asp Gly Lys Gly Tyr Phe Glu Asp Arg Arg
Pro 325 330 335Ala Ser Asn Met Asp Pro Tyr Leu Val Thr Ala Met Ile
Ala Glu Thr 340 345 350Thr Ile Leu Trp Glu Pro Ser His Gly His Gly
His Gly Gln Ser Asn 355 360 365Gly Lys 370351677DNAOryza sativa
35atcgacgtcg cctcctctcc tcctcctcct cgtcgctgca ttccggttga gtgagttggt
60gattatctgt agggggtgaa aatggcgcag gcggtggtgc cggcgatgca gtgccaggtc
120ggggccgtgc gggcgaggcc ggcggcggct gcggcggcgg cgggggggag
ggtgtgggga 180gtcaggagga ccgggcgcgg cacgtcgggg ttcagggtga
tggccgtgag cacggagacc 240accggggtgg tgacgcggat ggagcagctg
ctcaacatgg acaccacccc cttcaccgac 300aagatcatcg ccgagtacat
ctgggttgga ggaactggaa ttgacctcag aagcaaatca 360aggacaatat
caaaaccagt ggaggacccc tcggagctac caaaatggaa ctacgatgga
420tcaagcacag ggcaagctcc aggagaagat agtgaagtca tcttataccc
acaggctata 480ttcaaggacc catttcgagg tggcaacaac atattggtta
tgtgtgatac ctacacacca 540gctggggaac ccatccctac taacaaacgt
aacagggctg cacaagtatt cagtgatcca 600aaggttgtca gccaagtgcc
atggtttgga atagaacagg agtacacttt gctccagaga 660gacgtaaact
ggcctcttgg ctggcccgtt ggaggctacc ctgggcccca gggtccatac
720tactgcgctg taggatcgga caaatcgttt ggccgtgaca tatcagatgc
tcactacaag 780gcatgtcttt atgctggaat taacattagt ggaacaaatg
gagaggtcat gcctggtcag 840tgggagtacc aggttggacc tagtgtcggt
attgaagctg gagaccacat atggatttca 900agatatattc ttgagagaat
aacggagcag gctggtgtag tgcttaccct tgaccccaaa 960ccaattcagg
gagactggaa tggagctggg tgccacacaa actacagcac caagagtatg
1020cgtgaagatg gaggatttga ggtgatcaag aaggcaatcc taaacctatc
acttcgccat 1080gacttgcata taagtgcata tggtgaagga aatgaaagga
ggttgacagg tttacacgag 1140acagctagca ttgacaattt ctcatggggt
gtggcaaacc gtggatgctc tattcgggtg 1200gggcgagaca ccgaggcgaa
gggaaaaggc tacttggaag accgtcgccc ggcatcaaac 1260atggacccgt
acgtcgtgac agcgctattg gctgaaacca caattctttg ggagccaacc
1320ctcgaagcgg aggttcttgc tgctaagaag ttggccctga aggtatgaag
aacttggacg 1380atgaatcggg gcaaataaat cccagcaaaa tttgtttgct
gcccaccagt cttgatcttg 1440tatttcttct gtctggggat tggtctgtac
aaatctgcag tttctagaaa accacgccac 1500cttccattcg ccagttaaca
ttttggttga acaccacact tgatctgggt ctgtattttg 1560agtccatttg
tgagtgacag aacggatgat gaaacacatc agggacactt ttaagtttct
1620tcagtcctgc gtccttccct cgaaataaaa atgtttcctt gttttttatc ccgggct
167736428PRTOryza sativa 36Met Ala Gln Ala Val Val Pro Ala Met Gln
Cys Gln Val Gly Ala Val1 5 10 15Arg Ala Arg Pro Ala Ala Ala Ala Ala
Ala Ala Gly Gly Arg Val Trp 20 25 30Gly Val Arg Arg Thr Gly Arg Gly
Thr Ser Gly Phe Arg Val Met Ala 35 40 45Val Ser Thr Glu Thr Thr Gly
Val Val Thr Arg Met Glu Gln Leu Leu 50 55 60Asn Met Asp Thr Thr Pro
Phe Thr Asp Lys Ile Ile Ala Glu Tyr Ile65 70 75 80Trp Val Gly Gly
Thr Gly Ile Asp Leu Arg Ser Lys Ser Arg Thr Ile 85 90 95Ser Lys Pro
Val Glu Asp Pro Ser Glu Leu Pro Lys Trp Asn Tyr Asp 100 105 110Gly
Ser Ser Thr Gly Gln Ala Pro Gly Glu Asp Ser Glu Val Ile Leu 115 120
125Tyr Pro Gln Ala Ile Phe Lys Asp Pro Phe Arg Gly Gly Asn Asn Ile
130 135 140Leu Val Met Cys Asp Thr Tyr Thr Pro Ala Gly Glu Pro Ile
Pro Thr145 150 155 160Asn Lys Arg Asn Arg Ala Ala Gln Val Phe Ser
Asp Pro Lys Val Val 165 170 175Ser Gln Val Pro Trp Phe Gly Ile Glu
Gln Glu Tyr Thr Leu Leu Gln 180 185 190Arg Asp Val Asn Trp Pro Leu
Gly Trp Pro Val Gly Gly Tyr Pro Gly 195 200 205Pro Gln Gly Pro Tyr
Tyr Cys Ala Val Gly Ser Asp Lys Ser Phe Gly 210 215 220Arg Asp Ile
Ser Asp Ala His Tyr Lys Ala Cys Leu Tyr Ala Gly Ile225 230 235
240Asn Ile Ser Gly Thr Asn Gly Glu Val Met Pro Gly Gln Trp Glu Tyr
245 250 255Gln Val Gly Pro Ser Val Gly Ile Glu Ala Gly Asp His Ile
Trp Ile 260 265 270Ser Arg Tyr Ile Leu Glu Arg Ile Thr Glu Gln Ala
Gly Val Val Leu 275 280 285Thr Leu Asp Pro Lys Pro Ile Gln Gly Asp
Trp Asn Gly Ala Gly Cys 290 295 300His Thr Asn Tyr Ser Thr Lys Ser
Met Arg Glu Asp Gly Gly Phe Glu305 310 315 320Val Ile Lys Lys Ala
Ile Leu Asn Leu Ser Leu Arg His Asp Leu His 325 330 335Ile Ser Ala
Tyr Gly Glu Gly Asn Glu Arg Arg Leu Thr Gly Leu His 340 345 350Glu
Thr Ala Ser Ile Asp Asn Phe Ser Trp Gly Val Ala Asn Arg Gly 355 360
365Cys Ser Ile Arg Val Gly Arg Asp Thr Glu Ala Lys Gly Lys Gly Tyr
370 375 380Leu Glu Asp Arg Arg Pro Ala Ser Asn Met Asp Pro Tyr Val
Val Thr385 390 395 400Ala Leu Leu Ala Glu Thr Thr Ile Leu Trp Glu
Pro Thr Leu Glu Ala 405 410 415Glu Val Leu Ala Ala Lys Lys Leu Ala
Leu Lys Val 420 42537774DNASorghum bicolor 37atggccagcc tcaccgatct
cgttaacctc gacctgagtg attgcaccga caagatcatt 60gccgagtaca tctggattgg
aggatccggc atagacctca ggagcaaagc aaggacggtg 120aaaggcccca
tcaccgatcc gagccagctg ccaaaatgga actacgacgg ctccagcacc
180gggcaggctc ccggagagga cagcgaagtc atcctctacc ctcaagccat
tttcaaggac 240ccgttcagga agggcgacaa catccttgtg atgtgtgact
gctacacgcc acaaggcgag 300ccaatcccta ctaacaagag gtacaatgct
gccaaggttt tcagccaccc cgacgttgca 360gctgaggtgc catggtacgg
tattgagcag gagtacactc tccttcagaa ggatgtgaac 420tggccccttg
gctggcctgt tggtggatac cctggtcccc agggaccata ctactgcgct
480gccggtgccg ataaggcctt tgggcgcgat gtggtcgacg cccactacaa
agcctgcctc 540tacgccggca tcaacatcag cggcatcaac ggcgaagtca
tgcctggcca gtgggagttc 600caagttggcc cgtccgttgg gatatctgcc
ggtgacgaaa tatgggttgc ccgctacatt 660ctcgagaggg agggcaaggg
atacttcgag gaccgcaggc cggcatccaa catggacccc 720tacgtcgtca
ccggcatgat cgccgagacc accatcctgt ggaacggaaa ctga 77438357PRTSorghum
bicolor 38Met Ala Ser Leu Thr Asp Leu Val Asn Leu Asp Leu Ser Asp
Cys Thr1 5 10 15Asp Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser
Gly Ile Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Val Lys Gly Pro Ile
Thr Asp Pro Ser 35 40 45Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser
Thr Gly Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro
Gln Ala Ile Phe Lys Asp65 70 75 80Pro Phe Arg Lys Gly Asp Asn Ile
Leu Val Met Cys Asp Cys Tyr Thr 85 90 95Pro Gln Gly Glu Pro Ile Pro
Thr Asn Lys Arg Tyr Asn Ala Ala Lys 100 105 110Val Phe Ser His Pro
Asp Val Ala Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu
Tyr Thr Leu Leu Gln Lys Asp Val Asn Trp Pro Leu Gly 130 135 140Trp
Pro Val Gly Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala145 150
155 160Ala Gly Ala Asp Lys Ala Phe Gly Arg Asp Val Val Asp Ala His
Tyr 165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile
Asn Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly
Pro Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Ile Trp Val Ala
Arg Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Ile Val
Leu Ser Leu Asp Pro Lys Pro Ile Gln225 230 235 240Gly Asp Trp Asn
Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg
Glu Ala Gly Gly Tyr Glu Val Ile Lys Lys Ala Ile Glu Lys 260 265
270Leu Gly Lys Arg His Thr Glu His Ile Ala Ala Tyr Gly Glu Gly Asn
275 280 285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn
Thr Phe 290 295 300Lys Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg
Val Gly Arg Asp305 310 315 320Thr Glu Arg Glu Gly Lys Gly Tyr Phe
Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val
Thr Gly Met Ile Ala Glu Thr Thr Ile 340 345 350Leu Trp Asn Gly Asn
355391068DNASorghum bicolor 39atggcctccc tcaccgacct cgtcaacctc
agcctctcgg acaccaccga gaagatcatc 60gccgagtaca tatggatcgg tggatctggc
atggatctca ggagcaaagc caggaccctc 120tccggcccgg tgaccgatcc
cagcaagctg cccaagtgga actacgacgg ctccagcacc 180ggccaggccc
ccggcgagga cagtgaggtc atcctcccgc aggctatctt caaggaccca
240ttccggaggg gcaacaacat ccttgtcatg tgcgattgct acaccccagc
tggcgagcca 300attcccacca acaagaggca caacgccgcc aagatcttca
gcaaccctga ggtcgctgct 360gaggagccct ggtacggtat tgagcaggag
tacaccctcc ttcagaagga caccaactgg 420ccccttgggt ggcctcttgg
tggcttccct ggccctcagg gtccttacta ctgtggaatc 480ggtgcggaca
agtcattcgg gcgtgacata gttgatgccc actacaaggc ttgcatttat
540gcaggcatca acatcagtgg catcaacgga gaggtcatgc cagggcagtg
ggaattccaa 600gttggaccgt ccgtcggcat ttcttcaggt gatcaggtct
gggttgctcg ctacattctt 660gagaggatca ccgagatcgc cggtgtggtg
ttgacattcg acccaaagcc catccctggt 720gactggaacg gtgccggcgc
acacaccaac tacagcacca agtccatgag gaacgagggc 780gggtacgagg
tgatcaaggc cgccattgag aagctgaagt tgcggcacaa ggagcacatc
840gcggcctacg gcgagggcaa cgagcgccgc ctcaccggca ggcacgagac
cgccgacatc 900aacaccttca gctggggagt ggcaaaccgt ggcgcgtcag
tgcgcgtggg ccgggagacg 960gagcagaacg gcaagggcta cttcgaggac
cgccggccgg cgtccaacat ggacccatac 1020gtggtgacct ccatgatcgc
cgacaccacc atcctctgga agccctga 106840355PRTSorghum bicolor 40Met
Ala Ser Leu Thr Asp Leu Val Asn Leu Ser Leu Ser Asp Thr Thr1 5 10
15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met Asp
20 25 30Leu Arg Ser Lys Ala Arg Thr Leu Ser Gly Pro Val Thr Asp Pro
Ser 35 40 45Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln
Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Pro Gln Ala Ile Phe
Lys Asp Pro65 70 75 80Phe Arg Arg Gly Asn Asn Ile Leu Val Met Cys
Asp Cys Tyr Thr Pro 85 90 95Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg
His Asn Ala Ala Lys Ile 100 105 110Phe Ser Asn Pro Glu Val Ala Ala
Glu Glu Pro Trp Tyr Gly Ile Glu 115 120 125Gln Glu Tyr Thr Leu Leu
Gln Lys Asp Thr Asn Trp Pro Leu Gly Trp 130 135 140Pro Leu Gly Gly
Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly Ile145 150 155 160Gly
Ala Asp Lys Ser Phe Gly Arg Asp Ile Val Asp Ala His Tyr Lys 165 170
175Ala Cys Ile Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu Val
180 185 190Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly
Ile Ser 195 200 205Ser Gly Asp Gln Val Trp Val Ala Arg Tyr Ile Leu
Glu Arg Ile Thr 210 215 220Glu Ile Ala Gly Val Val Leu Thr Phe Asp
Pro Lys Pro Ile Pro Gly225 230 235 240Asp Trp Asn Gly Ala Gly Ala
His Thr Asn Tyr Ser Thr Lys Ser Met 245 250 255Arg Asn Glu Gly Gly
Tyr Glu Val Ile Lys Ala Ala Ile Glu Lys Leu 260 265 270Lys Leu Arg
His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn Glu 275 280 285Arg
Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe Ser 290 295
300Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly Arg Glu
Thr305 310 315 320Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp Arg Arg
Pro Ala Ser Asn 325 330 335Met Asp Pro Tyr
Val Val Thr Ser Met Ile Ala Asp Thr Thr Ile Leu 340 345 350Trp Lys
Pro 355411158DNASorghum bicolor 41atggcggcgc aggcggtggt gccggcgatg
cagtgccagg tcggagtgaa ggcggcggcg 60ggcgcccggg cgaggccggc ggcggcggga
ggcagggtgt ggggcgtcag gagtaggacc 120ggccgcggcg gcgcctcgcc
ggggttcaag gtcatggccg tcagcacggg cagcaccggg 180gtggtgccac
gcctggagca gctgctcaac atggacacca cgccctacac cgacaagatc
240atcgccgagt acatctgccc ccaggctatc ttcaaggacc cattccgagg
tggcaacaac 300attttggtta tctgtgatac ctacacgcca cagggtgaac
cccttcctac taacaaacgg 360cacagggctg cgcaaatttt tagtgaccca
aaggtcgttg aacaagtgcc atggtttggc 420atagagcaag agtacacttt
gctccagaaa gatgtgaatt ggcctcttgg ttggcctgtt 480ggaggctacc
ctggtcccca gggtccctac tactgtgctg taggagcaga caaatcattt
540ggccgtgaca tatcagatgc tcactacaag gcttgccttt atgctggaat
taacattagt 600ggaacaaacg gggaggtcat gcctggtcag tgggagtacc
aagttggacc tagtgttggc 660attgaagcag gagatcacat atggatttca
agatacattc tcgagagaat cacagagcaa 720gctggggttg tccttaccct
tgatccaaaa ccaattcagg gtgactggaa tggagctggc 780tgccacacaa
attacagcac aaagaccatg cgtgaagatg gaggatttga agatatcaag
840agagcaatcc tgaatctttc tctgcgccat gatttgcata ttagtgcata
cggagaagga 900aatgaaagaa gattgacagg gaagcatgag accgctagca
tcgagacctt ctcatggggt 960gtggcaaacc gtggctgctc tgttcgtgtg
gggcgagata ccgaggcaaa agggaaaggt 1020tacctagaag accgtcgccc
ggcatcaaac atggacccat acattgtgac ggggctactg 1080gctgaaacaa
caattctctg gcaaccaacc cttgaagcgg aggttcttgc cgccaagaag
1140ctggcgctga aggtatga 115842385PRTSorghum bicolor 42Met Ala Ala
Gln Ala Val Val Pro Ala Met Gln Cys Gln Val Gly Val1 5 10 15Lys Ala
Ala Ala Gly Ala Arg Ala Arg Pro Ala Ala Ala Gly Gly Arg 20 25 30Val
Trp Gly Val Arg Ser Arg Thr Gly Arg Gly Gly Ala Ser Pro Gly 35 40
45Phe Lys Val Met Ala Val Ser Thr Gly Ser Thr Gly Val Val Pro Arg
50 55 60Leu Glu Gln Leu Leu Asn Met Asp Thr Thr Pro Tyr Thr Asp Lys
Ile65 70 75 80Ile Ala Glu Tyr Ile Cys Pro Gln Ala Ile Phe Lys Asp
Pro Phe Arg 85 90 95Gly Gly Asn Asn Ile Leu Val Ile Cys Asp Thr Tyr
Thr Pro Gln Gly 100 105 110Glu Pro Leu Pro Thr Asn Lys Arg His Arg
Ala Ala Gln Ile Phe Ser 115 120 125Asp Pro Lys Val Val Glu Gln Val
Pro Trp Phe Gly Ile Glu Gln Glu 130 135 140Tyr Thr Leu Leu Gln Lys
Asp Val Asn Trp Pro Leu Gly Trp Pro Val145 150 155 160Gly Gly Tyr
Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala Val Gly Ala 165 170 175Asp
Lys Ser Phe Gly Arg Asp Ile Ser Asp Ala His Tyr Lys Ala Cys 180 185
190Leu Tyr Ala Gly Ile Asn Ile Ser Gly Thr Asn Gly Glu Val Met Pro
195 200 205Gly Gln Trp Glu Tyr Gln Val Gly Pro Ser Val Gly Ile Glu
Ala Gly 210 215 220Asp His Ile Trp Ile Ser Arg Tyr Ile Leu Glu Arg
Ile Thr Glu Gln225 230 235 240Ala Gly Val Val Leu Thr Leu Asp Pro
Lys Pro Ile Gln Gly Asp Trp 245 250 255Asn Gly Ala Gly Cys His Thr
Asn Tyr Ser Thr Lys Thr Met Arg Glu 260 265 270Asp Gly Gly Phe Glu
Asp Ile Lys Arg Ala Ile Leu Asn Leu Ser Leu 275 280 285Arg His Asp
Leu His Ile Ser Ala Tyr Gly Glu Gly Asn Glu Arg Arg 290 295 300Leu
Thr Gly Lys His Glu Thr Ala Ser Ile Glu Thr Phe Ser Trp Gly305 310
315 320Val Ala Asn Arg Gly Cys Ser Val Arg Val Gly Arg Asp Thr Glu
Ala 325 330 335Lys Gly Lys Gly Tyr Leu Glu Asp Arg Arg Pro Ala Ser
Asn Met Asp 340 345 350Pro Tyr Ile Val Thr Gly Leu Leu Ala Glu Thr
Thr Ile Leu Trp Gln 355 360 365Pro Thr Leu Glu Ala Glu Val Leu Ala
Ala Lys Lys Leu Ala Leu Lys 370 375 380Val385431359DNAZea mays
43gcccgagtga tggccagcct caccgacctc gtcaacctcg acctgagtga ctgcaccgac
60aggatcatcg ccgagtacat ctggattgga ggaaccggga tagacctcag gagcaaagcg
120aggacggtga aaggccccat caccgacccg atccagctgc cgaaatggaa
ctacgacggc 180tccagcaccg ggcaggctcc cggagaggac agcgaagtca
tcctctaccc tcaagccatt 240ttcaaggacc cgttcaggaa gggtaaccac
atccttgtga tgtgtgactg ctacacgcca 300caaggcgagc caatccccac
caacaagagg tacagcgccg ccaaggtttt cagccacccc 360gacgtcgcag
ctgaggtgcc gtggtacggt attgagcagg agtacaccct ccttcagaag
420gacgtgagct ggcccctcgg ctggcccgtt ggtggatacc ctggtcccca
gggaccatac 480tactgcgccg ccggtgccga caaggccttt gggcgcgacg
tggttgacgc ccactacaag 540gcctgcctct acgccggcat caacatcagc
ggcatcaacg gcgaagtcat gcctggacag 600tgggagttcc aagtggggcc
gtccgttggg atctctgccg gcgacgagat atgggtcgcc 660cgctacattc
tcgagaggat caccgagatg gccggaatcg tcctctccct cgacccgaag
720ccgatcaagg gcgactggaa cggcgccggc gcccacacca actacagcac
caagtcgatg 780agggaggccg ggggatacga ggtcatcaag gcggcgatcg
acaagctggg gaagaggcac 840aaggagcaca tcgccgcgta cggcgagggc
aacgagcgcc gcctcacggg ccgccacgag 900accgccgaca tcaacacctt
caaatggggc gtggcgaacc gcggcgcgtc catccgcgtc 960ggccgcgaca
ccgagaggga gggcaagggc tacttcgagg accgcaggcc ggcgtccaac
1020atggacccct acgtcgtcac cggcatgatc gccgagacca ccatcctgtg
gaatggaaac 1080tgatcaagca tgtgcattct cgagggagcc cactgttttt
cttctgcaca acgcatccgc 1140cgtggtgtcg ctttggtttt gaaatttaga
ttccgttgtc ctaaaattta tcactacggt 1200ctccagtgta ttgctcggga
acgaatgaat aacgactgcg atgtttgttt ttttttttgc 1260tggcgtagta
gatgtacgtt tggctgtgct tccagtttat tgggtaaatg aaaaaatgta
1320atggtctacc ggtcttaaaa tagtagtcat tttagctct 135944357PRTZea mays
44Met Ala Ser Leu Thr Asp Leu Val Asn Leu Asp Leu Ser Asp Cys Thr1
5 10 15Asp Arg Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Thr Gly Ile
Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Val Lys Gly Pro Ile Thr Asp
Pro Ile 35 40 45Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly
Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala
Ile Phe Lys Asp65 70 75 80Pro Phe Arg Lys Gly Asn His Ile Leu Val
Met Cys Asp Cys Tyr Thr 85 90 95Pro Gln Gly Glu Pro Ile Pro Thr Asn
Lys Arg Tyr Ser Ala Ala Lys 100 105 110Val Phe Ser His Pro Asp Val
Ala Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr
Leu Leu Gln Lys Asp Val Ser Trp Pro Leu Gly 130 135 140Trp Pro Val
Gly Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala145 150 155
160Ala Gly Ala Asp Lys Ala Phe Gly Arg Asp Val Val Asp Ala His Tyr
165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn
Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro
Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Ile Trp Val Ala Arg
Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Met Ala Gly Ile Val Leu
Ser Leu Asp Pro Lys Pro Ile Lys225 230 235 240Gly Asp Trp Asn Gly
Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Glu
Ala Gly Gly Tyr Glu Val Ile Lys Ala Ala Ile Asp Lys 260 265 270Leu
Gly Lys Arg His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280
285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300Lys Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val Gly
Arg Asp305 310 315 320Thr Glu Arg Glu Gly Lys Gly Tyr Phe Glu Asp
Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Gly
Met Ile Ala Glu Thr Thr Ile 340 345 350Leu Trp Asn Gly Asn
355451369DNAZea mays 45cgaaagcaca cacggatcaa tcacactcac tcgcggccat
tgtcctgccc gtgcgtgctc 60tgccttttca ggcgatcgac caaccaactt ctcgtcactg
ccatggctct gctctccgac 120ctcatcaacc tcgacctctc gggccgcacc
gggaagatca tcgccgagta catctgggtt 180ggcggttccg ggatggacgt
caggagcaaa gccaggacgc tgtccggacc tgttgatgac 240cccagcaagc
ttccgaagtg gaacttcgac ggctccagca ccggccaagc tccgggcgac
300gacagcgaag tcatcctttg ccctcgggcc atcttcaggg acccgttcag
gaaggggcag 360aacatactgg tcatgtgcga ctgctacgag ccgaacgggg
agccgatccc gagcaacaag 420cggcatgggg ccgcgaagat ctttagccac
cctgacgtca aggctgagga accatggttc 480gggattgagc aggagtacac
ccttctccag aaggacacca agtggcctct cggttggccg 540ctggcgtacc
ctggccctca gggaccttac tactgcgccg ccggagcgga caagtcctac
600gggcgggaca tcgtggactg cgcatacaag gcctgcctct acgccggcat
cgacatcagt 660ggcatcaacg gggaggtcat gccggggcag tgggagttcc
aggtggcccc tgccgtcggc 720gtctcggccg gcgaccagct ctgggtggct
cgctacattc ttgagaggat caccgagatc 780gccggcgtgg ttgtctcctt
cgaccccaag ccaattccgg gggactggaa tggcgctggt 840gcacacacca
actacagcac caagtcgatg aggagcgacg gcgggtacga ggtgatcaag
900aaggcgatcg gcaagctggg cctccggcac cgggagcaca tcgccgcgta
cggggacggc 960aacgagcgcc cgctcaccgg ccgccacgag accgccgaca
tcaacacctt cgtctggggc 1020gtgccgaacc gcggggcgtc ggtgcgggtg
ggccgagaca ccgagaagga aggcaaaggc 1080tacttcgagg accggaggcc
ggcgtccaac atggacccgt acgtggtgac ctgcctgatc 1140gcggagacaa
ccatgctgtg ggagcccagc cactccaacg gcgacggcaa gggcgccgcg
1200gctccttgat ttgattctgc ggagactgag ctctgtgtgt gagccggcct
gcgtagatgg 1260caaatgggac tgaccctgtc agaaacttga gatgaccata
ataatagctg cagtgtgctc 1320gttctggggt tggataagac ccaagaactt
tttttagctt tcttcgaac 136946368PRTZea mays 46Met Ala Leu Leu Ser Asp
Leu Ile Asn Leu Asp Leu Ser Gly Arg Thr1 5 10 15Gly Lys Ile Ile Ala
Glu Tyr Ile Trp Val Gly Gly Ser Gly Met Asp 20 25 30Val Arg Ser Lys
Ala Arg Thr Leu Ser Gly Pro Val Asp Asp Pro Ser 35 40 45Lys Leu Pro
Lys Trp Asn Phe Asp Gly Ser Ser Thr Gly Gln Ala Pro 50 55 60Gly Asp
Asp Ser Glu Val Ile Leu Cys Pro Arg Ala Ile Phe Arg Asp65 70 75
80Pro Phe Arg Lys Gly Gln Asn Ile Leu Val Met Cys Asp Cys Tyr Glu
85 90 95Pro Asn Gly Glu Pro Ile Pro Ser Asn Lys Arg His Gly Ala Ala
Lys 100 105 110Ile Phe Ser His Pro Asp Val Lys Ala Glu Glu Pro Trp
Phe Gly Ile 115 120 125Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Thr
Lys Trp Pro Leu Gly 130 135 140Trp Pro Leu Ala Tyr Pro Gly Pro Gln
Gly Pro Tyr Tyr Cys Ala Ala145 150 155 160Gly Ala Asp Lys Ser Tyr
Gly Arg Asp Ile Val Asp Cys Ala Tyr Lys 165 170 175Ala Cys Leu Tyr
Ala Gly Ile Asp Ile Ser Gly Ile Asn Gly Glu Val 180 185 190Met Pro
Gly Gln Trp Glu Phe Gln Val Ala Pro Ala Val Gly Val Ser 195 200
205Ala Gly Asp Gln Leu Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile Thr
210 215 220Glu Ile Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile
Pro Gly225 230 235 240Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr
Ser Thr Lys Ser Met 245 250 255Arg Ser Asp Gly Gly Tyr Glu Val Ile
Lys Lys Ala Ile Gly Lys Leu 260 265 270Gly Leu Arg His Arg Glu His
Ile Ala Ala Tyr Gly Asp Gly Asn Glu 275 280 285Arg Pro Leu Thr Gly
Arg His Glu Thr Ala Asp Ile Asn Thr Phe Val 290 295 300Trp Gly Val
Pro Asn Arg Gly Ala Ser Val Arg Val Gly Arg Asp Thr305 310 315
320Glu Lys Glu Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser Asn
325 330 335Met Asp Pro Tyr Val Val Thr Cys Leu Ile Ala Glu Thr Thr
Met Leu 340 345 350Trp Glu Pro Ser His Ser Asn Gly Asp Gly Lys Gly
Ala Ala Ala Pro 355 360 365471317DNAZea mays 47caatcccaca
ccaccaccac ctcctccggt ccccaacccc tgtcgcaccg cagccgccgg 60ccatggcctg
cctcaccgac ctcgtcaacc tcaacctctc ggacaccacc gagaagatca
120tcgcggaata catatggatc ggtggatctg gcatggatct caggagcaaa
gcaaggaccc 180tctccggccc ggtgaccgat cccagcaagc tgcccaagtg
gaactacgac ggctccagca 240cgggccaggc ccccggcgag gacagcgagg
tcatcctgta cccgcaggcc atcttcaagg 300acccattcag gaggggcaac
aacatccttg tgatgtgcga ttgctacacc ccagccggcg 360agccaatccc
caccaacaag aggtacaacg ccgccaagat cttcagcagc cctgaggtcg
420ccgccgagga gccgtggtat ggtattgagc aggagtacac cctcctccag
aaggacacca 480actggcccct tgggtggccc atcggtggct tccccggccc
tcagggtcct tactactgtg 540gaatcggcgc cgaaaagtcg ttcggccgcg
acatcgtgga cgcccactac aaggcctgct 600tgtatgcggg catcaacatc
agtggcatca acggggaggt gatgccaggg cagtgggagt 660tccaagtcgg
gccttccgtg ggtatttctt caggcgacca ggtctgggtc gctcgctaca
720ttcttgagag gatcacggag atcgccggtg tggtggtgac gttcgacccg
aagccgatcc 780cgggcgactg gaacggcgcc ggcgcgcaca ccaactacag
cacggagtcg atgaggaagg 840agggcgggta cgaggtgatc aaggcggcca
tcgagaagct gaagctgcgg cacagggagc 900acatcgcggc ctacggcgag
ggcaacgacg gccggctcac cggcaggcac gagaccgccg 960acatcaacac
gttcagctgg ggcgtggcca accgcggcgc gtcggtgcgc gtgggccggg
1020agacggagca gaacggcaag ggctacttcg aggaccgccg cccggcgtcc
aacatggacc 1080cctacgtggt cacctccatg atcgccgaga ccaccatcat
ctggaagccc tgagcgccgc 1140ggccgttgcg ttgcagggtc cccgaagcga
ttgcaaagcc actgttcctt ccgttctgtt 1200tgcttattat tgttattatc
tagctagatc atccggggtc aggtcgtcgt ggtgtgccaa 1260aacagaacac
agaaagagga agaagaaaaa aaaaacaaga cgtgtggcgt ttatgtt 131748356PRTZea
mays 48Met Ala Cys Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Asp Thr
Thr1 5 10 15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly
Met Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Leu Ser Gly Pro Val Thr
Asp Pro Ser 35 40 45Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr
Gly Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln
Ala Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu
Val Met Cys Asp Cys Tyr Thr 85 90 95Pro Ala Gly Glu Pro Ile Pro Thr
Asn Lys Arg Tyr Asn Ala Ala Lys 100 105 110Ile Phe Ser Ser Pro Glu
Val Ala Ala Glu Glu Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr
Thr Leu Leu Gln Lys Asp Thr Asn Trp Pro Leu Gly 130 135 140Trp Pro
Ile Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155
160Ile Gly Ala Glu Lys Ser Phe Gly Arg Asp Ile Val Asp Ala His Tyr
165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn
Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro
Ser Val Gly Ile 195 200 205Ser Ser Gly Asp Gln Val Trp Val Ala Arg
Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Val Val Val
Thr Phe Asp Pro Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn Gly
Ala Gly Ala His Thr Asn Tyr Ser Thr Glu Ser 245 250 255Met Arg Lys
Glu Gly Gly Tyr Glu Val Ile Lys Ala Ala Ile Glu Lys 260 265 270Leu
Lys Leu Arg His Arg Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280
285Asp Gly Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300Ser Trp Gly Val Ala Asn Arg Gly Ala Ser Val Arg Val Gly
Arg Glu305 310 315 320Thr Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp
Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Ser
Met Ile Ala Glu Thr Thr Ile 340 345 350Ile Trp Lys Pro
355491490DNAZea mays 49ccacatcctc ccctcattcc tccttgggtt cccagcccgt
gcgccccgcc tgtcgcagtg 60ccagtcgcgc cgcagccgcc ggccatggcc tgcctcaccg
acctcgtcaa cctcaacctc 120tcggacacca cagagaagat catcgccgag
tacatatgga tcggtggatc tggcatggat 180ctcaggagca aagccaggac
cctcccgggc ccggtgaccg atcccagcaa gctgcccaag 240tggaactacg
acggctccag caccggccag gcccccggcg aggacagcga ggtcatcctg
300tacccgcagg ccatcttcaa ggacccattc aggaggggca acaacatcct
tgtcatgtgc 360gattgctaca ccccagctgg cgagccaatt cccaccaaca
agaggtacag cgccgccaag 420atcttcagca gccctgaggt cgctgccgag
gagccctggt atggtatcga gcaggagtac 480accctccttc agaaggacac
caactggccc ctcgggtggc ctattggcgg cttccctggc 540cctcagggtc
cttactactg tggaatcggc gcggagaaat cgttcgggcg tgacatagtc
600gacgcccact acaaggcctg cctgtacgca ggcatcaaca tcagtggcat
caacggggag 660gtcatgccgg ggcagtggga gttccaggtc ggaccgtccg
tcggcatctc ttcgggcgat 720caggtgtggg ttgctcgcta cattcttgag
aggatcaccg agatcgccgg cgtggtggtg 780acgttcgacc cgaagccgat
cccgggcgac tggaacggcg cgggcgccca caccaactac 840agcaccgagt
ccatgaggaa ggagggcggg tacgaggtga tcaaggcggc catcgagaag
900ctgaagctgc ggcacaagga gcacatcgcg gcctacggcg agggcaacga
gcgccggctc 960accggcaggc acgagaccgc cgacatcaac accttcagct
ggggagtcgc caaccgtggc 1020gcgtcggtgg ccgtgggcca gacggagcag
aacggcaagg gctacttcga ggaccgccgg 1080ccggcgtcca acatggatcc
ctacgtggtc acctccatga tcgccgagac caccatcgtc 1140tggaagccct
gaggcatccc gtggccgtgt cgtgtcggtt tgctccgcgt acggcgctgg
1200ccgttgcatc gcagggccca gcggttgcgc aactattttc ccttccccgt
tccgtttgct 1260tgtactacta ctctaccgct agtcctgcat agcattttag
ctagaacaca acaacagcca 1320aaaaaaaaca ttgttgcttg cttcgacttc
gacgcttccc accactagtt ccattccatg 1380ccgtccgtcc acttccttcc
tgtgtaatcc tcctccaata atagacgtgt catgctgcat 1440cctctgcatt
gtataaaaga aagtggtgta atccttttgc tggcgcctcc 149050355PRTZea mays
50Met Ala Cys Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Asp Thr Thr1
5 10 15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Ser Gly Met
Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Thr Asp
Pro Ser 35 40 45Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly
Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala
Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu Val
Met Cys Asp Cys Tyr Thr 85 90 95Pro Ala Gly Glu Pro Ile Pro Thr Asn
Lys Arg Tyr Ser Ala Ala Lys 100 105 110Ile Phe Ser Ser Pro Glu Val
Ala Ala Glu Glu Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr
Leu Leu Gln Lys Asp Thr Asn Trp Pro Leu Gly 130 135 140Trp Pro Ile
Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155
160Ile Gly Ala Glu Lys Ser Phe Gly Arg Asp Ile Val Asp Ala His Tyr
165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn
Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro
Ser Val Gly Ile 195 200 205Ser Ser Gly Asp Gln Val Trp Val Ala Arg
Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Val Val Val
Thr Phe Asp Pro Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn Gly
Ala Gly Ala His Thr Asn Tyr Ser Thr Glu Ser 245 250 255Met Arg Lys
Glu Gly Gly Tyr Glu Val Ile Lys Ala Ala Ile Glu Lys 260 265 270Leu
Lys Leu Arg His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280
285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300Ser Trp Gly Val Ala Asn Arg Gly Ala Ser Val Ala Val Gly
Gln Thr305 310 315 320Glu Gln Asn Gly Lys Gly Tyr Phe Glu Asp Arg
Arg Pro Ala Ser Asn 325 330 335Met Asp Pro Tyr Val Val Thr Ser Met
Ile Ala Glu Thr Thr Ile Val 340 345 350Trp Lys Pro 355511298DNAZea
mays 51ctctctcttt ctctcttgtg ttcttgcctt ctgcctacta cgagtgatgg
ccagcctcac 60tgacctcgtc aacctcgacc tgagtgactg cacagacagg atcatcgccg
agtacatctg 120ggttggagga tccggcatag acctcaggag caaagcaagg
acggtgaaag gccccatcac 180cgatccgagc cagctgccaa aatggaacta
cgacggctcc agcaccgggc aggctcccgg 240agaggacagc gaagtcatcc
tctaccctca agccattttc aaggacccgt tcaggaaggg 300taacaacatc
cttgtgatgt gtgactgcta cacgccacaa ggcgagccaa tccccagtaa
360caagaggtac aaagctgcca cggttttcag ccaccccgat gttgcagctg
aggtgccatg 420gtacggtatt gagcaggagt acactctcct tcagaaggat
gtgagctggc cccttggctg 480gcctgttggt ggataccctg gtccccaggg
accatactac tgtgctgccg gtgccgataa 540ggcctttggg cgcgacgtgg
ttgacgccca ctacaaagcc tgcctctacg ccggcatcaa 600catcagcggc
atcaacggcg aagtcatgcc tggacagtgg gagttccaag tcgggccgtc
660cgttgggatc tctgccggcg acgagatatg ggtcgcccgc tacattctcg
agaggatcac 720tgagatggcc ggaatcgttc tctccctcga cccgaagccg
atcaagggtg actggaacgg 780cgccggcgct cacaccaact acagcaccaa
gtcgatgagg gaggccggtg gctacgaggt 840gatcaaggag gcgatcgaga
agctggggaa gaggcacagg gagcacatcg ccgcgtacgg 900cgagggcaac
gagcgccgcc tcacgggccg ccacgagacc gccgacatca acaccttcaa
960atggggcgtg gcgaaccgcg gcgcgtccat ccgcgtcggc cgcgacaccg
agaaggaggg 1020caagggatac ttcgaggacc gcaggccggc ttccaacatg
gacccctacg tcgtcaccgg 1080catgatcgcc gacaccacca tcctgtggaa
gggaaactga taaaaccact gttcttctcc 1140tgcacgcatg catccgcccc
gtgctgccac tttttgtttt tcaaatttcg attcccgtcc 1200taaagtttgt
tagcacttat tatttcgctc tccagtgtac tgctcggaaa gtccgaataa
1260aaacggctct aatgattttg tttaaaaaaa aaaaaaaa 129852357PRTZea mays
52Met Ala Ser Leu Thr Asp Leu Val Asn Leu Asp Leu Ser Asp Cys Thr1
5 10 15Asp Arg Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Ile
Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Val Lys Gly Pro Ile Thr Asp
Pro Ser 35 40 45Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly
Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala
Ile Phe Lys Asp65 70 75 80Pro Phe Arg Lys Gly Asn Asn Ile Leu Val
Met Cys Asp Cys Tyr Thr 85 90 95Pro Gln Gly Glu Pro Ile Pro Ser Asn
Lys Arg Tyr Lys Ala Ala Thr 100 105 110Val Phe Ser His Pro Asp Val
Ala Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr
Leu Leu Gln Lys Asp Val Ser Trp Pro Leu Gly 130 135 140Trp Pro Val
Gly Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala145 150 155
160Ala Gly Ala Asp Lys Ala Phe Gly Arg Asp Val Val Asp Ala His Tyr
165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn
Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro
Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Ile Trp Val Ala Arg
Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Met Ala Gly Ile Val Leu
Ser Leu Asp Pro Lys Pro Ile Lys225 230 235 240Gly Asp Trp Asn Gly
Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Glu
Ala Gly Gly Tyr Glu Val Ile Lys Glu Ala Ile Glu Lys 260 265 270Leu
Gly Lys Arg His Arg Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280
285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe
290 295 300Lys Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val Gly
Arg Asp305 310 315 320Thr Glu Lys Glu Gly Lys Gly Tyr Phe Glu Asp
Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Gly
Met Ile Ala Asp Thr Thr Ile 340 345 350Leu Trp Lys Gly Asn
355531483DNAZea mays 53gggcggcggc cggtccgtgt ccgtgtccgt cgacggttgg
ttcgggaatg gcgcaggcgg 60tggtgccggc gatgcagtgc cgggtcggag tgaaggcggc
ggcggggagg gtgtggagcg 120ccggcaggac taggaccggc cgcggcggcg
cctcgccggg gttcaaggtc atggccgtca 180gcacgggcag caccggggtg
gtgccgcgcc tcgagcagct gctcaacatg gacaccacgc 240cctacaccga
caaggtcatc gccgagtaca tctgggtcgg aggatctgga atcgacatca
300gaagcaaatc aaggacgatt tcgaaacccg tggaggatcc ctcagaacta
ccaaaatgga 360actacgatgg atctagcaca ggacaagccc cgggagaaga
cagtgaagtc attctatacc 420cccaggctat cttcaaggac ccattccgag
gtggcaacaa cgttttggtt atctgtgaca 480cctacacgcc acagggggaa
ccccttccaa ctaacaaacg ccacagggct gcgcaaattt 540tcagcgaccc
aaaggtcggt gaacaagtgc catggtttgg catagagcaa gagtacactt
600tgctccagaa agatgtaaat tggcctcttg gttggcctgt tggaggcttc
cctggtcccc 660agggtccata ctactgtgcc gtaggagccg acaaatcatt
tggccgtgac atatcagatg 720ctcactacaa ggcatgcctc tacgctggaa
tcaacattag tggaacaaac ggggaggtca 780tgcctggtca gtgggagtac
caagttggac ctagtgttgg tattgaagca ggagatcaca 840tatggatttc
gagatacatt ctcgagagaa tcacagagca agctggggtt gtccttaccc
900ttgatccaaa accaattcag ggtgactgga acggagctgg ctgccacaca
aattacagca 960caaagaccat gcgcgaagac ggcgggtttg aagagatcaa
gagagcaatc ctgaaccttt 1020ctctgcgcca tgatctgcat attagtgcat
acggagaagg aaatgaaaga agattgactg 1080ggaaacatga gactgcgagc
atcggaacct tctcatgggg tgtggcaaac cgcggctgct 1140ctatccgtgt
ggggcgggat accgaggcaa aagggaaagg ttacctggaa gaccgtcggc
1200cggcatcaaa catggacccg tacattgtga cggggctact ggccgagacc
acgatcctct 1260ggcagccatc cctcgaggcg gaggctcttg ccgccaagaa
gctggcgctg aaggtgtgaa 1320gcagctgaag gatggttcag gcaccaatat
aaaccggtcc gcgacaagat tgatctttgt 1380gtccatggcc gttgggtctt
gcgactctct gctcggcggt gccactctgt acaaaatcac 1440ggctgtcttt
gattcatcgg atattcggat acgtttgttt gtt 148354423PRTZea mays 54Met Ala
Gln Ala Val Val Pro Ala Met Gln Cys Arg Val Gly Val Lys1 5 10 15Ala
Ala Ala Gly Arg Val Trp Ser Ala Gly Arg Thr Arg Thr Gly Arg 20 25
30Gly Gly Ala Ser Pro Gly Phe Lys Val Met Ala Val Ser Thr Gly Ser
35 40 45Thr Gly Val Val Pro Arg Leu Glu Gln Leu Leu Asn Met Asp Thr
Thr 50 55 60Pro Tyr Thr Asp Lys Val Ile Ala Glu Tyr Ile Trp Val Gly
Gly Ser65 70 75 80Gly Ile Asp Ile Arg Ser Lys Ser Arg Thr Ile Ser
Lys Pro Val Glu 85 90 95Asp Pro Ser Glu Leu Pro Lys Trp Asn Tyr Asp
Gly Ser Ser Thr Gly 100 105 110Gln Ala Pro Gly Glu Asp Ser Glu Val
Ile Leu Tyr Pro Gln Ala Ile 115 120 125Phe Lys Asp Pro Phe Arg Gly
Gly Asn Asn Val Leu Val Ile Cys Asp 130 135 140Thr Tyr Thr Pro Gln
Gly Glu Pro Leu Pro Thr Asn Lys Arg His Arg145 150 155 160Ala Ala
Gln Ile Phe Ser Asp Pro Lys Val Gly Glu Gln Val Pro Trp 165 170
175Phe Gly Ile Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Val Asn Trp
180 185 190Pro Leu Gly Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly
Pro Tyr 195 200 205Tyr Cys Ala Val Gly Ala Asp Lys Ser Phe Gly Arg
Asp Ile Ser Asp 210 215 220Ala His Tyr Lys Ala Cys Leu Tyr Ala Gly
Ile Asn Ile Ser Gly Thr225 230 235 240Asn Gly Glu Val Met Pro Gly
Gln Trp Glu Tyr Gln Val Gly Pro Ser 245 250 255Val Gly Ile Glu Ala
Gly Asp His Ile Trp Ile Ser Arg Tyr Ile Leu 260 265 270Glu Arg Ile
Thr Glu Gln Ala Gly Val Val Leu Thr Leu Asp Pro Lys 275 280 285Pro
Ile Gln Gly Asp Trp Asn Gly Ala Gly Cys His Thr Asn Tyr Ser 290 295
300Thr Lys Thr Met Arg Glu Asp Gly Gly Phe Glu Glu Ile Lys Arg
Ala305 310 315 320Ile Leu Asn Leu Ser Leu Arg His Asp Leu His Ile
Ser Ala Tyr Gly 325 330 335Glu Gly Asn Glu Arg Arg Leu Thr Gly Lys
His Glu Thr Ala Ser Ile 340 345 350Gly Thr Phe Ser Trp Gly Val Ala
Asn Arg Gly Cys Ser Ile Arg Val 355 360 365Gly Arg Asp Thr Glu Ala
Lys Gly Lys Gly Tyr Leu Glu Asp Arg Arg 370 375 380Pro Ala Ser Asn
Met Asp Pro Tyr Ile Val Thr Gly Leu Leu Ala Glu385 390 395 400Thr
Thr Ile Leu Trp Gln Pro Ser Leu Glu Ala Glu Ala Leu Ala Ala 405 410
415Lys Lys Leu Ala Leu Lys Val 420
* * * * *