U.S. patent application number 14/929009 was filed with the patent office on 2016-05-05 for stress tolerant plants and methods thereof.
The applicant listed for this patent is Monsanto Technology LLC. Invention is credited to Santanu Dasgupta, Stanton B. Dotson, Targolli L. Jayaprakash, Kottaram Krishnadas Narayanan, Thomas G. Ruff, Carolyn J. Thai.
Application Number | 20160122778 14/929009 |
Document ID | / |
Family ID | 34710084 |
Filed Date | 2016-05-05 |
United States Patent
Application |
20160122778 |
Kind Code |
A1 |
Dasgupta; Santanu ; et
al. |
May 5, 2016 |
STRESS TOLERANT PLANTS AND METHODS THEREOF
Abstract
The present invention provides a method and DNA molecules that
when expressed in a plant produces transgenic plants with improved
abiotic stress tolerance. The invention includes plant expression
vectors comprising the DNA molecules, and plants containing such
DNA molecules.
Inventors: |
Dasgupta; Santanu;
(Bangalore, IN) ; Jayaprakash; Targolli L.;
(Bangalore, IN) ; Thai; Carolyn J.; (O'Fallon,
MO) ; Narayanan; Kottaram Krishnadas; (Bangalore,
IN) ; Ruff; Thomas G.; (Wildwood, MO) ;
Dotson; Stanton B.; (Chesterfield, MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Monsanto Technology LLC |
St. Louis |
MO |
US |
|
|
Family ID: |
34710084 |
Appl. No.: |
14/929009 |
Filed: |
October 30, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12896749 |
Oct 1, 2010 |
9200295 |
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14929009 |
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11961962 |
Dec 20, 2007 |
7851676 |
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12896749 |
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11007819 |
Dec 8, 2004 |
7807874 |
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11961962 |
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60528540 |
Dec 10, 2003 |
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Current U.S.
Class: |
800/289 ;
435/320.1; 800/278; 800/306; 800/312; 800/314; 800/320.1;
800/320.2; 800/320.3 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8271 20130101; C12N 15/8273 20130101 |
International
Class: |
C12N 15/82 20060101
C12N015/82 |
Claims
1. A method of generating a transgenic plant with enhanced
tolerance to environmental stress comprising the steps of: a)
transforming a plant cell with a DNA construct comprising a
promoter that functions in plants, operably linked to a DNA
polynucleotide molecule that encodes a protein substantially
homologous to a protein selected from the group consisting of SEQ
ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,
SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID
NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27,
SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID
NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45,
SEQ ID NO: 47 and SEQ ID NO: 51, and operably linked to a 3'
termination region; and b) regenerating said plant cell into a
fertile transgenic plant; and c) selecting said fertile transgenic
plant containing said DNA construct; wherein said fertile
transgenic plant exhibits enhanced stress tolerance compared to a
plant of a same plant species not transformed to contain said DNA
construct.
2. The method of claim 1, wherein said promoter is a plant virus
promoter.
3. The method of claim 1, wherein said promoter comprises a
heterologus plant promoter.
4. The method of claim 1, wherein said DNA molecule is
substantially homologous to a DNA molecule selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ
ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:
25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:
44, SEQ ID NO: 46 and SEQ ID NO: 50.
5. The method of claim 1, wherein said enhanced stress tolerance is
cold stress tolerance.
6. The method of claim 1, wherein said enhanced stress tolerance is
water stress tolerance.
7. The method of claim 1, wherein said crop plant cell is selected
from the group consisting of corn, soybean, wheat, cotton, rice and
rapeseed/canola.
8. A transgenic plant with enhanced stress tolerance compared to a
plant of a same plant species comprising a DNA construct, wherein
said DNA construct comprises a promoter that functions in plants,
operably linked to a DNA molecule that encodes a protein
substantially homologous to a protein selected from the group
consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 51 and operably
linked to a 3' termination region.
9. The transgenic plant of claim 8, wherein said enhanced stress
tolerance is cold tolerance.
10. The transgenic plant of claim 8, wherein said enhanced stress
tolerance is water stress tolerance.
11. The progeny of said transgenic plant of claim 8.
12. The transgenic plant of claim 8 wherein said transgenic plant
is selected from the group consisting of corn, soybean, wheat,
cotton, rice and rapeseed/canola.
13. A plant part produced by said transgenic plant of claim 8
comprising leaves, roots, stems, shoot, flowers, fibers, fruit, or
seed.
14. A DNA construct, wherein said DNA construct comprises a
promoter that functions in crop plants, operably linked to a DNA
molecule that encodes a protein substantially homologous to a
protein selected from the group consisting of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID
NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47
and SEQ ID NO: 51, and operably linked to a 3'termination
region.
15. The DNA construct of claim 14, wherein said protein comprises
at least a N-terminal substantial portion of a protein selected
from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID
NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14,
SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID
NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31,
SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID
NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO:
51.
16. The DNA construct of claim 14, wherein said protein comprises
at least a N-terminal 85% fragment of protein selected from the
group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ
ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:
16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ
ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 51.
17. The DNA construct of claim 14, wherein said protein comprises
at least a N-terminal 90% fragment of protein selected from the
group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ
ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:
16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ
ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 51.
18. The DNA construct of claim 14, wherein said protein comprises
at least a N-terminal 95% fragment of protein selected from the
group consisting of: SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ
ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:
16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ
ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47 and SEQ ID NO: 51.
19. The DNA construct of claim 14, wherein said DNA construct
comprises a promoter that functions in crop plants, operably linked
to a DNA molecule substantially homologous to DNA molecule selected
from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO:
5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42,
SEQ ID NO: 44, SEQ ID NO: 46 and SEQ ID NO: 50, and operably linked
to a 3'termination region.
20. A method of generating a transgenic crop plant with enhanced
tolerance to cold stress comprising the steps of: a) transforming a
plant cell with a DNA construct comprising a promoter that
functions in plants, operably linked to a DNA molecule that encodes
a protein substantially homologous to a protein selected from the
group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ
ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:
16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ
ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ
ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51 and
operably linked to a 3'termination region; and b) regenerating said
plant cell into a fertile transgenic crop plant; and c) selecting
said fertile transgenic crop plant containing said DNA construct;
wherein said crop plant exhibits enhanced cold stress tolerance
compared to a plant of a same plant species not transformed to
contain said DNA construct.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of priority under 35 U.S.C.
.sctn.119(e) to U.S. Provisional Application No. 60/528,540 filed
on Dec. 10, 2003, which is herein incorporated in its entirety by
reference.
INCORPORATION OF SEQUENCE LISTING
[0002] Two copies of the sequence listing (Seq. Listing Copy 1 and
Seq. Listing Copy 2) and a computer-readable form of the sequence
listing, all on CD_ROMs, each containing the file named
OsPK7Regular Filing.ST25.txt, which is 153, 600 bytes (measured in
MS-DOS) and was created on Dec. 7, 2004, are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0003] Described herein are inventions in the field of plant
molecular biology and plant genetic engineering. In particular, DNA
constructs encoding a polypeptide and transgenic plants containing
the DNA constructs are provided. The transgenic plants are
characterized by improved stress tolerance.
BACKGROUND OF THE INVENTION
[0004] One of the goals of plant genetic engineering is to produce
plants with agronomically, horticulturally or economically
important characteristics or traits. Traits of particular interest
include high yield, improved quality and yield stability. The yield
from a plant is greatly influenced by external environmental
factors including water availability and heat, of which tolerance
of extremes is in turn influenced by internal developmental
factors. Enhancement of plant yield may be achieved by genetically
modifying the plant to be tolerant to yield losses due to stressful
environmental conditions, such as heat and drought stress.
[0005] Seed and fruit production are both limited inherently due to
abiotic stress. Soybean (Glycine max), for instance, is a crop
species that suffers from loss of seed germination during storage
and fails to germinate when soil temperatures are cool (Zhang et
al., Plant Soil 188: (1997)). This is also true in corn and other
plants of agronomic importance. Improvement of abiotic stress
tolerance in plants would be an agronomic advantage to growers
allowing enhanced growth and/or germination in cold, drought,
flood, heat, UV stress, ozone increases, acid rain, pollution, salt
stress, heavy metals, mineralized soils, and other abiotic
stresses.
[0006] Traditional breeding (crossing specific alleles of one
genotype into another) has been used for centuries to increase
abiotic stress tolerance and yield. Traditional breeding is limited
inherently to the limited number of alleles present in the parental
plants. This in turn limits the amount of genetic variability that
can be added in this manner. Molecular biology has allowed the
inventors of the instant invention to look far and wide for genes
that will improve stress tolerance in plants. Protein
phosphorylation is one of the major mechanisms controlling cellular
functions in response to external signals in eukaryotes and kinases
represent a large and diverse protein family. Protein kinases in
plants have been shown to participate in a wide variety of
developmental processes. Protein kinases also respond to
environmental stresses
[0007] Members of the Snf1-related protein kinases play a major
role in phosphorylation cascades involved in carbon assimilation in
animals, fungi and plants. (Hardie D. G., Carling D. and Carlson
M.; Ann. Rev. Biochem. 67: 821-855, 1998). Members of the
AMP-activated/Snf1-related protein kinase subfamily are central
components of highly conserved protein kinase cascades that now
appear to be present in most, if not all, eukaryotic cells. Because
the downstream targets of the action of these enzymes are many and
varied, they have been discovered and rediscovered several times in
different guises and by different approaches. Alderson and
coworkers (Alderson A., et al. Proc. Natl. Acad. Sci. USA, 88:
8602-8605, 1991) cloned and sequenced a cDNA (RKIN1) encoding a
Snf1 homolog from the higher plant rye. Transformation of an Snf1
mutant strain of yeast with a low-copy RKIN1 plasmid restored the
ability to grow on nonfermentable carbon sources (Alderson A., et
al. Proc. Natl. Acad. Sci. USA, 88: 8602-8605, 1991), showing that
RKIN1 is functionally as well as structurally related to Snf1. Snf1
homologs were subsequently cloned from Arabidopsis thaliana (LeGuen
L., Thomas M., Bianchi M., Halford N.G., and Kreis M., Gene 120:
249-254, 1992), barley, (Hannappel U., Vincente-Carbajosa J., Baker
J. H. A., Shewery P. R., and Halford N. G., Plant Mol. Biol., 27:
1235-1240, 1995; Halford N. G., Vincente-Carbajosa J., Sabelli P.
A., Shewery P.R., Hannappel U., and Kreis M., Plant J., 2: 791-797,
1992), tobacco (Muranaka T., Banno H., Machida Y., Mol. Cell. Biol.
14: 2958-2965, 1994) rice and maize (Ohba H. et al. Mo Genet., 263:
359-366, 2000). Two Snf1-related protein kinases from rice, OsPK4
and OsPK7, which are structurally very similar and share more than
75% homology with the wheat homolog WPK4, exhibit very different
expression patterns as well as stress response in rice and maize
plants (Ohba H. et al. Mo Genet., 263: 359-366, 2000). Based on
yeast studies, Snf1 protein kinases including, OsPK4 and OsPK7, are
expected to play a central role in energy metabolism to provide
protection against environmental stress in the host organism. Very
little or no changes were observed in the expression pattern of
rice and maize OsPK7 genes in response to a variety of abiotic
stresses such as light, nutrients, cold, drought, and salt. (Ohba
H. et al. Mo Genet., 263: 359-366, 2000).
[0008] The current invention demonstrates and claims the
utilization of the OsPK7 gene and its homologs to produce plants
with enhanced abiotic stress tolerance, including response to
suboptimal growth temperatures and amounts of water required for
growth of natural plants.
SUMMARY OF THE INVENTION
[0009] In one embodiment, the present invention provides a method
of generating a transgenic plant with enhanced stress tolerance
comprising the steps of transforming a plant cell with a DNA
construct comprising a promoter that functions in the plant cell,
operably linked to a DNA molecule that encodes a protein
substantially homologous to a protein selected from the group
consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51 and operably
linked to a 3'termination region; and regenerating the plant cell
into a fertile transgenic plant; and selecting said fertile
transgenic plant containing the DNA construct; wherein the fertile
transgenic plant exhibits enhanced stress tolerance compared to a
plant of the same plant species not transformed to contain said DNA
construct.
[0010] In one preferred embodiment of the invention a DNA construct
is provided that contains a promoter that is a plant virus
promoter. In another preferred embodiment of the invention a DNA
construct is provided that contains a promoter that is a
heterologous plant promoter. In another preferred embodiment of the
invention the DNA construct contains a promoter that is a tissue
specific or tissue enhanced promoter. In one aspect of the
invention, the DNA construct contains a promoter that is a
constitutive promoter. In another aspect of the invention the DNA
construct contains a promoter that is a promoter that is found in
association with the native gene in the genome.
[0011] In another preferred embodiment, the DNA molecule is
substantially homologous to a DNA molecule selected from the group
consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:
7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ
ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO:
25, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ
ID NO: 36, SEQ ID NO:38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO:
44, SEQ ID NO: 46, and SEQ ID NO: 50.
[0012] In another aspect of the invention a transgenic plant
containing the DNA construct is provided wherein the transgenic
plant exhibits enhanced stress tolerance. The transgenic plant is
particularly tolerant to cold stress.
[0013] The transgenic plant is selected from the group consisting
of: Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula,
asparagus, avocado, banana, barley, beans, beet, blackberry,
blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe,
carrot, cassava, cauliflower, celery, cherry, cilantro, citrus,
clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant,
endive, escarole, eucalyptus, fennel, figs, forest tree, gourd,
grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks,
lemon, lime, loblolly pine, mango, melon, millet, mushroom, nut,
oat, okra, onion, orange, papaya, parsley, pea, peach, peanut,
pear, pepper, persimmon, pine, pineapple, plantain, plum,
pomegranate, poplar, potato, pumpkin, quince, radiata pine,
radicchio, radish, raspberry, rice, rye, sorghum, southern pine,
soybean, spinach, squash, strawberry, sugarbeet, sugarcane,
sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato,
turf, a vine, watermelon, wheat, yams, and zucchini.
[0014] The present invention also provides a transgenic plant with
enhanced stress tolerance compared to a plant of the same plant
species comprising a DNA construct wherein the DNA construct
comprises a promoter that functions in plants operably linked to a
DNA molecule that encodes a protein substantially homologous to a
protein selected from the group consisting of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID
NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,
and SEQ ID NO: 51 and operably linked to a 3'termination
region.
[0015] The present invention also provides a DNA construct wherein
the DNA construct comprises a promoter that functions in plants
operably linked to a DNA molecule that encodes a protein
substantially homologous to a protein selected from the group
consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO:
8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ
ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO:
26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ
ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO:
43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51, and operably
linked to, a 3'termination region.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 Plasmid map of plant expression vector pMON 72472
[0017] FIG. 2 Plasmid map of plant expression vector pMON 80878
[0018] FIG. 3 Plasmid map of plant expression vector pMON 53616
[0019] FIG. 4 Plasmid map of plant expression vector pMON 71709
[0020] FIG. 5 Plasmid map of plant expression vector pMON 71712
[0021] FIG. 6 Plasmid map of plant expression vector pMON 83200
[0022] FIG. 7 Plasmid map of plant expression vector pMON 71710
[0023] FIG. 8 Plasmid map of plant expression vector pMON 71713
[0024] FIG. 9 Plasmid map of plant expression vector pMON 83201
[0025] FIG. 10 Plasmid map of plant expression vector pMON
82629
[0026] The invention can be more fully understood from the
following detailed description and the accompanying Sequence
Listing that form a part of this application.
DETAILED DESCRIPTION OF THE INVENTION
[0027] The present invention is based, in part, on the
identification of polynucleic acid molecules encoding polypeptides
of the present invention from plants including maize, rice and
soybean and utilizing these molecules to enhance abiotic stress
tolerance in plants by ectopic expression of polypeptides of the
invention leading to potential enhancement in yield.
Isolated Polynucleic Acid Molecules of the Present Invention
[0028] The term "polynucleic acid molecule" as used herein means a
deoxyribonucleic acid (DNA) molecule or ribonucleic acid (RNA)
molecule. Both DNA and RNA molecules are constructed from
nucleotides linked end to end, wherein each of the nucleotides
contains a phosphate group, a sugar moiety, and either a purine or
a pyrimidine base. Polynucleic acid molecules can be single or
double-stranded polymers of nucleotides read from the 5' to the 3'
end. Polynucleic acid molecules may also optionally contain
synthetic, non-natural or altered nucleotide bases that permit
correct read through by a polymerase and do not alter expression of
a polypeptide encoded by that polynucleic acid molecule.
[0029] The term "an isolated polynucleic acid molecule" as used
herein, means a polynucleic acid molecule that is no longer
accompanied by some of materials with which it is associated in its
natural state, or to a polynucleic acid molecule for which the
structure of which is not identical to that of any naturally
occurring polynucleic acid molecule. It is also contemplated by the
inventors that the isolated polynucleic acid molecules of the
present invention also include known types of modifications.
[0030] The term "nucleotide sequence" as used herein means the
linear arrangement of nucleotides to form a polynucleotide of the
sense and complementary strands of a polynucleic acid molecule
either as individual single strands or in the duplex
[0031] As used herein both terms "a coding sequence" and "a
structural polynucleotide molecule" mean a polynucleotide molecule
that is translated into a polypeptide, usually via mRNA, when
placed under the control of appropriate regulatory molecules. The
boundaries of the coding sequence are determined by a translation
start codon at the 5'-terminus and a translation stop codon at the
3'-terminus. A coding sequence can include, but is not limited to,
genomic DNA, cDNA, and recombinant polynucleotide sequences.
[0032] The term " recombinant DNAs" as used herein means DNAs that
contains a genetically engineered modification through manipulation
via mutagenesis, restriction enzymes, or other methods known in the
art for manipulation of DNA molecules.
[0033] The term "synthetic DNAs" as used herein means DNAs
assembled from oligonucleotide building blocks that are chemically
synthesized using procedures known to those skilled in the art.
[0034] Both terms "polypeptide" and "protein", as used herein, mean
a polymer composed of amino acids connected by peptide bonds. An
amino acid unit in a polypeptide (or protein) is called a residue.
The terms "polypeptide" and "protein" also apply to any 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 any naturally occurring amino acid polymers.
The essential nature of such analogues of naturally occurring amino
acids is that, when incorporated into a polypeptide, that
polypeptide is specifically reactive to antibodies elicited to the
same polypeptide but consisting entirely of naturally occurring
amino acids. It is well known in the art that proteins or
polypeptides may undergo modification. Exemplary modifications are
described in most basic texts, such as, for example,
Proteins--Structure and Molecular Properties, 2nd ed., T. E.
Creighton, W. H. Freeman and Company, New York (1993. Many detailed
reviews are available on this subject, for example, those provided
by Wold, F., Post-translational Protein Modifications. Perspectives
and Prospects, pp.1-12 in Post-translational Covalent Modification
of Proteins, B. C. Johnson, Ed., Academic Press, New York (1983);
Seifter et al., Meth. Enzymol. 182:626-M (1990) and Rattan et al.,
Protein Synthesis: Post-translational Modifications and Aging, Ann.
N.Y. Acad. Sci. 663:48-62 (1992).
[0035] The term "amino acid sequence" means the sequence of amino
acids in a polypeptide (or protein) that is written starting with
the amino-terminal (N-terminal) residue and ending with the
carboxyl-terminal (C-terminal) residue.
[0036] "Percentage of sequence identity" is determined by comparing
two optimally aligned sequences over a comparison window, wherein
the portion of the polynucleotide or amino acid sequence in the
comparison window may comprise additions or deletions (i.e., gaps)
as compared to the reference sequence (that 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.
[0037] The terms "substantially identical", "substantially
homologous" and "substantial identity", used in reference to two
polypeptide sequences or two polynucleotide sequences, mean that
one polypeptide sequence or one polynucleotide sequence has at
least 75% sequence identity compared to the other polypeptide
sequence or polynucleotide sequence as a reference sequence using
the Gap program in the WISCONSIN PACKAGE version 10.0-UNIX from
Genetics Computer Group, Inc. based on the method of Needleman and
Wunsch (J. Mol. Biol. 48:443-453, 1970) using the set of default
parameters for pairwise comparison (for amino acid sequence
comparison: Gap Creation Penalty=8, Gap Extension Penalty=2; for
nucleotide sequence comparison: Gap Creation Penalty=50; Gap
Extension Penalty=3) or using the TBLASTN program in the BLAST
2.2.1 software suite (Altschul et al., Nucleic Acids Res.
25:3389-3402), using BLOSUM62 matrix (Henikoff and Henikoff, Proc.
Natl. Acad. Sci. U.S.A. 89:10915-10919, 1992) and the set of
default parameters for pair-wise comparison (gap creation cost=11,
gap extension cost=1.)
[0038] One aspect of the present invention provides an isolated
polynucleic acid molecule comprising a nucleotide sequence or
complement thereof, wherein the nucleotide sequence encodes a
polypeptide from a crop plant having an amino acid sequence that
has at least 75% sequence identity, or 80% sequence identity, or at
least 85% or 90% sequence identity, or at least 95% sequence
identity, or at least 98% sequence identity to a member selected
from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ
ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO:
41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO:
51.
[0039] Polypeptides that are "substantially similar" share
sequences as noted above except that residue positions that are not
identical may differ by conservative amino acid changes.
"Conservative amino acid changes" and "Conservative amino acid
substitution" are used synonymously to describe the invention.
Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains.
"Conservative amino acid substitutions" mean substitutions of one
or more amino acids in a native amino acid sequence with another
amino acid(s) having similar side chains, resulting in a silent
change. Conserved substitutes for an amino acid within a native
amino acid sequence can be selected from other members of the group
to which the naturally occurring amino acid belongs. For example, a
group of amino acids having aliphatic side chains is glycine,
alanine, valine, leucine, and isoleucine; a group of amino acids
having aliphatic-hydroxyl side chains is serine and threonine; a
group of amino acids having amide-containing side chains is
asparagine and glutamine; a group of amino acids having aromatic
side chains is phenylalanine, tyrosine, and tryptophan; a group of
amino acids having basic side chains is lysine, arginine, and
histidine; and a group of amino acids having sulfur-containing side
chains is cysteine and methionine. Preferred conservative amino
acids substitution groups are: valine-leucine, valine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic
acid-glutamic acid, and asparagine-glutamine.
[0040] One skilled in the art will recognize that the values of the
above substantial identity of nucleotide sequences can be
appropriately adjusted to determine the corresponding sequence
identity of two nucleotide sequences encoding the polypeptides of
the present invention by taking into account codon degeneracy,
conservative amino acid substitutions and reading frame
positioning. Substantial identity of nucleotide sequences for these
purposes normally means sequence identity of at least 75%.
[0041] The term "codon degeneracy" means divergence in the genetic
code permitting variation of the nucleotide sequence without
affecting the amino acid sequence of an encoded polypeptide. The
skilled artisan is well aware of the "codon-bias" exhibited by a
specific host cell in usage of nucleotide codons to specify a given
amino acid. Therefore, when synthesizing a gene for ectopic
expression in a host cell, it is desirable to design the gene such
that its frequency of codon usage approaches the frequency of codon
usage of the host cell as observed in a codon usage table.
[0042] The polynucleic acid molecules encoding a polypeptide of the
present invention may be combined with other non-native, or
"heterologous" sequences in a variety of ways. By "heterologous"
sequences it is meant any sequence that is not naturally found
joined to the nucleotide sequence encoding polypeptide of the
present invention, including, for example, combinations of
nucleotide sequences from the same plant that are not naturally
found joined together, or the two sequences originate from two
different species.
[0043] The term "operably linked", as used in reference to a
regulatory molecule and a structural polynucleotide molecule, means
that the regulatory molecule causes regulated expression of the
operably linked structural polynucleotide molecule. "Expression"
means the transcription and stable accumulation of sense or
antisense RNA derived from the polynucleic acid molecule of the
present invention. Expression may also refer to translation of mRNA
into a polypeptide. "Sense RNA" means RNA transcript that includes
the mRNA and so can be translated into polypeptide or protein by
the cell. "Antisense RNA" means a RNA transcript that is
complementary to all or part of a target primary transcript or
complementary to mRNA and that blocks the expression of a target
gene (U.S. Pat. No. 5,107,065, incorporated herein by reference).
The complementarity of an antisense RNA may be with any part of the
specific gene transcript, i.e., at the 5' non-coding sequence, 3'
non-translated sequence, introns, or the coding sequence. "RNA
transcript" means the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from post-transcriptional processing of the
primary transcript and is referred to as the mature RNA.
[0044] The DNA construct of the present invention can, in one
embodiment, contain a promoter which causes the over-expression of
the polypeptide of the present invention, where "over-expression"
means the expression of a polypeptide either not normally present
in the host cell, or present in said host cell at a higher level
than that normally expressed from the endogenous gene encoding said
polypeptide. Promoters that can cause the over-expression of the
polypeptide of the present invention are generally known in the
art.
[0045] The DNA construct of the present invention can, in another
embodiment, contain a promoter which causes the ectopic expression
of the polypeptide of the invention, where "ectopic expression"
means the expression of a polypeptide in a cell type other than a
cell type in which the polypeptide is normally expressed; at a time
other than a time at which the polypeptide is normally expressed;
or at a expression level other than the level at which the
polypeptide normally is expressed. Promoters that can cause ectopic
expression of the polypeptide of the present invention are
generally known in the art. The expression level or pattern of the
promoter of the DNA construct of the present invention may be
modified to enhance its expression. Methods known to those of skill
in the art can be used to insert enhancing elements (for example,
subdomains of the CaMV 35S promoter, Benfey et. al, 1990 EMBO J. 9:
1677-1684) into the 5' sequence of genes. In one embodiment,
enhancing elements may be added to create a promoter that
encompasses the temporal and spatial expression of the native
promoter of the gene of the present invention but have altered
levels of expression as compared to the native levels of
expression. Similarly, tissue specific expression of the promoter
can be accomplished through modifications of the 5' region of the
promoter with elements determined to specifically activate or
repress gene expression (for example, pollen specific elements,
Eyal et al., 1995 Plant Cell 7: 373-384).
[0046] The term "a gene" means the segment of DNA that is involved
in producing a polypeptide. Such segment of DNA includes regulatory
molecules preceding (5' non-coding DNA molecules) and following (3'
non-coding DNA molecules) the coding region, as well as intervening
sequences (introns) between individual coding segments (exons). A
"native gene" means a gene as found in nature with its own
regulatory DNA sequences. "Chimeric gene" means any gene that is
not a native gene, comprising regulatory and coding sequences that
are not found together in nature. Accordingly, a chimeric gene may
comprise regulatory sequences and coding sequences that are derived
from different sources, or regulatory sequences and coding
sequences derived from the same source, but arranged in a manner
different than that found in nature. "Endogenous gene" means a
native gene in its natural location in the genome of an organism. A
"foreign gene" means a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes. A "transgene" is a gene
that has been introduced into the genome by a transformation
procedure resulting in a transgenic organism.
[0047] "Regulatory sequences" means polynucleotide molecules
located upstream (5' non-coding sequences), within, or downstream
(3' non-translated sequences) of a structural polynucleotide
sequence, and that influence the transcription, RNA processing or
stability, or translation of the associated structural
polynucleotide sequence. Regulatory sequences may include
promoters, translation leader sequences, introns, and
polyadenylation recognition sequences.
[0048] The term promoter sequence or promoter means a
polynucleotide molecule that is capable of causing expression of
one or more genes when present in "cis" location of the structural
polynucleotide capable of expressing polypeptide. Such promoter
regions are typically found upstream of the trinucleotide, ATG, at
the start site of a polypeptide coding region. Promoter molecules
can also include DNA sequences from which transcription of transfer
RNA (tRNA) or ribosomal RNA (rRNA) sequences are initiated.
Transcription involves the synthesis of a RNA chain representing
one strand of a DNA duplex which provides the template for its
synthesis. Transcription takes place by the usual process of
complementary base pairing, catalyzed and scrutinized by the enzyme
RNA polymerase. The reaction can be di\ided into three stages
described as initiation, elongation and termination.
[0049] Initiation begins with the binding of RNA polymerase to the
double stranded (DS or ds) DNA. The polynucleotide sequence of DNA
required for the initiation reaction defines the promoter. The site
at which the first nucleotide is incorporated is called the
start-site or start-point of transcription. Elongation describes
the phase during which the enzyme moves along the DNA and extends
the growing RNA chain. Elongation involves the disruption of the
DNA double stranded structure in which a transiently unwound region
exists as a hybrid RNA-DNA duplex and a displaced single strand of
DNA. Termination involves recognition of the point at which no
further bases should be added to the chain. To terminate
transcription, the formation of phosphodiester bonds must cease and
the transcription complex must come apart. When the last base is
added to the RNA chain, the RNA-DNA hybrid is disrupted, the DNA
reforms into a duplex state, and the RNA polymerase enzyme and RNA
molecule are both released from the DNA. The sequence of DNA
required for the termination reaction is called the transcription
termination region.
[0050] The promoter sequence consists of proximal and more distal
upstream elements, the latter elements often referred to as
enhancers. Accordingly, an "enhancer" is a DNA sequence that can
stimulate promoter activity and may be an innate element of the
promoter or a heterologous element inserted to enhance the level or
tissue-specificity of a promoter. Promoters may be derived in their
entirety from a native gene, or be composed of different elements
derived from different promoters found in nature, or even comprise
synthetic DNA segments. It is understood by those skilled in the
art that different promoters may direct the expression of a gene in
different tissues or cell types, or at different stages of
development, or in response to different environmental
conditions.
[0051] Promoters that are known or are found to cause transcription
of DNA in plant cells can be used in the present invention. Such
promoters may be obtained from a variety of sources such as plants
and plant viruses. A number of promoters, including constitutive
promoters, inducible promoters and tissue-specific promoters, that
are active in plant cells have been described in the literature. It
is preferred that the particular promoter selected should be
capable of causing sufficient expression to result in the
production of an effective amount of a polypeptide to cause the
desired phenotype. In addition to promoters that are known to cause
transcription of DNA in plant cells, other promoters may be
identified for use in the current invention by screening a plant
cDNA library for genes that are selectively or preferably expressed
in the target tissues and then determine the promoter regions.
[0052] The term "constitutive promoter" means a regulatory sequence
that causes expression of a structural nucleotide sequence in most
cells or tissues at most times. Constitutive promoters are active
under most environmental conditions and states of development or
cell differentiation. A variety of constitutive promoters are well
known in the art. Examples of constitutive promoters that are
active in plant cells include but are not limited to the nopaline
synthase (NOS) promoters; the cauliflower mosaic virus (P-CaMV) 19S
and 35S (U.S. Pat. No. 5,858,642); the figwort mosaic virus
promoter (P-FMV, U.S. Pat. No. 6,051,753); and actin promoters,
such as the rice actin promoter (P-Os.Act1, U.S. Pat. No.
5,641,876).
[0053] The term "inducible promoter" means a regulatory sequence
that causes conditional expression of a structural nucleotide
sequence under the influence of changing environmental conditions
(U.S. Pat. Nos. 5,922,564 and 5,965,791), or developmental
conditions. The term "tissue-specific promoter" means a regulatory
sequence that causes transcriptions or enhanced transcriptions of
DNA in specific cells or tissues at specific times during plant
development, such as in vegetative tissues or reproductive tissues.
Examples of tissue-specific promoters under developmental control
include promoters that initiate transcription only (or primarily
only) in certain tissues, such as vegetative tissues, e.g., roots,
leaves or stems, or reproductive tissues, such as fruit, ovules,
seeds, pollen, pistils, flowers, or any embryonic tissue.
Reproductive tissue specific promoters may be, e.g.,
ovule-specific, embryo-specific, endosperm-specific,
integument-specific, seed coat-specific, pollen-specific,
petal-specific, sepal-specific, or some combination thereof. One
skilled in the art will recognize that a tissue-specific promoter
may drive expression of operably linked DNA molecules in tissues
other than the target tissue. Thus, as used herein a
tissue-specific promoter is one that drives expression
preferentially in the target tissue, but may also lead to some
expression in other tissues as well.
[0054] A variety of promoters specifically active in vegetative
tissues, such as leaves, stems, roots and tubers, can be used to
express the polynucleic acid molecules of the present invention.
Examples of tuber-specific promoters include, but are not limited
to the class I and II patatin promoters (Bevan et al., EMBO J.
8:1899-1906, 1986; Koster-Topfer et al., Mol Gen Genet.
219:390-396, 1989; Mignery et al., Gene. 62:27-44, 1988; Jefferson
et al., Plant Mol. Biol. 14: 995-1006, 1990). Examples of
leaf-specific promoters include but are not limited to the ribulose
biphosphate carboxylase (RBCS or RuBISCO) promoters (see, e.g.,
Matsuoka et al., Plant J. 6:311-319, 1994,); the light harvesting
chlorophyll a/b binding protein gene promoter (see, e.g., Shiina et
al., Plant Physiol. 115:477-483, 1997). Examples of root-specific
promoters include, but are not limited to, the promoter for the
acid chitinase gene (Samac et al., Plant Mol. Biol. 25:587-596,
1994); the root specific subdomains of the CaMV35S promoter that
have been identified (Lam et al., Proc. Natl. Acad. Sci. (U.S.A.)
86:7890-7894, 1989).
[0055] Promoters derived from genes encoding embryonic storage
proteins, which includes the gene encoding the 2S storage protein
from Brassica napus (Dasgupta et al., Gene 133:301-302, 1993); the
gene encoding oleosin 20 kD from Brassica napus (GenBank No.
M63985); the genes encoding oleosin A (GenBank No. U09118) and
oleosin B (GenBank No. U09119) from soybean; the gene encoding
oleosin 18 lD from maize (GenBank No. J05212, Lee, Plant Mol. Biol.
26:1981-1987, 1994); and the gene encoding low molecular weight
sulphur rich protein from soybean (Choi et al., Mol. Gen. Genet.
246:266-268, 1995), can also be used. Promoters derived from zein
encoding genes (including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD,
and gamma genes, Pedersen et al., Cell 29:1015-1026, 1982) can be
also used. The zeins are a group of storage proteins found in maize
endosperm.
[0056] It is recognized that additional promoters that may be
utilized are described, for example, in U.S. Pat. Nos. 5,378,619,
5,391,725, 5,428,147, 5,447,858, 5,608,144, 5,608,144, 5,614,399,
5,633,441, 5,633,435, and 4,633,436, all of which are herein
incorporated in their entirety. In addition, a tissue specific
enhancer may be used (Fromm et al., The Plant Cell 1:977-984,
1989). It is further recognized that the exact boundaries of
regulatory sequences may not be completely defined and DNA
fragments of different lengths may have identical promoter
activity.
[0057] The "translation leader sequence" means a DNA sequence
located between the promoter sequence of a gene and the coding
sequence. The translation leader sequence is present in the fully
processed mRNA upstream of the translation start sequence. The
translation leader sequence may affect processing of the primary
transcript to mRNA, mRNA stability or translation efficiency.
Examples of translation leader sequences include maize and petunia
heat shock protein leaders, plant virus coat protein leaders, and
plant rubisco gene leaders among others (Turner and Foster,
Molecular Biotechnology 3:225, 1995).
[0058] The "3' non-translated sequences" or "3' termination region"
means DNA sequences located downstream of a structural nucleotide
sequence and include sequences encoding polyadenylation and other
regulatory signals capable of affecting mRNA processing or gene
expression. The polyadenylation signal functions in plants to cause
the addition of polyadenylate nucleotides to the 3' end of the mRNA
precursor. The polyadenylation sequence can be derived from the
natural gene, from a variety of plant genes, or from T-DNA. An
example of the polyadenylation sequence is the nopaline synthase 3'
sequence (nos 3'; Fraley et al., Proc. Natl. Acad. Sci. USA 80:
4803-4807, 1983). Ingelbrecht et al. exemplify the use of different
3' non-translated sequences (Plant Cell 1:671-680, 1989).
[0059] The laboratory procedures in recombinant DNA technology used
herein are those well known and commonly employed in the art.
Standard techniques are used for cloning, DNA and RNA isolation,
amplification and purification. Generally enzymatic reactions
involving DNA ligase, DNA polymerase, restriction endonucleases and
the like are performed according to the manufacturer's
specifications. These techniques and various other techniques are
generally performed according to Sambrook et al., Molecular
Cloning--A Laboratory Manual, 2nd. ed., Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1989), herein referred to as
Sambrook et al., (1989).
[0060] A "substantial portion" of a polynucleotide sequence
comprises enough of the sequence to afford specific identification
and/or isolation of a polynucleic acid molecule comprising the
sequence. Polynucleotide sequences can be evaluated either manually
by one skilled in the art, or by using computer-based sequence
comparison and identification tools that employ algorithms such as
BLAST (Basic Local Alignment Search Tool; Altschul et al. J Mol.
Biol. 215:403-410, 1993). In general, a sequence of thirty or more
contiguous nucleotides is necessary in order to putatively identify
a nucleotide sequence as homologous to a gene. Moreover, with
respect to polynucleotide sequences, gene-specific oligonucleotide
probes comprising 30 or more contiguous nucleotides may be used in
sequence-dependent methods of gene identification (e.g., Southern
hybridization) and isolation (e.g., in situ hybridization of
bacterial colonies or bacteriophage plaques). In addition, short
oligonucleotides of 12 or more nucleotides may be used as
amplification primers in PCR in order to obtain a particular
polynucleic acid molecule comprising the primers. The skilled
artisan having the benefit of the polynucleic acid molecules as
reported herein, may now use all or a substantial portion of the
disclosed sequences for purposes known to those skilled in this
art. Accordingly, the instant invention comprises the complete
polynucleotide sequences as reported in the accompanying Sequence
Listing, as well as substantial portions of those sequences as
defined above.
[0061] Isolation of polynucleic acid molecules encoding homologous
polypeptides using polynucleotide sequence-dependent protocols is
well known in the art. Examples of polynucleotide
sequence-dependent protocols include, but are not limited to,
methods of polynucleic acid molecule hybridization, and methods of
DNA and RNA amplification as exemplified by various uses of
polynucleic acid molecule amplification technologies (e.g.,
polymerase chain reaction, ligase chain reaction).
[0062] For example, structural polynucleic acid molecules encoding
additional polypeptides of the present invention, either as cDNAs
or genomic DNAs, could be isolated directly by using all or a
substantial portion of the polynucleic acid molecules of the
present invention as DNA hybridization probes to screen cDNA or
genomic libraries from any desired plant employing methodology well
known to those skilled in the art. Methods for forming such
libraries are well known in the art. Specific oligonucleotide
probes based upon the polynucleic acid molecules of the present
invention can be designed and synthesized by methods known in the
art. Moreover, the entire sequences of the polynucleic acid
molecules can be used directly to synthesize DNA probes by methods
known to the skilled artisan such as random primer DNA labeling,
nick translation, or end-labeling techniques, or RNA probes using
available in vitro transcription systems. In addition, specific
primers can be designed and used to amplify a part or all of the
sequences. The resulting amplification products can be labeled
directly during amplification reactions or labeled after
amplification reactions, and used as probes to isolate full-length
cDNA or genomic DNAs under conditions of appropriate
stringency.
[0063] Alternatively, the polynucleic acid molecules of interest
can be isolated from a mixture of polynucleic acid molecules using
amplification techniques. For instance, the disclosed polynucleic
acid molecules may be used to define a pair of primers that can be
used with the polymerase chain reaction (Mullis, et al., Cold
Spring Harbor Symp. Quant. Biol. 51:263-273, 1986; EP 50,424; EP
84,796, EP 258,017, EP 237,362, EP 201,184; U.S. Pat. No.
4,683,202; Erlich, U.S. Pat. No. 4,582,788, and U.S. Pat. No.
4,683,194) to amplify and obtain any desired polynucleic acid
molecule directly from mRNA, from cDNA, from genomic libraries or
cDNA libraries. PCR and other in vitro amplification methods may
also be useful, for example, to clone nucleotide sequences that
encode for polypeptides to be expressed, to make polynucleic acid
molecules to use as probes for detecting the presence of the
desired mRNA in samples, for nucleic acid sequencing, or for other
purposes.
[0064] In addition, two short segments of the polynucleic acid
molecules of the present invention may be used in polymerase chain
reaction protocols to amplify longer polynucleic acid molecules
encoding homologs of a polypeptide of the invention from DNA or
RNA. For example, the skilled artisan can follow the RACE protocol
(Frohman et al., Proc. Natl. Acad.
[0065] Sci. USA 85:8998, 1988) to generate cDNAs by using PCR to
amplify copies of the region between a single point in the
transcript and the 3' or 5' end. Primers oriented in the 3' and 5'
directions can be designed from the polynucleic acid molecules of
the present invention. Using commercially available 3'RACE or
5'RACE systems (Gibco BRL, Life Technologies, Gaithersburg, Md.
U.S.A.), specific 3' or 5' cDNA fragments can be isolated. Products
generated by the 3' and 5' RACE procedures can be combined to
generate full-length cDNAs (Frohman and Martin, Techniques 1:165,
1989).
[0066] Polynucleic acid molecules of interest may also be
synthesized, either completely or in part, especially where it is
desirable to provide modifications in the polynucleotide sequences,
by well-known techniques as described in the technical literature,
see, e.g., Carruthers et al., Cold Spring Harbor Symp. Quant. Biol.
47:411-418 (1982), and Adams et al., J. Am. Chem. Soc. 105:661
(1983).Thus, all or a portion of the polynucleic acid molecules of
the present invention may be synthesized using a codon usage table
of a selected plant host. Other modifications of the coding gene
sequences may result in mutants having slightly altered
activity.
[0067] After transgenic plants are obtained by one of the methods
described above, it will be necessary to screen individual
transgenic plants for those that most effectively display the
desired phenotype. Accordingly, the skilled artisan will develop
methods for screening large numbers of transformants. The nature of
these screens will generally be chosen on practical grounds. For
example, one can screen by looking for changes in gene expression
by using antibodies specific for the polypeptide encoded by the
gene being expressed. Alternatively, one could establish assays
that specifically measure enzyme activity. A preferred method will
be one that allows large numbers of samples to be processed
rapidly, since it will be expected that a large number of
transformants will be negative for the desired phenotype.
[0068] All or a substantial portion of the polynucleic acid
molecules of the present invention may also be used as probes for
genetically and physically mapping the genes that they are a part
of, and as markers for traits linked to those genes. Such
information may be useful in plant breeding in order to develop
lines with desired phenotypes. For example, the polynucleic acid
molecules of the present invention may be used as restriction
fragment length polymorphism (RFLP) markers. Southern blots
(Sambrook et al., 1989) of restriction-digested plant genomic DNA
may be probed with the polynucleic acid fragments of the present
invention. The resulting banding patterns may then be subjected to
genetic analyses using computer programs such as MapMaker (Lander
et al., Genomics 1:174-181, 1987), in order to construct a genetic
map. In addition, the polynucleic acid fragments of the present
invention may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the polynucleotide sequence of the
present invention in the genetic map previously obtained using this
population (Botstein et al., Am. J. Hum. Genet. 32:314-331,
1980).
[0069] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bernatzky and Tanksley (Plant
Mol. Biol. Reporter 4:37-41, 1986). Numerous publications describe
genetic mapping of specific cDNA clones using the methodology
outlined above or variations thereof. For example, F2 intercross
populations, backcross populations, randomly mated populations,
near isogenic lines, exotic germplasms, and other sets of
individuals may be used for mapping. Such methodologies are well
known to those skilled in the art.
[0070] Polynucleic acid probes derived from the polynucleic acid
molecules of the present invention may also be used for physical
mapping (i.e., placement of sequences on physical maps; see
Hoheisel et al., In: Non-mammalian Genomic Analysis: A Practical
Guide, Academic press 1996, pp. 319-346).
[0071] In another embodiment, polynucleic acid probes derived from
the polynucleic acid molecules of the present invention may be used
in direct fluorescence in situ hybridization (FISH) mapping (Trask,
Trends Genet. 7:149-154, 1991). Although current methods of FISH
mapping favor use of large clones (several to several hundred
kilobases; see Laan et al., Genome Res. 5:13-20, 1995),
improvements in sensitivity may allow performance of FISH mapping
using shorter probes.
[0072] A variety of polynucleic acid amplification-based methods of
genetic and physical mapping may be carried out using the
nucleotide molecules of the present invention. Examples include
allele-specific amplification (Kazazian et al., J. Lab. Clin. Med.
11:95-96, 1989), polymorphism of PCR-amplified fragments (CAPS;
Sheffield et al., Genomics 16:325-332, 1993), allele-specific
ligation (Landegren et al., Science 241:1077-1080, 1988),
nucleotide extension reactions (Sokolov et al., Nucleic Acid Res.
18:3671, 1990), Radiation
[0073] Hybrid Mapping (Walter et al., Nat. Genet. 7:22-28, 1997)
and Happy Mapping (Dear and Cook, Nucleic Acid Res. 17:6795-6807,
1989). For these methods, the sequence of a polynucleic acid
fragment is used to design and produce primer pairs for use in the
amplification reaction or in primer extension reactions. The design
of such primers is well known to those skilled in the art. In
methods employing PCR-based genetic mapping, it may be necessary to
identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the nucleotide
sequence. However, this identification is generally not necessary
for mapping methods.
[0074] Isolated polynucleic acid molecules of the present invention
may find use in the identification of loss of function mutant
phenotypes of a plant, due to a mutation in one or more endogenous
genes encoding polypeptides of the present invention. This can be
accomplished either by using targeted gene disruption protocols or
by identifying specific mutants for these genes contained in a
population of plants carrying mutations in all possible genes
(Ballinger and Benzer, Proc. Natl. Acad Sci USA 86:9402-9406, 1989;
Koes et al., Proc. Natl. Acad. Sci. USA 92:8149-8153, 1995; Bensen
et al., Plant Cell 7:75-84, 1995). The latter approach may be
accomplished in two ways. First, short segments of the polynucleic
acid molecules of the present invention may be used in polymerase
chain reaction protocols in conjunction with a mutation tag
sequence primer on DNAs prepared from a population of plants in
which mutator transposons or some other mutation-causing DNA
element has been introduced. The amplification of a specific DNA
fragment with these primers indicates the insertion of the mutation
tag element in or near the plant gene encoding polypeptides.
Alternatively, the polynucleic acid molecules of the present
invention may be used as a hybridization probe against PCR
amplification products generated from the mutation population using
the mutation tag sequence primer in conjunction with an arbitrary
genomic site primer, such as that for a restriction enzyme
site-anchored synthetic adapter.
[0075] The polypeptides of the present invention may also include
fusion polypeptides. A polypeptide that comprises one or more
additional polypeptide regions not derived from that polypeptide is
a "fusion" polypeptide. Such molecules may be derivatized to
contain carbohydrate or other moieties (such as keyhole, limpet,
hemocyanin, etc.). Fusion polypeptides of the present invention are
preferably produced via recombinant means.
[0076] The polypeptide molecules of the present invention may also
include polypeptides encoded by all or a substantial portion of
polypeptide-encoding sequences set forth in SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID
NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,
and SEQ ID NO: 51 or complements thereof or, fragments or fusions
thereof in which conservative, non-essential, or not relevant,
amino acid residues have been added, replaced, or deleted. An
example of such a homolog is the homolog polypeptide (or protein)
from different species. Such a homolog can be obtained by any of a
variety of methods. For example, as indicated above, one or more of
the disclosed sequences, all or a substantial portion of a
polypeptide-encoding sequences selected from the group consisting
of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID
NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17,
SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID
NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36,
SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID
NO: 46, and SEQ ID NO: 50 and complements thereof will be used to
define a pair of primers that may be used to isolate the homolog
encoding polynucleic acid molecules from any desired species. Such
molecules can be expressed to yield homologs by recombinant
means.
[0077] Polynucleic acid molecules that encode all or part of the
polypeptides of the present invention can be expressed, via
recombinant means, to yield polypeptides that can in turn be used
to elicit antibodies that are capable of binding the expressed
polypeptides. It may be desirable to derivatize the obtained
antibodies, for example with a ligand group (such as biotin) or a
detectable marker group (such as a fluorescent group, a
radioisotope or an enzyme). Such antibodies may be used in
immunoassays for that polypeptide. In a preferred embodiment, such
antibodies can be used to screen cDNA expression libraries to
isolate full-length cDNA clones of the present invention (Lemer,
Adv. Immunol. 36:1, 1984; Sambrook et al., 1989).
Plant Recombinant DNA Constructs and Transformed Plants
[0078] The isolated polynucleic acid molecules of the present
invention can find particular use in creating transgenic crop
plants in which polypeptides of the present invention are
overexpressed. Overexpression of these polypeptides in a plant can
enhance plant stress tolerance and thereby lead to improvement in
the yield of the plant. It will be particularly desirable to
enhance plant drought and osmotic stress tolerance in crop plants
that undergo such stresses over the course of a normal growing
season. Crop plants are defined as plants which are cultivated to
produce one or more commercial products. Examples of such crops or
crop plants include soybean, canola, rape, cotton (cottonseeds),
sunflower, and grains such as corn, wheat, rice, rye, and the
like.
[0079] The term "transgenic crop plant" means a plant that contains
an exogenous polynucleic acid, which can be derived from the same
plant species or from a different species. By "exogenous" it is
meant that a polynucleic acid molecule originates from outside the
plant into which the polynucleic acid molecule is introduced. An
exogenous polynucleic acid molecule can have a naturally occurring
or non-naturally occurring nucleotide sequence. One skilled in the
art understands that an exogenous polynucleic acid molecule can be
a heterologous polynucleic acid molecule derived from a different
plant species than the plant into which the polynucleic acid
molecule is introduced or can be a polynucleic acid molecule
derived from the same plant species as the plant into which it is
introduced.
[0080] Crop plant cell, as used herein, includes without
limitation, seeds, suspension cultures, embryos, meristematic
regions, callus tissue, leaves, roots, shoots, gametophytes,
sporophytes, pollen and microspores.
[0081] The term "genome" as it applies to plant cells encompasses
not only chromosomal DNA found within the nucleus, but organelle
DNA found within subcellular components of the cell. DNAs of the
present invention introduced into plant cells can therefore be
either chromosomally integrated or organelle-localized. The term
"genome" as it applies to bacteria encompasses both the chromosome
and plasmids within a bacterial host cell. Encoding DNAs of the
present invention introduced into bacterial or microbial host cells
can therefore be either chromosomally integrated or
plasmid-localized.
[0082] Exogenous polynucleic acid molecules may be transferred into
a crop plant cell by the use of a recombinant DNA construct (or
vector) designed for such a purpose. The present invention also
provides a plant recombinant DNA construct (or vector) for
producing transgenic crop plants, wherein the plant recombinant DNA
construct comprises a structural nucleotide sequence encoding an
polypeptide of the present invention. Methods that are well known
to those skilled in the art may be used to prepare the crop plant
recombinant DNA construct (or vector) of the present invention.
These methods include in vitro recombinant DNA techniques,
synthetic techniques, and in vivo genetic recombination. Such
techniques are described in Sambrook et al., (1989). The GATEWAYTM
cloning technology (Invitrogen Life Technologies, Carlsbad, Calif.)
is also used for construction of a few vectors of the invention.
GATEWAYTM technology uses phage lambda base site-specific
recombination for vector construction, instead of restriction
endonucleases and ligases. Using the GATEWAYTM cloning technology,
a desired DNA sequence, such as a coding sequence, may be amplified
by PCR with the phage lambda attB 1 sequence added to the 5' primer
and the attB2 sequence added to the 3' primer. Alternatively,
nested primers comprising a set of attB 1 and attB2 specific
primers and a second set of primers specific for the selected DNA
sequence can be used. Sequences, such as coding sequences, flanked
by attB 1 and attB2 sequences can be readily inserted into plant
expression vectors using GATEWAY.TM. methods. Assembly of DNA
constructs are done by standard molecular biology techniques as
described in Sambrooks et al.
[0083] A plant recombinant DNA construct of the present invention
contains a structural nucleotide sequence encoding a polypeptide of
the present invention and operably linked to regulatory sequences.
Exemplary regulatory sequences include but are not limited to
promoters, translation leader sequences, introns and 3'
non-translated sequences. The promoters can be constitutive,
inducible, native, or tissue-specific promoters.
[0084] A plant recombinant DNA construct of the present invention
will typically comprise a selectable marker that confers a
selectable phenotype on plant cells. Selectable markers may also be
used to select for plants or plant cells that contain the exogenous
polynucleic acid molecules encoding polypeptides of the present
invention. The marker may encode biocide resistance, antibiotic
resistance (e.g, kanamycin, G418, bleomycin, hygromycin, etc.), or
herbicide resistance (e.g., glyphosate, glufosinate, etc.).
Examples of selectable markers include, but are not limited to, a
neo gene (Potrykus et al., Mol. Gen. Genet. 199:183-188 (1985) that
codes for kanamycin resistance and can be selected for using
kanamycin, G418, etc.; a bar gene that codes for bialaphos
resistance; a mutant EPSP synthase gene (Hinchee et al.,
Bio/Technology 6:915-922 (1988)) that encodes glyphosate
resistance; a nitrilase gene that confers resistance to bromoxynil
(Stalker et al., J. Biol. Chem. 263:6310-6314 (1988) a mutant
acetolactate synthase gene (ALS) that confers imidazolinone or
sulphonylurea resistance, and a methotrexate resistant DHFR gene
(Thillet et al., J. Biol. Chem. 263:12500-12508 (1988)).
[0085] A plant recombinant DNA construct of the present invention
may also include a screenable marker. Screenable markers may be
used to monitor expression. Exemplary screenable markers include a
.beta.-glucuronidase or uidA gene (GUS:1) that encodes an enzyme
for which various chromogenic substrates are known (Jefferson,
Plant Mol. Biol, Rep. 5:387-405 (1987)); an R-locus gene that
encodes a product that regulates the production of anthocyanin
pigments (red color) in plant tissues (Dellaporta et al., Stadler
Symposium 11:263-282 (1988)); a .beta.-lactamase gene (Sutcliffe et
al., Proc. Natl. Acad. Sci. (U.S.A.) 75:3737-3741 (1978)), a gene
that encodes an enzyme for which various chromogenic substrates are
known (e.g., PADAC, a chromogenic cephalosporin); a luciferase gene
(Ow et al., Science 234:856-859 (1986)); a xylE gene (Zukowsky et
al., Proc. Natl. Acad. Sci. (U.S.A.) 80:1101-1105 (1983)) that
encodes a catechol dioxygenase that can convert chromogenic
catechols; an a-amylase gene (Ikatu et al., Bio/Technol. 8:241-242
(1990)); a tyrosinase gene (Katz et al., J. Gen. Microbiol.
129:2703-2714 (1983)) that encodes an enzyme capable of oxidizing
tyrosine to DOPA and dopaquinone that in turn condenses to melanin;
and an a-galactosidase that will turn over a chromogenic
a-galactose substrate.
[0086] Included within the terms "selectable or screenable marker
genes" are also genes that encode a secretable marker whose
secretion can be detected as a means of identifying or selecting
for transformed cells. Examples include markers that encode a
secretable antigen that can be identified by antibody interaction,
or even secretable enzymes that can be detected catalytically.
Secretable proteins fall into a number of classes, including small,
diffusible proteins detectable, e.g., by ELISA, small active
enzymes detectable in extracellular solution (e.g., a-amylase,
p-lactamase, phosphinothricin transferase), or proteins that are
inserted or trapped in the cell wall (such as proteins that include
a leader sequence such as that found in the expression unit of
extension or tobacco PR-S). Other possible selectable and/or
screenable marker genes will be apparent to those of skill in the
art.
[0087] In addition to a selectable marker, it may be desirable to
use a reporter gene. In some instances a reporter gene may be used
with or without a selectable marker. Reporter genes are genes that
are typically not present in the recipient organism or tissue and
typically encode for proteins resulting in some phenotypic change
or enzymatic property. Examples of such genes are provided in K.
Wising et al. Ann. Rev. Genetics, 22, 421 (1988). Preferred
reporter genes include the beta-glucuronidase (GUS) of the uidA
locus of E. coli, the chloramphenicol acetyl transferase gene from
Tn9 of E. coli, the green fluorescent protein from the
bioluminescent jellyfish Aequorea victoria, and the luciferase
genes from firefly Photinus pyralis. An assay for detecting
reporter gene expression may then be performed at a suitable time
after said gene has been introduced into recipient cells. A
preferred such assay entails the use of the gene encoding
beta-glucuronidase (GUS) of the uidA locus of E. coli as described
by Jefferson et al., (Biochem. Soc. Trans. 15, 17-19 (1987) to
identify transformed cells, referred to herein as GUS:1.
[0088] In preparing the recombinant DNA constructs (vectors) of the
present invention, the various components of the construct or
fragments thereof will normally be inserted into a convenient
cloning vector, e.g., a plasmid that is capable of replication in a
bacterial host, e.g., E. coli. Numerous cloning vectors exist that
have been described in the literature, many of which are
commercially available. After each cloning, the cloning vector with
the desired insert may be isolated and subjected to further
manipulation, such as restriction digestion, insertion of new
fragments or nucleotides, ligation, deletion, mutation, resection,
etc. so as to tailor the components of the desired sequence. Once
the construct has been completed, it may then be transferred to an
appropriate vector for further manipulation in accordance with the
manner of transformation of the host cell.
[0089] The present invention also provides a transgenic plant
comprising in its genome an isolated polynucleic acid that
comprises: (a) a 5' non-coding sequence that functions in the cell
to cause the production of a mRNA molecule; that is operably linked
to (b) a structural polynucleotide sequence encoding a polypeptide
of this invention that is operably linked to (c) a 3'
non-translated sequence that functions in said cell to cause
termination of transcription. Preferably, the amino acid sequence
of the polypeptide has at least 75% sequence identity, about 85%
sequence identity, or about 95% or about 98% sequence identity to a
member selected from the group consisting of SEQ ID NO: 2, SEQ ID
NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,
SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID
NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID
NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47,
and SEQ ID NO: 51. The polypeptide can also have one of the
sequences set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID
NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41,
SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51 with
conservative amino acid substitutions.
[0090] Transgenic crop plants of the present invention have
incorporated into their genome, or transformed into their
chloroplast or plastid genomes, an exogenous polynucleic acid
molecule that comprises at least a structural nucleotide sequence
that encodes a polypeptide having an amino acid sequence selected
from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO:
6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ
ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO:
24, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ
ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO:
41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, and SEQ ID NO: 51.
Transgenic crop plants are also meant to comprise progeny
(descendant, offspring, etc.) of any generation of such a
transgenic plant. A seed of any generation of all such transgenic
crop plants wherein said seed comprises a DNA sequence encoding the
polypeptide of the present invention is also an important aspect of
the invention.
[0091] In one embodiment, the transgenic crop plants of the present
invention will have enhanced tolerance to environmental stress due
to the expression of an exogenous polynucleic acid molecule
encoding a polypeptide of the present invention. The transgenic
crop plants of the present invention will have tolerance to abiotic
stresses, for example, variations from optimal condition to
sub-optimal conditions for water, humidity, temperature, light or
other radiations, organic and inorganic nutrients, and salinity.
"Cold" is defined as sub-optimal thermal conditions needed for
normal growth of natural plants. As used herein, "cold germination"
is germination occurring at temperatures below (two or more degrees
Celsius below) those normal for a particular species or particular
strain of plant. As used herein, "cold tolerance" is defined as the
ability of a plant to continue growth for a significant period of
time after being placed at a temperature below that normally
encountered by a plant of that species at that growth stage. As
used herein "enhanced" is defined as to increase or improve in
value, quality, desirability, or attractiveness of one or more
desired traits in a transgenic plant as compared to a nontransgenic
plant of comparable variety. The transgenic plants of the present
invention will have higher tolerance to cold, higher germination in
cold temperature and a higher yield of agricultural products under
stressed conditions. Similarly "water stress" is defined as a
sub-optimal amount of water needed for normal growth of natural
plants. As used herein "water-stress" is a plant condition
characterized by water potential in a plant tissue of less than
about .about.0.5 megapascals (MPa). Water potential in maize is
conveniently measured by clamping a leaf segment in a pressurizable
container so that a cut cross section of leaf is open to
atmospheric pressure. Gauge pressure (above atmospheric pressure)
on the contained leaf section is increased until water begins to
exude from the atmospheric-pressure-exposed cross section. The
gauge pressure at incipient water exudation is reported as negative
water potential in the plant tissue. The transgenic plants of the
present invention will have a higher tolerance to water stress as
compared to natural plants of same species and will have a higher
yield of agricultural products under water stressed conditions.
[0092] The DNA construct of the present invention may be introduced
into the genome of a desired plant host by a variety of
conventional transformation techniques that are well known to those
skilled in the art. Methods of transformation of plant cells or
tissues include, but are not limited to the Agrobacterium mediated
transformation method and the Biolistics or particle-gun mediated
transformation method. Suitable plant transformation vectors for
the purpose of Agrobacterium mediated transformation include those
derived from a Ti plasmid of Agrobacterium tumefaciens, as well as
those disclosed, e.g., by Herrera-Estrella et al., Nature 303:209
(1983); Bevan, Nucleic Acids Res. 12: 8711-8721 (1984); Klee et
al., Bio-Technology 3(7): 637-642 (1985); and EP 120,516. In
addition to plant transformation vectors derived from the Ti or
root-inducing (Ri) plasmids of Agrobacterium, alternative methods
can be used to insert the DNA constructs of this invention into
plant cells. Such methods may involve, but are not limited to, for
example, the use of liposomes, electroporation, chemicals that
increase free DNA uptake, free DNA delivery via microprojectile
bombardment, and transformation using viruses or pollen. A plasmid
expression vector suitable for the introduction of a polynucleic
acid encoding a polypeptide of present invention in monocots using
electroporation or particle-gun mediated transformation is composed
of the following: a promoter that is constitutive or
tissue-specific; an intron that provides a splice site to
facilitate expression of the gene, such as the maize Hsp70 intron
(U.S. Pat. No. 5,593,874, herein incorporated by reference in its
entirety); and a 3' polyadenylation sequence such as the nopaline
synthase 3' sequence (nos 3; Fraley et al., Proc. Natl. Acad. Sci.
USA 80: 4803-4807, 1983). This expression cassette may be assembled
on high copy replicons suitable for the production of large
quantities of DNA.
[0093] An example of a useful Ti plasmid cassette vector for plant
transformation is pMON17227. This vector is described in U.S. Pat.
No. 5,633,435, herein incorporated by reference in its entirety,
and contains a gene encoding an EPSPS enzyme with glyphosate
resistance (herein referred to as aroA:CP4), that is an excellent
selection marker gene for many plants. The gene is fused to the
Arabidopsis EPSPS chloroplast transit peptide (At. EPSPS:CTP2) and
expressed from the Figwort mosaic virus (P-FMV) promoter as
described therein.
[0094] When adequate numbers of cells containing the exogenous
polynucleic acid molecule encoding polypeptides from the present
invention are obtained, the cells can be cultured, then regenerated
into whole plants. Such regeneration techniques rely on
manipulation of certain phytohormones in a tissue culture growth
medium, typically relying on a biocide and/or herbicide marker that
has been introduced together with the desired nucleotide sequences.
Regeneration techniques are described generally in Klee et al.,
Ann. Rev. Plant Phys. 38:467-486 (1987).
[0095] The development or regeneration of transgenic crop plants
containing the exogenous polynucleic acid molecule that encodes a
polypeptide of interest is well known in the art. Preferably, the
regenerated plants are self-pollinated to provide homozygous
transgenic crop plants, as discussed above. Otherwise, pollen
obtained from the regenerated plants is crossed to seed-grown
plants of agronomically important lines. Conversely, pollen from
plants of these important lines is used to pollinate regenerated
plants.
[0096] Plants that can be made to have enhanced stress tolerance by
practice of the present invention include, but are not limited to,
Acacia, alfalfa, aneth, apple, apricot, artichoke, arugula,
asparagus, avocado, banana, barley, beans, beet, blackberry,
blueberry, broccoli, brussels sprouts, cabbage, canola, cantaloupe,
carrot, cassava, cauliflower, celery, cherry, cilantro, citrus,
clementines, coffee, corn, cotton, cucumber, Douglas fir, eggplant,
endive, escarole, eucalyptus, fennel, figs, forest trees, gourd,
grape, grapefruit, honey dew, jicama, kiwifruit, lettuce, leeks,
lemon, lime, Loblolly pine, mango, melon, mushroom, nut, oat, okra,
onion, orange, an ornamental plant, papaya, parsley, pea, peach,
peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum,
pomegranate, poplar, potato, pumpkin, quince, radiata pine,
radicchio, radish, raspberry, rice, rye, sorghum, Southern pine,
soybean, spinach, squash, strawberry, sugarbeet, sugarcane,
sunflower, sweet potato, sweetgum, tangerine, tea, tobacco, tomato,
turf, a vine, watermelon, wheat, yams, and zucchini.
[0097] The following examples are provided to better elucidate the
practice of the present invention and should not be interpreted in
any way to limit the scope of the present invention. Those skilled
in the art will recognize that various modifications, additions,
substitutions, truncations, etc., can be made to the methods and
genes described herein while not departing from the spirit and
scope of the present invention.
EXAMPLES
Example 1
[0098] Stock Rice Plants and Growth Conditions
[0099] Rice seeds (Oryza sativa, "Kasalath" cultivar) were obtained
from the National Institute of Agrobiological Resources
MAFF1-2Kannondai2-Chome, Tsukuba Ibrai-3058602 Japan. For
increasing the stock size of initial seeds, seeds were planted to
raise seedling and seedlings were subsequently transplanted in 6
inch (6'') pots for obtaining mature plants bearing panicles and
mature seeds.
[0100] Seed Propagation
[0101] For obtaining mature seeds, rice plants, plant organs or
immature embryos at the desired developmental stage, approximately
100 seeds of each variety were soaked in distilled water for 30 to
60 minutes at room temperature. During the soaking period floating
chaff and impurities from the seeds were removed, water was
decanted and the seeds were placed in properly labeled
pre-irrigated 6'' pots filled with red soil. After placing 1-2
seed(s)/pot on top of the soil, the seeds were covered with fine
sand and then gently patted. Each seeded pot was covered with
newspaper and was irrigated regularly with rose-can tin in order to
maintain humidity in the soil. After 4-6 days, paper covers from
the pots were removed, exposing germinated seeds to the light. The
germinated seeds were allowed to grow 1''-2'' in height which
usually occurred 7-8 days after planting seeds. Pots were then
transferred to a water tray for proper water and nutrient
treatments. Initial fertilizer was prepared by mixing 10 grams (gm)
urea, 30 gm of 17:17:17 N:P:K fertilizer, 2.5 gm of Multiplex -a
micro nutrient (Karnataka Agro chemicals, Bangalore, India), 0.25
gm of FeSO.sub.4 in one liter of water and adjusting the pH to 6.2.
Approximately 1 liter of this solution was used to fertilize pots
placed on 1 square meter of water trays. Water level was maintained
in trays with potted seedlings. Seedlings were allowed to grow for
20 days under natural sunlight (400-800 .mu.mole
/m.sup.2/sec)/10-12 hr day. Day temperature was observed at
28.degree. C.-30.degree. C., night temperature at 19.degree.
C.-20.degree. C. with a relative humidity of 60-70% in the
greenhouses.
[0102] Transplanting of Rice Seedlings
[0103] For transplanting rice seedlings to generate mature plants,
a red and black soil mixture was used as potting mix in 6'' pots.
Red and black soils were mixed in 3:1 ratio to bring soil pH
between pH 6 and pH 7. Ten grams of farm yard manure, (Varsha Agro.
Industries, Bangalore, India, from now on referred to as FYM) was
added per 0.003 cubic meter of soil (which is roughly equivalent to
a full 6'' pot soil). This mixture of soil was used to fill 6''
pots for transplanting. Potted soil was saturated with water and
then allowed to drain before packing the soil to the desired
density. Then soil in the pot was drenched with the fungicide
"Carbendzim" at the concentration of lgm/L (Carbendzim, 50% WP,
BASF India Ltd. Mumbai) and the insecticide Monocrotophos
(Monocrotophos 36% SL , Bayer India Ltd, Mumbai India) lml/L for
disinfection. During or prior to the disinfection procedure, all
clumps of soil in pot were eliminated to maximize the
treatment.
[0104] For transplanting, entire growing rice seedlings along with
the old soil were carefully removed from the pots. Excess soil from
the seedlings was removed by gentle tapping. Two seedlings were
planted (3-6 cm deep) in pots with new soil mix. For the first 10
days approximately 1'' water level followed by 2'' water level was
maintained until 10 days before harvesting. Before harvesting ripe
panicles with seed, water was siphoned out of the trays. Siphoning
was done by draining all the water from the tray on the 30.sup.th
day of heading and 10 days before harvesting. Fertilizer
application for growing rice was done as per the following
table:
TABLE-US-00001 TABLE 1 Composition of Different plant growth medium
used for growing rice plants and seedlings. Micro Nutrient
Application Fertilizers (Multiplex) N P K 17:17:17 Urea S.
Phosphate M. Potash Time Total Doses 15 5 7.5 gm/m.sup.2 Basal
Doses 2.5 2.5 5 2.5 14.71 11.90 0 DOP + 1 = A gm/m.sup.2 gm/m.sup.2
2.5 2.5 14.71 A + 15 = B Doses at Active 2.5 2.5 2.5 5.4 4.17 B +
15 = C Tillering gm/m.sup.2 gm/m.sup.2 2.5 2.5 2.5 5.4 4.17 C + 15
= D Doses at 2.5 2.5 5.4 4.17 D + 15 = E Panicle Initiation Stage
gm/m.sup.2 Doses at 2.5 2.5 5.4 E + 15 Heading gm/m.sup.2 Table
legend: Micro Nutrient (Multiplex - (Karnataka Agro chemicals,
Bangalore, India),. N, P, K (Nitrogen, P.sub.2O.sub.5, K.sub.2O in
the form of complex fertilizer 17:17:17 Madras Fertilizers Ltd,
Chennai). Superphosphate. Phosphate(P.sub.2O.sub.5 16%, EID Parry
India Ltd, Chennai India), Muriate of Potash (K.sub.2O 60% Zuari
Agro industries, Goa, India) Date of sowing (DOS). Date of
transplanting (DOP).
[0105] Seeds from transgenic or non-transgenic rice plants were
kept segregated from the time of harvest until next use as per
standard practices well know in the art.
Examples 2
[0106] This example demonstrates how rice OsPK7 was cloned to
express in rice plants. OsPK7 cDNA specific primers were designed
based on the gene sequences as shown in SEQ ID NO: 1. DNASTAR
software (DNASTAR, Inc. Madison, Wis., USA) was used for primer
design. The sequences of the 5' and 3' primer were SEQ ID NO: 48
and SEQ ID NO: 49 respectively. Total RNA was purified from pooled
rice (var. Nipponbare) coleoptile tissue by using Trizol reagent
from Life Technologies (Gibco BRL, Life Technologies, Gaithersburg,
Md. U.S.A. from now on referred to as Gibco), essentially as
recommended by the manufacturer . Total RNA was used as the
template to synthesize rice cDNA molecules by using a RT-PCR kit
manufactured by Life Technologies as per the instrctions of
manufacturer of the kit. This cDNA was used as template DNA in a
PCR reaction to amplify cDNA molecules which were purified on a low
melting agarose gel by electrophoresis as described by Sambrook et
al. Purified cDNA molecules of Seq ID NO: 1 were cloned in pCRTOPO
2.1 vector as per the manufacturer's instructions (Invitrogen,
Carlsbad, Calif. 92008). After confirming the sequence, cloned
molecules were excised and re-cloned in the publicly available rice
binary expression vector pCAMBIA 1300 (CAMBIA, Canberra, Australia)
to generate rice transforming vector molecules. Restriction
analysis was performed to identify the transforming vector with SEQ
ID NO: 1 in proper orientation which would encode polypeptide
molecules as shown in SEQ ID NO 2.
Example 3
[0107] Identification of Homologs, Paralogs or Orthologs:
[0108] This example explains how to isolate homologs, orthologs, or
paralogs of SEQ ID NO: 1 by generating cDNA libraries, sequencing
cDNA clones to generate a database for identification of desired
clones from desired plant species.
[0109] For construction of cDNA libraries from plants, plant
tissues are harvested and immediately frozen in liquid nitrogen and
stored at -80.degree. C. until total RNA extraction. Total RNA is
purified from tissues using Trizol reagent from Life Technologies
(Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.),
essentially as recommended by the manufacturer. Poly A+RNA (mRNA)
is purified using magnetic oligo dT beads essentially as
recommended by the manufacturer (Dynabeads, Dynal Corporation, Lake
Success, New York U.S.A.).
[0110] Construction of plant cDNA libraries is well known in the
art and a number of cloning strategies exist. A number of cDNA
library construction kits are commercially available. The
Superscript.TM. Plasmid System for cDNA synthesis and Plasmid
Cloning (Gibco BRL, Life Technologies, Gaithersburg, Md. U.S.A.) is
used, following the conditions suggested by the manufacturer.
[0111] The cDNA libraries are plated on LB agar containing the
appropriate antibiotics for selection and incubated at 37.degree.
for sufficient time to allow the growth of individual colonies.
Single selective-media colonies are individually placed in each
well of 96-well microtiter plates containing LB liquid including
the selective antibiotics. The plates are incubated overnight at
approximately 37.degree. C. with gentle shaking to promote growth
of the cultures.
[0112] The plasmid DNA is isolated from each clone using Qiaprep
plasmid isolation kits, using the conditions recommended by the
manufacturer (Qiagen Inc., Santa Clara, Calif. U.S.A.).
[0113] The template plasmid DNA clones are used for subsequent
sequencing. For sequencing the cDNA libraries, a commercially
available sequencing kit, such as the ABI PRISM dRhodamine
Terminator Cycle Sequencing Ready Reaction Kit with AmpliTaq.RTM.
DNA Polymerase, FS, is used under the conditions recommended by the
manufacturer (PE Applied Biosystems, Foster City, Calif.). The
cDNAs of the present invention are generated by sequencing
initiated from the 5' end or 3' end of each cDNA clone. Entire
inserts or only part of the inserts (ESTs or expressed sequenced
tags) are sequenced.
[0114] A number of DNA sequencing techniques are known in the art,
including fluorescence-based sequencing methodologies. These
methods have the detection, automation and instrumentation
capability necessary for the analysis of large volumes of sequence
data. Currently, the 377 and 3700 DNA Sequencer (Perkin-Elmer
Corp., Applied Biosystems Div., Foster City, Calif.) allow the most
rapid electrophoresis and data collection. With these types of
automated systems, fluorescent dye-labeled sequence reaction
products are detected and data are entered directly into the
computer, producing a chromatogram that is subsequently viewed,
stored, and analyzed using the corresponding software programs.
These methods are known to those of skill in the art and have been
described and reviewed (Birren et al., Genome Analysis: Analyzing
DNA,1, Cold Spring Harbor, N.Y.).
[0115] The generated ESTs (including any full-length cDNA inserts
or complete coding sequences) are combined with ESTs and
full-length cDNA sequences in public databases such as GenBank.
Duplicate sequences are removed, and duplicate sequence
identification numbers are replaced. The combined dataset is then
clustered and assembled using Pangea Systems (DoubleTwist, 2001
Broadway, Oakland, Calif. 94612) tool identified as CAT v.3.2.
First, the EST sequences are screened and filtered, e.g. high
frequency words are masked to prevent spurious clustering; sequence
common to known contaminants such as cloning bacteria are masked;
high frequency repeated sequences and simple sequences are masked;
unmasked sequences of less than 100 base pairs are eliminated. The
thus-screened and filtered ESTs are combined and subjected to a
word-based clustering algorithm that calculates sequence pair
distances based on word frequencies and uses a single linkage
method to group like sequences into clusters of more than one
sequence, as appropriate. Clustered sequences are assembled
individually using an iterative method based on PHRAP/CRAW/MAP
providing one or more self-consistent consensus sequences and
inconsistent singleton sequences. The assembled clustered sequence
files are checked for completeness and parsed to create data
representing each consensus contiguous sequence (contig), the
initial EST sequences, and the relative position of each EST in a
respective contig. The sequence of the 5' most clone is identified
from each contig. The initial sequences that are not included in a
contig are separated out.
[0116] Above described databases with nucleotide and peptide
sequences are queried with sequences of present invention to get
the following homologs, orthologs or paralogs as shown in Table 2.
The BLAST 2.2.1 software (Altschul, et.al., Nucleic Acids Res. 25:
3389-3402 (1997), with BLOSUM62 matrix and "no Filter" options, is
used in the queries. When necessary, frame-shifts in the DNA
sequences of the homologs are detected by aligning the DNA sequence
of the homolog in question to the protein sequence of present
invention, using the "frame+_n2p" program with default parameters
in the GenCore software package (Compugen Inc., 25 Leek Crescent ,
Richmond Hill, Ontario, L4B 4B3, Canada, 1998). Such frame-shifts
are conceptually corrected to yield open reading frames. The
"translate" program with default parameters in the same package is
used to translate open reading frames to corresponding peptide
sequences based on standard genetic codes.
TABLE-US-00002 TABLE 2 Description of homologs, orthologs or
paralogs of SEQ ID NO: 1 SEQ ID NO Genus species 1 to 6 Oryza
sativa 7 to 14 Zea mays 15 to 20 Glycine max 21 and 22 Gossypium
hirsutum 23 to 27 Triticum aestivum 28 and 29 Hordeum vulgare 30 to
33 Allium porrum 34 and 35 Brassica napus 36 and 37 Pisum sativum
38 and 39 Medicago truncatula 40 to 47 Arabidopsis thaliana
Example 4
Isolation of polynucleotide Molecules of the Present Invention and
their Modification
[0117] For isolating polynucleotide molecules of the present
invention, total RNA is isolated from the appropriate crop and
other desired plant species by pooling tissues of different
developmental stages of all vegetative and reproductive organs. RNA
is prepared from pooled plant tissue by the Trizol method (Gibco
BRL, Life Technologies, Gaithersburg, Md. U.S.A.) essentially as
recommended by the manufacturer. Sequences are amplified out from
total RNA by using the Superscript II kit (Gibco BRL, Life
Technologies, Gaithersburg, Md. U.S.A.) according to the
manufacturer's directions. Design of appropriate PCR primers for
isolating sequences of present invention is based on the sequence
information provided in the sequence listing of this disclosure.
Design of primers and reaction conditions are determined as
described in the art. (PCR Strategies, Edited by Michael A. Innis;
David H. Gelfand; & Johm J. Sninsky; Academic Press 1995 and
PCR Protocols, A Guide to Method and Applications, Edited by
Michael A. Innis; David H. Gelfand; Johm J. Sninsky; & Thomas
J. White Academic Press 1990). All reagents for isolating sequences
of the invention can be procured from Gibco BRL, Life Technologies,
Gaithersburg, Md. U.S.A.
Example 5
[0118] This example explains transformation of rice plants to
generate plants of the present invention.
[0119] Transgenic rice plants were produced by an Agrobacterium
mediated transformation method. A disarmed Agrobacterium strain C58
(EHA105) harboring the plant transformation construct was produced
by the standard electroporation method (Bio-Rad) of transforming
bacteria. Transformed bacterial cells were grown overnight in LB
medium (Gibco) containing 5 gm/L hygromycin at 25.degree. C.,
centrifuged and suspended in Co-cultivation medium (Table 3 shown
as CC1 medium) supplemented with acetosyringone (100 uM) at an
OD.sub.600 of 1. This suspension was used for transforming rice
tissue.
[0120] Tissue Preparation for Rice Transformation:
[0121] Panicles of Kasalath rice were collected 10-15 days after
anthesis. First, panicles were thoroughly washed with deionized
water containing a few drops of Tween 20, surface-sterilized with
70% ethanol for 3 minutes, and washed again at room temperature
with deionized water before treating with 2% Sodium hypochlorite
for 10 minutes. Sterilized panicles were washed with water
repeatedly to remove all sodium hypochorite. The husk was manually
removed to isolate immature seed, washed again with deionized
sterile water before a second sterilization with 70% ethanol
followed by three washes with sterile deionized water. Finally,
immature seeds were surface-sterilized with 2% Sodium hypochlorite
for 30-40 minutes, washed with deionized water remove traces of
sterilant. Immature seeds remained in sterilized water during
entire subsequent operation. Immature embryos or immature seeds
were placed on MSAg medium (Table 3) until the co-cultivation.
TABLE-US-00003 TABLE 3 Describes composition of different media
used for examples of the invention. Plant Component/L MSAg CC-1
CC-2 Delay Selection Regeneration development MS Salts 4.2 g 4.2 g
4.2 g 4.2 g 4.2 g 4.2 g 2.1 g (Hi media, India) CaCl2.cndot.2H2O
440 mg 440 mg 440 mg 440 mg 440 mg 440 mg 0 Thiamine HCl 1.0 mg 0.5
mg 0.5 mg 0 1.0 mg 0 0 Glutamine 500 mg 0 0 0 500 mg 0 0
Myo-Inositol 0 0 0 0 0 100 mg 0 Magnesium chloride 750 0 0 0 750 0
0 Casein Hydrolysate 100 mg 0 0 0 100 mg 0 0 Sucrose 20 g 20 g 20 g
20 g 20 g 30 15 g Glucose 0 10 g 10 g 0 0 0 0 2,4-D 2 mg 2 mg 2 mg
1.5 mg 2 mg 0 0 Kinetin 0 0 0 0.2 mg 0 2.0 mg 0 NAA 0 0 0 0 0 2.0
mg 0 BAP 0 0 0 0 0 4.0 mg 0 Phytagel 2.0 g 0 2.0 g 2 g 2.0 g 0 0
L-Proline 0 115 mg 115 mg 500 0 0 0 Acetosyringone 0 0 0 0 0 0 0
Cefataxime 0 0 0 250 mg 250 250 250 mg Hygromycin 0 0 0 0 50 mg 25
mg 25 mg
[0122] Infection of Rice Plants
[0123] Freshly isolated embryos were incubated with bacterial
culture (100 per 10 embryos) for 10 minutes. Individual embryos
were handpicked and cultured on CC2 medium after removing bacterial
suspension. Embryos were incubated for three days in the dark,
washed with sterilized water supplemented with Cefotaxime (Sigma
Chemical Co Catalog No. 22128) and then blotted dry before
culturing on delay medium (Table 3). After one week, roots were
excised and scutellar calli were subcultured on selection medium
(Table 3)
[0124] Selection and Regeneration of Rice Plants
[0125] Putative calli were selected by culturing treated calli on
selection medium (7-10 day interval) for two to three months or
until calli attained 10 mm size. These were then transferred to
regeneration medium for a week under darkness. For shoot
regeneration, calli were transferred to light. Once plants attained
a size of 5-10 mm, they were transferred to bottles containing 1/2
X, Murashige and Skoog basal salts medium (Now on referred as MS
medium or MS. MS can be procured from Sigma Chemical Co. Saint
Louis, Mo., Catalog No. M8900). Selection pressure with hygromycin
was maintained in vitro throughout. Once plants attained a height
of 4-6 inches, they were transferred to the greenhouse for
hardening. These plants are referred to as R0 plants.
[0126] Acclimatization:.
[0127] Primary Acclimatization
R0 plants were acclimatized by placing plant in greenhouse under
covered tunnel for 3-4 days. At the end of this period plants were
removed from agar medium, and all adhering agar was carefully
removed from roots by washing with water to avoid future fungus and
other plant infection. Root were dipped in Bavistin (Carbendzim,
50% WP, BASF India Ltd. Mumbai) solution (1.0 gm/L) for Y2 -1
minute before transplanting in net pots containing "Soilrite Mix"
(Chougule Industries, Bangalore, India), or " Cocopeat" (Varsha
Agro Industries , Bangalore India) . A suitable number (50 or 98)
of plants in net pots were placed on portray (a plastic tray, of
dimension 52.5 cm length.times.25.25 cm width, containing 50 plug
holes and each plug hole, with a dimension of 5cm diameter & 5
cm depth, was fitted with a net pot (5cm diameter X 4.7 cm depth)
and drenched with fungicide solution Bavistin/Dithane M 45 (1.0 gm
/L) (Carbendzim, 50% WP, BASF India Ltd. Mumbai, India/Mancozeb 75%
WP Indofil Chemicals Ltd. Mumbai, India).
[0128] Newly transplanted R0 plants on tray were kept for 7-10 days
in a humid chamber with 80%-90% relative humidity, 24.degree. C.
-25.degree. C. temperature and 800-100 Lux light intensity. During
this period, every 3-4 days the plants were treated with Hoagland
nutrient solution ( Sigma Chemical Co. Catalog No. H2395). After
initial period of 7-10 days the relative humidity was dropped to
70%-80% and the light intensity was increased to 1100-1500 Lux.
Then the plants were treated with 10:52:10 (N:P:K) fertilizer
solution at 100 ppm N level and a mild spray of Bavistin (0.5
gm/L).
[0129] Secondary Acclimatization
[0130] After primary acclimatization plants were acclimatized for 7
days at a light intensity of 1200-1800 Lux, 65%-75% relative
humidity and a temperature between 25.degree. C. -26.degree. C.
After secondary acclimatization plants were transferred to 6'' pots
and were grown as described earlier.
[0131] Details on number of lines, total plants received and
survival status during acclimatization are shown in Table 4.
TABLE-US-00004 TABLE 4 Survival Status of transgenic rice plant
lines after the acclimatization. Survival status Date of Date of
No. of No. of Primary Secondary transplanting Batch no. receipt GOI
lines plants Acclimatization Acclimatization to pot B.N. 2001-9 May
28, 2001 Ospk7 11 33 31 31 Jun. 7, 2001 B.N. 2001-9 Jun. 1, 2001
Ospk7 8 22 22 22 Jun. 7, 2001 B.N. 2001- Jun. 6, 2002 Ospk7 1 3 3 3
Jun. 18, 2001 10
Example 6
[0132] This example describes a method of determining in planta
sequence of OsPK7 gene in a rice plant transformed with the OsPK7
gene or its homolog. The basic methodology presented in this
example can be used for determining in planta sequence in any plant
of the invention.
[0133] DNA Isolation
[0134] Rice plant DNA was prepared using the Phenol extraction
method, modified from Sambrook et al., (1989). 0.5 to 1.0 g leaf
tissue was grinded with liquid nitrogen into a fine powder, and
then was mixed with extraction buffer immediately (at 1:5 w/v
ratio, and buffer composition: 500 mM NaCl, 100 mM Tris--Hcl (PH
8.0), 0.5% SDS, 50 mM EDTA, 80mM Beta-Mercaptoethanol) and
incubated at 65.degree. C. for 10 minutes. Equal volume of phenol:
chloroform (1:1) was added and gently mixed for 3 to 5 minutes,
centrifuged at 10,000 rpm for 10 minutes and the aqueous phase was
transferred into a fresh tube. The aqueous phase was extracted one
more time using only chloroform, and then added with two volumes of
chilled ethanol and gently mixed. DNA precipitates were spooled
into a fresh 1.5 ml tube and dissolved in 800 ul Tris-EDTA (TE)
buffer at room temperature. 5 ul of RNAase (10 mg/ml) was added and
incubated at 37.degree. C. for 30 minutes. The DNA sample was then
extracted with Phenol: chloroform (1:1) twice and chloroform once,
and precipitated using one tenth volume of 3M sodium acetate (pH
5.4) and two volumes of ethanol. DNA was spooled into a fresh 1.5
ml microfuge tube and washed with 70% ethanol. DNA pellet was then
dissolved in 60 to 100 ul TE buffer pH 8.0.
Amplification of Gene from Isolated DNA:
[0135] Nested sets of PCR primers were designed based on the
expression cassette of the plant transformation construct.
Designing of primer pairs is well known in the art and is also
briefly described in example two of the present disclosure.
Approximately 10 ng of isolated genomic DNA from each transgenic
rice plant was used in a standard PCR reaction for amplification of
in planta gene. Reaction mixture with genomic DNA, appropriated
primer pairs, and enzyme in reaction buffer was subjected to
initial denaturation of DNA by heating the mixture at 94.degree.
C., 2 minutes in a PCR machine, followed by 40 cycles of reaction.
Each cycle consisted of denaturation at 94.degree. C. for 30
seconds, annealing at 61.degree. C. for 30 seconds followed by
primer extension at 72.degree. C. for 90 seconds. Amplified DNA was
isolated at the end of PCR reaction by using QlAquick Gel
extraction kit (Qiagen, Cat No. 28704, Qiagen Inc., Santa Clara,
California U.S.A.). The DNA was eluted in TE buffer pH 8.0 and
stored at -20.degree. C. till further use.
Sequencing of Isolated In-Planta Gene:
[0136] Amplified DNA was used as a template in standard sequencing
reaction. Standard method of sequencing is described in Example 3
of the present disclosure. The DNA was sequenced by using
sequencing primers designed on the basis of expression cassette of
the gene in rice plants. In planta gene sequences from two of the
events in rice plants were confirmed to be same and are presented
as SEQ ID No: 50 and its translation is presented as SEQ ID NO
51.
[0137] In some cases sequencing the in planta gene from different
events of transgenic plants demonstrates minor variation in gene
sequences. Minor sequence variation is capable of providing
variation in the level of the desired phenotype in plants. Some
sequence variations were observed when comparing the gene sequence
from the transformation construct isolated from agrobacterium and
the gene sequence isolated from transgenic rice events transformed
with the construct.
Example 7
[0138] This example describes the morphological assay and
observations performed on rice plants of the present invention.
[0139] Transgenic and non-transgenic isolines were segregated based
on the southern analysis of genomic DNA isolated from plants.
Southern analysis of plant genomic DNA was performed by standard
procedures as described in Molecular Cloning, A Laboratory Manual,
Sambrook et al., (1989) and using a hpt DNA fragment as a
non-radioactive probe (using material and protocol supplied in
AlkPhos Direct labeling and detection kit, Amersham pharmacia).
[0140] Morphological Assay on R1 Seeds
[0141] R1 seeds were germinated on MS medium with 50 mg /L
hygormycin to separate transgenic seeds from non-transgenic, and
for further physiological/phenotypical analysis. A subset of these
seeds with the transgene was allowed to mature for production of R2
seeds. 15-20 seeds from 10 independent lines with different copy
numbers of genes were de-husked, surface sterilized and inoculated
on MS medium in culture bottles. Bottles were incubated in the dark
for 2 days and later on transferred to light. At the end of the
incubation period (13 Days) the plants were removed from the
bottles and washed under a gentle flow of water and used for
transplanting. The first ten tallest seedlings were transplanted to
pots for further morphological analysis of R1 plants.
[0142] Morphological data on R1 plants were recorded. Results are
shown in Table 5.
TABLE-US-00005 TABLE 5 Morphological observation of R1 plants
NUMBER OF TILLERS PLANT PANICLE SEED WT. YIELD PER PLANT Plant ID
DOH TOTAL NO. PROD. HEIGHT LENGTH PER 1000 TOTAL Yield Seed Yield
WT 74 .+-. 0.00 13.00 .+-. 1.49 11.70 .+-. 0.95 149.29 .+-. 6.05
26.82 .+-. 0.54 16.13 .+-. 0.65 18.91 .+-. 4.36 18.08 .+-. 4.48
(Kasalath) 653-4-1 76.80 .+-. 4.08 23.40 .+-. 3.95 21.80 .+-. 3.85
141.65 .+-. 6.87 23.40 .+-. 1.28 16.76 .+-. 0.54 12.67 .+-. 8.77
10.66 .+-. 9.22 652-1-1 77.62 .+-. 5.41 13.62 .+-. 5.41 12.54 .+-.
4s.61 145.55 .+-. 11.55 23.16 .+-. 2.44 16.35 .+-. 0.52 14.94 .+-.
4.09 13.86 .+-. 3.89 652-5-1 78.50 .+-. 5.61 11.17 .+-. 1.17 10.33
.+-. 1.51 153.61 .+-. 7.25 22.02 .+-. 0.88 17.12 .+-. 0.94 9.71
.+-. 4.49 8.52 .+-. 4.75 652-6-1 82.30 .+-. 5.10 12.50 .+-. 2.17
11.40 .+-. 1.84 141.35 .+-. 5.97 25.08 .+-. 1.30 17.78 .+-. 2.25
8.21 .+-. 6.08 6.95 .+-. 5.99 610-1-1 87.00 .+-. 0.00 12.38 .+-.
3.85 11.13 .+-. 3.56 146.83 .+-. 8.04 24.98 .+-. 0.69 16.58 .+-.
1.52 5.68 .+-. 3.67 4.35 .+-. 3.99 610-2-3 76.44 .+-. 6.88 15.11
.+-. 2.89 13.44 .+-. 3.68 143.41 .+-. 6.06 25.14 .+-. 0.64 17.31
.+-. 0.43 8.07 .+-. 4.76 6.25 .+-. 5.22 612-1-1 76.63 .+-. 1.06
9.88 .+-. 1.46 9.00 .+-. 1.77 142.91 .+-. 6.24 25.40 .+-. 0.90
16.26 .+-. 0.42 11.15 .+-. 3.19 10.47 .+-. 3.31 647-1-1 80.88 .+-.
3.23 15.88 .+-. 2.30 14.75 .+-. 2.31 136.51 .+-. 5.60 24.39 .+-.
1.14 16.76 .+-. 0.41 6.42 .+-. 3.87 5.39 .+-. 3.85 Table legend:
DOH (Day of heading)- this explains how many days the plant has
taken for flowering after transplanting. Data given here is an
average of 8-12 plants from each event with standard deviation.
WT--Wild type is control set.
Example 8
[0143] This example explains the selection of homozygous rice line
for performing physiological experiments on transgenic plants of
the present invention.
[0144] Homozygosity Test for R2 Seeds
[0145] Rice is a self-pollinated crop. Hence the R1 seed pool from
a R0 transgenic plant with a single copy of the transgene will
harbor the transgene in 1:2:1 ratio i.e one homozygous, 2
heterozygous and one null segregant. R1 homozygous plants will
produce R2 seeds where all the seeds are transgenic and homozygous.
Therefore homozygous lines were identified in the R2 generation by
germinating 30 R2 seeds from individual clones from different
events on 1/2 strength MS medium supplemented with hygormycin as
described earlier. A line with more than 80% germination is
considered homozygous as germination is also affected by seed
quality. Seeds from these homozygous lines were used in different
physiological assays.
TABLE-US-00006 TABLE 6 Homozygosity test No. of No. of SI seeds
seeds No. Plant ID GOI Variety Inoculated Germinated 1
T.sub.1610-1-1-1 OSPK-7 41 30 30 2 T.sub.1610-1-1-2 OSPK-7 41 30 22
3 T.sub.1610-1-1-3 OSPK-7 41 30 28 4 T.sub.1610-2-3-1 OSPK-7 41 30
28 5 T.sub.1610-2-3-3 OSPK-7 41 30 19 6 T.sub.1610-2-3-4 OSPK-7 41
30 22 7 T.sub.1612-1-1-1 OSPK-7 41 30 20 8 T.sub.1612-1-1-2 OSPK-7
41 30 27 9 T.sub.1612-1-1-3 OSPK-7 41 30 19 10 T.sub.1647-1-1-1
OSPK-7 41 30 26 11 T.sub.1647-1-1-2 OSPK-7 41 30 19 12
T.sub.1647-1-1-3 OSPK-7 41 30 23 13 T.sub.1652-1-1-1 OSPK-7 41 30
28 14 T.sub.1652-1-1-5 OSPK-7 41 30 0 15 T.sub.1652-1-1-6 OSPK-7 41
30 0 16 T.sub.1652-3-1-1 OSPK-7 41 30 26 17 T.sub.1652-3-1-2 OSPK-7
41 30 0 18 T.sub.1652-3-1-3 OSPK-7 41 30 0 19 T.sub.1652-3-1-5
OSPK-7 41 30 5 20 T.sub.1652-5-1-1 OSPK-7 41 30 30 21
T.sub.1652-5-1-2 OSPK-7 41 30 29 22 T.sub.1652-5-1-3 OSPK-7 41 30
17 23 T.sub.1652-5-1-6 OSPK-7 41 30 30 24 T.sub.1652-6-1-1 OSPK-7
41 30 24 25 T.sub.1652-6-1-2 OSPK-7 41 30 23 26 T.sub.1652-6-1-3
OSPK-7 41 30 19 27 T.sub.1653-4-1-1 OSPK-7 41 30 23 28
T.sub.1653-4-1-2 OSPK-7 41 30 23 29 T.sub.1653-4-1-3 OSPK-7 41 30
25 30 T.sub.1653-4-1-5 OSPK-7 41 30 30 31 T.sub.1653-4-1-6 OSPK-7
41 35 34 32 T.sub.1653-4-1-7 OSPK-7 41 30 30 33 41 control OSPK-7
41 27 25 34 41 control OSPK-7 35 0
Example 9
[0146] This example explains the water stress test for analyzing
transgenic rice plants of the invention.
[0147] R2 Generation Water Stress Test--Rapid Stress:
[0148] Germinated seedlings were planted in portrays. For plating
seedlings each net pot was filled with 75 g of red sandy loam soil
(dry) and the entire tray was drenched to saturation level with
water containing fungicide Bavistin (1 gm/l). Excess water was
drained before weighing the entire tray as well as individual net
pots. Individual net pots with water-saturated soil weighing about
95 to 100 grams were considered at 100% field water capacity.
Germinated seedlings were further grown in the greenhouse with
conditions as described in example 1. Every day during the growth
period lost water was measured (by weighing pots) and replenished
to maintain 100% of field water capacity in the desired pots. Loss
of water in pots with plants was due to evaporation and
transpiration. Ten net pots were maintained without plants to
calculate the amount of water lost due to evaporation. Plants were
fertilized once every three days with a solution containing 3 gm
urea, 6 gm N:P:K (17:17:17), 0.5 gm FeSO.sub.4 and 2.5 gm
micronutrient mix/32 L. Fifteen-day-old seedlings were subjected to
water stress by withholding irrigation for 4 days. Subsequently net
pots were saturated with water and excess water was drained to
attain 100% field water capacity for alleviating stress. The plats
were maintained at 100% field capacity throughout the recovery
period by weighing the pot every day and replenishing the amount of
water lost through evaporation/transpiration. The plants were
allowed to recover for twelve days. At the end of recovery i.e.,
the 12.sup.th day, growth was measured by weighing only the shoot
(above soil, i.e without root). Growth was recorded as fresh weight
in milligrams as shown in Table 7. The transgenic lines of the
present invention were observed to have significantly higher
biomass at the end of recovery as compared to the wild type rice
line.
TABLE-US-00007 TABLE 7 Result of the R2 generation water stress
test. lines Fresh. Wt. (mg) R2-610-1-1-3 311.0 .+-. 68.4
R2-610-2-3-1 445.5 .+-. 95.5 R2-612-1-1-2 390.3 .+-. 71.3
R2-652-5-1-1 343.8 .+-. 53.8 R2-652-3-1-1 332.5 .+-. 51.8
R2-653-4-1-5 297.2 .+-. 41.8 WT-kasalath 170.4 .+-. 70.1 (wild type
non-tansgenic control
Example 10
[0149] This example demonstrates the rate of survival of transgenic
rice plants as compared to non-transgenic rice plants after the
water stress.
[0150] Three-leaf or 12 days old rice seedlings grown as per the
earlier description and were subjected to water stress by
withholding irrigation for two days and allowing the plant to
recover for 8 days . At the end of recovery, surviving seedling
were counted and expressed as percent seedling survival. For
determining percent survival of transgenic rice plants, five
different sets of experiments designated as 2a, 2b, 2c, 2d, and 2e,
were conducted as described above. Ten plants/set were used for
this experiment. The results of this experiment are shown in Table
8 indicating all transgenic lines except R2-610-2-3-1 exhibited a
significantly high rate of survival at the end of water stress
compared to that of wild type.
TABLE-US-00008 TABLE 8 Showing the survival of transgenic rice
seedlings as compared to non- transgenic rice seedlings after water
stress treatment. Survival at the end of recovery (%) Exp. Exp.
Exp. Exp. Exp. Line code Lines 2a 2b 2c 2d 2e 1 R2-610-1-1-3 30 27
40 ND ND 2 R2-610-2-3-1 0 20 0 ND ND 3 R2-612-1-1-2 * 100 100 100
50 30 4 R2-647-1-1-1 50 54 20 80 60 5 R2-652-1-1-1 ND ND 60 ND ND 6
R2-652-5-1-1 ND ND 60 80 80 7 R2-652-3-1-1 66 54 60 60 20 8
R2-653-4-1-5 83 63 80 70 10 9 WT- kasalath (wild type 16 41 0 0 0
non-tansgenic control)
Example 11
[0151] This example demonstrates the effect of water stress on
plant biomass in transgenic rice plants of the invention in
comparison with wild type rice plants.
[0152] Three-leaf or 12 day-old rice seedlings, grown as per the
description of Example 7, were subjected to water stress by
withholding irrigation for two days and allowing plants to recover
for 10 days. At the end of recovery, growth was measured in terms
of fresh weight. Results of this experiment are shown in Table 9.
The transgenic lines of the present invention maintained higher
average biomass at the end of recovery compared to that of the wild
type.
TABLE-US-00009 TABLE 9 Biomass of rice seedlings as compared to
non-transgenic rice seedlings after water stress treatment. Biomass
of plant is indicated as fresh weight in milligrams. WT Kasalath is
natural, wild type rice plant. Line code Lines Fresh weight (mg) 3
R2-612-1-1-3 243.89 .+-. 227.45 4 R2-647-1-1-2 438.44 .+-. 273.98 6
R2-652-5-1-1 582.00 .+-. 374.53 7 R2-652-3-1-1 417.44 .+-. 327.82 8
R2-653-4-1-5 318.22 .+-. 271.63 WT WT-Kasalath 152.89 .+-.
112.52
Example 12
[0153] This example demonstrates the effect of water stress on
plant biomass in older transgenic rice plants of the invention in
comparison with wild type rice plants.
[0154] Five-leaf or 20 day-old rice seedlings, grown as per the
description of Example 7 were subjected to water stress by
withholding irrigation for two days and allowing plants to recover
for 6 days. At the end of recovery, growth was measured in terms of
fresh weight.
[0155] Results of this experiment are shown in Table 10. The
transgenic lines of the present invention maintained higher average
biomass at the end of recovery compared to that of the wild
type.
TABLE-US-00010 TABLE 10 Biomass of older rice seedling as compared
to non-transgenic rice seedlings after water stress treatment.
Biomass of the plant is indicated as fresh weight in milligrams.
WT-kasalath is non-transgenic. Line code Lines Fresh weight (mg) 3
R2-612-1-1-3 153.7 .+-. 40.8 4 R2-647-1-1-2 363.4 .+-. 109.79 6
R2-652-5-1-1 484.5 .+-. 180.59 7 R2-652-3-1-1 266.5 .+-. 96.03 8
R2-653-4-1-5 215.4 .+-. 78.33 WT WT-Kasalath 252.9 .+-. 93.28
Example 13
[0156] This example describes the effect of long term stress on R2
plants of the present invention.
[0157] Germinated seedlings were transferred to plastic pots (10 cm
diameter x 4 cm depth) containing 100 g of red sandy loam soil with
two different levels of water content. The two levels are 25
percent field capacity (FC25), 9.3 ml/100 g soil and 100 percent
field capacity (FC100), 37.5 ml/100 g soil. The seedlings were
allowed to adapted in two different water regimes for 15 days. The
seedlings were adapted in the greenhouse. During the growth period
the water level was maintained at designated field capacity by
weighing the pots every day and replenishing the amount of water
lost through evaporation/transpiration. Ten pots were maintained
without plants to calculate the amount of water lost due to
evaporation. During this period plants were fertilized once every
three days with solution as described in Example 7. On the 15th day
the difference in growth rate between transgenic and wild type was
assessed in terms of leaf extension growth by measuring the length
of the 4th leaf. All transgenic lines were observed to have
significant leaf growth differences as compared to non-transgenic
lines under experimental stress conditions as described in this
example. Results are show below in table 11.
TABLE-US-00011 TABLE 11 Effect of Long term Stress on R2 rice
plants of present invention as compared to non transgenic
WT-kasalath rice plants. Stressed Non-stressed Line code Lines (FC
25) (FC-100) 1 R2-610-1-1-3 11.45 .+-. 3.5 36.48 .+-. 3.82 3
R2-612-1-1-2 9.,34 .+-. 2.51 33.55 .+-. 3.15 4 R2-652-5-1-1 10.25
.+-. 1.96 33.76 .+-. 2.03 7 R2-652-3-1-1 8.07 .+-. 2.89 32.18 .+-.
3.84 9 WT-kasalath 5.74 .+-. 1.86 33.52 .+-. 3.59
Example 14
[0158] This example demonstrates the effect of cold stress on rice
plants of the present invention.
[0159] Twelve-day-old or three leaf stage seedlings were grown
according to Example 7 and were exposed to cold temperature at
12.degree. C. for 24 hours in the presence of 1000 micro
mol/mt2/Sec.light. Subsequently, the plants were allowed to recover
in the greenhouse for 20 days. The growth observations such as the
length of the 4th leaf on the 7th day and plant height (pl. ht),
fresh weight and dry weight were recorded on the 20th day of
recovery. The cold stressed OSPK-7 transgenic lines exhibited
significantly higher initial recovery growth measured in terms of
the length of the 4th leaf at the end of recovery. Further, the
transgenic lines exhibited significantly higher plant height and
marginally higher total biomass at the end of recovery compared to
that of the wild type. Results are shown in Tables 12 and 13.
TABLE-US-00012 TABLE 12 Results of recovery growth in terms of the
length of the 4th leaf of the plant after exposure to cold
temperature. Lines code Lines Stress Non-stress 1 R2-610-1-1-3 18.2
.+-. 3.5 16.9 .+-. 4.8 2 R2-610-2-3-1 17.9 .+-. 2.1 21.6 .+-. 3.2 3
R2-612-1-1-2 22.1 .+-. 3.2 21.2 .+-. 3.2 4 R2-652-5-1-1 19.1 .+-.
2.0 19.4 .+-. 6.2 7 R2-652-3-1-1 30.9 .+-. 3.1 18.8 .+-. 6.2 8
R2-653-4-1-5 15.3 .+-. 2.5 21.0 .+-. 3.5 9 WT-kasalath 3.7 .+-. 2.2
23.3 .+-. 3.3
TABLE-US-00013 TABLE 13 Results of recovery in terms of plant
height, fresh weight, and dry weight after exposure to cold
temperature. Plant ht. Fresh weight Dry weight Lines (cm) (mg) (mg)
R2-610-1-1-3 32.5 .+-. 5.5 354.9 .+-. 77.0 86.9 .+-. 19.5
R2-610-2-3-1 35.3 .+-. 3.1 389.8 .+-. 26.8 87.8 .+-. 8.2
R2-612-1-1-2 37.2 .+-. 4.2 387.1 .+-. 46.0 104.8 .+-. 42.7
R2-652-5-1-1 35.3 .+-. 2.6 325.8 .+-. 56.6 86.8 .+-. 10.5
R2-652-3-1-1 39.0 .+-. 5.0 488.5 .+-. 51.1 113.2 .+-. 13.6
R2-653-4-1-5 37.7 .+-. 5.1 432.9 .+-. 94.5 91.5 .+-. 18.0
WT-kasalath 29.9 .+-. 2.9 364.6 .+-. 61.6 86.0 .+-. 13.6
Example 15
Genetic Elements of Plant Expression Vectors pMON 80878 (FIG. 2),
pMON 71709 (FIG. 4), pMON 71712 (FIG. 5), pMON 83200 (FIG. 6), pMON
71710 (FIG. 7), pMON 71713 (FIG. 8), pMON 83201 (FIG. 9), and pMON
82629 (FIG. 10)
[0160] The DNA constructs are double border plant transformation
constructs that also contain DNA segments that provide replication
function and antibiotic selection in bacterial cells, for example,
an E. coli origin of replication such as ori322, a broad host range
origin of replication such as oriV or oriRi, and a coding region
for a selectable marker such as Spc/Str that encodes for Tn7
aminoglycoside adenyltransferase (aadA) conferring resistance to
spectinomycin or streptomycin, or a gentamicin (Gm, Gent)
selectable marker gene. For plant transformation, the host
bacterial strain is Agrobacterium tumefaciens ABI or LBA4404.
[0161] The polylinker regions in these DNA constructs provide for
multiple restriction endonuclease cut sites that digest the DNA to
provide a cloning site. Examples of such cloning sites may include
BglII, NcoI, EcoRI, Sall, Notl, XhoI and other sites known to those
skilled in the art of molecular biology. pMON 72472 plant
expression vector (FIG. 1) is modified for cloning and expression
of SEQ ID NO: 1 from rice plants by changing multiple cloning sites
to accept a DNA fragment with Not 1 and Sall restriction
enonuclease fragment. SEQ ID NO:1 is in cloned pMON 72472 (FIG. 1)
or pMON53616 (FIG. 3) at a restriction site resulting in a plant
expression vector pMON 80878 (FIG. 2) pMON 71709 (FIG. 4). The
construct is used for transforming wild type corn plants to
generated transgenic corn plants. Orthologs of SEQ ID NO:1 are
cloned in vector pMON 53616 or pMON 72472 by replacing an existing
expression cassette of the construct with a desired expression
cassette containing a desired promoter, the polynucletide of the
present invention and desired 3' terminator resulting in constructs
pMON 71712, pMON 83200, pMON 71710, pMON 71713, pMON 83201 or pMON
71709 as shown in FIGS. 5 to 10 and Table 14.
TABLE-US-00014 TABLE 14 Construction of plant transforming vectors.
Gene/Homolog Plant of Construct Vector for Transformed Construct's
name origin name the construct Cloning sites plant Figure Promoter
OsPK7 Oryza sativa pMON80878 pMON 72472 attB1 and attB2 LH59 corn
FIG. 1 rACT (promoter leader, intron) OsPK7 Oryza sativa pMON71709
pMON 53616 5' BsiWI; 3' XhoI LH244 corn, FIG. 2 rACT (promoter
(destroyed by haploid LH244 leader, intron) ligation to Not1) corn
OsPK7 Oryza sativa pMON71712 pMON 53616 5' BsiWI; 3' XhoI LH244
corn FIG. 3 CVY-CIK1 (destroyed by (promoter, intron ligation to
SalI) leader) OsPK7 Oryza sativa pMON 83200 pMON 53616 5' BsiWI; 3'
XhoI LH244 corn FIG. 4 Rab17 (destroyed by ligation to SalI) ZmPK4
Zea mays pMON71710 pMON 53616 5' BsiWI; 3' XhoI LH244 corn FIG. 5
rACT (promoter (destroyed by leader, intron) ligation to SalI)
ZmPK4 Zea mays pMON71713 pMON 53616 5' BsiWI; 3' XhoI LH244 corn
FIG. 6 CVY-CIK1 (destroyed by (promoter, intron ligation to SalI)
leader) ZmPK4 Zea mays pMON83201 pMON 53616 5' BsiWI; 3' XhoI LH244
corn FIG. 7 Rab17 (destroyed by ligation to SalI) ZmPK4 Zea mays
pMON82629 pMON 72472 attB1 and attB2 LH59 corn FIG. 8 rACT
(promoter leader, intron)
[0162] The DNA constructs used in the method of the current
invention comprise any promoter known to function to cause
transcription in plant cells and any antibiotic or herbicide
tolerance encoding polynucleotide sequence known to confer
antibiotic or herbicide tolerance to plant cells. The antibiotic
tolerance polynucleotide sequences include, but are not limited to
polynucleotide sequences encoding for proteins involved in
tolerance to kanamycin, neomycin, hygromycin, and other antibiotics
known in the art. An antibiotic tolerance gene in such a vector can
be replaced by a herbicide tolerance gene encoding for
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS, described in
U.S. Pat. Nos. 5,627,061, and 5,633,435, herein incorporated by
reference in its entirety; Padgette et al. (1996) Herbicide
Resistant Crops, Lewis Publishers, 53-85, and in Penaloza-Vazquez,
et al. (1995) Plant Cell Reports 14:482-487), aroA (U.S. Pat. No.
5,094,945) for glyphosate tolerance, bromoxynil nitrilase (Bxn) for
Bromoxynil tolerance (U.S. Pat. No. 4,810,648), phytoene desaturase
(crtI) (Misawa et al, (1993) Plant Journal 4:833-840, and (1994)
Plant Jour 6:481-489) for tolerance to norflurazon,
acetohydroxyacid synthase (AHAS, Sathasiivan et al. (1990) Nucl.
Acids Res. 18:2188-2193) and the bar gene for tolerance to
glufosinate (DeBlock, et al. (1987) EMBO J. 6:2513-2519).
Herbicides for which transgenic plant tolerance has been
demonstrated and the method of the present invention can be applied
include, but are not limited to: glyphosate, glufosinate,
sulfonylureas, imidazolinones, bromoxynil, delapon,
cyclohezanedione, protoporphyrionogen oxidase inhibitors, and
isoxaslutole herbicides.
[0163] Genetic elements of transgene DNA constructs used for plant
transformation and expression of transgenes in plants include, but
are not limited to: plant virus promoters, e.g., P-CaMV.35S
promoter (U.S. Pat. No. 5,858,742, herein incorporated by reference
in its entirety), the CaMV 35S promoter with a duplicated enhancer
(U.S. Pat. No.5,539,142, herein incorporated by reference in its
entirety), the Figwort mosaic virus promoter, P-FMV, as described
in U.S. Pat. No. 5,378,619, herein incorporated by reference in its
entirety; or the P-AtEF1a (P-AtEF1 or EF1a), the sugarcane
bacilliform virus promoter, commelina yellow mottle virus or other
Badnavirus promoters; heterologous plant promoters, e.g., plant
actin promoters including the rice actin 1 promoter and intron
(U.S. Pat. No. 5,641,876) and rice actin 2 promoter and intron
(U.S. Pat. No. 6,429,357), Arabidopsis actin promoters, a promoter
region from the tomato elongation factor gene and Arabidopsis
thaliana elongation factor gene la; or the DC3 promoter region from
carrot (Seffens et al., Develop. Genet. 11:65-76); or the TP12
promoter (GenBank accession no. U68483).
[0164] The genetic elements of the DNA construct further comprise
5' leader polynucleotides for example, the Hsp70 non-translated
leader sequence from Petunia hybrida as described in U.S. Pat. No.
5,362,865, herein incorporated by reference in its entirety.
[0165] The genetic elements further comprise herbicide tolerance
genes that include, but are not limited to, for example, the
aroA:CP4 coding region for EPSPS, a glyphosate resistant enzyme
isolated from Agrobacterium tumefaciens (AGRTU) strain CP4 as
described in U.S. Pat. No. 5,633,435, herein incorporated by
reference in its entirety.
[0166] The genetic elements of the DNA construct further comprise
3' termination regions that include, but are not limited to, the E9
3' termination region of the pea RbcS gene that functions as a
polyadenylation signal; the nos3' is the 3` end of the Ti plasmid
nopaline synthase gene that functions as a polyadenylation signal;
or the TML is 3' of the end of the Ti plasmid octopine pTi15955
synthase gene (GenBank Accession AF 242881) that functions as a
polyadenylation signal . The genetic elements of the DNA construct
further comprise the right border (RB) and left borders (LB) of the
Ti plasmid of Agrobacterium tumefaciens octopine and nopaline
strains.
Example 16
[0167] The following example describes transformation of soy and
corn plants with constructs expressing genes of present invention.
Different plants were transformed with constructs in accordance
with Table 14.
[0168] Corn
[0169] Transgenic corn can be produced by particle bombardment
transformation methods as described in U.S. Pat. No. 5,424,412. The
vector DNA of plasmid pMON 71709, pMON 71710, pMON 71712, pMON
71713 or pMON 80878 is digested with suitable restriction
endonucleases to isolate a plant expression cassette that expresses
the polypeptides of the present invention in the plant. The desired
expression cassette is purified by agarose gel electrophoresis,
then bombarded into embryogenic corn tissue culture cells using a
Biolistic.RTM. (Dupont, Wilmington, Del.) particle gun with
purified isolated DNA fragments. Transformed cells are selected on
selection media such glyphosate (N-phosphonomethyl glycine and its
salts) containing media and whole plants are regenerated then grown
under greenhouse conditions. Fertile seed is collected, planted and
the glyphosate tolerant phenotype is back crossed into commercially
acceptable corn germplasm by methods known in the art of corn
breeding (Sprague et al., Corn and Corn Improvement 3.sup.rd
Edition, Am. Soc. Agron. Publ (1988).
[0170] Transgenic corn plants can be produced by an Agrobacterium
mediated transformation method. A disarmed Agrobacterium strain C58
(ABI) harboring DNA as described earlier in the example is used for
transforming plants. The construct is first transferred into
Agrobacterium by a triparental mating method (Ditta et al., Proc.
Natl. Acad. Sci. 77:7347-7351). Liquid cultures of Agrobacterium
are initiated from glycerol stocks or from a freshly streaked plate
and grown overnight at 26.degree. C-28.degree. C. with shaking
(approximately 150 rpm) to mid-log growth phase in liquid LB
medium, pH 7.0 containing 50 mg/l kanamycin, 50 mg/l streptomycin
and spectinomycin and 25 mg/l chloramphenicol with 200 .mu.M
acetosyringone (AS). The Agrobacterium cells are resuspended in the
inoculation medium (liquid CM4C) and the density is adjusted to
OD.sub.660 of 1. Freshly isolated Type II immature LH244 and
LH59corn embryos are inoculated with Agrobacterium containing a DNA
construct of the present invention and co-cultured 2-3 days in the
dark at 23 .degree. C. The embryos are then transferred to delay
media (N6 1-100-12/micro/Carb 500/20 p.M AgNO3) and incubated at
28.degree. C. for 4 to 5 days. All subsequent cultures are kept at
this temperature. Coleoptiles are removed one week after
inoculation. The embryos are transferred to the first selection
medium (N61-0-12/Carb 500/0.5 mM glyphosate). Two weeks later,
surviving tissues are transferred to the second selection medium
(N61-0-12/Carb 500/1.0 mM glyphosate). Subculture surviving callus
every 2 weeks until events can be identified. This will take 3
subcultures on 1.0 mM glyphosate. Once events are identified, bulk
up the tissue to regenerate. For regeneration, callus tissues are
transferred to the regeneration medium (MSOD, 0.1 .mu.M ABA) and
incubated for two weeks. The regenerating calli are transferred to
a high sucrose medium and incubated for two weeks. The plantlets
are transferred to MSOD media in culture vessel and kept for two
weeks. Then the plants with roots are transferred into soil.
[0171] Soy Transformation:
[0172] Soybean plants are transformed using an
Agrobacterium-mediated transformation method, as described by
Martinell (U.S. Pat. No. 6,384,301, herein incorporated by
reference). For this method, overnight cultures of Agrobacterium
tumefaciens containing the plasmid that includes a gene of
interest, are grown to log phase and then diluted to a final
optical density at 660 nm (OD.sub.660) of 0.3 to 0.6 using standard
methods known to one skilled in the art. These cultures are used to
inoculate the soybean embryo explants prepared as described
below.
[0173] Commercially available soybean seeds (e.g., Asgrow A3244)
are germinated overnight and the meristematic tissue is excised.
The excised tissue is placed in a wounding vessel and mixed with
theAgrobacterium culture described above. The entire tissue is
wounded using sonication. Following the wounding, explants are
placed in co-culture for 2-5 days, at which point they are
transferred to selection media, i.e., WPM (as described on page 19
of U.S. Pat. No. 6,211,430, incorporated herein by reference) with
75 mM glyphosate (plus antibiotics to control Agrobacterium
overgrowth), for 6-8 weeks to allow selection and growth of
transgenic shoots. Phenotype positive shoots are harvested
approximately 6-8 weeks post transformation and placed into
selective rooting media (BRM, as described in Table 3 of U.S. Pat.
No. 6,384,301) with 25mM glyphosate) for 3-5 weeks. Shoots
producing roots are transferred to the greenhouse and potted in
soil. Shoots that remain healthy on selection, but do not produce
roots are transferred to non-selective rooting media (BRM without
glyphosate) for up to two weeks. Roots from the shoots that
produced roots off selection are tested for expression of the plant
selectable marker before they are transferred to the greenhouse and
potted in soil. Plants are maintained under standard greenhouse
conditions until seed harvest (R1). The collected seeds are
analyzed for protein and oil as described in Example.
[0174] Plant Selection:
[0175] After transformation of crop plants, positive tranformants
can by selected by any one, or a combination of many know
techniques in the art. Plant can be selected based on the
resistance provided by the transforming constructs, which may
include antibiotic resistance, or herbicide resistance. Plants can
also be selected by screening DNA isolated from transformed plant
part with polymerase chain reaction for presence or absence of gene
itself, or part of the transforming constructs. Gene or protein
specific antibodies can also be utilized for selecting transformed
plant expressing desired protein.
Example 17
[0176] This example describes a cold germination assay for
transgenic corn seeds of the present invention.
[0177] Three sets of seeds are used for the experiment. The first
set consists of twelve different positive transgenic events where
the genes of the present invention are expressed in the seed. The
second set consists of negative segregants from the same transgenic
events as the positive seeds. The third seed set consists of two
cold tolerant and two cold sensitive wild-type lines of corn. A
number from one to fourteen is randomly assigned to each of the
twelve transgenic events, the cold tolerant wild-type lines, and
the cold-sensitive wild-type lines. Positive and negative
segregants of the same event are designated as "A" and "B"
randomly. Each member of the cold-tolerant or cold-sensitive pair
is also designated as "A" and "B" randomly. All seeds are treated
with a fungicide " Captan" (Arvesta Corporation, San Francisco,
Calif., USA). 0.43 mL Captan is applied per 45 g of corn seeds by
mixing it well and drying the fungicide prior to the experiment.
Incubations at or below 23 degrees Celsius are conducted in growth
chambers (Conviron Model PGV36, Controlled Environments, Winnipeg,
Canada).
[0178] Ten Petri plates for the cold assay and 5 plates for the
warm assay are used. Petri plates (Cat. #353003) can be procured
from Becton, Dickinson and Company (Franklin
[0179] Lakes, NJ USA, from now on referred to as BD Biosciences).
Each plate is prepared for the experiment by placing a Whatman No.
1 paper on the inner side of the lid (90 mm Catalog #1001090) and
on the bottom of the plate (85 mm Catalog #1001085) manufactured by
Whatman International Ltd. (Maidstone, England) and wetting them
with 2 and 3 ml of sterile water respectively. Ten desired seeds
per plate are placed on the bottom filter paper with the embryo
side touching the paper, each plate is labeled, the lid with the
moist paper is placed on the plate and plates are placed in a
growth chamber set at 9.7.degree. C. (for cold assay) or 25.degree.
C. (for warm assay) in the dark. Ten plates are laid across the
bottom of a plastic box and stacked up to six layers high before
placing them in growth chambers. Seeds are watered with 2 ml of
deionized sterile water on the 3.sup.rd and 10.sup.th days. Warm
control seeds are watered only on the 3.sup.rd day. Seeds are
considered germinated if the emerged radicle size is 1 cm. Warm
control seeds are scored for germination four days after planting
and cold seeds are scored from days 10 to 14, days 17, 19 and 24
after planting. Scoring is conducted until all seeds have
germinated or until the end of 24 days after planting. The order of
plates is reversed (top to bottom, and bottom to top) on every
watering and scoring day. Six radicles per set of plates are
harvested at random on the last day of the experiment for analysis
of
[0180] RNA Expression by Taqman Assay.
[0181] After 24 days of data collection, a germination index is
calculated for each set of seeds. The germination index is
calculated as per:
Germination
index=(.SIGMA.([T+1-n.sub.i]*[P.sub.i-P.sub.i-1]))/T
[0182] Where : T is the total number of days for which the
germination experiment is performed. The number of days after
planting is defined by n. The number of times the germination has
been counted, including the current day, is indicated by i. P is
the percentage of seeds germinated during any given rating.
Statistical differences are calculated between positive and
negative selections within an event. Additionally, the germination
rate is fitted to a model to determine the number of days to 50%
germination and confidence intervals are used to determine the
statistical significance between positive and negative selections
within an event. The Taqman assay confirms the expression of the
RNA of the present invention. Any event which achieved 85% or
better germination in the warm is used for the cold assay;
otherwise it is dropped from the cold assay.
Example 18
[0183] This example describes a cold shock assay for transgenic
corn seeds of the present invention.
[0184] Experimental set-up for the cold shock assay is the same as
described in above example's second paragraph, except seeds are
grown in potted media for the cold shock assay.
[0185] The desired number of 2.5'' square plastic pots are placed
on flats (n=32, 4.times.8). Pots are filled with Metro Mix 200
soilless media containing 19:6:12 fertilizer (6 lbs/cubic yard)
(Metro Mix, Pots and Flat are obtained from Hummert International,
Earth City, Mo.). After planting seeds, pots are placed in a growth
chamber set at 23.degree. C., relative humidity of 65% with 12 hour
day and night photoperiod (300 uE/m2-min). Planted seeds are
watered for 20 minute every other day by sub-irrigation and flats
are rotated every third day in a growth chamber for growing corn
seedlings.
[0186] Chlorophyll fluorescence of plants is measured on the
10.sup.th day during the dark period of growth by using a PAM-2000
portable fluorometer as per the manufacturer's instructions (Walz,
Germany). After chlorophyll measurements, leaf samples from each
event are collected for confirming the expression of genes of the
present invention. For expression analysis six V1 leaf tips from
each selection are randomly harvested. Expression analysis can be
done using a Taqman assay to estimate the RNA expression the 3'
termination sequence or any other part of expression cassette which
will be part of the transgenic plant genome. Plants are then
repositioned in one flat by alternating between the "A" and "B"
selection for a total of sixteen "A" plants and sixteen "B" plants
per flat (A & B are described earlier examples). The flats are
moved to a growth chamber set at 5.degree. C. The actual
temperature at canopy level is 5.degree. C. during the dark cycle
and 8.degree. C. during the light cycle. All other conditions such
as humidity, day/night cycle and light intensity are kept the same
in the growth chamber. The flats are sub-irrigated every day after
transfer to the cold temperature.
[0187] On the 4.sup.th day chlorophyll fluorescence is measured
again. Plants are transferred to normal growth conditions after six
days of cold shock treatment and allowed to recover for the next
two days. During this recovery period the length of the V3 leaf is
measured on the 1.sup.st and 3.sup.rd days. After two days of
recovery V2 leaf damage is visually estimated by estimating percent
of green V2 leaf.
[0188] Statistical differences in V3 leaf growth, V2 necrosis and
fluorescence during pre-shock and cold shock can be used for
estimation of cold shock damage on corn plants.
Example 19
[0189] This example describes the early seedling growth assay for
transgenic corn seeds of the present invention.
[0190] Experimental set-up for the cold shock assay is the same as
described in example 15 second paragraph, except seeds are grown in
germination paper for the early seedling growth assay.
[0191] Three pieces of 12''.times.18'' germination paper (Anchor
Paper #SD7606) are used for each entry in the test, "A" and "B".
For each entry the papers are numbered #1 to #3. A line is drawn
using a wax pencil across the long dimension of the paper at about
four inches from the top edge. Wet the papers in a solution of 0.5%
KNO.sub.3 and 0.1% Thyram. For each paper, eighteen seeds are
placed on the line evenly spaced down the length of the paper. The
eighteen seeds are positioned on the paper such that the radical
will grow downwards, e.g. longer distance to the paper's edge. The
wet paper is rolled up starting from one of the short ends. The
paper is rolled evenly and tight enough to hold the seeds in place.
The roll is secured into place with two large paper clips, one at
the top and one at the bottom. The rolls are placed on end in a
tall bucket containing about one inch of the KNO.sub.3/thyram
solution. The top of the bucket is covered with a plastic bag. The
bag is secured such that the rolls are protected from a direct
breeze or strong flow of air, but not too tight to inhibit free
exchange of oxygen to the rolls.
[0192] The buckets are incubated in the growth chamber at
23.degree. C. for three days. The chamber is set up for 65%
humidity with no light cycle. For the cold stress treatment the
buckets are then incubated in a growth chamber at 12.degree. C. for
fourteen days. The chamber is set up for 65% humidity with no light
cycle. For the warm treatment the buckets are incubated at
23.degree. C. for an additional three days.
[0193] After the appropriate treatment the germination papers are
unrolled and the seeds are repositioned on the wax pencil line, if
necessary. Seeds that did not germinate are discarded. The tip of
the radicle and coleoptile are marked on the germination paper. The
germination papers are allowed to dry and then the lengths of the
radicle and coleoptile for each seed are measured and the data is
recorded. This process can be facilitated using an automated
caliper for electronic data transfer to a PC. A coleoptile sample
is collected from six individual kernels of each entry for
confirming the expression of genes of the present invention.
[0194] Statistical differences in the length of radical and shoot
during pre-shock and cold shock are used for an estimation of the
effect of the cold treatment on corn plants. The analysis is
conducted independently for the warm and cold treatments.
Example 20
[0195] This example describes a wilt assay for transgenic plants of
the present invention. 150 seeds from each event and a control set
are imbibed by soaking in sterile water overnight. Imbibed seeds
are rolled in germination paper. The seeds are placed in 3 rows on
one piece of wet 38 lb 11.5''.times.30''seed paper (Anchor Paper,
St. Paul, Minn.) and overlayed with a second wet piece of seed
paper. The wet papers are then placed on a 12''.times.36'' piece of
wax paper from Anchor Paper, rolled up and fastened with a rubber
band. The roll is placed in a 5 Liter Nalgene Pitcher with
approximately 1 liter of water and allowed to germinate for 46-50
hours in a growth chamber or a greenhouse. The growth chamber is
set with a day/night cycle of 16 hrs/8 hrs and 26.degree. C.
daytime/20.degree. nighttime temperatures. The light intensity of
the growth chamber is kept at 500 uE/m2-min.
[0196] One day before planting, pots are prepared for planting
germinated seed. 5.25'' square pots (Hummert Cat. No 129300) are
filled with dry standard greenhouse media mix (peat moss mix) and
adjusted to 330.+-.5 grams by hand compacting the soil and hand
watered thoroughly. After watering 1 germinated seed/pot is
planted. Seedlings are allowed to grow for 1 week. During this
period pots are watered by a capillary matting watering system. A
capillary mat (Hummerts Cat. No. 18-4046) is placed on top of a
piece of plywood that overlays the greenhouse bench (6 ft..times.12
ft.). Watering is done every three hours, beginning at 7.00 AM,
five times a day for 12-minute interval using seven 2GPH (gallons
per hour) pressure compensating drippers (Hummerts Cat. #18-4046)
per bench. After one week of growth, the V 1 leaf is sampled by
taking a leaf tear of approximately 2 square centimeters.
[0197] This leaf sample from the plant is used to determine the
presence of the selectable marker, CP4. Water is turned off for
several days (usually over the weekend). After 10 days plants of
8-9 cm height are selected based on the presence and absence of the
CP4 gene using standard methods. For each transformation an equal
number (24) of transgenic and wild type plants are selected based
on matched height. These plants are placed alternating a gene
positive plant with a gene negative plant on the capillary mat in a
serpentine fashion and subjected to dry treatment as described.
After arranging plants as per above description, 8 wettest looking
pots are weighed to determine maximum current pot weight. This
"maximum current pot weight" is used to calibrate all other pots by
adding a desired amount of water to bring them all up to the same
weight. 8 random pots are weighed every day to monitor pot weight.
When the average pot weight is between 600 to 700 grams this is
defined as the first day of the experiment. The height of all
plants is taken as the length in cm from the top of the soil to the
tip of the longest leaf on the start day of the experiment.
[0198] After start of the experiment, 8 pots from different flats
are weighed. Plants are allowed to grow without any watering if the
average weight of pots is greater than 500 grams. If the average
weight is less than 500 grams but greater than 365 grams then 35 ml
of water/pot is added. If the average weight is less than 365 grams
then enough water is added to bring pot weight to 400 grams
assuming that each ml of water weighs 1 gram
[0199] The treatment ends when the pots have had an average weight
below 500 g for 7 days. On the 8.sup.th day when the plants weigh
less than 500 grams, all plants are measured for height in cm. The
difference between the height at the end of the dry treatment and
the height at the beginning of the dry treatment is the key
quantitative phenotype of interest for this experiment. After the
first dry treatment all plants are fully watered for three days and
measured again to document drought recovery.
[0200] For the second round of drought and recovery estimation
plants are allowed to dry by turning off the water system for seven
days. After seven days plants will develop severe drought stress
exhibited by 10-25% of the plants where leaves will lean to touch
the top of pots. At this stage all plants are measured and allowed
to recover from stress by fully watering and resuming normal growth
conditions. During the recovery phase all plants are daily
monitored for recovery signs indicated by a flattening of inner
whorl leaves. After 7 days of recovery all plants are measured and
sampled for protein expression analysis prior to harvesting.
Harvested plants are placed in vented cellophane bags and weighed
to determine the fresh weight of the plants. After determining
fresh weight, plants are dried for approximately four weeks in a
seed drier at .about.90.degree. F., 20-40% humidity and weighed to
determine the dry weight of plants.
Sequence CWU 1
1
5111563DNAOryza sativa 1atgctgatgg cgaccgtctc gccggcgcgg agggagccga
cgccgcaggc ggtgcgggcg 60tccccgatgc catcggcggc ggcggcgttg gtgaggagag
gcggtggtgg tagcgggggg 120acggtgctgg ggaagtacga gctggggcgc
gtcctgggac agggctcgtt cgcgaaggtg 180taccaggcga ggcacctgga
gaccgacgag tgcgtggcaa tcaaggtgct cgacaaggag 240aaggccgtga
agggcgggat ggtccacctc gtcaagcgcg agatcaacgt gctccgccgg
300gtgcgccacc cgaacatcgt gcagctgttc gaggtaatgg ccagcaagac
caagatctac 360ttcgtcatgg agtatgtccg cggcggcgag ctcttctccc
gcgtctccaa gggacgcctc 420agggaggaca ccgcgcggcg ctacttccag
cagcttgtct ccgccgtcga cttctgccac 480gcccgcggcg tgttccaccg
tgacctcaag cccgagaacc tcctcgtgga tgagaacggg 540gacttgaagg
tctcggactt cggcctcgcc gccggccccg accagttcga ccccgacggt
600ctgctccaca cgttctgcgg cacgccggct tacgtcgccc ccgaggtgct
caggcgccgc 660ggatacgacg gcgccaaggc ggacatatgg tcatgcggtg
tcatcctctt tgcgctcatg 720gccgggtacc tccctttcca tgaccacaac
atcatggttc tgtaccggaa gatctacaat 780ggggagttca ggtgtccaag
gtggttctcc aaggatttta ctagattgat aacgcgcctt 840cttgacgcaa
accccaaaac taggatcacc gtgccagaga tcattgagag cgattggttc
900aagaaaggat acaagccagt caagttttac attgaggatg acaagctcta
caacctgtct 960gatgacgtgc tgaacttgga gcctgctgat cctgttcccc
caccattggg tttggcacct 1020cctgttcctc cacctccaca aggggatgat
cctgatggtt cagggtctga gtcagattca 1080tcagtcgtat cctgcccggc
cacattgtca actggggaga gccagagagt ccgtgggtca 1140ctaccacgcc
cagcaagcct taatgcattt gatatcatat cattctcaaa aggattcaac
1200ttgtctgggc tgtttgagga gagggggaac gagatcaggt ttgtatctgg
tgagcccatg 1260tctgacattg taaaaaagct ggaggagatt gcaaaggtca
agagcttcac agtgcggagg 1320aaggactggc gggtgagcat agagggtaca
cgcgaaggag ttaaggggcc tctaaccata 1380ggcgcggaga tatttgagct
tacaccctcc cttgtagtag tggaagtaaa aagaaaggca 1440ggtgataatg
aagagtatga ggatttctgc aacatggagt tgaagccagg aatgcagcac
1500cttgtgcacc agatgctccc agctccaaat ggaactcctg tgagtgagaa
ggttgaaagg 1560taa 15632521PRTOryza sativa 2Met Leu Met Ala Thr Val
Ser Pro Ala Arg Arg Glu Pro Thr Pro Gln 1 5 10 15 Ala Val Arg Ala
Ser Pro Met Pro Ser Ala Ala Ala Ala Leu Val Arg 20 25 30 Arg Gly
Gly Gly Gly Ser Gly Gly Thr Val Leu Gly Lys Tyr Glu Leu 35 40 45
Gly Arg Val Leu Gly Gln Gly Ser Phe Ala Lys Val Tyr Gln Ala Arg 50
55 60 His Leu Glu Thr Asp Glu Cys Val Ala Ile Lys Val Leu Asp Lys
Glu 65 70 75 80 Lys Ala Val Lys Gly Gly Met Val His Leu Val Lys Arg
Glu Ile Asn 85 90 95 Val Leu Arg Arg Val Arg His Pro Asn Ile Val
Gln Leu Phe Glu Val 100 105 110 Met Ala Ser Lys Thr Lys Ile Tyr Phe
Val Met Glu Tyr Val Arg Gly 115 120 125 Gly Glu Leu Phe Ser Arg Val
Ser Lys Gly Arg Leu Arg Glu Asp Thr 130 135 140 Ala Arg Arg Tyr Phe
Gln Gln Leu Val Ser Ala Val Asp Phe Cys His 145 150 155 160 Ala Arg
Gly Val Phe His Arg Asp Leu Lys Pro Glu Asn Leu Leu Val 165 170 175
Asp Glu Asn Gly Asp Leu Lys Val Ser Asp Phe Gly Leu Ala Ala Gly 180
185 190 Pro Asp Gln Phe Asp Pro Asp Gly Leu Leu His Thr Phe Cys Gly
Thr 195 200 205 Pro Ala Tyr Val Ala Pro Glu Val Leu Arg Arg Arg Gly
Tyr Asp Gly 210 215 220 Ala Lys Ala Asp Ile Trp Ser Cys Gly Val Ile
Leu Phe Ala Leu Met 225 230 235 240 Ala Gly Tyr Leu Pro Phe His Asp
His Asn Ile Met Val Leu Tyr Arg 245 250 255 Lys Ile Tyr Asn Gly Glu
Phe Arg Cys Pro Arg Trp Phe Ser Lys Asp 260 265 270 Phe Thr Arg Leu
Ile Thr Arg Leu Leu Asp Ala Asn Pro Lys Thr Arg 275 280 285 Ile Thr
Val Pro Glu Ile Ile Glu Ser Asp Trp Phe Lys Lys Gly Tyr 290 295 300
Lys Pro Val Lys Phe Tyr Ile Glu Asp Asp Lys Leu Tyr Asn Leu Ser 305
310 315 320 Asp Asp Val Leu Asn Leu Glu Pro Ala Asp Pro Val Pro Pro
Pro Leu 325 330 335 Gly Leu Ala Pro Pro Val Pro Pro Pro Pro Gln Gly
Asp Asp Pro Asp 340 345 350 Gly Ser Gly Ser Glu Ser Asp Ser Ser Val
Val Ser Cys Pro Ala Thr 355 360 365 Leu Ser Thr Gly Glu Ser Gln Arg
Val Arg Gly Ser Leu Pro Arg Pro 370 375 380 Ala Ser Leu Asn Ala Phe
Asp Ile Ile Ser Phe Ser Lys Gly Phe Asn 385 390 395 400 Leu Ser Gly
Leu Phe Glu Glu Arg Gly Asn Glu Ile Arg Phe Val Ser 405 410 415 Gly
Glu Pro Met Ser Asp Ile Val Lys Lys Leu Glu Glu Ile Ala Lys 420 425
430 Val Lys Ser Phe Thr Val Arg Arg Lys Asp Trp Arg Val Ser Ile Glu
435 440 445 Gly Thr Arg Glu Gly Val Lys Gly Pro Leu Thr Ile Gly Ala
Glu Ile 450 455 460 Phe Glu Leu Thr Pro Ser Leu Val Val Val Glu Val
Lys Arg Lys Ala 465 470 475 480 Gly Asp Asn Glu Glu Tyr Glu Asp Phe
Cys Asn Met Glu Leu Lys Pro 485 490 495 Gly Met Gln His Leu Val His
Gln Met Leu Pro Ala Pro Asn Gly Thr 500 505 510 Pro Val Ser Glu Lys
Val Glu Arg Ala 515 520 32524DNAOryza sativa 3taattaatct gtcattagta
aatgtttact ataacactac gctatcaaat catggtgcaa 60ttagccttaa aagatttgtc
ttgcaattta catgtaatcc gtgtaattgt tttttcctac 120aatactccat
gtatatatta aacattcgat gtgacagcat ggaaattttt gttttaggaa
180ctaaataggg ccaaaataaa agttcacacc aaaattgaaa atttgattga
aattggaatg 240atgtgatgaa aaatttgaaa gtttgtgtgt gtagaaaagt
tttaatgtga tggaaaagtt 300ggaagtttga aagaaaaact ttggggagaa
aacatttcaa agcgaaagcg aaatgaaact 360ctctagagaa gagaagcccc
agccgcagat attattcacg atccgttaag ctgttccccc 420tcccttccaa
cgccggccac tcgtctcctc ctcctcccac ctcccgtttc ccccgcgcca
480tcctcctccg cctcgccgcc atggccgcga ccccgccgtc gtcgcagcac
cggcggccgc 540tgtcctcctc cgcctccgcc gcctccctcg ctggcaagcc
gcgggggggc gggctcctgc 600tcgggcggta cgagctcggc cgcctcctcg
gccacggcac cttcgccaag gtgtaccagg 660cgcggagcgc ggattccggg
gagccggtcg cgatcaaggt gctcgacaag gagaaggcga 720tgcggcacgg
cctcgtcccg cacatcaagc gggagatcgc catcctccgc cgcgtccgcc
780accccaacat cgtgaggctg ttcgaggtga tggccaccaa gtccaagatc
tacttcgtga 840tggagctcgt ccgcggcggg gagctgttcg gccgcgtcgc
caaggggcgg ctcaaggagg 900acaccgcgcg gcgctacttc cagcagctcg
tctccgccgt cgggttctgc cacgcgcgcg 960gcgtgttcca ccgcgacctc
aagcccgaga acctcctcgt cgacgagcac ggcgacctca 1020aggtctccga
cttcggcctc tccgccgtcg ccgaccagtt ccaccccgac ggcctcctcc
1080acaccttctg cggcacgccc tcctacgtcg cgcccgaggt gctcgcgcgc
cgcggctacg 1140acggcgccaa ggcggacata tggtcctgcg gcatcatcct
cttcgtgctc atggctggct 1200accttccgtt ccatgaccag aatctcatgg
ccatgtaccg aaagatttac aggggggaat 1260tccggtgccc gagatggttc
tccaaggatc tttccagtct actgaatcgc atccttgaca 1320cgaacccaga
gacaaggatc actgtcaaag aggtcatgga gagcaggtgg ttccagaagg
1380gattccggcc ggtcagattc tatgttgagg atgatcaggt tcacagcttg
gcagatggtg 1440ataatgatat gccggagttg gaacctagtg agcctcctcc
tcctcctccg tttccgccgc 1500cgccgccgca gcaagatgat gacggtgagg
agtcgggatg ggagtcggac tcatccgtgg 1560catcatgtcc tgccacattg
tcatctgagg agcgtcggca aagacctctc gggtctctca 1620cacggccagc
aagtcttaat gcgttcgata tcatatcgtt ctcaaaggga tttgatttgt
1680cggggttgtt tgaggagcga gggagtgaag tgaggttcat atcggcagag
cctatgcaaa 1740caatcatcac aaaattggag gagatcgcaa aggtgaagag
cttcttcgtt cggcgaaaag 1800actggcgagt gagcatagaa ggcacgaggg
aaggtttgaa gggtccattg acaatcggcg 1860ctgagatatt tgagctcaca
ccaagcctgg tggtagtgga ggtgaagaag aaggcagggg 1920ataaggaaga
atatgatgac ttctgtaaca gggagttgaa acctgggatg cagcatctcg
1980tacaccatat gggatcagtt ccaaatatac cttctgatac ggagtagttt
gaactaagaa 2040aggtagttct ctttcttgga ggggtataag gaaattttgg
attaaaagta tatgtctatg 2100caagcatgaa cacctgagag gcaaaatgat
acccaattcc tttagaccag tgtccatgtt 2160ttggtgctgt tcgtttcttc
aatcgaaatg atgtatgcta gtgtttgcat gctaatatca 2220gctatcaaat
gtctgttttt agctgttaca gtttaaagag agtgacaaat ctgagtatat
2280ggcatcagta tcaatgaagt ggactagact tttatgtatg ccgcagcagt
gcagccattt 2340gtatttctat gctgccagtt agttctctga atacatatga
catcaacact gaagaaatta 2400gctcgaagtg ctctaaagaa gttctgtttt
gggattaaaa ttgtaaatat agggtgaatg 2460aataaattag acaaagcgtt
agcattctaa gtatctagtt gtttattact tctgtgcatc 2520aatt
25244508PRTOryza sativa 4Met Ala Ala Thr Pro Pro Ser Ser Gln His
Arg Arg Pro Leu Ser Ser 1 5 10 15 Ser Ala Ser Ala Ala Ser Leu Ala
Gly Lys Pro Arg Gly Gly Gly Leu 20 25 30 Leu Leu Gly Arg Tyr Glu
Leu Gly Arg Leu Leu Gly His Gly Thr Phe 35 40 45 Ala Lys Val Tyr
Gln Ala Arg Ser Ala Asp Ser Gly Glu Pro Val Ala 50 55 60 Ile Lys
Val Leu Asp Lys Glu Lys Ala Met Arg His Gly Leu Val Pro 65 70 75 80
His Ile Lys Arg Glu Ile Ala Ile Leu Arg Arg Val Arg His Pro Asn 85
90 95 Ile Val Arg Leu Phe Glu Val Met Ala Thr Lys Ser Lys Ile Tyr
Phe 100 105 110 Val Met Glu Leu Val Arg Gly Gly Glu Leu Phe Gly Arg
Val Ala Lys 115 120 125 Gly Arg Leu Lys Glu Asp Thr Ala Arg Arg Tyr
Phe Gln Gln Leu Val 130 135 140 Ser Ala Val Gly Phe Cys His Ala Arg
Gly Val Phe His Arg Asp Leu 145 150 155 160 Lys Pro Glu Asn Leu Leu
Val Asp Glu His Gly Asp Leu Lys Val Ser 165 170 175 Asp Phe Gly Leu
Ser Ala Val Ala Asp Gln Phe His Pro Asp Gly Leu 180 185 190 Leu His
Thr Phe Cys Gly Thr Pro Ser Tyr Val Ala Pro Glu Val Leu 195 200 205
Ala Arg Arg Gly Tyr Asp Gly Ala Lys Ala Asp Ile Trp Ser Cys Gly 210
215 220 Ile Ile Leu Phe Val Leu Met Ala Gly Tyr Leu Pro Phe His Asp
Gln 225 230 235 240 Asn Leu Met Ala Met Tyr Arg Lys Ile Tyr Arg Gly
Glu Phe Arg Cys 245 250 255 Pro Arg Trp Phe Ser Lys Asp Leu Ser Ser
Leu Leu Asn Arg Ile Leu 260 265 270 Asp Thr Asn Pro Glu Thr Arg Ile
Thr Val Lys Glu Val Met Glu Ser 275 280 285 Arg Trp Phe Gln Lys Gly
Phe Arg Pro Val Arg Phe Tyr Val Glu Asp 290 295 300 Asp Gln Val His
Ser Leu Ala Asp Gly Asp Asn Asp Met Pro Glu Leu 305 310 315 320 Glu
Pro Ser Glu Pro Pro Pro Pro Pro Pro Phe Pro Pro Pro Pro Pro 325 330
335 Gln Gln Asp Asp Asp Gly Glu Glu Ser Gly Trp Glu Ser Asp Ser Ser
340 345 350 Val Ala Ser Cys Pro Ala Thr Leu Ser Ser Glu Glu Arg Arg
Gln Arg 355 360 365 Pro Leu Gly Ser Leu Thr Arg Pro Ala Ser Leu Asn
Ala Phe Asp Ile 370 375 380 Ile Ser Phe Ser Lys Gly Phe Asp Leu Ser
Gly Leu Phe Glu Glu Arg 385 390 395 400 Gly Ser Glu Val Arg Phe Ile
Ser Ala Glu Pro Met Gln Thr Ile Ile 405 410 415 Thr Lys Leu Glu Glu
Ile Ala Lys Val Lys Ser Phe Phe Val Arg Arg 420 425 430 Lys Asp Trp
Arg Val Ser Ile Glu Gly Thr Arg Glu Gly Leu Lys Gly 435 440 445 Pro
Leu Thr Ile Gly Ala Glu Ile Phe Glu Leu Thr Pro Ser Leu Val 450 455
460 Val Val Glu Val Lys Lys Lys Ala Gly Asp Lys Glu Glu Tyr Asp Asp
465 470 475 480 Phe Cys Asn Arg Glu Leu Lys Pro Gly Met Gln His Leu
Val His His 485 490 495 Met Gly Ser Val Pro Asn Ile Pro Ser Asp Thr
Glu 500 505 52530DNAOryza sativa 5atccatcatt agcaaatgtt tactataaca
ccacgctatt aaatcatggt gcaattagcc 60ttaaaagatt cgtcttgcaa ttgcaattta
catgtaatct gtgtaattgt tttttcctac 120aatactgcat gtatatatta
aacattcgat gtgacagcat gaaaattttt gttttaggaa 180ctaaacaggg
ccaaaataaa agttcacacc aaaattgaaa atttgattga aattgaaatg
240atgtgatgaa aaatttaaaa gttcgtgtgt gtaggaaagt tttaatgtga
tgaaaaagtt 300ggaagtttga aagaaaaact ttggggagaa aacatttcaa
agcgaaagcg aaatgaaact 360ctctagagaa gagaagcccc agccgcagat
attattcacg atccgttaag ctgttccccc 420tcccttccaa cgccggccac
tcgtctcctc ctcctcccac ctcccgtttc ctccgcgcca 480tcctcctccg
cctcggcgcc atggccgcga ccccgccgtc gtcgcgggac ccgtcgccgc
540agcaccggcg gccgctgtcc tcctccgcct ccctcgctgg caagccgcgg
gggggcgggc 600tcctgctcgg gcggtacgag ctcggccgcc tcctcggcca
cggcaccttc gccaaggtgt 660accaggcgcg gagcgcggat tccggggagc
cggtcgcgat caaggtgctc gacaaggaga 720aggcgatgcg gcacggcctc
gtcccgcaca tcaagcggga gatcgccatc ctccgccgcg 780tccgccaccc
caacatcgtg aggctgttcg aggtgatggc caccaagtcc aagatctact
840tcgtgatgga gctcgtccgc ggcggggagc tgttcggccg cgtcgccaag
gggcggctca 900aggaggacac cgcgcggcgc tacttccagc agctcgtctc
cgccgtcggg ttctgccacg 960cgcgcggcgt gttccaccgc gacctcaagc
ccgagaacct cctcgtcgac gagcacggcg 1020acctcaaggt ctccgacttc
ggcctctccg ccgtcgccga ccagttccac cccgacggcc 1080tcctccacac
cttctgcggc acgccctcct acgtcgcgcc cgaggtgctc gcgcgccgcg
1140gctacgacgg cgccaaggcg gacatatggt cctgcggcat catcctcttc
gtgctcatgg 1200ctggctacct tccgttccat gaccagaatc tcatggccat
gtaccgaaag atttacaggg 1260gggaattccg gtgcccgaga tggttctcca
aggatctttc cagtctactg aatcgcatcc 1320ttgacacgaa cccagagaca
aggatcactg tcaaagaggt catggagagc aggtggttcc 1380agaagggatt
ccggccggtc agattctatg ttgaggatga tcaggttcac agcttggcag
1440atggtgataa tgatatgccg gagttggaac ctagtgagcc tcctcctcct
cctccgtttc 1500cgccgccgcc gccgcagcaa gatgatgacg gtgaggagtc
gggatgggag tcggactcat 1560ccgtggcatc atgtcctgcc acattgtcat
ctgaggagcg tcggcaaaga cctctcgggt 1620ctctcacacg gccagcaagt
cttaatgcgt tcgatatcat atcgttctca aagggatttg 1680atttgtcggg
gttgtttgag gagcgaggga gtgaagtgag gttcatatcg gcagagccta
1740tgcaaacaat catcacaaaa ttggaggaga tcgcaaaggt gaagagcttc
ttcgttcggc 1800gaaaagactg gcgagtgagc atagaaggca cgagggaagg
tttgaagggt ccattgacaa 1860tcagcgctga gatatttgag ctcacaccaa
gcctggtggt agtggaggtg aagaagaagg 1920caggggataa ggaagaatat
gatgacttct gtaacaggga gttgaaacct gggatgcagc 1980atctcgtaca
ccatatggga tcagttccaa atataccttc tgatacggag tagtttgaac
2040taagaaaggt agttctcttt cttggagggg tataaggaaa ttttggatta
aaagtatatg 2100tctatgcaag catgaacacc tgagaggcaa aatgataccc
aattccttta gaccagtgtc 2160catgttttgg tgctgttcgt ttcttcaatc
gaaatgatgt atgctagtgt ttgcatgcta 2220atatcagcta tcaaatgtct
gtttttagct gttacagttt aaagagagtg acaaatctga 2280gtatatggca
tcagtatcaa tgaagtggac tagactttta tgtatgccgc agcagtgcag
2340ccatttgtat ttctatgctg ccagttagtt ctctgaatac atatgacatc
aacactgaag 2400aaattagctc gaagtgctct aaagaagttc tgttttggga
ttaaaattgt aaatataggg 2460tgaatgaata aattagacaa agcgttagca
ttctaagtat ctagttgttt attacttctg 2520tgcatcaatt 25306510PRTOryza
sativa 6Met Ala Ala Thr Pro Pro Ser Ser Arg Asp Pro Ser Pro Gln His
Arg 1 5 10 15 Arg Pro Leu Ser Ser Ser Ala Ser Leu Ala Gly Lys Pro
Arg Gly Gly 20 25 30 Gly Leu Leu Leu Gly Arg Tyr Glu Leu Gly Arg
Leu Leu Gly His Gly 35 40 45 Thr Phe Ala Lys Val Tyr Gln Ala Arg
Ser Ala Asp Ser Gly Glu Pro 50 55 60 Val Ala Ile Lys Val Leu Asp
Lys Glu Lys Ala Met Arg His Gly Leu 65 70 75 80 Val Pro His Ile Lys
Arg Glu Ile Ala Ile Leu Arg Arg Val Arg His 85 90 95 Pro Asn Ile
Val Arg Leu Phe Glu Val Met Ala Thr Lys Ser Lys Ile 100 105 110 Tyr
Phe Val Met Glu Leu Val Arg Gly Gly Glu Leu Phe Gly Arg Val 115 120
125 Ala Lys Gly Arg Leu Lys Glu Asp Thr Ala Arg Arg Tyr Phe Gln Gln
130 135 140 Leu Val Ser Ala Val Gly Phe Cys His Ala Arg Gly Val Phe
His Arg 145 150 155 160 Asp Leu Lys Pro Glu Asn Leu Leu Val Asp Glu
His Gly Asp Leu Lys 165 170 175 Val Ser Asp Phe Gly Leu Ser Ala Val
Ala Asp Gln Phe His Pro Asp 180 185 190 Gly Leu Leu His Thr Phe Cys
Gly Thr Pro Ser Tyr Val Ala Pro Glu 195 200 205 Val Leu Ala Arg Arg
Gly Tyr Asp Gly Ala Lys Ala Asp Ile Trp Ser 210 215 220
Cys Gly Ile Ile Leu Phe Val Leu Met Ala Gly Tyr Leu Pro Phe His 225
230 235 240 Asp Gln Asn Leu Met Ala Met Tyr Arg Lys Ile Tyr Arg Gly
Glu Phe 245 250 255 Arg Cys Pro Arg Trp Phe Ser Lys Asp Leu Ser Ser
Leu Leu Asn Arg 260 265 270 Ile Leu Asp Thr Asn Pro Glu Thr Arg Ile
Thr Val Lys Glu Val Met 275 280 285 Glu Ser Arg Trp Phe Gln Lys Gly
Phe Arg Pro Val Arg Phe Tyr Val 290 295 300 Glu Asp Asp Gln Val His
Ser Leu Ala Asp Gly Asp Asn Asp Met Pro 305 310 315 320 Glu Leu Glu
Pro Ser Glu Pro Pro Pro Pro Pro Pro Phe Pro Pro Pro 325 330 335 Pro
Pro Gln Gln Asp Asp Asp Gly Glu Glu Ser Gly Trp Glu Ser Asp 340 345
350 Ser Ser Val Ala Ser Cys Pro Ala Thr Leu Ser Ser Glu Glu Arg Arg
355 360 365 Gln Arg Pro Leu Gly Ser Leu Thr Arg Pro Ala Ser Leu Asn
Ala Phe 370 375 380 Asp Ile Ile Ser Phe Ser Lys Gly Phe Asp Leu Ser
Gly Leu Phe Glu 385 390 395 400 Glu Arg Gly Ser Glu Val Arg Phe Ile
Ser Ala Glu Pro Met Gln Thr 405 410 415 Ile Ile Thr Lys Leu Glu Glu
Ile Ala Lys Val Lys Ser Phe Phe Val 420 425 430 Arg Arg Lys Asp Trp
Arg Val Ser Ile Glu Gly Thr Arg Glu Gly Leu 435 440 445 Lys Gly Pro
Leu Thr Ile Ser Ala Glu Ile Phe Glu Leu Thr Pro Ser 450 455 460 Leu
Val Val Val Glu Val Lys Lys Lys Ala Gly Asp Lys Glu Glu Tyr 465 470
475 480 Asp Asp Phe Cys Asn Arg Glu Leu Lys Pro Gly Met Gln His Leu
Val 485 490 495 His His Met Gly Ser Val Pro Asn Ile Pro Ser Asp Thr
Glu 500 505 510 71700DNAZea mays 7taccctcgag gccggcctcc tccgcggggc
ccgccaagcg cgtgggcctt ctgctcggcc 60gctacgagct gggccgcctg ctcggccacg
gcaccttcgc caaggtctac cacgcccgcc 120aagccgacac cggcgagacc
gtcgccatca aggtgctcga caaggagaag gccctccgca 180acggcctcgt
cccgcacatc aagcgcgaga tcgccatcct ccgccgcgtg cgccacccca
240atatcgtccg cctcttcgag gtcatggcca ccaagtccaa gatctacttc
gtcatggagt 300tcgtccgcgg cggggagctc ttcgcgcgcg tcgccaaggg
ccgcctcaag gaggataccg 360cgcgaaggta cttccagcag cttatctccg
ccgtcggctt ctgccacgcc cggggcgtct 420tccaccgcga cctcaagccc
gagaatctgc tcgtcgacga gcgcggcgac ctcaaggtct 480ccgattttgg
cctctcggcg gtggccgatc agttccaccc cgacggcctc ctccacacct
540tctgtggcac gccctcctac gtcgccccgg aggtgctcgc gcgccgcggt
tatgacggcg 600ccaaggcgga catatggtcg tgtggtgtca tcctgttcgt
gctgatggct ggctaccttc 660cttttcatga ccagaacctc atggcgatgt
accgtaagat ctacaggggg gaattccggt 720gtccgaggtg gttttccaag
gatcttagca gtctattgat tcgacttctt gacacgaacc 780cagagaccag
gatcaccgtg gctcagataa tggagagcag gtggtttaag aaagggttcc
840gaccggtcag attctacgtc gaggatgacc aagtgcacag cttagcagac
ggtgaggatg 900aggtgccgga actggggcct agtgagcctc caactccacc
tcccccgcca ccaccgcaga 960aagaggacga cggtgatgat tctggttggg
aatcagactc gtctgtagca tcctgcccag 1020ccacattgtc atcagaggag
aggagacggc ctgctggatc gctcccacgg ccagtaagtc 1080taaatgcatt
tgatatcata tcattctcaa ggggattcaa tctgtcgggg ttgtttgagg
1140agcgaggcaa tgaagtgaga tttgtctcag cacatcccat gcaaacgatt
ataacaaaat 1200tgggggagat cgcgaaggtg aagagctttg cagttcggcg
gaaggactgg cgggttagct 1260tggaaggcac gagagaaagt gaaaagggtc
cattgacaat cggggctgaa gtatttgagc 1320tcacaccaag ccttgtggtc
gtggaggtga ggatgaaggc aggggacagg caagaatatg 1380aggatttttg
tgagagggag ttgaagcctg ggatgcagca cctggtgcac catacaacct
1440cggttccaga tataccttct gatactgatt agcttaaaag gtagtgtgct
cttgattgga 1500atgattgtgg tgaagaaatt tggattgaaa ggatgcacct
ttctgtttca gcgtaagcat 1560ctgtgcagga aaatgttatt catagatttc
cgtagttttt ttttgttaat attctttctg 1620caatccaaaa tgttttgcga
tagtagtttt gtgctaatac caatttacaa aaaaaaaaaa 1680aaaaaagcgg
ccctcgagct 17008489PRTZea mays 8Pro Ser Arg Pro Ala Ser Ser Ala Gly
Pro Ala Lys Arg Val Gly Leu 1 5 10 15 Leu Leu Gly Arg Tyr Glu Leu
Gly Arg Leu Leu Gly His Gly Thr Phe 20 25 30 Ala Lys Val Tyr His
Ala Arg Gln Ala Asp Thr Gly Glu Thr Val Ala 35 40 45 Ile Lys Val
Leu Asp Lys Glu Lys Ala Leu Arg Asn Gly Leu Val Pro 50 55 60 His
Ile Lys Arg Glu Ile Ala Ile Leu Arg Arg Val Arg His Pro Asn 65 70
75 80 Ile Val Arg Leu Phe Glu Val Met Ala Thr Lys Ser Lys Ile Tyr
Phe 85 90 95 Val Met Glu Phe Val Arg Gly Gly Glu Leu Phe Ala Arg
Val Ala Lys 100 105 110 Gly Arg Leu Lys Glu Asp Thr Ala Arg Arg Tyr
Phe Gln Gln Leu Ile 115 120 125 Ser Ala Val Gly Phe Cys His Ala Arg
Gly Val Phe His Arg Asp Leu 130 135 140 Lys Pro Glu Asn Leu Leu Val
Asp Glu Arg Gly Asp Leu Lys Val Ser 145 150 155 160 Asp Phe Gly Leu
Ser Ala Val Ala Asp Gln Phe His Pro Asp Gly Leu 165 170 175 Leu His
Thr Phe Cys Gly Thr Pro Ser Tyr Val Ala Pro Glu Val Leu 180 185 190
Ala Arg Arg Gly Tyr Asp Gly Ala Lys Ala Asp Ile Trp Ser Cys Gly 195
200 205 Val Ile Leu Phe Val Leu Met Ala Gly Tyr Leu Pro Phe His Asp
Gln 210 215 220 Asn Leu Met Ala Met Tyr Arg Lys Ile Tyr Arg Gly Glu
Phe Arg Cys 225 230 235 240 Pro Arg Trp Phe Ser Lys Asp Leu Ser Ser
Leu Leu Ile Arg Leu Leu 245 250 255 Asp Thr Asn Pro Glu Thr Arg Ile
Thr Val Ala Gln Ile Met Glu Ser 260 265 270 Arg Trp Phe Lys Lys Gly
Phe Arg Pro Val Arg Phe Tyr Val Glu Asp 275 280 285 Asp Gln Val His
Ser Leu Ala Asp Gly Glu Asp Glu Val Pro Glu Leu 290 295 300 Gly Pro
Ser Glu Pro Pro Thr Pro Pro Pro Pro Pro Pro Pro Gln Lys 305 310 315
320 Glu Asp Asp Gly Asp Asp Ser Gly Trp Glu Ser Asp Ser Ser Val Ala
325 330 335 Ser Cys Pro Ala Thr Leu Ser Ser Glu Glu Arg Arg Arg Pro
Ala Gly 340 345 350 Ser Leu Pro Arg Pro Val Ser Leu Asn Ala Phe Asp
Ile Ile Ser Phe 355 360 365 Ser Arg Gly Phe Asn Leu Ser Gly Leu Phe
Glu Glu Arg Gly Asn Glu 370 375 380 Val Arg Phe Val Ser Ala His Pro
Met Gln Thr Ile Ile Thr Lys Leu 385 390 395 400 Gly Glu Ile Ala Lys
Val Lys Ser Phe Ala Val Arg Arg Lys Asp Trp 405 410 415 Arg Val Ser
Leu Glu Gly Thr Arg Glu Ser Glu Lys Gly Pro Leu Thr 420 425 430 Ile
Gly Ala Glu Val Phe Glu Leu Thr Pro Ser Leu Val Val Val Glu 435 440
445 Val Arg Met Lys Ala Gly Asp Arg Gln Glu Tyr Glu Asp Phe Cys Glu
450 455 460 Arg Glu Leu Lys Pro Gly Met Gln His Leu Val His His Thr
Thr Ser 465 470 475 480 Val Pro Asp Ile Pro Ser Asp Thr Asp 485
91993DNAZea mays 9ccacgcgtcc gaagctgcct gcttccgctg ccggccgtgc
tacctaatcg ccgcgcttgt 60tttcccaccg cccgatggcc gccatcacgc cgccgacgca
gtcggagccg tcgccgcaga 120cggggcgccc ggcctcgtct gccgccgccg
cggccaagcg tggagggggc ggggctggtg 180ccgccggcgg gccgctgatg
gggaagtacg agctggggcg cctcctgggg cacggcacct 240tcgcgaaggt
gtaccacgcg cggcacgtcg acacggggga caacgttgcc atcaaggtgc
300tcgacaagga gaaggccgtg aagagcgggc tcgtcccgca catcaagcgc
gagatcgctg 360tgctacgccg cgtgcgccac ccgaacatcg tgcacctgtt
cgaggttatg gccacgaaga 420ctaagatcta cttcgtcatg gagctcgtcc
gcggcggcga gctcttctcc cgcgtctcca 480agggccgact cagggaggac
accgcgcgcc gctacttcca gcagctcgtc tccgccgtgg 540ggttctgcca
cgcccgcggc gtcttccacc gcgacctgaa gcccgagaat ctactcgtcg
600acgagcaggg gaacctcaag gtatcggatt ttgggctctc cgccgtcgcc
gagcagttcc 660gtcccgacgg cctgctccac accttctgcg gcacgccggc
ctatgtggcc cccgaagtgc 720tcggccgccg cgggtacgac ggcgccaagg
cagatgtgtg gtcgtgcggt gtcatcctct 780ttgtgctcat ggccggatat
ctccctttcc atgacaaaaa catcatggcc atgtacaaga 840agatttacaa
gggcgagttc cgctgtgcga ggtggttctc caaagacctt accagcttgc
900tgatgcgcat tcttcacact aatcccaaca ctcggatcac tttgccggag
atcatggagt 960cccgctggtt caagaaagga ttcaagcctg tcaagttcta
tatcgaggat gaccagctgc 1020ataacgttat agatgacgaa gatggcctgt
tagatatggg acctgctggt cctgttcctc 1080caccattgcc acctccaccg
ccacctctac ctccaccaaa ggttgatggt gatgaatcag 1140ggtcggactc
agactcgtcg atctcatcct gccctgcttc aatgttatct gatgagagcc
1200aaaggccccg tggctctcta ccacgtccag caagtcttaa tgcctttgat
atcatatcat 1260tttcaagggg atttaactta tcagggttat ttgaggagaa
aggggatgaa gtgaggttca 1320tctcggctga gcccatgtca gatatcataa
ccaaattgga ggacatagcg aagctgaaga 1380gcttcaagtt gcggaggaag
gactggcgca tctgcctgga gggtacaagg gaaggagtta 1440aggggccatt
aacaattggc gcggagatat ttgaactcac acctcccctt gtaatggtgg
1500aggtaaaaaa gaaggcaggg gataatgaag agtacgagaa cttctgtgac
aaggaattga 1560agccagggat gcagcacctt gtccaccata tggtccgagc
tccaagtatg ctgcttactg 1620atgccaagta gatcgaaagg ctttgaactt
aacaacagca cttcgcacgg agctactggt 1680aacaggcgtg acattcagag
cggcatgagg ctagaggaga cagttgagca cagcacagtt 1740gaccagaaga
gatagtcgtc ggaacaaaaa ccttgaccag ttccacagcg ctgtagtttc
1800gcagatgatg agcagctcgg catctcatga ctgaataaac gcaatgcccg
ccatggaggg 1860agactccggt gtctttcttg tacctgagat ggttaagttg
ttactcgaat gctgtatcac 1920gagtggtgta gtcctgctat tcgtaatatt
tcgattaacc atcaaaaaaa aaaaaaaaaa 1980aaagggcggc cgc 199310518PRTZea
mays 10Met Ala Ala Ile Thr Pro Pro Thr Gln Ser Glu Pro Ser Pro Gln
Thr 1 5 10 15 Gly Arg Pro Ala Ser Ser Ala Ala Ala Ala Ala Lys Arg
Gly Gly Gly 20 25 30 Gly Ala Gly Ala Ala Gly Gly Pro Leu Met Gly
Lys Tyr Glu Leu Gly 35 40 45 Arg Leu Leu Gly His Gly Thr Phe Ala
Lys Val Tyr His Ala Arg His 50 55 60 Val Asp Thr Gly Asp Asn Val
Ala Ile Lys Val Leu Asp Lys Glu Lys 65 70 75 80 Ala Val Lys Ser Gly
Leu Val Pro His Ile Lys Arg Glu Ile Ala Val 85 90 95 Leu Arg Arg
Val Arg His Pro Asn Ile Val His Leu Phe Glu Val Met 100 105 110 Ala
Thr Lys Thr Lys Ile Tyr Phe Val Met Glu Leu Val Arg Gly Gly 115 120
125 Glu Leu Phe Ser Arg Val Ser Lys Gly Arg Leu Arg Glu Asp Thr Ala
130 135 140 Arg Arg Tyr Phe Gln Gln Leu Val Ser Ala Val Gly Phe Cys
His Ala 145 150 155 160 Arg Gly Val Phe His Arg Asp Leu Lys Pro Glu
Asn Leu Leu Val Asp 165 170 175 Glu Gln Gly Asn Leu Lys Val Ser Asp
Phe Gly Leu Ser Ala Val Ala 180 185 190 Glu Gln Phe Arg Pro Asp Gly
Leu Leu His Thr Phe Cys Gly Thr Pro 195 200 205 Ala Tyr Val Ala Pro
Glu Val Leu Gly Arg Arg Gly Tyr Asp Gly Ala 210 215 220 Lys Ala Asp
Val Trp Ser Cys Gly Val Ile Leu Phe Val Leu Met Ala 225 230 235 240
Gly Tyr Leu Pro Phe His Asp Lys Asn Ile Met Ala Met Tyr Lys Lys 245
250 255 Ile Tyr Lys Gly Glu Phe Arg Cys Ala Arg Trp Phe Ser Lys Asp
Leu 260 265 270 Thr Ser Leu Leu Met Arg Ile Leu His Thr Asn Pro Asn
Thr Arg Ile 275 280 285 Thr Leu Pro Glu Ile Met Glu Ser Arg Trp Phe
Lys Lys Gly Phe Lys 290 295 300 Pro Val Lys Phe Tyr Ile Glu Asp Asp
Gln Leu His Asn Val Ile Asp 305 310 315 320 Asp Glu Asp Gly Leu Leu
Asp Met Gly Pro Ala Gly Pro Val Pro Pro 325 330 335 Pro Leu Pro Pro
Pro Pro Pro Pro Leu Pro Pro Pro Lys Val Asp Gly 340 345 350 Asp Glu
Ser Gly Ser Asp Ser Asp Ser Ser Ile Ser Ser Cys Pro Ala 355 360 365
Ser Met Leu Ser Asp Glu Ser Gln Arg Pro Arg Gly Ser Leu Pro Arg 370
375 380 Pro Ala Ser Leu Asn Ala Phe Asp Ile Ile Ser Phe Ser Arg Gly
Phe 385 390 395 400 Asn Leu Ser Gly Leu Phe Glu Glu Lys Gly Asp Glu
Val Arg Phe Ile 405 410 415 Ser Ala Glu Pro Met Ser Asp Ile Ile Thr
Lys Leu Glu Asp Ile Ala 420 425 430 Lys Leu Lys Ser Phe Lys Leu Arg
Arg Lys Asp Trp Arg Ile Cys Leu 435 440 445 Glu Gly Thr Arg Glu Gly
Val Lys Gly Pro Leu Thr Ile Gly Ala Glu 450 455 460 Ile Phe Glu Leu
Thr Pro Pro Leu Val Met Val Glu Val Lys Lys Lys 465 470 475 480 Ala
Gly Asp Asn Glu Glu Tyr Glu Asn Phe Cys Asp Lys Glu Leu Lys 485 490
495 Pro Gly Met Gln His Leu Val His His Met Val Arg Ala Pro Ser Met
500 505 510 Leu Leu Thr Asp Ala Lys 515 11863DNAZea
maysmisc_feature(635)..(635)n is a, c, g, or t 11cggacgcgtg
ggtggagagc aggtggtgta agaaagggtt ccgaccggtc agattctacg 60tcgaggatgt
ccgagtgcac agcttagcag actggtgacg atgaggcgcc ggaactgagg
120ctcactgtca ctgggcctcc acccccacct ctctctgtgg tggtggtggt
ggtggcgcgg 180gagggagagg acgacggcga tgattctggc tgggagtcag
actcctctgt agcatcctgc 240ccagccacat tgtcatcaga ggaaaggaga
cggcctgtcg gatcgctccc acggccagta 300agtctaaacg cgtttgatat
catctcattc tcaaggggat tcaatctgtc ggggttgttc 360gaggagcgag
gcaatgaagt gagatttgtc tcagcacatc ccatgcaaac gatcataacg
420aaactggagg agatcgcgaa ggtgaagagc tttgcagttc ggcggaagga
ctggcgggtt 480agcttggaag gcacgagaga aagtgaaaag ggtccattga
caatcggggc tgaagtattt 540gagctcacac caagccttgt ggtcgtggag
gtgaggatga aggcagggga caggcaagaa 600tatgaggatt tttgtgagag
ggagttgaaa cctgngatgc agcacctggt gcaccataca 660gcctcggttc
cagatatacc ttctgatact gattagctta naaggtagtg tgctcttgat
720tggaatgatt gtggtgaaga aatttggatt gaaaggatgc acctttctgt
ttcagcgtaa 780gcatctgtgc aggaaaatgt tattcataga tttccgnagn
tttttttttg taatattctt 840tctgcaatcc aaaatgtttt gcg 8631296PRTZea
maysMISC_FEATURE(78)..(78)Xaa can be any naturally occurring amino
acid 12Met Gln Thr Ile Ile Thr Lys Leu Glu Glu Ile Ala Lys Val Lys
Ser 1 5 10 15 Phe Ala Val Arg Arg Lys Asp Trp Arg Val Ser Leu Glu
Gly Thr Arg 20 25 30 Glu Ser Glu Lys Gly Pro Leu Thr Ile Gly Ala
Glu Val Phe Glu Leu 35 40 45 Thr Pro Ser Leu Val Val Val Glu Val
Arg Met Lys Ala Gly Asp Arg 50 55 60 Gln Glu Tyr Glu Asp Phe Cys
Glu Arg Glu Leu Lys Pro Xaa Met Gln 65 70 75 80 His Leu Val His His
Thr Ala Ser Val Pro Asp Ile Pro Ser Asp Thr 85 90 95 132023DNAZea
mays 13ccacgcgtcc gcgccgcgct tgttttccca ccgcccgatg gccgccatca
cgccgccgac 60gcagtcggag ccgtcgccgc agacggggcg cccggcctcg tctgccgccg
ccgcggccaa 120gcgtggaggg ggcggggctg gtgccgccgg cgggccgctg
atggggaagt acgagctggg 180gcgcctcctg gggcacggca ccttcgcgaa
ggtgtaccac gcgcggcacg tcgacacggg 240ggacaacgtt gccatcaagg
tgctcgacaa ggagaaggcc gtgaagagcg ggctcgtccc 300gcacatcaag
cgcgagatcg ctgtgctacg ccgcgtgcgc cacccgaaca tcgtgcacct
360gttcgaggtt atggccacaa agactaagat ctacttcgtc atggagctcg
tccgcggcgg 420cgagctcttc tcccgcgtct ccaagggccg actcagggag
gacaccgcgc gccgctactt 480ccagcagctc gtctccgccg tggggttctg
ccacgcccgc ggcgtcttcc accgcgacct 540gaagcccgag aatctactcg
tcgacgagca ggggaacctc aaggtatcgg attttgggct 600ctccgccgtc
gccgagcagt tccgtcccga cggcctgctc cacaccttct gcggcacgcc
660ggcctatgtg gcccccgaag tgctcggccg ccgcgggtac gacggcgcca
aggcagacgt 720gtggtcgtgc ggtgtcatcc tctttgtgct catggccgga
tatctccctt tccatgacaa 780aaacatcatg gccatgtaca agaagattta
caagggcgag ttccgctgtg cgaggtggtt 840ctccaaagac cttaccagct
tgctgatgcg cattcttcac actaatccca acactcggat 900cactttgccg
gagatcatgg agtcccgctg gttcaagaaa ggattcaagc ctgtcaagtt
960ctatatcgag gatgaccagc tgcataacgt tatagatgac gaagatggcc
tgttagatat 1020gggacctgct ggtcctgttc ctccaccatt gccacctcca
ccgccacctc tacctccacc 1080aaaggttgat
ggtgatgaat cagggtctga ctcagactcg tcgatctcat cctgccctgc
1140ttcaatgtta tctgatgaga gccaaaggcc ccgtggctct ctaccacgtc
cagcaagtct 1200taatgccttt gatatcatat cattttcaag gggatttaac
ttatcagggt tatttgagga 1260gaaaggggat gaagtgaggt tcatctcggc
tgagcccatg tcagatatca taaccaaatt 1320ggaggacata gcgaagctga
agagcttcaa gttgcggagg aaggactggc gcatctgcct 1380ggagggtaca
agggaaggag ttaaggggcc attaacaatt ggcgcggaga tatttgaact
1440cacacctccc cttgtaatgg tggaggtaaa aaagaaggca ggggataatg
aagagtacga 1500gaacttctgt gacaaggaat tgaagccagg gatgcagcac
cttgtccacc atatggtccg 1560agctccaagt atgctgctta ctgatgccaa
gtagatcgaa aggctttgaa cttaacaaca 1620gcacttcgca cggagctact
ggtaacaggc gtgacattct gagcggcatg aggctagagg 1680agacagttga
gcacagcaca gttgaccaga agagatagtc gccggaacaa aaaccttgac
1740cagttccaca gcgctgtagt ttcgcagatg atgagcagct cggcatctca
tgactgaata 1800aacgcaatgc ccgccatgga gggagactcc ggtgtctttc
ttgtacctga ggtggttaag 1860ttgttactcg aatgctgtat cacgagtggt
gtagtcctgc tattcgtaat atttcgatta 1920accatcaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1980aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaagggcggc cgc 202314518PRTZea mays 14Met Ala
Ala Ile Thr Pro Pro Thr Gln Ser Glu Pro Ser Pro Gln Thr 1 5 10 15
Gly Arg Pro Ala Ser Ser Ala Ala Ala Ala Ala Lys Arg Gly Gly Gly 20
25 30 Gly Ala Gly Ala Ala Gly Gly Pro Leu Met Gly Lys Tyr Glu Leu
Gly 35 40 45 Arg Leu Leu Gly His Gly Thr Phe Ala Lys Val Tyr His
Ala Arg His 50 55 60 Val Asp Thr Gly Asp Asn Val Ala Ile Lys Val
Leu Asp Lys Glu Lys 65 70 75 80 Ala Val Lys Ser Gly Leu Val Pro His
Ile Lys Arg Glu Ile Ala Val 85 90 95 Leu Arg Arg Val Arg His Pro
Asn Ile Val His Leu Phe Glu Val Met 100 105 110 Ala Thr Lys Thr Lys
Ile Tyr Phe Val Met Glu Leu Val Arg Gly Gly 115 120 125 Glu Leu Phe
Ser Arg Val Ser Lys Gly Arg Leu Arg Glu Asp Thr Ala 130 135 140 Arg
Arg Tyr Phe Gln Gln Leu Val Ser Ala Val Gly Phe Cys His Ala 145 150
155 160 Arg Gly Val Phe His Arg Asp Leu Lys Pro Glu Asn Leu Leu Val
Asp 165 170 175 Glu Gln Gly Asn Leu Lys Val Ser Asp Phe Gly Leu Ser
Ala Val Ala 180 185 190 Glu Gln Phe Arg Pro Asp Gly Leu Leu His Thr
Phe Cys Gly Thr Pro 195 200 205 Ala Tyr Val Ala Pro Glu Val Leu Gly
Arg Arg Gly Tyr Asp Gly Ala 210 215 220 Lys Ala Asp Val Trp Ser Cys
Gly Val Ile Leu Phe Val Leu Met Ala 225 230 235 240 Gly Tyr Leu Pro
Phe His Asp Lys Asn Ile Met Ala Met Tyr Lys Lys 245 250 255 Ile Tyr
Lys Gly Glu Phe Arg Cys Ala Arg Trp Phe Ser Lys Asp Leu 260 265 270
Thr Ser Leu Leu Met Arg Ile Leu His Thr Asn Pro Asn Thr Arg Ile 275
280 285 Thr Leu Pro Glu Ile Met Glu Ser Arg Trp Phe Lys Lys Gly Phe
Lys 290 295 300 Pro Val Lys Phe Tyr Ile Glu Asp Asp Gln Leu His Asn
Val Ile Asp 305 310 315 320 Asp Glu Asp Gly Leu Leu Asp Met Gly Pro
Ala Gly Pro Val Pro Pro 325 330 335 Pro Leu Pro Pro Pro Pro Pro Pro
Leu Pro Pro Pro Lys Val Asp Gly 340 345 350 Asp Glu Ser Gly Ser Asp
Ser Asp Ser Ser Ile Ser Ser Cys Pro Ala 355 360 365 Ser Met Leu Ser
Asp Glu Ser Gln Arg Pro Arg Gly Ser Leu Pro Arg 370 375 380 Pro Ala
Ser Leu Asn Ala Phe Asp Ile Ile Ser Phe Ser Arg Gly Phe 385 390 395
400 Asn Leu Ser Gly Leu Phe Glu Glu Lys Gly Asp Glu Val Arg Phe Ile
405 410 415 Ser Ala Glu Pro Met Ser Asp Ile Ile Thr Lys Leu Glu Asp
Ile Ala 420 425 430 Lys Leu Lys Ser Phe Lys Leu Arg Arg Lys Asp Trp
Arg Ile Cys Leu 435 440 445 Glu Gly Thr Arg Glu Gly Val Lys Gly Pro
Leu Thr Ile Gly Ala Glu 450 455 460 Ile Phe Glu Leu Thr Pro Pro Leu
Val Met Val Glu Val Lys Lys Lys 465 470 475 480 Ala Gly Asp Asn Glu
Glu Tyr Glu Asn Phe Cys Asp Lys Glu Leu Lys 485 490 495 Pro Gly Met
Gln His Leu Val His His Met Val Arg Ala Pro Ser Met 500 505 510 Leu
Leu Thr Asp Ala Lys 515 152022DNAGlycine max 15tgaagctcca
tcaccactag cgaacacttc cattgttttt atctcacagg atcgatcgat 60atcaccagca
tcaccatggc agaggtggcg ccgccgaaga aggaaaaccc gaaccttctc
120ctggggcggt tcgagctcgg gaagctcctc gggcacggaa ccttcgcgaa
ggtccaccac 180gcgcgcaaca tcaaaaccgg agaaggagtc gccatcaaga
tcatcaacaa ggagaaaatc 240ctaaaggggg gtttggtctc ccacataaag
cgcgagatct ccattctccg gcgcgtgcgc 300caccccaaca tcgtgcaact
cttcgaagtg atggccacca agaccaagat ctacttcgtc 360atggagtacg
tgcgtggcgg cgaactcttc aacaaggtcg caaagggaag attaaaagaa
420gaagttgcga gaaattactt tcagcagtta gtttccgcgg tggagttttg
ccacgcgcgc 480ggcgtgttcc acagggacct gaagcccgag aacctgttgc
tggacgagga tgggaacctt 540aaagtctccg actttggtct cagtgccgtg
tcggatcaga taaggcagga cgggctgttc 600cacacgtttt gtgggacacc
tgcgtatgtt gctcctgagg tcttgtcgcg gaaaggctac 660gatggtgcaa
aggttgatat ttggtcttgt ggggttgttt tgtttgttct gatggccggc
720tatttgccct tcaatgaccg taacgttatg gctatgtata agaagattta
caagggtgag 780tttcggtgtc ccaggtggtt ttcttctgaa cttacaagac
ttctctctag gcttcttgat 840actaaccctc agacaaggat ttctattcct
gaagtcatgg agaatcgctg gttcaagaag 900ggtttcaagc agattaagtt
ttatgtggag gatgatagag tttgtagttt tgacgagaaa 960ctgttacttc
atcatgatga tgatttggca acatcggatt ctgaggttga gattaggagg
1020aagaatagta atggttcgtt gccgaggcct gcgagtttga atgcgtttga
catcatatcg 1080ttttctcagg gctttgatct atcagggttg tttgaggaaa
agggtgatga ggcgaggttt 1140gtgtcatctg ctccggtgtc gaagattata
tcaaaattgg aggaggttgc tcagttggtt 1200agtttcagtg tgaggaagaa
agattgcagg gtgagcttgg aggggtgtag agaaggtgtg 1260aaggggcctt
tgactattgc tgctgaggtt tttgagttga caccttcctt ggtggtggtg
1320gaggtcaaga aaaagggagg ggataaggcc gagtatgaga agttttgtaa
ctctgagttg 1380agacccgcgt tggagaattt agggatggag gaatctgctt
cttcttcttc ttcttgtcat 1440caatctacac acactcaatc tgaattccaa
caacatcgaa cactttctga ctctgccctt 1500aacagacatt cagataatga
atgtttgttc gaacgagagt taggtctagc agatgagact 1560agtatctcac
aacatggtga atcaaagttc gaatgtcaac aggaaaatat ggccatgttt
1620actatctgac catgtcttga gcactatgat tgtttccaaa gaatgaaaac
aaaaaagata 1680atgaatgctt gcattaatta aggtagcagg agaatgacag
aagatgatag catactattt 1740ctctcttgtt gttttaggct gtgtgtaagt
taaattttac tttctttttc cctcgagaat 1800tttccggcat ttttaggttt
gctccttgac tggagcatta gattctactg tatttgtatg 1860tccaaatgtt
gtgtttctgt aaggctaatt taatttaaat atagtgaatg aagtgtacat
1920gtcaacagtt cacatgtctt ggtaaattgc tctgtaactg tatttatttc
cattctttat 1980tgcaagtaat gagaaaataa taatgcaact ttcttgtgat tc
202216517PRTGlycine max 16Met Ala Glu Val Ala Pro Pro Lys Lys Glu
Asn Pro Asn Leu Leu Leu 1 5 10 15 Gly Arg Phe Glu Leu Gly Lys Leu
Leu Gly His Gly Thr Phe Ala Lys 20 25 30 Val His His Ala Arg Asn
Ile Lys Thr Gly Glu Gly Val Ala Ile Lys 35 40 45 Ile Ile Asn Lys
Glu Lys Ile Leu Lys Gly Gly Leu Val Ser His Ile 50 55 60 Lys Arg
Glu Ile Ser Ile Leu Arg Arg Val Arg His Pro Asn Ile Val 65 70 75 80
Gln Leu Phe Glu Val Met Ala Thr Lys Thr Lys Ile Tyr Phe Val Met 85
90 95 Glu Tyr Val Arg Gly Gly Glu Leu Phe Asn Lys Val Ala Lys Gly
Arg 100 105 110 Leu Lys Glu Glu Val Ala Arg Asn Tyr Phe Gln Gln Leu
Val Ser Ala 115 120 125 Val Glu Phe Cys His Ala Arg Gly Val Phe His
Arg Asp Leu Lys Pro 130 135 140 Glu Asn Leu Leu Leu Asp Glu Asp Gly
Asn Leu Lys Val Ser Asp Phe 145 150 155 160 Gly Leu Ser Ala Val Ser
Asp Gln Ile Arg Gln Asp Gly Leu Phe His 165 170 175 Thr Phe Cys Gly
Thr Pro Ala Tyr Val Ala Pro Glu Val Leu Ser Arg 180 185 190 Lys Gly
Tyr Asp Gly Ala Lys Val Asp Ile Trp Ser Cys Gly Val Val 195 200 205
Leu Phe Val Leu Met Ala Gly Tyr Leu Pro Phe Asn Asp Arg Asn Val 210
215 220 Met Ala Met Tyr Lys Lys Ile Tyr Lys Gly Glu Phe Arg Cys Pro
Arg 225 230 235 240 Trp Phe Ser Ser Glu Leu Thr Arg Leu Leu Ser Arg
Leu Leu Asp Thr 245 250 255 Asn Pro Gln Thr Arg Ile Ser Ile Pro Glu
Val Met Glu Asn Arg Trp 260 265 270 Phe Lys Lys Gly Phe Lys Gln Ile
Lys Phe Tyr Val Glu Asp Asp Arg 275 280 285 Val Cys Ser Phe Asp Glu
Lys Leu Leu Leu His His Asp Asp Asp Leu 290 295 300 Ala Thr Ser Asp
Ser Glu Val Glu Ile Arg Arg Lys Asn Ser Asn Gly 305 310 315 320 Ser
Leu Pro Arg Pro Ala Ser Leu Asn Ala Phe Asp Ile Ile Ser Phe 325 330
335 Ser Gln Gly Phe Asp Leu Ser Gly Leu Phe Glu Glu Lys Gly Asp Glu
340 345 350 Ala Arg Phe Val Ser Ser Ala Pro Val Ser Lys Ile Ile Ser
Lys Leu 355 360 365 Glu Glu Val Ala Gln Leu Val Ser Phe Ser Val Arg
Lys Lys Asp Cys 370 375 380 Arg Val Ser Leu Glu Gly Cys Arg Glu Gly
Val Lys Gly Pro Leu Thr 385 390 395 400 Ile Ala Ala Glu Val Phe Glu
Leu Thr Pro Ser Leu Val Val Val Glu 405 410 415 Val Lys Lys Lys Gly
Gly Asp Lys Ala Glu Tyr Glu Lys Phe Cys Asn 420 425 430 Ser Glu Leu
Arg Pro Ala Leu Glu Asn Leu Gly Met Glu Glu Ser Ala 435 440 445 Ser
Ser Ser Ser Ser Cys His Gln Ser Thr His Thr Gln Ser Glu Phe 450 455
460 Gln Gln His Arg Thr Leu Ser Asp Ser Ala Leu Asn Arg His Ser Asp
465 470 475 480 Asn Glu Cys Leu Phe Glu Arg Glu Leu Gly Leu Ala Asp
Glu Thr Ser 485 490 495 Ile Ser Gln His Gly Glu Ser Lys Phe Glu Cys
Gln Gln Glu Asn Met 500 505 510 Ala Met Phe Thr Ile 515
171975DNAGlycine max 17caatgaagct ccatcaccac tagcgaacac ttccattgtt
tttatctcac aggatcgatt 60tatccccacc accaccacca tggcagaggt ggcggcgccg
aagaaggaaa acccgaatct 120tctccttggg cggttcgagc tcggaaagct
cctcgggcac ggaaccttcg cgaaggtcca 180ccacgcgcgc aacatcaaaa
ccggagaagg agtcgccatc aagatcatca acaaggagaa 240aatcctaaag
ggtggtttgg tctcccacat caagcgcgag atctccatcc tccgccgcgt
300gcgccacccc aacatcgtgc aactcttcga agtcatggcc acaaagacca
agatctactt 360cgtcatggaa ttcgtccgtg gcggcgaact cttcaacaag
gtcgcaaagg gaaggttaaa 420agaagaagtc gccagaaagt acttccaaca
gttggtttcc gcggtggagt tttgccacgc 480gcgcggcgtg ttccacaggg
atttaaagcc cgagaatttg ttgctggacg aggatgggaa 540ccttaaagtc
tccgactttg gactcagtgc cgtgtcggac cagataaggc atgacgggct
600gttccacacg ttttgcggaa cacccgcgta tgttgctcct gaggttttgg
cgcggaaagg 660gtacgatggt gcaaaggttg atatttggtc ttgtggggtt
gttttgtttg ttttgatggc 720gggttatttg cccttccatg accgtaacgt
tatggctatg tataagaaga tttacaaggg 780tgagtttcgg tgtcccaggt
ggttttcttc tgaacttaca agacttttct ctaggcttct 840cgatactaac
cctcagacaa ggatttctat tcccgaaatc atggagaatc gctggttcaa
900gaagggtttc aagcagatta agttttatgt ggaggatgat agagtttgta
gttttgatga 960gaaacagctg cagcatcatg atggcgatga ttatttggca
acatcggatt ctgaggttga 1020gattagaagg aagaatagta attgcaatag
tactagtaat ggtaattcgt tgccgaggcc 1080tgcgagtttg aatgcgtttg
acataatatc gttttctcaa ggctttgatc tatcagggtt 1140gtttgaggag
aagggtgatg aggcgaggtt tgtgtcttct gctccggtgt cgaagattat
1200atcgaaattg gaggaggttg ctcagttggt tagcttcact gtgaggaaga
aagattgcag 1260ggtgagcttg gaggggtgta gagaaggtgt gaaagggcct
ttgactattg ctgctgagat 1320ttttgagttg acaccttcct tggtggtggt
ggaggtgaag aaaaaaggag gggataaggc 1380agagtatgag aagttttgta
actctgagct gaaacccgcg ttggagaatt tggggatgga 1440ggattctgct
tcttcttctt cttcttgtca tcaatctaca cacactcaat ctgaattcca
1500acaacaacat cgaacatttt ctgactctgc ccttaacaga cattcagata
ataatgaatg 1560cttatatgat caagagttgg gtctagcaga agagactagt
atcccacaac ttggtgaacc 1620aaagttcgaa tttcaacagg aaaatgtgcc
catgtttact atttgactgt gtctacaaca 1680ctattgtttt caaagaatga
aacatgtgga aaaccaaaaa aagataatga atgtttgcat 1740taattaaggt
accaggagaa tgacagaaga tgacaacata ctatttctct cttgttattt
1800ttaggctgtg tgtaagttaa attttacttt ctttttccct caagaatttt
ccggcatttt 1860taggtttgct ccttgactgg agcattagat gctactgtat
ttctttgtcc aaatgttgta 1920ttattgtaag gctaatttaa ttttaaatat
agtgaatgaa gaagtttata tgtgt 197518528PRTGlycine max 18Met Ala Glu
Val Ala Ala Pro Lys Lys Glu Asn Pro Asn Leu Leu Leu 1 5 10 15 Gly
Arg Phe Glu Leu Gly Lys Leu Leu Gly His Gly Thr Phe Ala Lys 20 25
30 Val His His Ala Arg Asn Ile Lys Thr Gly Glu Gly Val Ala Ile Lys
35 40 45 Ile Ile Asn Lys Glu Lys Ile Leu Lys Gly Gly Leu Val Ser
His Ile 50 55 60 Lys Arg Glu Ile Ser Ile Leu Arg Arg Val Arg His
Pro Asn Ile Val 65 70 75 80 Gln Leu Phe Glu Val Met Ala Thr Lys Thr
Lys Ile Tyr Phe Val Met 85 90 95 Glu Phe Val Arg Gly Gly Glu Leu
Phe Asn Lys Val Ala Lys Gly Arg 100 105 110 Leu Lys Glu Glu Val Ala
Arg Lys Tyr Phe Gln Gln Leu Val Ser Ala 115 120 125 Val Glu Phe Cys
His Ala Arg Gly Val Phe His Arg Asp Leu Lys Pro 130 135 140 Glu Asn
Leu Leu Leu Asp Glu Asp Gly Asn Leu Lys Val Ser Asp Phe 145 150 155
160 Gly Leu Ser Ala Val Ser Asp Gln Ile Arg His Asp Gly Leu Phe His
165 170 175 Thr Phe Cys Gly Thr Pro Ala Tyr Val Ala Pro Glu Val Leu
Ala Arg 180 185 190 Lys Gly Tyr Asp Gly Ala Lys Val Asp Ile Trp Ser
Cys Gly Val Val 195 200 205 Leu Phe Val Leu Met Ala Gly Tyr Leu Pro
Phe His Asp Arg Asn Val 210 215 220 Met Ala Met Tyr Lys Lys Ile Tyr
Lys Gly Glu Phe Arg Cys Pro Arg 225 230 235 240 Trp Phe Ser Ser Glu
Leu Thr Arg Leu Phe Ser Arg Leu Leu Asp Thr 245 250 255 Asn Pro Gln
Thr Arg Ile Ser Ile Pro Glu Ile Met Glu Asn Arg Trp 260 265 270 Phe
Lys Lys Gly Phe Lys Gln Ile Lys Phe Tyr Val Glu Asp Asp Arg 275 280
285 Val Cys Ser Phe Asp Glu Lys Gln Leu Gln His His Asp Gly Asp Asp
290 295 300 Tyr Leu Ala Thr Ser Asp Ser Glu Val Glu Ile Arg Arg Lys
Asn Ser 305 310 315 320 Asn Cys Asn Ser Thr Ser Asn Gly Asn Ser Leu
Pro Arg Pro Ala Ser 325 330 335 Leu Asn Ala Phe Asp Ile Ile Ser Phe
Ser Gln Gly Phe Asp Leu Ser 340 345 350 Gly Leu Phe Glu Glu Lys Gly
Asp Glu Ala Arg Phe Val Ser Ser Ala 355 360 365 Pro Val Ser Lys Ile
Ile Ser Lys Leu Glu Glu Val Ala Gln Leu Val 370 375 380 Ser Phe Thr
Val Arg Lys Lys Asp Cys Arg Val Ser Leu Glu Gly Cys 385 390 395 400
Arg Glu Gly Val Lys Gly Pro Leu Thr Ile Ala Ala Glu Ile Phe Glu 405
410 415 Leu Thr Pro Ser Leu Val Val Val Glu Val Lys Lys Lys Gly Gly
Asp 420 425 430 Lys Ala Glu Tyr Glu Lys Phe Cys Asn Ser Glu Leu Lys
Pro Ala Leu 435 440 445 Glu Asn Leu Gly Met Glu Asp Ser Ala Ser Ser
Ser Ser Ser Cys His 450 455 460 Gln Ser Thr His Thr Gln Ser Glu Phe
Gln Gln Gln His Arg Thr Phe 465 470 475 480 Ser Asp Ser Ala Leu Asn
Arg His Ser Asp Asn Asn Glu Cys Leu Tyr 485 490
495 Asp Gln Glu Leu Gly Leu Ala Glu Glu Thr Ser Ile Pro Gln Leu Gly
500 505 510 Glu Pro Lys Phe Glu Phe Gln Gln Glu Asn Val Pro Met Phe
Thr Ile 515 520 525 191885DNAGlycine maxmisc_feature(23)..(23)n is
a, c, g, or t 19agagccatgc taggccttac agnacnncnc ttnnnnnnnt
nnngnnnann nncnnnnnnn 60nnnnnnnnnn nnnccaaggt gtactacgcg cgtaacatca
aaaccggcga aggcgtggcc 120atcaaggtaa tcgacaagga gaagatcctc
aaaggaggtt tggtggcgca catcaagcgt 180gagatctcta tcctgcgccg
tgttcgccac cctaacatcg ttcagctctt cgaagtcatg 240gccaccaaga
gcaagatcta tttcgtaatg gaatacgttc gcggcggcga gcttttcaac
300aaggtcgcca agggaaggct caaagaagag gtcgcgagaa agtactttca
gcaattaatc 360tctgctgtgg gattctgcca cgccagaggg gtgtaccaca
gagatctcaa gcctgaaaat 420ttgttgcttg atgagaatgg caatctcaaa
gtctctgatt ttggattgag tgcggtgtct 480gatcaaatcc gacaggatgg
tcttttccac actttttgtg ggacacctgc gtatgttgct 540cctgaggttt
tggcgaggaa agggtacgat ggtgctaagg tggatctttg gtcttgtggg
600gtggtgttgt ttgtgttgat ggcggggtat ttgccctttc atgaccagaa
tgtgatggca 660atgtataaga agatttatag aggggagttt cggtgtccga
ggtggttttc tcctgatttg 720tccaggcttc tcacaaggct tcttgatacc
aagcctgaaa cccggattgc gattcctgaa 780attatggaga ataagtggtt
caagaaaggg tttaagcaga tcaagtttta tgtggaggat 840gataggcttt
gcaatgtggt ggatgatgat ggccttatgg acaatgatga tgacactgct
900tcgattgttt ctgttgcttc gttttcggat tactcggttt ccgagtctga
ttctgagatt 960gagactagga ggaggatcaa tgctcccttg cctagacctc
ctagtttgaa tgcctttgac 1020attatatcgt tctcgccggg ctttaatctt
tcggggttgt ttgaggagaa agaggatgag 1080acaaggtttg tgactgctgc
accggttaac aggatcattt ccaagctgga ggagattgct 1140cagttggtta
ggttttcggt gaggaagaag gattgcaggg tgagtttgga gggtaccaga
1200gagggggtta gagggccttt gactattgct gctgagatat ttgagttgac
accttctttg 1260gttgtggtgg aggtgaagaa aaaaggaggg gatagagccg
agtatgagag gttttgtaac 1320gatgagttaa agcctggatt gcagaatttg
atggtggagg agtctgctac ttcttcagag 1380ttgtctacac ctattcaacc
ttccctacta cgtggccttt ctgaacctgt gccggatatt 1440tcttctgata
ttgaaacccc gctctgtata ccttctgatg attgaagact cagatataga
1500gaagaagaga aaaatggtta aggactttct ctctaatctc tgtatcacac
acactctttc 1560tttctctctc tctctttttt tttttatgtt atagattgtg
tatggaaatt ggtaaaaaaa 1620tttccacaca ggattgattg tcctgctttt
aggtttgctt cttgactgga gcgttaggtg 1680cctactgttt gtctaattgc
catacgagaa aaaaggctaa ttgaaatata gtgaatgagt 1740atgtatttat
tttctacttt tcttggctct gtatagcaag tgataataaa aataacaaaa
1800cggtttagtg ctaatccatg cggcattgca ctggctttgt gtttggctct
atattcaagt 1860taaataagat catttgaaat tggag 188520475PRTGlycine
maxMISC_FEATURE(6)..(6)Xaa can be any naturally occurring amino
acid 20Met Leu Gly Leu Thr Xaa Lys Val Tyr Tyr Ala Arg Asn Ile Lys
Thr 1 5 10 15 Gly Glu Gly Val Ala Ile Lys Val Ile Asp Lys Glu Lys
Ile Leu Lys 20 25 30 Gly Gly Leu Val Ala His Ile Lys Arg Glu Ile
Ser Ile Leu Arg Arg 35 40 45 Val Arg His Pro Asn Ile Val Gln Leu
Phe Glu Val Met Ala Thr Lys 50 55 60 Ser Lys Ile Tyr Phe Val Met
Glu Tyr Val Arg Gly Gly Glu Leu Phe 65 70 75 80 Asn Lys Val Ala Lys
Gly Arg Leu Lys Glu Glu Val Ala Arg Lys Tyr 85 90 95 Phe Gln Gln
Leu Ile Ser Ala Val Gly Phe Cys His Ala Arg Gly Val 100 105 110 Tyr
His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Asp Glu Asn Gly 115 120
125 Asn Leu Lys Val Ser Asp Phe Gly Leu Ser Ala Val Ser Asp Gln Ile
130 135 140 Arg Gln Asp Gly Leu Phe His Thr Phe Cys Gly Thr Pro Ala
Tyr Val 145 150 155 160 Ala Pro Glu Val Leu Ala Arg Lys Gly Tyr Asp
Gly Ala Lys Val Asp 165 170 175 Leu Trp Ser Cys Gly Val Val Leu Phe
Val Leu Met Ala Gly Tyr Leu 180 185 190 Pro Phe His Asp Gln Asn Val
Met Ala Met Tyr Lys Lys Ile Tyr Arg 195 200 205 Gly Glu Phe Arg Cys
Pro Arg Trp Phe Ser Pro Asp Leu Ser Arg Leu 210 215 220 Leu Thr Arg
Leu Leu Asp Thr Lys Pro Glu Thr Arg Ile Ala Ile Pro 225 230 235 240
Glu Ile Met Glu Asn Lys Trp Phe Lys Lys Gly Phe Lys Gln Ile Lys 245
250 255 Phe Tyr Val Glu Asp Asp Arg Leu Cys Asn Val Val Asp Asp Asp
Gly 260 265 270 Leu Met Asp Asn Asp Asp Asp Thr Ala Ser Ile Val Ser
Val Ala Ser 275 280 285 Phe Ser Asp Tyr Ser Val Ser Glu Ser Asp Ser
Glu Ile Glu Thr Arg 290 295 300 Arg Arg Ile Asn Ala Pro Leu Pro Arg
Pro Pro Ser Leu Asn Ala Phe 305 310 315 320 Asp Ile Ile Ser Phe Ser
Pro Gly Phe Asn Leu Ser Gly Leu Phe Glu 325 330 335 Glu Lys Glu Asp
Glu Thr Arg Phe Val Thr Ala Ala Pro Val Asn Arg 340 345 350 Ile Ile
Ser Lys Leu Glu Glu Ile Ala Gln Leu Val Arg Phe Ser Val 355 360 365
Arg Lys Lys Asp Cys Arg Val Ser Leu Glu Gly Thr Arg Glu Gly Val 370
375 380 Arg Gly Pro Leu Thr Ile Ala Ala Glu Ile Phe Glu Leu Thr Pro
Ser 385 390 395 400 Leu Val Val Val Glu Val Lys Lys Lys Gly Gly Asp
Arg Ala Glu Tyr 405 410 415 Glu Arg Phe Cys Asn Asp Glu Leu Lys Pro
Gly Leu Gln Asn Leu Met 420 425 430 Val Glu Glu Ser Ala Thr Ser Ser
Glu Leu Ser Thr Pro Ile Gln Pro 435 440 445 Ser Leu Leu Arg Gly Leu
Ser Glu Pro Val Pro Asp Ile Ser Ser Asp 450 455 460 Ile Glu Thr Pro
Leu Cys Ile Pro Ser Asp Asp 465 470 475 21947DNAGossypium hirsutum
21cccacgcgtc cgaattaatc tcggccgttc atttttgcca cgcgcgtggc gtttaccacc
60gtgacctgaa ggctggagaa tctacttctc gatgaaaatg gggatttgaa agtctctgat
120ttcgggttga gtgctgtatc ggatcagatc cggcaagacg gtttgtttca
cacgttttgt 180ggaaccccgg cttttgttgc gccggaagtt ttggcgagga
aaggatacga tgcggcgaaa 240gtagatatct ggtcttgtgg agtgatttta
tttgttctaa tggcagggta tttaccattt 300caagatcaga acattatggc
tatgtacaag aagatttaca agggtgagtt tcggtgtccg 360agatggtttt
cacccgagtt aattcggtta ctcaccaaac tcctagacac caacccggaa
420acaagaatta cgattccaga aatcatggag aaacgctggt tcaaaaaggg
gtttaaacat 480attaagttct acatcgaaga tgataagtta tgcagtgtcg
aagacgatga taatgatgtt 540gggccatgtt cagaccaatc atcaatgtct
gagtcagaaa cagagttgga aacgaggaaa 600cgagttggca cattgccaag
gccagctagt ttaaacgcgt tcgaccttat atctttctcc 660ccagggttca
acctatccgg gttgttcgag gaaggagaag aaggttcccg gtttgtttca
720ggggcaccgg tttcgacaat catatcgaaa ttggaggaga tagccaaggt
tgttagcttt 780actgtgagga aaaaggattg tagagtgagc ttggagggtt
ctagagaagg agctaaaggt 840ccattatcga ttgctgctga gatattcgaa
ttaacccctt cattagtcgt tgtggaagtg 900aagaagaaag gaggtgaacg
aggagagtat gaggattttt tgtaaca 94722313PRTGossypium
hirsutumMISC_FEATURE(24)..(24)Xaa can be any naturally occurring
amino acid 22His Ala Ser Glu Leu Ile Ser Ala Val His Phe Cys His
Ala Arg Gly 1 5 10 15 Val Tyr His Arg Asp Leu Lys Xaa Glu Asn Leu
Leu Leu Asp Glu Asn 20 25 30 Gly Asp Leu Lys Val Ser Asp Phe Gly
Leu Ser Ala Val Ser Asp Gln 35 40 45 Ile Arg Gln Asp Gly Leu Phe
His Thr Phe Cys Gly Thr Pro Ala Phe 50 55 60 Val Ala Pro Glu Val
Leu Ala Arg Lys Gly Tyr Asp Ala Ala Lys Val 65 70 75 80 Asp Ile Trp
Ser Cys Gly Val Ile Leu Phe Val Leu Met Ala Gly Tyr 85 90 95 Leu
Pro Phe Gln Asp Gln Asn Ile Met Ala Met Tyr Lys Lys Ile Tyr 100 105
110 Lys Gly Glu Phe Arg Cys Pro Arg Trp Phe Ser Pro Glu Leu Ile Arg
115 120 125 Leu Leu Thr Lys Leu Leu Asp Thr Asn Pro Glu Thr Arg Ile
Thr Ile 130 135 140 Pro Glu Ile Met Glu Lys Arg Trp Phe Lys Lys Gly
Phe Lys His Ile 145 150 155 160 Lys Phe Tyr Ile Glu Asp Asp Lys Leu
Cys Ser Val Glu Asp Asp Asp 165 170 175 Asn Asp Val Gly Pro Cys Ser
Asp Gln Ser Ser Met Ser Glu Ser Glu 180 185 190 Thr Glu Leu Glu Thr
Arg Lys Arg Val Gly Thr Leu Pro Arg Pro Ala 195 200 205 Ser Leu Asn
Ala Phe Asp Leu Ile Ser Phe Ser Pro Gly Phe Asn Leu 210 215 220 Ser
Gly Leu Phe Glu Glu Gly Glu Glu Gly Ser Arg Phe Val Ser Gly 225 230
235 240 Ala Pro Val Ser Thr Ile Ile Ser Lys Leu Glu Glu Ile Ala Lys
Val 245 250 255 Val Ser Phe Thr Val Arg Lys Lys Asp Cys Arg Val Ser
Leu Glu Gly 260 265 270 Ser Arg Glu Gly Ala Lys Gly Pro Leu Ser Ile
Ala Ala Glu Ile Phe 275 280 285 Glu Leu Thr Pro Ser Leu Val Val Val
Glu Val Lys Lys Lys Gly Gly 290 295 300 Glu Arg Gly Glu Tyr Glu Asp
Phe Leu 305 310 231838DNATriticum aestivum 23aaagccaaag ccaagccaac
ctgttcccgt tccgttcctc ccacccccgc tcaacccgcc 60tccctccccg cgcctccgcc
cgtccccatg gcggccaccc cgccgtcgtc gcgggacccg 120tcgccgcagc
cccgccggcc ggccgccgcc gcgggccggc ccgccgccag cggcaccggc
180accataggca acggcaagcg cggcgggctc ctgctcggcc gctacgagct
gggccgcgtc 240ctcggccacg gcaccttcgc caaggtctac cacgcccgcc
acgccgacac gggcgagacg 300gtcgccatca aggtgctcga caaggagaag
gcgctgcggg cgggcctcgt cccgcacatc 360aagcgcgaga tcaccatcct
ccgccgcgtc cgccacccca acatcgtgcg cctcttcgag 420gtcatggcca
ccaagtccaa gatctacttc gtcatggagt tcgtccgcgg cggcgagctc
480ttcgcgcgcg tcgccaaggg ccgcctcaag gaggacaccg cgcgccgcta
cttccagcag 540ctcatctccg ccgtcggctt ctgccacgcg cgcggcgtct
tccaccgcga cctcaagccc 600gagaacctcc tcgtcgacga gcgcggggac
ctcaaggtct ccgacttcgg cctctccgcc 660gtcgccgacc agttccaccc
cgacggcctc ctccacacct tctgcggcac cccctcctac 720gtcgcgccgg
agatgctcgc gcgccgcgga tacgacggcg ccaaggctga catatggtcc
780tgcggcgtca tcctcttcgt cctcatggcc ggctacctcc ctttccatga
ccagaacctc 840atggccatgt accgcaagat ttacagaggg gagttccggt
gtccgagatg gttctccaga 900gatctcacca gcctattgaa tcggcttctt
gacaccaacc cggagacaag gatcaccatg 960gcggaagtca tgcagagcag
gtggtttcag aaggggattt cggcccgtca ggttctatgt 1020tgaagacgat
cagctgcaca gcttagggga cagtgagagt gaggagctgg ggctggtcga
1080acctacggag cctcctcttc ctcctccact ttccgccgcc gctgccgcca
ccaccgcagc 1140aagaggatga tgactcaggg tgggagtcgg attcctctgt
cgcatcctgc cctgccacgc 1200tgtcgtgcga ggagcggcaa cggcctgccg
ggcgtctcac acggccagca agcctcaacg 1260ctttcgatat catatccttc
tccaagggat ttgatctatc agggctgttc gaggagcgag 1320ggagcgaagt
gagattcatc tcggcacaac ccatggaaac cattgttaca aaattggagg
1380agattgccaa gatgaagagc ttctccattc gccgcaagga ctggcgcgta
agcatagaag 1440gcaccaggga aggggagaag gggccattga cgattggggc
tgagatattt gagcttacac 1500caagcctctt ggtgttggag gtgaagaaga
aggcagggga taaggcagag tatgatgact 1560tctgcaacaa agagttgaaa
cctgggatgg agcctctcgt gcaccaccaa tctggttcgg 1620ctcgaaatgt
accttctgat actgagtagt tctaaaggta gctctcttgc ttgaaaggaa
1680tataaggaaa ttttggattg aaaggatgcg tcttttatat gtttattaag
catgggacct 1740gagcagaaaa acgctattca tattccttag tcccttttgt
gttagtatta ttcatttttg 1800caatccagaa tttttcatgc ttaaaaaaaa aaaaaaaa
183824519PRTTriticum aestivummisc_feature(302)..(302)Xaa can be any
naturally occurring amino acid 24Met Ala Ala Thr Pro Pro Ser Ser
Arg Asp Pro Ser Pro Gln Pro Arg 1 5 10 15 Arg Pro Ala Ala Ala Ala
Gly Arg Pro Ala Ala Ser Gly Thr Gly Thr 20 25 30 Ile Gly Asn Gly
Lys Arg Gly Gly Leu Leu Leu Gly Arg Tyr Glu Leu 35 40 45 Gly Arg
Val Leu Gly His Gly Thr Phe Ala Lys Val Tyr His Ala Arg 50 55 60
His Ala Asp Thr Gly Glu Thr Val Ala Ile Lys Val Leu Asp Lys Glu 65
70 75 80 Lys Ala Leu Arg Ala Gly Leu Val Pro His Ile Lys Arg Glu
Ile Thr 85 90 95 Ile Leu Arg Arg Val Arg His Pro Asn Ile Val Arg
Leu Phe Glu Val 100 105 110 Met Ala Thr Lys Ser Lys Ile Tyr Phe Val
Met Glu Phe Val Arg Gly 115 120 125 Gly Glu Leu Phe Ala Arg Val Ala
Lys Gly Arg Leu Lys Glu Asp Thr 130 135 140 Ala Arg Arg Tyr Phe Gln
Gln Leu Ile Ser Ala Val Gly Phe Cys His 145 150 155 160 Ala Arg Gly
Val Phe His Arg Asp Leu Lys Pro Glu Asn Leu Leu Val 165 170 175 Asp
Glu Arg Gly Asp Leu Lys Val Ser Asp Phe Gly Leu Ser Ala Val 180 185
190 Ala Asp Gln Phe His Pro Asp Gly Leu Leu His Thr Phe Cys Gly Thr
195 200 205 Pro Ser Tyr Val Ala Pro Glu Met Leu Ala Arg Arg Gly Tyr
Asp Gly 210 215 220 Ala Lys Ala Asp Ile Trp Ser Cys Gly Val Ile Leu
Phe Val Leu Met 225 230 235 240 Ala Gly Tyr Leu Pro Phe His Asp Gln
Asn Leu Met Ala Met Tyr Arg 245 250 255 Lys Ile Tyr Arg Gly Glu Phe
Arg Cys Pro Arg Trp Phe Ser Arg Asp 260 265 270 Leu Thr Ser Leu Leu
Asn Arg Leu Leu Asp Thr Asn Pro Glu Thr Arg 275 280 285 Ile Thr Met
Ala Glu Val Met Gln Ser Arg Trp Phe Gln Xaa Gly Phe 290 295 300 Arg
Pro Val Arg Phe Tyr Val Glu Asp Asp Gln Leu His Ser Leu Gly 305 310
315 320 Asp Ser Glu Ser Glu Glu Leu Gly Leu Val Glu Pro Thr Glu Pro
Pro 325 330 335 Leu Pro Pro Pro Xaa Pro Pro Pro Leu Pro Pro Pro Pro
Gln Gln Glu 340 345 350 Asp Asp Asp Ser Gly Trp Glu Ser Asp Ser Ser
Val Ala Ser Cys Pro 355 360 365 Ala Thr Leu Ser Cys Glu Glu Arg Gln
Arg Pro Ala Gly Arg Leu Thr 370 375 380 Arg Pro Ala Ser Leu Asn Ala
Phe Asp Ile Ile Ser Phe Ser Lys Gly 385 390 395 400 Phe Asp Leu Ser
Gly Leu Phe Glu Glu Arg Gly Ser Glu Val Arg Phe 405 410 415 Ile Ser
Ala Gln Pro Met Glu Thr Ile Val Thr Lys Leu Glu Glu Ile 420 425 430
Ala Lys Met Lys Ser Phe Ser Ile Arg Arg Lys Asp Trp Arg Val Ser 435
440 445 Ile Glu Gly Thr Arg Glu Gly Glu Lys Gly Pro Leu Thr Ile Gly
Ala 450 455 460 Glu Ile Phe Glu Leu Thr Pro Ser Leu Leu Val Leu Glu
Val Lys Lys 465 470 475 480 Lys Ala Gly Asp Lys Ala Glu Tyr Asp Asp
Phe Cys Asn Lys Glu Leu 485 490 495 Lys Pro Gly Met Glu Pro Leu Val
His His Gln Ser Gly Ser Ala Arg 500 505 510 Asn Val Pro Ser Asp Thr
Glu 515 252110DNATriticum aestivum 25gaaatagttt tcgcagagcc
gttaagctca cctccttcga ggccggctgc tccacctcca 60cctccaccta atccccattc
gcctcgcctc cccgcccacc gccaccaccc gtcgatggcg 120gccatcaagc
cgccgccgcc tgaccggccg ccgcaggccg cgcggctgcc gtccccttcc
180tcttcctcct cggcggtggc ggcggccaag cgaggcgcca caggctcccg
cgggctgctc 240atggggcgct acgagctggg ccgcgtcctg ggcaaaggca
ccttcgccaa ggtgtaccac 300gcgcggcacg tgcagaccgg cgagagcgtg
gccatcaagg tgctcgaccg ggagaaggcc 360gtgcggagcg gcctcgtctc
gcacatcaag cgcgagatcg ccgtgctccg ccgcgtgcgc 420caccccaaca
tcgtgcacct cttcgaggtc atggccacca agaccaagat ctacttcgtc
480atggagctcg tccgcggcgg cgagctcttc tcccgcgtct ccaagggccg
cctcaaggag 540gacattgcgc gccgctactt ccagcacctc atctccgccg
tcggcttctg ccacacccgc 600ggggtcttcc accgggacct caagccggag
aacctcctcg tcgacgaggc gggcaacctc 660aaggtgtccg acttcggcct
ctccgccgtc gccgagccgt tccagccaga gggtctcctc 720cacaccttct
gcggcacgcc ggcctacgtc gcgcccgaag tcctcgcccg ccgtggatac
780gaaggcgcca aggccgacat atggtcctgc ggtgtcatcc tctttgttct
catggccgga 840tacctccctt tccatgacca gaacctcatg gccatgtacc
gtaaggttta caagggagag 900ttccgatgtc caaggtggtt ctccaaggac
cttactagct tgatcatgcg ttttcttgac 960acaaacccaa gcaccaggat
caccttgccg gaggtcatgg agagccggtg gttcaagaaa 1020ggtttccggc
cagtcaagtt ctatattgaa gatgaccagc tgtacaacgt cattgatgcc
1080gagaatgata tgctcgactt gggtctccct gaccctcttc ctcaaccatt
gcttcctcca 1140ccttcatctc catctccgca agaagttgat ggagatgact
cagggtcaga atccgacgca 1200tcagtcgtgt cctgccctgc cacatcgtca
tttgaagagc gccacaggct ccgcgggcca 1260ctcccacgcc ccgcaagcct
taacgcgttt gatatcatat cattctcaag gggattcaac 1320ttgtcggggc
tgtttgagga aaaaggggac gaggtgagat tcatctcgag tgaacctatg
1380tcgggcatta taacgaaatt agaggagatc gcaaatgtga agagcttcgc
ggtgcggaag 1440aaggattggc gggtgagcct agagggcaca agggaagggg
ttaaggggcc actaacaatc 1500tgcgcggaga tatttgaact cacaccctcc
cttgtagtag tggaggtaaa aaagaaggcg 1560ggggataagg aagagtatga
tgatttctgc aacaaggaat tgaagccagg aatgcagcat 1620cttgtgcacc
agatggcccc agttccaatt acacctacca tttctgagta ggccgaaagg
1680ccttcaaggt aacaggcgcc accgccccta gagctaacgg ggataggggg
agcgactctc 1740caagctagaa agaaactgga gtcgggtgca actgacagga
gcaagagttc ttgtagtctc 1800gggcactgac gatgatgagc gcggttactt
ggttaactct ggagagcata cgtaatgtta 1860tccgagacgg gagctagatt
gtggctgtat atggtgtcac cctagctgct gtttaaatgt 1920ttgtactttt
ctctacttaa tttgctgatg atgattgtgt atgtactccc gctgttggtt
1980tatcagcaga accgaataat tttgggcaat cgttaattca aggaccaaac
tgattgagga 2040ataaattggg tgcaacatgc attgcatgca ccctttggcc
accaggcaca tgcagacgtg 2100cttggattcc 211026518PRTTriticum aestivum
26Met Ala Ala Ile Lys Pro Pro Pro Pro Asp Arg Pro Pro Gln Ala Ala 1
5 10 15 Arg Leu Pro Ser Pro Ser Ser Ser Ser Ser Ala Val Ala Ala Ala
Lys 20 25 30 Arg Gly Ala Thr Gly Ser Arg Gly Leu Leu Met Gly Arg
Tyr Glu Leu 35 40 45 Gly Arg Val Leu Gly Lys Gly Thr Phe Ala Lys
Val Tyr His Ala Arg 50 55 60 His Val Gln Thr Gly Glu Ser Val Ala
Ile Lys Val Leu Asp Arg Glu 65 70 75 80 Lys Ala Val Arg Ser Gly Leu
Val Ser His Ile Lys Arg Glu Ile Ala 85 90 95 Val Leu Arg Arg Val
Arg His Pro Asn Ile Val His Leu Phe Glu Val 100 105 110 Met Ala Thr
Lys Thr Lys Ile Tyr Phe Val Met Glu Leu Val Arg Gly 115 120 125 Gly
Glu Leu Phe Ser Arg Val Ser Lys Gly Arg Leu Lys Glu Asp Ile 130 135
140 Ala Arg Arg Tyr Phe Gln His Leu Ile Ser Ala Val Gly Phe Cys His
145 150 155 160 Thr Arg Gly Val Phe His Arg Asp Leu Lys Pro Glu Asn
Leu Leu Val 165 170 175 Asp Glu Ala Gly Asn Leu Lys Val Ser Asp Phe
Gly Leu Ser Ala Val 180 185 190 Ala Glu Pro Phe Gln Pro Glu Gly Leu
Leu His Thr Phe Cys Gly Thr 195 200 205 Pro Ala Tyr Val Ala Pro Glu
Val Leu Ala Arg Arg Gly Tyr Glu Gly 210 215 220 Ala Lys Ala Asp Ile
Trp Ser Cys Gly Val Ile Leu Phe Val Leu Met 225 230 235 240 Ala Gly
Tyr Leu Pro Phe His Asp Gln Asn Leu Met Ala Met Tyr Arg 245 250 255
Lys Val Tyr Lys Gly Glu Phe Arg Cys Pro Arg Trp Phe Ser Lys Asp 260
265 270 Leu Thr Ser Leu Ile Met Arg Phe Leu Asp Thr Asn Pro Ser Thr
Arg 275 280 285 Ile Thr Leu Pro Glu Val Met Glu Ser Arg Trp Phe Lys
Lys Gly Phe 290 295 300 Arg Pro Val Lys Phe Tyr Ile Glu Asp Asp Gln
Leu Tyr Asn Val Ile 305 310 315 320 Asp Ala Glu Asn Asp Met Leu Asp
Leu Gly Leu Pro Asp Pro Leu Pro 325 330 335 Gln Pro Leu Leu Pro Pro
Pro Ser Ser Pro Ser Pro Gln Glu Val Asp 340 345 350 Gly Asp Asp Ser
Gly Ser Glu Ser Asp Ala Ser Val Val Ser Cys Pro 355 360 365 Ala Thr
Ser Ser Phe Glu Glu Arg His Arg Leu Arg Gly Pro Leu Pro 370 375 380
Arg Pro Ala Ser Leu Asn Ala Phe Asp Ile Ile Ser Phe Ser Arg Gly 385
390 395 400 Phe Asn Leu Ser Gly Leu Phe Glu Glu Lys Gly Asp Glu Val
Arg Phe 405 410 415 Ile Ser Ser Glu Pro Met Ser Gly Ile Ile Thr Lys
Leu Glu Glu Ile 420 425 430 Ala Asn Val Lys Ser Phe Ala Val Arg Lys
Lys Asp Trp Arg Val Ser 435 440 445 Leu Glu Gly Thr Arg Glu Gly Val
Lys Gly Pro Leu Thr Ile Cys Ala 450 455 460 Glu Ile Phe Glu Leu Thr
Pro Ser Leu Val Val Val Glu Val Lys Lys 465 470 475 480 Lys Ala Gly
Asp Lys Glu Glu Tyr Asp Asp Phe Cys Asn Lys Glu Leu 485 490 495 Lys
Pro Gly Met Gln His Leu Val His Gln Met Ala Pro Val Pro Ile 500 505
510 Thr Pro Thr Ile Ser Glu 515 27527PRTTriticum aestivum 27Met Ser
Ala Ile Lys Pro Pro Pro Pro Asp Arg Pro Pro Gln Ala Ala 1 5 10 15
Arg Leu Pro Ser Pro Ser Ser Ser Ser Ser Ala Ala Ala Ala Ala Lys 20
25 30 Gln Gly Gly Thr Gly Ser Arg Gly Leu Leu Met Gly Arg Tyr Glu
Leu 35 40 45 Gly Arg Val Leu Gly Lys Gly Thr Phe Ala Lys Val Tyr
His Ala Arg 50 55 60 His Val Gln Thr Gly Glu Ser Val Ala Ile Lys
Val Leu Asp Arg Glu 65 70 75 80 Lys Ala Val Arg Ser Gly Leu Val Ser
His Ile Lys Arg Glu Ile Ala 85 90 95 Val Leu Arg Arg Val Arg His
Pro Asn Ile Val His Leu Phe Glu Val 100 105 110 Met Ala Thr Lys Thr
Lys Ile Tyr Phe Val Met Glu Leu Val Val Ala 115 120 125 Ala Leu Leu
Arg Phe Ser Lys Gly Arg Leu Lys Glu Asp Ile Ala Arg 130 135 140 Arg
Tyr Phe Gln His Leu Ile Ser Ala Val Gly Phe Cys His Thr Arg 145 150
155 160 Gly Val Phe His Arg Asp Leu Lys Pro Glu Asn Leu Leu Val Asp
Glu 165 170 175 Ala Gly Asn Leu Lys Val Ser Asp Phe Gly Leu Ser Ala
Val Ala Glu 180 185 190 Pro Phe Gln Pro Glu Gly Leu Leu His Thr Phe
Cys Gly Thr Arg Ala 195 200 205 Tyr Val Ala Pro Glu Val Leu Ala Arg
Arg Gly Tyr Glu Gly Ala Lys 210 215 220 Ala Asp Ile Trp Ser Cys Gly
Val Ile Leu Phe Val Leu Met Ala Gly 225 230 235 240 Tyr Leu Pro Phe
His Asp Gln Asn Leu Met Ala Met Tyr Arg Lys Phe 245 250 255 Thr Arg
Glu Ser Ser Met Ser Arg Trp Phe Ser Lys Asp Leu Thr Ser 260 265 270
Leu Ile Met Arg Phe Leu Asp Thr Asn Pro Ser Thr Arg Ile Thr Leu 275
280 285 Pro Glu Ser Trp Arg Ala Gly Gly Ser Arg Lys Val Ser Gly Gln
Ser 290 295 300 Ser Ser Ile Leu Lys Thr Asn Gln Leu Tyr Asn Val Ile
Asp Ala Glu 305 310 315 320 Asn Asp Met Leu Asp Leu Gly Leu Pro Asp
Pro Leu Pro Gln Pro Leu 325 330 335 Pro Pro Pro Pro Pro Ser Pro Ser
Pro Gln Gln Val Asp Gly Asp Asp 340 345 350 Ser Gly Ser Glu Ser Asp
Ala Ser Val Val Ser Cys Pro Ala Thr Ser 355 360 365 Ser Phe Glu Glu
Arg His Arg Leu Arg Gly Pro Leu Pro Arg Pro Ala 370 375 380 Ser Leu
Asn Ala Phe Asp Ile Ile Ser Phe Ser Arg Gly Phe Asn Leu 385 390 395
400 Ser Gly Leu Phe Glu Glu Lys Gly Asp Glu Val Arg Phe Ile Ser Gly
405 410 415 Glu Pro Met Pro Asp Ile Ile Thr Lys Leu Glu Glu Ile Ala
Asn Val 420 425 430 Lys Ser Phe Ala Cys Glu Glu Gly Leu Ala Gly Asp
Leu Glu Gly Thr 435 440 445 Arg Glu Gly Val Lys Gly Pro Leu Thr Ile
Cys Ala Glu Ile Phe Glu 450 455 460 Leu Thr Pro Ser Leu Val Val Val
Glu Val Lys Lys Lys Ala Gly Asp 465 470 475 480 Lys Glu Glu Tyr Asp
Asp Phe Cys Asn Lys Glu Leu Lys Pro Gly Met 485 490 495 Gln His Leu
Val His Gln Met Val Pro Val Pro Asn Thr Pro Thr Ile 500 505 510 Ser
Glu Leu Ala Glu Thr Val Gln Gly Asn Arg Arg His Arg Pro 515 520 525
28877DNAHordeum vulgare 28cccgggctgc aggaattcgg cacgaggtgc
tcgacttggg tctctctgat cctcttcctc 60aaccattgcc acctccacct ccacctccgc
aagaagttga tggaaatgac tcagggtcag 120aatcggactc atcagtcatg
tcctgccctg ccacatcgtc atttgaagag cgccagaggc 180tccgcgggcc
actcccacgc cccgcaagtc ttaatgcatt cgatatcata tcattctcaa
240ggggattcaa cttgtcgggg ctgtttgagg aaaaagggga cgaggtgaga
ttcatctcga 300gtgaacctat gtcggacatt ataacgaaat tggaggagat
cgcaaatgtg aagagctttg 360cggtgcggaa gaaggattgg cgggtgagcc
tagagggtac aagggaagga gttaaggggc 420cactaacaat cggcgcagag
atatttgaac tcacaccctc ccttgtagta gtggaggtaa 480aaaagaaggc
gggggataag gaagagtatg atgatttctg caacaaggaa ttgaagccag
540gaatggagca tcttgtgcac cagatggtcc cagttccaaa tacacctacc
atttctgagt 600aggccaaagg ccttgaaggt tactggcgcc actgccccta
gagctaacgg ggataggagg 660agcgactctc tccaagctag aaacaggccg
gagtcgtgtg gaactgacag gaggagcatc 720tcttgtagtg tgggacggga
gccccctgac cagctcgggc agggcactga tgatgagcgc 780ggtttactct
tacgagctcg cttctctgga gagcataaca caatgttgtc cgagacggag
840ctagattgtg gctgtagtac tgtatatggc gtcgccc 87729199PRTHordeum
vulgare 29Arg Ala Ala Gly Ile Arg His Glu Val Leu Asp Leu Gly Leu
Ser Asp 1 5 10 15 Pro Leu Pro Gln Pro Leu Pro Pro Pro Pro Pro Pro
Pro Gln Glu Val 20 25 30 Asp Gly Asn Asp Ser Gly Ser Glu Ser Asp
Ser Ser Val Met Ser Cys 35 40 45 Pro Ala Thr Ser Ser Phe Glu Glu
Arg Gln Arg Leu Arg Gly Pro Leu 50 55 60 Pro Arg Pro Ala Ser Leu
Asn Ala Phe Asp Ile Ile Ser Phe Ser Arg 65 70 75 80 Gly Phe Asn Leu
Ser Gly Leu Phe Glu Glu Lys Gly Asp Glu Val Arg 85 90 95 Phe Ile
Ser Ser Glu Pro Met Ser Asp Ile Ile Thr Lys Leu Glu Glu 100 105 110
Ile Ala Asn Val Lys Ser Phe Ala Val Arg Lys Lys Asp Trp Arg Val 115
120 125 Ser Leu Glu Gly Thr Arg Glu Gly Val Lys Gly Pro Leu Thr Ile
Gly 130 135 140 Ala Glu Ile Phe Glu Leu Thr Pro Ser Leu Val Val Val
Glu Val Lys 145 150 155 160 Lys Lys Ala Gly Asp Lys Glu Glu Tyr Asp
Asp Phe Cys Asn Lys Glu 165 170 175 Leu Lys Pro Gly Met Glu His Leu
Val His Gln Met Val Pro Val Pro 180 185 190 Asn Thr Pro Thr Ile Ser
Glu 195 30750DNAAllium porrum 30gcctgtgaaa tattacattg agaacgatag
atttcataag tggtgtagct tagatgaaga 60gaatgctaat gacgaggagg aggtagaatc
tggagatgaa tcgggactct tcagtttgct 120tcctgcccct gccgcgcttg
acgagggaaa agaaagaaaa aggacagggg aaactccaat 180aggcctttga
gtttgaatgc atttgacata atttcctttt caagaggatt tgatctttcg
240ggtttgtttg atgaaacagg agatgaaact agatttgtgt cgggtgaatc
gataccgaac 300atcatatcga aactagagga gattgcaaag gttgggagtt
ttacctttag gaagaaggat 360tgtagggtta gtttagaagg gacgcgggaa
ggagtgaagg gcccgcttac aattggtgct 420gagatatttg agctgacgcc
ttgtttggtt gttgttgagc ttaagaagaa agcaggagac 480aaagcagagt
atgaggagtt ttgtaacaag gagctgaaac ctgggttgct acatcttatg
540tttcctgatg gcggtgttcc ttccaacaca acttctgata cagagtaggc
agtgcaggga 600attctagttt tctaggtgtt ggcctcctgg gccccccggg
accttctgat tctcaattgt 660tatctgtatt atatagcagt gttttatgat
tcattttgtg ttagatttgt agtaagaaat 720ttatgttaac ttagatgaaa
atcaagtttc 75031148PRTAllium porrum 31Arg Gly Lys Arg Lys Lys Lys
Asp Arg Gly Asn Ser Asn Arg Pro Leu 1 5 10 15 Ser Leu Asn Ala Phe
Asp Ile Ile Ser Phe Ser Arg Gly Phe Asp Leu 20 25 30 Ser Gly Leu
Phe Asp Glu Thr Gly Asp Glu Thr Arg Phe Val Ser Gly 35 40 45 Glu
Ser Ile Pro Asn Ile Ile Ser Lys Leu Glu Glu Ile Ala Lys Val 50 55
60 Gly Ser Phe Thr Phe Arg Lys Lys Asp Cys Arg Val Ser Leu Glu Gly
65 70 75 80 Thr Arg Glu Gly Val Lys Gly Pro Leu Thr Ile Gly Ala Glu
Ile Phe 85 90 95 Glu Leu Thr Pro Cys Leu Val Val Val Glu Leu Lys
Lys Lys Ala Gly 100 105 110 Asp Lys Ala Glu Tyr Glu Glu Phe Cys Asn
Lys Glu Leu Lys Pro Gly 115 120 125 Leu Leu His Leu Met Phe Pro Asp
Gly Gly Val Pro Ser Asn Thr Thr 130 135 140 Ser Asp Thr Glu 145
321235DNAAllium porrum 32aattcggcac gagagacggt ccgattccaa
ttccgttctg ctgatccggc acgaggctgg 60gcaagctcct cggccatggc aacttcgcca
aggtctacct cgcgcgcaac ctcgcctcca 120acgaggaagt cgctatcaag
gtcttcgata aggagaaaat cctcaaatcc ggcctcgtca 180accacaccaa
acgcgagatc tcaatcctcc gccgtcttcg tcatcccaat gtcgtcgagc
240tcttcgaggt catggccacc aaatcaaaga tctatttcgt aatagagtac
gtccgaggtg 300gtgaattgtt cggcaaggta gccaaagggc gtctcaacga
gaacacggca agaaagtact 360ttcagcaatt gatttccgcc gttgatttct
gccacgccag aggcgtgtac caccgagatc 420tgaagccgga gaatttgttg
ttagacgata atggcgattt gaaggtgtcg gatttcgggt 480tgagcgctgt
atcggaccag atgaggcagg atggtttgtt tcacacgttt tgtggtactc
540cagcctacgt tgctccagag gttctcggaa ggaaagggta tgatggggct
aaatttgaca 600tttggtcatg tggtgttatt ttgtttttgt tgatggcagg
gtacttgccc tttcatgatc 660aaaacgtgat ggctatgtat aagaagattt
ataaagggga gtttaggtgt ccgagatggt 720tttcaaagga tttgacaagg
ttgctgatga ggcttcttga tacaaatccc aaaacccgga 780ttactattcc
gggggggatg gagaacagat ggttcaagaa tggattcgag cctgtgaaat
840attacattga gaatgataga tttcataagt ggtgtagctt agatgaagag
aacgctaatg 900acgaggagga ggtagaatct gctcgtgccg cggtctcttc
agttgcttcc tgccctgccg 960cgcttgatga gggaaagaag aaaaggacag
ggaaactcca taggccttta aggttgaatg 1020catttgacat aatttccttt
tcaagaggat ttgatctttc gggtttgttt gatgaaacag 1080gagacgaaac
tagatttgtg tcgggtgaat caatacccaa catcatattt cctcttccaa
1140aaccgtttta agcaatccgg agtttgtata ccttcccttc caaagccctt
gtctctaaat 1200cgccatcgct gcaccaatag ccgccactga ccacc
123533368PRTAllium porrum 33Ser Gly Thr Arg Leu Gly Lys Leu Leu Gly
His Gly Asn Phe Ala Lys 1 5 10 15 Val Tyr Leu Ala Arg Asn Leu Ala
Ser Asn Glu Glu Val Ala Ile Lys 20 25 30 Val Phe Asp Lys Glu Lys
Ile Leu Lys Ser Gly Leu Val Asn His Thr 35 40 45 Lys Arg Glu Ile
Ser Ile Leu Arg Arg Leu Arg His Pro Asn Val Val 50 55 60 Glu Leu
Phe Glu Val Met Ala Thr Lys Ser Lys Ile Tyr Phe Val Ile 65 70 75 80
Glu Tyr Val Arg Gly Gly Glu Leu Phe Gly Lys Val Ala Lys Gly Arg 85
90 95 Leu Asn Glu Asn Thr Ala Arg Lys Tyr Phe Gln Gln Leu Ile Ser
Ala 100 105 110 Val Asp Phe Cys His Ala Arg Gly Val Tyr His Arg Asp
Leu Lys Pro 115 120 125 Glu Asn Leu Leu Leu Asp Asp Asn Gly Asp Leu
Lys Val Ser Asp Phe 130 135 140 Gly Leu Ser Ala Val Ser Asp Gln Met
Arg Gln Asp Gly Leu Phe His 145 150 155 160 Thr Phe Cys Gly Thr Pro
Ala Tyr Val Ala Pro Glu Val Leu Gly Arg 165 170 175 Lys Gly Tyr Asp
Gly Ala Lys Phe Asp Ile Trp Ser Cys Gly Val Ile 180 185 190 Leu Phe
Leu Leu Met Ala Gly Tyr Leu Pro Phe His Asp Gln Asn Val 195 200 205
Met Ala Met Tyr Lys Lys Ile Tyr Lys Gly Glu Phe Arg Cys Pro Arg 210
215 220 Trp Phe Ser Lys Asp Leu Thr Arg Leu Leu Met Arg Leu Leu Asp
Thr 225 230 235 240 Asn Pro Lys Thr Arg Ile Thr Ile Pro Gly Gly Met
Glu Asn Arg Trp 245 250 255 Phe Lys Asn Gly Phe Glu Pro Val Lys Tyr
Tyr Ile Glu Asn Asp Arg 260 265 270 Phe His Lys Trp Cys Ser Leu Asp
Glu Glu Asn Ala Asn Asp Glu Glu 275 280 285 Glu
Val Glu Ser Ala Arg Ala Ala Val Ser Ser Val Ala Ser Cys Pro 290 295
300 Ala Ala Leu Asp Glu Gly Lys Lys Lys Arg Thr Gly Lys Leu His Arg
305 310 315 320 Pro Leu Arg Leu Asn Ala Phe Asp Ile Ile Ser Phe Ser
Arg Gly Phe 325 330 335 Asp Leu Ser Gly Leu Phe Asp Glu Thr Gly Asp
Glu Thr Arg Phe Val 340 345 350 Ser Gly Glu Ser Ile Pro Asn Ile Ile
Phe Pro Leu Pro Lys Pro Phe 355 360 365 341427DNABrassica napus
34gtcgacccac gcgtccggtc cgcggcggcg agctcttcaa caaagtcgcc aaagggcgcc
60tcaaggagga tgtcgcccgc aagtacttcc agcagctgat ctccgccgtc acgttctgcc
120acgcccgcgg cgtctaccac cgcgacatca agccggagaa tctcctcctc
gacgagaacg 180ggaacctcaa agtctccgac tttgggctca gcgctgtctc
cgatcagatt cgccaggacg 240ggcttttcca cacgttctgt gggacccctg
cttacgtggc gccagaggtt ttggctagga 300aggggtacga cgcgggtaaa
gttgatatct ggtcttgtgg tgttgtgttg tttgttttga 360tggctggtta
cctccctttt cacgaccgta acgttatggc tatgtacaag aagatttaca
420aaggagagtt taggtgtccg agttggttct ctcccgagct cacgaggttg
tgttctcgcc 480tcctcgagac gaatccggag aaacggttta cgttccctca
gattatggag aactcttggt 540tcaagaaagg gtttaagcat gttaagttct
acgtggaaga tgataagctt tgtaacgttg 600ttgatgatga cgatgagttg
gagactggtt ccgttgagtc tgatcggtct tctaccgttt 660ctgaatcgga
cgttgagttt ttcaagcccg cgaggagagt tggggggttg cctaggcctg
720cgagtttgaa tgcttttgat atatatcgtt ctcgcaaggg tttgatttgt
ctggtctgtt 780tgatgatgat ggggaagggt ctaggtttgt ctcgggagct
ccggtttcga agattatatc 840gaagctggaa gagattgcta aagttgtgag
ctttaccgtg aggaagaagg attgtagagt 900gagtcttgaa gggtcgagac
aaggagtgaa aggtcctttg actattgctg cggagatatt 960cgagctgacg
ccgtctttgg ttgttgtgga agttaagaag aaaggagggg atagaactga
1020gtatgaagag ttttgtaaca aggagttgaa accgaagttg cagaccttga
cggctgatga 1080agtagatgat cctgtggcgg tgtcagcggt ggttgatgaa
accgcgtctg gagtggcgaa 1140ttctccgccg gtttgtttct tgccttctga
cactgagtag aagatgagat catgaggggt 1200tttgttaacc gaactgatga
aactgcttag ggttggtgaa atgtagaacc gaagtatgta 1260acatgttatg
ttttacagtt ggagagatcg ttagagacgg actttgaatt atgtttttac
1320taacctttta gcagtttttt tgtgttcttg tgtgtgtgtg gaagagtttg
taaacagttt 1380cgtatcagat cttttaatat gtaaaaaaaa aaaaaaaggg cggccgc
142735392PRTBrassica napusmisc_feature(248)..(248)Xaa can be any
naturally occurring amino acid 35Arg Pro Thr Arg Pro Val Arg Gly
Gly Glu Leu Phe Asn Lys Val Ala 1 5 10 15 Lys Gly Arg Leu Lys Glu
Asp Val Ala Arg Lys Tyr Phe Gln Gln Leu 20 25 30 Ile Ser Ala Val
Thr Phe Cys His Ala Arg Gly Val Tyr His Arg Asp 35 40 45 Ile Lys
Pro Glu Asn Leu Leu Leu Asp Glu Asn Gly Asn Leu Lys Val 50 55 60
Ser Asp Phe Gly Leu Ser Ala Val Ser Asp Gln Ile Arg Gln Asp Gly 65
70 75 80 Leu Phe His Thr Phe Cys Gly Thr Pro Ala Tyr Val Ala Pro
Glu Val 85 90 95 Leu Ala Arg Lys Gly Tyr Asp Ala Gly Lys Val Asp
Ile Trp Ser Cys 100 105 110 Gly Val Val Leu Phe Val Leu Met Ala Gly
Tyr Leu Pro Phe His Asp 115 120 125 Arg Asn Val Met Ala Met Tyr Lys
Lys Ile Tyr Lys Gly Glu Phe Arg 130 135 140 Cys Pro Ser Trp Phe Ser
Pro Glu Leu Thr Arg Leu Cys Ser Arg Leu 145 150 155 160 Leu Glu Thr
Asn Pro Glu Lys Arg Phe Thr Phe Pro Gln Ile Met Glu 165 170 175 Asn
Ser Trp Phe Lys Lys Gly Phe Lys His Val Lys Phe Tyr Val Glu 180 185
190 Asp Asp Lys Leu Cys Asn Val Val Asp Asp Asp Asp Glu Leu Glu Thr
195 200 205 Gly Ser Val Glu Ser Asp Arg Ser Ser Thr Val Ser Glu Ser
Asp Val 210 215 220 Glu Phe Phe Lys Pro Ala Arg Arg Val Gly Gly Leu
Pro Arg Pro Ala 225 230 235 240 Ser Leu Asn Ala Phe Asp Ile Xaa Ser
Phe Ser Gln Gly Phe Asp Leu 245 250 255 Ser Gly Leu Phe Asp Asp Asp
Gly Glu Gly Ser Arg Phe Val Ser Gly 260 265 270 Ala Pro Val Ser Lys
Ile Ile Ser Lys Leu Glu Glu Ile Ala Lys Val 275 280 285 Val Ser Phe
Thr Val Arg Lys Lys Asp Cys Arg Val Ser Leu Glu Gly 290 295 300 Ser
Arg Gln Gly Val Lys Gly Pro Leu Thr Ile Ala Ala Glu Ile Phe 305 310
315 320 Glu Leu Thr Pro Ser Leu Val Val Val Glu Val Lys Lys Lys Gly
Gly 325 330 335 Asp Arg Thr Glu Tyr Glu Glu Phe Cys Asn Lys Glu Leu
Lys Pro Lys 340 345 350 Leu Gln Thr Leu Thr Ala Asp Glu Val Asp Asp
Pro Val Ala Val Ser 355 360 365 Ala Val Val Asp Glu Thr Ala Ser Gly
Val Ala Asn Ser Pro Pro Val 370 375 380 Cys Phe Leu Pro Ser Asp Thr
Glu 385 390 361840DNAPisum sativum 36gcacgaggtc catcacaaga
aactagagaa acctctcatc tccatccatg gcagtagtag 60cagctcccaa gaagaacaac
tcattcaaca agaaagacaa cccaaatctt ctattgggtc 120gtttcgaatt
aggaaaactc ctcggccatg gaaccttcgc caaggtccac ctagctaaaa
180acatcaaaac cggtgaagca gtagctataa agatcataag caaagacaaa
atccttaaaa 240gtggtttagt ttcacacatc aaacgagaaa tctccattct
ccgccgtgtc cgccacccca 300acatcgtcca gctgttcgaa gtcatggcga
caaagacaaa gatctacttc gtgatggaat 360atgtacgagg tggagagctt
ttcaataaag ttgctaaagg taggttgaaa gaagaggttg 420cgagaaaata
ttttcaacag ttaatatgtg cggttgaatt ttgtcatgct agaggtgttt
480ttcatagaga tataaagcct gagaatttgt tgcttgatga aaatggtaac
cttaaagttt 540ccgattttgg gttaagtgct gtgtcggatg agattaagca
agatgggttg tttcatactt 600tttgtggtac acctgcatat gttgctcctg
aggttttgtc taggaaaggt tatgatggtg 660gtaaggttga tatttggtct
tgtggtgttg ttttgtttgt tttaatggct ggttatttac 720cttttcatga
tcctaataat gttatggtta tgtataagaa gatttataaa ggtgatttta
780ggtgtcctag atggttttct cctgagcttg ttaaccttct tactaggctt
cttgatacta 840agcctcaaac taggatttcg attccggaga ttatggagaa
tcgttggttt aagataggtt 900ttaagcgtat taagttttat gttgaggatg
atgttgtttg taatcttgat tctcttggtc 960ttgatggtaa taatggtaat
gatggtaatg atgataagaa ggtgctaaac attgatgaac 1020accgtgatga
agcgttggaa tcggtatcag aatcagaatg ggattctgag gttgtgaata
1080gaaggaagaa tcgtcagctt ggttcattgc caaggcctgc gagtttgaat
gcttttgaca 1140ttatatcgtt ttcgcaaggc tttgatcttt ctggattgtt
tgaggaaaag ggcgacgaag 1200caaggtttgt gtctggtgcg tcggtgtcaa
agattatgac gaaattggag gaagttgctc 1260agttggttag tttcaaagtg
aggaagaaag attgcagggt tagcttcgag ggttcaagag 1320aaggggtaaa
agggccgttg agtatcgctg ctgaggtatt cgagttaacc ccgtctttgg
1380ttgttgttga agtgaagaaa aaaggagggg ataaagttga gtatgatagg
tttttaaaca 1440ctgaattgaa gtctgctttg catagtttaa ccatggaaga
atctgcaggt tcttcatgtc 1500aaaatacacc agatgaaact ttgcaacaac
gcgcgttttc tgattccgcc attgacaaac 1560attcagatag cattgaatct
ctgaacttag acacctgaag aatgaatgac ctataataag 1620ataaaaaggg
tattttattt tcaatgtttt tataggctgt gtatttatag tacttaaatt
1680tatgttactt ttttctccag gattgtcctg tttttttgtt ttgtgtgtgt
cctaatgttg 1740taagatgaat gccaattcaa ttttaatata ctcaataaaa
caaacatgtt atgttttggc 1800caaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 184037516PRTPisum sativum 37Met Ala Val Val Ala Ala Pro
Lys Lys Asn Asn Ser Phe Asn Lys Lys 1 5 10 15 Asp Asn Pro Asn Leu
Leu Leu Gly Arg Phe Glu Leu Gly Lys Leu Leu 20 25 30 Gly His Gly
Thr Phe Ala Lys Val His Leu Ala Lys Asn Ile Lys Thr 35 40 45 Gly
Glu Ala Val Ala Ile Lys Ile Ile Ser Lys Asp Lys Ile Leu Lys 50 55
60 Ser Gly Leu Val Ser His Ile Lys Arg Glu Ile Ser Ile Leu Arg Arg
65 70 75 80 Val Arg His Pro Asn Ile Val Gln Leu Phe Glu Val Met Ala
Thr Lys 85 90 95 Thr Lys Ile Tyr Phe Val Met Glu Tyr Val Arg Gly
Gly Glu Leu Phe 100 105 110 Asn Lys Val Ala Lys Gly Arg Leu Lys Glu
Glu Val Ala Arg Lys Tyr 115 120 125 Phe Gln Gln Leu Ile Cys Ala Val
Glu Phe Cys His Ala Arg Gly Val 130 135 140 Phe His Arg Asp Ile Lys
Pro Glu Asn Leu Leu Leu Asp Glu Asn Gly 145 150 155 160 Asn Leu Lys
Val Ser Asp Phe Gly Leu Ser Ala Val Ser Asp Glu Ile 165 170 175 Lys
Gln Asp Gly Leu Phe His Thr Phe Cys Gly Thr Pro Ala Tyr Val 180 185
190 Ala Pro Glu Val Leu Ser Arg Lys Gly Tyr Asp Gly Gly Lys Val Asp
195 200 205 Ile Trp Ser Cys Gly Val Val Leu Phe Val Leu Met Ala Gly
Tyr Leu 210 215 220 Pro Phe His Asp Pro Asn Asn Val Met Val Met Tyr
Lys Lys Ile Tyr 225 230 235 240 Lys Gly Asp Phe Arg Cys Pro Arg Trp
Phe Ser Pro Glu Leu Val Asn 245 250 255 Leu Leu Thr Arg Leu Leu Asp
Thr Lys Pro Gln Thr Arg Ile Ser Ile 260 265 270 Pro Glu Ile Met Glu
Asn Arg Trp Phe Lys Ile Gly Phe Lys Arg Ile 275 280 285 Lys Phe Tyr
Val Glu Asp Asp Val Val Cys Asn Leu Asp Ser Leu Gly 290 295 300 Leu
Asp Gly Asn Asn Gly Asn Asp Gly Asn Asp Asp Lys Lys Val Leu 305 310
315 320 Asn Ile Asp Glu His Arg Asp Glu Ala Leu Glu Ser Val Ser Glu
Ser 325 330 335 Glu Trp Asp Ser Glu Val Val Asn Arg Arg Lys Asn Arg
Gln Leu Gly 340 345 350 Ser Leu Pro Arg Pro Ala Ser Leu Asn Ala Phe
Asp Ile Ile Ser Phe 355 360 365 Ser Gln Gly Phe Asp Leu Ser Gly Leu
Phe Glu Glu Lys Gly Asp Glu 370 375 380 Ala Arg Phe Val Ser Gly Ala
Ser Val Ser Lys Ile Met Thr Lys Leu 385 390 395 400 Glu Glu Val Ala
Gln Leu Val Ser Phe Lys Val Arg Lys Lys Asp Cys 405 410 415 Arg Val
Ser Phe Glu Gly Ser Arg Glu Gly Val Lys Gly Pro Leu Ser 420 425 430
Ile Ala Ala Glu Val Phe Glu Leu Thr Pro Ser Leu Val Val Val Glu 435
440 445 Val Lys Lys Lys Gly Gly Asp Lys Val Glu Tyr Asp Arg Phe Leu
Asn 450 455 460 Thr Glu Leu Lys Ser Ala Leu His Ser Leu Thr Met Glu
Glu Ser Ala 465 470 475 480 Gly Ser Ser Cys Gln Asn Thr Pro Asp Glu
Thr Leu Gln Gln Arg Ala 485 490 495 Phe Ser Asp Ser Ala Ile Asp Lys
His Ser Asp Ser Ile Glu Ser Leu 500 505 510 Asn Leu Asp Thr 515
381124DNAMedicago truncatulamisc_feature(5)..(5)n is a, c, g, or t
38tgatnacgcc aagctcgaaa ttaaccctca ctaaagggaa caaaagctgg agctccaccg
60cggtggcggc cgctctagag ccagtgcctc ccccgcgcgg ctggaggtac atttccatca
120ctagaagaaa aaaaaatcat aacacctcca attccaatcc aatagaaccc
tttccactcc 180ggattatcca tccatggcag ttgtagctgc tcccaagaag
aacaactcaa tgaacaagaa 240agataatcca aatcttctat tgggacgttt
tgaattagga aaacttcttg gccatggaac 300ctttgcaaaa gtccaccttg
ccaagaacct caaaacaggt gaatccgtag ctataaagat 360cataagtaaa
gataaaatcc ttaaaagtgg tttagtttca catatcaaac gagaaatctc
420cattctgcgc cgtgttcgtc accccaacat tgttcaactc tttgaagtca
tggctacaaa 480gacaaagatt tactttgtga tggaatatgt acgaggtggt
gagcttttca acaaggttgc 540taaaggtagg ttgaaagaag aagttgcaag
gaaatatttt cagcagttaa tatgtgctgt 600tggattttgt catgctagag
gtgtttttca tagagatcta aagcctgaaa atttgttgct 660tgatgaaaaa
ggtaacctta aagtttcaga ttttggtctt agtgctgtgt cggatgaaat
720taagcaagat gggttgtttc atactttttg tggtacacct gcttatgttg
ctcctgaggt 780tttgtctagg aaaggttatg atggtgctaa ggttgatatt
tggtcttgtg gggttgtttt 840gtttgttttg atggctggtt atttaccttt
tcatgatcct aataatgtta tggctatgta 900taagaagatt tataaaggtg
aatttaggtg tcctagatgg ttttcaccag aacttgttag 960tcttcttact
aggcttcttg atattaaacc tcaaactagg atttctattc ctgagattat
1020ggagaatcgt tggtttaaga taggttttaa gcatattaaa ttttatgttg
aggatgatgt 1080tgtttgtgat cttgattcac ttgatcttga tggtgaggat aata
112439310PRTMedicago truncatula 39Met Ala Val Val Ala Ala Pro Lys
Lys Asn Asn Ser Met Asn Lys Lys 1 5 10 15 Asp Asn Pro Asn Leu Leu
Leu Gly Arg Phe Glu Leu Gly Lys Leu Leu 20 25 30 Gly His Gly Thr
Phe Ala Lys Val His Leu Ala Lys Asn Leu Lys Thr 35 40 45 Gly Glu
Ser Val Ala Ile Lys Ile Ile Ser Lys Asp Lys Ile Leu Lys 50 55 60
Ser Gly Leu Val Ser His Ile Lys Arg Glu Ile Ser Ile Leu Arg Arg 65
70 75 80 Val Arg His Pro Asn Ile Val Gln Leu Phe Glu Val Met Ala
Thr Lys 85 90 95 Thr Lys Ile Tyr Phe Val Met Glu Tyr Val Arg Gly
Gly Glu Leu Phe 100 105 110 Asn Lys Val Ala Lys Gly Arg Leu Lys Glu
Glu Val Ala Arg Lys Tyr 115 120 125 Phe Gln Gln Leu Ile Cys Ala Val
Gly Phe Cys His Ala Arg Gly Val 130 135 140 Phe His Arg Asp Leu Lys
Pro Glu Asn Leu Leu Leu Asp Glu Lys Gly 145 150 155 160 Asn Leu Lys
Val Ser Asp Phe Gly Leu Ser Ala Val Ser Asp Glu Ile 165 170 175 Lys
Gln Asp Gly Leu Phe His Thr Phe Cys Gly Thr Pro Ala Tyr Val 180 185
190 Ala Pro Glu Val Leu Ser Arg Lys Gly Tyr Asp Gly Ala Lys Val Asp
195 200 205 Ile Trp Ser Cys Gly Val Val Leu Phe Val Leu Met Ala Gly
Tyr Leu 210 215 220 Pro Phe His Asp Pro Asn Asn Val Met Ala Met Tyr
Lys Lys Ile Tyr 225 230 235 240 Lys Gly Glu Phe Arg Cys Pro Arg Trp
Phe Ser Pro Glu Leu Val Ser 245 250 255 Leu Leu Thr Arg Leu Leu Asp
Ile Lys Pro Gln Thr Arg Ile Ser Ile 260 265 270 Pro Glu Ile Met Glu
Asn Arg Trp Phe Lys Ile Gly Phe Lys His Ile 275 280 285 Lys Phe Tyr
Val Glu Asp Asp Val Val Cys Asp Leu Asp Ser Leu Asp 290 295 300 Leu
Asp Gly Glu Asp Asn 305 310 402083DNAArabidopsis thaliana
40taaacagaga cgcgttgcta tttttagtga tgaatctcta aaaacagtag agagagaaac
60cttcttcttc tttcttcttc ttctacaaaa tttcacaaaa cgagagagag gagagattca
120aacaaacgaa tcaacaggtg agaaattcga aatctttgcg agctcgtctc
gcccagaatc 180tcgatttctc cacctttcct cttcaattca tcttccaaat
ccctaaaaaa aagactcaaa 240ctttttaatt ttggtccaaa aaagactcaa
actttcttca tcaatggcgg agaaaatcac 300gagagagacg tcgttaccta
aagagagaag cagcccacaa gctctaatcc tgggacgata 360cgaaatgggt
aagcttctcg gccatggtac cttcgctaaa gtttacctcg cacgtaacgt
420gaaaacaaac gaaagcgtag caatcaaagt aatcgacaag gagaaagttc
tcaaaggagg 480tttaatcgca cacatcaaac gcgagatctc gattcttcga
cgtgttcgtc acccaaacat 540cgttcagcta ttcgaagtca tggcgacgaa
agctaagatc tatttcgtga tggagtatgt 600tcgtggaggt gagttattca
ataaagtagc taaaggtcgt cttaaagaag aagtagctcg 660caaatatttc
cagcaattga tctctgctgt tactttctgt cacgcgagag gtgtttatca
720tagagatctg aaacctgaga atcttttgtt agatgagaat ggtaatctta
aagtctctga 780ctttggactt agtgctgtct ctgatcagat tcgtcaagat
gggctttttc atacgttttg 840tggtactcct gcttatgttg ctcctgaggt
tttagctagg aaaggttatg atgctgctaa 900agttgatatt tggtcttgtg
gtgttatctt gtttgtgttg atggctggtt atttgccgtt 960tcatgatcgg
aatgttatgg ctatgtataa gaagatttac agaggggagt ttaggtgtcc
1020taggtggttt tctactgagc ttaccaggtt gttgtcgaag cttttggaga
cgaatccgga 1080gaaacggttc actttccctg agattatgga gaattcttgg
tttaagaaag ggtttaagca 1140tattaagttt tatgtggagg atgataagtt
gtgtaatgtt gttgatgatg atgaactgga 1200gtctgactcg gtggagtcgg
atagagattc cgcggcttct gagtcggaga ttgagtattt 1260ggagcctagg
aggagagttg gagggttgcc tagacctgcg agtttgaatg ctttcgatat
1320tatatcgttt tcgcaaggtt ttgatttatc gggtttgttt gatgacgatg
gggagggttc 1380taggtttgtt tcgggagctc cggtttcgaa gattatatcg
aagttggaag agattgctaa 1440agttgtgagc tttactgtga ggaagaagga
ttgtagggta agtcttgaag gttcaagaca 1500aggagtgaaa ggtccattga
cgattgcagc agagatattc gaattgacac catcgttggt 1560tgttgtggaa
gtcaagaaga aaggaggaga taaaacagag tatgaagatt tctgtaacaa
1620tgaattgaaa cccaagttgc aaaacttgac agctgatgat gtagtagctg
agcctgtcgc 1680ggtttcagcg gttgatgaaa ccgctatccc gaattctcca
accatttctt tcttgccgtc 1740tgacactgaa tagaaggact tgatggaaga
ccacaaagcc agagatcatg aggggtatgt 1800atgtacactg tatgttttgg
gttttgtaat ctggattggg aaagaaaaaa agctgcttac 1860ggttggtgaa
atttagaatc
gaattatatg taatacttat gtttctgttg gagaggatcg 1920ttagagaaat
tgagttatgt tttttactaa ccttttagca gttttttttt gtaatgggag
1980aattgtaaac agtttcgcat aatcagatct ttgatatgta taaaaacaat
gaaataaata 2040aaagaaagtt cctttcttct tagtgaactc tcgagagatc tat
208341489PRTArabidopsis thaliana 41Met Ala Glu Lys Ile Thr Arg Glu
Thr Ser Leu Pro Lys Glu Arg Ser 1 5 10 15 Ser Pro Gln Ala Leu Ile
Leu Gly Arg Tyr Glu Met Gly Lys Leu Leu 20 25 30 Gly His Gly Thr
Phe Ala Lys Val Tyr Leu Ala Arg Asn Val Lys Thr 35 40 45 Asn Glu
Ser Val Ala Ile Lys Val Ile Asp Lys Glu Lys Val Leu Lys 50 55 60
Gly Gly Leu Ile Ala His Ile Lys Arg Glu Ile Ser Ile Leu Arg Arg 65
70 75 80 Val Arg His Pro Asn Ile Val Gln Leu Phe Glu Val Met Ala
Thr Lys 85 90 95 Ala Lys Ile Tyr Phe Val Met Glu Tyr Val Arg Gly
Gly Glu Leu Phe 100 105 110 Asn Lys Val Ala Lys Gly Arg Leu Lys Glu
Glu Val Ala Arg Lys Tyr 115 120 125 Phe Gln Gln Leu Ile Ser Ala Val
Thr Phe Cys His Ala Arg Gly Val 130 135 140 Tyr His Arg Asp Leu Lys
Pro Glu Asn Leu Leu Leu Asp Glu Asn Gly 145 150 155 160 Asn Leu Lys
Val Ser Asp Phe Gly Leu Ser Ala Val Ser Asp Gln Ile 165 170 175 Arg
Gln Asp Gly Leu Phe His Thr Phe Cys Gly Thr Pro Ala Tyr Val 180 185
190 Ala Pro Glu Val Leu Ala Arg Lys Gly Tyr Asp Ala Ala Lys Val Asp
195 200 205 Ile Trp Ser Cys Gly Val Ile Leu Phe Val Leu Met Ala Gly
Tyr Leu 210 215 220 Pro Phe His Asp Arg Asn Val Met Ala Met Tyr Lys
Lys Ile Tyr Arg 225 230 235 240 Gly Glu Phe Arg Cys Pro Arg Trp Phe
Ser Thr Glu Leu Thr Arg Leu 245 250 255 Leu Ser Lys Leu Leu Glu Thr
Asn Pro Glu Lys Arg Phe Thr Phe Pro 260 265 270 Glu Ile Met Glu Asn
Ser Trp Phe Lys Lys Gly Phe Lys His Ile Lys 275 280 285 Phe Tyr Val
Glu Asp Asp Lys Leu Cys Asn Val Val Asp Asp Asp Glu 290 295 300 Leu
Glu Ser Asp Ser Val Glu Ser Asp Arg Asp Ser Ala Ala Ser Glu 305 310
315 320 Ser Glu Ile Glu Tyr Leu Glu Pro Arg Arg Arg Val Gly Gly Leu
Pro 325 330 335 Arg Pro Ala Ser Leu Asn Ala Phe Asp Ile Ile Ser Phe
Ser Gln Gly 340 345 350 Phe Asp Leu Ser Gly Leu Phe Asp Asp Asp Gly
Glu Gly Ser Arg Phe 355 360 365 Val Ser Gly Ala Pro Val Ser Lys Ile
Ile Ser Lys Leu Glu Glu Ile 370 375 380 Ala Lys Val Val Ser Phe Thr
Val Arg Lys Lys Asp Cys Arg Val Ser 385 390 395 400 Leu Glu Gly Ser
Arg Gln Gly Val Lys Gly Pro Leu Thr Ile Ala Ala 405 410 415 Glu Ile
Phe Glu Leu Thr Pro Ser Leu Val Val Val Glu Val Lys Lys 420 425 430
Lys Gly Gly Asp Lys Thr Glu Tyr Glu Asp Phe Cys Asn Asn Glu Leu 435
440 445 Lys Pro Lys Leu Gln Asn Leu Thr Ala Asp Asp Val Val Ala Glu
Pro 450 455 460 Val Ala Val Ser Ala Val Asp Glu Thr Ala Ile Pro Asn
Ser Pro Thr 465 470 475 480 Ile Ser Phe Leu Pro Ser Asp Thr Glu 485
422449DNAArabidopsis thaliana 42tattccattt ccattgtttc tatatctatg
gaaatgaaaa ataattcatt gatcttttct 60atctaaataa aaaaattctc cttcggtttc
aaattatttt ttattgtttg tattagaaac 120aatcaatttt tctaacatag
tattagtttt ttaagcattt aaagcaaaaa aaaaaaaaac 180agttgaccaa
taggctatat atatgtgttg gtggtataca aaaagtgaga tttatttgta
240taccaattct gaaacatttc caaatatacc acaagaaaaa tcctatttct
ggaaaaagcc 300ctaaaaacag aacagaggaa gacgagaaaa acagagaaag
agagagagag agagagagat 360cgtcttcttc tacaacctct caataatcaa
acaaaaaaac gtgttttttt tttttttgcg 420aattcgatct tcgatcaaga
agatcttgat ctcaaaatcc aaacttttct tcaccatttc 480atgagaatct
ctcgctttca atggcggatt tgttaagaaa agtgaaatcg ataaagaaga
540agcaggatca gagcaatcat caagctctga tccttggcaa atacgaaatg
ggtaggcttc 600ttggccacgg aaccttcgct aaagtctatc tcgcacgaaa
cgctcaatct ggagaaagcg 660tagcgatcaa ggtaattgac aaagagaaag
ttctcaaatc cggtttaatc gcacacatca 720aacgcgagat ctcgatcttg
cgccgtgttc gtcatcctaa catcgttcag ctattcgaag 780tcatggcgac
gaaatctaag atctatttcg taatggaata tgttaaagga ggtgaattgt
840tcaacaaggt agctaaagga aggttaaaag aagaaatggc acgtaaatat
tttcaacagt 900tgatctcagc cgtatcgttt tgtcacttcc gtggtgttta
tcatcgagat ttgaaaccgg 960agaatcttct tttagacgaa aatggaaacc
taaaagtctc tgattttggt cttagtgctg 1020tttctgatca gattcgacaa
gatgggttat ttcatacttt ttgtgggacc cctgcttacg 1080tggcaccgga
ggttcttgct cggaaaggct acgatggagc taaagtcgat atttggtctt
1140gtggagtgat cttgtttgtg ttaatggcag ggtttcttcc ttttcatgat
cggaatgtta 1200tggctatgta taagaagatt tacagaggag attttaggtg
tccgagatgg tttccggttg 1260agattaaccg gttattgatt cgaatgttgg
agactaaacc ggagagacgg tttacaatgc 1320cggatattat ggagactagt
tggttcaaga aaggttttaa gcatattaag ttttatgttg 1380aagatgatca
tcagctttgt aacgttgctg atgatgatga gatcgaatcg attgaatcgg
1440tttcggggag gtcttctacg gtttctgaac cggaagactt cgagtctttt
gatgggagga 1500gaagaggtgg ttcgatgcct agaccggcaa gtttgaatgc
tttcgatctc atttcgtttt 1560cgccaggttt tgatctttcg ggtttgtttg
aggatgatgg tgaaggatct aggtttgtgt 1620ctggtgctcc tgttggtcag
atcatttcta agttggagga aatcgcgagg attgtgagtt 1680ttactgtgcg
aaagaaggat tgtaaagtga gtcttgaagg ttcaagagaa ggaagtatga
1740aaggtccatt gtcaattgct gctgagatat ttgaactgac accagctttg
gttgttgttg 1800aagtgaagaa gaaaggaggt gataaaatgg agtatgatga
gttttgtaat aaggagttga 1860aacctaagtt gcagaatttg tcttccgaaa
atggccaacg ggtttctggt tcgcgttctt 1920tgccatcgtt tttactttct
gatactgatt aggaagatga aaaatgaagt ttttgtttct 1980gttttattag
ttttgtgact catatgtggg gttaacgact tgtaatgttc ttgttctttg
2040atggtgtgtg agagacatta gaatttagac ctaaagagag tggtgagata
tgaatcattg 2100atgtgtagaa aacacagatg gaataaacaa gtttctttat
gagtttgctt cttttttttc 2160tttctttttc cttcttttga attttaattc
tgttagtttg aaatatgaca gaaattcact 2220tagaacaaga ttgtgtaatt
tctgttggaa attttgtcta ctaacgtcaa ttaatatgac 2280ggtttatgat
atataattga acatgtagag tttacaaaaa caaaatcttg agaagaaagt
2340ttagcattat aatccaagcc acaccattag ctaatccaaa tttgtgttgt
tcttttaaat 2400atgttatatt ctagtcatgc acctttaacc ataaacaatt
tattaatcc 244943483PRTArabidopsis thaliana 43Met Ala Asp Leu Leu
Arg Lys Val Lys Ser Ile Lys Lys Lys Gln Asp 1 5 10 15 Gln Ser Asn
His Gln Ala Leu Ile Leu Gly Lys Tyr Glu Met Gly Arg 20 25 30 Leu
Leu Gly His Gly Thr Phe Ala Lys Val Tyr Leu Ala Arg Asn Ala 35 40
45 Gln Ser Gly Glu Ser Val Ala Ile Lys Val Ile Asp Lys Glu Lys Val
50 55 60 Leu Lys Ser Gly Leu Ile Ala His Ile Lys Arg Glu Ile Ser
Ile Leu 65 70 75 80 Arg Arg Val Arg His Pro Asn Ile Val Gln Leu Phe
Glu Val Met Ala 85 90 95 Thr Lys Ser Lys Ile Tyr Phe Val Met Glu
Tyr Val Lys Gly Gly Glu 100 105 110 Leu Phe Asn Lys Val Ala Lys Gly
Arg Leu Lys Glu Glu Met Ala Arg 115 120 125 Lys Tyr Phe Gln Gln Leu
Ile Ser Ala Val Ser Phe Cys His Phe Arg 130 135 140 Gly Val Tyr His
Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Asp Glu 145 150 155 160 Asn
Gly Asn Leu Lys Val Ser Asp Phe Gly Leu Ser Ala Val Ser Asp 165 170
175 Gln Ile Arg Gln Asp Gly Leu Phe His Thr Phe Cys Gly Thr Pro Ala
180 185 190 Tyr Val Ala Pro Glu Val Leu Ala Arg Lys Gly Tyr Asp Gly
Ala Lys 195 200 205 Val Asp Ile Trp Ser Cys Gly Val Ile Leu Phe Val
Leu Met Ala Gly 210 215 220 Phe Leu Pro Phe His Asp Arg Asn Val Met
Ala Met Tyr Lys Lys Ile 225 230 235 240 Tyr Arg Gly Asp Phe Arg Cys
Pro Arg Trp Phe Pro Val Glu Ile Asn 245 250 255 Arg Leu Leu Ile Arg
Met Leu Glu Thr Lys Pro Glu Arg Arg Phe Thr 260 265 270 Met Pro Asp
Ile Met Glu Thr Ser Trp Phe Lys Lys Gly Phe Lys His 275 280 285 Ile
Lys Phe Tyr Val Glu Asp Asp His Gln Leu Cys Asn Val Ala Asp 290 295
300 Asp Asp Glu Ile Glu Ser Ile Glu Ser Val Ser Gly Arg Ser Ser Thr
305 310 315 320 Val Ser Glu Pro Glu Asp Phe Glu Ser Phe Asp Gly Arg
Arg Arg Gly 325 330 335 Gly Ser Met Pro Arg Pro Ala Ser Leu Asn Ala
Phe Asp Leu Ile Ser 340 345 350 Phe Ser Pro Gly Phe Asp Leu Ser Gly
Leu Phe Glu Asp Asp Gly Glu 355 360 365 Gly Ser Arg Phe Val Ser Gly
Ala Pro Val Gly Gln Ile Ile Ser Lys 370 375 380 Leu Glu Glu Ile Ala
Arg Ile Val Ser Phe Thr Val Arg Lys Lys Asp 385 390 395 400 Cys Lys
Val Ser Leu Glu Gly Ser Arg Glu Gly Ser Met Lys Gly Pro 405 410 415
Leu Ser Ile Ala Ala Glu Ile Phe Glu Leu Thr Pro Ala Leu Val Val 420
425 430 Val Glu Val Lys Lys Lys Gly Gly Asp Lys Met Glu Tyr Asp Glu
Phe 435 440 445 Cys Asn Lys Glu Leu Lys Pro Lys Leu Gln Asn Leu Ser
Ser Glu Asn 450 455 460 Gly Gln Arg Val Ser Gly Ser Arg Ser Leu Pro
Ser Phe Leu Leu Ser 465 470 475 480 Asp Thr Asp
441788DNAArabidopsis thaliana 44accactttct ttggacacgg atggctcaag
ccttggctca accaccactg gtggtcacca 60ccgtcgtccc agacccgccg ccgccgccac
caccaccgca cccaaagccg tatgctctac 120gatacatggc ggatcttctt
ggccggattg gtataatgga tacagacaaa gatggtaaca 180tcagcccaca
gagtccgagg agtcctagga gcccaagaaa caacattctc atggggaagt
240acgagcttgg gaagcttctc ggccacggaa cctttgcaaa ggtttattta
gctcaaaaca 300tcaaatctgg agataaagtc gccattaaag tcatcgacaa
ggagaagatt atgaagagtg 360gtttggttgc tcacatcaaa cgggaaatct
ctatcctccg ccgtgtccgt cacccttaca 420tcgttcatct attcgaggtt
atggcgacga agtccaagat ttactttgtg atggagtacg 480ttggaggcgg
cgagttgttc aacacggttg ctaaaggtcg attgcccgag gaaactgctc
540ggagatattt ccagcagctg atctcctctg tttcgttctg ccatggccgc
ggtgtttacc 600accgtgacct taaaccagag aatctgcttt tagacaacaa
agggaacctt aaagtatctg 660actttggtct cagcgcggtg gcagagcagc
ttcgtcaaga cgggctctgc cacacgtttt 720gcgggactcc agcgtatatt
gcacccgagg ttttgactag aaaagggtac gatgcagcga 780aagccgatgt
ttggtcatgt ggagtgatct tattcgtgtt gatggctggt cacattccgt
840tctacgacaa gaacataatg gttatgtaca agaagattta caaaggggaa
tttaggtgtc 900ctcgttggtt ttcatcggat cttgttcggt tattgactcg
gcttcttgat acgaatccgg 960atactcggat tacaataccc gagatcatga
agaacagatg gttcaagaaa ggattcaaac 1020atgttaaatt ctacatcgaa
gatgataaac tgtgtaggga agatgaagat gaggaggaag 1080aggcatcatc
atcaggccgc tcttcgacag tttcagagag cgatgcagag ttcgatgtaa
1140aacggatggg aataggttca atgccaagac cctcgagctt aaacgcgttt
gacattatat 1200ctttctcttc agggtttgat ctgtctggtt tgtttgagga
agaaggagga gaagggacga 1260ggtttgtgtc aggtgctcct gtttcaaaga
tcatatcgaa gctggaagag attgcgaaaa 1320tcgtgagctt tactgtgagg
aagaaagaat ggagtttgag attagaaggt tgtagagaag 1380gagcaaaagg
accgttgaca attgcggctg agatatttga gctgactcca tctctagtgg
1440tggtggaggt gaagaagaaa ggaggagaca gagaagagta tgaagagttt
tgcaacaagg 1500aactcagacc agagctggag aaactaatcc atgaagaagt
tgtagtagaa gaagcattgt 1560atttgccatc tgatactgaa tagtataaac
caaggaaggc tgataccaag aatatccaag 1620aaacaagatt gtgttacatt
cttttgttac tattgattat ttattcgtta ttcttgttct 1680atgttaatgt
tgatgttggt gtaaaactga gagatttcgg agatcttcac gatttgttgt
1740gatctccgaa atctcccagt gtgtgtttat gtatataagt ggtattgt
178845520PRTArabidopsis thaliana 45Met Ala Gln Ala Leu Ala Gln Pro
Pro Leu Val Val Thr Thr Val Val 1 5 10 15 Pro Asp Pro Pro Pro Pro
Pro Pro Pro Pro His Pro Lys Pro Tyr Ala 20 25 30 Leu Arg Tyr Met
Ala Asp Leu Leu Gly Arg Ile Gly Ile Met Asp Thr 35 40 45 Asp Lys
Asp Gly Asn Ile Ser Pro Gln Ser Pro Arg Ser Pro Arg Ser 50 55 60
Pro Arg Asn Asn Ile Leu Met Gly Lys Tyr Glu Leu Gly Lys Leu Leu 65
70 75 80 Gly His Gly Thr Phe Ala Lys Val Tyr Leu Ala Gln Asn Ile
Lys Ser 85 90 95 Gly Asp Lys Val Ala Ile Lys Val Ile Asp Lys Glu
Lys Ile Met Lys 100 105 110 Ser Gly Leu Val Ala His Ile Lys Arg Glu
Ile Ser Ile Leu Arg Arg 115 120 125 Val Arg His Pro Tyr Ile Val His
Leu Phe Glu Val Met Ala Thr Lys 130 135 140 Ser Lys Ile Tyr Phe Val
Met Glu Tyr Val Gly Gly Gly Glu Leu Phe 145 150 155 160 Asn Thr Val
Ala Lys Gly Arg Leu Pro Glu Glu Thr Ala Arg Arg Tyr 165 170 175 Phe
Gln Gln Leu Ile Ser Ser Val Ser Phe Cys His Gly Arg Gly Val 180 185
190 Tyr His Arg Asp Leu Lys Pro Glu Asn Leu Leu Leu Asp Asn Lys Gly
195 200 205 Asn Leu Lys Val Ser Asp Phe Gly Leu Ser Ala Val Ala Glu
Gln Leu 210 215 220 Arg Gln Asp Gly Leu Cys His Thr Phe Cys Gly Thr
Pro Ala Tyr Ile 225 230 235 240 Ala Pro Glu Val Leu Thr Arg Lys Gly
Tyr Asp Ala Ala Lys Ala Asp 245 250 255 Val Trp Ser Cys Gly Val Ile
Leu Phe Val Leu Met Ala Gly His Ile 260 265 270 Pro Phe Tyr Asp Lys
Asn Ile Met Val Met Tyr Lys Lys Ile Tyr Lys 275 280 285 Gly Glu Phe
Arg Cys Pro Arg Trp Phe Ser Ser Asp Leu Val Arg Leu 290 295 300 Leu
Thr Arg Leu Leu Asp Thr Asn Pro Asp Thr Arg Ile Thr Ile Pro 305 310
315 320 Glu Ile Met Lys Asn Arg Trp Phe Lys Lys Gly Phe Lys His Val
Lys 325 330 335 Phe Tyr Ile Glu Asp Asp Lys Leu Cys Arg Glu Asp Glu
Asp Glu Glu 340 345 350 Glu Glu Ala Ser Ser Ser Gly Arg Ser Ser Thr
Val Ser Glu Ser Asp 355 360 365 Ala Glu Phe Asp Val Lys Arg Met Gly
Ile Gly Ser Met Pro Arg Pro 370 375 380 Ser Ser Leu Asn Ala Phe Asp
Ile Ile Ser Phe Ser Ser Gly Phe Asp 385 390 395 400 Leu Ser Gly Leu
Phe Glu Glu Glu Gly Gly Glu Gly Thr Arg Phe Val 405 410 415 Ser Gly
Ala Pro Val Ser Lys Ile Ile Ser Lys Leu Glu Glu Ile Ala 420 425 430
Lys Ile Val Ser Phe Thr Val Arg Lys Lys Glu Trp Ser Leu Arg Leu 435
440 445 Glu Gly Cys Arg Glu Gly Ala Lys Gly Pro Leu Thr Ile Ala Ala
Glu 450 455 460 Ile Phe Glu Leu Thr Pro Ser Leu Val Val Val Glu Val
Lys Lys Lys 465 470 475 480 Gly Gly Asp Arg Glu Glu Tyr Glu Glu Phe
Cys Asn Lys Glu Leu Arg 485 490 495 Pro Glu Leu Glu Lys Leu Ile His
Glu Glu Val Val Val Glu Glu Ala 500 505 510 Leu Tyr Leu Pro Ser Asp
Thr Glu 515 520 462506DNAArabidopsis thaliana 46tctataccca
attcaaaccc aattaactgt tggaagtttt ttcaagctaa ctgtttccat 60tcaggtgaag
gttaccagga ctacaaggca gcaaagtcta caggtaacat ttacacattt
120cagtttatca tatagtctct ctgatgaagc ataaatatgt gttagcttag
gatgaacaag 180acagtgttat aggggaaggc cgagaaaaaa attccattaa
gctcgtctct ttgtagagat 240acatgtacaa catattagca ataaacgaaa
aactagccat ttaatcgcca gcaaaaaccg 300tctaactgcc ttattaagat
ctcactctta atttcttttt ttctctgatc ttcctaatca 360ctctcattac
aactctcact ttcatatata tacacaaaac aaattaagta tagtaacaaa
420gaatgttaat attcgtttct atgatacctt ctcttgttat gtcttcttct
ctcgaccttc 480ctgttttctt aactttgtca ctgttcaatt tcaggtggca
aacacattac ttcttcaact 540tcatctgctt ggtaatgcat cagtttctcc
agctgtggtc taagttcctt gttgcaaaac 600tcttcatact cttctatatt
tcctcctttc ttcttcactt caaccaccac aagagatggc
660gtcagctcaa agatctcgac tctaattgtc aacggtcctt tagctccttc
tctacaacct 720tctagcctca cgctccaatc cttcttcctc accataaatt
tcacctcttt ggcaatctct 780tccaatttcg atatgatctt tgtcatagga
gcagcagata caaaccttgc tccttgtcca 840ccttcttcaa acaaacccga
aagatctgag aacgataaga tgtcaaatgc gtttagactc 900gcgggtctcg
gcattgaatc aaccctttta atatcaaact ctgcatctcc ttctgaagca
960gtcgatgatc ggcctgatga caatgatgat gaatcgtcat catcattgtc
atcatcctcc 1020ctacataact tatcgttttc aatatagaat ttgacatgtt
tgaacccttt cttgaaccat 1080ctatgcttca tgatctccgg tattgtgatt
cgggtatctg gattcgtgtc tagcatccgg 1140gtcacaagcc ttgcaagctc
aggagaaaac catttaggac acttaaactg ccctttatat 1200atctttgtat
acataaccaa tatgttcttg tcatcaaatg gaagataacc agccatcaat
1260acaaacaaga tcactccaca agaccaaata tcggcttttg caccttcata
acctttcctt 1320gtcaaaacct caggcgccaa ataagctggt gtcccgcaaa
acgtttgaca gattccttct 1380tgcttgagct gctccgagac aacgctgagc
ccaaagtcag agactttcac gttccccttg 1440tcgtccaaaa gcagattctc
aagcttgaga tcgcggtggt aaacaccgcg gctgtggcag 1500aaagcaacgg
atgagatcaa ctgctggaaa tatctcctcg cggttccttc tcgaagccgt
1560cctctagcca ccgtattata aagctctccg cctcgaacgt actccatcac
aatgtaaatc 1620tttgtcttcg tagccataac ctcgagtagg tgtacaatgt
aagggtggcg gacacggcgg 1680aggattgaaa tctcccgttt aatatgaccg
gccaatccac tcttcactat cttctccttg 1740tcaatgactt tgatggcaac
atcctcgcca gaatgaatgt tccgtgctaa atagacctta 1800gcgaagcttc
cgtggccaag aagctttcct atttcgtact tgtccatgag aatagagcct
1860tgtggagtcc gcggactcct cgggctctct ggagtactgg tctctttgtt
cgtatttttt 1920gtaacgattc gagcaagaag acccgccatg aattgtattg
gcgttgggcc ggggatggcc 1980aacggtgtag atagtacttg agccatccgt
aggctgagac ttttatttag ttctggttgc 2040tctctaagtg taaatgtaac
tgttgtttgt tgattccgac acggttttac cgggaaacga 2100accaaaacaa
gaaaatgaaa tgaagaaacg gacaaaaata agatatggtg gggttgttgt
2160ttcggttgtg atgttgtctt aacttggcct ttttcgtgtt cgttttataa
cagttttcga 2220gttgacttta tcttatgttt cgagaagctg aaaagtcatt
tgattttaaa atattgctat 2280ttgatgttga agttttatcc taatccaaat
attttgccaa cagaataaca cgttggacgg 2340attttcaaat tataaaaggc
aaacttatat gttctatcca tacgcaatgt caactttgga 2400atacatttaa
gctttcttaa aggacagata ataaggttga cttatcaatg aggctgatag
2460ataagcagat catggttcgt taagatgtca tcacacattt tattta
250647502PRTArabidopsis thaliana 47Met Ala Gln Val Leu Ser Thr Pro
Leu Ala Ile Pro Gly Pro Thr Pro 1 5 10 15 Ile Gln Phe Met Ala Gly
Leu Leu Ala Arg Ile Val Thr Lys Asn Thr 20 25 30 Asn Lys Glu Thr
Ser Thr Pro Glu Ser Pro Arg Ser Pro Arg Thr Pro 35 40 45 Gln Gly
Ser Ile Leu Met Asp Lys Tyr Glu Ile Gly Lys Leu Leu Gly 50 55 60
His Gly Ser Phe Ala Lys Val Tyr Leu Ala Arg Asn Ile His Ser Gly 65
70 75 80 Glu Asp Val Ala Ile Lys Val Ile Asp Lys Glu Lys Ile Val
Lys Ser 85 90 95 Gly Leu Ala Gly His Ile Lys Arg Glu Ile Ser Ile
Leu Arg Arg Val 100 105 110 Arg His Pro Tyr Ile Val His Leu Leu Glu
Val Met Ala Thr Lys Thr 115 120 125 Lys Ile Tyr Ile Val Met Glu Tyr
Val Arg Gly Gly Glu Leu Tyr Asn 130 135 140 Thr Val Ala Arg Gly Arg
Leu Arg Glu Gly Thr Ala Arg Arg Tyr Phe 145 150 155 160 Gln Gln Leu
Ile Ser Ser Val Ala Phe Cys His Ser Arg Gly Val Tyr 165 170 175 His
Arg Asp Leu Lys Leu Glu Asn Leu Leu Leu Asp Asp Lys Gly Asn 180 185
190 Val Lys Val Ser Asp Phe Gly Leu Ser Val Val Ser Glu Gln Leu Lys
195 200 205 Gln Glu Gly Ile Cys Gln Thr Phe Cys Gly Thr Pro Ala Tyr
Leu Ala 210 215 220 Pro Glu Val Leu Thr Arg Lys Gly Tyr Glu Gly Ala
Lys Ala Asp Ile 225 230 235 240 Trp Ser Cys Gly Val Ile Leu Phe Val
Leu Met Ala Gly Tyr Leu Pro 245 250 255 Phe Asp Asp Lys Asn Ile Leu
Val Met Tyr Thr Lys Ile Tyr Lys Gly 260 265 270 Gln Phe Lys Cys Pro
Lys Trp Phe Ser Pro Glu Leu Ala Arg Leu Val 275 280 285 Thr Arg Met
Leu Asp Thr Asn Pro Asp Thr Arg Ile Thr Ile Pro Glu 290 295 300 Ile
Met Lys His Arg Trp Phe Lys Lys Gly Phe Lys His Val Lys Phe 305 310
315 320 Tyr Ile Glu Asn Asp Lys Leu Cys Arg Glu Asp Asp Asp Asn Asp
Asp 325 330 335 Asp Asp Ser Ser Ser Leu Ser Ser Gly Arg Ser Ser Thr
Ala Ser Glu 340 345 350 Gly Asp Ala Glu Phe Asp Ile Lys Arg Val Asp
Ser Met Pro Arg Pro 355 360 365 Ala Ser Leu Asn Ala Phe Asp Ile Leu
Ser Phe Ser Asp Leu Ser Gly 370 375 380 Leu Phe Glu Glu Gly Gly Gln
Gly Ala Arg Phe Val Ser Ala Ala Pro 385 390 395 400 Met Thr Lys Ile
Ile Ser Lys Leu Glu Glu Ile Ala Lys Glu Val Lys 405 410 415 Phe Met
Val Arg Lys Lys Asp Trp Ser Val Arg Leu Glu Gly Cys Arg 420 425 430
Glu Gly Ala Lys Gly Pro Leu Thr Ile Arg Val Glu Ile Phe Glu Leu 435
440 445 Thr Pro Ser Leu Val Val Val Glu Val Lys Lys Lys Gly Gly Asn
Ile 450 455 460 Glu Glu Tyr Glu Glu Phe Cys Asn Lys Glu Leu Arg Pro
Gln Leu Glu 465 470 475 480 Lys Leu Met His Tyr Gln Ala Asp Glu Val
Glu Glu Val Met Cys Leu 485 490 495 Pro Pro Glu Ile Glu Gln 500
4829DNAArtificial sequence3' PCR Primer for Amplification of DNA
Molecule embedding coding region as shown in SEQ ID NO 1
48gacccgggat gctgatggcg accgtctcg 294929DNAArtificial Sequence3'
PCR Primer for Amplification of DNA Molecule embedding coding
region as shown in SEQ ID NO 1 49ctaagcttac ctttcaacct tctcactca
29501766DNAOryza sativa 50tttcatttgg agaggacaca gaaaaatttg
ctacattgtt tcacaaactt caaatattat 60tcatttattt gtcagctttc aaactctttg
tttcttgttt gttgattaga tcaattcgcc 120cttgacccgg gatgctgatg
gcgaccgtct cgccggcgcg gagggagccg acgccgcagg 180cggtgcgggc
gtccccgatg ccatcggcgg cggcggcgtt ggtgaggaga ggcggtggtg
240gtagcggggg gacggtgctg gggaagtacg agctggggcg cgtcctggga
cagggctcgt 300tcgcgaaggt gtaccaggcg aggcacctgg agaccgacga
gtgcgtggca atcaaggtgc 360tcgacaagga gaaggccgtg aagggcggga
tggtccacct cgtcaagcgc gagatcaacg 420tgctccgccg ggtgcgccac
ccgaacatcg tgcagctgtt cgaggtaatg gccagcaaga 480ccaagatcta
cttcgtcatg gagtatgtcc ccggcggcga gctcttctcc cgcgtctcca
540agggacgcct cagggaggac accgcgcggc gctactccca gcagcttgtc
tccgccgtcg 600acttctgcca cgcccgcggc gtgttccacc gtgacctcaa
gcccgagaac ctcctcgtgg 660atgagaacgg ggacttgaag gtctcggact
tcggcctcgc cgccggcccc gaccagttcg 720accccgacgg tctgctccac
acgttctgcg gcacgccggc ctacgtcgcc cccgaggtgc 780tcaggcgccg
cggatacgac ggcgccaagg cggacatatg gtcatgcggt gtcatcctct
840ttgcgctcat ggccgggtac ctccctttcc atgaccacaa catcatggtt
ctgtaccgga 900agatctacaa tggggagttc aggtgtccaa ggtggttctc
caaggatttt actagattga 960taacgcgcct tcttgacgca aaccccaaaa
ctaggatcac cgtgccagag atcattgaga 1020gcgattggtt caagaaagga
tacaagccag tcaagtttta cattgaggat gacaagctct 1080acaacctgtc
tgatgacgtg ctgaacttgg agcctgctga tcctgttccc ccaccattgg
1140gtttggcacc tcctgttcct ccacctccac aaggggatga tcctgatggt
tcagggtctg 1200agtcagattc atcagtcgta tcctgcccgg ccacattgtc
aactggggag agccagagag 1260tccgtgggtc actaccacgc ccagcaagcc
ttaatgcatt tgatatcata tcattctcaa 1320aaggattcaa cttgtctggg
ctgtttgagg agagggggaa cgagatcagg tttgtatctg 1380gtgagcccat
gtctgacatt gtaaaaaagc tggaggagat tgcaaaggtc aagagcttca
1440cagtgcggag gaaggactgg cgggtgagca tagagggtac acgcgaagga
gttaaggggc 1500ctctaaccat aggcgcggag atatttgagc ttacactctc
ccttgtagta gtggaagtaa 1560aaagaaaggc aggtgataat gaagagtatg
aggatttctg caacatggag ttgaagccag 1620gaatgcagca ccttgtgcac
cagatgctcc cagctccaaa tggaactcct gtgagtgaga 1680aggttgaaag
gtaagcttag aagggcgaat taattcctcg agcgattagg atgatgataa
1740gtaagtcgac ctagttagtt aattca 176651520PRTOryza sativa 51Met Leu
Met Ala Thr Val Ser Pro Ala Arg Arg Glu Pro Thr Pro Gln 1 5 10 15
Ala Val Arg Ala Ser Pro Met Pro Ser Ala Ala Ala Ala Leu Val Arg 20
25 30 Arg Gly Gly Gly Gly Ser Gly Gly Thr Val Leu Gly Lys Tyr Glu
Leu 35 40 45 Gly Arg Val Leu Gly Gln Gly Ser Phe Ala Lys Val Tyr
Gln Ala Arg 50 55 60 His Leu Glu Thr Asp Glu Cys Val Ala Ile Lys
Val Leu Asp Lys Glu 65 70 75 80 Lys Ala Val Lys Gly Gly Met Val His
Leu Val Lys Arg Glu Ile Asn 85 90 95 Val Leu Arg Arg Val Arg His
Pro Asn Ile Val Gln Leu Phe Glu Val 100 105 110 Met Ala Ser Lys Thr
Lys Ile Tyr Phe Val Met Glu Tyr Val Pro Gly 115 120 125 Gly Glu Leu
Phe Ser Arg Val Ser Lys Gly Arg Leu Arg Glu Asp Thr 130 135 140 Ala
Arg Arg Tyr Ser Gln Gln Leu Val Ser Ala Val Asp Phe Cys His 145 150
155 160 Ala Arg Gly Val Phe His Arg Asp Leu Lys Pro Glu Asn Leu Leu
Val 165 170 175 Asp Glu Asn Gly Asp Leu Lys Val Ser Asp Phe Gly Leu
Ala Ala Gly 180 185 190 Pro Asp Gln Phe Asp Pro Asp Gly Leu Leu His
Thr Phe Cys Gly Thr 195 200 205 Pro Ala Tyr Val Ala Pro Glu Val Leu
Arg Arg Arg Gly Tyr Asp Gly 210 215 220 Ala Lys Ala Asp Ile Trp Ser
Cys Gly Val Ile Leu Phe Ala Leu Met 225 230 235 240 Ala Gly Tyr Leu
Pro Phe His Asp His Asn Ile Met Val Leu Tyr Arg 245 250 255 Lys Ile
Tyr Asn Gly Glu Phe Arg Cys Pro Arg Trp Phe Ser Lys Asp 260 265 270
Phe Thr Arg Leu Ile Thr Arg Leu Leu Asp Ala Asn Pro Lys Thr Arg 275
280 285 Ile Thr Val Pro Glu Ile Ile Glu Ser Asp Trp Phe Lys Lys Gly
Tyr 290 295 300 Lys Pro Val Lys Phe Tyr Ile Glu Asp Asp Lys Leu Tyr
Asn Leu Ser 305 310 315 320 Asp Asp Val Leu Asn Leu Glu Pro Ala Asp
Pro Val Pro Pro Pro Leu 325 330 335 Gly Leu Ala Pro Pro Val Pro Pro
Pro Pro Gln Gly Asp Asp Pro Asp 340 345 350 Gly Ser Gly Ser Glu Ser
Asp Ser Ser Val Val Ser Cys Pro Ala Thr 355 360 365 Leu Ser Thr Gly
Glu Ser Gln Arg Val Arg Gly Ser Leu Pro Arg Pro 370 375 380 Ala Ser
Leu Asn Ala Phe Asp Ile Ile Ser Phe Ser Lys Gly Phe Asn 385 390 395
400 Leu Ser Gly Leu Phe Glu Glu Arg Gly Asn Glu Ile Arg Phe Val Ser
405 410 415 Gly Glu Pro Met Ser Asp Ile Val Lys Lys Leu Glu Glu Ile
Ala Lys 420 425 430 Val Lys Ser Phe Thr Val Arg Arg Lys Asp Trp Arg
Val Ser Ile Glu 435 440 445 Gly Thr Arg Glu Gly Val Lys Gly Pro Leu
Thr Ile Gly Ala Glu Ile 450 455 460 Phe Glu Leu Thr Leu Ser Leu Val
Val Val Glu Val Lys Arg Lys Ala 465 470 475 480 Gly Asp Asn Glu Glu
Tyr Glu Asp Phe Cys Asn Met Glu Leu Lys Pro 485 490 495 Gly Met Gln
His Leu Val His Gln Met Leu Pro Ala Pro Asn Gly Thr 500 505 510 Pro
Val Ser Glu Lys Val Glu Arg 515 520
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