U.S. patent application number 12/045098 was filed with the patent office on 2008-09-11 for manipulation of ammonium transporters (amts) to improve nitrogen use efficiency in higher plants.
This patent application is currently assigned to PIONEER HI-BRED INTERNATIONAL, INC.. Invention is credited to Kanwarpal S. Dhugga, Rajeev Gupta, Juan Liu, Carl R. Simmons.
Application Number | 20080222753 12/045098 |
Document ID | / |
Family ID | 39615696 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080222753 |
Kind Code |
A1 |
Gupta; Rajeev ; et
al. |
September 11, 2008 |
Manipulation of Ammonium Transporters (AMTS) to Improve Nitrogen
Use Efficiency in Higher Plants
Abstract
The present invention provides polynucleotides and related
polypeptides of the protein AMT. The invention provides genomic
sequence for the AMT gene. AMT is responsible for controlling
nitrogen utilization efficiency in plants.
Inventors: |
Gupta; Rajeev; (Johnston,
IA) ; Liu; Juan; (Johnston, IA) ; Dhugga;
Kanwarpal S.; (Johnston, IA) ; Simmons; Carl R.;
(Des Moines, IA) |
Correspondence
Address: |
PIONEER HI-BRED INTERNATIONAL, INC.
7250 N.W. 62ND AVENUE, P.O. BOX 552
JOHNSTON
IA
50131-0552
US
|
Assignee: |
PIONEER HI-BRED INTERNATIONAL,
INC.
Johnston
IA
|
Family ID: |
39615696 |
Appl. No.: |
12/045098 |
Filed: |
March 10, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60893901 |
Mar 9, 2007 |
|
|
|
Current U.S.
Class: |
800/278 ;
435/358; 435/468; 536/23.6; 800/298; 800/312; 800/314; 800/320;
800/320.1; 800/320.2; 800/320.3; 800/322 |
Current CPC
Class: |
C12N 15/8261 20130101;
C07K 14/415 20130101; Y02A 40/146 20180101 |
Class at
Publication: |
800/278 ;
536/23.6; 435/358; 800/298; 800/320.1; 800/312; 800/322; 800/320;
800/320.3; 800/314; 800/320.2; 435/468 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C12N 15/29 20060101 C12N015/29; C12N 5/10 20060101
C12N005/10; A01H 5/00 20060101 A01H005/00 |
Claims
1. An isolated polynucleotide selected from the group consisting
of: a. a polynucleotide having at least 70% sequence identity, as
determined by the GAP algorithm under default parameters, to the
full length sequence of a polynucleotide selected from the group
consisting of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21,
23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55,
57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79 or 81; wherein the
polynucleotide encodes a polypeptide that functions as a modifier
of AMT; b. a polynucleotide encoding a polypeptide selected from
the group consisting of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52,
54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80 or 82; c. a
polynucleotide selected from the group consisting of SEQ ID NOS: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,
73, 75, 77, 79 or 81; and d. A polynucleotide which is
complementary to the polynucleotide of (a), (b) or (c).
2. A recombinant expression cassette, comprising the polynucleotide
of claim 1, wherein the polynucleotide is operably linked, in sense
or anti-sense orientation, to a promoter.
3. A host cell comprising the expression cassette of claim 2.
4. A transgenic plant comprising the recombinant expression
cassette of claim 2.
5. The transgenic plant of claim 4, wherein said plant is a
monocot.
6. The transgenic plant of claim 4, wherein said plant is a
dicot.
7. The transgenic plant of claim 4, wherein said plant is selected
from the group consisting of: maize, soybean, sunflower, sorghum,
canola, wheat, alfalfa, cotton, rice, barley, millet, peanut,
switchgrass, myscanthus, triticale and cocoa.
8. A transgenic seed from the transgenic plant of claim 4.
9. A method of modulating the AMT in plants, comprising: a.
introducing into a plant cell a recombinant expression cassette
comprising the polynucleotide of claim 1 operably linked to a
promoter; and b. culturing the plant under plant cell growing
conditions; wherein the AMT in said plant cell is modulated.
10. The method of claim 9, wherein the plant cell is from a plant
selected from the group consisting of: maize, soybean, sunflower,
sorghum, canola, wheat, alfalfa, cotton, rice, barley, millet,
peanut, switchgrass, myscanthus, triticale and cocoa.
11. A method of modulating the AMT in a plant, comprising: a.
introducing into a plant cell a recombinant expression cassette
comprising the polynucleotide of claim 1 operably linked to a
promoter; b. culturing the plant cell under plant cell growing
conditions; and c. regenerating a plant form said plant cell;
wherein the AMT in said plant is modulated.
12. The method of claim 11, wherein the plant is selected from the
group consisting of: maize, soybean, sorghum, canola, wheat,
alfalfa, cotton, rice, barley, millet, peanut, switchgrass,
myscanthus, triticale and cocoa.
13. A method of decreasing the AMT transporter polypeptide activity
in a plant cell, comprising: a. providing a nucleotide sequence
comprising at least 15 consecutive nucleotides of the complement of
SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29,
31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63,
65, 67, 69, 71, 73, 75, 77, 79 or 81; b. providing a plant cell
comprising an mRNA having the sequence set forth in SEQ ID NO: 1,
3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37,
39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71,
73, 75, 77, 79 or 81; and c. introducing the nucleotide sequence of
step (a) into the plant cell, wherein the nucleotide sequence
inhibits expression of the mRNA in the plant cell.
14. The method of claim 13, wherein said plant cell is from a
monocot.
15. The method of claim 14, wherein said monocot is maize, wheat,
rice, barley, sorghum, switchgrass, myscanthus, triticale or
rye.
16. The method of claim 13, wherein said plant cell is from a
dicot.
17. The transgenic plant of claim 4, wherein the AMT transporter
activity in said plant is decreased.
18. The transgenic plant of claim 17, wherein the plant has
enhanced root growth.
19. The transgenic plant of claim 17, wherein the plant has
increased seed size.
20. The transgenic plant of claim 17, wherein the plant has
increased seed weight.
21. The transgenic plant of claim 17, wherein the plant has seed
with increased embryo size.
22. The transgenic plant of claim 17, wherein the plant has
increased leaf size.
23. The transgenic plant of claim 17, wherein the plant has
increased seedling vigor.
24. The transgenic plant of claim 17, wherein the plant has
enhanced silk emergence.
25. The transgenic plant of claim 17, wherein the plant has
increased ear size.
26. The transgenic plant of claim 4, wherein the AMT transporter
activity in said plant is increased.
Description
CROSS REFERENCE
[0001] This utility application claims the benefit U.S. Provisional
Application No. 60/893,901, filed Mar. 9, 2007, which is
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention relates generally to the field of molecular
biology.
BACKGROUND OF THE INVENTION
[0003] Nitrogen (N) is the most abundant inorganic nutrient taken
up from the soil by plants for growth and development. Maize roots
absorb most of the N from the soil in the form of nitrate, the
majority of which is transported to the leaf for reduction and
assimilation. Nitrate is reduced to nitrite by nitrate reductase
(NR) in the cytosol and then nitrite is transported into
chloroplast where it is reduced by nitrite reductase (NiR) to
ammonium. Ammonium is assimilated into glutamine by the glutamine
synthase-glutamate synthase system (Crawford and Glass, (1998)
Trends in Plant Science 3:389-395.). Also, it has long been known
that significant amounts of N are lost from the plant aerial parts
by volatilization (Glyan'ko, et al., (1980) "Effect of autumn frost
and forms of nitrogen on translocation of nitrogen compounds to
spring wheat grain", Agrokhimiya 8:19-26; Hooker, et al., (1980)
"Gaseous N losses from winter wheat", Agronomy Journal
72(5):789-792; Silva, et al., (1981) "Nitrogen volatilization from
rice leaves. II. Effects of source of applied nitrogen in nutrient
culture solution", Crop Science 21(6): 913-916; Stutte, et al.,
(1981) "Nitrogen volatilization from rice leaves. I. Effects of
genotype and air temperature", Crop Science 21(4):596-600; Foster,
et al., (1986) "Glutamine synthetase activity and foliar nitrogen
volatilization in response to temperature and inhibitor chemicals"
Annals of Botany 57(3):305-307; Parton, et al., (1988) "Ammonia
volatilization from spring wheat plants" Agronomy Journal
80(3):419-425; Kamiji, et al., (1989) "Measurement of ammonium
nitrogen volatilization rates from rice leaves during the ripening
period." Japanese Journal of Crop Science 58(1):140-142; Morgan, et
al., (1989) "Characteristics of ammonia volatilization from spring
wheat", Crop Science 29(3):726-731; O'Deen, (1989) "Wheat
volatilized ammonia and resulting nitrogen isotopic fractionation."
Agronomy Journal 81(6):980-985; Guindo, et al., (1994) "Nitrogen
loss from rice plants during grain fill and oven drying", Arkansas
Farm Research 43(1):12-13; Heckathorn, et al., (1995) "Ammonia
volatilization during drought in perennial C4 grasses of tallgrass
prairie." Oecologia 101(3):361-365; Cabezas, et al., (1997). "NH3-N
volatilization in a maize crop: I Effect of irrigation and partial
substitution of urea by ammonium sulphate", Revista Brasileira de
Ciencia do Solo 21(3):481-487). Experimental evidence supports the
loss of N through ammonium and not through N oxides (Hooker, et
al., 1980). Treatment with chemicals that inhibit glutamine or
glutamate synthase activities led to increased loss of ammonium
through volatilization (Foster, et al., 1986). Loss of N is not
only limited to C-3 species as C-4 plants have also been reported
to lose N through volatilization (Heckathorn, et al., 1995).
[0004] Manipulation of AMTs can be utilized to improve NUE by
causing increased dry matter, thereby contributing to an increase
in plant yield. Two of the ways to improved dry matter accumulation
are: 1) reduce N loss through volatilization and 2) reduce N
content of the plant so that more dry matter can be accumulated in
the form of low-energy constituents, e.g., starch or cellulose.
[0005] For ammonium to be lost from the leaf, it must first pass
through a facilitated channel since it is highly hydrophilic.
Ammonium transporters (AMTs) were originally discovered as ammonium
transporters but some recent studies have shown that at least in
some cases AMTs can act as gas channels (Soupene, et al., (2002)
Proc Natl Acad Sci USA 99:3926-3931; Kustu and Inwood, (2006)
Transfus Clin Biol 13:103-110). An amtB knock-out mutant of
Salmonella grows better on poor N source, apparently because it can
sequester more N by keeping it from leaking back out (Soupene, et
al., 2002). This application details an invention which is used to
manipulate AMTs in higher plants to improve NUE. The inventors
identified chloroplast-specific and/or leaf-preferred AMT(s) and
knocked them out/down to minimize the loss of ammonium, which
resulting in better N assimilation/NUE. In addition, work was not
limited only to the chloroplast-localized AMTs but will also
down-regulation of the AMTs that are localized to other
organelles/membranes.
SUMMARY OF THE INVENTION
[0006] The present invention provides polynucleotides, related
polypeptides and all conservatively modified variants of the
present AMT sequences. The invention provides sequences for the AMT
genes. Six Arabidopsis, 7 maize, 17 rice, and 11 soybean AMT genes
were identified. Table 1 lists these genes and their seq id
numbers.
TABLE-US-00001 TABLE 1 SEQUENCE ID NUMBER IDENTITY SEQ ID NOS: 1
AtAMT 1 polynucleotide SEQ ID NOS: 2 AtAMT 1 polypeptide SEQ ID NO:
3 AtAMT 1;2 polynucleotide SEQ ID NO: 4 AtAMT 1;2 polypeptide SEQ
ID NO: 5 AtAMT 1;3 polynucleotide SEQ ID NO: 6 AtAMT 1;3
polypeptide SEQ ID NO: 7 AtAMT 2 polynucleotide SEQ ID NO: 8 AtAMT
2 polypeptide SEQ ID NO: 9 AtAMT 3 polynucleotide SEQ ID NO: 10
AtAMT 3 polypeptide SEQ ID NO: 11 AtAMT 4 polynucleotide SEQ ID NO:
12 AtAMT 4 polypeptide SEQ ID NO: 13 ZmAMT 1 polynucleotide SEQ ID
NO: 14 ZmAMT 1 polypeptide SEQ ID NO: 15 ZmAMT 2 polynucleotide SEQ
ID NO: 16 ZmAMT 2 polypeptide SEQ ID NO: 17 ZmAMT 3 polynucleotide
SEQ ID NO: 18 ZmAMT 3 polypeptide SEQ ID NO: 19 ZmAMT 4
polynucleotide SEQ ID NO: 20 ZmAMT 4 polypeptide SEQ ID NO: 21
ZmAMT 5 polynucleotide SEQ ID NO: 22 ZmAMT 5 polypeptide SEQ ID NO:
23 ZmAMT 6 polynucleotide SEQ ID NO: 24 ZmAMT 6 polypeptide SEQ ID
NO: 25 ZmAMT 7 polynucleotide SEQ ID NO: 26 ZmAMT 7 polypeptide SEQ
ID NO: 27 OsAMT 1 polynucleotide SEQ ID NO: 28 OsAMT 1 polypeptide
SEQ ID NO: 29 OsAMT 2 polynucleotide SEQ ID NO: 30 OsAMT 2
polypeptide SEQ ID NO: 31 OsAMT 3 polynucleotide SEQ ID NO: 32
OsAMT 3 polypeptide SEQ ID NO: 33 OsAMT 4 polynucleotide SEQ ID NO:
34 OsAMT 4 polypeptide SEQ ID NO: 35 OsAMT 5 polynucleotide SEQ ID
NO: 36 OsAMT 5 polypeptide SEQ ID NO: 37 OsAMT 6 polynucleotide SEQ
ID NO: 38 OsAMT 6 polypeptide SEQ ID NO: 39 OsAMT 7 polynucleotide
SEQ ID NO: 40 OsAMT 7 polypeptide SEQ ID NO: 41 OsAMT 8
polynucleotide SEQ ID NO: 42 OsAMT 8 polypeptide SEQ ID NO: 43
OsAMT 9 polynucleotide SEQ ID NO: 44 OsAMT 9 polypeptide SEQ ID NO:
45 OsAMT 10 polynucleotide SEQ ID NO: 46 OsAMT 10 polypeptide SEQ
ID NO: 47 OsAMT 11 polynucleotide SEQ ID NO: 48 OsAMT 11
polypeptide SEQ ID NO: 49 OsAMT 12 polynucleotide SEQ ID NO: 50
OsAMT 12 polypeptide SEQ ID NO: 51 OsAMT 13 polynucleotide SEQ ID
NO: 52 OsAMT 13 polypeptide SEQ ID NO: 53 OsAMT 14 polynucleotide
SEQ ID NO: 54 OsAMT 14 polypeptide SEQ ID NO: 55 OsAMT 15
polynucleotide SEQ ID NO: 56 OsAMT 15 polypeptide SEQ ID NO: 57
OsAMT 16 polynucleotide SEQ ID NO: 58 OsAMT 16 polypeptide SEQ ID
NO: 59 OsAMT 17 polynucleotide SEQ ID NO: 60 OsAMT 17
polynucleotide SEQ ID NO: 61 GmAMT 1 polynucleotide SEQ ID NO: 62
GmAMT 1 polypeptide SEQ ID NO: 63 GmAMT 2 polynucleotide SEQ ID NO:
64 GmAMT 2 polypeptide SEQ ID NO: 65 GmAMT 3 polynucleotide SEQ ID
NO: 66 GmAMT 3 polypeptide SEQ ID NO: 67 GmAMT 4 polynucleotide SEQ
ID NO: 68 GmAMT 4 polypeptide SEQ ID NO: 69 GmAMT 5 polynucleotide
SEQ ID NO: 70 GmAMT 5 polypeptide SEQ ID NO: 71 GmAMT 6
polynucleotide SEQ ID NO: 72 GmAMT 6 polypeptide SEQ ID NO: 73
GmAMT 7 polynucleotide SEQ ID NO: 74 GmAMT 7 polypeptide SEQ ID NO:
75 GmAMT 8 polynucleotide SEQ ID NO: 76 GmAMT 8 polypeptide SEQ ID
NO: 77 GmAMT 9 polynucleotide SEQ ID NO: 78 GmAMT 9 polypeptide SEQ
ID NO: 79 GmAMT 10 polynucleotide SEQ ID NO: 80 GmAMT 10
polypeptide SEQ ID NO: 81 GmAMT 11 polynucleotide SEQ ID NO: 82
GmAMT 11 polypeptide
[0007] Therefore, in one aspect, the present invention relates to
an isolated nucleic acid comprising an isolated polynucleotide
sequence encoding an AMT protein. One embodiment of the invention
is an isolated polynucleotide comprising a nucleotide sequence
selected from the group consisting of: (a) the nucleotide sequence
comprising SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57,
59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79 or 81; (b) the
nucleotide sequence encoding an amino acid sequence comprising SEQ
ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, 72, 74, 76, 78, 80 or 82; and (c) the nucleotide sequence
comprising at least 70% sequence identity to SEQ ID NO: 1, 3, 5, 7,
9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75,
77, 79 or 81, wherein said polynucleotide encodes a polypeptide
having AMT transporter activity.
[0008] Compositions of the invention include an isolated
polypeptide comprising an amino acid sequence selected from the
group consisting of: (a) the amino acid sequence comprising SEQ ID
NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34,
36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, 78, 80 or 82; and (b) the amino acid sequence
comprising at least 70% sequence identity to SEQ ID NO: 2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42,
44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76,
78, 80 or 82, wherein said polypeptide has AMT transporter
activity.
[0009] In another aspect, the present invention relates to a
recombinant expression cassette comprising a nucleic acid as
described. Additionally, the present invention relates to a vector
containing the recombinant expression cassette. Further, the vector
containing the recombinant expression cassette can facilitate the
transcription and translation of the nucleic acid in a host cell.
The present invention also relates to the host cells able to
express the polynucleotide of the present invention. A number of
host cells could be used, such as but not limited to, microbial,
mammalian, plant, or insect.
[0010] In yet another embodiment, the present invention is directed
to a transgenic plant or plant cells, containing the nucleic acids
of the present invention. Preferred plants containing the
polynucleotides of the present invention include but are not
limited to maize, soybean, sunflower, sorghum, canola, wheat,
alfalfa, cotton, rice, barley, tomato, switchgrass, myscanthus,
triticale and millet. In another embodiment, the transgenic plant
is a maize plant or plant cells. Another embodiment is the
transgenic seeds from the transgenic plant. Another embodiment of
the invention includes plants comprising an amt polypeptide of the
invention operably linked to a promoter that drives expression in
the plant. The plants of the invention can have altered AMT as
compared to a control plant. In some plants, the AMT is altered in
a vegetative tissue, a reproductive tissue, or a vegetative tissue
and a reproductive tissue. Plants of the invention can have at
least one of the following phenotypes including but not limited to:
increased leaf size, increased ear size, increased seed size,
increased endosperm size, alterations in the relative size of
embryos and endosperms leading to changes in the relative levels of
protein, oil, and/or starch in the seeds, absence of tassels,
absence of functional pollen bearing tassels, or increased plant
size.
[0011] Another embodiment of the invention would be plants that
have been genetically modified at a genomic locus, wherein the
genomic locus encodes an amt polypeptide of the invention.
[0012] Methods for increasing the activity of an amt polypeptide in
a plant are provided. The method can comprise introducing into the
plant an amt polynucleotide of the invention. Providing the
polypeptide can decrease the number of cells in plant tissue,
modulating the tissue growth and size.
[0013] Methods for reducing or eliminating the level of an amt
polypeptide in the plant are provided. The level or activity of the
polypeptide could also be reduced or eliminated in specific
tissues, causing increased AMT in said tissues. Reducing the level
and/or activity of the AMT polypeptide increases the number of
cells produced in the associated tissue.
[0014] Compositions further include plants and seed having a DNA
construct comprising a nucleotide sequence of interest operably
linked to a promoter of the current invention. In specific
embodiments, the DNA construct is stably integrated into the genome
of the plant. The method comprises introducing into a plant a
nucleotide sequence of interest operably linked to a promoter of
the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1: Phylogentic tree of AMTs from Arabidopsis, rice,
soybean and maize
[0016] Phylogenetic analyses of all the AMTs from Arabidopsis,
rice, maize and soybean are shown in FIG. 1. The length of the line
at the base of the figure represents an equivalent of 10 amino acid
differences and could be used to approximate the amino acid
differences between different ammonium transporter proteins from
the individual branch lengths.
[0017] FIG. 2: Expression analysis of ZM-AMTs
[0018] In order to identify leaf specific/preferred/expressed
AMT(s) in maize, Lynx MPSS expression analyses in .about.300
libraries reveal that ZmAMT1 (SEQ ID NO: 14), 2, 7 are expressed
both in roots and leaves whereas ZmAMT4 (SEQ ID NO: 20) is a root
preferred AMT. ZmAMT6 (SEQ ID NO: 24) expresses at very low level
in comparison to other Zm-AMTs. In case of ZmAMT5 there was no
specific Lynx tag available.
[0019] FIG. 3: Characterization of atamt1;2 T-DNA knock-out
mutant
[0020] In cTP prediction analyses, AtAMT1;2 (SEQ ID NO: 4) posses a
putative cTP. For functional analyses of AtAMT1;2 (SEQ ID NO: 4)
and to determine it's role in N-assimilation, analyses identified a
T-DNA mutant line (SM.sub.--3.15680) from the Arabidopsis T-DNA
mutant data base. In this mutant line T-DNA was inserted in
c-terminal of AtAMT1;2 (SEQ ID NO: 4) gene (FIG. 4A). Genomic PCRs
using AtAMT1;2 (SEQ ID NO: 4) gene and T-DNA specific primers show
that T-DNA is indeed inserted in the AtAMT1;2 (SEQ ID NO: 4) (FIG.
4B). AtAMT1;2 (SEQ ID NO: 4) gene specific primers flanking the
T-DNA insert couldn't amplify any DNA region in mutant plants where
as an expected PCR product was detected in wild type plant (FIG.
4B, upper panel). Similarly, genomic PCR with AtAMT1;2 (SEQ ID NO:
4) specific forward primer and T-DNA specific reverse primers
amplify an expected product in mutant lines and nothing in wild
type plants as expected (FIG. 4B, lower panel). Saturated RT-PCRs
(35 cycles) analyses couldn't detect a full length atamt1;2 mRNA in
mutant (FIG. 4C, upper panel) suggesting that AtAMT1;2 (SEQ ID NO:
4) is completely knocked out in this T-DNA mutant. Actin control
RT-PCR worked fine in both mutant and wild type plants (FIG. 4C,
lower panel).
[0021] FIG. 4: Knock-out of multiple AMTs in Arabidopsis by single
RNAi vector
[0022] Six AMT genes are present in Arabidopsis genome. Hence, it
is very likely that due to functional redundancy one might need to
manipulate the expression of multiple AMTs simultaneously. Analyses
of the DNA sequence of all these AMTs was performed which
identified the high homology regions among them. There is a stretch
of .about.200 bp among AtAMT1;2 (SEQ ID NO: 4), AtAMT1 (SEQ ID NO:
2), AMT1;3 (SEQ ID NO: 6), At3g24290 (SEQ ID NO: 10) and At4g28700
(SEQ ID NO: 12) where as AMT2 (SEQ ID NO: 8) stood independent.
Amplification of these regions was accomplished (bold and
underlined in FIG. 4) by PCR from AtAMT1;2 (SEQ ID NO: 4) and
AtAMT2 (SEQ ID NO: 8) and a multi-way ligation was performed to
make an inverted repeat using ADH-intron as a spacer. The RNAi
cassette of these hybrid inverted repeats is driven by constitutive
or root specific or leaf specific promoter.
[0023] FIG. 5: Knock-out/down of multiple AMTs in Maize by single
RNAi vector
[0024] Detailed analyses of all 7 maize AMTs were performed to
identify the DNA regions showing high homology among different
ZmAMTs. This analysis reveals that ZmAMT1 (SEQ ID NO: 14) and
ZmAMT5 (SEQ ID NO: 22), ZmAMT3 (SEQ ID NO: 18) and ZmAMT4 (SEQ ID
NO: 20) and ZmAMT2 (SEQ ID NO: 16), ZmAMT6 (SEQ ID NO: 24) and
ZmAMT7 (SEQ ID NO: 26) form three separate groups and there is a
very high homology in stretches of DNA sequences with in each
group. Three DNA fragments (bold and underlined in FIG. 5) from
ZmAMT 1, 4 and 7 (SEQ ID NOS: 14, 20 and 26) representing each of
the different groups were amplified by PCR. Multi-way ligations
were performed to make inverted repeats with hybrid of these 3
fragments and ADH intron as a spacer to facilitate the formation of
stem-loop structure. This RNAi cassette of `ZmAMT1 (SEQ ID NO:
14):ZmAMT4 (SEQ ID NO: 20):ZmAMT7 (SEQ ID NO: 26)` inverted repeats
was driven by a constitutive (Zm-UBI promoter) or leaf-specific
promoter. MOPAT driven by Zm-UBI promoter was used as herbicide
resistance marker for selected. In addition to that RFP driven by a
pericarp specific promoter LTP2 was also used to sort out the
transgenic seeds (red) from there segregating non-transgenic
seeds.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Unless
mentioned otherwise, the techniques employed or contemplated herein
are standard methodologies well known to one of ordinary skill in
the art. The materials, methods and examples are illustrative only
and not limiting. The following is presented by way of illustration
and is not intended to limit the scope of the invention.
[0026] The present inventions now will be described more fully
hereinafter with reference to the accompanying drawings, in which
some, but not all embodiments of the invention are shown. Indeed,
these inventions may be embodied in many different forms and should
not be construed as limited to the embodiments set forth herein;
rather, these embodiments are provided so that this disclosure will
satisfy applicable legal requirements. Like numbers refer to like
elements throughout.
[0027] Many modifications and other embodiments of the inventions
set forth herein will come to mind to one skilled in the art to
which these inventions pertain having the benefit of the teachings
presented in the foregoing descriptions and the associated
drawings. Therefore, it is to be understood that the inventions are
not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
[0028] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of botany,
microbiology, tissue culture, molecular biology, chemistry,
biochemistry and recombinant DNA technology, which are within the
skill of the art. Such techniques are explained fully in the
literature. See, e.g., Langenheim and Thimann, BOTANY: PLANT
BIOLOGY AND ITS RELATION TO HUMAN AFFAIRS, John Wiley (1982); CELL
CULTURE AND SOMATIC CELL GENETICS OF PLANTS, vol. 1, Vasil, ed.
(1984); Stanier, et al., THE MICROBIAL WORLD, 5.sup.th ed.,
Prentice-Hall (1986); Dhringra and Sinclair, BASIC PLANT PATHOLOGY
METHODS, CRC Press (1985); Maniatis, et al., MOLECULAR CLONING: A
LABORATORY MANUAL (1982); DNA CLONING, vols. I and II, Glover, ed.
(1985); OLIGONUCLEOTIDE SYNTHESIS, Gait, ed. (1984); NUCLEIC ACID
HYBRIDIZATION, Hames and Higgins, eds. (1984); and the series
METHODS IN ENZYMOLOGY, Colowick and Kaplan, eds, Academic Press,
Inc., San Diego, Calif.
[0029] Units, prefixes, and symbols may be denoted in their SI
accepted form. Unless otherwise indicated, nucleic acids are
written left to right in 5' to 3' orientation; amino acid sequences
are written left to right in amino to carboxy orientation,
respectively. Numeric ranges are inclusive of the numbers defining
the range. Amino acids may be referred to herein by either their
commonly known three letter symbols or by the one-letter symbols
recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
Nucleotides, likewise, may be referred to by their commonly
accepted single-letter codes. The terms defined below are more
fully defined by reference to the specification as a whole.
[0030] In describing the present invention, the following terms
will be employed, and are intended to be defined as indicated
below.
[0031] By "microbe" is meant any microorganism (including both
eukaryotic and prokaryotic microorganisms), such as fungi, yeast,
bacteria, actinomycetes, algae and protozoa, as well as other
unicellular structures.
[0032] By "amplified" is meant the construction of multiple copies
of a nucleic acid sequence or multiple copies complementary to the
nucleic acid sequence using at least one of the nucleic acid
sequences as a template. Amplification systems include the
polymerase chain reaction (PCR) system, ligase chain reaction (LCR)
system, nucleic acid sequence based amplification (NASBA, Cangene,
Mississauga, Ontario), Q-Beta Replicase systems,
transcription-based amplification system (TAS), and strand
displacement amplification (SDA). See, e.g., DIAGNOSTIC MOLECULAR
MICROBIOLOGY: PRINCIPLES AND APPLICATIONS, Persing, et al., eds.,
American Society for Microbiology, Washington, D.C. (1993). The
product of amplification is termed an amplicon.
[0033] The term "conservatively modified variants" applies to both
amino acid and nucleic acid sequences. With respect to particular
nucleic acid sequences, conservatively modified variants refer to
those nucleic acids that encode identical or conservatively
modified variants of the amino acid sequences. Because of the
degeneracy of the genetic code, a large number of functionally
identical nucleic acids encode any given protein. For instance, the
codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
Thus, at every position where an alanine is specified by a codon,
the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations" and represent one species
of conservatively modified variation. Every nucleic acid sequence
herein that encodes a polypeptide also describes every possible
silent variation of the nucleic acid. One of ordinary skill will
recognize that each codon in a nucleic acid (except AUG, which is
ordinarily the only codon for methionine; one exception is
Micrococcus rubens, for which GTG is the methionine codon
(Ishizuka, et al., (1993) J. Gen. Microbiol. 139:425-32) can be
modified to yield a functionally identical molecule. Accordingly,
each silent variation of a nucleic acid, which encodes a
polypeptide of the present invention, is implicit in each described
polypeptide sequence and incorporated herein by reference.
[0034] As to amino acid sequences, one of skill will recognize that
individual substitutions, deletions or additions to a nucleic acid,
peptide, polypeptide, or protein sequence which alters, adds or
deletes a single amino acid or a small percentage of amino acids in
the encoded sequence is a "conservatively modified variant" when
the alteration results in the substitution of an amino acid with a
chemically similar amino acid. Thus, any number of amino acid
residues selected from the group of integers consisting of from 1
to 15 can be so altered. Thus, for example, 1, 2, 3, 4, 5, 7 or 10
alterations can be made. Conservatively modified variants typically
provide similar biological activity as the unmodified polypeptide
sequence from which they are derived. For example, substrate
specificity, enzyme activity, or ligand/receptor binding is
generally at least 30%, 40%, 50%, 60%, 70%, 80% or 90%, preferably
60-90% of the native protein for it's native substrate.
Conservative substitution tables providing functionally similar
amino acids are well known in the art.
[0035] The following six groups each contain amino acids that are
conservative substitutions for one another:
[0036] 1) Alanine (A), Serine (S), Threonine (T);
[0037] 2) Aspartic acid (D), Glutamic acid (E);
[0038] 3) Asparagine (N), Glutamine (Q);
[0039] 4) Arginine (R), Lysine (K);
[0040] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0041] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
See also, Creighton, PROTEINS, W.H. Freeman and Co. (1984).
[0042] As used herein, "consisting essentially of" means the
inclusion of additional sequences to an object polynucleotide where
the additional sequences do not selectively hybridize, under
stringent hybridization conditions, to the same cDNA as the
polynucleotide and where the hybridization conditions include a
wash step in 0.1.times.SSC and 0.1% sodium dodecyl sulfate at
65.degree. C.
[0043] By "encoding" or "encoded," with respect to a specified
nucleic acid, is meant comprising the information for translation
into the specified protein. A nucleic acid encoding a protein may
comprise non-translated sequences (e.g., introns) within translated
regions of the nucleic acid, or may lack such intervening
non-translated sequences (e.g., as in cDNA). The information by
which a protein is encoded is specified by the use of codons.
Typically, the amino acid sequence is encoded by the nucleic acid
using the "universal" genetic code. However, variants of the
universal code, such as is present in some plant, animal, and
fungal mitochondria, the bacterium Mycoplasma capricolum (Yamao, et
al., (1985) Proc. Natl. Acad. Sci. USA 82:2306-9), or the ciliate
Macronucleus, may be used when the nucleic acid is expressed using
these organisms.
[0044] When the nucleic acid is prepared or altered synthetically,
advantage can be taken of known codon preferences of the intended
host where the nucleic acid is to be expressed. For example,
although nucleic acid sequences of the present invention may be
expressed in both monocotyledonous and dicotyledonous plant
species, sequences can be modified to account for the specific
codon preferences and GC content preferences of monocotyledonous
plants or dicotyledonous plants as these preferences have been
shown to differ (Murray, et al., (1989) Nucleic Acids Res.
17:477-98 and herein incorporated by reference). Thus, the maize
preferred codon for a particular amino acid might be derived from
known gene sequences from maize. Maize codon usage for 28 genes
from maize plants is listed in Table 4 of Murray, et al.,
supra.
[0045] As used herein, "heterologous" in reference to a nucleic
acid is a nucleic acid that originates from a foreign species, or,
if from the same species, is substantially modified from its native
form in composition and/or genomic locus by deliberate human
intervention. For example, a promoter operably linked to a
heterologous structural gene is from a species different from that
from which the structural gene was derived or, if from the same
species, one or both are substantially modified from their original
form. A heterologous protein may originate from a foreign species
or, if from the same species, is substantially modified from its
original form by deliberate human intervention.
[0046] By "host cell" is meant a cell, which comprises a
heterologous nucleic acid sequence of the invention, which contains
a vector and supports the replication and/or expression of the
expression vector. Host cells may be prokaryotic cells such as E.
coli, or eukaryotic cells such as yeast, insect, plant, amphibian,
or mammalian cells. Preferably, host cells are monocotyledonous or
dicotyledonous plant cells, including but not limited to maize,
sorghum, sunflower, soybean, wheat, alfalfa, rice, cotton, canola,
barley, millet, switchgrass, myscanthus, triticale, and tomato. A
particularly preferred monocotyledonous host cell is a maize host
cell.
[0047] The term "hybridization complex" includes reference to a
duplex nucleic acid structure formed by two single-stranded nucleic
acid sequences selectively hybridized with each other.
[0048] The term "introduced" in the context of inserting a nucleic
acid into a cell, means "transfection" or "transformation" or
"transduction" and includes reference to the incorporation of a
nucleic acid into a eukaryotic or prokaryotic cell where the
nucleic acid may be incorporated into the genome of the cell (e.g.,
chromosome, plasmid, plastid or mitochondrial DNA), converted into
an autonomous replicon, or transiently expressed (e.g., transfected
mRNA).
[0049] The terms "isolated" refers to material, such as a nucleic
acid or a protein, which is substantially or essentially free from
components which normally accompany or interact with it as found in
its naturally occurring environment. The isolated material
optionally comprises material not found with the material in its
natural environment. Nucleic acids, which are "isolated", as
defined herein, are also referred to as "heterologous" nucleic
acids. Unless otherwise stated, the term "AMT nucleic acid" means a
nucleic acid comprising a polynucleotide ("AMT polynucleotide")
encoding a full length or partial length AMT polypeptide.
[0050] As used herein, "nucleic acid" includes reference to a
deoxyribonucleotide or ribonucleotide polymer in either single- or
double-stranded form, and unless otherwise limited, encompasses
known analogues having the essential nature of natural nucleotides
in that they hybridize to single-stranded nucleic acids in a manner
similar to naturally occurring nucleotides (e.g., peptide nucleic
acids).
[0051] By "nucleic acid library" is meant a collection of isolated
DNA or RNA molecules, which comprise and substantially represent
the entire transcribed fraction of a genome of a specified
organism. Construction of exemplary nucleic acid libraries, such as
genomic and cDNA libraries, is taught in standard molecular biology
references such as Berger and Kimmel, GUIDE TO MOLECULAR CLONING
TECHNIQUES, from the series METHODS IN ENZYMOLOGY, vol. 152,
Academic Press, Inc., San Diego, Calif. (1987); Sambrook, et al.,
MOLECULAR CLONING: A LABORATORY MANUAL, 2.sup.nd ed., vols. 1-3
(1989); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, et
al., eds, Current Protocols, a joint venture between Greene
Publishing Associates, Inc. and John Wiley & Sons, Inc. (1994
Supplement).
[0052] As used herein "operably linked" includes reference to a
functional linkage between a first sequence, such as a promoter,
and a second sequence, wherein the promoter sequence initiates and
mediates transcription of the DNA corresponding to the second
sequence. Generally, operably linked means that the nucleic acid
sequences being linked are contiguous and, where necessary to join
two protein coding regions, contiguous and in the same reading
frame.
[0053] As used herein, the term "plant" includes reference to whole
plants, plant organs (e.g., leaves, stems, roots, etc.), seeds and
plant cells and progeny of same. Plant cell, as used herein
includes, without limitation, seeds, suspension cultures, embryos,
meristematic regions, callus tissue, leaves, roots, shoots,
gametophytes, sporophytes, pollen, and microspores. The class of
plants, which can be used in the methods of the invention, is
generally as broad as the class of higher plants amenable to
transformation techniques, including both monocotyledonous and
dicotyledonous plants including species from the genera: Cucurbita,
Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis,
Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot,
Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum,
Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia,
Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus,
Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium,
Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis,
Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Avena,
Hordeum, Secale, Allium, and Triticum. A particularly preferred
plant is Zea mays.
[0054] As used herein, "yield" may include reference to bushels per
acre of a grain crop at harvest, as adjusted for grain moisture
(15% typically for maize, for example). Grain moisture is measured
in the grain at harvest. The adjusted test weight of grain is
determined to be the weight in pounds per bushel, adjusted for
grain moisture level at harvest.
[0055] As used herein, "polynucleotide" includes reference to a
deoxyribopolynucleotide, ribopolynucleotide, or analogs thereof
that have the essential nature of a natural ribonucleotide in that
they hybridize, under stringent hybridization conditions, to
substantially the same nucleotide sequence as naturally occurring
nucleotides and/or allow translation into the same amino acid(s) as
the naturally occurring nucleotide(s). A polynucleotide can be
full-length or a subsequence of a native or heterologous structural
or regulatory gene. Unless otherwise indicated, the term includes
reference to the specified sequence as well as the complementary
sequence thereof. Thus, DNAs or RNAs with backbones modified for
stability or for other reasons are "polynucleotides" as that term
is intended herein. Moreover, DNAs or RNAs comprising unusual
bases, such as inosine, or modified bases, such as tritylated
bases, to name just two examples, are polynucleotides as the term
is used herein. It will be appreciated that a great variety of
modifications have been made to DNA and RNA that serve many useful
purposes known to those of skill in the art. The term
polynucleotide as it is employed herein embraces such chemically,
enzymatically or metabolically modified forms of polynucleotides,
as well as the chemical forms of DNA and RNA characteristic of
viruses and cells, including inter alia, simple and complex
cells.
[0056] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0057] As used herein "promoter" includes reference to a region of
DNA upstream from the start of transcription and involved in
recognition and binding of RNA polymerase and other proteins to
initiate transcription. A "plant promoter" is a promoter capable of
initiating transcription in plant cells. Exemplary plant promoters
include, but are not limited to, those that are obtained from
plants, plant viruses, and bacteria which comprise genes expressed
in plant cells such Agrobacterium or Rhizobium. Examples are
promoters that preferentially initiate transcription in certain
tissues, such as leaves, roots, seeds, fibres, xylem vessels,
tracheids, or sclerenchyma. Such promoters are referred to as
"tissue preferred." A "cell type" specific promoter primarily
drives expression in certain cell types in one or more organs, for
example, vascular cells in roots or leaves. An "inducible" or
"regulatable" promoter is a promoter, which is under environmental
control. Examples of environmental conditions that may effect
transcription by inducible promoters include anaerobic conditions
or the presence of light. Another type of promoter is a
developmentally regulated promoter, for example, a promoter that
drives expression during pollen development. Tissue preferred, cell
type specific, developmentally regulated, and inducible promoters
constitute the class of "non-constitutive" promoters. A
"constitutive" promoter is a promoter, which is active under most
environmental conditions.
[0058] The term "AMT polypeptide" refers to one or more amino acid
sequences. The term is also inclusive of fragments, variants,
homologs, alleles or precursors (e.g., preproproteins or
proproteins) thereof. A "AMT protein" comprises an amt polypeptide.
Unless otherwise stated, the term "AMT nucleic acid" means a
nucleic acid comprising a polynucleotide ("AMT polynucleotide")
encoding an amt polypeptide.
[0059] As used herein "recombinant" includes reference to a cell or
vector, that has been modified by the introduction of a
heterologous nucleic acid or that the cell is derived from a cell
so modified. Thus, for example, recombinant cells express genes
that are not found in identical form within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all as a result of deliberate human intervention; or may have
reduced or eliminated expression of a native gene. The term
"recombinant" as used herein does not encompass the alteration of
the cell or vector by naturally occurring events (e.g., spontaneous
mutation, natural transformation/transduction/transposition) such
as those occurring without deliberate human intervention.
[0060] As used herein, a "recombinant expression cassette" is a
nucleic acid construct, generated recombinantly or synthetically,
with a series of specified nucleic acid elements, which permit
transcription of a particular nucleic acid in a target cell. The
recombinant expression cassette can be incorporated into a plasmid,
chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid
fragment. Typically, the recombinant expression cassette portion of
an expression vector includes, among other sequences, a nucleic
acid to be transcribed, and a promoter.
[0061] The term "residue" or "amino acid residue" or "amino acid"
are used interchangeably herein to refer to an amino acid that is
incorporated into a protein, polypeptide, or peptide (collectively
"protein"). The amino acid may be a naturally occurring amino acid
and, unless otherwise limited, may encompass known analogs of
natural amino acids that can function in a similar manner as
naturally occurring amino acids.
[0062] The term "selectively hybridizes" includes reference to
hybridization, under stringent hybridization conditions, of a
nucleic acid sequence to a specified nucleic acid target sequence
to a detectably greater degree (e.g., at least 2-fold over
background) than its hybridization to non-target nucleic acid
sequences and to the substantial exclusion of non-target nucleic
acids. Selectively hybridizing sequences typically have about at
least 40% sequence identity, preferably 60-90% sequence identity,
and most preferably 100% sequence identity (i.e., complementary)
with each other.
[0063] The terms "stringent conditions" or "stringent hybridization
conditions" include reference to conditions under which a probe
will hybridize to its target sequence, to a detectably greater
degree than other sequences (e.g., at least 2-fold over
background). Stringent conditions are sequence-dependent and will
be different in different circumstances. By controlling the
stringency of the hybridization and/or washing conditions, target
sequences can be identified which can be up to 100% complementary
to the probe (homologous probing). Alternatively, stringency
conditions can be adjusted to allow some mismatching in sequences
so that lower degrees of similarity are detected (heterologous
probing). Optimally, the probe is approximately 500 nucleotides in
length, but can vary greatly in length from less than 500
nucleotides to equal to the entire length of the target
sequence.
[0064] Typically, stringent conditions will be those in which the
salt concentration is less than about 1.5 M Na ion, typically about
0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to
8.3 and the temperature is at least about 30.degree. C. for short
probes (e.g., 10 to 50 nucleotides) and at least about 60.degree.
C. for long probes (e.g., greater than 50 nucleotides). Stringent
conditions may also be achieved with the addition of destabilizing
agents such as formamide or Denhardt's. Exemplary low stringency
conditions include hybridization with a buffer solution of 30 to
35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at
37.degree. C., and a wash in 1.times. to 2.times.SSC
(20.times.SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to
55.degree. C. Exemplary moderate stringency conditions include
hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at
37.degree. C., and a wash in 0.5.times. to 1.times.SSC at 55 to
60.degree. C. Exemplary high stringency conditions include
hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37.degree. C.,
and a wash in 0.1.times.SSC at 60 to 65.degree. C. Specificity is
typically the function of post-hybridization washes, the critical
factors being the ionic strength and temperature of the final wash
solution. For DNA-DNA hybrids, the T.sub.m can be approximated from
the equation of Meinkoth and Wahl, (1984) Anal. Biochem.,
138:267-84: T.sub.m=81.5.degree. C.+16.6 (log M)+0.41 (% GC)-0.61
(% form)-500/L; where M is the molarity of monovalent cations, % GC
is the percentage of guanosine and cytosine nucleotides in the DNA,
% form is the percentage of formamide in the hybridization
solution, and L is the length of the hybrid in base pairs. The
T.sub.m is the temperature (under defined ionic strength and pH) at
which 50% of a complementary target sequence hybridizes to a
perfectly matched probe. T.sub.m is reduced by about 1.degree. C.
for each 1% of mismatching; thus, T.sub.m, hybridization and/or
wash conditions can be adjusted to hybridize to sequences of the
desired identity. For example, if sequences with .gtoreq.90%
identity are sought, the T.sub.m can be decreased 10.degree. C.
Generally, stringent conditions are selected to be about 5.degree.
C. lower than the thermal melting point (T.sub.m) for the specific
sequence and its complement at a defined ionic strength and pH.
However, severely stringent conditions can utilize a hybridization
and/or wash at 1, 2, 3 or 4.degree. C. lower than the thermal
melting point (T.sub.m); moderately stringent conditions can
utilize a hybridization and/or wash at 6, 7, 8, 9 or 10.degree. C.
lower than the thermal melting point (T.sub.m); low stringency
conditions can utilize a hybridization and/or wash at 11, 12, 13,
14, 15 or 20.degree. C. lower than the thermal melting point
(T.sub.m). Using the equation, hybridization and wash compositions,
and desired T.sub.m, those of ordinary skill will understand that
variations in the stringency of hybridization and/or wash solutions
are inherently described. If the desired degree of mismatching
results in a T.sub.m of less than 45.degree. C. (aqueous solution)
or 32.degree. C. (formamide solution) it is preferred to increase
the SSC concentration so that a higher temperature can be used. An
extensive guide to the hybridization of nucleic acids is found in
Tijssen, LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR
BIOLOGY--HYBRIDIZATION WITH NUCLEIC ACID PROBES, part 1, chapter 2,
"Overview of principles of hybridization and the strategy of
nucleic acid probe assays," Elsevier, N.Y. (1993); and CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, chapter 2, Ausubel, et al., eds,
Greene Publishing and Wiley-Interscience, New York (1995). Unless
otherwise stated, in the present application high stringency is
defined as hybridization in 4.times.SSC, 5.times.Denhardt's (5 g
Ficoll, 5 g polyvinylpyrrolidone, 5 g bovine serum albumin in 500
ml of water), 0.1 mg/ml boiled salmon sperm DNA, and 25 mM Na
phosphate at 65.degree. C., and a wash in 0.1.times.SSC, 0.1% SDS
at 65.degree. C.
[0065] As used herein, "transgenic plant" includes reference to a
plant, which comprises within its genome a heterologous
polynucleotide. Generally, the heterologous polynucleotide is
stably integrated within the genome such that the polynucleotide is
passed on to successive generations. The heterologous
polynucleotide may be integrated into the genome alone or as part
of a recombinant expression cassette. "Transgenic" is used herein
to include any cell, cell line, callus, tissue, plant part or
plant, the genotype of which has been altered by the presence of
heterologous nucleic acid including those transgenics initially so
altered as well as those created by sexual crosses or asexual
propagation from the initial transgenic. The term "transgenic" as
used herein does not encompass the alteration of the genome
(chromosomal or extra-chromosomal) by conventional plant breeding
methods or by naturally occurring events such as random
cross-fertilization, non-recombinant viral infection,
non-recombinant bacterial transformation, non-recombinant
transposition, or spontaneous mutation.
[0066] As used herein, "vector" includes reference to a nucleic
acid used in transfection of a host cell and into which can be
inserted a polynucleotide. Vectors are often replicons. Expression
vectors permit transcription of a nucleic acid inserted
therein.
[0067] The following terms are used to describe the sequence
relationships between two or more nucleic acids or polynucleotides
or polypeptides: (a) "reference sequence," (b) "comparison window,"
(c) "sequence identity," (d) "percentage of sequence identity," and
(e) "substantial identity."
[0068] As used herein, "reference sequence" is a defined sequence
used as a basis for sequence comparison. A reference sequence may
be a subset or the entirety of a specified sequence; for example,
as a segment of a full-length cDNA or gene sequence, or the
complete cDNA or gene sequence.
[0069] As used herein, "comparison window" means includes reference
to a contiguous and specified segment of a polynucleotide sequence,
wherein the polynucleotide sequence may be compared to a reference
sequence and wherein the portion of the polynucleotide sequence in
the comparison window may comprise additions or deletions (i.e.,
gaps) compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Generally, the comparison window is at least 20 contiguous
nucleotides in length, and optionally can be 30, 40, 50, 100 or
longer. Those of skill in the art understand that to avoid a high
similarity to a reference sequence due to inclusion of gaps in the
polynucleotide sequence a gap penalty is typically introduced and
is subtracted from the number of matches.
[0070] Methods of alignment of nucleotide and amino acid sequences
for comparison are well known in the art. The local homology
algorithm (BESTFIT) of Smith and Waterman, (1981) Adv. Appl. Math
2:482, may conduct optimal alignment of sequences for comparison;
by the homology alignment algorithm (GAP) of Needleman and Wunsch,
(1970) J. Mol. Biol. 48:443-53; by the search for similarity method
(Tfasta and Fasta) of Pearson and Lipman, (1988) Proc. Natl. Acad.
Sci. USA 85:2444; by computerized implementations of these
algorithms, including, but not limited to: CLUSTAL in the PC/Gene
program by Intelligenetics, Mountain View, Calif., GAP, BESTFIT,
BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Version 8 (available from Genetics Computer Group
(GCG.RTM. programs (Accelrys, Inc., San Diego, Calif.).). The
CLUSTAL program is well described by Higgins and Sharp, (1988) Gene
73:237-44; Higgins and Sharp, (1989) CABIOS 5:151-3; Corpet, et
al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al., (1992)
Computer Applications in the Biosciences 8:155-65, and Pearson, et
al., (1994) Meth. Mol. Biol. 24:307-31. The preferred program to
use for optimal global alignment of multiple sequences is PileUp
(Feng and Doolittle, (1987) J. Mol. Evol., 25:351-60 which is
similar to the method described by Higgins and Sharp, (1989) CABIOS
5:151-53 and hereby incorporated by reference). The BLAST family of
programs which can be used for database similarity searches
includes: BLASTN for nucleotide query sequences against nucleotide
database sequences; BLASTX for nucleotide query sequences against
protein database sequences; BLASTP for protein query sequences
against protein database sequences; TBLASTN for protein query
sequences against nucleotide database sequences; and TBLASTX for
nucleotide query sequences against nucleotide database sequences.
See, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Chapter 19, Ausubel,
et al., eds., Greene Publishing and Wiley-Interscience, New York
(1995).
[0071] GAP uses the algorithm of Needleman and Wunsch, supra, to
find the alignment of two complete sequences that maximizes the
number of matches and minimizes the number of gaps. GAP considers
all possible alignments and gap positions and creates the alignment
with the largest number of matched bases and the fewest gaps. It
allows for the provision of a gap creation penalty and a gap
extension penalty in units of matched bases. GAP must make a profit
of gap creation penalty number of matches for each gap it inserts.
If a gap extension penalty greater than zero is chosen, GAP must,
in addition, make a profit for each gap inserted of the length of
the gap times the gap extension penalty. Default gap creation
penalty values and gap extension penalty values in Version 10 of
the Wisconsin Genetics Software Package are 8 and 2, respectively.
The gap creation and gap extension penalties can be expressed as an
integer selected from the group of integers consisting of from 0 to
100. Thus, for example, the gap creation and gap extension
penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40,
50 or greater.
[0072] GAP presents one member of the family of best alignments.
There may be many members of this family, but no other member has a
better quality. GAP displays four figures of merit for alignments:
Quality, Ratio, Identity, and Similarity. The Quality is the metric
maximized in order to align the sequences. Ratio is the quality
divided by the number of bases in the shorter segment. Percent
Identity is the percent of the symbols that actually match. Percent
Similarity is the percent of the symbols that are similar. Symbols
that are across from gaps are ignored. A similarity is scored when
the scoring matrix value for a pair of symbols is greater than or
equal to 0.50, the similarity threshold. The scoring matrix used in
Version 10 of the Wisconsin Genetics Software Package is BLOSUM62
(see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci. USA
89:10915).
[0073] Unless otherwise stated, sequence identity/similarity values
provided herein refer to the value obtained using the BLAST 2.0
suite of programs using default parameters (Altschul, et al.,
(1997) Nucleic Acids Res. 25:3389-402).
[0074] As those of ordinary skill in the art will understand, BLAST
searches assume that proteins can be modeled as random sequences.
However, many real proteins comprise regions of nonrandom
sequences, which may be homopolymeric tracts, short-period repeats,
or regions enriched in one or more amino acids. Such low-complexity
regions may be aligned between unrelated proteins even though other
regions of the protein are entirely dissimilar. A number of
low-complexity filter programs can be employed to reduce such
low-complexity alignments. For example, the SEG (Wooten and
Federhen, (1993) Comput. Chem. 17:149-63) and XNU (Claverie and
States, (1993) Comput. Chem. 17:191-201) low-complexity filters can
be employed alone or in combination.
[0075] As used herein, "sequence identity" or "identity" in the
context of two nucleic acid or polypeptide sequences includes
reference to the residues in the two sequences, which are the same
when aligned for maximum correspondence over a specified comparison
window. When percentage of sequence identity is used in reference
to proteins it is recognized that residue positions which are not
identical often differ by conservative amino acid substitutions,
where amino acid residues are substituted for other amino acid
residues with similar chemical properties (e.g., charge or
hydrophobicity) and therefore do not change the functional
properties of the molecule. Where sequences differ in conservative
substitutions, the percent sequence identity may be adjusted
upwards to correct for the conservative nature of the substitution.
Sequences, which differ by such conservative substitutions, are
said to have "sequence similarity" or "similarity." Means for
making this adjustment are well known to those of skill in the art.
Typically this involves scoring a conservative substitution as a
partial rather than a full mismatch, thereby increasing the
percentage sequence identity. Thus, for example, where an identical
amino acid is given a score of 1 and a non-conservative
substitution is given a score of zero, a conservative substitution
is given a score between zero and 1. The scoring of conservative
substitutions is calculated, e.g., according to the algorithm of
Meyers and Miller, (1988) Computer Applic. Biol. Sci. 4:11-17,
e.g., as implemented in the program PC/GENE (Intelligenetics,
Mountain View, Calif., USA).
[0076] As used herein, "percentage of sequence identity" means the
value determined by comparing two optimally aligned sequences over
a comparison window, wherein the portion of the polynucleotide
sequence in the comparison window may comprise additions or
deletions (i.e., gaps) as compared to the reference sequence (which
does not comprise additions or deletions) for optimal alignment of
the two sequences. The percentage is calculated by determining the
number of positions at which the identical nucleic acid base or
amino acid residue occurs in both sequences to yield the number of
matched positions, dividing the number of matched positions by the
total number of positions in the window of comparison and
multiplying the result by 100 to yield the percentage of sequence
identity.
[0077] The term "substantial identity" of polynucleotide sequences
means that a polynucleotide comprises a sequence that has between
50-100% sequence identity, preferably at least 50% sequence
identity, preferably at least 60% sequence identity, preferably at
least 70%, more preferably at least 80%, more preferably at least
90%, and most preferably at least 95%, compared to a reference
sequence using one of the alignment programs described using
standard parameters. One of skill will recognize that these values
can be appropriately adjusted to determine corresponding identity
of proteins encoded by two nucleotide sequences by taking into
account codon degeneracy, amino acid similarity, reading frame
positioning and the like. Substantial identity of amino acid
sequences for these purposes normally means sequence identity of
between 55-100%, preferably at least 55%, preferably at least 60%,
more preferably at least 70%, 80%, 90%, and most preferably at
least 95%.
[0078] Another indication that nucleotide sequences are
substantially identical is if two molecules hybridize to each other
under stringent conditions. The degeneracy of the genetic code
allows for many amino acids substitutions that lead to variety in
the nucleotide sequence that code for the same amino acid, hence it
is possible that the DNA sequence could code for the same
polypeptide but not hybridize to each other under stringent
conditions. This may occur, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code. One indication that two nucleic acid sequences are
substantially identical is that the polypeptide, which the first
nucleic acid encodes, is immunologically cross reactive with the
polypeptide encoded by the second nucleic acid.
[0079] The terms "substantial identity" in the context of a peptide
indicates that a peptide comprises a sequence with between 55-100%
sequence identity to a reference sequence preferably at least 55%
sequence identity, preferably 60% preferably 70%, more preferably
80%, most preferably at least 90% or 95% sequence identity to the
reference sequence over a specified comparison window. Preferably,
optimal alignment is conducted using the homology alignment
algorithm of Needleman and Wunsch, supra. An indication that two
peptide sequences are substantially identical is that one peptide
is immunologically reactive with antibodies raised against the
second peptide. Thus, a peptide is substantially identical to a
second peptide, for example, where the two peptides differ only by
a conservative substitution. In addition, a peptide can be
substantially identical to a second peptide when they differ by a
non-conservative change if the epitope that the antibody recognizes
is substantially identical. Peptides, which are "substantially
similar" share sequences as, noted above except that residue
positions, which are not identical, may differ by conservative
amino acid changes.
[0080] The invention discloses AMT polynucleotides and
polypeptides. The novel nucleotides and proteins of the invention
have an expression pattern which indicates that they regulate
ammonium transport and thus play an important role in plant
development. The polynucleotides are expressed in various plant
tissues. The polynucleotides and polypeptides thus provide an
opportunity to manipulate plant development to alter seed and
vegetative tissue development, timing or composition. This may be
used to create a plant with altered N composition in source and
sink.
Nucleic Acids
[0081] The present invention provides, inter alia, isolated nucleic
acids of RNA, DNA, and analogs and/or chimeras thereof, comprising
an amt polynucleotide.
[0082] The present invention also includes polynucleotides
optimized for expression in different organisms. For example, for
expression of the polynucleotide in a maize plant, the sequence can
be altered to account for specific codon preferences and to alter
GC content as according to Murray, et al., supra. Maize codon usage
for 28 genes from maize plants is listed in Table 4 of Murray et
al., supra.
[0083] The AMT nucleic acids of the present invention comprise
isolated AMT polynucleotides which are inclusive of: [0084] (a) a
polynucleotide encoding an AMT polypeptide and conservatively
modified and polymorphic variants thereof; [0085] (b) a
polynucleotide having at least 70% sequence identity with
polynucleotides of (a) or (b); [0086] (c) complementary sequences
of polynucleotides of (a) or (b).
Construction of Nucleic Acids
[0087] The isolated nucleic acids of the present invention can be
made using (a) standard recombinant methods, (b) synthetic
techniques, or combinations thereof. In some embodiments, the
polynucleotides of the present invention will be cloned, amplified,
or otherwise constructed from a fungus or bacteria.
[0088] The nucleic acids may conveniently comprise sequences in
addition to a polynucleotide of the present invention. For example,
a multi-cloning site comprising one or more endonuclease
restriction sites may be inserted into the nucleic acid to aid in
isolation of the polynucleotide. Also, translatable sequences may
be inserted to aid in the isolation of the translated
polynucleotide of the present invention. For example, a
hexa-histidine marker sequence provides a convenient means to
purify the proteins of the present invention. The nucleic acid of
the present invention--excluding the polynucleotide sequence--is
optionally a vector, adapter, or linker for cloning and/or
expression of a polynucleotide of the present invention. Additional
sequences may be added to such cloning and/or expression sequences
to optimize their function in cloning and/or expression, to aid in
isolation of the polynucleotide, or to improve the introduction of
the polynucleotide into a cell. Typically, the length of a nucleic
acid of the present invention less the length of its polynucleotide
of the present invention is less than 20 kilobase pairs, often less
than 15 kb, and frequently less than 10 kb. Use of cloning vectors,
expression vectors, adapters, and linkers is well known in the art.
Exemplary nucleic acids include such vectors as: M13, lambda ZAP
Express, lambda ZAP II, lambda gt10, lambda gt11, pBK-CMV, pBK-RSV,
pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL 4,
pWE15, SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/-, pSG5, pBK,
pCR-Script, pET, pSPUTK, p3'SS, pGEM, pSK+/-, pGEX, pSPORTI and II,
pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac, pMC1neo, pOG44,
pOG45, pFRT.beta.GAL, pNEO.beta.GAL, pRS403, pRS404, pRS405,
pRS406, pRS413, pRS414, pRS415, pRS416, lambda MOSSlox, and lambda
MOSElox. Optional vectors for the present invention, include but
are not limited to, lambda ZAP II, and pGEX. For a description of
various nucleic acids see, e.g., Stratagene Cloning Systems,
Catalogs 1995, 1996, 1997 (La Jolla, Calif.); and, Amersham Life
Sciences, Inc, Catalog '97 (Arlington Heights, Ill.).
Synthetic Methods for Constructing Nucleic Acids
[0089] The isolated nucleic acids of the present invention can also
be prepared by direct chemical synthesis by methods such as the
phosphotriester method of Narang, et al., (1979) Meth. Enzymol.
68:90-9; the phosphodiester method of Brown, et al., (1979) Meth.
Enzymol. 68:109-51; the diethylphosphoramidite method of Beaucage,
et al., (1981) Tetra. Letts. 22(20):1859-62; the solid phase
phosphoramidite triester method described by Beaucage, et al.,
supra, e.g., using an automated synthesizer, e.g., as described in
Needham-VanDevanter, et al., (1984) Nucleic Acids Res. 12:6159-68;
and, the solid support method of U.S. Pat. No. 4,458,066. Chemical
synthesis generally produces a single stranded oligonucleotide.
This may be converted into double stranded DNA by hybridization
with a complementary sequence or by polymerization with a DNA
polymerase using the single strand as a template. One of skill will
recognize that while chemical synthesis of DNA is limited to
sequences of about 100 bases, longer sequences may be obtained by
the ligation of shorter sequences.
UTRs and Codon Preference
[0090] In general, translational efficiency has been found to be
regulated by specific sequence elements in the 5' non-coding or
untranslated region (5' UTR) of the RNA. Positive sequence motifs
include translational initiation consensus sequences (Kozak, (1987)
Nucleic Acids Res. 15:8125) and the 5<G> 7 methyl GpppG RNA
cap structure (Drummond, et al., (1985) Nucleic Acids Res.
13:7375). Negative elements include stable intramolecular 5' UTR
stem-loop structures (Muesing, et al., (1987) Cell 48:691) and AUG
sequences or short open reading frames preceded by an appropriate
AUG in the 5' UTR (Kozak, supra, Rao, et al., (1988) Mol. and Cell.
Biol. 8:284). Accordingly, the present invention provides 5' and/or
3' UTR regions for modulation of translation of heterologous coding
sequences.
[0091] Further, the polypeptide-encoding segments of the
polynucleotides of the present invention can be modified to alter
codon usage. Altered codon usage can be employed to alter
translational efficiency and/or to optimize the coding sequence for
expression in a desired host or to optimize the codon usage in a
heterologous sequence for expression in maize. Codon usage in the
coding regions of the polynucleotides of the present invention can
be analyzed statistically using commercially available software
packages such as "Codon Preference" available from the University
of Wisconsin Genetics Computer Group. See, Devereaux, et al.,
(1984) Nucleic Acids Res. 12:387-395; or MacVector 4.1 (Eastman
Kodak Co., New Haven, Conn.). Thus, the present invention provides
a codon usage frequency characteristic of the coding region of at
least one of the polynucleotides of the present invention. The
number of polynucleotides (3 nucleotides per amino acid) that can
be used to determine a codon usage frequency can be any integer
from 3 to the number of polynucleotides of the present invention as
provided herein. Optionally, the polynucleotides will be
full-length sequences. An exemplary number of sequences for
statistical analysis can be at least 1, 5, 10, 20, 50 or 100.
Sequence Shuffling
[0092] The present invention provides methods for sequence
shuffling using polynucleotides of the present invention, and
compositions resulting therefrom. Sequence shuffling is described
in PCT publication No. 96/19256. See also, Zhang, et al., (1997)
Proc. Natl. Acad. Sci. USA 94:4504-9; and Zhao, et al., (1998)
Nature Biotech 16:258-61. Generally, sequence shuffling provides a
means for generating libraries of polynucleotides having a desired
characteristic, which can be selected or screened for. Libraries of
recombinant polynucleotides are generated from a population of
related sequence polynucleotides, which comprise sequence regions,
which have substantial sequence identity and can be homologously
recombined in vitro or in vivo. The population of
sequence-recombined polynucleotides comprises a subpopulation of
polynucleotides which possess desired or advantageous
characteristics and which can be selected by a suitable selection
or screening method. The characteristics can be any property or
attribute capable of being selected for or detected in a screening
system, and may include properties of: an encoded protein, a
transcriptional element, a sequence controlling transcription, RNA
processing, RNA stability, chromatin conformation, translation, or
other expression property of a gene or transgene, a replicative
element, a protein-binding element, or the like, such as any
feature which confers a selectable or detectable property. In some
embodiments, the selected characteristic will be an altered K.sub.m
and/or K.sub.cat over the wild-type protein as provided herein. In
other embodiments, a protein or polynucleotide generated from
sequence shuffling will have a ligand binding affinity greater than
the non-shuffled wild-type polynucleotide. In yet other
embodiments, a protein or polynucleotide generated from sequence
shuffling will have an altered pH optimum as compared to the
non-shuffled wild-type polynucleotide. The increase in such
properties can be at least 110%, 120%, 130%, 140% or greater than
150% of the wild-type value.
Recombinant Expression Cassettes
[0093] The present invention further provides recombinant
expression cassettes comprising a nucleic acid of the present
invention. A nucleic acid sequence coding for the desired
polynucleotide of the present invention, for example a cDNA or a
genomic sequence encoding a polypeptide long enough to code for an
active protein of the present invention, can be used to construct a
recombinant expression cassette which can be introduced into the
desired host cell. A recombinant expression cassette will typically
comprise a polynucleotide of the present invention operably linked
to transcriptional initiation regulatory sequences which will
direct the transcription of the polynucleotide in the intended host
cell, such as tissues of a transformed plant.
[0094] For example, plant expression vectors may include (1) a
cloned plant gene under the transcriptional control of 5' and 3'
regulatory sequences and (2) a dominant selectable marker. Such
plant expression vectors may also contain, if desired, a promoter
regulatory region (e.g., one conferring inducible or constitutive,
environmentally- or developmentally-regulated, or cell- or
tissue-specific/selective expression), a transcription initiation
start site, a ribosome binding site, an RNA processing signal, a
transcription termination site, and/or a polyadenylation
signal.
[0095] A plant promoter fragment can be employed which will direct
expression of a polynucleotide of the present invention in all
tissues of a regenerated plant. Such promoters are referred to
herein as "constitutive" promoters and are active under most
environmental conditions and states of development or cell
differentiation. Examples of constitutive promoters include the 1'-
or 2'-promoter derived from T-DNA of Agrobacterium tumefaciens, the
Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S.
Pat. No. 5,683,439), the Nos promoter, the rubisco promoter, the
GRP1-8 promoter, the 35S promoter from cauliflower mosaic virus
(CaMV), as described in Odell, et al., (1985) Nature 313:810-2;
rice actin (McElroy, et al., (1990) Plant Cell 163-171); ubiquitin
(Christensen, et al., (1992) Plant Mol. Biol. 12:619-632 and
Christensen, et al., (1992) Plant Mol. Biol. 18:675-89); PEMU
(Last, et al., (1991) Theor. Appl. Genet. 81:581-8); MAS (Velten,
et al., (1984) EMBO J. 3:2723-30); and maize H3 histone (Lepetit,
et al., (1992) Mol. Gen. Genet. 231:276-85; and Atanassvoa, et al.,
(1992) Plant Journal 2(3):291-300); ALS promoter, as described in
PCT Application Number WO 96/30530; and other transcription
initiation regions from various plant genes known to those of
skill. For the present invention ubiquitin is the preferred
promoter for expression in monocot plants.
[0096] Alternatively, the plant promoter can direct expression of a
polynucleotide of the present invention in a specific tissue or may
be otherwise under more precise environmental or developmental
control. Such promoters are referred to here as "inducible"
promoters. Environmental conditions that may effect transcription
by inducible promoters include pathogen attack, anaerobic
conditions, or the presence of light. Examples of inducible
promoters are the Adh1 promoter, which is inducible by hypoxia or
cold stress, the Hsp70 promoter, which is inducible by heat stress,
and the PPDK promoter, which is inducible by light.
[0097] Examples of promoters under developmental control include
promoters that initiate transcription only, or preferentially, in
certain tissues, such as leaves, roots, fruit, seeds, or flowers.
The operation of a promoter may also vary depending on its location
in the genome. Thus, an inducible promoter may become fully or
partially constitutive in certain locations.
[0098] If polypeptide expression is desired, it is generally
desirable to include a polyadenylation region at the 3'-end of a
polynucleotide coding region. The polyadenylation region can be
derived from a variety of plant genes, or from T-DNA. The 3' end
sequence to be added can be derived from, for example, the nopaline
synthase or octopine synthase genes, or alternatively from another
plant gene, or less preferably from any other eukaryotic gene.
Examples of such regulatory elements include, but are not limited
to, 3' termination and/or polyadenylation regions such as those of
the Agrobacterium tumefaciens nopaline synthase (nos) gene (Bevan,
et al., (1983) Nucleic Acids Res. 12:369-85); the potato proteinase
inhibitor II (PINII) gene (Keil, et al., (1986) Nucleic Acids Res.
14:5641-50; and An, et al., (1989) Plant Cell 1:115-22); and the
CaMV 19S gene (Mogen, et al., (1990) Plant Cell 2:1261-72).
[0099] An intron sequence can be added to the 5' untranslated
region or the coding sequence of the partial coding sequence to
increase the amount of the mature message that accumulates in the
cytosol. Inclusion of a spliceable intron in the transcription unit
in both plant and animal expression constructs has been shown to
increase gene expression at both the mRNA and protein levels up to
1000-fold (Buchman and Berg, (1988) Mol. Cell Biol. 8:4395-4405;
Callis, et al., (1987) Genes Dev. 1:1183-200). Such intron
enhancement of gene expression is typically greatest when placed
near the 5' end of the transcription unit. Use of maize introns
Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the
art. See generally, THE MAIZE HANDBOOK, Chapter 116, Freeling and
Walbot, eds., Springer, N.Y. (1994).
[0100] Plant signal sequences, including, but not limited to,
signal-peptide encoding DNA/RNA sequences which target proteins to
the extracellular matrix of the plant cell (Dratewka-Kos, et al.,
(1989) J. Biol. Chem. 264:4896-900), such as the Nicotiana
plumbaginifolia extension gene (DeLoose, et al., (1991) Gene
99:95-100); signal peptides which target proteins to the vacuole,
such as the sweet potato sporamin gene (Matsuka, et al., (1991)
Proc. Natl. Acad. Sci. USA 88:834) and the barley lectin gene
(Wilkins, et al., (1990) Plant Cell, 2:301-13); signal peptides
which cause proteins to be secreted, such as that of PRIb (Lind, et
al., (1992) Plant Mol. Biol. 18:47-53) or the barley alpha amylase
(BAA) (Rahmatullah, et al., (1989) Plant Mol. Biol. 12:119, and
hereby incorporated by reference), or signal peptides which target
proteins to the plastids such as that of rapeseed enoyl-Acp
reductase (Verwaert, et al., (1994) Plant Mol. Biol. 26:189-202)
are useful in the invention. The barley alpha amylase signal
sequence fused to the AMT polynucleotide is the preferred construct
for expression in maize for the present invention.
[0101] The vector comprising the sequences from a polynucleotide of
the present invention will typically comprise a marker gene, which
confers a selectable phenotype on plant cells. Usually, the
selectable marker gene will encode antibiotic resistance, with
suitable genes including genes coding for resistance to the
antibiotic spectinomycin (e.g., the aada gene), the streptomycin
phosphotransferase (SPT) gene coding for streptomycin resistance,
the neomycin phosphotransferase (NPTII) gene encoding kanamycin or
geneticin resistance, the hygromycin phosphotransferase (HPT) gene
coding for hygromycin resistance, genes coding for resistance to
herbicides which act to inhibit the action of acetolactate synthase
(ALS), in particular the sulfonylurea-type herbicides (e.g., the
acetolactate synthase (ALS) gene containing mutations leading to
such resistance in particular the S4 and/or Hra mutations), genes
coding for resistance to herbicides which act to inhibit action of
glutamine synthase, such as phosphinothricin or basta (e.g., the
bar gene), or other such genes known in the art. The bar gene
encodes resistance to the herbicide basta, and the ALS gene encodes
resistance to the herbicide chlorsulfuron.
[0102] Typical vectors useful for expression of genes in higher
plants are well known in the art and include vectors derived from
the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens
described by Rogers, et al., (1987) Meth. Enzymol. 153:253-77.
These vectors are plant integrating vectors in that on
transformation, the vectors integrate a portion of vector DNA into
the genome of the host plant. Exemplary A. tumefaciens vectors
useful herein are plasmids pKYLX6 and pKYLX7 of Schardl, et al.,
(1987) Gene 61:1-11, and Berger, et al., (1989) Proc. Natl. Acad.
Sci. USA, 86:8402-6. Another useful vector herein is plasmid
pBI101.2 that is available from CLONTECH Laboratories, Inc. (Palo
Alto, Calif.).
Expression of Proteins in Host Cells
[0103] Using the nucleic acids of the present invention, one may
express a protein of the present invention in a recombinantly
engineered cell such as bacteria, yeast, insect, mammalian, or
preferably plant cells. The cells produce the protein in a
non-natural condition (e.g., in quantity, composition, location,
and/or time), because they have been genetically altered through
human intervention to do so.
[0104] It is expected that those of skill in the art are
knowledgeable in the numerous expression systems available for
expression of a nucleic acid encoding a protein of the present
invention. No attempt to describe in detail the various methods
known for the expression of proteins in prokaryotes or eukaryotes
will be made.
[0105] In brief summary, the expression of isolated nucleic acids
encoding a protein of the present invention will typically be
achieved by operably linking, for example, the DNA or cDNA to a
promoter (which is either constitutive or inducible), followed by
incorporation into an expression vector. The vectors can be
suitable for replication and integration in either prokaryotes or
eukaryotes. Typical expression vectors contain transcription and
translation terminators, initiation sequences, and promoters useful
for regulation of the expression of the DNA encoding a protein of
the present invention. To obtain high level expression of a cloned
gene, it is desirable to construct expression vectors which
contain, at the minimum, a strong promoter, such as ubiquitin, to
direct transcription, a ribosome binding site for translational
initiation, and a transcription/translation terminator.
Constitutive promoters are classified as providing for a range of
constitutive expression. Thus, some are weak constitutive
promoters, and others are strong constitutive promoters. Generally,
by "weak promoter" is intended a promoter that drives expression of
a coding sequence at a low level. By "low level" is intended at
levels of about 1/10,000 transcripts to about 1/100,000 transcripts
to about 1/500,000 transcripts. Conversely, a "strong promoter"
drives expression of a coding sequence at a "high level," or about
1/10 transcripts to about 1/100 transcripts to about 1/1,000
transcripts.
[0106] One of skill would recognize that modifications could be
made to a protein of the present invention without diminishing its
biological activity. Some modifications may be made to facilitate
the cloning, expression, or incorporation of the targeting molecule
into a fusion protein. Such modifications are well known to those
of skill in the art and include, for example, a methionine added at
the amino terminus to provide an initiation site, or additional
amino acids (e.g., poly His) placed on either terminus to create
conveniently located restriction sites or termination codons or
purification sequences.
Expression in Prokaryotes
[0107] Prokaryotic cells may be used as hosts for expression.
Prokaryotes most frequently are represented by various strains of
E. coli; however, other microbial strains may also be used.
Commonly used prokaryotic control sequences which are defined
herein to include promoters for transcription initiation,
optionally with an operator, along with ribosome binding site
sequences, include such commonly used promoters as the beta
lactamase (penicillinase) and lactose (lac) promoter systems
(Chang, et al., (1977) Nature 198:1056), the tryptophan (trp)
promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057)
and the lambda derived P L promoter and N-gene ribosome binding
site (Shimatake, et al., (1981) Nature 292:128). The inclusion of
selection markers in DNA vectors transfected in E. coli is also
useful. Examples of such markers include genes specifying
resistance to ampicillin, tetracycline, or chloramphenicol.
[0108] The vector is selected to allow introduction of the gene of
interest into the appropriate host cell. Bacterial vectors are
typically of plasmid or phage origin. Appropriate bacterial cells
are infected with phage vector particles or transfected with naked
phage vector DNA. If a plasmid vector is used, the bacterial cells
are transfected with the plasmid vector DNA. Expression systems for
expressing a protein of the present invention are available using
Bacillus sp. and Salmonella (Palva, et al., (1983) Gene 22:229-35;
Mosbach, et al., (1983) Nature 302:543-5). The pGEX-4T-1 plasmid
vector from Pharmacia is the preferred E. coli expression vector
for the present invention.
Expression in Eukaryotes
[0109] A variety of eukaryotic expression systems such as yeast,
insect cell lines, plant and mammalian cells, are known to those of
skill in the art. As explained briefly below, the present invention
can be expressed in these eukaryotic systems. In some embodiments,
transformed/transfected plant cells, as discussed infra, are
employed as expression systems for production of the proteins of
the instant invention.
[0110] Synthesis of heterologous proteins in yeast is well known.
Sherman, et al., METHODS IN YEAST GENETICS, Cold Spring Harbor
Laboratory (1982) is a well recognized work describing the various
methods available to produce the protein in yeast. Two widely
utilized yeasts for production of eukaryotic proteins are
Saccharomyces cerevisiae and Pichia pastoris. Vectors, strains, and
protocols for expression in Saccharomyces and Pichia are known in
the art and available from commercial suppliers (e.g., Invitrogen).
Suitable vectors usually have expression control sequences, such as
promoters, including 3-phosphoglycerate kinase or alcohol oxidase,
and an origin of replication, termination sequences and the like as
desired.
[0111] A protein of the present invention, once expressed, can be
isolated from yeast by lysing the cells and applying standard
protein isolation techniques to the lysates or the pellets. The
monitoring of the purification process can be accomplished by using
Western blot techniques or radioimmunoassay of other standard
immunoassay techniques.
[0112] The sequences encoding proteins of the present invention can
also be ligated to various expression vectors for use in
transfecting cell cultures of, for instance, mammalian, insect, or
plant origin. Mammalian cell systems often will be in the form of
monolayers of cells although mammalian cell suspensions may also be
used. A number of suitable host cell lines capable of expressing
intact proteins have been developed in the art, and include the
HEK293, BHK21, and CHO cell lines. Expression vectors for these
cells can include expression control sequences, such as an origin
of replication, a promoter (e.g., the CMV promoter, a HSV tk
promoter or pgk (phosphoglycerate kinase) promoter), an enhancer
(Queen, et al., (1986) Immunol. Rev. 89:49), and necessary
processing information sites, such as ribosome binding sites, RNA
splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly
A addition site), and transcriptional terminator sequences. Other
animal cells useful for production of proteins of the present
invention are available, for instance, from the American Type
Culture Collection Catalogue of Cell Lines and Hybridomas (7.sup.th
ed., 1992).
[0113] Appropriate vectors for expressing proteins of the present
invention in insect cells are usually derived from the SF9
baculovirus. Suitable insect cell lines include mosquito larvae,
silkworm, armyworm, moth, and Drosophila cell lines such as a
Schneider cell line (see, e.g., Schneider, (1987) J. Embryol. Exp.
Morphol. 27:353-65).
[0114] As with yeast, when higher animal or plant host cells are
employed, polyadenylation or transcription terminator sequences are
typically incorporated into the vector. An example of a terminator
sequence is the polyadenylation sequence from the bovine growth
hormone gene. Sequences for accurate splicing of the transcript may
also be included. An example of a splicing sequence is the VP1
intron from SV40 (Sprague, et al., (1983) J. Virol. 45:773-81).
Additionally, gene sequences to control replication in the host
cell may be incorporated into the vector such as those found in
bovine papilloma virus type-vectors (Saveria-Campo, "Bovine
Papilloma Virus DNA a Eukaryotic Cloning Vector," in DNA CLONING: A
PRACTICAL APPROACH, vol. II, Glover, ed., IRL Press, Arlington,
Va., pp. 213-38 (1985)).
[0115] In addition, the gene for AMT placed in the appropriate
plant expression vector can be used to transform plant cells. The
polypeptide can then be isolated from plant callus or the
transformed cells can be used to regenerate transgenic plants. Such
transgenic plants can be harvested, and the appropriate tissues
(seed or leaves, for example) can be subjected to large scale
protein extraction and purification techniques.
Plant Transformation Methods
[0116] Numerous methods for introducing foreign genes into plants
are known and can be used to insert an amt polynucleotide into a
plant host, including biological and physical plant transformation
protocols. See, e.g., Miki, et al., "Procedure for Introducing
Foreign DNA into Plants," in METHODS IN PLANT MOLECULAR BIOLOGY AND
BIOTECHNOLOGY, Glick and Thompson, eds., CRC Press, Inc., Boca
Raton, pp. 67-88 (1993). The methods chosen vary with the host
plant, and include chemical transfection methods such as calcium
phosphate, microorganism-mediated gene transfer such as
Agrobacterium (Horsch, et al., (1985) Science 227:1229-31),
electroporation, micro-injection, and biolistic bombardment.
[0117] Expression cassettes and vectors and in vitro culture
methods for plant cell or tissue transformation and regeneration of
plants are known and available. See, e.g., Gruber, et al., "Vectors
for Plant Transformation," in METHODS IN PLANT MOLECULAR BIOLOGY
AND BIOTECHNOLOGY, supra, pp. 89-119.
[0118] The isolated polynucleotides or polypeptides may be
introduced into the plant by one or more techniques typically used
for direct delivery into cells. Such protocols may vary depending
on the type of organism, cell, plant or plant cell, i.e. monocot or
dicot, targeted for gene modification. Suitable methods of
transforming plant cells include microinjection (Crossway, et al.,
(1986) Biotechniques 4:320-334; and U.S. Pat. No. 6,300,543),
electroporation (Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA
83:5602-5606, direct gene transfer (Paszkowski, et al., (1984) EMBO
J. 3:2717-2722), and ballistic particle acceleration (see, for
example, Sanford, et al., U.S. Pat. No. 4,945,050; WO 91/10725; and
McCabe, et al., (1988) Biotechnology 6:923-926). Also see, Tomes,
et al., Direct DNA Transfer into Intact Plant Cells Via
Microprojectile Bombardment. pp. 197-213 in Plant Cell, Tissue and
Organ Culture, Fundamental Methods. eds. O. L. Gamborg & G. C.
Phillips. Springer-Verlag Berlin Heidelberg N.Y., 1995; U.S. Pat.
No. 5,736,369 (meristem); Weissinger, et al., (1988) Ann. Rev.
Genet. 22:421-477; Sanford, et al., (1987) Particulate Science and
Technology 5:27-37 (onion); Christou, et al., (1988) Plant Physiol.
87:671-674 (soybean); Datta, et al., (1990) Biotechnology 8:736-740
(rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA
85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563
(maize); WO 91/10725 (maize); Klein, et al., (1988) Plant Physiol.
91:440-444 (maize); Fromm, et al., (1990) Biotechnology 8:833-839;
and Gordon-Kamm, et al., (1990) Plant Cell 2:603-618 (maize);
Hooydaas-Van Slogteren and Hooykaas (1984) Nature (London)
311:763-764; Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA
84:5345-5349 (Liliaceae); De Wet, et al., (1985) In The
Experimental Manipulation of Ovule Tissues, ed. G. P. Chapman, et
al., pp. 197-209 Longman, N.Y. (pollen); Kaeppler, et al., (1990)
Plant Cell Reports 9:415-418; and Kaeppler, et al., (1992) Theor.
Appl. Genet. 84:560-566 (whisker-mediated transformation); U.S.
Pat. No. 5,693,512 (sonication); D'Halluin, et al., (1992) Plant
Cell 4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell
Reports 12:250-255; and Christou and Ford (1995) Annals of Botany
75:407-413 (rice); Osjoda, et al., (1996) Nature Biotech.
14:745-750; Agrobacterium mediated maize transformation (U.S. Pat.
No. 5,981,840); silicon carbide whisker methods (Frame, et al.,
(1994) Plant J. 6:941-948); laser methods (Guo, et al., (1995)
Physiologia Plantarum 93:19-24); sonication methods (Bao, et al.,
(1997) Ultrasound in Medicine & Biology 23:953-959; Finer and
Finer (2000) Lett Appl Microbiol. 30:406-10; Amoah, et al., (2001)
J Exp Bot 52:1135-42); polyethylene glycol methods (Krens, et al.,
(1982) Nature 296:72-77); protoplasts of monocot and dicot cells
can be transformed using electroporation (Fromm, et al., (1985)
Proc. Natl. Acad. Sci. USA 82:5824-5828) and microinjection
(Crossway, et al., (1986) Mol. Gen. Genet. 202:179-185); all of
which are herein incorporated by reference.
Agrobacterium-Mediated Transformation
[0119] The most widely utilized method for introducing an
expression vector into plants is based on the natural
transformation system of Agrobacterium. A. tumefaciens and A.
rhizogenes are plant pathogenic soil bacteria, which genetically
transform plant cells. The Ti and Ri plasmids of A. tumefaciens and
A. rhizogenes, respectively, carry genes responsible for genetic
transformation of plants. See, e.g., Kado, (1991) Crit. Rev. Plant
Sci. 10:1. Descriptions of the Agrobacterium vector systems and
methods for Agrobacterium-mediated gene transfer are provided in
Gruber, et al., supra; Miki, et al., supra; and Moloney, et al.,
(1989) Plant Cell Reports 8:238.
[0120] Similarly, the gene can be inserted into the T-DNA region of
a Ti or Ri plasmid derived from A. tumefaciens or A. rhizogenes,
respectively. Thus, expression cassettes can be constructed as
above, using these plasmids. Many control sequences are known which
when coupled to a heterologous coding sequence and transformed into
a host organism show fidelity in gene expression with respect to
tissue/organ specificity of the original coding sequence. See,
e.g., Benfey and Chua, (1989) Science 244:174-81. Particularly
suitable control sequences for use in these plasmids are promoters
for constitutive leaf-specific expression of the gene in the
various target plants. Other useful control sequences include a
promoter and terminator from the nopaline synthase gene (NOS). The
NOS promoter and terminator are present in the plasmid pARC2,
available from the American Type Culture Collection and designated
ATCC 67238. If such a system is used, the virulence (vir) gene from
either the Ti or Ri plasmid must also be present, either along with
the T-DNA portion, or via a binary system where the vir gene is
present on a separate vector. Such systems, vectors for use
therein, and methods of transforming plant cells are described in
U.S. Pat. No. 4,658,082; US Patent Application Number 913,914,
filed Oct. 1, 1986, as referenced in U.S. Pat. No. 5,262,306,
issued Nov. 16, 1993; and Simpson, et al., (1986) Plant Mol. Biol.
6:403-15 (also referenced in the '306 patent); all incorporated by
reference in their entirety.
[0121] Once constructed, these plasmids can be placed into A.
rhizogenes or A. tumefaciens and these vectors used to transform
cells of plant species, which are ordinarily susceptible to
Fusarium or Alternaria infection. Several other transgenic plants
are also contemplated by the present invention including but not
limited to soybean, corn, sorghum, alfalfa, rice, clover, cabbage,
banana, coffee, celery, tobacco, cowpea, cotton, melon,
switchgrass, myscanthus, triticale and pepper. The selection of
either A. tumefaciens or A. rhizogenes will depend on the plant
being transformed thereby. In general A. tumefaciens is the
preferred organism for transformation. Most dicotyledonous plants,
some gymnosperms, and a few monocotyledonous plants (e.g., certain
members of the Liliales and Arales) are susceptible to infection
with A. tumefaciens. A. rhizogenes also has a wide host range,
embracing most dicots and some gymnosperms, which includes members
of the Leguminosae, Compositae, and Chenopodiaceae. Monocot plants
can now be transformed with some success. EP Patent Application
Number 604 662 A1 discloses a method for transforming monocots
using Agrobacterium. EP Application Number 672 752 A1 discloses a
method for transforming monocots with Agrobacterium using the
scutellum of immature embryos. Ishida, et al., discuss a method for
transforming maize by exposing immature embryos to A. tumefaciens
(Nature Biotechnology 14:745-50 (1996)).
[0122] Once transformed, these cells can be used to regenerate
transgenic plants. For example, whole plants can be infected with
these vectors by wounding the plant and then introducing the vector
into the wound site. Any part of the plant can be wounded,
including leaves, stems and roots. Alternatively, plant tissue, in
the form of an explant, such as cotyledonary tissue or leaf disks,
can be inoculated with these vectors, and cultured under
conditions, which promote plant regeneration. Roots or shoots
transformed by inoculation of plant tissue with A. rhizogenes or A.
tumefaciens, containing the gene coding for the fumonisin
degradation enzyme, can be used as a source of plant tissue to
regenerate fumonisin-resistant transgenic plants, either via
somatic embryogenesis or organogenesis. Examples of such methods
for regenerating plant tissue are disclosed in Shahin, (1985)
Theor. Appl. Genet. 69:235-40; U.S. Pat. No. 4,658,082; Simpson, et
al., supra; and US Patent Application Numbers 913,913 and 913,914,
both filed Oct. 1, 1986, as referenced in U.S. Pat. No. 5,262,306,
issued Nov. 16, 1993, the entire disclosures therein incorporated
herein by reference.
Direct Gene Transfer
[0123] Despite the fact that the host range for
Agrobacterium-mediated transformation is broad, some major cereal
crop species and gymnosperms have generally been recalcitrant to
this mode of gene transfer, even though some success has recently
been achieved in rice (Hiei, et al., (1994) The Plant Journal
6:271-82). Several methods of plant transformation, collectively
referred to as direct gene transfer, have been developed as an
alternative to Agrobacterium-mediated transformation.
[0124] A generally applicable method of plant transformation is
microprojectile-mediated transformation, where DNA is carried on
the surface of microprojectiles measuring about 1 to 4 .mu.m. The
expression vector is introduced into plant tissues with a biolistic
device that accelerates the microprojectiles to speeds of 300 to
600 m/s which is sufficient to penetrate the plant cell walls and
membranes (Sanford, et al., (1987) Part. Sci. Technol. 5:27;
Sanford, (1988) Trends Biotech 6:299; Sanford, (1990) Physiol.
Plant 79:206; and Klein, et al., (1992) Biotechnology 10:268).
[0125] Another method for physical delivery of DNA to plants is
sonication of target cells as described in Zang, et al., (1991)
BioTechnology 9:996. Alternatively, liposome or spheroplast fusions
have been used to introduce expression vectors into plants. See,
e.g., Deshayes, et al., (1985) EMBO J. 4:2731; and Christou, et
al., (1987) Proc. Natl. Acad. Sci. USA 84:3962. Direct uptake of
DNA into protoplasts using CaCl.sub.2 precipitation, polyvinyl
alcohol, or poly-L-ornithine has also been reported. See, e.g.,
Hain, et al., (1985) Mol. Gen. Genet. 199:161; and Draper, et al.,
(1982) Plant Cell Physiol. 23:451.
[0126] Electroporation of protoplasts and whole cells and tissues
has also been described. See, e.g., Donn, et al., (1990) in
Abstracts of the VIIth Int'l. Congress on Plant Cell and Tissue
Culture IAPTC, A2-38, p. 53; D'Halluin, et al., (1992) Plant Cell
4:1495-505; and Spencer, et al., (1994) Plant Mol. Biol.
24:51-61.
Increasing the Activity and/or Level of an amt Polypeptide
[0127] Methods are provided to increase the activity and/or level
of the AMT polypeptide of the invention. An increase in the level
and/or activity of the AMT polypeptide of the invention can be
achieved by providing to the plant an amt polypeptide. The AMT
polypeptide can be provided by introducing the amino acid sequence
encoding the AMT polypeptide into the plant, introducing into the
plant a nucleotide sequence encoding an amt polypeptide or
alternatively by modifying a genomic locus encoding the AMT
polypeptide of the invention.
[0128] As discussed elsewhere herein, many methods are known the
art for providing a polypeptide to a plant including, but not
limited to, direct introduction of the polypeptide into the plant,
introducing into the plant (transiently or stably) a polynucleotide
construct encoding a polypeptide having AMT transporter activity.
It is also recognized that the methods of the invention may employ
a polynucleotide that is not capable of directing, in the
transformed plant, the expression of a protein or an RNA. Thus, the
level and/or activity of an amt polypeptide may be increased by
altering the gene encoding the AMT polypeptide or its promoter.
See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarling, et al.,
PCT/US93/03868. Therefore mutagenized plants that carry mutations
in AMT genes, where the mutations increase expression of the AMT
gene or increase the AMT transporter activity of the encoded AMT
polypeptide are provided.
Reducing the Activity and/or Level of an amt Polypeptide
[0129] Methods are provided to reduce or eliminate the activity of
an amt polypeptide of the invention by transforming a plant cell
with an expression cassette that expresses a polynucleotide that
inhibits the expression of the AMT polypeptide. The polynucleotide
may inhibit the expression of the AMT polypeptide directly, by
preventing transcription or translation of the AMT messenger RNA,
or indirectly, by encoding a polypeptide that inhibits the
transcription or translation of an amt gene encoding an amt
polypeptide. Methods for inhibiting or eliminating the expression
of a gene in a plant are well known in the art, and any such method
may be used in the present invention to inhibit the expression of
an amt polypeptide.
[0130] In accordance with the present invention, the expression of
an amt polypeptide is inhibited if the protein level of the AMT
polypeptide is less than 70% of the protein level of the same AMT
polypeptide in a plant that has not been genetically modified or
mutagenized to inhibit the expression of that AMT polypeptide. In
particular embodiments of the invention, the protein level of the
AMT polypeptide in a modified plant according to the invention is
less than 60%, less than 50%, less than 40%, less than 30%, less
than 20%, less than 10%, less than 5% or less than 2% of the
protein level of the same AMT polypeptide in a plant that is not a
mutant or that has not been genetically modified to inhibit the
expression of that AMT polypeptide. The expression level of the AMT
polypeptide may be measured directly, for example, by assaying for
the level of AMT polypeptide expressed in the plant cell or plant,
or indirectly, for example, by measuring the AMT transporter
activity of the AMT polypeptide in the plant cell or plant, or by
measuring the AMT in the plant. Methods for performing such assays
are described elsewhere herein.
[0131] In other embodiments of the invention, the activity of the
AMT polypeptides is reduced or eliminated by transforming a plant
cell with an expression cassette comprising a polynucleotide
encoding a polypeptide that inhibits the activity of an amt
polypeptide. The AMT transporter activity of an amt polypeptide is
inhibited according to the present invention if the AMT transporter
activity of the AMT polypeptide is less than 70% of the AMT
transporter activity of the same AMT polypeptide in a plant that
has not been modified to inhibit the AMT transporter activity of
that AMT polypeptide. In particular embodiments of the invention,
the AMT transporter activity of the AMT polypeptide in a modified
plant according to the invention is less than 60%, less than 50%,
less than 40%, less than 30%, less than 20%, less than 10% or less
than 5% of the AMT transporter activity of the same AMT polypeptide
in a plant that that has not been modified to inhibit the
expression of that AMT polypeptide. The AMT transporter activity of
an amt polypeptide is "eliminated" according to the invention when
it is not detectable by the assay methods described elsewhere
herein. Methods of determining the AMT transporter activity of an
amt polypeptide are described elsewhere herein.
[0132] In other embodiments, the activity of an amt polypeptide may
be reduced or eliminated by disrupting the gene encoding the AMT
polypeptide. The invention encompasses mutagenized plants that
carry mutations in AMT genes, where the mutations reduce expression
of the AMT gene or inhibit the AMT transporter activity of the
encoded AMT polypeptide.
[0133] Thus, many methods may be used to reduce or eliminate the
activity of an amt polypeptide. In addition, more than one method
may be used to reduce the activity of a single AMT polypeptide.
Non-limiting examples of methods of reducing or eliminating the
expression of AMT polypeptides are given below.
[0134] 1. Polynucleotide-Based Methods:
[0135] In some embodiments of the present invention, a plant is
transformed with an expression cassette that is capable of
expressing a polynucleotide that inhibits the expression of an amt
polypeptide of the invention. The term "expression" as used herein
refers to the biosynthesis of a gene product, including the
transcription and/or translation of said gene product. For example,
for the purposes of the present invention, an expression cassette
capable of expressing a polynucleotide that inhibits the expression
of at least one AMT polypeptide is an expression cassette capable
of producing an RNA molecule that inhibits the transcription and/or
translation of at least one AMT polypeptide of the invention. The
"expression" or "production" of a protein or polypeptide from a DNA
molecule refers to the transcription and translation of the coding
sequence to produce the protein or polypeptide, while the
"expression" or "production" of a protein or polypeptide from an
RNA molecule refers to the translation of the RNA coding sequence
to produce the protein or polypeptide.
[0136] Examples of polynucleotides that inhibit the expression of
an amt polypeptide are given below.
[0137] i. Sense Suppression/Cosuppression
[0138] In some embodiments of the invention, inhibition of the
expression of an amt polypeptide may be obtained by sense
suppression or cosuppression. For cosuppression, an expression
cassette is designed to express an RNA molecule corresponding to
all or part of a messenger RNA encoding an amt polypeptide in the
"sense" orientation. Over expression of the RNA molecule can result
in reduced expression of the native gene. Accordingly, multiple
plant lines transformed with the cosuppression expression cassette
are screened to identify those that show the greatest inhibition of
AMT polypeptide expression.
[0139] The polynucleotide used for cosuppression may correspond to
all or part of the sequence encoding the AMT polypeptide, all or
part of the 5' and/or 3' untranslated region of an amt polypeptide
transcript, or all or part of both the coding sequence and the
untranslated regions of a transcript encoding an amt polypeptide.
In some embodiments where the polynucleotide comprises all or part
of the coding region for the AMT polypeptide, the expression
cassette is designed to eliminate the start codon of the
polynucleotide so that no protein product will be translated.
[0140] Cosuppression may be used to inhibit the expression of plant
genes to produce plants having undetectable protein levels for the
proteins encoded by these genes. See, for example, Broin, et al.,
(2002) Plant Cell 14:1417-1432. Cosuppression may also be used to
inhibit the expression of multiple proteins in the same plant. See,
for example, U.S. Pat. No. 5,942,657. Methods for using
cosuppression to inhibit the expression of endogenous genes in
plants are described in Flavell, et al., (1994) Proc. Natl. Acad.
Sci. USA 91:3490-3496; Jorgensen, et al., (1996) Plant Mol. Biol.
31:957-973; Johansen and Carrington (2001) Plant Physiol.
126:930-938; Broin, et al., (2002) Plant Cell 14:1417-1432;
Stoutjesdijk, et al., (2002) Plant Physiol. 129:1723-1731; Yu, et
al., (2003) Phytochemistry 63:753-763; and U.S. Pat. Nos.
5,034,323, 5,283,184, and 5,942,657; each of which is herein
incorporated by reference. The efficiency of cosuppression may be
increased by including a poly-dT region in the expression cassette
at a position 3' to the sense sequence and 5' of the
polyadenylation signal. See, US Patent Application Publication
Number 20020048814, herein incorporated by reference. Typically,
such a nucleotide sequence has substantial sequence identity to the
sequence of the transcript of the endogenous gene, optimally
greater than about 65% sequence identity, more optimally greater
than about 85% sequence identity, most optimally greater than about
95% sequence identity. See, U.S. Pat. Nos. 5,283,184 and 5,034,323;
herein incorporated by reference.
[0141] ii. Antisense Suppression
[0142] In some embodiments of the invention, inhibition of the
expression of the AMT polypeptide may be obtained by antisense
suppression. For antisense suppression, the expression cassette is
designed to express an RNA molecule complementary to all or part of
a messenger RNA encoding the AMT polypeptide. Over expression of
the antisense RNA molecule can result in reduced expression of the
native gene. Accordingly, multiple plant lines transformed with the
antisense suppression expression cassette are screened to identify
those that show the greatest inhibition of AMT polypeptide
expression.
[0143] The polynucleotide for use in antisense suppression may
correspond to all or part of the complement of the sequence
encoding the AMT polypeptide, all or part of the complement of the
5' and/or 3' untranslated region of the AMT transcript, or all or
part of the complement of both the coding sequence and the
untranslated regions of a transcript encoding the AMT polypeptide.
In addition, the antisense polynucleotide may be fully
complementary (i.e., 100% identical to the complement of the target
sequence) or partially complementary (i.e., less than 100%
identical to the complement of the target sequence) to the target
sequence. Antisense suppression may be used to inhibit the
expression of multiple proteins in the same plant. See, for
example, U.S. Pat. No. 5,942,657. Furthermore, portions of the
antisense nucleotides may be used to disrupt the expression of the
target gene. Generally, sequences of at least 50 nucleotides, 100
nucleotides, 200 nucleotides, 300, 400, 450, 500, 550 or greater
may be used. Methods for using antisense suppression to inhibit the
expression of endogenous genes in plants are described, for
example, in Liu, et al., (2002) Plant Physiol. 129:1732-1743 and
U.S. Pat. Nos. 5,759,829 and 5,942,657, each of which is herein
incorporated by reference. Efficiency of antisense suppression may
be increased by including a poly-dT region in the expression
cassette at a position 3' to the antisense sequence and 5' of the
polyadenylation signal. See, US Patent Application Publication
Number 20020048814, herein incorporated by reference.
[0144] iii. Double-Stranded RNA Interference
[0145] In some embodiments of the invention, inhibition of the
expression of an amt polypeptide may be obtained by double-stranded
RNA (dsRNA) interference. For dsRNA interference, a sense RNA
molecule like that described above for cosuppression and an
antisense RNA molecule that is fully or partially complementary to
the sense RNA molecule are expressed in the same cell, resulting in
inhibition of the expression of the corresponding endogenous
messenger RNA.
[0146] Expression of the sense and antisense molecules can be
accomplished by designing the expression cassette to comprise both
a sense sequence and an antisense sequence. Alternatively, separate
expression cassettes may be used for the sense and antisense
sequences. Multiple plant lines transformed with the dsRNA
interference expression cassette or expression cassettes are then
screened to identify plant lines that show the greatest inhibition
of AMT polypeptide expression. Methods for using dsRNA interference
to inhibit the expression of endogenous plant genes are described
in Waterhouse, et al., (1998) Proc. Natl. Acad. Sci. USA
95:13959-13964, Liu, et al., (2002) Plant Physiol. 129:1732-1743,
and WO 99/49029, WO 99/53050, WO 99/61631, and WO 00/49035; each of
which is herein incorporated by reference.
[0147] iv. Hairpin RNA Interference and Intron-Containing Hairpin
RNA Interference
[0148] In some embodiments of the invention, inhibition of the
expression of an amt polypeptide may be obtained by hairpin RNA
(hpRNA) interference or intron-containing hairpin RNA (ihpRNA)
interference. These methods are highly efficient at inhibiting the
expression of endogenous genes. See, Waterhouse and Helliwell
(2003) Nat. Rev. Genet. 4:29-38 and the references cited
therein.
[0149] For hpRNA interference, the expression cassette is designed
to express an RNA molecule that hybridizes with itself to form a
hairpin structure that comprises a single-stranded loop region and
a base-paired stem. The base-paired stem region comprises a sense
sequence corresponding to all or part of the endogenous messenger
RNA encoding the gene whose expression is to be inhibited, and an
antisense sequence that is fully or partially complementary to the
sense sequence. Alternatively, the base-paired stem region may
correspond to a portion of a promoter sequence controlling
expression of the gene to be inhibited. Thus, the base-paired stem
region of the molecule generally determines the specificity of the
RNA interference. hpRNA molecules are highly efficient at
inhibiting the expression of endogenous genes, and the RNA
interference they induce is inherited by subsequent generations of
plants. See, for example, Chuang and Meyerowitz (2000) Proc. Natl.
Acad. Sci. USA 97:4985-4990; Stoutjesdijk, et al., (2002) Plant
Physiol. 129:1723-1731; and Waterhouse and Helliwell (2003) Nat.
Rev. Genet. 4:29-38. Methods for using hpRNA interference to
inhibit or silence the expression of genes are described, for
example, in Chuang and Meyerowitz (2000) Proc. Natl. Acad. Sci. USA
97:4985-4990; Stoutjesdijk, et al., (2002) Plant Physiol.
129:1723-1731; Waterhouse and Helliwell (2003) Nat. Rev. Genet.
4:29-38; Pandolfini, et al., BMC Biotechnology 3:7, and US Patent
Application Publication Number 20030175965; each of which is herein
incorporated by reference. A transient assay for the efficiency of
hpRNA constructs to silence gene expression in vivo has been
described by Panstruga, et al., (2003) Mol. Biol. Rep. 30:135-140,
herein incorporated by reference.
[0150] For ihpRNA, the interfering molecules have the same general
structure as for hpRNA, but the RNA molecule additionally comprises
an intron that is capable of being spliced in the cell in which the
ihpRNA is expressed. The use of an intron minimizes the size of the
loop in the hairpin RNA molecule following splicing, and this
increases the efficiency of interference. See, for example, Smith,
et al., (2000) Nature 407:319-320. In fact, Smith, et al., show
100% suppression of endogenous gene expression using
ihpRNA-mediated interference. Methods for using ihpRNA interference
to inhibit the expression of endogenous plant genes are described,
for example, in Smith, et al., (2000) Nature 407:319-320; Wesley,
et al., (2001) Plant J. 27:581-590; Wang and Waterhouse (2001)
Curr. Opin. Plant Biol. 5:146-150; Waterhouse and Helliwell (2003)
Nat. Rev. Genet. 4:29-38; Helliwell and Waterhouse (2003) Methods
30:289-295, and US Patent Application Publication Number
20030180945, each of which is herein incorporated by reference.
[0151] The expression cassette for hpRNA interference may also be
designed such that the sense sequence and the antisense sequence do
not correspond to an endogenous RNA. In this embodiment, the sense
and antisense sequence flank a loop sequence that comprises a
nucleotide sequence corresponding to all or part of the endogenous
messenger RNA of the target gene. Thus, it is the loop region that
determines the specificity of the RNA interference. See, for
example, WO 02/00904, Mette, et al., (2000) EMBO J. 19:5194-5201;
Matzke, et al., (2001) Curr. Opin. Genet. Devel. 11:221-227;
Scheid, et al., (2002) Proc. Natl. Acad. Sci., USA 99:13659-13662;
Aufsaftz, et al., (2002) Proc. Nat'l. Acad. Sci. 99(4):16499-16506;
Sijen, et al., Curr. Biol. (2001) 11:436-440), herein incorporated
by reference.
[0152] v. Amplicon-Mediated Interference
[0153] Amplicon expression cassettes comprise a plant virus-derived
sequence that contains all or part of the target gene but generally
not all of the genes of the native virus. The viral sequences
present in the transcription product of the expression cassette
allow the transcription product to direct its own replication. The
transcripts produced by the amplicon may be either sense or
antisense relative to the target sequence (i.e., the messenger RNA
for the AMT polypeptide). Methods of using amplicons to inhibit the
expression of endogenous plant genes are described, for example, in
Angell and Baulcombe (1997) EMBO J. 16:3675-3684, Angell and
Baulcombe (1999) Plant J. 20:357-362, and U.S. Pat. No. 6,646,805,
each of which is herein incorporated by reference.
[0154] vi. Ribozymes
[0155] In some embodiments, the polynucleotide expressed by the
expression cassette of the invention is catalytic RNA or has
ribozyme activity specific for the messenger RNA of the AMT
polypeptide. Thus, the polynucleotide causes the degradation of the
endogenous messenger RNA, resulting in reduced expression of the
AMT polypeptide. This method is described, for example, in U.S.
Pat. No. 4,987,071, herein incorporated by reference.
[0156] vii. Small Interfering RNA or Micro RNA
[0157] In some embodiments of the invention, inhibition of the
expression of an amt polypeptide may be obtained by RNA
interference by expression of a gene encoding a micro RNA (miRNA).
miRNAs are regulatory agents consisting of about 22
ribonucleotides. miRNA are highly efficient at inhibiting the
expression of endogenous genes. See, for example, Javier, et al.,
(2003) Nature 425:257-263, herein incorporated by reference.
[0158] For miRNA interference, the expression cassette is designed
to express an RNA molecule that is modeled on an endogenous miRNA
gene. The miRNA gene encodes an RNA that forms a hairpin structure
containing a 22-nucleotide sequence that is complementary to
another endogenous gene (target sequence). For suppression of AMT
expression, the 22-nucleotide sequence is selected from an amt
transcript sequence and contains 22 nucleotides of said AMT
sequence in sense orientation and 21 nucleotides of a corresponding
antisense sequence that is complementary to the sense sequence.
miRNA molecules are highly efficient at inhibiting the expression
of endogenous genes, and the RNA interference they induce is
inherited by subsequent generations of plants.
[0159] 2. Polypeptide-Based Inhibition of Gene Expression
[0160] In one embodiment, the polynucleotide encodes a zinc finger
protein that binds to a gene encoding an amt polypeptide, resulting
in reduced expression of the gene. In particular embodiments, the
zinc finger protein binds to a regulatory region of an amt gene. In
other embodiments, the zinc finger protein binds to a messenger RNA
encoding an amt polypeptide and prevents its translation. Methods
of selecting sites for targeting by zinc finger proteins have been
described, for example, in U.S. Pat. No. 6,453,242, and methods for
using zinc finger proteins to inhibit the expression of genes in
plants are described, for example, in US Patent Application
Publication Number 20030037355; each of which is herein
incorporated by reference.
[0161] 3. Polypeptide-Based Inhibition of Protein Activity
[0162] In some embodiments of the invention, the polynucleotide
encodes an antibody that binds to at least one AMT polypeptide, and
reduces the AMT transporter activity of the AMT polypeptide. In
another embodiment, the binding of the antibody results in
increased turnover of the antibody-AMT complex by cellular quality
control mechanisms. The expression of antibodies in plant cells and
the inhibition of molecular pathways by expression and binding of
antibodies to proteins in plant cells are well known in the art.
See, for example, Conrad and Sonnewald (2003) Nature Biotech.
21:35-36, incorporated herein by reference.
[0163] 4. Gene Disruption
[0164] In some embodiments of the present invention, the activity
of an amt polypeptide is reduced or eliminated by disrupting the
gene encoding the AMT polypeptide. The gene encoding the AMT
polypeptide may be disrupted by any method known in the art. For
example, in one embodiment, the gene is disrupted by transposon
tagging. In another embodiment, the gene is disrupted by
mutagenizing plants using random or targeted mutagenesis, and
selecting for plants that have reduced AMT transporter
activity.
[0165] i. Transposon Tagging
[0166] In one embodiment of the invention, transposon tagging is
used to reduce or eliminate the AMT activity of one or more AMT
polypeptide. Transposon tagging comprises inserting a transposon
within an endogenous AMT gene to reduce or eliminate expression of
the AMT polypeptide. "AMT gene" is intended to mean the gene that
encodes an amt polypeptide according to the invention.
[0167] In this embodiment, the expression of one or more AMT
polypeptide is reduced or eliminated by inserting a transposon
within a regulatory region or coding region of the gene encoding
the AMT polypeptide. A transposon that is within an exon, intron,
5' or 3' untranslated sequence, a promoter, or any other regulatory
sequence of an amt gene may be used to reduce or eliminate the
expression and/or activity of the encoded AMT polypeptide.
[0168] Methods for the transposon tagging of specific genes in
plants are well known in the art. See, for example, Maes, et al.,
(1999) Trends Plant Sci. 4:90-96; Dharmapuri and Sonti (1999) FEMS
Microbiol. Lett. 179:53-59; Meissner, et al., (2000) Plant J.
22:265-274; Phogat, et al., (2000) J. Biosci. 25:57-63; Walbot
(2000) Curr. Opin. Plant Biol. 2:103-107; Gai, et al., (2000)
Nucleic Acids Res. 28:94-96; Fitzmaurice, et al., (1999) Genetics
153:1919-1928). In addition, the TUSC process for selecting Mu
insertions in selected genes has been described in Bensen, et al.,
(1995) Plant Cell 7:75-84; Mena, et al., (1996) Science
274:1537-1540; and U.S. Pat. No. 5,962,764; each of which is herein
incorporated by reference.
[0169] ii. Mutant Plants with Reduced Activity
[0170] Additional methods for decreasing or eliminating the
expression of endogenous genes in plants are also known in the art
and can be similarly applied to the instant invention. These
methods include other forms of mutagenesis, such as ethyl
methanesulfonate-induced mutagenesis, deletion mutagenesis, and
fast neutron deletion mutagenesis used in a reverse genetics sense
(with PCR) to identify plant lines in which the endogenous gene has
been deleted. For examples of these methods see, Ohshima, et al.,
(1998) Virology 243:472-481; Okubara, et al., (1994) Genetics
137:867-874; and Quesada, et al., (2000) Genetics 154:421-436; each
of which is herein incorporated by reference. In addition, a fast
and automatable method for screening for chemically induced
mutations, TILLING (Targeting Induced Local Lesions In Genomes),
using denaturing HPLC or selective endonuclease digestion of
selected PCR products is also applicable to the instant invention.
See, McCallum, et al., (2000) Nat. Biotechnol. 18:455-457, herein
incorporated by reference.
[0171] Mutations that impact gene expression or that interfere with
the function (AMT transporter activity) of the encoded protein are
well known in the art. Insertional mutations in gene exons usually
result in null-mutants. Mutations in conserved residues are
particularly effective in inhibiting the AMT transporter activity
of the encoded protein. Conserved residues of plant AMT
polypeptides suitable for mutagenesis with the goal to eliminate
AMT transporter activity have been described. Such mutants can be
isolated according to well-known procedures, and mutations in
different AMT loci can be stacked by genetic crossing. See, for
example, Gruis, et al., (2002) Plant Cell 14:2863-2882.
[0172] In another embodiment of this invention, dominant mutants
can be used to trigger RNA silencing due to gene inversion and
recombination of a duplicated gene locus. See, for example, Kusaba,
et al., (2003) Plant Cell 15:1455-1467.
[0173] The invention encompasses additional methods for reducing or
eliminating the activity of one or more AMT polypeptide. Examples
of other methods for altering or mutating a genomic nucleotide
sequence in a plant are known in the art and include, but are not
limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors,
RNA:DNA repair vectors, mixed-duplex oligonucleotides,
self-complementary RNA:DNA oligonucleotides, and recombinogenic
oligonucleobases. Such vectors and methods of use are known in the
art. See, for example, U.S. Pat. Nos. 5,565,350; 5,731,181;
5,756,325; 5,760,012; 5,795,972; and 5,871,984; each of which are
herein incorporated by reference. See also, WO 98/49350, WO
99/07865, WO 99/25821, and Beetham, et al., (1999) Proc. Natl.
Acad. Sci. USA 96:8774-8778; each of which is herein incorporated
by reference.
[0174] iii. Modulating AMT Transporter Activity
[0175] In specific methods, the level and/or activity of an amt
regulator in a plant is decreased by increasing the level or
activity of the AMT polypeptide in the plant. Methods for
increasing the level and/or activity of AMT polypeptides in a plant
are discussed elsewhere herein. Briefly, such methods comprise
providing an amt polypeptide of the invention to a plant and
thereby increasing the level and/or activity of the AMT
polypeptide. In other embodiments, an amt nucleotide sequence
encoding an amt polypeptide can be provided by introducing into the
plant a polynucleotide comprising an amt nucleotide sequence of the
invention, expressing the AMT sequence, increasing the activity of
the AMT polypeptide, and thereby decreasing the ammonium uptake or
transport in the plant or plant part. In other embodiments, the AMT
nucleotide construct introduced into the plant is stably
incorporated into the genome of the plant.
[0176] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate the level/activity of an
amt transporter in the plant. Exemplary promoters for this
embodiment have been disclosed elsewhere herein.
[0177] Accordingly, the present invention further provides plants
having a modified number of cells when compared to the number of
cells of a control plant tissue. In one embodiment, the plant of
the invention has an increased level/activity of the AMT
polypeptide of the invention and thus has an increased Ammonium
transport in the plant tissue. In other embodiments, the plant of
the invention has a reduced or eliminated level of the AMT
polypeptide of the invention and thus has an increased NUE in the
plant tissue. In other embodiments, such plants have stably
incorporated into their genome a nucleic acid molecule comprising
an amt nucleotide sequence of the invention operably linked to a
promoter that drives expression in the plant cell.
[0178] iv. Modulating Root Development
[0179] Methods for modulating root development in a plant are
provided. By "modulating root development" is intended any
alteration in the development of the plant root when compared to a
control plant. Such alterations in root development include, but
are not limited to, alterations in the growth rate of the primary
root, the fresh root weight, the extent of lateral and adventitious
root formation, the vasculature system, meristem development, or
radial expansion.
[0180] Methods for modulating root development in a plant are
provided. The methods comprise modulating the level and/or activity
of the AMT polypeptide in the plant. In one method, an amt sequence
of the invention is provided to the plant. In another method, the
AMT nucleotide sequence is provided by introducing into the plant a
polynucleotide comprising an amt nucleotide sequence of the
invention, expressing the AMT sequence, and thereby modifying root
development. In still other methods, the AMT nucleotide construct
introduced into the plant is stably incorporated into the genome of
the plant.
[0181] In other methods, root development is modulated by altering
the level or activity of the AMT polypeptide in the plant. A
decrease in AMT activity can result in at least one or more of the
following alterations to root development, including, but not
limited to, larger root meristems, increased in root growth,
enhanced radial expansion, an enhanced vasculature system,
increased root branching, more adventitious roots, and/or an
increase in fresh root weight when compared to a control plant.
[0182] As used herein, "root growth" encompasses all aspects of
growth of the different parts that make up the root system at
different stages of its development in both monocotyledonous and
dicotyledonous plants. It is to be understood that enhanced root
growth can result from enhanced growth of one or more of its parts
including the primary root, lateral roots, adventitious roots,
etc.
[0183] Methods of measuring such developmental alterations in the
root system are known in the art. See, for example, US Patent
Application Publication Number 2003/0074698 and Werner, et al.,
(2001) PNAS 18:10487-10492, both of which are herein incorporated
by reference.
[0184] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate root development in the
plant. Exemplary promoters for this embodiment include constitutive
promoters and root-preferred promoters. Exemplary root-preferred
promoters have been disclosed elsewhere herein.
[0185] Stimulating root growth and increasing root mass by
decreasing the activity and/or level of the AMT polypeptide also
finds use in improving the standability of a plant. The term
"resistance to lodging" or "standability" refers to the ability of
a plant to fix itself to the soil. For plants with an erect or
semi-erect growth habit, this term also refers to the ability to
maintain an upright position under adverse (environmental)
conditions. This trait relates to the size, depth and morphology of
the root system. In addition, stimulating root growth and
increasing root mass by decreasing the level and/or activity of the
AMT polypeptide also finds use in promoting in vitro propagation of
explants.
[0186] Furthermore, higher root biomass production due to an
decreased level and/or activity of AMT activity has a direct effect
on the yield and an indirect effect of production of compounds
produced by root cells or transgenic root cells or cell cultures of
said transgenic root cells. One example of an interesting compound
produced in root cultures is shikonin, the yield of which can be
advantageously enhanced by said methods.
[0187] Accordingly, the present invention further provides plants
having modulated root development when compared to the root
development of a control plant. In some embodiments, the plant of
the invention has an increased level/activity of the AMT
polypeptide of the invention and has enhanced root growth and/or
root biomass. In other embodiments, such plants have stably
incorporated into their genome a nucleic acid molecule comprising
an amt nucleotide sequence of the invention operably linked to a
promoter that drives expression in the plant cell.
[0188] v. Modulating Shoot and Leaf Development
[0189] Methods are also provided for modulating shoot and leaf
development in a plant. By "modulating shoot and/or leaf
development" is intended any alteration in the development of the
plant shoot and/or leaf. Such alterations in shoot and/or leaf
development include, but are not limited to, alterations in shoot
meristem development, in leaf number, leaf size, leaf and stem
vasculature, internode length, and leaf senescence. As used herein,
"leaf development" and "shoot development" encompasses all aspects
of growth of the different parts that make up the leaf system and
the shoot system, respectively, at different stages of their
development, both in monocotyledonous and dicotyledonous plants.
Methods for measuring such developmental alterations in the shoot
and leaf system are known in the art. See, for example, Werner, et
al., (2001) PNAS 98:10487-10492 and US Patent Application
Publication Number 2003/0074698, each of which is herein
incorporated by reference.
[0190] The method for modulating shoot and/or leaf development in a
plant comprises modulating the activity and/or level of an AMT
polypeptide of the invention. In one embodiment, an amt sequence of
the invention is provided. In other embodiments, the AMT nucleotide
sequence can be provided by introducing into the plant a
polynucleotide comprising an amt nucleotide sequence of the
invention, expressing the AMT sequence, and thereby modifying shoot
and/or leaf development. In other embodiments, the AMT nucleotide
construct introduced into the plant is stably incorporated into the
genome of the plant.
[0191] In specific embodiments, shoot or leaf development is
modulated by increasing the level and/or activity of the AMT
polypeptide in the plant. An increase in AMT activity can result in
at least one or more of the following alterations in shoot and/or
leaf development, including, but not limited to, reduced leaf
number, reduced leaf surface, reduced vascular, shorter internodes
and stunted growth, and retarded leaf senescence, when compared to
a control plant.
[0192] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate shoot and leaf development
of the plant. Exemplary promoters for this embodiment include
constitutive promoters, shoot-preferred promoters, shoot
meristem-preferred promoters, and leaf-preferred promoters.
Exemplary promoters have been disclosed elsewhere herein.
[0193] Increasing AMT activity and/or level in a plant results in
shorter internodes and stunted growth. Thus, the methods of the
invention find use in producing dwarf plants. In addition, as
discussed above, modulation AMT activity in the plant modulates
both root and shoot growth. Thus, the present invention further
provides methods for altering the root/shoot ratio. Shoot or leaf
development can further be modulated by decreasing the level and/or
activity of the AMT polypeptide in the plant.
[0194] Accordingly, the present invention further provides plants
having modulated shoot and/or leaf development when compared to a
control plant. In some embodiments, the plant of the invention has
an increased level/activity of the AMT polypeptide of the
invention. In other embodiments, the plant of the invention has a
decreased level/activity of the AMT polypeptide of the
invention.
[0195] vi Modulating Reproductive Tissue Development
[0196] Methods for modulating reproductive tissue development are
provided. In one embodiment, methods are provided to modulate
floral development in a plant. By "modulating floral development"
is intended any alteration in a structure of a plant's reproductive
tissue as compared to a control plant in which the activity or
level of the AMT polypeptide has not been modulated. "Modulating
floral development" further includes any alteration in the timing
of the development of a plant's reproductive tissue (i.e., a
delayed or a accelerated timing of floral development) when
compared to a control plant in which the activity or level of the
AMT polypeptide has not been modulated. Macroscopic alterations may
include changes in size, shape, number, or location of reproductive
organs, the developmental time period that these structures form,
or the ability to maintain or proceed through the flowering process
in times of environmental stress. Microscopic alterations may
include changes to the types or shapes of cells that make up the
reproductive organs.
[0197] The method for modulating floral development in a plant
comprises modulating AMT activity in a plant. In one method, an AMT
sequence of the invention is provided. AN AMT nucleotide sequence
can be provided by introducing into the plant a polynucleotide
comprising an amt nucleotide sequence of the invention, expressing
the AMT sequence, and thereby modifying floral development. In
other embodiments, the AMT nucleotide construct introduced into the
plant is stably incorporated into the genome of the plant.
[0198] In specific methods, floral development is modulated by
increasing the level or activity of the AMT polypeptide in the
plant. An increase in AMT activity can result in at least one or
more of the following alterations in floral development, including,
but not limited to, retarded flowering, reduced number of flowers,
partial male sterility, and reduced seed set, when compared to a
control plant. Inducing delayed flowering or inhibiting flowering
can be used to enhance yield in forage crops such as alfalfa.
Methods for measuring such developmental alterations in floral
development are known in the art. See, for example, Mouradov, et
al., (2002) The Plant Cell S11-S130, herein incorporated by
reference.
[0199] As discussed above, one of skill will recognize the
appropriate promoter to use to modulate floral development of the
plant. Exemplary promoters for this embodiment include constitutive
promoters, inducible promoters, shoot-preferred promoters, and
inflorescence-preferred promoters.
[0200] In other methods, floral development is modulated by
decreasing the level and/or activity of the AMT sequence of the
invention. Such methods can comprise introducing an amt nucleotide
sequence into the plant and decreasing the activity of the AMT
polypeptide. In other methods, the AMT nucleotide construct
introduced into the plant is stably incorporated into the genome of
the plant. Decreasing expression of the AMT sequence of the
invention can modulate floral development during periods of stress.
Such methods are described elsewhere herein. Accordingly, the
present invention further provides plants having modulated floral
development when compared to the floral development of a control
plant. Compositions include plants having a decreased
level/activity of the AMT polypeptide of the invention and having
an altered floral development. Compositions also include plants
having a decreased level/activity of the AMT polypeptide of the
invention wherein the plant maintains or proceeds through the
flowering process in times of stress.
[0201] Methods are also provided for the use of the AMT sequences
of the invention to increase nitrogen use efficiency. The method
comprises decreasing or increasing the activity of the AMT
sequences in a plant or plant part, such as the roots, shoot,
epidermal cells, etc.
[0202] As discussed above, one of skill will recognize the
appropriate promoter to use to manipulate the expression of AMTs.
Exemplary promoters of this embodiment include constitutive
promoters, inducible promoters, and root or shoot or leaf preferred
promoters.
[0203] vii. Method of Use for AMT Promoter Polynucleotides
[0204] The polynucleotides comprising the AMT promoters disclosed
in the present invention, as well as variants and fragments
thereof, are useful in the genetic manipulation of any host cell,
preferably plant cell, when assembled with a DNA construct such
that the promoter sequence is operably linked to a nucleotide
sequence comprising a polynucleotide of interest. In this manner,
the AMT promoter polynucleotides of the invention are provided in
expression cassettes along with a polynucleotide sequence of
interest for expression in the host cell of interest. As discussed
in Example XX below, the AMT promoter sequences of the invention
are expressed in a variety of tissues and thus the promoter
sequences can find use in regulating the temporal and/or the
spatial expression of polynucleotides of interest.
[0205] Synthetic hybrid promoter regions are known in the art. Such
regions comprise upstream promoter elements of one polynucleotide
operably linked to the promoter element of another polynucleotide.
In an embodiment of the invention, heterologous sequence expression
is controlled by a synthetic hybrid promoter comprising the AMT
promoter sequences of the invention, or a variant or fragment
thereof, operably linked to upstream promoter element(s) from a
heterologous promoter. Upstream promoter elements that are involved
in the plant defense system have been identified and may be used to
generate a synthetic promoter. See, for example, Rushton, et al.,
(1998) Curr. Opin. Plant Biol. 1:311-315. Alternatively, a
synthetic AMT promoter sequence may comprise duplications of the
upstream promoter elements found within the AMT promoter
sequences.
[0206] It is recognized that the promoter sequence of the invention
may be used with its native AMT coding sequences. A DNA construct
comprising the AMT promoter operably linked with its native AMT
gene may be used to transform any plant of interest to bring about
a desired phenotypic change, such as, modulating root, shoot, leaf,
floral, and embryo development, stress tolerance, and any other
phenotype described elsewhere herein.
[0207] The promoter nucleotide sequences and methods disclosed
herein are useful in regulating expression of any heterologous
nucleotide sequence in a host plant in order to vary the phenotype
of a plant. Various changes in phenotype are of interest including
modifying the fatty acid composition in a plant, altering the amino
acid content of a plant, altering a plant's pathogen defense
mechanism, and the like. These results can be achieved by providing
expression of heterologous products or increased expression of
endogenous products in plants. Alternatively, the results can be
achieved by providing for a reduction of expression of one or more
endogenous products, particularly enzymes or cofactors in the
plant. These changes result in a change in phenotype of the
transformed plant.
[0208] Genes of interest are reflective of the commercial markets
and interests of those involved in the development of the crop.
Crops and markets of interest change, and as developing nations
open up world markets, new crops and technologies will emerge also.
In addition, as our understanding of agronomic traits and
characteristics such as yield and heterosis increase, the choice of
genes for transformation will change accordingly. General
categories of genes of interest include, for example, those genes
involved in information, such as zinc fingers, those involved in
communication, such as kinases, and those involved in housekeeping,
such as heat shock proteins. More specific categories of
transgenes, for example, include genes encoding important traits
for agronomics, insect resistance, disease resistance, herbicide
resistance, sterility, grain characteristics, and commercial
products. Genes of interest include, generally, those involved in
oil, starch, carbohydrate, or nutrient metabolism as well as those
affecting kernel size, sucrose loading, and the like.
[0209] In certain embodiments the nucleic acid sequences of the
present invention can be used in combination ("stacked") with other
polynucleotide sequences of interest in order to create plants with
a desired phenotype. The combinations generated can include
multiple copies of any one or more of the polynucleotides of
interest. The polynucleotides of the present invention may be
stacked with any gene or combination of genes to produce plants
with a variety of desired trait combinations, including but not
limited to traits desirable for animal feed such as high oil genes
(e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g.,
hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and
5,703,409); barley high lysine (Williamson, et al., (1987) Eur. J.
Biochem. 165:99-106; and WO 98/20122); and high methionine proteins
(Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et
al., (1988) Gene 71:359; and Musumura, et al., (1989) Plant Mol.
Biol. 12: 123)); increased digestibility (e.g., modified storage
proteins (U.S. patent application Ser. No. 10/053,410, filed Nov.
7, 2001); and thioredoxins (U.S. patent application Ser. No.
10/005,429, filed Dec. 3, 2001)), the disclosures of which are
herein incorporated by reference. The polynucleotides of the
present invention can also be stacked with traits desirable for
insect, disease or herbicide resistance (e.g., Bacillus
thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450;
5,737,514; 5,723,756; 5,593,881; Geiser, et al., (1986) Gene
48:109); lectins (Van Damme, et al., (1994) Plant Mol. Biol.
24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931);
avirulence and disease resistance genes (Jones, et al., (1994)
Science 266:789; Martin, et al., (1993) Science 262:1432;
Mindrinos, et al., (1994) Cell 78:1089); acetolactate synthase
(ALS) mutants that lead to herbicide resistance such as the S4
and/or Hra mutations; inhibitors of glutamine synthase such as
phosphinothricin or basta (e.g., bar gene); and glyphosate
resistance (EPSPS gene)); and traits desirable for processing or
process products such as high oil (e.g., U.S. Pat. No. 6,232,529);
modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No.
5,952,544; WO 94/11516)); modified starches (e.g., ADPG
pyrophosphorylases (AGPase), starch synthases (SS), starch
branching enzymes (SBE) and starch debranching enzymes (SDBE)); and
polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;
beta-ketothiolase, polyhydroxybutyrate synthase, and
acetoacetyl-CoA reductase (Schubert, et al., (1988) J. Bacteriol.
170:5837-5847) facilitate expression of polyhydroxyalkanoates
(PHAs)), the disclosures of which are herein incorporated by
reference. One could also combine the polynucleotides of the
present invention with polynucleotides affecting agronomic traits
such as male sterility (e.g., see, U.S. Pat. No. 5,583,210), stalk
strength, flowering time, or transformation technology traits such
as cell cycle regulation or gene targeting (e.g., WO 99/61619; WO
00/17364; WO 99/25821), the disclosures of which are herein
incorporated by reference.
[0210] In one embodiment, sequences of interest improve plant
growth and/or crop yields. For example, sequences of interest
include agronomically important genes that result in improved
primary or lateral root systems. Such genes include, but are not
limited to, nutrient/water transporters and growth induces.
Examples of such genes, include but are not limited to, maize
plasma membrane H.sup.+-ATPase (MHA2) (Frias, et al., (1996) Plant
Cell 8:1533-44); AKT1, a component of the potassium uptake
apparatus in Arabidopsis, (Spalding, et al., (1999) J Gen Physiol
113:909-18); RML genes which activate cell division cycle in the
root apical cells (Cheng, et al., (1995) Plant Physiol 108:881);
maize glutamine synthetase genes (Sukanya, et al., (1994) Plant Mol
Biol 26:1935-46) and hemoglobin (Duff, et al., (1997) J. Biol.
Chem. 27:16749-16752, Arredondo-Peter, et al., (1997) Plant
Physiol. 115:1259-1266; Arredondo-Peter, et al., (1997) Plant
Physiol 114:493-500 and references sited therein). The sequence of
interest may also be useful in expressing antisense nucleotide
sequences of genes that that negatively affects root
development.
[0211] Additional, agronomically important traits such as oil,
starch, and protein content can be genetically altered in addition
to using traditional breeding methods. Modifications include
increasing content of oleic acid, saturated and unsaturated oils,
increasing levels of lysine and sulfur, providing essential amino
acids, and also modification of starch. Hordothionin protein
modifications are described in U.S. Pat. Nos. 5,703,049, 5,885,801,
5,885,802, and 5,990,389, herein incorporated by reference. Another
example is lysine and/or sulfur rich seed protein encoded by the
soybean 2S albumin described in U.S. Pat. No. 5,850,016, and the
chymotrypsin inhibitor from barley, described in Williamson, et
al., (1987) Eur. J. Biochem. 165:99-106, the disclosures of which
are herein incorporated by reference.
[0212] Derivatives of the coding sequences can be made by
site-directed mutagenesis to increase the level of preselected
amino acids in the encoded polypeptide. For example, the gene
encoding the barley high lysine polypeptide (BHL) is derived from
barley chymotrypsin inhibitor, U.S. patent application Ser. No.
08/740,682, filed Nov. 1, 1996, and WO 98/20133, the disclosures of
which are herein incorporated by reference. Other proteins include
methionine-rich plant proteins such as from sunflower seed (Lilley,
et al., (1989) Proceedings of the World Congress on Vegetable
Protein Utilization in Human Foods and Animal Feedstuffs, ed.
Applewhite (American Oil Chemists Society, Champaign, Ill.), pp.
497-502; herein incorporated by reference); corn (Pedersen, et al.,
(1986) J. Biol. Chem. 261:6279; Kirihara, et al., (1988) Gene
71:359; both of which are herein incorporated by reference); and
rice (Musumura, et al., (1989) Plant Mol. Biol. 12:123, herein
incorporated by reference). Other agronomically important genes
encode latex, Floury 2, growth factors, seed storage factors, and
transcription factors.
[0213] Insect resistance genes may encode resistance to pests that
have great yield drag such as rootworm, cutworm, European Corn
Borer, and the like. Such genes include, for example, Bacillus
thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892;
5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser, et al.,
(1986) Gene 48:109); and the like.
[0214] Genes encoding disease resistance traits include
detoxification genes, such as against fumonosin (U.S. Pat. No.
5,792,931); avirulence (avr) and disease resistance (R) genes
(Jones, et al., (1994) Science 266:789; Martin, et al., (1993)
Science 262:1432; and Mindrinos, et al., (1994) Cell 78:1089); and
the like.
[0215] Herbicide resistance traits may include genes coding for
resistance to herbicides that act to inhibit the action of
acetolactate synthase (ALS), in particular the sulfonylurea-type
herbicides (e.g., the acetolactate synthase (ALS) gene containing
mutations leading to such resistance, in particular the S4 and/or
Hra mutations), genes coding for resistance to herbicides that act
to inhibit action of glutamine synthase, such as phosphinothricin
or basta (e.g., the bar gene), or other such genes known in the
art. The bar gene encodes resistance to the herbicide basta, the
nptII gene encodes resistance to the antibiotics kanamycin and
geneticin, and the ALS-gene mutants encode resistance to the
herbicide chlorsulfuron.
[0216] Sterility genes can also be encoded in an expression
cassette and provide an alternative to physical detasseling.
Examples of genes used in such ways include male tissue-preferred
genes and genes with male sterility phenotypes such as QM,
described in U.S. Pat. No. 5,583,210. Other genes include kinases
and those encoding compounds toxic to either male or female
gametophytic development.
[0217] The quality of grain is reflected in traits such as levels
and types of oils, saturated and unsaturated, quality and quantity
of essential amino acids, and levels of cellulose. In corn,
modified hordothionin proteins are described in U.S. Pat. Nos.
5,703,049, 5,885,801, 5,885,802 and 5,990,389.
[0218] Commercial traits can also be encoded on a gene or genes
that could increase for example, starch for ethanol production, or
provide expression of proteins. Another important commercial use of
transformed plants is the production of polymers and bioplastics
such as described in U.S. Pat. No. 5,602,321. Genes such as
.beta.-Ketothiolase, PHBase (polyhydroxyburyrate synthase), and
acetoacetyl-CoA reductase (see, Schubert, et al., (1988) J.
Bacteriol. 170:5837-5847) facilitate expression of
polyhyroxyalkanoates (PHAs).
[0219] Exogenous products include plant enzymes and products as
well as those from other sources including procaryotes and other
eukaryotes. Such products include enzymes, cofactors, hormones, and
the like. The level of proteins, particularly modified proteins
having improved amino acid distribution to improve the nutrient
value of the plant, can be increased. This is achieved by the
expression of such proteins having enhanced amino acid content.
[0220] This invention can be better understood by reference to the
following non-limiting examples. It will be appreciated by those
skilled in the art that other embodiments of the invention may be
practiced without departing from the spirit and the scope of the
invention as herein disclosed and claimed.
EXAMPLES
Example 1
Isolation of AMT Sequences
[0221] A routine for identifying all members of a given species'
ammonium transporter (AMT) gene family was employed. First, a
diverse set of all the known available members of the gene family
as protein sequences was prepared from public and proprietary
sources. This data could include orthologous sequences from other
species besides these four. Then, as in the example of maize, these
protein query sequences were BLAST algorithm searched against a
combination of proprietary and public maize, genomic or transcript,
nucleotide sequence datasets, and a non-redundant set of candidate
AMTs or `hits` was identified. These sequences were combined with
any existing maize gene family sequences, and then curated and
edited to arrive at a new working set of unique maize AMT gene or
transcript sequences and their translations. This search for gene
family members was repeated. If there were recovered new sequences
whose nucleotide sequences were unique (not same-gene matches), the
process repeated until completion, that is until no new and
distinct nucleotide sequences were found. In this way it was
determined that the maize AMT family of genes consisted of at least
seven members. Eleven distinct soybean sequences were found.
Without the complete genome sequences of maize or soybean
available, researchers were less certain of the exact gene family
size, than they were for Arabidopsis (6 members) and rice (17
members). The availability of complete genome sequences for
Arabidopsis and rice simplified the search, aided also by
availability of fairly mature gene models and annotations for these
species.
Example 2
Transformation and Regeneration of Transgenic Plants
[0222] Immature maize embryos from greenhouse donor plants are
bombarded with a plasmid containing the AMT sequence operably
linked to the drought-inducible promoter RAB17 promoter (Vilardell,
et al., (1990) Plant Mol Biol 14:423-432) and the selectable marker
gene PAT, which confers resistance to the herbicide Bialaphos.
Alternatively, the selectable marker gene is provided on a separate
plasmid. Transformation is performed as follows. Media recipes
follow below.
[0223] Preparation of Target Tissue:
[0224] The ears are husked and surface sterilized in 30% Clorox
bleach plus 0.5% Micro detergent for 20 minutes, and rinsed two
times with sterile water. The immature embryos are excised and
placed embryo axis side down (scutellum side up), 25 embryos per
plate, on 560Y medium for 4 hours and then aligned within the
2.5-cm target zone in preparation for bombardment.
[0225] Preparation of DNA:
[0226] A plasmid vector comprising the AMT sequence operably linked
to an ubiquitin promoter is made. This plasmid DNA plus plasmid DNA
containing a PAT selectable marker is precipitated onto 1.1 .mu.m
(average diameter) tungsten pellets using a CaCl.sub.2
precipitation procedure as follows:
[0227] 100 .mu.l prepared tungsten particles in water
[0228] 10 .mu.l (1 .mu.g) DNA in Tris EDTA buffer (1 .mu.g total
DNA)
[0229] 100 .mu.l 2.5 M CaC1.sub.2
[0230] 10 .mu.l 0.1 M spermidine
[0231] Each reagent is added sequentially to the tungsten particle
suspension, while maintained on the multitube vortexer. The final
mixture is sonicated briefly and allowed to incubate under constant
vortexing for 10 minutes. After the precipitation period, the tubes
are centrifuged briefly, liquid removed, washed with 500 ml 100%
ethanol, and centrifuged for 30 seconds. Again the liquid is
removed, and 105 .mu.l 100% ethanol is added to the final tungsten
particle pellet. For particle gun bombardment, the tungsten/DNA
particles are briefly sonicated and 10 .mu.l spotted onto the
center of each macrocarrier and allowed to dry about 2 minutes
before bombardment.
[0232] Particle Gun Treatment:
[0233] The sample plates are bombarded at level #4 in particle gun
#HE34-1 or #HE34-2. All samples receive a single shot at 650 PSI,
with a total of ten aliquots taken from each tube of prepared
particles/DNA.
[0234] Subsequent Treatment:
[0235] Following bombardment, the embryos are kept on 560Y medium
for 2 days, then transferred to 560R selection medium containing 3
mg/liter Bialaphos, and subcultured every 2 weeks. After
approximately 10 weeks of selection, selection-resistant callus
clones are transferred to 288J medium to initiate plant
regeneration. Following somatic embryo maturation (2-4 weeks),
well-developed somatic embryos are transferred to medium for
germination and transferred to the lighted culture room.
Approximately 7-10 days later, developing plantlets are transferred
to 272V hormone-free medium in tubes for 7-10 days until plantlets
are well established. Plants are then transferred to inserts in
flats (equivalent to 2.5'' pot) containing potting soil and grown
for 1 week in a growth chamber, subsequently grown an additional
1-2 weeks in the greenhouse, then transferred to classic 600 pots
(1.6 gallon) and grown to maturity. Plants are monitored and scored
for increased drought tolerance. Assays to measure improved drought
tolerance are routine in the art and include, for example,
increased kernel-earring capacity yields under drought conditions
when compared to control maize plants under identical environmental
conditions. Alternatively, the transformed plants can be monitored
for a modulation in meristem development (i.e., a decrease in
spikelet formation on the ear). See, for example, Bruce, et al.,
(2002) Journal of Experimental Botany 53:1-13.
[0236] Bombardment and Culture Media:
[0237] Bombardment medium (560Y) comprises 4.0 g/l N6 basal salts
(SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 120.0 g/l sucrose,
1.0 mg/l 2,4-D, and 2.88 g/l L-proline (brought to volume with D-1
H.sub.2O following adjustment to pH 5.8 with KOH); 2.0 g/l Gelrite
(added after bringing to volume with D-I H.sub.2O); and 8.5 mg/l
silver nitrate (added after sterilizing the medium and cooling to
room temperature). Selection medium (560R) comprises 4.0 g/l N6
basal salts (SIGMA C-1416), 1.0 ml/l Eriksson's Vitamin Mix
(1000.times.SIGMA-1511), 0.5 mg/l thiamine HCl, 30.0 g/l sucrose,
and 2.0 mg/l 2,4-D (brought to volume with D-I H.sub.2O following
adjustment to pH 5.8 with KOH); 3.0 g/l Gelrite (added after
bringing to volume with D-I H.sub.2O); and 0.85 mg/l silver nitrate
and 3.0 mg/l bialaphos (both added after sterilizing the medium and
cooling to room temperature).
[0238] Plant regeneration medium (288J) comprises 4.3 g/l MS salts
(GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100 g
nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL, and
0.40 g/l glycine brought to volume with polished D-I H.sub.2O)
(Murashige and Skoog (1962) Physiol. Plant. 15:473), 100 mg/l
myo-inositol, 0.5 mg/l zeatin, 60 g/l sucrose, and 1.0 ml/l of 0.1
mM abscisic acid (brought to volume with polished D-I H.sub.2O
after adjusting to pH 5.6); 3.0 g/l Gelrite (added after bringing
to volume with D-I H.sub.2O); and 1.0 mg/l indoleacetic acid and
3.0 mg/l bialaphos (added after sterilizing the medium and cooling
to 60.degree. C.). Hormone-free medium (272V) comprises 4.3 g/l MS
salts (GIBCO 11117-074), 5.0 ml/l MS vitamins stock solution (0.100
g/l nicotinic acid, 0.02 g/l thiamine HCL, 0.10 g/l pyridoxine HCL,
and 0.40 g/l glycine brought to volume with polished D-I H.sub.2O),
0.1 g/1 myo-inositol, and 40.0 g/l sucrose (brought to volume with
polished D-I H.sub.2O after adjusting pH to 5.6); and 6 g/l
bacto-agar (added after bringing to volume with polished D-I
H.sub.2O), sterilized and cooled to 60.degree. C.
Example 3
Agrobacterium-Mediated Transformation
[0239] For Agrobacterium-mediated transformation of maize with an
antisense sequence of the AMT sequence of the present invention,
preferably the method of Zhao is employed (U.S. Pat. No. 5,981,840,
and PCT patent publication WO98/32326; the contents of which are
hereby incorporated by reference). Briefly, immature embryos are
isolated from maize and the embryos contacted with a suspension of
Agrobacterium, where the bacteria are capable of transferring the
antisense AMT sequences to at least one cell of at least one of the
immature embryos (step 1: the infection step). In this step the
immature embryos are preferably immersed in an Agrobacterium
suspension for the initiation of inoculation. The embryos are
co-cultured for a time with the Agrobacterium (step 2: the
co-cultivation step). Preferably the immature embryos are cultured
on solid medium following the infection step. Following this
co-cultivation period an optional "resting" step is contemplated.
In this resting step, the embryos are incubated in the presence of
at least one antibiotic known to inhibit the growth of
Agrobacterium without the addition of a selective agent for plant
transformants (step 3: resting step). Preferably the immature
embryos are cultured on solid medium with antibiotic, but without a
selecting agent, for elimination of Agrobacterium and for a resting
phase for the infected cells. Next, inoculated embryos are cultured
on medium containing a selective agent and growing transformed
callus is recovered (step 4: the selection step). Preferably, the
immature embryos are cultured on solid medium with a selective
agent resulting in the selective growth of transformed cells. The
callus is then regenerated into plants (step 5: the regeneration
step), and preferably calli grown on selective medium are cultured
on solid medium to regenerate the plants. Plants are monitored and
scored for a modulation in tissue development.
Example 4
Soybean Embryo Transformation
[0240] Soybean embryos are bombarded with a plasmid containing an
antisense AMT sequences operably linked to an ubiquitin promoter as
follows. To induce somatic embryos, cotyledons, 3-5 mm in length
dissected from surface-sterilized, immature seeds of the soybean
cultivar A2872, are cultured in the light or dark at 26.degree. C.
on an appropriate agar medium for six to ten weeks. Somatic embryos
producing secondary embryos are then excised and placed into a
suitable liquid medium. After repeated selection for clusters of
somatic embryos that multiplied as early, globular-staged embryos,
the suspensions are maintained as described below.
[0241] Soybean embryogenic suspension cultures can be maintained in
35 ml liquid media on a rotary shaker, 150 rpm, at 26.degree. C.
with florescent lights on a 16:8 hour day/night schedule. Cultures
are subcultured every two weeks by inoculating approximately 35 mg
of tissue into 35 ml of liquid medium.
[0242] Soybean embryogenic suspension cultures may then be
transformed by the method of particle gun bombardment (Klein, et
al., (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A
Du Pont Biolistic PDS1000/HE instrument (helium retrofit) can be
used for these transformations.
[0243] A selectable marker gene that can be used to facilitate
soybean transformation is a transgene composed of the 35S promoter
from Cauliflower Mosaic Virus (Odell, et al., (1985) Nature
313:810-812), the hygromycin phosphotransferase gene from plasmid
pJR225 (from E. coli; Gritz, et al., (1983) Gene 25:179-188), and
the 3' region of the nopaline synthase gene from the T-DNA of the
Ti plasmid of Agrobacterium tumefaciens. The expression cassette
comprising an antisense AMT sequence operably linked to the
ubiquitin promoter can be isolated as a restriction fragment. This
fragment can then be inserted into a unique restriction site of the
vector carrying the marker gene.
[0244] To 50 .mu.l of a 60 mg/ml 1 .mu.m gold particle suspension
is added (in order): 5 .mu.l DNA (1 .mu.g/.mu.l), 20 .mu.l
spermidine (0.1 M), and 50 .mu.l CaCl.sub.2 (2.5 M). The particle
preparation is then agitated for three minutes, spun in a microfuge
for 10 seconds and the supernatant removed. The DNA-coated
particles are then washed once in 400 .mu.l 70% ethanol and
resuspended in 40 .mu.l of anhydrous ethanol. The DNA/particle
suspension can be sonicated three times for one second each. Five
microliters of the DNA-coated gold particles are then loaded on
each macro carrier disk.
[0245] Approximately 300-400 mg of a two-week-old suspension
culture is placed in an empty 60.times.15 mm petri dish and the
residual liquid removed from the tissue with a pipette. For each
transformation experiment, approximately 5-10 plates of tissue are
normally bombarded. Membrane rupture pressure is set at 1100 psi,
and the chamber is evacuated to a vacuum of 28 inches mercury. The
tissue is placed approximately 3.5 inches away from the retaining
screen and bombarded three times. Following bombardment, the tissue
can be divided in half and placed back into liquid and cultured as
described above.
[0246] Five to seven days post bombardment, the liquid media may be
exchanged with fresh media, and eleven to twelve days
post-bombardment with fresh media containing 50 mg/ml hygromycin.
This selective media can be refreshed weekly. Seven to eight weeks
post-bombardment, green, transformed tissue may be observed growing
from untransformed, necrotic embryogenic clusters. Isolated green
tissue is removed and inoculated into individual flasks to generate
new, clonally propagated, transformed embryogenic suspension
cultures. Each new line may be treated as an independent
transformation event. These suspensions can then be subcultured and
maintained as clusters of immature embryos or regenerated into
whole plants by maturation and germination of individual somatic
embryos.
Example 5
Sunflower Meristem Tissue Transformation
[0247] Sunflower meristem tissues are transformed with an
expression cassette containing an antisense AMT sequences operably
linked to a ubiquitin promoter as follows (see also, European
Patent Number EP 0 486233, herein incorporated by reference, and
Malone-Schoneberg, et al., (1994) Plant Science 103:199-207).
Mature sunflower seed (Helianthus annuus L.) are dehulled using a
single wheat-head thresher. Seeds are surface sterilized for 30
minutes in a 20% Clorox bleach solution with the addition of two
drops of Tween 20 per 50 ml of solution. The seeds are rinsed twice
with sterile distilled water.
[0248] Split embryonic axis explants are prepared by a modification
of procedures described by Schrammeijer, et al. (Schrammeijer, et
al., (1990) Plant Cell Rep. 9:55-60). Seeds are imbibed in
distilled water for 60 minutes following the surface sterilization
procedure. The cotyledons of each seed are then broken off,
producing a clean fracture at the plane of the embryonic axis.
Following excision of the root tip, the explants are bisected
longitudinally between the primordial leaves. The two halves are
placed, cut surface up, on GBA medium consisting of Murashige and
Skoog mineral elements (Murashige, et al., (1962) Physiol. Plant.,
15:473-497), Shepard's vitamin additions (Shepard (1980) in
Emergent Techniques for the Genetic Improvement of Crops
(University of Minnesota Press, St. Paul, Minn.), 40 mg/l adenine
sulfate, 30 g/l sucrose, 0.5 mg/l 6-benzyl-aminopurine (BAP), 0.25
mg/l indole-3-acetic acid (IAA), 0.1 mg/l gibberellic acid
(GA.sub.3), pH 5.6, and 8 g/l Phytagar.
[0249] The explants are subjected to microprojectile bombardment
prior to Agrobacterium treatment (Bidney, et al., (1992) Plant Mol.
Biol. 18:301-313). Thirty to forty explants are placed in a circle
at the center of a 60.times.20 mm plate for this treatment.
Approximately 4.7 mg of 1.8 mm tungsten microprojectiles are
resuspended in 25 ml of sterile TE buffer (10 mM Tris HCl, 1 mM
EDTA, pH 8.0) and 1.5 ml aliquots are used per bombardment. Each
plate is bombarded twice through a 150 mm nytex screen placed 2 cm
above the samples in a PDS 1000.RTM. particle acceleration
device.
[0250] Disarmed Agrobacterium tumefaciens strain EHA105 is used in
all transformation experiments. A binary plasmid vector comprising
the expression cassette that contains the AMT gene operably linked
to the ubiquitin promoter is introduced into Agrobacterium strain
EHA105 via freeze-thawing as described by Holsters, et al., (1978)
Mol. Gen. Genet. 163:181-187. This plasmid further comprises a
kanamycin selectable marker gene (i.e., nptII). Bacteria for plant
transformation experiments are grown overnight (28.degree. C. and
100 RPM continuous agitation) in liquid YEP medium (10 gm/l yeast
extract, 10 gm/l Bactopeptone, and 5 gm/l NaCl, pH 7.0) with the
appropriate antibiotics required for bacterial strain and binary
plasmid maintenance. The suspension is used when it reaches an
OD.sub.600 of about 0.4 to 0.8. The Agrobacterium cells are
pelleted and resuspended at a final OD.sub.600 of 0.5 in an
inoculation medium comprised of 12.5 mM MES pH 5.7, 1 gm/l
NH.sub.4Cl, and 0.3 gm/l MgSO.sub.4.
[0251] Freshly bombarded explants are placed in an Agrobacterium
suspension, mixed, and left undisturbed for 30 minutes. The
explants are then transferred to GBA medium and co-cultivated, cut
surface down, at 26.degree. C. and 18-hour days. After three days
of co-cultivation, the explants are transferred to 374B (GBA medium
lacking growth regulators and a reduced sucrose level of 1%)
supplemented with 250 mg/l cefotaxime and 50 mg/l kanamycin
sulfate. The explants are cultured for two to five weeks on
selection and then transferred to fresh 374B medium lacking
kanamycin for one to two weeks of continued development. Explants
with differentiating, antibiotic-resistant areas of growth that
have not produced shoots suitable for excision are transferred to
GBA medium containing 250 mg/l cefotaxime for a second 3-day
phytohormone treatment. Leaf samples from green,
kanamycin-resistant shoots are assayed for the presence of NPTII by
ELISA and for the presence of transgene expression by assaying for
a modulation in meristem development (i.e., an alteration of size
and appearance of shoot and floral meristems).
[0252] NPTII-positive shoots are grafted to Pioneer.RTM. hybrid
6440 in vitro-grown sunflower seedling rootstock. Surface
sterilized seeds are germinated in 48-0 medium (half-strength
Murashige and Skoog salts, 0.5% sucrose, 0.3% gelrite, pH 5.6) and
grown under conditions described for explant culture. The upper
portion of the seedling is removed, a 1 cm vertical slice is made
in the hypocotyl, and the transformed shoot inserted into the cut.
The entire area is wrapped with parafilm to secure the shoot.
Grafted plants can be transferred to soil following one week of in
vitro culture. Grafts in soil are maintained under high humidity
conditions followed by a slow acclimatization to the greenhouse
environment. Transformed sectors of T.sub.0 plants (parental
generation) maturing in the greenhouse are identified by NPTII
ELISA and/or by AMT activity analysis of leaf extracts while
transgenic seeds harvested from NPTII-positive T.sub.0 plants are
identified by AMT activity analysis of small portions of dry seed
cotyledon.
[0253] An alternative sunflower transformation protocol allows the
recovery of transgenic progeny without the use of chemical
selection pressure. Seeds are dehulled and surface-sterilized for
20 minutes in a 20% Clorox bleach solution with the addition of two
to three drops of Tween 20 per 100 ml of solution, then rinsed
three times with distilled water. Sterilized seeds are imbibed in
the dark at 26.degree. C. for 20 hours on filter paper moistened
with water. The cotyledons and root radical are removed, and the
meristem explants are cultured on 374E (GBA medium consisting of MS
salts, Shepard vitamins, 40 mg/l adenine sulfate, 3% sucrose, 0.5
mg/l 6-BAP, 0.25 mg/l IAA, 0.1 mg/l GA, and 0.8% Phytagar at pH
5.6) for 24 hours under the dark. The primary leaves are removed to
expose the apical meristem, around 40 explants are placed with the
apical dome facing upward in a 2 cm circle in the center of 374M
(GBA medium with 1.2% Phytagar), and then cultured on the medium
for 24 hours in the dark.
[0254] Approximately 18.8 mg of 1.8 .mu.m tungsten particles are
resuspended in 150 .mu.l absolute ethanol. After sonication, 8
.mu.l of it is dropped on the center of the surface of
macrocarrier. Each plate is bombarded twice with 650 psi rupture
discs in the first shelf at 26 mm of Hg helium gun vacuum.
[0255] The plasmid of interest is introduced into Agrobacterium
tumefaciens strain EHA105 via freeze thawing as described
previously. The pellet of overnight-grown bacteria at 28.degree. C.
in a liquid YEP medium (10 g/l yeast extract, 10 g/l Bactopeptone,
and 5 g/l NaCl, pH 7.0) in the presence of 50 .mu.g/l kanamycin is
resuspended in an inoculation medium (12.5 mM 2-mM 2-(N-morpholino)
ethanesulfonic acid, MES, 1 g/l NH.sub.4Cl and 0.3 g/l MgSO.sub.4
at pH 5.7) to reach a final concentration of 4.0 at OD 600.
Particle-bombarded explants are transferred to GBA medium (374E),
and a droplet of bacteria suspension is placed directly onto the
top of the meristem. The explants are co-cultivated on the medium
for 4 days, after which the explants are transferred to 374C medium
(GBA with 1% sucrose and no BAP, IAA, GA3 and supplemented with 250
.mu.g/ml cefotaxime). The plantlets are cultured on the medium for
about two weeks under 16-hour day and 26.degree. C. incubation
conditions.
[0256] Explants (around 2 cm long) from two weeks of culture in
374C medium are screened for a modulation in meristem development
(i.e., an alteration of size and appearance of shoot and floral
meristems). After positive (i.e., a decrease in AMT expression)
explants are identified, those shoots that fail to exhibit a
decrease in AMT activity are discarded, and every positive explant
is subdivided into nodal explants. One nodal explant contains at
least one potential node. The nodal segments are cultured on GBA
medium for three to four days to promote the formation of auxiliary
buds from each node. Then they are transferred to 374C medium and
allowed to develop for an additional four weeks. Developing buds
are separated and cultured for an additional four weeks on 374C
medium. Pooled leaf samples from each newly recovered shoot are
screened again by the appropriate protein activity assay. At this
time, the positive shoots recovered from a single node will
generally have been enriched in the transgenic sector detected in
the initial assay prior to nodal culture.
[0257] Recovered shoots positive for a decreased AMT expression are
grafted to Pioneer hybrid 6440 in vitro-grown sunflower seedling
rootstock. The rootstocks are prepared in the following manner.
Seeds are dehulled and surface-sterilized for 20 minutes in a 20%
Clorox bleach solution with the addition of two to three drops of
Tween 20 per 100 ml of solution, and are rinsed three times with
distilled water. The sterilized seeds are germinated on the filter
moistened with water for three days, then they are transferred into
48 medium (half-strength MS salt, 0.5% sucrose, 0.3% gelrite pH
5.0) and grown at 26.degree. C. under the dark for three days, then
incubated at 16-hour-day culture conditions. The upper portion of
selected seedling is removed, a vertical slice is made in each
hypocotyl, and a transformed shoot is inserted into a V-cut. The
cut area is wrapped with parafilm. After one week of culture on the
medium, grafted plants are transferred to soil. In the first two
weeks, they are maintained under high humidity conditions to
acclimatize to a greenhouse environment.
Example 6
Identification, Phylogenetic Analysis and Chloroplast Targeting
Peptide (cTP) Predictions of AMTs in Arabidopsis, Rice, Soybean and
Maize
[0258] Taking a `genomic` approach AMTs were identified in several
higher plants. In Arabidopsis 6 AMTs have been identified, and
phylogenetic analyses reveals that AtAMT1 (SEQ ID NO: 2) AtAMT1;2
(SEQ ID NO: 4), AtAMT1;3 (SEQ ID NO: 6) and At3g24290 (SEQ ID NO:
10) cluster in one group where as AtAMT2 (SEQ ID NO: 8) and
At4g28700 (SEQ ID NO: 12) are independent. Chloroplast targeting
peptide (cTP) prediction by ChloroP program reveals that AtAMT1;2
(SEQ ID NO: 4) have a putative cTP (with 55% probability) where as
all other AtAMTs did not contain any predicted cTP In rice, soybean
and maize, 17, 11, 7 AMTs have been identified, respectively. cTP
prediction in AMTs proteins from maize and soybean didn't identify
any AMT candidate with a putative cTP, however in rice one AMT has
putative cTP with more than 50% probability. Phylogenetic analyses
of all the AMTs from Arabidopsis, rice, maize and soybean are shown
in FIG. 1.
Example 7
Expression Analysis of AMTs in Maize
[0259] In order to identify leaf specific/preferred/expressed
AMT(s) in maize, Lynx MPSS expression analyses in .about.300
libraries reveal that ZmAMT1 (SEQ ID NO: 14), 2, 7 are expressed
both in roots and leaves (FIG. 2) whereas ZmAMT4 (SEQ ID NO: 20) is
a root preferred AMT. ZmAMT6 (SEQ ID NO: 24) expresses at very low
level in comparison to other ZmAMTs. In case of ZmAMT5 there was no
specific Lynx tag available. Researchers also performed RT-PCR on
leaf and roots of B73 maize and the results confirm Lynx analysis
results that there is no leaf specific AMT in maize, although
ZmAMT1, 2, 7 (SEQ ID NOS: 14, 16 and 26) are expressed in leaves
and roots.
Example 8
CTP Predictions in Chloroplast Outer Envelope Proteins
[0260] Initial cTP prediction couldn't detect a putative cTP in
most of the higher plant AMTs analyzed. The chloroplast localized
AMT (if any) has to be in the outer envelope of the chloroplast. In
order to determine whether proteins localized in outer envelop of
the chloroplast have any predicted cTP, researchers searched the
NCBI database using `chloroplast outer envelop/membrane` as keyword
and identified the 14, 14, and 5 proteins from Arabidopsis, rice
and maize, respectively that are suppose to be localized in outer
envelop of chloroplast. Some of these are well characterized
proteins and known to be localized in the outer membrane of
chloroplast. ChloroP program was used to identify putative cTP in
these 33 candidate proteins and interestingly none of these
proteins show any putative cTP with high probability. These
observations suggest that either a cTP is not required or not
identified/characterized for these proteins so far. This also
suggests that although most of the AMTs don't have a predicted cTP
but some of them might be localized in the chloroplast outer
membrane.
Example 9
Isolation and Characterization of AtAMT1;2 (SEQ ID NO: 4) T-DNA
Mutant
[0261] In cTP prediction analyses, AtAMT1;2 (SEQ ID NO: 4) posses a
putative cTP. For functional analyses of AtAMT1;2 (SEQ ID NO: 4)
and to determine it's role in N-assimilation, researchers
identified a T-DNA mutant line (SM.sub.--3.15680) from the
Arabidopsis T-DNA mutant data base. The T-DNA mutant line was
ordered from ABRC and the homozygous plants were subjected to
molecular analyses. In this mutant line T-DNA was inserted in
c-terminal of AtAMT1;2 (SEQ ID NO: 4) gene (FIG. 3A). Genomic PCRs
using AtAMT1;2 (SEQ ID NO: 4) gene and T-DNA specific primers show
that T-DNA is indeed inserted in the AtAMT1;2 (SEQ ID NO: 4) (FIG.
3B). AtAMT1;2 (SEQ ID NO: 4) gene specific primers flanking the
T-DNA insert couldn't amplify any DNA region in mutant plants where
as an expected PCR product was detected in wild type plant (FIG.
4B, upper panel). Similarly, genomic PCR with AtAMT1;2 (SEQ ID NO:
4) specific forward primer and T-DNA specific reverse primers
amplify an expected product in mutant lines and nothing in wild
type plants as expected (FIG. 4B, lower panel). Saturated RT-PCRs
(35 cycles) analyses couldn't detect a full length atamt1;2 mRNA in
mutant (FIG. 4C, upper panel) suggesting that AtAMT1;2 (SEQ ID NO:
4) is completely knocked out in this T-DNA mutant. Actin control
RT-PCR worked fine in both mutant and wild type plants (FIG. 3C,
lower panel).
Example 10
Generation and Molecular Characterization of AtAMT1;2 (SEQ ID NO:
4) RNAi Lines
[0262] In addition to T-DNA mutant, another parallel approach was
also undertaken for functional analysis of AtAMT1;2 (SEQ ID NO: 4).
A RNAi vector containing ZM-UBI promoter driven RNAi cassette
consisting of inverted repeats of AtAMT1;2 (SEQ ID NO: 4) specific
DNA regions and ADH intron as a spacer was constructed. Wild type
Arabidopsis (Columbia-0) was transformed with this RNAi vector by
Agrobacterium mediated `floral-dip` method. Several transgenic
lines were identified by selecting the T0 seeds for herbicide
resistance in soil. Molecular characterization of these transgenic
lines were performed by RT-PCR for Actin, AtAMT1;2 (SEQ ID NO: 4)
RNAi cassette, endogenous AtAMT1;2 (SEQ ID NO: 4) and presence of
gDNA in RNA preparations. Several lines with a significant reduced
levels of AtAMT1;2 (SEQ ID NO: 4) were identified after molecular
analysis.
Example 11
Sub-Cellular Localization and Regulation of Expression of AtAMT1;2
(SEQ ID NO: 4)
[0263] cTP prediction analyses indicate that AtAMT1;2 (SEQ ID NO:
4) contains a putative predicted cTP (but with only 55%
probability). The objectives of the experiments described in this
example are to determine sub-cellular localization and regulation
of expression the endogenous AtAMT1;2 (SEQ ID NO: 4). The coding
sequence of AtAMT1;2 (SEQ ID NO: 4) was tagged with green
fluorescent protein (GFP) as an in-frame C-terminal fusion under
the control of AtAMT1;2 (SEQ ID NO: 4) native promoter and a strong
constitutive (ZM-UBI) promoter. Arabidopsis transgenic lines were
generated and analyzed for GFP expression by confocal microscopy.
Analyses show that AtAMT1;2:GFP is localized in the plasma membrane
of endodermis and the cortex in roots.
Example 12
Knock-Out/Knock-Down of Zm-AMTs in Maize
[0264] ESTs corresponding to all seven maize AMTs were identified
and annotated and full length cDNA clones were obtained.
Experiments to knock-out/knock-down of all these individual ZmAMTs
by RNAi are in progress. TUSC screening experiments were used to
identify knock-out mutants for three leaf expressed ZmAMT1 (SEQ ID
NO: 14), ZmAMT2 (SEQ ID NO: 16) and ZmAMT7 (SEQ ID NO: 26).
Example 13
Knock-Out/Knock-Down of Multiple AtAMTs with Single RNAi Vector in
Arabidopsis
[0265] Six AMT genes are present in Arabidopsis genome. Hence, it
is very likely that due to functional redundancy one might need to
manipulate the expression of multiple AMTs simultaneously. The DNA
sequence of all these AMTs was analyzed and identified the high
homology regions among them. For example there is such a stretch of
.about.200 bp among AtAMT1;2 (SEQ ID NO: 4), AtAMT1 (SEQ ID NO: 2),
AMT1;3 (SEQ ID NO: 6), At3g24290 (SEQ ID NO: 10) and At4g28700 (SEQ
ID NO: 12) where as AMT2 (SEQ ID NO: 8) stood independent (FIG. 4).
These regions were amplified (bold and underlined in FIG. 4) by PCR
from AtAMT1;2 (SEQ ID NO: 4) and AtAMT2 (SEQ ID NO: 8) and
performed a multi-way ligation to make an inverted repeat using
ADH-intron as a spacer. The RNAi cassette of these hybrid inverted
repeats is driven by a constitutive or root-specific or
leaf-specific promoter. Several transgenic Arabidopsis lines were
generated for these three constructs. Molecular analyses of these
lines were performed by genomic and RT-PCR. Several lines were
identified that expressed significantly reduced levels of multiple
AtAMTs. These transgenic lines show a methyl ammonium (ammonium
analog toxic to plants) tolerant/better growth phenotype as
compared to wild type control when grown on MS media supplemented
with 10-30 mM of methyl ammonium. These results indicate multiple
AMTs were knocked-down in these lines, resulting in reduced uptake
of methyl ammonium.
Example 14
Knock-Out/Knock-Down of Multiple ZmAMTs in Maize by Single RNAi
Vector
[0266] In maize at least 7 AMT like genes were identified and at
least 3 of them are expressed both in leaf and root (see, Example
2). For improving NUE by reducing loss of ammonia by
volatilization, one might have to knock-out/knock-down multiple
AMTs. Detailed analyses of all 7 maize AMTs were performed to
identify the DNA regions showing high homology among different
ZmAMTs. This analysis reveals that ZmAMT1 (SEQ ID NO: 14) and
ZmAMT5 (SEQ ID NO: 22), ZmAMT3 (SEQ ID NO: 18) and ZmAMT4 (SEQ ID
NO: 20) and ZmAMT2 (SEQ ID NO: 16), ZmAMT6 (SEQ ID NO: 24) and
ZmAMT7 (SEQ ID NO: 26) form three separate groups and there is a
very high homology in stretches of DNA sequences with in each group
(FIG. 5). Three DNA fragments (bold and underlined in FIG. 5) from
ZmAMT 1, 4 and 7 (SEQ ID NOS: 14, 20 and 26) representing each of
the different groups were amplified by PCR. Multi-way ligations
were performed to make inverted repeats with hybrid of these 3
fragments and ADH intron as a spacer to facilitate the formation of
stem-loop structure. This hybrid RNAi cassette of `ZmAMT1 (SEQ ID
NO: 14):ZmAMT4 (SEQ ID NO: 20):ZmAMT7 (SEQ ID NO: 26)` inverted
repeats was driven by Zm-UBI promoter and a leaf-specific promoter.
MOPAT driven by Zm-UBI promoter was used as herbicide resistance
marker for selected. In addition to that RFP driven by a pericarp
specific promoter LTP2 was also used to sort out the transgenic
seeds (red) from there segregating non-transgenic seeds. Transgenic
lines for the constructs were generated, with molecular analyses of
the T0 events performed by genomic and RT-PCR. Several lines with
significantly reduced expression of individual/multiple ZmAMTs have
been identified and characterized.
Example 15
Variants of AMT Sequences
[0267] A. Variant Nucleotide Sequences of AMT that do not Alter the
Encoded Amino Acid Sequence
[0268] The AMT nucleotide sequences are used to generate variant
nucleotide sequences having the nucleotide sequence of the open
reading frame with about 70%, 75%, 80%, 85%, 90% and 95% nucleotide
sequence identity when compared to the starting unaltered ORF
nucleotide sequence of the corresponding SEQ ID NO. These
functional variants are generated using a standard codon table.
While the nucleotide sequence of the variants are altered, the
amino acid sequence encoded by the open reading frames do not
change.
[0269] B. Variant Amino Acid Sequences of AMT Polypeptides
[0270] Variant amino acid sequences of the AMT polypeptides are
generated. In this example, one amino acid is altered.
Specifically, the open reading frames are reviewed to determine the
appropriate amino acid alteration. The selection of the amino acid
to change is made by consulting the protein alignment (with the
other orthologs and other gene family members from various
species). An amino acid is selected that is deemed not to be under
high selection pressure (not highly conserved) and which is rather
easily substituted by an amino acid with similar chemical
characteristics (i.e., similar functional side-chain). Using the
protein alignment set forth in FIG. 2, an appropriate amino acid
can be changed. Once the targeted amino acid is identified, the
procedure outlined in the following section C is followed. Variants
having about 70%, 75%, 80%, 85%, 90% and 95% nucleic acid sequence
identity are generated using this method.
[0271] C. Additional Variant Amino Acid Sequences of AMT
Polypeptides
[0272] In this example, artificial protein sequences are created
having 80%, 85%, 90% and 95% identity relative to the reference
protein sequence. This latter effort requires identifying conserved
and variable regions from the alignment set forth in FIG. 2 and
then the judicious application of an amino acid substitutions
table. These parts will be discussed in more detail below.
[0273] Largely, the determination of which amino acid sequences are
altered is made based on the conserved regions among AMT protein or
among the other AMT polypeptides. Based on the sequence alignment,
the various regions of the AMT polypeptide that can likely be
altered are represented in lower case letters, while the conserved
regions are represented by capital letters. It is recognized that
conservative substitutions can be made in the conserved regions
below without altering function. In addition, one of skill will
understand that functional variants of the AMT sequence of the
invention can have minor non-conserved amino acid alterations in
the conserved domain.
[0274] Artificial protein sequences are then created that are
different from the original in the intervals of 80-85%, 85-90%,
90-95% and 95-100% identity. Midpoints of these intervals are
targeted, with liberal latitude of plus or minus 1%, for example.
The amino acids substitutions will be effected by a custom Perl
script. The substitution table is provided below in Table 2.
TABLE-US-00002 TABLE 2 Substitution Table Strongly Rank of Similar
and Order Optimal to Amino Acid Substitution Change Comment I L, V
1 50:50 substitution L I, V 2 50:50 substitution V I, L 3 50:50
substitution A G 4 G A 5 D E 6 E D 7 W Y 8 Y W 9 S T 10 T S 11 K R
12 R K 13 N Q 14 Q N 15 F Y 16 M L 17 First methionine cannot
change H Na No good substitutes C Na No good substitutes P Na No
good substitutes
[0275] First, any conserved amino acids in the protein that should
not be changed is identified and "marked off" for insulation from
the substitution. The start methionine will of course be added to
this list automatically. Next, the changes are made. H, C, and P
are not changed in any circumstance. The changes will occur with
isoleucine first, sweeping N-terminal to C-terminal. Then leucine,
and so on down the list until the desired target it reached.
Interim number substitutions can be made so as not to cause
reversal of changes. The list is ordered 1-17, so start with as
many isoleucine changes as needed before leucine, and so on down to
methionine. Clearly many amino acids will in this manner not need
to be changed. L, I and V will involve a 50:50 substitution of the
two alternate optimal substitutions.
[0276] The variant amino acid sequences are written as output. Perl
script is used to calculate the percent identities. Using this
procedure, variants of the AMT polypeptides are generating having
about 80%, 85%, 90%, and 95% amino acid identity to the starting
unaltered ORF nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45,
47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79
or 81.
Example 16
Over-Expression of AMTs in Plants to Improve NUE
[0277] The over-expression of AMTs has been demonstrated with
strong constitutively or organ-specific (e.g. in roots) expression
which improves ammonium uptake (especially in low ammonium soils in
anaerobic conditions typical of rice field conditions) leading to
improved nitrogen use efficiency. In other plants, such as maize,
typically most of the N is absorbed by roots in the form of
nitrate, the available source in most soil, however there is still
a considerable proportion of N available as ammonium.
Over-expression of AMTs in these conditions leads to improved
nitrogen utilization. Since nitrate needs to be reduced to ammonium
by an energy expensive reaction before it is assimilated, ammonium
is a preferable source of N when available to the plant.
[0278] All publications and patent applications in this
specification are indicative of the level of ordinary skill in the
art to which this invention pertains. All publications and patent
applications are herein incorporated by reference to the same
extent as if each individual publication or patent application was
specifically and individually indicated by reference.
[0279] The invention has been described with reference to various
specific and preferred embodiments and techniques. However, it
should be understood that many variations and modifications may be
made while remaining within the spirit and scope of the invention.
Sequence CWU 1
1
8211736DNAArabidopsis thaliana 1agcctctctg tttcatcttc ttctctaaac
tctcaacatg tcttgctcgg ccaccgatct 60cgctgtcctg ttgggtccta atgccacggc
ggcggccaac tacatctgtg gccagttagg 120cgacgtcaac aacaaattta
tcgacaccgc tttcgctata gacaacactt accttctctt 180ctccgcctac
cttgtcttct ctatgcagct tggcttcgct atgctctgtg ccggttccgt
240gagagccaag aatactatga acatcatgct taccaacgtc cttgacgctg
cagccggtgg 300tctcttctat tatctgtttg gctacgcctt tgcctttgga
tctccgtcca atggtttcat 360cggtaaacac tactttggtc tcaaagacat
ccccacggcc tctgctgact actccaactt 420tctctaccaa tgggcctttg
caatcgctgc ggctggaatc acaagtggct cgatcgctga 480acggacacag
ttcgtggctt acctaatcta ttcctctttc ttaaccgggt ttgtttaccc
540ggtcgtctct cactggttct ggtcagttga tggatgggcc agcccgttcc
gtaccgatgg 600agatttgctt ttcagcaccg gagcgataga tttcgctggg
tccggtgttg ttcatatggt 660cggaggtatc gctggactct ggggtgcgct
catcgaaggt ccacgacttg gccggttcga 720taacggaggc cgtgccatcg
ctcttcgtgg ccactcggcg tcacttgttg tccttggaac 780attcctcctc
tggtttggat ggtacggatt taaccccggt tccttcaaca agatcctagt
840cacgtacgag acaggcacat acaacggcca gtggagcgcg gtcggacgga
cagctgtcac 900aacaacgtta gctggctgca ccgcggcgct gacaacccta
tttgggaaac gtctactctc 960gggacattgg aacgtcactg atgtatgcaa
cggcctcctc ggagggtttg cagccataac 1020tggtggctgc tctgtcgttg
agccatgggc tgcgatcatc tgcgggttcg tggcggccct 1080agtcctcctc
ggatgcaaca agctcgctga gaagctcaaa tacgacgacc ctcttgaggc
1140agcacaacta cacggtggtt gcggtgcgtg gggactaata ttcacggctc
tcttcgctca 1200agaaaagtac ttgaaccaga tttacggcaa caaacccgga
aggccacacg gtttgtttat 1260gggcggtgga ggaaaactac ttggagctca
gctgattcag atcattgtga tcacgggttg 1320ggtaagtgcg accatgggga
cacttttctt catcctcaag aaaatgaaat tgttgcggat 1380atcgtccgag
gatgagatgg ccggtatgga tatgaccagg cacggtggtt ttgcttatat
1440gtactttgat gatgatgagt ctcacaaagc cattcagctt aggagagttg
agccacgatc 1500tccttctcct tctggtgcta atactacacc tactccggtt
tgatttggat ttttactttt 1560attctctatt ttctagagta ttattttaaa
tgatgttttg tgatacttaa atattgtttt 1620ggatattttt ttgcatttca
gtaatgtttt agatgtacag tttcatgggg ttgtgatgat 1680aatatctatg
tggtcatttg tgttctcttt ggagtttttt ctataacgct tttttc
17362501PRTArabidopsis thaliana 2Met Ser Cys Ser Ala Thr Asp Leu
Ala Val Leu Leu Gly Pro Asn Ala1 5 10 15Thr Ala Ala Ala Asn Tyr Ile
Cys Gly Gln Leu Gly Asp Val Asn Asn20 25 30Lys Phe Ile Asp Thr Ala
Phe Ala Ile Asp Asn Thr Tyr Leu Leu Phe35 40 45Ser Ala Tyr Leu Val
Phe Ser Met Gln Leu Gly Phe Ala Met Leu Cys50 55 60Ala Gly Ser Val
Arg Ala Lys Asn Thr Met Asn Ile Met Leu Thr Asn65 70 75 80Val Leu
Asp Ala Ala Ala Gly Gly Leu Phe Tyr Tyr Leu Phe Gly Tyr85 90 95Ala
Phe Ala Phe Gly Ser Pro Ser Asn Gly Phe Ile Gly Lys His Tyr100 105
110Phe Gly Leu Lys Asp Ile Pro Thr Ala Ser Ala Asp Tyr Ser Asn
Phe115 120 125Leu Tyr Gln Trp Ala Phe Ala Ile Ala Ala Ala Gly Ile
Thr Ser Gly130 135 140Ser Ile Ala Glu Arg Thr Gln Phe Val Ala Tyr
Leu Ile Tyr Ser Ser145 150 155 160Phe Leu Thr Gly Phe Val Tyr Pro
Val Val Ser His Trp Phe Trp Ser165 170 175Val Asp Gly Trp Ala Ser
Pro Phe Arg Thr Asp Gly Asp Leu Leu Phe180 185 190Ser Thr Gly Ala
Ile Asp Phe Ala Gly Ser Gly Val Val His Met Val195 200 205Gly Gly
Ile Ala Gly Leu Trp Gly Ala Leu Ile Glu Gly Pro Arg Leu210 215
220Gly Arg Phe Asp Asn Gly Gly Arg Ala Ile Ala Leu Arg Gly His
Ser225 230 235 240Ala Ser Leu Val Val Leu Gly Thr Phe Leu Leu Trp
Phe Gly Trp Tyr245 250 255Gly Phe Asn Pro Gly Ser Phe Asn Lys Ile
Leu Val Thr Tyr Glu Thr260 265 270Gly Thr Tyr Asn Gly Gln Trp Ser
Ala Val Gly Arg Thr Ala Val Thr275 280 285Thr Thr Leu Ala Gly Cys
Thr Ala Ala Leu Thr Thr Leu Phe Gly Lys290 295 300Arg Leu Leu Ser
Gly His Trp Asn Val Thr Asp Val Cys Asn Gly Leu305 310 315 320Leu
Gly Gly Phe Ala Ala Ile Thr Gly Gly Cys Ser Val Val Glu Pro325 330
335Trp Ala Ala Ile Ile Cys Gly Phe Val Ala Ala Leu Val Leu Leu
Gly340 345 350Cys Asn Lys Leu Ala Glu Lys Leu Lys Tyr Asp Asp Pro
Leu Glu Ala355 360 365Ala Gln Leu His Gly Gly Cys Gly Ala Trp Gly
Leu Ile Phe Thr Ala370 375 380Leu Phe Ala Gln Glu Lys Tyr Leu Asn
Gln Ile Tyr Gly Asn Lys Pro385 390 395 400Gly Arg Pro His Gly Leu
Phe Met Gly Gly Gly Gly Lys Leu Leu Gly405 410 415Ala Gln Leu Ile
Gln Ile Ile Val Ile Thr Gly Trp Val Ser Ala Thr420 425 430Met Gly
Thr Leu Phe Phe Ile Leu Lys Lys Met Lys Leu Leu Arg Ile435 440
445Ser Ser Glu Asp Glu Met Ala Gly Met Asp Met Thr Arg His Gly
Gly450 455 460Phe Ala Tyr Met Tyr Phe Asp Asp Asp Glu Ser His Lys
Ala Ile Gln465 470 475 480Leu Arg Arg Val Glu Pro Arg Ser Pro Ser
Pro Ser Gly Ala Asn Thr485 490 495Thr Pro Thr Pro
Val50031860DNAArabidopsis thaliana 3acttaagcaa acacgttcca
caatcaagta ccctctctct atctctccct ccctccctct 60ccaccatgga caccgcaacc
accacatgct ctgccgtaga tctatctgcc ctcctatcct 120cttcttctaa
ctcaacatct tccctcgccg cggcaacctt tttatgttcc caaatttcaa
180acatctccaa caaactctcc gacacaactt atgccgtcga caacacgtat
ctcctcttct 240ccgcctacct tgtctttgcc atgcagctcg gtttcgctat
gctttgtgct ggatcagtcc 300gagccaagaa cactatgaac atcatgctta
ccaatgtcct tgatgctgcc gctggagcca 360tctcttacta cctcttcgga
ttcgcattcg cctttggtac accttccaac ggattcatcg 420gtcgccacca
tagcttcttc gctttaagct cttaccctga acgccccggc tccgacttca
480gctttttcct ctaccaatgg gcttttgcca tagccgcggc cggaatcact
agcggttcca 540tcgccgagcg aacgcaattc gttgcttacc ttatctactc
tactttcttg accggttttg 600tttacccgac agtctcgcac tggttctggt
caagtgatgg atgggctagc gcgtcccggt 660ctgacaacaa tctcttgttt
ggctcaggtg ctattgattt cgcaggttca ggagttgttc 720acatggtagg
tggaattgcc ggtttatgtg gagcgttagt tgaaggacca agaataggta
780gatttgaccg gtcaggccgg tccgtggctt tacgtggtca cagtgcatcc
cttgtcgtgc 840ttggtacctt cttgttgtgg tttggatggt atgggtttaa
ccctggttcc tttttaacca 900ttcttaaagg ctacgacaag tctcggccat
attatggtca atggagcgct gtaggtcgca 960ccgcggtcac cacaacgctt
tctggctgca ccgctgcgtt gactactcta ttcagtaaac 1020ggcttttagc
aggtcattgg aacgttattg acgtatgcaa cggacttcta ggcggctttg
1080cagctataac ctccggatgt gccgtggtgg agccgtgggc tgctatagta
tgtggctttg 1140tggcatcatg ggttttaatc ggatttaact tgcttgccaa
gaaacttaaa tatgatgacc 1200cactcgaggc tgctcagctc cacggtggat
gtggagcatg gggattaatc tttaccgggc 1260tgttcgcaag gaaagaatac
gttaacgaga tttactccgg tgataggcct tacggactgt 1320tcatgggcgg
gggaggaaaa ctgctcgccg cgcagatcgt tcagattatt gtgatcgttg
1380ggtgggtgac ggtaactatg ggaccgttgt tttatgggtt acataagatg
aatcttttga 1440ggatatcagc agaagatgag atggcaggaa tggacatgac
acgtcatgga ggatttgctt 1500acgcatacaa tgacgaagac gacgtgtcga
ctaaaccatg gggtcatttc gctggaagag 1560tggagcctac aagccggagc
tcgactccta caccgacctt gactgtttga tactttgatt 1620ggagaattga
gtggtcccaa acgagtcagt tttaatgtgg tgaagacaag agttcgggca
1680ccaaacatgt tggacgcatc tttgtgtatt attggtcttc ttcttcttct
ttttttttct 1740cttggttatc gctctgttgt ggacagatag tgtggaactg
ttaacaataa catgatcagt 1800atgtcttttt aattaaagtg aacgtttggt
atcaaaatta aacattggaa tttgagcggt 18604514PRTArabidopsis thaliana
4Met Asp Thr Ala Thr Thr Thr Cys Ser Ala Val Asp Leu Ser Ala Leu1 5
10 15Leu Ser Ser Ser Ser Asn Ser Thr Ser Ser Leu Ala Ala Ala Thr
Phe20 25 30Leu Cys Ser Gln Ile Ser Asn Ile Ser Asn Lys Leu Ser Asp
Thr Thr35 40 45Tyr Ala Val Asp Asn Thr Tyr Leu Leu Phe Ser Ala Tyr
Leu Val Phe50 55 60Ala Met Gln Leu Gly Phe Ala Met Leu Cys Ala Gly
Ser Val Arg Ala65 70 75 80Lys Asn Thr Met Asn Ile Met Leu Thr Asn
Val Leu Asp Ala Ala Ala85 90 95Gly Ala Ile Ser Tyr Tyr Leu Phe Gly
Phe Ala Phe Ala Phe Gly Thr100 105 110Pro Ser Asn Gly Phe Ile Gly
Arg His His Ser Phe Phe Ala Leu Ser115 120 125Ser Tyr Pro Glu Arg
Pro Gly Ser Asp Phe Ser Phe Phe Leu Tyr Gln130 135 140Trp Ala Phe
Ala Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala145 150 155
160Glu Arg Thr Gln Phe Val Ala Tyr Leu Ile Tyr Ser Thr Phe Leu
Thr165 170 175Gly Phe Val Tyr Pro Thr Val Ser His Trp Phe Trp Ser
Ser Asp Gly180 185 190Trp Ala Ser Ala Ser Arg Ser Asp Asn Asn Leu
Leu Phe Gly Ser Gly195 200 205Ala Ile Asp Phe Ala Gly Ser Gly Val
Val His Met Val Gly Gly Ile210 215 220Ala Gly Leu Cys Gly Ala Leu
Val Glu Gly Pro Arg Ile Gly Arg Phe225 230 235 240Asp Arg Ser Gly
Arg Ser Val Ala Leu Arg Gly His Ser Ala Ser Leu245 250 255Val Val
Leu Gly Thr Phe Leu Leu Trp Phe Gly Trp Tyr Gly Phe Asn260 265
270Pro Gly Ser Phe Leu Thr Ile Leu Lys Gly Tyr Asp Lys Ser Arg
Pro275 280 285Tyr Tyr Gly Gln Trp Ser Ala Val Gly Arg Thr Ala Val
Thr Thr Thr290 295 300Leu Ser Gly Cys Thr Ala Ala Leu Thr Thr Leu
Phe Ser Lys Arg Leu305 310 315 320Leu Ala Gly His Trp Asn Val Ile
Asp Val Cys Asn Gly Leu Leu Gly325 330 335Gly Phe Ala Ala Ile Thr
Ser Gly Cys Ala Val Val Glu Pro Trp Ala340 345 350Ala Ile Val Cys
Gly Phe Val Ala Ser Trp Val Leu Ile Gly Phe Asn355 360 365Leu Leu
Ala Lys Lys Leu Lys Tyr Asp Asp Pro Leu Glu Ala Ala Gln370 375
380Leu His Gly Gly Cys Gly Ala Trp Gly Leu Ile Phe Thr Gly Leu
Phe385 390 395 400Ala Arg Lys Glu Tyr Val Asn Glu Ile Tyr Ser Gly
Asp Arg Pro Tyr405 410 415Gly Leu Phe Met Gly Gly Gly Gly Lys Leu
Leu Ala Ala Gln Ile Val420 425 430Gln Ile Ile Val Ile Val Gly Trp
Val Thr Val Thr Met Gly Pro Leu435 440 445Phe Tyr Gly Leu His Lys
Met Asn Leu Leu Arg Ile Ser Ala Glu Asp450 455 460Glu Met Ala Gly
Met Asp Met Thr Arg His Gly Gly Phe Ala Tyr Ala465 470 475 480Tyr
Asn Asp Glu Asp Asp Val Ser Thr Lys Pro Trp Gly His Phe Ala485 490
495Gly Arg Val Glu Pro Thr Ser Arg Ser Ser Thr Pro Thr Pro Thr
Leu500 505 510Thr Val51758DNAArabidopsis thaliana 5gtatctctct
ttctctctct cagctctctc aaacatgtca ggagcaataa catgctctgc 60ggccgatctc
gccaccctac ttggccccaa cgccacggcg gcggccgact acatttgcgg
120ccaattaggc accgttaaca acaagttcac cgatgcagcc ttcgccatag
acaacaccta 180cctcctcttc tctgcctacc ttgtcttcgc catgcagctc
ggcttcgcta tgctttgtgc 240tggttctgtt agagccaaga atacgatgaa
catcatgctt accaatgtcc ttgacgctgc 300agccggagga ctcttctact
atctctttgg ttacgccttt gcctttggag gatcctccga 360agggttcatt
ggaagacaca actttgctct tagagacttt ccgactccca cagctgatta
420ctctttcttc ctctaccaat gggcgttcgc aatcgcggcc gctggaatca
caagtggttc 480gatcgcagag aggactcagt tcgtggctta cttgatatac
tcttctttct taaccggatt 540tgtttacccg gttgtctctc actggttttg
gtccccggat ggatgggcca gtccctttcg 600ttcagcggat gatcgtttgt
ttagcaccgg agccattgac tttgctggct ccggtgttgt 660tcacatggtt
ggtggcatag caggtttatg gggtgctctt attgaaggtc ctcgtcgtgg
720tcggttcgag aaaggtggtc gcgctattgc tctgcgcggc cactctgcct
cgctagtagt 780cttaggaacc ttcctcctat ggtttggatg gtatggtttc
aaccccggtt ccttcactaa 840gatactcgtt ccgtataatt ctggttccaa
ctacggccaa tggagcggaa tcggccgtac 900agcggttaac accacactct
caggatgcac agcagctcta accacactct ttggtaaacg 960tctcctatca
ggccactgga acgtaacgga cgtttgcaac gggttactcg gtgggtttgc
1020ggccataacc gcaggttgct ccgtcgtaga gccatgggca gcgattgtgt
gcggcttcat 1080ggcttctgtc gtccttatcg gatgcaacaa gctcgcggag
cttgtacaat atgatgatcc 1140actcgaggca gcccaactac atggagggtg
tggcgcgtgg gggttgatat tcgtaggatt 1200gtttgccaaa gagaagtatc
taaacgaggt ttatggcgcc accccgggaa ggccatatgg 1260actatttatg
ggcggaggag ggaagctgtt gggagcacaa ttggttcaaa tacttgtgat
1320tgtaggatgg gttagtgcca caatgggaac actcttcttc atcctcaaaa
ggctcaatct 1380gcttaggatc tcggagcagc atgaaatgca agggatggat
atgacacgtc acggtggctt 1440tgcttatatc taccatgata atgatgatga
gtctcataga gtggatcctg gatctccttt 1500ccctcgatca gctactcctc
ctcgcgttta attttcaact ttttggtaat ttattaccgt 1560ttaagtattg
tttgggtttt ggttttgaaa tataaatatt tggatgtttt ggtttgtttt
1620aagtgaccta tcgtcttttt gtgtttataa gtgttttagt ttatgttttt
tttttttttc 1680ttgaatttta attttacatg cctcggctaa tgtttatgct
atttcttaga aatttatata 1740tacaactttt ggtgatcc
17586498PRTArabidopsis thaliana 6Met Ser Gly Ala Ile Thr Cys Ser
Ala Ala Asp Leu Ala Thr Leu Leu1 5 10 15Gly Pro Asn Ala Thr Ala Ala
Ala Asp Tyr Ile Cys Gly Gln Leu Gly20 25 30Thr Val Asn Asn Lys Phe
Thr Asp Ala Ala Phe Ala Ile Asp Asn Thr35 40 45Tyr Leu Leu Phe Ser
Ala Tyr Leu Val Phe Ala Met Gln Leu Gly Phe50 55 60Ala Met Leu Cys
Ala Gly Ser Val Arg Ala Lys Asn Thr Met Asn Ile65 70 75 80Met Leu
Thr Asn Val Leu Asp Ala Ala Ala Gly Gly Leu Phe Tyr Tyr85 90 95Leu
Phe Gly Tyr Ala Phe Ala Phe Gly Gly Ser Ser Glu Gly Phe Ile100 105
110Gly Arg His Asn Phe Ala Leu Arg Asp Phe Pro Thr Pro Thr Ala
Asp115 120 125Tyr Ser Phe Phe Leu Tyr Gln Trp Ala Phe Ala Ile Ala
Ala Ala Gly130 135 140Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr Gln
Phe Val Ala Tyr Leu145 150 155 160Ile Tyr Ser Ser Phe Leu Thr Gly
Phe Val Tyr Pro Val Val Ser His165 170 175Trp Phe Trp Ser Pro Asp
Gly Trp Ala Ser Pro Phe Arg Ser Ala Asp180 185 190Asp Arg Leu Phe
Ser Thr Gly Ala Ile Asp Phe Ala Gly Ser Gly Val195 200 205Val His
Met Val Gly Gly Ile Ala Gly Leu Trp Gly Ala Leu Ile Glu210 215
220Gly Pro Arg Arg Gly Arg Phe Glu Lys Gly Gly Arg Ala Ile Ala
Leu225 230 235 240Arg Gly His Ser Ala Ser Leu Val Val Leu Gly Thr
Phe Leu Leu Trp245 250 255Phe Gly Trp Tyr Gly Phe Asn Pro Gly Ser
Phe Thr Lys Ile Leu Val260 265 270Pro Tyr Asn Ser Gly Ser Asn Tyr
Gly Gln Trp Ser Gly Ile Gly Arg275 280 285Thr Ala Val Asn Thr Thr
Leu Ser Gly Cys Thr Ala Ala Leu Thr Thr290 295 300Leu Phe Gly Lys
Arg Leu Leu Ser Gly His Trp Asn Val Thr Asp Val305 310 315 320Cys
Asn Gly Leu Leu Gly Gly Phe Ala Ala Ile Thr Ala Gly Cys Ser325 330
335Val Val Glu Pro Trp Ala Ala Ile Val Cys Gly Phe Met Ala Ser
Val340 345 350Val Leu Ile Gly Cys Asn Lys Leu Ala Glu Leu Val Gln
Tyr Asp Asp355 360 365Pro Leu Glu Ala Ala Gln Leu His Gly Gly Cys
Gly Ala Trp Gly Leu370 375 380Ile Phe Val Gly Leu Phe Ala Lys Glu
Lys Tyr Leu Asn Glu Val Tyr385 390 395 400Gly Ala Thr Pro Gly Arg
Pro Tyr Gly Leu Phe Met Gly Gly Gly Gly405 410 415Lys Leu Leu Gly
Ala Gln Leu Val Gln Ile Leu Val Ile Val Gly Trp420 425 430Val Ser
Ala Thr Met Gly Thr Leu Phe Phe Ile Leu Lys Arg Leu Asn435 440
445Leu Leu Arg Ile Ser Glu Gln His Glu Met Gln Gly Met Asp Met
Thr450 455 460Arg His Gly Gly Phe Ala Tyr Ile Tyr His Asp Asn Asp
Asp Glu Ser465 470 475 480His Arg Val Asp Pro Gly Ser Pro Phe Pro
Arg Ser Ala Thr Pro Pro485 490 495Arg Val71428DNAArabidopsis
thaliana 7atggccggag cttacgatcc aagcttgccg gaggttcctg aatggctcaa
caaaggagac 60aacgcgtggc agctcacggc agcgactctg gttggtctac agagtatgcc
aggtcttgtt 120atcctctatg cctccatcgt caagaagaaa tgggctgtga
attcagcttt tatggctctt 180tacgctttcg ccgccgttct tctctgttgg
gttctcctct gttacaaaat ggcttttgga 240gaagagcttt tgccgttttg
gggcaaaggt ggtccagctt tcgaccaagg ataccttaag 300ggacaagcaa
agatcccaaa tagtaatgtg gcggcgccgt attttccgat ggcgacgttg
360gtgtattttc agttcacatt cgcggcgata acgacgatac ttgtggcggg
atctgtgttg 420gggaggatga atattaaagc atggatggct tttgtgccat
tgtggttgat ctttagctac 480acagttggag cttatagtat atggggaggt
gggtttctgt atcagtgggg agttattgat 540tattccggcg gttatgttat
tcatctctcc tccggtgttg ccggtttcgt cgctgcttac 600tgggtaggac
caaggcctaa ggctgacaga gagagattcc caccgaacaa tgttcttcta
660atgcttgctg gagctggact tttatggatg ggatggtccg gttttaacgg
tggtgctcct 720tacgcggcca acttaacctc ctctatcgcc gtgttaaaca
ccaacctctc ggccgccaca 780agcctccttg tatggactac acttgatgtc
atcttctttg gcaaaccttc tgtcatcgga 840gcaattcaag gcatggttac
tggcttagcc ggcgtcactc ccggagcagg tttgatccaa 900acatgggcag
ctataataat tggagtagtc tcaggaacag ctccatgggc ctctatgatg
960atcattcaca agaaatccgc tctccttcaa aaggtggatg atacattagc
ggtgttttac 1020acacacgccg tggctggttt acttggtgga ataatgacag
ggttgtttgc acaccctgat 1080ctctgcgttt tggtacttcc tctcccagcg
accagaggag ctttctacgg tggcaatggc 1140ggcaaacagc ttttgaaaca
gttggctgga gctgccttca ttgccgtctg gaatgtggtg 1200tcgactacta
tcattctact cgctattagg gtgttcatac cattgagaat ggctgaggaa
1260gagctcggga ttggagacga cgcagcacat ggggaagaag cttatgctct
ttggggagat 1320ggagagaagt ttgatgctac aaggcatgtg caacagtttg
agagagatca agaagctgct 1380catccttctt atgttcatgg tgctagaggt
gtcaccattg ttctatga 14288475PRTArabidopsis thaliana 8Met Ala Gly
Ala Tyr Asp Pro Ser Leu Pro Glu Val Pro Glu Trp Leu1 5 10 15Asn Lys
Gly Asp Asn Ala Trp Gln Leu Thr Ala Ala Thr Leu Val Gly20 25 30Leu
Gln Ser Met Pro Gly Leu Val Ile Leu Tyr Ala Ser Ile Val Lys35 40
45Lys Lys Trp Ala Val Asn Ser Ala Phe Met Ala Leu Tyr Ala Phe Ala50
55 60Ala Val Leu Leu Cys Trp Val Leu Leu Cys Tyr Lys Met Ala Phe
Gly65 70 75 80Glu Glu Leu Leu Pro Phe Trp Gly Lys Gly Gly Pro Ala
Phe Asp Gln85 90 95Gly Tyr Leu Lys Gly Gln Ala Lys Ile Pro Asn Ser
Asn Val Ala Ala100 105 110Pro Tyr Phe Pro Met Ala Thr Leu Val Tyr
Phe Gln Phe Thr Phe Ala115 120 125Ala Ile Thr Thr Ile Leu Val Ala
Gly Ser Val Leu Gly Arg Met Asn130 135 140Ile Lys Ala Trp Met Ala
Phe Val Pro Leu Trp Leu Ile Phe Ser Tyr145 150 155 160Thr Val Gly
Ala Tyr Ser Ile Trp Gly Gly Gly Phe Leu Tyr Gln Trp165 170 175Gly
Val Ile Asp Tyr Ser Gly Gly Tyr Val Ile His Leu Ser Ser Gly180 185
190Val Ala Gly Phe Val Ala Ala Tyr Trp Val Gly Pro Arg Pro Lys
Ala195 200 205Asp Arg Glu Arg Phe Pro Pro Asn Asn Val Leu Leu Met
Leu Ala Gly210 215 220Ala Gly Leu Leu Trp Met Gly Trp Ser Gly Phe
Asn Gly Gly Ala Pro225 230 235 240Tyr Ala Ala Asn Leu Thr Ser Ser
Ile Ala Val Leu Asn Thr Asn Leu245 250 255Ser Ala Ala Thr Ser Leu
Leu Val Trp Thr Thr Leu Asp Val Ile Phe260 265 270Phe Gly Lys Pro
Ser Val Ile Gly Ala Ile Gln Gly Met Val Thr Gly275 280 285Leu Ala
Gly Val Thr Pro Gly Ala Gly Leu Ile Gln Thr Trp Ala Ala290 295
300Ile Ile Ile Gly Val Val Ser Gly Thr Ala Pro Trp Ala Ser Met
Met305 310 315 320Ile Ile His Lys Lys Ser Ala Leu Leu Gln Lys Val
Asp Asp Thr Leu325 330 335Ala Val Phe Tyr Thr His Ala Val Ala Gly
Leu Leu Gly Gly Ile Met340 345 350Thr Gly Leu Phe Ala His Pro Asp
Leu Cys Val Leu Val Leu Pro Leu355 360 365Pro Ala Thr Arg Gly Ala
Phe Tyr Gly Gly Asn Gly Gly Lys Gln Leu370 375 380Leu Lys Gln Leu
Ala Gly Ala Ala Phe Ile Ala Val Trp Asn Val Val385 390 395 400Ser
Thr Thr Ile Ile Leu Leu Ala Ile Arg Val Phe Ile Pro Leu Arg405 410
415Met Ala Glu Glu Glu Leu Gly Ile Gly Asp Asp Ala Ala His Gly
Glu420 425 430Glu Ala Tyr Ala Leu Trp Gly Asp Gly Glu Lys Phe Asp
Ala Thr Arg435 440 445His Val Gln Gln Phe Glu Arg Asp Gln Glu Ala
Ala His Pro Ser Tyr450 455 460Val His Gly Ala Arg Gly Val Thr Ile
Val Leu465 470 47591491DNAArabidopsis thaliana 9atgtcaggag
ctattacttg ctctgcggct gatctctcag ccctactcgg cccaaatgcc 60acggcagcgg
ctgactacat ttgcggccag ttgggttccg ttaacaacaa gtttaccgat
120gcagcctacg ctatagacaa cacgtacctc ctcttctctg cctatcttgt
ctttgcgatg 180cagctcggct tcgctatgct ttgtgctggc tccgttagag
ctaagaacac gatgaacatc 240atgctcacta atgtccttga tgctgcagcc
ggaggactct tctactacct ctttggttat 300gcatttgcct ttggtgaatc
ctccgatgga ttcattggaa gacacaactt tggtcttcaa 360aactttccga
ctctcacctc ggattactcc ttcttcctct accaatgggc gtttgcaatc
420gcagccgctg gaatcaccag cggctccatt gccgagagga ctaagttcgt
ggcgtatttg 480atatactctt cttttttgac cgggtttgtt tacccagttg
tctctcactg gttctggtct 540ccggatggat gggctagtcc cttccgttca
gaagaccgtt tgtttggcac tggagccatc 600gactttgctg ggtcaggtgt
tgttcacatg gttggtggta tcgcaggatt atggggtgcc 660cttattgaag
gccctcggat tggtcggttt cctgatgggg gtcatgctat tgctctgcga
720ggccactctg cctcactcgt cgtcttaggg accttccttc tctggtttgg
ttggtacggg 780ttcaaccctg gttccttcac caagatactc attccctaca
attctggttc caactatggc 840caatggagtg gaataggccg caccgcggtt
acaactacac tctcgggatg cacagcggct 900ctaaccacac tcttcggaaa
acgtctccta tcaggccact ggaacgtaac tgacgtttgc 960aacgggttac
tcggagggtt tgcggccata acggcaggtt gctctgtggt tgatccatgg
1020gcagcgatcg tatgtggctt cgtggcttcc ctcgtcctta tcggatgcaa
caagctcgca 1080gagctcttaa aatatgacga tccacttgag gccgcacaac
tacacggagg gtgtggtgct 1140tggggtttga tatttgtagg actgtttgca
aaagagaagt atataaatga ggtttacggc 1200gcgagcccag gaaggcacta
cgggctattt atgggcggag gagggaagct attgggagca 1260caactggttc
aaataattgt gattgttgga tgggttagtg ccacaatggg aacactcttc
1320ttcatcctca aaaagctcaa tttgcttagg atctcggagc agcatgaaat
gcgaggaatg 1380gatttagcag gtcatggtgg ttttgcttat atctaccatg
ataatgatga tgattccatt 1440ggagtgcctg gatctccagt acctcgtgcg
cctaaccctc cagccgtttg a 149110496PRTArabidopsis thaliana 10Met Ser
Gly Ala Ile Thr Cys Ser Ala Ala Asp Leu Ser Ala Leu Leu1 5 10 15Gly
Pro Asn Ala Thr Ala Ala Ala Asp Tyr Ile Cys Gly Gln Leu Gly20 25
30Ser Val Asn Asn Lys Phe Thr Asp Ala Ala Tyr Ala Ile Asp Asn Thr35
40 45Tyr Leu Leu Phe Ser Ala Tyr Leu Val Phe Ala Met Gln Leu Gly
Phe50 55 60Ala Met Leu Cys Ala Gly Ser Val Arg Ala Lys Asn Thr Met
Asn Ile65 70 75 80Met Leu Thr Asn Val Leu Asp Ala Ala Ala Gly Gly
Leu Phe Tyr Tyr85 90 95Leu Phe Gly Tyr Ala Phe Ala Phe Gly Glu Ser
Ser Asp Gly Phe Ile100 105 110Gly Arg His Asn Phe Gly Leu Gln Asn
Phe Pro Thr Leu Thr Ser Asp115 120 125Tyr Ser Phe Phe Leu Tyr Gln
Trp Ala Phe Ala Ile Ala Ala Ala Gly130 135 140Ile Thr Ser Gly Ser
Ile Ala Glu Arg Thr Lys Phe Val Ala Tyr Leu145 150 155 160Ile Tyr
Ser Ser Phe Leu Thr Gly Phe Val Tyr Pro Val Val Ser His165 170
175Trp Phe Trp Ser Pro Asp Gly Trp Ala Ser Pro Phe Arg Ser Glu
Asp180 185 190Arg Leu Phe Gly Thr Gly Ala Ile Asp Phe Ala Gly Ser
Gly Val Val195 200 205His Met Val Gly Gly Ile Ala Gly Leu Trp Gly
Ala Leu Ile Glu Gly210 215 220Pro Arg Ile Gly Arg Phe Pro Asp Gly
Gly His Ala Ile Ala Leu Arg225 230 235 240Gly His Ser Ala Ser Leu
Val Val Leu Gly Thr Phe Leu Leu Trp Phe245 250 255Gly Trp Tyr Gly
Phe Asn Pro Gly Ser Phe Thr Lys Ile Leu Ile Pro260 265 270Tyr Asn
Ser Gly Ser Asn Tyr Gly Gln Trp Ser Gly Ile Gly Arg Thr275 280
285Ala Val Thr Thr Thr Leu Ser Gly Cys Thr Ala Ala Leu Thr Thr
Leu290 295 300Phe Gly Lys Arg Leu Leu Ser Gly His Trp Asn Val Thr
Asp Val Cys305 310 315 320Asn Gly Leu Leu Gly Gly Phe Ala Ala Ile
Thr Ala Gly Cys Ser Val325 330 335Val Asp Pro Trp Ala Ala Ile Val
Cys Gly Phe Val Ala Ser Leu Val340 345 350Leu Ile Gly Cys Asn Lys
Leu Ala Glu Leu Leu Lys Tyr Asp Asp Pro355 360 365Leu Glu Ala Ala
Gln Leu His Gly Gly Cys Gly Ala Trp Gly Leu Ile370 375 380Phe Val
Gly Leu Phe Ala Lys Glu Lys Tyr Ile Asn Glu Val Tyr Gly385 390 395
400Ala Ser Pro Gly Arg His Tyr Gly Leu Phe Met Gly Gly Gly Gly
Lys405 410 415Leu Leu Gly Ala Gln Leu Val Gln Ile Ile Val Ile Val
Gly Trp Val420 425 430Ser Ala Thr Met Gly Thr Leu Phe Phe Ile Leu
Lys Lys Leu Asn Leu435 440 445Leu Arg Ile Ser Glu Gln His Glu Met
Arg Gly Met Asp Leu Ala Gly450 455 460His Gly Gly Phe Ala Tyr Ile
Tyr His Asp Asn Asp Asp Asp Ser Ile465 470 475 480Gly Val Pro Gly
Ser Pro Val Pro Arg Ala Pro Asn Pro Pro Ala Val485 490
495111515DNAArabidopsis thaliana 11atggcgtcgg ctctctcttg ctctgcctct
gatctgattc cattactatc aggtggagcc 60aacgccaccg cagcagcagc cgccgctgaa
tacatctgcg ggagattcga cacagtcgcc 120gggaaattca ctgatgcggc
ttacgcaatc gacaacactt accttctctt ctctgcttac 180ctcgttttcg
cgatgcagct cggtttcgcc atgctctgtg ccggatccgt acgtgcaaaa
240aacacgatga acattatgct cacgaacgtc atcgacgctg cagccggagg
tctcttctat 300tatctcttcg gtttcgcttt tgcttttgga tctccttcta
atggattcat cggaaaacat 360ttctttggaa tgtatgattt tcctcaacct
acgtttgatt atccttattt tctatatcaa 420tggactttcg ctatcgccgc
cgctggaatc acgagtggtt cgatagcgga gaggactcag 480ttcgttgcgt
atttgatcta ttcttctttc ttgacgggtc ttgtttaccc gattgtgtcg
540cattggtttt ggtcttctga tggttgggcg tctccggcta gatctgagaa
ccttctgttt 600caatcaggtg tgattgattt cgctggctct ggtgttgttc
atatggttgg tggtattgct 660ggtttatggg gagctttaat tgaaggacct
aggattggtc ggtttggagt tgggggtaaa 720ccggttacgt tgcgtggtca
tagtgctacg ttggttgttc ttggaacgtt tttgttatgg 780ttcggatggt
acgggtttaa cccgggctcg tttgcaacta tttttaaggc gtatggggag
840actccaggga gctcgtttta cggacaatgg agcgcagttg ggagaaccgc
ggtaacaact 900acgttagctg gttgcacggc ggcgttaacg actctgtttg
ggaaaagact tattgatggg 960tattggaatg taactgatgt ttgcaatggt
ttgttaggcg ggtttgcggc tataactagc 1020ggatgttcgg ttgtggaacc
gtgggctgcg cttgtatgtg ggtttgtagc cgcatgggtg 1080ctgatgggat
gcaatagact agcggaaaag ctccaatttg atgatccgtt ggaagcggct
1140cagcttcacg gtggttgtgg tgcgtggggg attattttca ccgggttgtt
cgcggagaaa 1200agatacattg ccgagatctt tggaggcgac ccgaataggc
ctttcggatt gctaatggga 1260ggaggaggta ggttgcttgc ggcgcacgtc
gttcagattt tggtgattac gggttgggtt 1320agtgtgacaa tggggactct
gttttttatt ttgcataagc tgaaactgtt gaggataccg 1380gcggaggatg
agatagctgg ggtggatccg acgagtcacg gagggttggc ttatatgtac
1440acagaagatg agattaggaa tgggatcatg gttaggagag tgggtggtga
taatgatccc 1500aatgtaggtg tttga 151512504PRTArabidopsis thaliana
12Met Ala Ser Ala Leu Ser Cys Ser Ala Ser Asp Leu Ile Pro Leu Leu1
5 10 15Ser Gly Gly Ala Asn Ala Thr Ala Ala Ala Ala Ala Ala Glu Tyr
Ile20 25 30Cys Gly Arg Phe Asp Thr Val Ala Gly Lys Phe Thr Asp Ala
Ala Tyr35 40 45Ala Ile Asp Asn Thr Tyr Leu Leu Phe Ser Ala Tyr Leu
Val Phe Ala50 55 60Met Gln Leu Gly Phe Ala Met Leu Cys Ala Gly Ser
Val Arg Ala Lys65 70 75 80Asn Thr Met Asn Ile Met Leu Thr Asn Val
Ile Asp Ala Ala Ala Gly85 90 95Gly Leu Phe Tyr Tyr Leu Phe Gly Phe
Ala Phe Ala Phe Gly Ser Pro100 105 110Ser Asn Gly Phe Ile Gly Lys
His Phe Phe Gly Met Tyr Asp Phe Pro115 120 125Gln Pro Thr Phe Asp
Tyr Pro Tyr Phe Leu Tyr Gln Trp Thr Phe Ala130 135 140Ile Ala Ala
Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr Gln145 150 155
160Phe Val Ala Tyr Leu Ile Tyr Ser Ser Phe Leu Thr Gly Leu Val
Tyr165 170 175Pro Ile Val Ser His Trp Phe Trp Ser Ser Asp Gly Trp
Ala Ser Pro180 185 190Ala Arg Ser Glu Asn Leu Leu Phe Gln Ser Gly
Val Ile Asp Phe Ala195 200 205Gly Ser Gly Val Val His Met Val Gly
Gly Ile Ala Gly Leu Trp Gly210 215 220Ala Leu Ile Glu Gly Pro Arg
Ile Gly Arg Phe Gly Val Gly Gly Lys225 230 235 240Pro Val Thr Leu
Arg Gly His Ser Ala Thr Leu Val Val Leu Gly Thr245 250 255Phe Leu
Leu Trp Phe Gly Trp Tyr Gly Phe Asn Pro Gly Ser Phe Ala260 265
270Thr Ile Phe Lys Ala Tyr Gly Glu Thr Pro Gly Ser Ser Phe Tyr
Gly275 280 285Gln Trp Ser Ala Val Gly Arg Thr Ala Val Thr Thr Thr
Leu Ala Gly290 295 300Cys Thr Ala Ala Leu Thr Thr Leu Phe Gly Lys
Arg Leu Ile Asp Gly305 310 315 320Tyr Trp Asn Val Thr Asp Val Cys
Asn Gly Leu Leu Gly Gly Phe Ala325 330 335Ala Ile Thr Ser Gly Cys
Ser Val Val Glu Pro Trp Ala Ala Leu Val340 345 350Cys Gly Phe Val
Ala Ala Trp Val Leu Met Gly Cys Asn Arg Leu Ala355 360 365Glu Lys
Leu Gln Phe Asp Asp Pro Leu Glu Ala Ala Gln Leu His Gly370 375
380Gly Cys Gly Ala Trp Gly Ile Ile Phe Thr Gly Leu Phe Ala Glu
Lys385 390 395 400Arg Tyr Ile Ala Glu Ile Phe Gly Gly Asp Pro Asn
Arg Pro Phe Gly405 410 415Leu Leu Met Gly Gly Gly Gly Arg Leu Leu
Ala Ala His Val Val Gln420 425 430Ile Leu Val Ile Thr Gly Trp Val
Ser Val Thr Met Gly Thr Leu Phe435 440 445Phe Ile Leu His Lys Leu
Lys Leu Leu Arg Ile Pro Ala Glu Asp Glu450 455 460Ile Ala Gly Val
Asp Pro Thr Ser His Gly Gly Leu Ala Tyr Met Tyr465 470 475 480Thr
Glu Asp Glu Ile Arg Asn Gly Ile Met Val Arg Arg Val Gly Gly485 490
495Asp Asn Asp Pro Asn Val Gly Val500132073DNAZea mays 13atccgcgcca
caccctccca atcccctccc cctcgcgtat ccacactttt cacacgcgac 60gccggagaga
cagagcgcgc gcgcgcccga aagatgtcga cgtgcgcggc ggacctggcg
120ccgctgctcg gcccggcggc ggcgaacgcc acggactacc tgtgcgggca
gttcgcggac 180acggcctccg cggtggacgc cacgtacctg ctcttctcgg
cctacctcgt gttcgccatg 240cagctcggct tcgccatgct gtgcgccggc
tccgtccgcg ccaagaacac catgaacatc 300atgctcacca acgtgctcga
cgccgccgcg ggggcgctct tctactacct cttcggcttc 360gccttcgcct
tcggcacgcc ctccaacggc ttcatcggca agcagttctt cgggctcaag
420cacctgccca ggaccggctt cgactacgac ttcttcctct accagtgggc
cttcgccatc 480gccgccgcgg gcatcacgtc gggctccatc gccgagcgga
cccagttcgt cgcctacctc 540atctactccg cgttcctgac ggggttcgtc
taccccgtgg tgtcgcactg gttctggtcc 600gccgacggct gggccggcgc
cagccgcacg tccggcccgc tgctcttcgg gtccggcgtc 660atcgacttcg
ccggctccgg cgtcgtccac atggtcggcg gcatcgcggg gctgtggggc
720gcgctcatcg agggcccccg catcgggcgc ttcgaccacg ccggccgctc
cgtggcgctc 780aagggccaca gcgcgtcgct cgtggtgctc ggcaccttcc
tgctgtggtt cggctggtac 840gggttcaacc ccgggtcctt caccaccatc
ctcaagtcgt acggccccgc cgggaccgtc 900cacgggcagt ggtcggccgt
gggccgcacc gccgtcacca ccaccctcgc cggcagcgtc 960gccgcgctca
ccacgctgtt cgggaagcgg ctccagacgg gccactggaa cgtggtggac
1020gtctgcaacg gcctcctcgg cgggttcgcg gccatcacgg ccgggtgcag
cgtggtggag 1080ccgtgggcgg ccgtcatctg cgggttcgtg tccgcgtggg
tgctcatcgg cgccaacgcc 1140ctcgcggcgc gcttcaggtt cgacgacccg
ctggaggcgg cgcagctgca cggcgggtgt 1200ggcgcctggg gcgtcctctt
cacggggctc ttcgcgaggc gaaagtacgt ggaggagatc 1260tacggcgccg
ggaggcccta cgggctgttc atgggcggcg gcgggaagct cctcgccgcg
1320cagatcatcc agatcctggt gatcgccggg tgggtgagct gcaccatggg
cccgctcttc 1380tacgcgctca agaagctggg cctgctgcgc atctcggccg
acgacgagat gtccggcatg 1440gacctgaccc ggcacggcgg cttcgcctac
gtctaccacg acgaggaccc tggcgacaag 1500gccggggttg gtgggttcat
gctcaagtcc gcgcagaacc gtgtcgagcc ggcggcggcg 1560gtggcggcgg
cgaccagcag ccaggtgtaa aaaaaaaatc aggagcaaat tgaaaccgag
1620ctgaagttac gtgcttgcct ttttcagtat gttgtcgcgt atcacgtttg
aggtggatcg 1680tatctgccgg tcagtacgca gtgtttgggc aaatacttgg
ctacttggga gtcgcaagaa 1740attgtgtaaa ttatatagag gaggatggcg
acgaagcacg catgtgttac gtagttgggg 1800tttgtgtgca catggtggtg
ggcaggggct aggagagggt ttatctttag gttattttcg 1860tagtggaatg
aatcttatga tcggatatcc atcgtcggaa ggtgtggcgg gctgctggtc
1920aagataggtg gcttctatga ctatgagggt tgaaacaaca agtggacgat
tctgtcctgt 1980ggtcactgct catcatccaa tctagcggct ttgacggtcg
tgccttttta gtatcaataa 2040tattattcca agtttaaaaa aaaaaaaaaa aaa
207314498PRTZea mays 14Met Ser Thr Cys Ala Ala Asp Leu Ala Pro Leu
Leu Gly Pro Ala Ala1 5 10 15Ala Asn Ala Thr Asp Tyr Leu Cys Gly Gln
Phe Ala Asp Thr Ala Ser20 25 30Ala Val Asp Ala Thr Tyr Leu Leu Phe
Ser Ala Tyr Leu Val Phe Ala35 40 45Met Gln Leu Gly Phe Ala Met Leu
Cys Ala Gly Ser Val Arg Ala Lys50 55 60Asn Thr Met Asn Ile Met Leu
Thr Asn Val Leu Asp Ala Ala Ala Gly65 70 75 80Ala Leu Phe Tyr Tyr
Leu Phe Gly Phe Ala Phe Ala Phe Gly Thr Pro85 90 95Ser Asn Gly Phe
Ile Gly Lys Gln Phe Phe Gly Leu Lys His Leu Pro100 105 110Arg Thr
Gly Phe Asp Tyr Asp Phe Phe Leu Tyr Gln Trp Ala Phe Ala115 120
125Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr
Gln130 135 140Phe Val Ala Tyr Leu Ile Tyr Ser Ala Phe Leu Thr Gly
Phe
Val Tyr145 150 155 160Pro Val Val Ser His Trp Phe Trp Ser Ala Asp
Gly Trp Ala Gly Ala165 170 175Ser Arg Thr Ser Gly Pro Leu Leu Phe
Gly Ser Gly Val Ile Asp Phe180 185 190Ala Gly Ser Gly Val Val His
Met Val Gly Gly Ile Ala Gly Leu Trp195 200 205Gly Ala Leu Ile Glu
Gly Pro Arg Ile Gly Arg Phe Asp His Ala Gly210 215 220Arg Ser Val
Ala Leu Lys Gly His Ser Ala Ser Leu Val Val Leu Gly225 230 235
240Thr Phe Leu Leu Trp Phe Gly Trp Tyr Gly Phe Asn Pro Gly Ser
Phe245 250 255Thr Thr Ile Leu Lys Ser Tyr Gly Pro Ala Gly Thr Val
His Gly Gln260 265 270Trp Ser Ala Val Gly Arg Thr Ala Val Thr Thr
Thr Leu Ala Gly Ser275 280 285Val Ala Ala Leu Thr Thr Leu Phe Gly
Lys Arg Leu Gln Thr Gly His290 295 300Trp Asn Val Val Asp Val Cys
Asn Gly Leu Leu Gly Gly Phe Ala Ala305 310 315 320Ile Thr Ala Gly
Cys Ser Val Val Glu Pro Trp Ala Ala Val Ile Cys325 330 335Gly Phe
Val Ser Ala Trp Val Leu Ile Gly Ala Asn Ala Leu Ala Ala340 345
350Arg Phe Arg Phe Asp Asp Pro Leu Glu Ala Ala Gln Leu His Gly
Gly355 360 365Cys Gly Ala Trp Gly Val Leu Phe Thr Gly Leu Phe Ala
Arg Arg Lys370 375 380Tyr Val Glu Glu Ile Tyr Gly Ala Gly Arg Pro
Tyr Gly Leu Phe Met385 390 395 400Gly Gly Gly Gly Lys Leu Leu Ala
Ala Gln Ile Ile Gln Ile Leu Val405 410 415Ile Ala Gly Trp Val Ser
Cys Thr Met Gly Pro Leu Phe Tyr Ala Leu420 425 430Lys Lys Leu Gly
Leu Leu Arg Ile Ser Ala Asp Asp Glu Met Ser Gly435 440 445Met Asp
Leu Thr Arg His Gly Gly Phe Ala Tyr Val Tyr His Asp Glu450 455
460Asp Pro Gly Asp Lys Ala Gly Val Gly Gly Phe Met Leu Lys Ser
Ala465 470 475 480Gln Asn Arg Val Glu Pro Ala Ala Ala Val Ala Ala
Ala Thr Ser Ser485 490 495Gln Val151597DNAZea mays 15tttgctagcg
aagtccagta gtgcaactca ccccttcctg gtcctgctgc tccgccctct 60ccacctagct
accactccct tagagcgcca ctgccaagcc atggcgggag gaggggcggc
120ctaccagagc tcgtcggcgt cgccggactg gctgaacaag ggcgacaatg
cgtggcagat 180gacgtccgcg acgctggtgg gcctgcagag catgcccggg
ctggtgatcc tgtacggcag 240catcgtgaag aagaagtggg ccatcaactc
ggcgttcatg gcgctgtacg ccttcgccgc 300cgtctggctc tgctgggtgg
tgtgggccta caacatgtcg ttcggcgacc ggctgctgcc 360cttctggggc
aaggcgaggc cggcgctcgg gcagcgcttc ctggtggcgc agtcccagct
420cacggccacc gccgtgcggt accgcgacgg gtcgctcgag gcggagatgc
tccacccctt 480ctacccggcc gccaccatgg tgtacttcca gtgcgtgttc
gccagcatca ccgtcatcat 540cctcgccggc tcgctgctgg gccgcatgga
catcaaggcc tggatggcct tcgtcccgct 600ctggatcacc ttctcctaca
ccgtctccgc cttctcgctc tggggcggcg gcttcctctt 660ccagtggggc
gtcatcgact actccggcgg ctacgtcatc cacctctcct cgggaatcgc
720cggcctcacc gccgcttact gggtagggcc aaggtcggcg tcggacaggg
agcggttccc 780tcccaacaac atactgctgg tgctggcggg ggcaggcctg
ctgtggctcg gatggactgg 840cttcaacggc ggcgacccgt actcggccaa
catcgactcg tccatggcgg tgctcaacac 900gcacatctgc gcctccacca
gcctcctcat gtggaccctc cttgacgtct tcttcttcgg 960gaagccgtcg
gtgatcggtg ctgtgcaggg catgatcacc ggccttgtgt gcatcacgcc
1020tggcgcaggc ctggtgcaag ggtgggcagc cattgtcatg ggaattctct
caggtagcat 1080cccctggtac actatgatgg tactgcacaa gaaatggtcc
ttcatgcaga ggatcgacga 1140caccctcggc gtattccaca cccatgcggt
cgctgggctc ctcggcggcg ccactactgg 1200actctttgct gagcctgtcc
tctgcaacct cttcctcgcc atcccggact ccagaggtgc 1260attttatggt
ggtggtggat cacagtttgg gaagcagatc gctggcgcac tcttcgtcat
1320tggctggaac attgttatca cttccataat ctgtgttctt attggcctag
tcctgcccct 1380ccgaattcct gatgcacagc tgcttatcgg ggatgatgct
gtacatggtg aggaggcgta 1440tgctatatgg gcagaaggcg agctcaacga
tgtaacccgc caagatgaaa gcaggcatgg 1500cagcgtcgct gtaggagtca
cacaatgttt gagcatagtt cttgtaaggt tgaaagaaag 1560aaaaatacaa
gtgcatttgt ttgctaattg ctattaa 159716498PRTZea mays 16Met Ala Gly
Gly Gly Ala Ala Tyr Gln Ser Ser Ser Ala Ser Pro Asp1 5 10 15Trp Leu
Asn Lys Gly Asp Asn Ala Trp Gln Met Thr Ser Ala Thr Leu20 25 30Val
Gly Leu Gln Ser Met Pro Gly Leu Val Ile Leu Tyr Gly Ser Ile35 40
45Val Lys Lys Lys Trp Ala Ile Asn Ser Ala Phe Met Ala Leu Tyr Ala50
55 60Phe Ala Ala Val Trp Leu Cys Trp Val Val Trp Ala Tyr Asn Met
Ser65 70 75 80Phe Gly Asp Arg Leu Leu Pro Phe Trp Gly Lys Ala Arg
Pro Ala Leu85 90 95Gly Gln Arg Phe Leu Val Ala Gln Ser Gln Leu Thr
Ala Thr Ala Val100 105 110Arg Tyr Arg Asp Gly Ser Leu Glu Ala Glu
Met Leu His Pro Phe Tyr115 120 125Pro Ala Ala Thr Met Val Tyr Phe
Gln Cys Val Phe Ala Ser Ile Thr130 135 140Val Ile Ile Leu Ala Gly
Ser Leu Leu Gly Arg Met Asp Ile Lys Ala145 150 155 160Trp Met Ala
Phe Val Pro Leu Trp Ile Thr Phe Ser Tyr Thr Val Ser165 170 175Ala
Phe Ser Leu Trp Gly Gly Gly Phe Leu Phe Gln Trp Gly Val Ile180 185
190Asp Tyr Ser Gly Gly Tyr Val Ile His Leu Ser Ser Gly Ile Ala
Gly195 200 205Leu Thr Ala Ala Tyr Trp Val Gly Pro Arg Ser Ala Ser
Asp Arg Glu210 215 220Arg Phe Pro Pro Asn Asn Ile Leu Leu Val Leu
Ala Gly Ala Gly Leu225 230 235 240Leu Trp Leu Gly Trp Thr Gly Phe
Asn Gly Gly Asp Pro Tyr Ser Ala245 250 255Asn Ile Asp Ser Ser Met
Ala Val Leu Asn Thr His Ile Cys Ala Ser260 265 270Thr Ser Leu Leu
Met Trp Thr Leu Leu Asp Val Phe Phe Phe Gly Lys275 280 285Pro Ser
Val Ile Gly Ala Val Gln Gly Met Ile Thr Gly Leu Val Cys290 295
300Ile Thr Pro Gly Ala Gly Leu Val Gln Gly Trp Ala Ala Ile Val
Met305 310 315 320Gly Ile Leu Ser Gly Ser Ile Pro Trp Tyr Thr Met
Met Val Leu His325 330 335Lys Lys Trp Ser Phe Met Gln Arg Ile Asp
Asp Thr Leu Gly Val Phe340 345 350His Thr His Ala Val Ala Gly Leu
Leu Gly Gly Ala Thr Thr Gly Leu355 360 365Phe Ala Glu Pro Val Leu
Cys Asn Leu Phe Leu Ala Ile Pro Asp Ser370 375 380Arg Gly Ala Phe
Tyr Gly Gly Gly Gly Ser Gln Phe Gly Lys Gln Ile385 390 395 400Ala
Gly Ala Leu Phe Val Ile Gly Trp Asn Ile Val Ile Thr Ser Ile405 410
415Ile Cys Val Leu Ile Gly Leu Val Leu Pro Leu Arg Ile Pro Asp
Ala420 425 430Gln Leu Leu Ile Gly Asp Asp Ala Val His Gly Glu Glu
Ala Tyr Ala435 440 445Ile Trp Ala Glu Gly Glu Leu Asn Asp Val Thr
Arg Gln Asp Glu Ser450 455 460Arg His Gly Ser Val Ala Val Gly Val
Thr Gln Cys Leu Ser Ile Val465 470 475 480Leu Val Arg Leu Lys Glu
Arg Lys Ile Gln Val His Leu Phe Ala Asn485 490 495Cys
Tyr17964DNAZea mays 17cgttgtccac atggtgggcg gaatcgccgg cctctggggc
gccctcatcg agggcccccg 60cattggccgg ttcgaccacg ccggccgctc ggtggcgctg
cgcggccaca gcgcgtcgct 120cgtcgtgctc ggcactttcc tgctgtggtt
cggctggttc gggttcaacc ccgggtcgtt 180cctcaccatc ctcaagagct
acggcccggc cggcagcatc cacgggcagt ggtcggccgt 240gggccgcacg
gccgtgacca ccaccctcgc cggcagcacg gcggcgctca cgacgctctt
300cgggaagagg ctccagacgg ggcactggaa cgtggtcgac gtctgcaacg
gcctcctcgg 360cggcttcgcg gcgatcaccg cgggctgctc cgtggtcgac
ccctgggcgg ccatcatatg 420cgggttcgtg tcggcgtggg tgctcatcgg
gctcaacgcg ctggccgcga ggctccggtt 480cgacgacccg ctggaggccg
cgcagttgca cggtgggtgc ggcgcgtggg gggtcctctt 540cacgggcctg
ttcgcgcgca gggagtacgt ggagcagatc tacggcacgc cggggcggcc
600gtacggcctg ttcatgggcg gcggcgggag gctgctggcc gcgaacgtgg
tgatgatcct 660ggtgatcgcc gcgtgggtta gcgtcaccat ggctccgctg
ttcctggcgc tcaacaagat 720ggggctgctc cgagtctcgg ccgaggacga
gatggccggc atggaccaga cgcggcacgg 780cgggttcgcg tacgcgtacc
acgacgacga cttgagcttg agcagcaggc ccaaggggat 840gcagagcacg
cagatcgcgg acgcggccag cggcgagttc tagtgtgttg gatcacaaat
900ctcagtatgc tagtcctaca tcatgattgt caatagggcc attttaaaac
cccttctttt 960gggt 96418293PRTZea mays 18Val Val His Met Val Gly
Gly Ile Ala Gly Leu Trp Gly Ala Leu Ile1 5 10 15Glu Gly Pro Arg Ile
Gly Arg Phe Asp His Ala Gly Arg Ser Val Ala20 25 30Leu Arg Gly His
Ser Ala Ser Leu Val Val Leu Gly Thr Phe Leu Leu35 40 45Trp Phe Gly
Trp Phe Gly Phe Asn Pro Gly Ser Phe Leu Thr Ile Leu50 55 60Lys Ser
Tyr Gly Pro Ala Gly Ser Ile His Gly Gln Trp Ser Ala Val65 70 75
80Gly Arg Thr Ala Val Thr Thr Thr Leu Ala Gly Ser Thr Ala Ala Leu85
90 95Thr Thr Leu Phe Gly Lys Arg Leu Gln Thr Gly His Trp Asn Val
Val100 105 110Asp Val Cys Asn Gly Leu Leu Gly Gly Phe Ala Ala Ile
Thr Ala Gly115 120 125Cys Ser Val Val Asp Pro Trp Ala Ala Ile Ile
Cys Gly Phe Val Ser130 135 140Ala Trp Val Leu Ile Gly Leu Asn Ala
Leu Ala Ala Arg Leu Arg Phe145 150 155 160Asp Asp Pro Leu Glu Ala
Ala Gln Leu His Gly Gly Cys Gly Ala Trp165 170 175Gly Val Leu Phe
Thr Gly Leu Phe Ala Arg Arg Glu Tyr Val Glu Gln180 185 190Ile Tyr
Gly Thr Pro Gly Arg Pro Tyr Gly Leu Phe Met Gly Gly Gly195 200
205Gly Arg Leu Leu Ala Ala Asn Val Val Met Ile Leu Val Ile Ala
Ala210 215 220Trp Val Ser Val Thr Met Ala Pro Leu Phe Leu Ala Leu
Asn Lys Met225 230 235 240Gly Leu Leu Arg Val Ser Ala Glu Asp Glu
Met Ala Gly Met Asp Gln245 250 255Thr Arg His Gly Gly Phe Ala Tyr
Ala Tyr His Asp Asp Asp Leu Ser260 265 270Leu Ser Ser Arg Pro Lys
Gly Met Gln Ser Thr Gln Ile Ala Asp Ala275 280 285Ala Ser Gly Glu
Phe290191587DNAZea mays 19atggcgacgt gcgctacgac cctcgcacct
cttctgggcc cggcggcaaa cgcgacggag 60tacctttgca accaattcgc ggacaccacg
tcggcggtgg actcgacgta cctgctcttc 120tcggcctacc tcgtcttcgc
catgcagctc gggttcgcca tgctctgcgc gggctccgtc 180cgcgccaaga
acaccatgaa catcatgctc accaacgtgc tcgacgccgc cgccggcgcg
240ctcttctact acctattcgg cttcgccttc gcgtacggga ccccgtccaa
cggcttcatc 300ggcaagcact tcttcggcct caagcggctt ccccaggtcg
ggttcgacta cgacttcttc 360ctcttccagt gggctttcgc catcgccgcc
gccgggatca cgtccggctc catcgccgag 420cgcacgcagt tcgtggcgta
cctcatctac tccgccttcc tcaccggctt cgtgtacccg 480gtggtgtccc
actgggtctg gtccgccgac ggctgggcct cgccgtcacg gacgtcgggg
540aagctcctct tcggctccgg catcatcgac ttcgccgggt ccagcgttgt
ccacatggtg 600ggcggaatcg ccggcctctg gggcgccctc atcgagggcc
cccgcattgg ccggttcgac 660cacgccggcc gctcggtggc gctgcgcggc
cacagcgcgt cgctcgtcgt gctcggcact 720ttcctgctgt ggttcggctg
gttcgggttc aaccccgggt cgttcctcac catcctcaag 780agctacggcc
cggccggcag catccacggg cagtggtcgg ccgtgggccg cacggccgtg
840accaccaccc tcgccggcag cacggcggcg ctcacgacgc tcttcgggaa
gaggctccag 900acggggcact ggaacgtggt cgacgtctgc aacggcctcc
tcggcggctt cgcggcgatc 960accgcgggct gctccgtggt cgacccctgg
gcggccatca tatgcgggtt cgtgtcggcg 1020tgggtgctca tcgggctcaa
cctggccgcg aggctccggt tcgacgaccc ccgggaggcc 1080gcgcagttgc
acggtgggtg cggcgcgtgg ggggtcctct tcacgggcct gttcgcgcgc
1140agggagtacg tggagcagag cacgccgggg cggccgtacg gcctgttcat
gggcggcggc 1200aggctgctgg ccgcgaacgt ggtgatgatc ctggtgatcg
ccgcgtgggt tagcgtcacc 1260atggctccgc tgttcctggc gctcaacaag
atggggctgc tccgagtctc ggccgaggac 1320gagatggccg gcatggacca
gacgcggcac ggcgggttcg cgtacgcgta ccacgacgac 1380gacttgagct
tgagcagcag gcccaagggg atgcgagcac gcagatcgcg gacgcggcca
1440gcggcgagtt ctagtgtgtt ggatcacaaa tctcagtatg ctagtcctac
atcatgattg 1500tacaataaca accatgagta tactcccttc gttctaagga
ttactttgac gaagtatcta 1560gttaatttaa agataaagaa aatttaa
158720498PRTZea mays 20Met Ala Thr Cys Ala Thr Thr Leu Ala Pro Leu
Leu Gly Pro Ala Ala1 5 10 15Asn Ala Thr Glu Tyr Leu Cys Asn Gln Phe
Ala Asp Thr Thr Ser Ala20 25 30Val Asp Ser Thr Tyr Leu Leu Phe Ser
Ala Tyr Leu Val Phe Ala Met35 40 45Gln Leu Gly Phe Ala Met Leu Cys
Ala Gly Ser Val Arg Ala Lys Asn50 55 60Thr Met Asn Ile Met Leu Thr
Asn Val Leu Asp Ala Ala Ala Gly Ala65 70 75 80Leu Phe Tyr Tyr Leu
Phe Gly Phe Ala Phe Ala Tyr Gly Thr Pro Ser85 90 95Asn Gly Phe Ile
Gly Lys His Phe Phe Gly Leu Lys Arg Leu Pro Gln100 105 110Val Gly
Phe Asp Tyr Asp Phe Phe Leu Phe Gln Trp Ala Phe Ala Ile115 120
125Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr Gln
Phe130 135 140Val Ala Tyr Leu Ile Tyr Ser Ala Phe Leu Thr Gly Phe
Val Tyr Pro145 150 155 160Val Val Ser His Trp Val Trp Ser Ala Asp
Gly Trp Ala Ser Pro Ser165 170 175Arg Thr Ser Gly Lys Leu Leu Phe
Gly Ser Gly Ile Ile Asp Phe Ala180 185 190Gly Ser Ser Val Val His
Met Val Gly Gly Ile Ala Gly Leu Trp Gly195 200 205Ala Leu Ile Glu
Gly Pro Arg Ile Gly Arg Phe Asp His Ala Gly Arg210 215 220Ser Val
Ala Leu Arg Gly His Ser Ala Ser Leu Val Val Leu Gly Thr225 230 235
240Phe Leu Leu Trp Phe Gly Trp Phe Gly Phe Asn Pro Gly Ser Phe
Leu245 250 255Thr Ile Leu Lys Ser Tyr Gly Pro Ala Gly Ser Ile His
Gly Gln Trp260 265 270Ser Ala Val Gly Arg Thr Ala Val Thr Thr Thr
Leu Ala Gly Ser Thr275 280 285Ala Ala Leu Thr Thr Leu Phe Gly Lys
Arg Leu Gln Thr Gly His Trp290 295 300Asn Val Val Asp Val Cys Asn
Gly Leu Leu Gly Gly Phe Ala Ala Ile305 310 315 320Thr Ala Gly Cys
Ser Val Val Asp Pro Trp Ala Ala Ile Ile Cys Gly325 330 335Phe Val
Ser Ala Trp Val Leu Ile Gly Leu Asn Leu Ala Ala Arg Leu340 345
350Arg Phe Asp Asp Pro Arg Glu Ala Ala Gln Leu His Gly Gly Cys
Gly355 360 365Ala Trp Gly Val Leu Phe Thr Gly Leu Phe Ala Arg Arg
Glu Tyr Val370 375 380Glu Gln Ser Thr Pro Gly Arg Pro Tyr Gly Leu
Phe Met Gly Gly Gly385 390 395 400Arg Leu Leu Ala Ala Asn Val Val
Met Ile Leu Val Ile Ala Ala Trp405 410 415Val Ser Val Thr Met Ala
Pro Leu Phe Leu Ala Leu Asn Lys Met Gly420 425 430Leu Leu Arg Val
Ser Ala Glu Asp Glu Met Ala Gly Met Asp Gln Thr435 440 445Arg His
Gly Gly Phe Ala Tyr Ala Tyr His Asp Asp Asp Leu Ser Leu450 455
460Ser Ser Arg Pro Lys Gly Met Arg Ala Arg Arg Ser Arg Thr Arg
Pro465 470 475 480Ala Ala Ser Ser Ser Val Leu Asp His Lys Ser Gln
Tyr Ala Ser Pro485 490 495Thr Ser21744DNAZea maysmisc_feature715n =
A,T,C or G 21tcccaatccc ctccccctcg cgtatccaca cttttcacac gcgacgccgg
agagacagag 60cgcgcgcgcg cccgaaagat ggcgacgtgc gcgacggacc tggcgccgct
gctcggcccg 120gcggcggcaa acgccacgga ctacctctgc aaccaattcg
cggacaccac ctccgcggtg 180gacgccacgt acctgctctt ctcggcctac
ctcgtcttcg ccatgcagct cggcttcgcc 240atgctctgcg ccggctccgt
ccgcgccaag aacaccatga acatcatgct caccaacgtg 300ctcgacgccg
ccgccggcgc gctcttctac tacctattcg gcttcgcctt cgcctacggc
360accccgtcca acggcttcat cggcaagcac ttcttcggcc tcaagcgcct
gcccaagacc 420ggcttcgact acgacttctt cctataccag tgggccttcg
ccatcgccgc cgccggcatc 480acgtccggct ccatcgccga gagcacccag
ttcgtcgcct acctcatcta ctccgccttc 540ctcaccggct tcgtgtaccc
cgtggcgtcc cactgggtct ggtccgccga cggctgggcc 600gccgccggcc
gcacgtccgg cccgctgctc ttcgggtccg gcgccatcga cttcgccggc
660tccggcgtgg tccacatggt cggcggcatc gcggggttct ggggcgcgct
cgtcnagggc 720ccccgtatcg ggcgcttcga ccac 74422222PRTZea
maysVARIANT213Xaa = Any Amino Acid 22Met Ala Thr Cys Ala Thr Asp
Leu Ala Pro Leu Leu Gly Pro Ala Ala1 5 10 15Ala Asn Ala Thr Asp Tyr
Leu Cys Asn Gln Phe Ala Asp Thr Thr Ser20 25 30Ala Val Asp Ala Thr
Tyr Leu Leu Phe Ser Ala Tyr Leu Val Phe Ala35 40 45Met Gln Leu Gly
Phe Ala Met Leu Cys Ala Gly Ser Val Arg Ala Lys50 55 60Asn Thr Met
Asn Ile Met Leu Thr Asn Val Leu Asp Ala Ala Ala Gly65 70 75 80Ala
Leu Phe Tyr Tyr Leu Phe Gly Phe Ala Phe Ala Tyr Gly Thr Pro85 90
95Ser Asn Gly Phe Ile Gly Lys His Phe Phe Gly Leu Lys Arg Leu
Pro100
105 110Lys Thr Gly Phe Asp Tyr Asp Phe Phe Leu Tyr Gln Trp Ala Phe
Ala115 120 125Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu
Ser Thr Gln130 135 140Phe Val Ala Tyr Leu Ile Tyr Ser Ala Phe Leu
Thr Gly Phe Val Tyr145 150 155 160Pro Val Ala Ser His Trp Val Trp
Ser Ala Asp Gly Trp Ala Ala Ala165 170 175Gly Arg Thr Ser Gly Pro
Leu Leu Phe Gly Ser Gly Ala Ile Asp Phe180 185 190Ala Gly Ser Gly
Val Val His Met Val Gly Gly Ile Ala Gly Phe Trp195 200 205Gly Ala
Leu Val Xaa Gly Pro Arg Ile Gly Arg Phe Asp His210 215
220231024DNAZea mays 23gaggtcgtcg tctctagcta gctgctaaga gagagagaga
gagagaggta tacgtaggac 60cgccggcaac tagctaacta acatgtcgtc gtcgtccggg
acgacgatgc cgctggcgta 120ccagacgtcg gcgtcgtctc ccgagtggct
gaacaagggc gacaacgcgt ggcagctgac 180ggcggcgacg ctggtggggc
tgcagagctt cccgggtctg gtggtcctgt acggcggcgt 240ggtgaagaag
aagtgggccg tgaactcggc cttcatggcg ctgtacgcgt tcgcggcggt
300gtggatctgc tgggtgacct gggcctacaa catgtccttc ggcgacaggc
tgctgccgct 360gtggggcaag gcgcggccgg cgctgagcca gggcgggctg
gtggggcagg ccggcctccc 420cgccacggcg caccacttcg ccagcggcgc
cctggagacc ccggccgcgg agccgctgta 480cccgatggcc acggtggtgt
acttccagtg cgtgttcgcg gccatcaccc tggtgctggt 540cgccgggtcg
ctgctgggcc ggatgagctt cgccgcgtgg atgctgttcg tgccgctctg
600gctcaccttc tcctacaccg tcggcgcctt ctccgtatgg ggcggcgggt
tcctcttcca 660gtggggcgtc atcgactact gcggcggcta cgtcatccac
ctctccgctg gcttcgccgg 720gttcacggca gcctactggg tggggccccg
ggcgcagaag gacagggaga ggttcccgcc 780gaacaacatc ctgttcacgc
tcaccggcgc gggcctgctg tggatggggt gggccggctt 840caacggcggc
gggccgtacg ccgccaacgt ggtggcgtcc atgtcggtgc tcaacaccaa
900catctgcacc gccatgagcc tcctcgtctg gacctgcctc gacgtcgtct
tcttcaagaa 960gccctccgtc gtgggcgccg tccagggcat gatcaccgga
ctcgtctgca tcacgcccgc 1020cgca 102424314PRTZea mays 24Met Ser Ser
Ser Ser Gly Thr Thr Met Pro Leu Ala Tyr Gln Thr Ser1 5 10 15Ala Ser
Ser Pro Glu Trp Leu Asn Lys Gly Asp Asn Ala Trp Gln Leu20 25 30Thr
Ala Ala Thr Leu Val Gly Leu Gln Ser Phe Pro Gly Leu Val Val35 40
45Leu Tyr Gly Gly Val Val Lys Lys Lys Trp Ala Val Asn Ser Ala Phe50
55 60Met Ala Leu Tyr Ala Phe Ala Ala Val Trp Ile Cys Trp Val Thr
Trp65 70 75 80Ala Tyr Asn Met Ser Phe Gly Asp Arg Leu Leu Pro Leu
Trp Gly Lys85 90 95Ala Arg Pro Ala Leu Ser Gln Gly Gly Leu Val Gly
Gln Ala Gly Leu100 105 110Pro Ala Thr Ala His His Phe Ala Ser Gly
Ala Leu Glu Thr Pro Ala115 120 125Ala Glu Pro Leu Tyr Pro Met Ala
Thr Val Val Tyr Phe Gln Cys Val130 135 140Phe Ala Ala Ile Thr Leu
Val Leu Val Ala Gly Ser Leu Leu Gly Arg145 150 155 160Met Ser Phe
Ala Ala Trp Met Leu Phe Val Pro Leu Trp Leu Thr Phe165 170 175Ser
Tyr Thr Val Gly Ala Phe Ser Val Trp Gly Gly Gly Phe Leu Phe180 185
190Gln Trp Gly Val Ile Asp Tyr Cys Gly Gly Tyr Val Ile His Leu
Ser195 200 205Ala Gly Phe Ala Gly Phe Thr Ala Ala Tyr Trp Val Gly
Pro Arg Ala210 215 220Gln Lys Asp Arg Glu Arg Phe Pro Pro Asn Asn
Ile Leu Phe Thr Leu225 230 235 240Thr Gly Ala Gly Leu Leu Trp Met
Gly Trp Ala Gly Phe Asn Gly Gly245 250 255Gly Pro Tyr Ala Ala Asn
Val Val Ala Ser Met Ser Val Leu Asn Thr260 265 270Asn Ile Cys Thr
Ala Met Ser Leu Leu Val Trp Thr Cys Leu Asp Val275 280 285Val Phe
Phe Lys Lys Pro Ser Val Val Gly Ala Val Gln Gly Met Ile290 295
300Thr Gly Leu Val Cys Ile Thr Pro Ala Ala305 310251798DNAZea mays
25ttatgatcca cttggttaac tagcataatt aatcgcagat gaagcagcag ttcatgaagg
60caggaagcag ctaaatcacc catataaatg gtcgcgcgcg ctagcatagc atagtagcga
120tagccaccac cgatcgaagc atgatggcgg cgtcgggcgc gtacgcggcg
caactcccgg 180cggtgccgga gtggctgaac aagggcgaca acgcgtggca
gctgacggcg gcgacgctgg 240tgggcatcca gtcgatgccg gggctggtgg
tgctgtacgg cagcatcgtg aagaagaagt 300gggcggtgaa ctcggcgttc
atggcgctgt acgcctacgc gtcgtcgctg ctggtgtggg 360tgctggcggg
gttccgcatg gcgttcgggg agcggctgct cccgttctgg ggcaaggccg
420gggtggcgct ctcccagggc tacctggtcc ggcgcgcctc gctctcggcg
accgcgcacg 480gggccacgcc ccgcaccgag cccctgtacc cggaggcgac
gctggtgctg ttccagttcg 540agttcgccgc catcacgctg gtgctcctgg
ccggctccgt gcttggccgc atgaacatca 600aggcctggat ggccttcacc
ccgctctggc tcctcttctc ctacaccgtc ggcgccttca 660gcatctgggg
cggcggcttc ctctaccact ggggcgtcat cgactactcc ggcggatacg
720tcatccacct ctcctccggc atcgccggct tcaccgccgc atactgggtg
ggcccgaggc 780tgaagagcga ccgggagcgc ttctccccga acaacatcct
gctgatgatc gcgggcggcg 840ggctgctgtg gatgggctgg gccgggttca
acggcggcgc gccctacgcc gccaacatcg 900cggcgtccgt ggccgtgctc
aacaccaacg tctccgccgc caccagcctc ctcacctgga 960cctgcctcga
cgtcatcttc ttcggcaagc cgtccgtgat cggcgccgtg cagggcatga
1020tgacggggct cgtctgcatc acccccggag cagggctggt gcagacctgg
gcggcggtga 1080tcatgggcgt gttcgcgggc agcgtgccgt ggttcaccat
gatgatcctg cacaagaagg 1140tggcgctgct gacgagggtg gacgacacgc
tgggcgtctt ccacacgcac gccgtcgcgg 1200gcctgctggg cggcgtcctc
acggggctgc tggccacgcc ggagctgctg gagatcgagt 1260cccccgtgcc
gggcctccgc ggcgcgttct acggcggagg gatccgccag gtcggcaagc
1320agctggcggg ggccgccttc gtggtggcgt ggaacgtcgt ggtcacgtcg
ctcatcctgc 1380tggccatcgg cctgctggtg cccctgcgga tgcccgagga
ccagctcatg atcggcgacg 1440acgccgcgca cggggaggag gcctacgcgc
tctggggcga cggggagaag ttcgatgcca 1500ccaggcacga cgcggtcagg
gtcgccggcg tcatggatag agaagggtcc gcggagcagc 1560ggctatcagg
gggcgtcacc attcagctgt aggcgcacgc ccgacggtcc ataagacacg
1620actttttagc ggacattttt ttttcatggg agaagagcag tgttttaggc
tttttattat 1680tagcatgaaa ggttgtccat gtatcatatt tggcccagag
cacgtagtct ctgctagttt 1740ataaagaaat taggtcatgt atttttcctc
ttaatctagt ctacccgcaa catgtact 179826483PRTZea mays 26Met Met Ala
Ala Ser Gly Ala Tyr Ala Ala Gln Leu Pro Ala Val Pro1 5 10 15Glu Trp
Leu Asn Lys Gly Asp Asn Ala Trp Gln Leu Thr Ala Ala Thr20 25 30Leu
Val Gly Ile Gln Ser Met Pro Gly Leu Val Val Leu Tyr Gly Ser35 40
45Ile Val Lys Lys Lys Trp Ala Val Asn Ser Ala Phe Met Ala Leu Tyr50
55 60Ala Tyr Ala Ser Ser Leu Leu Val Trp Val Leu Ala Gly Phe Arg
Met65 70 75 80Ala Phe Gly Glu Arg Leu Leu Pro Phe Trp Gly Lys Ala
Gly Val Ala85 90 95Leu Ser Gln Gly Tyr Leu Val Arg Arg Ala Ser Leu
Ser Ala Thr Ala100 105 110His Gly Ala Thr Pro Arg Thr Glu Pro Leu
Tyr Pro Glu Ala Thr Leu115 120 125Val Leu Phe Gln Phe Glu Phe Ala
Ala Ile Thr Leu Val Leu Leu Ala130 135 140Gly Ser Val Leu Gly Arg
Met Asn Ile Lys Ala Trp Met Ala Phe Thr145 150 155 160Pro Leu Trp
Leu Leu Phe Ser Tyr Thr Val Gly Ala Phe Ser Ile Trp165 170 175Gly
Gly Gly Phe Leu Tyr His Trp Gly Val Ile Asp Tyr Ser Gly Gly180 185
190Tyr Val Ile His Leu Ser Ser Gly Ile Ala Gly Phe Thr Ala Ala
Tyr195 200 205Trp Val Gly Pro Arg Leu Lys Ser Asp Arg Glu Arg Phe
Ser Pro Asn210 215 220Asn Ile Leu Leu Met Ile Ala Gly Gly Gly Leu
Leu Trp Met Gly Trp225 230 235 240Ala Gly Phe Asn Gly Gly Ala Pro
Tyr Ala Ala Asn Ile Ala Ala Ser245 250 255Val Ala Val Leu Asn Thr
Asn Val Ser Ala Ala Thr Ser Leu Leu Thr260 265 270Trp Thr Cys Leu
Asp Val Ile Phe Phe Gly Lys Pro Ser Val Ile Gly275 280 285Ala Val
Gln Gly Met Met Thr Gly Leu Val Cys Ile Thr Pro Gly Ala290 295
300Gly Leu Val Gln Thr Trp Ala Ala Val Ile Met Gly Val Phe Ala
Gly305 310 315 320Ser Val Pro Trp Phe Thr Met Met Ile Leu His Lys
Lys Val Ala Leu325 330 335Leu Thr Arg Val Asp Asp Thr Leu Gly Val
Phe His Thr His Ala Val340 345 350Ala Gly Leu Leu Gly Gly Val Leu
Thr Gly Leu Leu Ala Thr Pro Glu355 360 365Leu Leu Glu Ile Glu Ser
Pro Val Pro Gly Leu Arg Gly Ala Phe Tyr370 375 380Gly Gly Gly Ile
Arg Gln Val Gly Lys Gln Leu Ala Gly Ala Ala Phe385 390 395 400Val
Val Ala Trp Asn Val Val Val Thr Ser Leu Ile Leu Leu Ala Ile405 410
415Gly Leu Leu Val Pro Leu Arg Met Pro Glu Asp Gln Leu Met Ile
Gly420 425 430Asp Asp Ala Ala His Gly Glu Glu Ala Tyr Ala Leu Trp
Gly Asp Gly435 440 445Glu Lys Phe Asp Ala Thr Arg His Asp Ala Val
Arg Val Ala Gly Val450 455 460Met Asp Arg Glu Gly Ser Ala Glu Gln
Arg Leu Ser Gly Gly Val Thr465 470 475 480Ile Gln Leu27330DNAOryza
sativa 27atggtgccgg gactccgcgg cgcgttctac ggcggcggca tcaagcagat
cagcaagcag 60ctcggcggcg ctgcgtttgt gatcgcgtgg aacctcgtgg tcaccacggc
catcctcctt 120ggcatcggcc tgttcatccc gctgcggatg cccgacgagc
agctcatgat cggcgacgac 180gcggcgcacg gcgaggaggc ctacgcgttg
tggggcgacg gcgagaagtt caacgcgaca 240cagcacgacc tatcgagggg
tggcggcggc ggcgacaggg acggccccga gcggctctcc 300atcctaggcg
ccaggggcgt caccatctag 33028186PRTOryza sativa 28Met Thr Pro Pro Arg
Gly Pro Ser Pro Ser Thr Asn Ala Ala Arg Arg1 5 10 15Cys Arg Leu Thr
Lys His Arg His Gly Arg Ala Thr Pro Ser Pro Pro20 25 30Ile Thr Cys
Ala Ser Ser Arg Arg Pro Pro Arg Glu Thr Thr Leu Pro35 40 45His Pro
Arg Cys Gly Gly Ala Pro Arg Arg His Pro His Gly Pro Pro50 55 60Gly
His Pro Gly Ala Leu Leu Pro Arg Gly Leu Glu Ser Met Val Pro65 70 75
80Gly Leu Arg Gly Ala Phe Tyr Gly Gly Gly Ile Lys Gln Ile Ser Lys85
90 95Gln Leu Gly Gly Ala Ala Phe Val Ile Ala Trp Asn Leu Val Val
Thr100 105 110Thr Ala Ile Leu Leu Gly Ile Gly Leu Phe Ile Pro Leu
Arg Met Pro115 120 125Asp Glu Gln Leu Met Ile Gly Asp Asp Ala Ala
His Gly Glu Glu Ala130 135 140Tyr Ala Leu Trp Gly Asp Gly Glu Lys
Phe Asn Ala Thr Gln His Asp145 150 155 160Leu Ser Arg Gly Gly Gly
Gly Gly Asp Arg Asp Gly Pro Glu Arg Leu165 170 175Ser Ile Leu Gly
Ala Arg Gly Val Thr Ile180 185294123DNAOryza sativa 29gataaccaaa
tcggacgctg accttgctgg gcgaactggg tgatcatcga tggcgatgcg 60agacatcacc
caactgcgtc gggtctccac aggagggagt tgcttgtgct tggtccgttt
120ggggatcgtt aacttaaaca cttttacggc gacctcgaca cagctaaacc
ctaaactaat 180tgcgagttag aggcttatct cgatctcttc tatgcagatg
tttgacaact tgggagtagt 240ttactgctgg tttggagtat cttctcaact
tgcaatttga ttatgtttaa acggggagtg 300catgattggt gttcgcatgg
ttttaaatca gattttataa actgatgctc gtcaagagac 360gacaaggggc
cagattaggg cagcagagta cgtgcttgct tgaattctga agcatgtacg
420aaataaatac gatagaaatt tcttaagaaa ttaggtattt ttctgaccct
ccaataagat 480cgcgtggttg ccagtattgc acgtcgacta ctacatatct
gaattcagaa caatccaaaa 540gagaagttac tgttgatatt tctacgtata
aaaaaaacat caaaatgctt tgtatattac 600gaaaacagag cgagttccct
tattgaccag agcaaaaagg ttgagcctga ttaaacaaag 660tctatgagct
tgcaggatgc gtctcttccc aaatttattc acaccaaagt cctcttcgat
720gacatcgccc tatttgaatc ttatcgttga cattgctcat tttgctcttt
agttaatctg 780ggcaaatgat tggcggtggt acttcgtgat gtggaacagt
gaaactgttt gtcaatctgt 840gcgctcgagg tacaaccagg tcggttcctt
tgctgtttta ttaataaaag gagcataaat 900tagcgccaaa actcaagttt
taccacaaaa aaacagtcag ttttaataaa gattaagcaa 960acccttgaat
tgcactctgt aaaatgtttg tttcccctca aaagctgata aggacggacg
1020ccgatgtgaa acgaaacctg ctatttcaac catgtacata tataatcaag
aatttcctac 1080acgacttcca ttttttgtgt gttgactagt ttctctcctt
cctggaggtg ttaaaagagt 1140tccgattctg tcaaaacttc catacagata
aatccaacct gtcaaactac cagctgttta 1200attattcctt ttcccatttt
gttatggtac acaaaggcac ataaccattt acacggagca 1260gaacagaata
ggatatgtat taaaaaaaca gaatggaaga aaaatcctga gtcacaagca
1320cgaaaaatga aggcgagatt aattcgaaac catacacatc atcatccaca
tctcgtcgtt 1380tgtctcacag gacatgacac agggagcgaa aaccacatca
ttaatcgcgg cctacagcta 1440cacatccaga ttctcccggg atccccgaaa
cggctcccac cccgcaaccg ccgcaagccg 1500acccagccaa aggagatccc
cctccaccac ggaagattca ctgcgcggtg ggccccgccg 1560ccaaaaacca
aaacgacgaa accattccgc gtcatctctc ccgcacggcg agcgagcgag
1620cgagcgacct gacctcctcc tcctataaat ccggcgccag cgtatctccc
caacctccca 1680cgcccaatcc tgccgccgtt tcagcagctc tagtttgaac
gagggatcgt agagaggagg 1740gtttggtgag ggagggagga agatggcgac
gtgcgcggcg gacctggcgc cgctgctggg 1800gccggtggcg gcgaacgcga
cggactacct gtgcaaccgg ttcgccgaca cgacgtcggc 1860ggtggacgcg
acgtacctgc tcttctcggc gtacctcgtg ttcgccatgc agctcgggtt
1920cgcgatgctc tgcgccgggt cggtgcgggc caagaacacg atgaacatca
tgctcaccaa 1980cgtgctcgac gccgcggccg gggcgctctt ctactacctc
ttcggcttcg ccttcgcctt 2040cggcacgccg tccaacggct tcatcgggaa
gcagttcttc ggcctcaagc acatgccgca 2100gaccgggttc gactacgact
tcttcctctt ccagtgggcc ttcgccatcg ccgccgccgg 2160gatcacgtcg
ggctccatcg ccgagaggac gcagttcgtc gcctacctca tctactccgc
2220cttcctcacc gggttcgtct acccggtggt gtcccactgg atctggtccg
ccgatgggtg 2280ggcctctgcc tcccgcacgt ccggacctct gctgttcggc
tccggtgtca tcgacttcgc 2340cggctccggc gtcgtccaca tggtcggcgg
tgtcgccggg ctctggggcg cgctcatcga 2400gggcccccgc atcgggaggt
tcgaccacgc cggccgatcg gtggcgctca agggccacag 2460cgcgtcgctc
gtcgtgcttg gcaccttcct gctgtggttc ggctggtacg gattcaaccc
2520cgggtcgttc accaccatcc tcaagacgta cggcccggcc ggcggcatca
acgggcagtg 2580gtccggagtc ggccgcaccg ccgtgacgac gaccctggcc
ggcagcgtgg cggcgctcac 2640cacgctgttc gggaagcggc tccagacggg
gcactggaac gtggtcgacg tctgcaacgg 2700cctcctcggc gggttcgccg
ccatcaccgc cgggtgcagc gtcgtcgacc cgtgggccgc 2760gatcatctgc
gggttcgtct cggcgtgggt gctcatcggc ctcaacgcgc tcgccgcgcg
2820cctcaagttc gacgacccgc tcgaggccgc ccagctccac ggcgggtgcg
gcgcgtgggg 2880gatcctcttc accgcgctct tcgcgaggca gaagtacgtc
gaggagatct acggcgccgg 2940ccggccgtac ggcctgttca tgggcggcgg
cggcaagctg ctcgccgcgc acgtcatcca 3000gatcctggtc atcttcgggt
gggtcagctg caccatggga cctctcttct acgggctcaa 3060gaagctcggc
ctgctccgca tctccgccga ggacgagacg tccggcatgg acctgacacg
3120gcacggcggg ttcgcgtacg tctaccacga cgaggacgag cacgacaagt
ctggggttgg 3180tgggttcatg ctccggtccg cgcagacccg cgtcgagccg
gcggcggccg gctgcctcca 3240acagcaacaa ccaagtgtaa ccaatccaga
acgaacgacg tcacagcgaa ggaagaaatc 3300acgggtttct ctccctctcc
gatctcgatc gtcacgtcat aaatttgatc cccatatttg 3360attgccagtt
tctgtttggg ccaaatgctt ttgccgctct ctctggtgtt gcaagactgt
3420aaaaacactg taggatggac gagtgtcttt cacttttgct gggcttctct
tgtgtacagg 3480catgcgtacg tgtcttagaa tgtgtggtgt gaaggtggga
agaatcagag gttagggttt 3540aattttcttt tgcacaatgg ttactgctat
tattgtttta ttttgtggtc gaattttatc 3600gtcataaggg tgtggtggaa
tggtggtcaa gataggtggc tgtgcagggc tcaaagactt 3660tgcgtgggtc
cttttgtcct gcagtgctct acctctctat caaaactttg gcttatttcc
3720tggaatctag tggtttgaga gtgtttgttt tatactcagt tctgcattat
gtttacgata 3780tatttttttt tttaccaaaa gcatttcatt taaactctac
cgagagtact tgtttatgct 3840gaatcagtac atctacactg agtgatattt
agagccttat actaattgaa gattaaatag 3900tcaaagtcca tgtgcacatt
tctactcgcc agttagtctg aaagaaaaga ttcctgtgtg 3960caattgtgca
tatcagcata tgccacctgg cgataaagta aacatactat agttgtgaac
4020tgtgcgatga caacgaccaa attaagcagc ctgatcttta caacgaccgc
tgtatagaga 4080acagactata tcaaggtttt gggtccgtgg tcttcttttt ggg
412330532PRTOryza sativa 30Met Ala Thr Cys Ala Ala Asp Leu Ala Pro
Leu Leu Gly Pro Val Ala1 5 10 15Ala Asn Ala Thr Asp Tyr Leu Cys Asn
Arg Phe Ala Asp Thr Thr Ser20 25 30Ala Val Asp Ala Thr Tyr Leu Leu
Phe Ser Ala Tyr Leu Val Phe Ala35 40 45Met Gln Leu Gly Phe Ala Met
Leu Cys Ala Gly Ser Val Arg Ala Lys50 55 60Asn Thr Met Asn Ile Met
Leu Thr Asn Val Leu Asp Ala Ala Ala Gly65 70 75 80Ala Leu Phe Tyr
Tyr Leu Phe Gly Phe Ala Phe Ala Phe Gly Thr Pro85 90 95Ser Asn Gly
Phe Ile Gly Lys Gln Phe Phe Gly Leu Lys His Met Pro100 105 110Gln
Thr Gly Phe Asp Tyr Asp Phe Phe Leu Phe Gln Trp Ala Phe Ala115 120
125Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr
Gln130 135 140Phe Val Ala Tyr Leu Ile Tyr Ser Ala Phe Leu Thr Gly
Phe Val Tyr145 150 155 160Pro Val Val Ser His Trp Ile Trp Ser Ala
Asp Gly Trp Ala Ser Ala165 170 175Ser Arg Thr Ser Gly Pro Leu Leu
Phe Gly Ser Gly Val Ile Asp Phe180 185 190Ala Gly Ser Gly Val Val
His Met Val Gly Gly Val Ala Gly Leu Trp195 200 205Gly Ala Leu Ile
Glu Gly Pro Arg Ile Gly Arg Phe Asp His Ala Gly210 215 220Arg Ser
Val Ala Leu Lys Gly His Ser Ala Ser Leu Val Val Leu Gly225 230
235 240Thr Phe Leu Leu Trp Phe Gly Trp Tyr Gly Phe Asn Pro Gly Ser
Phe245 250 255Thr Thr Ile Leu Lys Thr Tyr Gly Pro Ala Gly Gly Ile
Asn Gly Gln260 265 270Trp Ser Gly Val Gly Arg Thr Ala Val Thr Thr
Thr Leu Ala Gly Ser275 280 285Val Ala Ala Leu Thr Thr Leu Phe Gly
Lys Arg Leu Gln Thr Gly His290 295 300Trp Asn Val Val Asp Val Cys
Asn Gly Leu Leu Gly Gly Phe Ala Ala305 310 315 320Ile Thr Ala Gly
Cys Ser Val Val Asp Pro Trp Ala Ala Ile Ile Cys325 330 335Gly Phe
Val Ser Ala Trp Val Leu Ile Gly Leu Asn Ala Leu Ala Ala340 345
350Arg Leu Lys Phe Asp Asp Pro Leu Glu Ala Ala Gln Leu His Gly
Gly355 360 365Cys Gly Ala Trp Gly Ile Leu Phe Thr Ala Leu Phe Ala
Arg Gln Lys370 375 380Tyr Val Glu Glu Ile Tyr Gly Ala Gly Arg Pro
Tyr Gly Leu Phe Met385 390 395 400Gly Gly Gly Gly Lys Leu Leu Ala
Ala His Val Ile Gln Ile Leu Val405 410 415Ile Phe Gly Trp Val Ser
Cys Thr Met Gly Pro Leu Phe Tyr Gly Leu420 425 430Lys Lys Leu Gly
Leu Leu Arg Ile Ser Ala Glu Asp Glu Thr Ser Gly435 440 445Met Asp
Leu Thr Arg His Gly Gly Phe Ala Tyr Val Tyr His Asp Glu450 455
460Asp Glu His Asp Lys Ser Gly Val Gly Gly Phe Met Leu Arg Ser
Ala465 470 475 480Gln Thr Arg Val Glu Pro Ala Ala Ala Gly Cys Leu
Gln Gln Gln Gln485 490 495Pro Ser Val Thr Asn Pro Glu Arg Thr Thr
Ser Gln Arg Arg Lys Lys500 505 510Ser Arg Val Ser Leu Pro Leu Arg
Ser Arg Ser Ser Arg His Lys Phe515 520 525Asp Pro His
Ile530314654DNAOryza sativa 31gagctccact cagctaccgg atcttgaccg
ggaacctgtt tgtctacgta ctccaacgcc 60ttgaatgatg ccgccgtgca gccaatttta
accagctgct gcgcaaccgg ccaaccgccc 120agccgtgcag ctgtggtgga
gtgaccacgg ccacgactcc gtgcgcgcgg gtggacgtaa 180gcgttgggcc
ctcggctcgc gcgcgcggcc gcatccggcg atgcatcggt cgcgttcgcg
240gtttgtggct tcgcgtcatc gccgatgcga acagaggctg ctttgcgttg
tcgtcatcgg 300cttgttgacg tccacgagtt ggcgagttgc tctgttcctc
tctcgcgcgc gccgcagata 360tccgaggtgg aaaaaatata ctacatatga
acagatgtgg cccagctgtg agcaagacgc 420caagaccaaa gataagtgca
gttcaaatgg gcctgaaatt ggccttcatc aattacaaag 480cccgtgaaaa
gtttcagaaa agcattacaa agcttcagat aagttcaggg gtgactgaaa
540tacacataca acaagtaacg tagagagatc cccaaatcag ctgcggcaga
aggcagaaac 600cgtgactagt acatctcata aacttaacga gcagtacaat
ttctgtacat tggtttatca 660ataagtcaag agtagcattt gggtaagaag
agaaaaaaaa tcttttacgg tggcgtttat 720tgacatttga tccctggagc
cgagaagact agtttatctc atccgtgaaa actatttgtc 780actagacatc
aacgtctcgc tgaggacacc cggtttgcaa tttgctaata agaaacactc
840gtttccgtcc aatggcgatt cgtttactag agatccgtcc attctctgaa
cttctgaagg 900tcaaccttct gatatgcata caggtgtggt agcaggcacg
acaaaagtat aaaacaatag 960gtatttaatc gcatcagcgt gatctatctc
cagagtgtaa aaattagata cgcagcatct 1020gcaagtcata cttgcaagga
agaagcaatt gcgtccctat ccaccacatc ttatccagtc 1080ttgtcagagt
tttgacctaa ggaattgctt catcatctga atttattcta ctggaagaaa
1140ttacctactg ctatgccaag aagtaagaat acatggttaa tctatgttga
caccctcatt 1200tctgaagaca aacaacataa atttgcagtg agttaaaaca
tatagtttca gtgaaaatat 1260cgtacaggtt aatgtagcca gaccaacaca
gagatctgat tgacaattac agagtactca 1320gtagtcagca agcaggttta
gcatggacta cctgttgatt acatggtttc agtcagacac 1380gagttcttca
gggaggcaaa ttaatcacaa ggttcttcca agacagaagc cgccggtaag
1440gtatggaagc aaatgggttt atctccgttt gtgccaataa ccttatcgaa
tatctaattg 1500ttcgctaagt acgcattgca cacatcatat accatctcga
ttgatgagtt ttgtcgccat 1560gttctgctag gtacgaccta tccgctgctg
ggtttacatg catgcctgaa gaacttaaac 1620agttaatcaa caaagtcaat
ggatattacg catacaagta tatggttgta tatatgcaac 1680ttcatgacac
aacagtatgc gtatagtcgg acgtgacgac aagcaactac ctcgtgcaag
1740gatgcgagga gataccagat taaaacctgt aatacttgaa gttgacaaaa
tgcgattctt 1800cagacgatat aataacaaga acatttctga atcttccttt
aaaaaatgtt gaatgcataa 1860aagaatctta gctgtgatgg caacaacacg
actttctgat agtgacattg gatcttagtt 1920gaatctggca tcttgcgtat
gcgaccttgc ttggatctgt cggatactcc caacccagca 1980taaattactg
atgtctgaaa ctttctgagc aaagcgggaa ctcaggctag ttgagtcgct
2040catcatcaaa agtcaagaca atacttaagt aaaaacaaaa caaatatcac
tgtcgcaaaa 2100ccagtgtaaa cctattggga taattttaac agtcttacaa
caccagcgcc gagacgtcta 2160ggtaacaaat taaatcatca ctgacaatat
ttgaagataa gtgaatcaca tgtctttctc 2220aatgaactta aatttatgaa
tgaaccaacc tatacatgca actaaatatg atatcaagat 2280cagttaaaaa
tcttgttttg tgaaatttca aaacaaaaaa ataaaattgc agcgtacctt
2340tatttcaaga gaaatttaat tcactaaaaa aaagtaattg tatgtgccaa
attttacatt 2400aaaaaatggt atctgcaatg tattcttaag gatgataaaa
ttatctcagt aatattcaat 2460cattcattaa tagaggtgaa ctgctcgttc
ttttactcat cacacctgtt ggatgaaaag 2520attggttgct gccactaaaa
aaattaatca actcattgca gttggcaaaa aagaaaaaaa 2580aaggttttca
ggcactagta tatgtgatgt tagaaggaat ccaaaacagt atacatgcat
2640ccacgcgcac ctcggttttg catttcccgc tgtctgtgat catgaatcat
ccaattaaaa 2700acaaccatca accatggatt ccaatgtgtt ctgcccctat
aaatagtcca gcatctcatt 2760ctctcgtcta cttcaaagaa tcacacacca
aaacctccat tagttcctaa accctagcaa 2820gaagcagcac aaaaccttgc
cacacttggc tagtgacact gagacacacc atggcgacgt 2880gcttggacag
cctcgggccg cttctcggcg gcgcggcgaa ctccaccgac gcggccaact
2940acatctgcaa caggttcacg gacacctcct ccgcggtgga cgcgacgtac
ctgctcttct 3000cggcctacct cgtgttcgcc atgcagctcg ggttcgccat
gctctgcgcg ggctccgtcc 3060gcgccaagaa ctcaatgaac atcatgctca
ccaacgtgtt cgacgccgcc gccggcgcgc 3120tcttctacta cctcttcggc
ttcgcttcgc gtcggacgcc gtccaagggc ttcatcggga 3180agcagttctt
cgggctgaag cacatgccgc agacagggta cgactacgac ttcttcctct
3240tccagtgggc cttcgccatc gccgccgccg gcatcacgtc cggttccatc
gccgaacgga 3300cgcgcttcag cgcgtatctc atctactccg ccttcctcac
cgggttcgtg tacccggtgg 3360tgtcgcactg gttctggtcc accgacgggt
gggcttcggc cggccggctg acgggtccgt 3420tgctgttcaa gtcgggcgtc
atcgacttcg ccggctccgg cgtcgtccat ctggtcggtg 3480gcattgctgg
cctgtggggt gccttcatcg agggcccccg catcgggcgc ttcgacgccg
3540ccggccgcac ggtggcgatg aaagggcaca gcgcctcact ggtcgtgctc
ggcaccttcc 3600tgctgtggtt cgggtggttc ggcttcaacc cggggtcctt
caccaccatc tccaagatct 3660acggcgagtc gggcacgatc gacgggcagt
ggtcggcggt gggccgcacc gccgtgacga 3720cgtcgctggc gggcagcgtc
gccgcgctta accacgctgt acggcaagag atggctgacg 3780gggcactgga
acgtgaccga cgtctgcaac ggtctcctcg gcgggttcgc gcgatcaccg
3840cgggctgctc cgtggtcgac ccgtgggcgt cggtgatctg cgggttcgtg
tcggcgtggg 3900tcctcatcgg ctgcaacaag ctggcgctga tgctcaagtt
cgatgacccg ctggaggcga 3960cgcagctgca cggcgggtgc ggcgcgtggg
ggatcatctt caccgcgctg ttcgcgcgca 4020aggagtacgt cgagctgatc
tacggggtgc cggggaggcc gtacgggctg ttcatgggcg 4080gcggcgggag
gcttctcgcg gcgcacatcg tgcagatcct ggtgatcgtc gggtgggtca
4140gcgccaccat ggggacgctc ttctacgtgc tgcacaggtt cgggctgctc
cgcgtctcga 4200cctcgacaga gatggaaggc atggacccgt cgtgccacgg
cgggttcggg tacgtggacg 4260aggacgaagg ccagcgccgc gtcagggcca
agtcggcggc ggagacggct cgcgtggagc 4320ccagaaagtc gccggagcaa
gccgcggcgg gccagttggt gtagtaggat catatcgatc 4380gtgtccgttc
ggggaaagtg ttttgtgaag tgtgcatata taagctgagg cagtcagtcg
4440tgtgggcgtg gtggcacttc agcccatggt ggttgtggct ttcttttgat
atttgcttcc 4500tttcttctct gcatttgcat ctgtatggat ttttgtggct
ttcaatcttt tatgcttttc 4560tttaggtatt cagtctttta tgctttcttg
tacatgttta gacgtgtcca gtttgtatca 4620gtatttaggt cattatgatg
ttaacgtgga gctc 465432497PRTOryza sativa 32Met Ala Thr Cys Leu Asp
Ser Leu Gly Pro Leu Leu Gly Gly Ala Ala1 5 10 15Asn Ser Thr Asp Ala
Ala Asn Tyr Ile Cys Asn Arg Phe Thr Asp Thr20 25 30Ser Ser Ala Val
Asp Ala Thr Tyr Leu Leu Phe Ser Ala Tyr Leu Val35 40 45Phe Ala Met
Gln Leu Gly Phe Ala Met Leu Cys Ala Gly Ser Val Arg50 55 60Ala Lys
Asn Ser Met Asn Ile Met Leu Thr Asn Val Phe Asp Ala Ala65 70 75
80Ala Gly Ala Leu Phe Tyr Tyr Leu Phe Gly Phe Ala Ser Arg Arg Thr85
90 95Pro Ser Lys Gly Phe Ile Gly Lys Gln Phe Phe Gly Leu Lys His
Met100 105 110Pro Gln Thr Gly Tyr Asp Tyr Asp Phe Phe Leu Phe Gln
Trp Ala Phe115 120 125Ala Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser
Ile Ala Glu Arg Thr130 135 140Arg Phe Ser Ala Tyr Leu Ile Tyr Ser
Ala Phe Leu Thr Gly Phe Val145 150 155 160Tyr Pro Val Val Ser His
Trp Phe Trp Ser Thr Asp Gly Trp Ala Ser165 170 175Ala Gly Arg Leu
Thr Gly Pro Leu Leu Phe Lys Ser Gly Val Ile Asp180 185 190Phe Ala
Gly Ser Gly Val Val His Leu Val Gly Gly Ile Ala Gly Leu195 200
205Trp Gly Ala Phe Ile Glu Gly Pro Arg Ile Gly Arg Phe Asp Ala
Ala210 215 220Gly Arg Thr Val Ala Met Lys Gly His Ser Ala Ser Leu
Val Val Leu225 230 235 240Gly Thr Phe Leu Leu Trp Phe Gly Trp Phe
Gly Phe Asn Pro Gly Ser245 250 255Phe Thr Thr Ile Ser Lys Ile Tyr
Gly Glu Ser Gly Thr Ile Asp Gly260 265 270Gln Trp Ser Ala Val Gly
Arg Thr Ala Val Thr Thr Ser Leu Ala Gly275 280 285Ser Val Ala Ala
Leu Asn His Ala Val Arg Gln Glu Met Ala Asp Gly290 295 300Ala Leu
Glu Arg Asp Arg Arg Leu Gln Arg Ser Pro Arg Arg Val Arg305 310 315
320Ala Ile Thr Ala Gly Cys Ser Val Val Asp Pro Trp Ala Ser Val
Ile325 330 335Cys Gly Phe Val Ser Ala Trp Val Leu Ile Gly Cys Asn
Lys Leu Ala340 345 350Leu Met Leu Lys Phe Asp Asp Pro Leu Glu Ala
Thr Gln Leu His Gly355 360 365Gly Cys Gly Ala Trp Gly Ile Ile Phe
Thr Ala Leu Phe Ala Arg Lys370 375 380Glu Tyr Val Glu Leu Ile Tyr
Gly Val Pro Gly Arg Pro Tyr Gly Leu385 390 395 400Phe Met Gly Gly
Gly Gly Arg Leu Leu Ala Ala His Ile Val Gln Ile405 410 415Leu Val
Ile Val Gly Trp Val Ser Ala Thr Met Gly Thr Leu Phe Tyr420 425
430Val Leu His Arg Phe Gly Leu Leu Arg Val Ser Thr Ser Thr Glu
Met435 440 445Glu Gly Met Asp Pro Ser Cys His Gly Gly Phe Gly Tyr
Val Asp Glu450 455 460Asp Glu Gly Gln Arg Arg Val Arg Ala Lys Ser
Ala Ala Glu Thr Ala465 470 475 480Arg Val Glu Pro Arg Lys Ser Pro
Glu Gln Ala Ala Ala Gly Gln Leu485 490 495Val332987DNAOryza
sativamisc_feature212n = A,T,C or G 33attatctcca atgatttcat
agctaatcca tatgctggaa gggttaggaa ttcaagccat 60ttcaaattcc aaaaaattac
ctatactaaa gtaaaaaaaa acctatgacc taccctcaat 120gtttgttaac
caatttaggc cttgtttgat tccacttaga attattataa tcctgattat
180tattaggagt aagctgaaac aaacagataa cntattatga tagattatta
taatctataa 240gccagattac tataatctgg taatccactc taaaggtgct
ttttttaatt attggatagc 300taataactag caaacagcta ataatccaga
taaacaaaca gctaacaact tattctatat 360cggcttatta taatcttatt
ataatccaat ttatagtaat ctagctcaat aatatatatt 420ataataatct
taaactgaaa caaacagggc tttagaaatt catatgtttt gaaatggaga
480tagtaccact cagaaagctt gaaggatttc atgtgttttg gttaacatat
tcatgtgtgt 540cttttcgtgc aaccaaaatt ttctttagaa acatggtgaa
ccaattagat ttagaaatta 600taaaatattt ccaagtgtta caagtggaaa
tataataaaa ataatattgt taaaaaagta 660aagaaagttt aagtacaaac
tgaggaggaa atataacaag tgcttcacta tagacaaata 720tagaggtgga
cgaaatgtac aaacagtcgt ttttaaaaat acaaaccacc gtattgcgac
780tcaggccttg tttagatccc aaaaaatttt agccaaaaat atcacatcaa
atgtttggac 840acatgcatag gatattaaat atggggaaaa aaaatcaatt
acacagtttg caggtaaatt 900gcgagacaaa tctttttagt ctaattacgt
catgatttga ccatgtgatg ctatagtaaa 960catttactaa tgacagattg
attaggctta ataaattcat ctcgcaattt acaaacagaa 1020tctataattt
attttattat tagtctatat ttaatatttt aaatatatat ccgtgtagtt
1080caaaaacttt atatcaaaag aactaaacac agcctccagg ccgcagccta
cagtaggcct 1140atagagagat tccacgggat tcgatgaact acgaccacga
acaggagggg gacaaatcaa 1200caagcaaatc ataggggtcg cacatttcag
aggtagccaa agattcactg gcaggtgggc 1260ccttcacact ttgaaggaat
caacaacgac accccccaag tcatggattc cttctcgctc 1320cctctccacg
tcgcctataa atccgacgcg ggccgctccc cactccaccc acagcccaca
1380cttccattgc tcctcccctc tcctctacag tctgtgttga gcgcgcgtcg
agcggcgagg 1440atggcaacgt gcgcggatac cctcggcccg ctgctgggca
cggcggcggc gaacgcgacg 1500gactacctgt gcaaccagtt cgcggacacc
acgtcggccg tggactcgac gtacctgctc 1560ttctcggcgt acctcgtgtt
cgccatgcag ctcggcttcg ccatgctctg cgccgggtcc 1620gtccgcgcca
agaacaccat gaacatcatg cttaccaacg tgctcgacgc cgccgccggc
1680gcgctcttct actacctctt cggcttcgcc ttcgccttcg gggcgccgtc
caacggcttc 1740atcgggaagc acttcttcgg cctcaagcag gtcccacagg
tcggcttcga ctacagcttc 1800ttcctcttcc agtgggcctt cgccatcgcc
gccgcgggca tcacgtccgg ctccatcgcc 1860gagcggaccc agttcgtggc
gtacctcatc tactccgcct tcctcaccgg cttcgtctac 1920ccggtggtgt
cccactggat ctggtccgcc gacgggtggg cctcggcttc ccgaacgtcg
1980gggtcgctgc tcttcgggtc cggcgtcatc gacttcgccg ggtcaggggt
tgtccacatg 2040gtggcggcgt gccggactct ggggcgccct catcgagggc
ccccgcattg gcggttcgac 2100cacgccggcc gctcggtggc gctgcgcggc
cacagcgcgt cgctcgtcgt gctcggcagc 2160ttcctgctgt ggttcgggtg
gtacgggttt aaccccggct cgttcctcac catcctcaaa 2220tcctacggcc
cgcccggtag catccacggg cagtggtcgg cggtgggacg caccgccgtg
2280accaccaccc tcgccggcag cacggcggcg ctcacgacgc tcttcgggaa
gaggctccag 2340acggggcact ggaacgtgat cgacgtctgc aacggcctcc
tcggcggctt cgcggcgatc 2400accgccggtt gctccgtcgt cgacccgtgg
gccgcgatca tctgcgggtt cgtctcggcg 2460tgggtgctca tcggcctcaa
cgcgctggcg gcgaggctca agttcgacga cccgctcgag 2520gcggcgcagc
tgcacggcgg gtgcggcgcg tggggggtca tcttcacggc gctgttcgcg
2580cgcaaggagt acgtggacca gatcttcggc cagcccgggc gcccgtatgg
gctgttcatg 2640ggcggcggcg gccggctgct cggggcgcac atagtggtaa
tcctggtcat cgcggcgtgg 2700gtgagcttca ccatggcgcc gctgttcctg
gtgctcaaca agctgggatt gctgcgcatc 2760tcggccgagg acgagatggc
cggcatggac cagacgcgcc acggcgggtt cgcgtacgcg 2820taccacgacg
acgacgcgag cggcaagccg gaccgcagct tcggcgggtt catgctcaag
2880tcggcgcacg gcacgcaggt cgccgccgag atgggaggcc atgtctagtg
gaaccggagg 2940agctgagcta gtagtacata catgcagcat catcgatcct cgagctc
298734495PRTOryza sativa 34Met Ala Thr Cys Ala Asp Thr Leu Gly Pro
Leu Leu Gly Thr Ala Ala1 5 10 15Ala Asn Ala Thr Asp Tyr Leu Cys Asn
Gln Phe Ala Asp Thr Thr Ser20 25 30Ala Val Asp Ser Thr Tyr Leu Leu
Phe Ser Ala Tyr Leu Val Phe Ala35 40 45Met Gln Leu Gly Phe Ala Met
Leu Cys Ala Gly Ser Val Arg Ala Lys50 55 60Asn Thr Met Asn Ile Met
Leu Thr Asn Val Leu Asp Ala Ala Ala Gly65 70 75 80Ala Leu Phe Tyr
Tyr Leu Phe Gly Phe Ala Phe Ala Phe Gly Ala Pro85 90 95Ser Asn Gly
Phe Ile Gly Lys His Phe Phe Gly Leu Lys Gln Val Pro100 105 110Gln
Val Gly Phe Asp Tyr Ser Phe Phe Leu Phe Gln Trp Ala Phe Ala115 120
125Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr
Gln130 135 140Phe Val Ala Tyr Leu Ile Tyr Ser Ala Phe Leu Thr Gly
Phe Val Tyr145 150 155 160Pro Val Val Ser His Trp Ile Trp Ser Ala
Asp Gly Trp Ala Ser Ala165 170 175Ser Arg Thr Ser Gly Ser Leu Leu
Phe Gly Ser Gly Val Ile Asp Phe180 185 190Ala Gly Ser Gly Val Val
His Met Val Ala Ala Cys Arg Thr Leu Gly195 200 205Arg Pro His Arg
Gly Pro Pro His Trp Arg Phe Asp His Ala Gly Arg210 215 220Ser Val
Ala Leu Arg Gly His Ser Ala Ser Leu Val Val Leu Gly Ser225 230 235
240Phe Leu Leu Trp Phe Gly Trp Tyr Gly Phe Asn Pro Gly Ser Phe
Leu245 250 255Thr Ile Leu Lys Ser Tyr Gly Pro Pro Gly Ser Ile His
Gly Gln Trp260 265 270Ser Ala Val Gly Arg Thr Ala Val Thr Thr Thr
Leu Ala Gly Ser Thr275 280 285Ala Ala Leu Thr Thr Leu Phe Gly Lys
Arg Leu Gln Thr Gly His Trp290 295 300Asn Val Ile Asp Val Cys Asn
Gly Leu Leu Gly Gly Phe Ala Ala Ile305 310 315 320Thr Ala Gly Cys
Ser Val Val Asp Pro Trp Ala Ala Ile Ile Cys Gly325 330 335Phe Val
Ser Ala Trp Val Leu Ile Gly Leu Asn Ala Leu Ala Ala Arg340 345
350Leu Lys Phe Asp Asp Pro Leu Glu Ala Ala Gln Leu His Gly Gly
Cys355 360 365Gly Ala Trp Gly Val Ile Phe Thr Ala Leu Phe Ala Arg
Lys Glu Tyr370 375 380Val Asp Gln Ile Phe Gly Gln Pro Gly Arg Pro
Tyr Gly Leu Phe Met385 390 395 400Gly Gly Gly Gly Arg Leu Leu Gly
Ala His Ile Val Val Ile Leu Val405 410 415Ile Ala Ala Trp Val Ser
Phe Thr Met Ala Pro Leu Phe Leu Val Leu420 425 430Asn Lys Leu Gly
Leu Leu Arg Ile Ser Ala Glu Asp Glu Met Ala Gly435 440 445Met Asp
Gln Thr Arg His Gly Gly Phe Ala Tyr Ala Tyr His Asp Asp450 455
460Asp Ala Ser Gly Lys Pro Asp Arg Ser Phe Gly Gly Phe Met Leu
Lys465 470 475 480Ser Ala His Gly Thr Gln Val Ala Ala Glu Met Gly
Gly His Val485 490
49535900DNAOryza sativa 35atggcggcgg aggcggcgcc ggagtgggtg
gagaaggggg acaacgcgtg gccgctagcg 60gcggcgacgc tggtggggct gcagagcgtg
ccgaggctgg tgatcctgta cggcgactgc 120ggcgcggtcg gtccgaggac
ggagaaggac agggaggcgt tcccgccgaa caacgtcctg 180ctcacgctcg
ccggagcggg gctgctgctg tggatggggt ggacggggtt caacggcggc
240gcgccgtacg ccgccaacgt cgacgcgtcg gtcaccgtcg tgaacacgca
cctctgcacg 300gcgacgagcc tcctggtgtg gctcctcctc gacagcttcg
tcttcggccg cctctccgtc 360atcagcgccg tgcagggcat gatcaccggc
ctcgtctgcg tcaccccggc ggccaggctg 420gtgctgcaca agcggagccg
cctcctggcg cgcgtcgacg acacgctcgc cgtgctccac 480acccacggcg
tcgccggcag cctcagcggc gtcctgacgg ggctcctgct cctcgccgag
540ccgcgcttcg ccaggctctt cttcggcgac gacccgcgct acgtcggcct
cgcgtacgct 600gtcagggacg gccgcgccgg ctcggggctc cggcaggtcg
gcgtgcagct ggccgggatc 660gcgttcgtgg tggcgctcaa cgtcgccgtg
acgagcgccg tgtgcctggc cgtcagggtg 720gccgtgccgc agctcgccgg
cggcggcgac gccatacacg gcgaggacgc gtacgcggtg 780tggggcgacg
gcgagacgta cgagcagtac tccgtgcacg gcggcggcag caaccacggc
840ggcttcccca tgacggccaa tcccgtggcg tccaaagccg acgagatgat
atggatataa 90036299PRTOryza sativa 36Met Ala Ala Glu Ala Ala Pro
Glu Trp Val Glu Lys Gly Asp Asn Ala1 5 10 15Trp Pro Leu Ala Ala Ala
Thr Leu Val Gly Leu Gln Ser Val Pro Arg20 25 30Leu Val Ile Leu Tyr
Gly Asp Cys Gly Ala Val Gly Pro Arg Thr Glu35 40 45Lys Asp Arg Glu
Ala Phe Pro Pro Asn Asn Val Leu Leu Thr Leu Ala50 55 60Gly Ala Gly
Leu Leu Leu Trp Met Gly Trp Thr Gly Phe Asn Gly Gly65 70 75 80Ala
Pro Tyr Ala Ala Asn Val Asp Ala Ser Val Thr Val Val Asn Thr85 90
95His Leu Cys Thr Ala Thr Ser Leu Leu Val Trp Leu Leu Leu Asp
Ser100 105 110Phe Val Phe Gly Arg Leu Ser Val Ile Ser Ala Val Gln
Gly Met Ile115 120 125Thr Gly Leu Val Cys Val Thr Pro Ala Ala Arg
Leu Val Leu His Lys130 135 140Arg Ser Arg Leu Leu Ala Arg Val Asp
Asp Thr Leu Ala Val Leu His145 150 155 160Thr His Gly Val Ala Gly
Ser Leu Ser Gly Val Leu Thr Gly Leu Leu165 170 175Leu Leu Ala Glu
Pro Arg Phe Ala Arg Leu Phe Phe Gly Asp Asp Pro180 185 190Arg Tyr
Val Gly Leu Ala Tyr Ala Val Arg Asp Gly Arg Ala Gly Ser195 200
205Gly Leu Arg Gln Val Gly Val Gln Leu Ala Gly Ile Ala Phe Val
Val210 215 220Ala Leu Asn Val Ala Val Thr Ser Ala Val Cys Leu Ala
Val Arg Val225 230 235 240Ala Val Pro Gln Leu Ala Gly Gly Gly Asp
Ala Ile His Gly Glu Asp245 250 255Ala Tyr Ala Val Trp Gly Asp Gly
Glu Thr Tyr Glu Gln Tyr Ser Val260 265 270His Gly Gly Gly Ser Asn
His Gly Gly Phe Pro Met Thr Ala Asn Pro275 280 285Val Ala Ser Lys
Ala Asp Glu Met Ile Trp Ile290 295372040DNAOryza sativa
37ggaggctttg gctaccctgc tcccctcgcc atttcattgg ccgtttcgtg gccatccatc
60acgaactcga tcgattcccc tcttcgagcc cgtaccaatt attagctagt ttaactcgta
120cgatgaatca cgccgaaaca caatataaat ggtggagtcg gctcgctgtc
aaacgcgcgg 180gagctcgcgc cacttgtaat ttttcgcgtc tcctctcgtc
cggcacagca caggagcgcg 240gacttgaaga cctcaagtag cgattcgtcc
gtgcggcgcg gcgcaagaag ggaagggaag 300gggactaggg gagggcgaga
tggcggcggc gggggcgtac tcggcgagcc taccggcggt 360gccggactgg
ctgaacaagg gggacaacgc gtggcagctg acggcgtcga cgctggtggg
420gatccagtcg atgcccgggc tggtggtgct gtacggcagc atcgtgaaga
agaagtgggc 480ggtgaactcg gcgttcatgg cgctctacgc ctacgcgtcg
tcgctgctgg tgtgggtgct 540ggtcggcttc cgcatggcgt tcggcgacca
gctgctgccg ttctggggca aggccggcgt 600ggcgctgacc cagagctacc
tcgtcggccg cgccacgctg ccggccaccg cgcacggcgc 660catcccgcgc
accgagccct tctacccgga ggccacgctg gtgctcttcc agttcgagtt
720cgccgccatc acgctcgtcc tcctcgccgg ctccgtcctc ggccgcatga
acatcaaggc 780ctggatggcc ttcaccccgc tctggctcct cctctcctac
accgtcggcg ccttcagcct 840ctggggcggc ggcttcctct accgctgggg
cgtcatcgac tactccggcg gctacgtcat 900ccacctctcc tccggcatcg
ccggcttcac cgccgcctac tgggtggggc caaggctgaa 960gagcgaccgt
gagcggttct caccgaacaa catcctgctg atgatcgcgg gcggcgggct
1020gctgtggatg gggtgggccg ggttcaacgg cggcgcgccg tacgccgcca
acatcgcggc 1080gtcggtcgcc gtgctcaaca ccaacgtctg cgccgccacc
agcctcctca tgtggacctg 1140cctcgacgtc atcttcttcc gcaagccgtc
cgtcatcggc gccgtgcagg gcatgatgac 1200cggcctcgtc tgcatcaccc
ccggcgcagg gctggtgcag acctgggcgg ccgtggtaat 1260gggcatcttc
gccggcagcg tgccgtggtt caccatgatg atcctgcaca agaagtcagc
1320gctgctgatg aaggtggacg acacgctcgc cgtgttccac acccacgccg
tggcggggct 1380cctcggcggc atcctcacgg gcctcctggc caccccggag
ctcttctccc tcgagtccac 1440ggtgccggga ctccgcggcg cgttctacgg
cggcggtatc aagcagatcg gcaagcagct 1500cggcggcgcc gcgttcgtga
tcgcgtggaa cctcgtggtc accacggcca tcctcctcgg 1560catcggcctg
ttcatcccgc tgcggatgcc cgacgagcag ctcatgatcg gcgacgacgc
1620ggcgcacggc gaggaggcct acgcgctgtg gggcgacggc gagaagttcg
acgcgacgcg 1680gcacgacctg tcgaggggcg gcggaggcgg cgacagggac
ggccccgccg gcgagcgcct 1740ctccgcccta ggcgccaggg gcgtcaccat
ccagctctag gcgcgccacg ccacgccacg 1800ccgcgccgcg ccgcggcctg
gcctctaatt acacgcgcgt ttgtactgtt tttggacgtg 1860ttattgttta
ggagtagtga agtgaaccaa cgattgactg caaggtgaag ggtgagaacg
1920cgagagacca gaccactata gtctatagta catatggatg ctgtaatgat
gttgatccga 1980gttcgttttt ccaacacgat aaaggccgac atgcctatta
aatttaaaaa aaaaaaaaaa 204038486PRTOryza sativa 38Met Ala Ala Ala
Gly Ala Tyr Ser Ala Ser Leu Pro Ala Val Pro Asp1 5 10 15Trp Leu Asn
Lys Gly Asp Asn Ala Trp Gln Leu Thr Ala Ser Thr Leu20 25 30Val Gly
Ile Gln Ser Met Pro Gly Leu Val Val Leu Tyr Gly Ser Ile35 40 45Val
Lys Lys Lys Trp Ala Val Asn Ser Ala Phe Met Ala Leu Tyr Ala50 55
60Tyr Ala Ser Ser Leu Leu Val Trp Val Leu Val Gly Phe Arg Met Ala65
70 75 80Phe Gly Asp Gln Leu Leu Pro Phe Trp Gly Lys Ala Gly Val Ala
Leu85 90 95Thr Gln Ser Tyr Leu Val Gly Arg Ala Thr Leu Pro Ala Thr
Ala His100 105 110Gly Ala Ile Pro Arg Thr Glu Pro Phe Tyr Pro Glu
Ala Thr Leu Val115 120 125Leu Phe Gln Phe Glu Phe Ala Ala Ile Thr
Leu Val Leu Leu Ala Gly130 135 140Ser Val Leu Gly Arg Met Asn Ile
Lys Ala Trp Met Ala Phe Thr Pro145 150 155 160Leu Trp Leu Leu Leu
Ser Tyr Thr Val Gly Ala Phe Ser Leu Trp Gly165 170 175Gly Gly Phe
Leu Tyr Arg Trp Gly Val Ile Asp Tyr Ser Gly Gly Tyr180 185 190Val
Ile His Leu Ser Ser Gly Ile Ala Gly Phe Thr Ala Ala Tyr Trp195 200
205Val Gly Pro Arg Leu Lys Ser Asp Arg Glu Arg Phe Ser Pro Asn
Asn210 215 220Ile Leu Leu Met Ile Ala Gly Gly Gly Leu Leu Trp Met
Gly Trp Ala225 230 235 240Gly Phe Asn Gly Gly Ala Pro Tyr Ala Ala
Asn Ile Ala Ala Ser Val245 250 255Ala Val Leu Asn Thr Asn Val Cys
Ala Ala Thr Ser Leu Leu Met Trp260 265 270Thr Cys Leu Asp Val Ile
Phe Phe Arg Lys Pro Ser Val Ile Gly Ala275 280 285Val Gln Gly Met
Met Thr Gly Leu Val Cys Ile Thr Pro Gly Ala Gly290 295 300Leu Val
Gln Thr Trp Ala Ala Val Val Met Gly Ile Phe Ala Gly Ser305 310 315
320Val Pro Trp Phe Thr Met Met Ile Leu His Lys Lys Ser Ala Leu
Leu325 330 335Met Lys Val Asp Asp Thr Leu Ala Val Phe His Thr His
Ala Val Ala340 345 350Gly Leu Leu Gly Gly Ile Leu Thr Gly Leu Leu
Ala Thr Pro Glu Leu355 360 365Phe Ser Leu Glu Ser Thr Val Pro Gly
Leu Arg Gly Ala Phe Tyr Gly370 375 380Gly Gly Ile Lys Gln Ile Gly
Lys Gln Leu Gly Gly Ala Ala Phe Val385 390 395 400Ile Ala Trp Asn
Leu Val Val Thr Thr Ala Ile Leu Leu Gly Ile Gly405 410 415Leu Phe
Ile Pro Leu Arg Met Pro Asp Glu Gln Leu Met Ile Gly Asp420 425
430Asp Ala Ala His Gly Glu Glu Ala Tyr Ala Leu Trp Gly Asp Gly
Glu435 440 445Lys Phe Asp Ala Thr Arg His Asp Leu Ser Arg Gly Gly
Gly Gly Gly450 455 460Asp Arg Asp Gly Pro Ala Gly Glu Arg Leu Ser
Ala Leu Gly Ala Arg465 470 475 480Gly Val Thr Ile Gln
Leu485391494DNAOryza sativa 39atggcgtcgc cgacccggcc ggggccgtac
atgccgcgcc caccggcggt gccggagtgg 60ctgaacaccg gggacaacgg gtggcagctc
gcggcggcga cgttcgtcgg gctccagtcg 120atgcctgggc tggtggtgct
gtacggcagc atcgtgaaga agaagtgggc cgtcaactcg 180gccttcatgg
cgctgtacgc gtacgcgtcc acgctcatcg tgtgggtgct ggtcggcttc
240cgcatggcgt tcggcgaccg gctgctcccg ttctggggga aggccggcgc
ggcgctgacg 300gaggggttcc tcgtggcgcg cgcgtcggtc ccggccacgg
cgcactacgg gaaggacggc 360gccctggagt cgccgcgcac cgagccgttc
tacccggagg cgtccatggt gctgttccag 420ttcgagctcg ccgccatcac
gctggtgctg ctcgccgggt cgctcctcgg gaggatgaac 480atcaaggcgt
ggatggcgtt cactccgctc tggctcctct tctcctacac cgtctgcgcc
540ttcagcctct ggggcggcgg cttcctctac cagtggggcg tcatcgacta
ctccggcgga 600tacgtcatcc acctctcctc cggcatcgcc ggcttcaccg
ccgcctactg ggtggggccg 660aggctgaaga gcgacaggga gcggttctcg
ccgaacaaca tcctcctcat gatcgccggc 720ggcgggctgc tgtggctggg
ctgggccggg ttcaacggcg gcgcgccgta cgccccaaac 780atcaccgcgt
ccatcgccgt gctcaacacc aacgtcagcg ccgcggcgag cctcctcacc
840tggacctgcc tcgacgtcat cttcttcggc aagccctccg tcatcggcgc
cgtgcagggc 900atgatgaccg gtctcgtctg catcaccccc ggcgcaggtc
tggtgcacac gtgggcggcc 960atactgatgg gcatctgtgg cggcagcttg
ccgtggttct ccatgatgat cctccacaag 1020agatcggcgc tgctccagaa
ggtggacgac accctcgccg tcttccacac ccacgccgtc 1080gcgggcctcc
tcggcggctt cctcacgggc ctgttcgcct tgccggacct caccgccgtc
1140cacacccaca tccctggcgc gcgcggcgcg ttctacggcg gcggcatcgc
ccaggtgggg 1200aagcagatcg ccggcgcgct cttcgtcgtc gtgtggaacg
tcgtggccac caccgtcatc 1260ctgctcggcg tcggcctcgt cgtcccgctc
cgcatgcccg acgagcagct caagatcggc 1320gacgacgcgg cgcacgggga
ggaggcctac gcgctatggg gagacggcga gaggttcgac 1380gtgacgcgcc
atgagggggc gaggggcggc gcgtggggcg ccgcggtcgt ggacgaggcg
1440atggatcacc ggctggccgg aatgggagcg agaggagtca cgattcagct gtag
149440497PRTOryza sativa 40Met Ala Ser Pro Thr Arg Pro Gly Pro Tyr
Met Pro Arg Pro Pro Ala1 5 10 15Val Pro Glu Trp Leu Asn Thr Gly Asp
Asn Gly Trp Gln Leu Ala Ala20 25 30Ala Thr Phe Val Gly Leu Gln Ser
Met Pro Gly Leu Val Val Leu Tyr35 40 45Gly Ser Ile Val Lys Lys Lys
Trp Ala Val Asn Ser Ala Phe Met Ala50 55 60Leu Tyr Ala Tyr Ala Ser
Thr Leu Ile Val Trp Val Leu Val Gly Phe65 70 75 80Arg Met Ala Phe
Gly Asp Arg Leu Leu Pro Phe Trp Gly Lys Ala Gly85 90 95Ala Ala Leu
Thr Glu Gly Phe Leu Val Ala Arg Ala Ser Val Pro Ala100 105 110Thr
Ala His Tyr Gly Lys Asp Gly Ala Leu Glu Ser Pro Arg Thr Glu115 120
125Pro Phe Tyr Pro Glu Ala Ser Met Val Leu Phe Gln Phe Glu Leu
Ala130 135 140Ala Ile Thr Leu Val Leu Leu Ala Gly Ser Leu Leu Gly
Arg Met Asn145 150 155 160Ile Lys Ala Trp Met Ala Phe Thr Pro Leu
Trp Leu Leu Phe Ser Tyr165 170 175Thr Val Cys Ala Phe Ser Leu Trp
Gly Gly Gly Phe Leu Tyr Gln Trp180 185 190Gly Val Ile Asp Tyr Ser
Gly Gly Tyr Val Ile His Leu Ser Ser Gly195 200 205Ile Ala Gly Phe
Thr Ala Ala Tyr Trp Val Gly Pro Arg Leu Lys Ser210 215 220Asp Arg
Glu Arg Phe Ser Pro Asn Asn Ile Leu Leu Met Ile Ala Gly225 230 235
240Gly Gly Leu Leu Trp Leu Gly Trp Ala Gly Phe Asn Gly Gly Ala
Pro245 250 255Tyr Ala Pro Asn Ile Thr Ala Ser Ile Ala Val Leu Asn
Thr Asn Val260 265 270Ser Ala Ala Ala Ser Leu Leu Thr Trp Thr Cys
Leu Asp Val Ile Phe275 280 285Phe Gly Lys Pro Ser Val Ile Gly Ala
Val Gln Gly Met Met Thr Gly290 295 300Leu Val Cys Ile Thr Pro Gly
Ala Gly Leu Val His Thr Trp Ala Ala305 310 315 320Ile Leu Met Gly
Ile Cys Gly Gly Ser Leu Pro Trp Phe Ser Met Met325 330 335Ile Leu
His Lys Arg Ser Ala Leu Leu Gln Lys Val Asp Asp Thr Leu340 345
350Ala Val Phe His Thr His Ala Val Ala Gly Leu Leu Gly Gly Phe
Leu355 360 365Thr Gly Leu Phe Ala Leu Pro Asp Leu Thr Ala Val His
Thr His Ile370 375 380Pro Gly Ala Arg Gly Ala Phe Tyr Gly Gly Gly
Ile Ala Gln Val Gly385 390 395 400Lys Gln Ile Ala Gly Ala Leu Phe
Val Val Val Trp Asn Val Val Ala405 410 415Thr Thr Val Ile Leu Leu
Gly Val Gly Leu Val Val Pro Leu Arg Met420 425 430Pro Asp Glu Gln
Leu Lys Ile Gly Asp Asp Ala Ala His Gly Glu Glu435 440 445Ala Tyr
Ala Leu Trp Gly Asp Gly Glu Arg Phe Asp Val Thr Arg His450 455
460Glu Gly Ala Arg Gly Gly Ala Trp Gly Ala Ala Val Val Asp Glu
Ala465 470 475 480Met Asp His Arg Leu Ala Gly Met Gly Ala Arg Gly
Val Thr Ile Gln485 490 495Leu411494DNAOryza sativa 41atggcgtcgc
cgccgcagcc cgggccgtac atgccggacc tgccggcggt gccggcgtgg 60ctgaacaagg
gcgacaccgc gtggcagctg gtggcggcga cgttcgtcgg catccagtcg
120atgcctgggc tggtggtgat ctacggcagc atcgtgaaga agaagtgggc
cgtcaactcc 180gccttcatgg cgctgtacgc ctacgcgtcc acgcttatcg
tgtgggtgct cgtcggcttc 240cgcatggcgt tcggcgaccg gctgctcccg
ttctgggcca aggccgggcc ggcgctgacg 300caggacttcc tggtgcaacg
cgcggtgttc ccggcgacgg cgcactacgg cagcgacggc 360acgctcgaga
cgccgcgcac cgagccgttc tacgcggagg cggcgctggt gctgttcgag
420ttcgagttcg cggccatcac gctggtgctg ctcgccgggt cgctcctggg
gcggatgaac 480atcaaggcgt ggatggcgtt caccccgctc tggctcctct
tctcctacac cgtcggcgcg 540ttcagcctct ggggcggcgg cttcctctac
cagtggggcg tcatcgacta ctccggcgga 600tacgtcatcc acctctcctc
cggcgtcgcc ggcttcaccg ccgcctactg ggtgggcccg 660aggctgaaga
gcgacaggga gcggttctcg ccgaacaaca tcctgctcat gatcgccggc
720ggcgggctgc tgtggttggg ctgggccggg ttcaacggcg gcgcgccgta
cgcccccaac 780gtcaccgcca cggtcgccgt gctcaacacc aacgtcagcg
ccgcgacgag cctcctcacc 840tggacctgcc tcgacgtcat cttcttcggc
aagccctccg tcatcggcgc cgtgcagggt 900atgatgacgg ggctcgtctg
catcacgccc ggcgccgggc tggtgcacac gtggtcagcg 960atgctgatgg
gcatgttcgc cggcagcgtc ccgtggttca cgatgatgat cctgcacaag
1020aaatccacct tcctcatgaa ggtcgacgac accctcgccg tcttccacac
ccacgccgtc 1080gccggcatcc tgggcggcgt cctcacgggc ctcctcgcca
cgccggagct ctgcgctctc 1140gattgcccga tcccgaacat gcgcggcgtc
ttctacggca gcggcatcgg ccagctcggg 1200aagcagctcg gcggcgcgct
gttcgtcacc gtctggaacc tcatcgtcac cagcgccatt 1260ctcctctgca
tcggcctctt catcccgctc cgcatgtccg acgaccagct catgatcggc
1320gacgacgcgg cgcacgggga ggaggcctac gctctgtggg gggacggtga
gaagttcgac 1380gtgacgcggc cggagacgac gaggacggga ggtgcaggcg
gcgcgggcag ggaggacacc 1440atggagcaga ggctgaccaa catgggagcc
aggggtgtca ccattcagtt gtag 149442497PRTOryza sativa 42Met Ala Ser
Pro Pro Gln Pro Gly Pro Tyr Met Pro Asp Leu Pro Ala1 5 10 15Val Pro
Ala Trp Leu Asn Lys Gly Asp Thr Ala Trp Gln Leu Val Ala20 25 30Ala
Thr Phe Val Gly Ile Gln Ser Met Pro Gly Leu Val Val Ile Tyr35 40
45Gly Ser Ile Val Lys Lys Lys Trp Ala Val Asn Ser Ala Phe Met Ala50
55 60Leu Tyr Ala Tyr Ala Ser Thr Leu Ile Val Trp Val Leu Val Gly
Phe65 70 75 80Arg Met Ala Phe Gly Asp Arg Leu Leu Pro Phe Trp Ala
Lys Ala Gly85 90 95Pro Ala Leu Thr Gln Asp Phe Leu Val Gln Arg Ala
Val Phe Pro Ala100 105 110Thr Ala His Tyr Gly Ser Asp Gly Thr Leu
Glu Thr Pro Arg Thr Glu115 120 125Pro Phe Tyr Ala Glu Ala Ala Leu
Val Leu Phe Glu Phe Glu Phe Ala130 135 140Ala Ile Thr Leu Val Leu
Leu Ala Gly Ser Leu Leu Gly Arg Met Asn145 150 155 160Ile Lys Ala
Trp Met Ala Phe Thr Pro Leu Trp Leu Leu Phe Ser Tyr165 170 175Thr
Val Gly Ala Phe Ser Leu Trp Gly Gly Gly Phe Leu Tyr Gln Trp180 185
190Gly Val Ile Asp Tyr Ser Gly Gly Tyr Val Ile His Leu Ser Ser
Gly195 200 205Val Ala Gly Phe Thr Ala Ala Tyr Trp Val Gly Pro Arg
Leu Lys Ser210 215 220Asp Arg Glu Arg Phe Ser Pro Asn Asn Ile Leu
Leu Met Ile Ala Gly225 230 235 240Gly Gly Leu Leu Trp Leu Gly Trp
Ala Gly Phe Asn Gly Gly Ala Pro245 250 255Tyr Ala Pro Asn Val Thr
Ala Thr Val Ala Val Leu Asn Thr Asn Val260 265 270Ser Ala Ala Thr
Ser Leu Leu Thr Trp Thr Cys Leu Asp Val Ile Phe275 280 285Phe Gly
Lys Pro Ser Val Ile Gly Ala Val Gln Gly Met Met Thr Gly290
295 300Leu Val Cys Ile Thr Pro Gly Ala Gly Leu Val His Thr Trp Ser
Ala305 310 315 320Met Leu Met Gly Met Phe Ala Gly Ser Val Pro Trp
Phe Thr Met Met325 330 335Ile Leu His Lys Lys Ser Thr Phe Leu Met
Lys Val Asp Asp Thr Leu340 345 350Ala Val Phe His Thr His Ala Val
Ala Gly Ile Leu Gly Gly Val Leu355 360 365Thr Gly Leu Leu Ala Thr
Pro Glu Leu Cys Ala Leu Asp Cys Pro Ile370 375 380Pro Asn Met Arg
Gly Val Phe Tyr Gly Ser Gly Ile Gly Gln Leu Gly385 390 395 400Lys
Gln Leu Gly Gly Ala Leu Phe Val Thr Val Trp Asn Leu Ile Val405 410
415Thr Ser Ala Ile Leu Leu Cys Ile Gly Leu Phe Ile Pro Leu Arg
Met420 425 430Ser Asp Asp Gln Leu Met Ile Gly Asp Asp Ala Ala His
Gly Glu Glu435 440 445Ala Tyr Ala Leu Trp Gly Asp Gly Glu Lys Phe
Asp Val Thr Arg Pro450 455 460Glu Thr Thr Arg Thr Gly Gly Ala Gly
Gly Ala Gly Arg Glu Asp Thr465 470 475 480Met Glu Gln Arg Leu Thr
Asn Met Gly Ala Arg Gly Val Thr Ile Gln485 490 495Leu431440DNAOryza
sativa 43atgtcgtcgt cggcgacggt ggtgccgctg gcgtaccagg ggaacacgtc
ggcgtcggtg 60gcggactggc tgaacaaggg cgacaacgcg tggcagctgg tggcggcgac
ggtggtgggg 120ctgcagagcg tgccgggctt ggtggtgctg tacggcggcg
tggtgaagaa gaagtgggcg 180gtgaactcgg cgttcatggc gctctacgcc
ttcgccgccg tgtggatctg ctgggtcacc 240tgggcgtaca acatgtcgtt
cggggagaag ctcctcccga tctgggggaa ggcgcggccg 300gcgctggacc
agggcctcct cgtcggccgc gccgcgctgc cggcgacggt ccactaccgc
360gccgacggca gcgtggagac ggcggcggtg gagccgctgt acccgatggc
gacggtggtg 420tacttccagt gcgtgttcgc cgccatcacc ctcatcctcg
tcgccggctc cctcctcggc 480cgcatgagct tcctcgcctg gatgatcttc
gtcccgctct ggctcacctt ctcctacacc 540gtcggcgcct tctccctctg
gggcggcggc ttcctcttcc actggggcgt catcgactac 600tgcggcggct
acgtcatcca cgtctccgcc ggcatcgccg gcttcaccgc cgcctactgg
660gtggggccaa gggcacagaa ggacagggag aggttcccgc cgaacaacat
actgttcacg 720ctgacggggg cagggttact atggatgggg tgggcagggt
tcaacggcgg tggtccgtac 780gccgccaact ccgtcgcctc catggccgtc
ctcaacacca acatctgcac cgccatgagc 840ctcatcgtct ggacatgcct
cgacgtcatc ttcttcaaga agccctccgt cgtcggcgcc 900gtccagggca
tgatcaccgg cctcgtctgc atcacccccg ctgcaggggt ggtgcagggg
960tgggcggcgc tggtgatggg ggtgctcgcc ggcagcatcc cgtggtacac
catgatgatc 1020ctccacaagc gctccaagat cctgcagcgc gtcgacgaca
ccctcggcgt cttccacacc 1080cacggcgtcg ccggcctcct cggcggcctc
ctcaccggcc tcttcgccga gcccaccctc 1140tgcaacctct tcctccccgt
cgccgactcc cggggcgcct tctacggcgg cgccggcggc 1200gcccagttcg
gcaagcagat cgccggtggc ctcttcgtcg tcgcctggaa cgtcgccgtc
1260acctccctca tctgcctcgc catcaacctc ctcgtcccgc tccgcatgcc
cgacgacaag 1320ctcgaggtcg gcgacgacgc cgtccacggc gaggaggcct
acgcgctctg gggcgacggc 1380gagatgtacg acgtcaccaa gcacggctcc
gacgccgccg ttgcgcccgt cgtcgtatga 144044479PRTOryza sativa 44Met Ser
Ser Ser Ala Thr Val Val Pro Leu Ala Tyr Gln Gly Asn Thr1 5 10 15Ser
Ala Ser Val Ala Asp Trp Leu Asn Lys Gly Asp Asn Ala Trp Gln20 25
30Leu Val Ala Ala Thr Val Val Gly Leu Gln Ser Val Pro Gly Leu Val35
40 45Val Leu Tyr Gly Gly Val Val Lys Lys Lys Trp Ala Val Asn Ser
Ala50 55 60Phe Met Ala Leu Tyr Ala Phe Ala Ala Val Trp Ile Cys Trp
Val Thr65 70 75 80Trp Ala Tyr Asn Met Ser Phe Gly Glu Lys Leu Leu
Pro Ile Trp Gly85 90 95Lys Ala Arg Pro Ala Leu Asp Gln Gly Leu Leu
Val Gly Arg Ala Ala100 105 110Leu Pro Ala Thr Val His Tyr Arg Ala
Asp Gly Ser Val Glu Thr Ala115 120 125Ala Val Glu Pro Leu Tyr Pro
Met Ala Thr Val Val Tyr Phe Gln Cys130 135 140Val Phe Ala Ala Ile
Thr Leu Ile Leu Val Ala Gly Ser Leu Leu Gly145 150 155 160Arg Met
Ser Phe Leu Ala Trp Met Ile Phe Val Pro Leu Trp Leu Thr165 170
175Phe Ser Tyr Thr Val Gly Ala Phe Ser Leu Trp Gly Gly Gly Phe
Leu180 185 190Phe His Trp Gly Val Ile Asp Tyr Cys Gly Gly Tyr Val
Ile His Val195 200 205Ser Ala Gly Ile Ala Gly Phe Thr Ala Ala Tyr
Trp Val Gly Pro Arg210 215 220Ala Gln Lys Asp Arg Glu Arg Phe Pro
Pro Asn Asn Ile Leu Phe Thr225 230 235 240Leu Thr Gly Ala Gly Leu
Leu Trp Met Gly Trp Ala Gly Phe Asn Gly245 250 255Gly Gly Pro Tyr
Ala Ala Asn Ser Val Ala Ser Met Ala Val Leu Asn260 265 270Thr Asn
Ile Cys Thr Ala Met Ser Leu Ile Val Trp Thr Cys Leu Asp275 280
285Val Ile Phe Phe Lys Lys Pro Ser Val Val Gly Ala Val Gln Gly
Met290 295 300Ile Thr Gly Leu Val Cys Ile Thr Pro Ala Ala Gly Val
Val Gln Gly305 310 315 320Trp Ala Ala Leu Val Met Gly Val Leu Ala
Gly Ser Ile Pro Trp Tyr325 330 335Thr Met Met Ile Leu His Lys Arg
Ser Lys Ile Leu Gln Arg Val Asp340 345 350Asp Thr Leu Gly Val Phe
His Thr His Gly Val Ala Gly Leu Leu Gly355 360 365Gly Leu Leu Thr
Gly Leu Phe Ala Glu Pro Thr Leu Cys Asn Leu Phe370 375 380Leu Pro
Val Ala Asp Ser Arg Gly Ala Phe Tyr Gly Gly Ala Gly Gly385 390 395
400Ala Gln Phe Gly Lys Gln Ile Ala Gly Gly Leu Phe Val Val Ala
Trp405 410 415Asn Val Ala Val Thr Ser Leu Ile Cys Leu Ala Ile Asn
Leu Leu Val420 425 430Pro Leu Arg Met Pro Asp Asp Lys Leu Glu Val
Gly Asp Asp Ala Val435 440 445His Gly Glu Glu Ala Tyr Ala Leu Trp
Gly Asp Gly Glu Met Tyr Asp450 455 460Val Thr Lys His Gly Ser Asp
Ala Ala Val Ala Pro Val Val Val465 470 475451497DNAOryza sativa
45atgtcggggg acgcgttcaa catgtcggtg gcgtaccagc cgtcggggat ggcggtgccg
60gagtggctga acaagggcga caacgcgtgg cagatgatct cggcgacgct ggtggggatg
120cagagcgtgc cggggctggt gatcctgtac ggcagcatcg tgaagaagaa
gtgggcggtg 180aactcggcgt tcatggcgct ctacgccttc gccgccgtgt
ggctgtgctg ggtcacctgg 240ggctacaaca tgtcgttcgg ccacaagctc
ctcccgttct ggggcaaggc gcggccggcg 300ctgggccaga gcttcctcct
cgcccaggcc gtgctcccgc agacgacgca gttctacaag 360ggcggcggcg
gcgccgacgc cgtggtggag acgccatggg tgaacccgct ctacccgatg
420gccaccatgg tgtacttcca gtgcgtgttc gccgccatca cgctcatcct
cctcgccggc 480tcgctgctgg ggcggatgaa catcaaggcg tggatgctgt
tcgtcccgct ctggctcacc 540ttctcctaca ccgtcggcgc cttctcgctg
tggggcggcg gcttcctctt ccactggggg 600gtcatggact actccggcgg
ctacgtcatc cacctctcgt cgggtgtcgc cggcttcacc 660gcggcgtact
gggtggggcc caggtcgacc aaggacaggg agaggttccc gccaaacaac
720gtgctgctca tgctcaccgg cgccggcata ctgtggatgg ggtgggcggg
gttcaacggc 780ggcgacccgt actccgccaa catcgactcc tcgctcgccg
tgctcaacac caacatctgc 840gccgccacca gcctcctcgt ctggacttgc
ctcgacgtca tcttcttcaa gaagccgtcc 900gtcatcggcg ccgtccaggg
catgatcacc ggcctcgtct gcatcactcc cggcgcaggc 960ctggtgcagg
gttgggcggc gatcgtgatg ggcatcctct ccggcagcat cccgtggttc
1020acgatgatgg tggtgcacaa gcggtcgcgc ctcctgcagc aggtggacga
caccctgggc 1080gtcttccaca cccacgccgt cgccggattc ctcggcggcg
ccaccacggg cctcttcgcc 1140gagcccgtcc tctgctccct cttcctcccc
gtcaccaact cccgcggcgc cttctacccc 1200ggccgcggcg gcggcctcca
gttcgtccgc caggtggccg gcgccctctt catcatctgc 1260tggaacgtgg
tggtcaccag cctcgtctgc ctcgccgtcc gcgccgtggt tcccctccgg
1320atgcccgagg aggagctcgc catcggcgac gacgccgtgc acggggagga
ggcgtacgcg 1380ctgtggggcg acggcgagaa gtacgactcc accaagcacg
gatggtactc cgacaacaac 1440gacacgcacc acaacaacaa caaggccgcg
cccagcggcg tcacgcagaa cgtctga 149746498PRTOryza sativa 46Met Ser
Gly Asp Ala Phe Asn Met Ser Val Ala Tyr Gln Pro Ser Gly1 5 10 15Met
Ala Val Pro Glu Trp Leu Asn Lys Gly Asp Asn Ala Trp Gln Met20 25
30Ile Ser Ala Thr Leu Val Gly Met Gln Ser Val Pro Gly Leu Val Ile35
40 45Leu Tyr Gly Ser Ile Val Lys Lys Lys Trp Ala Val Asn Ser Ala
Phe50 55 60Met Ala Leu Tyr Ala Phe Ala Ala Val Trp Leu Cys Trp Val
Thr Trp65 70 75 80Gly Tyr Asn Met Ser Phe Gly His Lys Leu Leu Pro
Phe Trp Gly Lys85 90 95Ala Arg Pro Ala Leu Gly Gln Ser Phe Leu Leu
Ala Gln Ala Val Leu100 105 110Pro Gln Thr Thr Gln Phe Tyr Lys Gly
Gly Gly Gly Ala Asp Ala Val115 120 125Val Glu Thr Pro Trp Val Asn
Pro Leu Tyr Pro Met Ala Thr Met Val130 135 140Tyr Phe Gln Cys Val
Phe Ala Ala Ile Thr Leu Ile Leu Leu Ala Gly145 150 155 160Ser Leu
Leu Gly Arg Met Asn Ile Lys Ala Trp Met Leu Phe Val Pro165 170
175Leu Trp Leu Thr Phe Ser Tyr Thr Val Gly Ala Phe Ser Leu Trp
Gly180 185 190Gly Gly Phe Leu Phe His Trp Gly Val Met Asp Tyr Ser
Gly Gly Tyr195 200 205Val Ile His Leu Ser Ser Gly Val Ala Gly Phe
Thr Ala Ala Tyr Trp210 215 220Val Gly Pro Arg Ser Thr Lys Asp Arg
Glu Arg Phe Pro Pro Asn Asn225 230 235 240Val Leu Leu Met Leu Thr
Gly Ala Gly Ile Leu Trp Met Gly Trp Ala245 250 255Gly Phe Asn Gly
Gly Asp Pro Tyr Ser Ala Asn Ile Asp Ser Ser Leu260 265 270Ala Val
Leu Asn Thr Asn Ile Cys Ala Ala Thr Ser Leu Leu Val Trp275 280
285Thr Cys Leu Asp Val Ile Phe Phe Lys Lys Pro Ser Val Ile Gly
Ala290 295 300Val Gln Gly Met Ile Thr Gly Leu Val Cys Ile Thr Pro
Gly Ala Gly305 310 315 320Leu Val Gln Gly Trp Ala Ala Ile Val Met
Gly Ile Leu Ser Gly Ser325 330 335Ile Pro Trp Phe Thr Met Met Val
Val His Lys Arg Ser Arg Leu Leu340 345 350Gln Gln Val Asp Asp Thr
Leu Gly Val Phe His Thr His Ala Val Ala355 360 365Gly Phe Leu Gly
Gly Ala Thr Thr Gly Leu Phe Ala Glu Pro Val Leu370 375 380Cys Ser
Leu Phe Leu Pro Val Thr Asn Ser Arg Gly Ala Phe Tyr Pro385 390 395
400Gly Arg Gly Gly Gly Leu Gln Phe Val Arg Gln Val Ala Gly Ala
Leu405 410 415Phe Ile Ile Cys Trp Asn Val Val Val Thr Ser Leu Val
Cys Leu Ala420 425 430Val Arg Ala Val Val Pro Leu Arg Met Pro Glu
Glu Glu Leu Ala Ile435 440 445Gly Asp Asp Ala Val His Gly Glu Glu
Ala Tyr Ala Leu Trp Gly Asp450 455 460Gly Glu Lys Tyr Asp Ser Thr
Lys His Gly Trp Tyr Ser Asp Asn Asn465 470 475 480Asp Thr His His
Asn Asn Asn Lys Ala Ala Pro Ser Gly Val Thr Gln485 490 495Asn
Val471497DNAOryza sativa 47atggcgacgt gcgcggcgga cctggcgccg
ctgctggggc cggtggcggc gaacgcgacg 60gactacctgt gcaaccggtt cgccgacacg
acgtcggcgg tggacgcgac gtacctgctc 120ttctcggcgt acctcgtgtt
cgccatgcag ctcgggttcg cgatgctctg cgccgggtcg 180gtgcgggcca
agaacacgat gaacatcatg ctcaccaacg tgctcgacgc cgcggccggg
240gcgctcttct actacctctt cggcttcgcc ttcgccttcg gcacgccgtc
caacggcttc 300atcgggaagc agttcttcgg cctcaagcac atgccgcaga
ccgggttcga ctacgacttc 360ttcctcttcc agtgggcctt cgccatcgcc
gccgccggga tcacgtcggg ctccatcgcc 420gagaggacgc agttcgtcgc
ctacctcatc tactccgcct tcctcaccgg gttcgtctac 480ccggtggtgt
cccactggat ctggtccgcc gatgggtggg cctctgcctc ccgcacgtcc
540ggacctctgc tgttcggctc cggtgtcatc gacttcgccg gctccggcgt
cgtccacatg 600gtcggcggtg tcgccgggct ctggggcgcg ctcatcgagg
gcccccgcat cgggaggttc 660gaccacgccg gccgatcggt ggcgctcaag
ggccacagcg cgtcgctcgt cgtgcttggc 720accttcctgc tgtggttcgg
ctggtacgga ttcaaccccg ggtcgttcac caccatcctc 780aagacgtacg
gcccggccgg cggcatcaac gggcagtggt ccggagtcgg ccgcaccgcc
840gtgacgacga ccctggccgg cagcgtggcg gcgctcacca cgctgttcgg
gaagcggctc 900cagacggggc actggaacgt ggtcgacgtc tgcaacggcc
tcctcggcgg gttcgccgcc 960atcaccgccg ggtgcagcgt cgtcgacccg
tgggccgcga tcatctgcgg gttcgtctcg 1020gcgtgggtgc tcatcggcct
caacgcgctc gccgcgcgcc tcaagttcga cgacccgctc 1080gaggccgccc
agctccacgg cgggtgcggc gcgtggggga tcctcttcac cgcgctcttc
1140gcgaggcaga agtacgtcga ggagatctac ggcgccggcc ggccgtacgg
cctgttcatg 1200ggcggcggcg gcaagctgct cgccgcgcac gtcatccaga
tcctggtcat cttcgggtgg 1260gtcagctgca ccatgggacc tctcttctac
gggctcaaga agctcggcct gctccgcatc 1320tccgccgagg acgagacgtc
cggcatggac ctgacacggc acggcgggtt cgcgtacgtc 1380taccacgacg
aggacgagca cgacaagtct ggggttggtg ggttcatgct ccggtccgcg
1440cagacccgcg tcgagccggc ggcggcggct gcctccaaca gcaacaacca agtgtaa
149748498PRTOryza sativa 48Met Ala Thr Cys Ala Ala Asp Leu Ala Pro
Leu Leu Gly Pro Val Ala1 5 10 15Ala Asn Ala Thr Asp Tyr Leu Cys Asn
Arg Phe Ala Asp Thr Thr Ser20 25 30Ala Val Asp Ala Thr Tyr Leu Leu
Phe Ser Ala Tyr Leu Val Phe Ala35 40 45Met Gln Leu Gly Phe Ala Met
Leu Cys Ala Gly Ser Val Arg Ala Lys50 55 60Asn Thr Met Asn Ile Met
Leu Thr Asn Val Leu Asp Ala Ala Ala Gly65 70 75 80Ala Leu Phe Tyr
Tyr Leu Phe Gly Phe Ala Phe Ala Phe Gly Thr Pro85 90 95Ser Asn Gly
Phe Ile Gly Lys Gln Phe Phe Gly Leu Lys His Met Pro100 105 110Gln
Thr Gly Phe Asp Tyr Asp Phe Phe Leu Phe Gln Trp Ala Phe Ala115 120
125Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr
Gln130 135 140Phe Val Ala Tyr Leu Ile Tyr Ser Ala Phe Leu Thr Gly
Phe Val Tyr145 150 155 160Pro Val Val Ser His Trp Ile Trp Ser Ala
Asp Gly Trp Ala Ser Ala165 170 175Ser Arg Thr Ser Gly Pro Leu Leu
Phe Gly Ser Gly Val Ile Asp Phe180 185 190Ala Gly Ser Gly Val Val
His Met Val Gly Gly Val Ala Gly Leu Trp195 200 205Gly Ala Leu Ile
Glu Gly Pro Arg Ile Gly Arg Phe Asp His Ala Gly210 215 220Arg Ser
Val Ala Leu Lys Gly His Ser Ala Ser Leu Val Val Leu Gly225 230 235
240Thr Phe Leu Leu Trp Phe Gly Trp Tyr Gly Phe Asn Pro Gly Ser
Phe245 250 255Thr Thr Ile Leu Lys Thr Tyr Gly Pro Ala Gly Gly Ile
Asn Gly Gln260 265 270Trp Ser Gly Val Gly Arg Thr Ala Val Thr Thr
Thr Leu Ala Gly Ser275 280 285Val Ala Ala Leu Thr Thr Leu Phe Gly
Lys Arg Leu Gln Thr Gly His290 295 300Trp Asn Val Val Asp Val Cys
Asn Gly Leu Leu Gly Gly Phe Ala Ala305 310 315 320Ile Thr Ala Gly
Cys Ser Val Val Asp Pro Trp Ala Ala Ile Ile Cys325 330 335Gly Phe
Val Ser Ala Trp Val Leu Ile Gly Leu Asn Ala Leu Ala Ala340 345
350Arg Leu Lys Phe Asp Asp Pro Leu Glu Ala Ala Gln Leu His Gly
Gly355 360 365Cys Gly Ala Trp Gly Ile Leu Phe Thr Ala Leu Phe Ala
Arg Gln Lys370 375 380Tyr Val Glu Glu Ile Tyr Gly Ala Gly Arg Pro
Tyr Gly Leu Phe Met385 390 395 400Gly Gly Gly Gly Lys Leu Leu Ala
Ala His Val Ile Gln Ile Leu Val405 410 415Ile Phe Gly Trp Val Ser
Cys Thr Met Gly Pro Leu Phe Tyr Gly Leu420 425 430Lys Lys Leu Gly
Leu Leu Arg Ile Ser Ala Glu Asp Glu Thr Ser Gly435 440 445Met Asp
Leu Thr Arg His Gly Gly Phe Ala Tyr Val Tyr His Asp Glu450 455
460Asp Glu His Asp Lys Ser Gly Val Gly Gly Phe Met Leu Arg Ser
Ala465 470 475 480Gln Thr Arg Val Glu Pro Ala Ala Ala Ala Ala Ser
Asn Ser Asn Asn485 490 495Gln Val49438DNAOryza sativa 49atggcgtggg
tgagcttcac catggcgctg ctgttcctgg tgctcaacaa gctgggcttg 60ctgcgcatct
cggccgagga caagatggcc ggcatggacc agacgcgcca cggcgggtta
120ccacgacgac gacgcgagcg gcaagccaga ccttggcatt ggcgggttca
tgctcaagtc 180ggtgcacggc acgcaggttc gtcggtgtcg acggaggcga
cgactgcggg gatggtggcc 240gcgagggccg tgcaggagtt gtggaacggt
tcggacaccg agcagaagag gacataccca 300ccggttctgc tcgccggaga
gggaggggac aacgactgcg gtgtccatca ctggctgcgc 360ttgccactac
catcgctggt ctcgtggaag agaggagagg ggagaaagag gaagaagaag
420gaaagaaggg caatttga 43850145PRTOryza sativa 50Met Ala Trp Val
Ser Phe Thr Met Ala Leu Leu Phe Leu Val Leu Asn1 5 10 15Lys Leu Gly
Leu Leu Arg Ile Ser Ala Glu Asp Lys Met Ala Gly Met20 25 30Asp Gln
Thr Arg His Gly Gly Leu Pro Arg Arg Arg Arg Glu Arg Gln35 40 45Ala
Arg Pro Trp His Trp Arg Val His Ala Gln Val Gly Ala Arg His50 55
60Ala Gly Ser Ser Val Ser Thr Glu Ala Thr Thr Ala Gly Met Val Ala65
70 75 80Ala Arg Ala Val Gln Glu Leu Trp Asn Gly Ser Asp Thr Glu Gln
Lys85
90 95Arg Thr Tyr Pro Pro Val Leu Leu Ala Gly Glu Gly Gly Asp Asn
Asp100 105 110Cys Gly Val His His Trp Leu Arg Leu Pro Leu Pro Ser
Leu Val Ser115 120 125Trp Lys Arg Gly Glu Gly Arg Lys Arg Lys Lys
Lys Glu Arg Arg Ala130 135 140Ile145511497DNAOryza sativa
51atggcgacgt gcttggacag cctcgggccg ctgctcggcg gcgcggcgaa ctccaccgac
60gcggccaact acatctgcaa caggttcacg gacacctcct ccgcggtgga cgcgacgtac
120ctgctcttct cggcctacct cgtgttcgcc atgcagctcg ggttcgccat
gctctgcgcg 180ggctccgtcc gcgccaagaa ctccatgaac atcatgctca
ccaacgtgct cgacgccgcc 240gccggcgcgc tcttctacta cctcttcggc
ttcgccttcg cgttcgggac gccgtccaag 300ggcttcatcg ggaagcagtt
cttcgggctg aagcacatgc cgcagacagg gtacgactac 360gacttcttcc
tcttccagtg ggccttcgcc atcgccgccg ccggcatcac gtccggttcc
420atcgccgagc ggacgcgctt cagcgcgtat ctcatctact ccgccttcct
caccgggttc 480gtgtacccgg tggtgtcgca ctggttctgg tccaccgacg
ggtgggcttc ggccggccgg 540ttgacgggtc cgttgctgtt caagtcgggc
gtcatcgact tcgccggctc cggcgtcgtc 600catctggtcg gtggcattgc
tggcctgtgg ggtgccttca tcgagggccc tcgcatcggg 660cggttcgacg
ccgccggccg cacggtggcg atgaaagggc acagcgcctc actggtcgtg
720ctcggcacct tcctgctgtg gttcgggtgg ttcggcttca acccggggtc
cttcaccacc 780atctccaaga tctacggcga gtcgggcacg atcgacgggc
agtggtcggc ggtgggccgc 840accgccgtga cgacgtcgct ggcggggagc
gtcgccgcgc tgacgacgct ctacggcaag 900agatggctga cggggcactg
gaacgtgacc gacgtctgca acggtctcct cggcggcttc 960gccgcgatca
ccgcgggctg ctccgtggtc gacccgtggg cgtcggtgat ctgcgggttc
1020gtgtcggcgt gggtcctcat cggctgcaac aagctgtcgc tgattctcaa
gttcgacgac 1080ccgctggagg cgacgcagct gcacgccggg tgcggcgcgt
gggggatcat cttcaccgcg 1140ctgttcgcgc gcagggagta cgtcgagctg
atctacgggg tgccggggag gccgtacggg 1200ctgttcatgg gcggcggcgg
gaggcttctc gcggcgcaca tcgtgcagat cctggtgatc 1260gtcgggtggg
tcagcgccac catggggacg ctcttctacg tgctgcacag gttcgggctg
1320ctccgcgtct cgcccgcgac agagatggaa ggcatggacc cgacgtgcca
cggcgggttc 1380gggtacgtgg acgaggacga aggcgagcgc cgcgtcaggg
ccaagtcggc ggcggagacg 1440gctcgcgtgg agcccagaaa gtcgccggag
caagccgcgg cgggccagtt tgtgtag 149752498PRTOryza sativa 52Met Ala
Thr Cys Leu Asp Ser Leu Gly Pro Leu Leu Gly Gly Ala Ala1 5 10 15Asn
Ser Thr Asp Ala Ala Asn Tyr Ile Cys Asn Arg Phe Thr Asp Thr20 25
30Ser Ser Ala Val Asp Ala Thr Tyr Leu Leu Phe Ser Ala Tyr Leu Val35
40 45Phe Ala Met Gln Leu Gly Phe Ala Met Leu Cys Ala Gly Ser Val
Arg50 55 60Ala Lys Asn Ser Met Asn Ile Met Leu Thr Asn Val Leu Asp
Ala Ala65 70 75 80Ala Gly Ala Leu Phe Tyr Tyr Leu Phe Gly Phe Ala
Phe Ala Phe Gly85 90 95Thr Pro Ser Lys Gly Phe Ile Gly Lys Gln Phe
Phe Gly Leu Lys His100 105 110Met Pro Gln Thr Gly Tyr Asp Tyr Asp
Phe Phe Leu Phe Gln Trp Ala115 120 125Phe Ala Ile Ala Ala Ala Gly
Ile Thr Ser Gly Ser Ile Ala Glu Arg130 135 140Thr Arg Phe Ser Ala
Tyr Leu Ile Tyr Ser Ala Phe Leu Thr Gly Phe145 150 155 160Val Tyr
Pro Val Val Ser His Trp Phe Trp Ser Thr Asp Gly Trp Ala165 170
175Ser Ala Gly Arg Leu Thr Gly Pro Leu Leu Phe Lys Ser Gly Val
Ile180 185 190Asp Phe Ala Gly Ser Gly Val Val His Leu Val Gly Gly
Ile Ala Gly195 200 205Leu Trp Gly Ala Phe Ile Glu Gly Pro Arg Ile
Gly Arg Phe Asp Ala210 215 220Ala Gly Arg Thr Val Ala Met Lys Gly
His Ser Ala Ser Leu Val Val225 230 235 240Leu Gly Thr Phe Leu Leu
Trp Phe Gly Trp Phe Gly Phe Asn Pro Gly245 250 255Ser Phe Thr Thr
Ile Ser Lys Ile Tyr Gly Glu Ser Gly Thr Ile Asp260 265 270Gly Gln
Trp Ser Ala Val Gly Arg Thr Ala Val Thr Thr Ser Leu Ala275 280
285Gly Ser Val Ala Ala Leu Thr Thr Leu Tyr Gly Lys Arg Trp Leu
Thr290 295 300Gly His Trp Asn Val Thr Asp Val Cys Asn Gly Leu Leu
Gly Gly Phe305 310 315 320Ala Ala Ile Thr Ala Gly Cys Ser Val Val
Asp Pro Trp Ala Ser Val325 330 335Ile Cys Gly Phe Val Ser Ala Trp
Val Leu Ile Gly Cys Asn Lys Leu340 345 350Ser Leu Ile Leu Lys Phe
Asp Asp Pro Leu Glu Ala Thr Gln Leu His355 360 365Ala Gly Cys Gly
Ala Trp Gly Ile Ile Phe Thr Ala Leu Phe Ala Arg370 375 380Arg Glu
Tyr Val Glu Leu Ile Tyr Gly Val Pro Gly Arg Pro Tyr Gly385 390 395
400Leu Phe Met Gly Gly Gly Gly Arg Leu Leu Ala Ala His Ile Val
Gln405 410 415Ile Leu Val Ile Val Gly Trp Val Ser Ala Thr Met Gly
Thr Leu Phe420 425 430Tyr Val Leu His Arg Phe Gly Leu Leu Arg Val
Ser Pro Ala Thr Glu435 440 445Met Glu Gly Met Asp Pro Thr Cys His
Gly Gly Phe Gly Tyr Val Asp450 455 460Glu Asp Glu Gly Glu Arg Arg
Val Arg Ala Lys Ser Ala Ala Glu Thr465 470 475 480Ala Arg Val Glu
Pro Arg Lys Ser Pro Glu Gln Ala Ala Ala Gly Gln485 490 495Phe
Val531853DNAOryza sativa 53acagcccaca cttccattgc tcctcccctc
tcctctacag tctgtgttga gcgcgcgtcg 60aggcggcgag gatggcaacg tgcgcggata
ccctcggccc gctgctgggc acggcggcgg 120cgaacgcgac ggactacctg
tgcaaccagt tcgcggacac gacgtcggcc gtggactcga 180cgtacctgct
cttctcggcg tacctcgtgt tcgccatgca gctcggcttc gccatgctct
240gcgccgggtc cgtccgcgcc aagaacacca tgaacatcat gcttaccaac
gtgctcgacg 300ccgccgccgg cgcgctcttc tactacctct tcggcttcgc
cttcgccttc ggggcgccgt 360ccaacggctt catcgggaag cacttcttcg
gcctcaagca ggtcccacag gtcggcttcg 420actacagctt cttcctcttc
cagtgggcct tcgccatcgc cgccgcgggc atcacgtccg 480gctccatcgc
cgagcggacc cagttcgtgg cgtacctcat ctactccgcc ttcctcaccg
540gcttcgtcta cccggtggtg tcccactgga tctggtccgc cgacgggtgg
gcctcggctt 600cccggacgtc ggggtcgctg ctcttcgggt ccggcgtcat
cgacttcgcc gggtcagggg 660ttgtccacat ggtgggcggc gtggccggac
tctggggcgc cctcatcgag ggcccccgca 720ttgggcggtt cgaccacgcc
ggccgctcgg tggcgctgcg cggccacagc gcgtcgctcg 780tcgtgctcgg
cagcttcctt ctgtggttcg ggtggtacgg gtttaacccc ggctcgttcc
840tcaccatcct caaatcctac ggcccgcccg gtagcatcca cgggcagtgg
tcggcggtgg 900gacgcaccgc cgtgaccacc accctcgccg gcagcacggc
ggcgctcacg acgctcttcg 960ggaagaggct ccagacgggg cactggaacg
tgatcgacgt ctgcaacggc ctcctcggcg 1020gcttcgcggc gatcaccgcc
ggttgctccg tcgtcgaccc gtgggccgcg atcatctgcg 1080ggttcgtctc
ggcgtgggtg ctcatcggcc tcaacgcgct ggcggcgagg ctcaagttcg
1140acgacccgct cgaggcggcg cagctgcacg gcgggtgcgg cgcgtggggg
gtcatcttca 1200cggcgctgtt cgcgcgcaag gagtacgtgg accagatctt
cggccagccc gggcgcccgt 1260acgggctgtt catgggcggc ggcggccggc
tgctcggggc gcacatagtg gtcatcctgg 1320tcatcgcggc gtgggtgagc
ttcaccatgg cgccgctgtt cctggtgctc aacaagctgg 1380gcttgctgcg
catctcggcc gaggacgaga tggccggcat ggaccagacg cgccacggcg
1440ggttcgcgta cgcgtaccac gacgacgacg cgagcggcaa gccggaccgc
agcgtcggcg 1500ggttcatgct caagtcggcg cacggcacgc aggtcgccgc
cgagatggga ggccatgtct 1560agtggaaccg gaggagctga gctagtagta
catacatgca gcatcatcga tcgaacgaaa 1620tgcatataag cgtttttcaa
ggttgatctg atgctgcagg tttcgtgatt gtataatagg 1680aagaaaaaga
tagtagtatt ttttatctga gatcatctgt ttggaacagg ggatttgact
1740aagatttgat ataaatttac acaaaatctt agcaaaaatc cctttatctc
aactctcaag 1800tagagctttg ctttgtacaa caaagtatca tgtgtgatat
aattgtcagg tgg 185354496PRTOryza sativa 54Met Ala Thr Cys Ala Asp
Thr Leu Gly Pro Leu Leu Gly Thr Ala Ala1 5 10 15Ala Asn Ala Thr Asp
Tyr Leu Cys Asn Gln Phe Ala Asp Thr Thr Ser20 25 30Ala Val Asp Ser
Thr Tyr Leu Leu Phe Ser Ala Tyr Leu Val Phe Ala35 40 45Met Gln Leu
Gly Phe Ala Met Leu Cys Ala Gly Ser Val Arg Ala Lys50 55 60Asn Thr
Met Asn Ile Met Leu Thr Asn Val Leu Asp Ala Ala Ala Gly65 70 75
80Ala Leu Phe Tyr Tyr Leu Phe Gly Phe Ala Phe Ala Phe Gly Ala Pro85
90 95Ser Asn Gly Phe Ile Gly Lys His Phe Phe Gly Leu Lys Gln Val
Pro100 105 110Gln Val Gly Phe Asp Tyr Ser Phe Phe Leu Phe Gln Trp
Ala Phe Ala115 120 125Ile Ala Ala Ala Gly Ile Thr Ser Gly Ser Ile
Ala Glu Arg Thr Gln130 135 140Phe Val Ala Tyr Leu Ile Tyr Ser Ala
Phe Leu Thr Gly Phe Val Tyr145 150 155 160Pro Val Val Ser His Trp
Ile Trp Ser Ala Asp Gly Trp Ala Ser Ala165 170 175Ser Arg Thr Ser
Gly Ser Leu Leu Phe Gly Ser Gly Val Ile Asp Phe180 185 190Ala Gly
Ser Gly Val Val His Met Val Gly Gly Val Ala Gly Leu Trp195 200
205Gly Ala Leu Ile Glu Gly Pro Arg Ile Gly Arg Phe Asp His Ala
Gly210 215 220Arg Ser Val Ala Leu Arg Gly His Ser Ala Ser Leu Val
Val Leu Gly225 230 235 240Ser Phe Leu Leu Trp Phe Gly Trp Tyr Gly
Phe Asn Pro Gly Ser Phe245 250 255Leu Thr Ile Leu Lys Ser Tyr Gly
Pro Pro Gly Ser Ile His Gly Gln260 265 270Trp Ser Ala Val Gly Arg
Thr Ala Val Thr Thr Thr Leu Ala Gly Ser275 280 285Thr Ala Ala Leu
Thr Thr Leu Phe Gly Lys Arg Leu Gln Thr Gly His290 295 300Trp Asn
Val Ile Asp Val Cys Asn Gly Leu Leu Gly Gly Phe Ala Ala305 310 315
320Ile Thr Ala Gly Cys Ser Val Val Asp Pro Trp Ala Ala Ile Ile
Cys325 330 335Gly Phe Val Ser Ala Trp Val Leu Ile Gly Leu Asn Ala
Leu Ala Ala340 345 350Arg Leu Lys Phe Asp Asp Pro Leu Glu Ala Ala
Gln Leu His Gly Gly355 360 365Cys Gly Ala Trp Gly Val Ile Phe Thr
Ala Leu Phe Ala Arg Lys Glu370 375 380Tyr Val Asp Gln Ile Phe Gly
Gln Pro Gly Arg Pro Tyr Gly Leu Phe385 390 395 400Met Gly Gly Gly
Gly Arg Leu Leu Gly Ala His Ile Val Val Ile Leu405 410 415Val Ile
Ala Ala Trp Val Ser Phe Thr Met Ala Pro Leu Phe Leu Val420 425
430Leu Asn Lys Leu Gly Leu Leu Arg Ile Ser Ala Glu Asp Glu Met
Ala435 440 445Gly Met Asp Gln Thr Arg His Gly Gly Phe Ala Tyr Ala
Tyr His Asp450 455 460Asp Asp Ala Ser Gly Lys Pro Asp Arg Ser Val
Gly Gly Phe Met Leu465 470 475 480Lys Ser Ala His Gly Thr Gln Val
Ala Ala Glu Met Gly Gly His Val485 490 495551870DNAOryza sativa
55cactagtact ccctccgtcc caatataagt gcatttagga caggatgtga tatatcctag
60gactacaaat ctggacagtt gtctgttcat attcgtagtc ctaggatatg tcacatacta
120tactaggtgt atttatattg ggacggaggg agcagtactt aaagtatatt
tgcaactttt 180tactgaactt ggtgtgctgt gtcaggcgac tactccagag
gattgattac ttcatgcctt 240gacaatgatg tgaagtagca tgaccttgcg
attcatatgg tcggggatcg aggcatatat 300acacccaacc cagttcattg
agtgatcagt agagagattc ttcccctctt ctcctgccag 360ctcttccagg
ttctgagttc tgaccatggc ggctggagcg attccaatgg cgtaccagac
420cactccgtca tcgccagact ggctgaacaa gggcgacaac gcatggcaga
tgacatcggc 480gaccctcgtc ggcctgcaga gcatgccagg gctggtgatc
ctgtacggca gcattgtcaa 540gaagaagtgg gctatcaact cggcgttcat
ggcgctgtat gccttcgctg ctgtctggat 600ctgctgggtt gtctgggcat
acaacatgtc gttcggcgac cgcctcctgc cattctgggg 660taaggcacgg
ccagcgctcg ggcagagctt cctcgtggcg cagtctgagc tcactgctac
720cgctattcgc taccacaatg ggtcagctga ggcgcccatg ctcaagccgt
tgtacccagt 780cgccaccatg gtgtacttcc agtgcatgtt tgcgagcatc
accatcatca tcctcgcagg 840ctcactgctt gggcgcatga acatcaaggc
gtggatggcc tttgtgccgc tctggatcac 900cttctcttac acggtctgcg
ccttctcgct ctggggtggc ggtttcctct tccagtgggg 960tgtcatagac
tactctggtg gctatgtcat ccatctctct tctggcatcg caggcctcac
1020tgctgcctac tgggttggac caaggtcagc atcagatagg gagagattcc
cgcccaacaa 1080catactgctg gtgctagcag gggcggggct gctgtggctt
gggtggacag gtttcaatgg 1140aggagaccca tattcagcca atattgattc
atccatggca gtgctcaaca cacatatctg 1200cgcatccacc agcctactcg
tgtggacaat cctggatgtc ttcttcttcg ggaagccatc 1260ggtaattggc
gcggtgcagg gcatgatcac tggcctggta tgcatcaccc ctggtgcagg
1320cctggtgcaa ggttgggcag ctattgtgat gggaattctc tctggtagca
ttccatggta 1380caccatgatg gtgctgcaca agaaatggtc attcatgcag
aggattgatg acacgcttgg 1440tgtcttccac acccatgcgg tggctgggtt
ccttggtggc gccaccactg gactcttcgc 1500cgagcccatc ctatgcagtc
tcttcctatc tatcccagat tctaaaggtg cattctacgg 1560tggccccggt
ggatcacagt tcgggaagca gattgctggc gcactatttg tcactgcctg
1620gaatattgtt atcacctcca tcatctgtgt catcatcagc ctaatcctgc
ccctccgtat 1680agctgatcaa gaactgctta ttggagatga tgctgtacac
ggtgaggagg catatgctat 1740ctgggcagag ggagagctca atgacatgac
ccaccacaat gagagcacac atagtggtgt 1800ctctgtagga gtgacacaga
atgtttgaac agtacccact ttattgagga aaaagaaata 1860taattgtctt
187056480PRTOryza sativa 56Met Ala Ala Gly Ala Ile Pro Met Ala Tyr
Gln Thr Thr Pro Ser Ser1 5 10 15Pro Asp Trp Leu Asn Lys Gly Asp Asn
Ala Trp Gln Met Thr Ser Ala20 25 30Thr Leu Val Gly Leu Gln Ser Met
Pro Gly Leu Val Ile Leu Tyr Gly35 40 45Ser Ile Val Lys Lys Lys Trp
Ala Ile Asn Ser Ala Phe Met Ala Leu50 55 60Tyr Ala Phe Ala Ala Val
Trp Ile Cys Trp Val Val Trp Ala Tyr Asn65 70 75 80Met Ser Phe Gly
Asp Arg Leu Leu Pro Phe Trp Gly Lys Ala Arg Pro85 90 95Ala Leu Gly
Gln Ser Phe Leu Val Ala Gln Ser Glu Leu Thr Ala Thr100 105 110Ala
Ile Arg Tyr His Asn Gly Ser Ala Glu Ala Pro Met Leu Lys Pro115 120
125Leu Tyr Pro Val Ala Thr Met Val Tyr Phe Gln Cys Met Phe Ala
Ser130 135 140Ile Thr Ile Ile Ile Leu Ala Gly Ser Leu Leu Gly Arg
Met Asn Ile145 150 155 160Lys Ala Trp Met Ala Phe Val Pro Leu Trp
Ile Thr Phe Ser Tyr Thr165 170 175Val Cys Ala Phe Ser Leu Trp Gly
Gly Gly Phe Leu Phe Gln Trp Gly180 185 190Val Ile Asp Tyr Ser Gly
Gly Tyr Val Ile His Leu Ser Ser Gly Ile195 200 205Ala Gly Leu Thr
Ala Ala Tyr Trp Val Gly Pro Arg Ser Ala Ser Asp210 215 220Arg Glu
Arg Phe Pro Pro Asn Asn Ile Leu Leu Val Leu Ala Gly Ala225 230 235
240Gly Leu Leu Trp Leu Gly Trp Thr Gly Phe Asn Gly Gly Asp Pro
Tyr245 250 255Ser Ala Asn Ile Asp Ser Ser Met Ala Val Leu Asn Thr
His Ile Cys260 265 270Ala Ser Thr Ser Leu Leu Val Trp Thr Ile Leu
Asp Val Phe Phe Phe275 280 285Gly Lys Pro Ser Val Ile Gly Ala Val
Gln Gly Met Ile Thr Gly Leu290 295 300Val Cys Ile Thr Pro Gly Ala
Gly Leu Val Gln Gly Trp Ala Ala Ile305 310 315 320Val Met Gly Ile
Leu Ser Gly Ser Ile Pro Trp Tyr Thr Met Met Val325 330 335Leu His
Lys Lys Trp Ser Phe Met Gln Arg Ile Asp Asp Thr Leu Gly340 345
350Val Phe His Thr His Ala Val Ala Gly Phe Leu Gly Gly Ala Thr
Thr355 360 365Gly Leu Phe Ala Glu Pro Ile Leu Cys Ser Leu Phe Leu
Ser Ile Pro370 375 380Asp Ser Lys Gly Ala Phe Tyr Gly Gly Pro Gly
Gly Ser Gln Phe Gly385 390 395 400Lys Gln Ile Ala Gly Ala Leu Phe
Val Thr Ala Trp Asn Ile Val Ile405 410 415Thr Ser Ile Ile Cys Val
Ile Ile Ser Leu Ile Leu Pro Leu Arg Ile420 425 430Ala Asp Gln Glu
Leu Leu Ile Gly Asp Asp Ala Val His Gly Glu Glu435 440 445Ala Tyr
Ala Ile Trp Ala Glu Gly Glu Leu Asn Asp Met Thr His His450 455
460Asn Glu Ser Thr His Ser Gly Val Ser Val Gly Val Thr Gln Asn
Val465 470 475 48057981DNAOryza sativa 57atggcgtcgg cggcggtgcc
ggagtggctg aacaagggcg acaatgcctg gcagatgctc 60tccgccacgc tcgtcgccct
tcagggcttc ccgggcctcg ccctcttcta cgtcggtgcc 120gtcccccgca
agtgggcgct cacctccgca ttcatggcgc tctacgccat ggccgccacc
180atgccgtgct gggcgctctg ggcgcacaac atggccttcg gccgccgcct
cctccccttc 240gtcggccgcc ccgccccggc gctcgcccag gactacatgc
tcagccaggc gctgctcccc 300tccaccctcc acctccgctc caacggcgag
gttgagacgg ccgcggtggc gccgctgtac 360ccgtcggcga gcatggtgtt
cttccagtgg gccttcgccg gcgtcaccgt ggggctggtc 420gccggcgccg
tgctcgggcg catgagcgtc aaggcgtgga tggcgttcgt gccgctgtgg
480acgacgctgt cctacacggt gggagcgtac agcatctggg gcggaggctt
cctcttccac 540tggggcgtca tggactactc cggcggctac gtcgtgctcc
tcgccgccgg cgtctccggc 600tacacggccg cgtactgggt gggacccagg
aggaaggagg aggacgagga ggaaatggca 660acggcgagtg gtggcaacct
ggtggtgatg gtggccggcg cgggcatcct gtggatgggg 720tggaccggct
tcaacggcgg cgaccccttc tccgccaaca ccgactcgtc ggtggcggtg
780ctcaacacgc acatctgcgc caccaccagc atcgtcgctt gggtttgctg
cgacgtcgcc 840gtccgcggga ggccgtcggt ggtgggcgcg gtgcagggca
tgatcaccgg cctggtgtgc 900atcactccaa ggtcaaacat caagtacagc
tttcttctag tagtaatttc tgatgagatg 960cctgttcctg atctgagcta g
98158326PRTOryza sativa 58Met Ala Ser Ala Ala Val Pro Glu Trp Leu
Asn Lys Gly Asp Asn Ala1 5 10 15Trp Gln Met Leu Ser Ala Thr Leu Val
Ala Leu Gln Gly Phe Pro Gly20 25 30Leu Ala Leu Phe Tyr Val Gly Ala
Val Pro Arg Lys Trp Ala Leu Thr35 40 45Ser Ala Phe Met Ala Leu Tyr
Ala Met Ala Ala Thr Met Pro Cys Trp50 55 60Ala Leu Trp Ala His Asn
Met Ala Phe Gly Arg Arg Leu Leu Pro Phe65 70 75 80Val Gly Arg Pro
Ala Pro Ala Leu Ala Gln Asp Tyr Met Leu Ser Gln85 90 95Ala Leu Leu
Pro Ser Thr Leu His Leu Arg Ser Asn Gly Glu Val Glu100 105 110Thr
Ala Ala Val Ala Pro Leu Tyr Pro Ser Ala Ser Met Val Phe Phe115 120
125Gln Trp Ala Phe Ala Gly Val Thr Val Gly Leu Val Ala Gly Ala
Val130 135 140Leu Gly Arg Met Ser Val Lys Ala Trp Met Ala Phe Val
Pro Leu Trp145 150 155 160Thr Thr Leu Ser Tyr Thr Val Gly Ala Tyr
Ser Ile Trp Gly Gly Gly165 170 175Phe Leu Phe His Trp Gly Val Met
Asp Tyr Ser Gly Gly Tyr Val Val180 185 190Leu Leu Ala Ala Gly Val
Ser Gly Tyr Thr Ala Ala Tyr Trp Val Gly195 200 205Pro Arg Arg Lys
Glu Glu Asp Glu Glu Glu Met Ala Thr Ala Ser Gly210 215 220Gly Asn
Leu Val Val Met Val Ala Gly Ala Gly Ile Leu Trp Met Gly225 230 235
240Trp Thr Gly Phe Asn Gly Gly Asp Pro Phe Ser Ala Asn Thr Asp
Ser245 250 255Ser Val Ala Val Leu Asn Thr His Ile Cys Ala Thr Thr
Ser Ile Val260 265 270Ala Trp Val Cys Cys Asp Val Ala Val Arg Gly
Arg Pro Ser Val Val275 280 285Gly Ala Val Gln Gly Met Ile Thr Gly
Leu Val Cys Ile Thr Pro Arg290 295 300Ser Asn Ile Lys Tyr Ser Phe
Leu Leu Val Val Ile Ser Asp Glu Met305 310 315 320Pro Val Pro Asp
Leu Ser325591377DNAOryza sativa 59atggcgtcgg tggcggtgcc ggagtggctg
aacaagggcg acaacgcctg gcagatgctc 60tccgccacgc tcgtcgccct gcagggcttc
cccggtctcg ccctcttcta cgccggcgcc 120gtcacccgca agtgcgcgct
cacctccgca ttcatggcgc tctacgccat ggccgccacc 180atgccgtgct
gggcgctctg ggcgcacaac atggccttcg gccaccgcct cctgcccttc
240gtcggccgcc ccgccccggc gctcgcccag cactacatgc tcacccaggc
gctgctcccc 300ttcaccctcc acctccactc caacggcgag gtggagacgg
ccgcggtggc gccgctgtac 360ccgtcggcga gcatggtgtt cttccagtgg
gcctccgccg gcgtcaccgt ggggctggtc 420gccggcgccg tgctcgggcg
catgagcgtc aaggcgtgga tggcgttcgt gccgctgtgg 480acgacgctgt
cctatacggt gggagcgtac agcatttggg gcgggggctt cctcttccac
540tggggcgtca tggactactc cggcggctac gtcgttcacc tcgccgccgg
cgtctccggc 600tacacggccg cgtactgggt gggaccaagg aggaaggagg
aggaggaaat gacaatggcg 660ggtggtggca acctggtggc gatggtggcc
ggcgcgggca tcctgtggat ggggtggacc 720ggcttcaacg gcggcgaccc
cttctccgcc aacaccgact cgtcggtggc ggtgctcaac 780acgcacatct
gcaccaccac cagcatcctc gcttgggttt gctgcgacat cgccgtccgc
840gggaggccgt cggtggtggg cgcggtgcag ggcatgatca ccggcctggt
gtgcataact 900ccggcggcag ggctggtgca ggggtgggca gctctgctaa
tgggcgtcgc gtcggggaca 960ctgccatgct acaccatgaa cgccgccatg
tcgttcaagg tagacgacac gctgggcatc 1020ctgcacaccc acgcggtgtc
cggtgttctg ggcggcgtcc tcaccggcgt tttcgcgcac 1080cctactctct
gtgacatgtt ccttccggtg accggctcaa ggggcctcgt ctacggcgtc
1140cgcgccggcg gggtgcaggt gttgaagcag gtcgccgccg cattgttcgt
tgccgcatgg 1200aacgtggccg ccacgtccat catcttggtc gtcgtcaggg
cgttcgtgcc gctgaggatg 1260acggaagatg agctgctcgc cggagacatt
gccgtacatg gggaacaagc ttattatttt 1320tcgagtggca ccaattgtag
tttaagccat gagaccattg aggtcggaaa ttcataa 137760458PRTOryza sativa
60Met Ala Ser Val Ala Val Pro Glu Trp Leu Asn Lys Gly Asp Asn Ala1
5 10 15Trp Gln Met Leu Ser Ala Thr Leu Val Ala Leu Gln Gly Phe Pro
Gly20 25 30Leu Ala Leu Phe Tyr Ala Gly Ala Val Thr Arg Lys Cys Ala
Leu Thr35 40 45Ser Ala Phe Met Ala Leu Tyr Ala Met Ala Ala Thr Met
Pro Cys Trp50 55 60Ala Leu Trp Ala His Asn Met Ala Phe Gly His Arg
Leu Leu Pro Phe65 70 75 80Val Gly Arg Pro Ala Pro Ala Leu Ala Gln
His Tyr Met Leu Thr Gln85 90 95Ala Leu Leu Pro Phe Thr Leu His Leu
His Ser Asn Gly Glu Val Glu100 105 110Thr Ala Ala Val Ala Pro Leu
Tyr Pro Ser Ala Ser Met Val Phe Phe115 120 125Gln Trp Ala Ser Ala
Gly Val Thr Val Gly Leu Val Ala Gly Ala Val130 135 140Leu Gly Arg
Met Ser Val Lys Ala Trp Met Ala Phe Val Pro Leu Trp145 150 155
160Thr Thr Leu Ser Tyr Thr Val Gly Ala Tyr Ser Ile Trp Gly Gly
Gly165 170 175Phe Leu Phe His Trp Gly Val Met Asp Tyr Ser Gly Gly
Tyr Val Val180 185 190His Leu Ala Ala Gly Val Ser Gly Tyr Thr Ala
Ala Tyr Trp Val Gly195 200 205Pro Arg Arg Lys Glu Glu Glu Glu Met
Thr Met Ala Gly Gly Gly Asn210 215 220Leu Val Ala Met Val Ala Gly
Ala Gly Ile Leu Trp Met Gly Trp Thr225 230 235 240Gly Phe Asn Gly
Gly Asp Pro Phe Ser Ala Asn Thr Asp Ser Ser Val245 250 255Ala Val
Leu Asn Thr His Ile Cys Thr Thr Thr Ser Ile Leu Ala Trp260 265
270Val Cys Cys Asp Ile Ala Val Arg Gly Arg Pro Ser Val Val Gly
Ala275 280 285Val Gln Gly Met Ile Thr Gly Leu Val Cys Ile Thr Pro
Ala Ala Gly290 295 300Leu Val Gln Gly Trp Ala Ala Leu Leu Met Gly
Val Ala Ser Gly Thr305 310 315 320Leu Pro Cys Tyr Thr Met Asn Ala
Ala Met Ser Phe Lys Val Asp Asp325 330 335Thr Leu Gly Ile Leu His
Thr His Ala Val Ser Gly Val Leu Gly Gly340 345 350Val Leu Thr Gly
Val Phe Ala His Pro Thr Leu Cys Asp Met Phe Leu355 360 365Pro Val
Thr Gly Ser Arg Gly Leu Val Tyr Gly Val Arg Ala Gly Gly370 375
380Val Gln Val Leu Lys Gln Val Ala Ala Ala Leu Phe Val Ala Ala
Trp385 390 395 400Asn Val Ala Ala Thr Ser Ile Ile Leu Val Val Val
Arg Ala Phe Val405 410 415Pro Leu Arg Met Thr Glu Asp Glu Leu Leu
Ala Gly Asp Ile Ala Val420 425 430His Gly Glu Gln Ala Tyr Tyr Phe
Ser Ser Gly Thr Asn Cys Ser Leu435 440 445Ser His Glu Thr Ile Glu
Val Gly Asn Ser450 455611750DNAGlycine max 61atttcatata tgtatatata
gcatcagaga gagaacaatt ctttgaaggg tgaaaaacct 60tgatcaagaa ttgaagcatt
aatcttcaac catggccaca cccttggcct accaagagca 120ccttccggcg
gcacccggtt ggctgaacaa aggtgacaac gcatggcagt taacagcagc
180caccctcgtt ggtcttcaaa gcatgccggg tctcgtgatc ctctacgcaa
gcatagtgaa 240gaagaaatgg gcagtgaatt cagctttcat ggctctctat
gcctttgcag cagttctaat 300atgttgggtg cttgtgtgtt accgcatggc
ctttggagaa gaacttttac ccttctgggg 360taagggtgct ccagcactag
gccagaagtt cctcacaaaa cgagccgtag tcaatgaaac 420catccaccac
tttgataatg gcactgttga atcacctcct gaggaaccct tttaccctat
480ggcctcgctt gtgtatttcc aattcacttt tgctgctatt actcttattt
tgttggctgg 540ctctgtcctt ggccgaatga acatcaaggc ttggatggct
tttgtgcctc tttggttgat 600cttttcctac acagtcgggg cttttagtct
ttggggtggt ggctttctct accaatgggg 660cgttattgat tattctggcg
gctatgtcat acacctttct tctggaatcg ctggcttcac 720tgctgcttac
tgggttggac caaggttgaa gagtgatagg gagaggttcc caccaaacaa
780tgtgcttctc atgcttgctg gtgctgggtt gttgtggatg ggttggtcag
ggttcaacgg 840tggagcacca tatgctgcaa acattgcatc ttcaattgcg
gtgttgaaca caaacatttg 900tgcagccact agcttccttg tgtggacaac
tttggatgtc attttttttg ggaaaccttc 960ggtgattgga gctgtgcagg
gcatgatgac tggacttgta tgcatcaccc caggggcagg 1020gcttgtgcat
tcatgggctg ttatagtgat gggaatatta tttgggagca ttccatgggt
1080gactatgatg attttgcata aaaagtcaac tttgctacag aaggtagatg
acacccttgg 1140tgtgtttcac acacatgctg tggctggcct tttgggtggt
ctcctcacag gtctattagc 1200agaaccagcc ctttgtagac ttctattgcc
agtaacaaat tcaaggggtg cattctatgg 1260tggaggtggt ggtgtgcagt
tcttcaagca attggtggcg gccatgtttg ttattggatg 1320gaacttggtg
tccaccacca ttattctcct tgtcataaaa ttgttcatac ccttgaggat
1380gccggacgag cagctggaaa tcggtgacga cgccgtccac ggtgaggaag
cttatgccct 1440ttggggtgat ggagaaaaat atgacccaac taggcatggt
tccttgcaaa gtggcaacac 1500tactgtctca ccttatgtta atggtgcaag
aggggtgact ataaacttat gagtcaagaa 1560attaggctgt gccttgctca
cacatgcatg tgtataaatt tatatgatta acaaatgtga 1620tgaatccgtg
agtggtataa gtagatattt gattttgtca tgaaagaaaa tttccaaatt
1680ttgagatgtg atgttcctct ggtcatcttg cattcgaaga ctctggtcat
atatttctgg 1740cacagaatgt 175062486PRTGlycine max 62Met Ala Thr Pro
Leu Ala Tyr Gln Glu His Leu Pro Ala Ala Pro Gly1 5 10 15Trp Leu Asn
Lys Gly Asp Asn Ala Trp Gln Leu Thr Ala Ala Thr Leu20 25 30Val Gly
Leu Gln Ser Met Pro Gly Leu Val Ile Leu Tyr Ala Ser Ile35 40 45Val
Lys Lys Lys Trp Ala Val Asn Ser Ala Phe Met Ala Leu Tyr Ala50 55
60Phe Ala Ala Val Leu Ile Cys Trp Val Leu Val Cys Tyr Arg Met Ala65
70 75 80Phe Gly Glu Glu Leu Leu Pro Phe Trp Gly Lys Gly Ala Pro Ala
Leu85 90 95Gly Gln Lys Phe Leu Thr Lys Arg Ala Val Val Asn Glu Thr
Ile His100 105 110His Phe Asp Asn Gly Thr Val Glu Ser Pro Pro Glu
Glu Pro Phe Tyr115 120 125Pro Met Ala Ser Leu Val Tyr Phe Gln Phe
Thr Phe Ala Ala Ile Thr130 135 140Leu Ile Leu Leu Ala Gly Ser Val
Leu Gly Arg Met Asn Ile Lys Ala145 150 155 160Trp Met Ala Phe Val
Pro Leu Trp Leu Ile Phe Ser Tyr Thr Val Gly165 170 175Ala Phe Ser
Leu Trp Gly Gly Gly Phe Leu Tyr Gln Trp Gly Val Ile180 185 190Asp
Tyr Ser Gly Gly Tyr Val Ile His Leu Ser Ser Gly Ile Ala Gly195 200
205Phe Thr Ala Ala Tyr Trp Val Gly Pro Arg Leu Lys Ser Asp Arg
Glu210 215 220Arg Phe Pro Pro Asn Asn Val Leu Leu Met Leu Ala Gly
Ala Gly Leu225 230 235 240Leu Trp Met Gly Trp Ser Gly Phe Asn Gly
Gly Ala Pro Tyr Ala Ala245 250 255Asn Ile Ala Ser Ser Ile Ala Val
Leu Asn Thr Asn Ile Cys Ala Ala260 265 270Thr Ser Phe Leu Val Trp
Thr Thr Leu Asp Val Ile Phe Phe Gly Lys275 280 285Pro Ser Val Ile
Gly Ala Val Gln Gly Met Met Thr Gly Leu Val Cys290 295 300Ile Thr
Pro Gly Ala Gly Leu Val His Ser Trp Ala Val Ile Val Met305 310 315
320Gly Ile Leu Phe Gly Ser Ile Pro Trp Val Thr Met Met Ile Leu
His325 330 335Lys Lys Ser Thr Leu Leu Gln Lys Val Asp Asp Thr Leu
Gly Val Phe340 345 350His Thr His Ala Val Ala Gly Leu Leu Gly Gly
Leu Leu Thr Gly Leu355 360 365Leu Ala Glu Pro Ala Leu Cys Arg Leu
Leu Leu Pro Val Thr Asn Ser370 375 380Arg Gly Ala Phe Tyr Gly Gly
Gly Gly Gly Val Gln Phe Phe Lys Gln385 390 395 400Leu Val Ala Ala
Met Phe Val Ile Gly Trp Asn Leu Val Ser Thr Thr405 410 415Ile Ile
Leu Leu Val Ile Lys Leu Phe Ile Pro Leu Arg Met Pro Asp420 425
430Glu Gln Leu Glu Ile Gly Asp Asp Ala Val His Gly Glu Glu Ala
Tyr435 440 445Ala Leu Trp Gly Asp Gly Glu Lys Tyr Asp Pro Thr Arg
His Gly Ser450 455 460Leu Gln Ser Gly Asn Thr Thr Val Ser Pro Tyr
Val Asn Gly Ala Arg465 470 475 480Gly Val Thr Ile Asn
Leu485632191DNAGlycine max 63cgtaatacac taaccaaccc accatgtcgc
tgcctgcttg tcccgccgaa caactggccc 60aacttctcgg cccaaacacc acagacgcct
ccgccgccgc ctcccttatc tgcggccatt 120tcgccgccgt ggacagcaag
ttcgtcgaca cggccttcgc cgtcgacaac acctacctcc 180tcttttccgc
ctacctcgtt ttttctatgc agctcggctt cgccatgctc tgcgccggct
240ccgtccgcgc caagaacacc atgaacatca tgctcaccaa cgtcctggac
gctgccgccg 300gcggcctctt ctactacctc ttcggcttcg ccttcgcttt
cggctccccc tccaacggct 360tcatcggtaa acatttcttc ggcctcaagg
acatcccttc atcctcctac gactacagct 420acttcctcta ccaatgggcc
ttcgccatcg ccgccgccgg catcaccagc ggaagcatcg 480ccgaacgcac
acagttcgtg gcctatctca tctactcctc cttcctcacc ggcttcgtct
540atccggtggt ctcccactgg ttctggtccc cagacggctg ggcctctgcc
tttaagatca 600ccgaccggct attttccacc ggcgtaatag acttcgccgg
ttccggcgta gtccacatgg 660tcggcggaat agccggccta tggggagcgc
tgatcgaagg cccaagaatg ggacgtttcg 720atcatgcagg acgagctgtg
gccttgcgag gccacagcgc gtccttagta gtcctgggaa 780ccttcttgct
ttggttcggt tggtacggat ttaaccccgg ttcatttaac aaaatcctac
840ttacttacgg taactcagga aattactacg gtcaatggag cgcggttggc
agaaccgcgg 900tcaccactac cctagcgggg tcaacagctg ccttgaccac
gctattcggt aaacgggtga 960tatccggtca ctggaacgtg accgatgtct
gcaacgggct gttaggcggt ttcgcggcga 1020taacagccgg ttgctccgtg
gttgagccat gggcagccat cgtatgcggt tttgttgctt 1080ctatagtatt
aatagcttgc aacaaattag cagagaaggt taagttcgac gatcctctgg
1140aggcggcgca gttgcacggt gggtgtggca cgtggggggt gatattcacg
gcgttgttcg 1200caaaaaagga gtatgtgaag gaggtttacg ggttggggag
ggcgcacggg ttgctcatgg 1260ggggtggtgg gaagttgctg gcggcgcacg
tgattcagat tctggtgatt gctgggtggg 1320ttagtgcgac catgggaccc
ttgttttggg ggttgaataa actgaagctg ttgaggattt 1380cttcagagga
tgagcttgcg gggatggaca tgactcgcca tggaggcttt gcttatgctt
1440atgaggatga tgagacgcac aagcatggga tgcagttgag gagggttggg
cccaacgcgt 1500cttccacacc caccactgat gaatgatctt tttttcccat
atgcatgtct cattagtcaa 1560acattaaatt tggatacata ttccttgtaa
ggattcaaac cttggttact tgttacttct 1620gttagatcca actccggttg
atactcatga ctttttactt cttttttttt tatttgtctt 1680gggtcttctt
ttttcgtaga tttttctttt tatgatgatg ggcaattagg gattttgatt
1740tgtaattgtc attggtcgtg cattggtgga tgctggaagt taaagattct
ggtggaagat 1800gcgtacgttt ctgtgggggg tggttgttga ctaaggcatg
ttggtcctgg aaatgacaga 1860tggctgtgga aaatggaaat ttgtgggatt
tatttttgta gttttcacca aaaaagaagg 1920aagaagattg gtatatagta
gaaatactac tgtttggccg tgaggcatat agtttttttt 1980tcttttcctt
aatttgagac ttttatgtta aactttttca ttatgtctaa tgtaaatata
2040tggaagtagt ttttatattt tactgcctga atgtttgttt tttgtgttat
atgtttttgt 2100ttatatggaa ttgaaatcga ttgtaatatg ttacgtggaa
gtaatgtaag ttaaaagatg 2160atgtaggtag tgttatttag tgtttttttt t
219164500PRTGlycine max 64Met Ser Leu Pro Ala Cys Pro Ala Glu Gln
Leu Ala Gln Leu Leu Gly1 5 10 15Pro Asn Thr Thr Asp Ala Ser Ala Ala
Ala Ser Leu Ile Cys Gly His20 25 30Phe Ala Ala Val Asp Ser Lys Phe
Val Asp Thr Ala Phe Ala Val Asp35 40 45Asn Thr Tyr Leu Leu Phe Ser
Ala Tyr Leu Val Phe Ser Met Gln Leu50 55 60Gly Phe Ala Met Leu Cys
Ala Gly Ser Val Arg Ala Lys Asn Thr Met65 70 75 80Asn Ile Met Leu
Thr Asn Val Leu Asp Ala Ala Ala Gly Gly Leu Phe85 90 95Tyr Tyr Leu
Phe Gly Phe Ala Phe Ala Phe Gly Ser Pro Ser Asn Gly100 105 110Phe
Ile Gly Lys His Phe Phe Gly Leu Lys Asp Ile Pro Ser Ser Ser115 120
125Tyr Asp Tyr Ser Tyr Phe Leu Tyr Gln Trp Ala Phe Ala Ile Ala
Ala130 135 140Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg Thr Gln
Phe Val Ala145 150 155 160Tyr Leu Ile Tyr Ser Ser Phe Leu Thr Gly
Phe Val Tyr Pro Val Val165 170 175Ser His Trp Phe Trp Ser Pro Asp
Gly Trp Ala Ser Ala Phe Lys Ile180 185 190Thr Asp Arg Leu Phe Ser
Thr Gly Val Ile Asp Phe Ala Gly Ser Gly195 200 205Val Val His Met
Val Gly Gly Ile Ala Gly Leu Trp Gly Ala Leu Ile210 215 220Glu Gly
Pro Arg Met Gly Arg Phe Asp His Ala Gly Arg Ala Val Ala225 230 235
240Leu Arg Gly His Ser Ala Ser Leu Val Val Leu Gly Thr Phe Leu
Leu245 250 255Trp Phe Gly Trp Tyr Gly Phe Asn Pro Gly Ser Phe Asn
Lys Ile Leu260 265 270Leu Thr Tyr Gly Asn Ser Gly Asn Tyr Tyr Gly
Gln Trp Ser Ala Val275 280 285Gly Arg Thr Ala Val Thr Thr Thr Leu
Ala Gly Ser Thr Ala Ala Leu290 295 300Thr Thr Leu Phe Gly Lys Arg
Val Ile Ser Gly His Trp Asn Val Thr305 310 315 320Asp Val Cys Asn
Gly Leu Leu Gly Gly Phe Ala Ala Ile Thr Ala Gly325 330 335Cys Ser
Val Val Glu Pro Trp Ala Ala Ile Val Cys Gly Phe Val Ala340 345
350Ser Ile Val Leu Ile Ala Cys Asn Lys Leu Ala Glu Lys Val Lys
Phe355 360 365Asp Asp Pro Leu Glu Ala Ala Gln Leu His Gly Gly Cys
Gly Thr Trp370 375 380Gly Val Ile Phe Thr Ala Leu Phe Ala Lys Lys
Glu Tyr Val Lys Glu385 390 395 400Val Tyr Gly Leu Gly Arg Ala His
Gly Leu Leu Met Gly Gly Gly
Gly405 410 415Lys Leu Leu Ala Ala His Val Ile Gln Ile Leu Val Ile
Ala Gly Trp420 425 430Val Ser Ala Thr Met Gly Pro Leu Phe Trp Gly
Leu Asn Lys Leu Lys435 440 445Leu Leu Arg Ile Ser Ser Glu Asp Glu
Leu Ala Gly Met Asp Met Thr450 455 460Arg His Gly Gly Phe Ala Tyr
Ala Tyr Glu Asp Asp Glu Thr His Lys465 470 475 480His Gly Met Gln
Leu Arg Arg Val Gly Pro Asn Ala Ser Ser Thr Pro485 490 495Thr Thr
Asp Glu50065800DNAGlycine max 65gcttctccca cctcaaacgc cgtcgtttcg
accaccttct tcggtcgcgg cacaaccaat 60aaccatgtcg ctgccagatt gtcccgccgt
ccaacttgcc caactcctgg gcccaaatac 120cacaaacgct gccgccgccg
cctccttcat ctgcgaccgg ttcaccgccg tggacaacaa 180gttcgtcgac
acggccttcg cggtcgacaa cacttacctc ctcttctccg cctacctcgt
240cttctcgatg cagctcggct tcgccatgct ctgcgccggc tccgtccgcg
ccaagaacac 300catgaacatc atgctcacca acgtcctcga cgccgccgcc
ggcggcctct tctactacct 360cttcggcttc gccttcgcct tcggctcccc
ctccaacggc ttcattggca aacacttctt 420cggcctcaag gaactcccct
cccaaagctt cgactacagc aactttctct atcaatgggc 480cttcgccatc
gccgccgccg gcatcaccag cggctccatc gccgaacgca cacagttcgt
540ggcctatctc atctactcct ccttcctcac cggcttcgtc taccccgtcg
tctcccactg 600gttctggtcc gcagacggct gggcttctgc catttccccc
ggagaccggc tattttccac 660cggcgtgata gacttcgccg gctccggcgt
agtccacatg gttggtggag tagccggctt 720ctggggcgca ctgatagaag
gcccgagaat cggacgcttc gaccacgcgg gacgcgccgt 780tgccctcaga
ggccacagcg 80066245PRTGlycine max 66Met Ser Leu Pro Asp Cys Pro Ala
Val Gln Leu Ala Gln Leu Leu Gly1 5 10 15Pro Asn Thr Thr Asn Ala Ala
Ala Ala Ala Ser Phe Ile Cys Asp Arg20 25 30Phe Thr Ala Val Asp Asn
Lys Phe Val Asp Thr Ala Phe Ala Val Asp35 40 45Asn Thr Tyr Leu Leu
Phe Ser Ala Tyr Leu Val Phe Ser Met Gln Leu50 55 60Gly Phe Ala Met
Leu Cys Ala Gly Ser Val Arg Ala Lys Asn Thr Met65 70 75 80Asn Ile
Met Leu Thr Asn Val Leu Asp Ala Ala Ala Gly Gly Leu Phe85 90 95Tyr
Tyr Leu Phe Gly Phe Ala Phe Ala Phe Gly Ser Pro Ser Asn Gly100 105
110Phe Ile Gly Lys His Phe Phe Gly Leu Lys Glu Leu Pro Ser Gln
Ser115 120 125Phe Asp Tyr Ser Asn Phe Leu Tyr Gln Trp Ala Phe Ala
Ile Ala Ala130 135 140Ala Gly Ile Thr Ser Gly Ser Ile Ala Glu Arg
Thr Gln Phe Val Ala145 150 155 160Tyr Leu Ile Tyr Ser Ser Phe Leu
Thr Gly Phe Val Tyr Pro Val Val165 170 175Ser His Trp Phe Trp Ser
Ala Asp Gly Trp Ala Ser Ala Ile Ser Pro180 185 190Gly Asp Arg Leu
Phe Ser Thr Gly Val Ile Asp Phe Ala Gly Ser Gly195 200 205Val Val
His Met Val Gly Gly Val Ala Gly Phe Trp Gly Ala Leu Ile210 215
220Glu Gly Pro Arg Ile Gly Arg Phe Asp His Ala Gly Arg Ala Val
Ala225 230 235 240Leu Arg Gly His Ser24567644DNAGlycine max
67cggtgcttaa caccaacatt tgcgccgcca ccagcctcct cgtatggacg tggttggacg
60ttattttctt caagaaaccc tcagttattg gagccgttca gggcatgata actggccttg
120tttgcatcac tcccggagct ggtctggttc aaggatgggc tgccatagtg
atgggacttc 180tttcaggcag tgtcccatgg ttcagcatga tggtattagg
gaaaaagctg aaattgtttc 240aaatggttga tgacaccctt gctgtgttcc
acactcatgc tgtggctggc cttcttggag 300gcatactcac tggcctattt
gccgaacctc gtctgtgtgc actctttcta cctgtcacca 360actccaaaag
aggagtctat ggaggccctg gtggagtcca aatccttaaa caaatcgtgg
420gagctttgtt catcattggg tggaaccttg tggtcacttc aattatttgt
gtggttatta 480gtttcatagt tccacttaga atgacagagg aagagcttct
cattggagat gatgcggttc 540atggggaaga ggcttatgct ctgtggggtg
atggagagaa acttagcatc tacaaagatg 600ataccactca ccatggagtt
gtgtctagtg gtgccactca agtg 64468204PRTGlycine max 68Val Leu Asn Thr
Asn Ile Cys Ala Ala Thr Ser Leu Leu Val Trp Thr1 5 10 15Trp Leu Asp
Val Ile Phe Phe Lys Lys Pro Ser Val Ile Gly Ala Val20 25 30Gln Gly
Met Ile Thr Gly Leu Val Cys Ile Thr Pro Gly Ala Gly Leu35 40 45Val
Gln Gly Trp Ala Ala Ile Val Met Gly Leu Leu Ser Gly Ser Val50 55
60Pro Trp Phe Ser Met Met Val Leu Gly Lys Lys Leu Lys Leu Phe Gln65
70 75 80Met Val Asp Asp Thr Leu Ala Val Phe His Thr His Ala Val Ala
Gly85 90 95Leu Leu Gly Gly Ile Leu Thr Gly Leu Phe Ala Glu Pro Arg
Leu Cys100 105 110Ala Leu Phe Leu Pro Val Thr Asn Ser Lys Arg Gly
Val Tyr Gly Gly115 120 125Pro Gly Gly Val Gln Ile Leu Lys Gln Ile
Val Gly Ala Leu Phe Ile130 135 140Ile Gly Trp Asn Leu Val Val Thr
Ser Ile Ile Cys Val Val Ile Ser145 150 155 160Phe Ile Val Pro Leu
Arg Met Thr Glu Glu Glu Leu Leu Ile Gly Asp165 170 175Asp Ala Val
His Gly Glu Glu Ala Tyr Ala Leu Trp Gly Asp Gly Glu180 185 190Lys
Leu Ser Ile Tyr Lys Asp Asp Thr Thr His His195 20069749DNAGlycine
max 69gccacaaaca attcatcagc tcatacacgt aatttctttt cctcttttcc
tcttatccaa 60ttctaatcac gatcagacat taaatgtaaa cacttctcta tcaaaaattt
gaacttagtt 120cgcctcacac ttttgttttg tcaccttgtg agagactaat
tccctctaat aaacgcaacg 180ttgttcatca gtggcacata catatacagc
atcacaattc tttgaagggt gaaaaagctt 240gatcaagaat tgaagcatat
tgatcttcag ccatggctac acccttggcc taccaagagc 300accttccggc
ggcacccgaa tggctgaaca aaggtgacaa cgcatggcag ctaacagcag
360ccaccctcgt cggtcttcaa agcatgccgg gtctcgtgat cctctacgcc
agcatagtga 420agaaaaaatg ggcagtgaac tcagctttca tggctctcta
cgcctttgcg gcggttctaa 480tatgttgggt gcttgtgtgt taccgcatgg
cctttggaga aaaactttta cccttctggg 540ggaagggtgc tcccagactt
aggccagaat tcgtcacaaa acgagccgga gtcaatgaaa 600cgctgcacca
ctttgatagt ggcactgtag aatcccctcg cgaagagcca ctttacccta
660atggcgtact tgtgtatgtc cgattgactt ttgctgctat gtaccatata
gtgatggctg 720gctctgtgct gccacgaaga acatcgaag 74970159PRTGlycine
max 70Met Ala Thr Pro Leu Ala Tyr Gln Glu His Leu Pro Ala Ala Pro
Glu1 5 10 15Trp Leu Asn Lys Gly Asp Asn Ala Trp Gln Leu Thr Ala Ala
Thr Leu20 25 30Val Gly Leu Gln Ser Met Pro Gly Leu Val Ile Leu Tyr
Ala Ser Ile35 40 45Val Lys Lys Lys Trp Ala Val Asn Ser Ala Phe Met
Ala Leu Tyr Ala50 55 60Phe Ala Ala Val Leu Ile Cys Trp Val Leu Val
Cys Tyr Arg Met Ala65 70 75 80Phe Gly Glu Lys Leu Leu Pro Phe Trp
Gly Lys Gly Ala Pro Arg Leu85 90 95Arg Pro Glu Phe Val Thr Lys Arg
Ala Gly Val Asn Glu Thr Leu His100 105 110His Phe Asp Ser Gly Thr
Val Glu Ser Pro Arg Glu Glu Pro Leu Tyr115 120 125Pro Asn Gly Val
Leu Val Tyr Val Arg Leu Thr Phe Ala Ala Met Tyr130 135 140His Ile
Val Met Ala Gly Ser Val Leu Pro Arg Arg Thr Ser Lys145 150
155711871DNAGlycine max 71ctctaacagc caaagcatgg cttctctctc
ttgctccgcc aacgaccttg ccccactctt 60caacgacacc gccgccgcca actacctctg
cgcccaattc gattccattt ctagaaagct 120cgccgaaaca acctacgccg
tcgacaacac ctaccttctg ttttcagcgt atcttgtctt 180cgccatgcag
ctcggcttcg ccatgctctg cgccggctcc gtcagagcca aaaacaccat
240gaacatcatg ctcaccaacg tcctcgacgc cgccgccggc ggtctctcct
actacctatt 300cggctttgca ttcgccttcg gcggcccctc caacggcttc
atcggccgcc acttcttcgg 360cctacgagat tacccaatgg gctcctctcc
ctccggcgac tacagcttct tcctctacca 420gtgggccttc gccatcgccg
ccgcaggaat caccagcggc tccatcgccg agagaacaca 480gttcgtggct
taccttatct actcttcttt cttaaccggt ttcgtttacc ccatcgtttc
540gcattggttc tggtcctcag acggttgggc cagcgcgact cgtagccacg
gaaatgtttt 600attcgggtct ggagtcatcg acttcgcggg ctcaggcgtt
gttcacatgg ttggcgggat 660agcgggcctg tggggggctt taattgaagg
cccgagaatc ggccggttcg accgttcggg 720ccggtcggtt gctttacgtg
gccacagcgc gtctttagtt gtgcttggta cgtttttgtt 780atggttcggc
tggtacggct tcaaccctgg ttcgtttgtg acaatagaca aggggtatga
840aagtggaggg tattatggtc aatggagcgc tatagggagg acagctgtca
cgacgacatt 900ggctgggagc actgcggctc tgacgacgtt gttcagcaag
cggttattgg ttggccactg 960gaacgtgatt gacgtgtgta acggcctgct
tggcgggttc gctgccatta catcgggctg 1020tgccgttgtg gaaccgtggg
ccgcgattgt gtgtgggttt gtggcggcgt gggttttgat 1080tgggcttaat
aagcttgccg cgaaggtaga gtacgatgat ccgttggagg cggcgcagct
1140tcacggcggg tgcggcgcgt ggggtgtttt cttcacggga ttgtttgcga
agaaagtgta 1200cgtggaggag atttacggtg ttggaaggcc gttcggggct
ttgatgggtg gcggagggag 1260gctgctggcg gcgcaggtga ttcagatatt
ggtggtgtgc gggtgggtta cggcgaccat 1320ggcgccgttg ttctatgggc
ttcataagat gaaactgttg agaatttcga gggatgatga 1380gactgcgggg
atggatttga cgaggcatgg tgggtttgct tatgcatacc atgatgatga
1440agatggttca agcaggggag tagggttcat gctgcgtaga attgagcctg
ctgctagtac 1500cactccctct ccccccgctg caccacaagt ttaatcaaaa
tgtggtttat gattttcaag 1560cgttttttag tttcgtacct gcacatagct
atgggcaaag ctagccttgt caaaaccata 1620tacaagcaag acacgaggga
tgcatatatg aagtataaaa attaatgcgt gggggtcaac 1680atttaggaaa
tgtcttctag agttactgta cattttaaaa tgtttgttgg cttggtttat
1740tattttcatc tttgaattcc aagactagtt tggtcgactg ttgtcacgtt
agtttgtatc 1800ctgctgcaga ataacttgct tgtaattgta tactgattag
ttggtatata gtgatatatt 1860atatatacta a 187172505PRTGlycine max
72Met Ala Ser Leu Ser Cys Ser Ala Asn Asp Leu Ala Pro Leu Phe Asn1
5 10 15Asp Thr Ala Ala Ala Asn Tyr Leu Cys Ala Gln Phe Asp Ser Ile
Ser20 25 30Arg Lys Leu Ala Glu Thr Thr Tyr Ala Val Asp Asn Thr Tyr
Leu Leu35 40 45Phe Ser Ala Tyr Leu Val Phe Ala Met Gln Leu Gly Phe
Ala Met Leu50 55 60Cys Ala Gly Ser Val Arg Ala Lys Asn Thr Met Asn
Ile Met Leu Thr65 70 75 80Asn Val Leu Asp Ala Ala Ala Gly Gly Leu
Ser Tyr Tyr Leu Phe Gly85 90 95Phe Ala Phe Ala Phe Gly Gly Pro Ser
Asn Gly Phe Ile Gly Arg His100 105 110Phe Phe Gly Leu Arg Asp Tyr
Pro Met Gly Ser Ser Pro Ser Gly Asp115 120 125Tyr Ser Phe Phe Leu
Tyr Gln Trp Ala Phe Ala Ile Ala Ala Ala Gly130 135 140Ile Thr Ser
Gly Ser Ile Ala Glu Arg Thr Gln Phe Val Ala Tyr Leu145 150 155
160Ile Tyr Ser Ser Phe Leu Thr Gly Phe Val Tyr Pro Ile Val Ser
His165 170 175Trp Phe Trp Ser Ser Asp Gly Trp Ala Ser Ala Thr Arg
Ser His Gly180 185 190Asn Val Leu Phe Gly Ser Gly Val Ile Asp Phe
Ala Gly Ser Gly Val195 200 205Val His Met Val Gly Gly Ile Ala Gly
Leu Trp Gly Ala Leu Ile Glu210 215 220Gly Pro Arg Ile Gly Arg Phe
Asp Arg Ser Gly Arg Ser Val Ala Leu225 230 235 240Arg Gly His Ser
Ala Ser Leu Val Val Leu Gly Thr Phe Leu Leu Trp245 250 255Phe Gly
Trp Tyr Gly Phe Asn Pro Gly Ser Phe Val Thr Ile Asp Lys260 265
270Gly Tyr Glu Ser Gly Gly Tyr Tyr Gly Gln Trp Ser Ala Ile Gly
Arg275 280 285Thr Ala Val Thr Thr Thr Leu Ala Gly Ser Thr Ala Ala
Leu Thr Thr290 295 300Leu Phe Ser Lys Arg Leu Leu Val Gly His Trp
Asn Val Ile Asp Val305 310 315 320Cys Asn Gly Leu Leu Gly Gly Phe
Ala Ala Ile Thr Ser Gly Cys Ala325 330 335Val Val Glu Pro Trp Ala
Ala Ile Val Cys Gly Phe Val Ala Ala Trp340 345 350Val Leu Ile Gly
Leu Asn Lys Leu Ala Ala Lys Val Glu Tyr Asp Asp355 360 365Pro Leu
Glu Ala Ala Gln Leu His Gly Gly Cys Gly Ala Trp Gly Val370 375
380Phe Phe Thr Gly Leu Phe Ala Lys Lys Val Tyr Val Glu Glu Ile
Tyr385 390 395 400Gly Val Gly Arg Pro Phe Gly Ala Leu Met Gly Gly
Gly Gly Arg Leu405 410 415Leu Ala Ala Gln Val Ile Gln Ile Leu Val
Val Cys Gly Trp Val Thr420 425 430Ala Thr Met Ala Pro Leu Phe Tyr
Gly Leu His Lys Met Lys Leu Leu435 440 445Arg Ile Ser Arg Asp Asp
Glu Thr Ala Gly Met Asp Leu Thr Arg His450 455 460Gly Gly Phe Ala
Tyr Ala Tyr His Asp Asp Glu Asp Gly Ser Ser Arg465 470 475 480Gly
Val Gly Phe Met Leu Arg Arg Ile Glu Pro Ala Ala Ser Thr Thr485 490
495Pro Ser Pro Pro Ala Ala Pro Gln Val500 505731053DNAGlycine max
73tttcacacac atgctgtggc tggccttttg ggtggtctcc tcacaggtct attagcagaa
60ccagcccttt gtagactact attgccagtt accaactcaa ggggtgcatt ctatggtggt
120ggtggtggta tgcagttctt caagcaattg gtggcggcca tgtttgtcat
tggatggaac 180ttggtgtcca ccaccatcat tctccttgtc ataaaattgt
tcataccctt gaggatgccg 240gatgagcagc tggaaatcgg cgacgacgcc
gtccacggcg aggaagctta tgccctctgg 300ggtgatggag aaaaatatga
cccaactagg catggttcct tgcaaagtgg caacactttt 360gtgtcacctt
atgttaatgg tgcaagaggg gtgaccataa acttatgagt caagaaattc
420ggctgtgctt tgctcacaca tatgtataaa gttatgtgat gaatccgtga
gtggtgtaag 480tagaaatttg attttgtcat gaaagaaaat tcaagttttg
agatctgatg ttcctctggc 540catccagcat tcgaagacct gatcatatat
ttctggcaca gattgtgttg acatgtttat 600aaaatttaga tttgtcaatt
tttgaaggag cttgtgatta gttttctttt ccacttatat 660gttttaatta
ctagaagaat atcaaatttt ctttttacga aatgcttagt acataattgt
720taaaaaaaat catcatgtaa tgggtacgaa atatttatca attctatgaa
tgagtatttt 780tttcttagat aacttcagtg accactttta gaaaatttat
cctatgtata aattttaaaa 840gaatggtttt aactccaaaa ttttcaccta
gtccttgtca aacaaatttt attttggctc 900acttaaaggt aaaattattt
agttatgcat ttcagaatga agtttggttc gaaatatttt 960gacagtgtgt
caaatataaa ttcttcaaaa gaaaaagcca agactacttt acaacaaaat
1020agataagttt ctcataaact gagcacaagt ttt 105374135PRTGlycine max
74Phe His Thr His Ala Val Ala Gly Leu Leu Gly Gly Leu Leu Thr Gly1
5 10 15Leu Leu Ala Glu Pro Ala Leu Cys Arg Leu Leu Leu Pro Val Thr
Asn20 25 30Ser Arg Gly Ala Phe Tyr Gly Gly Gly Gly Gly Met Gln Phe
Phe Lys35 40 45Gln Leu Val Ala Ala Met Phe Val Ile Gly Trp Asn Leu
Val Ser Thr50 55 60Thr Ile Ile Leu Leu Val Ile Lys Leu Phe Ile Pro
Leu Arg Met Pro65 70 75 80Asp Glu Gln Leu Glu Ile Gly Asp Asp Ala
Val His Gly Glu Glu Ala85 90 95Tyr Ala Leu Trp Gly Asp Gly Glu Lys
Tyr Asp Pro Thr Arg His Gly100 105 110Ser Leu Gln Ser Gly Asn Thr
Phe Val Ser Pro Tyr Val Asn Gly Ala115 120 125Arg Gly Val Thr Ile
Asn Leu130 13575799DNAGlycine max 75gtgtgtggtt ttgtcgcttc
agtgtttctg atagcgtgca acaaattagc agagaaggtt 60aagttcgatg atcctttgga
agcggcgcag ttacacggtg ggtgtggcgc gtggggggtg 120atattcacgg
cgctgttcgc gaaaaaggag tatgtgagcc aggtttatgg ggaggggagg
180gcgcacgggt tgttcatgag gggtggaggg aagttgctgg cggcgcacgt
gattcagatt 240ttggttattg ttgggtgggt gagtgcgacc atgggaccct
tgttttgggg gttgaataaa 300ttgaaattgt tgaggatttc ttccgaggat
gagcttgcgg ggatggatct tacccgtcat 360ggaggatttg cttatgctta
tgaggatgat gagtcgcaca agcatgggat tcagctgagg 420aaggttgggc
ccaacgcgtc gtccacaccc accactgatg aatgattacg atcacgatta
480attcggcccc gacagtatta tcttcaattg aaattacgtg tgacttagaa
gaagaaaaaa 540agatgatgat gattttgttt gtaatttatt ttatttgttt
tgggtttttt ttttaatttt 600gtagattttt ctttttatga tgggtaagta
gggattttaa tttgtaattg ttattggccg 660tatattggta gatgctggaa
attgaagatt ctgctggaag atgcgaacgt ttctgaaaat 720gatagatggc
tgtggaaaat gaaaatattt tatttgtggg atttaatttt cgtagttttc
780gccaaaaaag aaggaagag 79976154PRTGlycine max 76Val Cys Gly Phe
Val Ala Ser Val Phe Leu Ile Ala Cys Asn Lys Leu1 5 10 15Ala Glu Lys
Val Lys Phe Asp Asp Pro Leu Glu Ala Ala Gln Leu His20 25 30Gly Gly
Cys Gly Ala Trp Gly Val Ile Phe Thr Ala Leu Phe Ala Lys35 40 45Lys
Glu Tyr Val Ser Gln Val Tyr Gly Glu Gly Arg Ala His Gly Leu50 55
60Phe Met Arg Gly Gly Gly Lys Leu Leu Ala Ala His Val Ile Gln Ile65
70 75 80Leu Val Ile Val Gly Trp Val Ser Ala Thr Met Gly Pro Leu Phe
Trp85 90 95Gly Leu Asn Lys Leu Lys Leu Leu Arg Ile Ser Ser Glu Asp
Glu Leu100 105 110Ala Gly Met Asp Leu Thr Arg His Gly Gly Phe Ala
Tyr Ala Tyr Glu115 120 125Asp Asp Glu Ser His Lys His Gly Ile Gln
Leu Arg Lys Val Gly Pro130 135 140Asn Ala Ser Ser Thr Pro Thr Thr
Asp Glu145 1507790DNAGlycine max 77tttctctacc aatggggggt tattgactat
tctggcggct atgtcatcca cctttcttct 60ggaatcgctg gtttaactgc tgcttactgg
907830PRTGlycine max 78Phe Leu Tyr Gln Trp Gly Val Ile Asp Tyr Ser
Gly Gly Tyr Val Ile1 5 10 15His Leu Ser Ser Gly Ile Ala Gly Leu Thr
Ala Ala Tyr
Trp20 25 3079459DNAGlycine max 79caaattcgct ttacatacag tatggtaatt
gtccaaattt ttacgaccga tttgtcaggt 60acatcattta atgcatggca acatacatga
taagatgaat caataaatac attccagctt 120ccacgtacgt acgtctgcca
acatagccgg cctcataatg tctcatccaa gtaaataaaa 180cgacaaaatg
attgattgta taaacctgct gcaaataact cagtatcata aagccttggc
240cttgaacacc ctcactcgag ttttcagcca attaaccaaa tcacactgaa
acactgaagt 300actagttatt caactactag taataagcat aattaaatat
agaggagccg aagacgaagc 360aagcccagaa aggttgaaca aaggagacaa
cgcatggcag ttaatggcag ccacagtggt 420gggtatggtg attctctatg
gaagcctaga gtgaaaaag 4598028PRTGlycine max 80Pro Glu Arg Leu Asn
Lys Gly Asp Asn Ala Trp Gln Leu Met Ala Ala1 5 10 15Thr Val Val Gly
Met Val Ile Leu Tyr Gly Ser Leu20 2581451DNAGlycine max
81acttgtgcta cccatggcca ctcccacagc ataccaagaa cacctccctg catcccccca
60ctggctaaac aaaggggaca acgcatggca gctgacagca gccactctcg taggtctcca
120aagcatgccg ggtctggtga tcctctacgc cagcatggtg aagaagaaat
gggccgtgaa 180ctctgcattc atggccctct acgcctttgc agcagtccta
atatgctggg tgctcgtttg 240tcaccgaatg gccttcggtg acaaactcct
tcccttctgg gggaagggcg ccccagcact 300aggccagaag tttttaacac
accgcgccaa agtccccgaa agcacgcact attataacaa 360tggtacggtc
gaaagcgcga cttcggaacc gttgtttgcc acggcttctc ttgtgtattt
420tcaattcacg tttgcggcta tcacgcttat c 45182146PRTGlycine max 82Met
Ala Thr Pro Thr Ala Tyr Gln Glu His Leu Pro Ala Ser Pro His1 5 10
15Trp Leu Asn Lys Gly Asp Asn Ala Trp Gln Leu Thr Ala Ala Thr Leu20
25 30Val Gly Leu Gln Ser Met Pro Gly Leu Val Ile Leu Tyr Ala Ser
Met35 40 45Val Lys Lys Lys Trp Ala Val Asn Ser Ala Phe Met Ala Leu
Tyr Ala50 55 60Phe Ala Ala Val Leu Ile Cys Trp Val Leu Val Cys His
Arg Met Ala65 70 75 80Phe Gly Asp Lys Leu Leu Pro Phe Trp Gly Lys
Gly Ala Pro Ala Leu85 90 95Gly Gln Lys Phe Leu Thr His Arg Ala Lys
Val Pro Glu Ser Thr His100 105 110Tyr Tyr Asn Asn Gly Thr Val Glu
Ser Ala Thr Ser Glu Pro Leu Phe115 120 125Ala Thr Ala Ser Leu Val
Tyr Phe Gln Phe Thr Phe Ala Ala Ile Thr130 135 140Leu Ile145
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