U.S. patent application number 14/497793 was filed with the patent office on 2015-01-29 for plants having enhanced yield-related traits and a method for making the same.
The applicant listed for this patent is Crop Functional Genomics Center, CropDesign N.V.. Invention is credited to Yang Do Choi, Ho Hee Jang, Sang Yeol Lee, Ohkmae K. Park, Huh Sun Mi.
Application Number | 20150033412 14/497793 |
Document ID | / |
Family ID | 39735038 |
Filed Date | 2015-01-29 |
United States Patent
Application |
20150033412 |
Kind Code |
A1 |
Lee; Sang Yeol ; et
al. |
January 29, 2015 |
PLANTS HAVING ENHANCED YIELD-RELATED TRAITS AND A METHOD FOR MAKING
THE SAME
Abstract
The present invention relates generally to the field of
molecular biology and concerns a method for enhancing plant
yield-related traits relative to control plants. More specifically,
the present invention concerns a method for enhancing yield related
traits in plants relative to control plants, by modulating,
preferably increasing, expression in the roots of a plant, of a
nucleic acid sequence encoding a 2-cysteine peroxiredoxin (2-Cys
PRX); or by modulating expression of a nucleic acid encoding an ANN
polypeptide in a plant. The present invention also concerns plants
having modulated, preferably increased, expression in the roots, of
a nucleic acid sequence encoding a 2-Cys PRX, or having modulated
expression of a nucleic acid encoding an ANN polypeptide, which
plants have enhanced yield-related traits relative to control
plants. The invention also provides constructs useful in the
methods of the invention.
Inventors: |
Lee; Sang Yeol; (Seoul,
KR) ; Choi; Yang Do; (Seoul, KR) ; Jang; Ho
Hee; (Seoul, KR) ; Park; Ohkmae K.; (Seoul,
KR) ; Sun Mi; Huh; (Seoul, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CropDesign N.V.
Crop Functional Genomics Center |
Zwijnaarde
Seoul |
|
BE
KR |
|
|
Family ID: |
39735038 |
Appl. No.: |
14/497793 |
Filed: |
September 26, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12601023 |
Nov 20, 2009 |
8878006 |
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PCT/EP2008/056381 |
May 23, 2008 |
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14497793 |
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60932226 |
May 29, 2007 |
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60937994 |
Jun 29, 2007 |
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Current U.S.
Class: |
800/287 ;
435/320.1; 435/419; 435/468; 800/290; 800/298; 800/320; 800/320.1;
800/320.2; 800/320.3 |
Current CPC
Class: |
C07K 14/415 20130101;
C12N 15/8223 20130101; C12N 15/8216 20130101; C12N 15/8261
20130101; Y02A 40/146 20180101 |
Class at
Publication: |
800/287 ;
800/290; 800/298; 435/320.1; 435/468; 435/419; 800/320; 800/320.2;
800/320.1; 800/320.3 |
International
Class: |
C12N 15/82 20060101
C12N015/82; C07K 14/415 20060101 C07K014/415 |
Foreign Application Data
Date |
Code |
Application Number |
May 23, 2007 |
EP |
07108768.8 |
Jun 12, 2007 |
EP |
07110086.1 |
Claims
1. A method for enhancing a yield-related trait in a plant relative
to a control plant, comprising introducing and expressing in a
plant, plant part, or plant cell a nucleic acid encoding an
Annexin-like (ANN) polypeptide, wherein said ANN polypeptide
comprises at least an annexin domain and one or more signature
sequences of SEQ ID NO: 87, 88, 89, 90, 91, 92 and 93.
2. The method of claim 1, wherein said nucleic acid comprises: i)
any one of the nucleic acids listed in Table B1; ii) a nucleotide
sequence capable of hybridizing with any one of the nucleic acids
listed in Table B1 under conditions comprising hybridization at
50.degree. C. in 4.times.SSC or at 40.degree. C. in 6.times.SSC and
50% formamide, followed by washing at 50.degree. C. in 2.times.SSC;
or iii) a nucleotide sequence encoding any one of the proteins
listed in Table B1.
3. The method of claim 1, wherein said nucleic acid comprises: i)
the nucleotide sequence of SEQ ID NO: 83; ii) a nucleotide sequence
encoding a polypeptide comprising the amino acid sequence of SEQ ID
NO: 84; or iii) a nucleotide sequence capable of hybridizing with
the nucleotide sequence of SEQ ID NO: 83 under conditions
comprising hybridisation at 50.degree. C. in 4.times.SSC or at
40.degree. C. in 6.times.SSC and 50% formamide, followed by washing
at 50.degree. C. in 2.times.SSC.
4. The method of claim 1, wherein the enhanced yield-related trait
comprises increased yield and/or increased seed yield relative to a
control plant.
5. The method of claim 1, wherein the enhanced yield-related trait
is obtained under non-stress conditions.
6. The method of claim 1, wherein the enhanced yield-related trait
is obtained under conditions of drought.
7. The method of claim 1, wherein said nucleic acid is operably
linked to a constitutive promoter, a GOS2 promoter, or a GOS2
promoter from rice.
8. The method of claim 1, wherein said nucleic acid is operably
linked to a green-tissue specific promoter, an expansin promoter,
or an expansin promoter from rice.
9. The method of claim 1, wherein said nucleic acid is of plant
origin, from a dicotyledonous plant, from a plant of the family
Brassicaceae, from a plant of the genus Arabidopsis, or from an
Arabidopsis thaliana plant.
10. A plant obtained by the method of claim 1, or a plant part,
seed, or progeny of said plant, wherein said plant, or said plant
part, seed, or progeny, comprises a recombinant nucleic acid
encoding said ANN polypeptide.
11. A construct comprising: a) a nucleic acid sequence encoding an
ANN polypeptide as defined in claim 1; b) one or more control
sequences capable of driving expression of the nucleic acid
sequence of a); and optionally c) a transcription termination
sequence.
12. The construct of claim 11, wherein one of the control sequences
is a constitutive promoter, a GOS2 promoter, or a GOS2 promoter
from rice.
13. The construct of claim 11, wherein one of the control sequences
is a green-tissue specific promoter, an expansin promoter, or an
expansin promoter from rice.
14. A method for making a plant having increased yield, increased
biomass, and/or increased seed yield relative to a control plant,
comprising transforming the construct of claim 11 into a plant,
plant part, or plant cell.
15. A plant, plant part, or plant cell comprising the construct of
claim 11.
16. A method for the production of a transgenic plant having
increased yield and/or increased seed yield relative to a control
plant, comprising: a) introducing and expressing in a plant a
nucleic acid encoding an ANN polypeptide as defined in claim 1; and
b) cultivating the plant under conditions promoting plant growth
and development.
17. A transgenic plant having increased yield and/or increased seed
yield relative to a control plant, resulting from increased
expression of a nucleic acid encoding an ANN polypeptide as defined
in claim 1, or a transgenic plant cell derived from said transgenic
plant.
18. The transgenic plant of claim 17, wherein said plant is a crop
plant, a monocot, or a cereal, or wherein said plant is rice,
maize, wheat, barley, millet, rye, triticale, sorghum, or oats.
19. Harvestable parts of the transgenic plant of claim 17, wherein
said harvestable parts are preferably seeds.
20. Products derived from the transgenic plant of claim 17 or
harvestable parts of said transgenic plant.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of patent application Ser.
No. 12/601,023 filed Nov. 20, 2009, which is a national stage
application (under 35 U.S.C. .sctn.371) of PCT/EP2008/056381, filed
May 23, 2008, which claims benefit of European Application
07108768.8, filed May 23, 2007; U.S. Provisional Application
60/932,226, filed May 29, 2007, European Application 07110086.1,
filed Jun. 12, 2007, and U.S. Provisional Application 60/937,994,
filed Jun. 29, 2007. The entire content of each aforementioned
application is hereby incorporated by reference in its
entirety.
SUBMISSION OF SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is
Sequence_Listing.sub.--074029.sub.--0054.sub.--01. The size of the
text file is 335 KB, and the text file was created on Sep. 26,
2014.
[0003] The present invention relates generally to the field of
molecular biology and concerns a method for enhancing plant
yield-related traits relative to control plants. More specifically,
the present invention concerns a method for enhancing yield related
traits in plants relative to control plants, by modulating,
preferably increasing, expression in the roots of a plant, of a
nucleic acid sequence encoding a 2-cysteine peroxiredoxin (2-Cys
PRX); or by modulating expression of a nucleic acid encoding an ANN
polypeptide in a plant. The present invention also concerns plants
having modulated, preferably increased, expression in the roots, of
a nucleic acid sequence encoding a 2-Cys PRX, or having modulated
expression of a nucleic acid encoding an ANN polypeptide, which
plants have enhanced yield-related traits relative to control
plants. The invention also provides constructs useful in the
methods of the invention.
[0004] The ever-increasing world population and the dwindling
supply of arable land available for agriculture fuels research
towards increasing the efficiency of agriculture. Conventional
means for crop and horticultural improvements utilise selective
breeding techniques to identify plants having desirable
characteristics. However, such selective breeding techniques have
several drawbacks, namely that these techniques are typically
labour intensive and result in plants that often contain
heterogeneous genetic components that may not always result in the
desirable trait being passed on from parent plants. Advances in
molecular biology have allowed mankind to modify the germplasm of
animals and plants. Genetic engineering of plants entails the
isolation and manipulation of genetic material (typically in the
form of DNA or RNA) and the subsequent introduction of that genetic
material into a plant. Such technology has the capacity to deliver
crops or plants having various improved economic, agronomic or
horticultural traits.
[0005] A trait of particular economic interest is increased yield.
Yield is normally defined as the measurable produce of economic
value from a crop. This may be defined in terms of quantity and/or
quality. Yield is directly dependent on several factors, for
example, the number and size of the organs, plant architecture (for
example, the number of branches), seed production, leaf senescence
and more. Root development, nutrient uptake, stress tolerance and
early vigour may also be important factors in determining yield.
Optimizing the abovementioned factors may therefore contribute to
increasing crop yield.
[0006] Seed yield is a particularly important trait, since the
seeds of many plants are important for human and animal nutrition.
Crops such as corn, rice, wheat, canola and soybean account for
over half the total human caloric intake, whether through direct
consumption of the seeds themselves or through consumption of meat
products raised on processed seeds. They are also a source of
sugars, oils and many kinds of metabolites used in industrial
processes. Seeds contain an embryo (the source of new shoots and
roots) and an endosperm (the source of nutrients for embryo growth
during germination and during early growth of seedlings). The
development of a seed involves many genes, and requires the
transfer of metabolites from the roots leaves and stems into the
growing seed. The endosperm, in particular, assimilates the
metabolic precursors of carbohydrates, oils and proteins and
synthesizes them into storage macromolecules to fill out the
grain.
[0007] Another important trait for many crops is early vigour.
Improving early vigour is an important objective of modern rice
breeding programs in both temperate and tropical rice cultivars.
Long roots are important for proper soil anchorage in water-seeded
rice. Where rice is sown directly into flooded fields, and where
plants must emerge rapidly through water, longer shoots are
associated with vigour. Where drill-seeding is practiced, longer
mesocotyls and coleoptiles are important for good seedling
emergence. The ability to engineer early vigour into plants would
be of great importance in agriculture. For example, poor early
vigour has been a limitation to the introduction of maize (Zea mays
L.) hybrids based on Corn Belt germplasm in the European
Atlantic.
[0008] Plant biomass is yield for forage crops like alfalfa, silage
corn and hay. Many proxies for yield have been used in grain crops.
Chief amongst these are estimates of plant size. Plant size can be
measured in many ways depending on species and developmental stage,
but include total plant dry weight, above-ground dry weight,
above-ground fresh weight, leaf area, stem volume, plant height,
rosette diameter, leaf length, root length, root mass, tiller
number and leaf number. Many species maintain a conservative ratio
between the size of different parts of the plant at a given
developmental stage. These allometric relationships are used to
extrapolate from one of these measures of size to another (e.g.
Tittonell et al 2005 Agric Ecosys & Environ 105: 213). Plant
size at an early developmental stage will typically correlate with
plant size later in development. A larger plant with a greater leaf
area can typically absorb more light and carbon dioxide than a
smaller plant and therefore will likely gain a greater weight
during the same period (Fasoula & Tollenaar 2005 Maydica
50:39). This is in addition to the potential continuation of the
micro-environmental or genetic advantage that the plant had to
achieve the larger size initially. There is a strong genetic
component to plant size and growth rate (e.g. ter Steege et al 2005
Plant Physiology 139:1078), and so for a range of diverse genotypes
plant size under one environmental condition is likely to correlate
with size under another (Hittalmani et al 2003 Theoretical Applied
Genetics 107:679). In this way a standard environment is used as a
proxy for the diverse and dynamic environments encountered at
different locations and times by crops in the field.
[0009] Harvest index, the ratio of seed yield to aboveground dry
weight, is relatively stable under many environmental conditions
and so a robust correlation between plant size and grain yield can
often be obtained (e.g. Rebetzke et al 2002 Crop Science 42:739).
These processes are intrinsically linked because the majority of
grain biomass is dependent on current or stored photosynthetic
productivity by the leaves and stem of the plant (Gardener et al
1985 Physiology of Crop Plants. Iowa State University Press, pp
68-73). Therefore, selecting for plant size, even at early stages
of development, has been used as an indicator for future potential
yield (e.g. Tittonell et al 2005 Agric Ecosys & Environ 105:
213). When testing for the impact of genetic differences on stress
tolerance, the ability to standardize soil properties, temperature,
water and nutrient availability and light intensity is an intrinsic
advantage of greenhouse or plant growth chamber environments
compared to the field. However, artificial limitations on yield due
to poor pollination due to the absence of wind or insects, or
insufficient space for mature root or canopy growth, can restrict
the use of these controlled environments for testing yield
differences. Therefore, measurements of plant size in early
development, under standardized conditions in a growth chamber or
greenhouse, are standard practices to provide indication of
potential genetic yield advantages.
[0010] Another trait of importance is that of improved abiotic
stress tolerance. Abiotic stress is a primary cause of crop loss
worldwide, reducing average yields for most major crop plants by
more than 50% (Wang et al. (2003) Planta 218: 1-14). Abiotic
stresses may be caused by drought, salinity, extremes of
temperature, chemical toxicity, excess or lack of nutrients
(macroelements and/or microelements), radiation and oxidative
stress. The ability to increase plant tolerance to abiotic stress
would be of great economic advantage to farmers worldwide and would
allow for the cultivation of crops during adverse conditions and in
territories where cultivation of crops may not otherwise be
possible.
[0011] Crop yield may therefore be increased by optimising one of
the above-mentioned factors.
[0012] Depending on the end use, the modification of certain yield
traits may be favoured over others. For example for applications
such as forage or wood production, or bio-fuel resource, an
increase in the vegetative parts of a plant may be desirable, and
for applications such as flour, starch or oil production, an
increase in seed parameters may be particularly desirable. Even
amongst the seed parameters, some may be favoured over others,
depending on the application. Various mechanisms may contribute to
increasing seed yield, whether that is in the form of increased
seed size or increased seed number.
[0013] One approach to enhance yield-related traits (for example
increasing yield, in particular seed yield and/or biomass) in
plants may be through modification of the inherent growth
mechanisms of a plant, such as the cell cycle or various signalling
pathways involved in plant growth or in defense mechanisms.
[0014] Surprisingly, it has now been found that various plant
yield-related traits may be enhanced relative to control plants, by
modulating, preferably increasing, expression in the roots of a
plant, of a nucleic acid sequence encoding a 2-cysteine
peroxiredoxin (2-Cys PRX), or by modulating expression of a nucleic
acid encoding an ANN polypeptide in a plant.
[0015] According to one embodiment, there is provided a method for
enhancing various yield-related traits relative to control plants,
comprising modulating, preferably increasing, expression in the
roots of a plant, of a nucleic acid sequence encoding a 2-cysteine
peroxiredoxin (2-Cys PRX), or by modulating expression of a nucleic
acid encoding an ANN polypeptide, in a plant.
BACKGROUND
1. 2-Cysteine Peroxiredoxin (2-Cys PRX)
[0016] Thiol peroxidases (PRX) are ubiquitous heme-free
peroxidases, which catalyze the reduction of peroxynitrites and of
various peroxides by catalytic cysteine residues and
thiol-containing proteins as reductants. In plants, five different
classes can be distinguished, according to the number and the
position of conserved catalytic cysteines. Four classes are defined
as peroxiredoxins and were already identified by phylogenetic
sequence analysis, 1-Cys, 2-Cys, type II, and type Q
peroxiredoxins, and the fifth is represented by glutathione
peroxidases, which were recently shown to possess a
thioredoxin-dependent activity in plants (Rouhier & Jacquot
(2005) Free Radic Biol Med 38(11): 1413-21). The analysis of the
Arabidopsis thaliana genome indicates that at least 17 isoforms of
thioredoxin-dependent peroxidases are expressed in various plant
compartments.
[0017] 2-Cysteine peroxiredoxin (2-Cys PRX) are a group of proteins
that participate in cell proliferation, differentiation, apoptosis,
and photosynthesis. These enzymes reduce H.sub.2O.sub.2,
peroxinitrite and alkyl hydroperoxide to water or alcohol,
respectively (Netto et al., (1996) J Biol Chem 271(26):
15315-15321) with thioredoxin (Trx) as electron donor. By doing so,
2-Cys PRXs regulate signal transduction pathways or protect
macromolecules against oxidative damage. These proteins are
homodimers and each subunit has the two conserved cysteines (Choi
et al., (1998) Nature Struct Biol 5:400-406). The peroxide oxidizes
the N-terminal cysteine of one subunit to sulphenic acid, which
reacts with the C-terminal cysteine of the other subunit to form an
intermolecular disulphide. To complete the catalytic cycle the
enzyme is reduced via a thiol/disulphide redox interchange (Chae et
al., (1994) Proc Natl Acad Sci USA 91: 7017-7021).
[0018] Transgenic Arabidopsis thaliana plants (Baier et al. (2000)
Plant Physiol 124(2): 823-32) with reduced levels of 2-Cys PRX were
generated by antisense suppression. The suppression of 2-Cys PRX
expression lead to increased expression of other anti-oxidative
genes, demonstrating that the enzyme forms an integral part of the
anti-oxidant network of chloroplasts and is functionally
interconnected with other defense systems.
[0019] International patent application WO05/116082 describes the
obtention of transgenic Arabidopsis plants overexpressing an
Arabidopsis 2-Cys PRX (named BAS1) using the constitutive
cauliflower mosaic virus 35S promoter. The transgenic plants are
described as having more potential resistance to heat shock and
pathogens than the wild type plants.
[0020] Surprisingly, it has now been found that modulating,
preferably increasing, expression in the roots of a plant, of a
nucleic acid sequence encoding a 2-Cys PRX polypeptide gives plants
having enhanced yield-related traits relative to control
plants.
[0021] According one embodiment, there is provided a method for
enhancing yield related traits of a plant relative to control
plants, comprising modulating, preferably increasing, expression in
the roots of a plant, of a nucleic acid sequence encoding a 2-Cys
PRX polypeptide. The enhanced yield related traits comprise one or
more of: (i) improved early vigour; (ii) increased aboveground
biomass; (iii) increased root (thick and thin) biomass; (iv)
increase number of flowers per panicle; (v) increased seed fill
rate; (vi) increased total seed yield per plant; (vii) increased
number of (filled) seeds; (viii) increased harvest index; or (ix)
increased thousand kernel weight (TKW).
2. Annexin-Like (ANN)
[0022] Annexins form a family of calcium dependent phospholipid
binding proteins and are found in plants and animals. In all plant
species tested, the presence of at least two different annexins has
been demonstrated. Structurally, plant annexins are less divergent
than animal annexins. Comparative studies revealed that plant
annexins share significant homology in a core domain which
comprises at least one, usually four or more conserved repeats
which are approximately 70 amino acids in length. As
calcium-binding proteins, annexins are postulated to play a role in
calcium signalling pathways. Although the structure of annexins is
well known nowadays, functionally they are not well characterised.
In plants, annexins are reported to be involved in Golgi-mediated
secretion, cell expansion, vacuole biogenesis, chloroplast membrane
binding, cell cycle, nodulation signalling, stress signalling.
[0023] US20050089872 describes T-DNA insertion mutants (anx1 and
anx4-1) for respectively the Annexin 1 and Annexin 4 encoding genes
from Arabidopsis thaliana. The mutants were sensitive to salt
stress and osmotic stress. Also abscisic acid had a negative effect
on germination and growth of the anx1 and anx4-1 mutants.
Expression analysis revealed that the ANX1 protein was
predominantly expressed in the root, but not in flower, stem or
leaf tissues. It is postulated that the ANX1 and ANX4 proteins play
a role in the transduction of osmotic stress and ABA signals.
[0024] Surprisingly, it has now been found that modulating
expression of a nucleic acid encoding an ANN polypeptide gives
plants having enhanced yield-related traits, in particular
increased yield relative to control plants.
[0025] According one embodiment, there is provided a method for
enhancing yield-related traits of a plant relative to control
plants, comprising modulating expression of a nucleic acid encoding
an ANN polypeptide in a plant. The improved yield related traits
comprise increased seed yield.
DEFINITIONS
Polypeptide(s)/Protein(s)
[0026] The terms "polypeptide" and "protein" are used
interchangeably herein and refer to amino acids in a polymeric form
of any length, linked together by peptide bonds.
Polynucleotide(s)/Nucleic Acid(s)/Nucleic Acid
Sequence(s)/Nucleotide Sequence(s)
[0027] The terms "polynucleotide(s)", "nucleic acid sequence(s)",
"nucleotide sequence(s)", "nucleic acid(s)", "nucleic acid
molecule(s)" are used interchangeably herein and refer to
nucleotides, either ribonucleotides or deoxyribonucleotides or a
combination of both, in a polymeric unbranched form of any
length.
Control Plant(s)
[0028] The choice of suitable control plants is a routine part of
an experimental setup and may include corresponding wild type
plants or corresponding plants without the gene of interest. The
control plant is typically of the same plant species or even of the
same variety as the plant to be assessed. The control plant may
also be a nullizygote of the plant to be assessed. A "control
plant" as used herein refers not only to whole plants, but also to
plant parts, including seeds and seed parts.
Homologue(s)
[0029] "Homologues" of a protein encompass peptides, oligopeptides,
polypeptides, proteins and enzymes having amino acid substitutions,
deletions and/or insertions relative to the unmodified protein in
question and having similar biological and functional activity as
the unmodified protein from which they are derived.
[0030] A deletion refers to removal of one or more amino acids from
a protein.
[0031] An insertion refers to one or more amino acid residues being
introduced into a predetermined site in a protein. Insertions may
comprise N-terminal and/or C-terminal fusions as well as
intra-sequence insertions of single or multiple amino acids.
Generally, insertions within the amino acid sequence will be
smaller than N- or C-terminal fusions, of the order of about 1 to
10 residues. Examples of N- or C-terminal fusion proteins or
peptides include the binding domain or activation domain of a
transcriptional activator as used in the yeast two-hybrid system,
phage coat proteins, (histidine)-6-tag, glutathione
S-transferase-tag, protein A, maltose-binding protein,
dihydrofolate reductase, Tag.100 epitope, c-myc epitope,
FLAG.RTM.-epitope, lacZ, CMP (calmodulin-binding peptide), HA
epitope, protein C epitope and VSV epitope.
[0032] A substitution refers to replacement of amino acids of the
protein with other amino acids having similar properties (such as
similar hydrophobicity, hydrophilicity, antigenicity, propensity to
form or break .alpha.-helical structures or .beta.-sheet
structures). Amino acid substitutions are typically of single
residues, but may be clustered depending upon functional
constraints placed upon the polypeptide; insertions will usually be
of the order of about 1 to 10 amino acid residues. The amino acid
substitutions are preferably conservative amino acid substitutions.
Conservative substitution tables are well known in the art (see for
example Creighton (1984) Proteins. W.H. Freeman and Company (Eds)
and Table 1 below).
TABLE-US-00001 TABLE 1 Examples of conserved amino acid
substitutions Residue Conservative Substitutions Ala Ser Arg Lys
Asn Gln; His Asp Glu Gln Asn Cys Ser Glu Asp Gly Pro His Asn; Gln
Ile Leu, Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu;
Tyr Ser Thr; Gly Thr Ser; Val Trp Tyr Tyr Trp; Phe Val Ile; Leu
[0033] Amino acid substitutions, deletions and/or insertions may
readily be made using peptide synthetic techniques well known in
the art, such as solid phase peptide synthesis and the like, or by
recombinant DNA manipulation. Methods for the manipulation of DNA
sequences to produce substitution, insertion or deletion variants
of a protein are well known in the art. For example, techniques for
making substitution mutations at predetermined sites in DNA are
well known to those skilled in the art and include M13 mutagenesis,
T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange
Site Directed mutagenesis (Stratagene, San Diego, Calif.),
PCR-mediated site-directed mutagenesis or other site-directed
mutagenesis protocols.
Derivatives
[0034] "Derivatives" include peptides, oligopeptides, polypeptides
which may, compared to the amino acid sequence of the
naturally-occurring form of the protein, such as the protein of
interest, comprise substitutions of amino acids with non-naturally
occurring amino acid residues, or additions of non-naturally
occurring amino acid residues. "Derivatives" of a protein also
encompass peptides, oligopeptides, polypeptides which comprise
naturally occurring altered (glycosylated, acylated, prenylated,
phosphorylated, myristoylated, sulphated etc.) or non-naturally
altered amino acid residues compared to the amino acid sequence of
a naturally-occurring form of the polypeptide. A derivative may
also comprise one or more non-amino acid substituents or additions
compared to the amino acid sequence from which it is derived, for
example a reporter molecule or other ligand, covalently or
non-covalently bound to the amino acid sequence, such as a reporter
molecule which is bound to facilitate its detection, and
non-naturally occurring amino acid residues relative to the amino
acid sequence of a naturally-occurring protein.
Orthologue(s)/Paralogue(s)
[0035] Orthologues and paralogues encompass evolutionary concepts
used to describe the ancestral relationships of genes. Paralogues
are genes within the same species that have originated through
duplication of an ancestral gene; orthologues are genes from
different organisms that have originated through speciation, and
are also derived from a common ancestral gene.
Domain
[0036] The term "domain" refers to a set of amino acids conserved
at specific positions along an alignment of sequences of
evolutionarily related proteins. While amino acids at other
positions can vary between homologues, amino acids that are highly
conserved at specific positions indicate amino acids that are
likely essential in the structure, stability or function of a
protein. Identified by their high degree of conservation in aligned
sequences of a family of protein homologues, they can be used as
identifiers to determine if any polypeptide in question belongs to
a previously identified polypeptide family.
Motif/Consensus Sequence/Signature
[0037] The term "motif" or "consensus sequence" or "signature"
refers to a short conserved region in the sequence of
evolutionarily related proteins. Motifs are frequently highly
conserved parts of domains, but may also include only part of the
domain, or be located outside of conserved domain (if all of the
amino acids of the motif fall outside of a defined domain).
Hybridisation
[0038] The term "hybridisation" as defined herein is a process
wherein substantially homologous complementary nucleotide sequences
anneal to each other. The hybridisation process can occur entirely
in solution, i.e. both complementary nucleic acid molecules are in
solution. The hybridisation process can also occur with one of the
complementary nucleic acid molecules immobilised to a matrix such
as magnetic beads, Sepharose beads or any other resin. The
hybridisation process can furthermore occur with one of the
complementary nucleic acid molecules immobilised to a solid support
such as a nitro-cellulose or nylon membrane or immobilised by e.g.
photolithography to, for example, a siliceous glass support (the
latter known as nucleic acid arrays or microarrays or as nucleic
acid chips). In order to allow hybridisation to occur, the nucleic
acid molecules are generally thermally or chemically denatured to
melt a double strand into two single strands and/or to remove
hairpins or other secondary structures from single stranded nucleic
acid molecules.
[0039] The term "stringency" refers to the conditions under which a
hybridisation takes place. The stringency of hybridisation is
influenced by conditions such as temperature, salt concentration,
ionic strength and hybridisation buffer composition. Generally, low
stringency conditions are selected to be about 30.degree. C. lower
than the thermal melting 2-Cys PRXnt (T.sub.m) for the specific
sequence at a defined ionic strength and pH. Medium stringency
conditions are when the temperature is 20.degree. C. below T.sub.m,
and high stringency conditions are when the temperature is
10.degree. C. below T.sub.m. High stringency hybridisation
conditions are typically used for isolating hybridising sequences
that have high sequence similarity to the target nucleic acid
sequence. However, nucleic acid sequences may deviate in sequence
composition and still encode a substantially identical polypeptide,
due to the degeneracy of the genetic code. Therefore medium
stringency hybridisation conditions may sometimes be needed to
identify such nucleic acid molecules.
[0040] The Tm is the temperature under defined ionic strength and
pH, at which 50% of the target sequence hybridises to a perfectly
matched probe. The T.sub.m is dependent upon the solution
conditions and the base composition and length of the probe. For
example, longer sequences hybridise specifically at higher
temperatures. The maximum rate of hybridisation is obtained from
about 16.degree. C. up to 32.degree. C. below T.sub.m. The presence
of monovalent cations in the hybridisation solution reduce the
electrostatic repulsion between the two nucleic acid strands
thereby promoting hybrid formation; this effect is visible for
sodium concentrations of up to 0.4M (for higher concentrations,
this effect may be ignored). Formamide reduces the melting
temperature of DNA-DNA and DNA-RNA duplexes with 0.6 to 0.7.degree.
C. for each percent formamide, and addition of 50% formamide allows
hybridisation to be performed at 30 to 45.degree. C., though the
rate of hybridisation will be lowered. Base pair mismatches reduce
the hybridisation rate and the thermal stability of the duplexes.
On average and for large probes, the Tm decreases about 1.degree.
C. per % base mismatch. The Tm may be calculated using the
following equations, depending on the types of hybrids:
1) DNA-DNA hybrids (Meinkoth and Wahl, Anal. Biochem., 138:
267-284, 1984):
T.sub.m=81.5.degree. C.+16.6.times.log.sub.10
[Na.sup.+].sup.a+0.41.times.%[G/C.sup.b]-500.times.[L.sup.c].sup.-1-0.61.-
times.% formamide
2) DNA-RNA or RNA-RNA hybrids:
Tm=79.8+18.5 (log.sub.10[Na.sup.+].sup.a)+0.58(% G/C.sup.b)+11.8(%
G/C.sup.b).sup.2-820/L.sup.c
3) oligo-DNA or oligo-RNA.sup.d hybrids: [0041] For <20
nucleotides: T.sub.m=2 (I.sub.n) [0042] For 20-35 nucleotides:
T.sub.m=22+1.46 (I.sub.n) [0043] .sup.a or for other monovalent
cation, but only accurate in the 0.01-0.4 M range. [0044] .sup.b
only accurate for % GC in the 30% to 75% range. [0045] .sup.c
L=length of duplex in base pairs. [0046] .sup.d oligo,
oligonucleotide; I.sub.n,=effective length of primer=2.times.(no.
of G/C)+(no. of NT).
[0047] Non-specific binding may be controlled using any one of a
number of known techniques such as, for example, blocking the
membrane with protein containing solutions, additions of
heterologous RNA, DNA, and SDS to the hybridisation buffer, and
treatment with Rnase. For non-homologous probes, a series of
hybridizations may be performed by varying one of (i) progressively
lowering the annealing temperature (for example from 68.degree. C.
to 42.degree. C.) or (ii) progressively lowering the formamide
concentration (for example from 50% to 0%). The skilled artisan is
aware of various parameters which may be altered during
hybridisation and which will either maintain or change the
stringency conditions.
[0048] Besides the hybridisation conditions, specificity of
hybridisation typically also depends on the function of
post-hybridisation washes. To remove background resulting from
non-specific hybridisation, samples are washed with dilute salt
solutions. Critical factors of such washes include the ionic
strength and temperature of the final wash solution: the lower the
salt concentration and the higher the wash temperature, the higher
the stringency of the wash. Wash conditions are typically performed
at or below hybridisation stringency. A positive hybridisation
gives a signal that is at least twice of that of the background.
Generally, suitable stringent conditions for nucleic acid
hybridisation assays or gene amplification detection procedures are
as set forth above. More or less stringent conditions may also be
selected. The skilled artisan is aware of various parameters which
may be altered during washing and which will either maintain or
change the stringency conditions.
[0049] For example, typical high stringency hybridisation
conditions for DNA hybrids longer than 50 nucleotides encompass
hybridisation at 65.degree. C. in 1.times.SSC or at 42.degree. C.
in 1.times.SSC and 50% formamide, followed by washing at 65.degree.
C. in 0.3.times.SSC. Examples of medium stringency hybridisation
conditions for DNA hybrids longer than 50 nucleotides encompass
hybridisation at 50.degree. C. in 4.times.SSC or at 40.degree. C.
in 6.times.SSC and 50% formamide, followed by washing at 50.degree.
C. in 2.times.SSC. The length of the hybrid is the anticipated
length for the hybridising nucleic acid molecule. When nucleic acid
molecules of known sequence are hybridised, the hybrid length may
be determined by aligning the sequences and identifying the
conserved regions described herein. 1.times.SSC is 0.15M NaCl and
15 mM sodium citrate; the hybridisation solution and wash solutions
may additionally include 5.times.Denhardt's reagent, 0.5-1.0% SDS,
100 .mu.g/ml denatured, fragmented salmon sperm DNA, 0.5% sodium
pyrophosphate.
[0050] For the purposes of defining the level of stringency,
reference can be made to Sambrook et al. (2001) Molecular Cloning:
a laboratory manual, 3.sup.rd Edition, Cold Spring Harbor
Laboratory Press, CSH, New York or to Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989 and yearly
updates).
Splice Variant
[0051] The term "splice variant" as used herein encompasses
variants of a nucleic acid sequence in which selected introns
and/or exons have been excised, replaced, displaced or added, or in
which introns have been shortened or lengthened. Such variants will
be ones in which the biological activity of the protein is
substantially retained; this may be achieved by selectively
retaining functional segments of the protein. Such splice variants
may be found in nature or may be manmade. Methods for predicting
and isolating such splice variants are well known in the art (see
for example Foissac and Schiex (2005) BMC Bioinformatics 6:
25).
Allelic Variant
[0052] Alleles or allelic variants are alternative forms of a given
gene, located at the same chromosomal position. Allelic variants
encompass Single Nucleotide Polymorphisms (SNPs), as well as Small
Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is
usually less than 100 bp. SNPs and INDELs form the largest set of
sequence variants in naturally occurring polymorphic strains of
most organisms.
Gene Shuffling/Directed Evolution
[0053] Gene shuffling or directed evolution consists of iterations
of DNA shuffling followed by appropriate screening and/or selection
to generate variants of nucleic acid sequences or portions thereof
encoding proteins having a modified biological activity (Castle et
al., (2004) Science 304(5674): 1151-4; U.S. Pat. Nos. 5,811,238 and
6,395,547).
Regulatory Element/Control Sequence/Promoter
[0054] The terms "regulatory element", "control sequence" and
"promoter" are all used interchangeably herein and are to be taken
in a broad context to refer to regulatory nucleic acid sequences
capable of effecting expression of the sequences to which they are
ligated. The term "promoter" typically refers to a nucleic acid
control sequence located upstream from the transcriptional start of
a gene and which is involved in recognising and binding of RNA
polymerase and other proteins, thereby directing transcription of
an operably linked nucleic acid sequence. Encompassed by the
aforementioned terms are transcriptional regulatory sequences
derived from a classical eukaryotic genomic gene (including the
TATA box which is required for accurate transcription initiation,
with or without a CCAAT box sequence) and additional regulatory
elements (i.e. upstream activating sequences, enhancers and
silencers) which alter gene expression in response to developmental
and/or external stimuli, or in a tissue-specific manner. Also
included within the term is a transcriptional regulatory sequence
of a classical prokaryotic gene, in which case it may include a -35
box sequence and/or -10 box transcriptional regulatory sequences.
The term "regulatory element" also encompasses a synthetic fusion
molecule or derivative that confers, activates or enhances
expression of a nucleic acid sequence in a cell, tissue or
organ.
[0055] A "plant promoter" comprises regulatory elements, which
mediate the expression of a coding sequence segment in plant cells.
Accordingly, a plant promoter need not be of plant origin, but may
originate from viruses or micro-organisms, for example from viruses
which attack plant cells. The "plant promoter" can also originate
from a plant cell, e.g. from the plant which is transformed with
the nucleic acid sequence to be expressed in the inventive process
and described herein. This also applies to other "plant" regulatory
signals, such as "plant" terminators. The promoters upstream of the
nucleotide sequences useful in the methods of the present invention
can be modified by one or more nucleotide substitution(s),
insertion(s) and/or deletion(s) without interfering with the
functionality or activity of either the promoters, the open reading
frame (ORF) or the 3'-regulatory region such as terminators or
other 3' regulatory regions which are located away from the ORF. It
is furthermore possible that the activity of the promoters is
increased by modification of their sequence, or that they are
replaced completely by more active promoters, even promoters from
heterologous organisms. For expression in plants, the nucleic acid
sequence must, as described above, be linked operably to or
comprise a suitable promoter which expresses the gene at the right
moment in time and with the required spatial expression
pattern.
[0056] For the identification of functionally equivalent promoters,
the promoter strength and/or expression pattern of a candidate
promoter may be analysed for example by operably linking the
promoter to a reporter gene and assaying the expression level and
pattern of the reporter gene in various tissues of the plant.
Suitable well-known reporter genes include for example
beta-glucuronidase or beta-galactosidase. The promoter activity is
assayed by measuring the enzymatic activity of the
beta-glucuronidase or beta-galactosidase. The promoter strength
and/or expression pattern may then be compared to that of a
reference promoter (such as the one used in the methods of the
present invention). Alternatively, promoter strength may be assayed
by quantifying mRNA levels or by comparing mRNA levels of the
nucleic acid sequence used in the methods of the present invention,
with mRNA levels of housekeeping genes such as 18S rRNA, using
methods known in the art, such as Northern blotting with
densitometric analysis of autoradiograms, quantitative real-time
PCR or RT-PCR (Heid et al., 1996 Genome Methods 6: 986-994).
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 1/500,0000 transcripts per cell. Conversely,
a "strong promoter" drives expression of a coding sequence at high
level, or at about 1/10 transcripts to about 1/100 transcripts to
about 1/1000 transcripts per cell.
Operably Linked
[0057] The term "operably linked" as used herein refers to a
functional linkage between the promoter sequence and the gene of
interest, such that the promoter sequence is able to initiate
transcription of the gene of interest.
Constitutive Promoter
[0058] A "constitutive promoter" refers to a promoter that is
transcriptionally active during most, but not necessarily all,
phases of growth and development and under most environmental
conditions, in at least one cell, tissue or organ. Table 2a below
gives examples of constitutive promoters.
TABLE-US-00002 TABLE 2a Examples of constitutive promoters Gene
Source Reference Actin McElroy et al, Plant Cell, 2: 163-171, 1990
HMGB WO 2004/070039 CAMV 35S Odell et al, Nature, 313: 810-812,
1985 CaMV 19S Nilsson et al., Physiol. Plant. 100: 456-462, 1997
GOS2 de Pater et al, Plant J Nov;2(6): 837-44, 1992, WO 2004/065596
Ubiquitin Christensen et al, Plant Mol. Biol. 18: 675-689, 1992
Rice cyclophilin Buchholz et al, Plant Mol Biol. 25(5): 837-43,
1994 Maize H3 histone Lepetit et al, Mol. Gen. Genet. 231: 276-285,
1992 Alfalfa H3 histone Wu et al. Plant Mol. Biol. 11: 641-649,
1988 Actin 2 An et al, Plant J. 10(1); 107-121, 1996 34S FMV Sanger
et al., Plant. Mol. Biol., 14, 1990: 433-443 Rubisco small U.S.
Pat. No. 4,962,028 subunit OCS Leisner (1988) Proc Natl Acad Sci
USA 85(5): 2553 SAD1 Jain et al., Crop Science, 39 (6), 1999: 1696
SAD2 Jain et al., Crop Science, 39 (6), 1999: 1696 nos Shaw et al.
(1984) Nucleic Acids Res. 12(20): 7831-7846 V-ATPase WO 01/14572
Super promoter WO 95/14098 G-box proteins WO 94/12015
Ubiquitous Promoter
[0059] A ubiquitous promoter is active in substantially all tissues
or cells of an organism.
Developmentally-Regulated Promoter
[0060] A developmentally-regulated promoter is active during
certain developmental stages or in parts of the plant that undergo
developmental changes.
Inducible Promoter
[0061] An inducible promoter has induced or increased transcription
initiation in response to a chemical (for a review see Gatz 1997,
Annu. Rev. Plant Physiol. Plant Mol. Biol., 48:89-108),
environmental or physical stimulus, or may be "stress-inducible",
i.e. activated when a plant is exposed to various stress
conditions, or a "pathogen-inducible" i.e. activated when a plant
is exposed to exposure to various pathogens.
Organ-Specific/Tissue-Specific Promoter
[0062] An organ-specific or tissue-specific promoter is one that is
capable of preferentially initiating transcription in certain
organs or tissues, such as the leaves, roots, seed tissue etc. For
example, a "root-specific promoter" is a promoter that is
transcriptionally active predominantly in plant roots,
substantially to the exclusion of any other parts of a plant,
whilst still allowing for any leaky expression in these other plant
parts. Promoters able to initiate transcription in certain cells
only are referred to herein as "cell-specific".
[0063] Examples of root-specific promoters are listed in Table 2b
below:
TABLE-US-00003 TABLE 2b Examples of root-specific promoters Gene
Source Reference Rice RCc3 Xu et al (1995) Plant Mol Biol 27(2):
237-48 Arabidopsis phosphate transporter PHT1 Kovama et al., 2005
Medicago phosphate transporter Xiao et al., 2006 Arabidopsis Pyk10
Nitz et al. (2001) Plant Sci 161(2): 337-346 Tobacco root-specific
genes RB7, RD2, RD5, Conkling et al. (1990) Plant Phys 93(3): RH12
1203-1211 Barley root-specific lectin Lerner & Raikhel (1989)
Plant Phys 91: 124- 129 Root-specific hydroxy-proline rich protein
Keller & Lamb (1989) Genes & Dev 3: 1639- 1646 Arabidopsis
CDC27B/hobbit Blilou et al. (2002) Genes & Dev 16: 2566-
2575
[0064] A seed-specific promoter is transcriptionally active
predominantly in seed tissue, but not necessarily exclusively in
seed tissue (in cases of leaky expression). The seed-specific
promoter may be active during seed development and/or during
germination. The seed specific promoter may be specific to one or
more of: endosperm, aleurone, or embryo specific. Examples of
seed-specific promoters are shown in Tables 2c, 2d, 2e, 2f below.
Further examples of seed-specific promoters are given in Qing Qu
and Takaiwa (Plant Biotechnol. J. 2, 113-125, 2004), which
disclosure is incorporated by reference herein as if fully set
forth.
TABLE-US-00004 TABLE 2c Examples of seed-specific promoters Gene
source Reference seed-specific genes Simon et al., Plant Mol. Biol.
5: 191, 1985; Scofield et al., J. Biol. Chem. 262: 12202, 1987.;
Baszczynski et al., Plant Mol. Biol. 14: 633, 1990. Brazil Nut
albumin Pearson et al., Plant Mol. Biol. 18: 235-245, 1992. Legumin
Ellis et al., Plant Mol. Biol. 10: 203-214, 1988. glutelin (rice)
Takaiwa et al., Mol. Gen. Genet. 208: 15-22, 1986; Takaiwa et al.,
FEBS Letts. 221: 43-47, 1987. Zein Matzke et al Plant Mol Biol,
14(3): 323-32 1990 NapA Stalberg et al, Planta 199: 515-519, 1996.
wheat LMW and HMW glutenin-1 Mol Gen Genet 216: 81-90, 1989; NAR
17: 461-2, 1989 wheat SPA Albani et al, Plant Cell, 9: 171-184,
1997 wheat .alpha., .beta., .gamma.-gliadins EMBO J. 3: 1409-15,
1984 barley Itr1 promoter Diaz et al. (1995) Mol Gen Genet 248(5):
592-8 barley B1, C, D, hordein Theor Appl Gen 98: 1253-62, 1999;
Plant J 4: 343-55, 1993; Mol Gen Genet 250: 750-60, 1996 barley DOF
Mena et al, The Plant Journal, 116(1): 53-62, 1998 blz2
EP99106056.7 Synthetic promoter Vicente-Carbajosa et al., Plant J.
13: 629-640, 1998. rice prolamin NRP33 Wu et al, Plant Cell
Physiology 39(8) 885-889, 1998 rice a-globulin Glb-1 Wu et al,
Plant Cell Physiology 39(8) 885-889, 1998 rice OSH1 Sato et al,
Proc. Natl. Acad. Sci. USA, 93: 8117-8122, 1996 rice
.alpha.-globulin REB/OHP-1 Nakase et al. Plant Mol. Biol. 33:
513-522, 1997 rice ADP-glucose pyrophos- Trans Res 6: 157-68, 1997
phorylase maize ESR gene family Plant J 12: 235-46, 1997 Sorghum
.alpha.-kafirin DeRose et al., Plant Mol. Biol 32: 1029-35, 1996
KNOX Postma-Haarsma et al, Plant Mol. Biol. 39: 257-71, 1999 rice
oleosin Wu et al, J. Biochem. 123: 386, 1998 sunflower oleosin
Cummins et al., Plant Mol. Biol. 19: 873-876, 1992 PRO0117,
putative rice 40S WO 2004/070039 ribosomal protein PRO0136, rice
alanine unpublished aminotransferase PRO0147, trypsin inhibitor
ITR1 Unpublished (barley) PRO0151, rice WSI18 WO 2004/070039
PRO0175, rice RAB21 WO 2004/070039 PRO005 WO 2004/070039 PRO0095 WO
2004/070039 .alpha.-amylase (Amy32b) Lanahan et al, Plant Cell 4:
203-211, 1992; Skriver et al, Proc Natl Acad Sci USA 88: 7266-7270,
1991 cathepsin .beta.-like gene Cejudo et al, Plant Mol Biol 20:
849-856, 1992 Barley Ltp2 Kalla et al., Plant J. 6: 849-60, 1994
Chi26 Leah et al., Plant J. 4: 579-89, 1994 Maize B-Peru Selinger
et al., Genetics 149; 1125-38, 1998
TABLE-US-00005 TABLE 2d examples of endosperm-specific promoters
Gene source Reference glutelin (rice) Takaiwa et al. (1986) Mol Gen
Genet 208: 15-22; Takaiwa et al. (1987) FEBS Letts. 221: 43-47 Zein
Matzke et al., (1990) Plant Mol Biol 14(3): 323-32 wheat LMW and
HMW glutenin-1 Colot et al. (1989) Mol Gen Genet 216: 81-90;
Anderson et al. (1989) NAR 17: 461-2 wheat SPA Albani et al. (1997)
Plant Cell 9: 171-184 wheat gliadins Rafalski et al. (1984) EMBO 3:
1409-15 barley Itr1 promoter Diaz et al. (1995) Mol Gen Genet
248(5): 592-8 barley B1, C, D, hordein Cho et al. (1999) Theor Appl
Genet 98: 1253-62; Muller et al. (1993) Plant J 4: 343-55; Sorenson
et al. (1996) Mol Gen Genet 250: 750-60 barley DOF Mena et al,
(1998) Plant J 116(1): 53-62 blz2 Onate et al. (1999) J Biol Chem
274(14): 9175-82 synthetic promoter Vicente-Carbajosa et al. (1998)
Plant J 13: 629-640 rice prolamin NRP33 Wu et al, (1998) Plant Cell
Physiol 39(8) 885-889 rice globulin Glb-1 Wu et al. (1998) Plant
Cell Physiol 39(8) 885-889 rice globulin REB/OHP-1 Nakase et al.
(1997) Plant Molec Biol 33: 513-522 rice ADP-glucose
pyrophosphorylase Russell et al. (1997) Trans Res 6: 157-68 maize
ESR gene family Opsahl-Ferstad et al. (1997) Plant J 12: 235-46
Sorghum kafirin DeRose et al. (1996) Plant Mol Biol 32: 1029-35
TABLE-US-00006 TABLE 2e Examples of embryo specific promoters: Gene
source Reference KNOX Postma-Haarsma et al, Plant Mol. Biol. 39:
257-71, 1999 PRO0151 WO 2004/070039 PRO0175 WO 2004/070039 PRO005
WO 2004/070039 PRO0095 WO 2004/070039
TABLE-US-00007 TABLE 2f Examples of aleurone-specific promoters:
Gene source Reference .alpha.-amylase Lanahan et al, Plant Cell 4:
203-211, 1992; (Amy32b) Skriver et al, Proc Natl Acad Sci USA 88:
7266-7270, 1991 cathepsin Cejudo et al, Plant Mol Biol 20: 849-856,
1992 .beta.-like gene Barley Ltp2 Kalla et al., Plant J. 6: 849-60,
1994 Chi26 Leah et al., Plant J. 4: 579-89, 1994 Maize B-Peru
Selinger et al., Genetics 149; 1125-38, 1998
[0065] A green tissue-specific promoter as defined herein is a
promoter that is transcriptionally active predominantly in green
tissue, substantially to the exclusion of any other parts of a
plant, whilst still allowing for any leaky expression in these
other plant parts.
[0066] Examples of green tissue-specific promoters which may be
used to perform the methods of the invention are shown in Table 2g
below.
TABLE-US-00008 TABLE 2g Examples of green tissue-specific promoters
Gene Expression Reference Maize Orthophosphate dikinase Leaf
specific Fukavama et al., 2001 Maize Phosphoenolpyruvate Leaf
specific Kausch et al., 2001 carboxylase Rice Phosphoenolpyruvate
Leaf specific Liu et al., 2003 carboxylase Rice small subunit
Rubisco Leaf specific Nomura et al., 2000 rice beta expansin EXBP9
Shoot specific WO 2004/070039 Pigeonpea small subunit Rubisco Leaf
specific Panguluri et al., 2005 Pea RBCS3A Leaf specific
[0067] Another example of a tissue-specific promoter is a
meristem-specific promoter, which is transcriptionally active
predominantly in meristematic tissue, substantially to the
exclusion of any other parts of a plant, whilst still allowing for
any leaky expression in these other plant parts. Examples of green
meristem-specific promoters which may be used to perform the
methods of the invention are shown in Table 2h below.
TABLE-US-00009 TABLE 2h Examples of meristem-specific promoters
Gene source Expression pattern Reference rice OSH1 Shoot apical
meristem, from Sato et al. (1996) Proc. Natl. embryo globular stage
to Acad. Sci. USA, 93: 8117- seedling stage 8122 Rice Meristem
specific BAD87835.1 metallothionein WAK1 & Shoot and root
apical Wagner & Kohorn (2001) WAK 2 meristems, and in expanding
Plant Cell 13(2): 303-318 leaves and sepals
Terminator
[0068] The term "terminator" encompasses a control sequence which
is a DNA sequence at the end of a transcriptional unit which
signals 3' processing and polyadenylation of a primary transcript
and termination of transcription. The terminator can be derived
from the natural gene, from a variety of other plant genes, or from
T-DNA. The terminator to be added may 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.
Modulation
[0069] The term "modulation" means in relation to expression or
gene expression, a process in which the expression level is changed
by said gene expression in comparison to the control plant,
preferably the expression level is increased. The original,
unmodulated expression may be of any kind of expression of a
structural RNA (rRNA, tRNA) or mRNA with subsequent translation.
The term "modulating the activity" shall mean any change of the
expression of the inventive nucleic acid sequences or encoded
proteins, which leads to enhanced yield-related traits (for
example, increased yield and/or increased growth) of the
plants.
Increased Expression/Overexpression
[0070] The term "increased expression" or "overexpression" as used
herein means any form of expression that is additional to the
original wild-type expression level.
[0071] Methods for increasing expression of genes or gene products
are well documented in the art and include, for example,
overexpression driven by appropriate promoters, the use of
transcription enhancers or translation enhancers. Isolated nucleic
acid sequences which serve as promoter or enhancer elements may be
introduced in an appropriate position (typically upstream) of a
non-heterologous form of a polynucleotide so as to upregulate
expression of a nucleic acid sequence encoding the polypeptide of
interest. For example, endogenous promoters may be altered in vivo
by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat.
No. 5,565,350; Zarling et al., WO9322443), or isolated promoters
may be introduced into a plant cell in the proper orientation and
distance from a gene of the present invention so as to control the
expression of the gene.
[0072] 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 the natural gene, from a variety of other plant genes,
or from T-DNA. The 3' end sequence to be added may 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.
[0073] An intron sequence may also be added to the 5' untranslated
region (UTR) 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 1:1183-1200). Such intron
enhancement of gene expression is typically greatest when placed
near the 5' end of the transcription unit. Use of the maize introns
Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the
art. For general information see: The Maize Handbook, Chapter 116,
Freeling and Walbot, Eds., Springer, N.Y. (1994).
Endogenous Gene
[0074] Reference herein to an "endogenous" gene not only refers to
the gene in question as found in a plant in its natural form (i.e.,
without there being any human intervention), but also refers to
that same gene (or a substantially homologous gene/nucleic acid
sequence) in an isolated form subsequently (re)introduced into a
plant (a transgene). For example, a transgenic plant containing
such a transgene may encounter a substantial reduction of the
transgene expression and/or substantial reduction of expression of
the endogenous gene. The isolated gene may be isolated from an
organism or may be manmade, for example by chemical synthesis.
Decreased Expression
[0075] Reference herein to "decreased expression" or "reduction or
substantial elimination" of expression is taken to mean a decrease
in endogenous gene expression and/or polypeptide levels and/or
polypeptide activity relative to control plants. The reduction or
substantial elimination is in increasing order of preference at
least 10%, 20%, 30%, 40% or 50%, 60%, 70%, 80%, 85%, 90%, or 95%,
96%, 97%, 98%, 99% or more reduced compared to that of control
plants.
[0076] For the reduction or substantial elimination of expression
an endogenous gene in a plant, a sufficient length of substantially
contiguous nucleotides of a nucleic acid sequence is required. In
order to perform gene silencing, this may be as little as 20, 19,
18, 17, 16, 15, 14, 13, 12, 11, 10 or fewer nucleotides,
alternatively this may be as much as the entire gene (including the
5' and/or 3' UTR, either in part or in whole). The stretch of
substantially contiguous nucleotides may be derived from the
nucleic acid sequence encoding the protein of interest (target
gene), or from any nucleic acid sequence capable of encoding an
orthologue, paralogue or homologue of the protein of interest.
Preferably, the stretch of substantially contiguous nucleotides is
capable of forming hydrogen bonds with the target gene (either
sense or antisense strand), more preferably, the stretch of
substantially contiguous nucleotides has, in increasing order of
preference, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,
100% sequence identity to the target gene (either sense or
antisense strand). A nucleic acid sequence encoding a (functional)
polypeptide is not a requirement for the various methods discussed
herein for the reduction or substantial elimination of expression
of an endogenous gene.
[0077] This reduction or substantial elimination of expression may
be achieved using routine tools and techniques. A method for the
reduction or substantial elimination of endogenous gene expression
is by RNA-mediated silencing using an inverted repeat of a nucleic
acid sequence or a part thereof (in this case a stretch of
substantially contiguous nucleotides derived from the gene of
interest, or from any nucleic acid sequence capable of encoding an
orthologue, paralogue or homologue of the protein of interest),
preferably capable of forming a hairpin structure. Another example
of an RNA silencing method involves the introduction of nucleic
acid sequences or parts thereof (in this case a stretch of
substantially contiguous nucleotides derived from the gene of
interest, or from any nucleic acid sequence capable of encoding an
orthologue, paralogue or homologue of the protein of interest) in a
sense orientation into a plant. Another example of an RNA silencing
method involves the use of antisense nucleic acid sequences. Gene
silencing may also be achieved by insertion mutagenesis (for
example, T-DNA insertion or transposon insertion) or by strategies
as described by, among others, Angell and Baulcombe ((1999) Plant J
20(3): 357-62), (Amplicon VIGS WO 98/36083), or Baulcombe (WO
99/15682). Other methods, such as the use of antibodies directed to
an endogenous polypeptide for inhibiting its function in planta, or
interference in the signalling pathway in which a polypeptide is
involved, will be well known to the skilled man. Artificial and/or
natural microRNAs (miRNAs) may be used to knock out gene expression
and/or mRNA translation. Endogenous miRNAs are single stranded
small RNAs of typically 19-24 nucleotides long. Artificial
microRNAs (amiRNAs), which are typically 21 nucleotides in length,
can be genetically engineered specifically to negatively regulate
gene expression of single or multiple genes of interest.
Determinants of plant microRNA target selection are well known in
the art. Empirical parameters for target recognition have been
defined and can be used to aid in the design of specific amiRNAs
(Schwab et al., (2005) Dev Cell 8(4):517-27). Convenient tools for
design and generation of amiRNAs and their precursors are also
available to the public (Schwab et al., (2006) Plant Cell
18(5):1121-33).
[0078] For optimal performance, the gene silencing techniques used
for reducing expression in a plant of an endogenous gene requires
the use of nucleic acid sequences from monocotyledonous plants for
transformation of monocotyledonous plants, and from dicotyledonous
plants for transformation of dicotyledonous plants. Preferably, a
nucleic acid sequence from any given plant species is introduced
into that same species. For example, a nucleic acid sequence from
rice is transformed into a rice plant. However, it is not an
absolute requirement that the nucleic acid sequence to be
introduced originates from the same plant species as the plant in
which it will be introduced. It is sufficient that there is
substantial homology between the endogenous target gene and the
nucleic acid sequence to be introduced.
[0079] Described above are examples of various methods for the
reduction or substantial elimination of expression in a plant of an
endogenous gene. A person skilled in the art would readily be able
to adapt the aforementioned methods for silencing so as to achieve
reduction of expression of an endogenous gene in a whole plant or
in parts thereof through the use of an appropriate promoter, for
example.
Selectable Marker (Gene)/Reporter Gene
[0080] "Selectable marker", "selectable marker gene" or "reporter
gene" includes any gene that confers a phenotype on a cell in which
it is expressed to facilitate the identification and/or selection
of cells that are transfected or transformed with a nucleic acid
construct of the invention. These marker genes enable the
identification of a successful transfer of the nucleic acid
molecules via a series of different principles. Suitable markers
may be selected from markers that confer antibiotic or herbicide
resistance, that introduce a new metabolic trait or that allow
visual selection. Examples of selectable marker genes include genes
conferring resistance to antibiotics (such as nptII that
phosphorylates neomycin and kanamycin, or hpt, phosphorylating
hygromycin, or genes conferring resistance to, for example,
bleomycin, streptomycin, tetracyclin, chloramphenicol, ampicillin,
gentamycin, geneticin (G418), spectinomycin or blasticidin), to
herbicides (for example bar which provides resistance to
Basta.RTM.; aroA or gox providing resistance against glyphosate, or
the genes conferring resistance to, for example, imidazolinone,
phosphinothricin or sulfonylurea), or genes that provide a
metabolic trait (such as manA that allows plants to use mannose as
sole carbon source or xylose isomerase for the utilisation of
xylose, or antinutritive markers such as the resistance to
2-deoxyglucose). Expression of visual marker genes results in the
formation of colour (for example .beta.-glucuronidase, GUS or
.beta.-galactosidase with its coloured substrates, for example
X-Gal), luminescence (such as the luciferin/luceferase system) or
fluorescence (Green Fluorescent Protein, GFP, and derivatives
thereof). This list represents only a small number of possible
markers. The skilled worker is familiar with such markers.
Different markers are preferred, depending on the organism and the
selection method.
[0081] It is known that upon stable or transient integration of
nucleic acid sequences into plant cells, only a minority of the
cells takes up the foreign DNA and, if desired, integrates it into
its genome, depending on the expression vector used and the
transfection technique used. To identify and select these
integrants, a gene coding for a selectable marker (such as the ones
described above) is usually introduced into the host cells together
with the gene of interest. These markers can for example be used in
mutants in which these genes are not functional by, for example,
deletion by conventional methods. Furthermore, nucleic acid
molecules encoding a selectable marker can be introduced into a
host cell on the same vector that comprises the sequence encoding
the polypeptides of the invention or used in the methods of the
invention, or else in a separate vector. Cells which have been
stably transfected with the introduced nucleic acid sequence can be
identified for example by selection (for example, cells which have
integrated the selectable marker survive whereas the other cells
die).
[0082] Since the marker genes, particularly genes for resistance to
antibiotics and herbicides, are no longer required or are undesired
in the transgenic host cell once the nucleic acid sequences have
been introduced successfully, the process according to the
invention for introducing the nucleic acid sequences advantageously
employs techniques which enable the removal or excision of these
marker genes. One such a method is what is known as
co-transformation. The co-transformation method employs two vectors
simultaneously for the transformation, one vector bearing the
nucleic acid sequence according to the invention and a second
bearing the marker gene(s). A large proportion of transformants
receives or, in the case of plants, comprises (up to 40% or more of
the transformants), both vectors. In case of transformation with
Agrobacteria, the transformants usually receive only a part of the
vector, i.e. the sequence flanked by the T-DNA, which usually
represents the expression cassette. The marker genes can
subsequently be removed from the transformed plant by performing
crosses. In another method, marker genes integrated into a
transposon are used for the transformation together with desired
nucleic acid sequence (known as the Ac/Ds technology). The
transformants can be crossed with a transposase source or the
transformants are transformed with a nucleic acid construct
conferring expression of a transposase, transiently or stable. In
some cases (approx. 10%), the transposon jumps out of the genome of
the host cell once transformation has taken place successfully and
is lost. In a further number of cases, the transposon jumps to a
different location. In these cases the marker gene must be
eliminated by performing crosses. In microbiology, techniques were
developed which make possible, or facilitate, the detection of such
events. A further advantageous method relies on what is known as
recombination systems; whose advantage is that elimination by
crossing can be dispensed with. The best-known system of this type
is what is known as the Cre/lox system. Cre1 is a recombinase that
removes the sequences located between the loxP sequences. If the
marker gene is integrated between the loxP sequences, it is removed
once transformation has taken place successfully, by expression of
the recombinase. Further recombination systems are the HIN/HIX,
FLP/FRT and REP/STB system (Tribble et al., J. Biol. Chem., 275,
2000: 22255-22267; Velmurugan et al., J. Cell Biol., 149, 2000:
553-566). A site-specific integration into the plant genome of the
nucleic acid sequences according to the invention is possible.
Naturally, these methods can also be applied to microorganisms such
as yeast, fungi or bacteria.
Transgenic/Transgene/Recombinant
[0083] For the purposes of the invention, "transgenic", "transgene"
or "recombinant" means with regard to, for example, a nucleic acid
sequence, an expression cassette, gene construct or a vector
comprising the nucleic acid sequence or an organism transformed
with the nucleic acid sequences, expression cassettes or vectors
according to the invention, all those constructions brought about
by recombinant methods in which either [0084] (a) the nucleic acid
sequences encoding proteins useful in the methods of the invention,
or [0085] (b) genetic control sequence(s) which is operably linked
with the nucleic acid sequence according to the invention, for
example a promoter, or [0086] (c) a) and b) are not located in
their natural genetic environment or have been modified by
recombinant methods, it being possible for the modification to take
the form of, for example, a substitution, addition, deletion,
inversion or insertion of one or more nucleotide residues. The
natural genetic environment is understood as meaning the natural
genomic or chromosomal locus in the original plant or the presence
in a genomic library. In the case of a genomic library, the natural
genetic environment of the nucleic acid sequence is preferably
retained, at least in part. The environment flanks the nucleic acid
sequence at least on one side and has a sequence length of at least
50 bp, preferably at least 500 bp, especially preferably at least
1000 bp, most preferably at least 5000 bp. A naturally occurring
expression cassette--for example the naturally occurring
combination of the natural promoter of the nucleic acid sequences
with the corresponding nucleic acid sequence encoding a polypeptide
useful in the methods of the present invention, as defined
above--becomes a transgenic expression cassette when this
expression cassette is modified by non-natural, synthetic
("artificial") methods such as, for example, mutagenic treatment.
Suitable methods are described, for example, in U.S. Pat. No.
5,565,350 or WO 00/15815.
[0087] A transgenic plant for the purposes of the invention is thus
understood as meaning, as above, that the nucleic acid sequences
used in the method of the invention are not at their natural locus
in the genome of said plant, it being possible for the nucleic acid
sequences to be expressed homologously or heterologously. However,
as mentioned, transgenic also means that, while the nucleic acid
sequences according to the invention or used in the inventive
method are at their natural position in the genome of a plant, the
sequence has been modified with regard to the natural sequence,
and/or that the regulatory sequences of the natural sequences have
been modified. Transgenic is preferably understood as meaning the
expression of the nucleic acid sequences according to the invention
at an unnatural locus in the genome, i.e. homologous or,
preferably, heterologous expression of the nucleic acid sequences
takes place. Preferred transgenic plants are mentioned herein.
Transformation
[0088] The term "introduction" or "transformation" as referred to
herein encompasses the transfer of an exogenous polynucleotide into
a host cell, irrespective of the method used for transfer. Plant
tissue capable of subsequent clonal propagation, whether by
organogenesis or embryogenesis, may be transformed with a genetic
construct of the present invention and a whole plant regenerated
there from. The particular tissue chosen will vary depending on the
clonal propagation systems available for, and best suited to, the
particular species being transformed. Exemplary tissue targets
include leaf disks, pollen, embryos, cotyledons, hypocotyls,
megagametophytes, callus tissue, existing meristematic tissue
(e.g., apical meristem, axillary buds, and root meristems), and
induced meristem tissue (e.g., cotyledon meristem and hypocotyl
meristem). The polynucleotide may be transiently or stably
introduced into a host cell and may be maintained non-integrated,
for example, as a plasmid. Alternatively, it may be integrated into
the host genome. The resulting transformed plant cell may then be
used to regenerate a transformed plant in a manner known to persons
skilled in the art.
[0089] The transfer of foreign genes into the genome of a plant is
called transformation. Transformation of plant species is now a
fairly routine technique. Advantageously, any of several
transformation methods may be used to introduce the gene of
interest into a suitable ancestor cell. The methods described for
the transformation and regeneration of plants from plant tissues or
plant cells may be utilized for transient or for stable
transformation. Transformation methods include the use of
liposomes, electroporation, chemicals that increase free DNA
uptake, injection of the DNA directly into the plant, particle gun
bombardment, transformation using viruses or pollen and
microprojection. Methods may be selected from the
calcium/polyethylene glycol method for protoplasts (Krens, F. A. et
al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol
Biol 8: 363-373); electroporation of protoplasts (Shillito R. D. et
al. (1985) Bio/Technol 3, 1099-1102); microinjection into plant
material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185);
DNA or RNA-coated particle bombardment (Klein T M et al., (1987)
Nature 327: 70) infection with (non-integrative) viruses and the
like. Transgenic plants, including transgenic crop plants, are
preferably produced via Agrobacterium-mediated transformation. An
advantageous transformation method is the transformation in planta.
To this end, it is possible, for example, to allow the agrobacteria
to act on plant seeds or to inoculate the plant meristem with
agrobacteria. It has proved particularly expedient in accordance
with the invention to allow a suspension of transformed
agrobacteria to act on the intact plant or at least on the flower
primordia. The plant is subsequently grown on until the seeds of
the treated plant are obtained (Clough and Bent, Plant J. (1998)
16, 735-743). Methods for Agrobacterium-mediated transformation of
rice include well known methods for rice transformation, such as
those described in any of the following: European patent
application EP 1198985 A1, Aldemita and Hodges (Planta 199:
612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491-506, 1993),
Hiei et al. (Plant J 6 (2): 271-282, 1994), which disclosures are
incorporated by reference herein as if fully set forth. In the case
of corn transformation, the preferred method is as described in
either Ishida et al. (Nat. Biotechnol 14(6): 745-50, 1996) or Frame
et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are
incorporated by reference herein as if fully set forth. Said
methods are further described by way of example in B. Jenes et al.,
Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1,
Engineering and Utilization, eds. S. D. Kung and R. Wu, Academic
Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol.
Plant Molec. Biol. 42 (1991) 205-225). The nucleic acid sequences
or the construct to be expressed is preferably cloned into a
vector, which is suitable for transforming Agrobacterium
tumefaciens, for example pBin19 (Bevan et al., Nucl. Acids Res. 12
(1984) 8711). Agrobacteria transformed by such a vector can then be
used in known manner for the transformation of plants, such as
plants used as a model, like Arabidopsis (Arabidopsis thaliana is
within the scope of the present invention not considered as a crop
plant), or crop plants such as, by way of example, tobacco plants,
for example by immersing bruised leaves or chopped leaves in an
agrobacterial solution and then culturing them in suitable media.
The transformation of plants by means of Agrobacterium tumefaciens
is described, for example, by Hofgen and Willmitzer in Nucl. Acid
Res. (1988) 16, 9877 or is known inter alia from F. F. White,
Vectors for Gene Transfer in Higher Plants; in Transgenic Plants,
Vol. 1, Engineering and Utilization, eds. S. D. Kung and R. Wu,
Academic Press, 1993, pp. 15-38.
[0090] In addition to the transformation of somatic cells, which
then have to be regenerated into intact plants, it is also possible
to transform the cells of plant meristems and in particular those
cells which develop into gametes. In this case, the transformed
gametes follow the natural plant development, giving rise to
transgenic plants. Thus, for example, seeds of Arabidopsis are
treated with agrobacteria and seeds are obtained from the
developing plants of which a certain proportion is transformed and
thus transgenic [Feldman, K A and Marks M D (1987). Mol Gen Genet
208:274-289; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell,
eds, Methods in Arabidopsis Research. Word Scientific, Singapore,
pp. 274-289]. Alternative methods are based on the repeated removal
of the inflorescences and incubation of the excision site in the
center of the rosette with transformed agrobacteria, whereby
transformed seeds can likewise be obtained at a later moment in
time (Chang (1994). Plant J. 5: 551-558; Katavic (1994). Mol Gen
Genet, 245: 363-370). However, an especially effective method is
the vacuum infiltration method with its modifications such as the
"floral dip" method. In the case of vacuum infiltration of
Arabidopsis, intact plants under reduced pressure are treated with
an agrobacterial suspension [Bechthold, N (1993). C R Acad Sci
Paris Life Sci, 316: 1194-1199], while in the case of the "floral
dip" method the developing floral tissue is incubated briefly with
a surfactant-treated agrobacterial suspension [Clough, S J and Bent
A F (1998) The Plant J. 16, 735-743]. A certain proportion of
transgenic seeds are harvested in both cases, and these seeds can
be distinguished from non-transgenic seeds by growing under the
above-described selective conditions. In addition the stable
transformation of plastids is of advantages because plastids are
inherited maternally is most crops reducing or eliminating the risk
of transgene flow through pollen. The transformation of the
chloroplast genome is generally achieved by a process which has
been schematically displayed in Klaus et al., 2004 [Nature
Biotechnology 22 (2), 225-229]. Briefly the sequences to be
transformed are cloned together with a selectable marker gene
between flanking sequences homologous to the chloroplast genome.
These homologous flanking sequences direct site specific
integration into the plastome. Plastidal transformation has been
described for many different plant species and an overview is given
in Bock (2001) Transgenic plastids in basic research and plant
biotechnology. J Mol Biol. 2001 Sep. 21; 312 (3):425-38 or Maliga,
P (2003) Progress towards commercialization of plastid
transformation technology. Trends Biotechnol. 21, 20-28. Further
biotechnological progress has recently been reported in form of
marker free plastid transformants, which can be produced by a
transient co-integrated maker gene (Klaus et al., 2004, Nature
Biotechnology 22(2), 225-229).
T-DNA Activation Taming
[0091] T-DNA activation tagging (Hayashi et al. Science (1992)
1350-1353), involves insertion of T-DNA, usually containing a
promoter (may also be a translation enhancer or an intron), in the
genomic region of the gene of interest or 10 kb up- or downstream
of the coding region of a gene in a configuration such that the
promoter directs expression of the targeted gene. Typically,
regulation of expression of the targeted gene by its natural
promoter is disrupted and the gene falls under the control of the
newly introduced promoter. The promoter is typically embedded in a
T-DNA. This T-DNA is randomly inserted into the plant genome, for
example, through Agrobacterium infection and leads to modified
expression of genes near the inserted T-DNA. The resulting
transgenic plants show dominant phenotypes due to modified
expression of genes close to the introduced promoter.
Tilling
[0092] The term "TILLING" is an abbreviation of "Targeted Induced
Local Lesions In Genomes" and refers to a mutagenesis technology
useful to generate and/or identify nucleic acid sequences encoding
proteins with modified expression and/or activity. TILLING also
allows selection of plants carrying such mutant variants. These
mutant variants may exhibit modified expression, either in strength
or in location or in timing (if the mutations affect the promoter
for example). These mutant variants may exhibit higher activity
than that exhibited by the gene in its natural form. TILLING
combines high-density mutagenesis with high-throughput screening
methods. The steps typically followed in TILLING are: (a) EMS
mutagenesis (Redei G P and Koncz C (1992) In Methods in Arabidopsis
Research, Koncz C, Chua N H, Schell J, eds. Singapore, World
Scientific Publishing Co, pp. 16-82; Feldmann et al., (1994) In
Meyerowitz E M, Somerville C R, eds, Arabidopsis. Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., pp 137-172;
Lightner J and Caspar T (1998) In J Martinez-Zapater, J Salinas,
eds, Methods on Molecular Biology, Vol. 82. Humana Press, Totowa,
N.J., pp 91-104); (b) DNA preparation and pooling of individuals;
(c) PCR amplification of a region of interest; (d) denaturation and
annealing to allow formation of heteroduplexes; (e) DHPLC, where
the presence of a heteroduplex in a pool is detected as an extra
peak in the chromatogram; (f) identification of the mutant
individual; and (g) sequencing of the mutant PCR product. Methods
for TILLING are well known in the art (McCallum et al., (2000) Nat
Biotechnol 18: 455-457; reviewed by Stemple (2004) Nat Rev Genet.
5(2): 145-50).
Homologous Recombination
[0093] Homologous recombination allows introduction in a genome of
a selected nucleic acid sequence at a defined selected position.
Homologous recombination is a standard technology used routinely in
biological sciences for lower organisms such as yeast or the moss
Physcomitrella. Methods for performing homologous recombination in
plants have been described not only for model plants (Offring a et
al. (1990) EMBO J 9(10): 3077-84) but also for crop plants, for
example rice (Terada et al. (2002) Nat Biotech 20(10): 1030-4; Iida
and Terada (2004) Curr Opin Biotech 15(2): 132-8); Terada et al.,
(2007) Plant Physiol).
Yield
[0094] The term "yield" in general means a measurable produce of
economic value, typically related to a specified crop, to an area,
and to a period of time. Individual plant parts directly contribute
to yield based on their number, size and/or weight, or the actual
yield is the yield per acre for a crop and year, which is
determined by dividing total production (includes both harvested
and appraised production) by planted acres. The term "yield" of a
plant may relate to vegetative biomass (root and/or shoot biomass),
to reproductive organs, and/or to propagules (such as seeds) of
that plant.
Early Vigour
[0095] "Early vigour" refers to active healthy well-balanced growth
especially during early stages of plant growth, and may result from
increased plant fitness due to, for example, the plants being
better adapted to their environment (i.e. optimizing the use of
energy resources and partitioning between shoot and root). Plants
having early vigour also show increased seedling survival and a
better establishment of the crop, which often results in highly
uniform fields (with the crop growing in uniform manner, i.e. with
the majority of plants reaching the various stages of development
at substantially the same time), and often better and higher yield.
Therefore, early vigour may be determined by measuring various
factors, such as thousand kernel weight, percentage germination,
percentage emergence, seedling growth, seedling height, root
length, root and shoot biomass and many more.
Increase/Improve/Enhance
[0096] The terms "increase", "improve" or "enhance" are
interchangeable and shall mean in the sense of the application at
least a 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%,
more preferably 25%, 30%, 35% or 40% more yield and/or growth in
comparison to control plants as defined herein.
Seed Yield
[0097] Increased seed yield may manifest itself as one or more of
the following: a) an increase in seed biomass (total seed weight)
which may be on an individual seed basis and/or per plant and/or
per hectare or acre; b) increased number of flowers per plant; c)
increased number of (filled) seeds; d) increased seed filling rate
(which is expressed as the ratio between the number of filled seeds
divided by the total number of seeds); e) increased harvest index,
which is expressed as a ratio of the yield of harvestable parts,
such as seeds, divided by the total biomass; and f) increased
thousand kernel weight (TKW), which is extrapolated from the number
of filled seeds counted and their total weight. An increased TKW
may result from an increased seed size and/or seed weight, and may
also result from an increase in embryo and/or endosperm size.
[0098] An increase in seed yield may also be manifested as an
increase in seed size and/or seed volume. Furthermore, an increase
in seed yield may also manifest itself as an increase in seed area
and/or seed length and/or seed width and/or seed perimeter.
Increased yield may also result in modified architecture, or may
occur because of modified architecture.
Greenness Index
[0099] The "greenness index" as used herein is calculated from
digital images of plants. For each pixel belonging to the plant
object on the image, the ratio of the green value versus the red
value (in the RGB model for encoding color) is calculated. The
greenness index is expressed as the percentage of pixels for which
the green-to-red ratio exceeds a given threshold. Under normal
growth conditions, under salt stress growth conditions, and under
reduced nutrient availability growth conditions, the greenness
index of plants is measured in the last imaging before flowering.
In contrast, under drought stress growth conditions, the greenness
index of plants is measured in the first imaging after drought.
Plant
[0100] The term "plant" as used herein encompasses whole plants,
ancestors and progeny of the plants and plant parts, including
seeds, shoots, stems, leaves, roots (including tubers), flowers,
and tissues and organs, wherein each of the aforementioned comprise
the gene/nucleic acid sequence of interest. The term "plant" also
encompasses plant cells, suspension cultures, callus tissue,
embryos, meristematic regions, gametophytes, sporophytes, pollen
and microspores, again wherein each of the aforementioned comprises
the gene/nucleic acid sequence of interest.
[0101] Plants that are particularly useful in the methods of the
invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous
plants including fodder or forage legumes, ornamental plants, food
crops, trees or shrubs selected from the list comprising Acer spp.,
Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp.,
Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila
arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis
spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena
sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa,
Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida,
Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica
napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]),
Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa,
Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa,
Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra,
Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp.,
Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus
sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus
sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota,
Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp.,
Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera),
Eleusine coracana, Erianthus sp., Eriobotrya japonica, Eucalyptus
sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca
arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo
biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max),
Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus),
Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum
vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus
spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus
spp., Luffa acutangula, Lupinus spp., Luzula sylvatica,
Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon
lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus
spp., Malpighia emarginata, Mammea americana, Mangifera indica,
Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp.,
Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa
spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus spp.,
Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum,
Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum
sp., Persea spp., Petroselinum crispum, Phalaris arundinacea,
Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites
australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp.,
Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp.,
Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus,
Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp.,
Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum
spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum
integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia
spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma
cacao, Trifolium spp., Triticosecale rimpaui, Triticale (Triticum
secale), Triticum spp. (e.g. Triticum aestivum, Triticum durum,
Triticum turgidum, Triticum hybernum, Triticum macha, Triticum
sativum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus,
Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp.,
Zea mays, Zizania palustris, Ziziphus spp., amongst others.
DETAILED DESCRIPTION OF THE INVENTION
1. 2-Cysteine Peroxiredoxin (2-Cys PRX)
[0102] Surprisingly, it has now been found that modulating,
preferably increasing, expression in the roots of a plant, of a
nucleic acid sequence encoding a 2-Cys PRX polypeptide gives plants
having enhanced yield-related traits relative to control plants.
According to a first embodiment, the present invention provides a
method for enhancing yield-related traits in plants relative to
control plants, comprising modulating, preferably increasing,
expression in the roots of a plant, of a nucleic acid sequence
encoding a 2-Cys PRX polypeptide.
[0103] A preferred method for modulating, preferably increasing,
expression of a nucleic acid sequence encoding a 2-Cys PRX
polypeptide is by introducing and expressing in the roots of a
plant, a nucleic acid sequence encoding a 2-Cys PRX
polypeptide.
[0104] Any reference hereinafter to a "protein useful in the
methods of the invention" is taken to mean a 2-Cys PRX polypeptide
as defined herein. Any reference hereinafter to a "nucleic acid
sequence useful in the methods of the invention" is taken to mean a
nucleic acid sequence capable of encoding such a 2-Cys PRX
polypeptide. The nucleic acid sequence to be introduced into a
plant (and therefore useful in performing the methods of the
invention) is any nucleic acid sequence encoding the type of
polypeptide, which will now be described, hereafter also named
"2-Cys PRX nucleic acid sequence" or "2-Cys PRX gene".
[0105] A "2-Cys PRX polypeptide" as defined herein refers to any
polypeptide comprising from N-terminus to C-terminus: (1) a
plastidic transit peptide; and (2) a 2-Cys PRX conserved
domain.
[0106] Additionally, a "2-Cys PRX polypeptide" comprises one or
both of: (i) Motif1 as represented by SEQ ID NO: 77, or a motif
having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98%, 99% or more amino acid sequence identity to SEQ ID NO: 77; or
(ii) Motif 2 as represented by SEQ ID NO: 78, or a motif having at
least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or
more amino acid sequence identity to SEQ ID NO: 78.
[0107] Alternatively or additionally, a "2-Cys PRX polypeptide" as
defined herein refers to any polypeptide sequence which when used
in the construction of a phylogenetic tree, such as the one
depicted in FIG. 3, tends to cluster with the 2-Cys PRX clade of
polypeptides comprising the polypeptide sequence as represented by
SEQ ID NO: 2, rather than with any other PRX clade.
[0108] Alternatively or additionally, a "2-Cys PRX polypeptide" as
defined herein refers to any polypeptide having in increasing order
of preference at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 98%, 99% or more sequence identity to the 2-Cys
PRX polypeptide as represented by SEQ ID NO: 2 or to any of the
polypeptide sequences given in Table A1 herein.
[0109] The term "domain" and "motif" is defined in the
"definitions" section herein. Specialist databases exist for the
identification of domains, for example, SMART (Schultz et al.
(1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al.
(2002) Nucleic Acids Res 30, 242-244, InterPro (Mulder et al.,
(2003) Nucl. Acids. Res. 31, 315-318, Prosite (Bucher and Bairoch
(1994), A generalized profile syntax for biomolecular sequences
motifs and its function in automatic sequence interpretation. (In)
ISMB-94; Proceedings 2nd International Conference on Intelligent
Systems for Molecular Biology. Altman R., Brutlag D., Karp P.,
Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo
et al., Nucl. Acids. Res. 32:D134-D137, (2004), or Pfam (Bateman et
al., Nucleic Acids Research 30(1): 276-280 (2002). A set of tools
for in silico analysis of protein sequences is available on the
ExPASy proteomics server (Swiss Institute of Bioinformatics
(Gasteiger et al., ExPASy: the proteomics server for in-depth
protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788
(2003)). Domains may also be identified using routine techniques,
such as by sequence alignment. Analysis of the polypeptide sequence
of SEQ ID NO: 2 is presented below in Examples 2 and 4.
[0110] Methods for the alignment of sequences for comparison are
well known in the art, such methods include GAP, BESTFIT, BLAST,
FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch
((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning
the complete sequences) alignment of two sequences that maximizes
the number of matches and minimizes the number of gaps. The BLAST
algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10)
calculates percent sequence identity and performs a statistical
analysis of the similarity between the two sequences. The software
for performing BLAST analysis is publicly available through the
National Centre for Biotechnology Information (NCBI). Homologues
may readily be identified using, for example, the ClustalW multiple
sequence alignment algorithm (version 1.83), with the default
pairwise alignment parameters, and a scoring method in percentage.
Global percentages of similarity and identity may also be
determined using one of the methods available in the MatGAT
software package (Campanella et al., BMC Bioinformatics. 2003 Jul.
10; 4:29. MatGAT: an application that generates similarity/identity
matrices using protein or DNA sequences.). Minor manual editing may
be performed to optimise alignment between conserved motifs, as
would be apparent to a person skilled in the art. Furthermore,
instead of using full-length sequences for the identification of
homologues, specific domains may also be used. The sequence
identity values, which are indicated below in Example 3 as a
percentage were determined over the entire nucleic acid sequence or
polypeptide sequence (Table A2 herein), but may also be determined
over selected domains or conserved motif(s) (such as Motif 1 as
represented by SEQ ID NO: 77, and such as Motif 2 as represented by
SEQ ID NO: 78, both Motif1 and Motif2 comprised in SEQ ID NO: 2),
using the programs mentioned above using the default
parameters.
[0111] The task of protein subcellular localisation prediction is
important and well studied. Knowing a protein's localisation helps
elucidate its function. Experimental methods for protein
localization range from immunolocalization to tagging of proteins
using green fluorescent protein (GFP). Such methods are accurate
although labor-intensive compared with computational methods.
Recently much progress has been made in computational prediction of
protein localisation from sequence data. Among algorithms well
known to a person skilled in the art are available at the ExPASy
Proteomics tools hosted by the Swiss Institute for Bioinformatics,
for example, PSort, TargetP, ChloroP, Predotar, LipoP, MITOPROT,
PATS, PTS1, SignalP and others. The identification of subcellular
localisation of the polypeptide of the invention is shown in
Example 5. In particular SEQ ID NO: 2 of the present invention is
assigned to the plastidic (chloroplastic) compartment of
photosynthetic (autotrophic) cells.
[0112] Methods for targeting proteins to plastids are well known in
the art and include the use of transit peptides. Table 3 below
shows examples of transit peptides which can be used to target any
2-Cys PRX polypeptide to a plastid, which 2-Cys PRX polypeptide is
not, in its natural form, normally targeted to a plastid, or which
2-Cys PRX polypeptide in its natural form is targeted to a plastid
by virtue of a different transit peptide (for example, its natural
transit peptide). For example, a nucleic acid sequence encoding a
cyanobacterial 2-Cys PRX polypeptide may also be suitable for use
in the methods of the invention as long as the 2-Cys PRX
polypeptide is targeted to a plastid, preferably to a
chloroplast.
TABLE-US-00010 TABLE 3 Examples of transit peptide sequences useful
in targeting polypeptides to plastids NCBI Accession Number Source
Organism Protein Function Transit Peptide Sequence SEQ ID NO P07839
Chlamydomonas Ferredoxin MAMAMRSTFAARVGAKPAVRGARPA 156 SRMSCMA
AAR23425 Chlamydomonas Rubisco activase MQVTMKSSAVSGQRVGGARVATRSV
157 RRAQLQV CAA56932 Arabidopsis thaliana Aspartate amino
MASLMLSLGSTSLLPREINKDKLKLGTS 158 transferase
ASNPFLKAKSFSRVTMTVAVKPSR CAA31991 Arabidopsis thaliana Acyl carrier
protein1 MATQFSASVSLQTSCLATTRISFQKPA 159
LISNHGKTNLSFNLRRSIPSRRLSVSC CAB63798 Arabidopsis thaliana Acyl
carrier protein2 MASIAASASISLQARPRQLAIAASQVKS 160
FSNGRRSSLSFNLRQLPTRLTVSCAAK PETVDKVCAVVRKQL CAB63799 Arabidopsis
thaliana Acyl carrier protein3 MASIATSASTSLQARPRQLVIGAKQVK 161
SFSYGSRSNLSFNLRQLPTRLTVYCAA KPETVDKVCAWRKQLSLKE
[0113] A 2-Cys PRX polypeptide is targeted and active in the
plastid, i.e., the 2-Cys PRX polypeptide (at least in its native
form) is capable of catalyzing the removal of H.sub.2O.sub.2, in
the chloroplast. Assays for testing this activity are well known in
the art. Further details are provided in Example 6.
[0114] The present invention is illustrated by transforming plants
with the nucleic acid sequence represented by SEQ ID NO: 1,
encoding the polypeptide sequence of SEQ ID NO: 2. However,
performance of the invention is not restricted to these sequences;
the methods of the invention may advantageously be performed using
any 2-Cys PRX-encoding nucleic acid sequence or 2-Cys PRX
polypeptide sequence as defined herein.
[0115] Examples of nucleic acid sequences encoding 2-Cys PRX
polypeptides are given in Table A1 of Example 1 herein. Such
nucleic acid sequences are useful in performing the methods of the
invention. The polypeptide sequences given in Table A1 of Example 1
are example sequences of orthologues and paralogues of the 2-Cys
PRX polypeptide represented by SEQ ID NO: 2, the terms
"orthologues" and "paralogues" being as defined herein. Further
orthologues and paralogues may readily be identified by performing
a so-called reciprocal blast search. Typically, this involves a
first BLAST involving BLASTing a query sequence (for example using
any of the sequences listed in Table A1 of Example 1) against any
sequence database, such as the publicly available NCBI database.
BLASTN or TBLASTX (using standard default values) are generally
used when starting from a nucleotide sequence, and BLASTP or
TBLASTN (using standard default values) when starting from a
protein sequence. The BLAST results may optionally be filtered. The
full-length sequences of either the filtered results or
non-filtered results are then BLASTed back (second BLAST) against
sequences from the organism from which the query sequence is
derived (where the query sequence is SEQ ID NO: 1 or SEQ ID NO: 2,
the second BLAST would therefore be against Brassica sequences).
The results of the first and second BLASTs are then compared. A
paralogue is identified if a high-ranking hit from the first blast
is from the same species as from which the query sequence is
derived, a BLAST back then ideally results in the query sequence
amongst the highest hits; an orthologue is identified if a
high-ranking hit in the first BLAST is not from the same species as
from which the query sequence is derived, and preferably results
upon BLAST back in the query sequence being among the highest
hits.
[0116] High-ranking hits are those having a low E-value. The lower
the E-value, the more significant the score (or in other words the
lower the chance that the hit was found by chance). Computation of
the E-value is well known in the art. In addition to E-values,
comparisons are also scored by percentage identity. Percentage
identity refers to the number of identical nucleotides (or amino
acids) between the two compared nucleic acid (or polypeptide)
sequences over a particular length. In the case of large families,
ClustalW may be used, followed by a neighbour joining tree, to help
visualize clustering of related genes and to identify orthologues
and paralogues (see FIG. 3).
[0117] Nucleic acid variants may also be useful in practising the
methods of the invention. Examples of such variants include nucleic
acid sequences encoding homologues and derivatives of any one of
the polypeptide sequences given in Table A1 of Example 1, the terms
"homologue" and "derivative" being as defined herein. Also useful
in the methods of the invention are nucleic acid sequences encoding
homologues and derivatives of orthologues or paralogues of any one
of the polypeptide sequences given in Table A1 of Example 1.
Homologues and derivatives useful in the methods of the present
invention have substantially the same biological and functional
activity as the unmodified protein from which they are derived.
[0118] Further nucleic acid variants useful in practising the
methods of the invention include portions of nucleic acid sequences
encoding 2-Cys PRX polypeptides, nucleic acid sequences hybridising
to nucleic acid sequences encoding 2-Cys PRX polypeptides, splice
variants of nucleic acid sequences encoding 2-Cys PRX polypeptides,
allelic variants of nucleic acid sequences encoding 2-Cys PRX
polypeptides and variants of nucleic acid sequences encoding 2-Cys
PRX polypeptides obtained by gene shuffling. The terms hybridising
sequence, splice variant, allelic variant and gene shuffling are as
described herein.
[0119] Nucleic acid sequences encoding 2-Cys PRX polypeptides need
not be full-length nucleic acid sequences, since performance of the
methods of the invention does not rely on the use of full-length
nucleic acid sequences. According to the present invention, there
is provided a method for enhancing yield-related traits in plants,
comprising introducing and expressing in the roots of a plant, a
portion of any one of the nucleic acid sequences given in Table A1
of Example 1, or a portion of a nucleic acid sequence encoding an
orthologue, paralogue or homologue of any of the polypeptide
sequences given in Table A1 of Example 1.
[0120] A portion of a nucleic acid sequence may be prepared, for
example, by making one or more deletions to the nucleic acid
sequence. The portions may be used in isolated form or they may be
fused to other coding (or non-coding) sequences in order to, for
example, produce a protein that combines several activities. When
fused to other coding sequences, the resultant polypeptide produced
upon translation may be bigger than that predicted for the protein
portion.
[0121] Portions useful in the methods of the invention, encode a
2-Cys PRX polypeptide as defined herein, and have substantially the
same biological activity as the polypeptide sequences given in
Table A1 of Example 1. Preferably, the portion is a portion of any
one of the nucleic acid sequences given in Table A1 of Example 1,
or is a portion of a nucleic acid sequence encoding an orthologue
or paralogue of any one of the polypeptide sequences given in Table
A1 of Example 1. Preferably the portion is at least 500, 550, 600,
650, 700, 750, 800, consecutive nucleotides in length, the
consecutive nucleotides being of any one of the nucleic acid
sequences given in Table A1 of Example 1, or of a nucleic acid
sequence encoding an orthologue or paralogue of any one of the
polypeptide sequences given in Table A1 of Example 1. Most
preferably the portion is a portion of the nucleic acid sequence of
SEQ ID NO: 1. Preferably, the portion encodes a polypeptide
sequence which when used in the construction of a phylogenetic
tree, such as the one depicted in FIG. 3, tends to cluster with the
group of 2-Cys PRX polypeptides comprising the polypeptide sequence
represented by SEQ ID NO: 2 rather than with any other group.
[0122] Another nucleic acid variant useful in the methods of the
invention is a nucleic acid sequence capable of hybridising, under
reduced stringency conditions, preferably under stringent
conditions, with a nucleic acid sequence encoding a 2-Cys PRX
polypeptide as defined herein, or with a portion as defined
herein.
[0123] According to the present invention, there is provided a
method for enhancing yield-related traits in plants, comprising
introducing and expressing in the roots of a plant, a nucleic acid
sequence capable of hybridizing to any one of the nucleic acid
sequences given in Table A1 of Example 1, or comprising introducing
and expressing in the roots of a plant, a nucleic acid sequence
capable of hybridising to a nucleic acid sequence encoding an
orthologue, paralogue or homologue of any of the nucleic acid
sequences given in Table A1 of Example 1.
[0124] Hybridising sequences useful in the methods of the invention
encode a 2-Cys PRX polypeptide as defined herein, having
substantially the same biological activity as the polypeptide
sequences given in Table A1 of Example 1. Preferably, the
hybridising sequence is capable of hybridising to any one of the
nucleic acid sequences given in Table A1 of Example 1, or to a
portion of any of these sequences, a portion being as defined
above, or the hybridising sequence is capable of hybridising to a
nucleic acid sequence encoding an orthologue or paralogue of any
one of the polypeptide sequences given in Table A1 of Example 1.
Most preferably, the hybridising sequence is capable of hybridising
to a nucleic acid sequence as represented by SEQ ID NO: 1 or to a
portion thereof.
[0125] Preferably, the hybridising sequence encodes a polypeptide
sequence which when used in the construction of a phylogenetic
tree, such as the one depicted in FIG. 3, tends to cluster with the
group of 2-Cys PRX polypeptides comprising the polypeptide sequence
represented by SEQ ID NO: 2 rather than with any other group.
[0126] Another nucleic acid variant useful in the methods of the
invention is a splice variant encoding a 2-Cys PRX polypeptide as
defined hereinabove, a splice variant being as defined herein.
[0127] According to the present invention, there is provided a
method for enhancing yield-related traits in plants, comprising
introducing and expressing in the roots of a plant, a splice
variant of any one of the nucleic acid sequences given in Table A1
of Example 1, or a splice variant of a nucleic acid sequence
encoding an orthologue, paralogue or homologue of any of the
polypeptide sequences given in Table A1 of Example 1.
[0128] Preferred splice variants are splice variants of a nucleic
acid sequence represented by SEQ ID NO: 1, or a splice variant of a
nucleic acid sequence encoding an orthologue or paralogue of SEQ ID
NO: 2. Preferably, the polypeptide sequence encoded by the splice
variant, when used in the construction of a phylogenetic tree, such
as the one depicted in FIG. 3, tends to cluster with the group of
2-Cys PRX polypeptides comprising the polypeptide sequence
represented by SEQ ID NO: 2 rather than with any other group.
[0129] Another nucleic acid variant useful in performing the
methods of the invention is an allelic variant of a nucleic acid
sequence encoding a 2-Cys PRX polypeptide as defined hereinabove,
an allelic variant being as defined herein.
[0130] According to the present invention, there is provided a
method for enhancing yield-related traits in plants, comprising
introducing and expressing in the roots of a plant, an allelic
variant of any one of the nucleic acid sequences given in Table A1
of Example 1, or comprising introducing and expressing in the roots
of a plant, an allelic variant of a nucleic acid sequence encoding
an orthologue, paralogue or homologue of any of the polypeptide
sequences given in Table A1 of Example 1.
[0131] The allelic variants useful in the methods of the present
invention have substantially the same biological activity as the
2-Cys PRX polypeptide of SEQ ID NO: 2 and any of the polypeptide
sequences depicted in Table A1 of Example 1. Allelic variants exist
in nature, and encompassed within the methods of the present
invention is the use of these natural alleles. Preferably, the
allelic variant is an allelic variant of SEQ ID NO: 1 or an allelic
variant of a nucleic acid sequence encoding an orthologue or
paralogue of SEQ ID NO: 2. Preferably, the polypeptide sequence
encoded by the allelic variant, when used in the construction of a
phylogenetic tree, such as the one depicted in FIG. 3, tends to
cluster with the 2-Cys PRX polypeptides comprising the polypeptide
sequence represented by SEQ ID NO: 2 rather than with any other
group.
[0132] Gene shuffling or directed evolution may also be used to
generate variants of nucleic acid sequences encoding 2-Cys PRX
polypeptides as defined above; the term "gene shuffling" being as
defined herein.
[0133] According to the present invention, there is provided a
method for enhancing yield-related traits in plants, comprising
introducing and expressing in the roots of a plant, a variant of
any one of the nucleic acid sequences given in Table A1 of Example
1, or comprising introducing and expressing in the roots of a
plant, a variant of a nucleic acid sequence encoding an orthologue,
paralogue or homologue of any of the polypeptide sequences given in
Table A1 of Example 1, which variant nucleic acid sequence is
obtained by gene shuffling.
[0134] Preferably, the polypeptide sequence encoded by the variant
nucleic acid sequence obtained by gene shuffling, when used in the
construction of a phylogenetic tree such as the one depicted in
FIG. 3, tends to cluster with the group of 2-Cys PRX polypeptides
comprising the polypeptide sequence represented by SEQ ID NO: 2
rather than with any other group.
[0135] Furthermore, nucleic acid variants may also be obtained by
site-directed mutagenesis. Several methods are available to achieve
site-directed mutagenesis, the most common being PCR based methods
(Current Protocols in Molecular Biology, Wiley Eds.).
[0136] Nucleic acid sequences encoding 2-Cys PRX polypeptides may
be from a natural source, such as from eubacteria and eukaryotes
(fungi, plants, or animals). The nucleic acid sequence derived from
any artificial source, or may be modified from its native form in
composition and/or genomic environment through deliberate human
manipulation. Preferably the 2-Cys PRX polypeptide-encoding nucleic
acid sequence is from a plant, further preferably from a
dicotyledonous plant, more preferably from the family Brassicaceae,
most preferably the nucleic acid sequence is from Brassica
rapa.
[0137] Performance of the methods of the invention gives plants
having enhanced yield-related traits. In particular performance of
the methods of the invention gives plants having increased yield,
especially increased seed yield relative to control plants. The
terms "yield" and "seed yield" are described in more detail in the
"definitions" section herein.
[0138] Reference herein to enhanced yield-related traits is taken
to mean an increase in biomass (weight) of one or more parts of a
plant, which may include aboveground (harvestable) parts and/or
(harvestable) parts below ground. In particular, such harvestable
parts are seeds, and performance of the methods of the invention
results in plants having increased seed yield relative to the seed
yield of control plants.
[0139] Taking corn as an example, a yield increase may be
manifested as one or more of the following: increase in the number
of plants established per hectare or acre, an increase in the
number of ears per plant, an increase in the number of rows, number
of kernels per row, kernel weight, thousand kernel weight, ear
length/diameter, increase in the seed filling rate (which is the
number of filled seeds divided by the total number of seeds and
multiplied by 100), among others. Taking rice as an example, a
yield increase may manifest itself as an increase in one or more of
the following: number of plants per hectare or acre, number of
panicles per plant, number of spikelets per panicle, number of
flowers (florets) per panicle (which is expressed as a ratio of the
number of filled seeds over the number of primary panicles),
increase in the seed filling rate (which is the number of filled
seeds divided by the total number of seeds and multiplied by 100),
increase in thousand kernel weight, among others.
[0140] The present invention provides a method for enhancing
yield-related traits in plants, especially seed yield of plants,
relative to control plants, which method comprises modulating,
preferably increasing, expression in the roots of a plant, of a
nucleic acid sequence encoding a 2-Cys PRX polypeptide as defined
herein.
[0141] Since the transgenic plants according to the present
invention have enhanced yield-related traits, it is likely that
these plants exhibit an increased growth rate (during at least part
of their life cycle), relative to the growth rate of control plants
at a corresponding stage in their life cycle.
[0142] The increased growth rate may be specific to one or more
parts of a plant (including seeds), or may be throughout
substantially the whole plant. Plants having an increased growth
rate may have a shorter life cycle. The life cycle of a plant may
be taken to mean the time needed to grow from a dry mature seed up
to the stage where the plant has produced dry mature seeds, similar
to the starting material. This life cycle may be influenced by
factors such as early vigour, growth rate, greenness index,
flowering time and speed of seed maturation. The increase in growth
rate may take place at one or more stages in the life cycle of a
plant or during substantially the whole plant life cycle. Increased
growth rate during the early stages in the life cycle of a plant
may reflect enhanced vigour. The increase in growth rate may alter
the harvest cycle of a plant allowing plants to be sown later
and/or harvested sooner than would otherwise be possible (a similar
effect may be obtained with earlier flowering time). If the growth
rate is sufficiently increased, it may allow for the further sowing
of seeds of the same plant species (for example sowing and
harvesting of rice plants followed by sowing and harvesting of
further rice plants all within one conventional growing period).
Similarly, if the growth rate is sufficiently increased, it may
allow for the further sowing of seeds of different plants species
(for example the sowing and harvesting of corn plants followed by,
for example, the sowing and optional harvesting of soybean, potato
or any other suitable plant). Harvesting additional times from the
same rootstock in the case of some crop plants may also be
possible. Altering the harvest cycle of a plant may lead to an
increase in annual biomass production per acre (due to an increase
in the number of times (say in a year) that any particular plant
may be grown and harvested). An increase in growth rate may also
allow for the cultivation of transgenic plants in a wider
geographical area than their wild-type counterparts, since the
territorial limitations for growing a crop are often determined by
adverse environmental conditions either at the time of planting
(early season) or at the time of harvesting (late season). Such
adverse conditions may be avoided if the harvest cycle is
shortened. The growth rate may be determined by deriving various
parameters from growth curves, such parameters may be: T-Mid (the
time taken for plants to reach 50% of their maximal size) and T-90
(time taken for plants to reach 90% of their maximal size), amongst
others.
[0143] According to a preferred feature of the present invention,
performance of the methods of the invention gives plants having an
increased growth rate relative to control plants. Therefore,
according to the present invention, there is provided a method for
increasing the growth rate of plants, which method comprises
modulating, preferably increasing, expression in the roots of a
plant, of a nucleic acid sequence encoding a 2-Cys PRX polypeptide
as defined herein.
[0144] An increase in yield and/or growth rate occurs whether the
plant is under non-stress conditions or whether the plant is
exposed to various stresses compared to control plants. Plants
typically respond to exposure to stress by growing more slowly. In
conditions of severe stress, the plant may even stop growing
altogether. Mild stress on the other hand is defined herein as
being any stress to which a plant is exposed which does not result
in the plant ceasing to grow altogether without the capacity to
resume growth. Mild stress in the sense of the invention leads to a
reduction in the growth of the stressed plants of less than 40%,
35% or 30%, preferably less than 25%, 20% or 15%, more preferably
less than 14%, 13%, 12%, 11% or 10% or less in comparison to the
control plant under non-stress conditions. Due to advances in
agricultural practices (irrigation, fertilization, pesticide
treatments) severe stresses are not often encountered in cultivated
crop plants. As a consequence, the compromised growth induced by
mild stress is often an undesirable feature for agriculture. Mild
stresses are the everyday biotic and/or abiotic (environmental)
stresses to which a plant is exposed. Abiotic stresses may be due
to drought or excess water, anaerobic stress, salt stress, chemical
toxicity, oxidative stress and hot, cold or freezing temperatures.
The abiotic stress may be an osmotic stress caused by a water
stress (particularly due to drought), salt stress, oxidative stress
or an ionic stress. Biotic stresses are typically those stresses
caused by pathogens, such as bacteria, viruses, fungi and
insects.
[0145] In particular, the methods of the present invention may be
performed under non-stress conditions or under conditions of mild
drought to give plants having enhanced yield-related tarits
relative to control plants grown under comparable conditions. As
reported in Wang et al. (Planta (2003) 218: 1-14), abiotic stress
leads to a series of morphological, physiological, biochemical and
molecular changes that adversely affect plant growth and
productivity. Drought, salinity, extreme temperatures and oxidative
stress are known to be interconnected and may induce growth and
cellular damage through similar mechanisms. Rabbani et al. (Plant
Physiol (2003) 133: 1755-1767) describes a particularly high degree
of "cross talk" between drought stress and high-salinity stress.
For example, drought and/or salinisation are manifested primarily
as osmotic stress, resulting in the disruption of homeostasis and
ion distribution in the cell. Oxidative stress, which frequently
accompanies high or low temperature, salinity or drought stress,
may cause denaturing of functional and structural proteins. As a
consequence, these diverse environmental stresses often activate
similar cell signalling pathways and cellular responses, such as
the production of stress proteins, up-regulation of anti-oxidants,
accumulation of compatible solutes and growth arrest. The term
"non-stress" conditions as used herein are those environmental
conditions that allow optimal growth of plants. Persons skilled in
the art are aware of normal soil conditions and climatic conditions
for a given location.
[0146] Performance of the methods of the invention gives plants
grown under non-stress conditions or under mild drought conditions
enhanced yield-related traits relative to control plants grown
under comparable conditions. Therefore, according to the present
invention, there is provided a method for enhanced yield-related
traits in plants grown under non-stress conditions or under mild
drought conditions, which method comprises modulating, preferably
increasing, expression in the roots a plant of a nucleic acid
sequence encoding a 2-Cys PRX polypeptide.
[0147] Performance of the methods according to the present
invention results in plants grown under abiotic stress conditions
having enhanced yield-related traits relative to control plants
grown under comparable stress conditions. As reported in Wang et
al. (Planta (2003) 218: 1-14), abiotic stress leads to a series of
morphological, physiological, biochemical and molecular changes
that adversely affect plant growth and productivity. Drought,
salinity, extreme temperatures and oxidative stress are known to be
interconnected and may induce growth and cellular damage through
similar mechanisms. For example, drought and/or salinisation are
manifested primarily as osmotic stress, resulting in the disruption
of homeostasis and ion distribution in the cell. Oxidative stress,
which frequently accompanies high or low temperature, salinity or
drought stress may cause denaturation of functional and structural
proteins. As a consequence, these diverse environmental stresses
often activate similar cell signaling pathways and cellular
responses, such as the production of stress proteins, up-regulation
of anti-oxidants, accumulation of compatible solutes and growth
arrest. Since diverse environmental stresses activate similar
pathways, the exemplification of the present invention with drought
stress should not be seen as a limitation to drought stress, but
more as a screen to indicate the involvement of 2-Cys PRX
polypeptides as defined above, in enhancing yield-related traits
relative to control plants grown in comparable stress conditions,
in abiotic stresses in general.
[0148] A particularly high degree of "cross talk" is reported
between drought stress and high-salinity stress (Rabbani et al.
(2003) Plant Physiol 133: 1755-1767). Therefore, it would be
apparent that a 2-Cys PRX polypeptides would, along with its
usefulness in enhancing yield-related traits in plants, relative to
control plants grown under drought stress conditions, also find use
in enhancing yield-related traits in plants, relative to control
plants grown under various other abiotic stress conditions.
[0149] The term "abiotic stress" as defined herein is taken to mean
any one or more of: water stress (due to drought or excess water),
anaerobic stress, salt stress, temperature stress (due to hot, cold
or freezing temperatures), chemical toxicity stress and oxidative
stress. According to one aspect of the invention, the abiotic
stress is an osmotic stress, selected from water stress, salt
stress, oxidative stress and ionic stress. Preferably, the water
stress is drought stress. The term salt stress is not restricted to
common salt (NaCl), but may be any stress caused by one or more of:
NaCl, KCl, LiCl, MgCl.sub.2, CaCl.sub.2, amongst others.
[0150] In particular, the enhanced yield-related traits in plants
grown under abiotic stress conditions (preferably under drought
stress conditions) relative to control plants grown in comparable
stress conditions, may include one or more of the following: (i)
improved early vigour; (ii) increased aboveground biomass; (iii)
increased root (thick and thin) biomass; (iv) increase number of
flowers per panicle; (v) increased seed fill rate; (vi) increased
total seed yield per plant; (vii) increased number of (filled)
seeds; (viii) increased harvest index; or (ix) increased thousand
kernel weight (TKW).
[0151] Performance of the methods of the invention gives plants
having enhanced yield-related traits under abiotic stress
conditions relative to control plants grown in comparable stress
conditions. Therefore, according to the present invention, there is
provided a method for enhancing yield-related traits in plants
grown under abiotic stress conditions, which method comprises
modulating, preferably increasing, expression in the roots of a
plant, of a nucleic acid sequence encoding a 2-Cys PRX polypeptide.
According to one aspect of the invention, the abiotic stress is an
osmotic stress, selected from one or more of the following: water
stress, salt stress, oxidative stress and ionic stress. Preferably,
the water stress is drought stress.
[0152] Another example of abiotic environmental stress is the
reduced availability of one or more nutrients that need to be
assimilated by the plants for growth and development. Because of
the strong influence of nutrition utilization efficiency on plant
yield and product quality, a huge amount of fertilizer is poured
onto fields to optimize plant growth and quality. Productivity of
plants ordinarily is limited by three primary nutrients,
phosphorous, potassium and nitrogen, which is usually the
rate-limiting element in plant growth of these three. Therefore the
major nutritional element required for plant growth is nitrogen
(N). It is a constituent of numerous important compounds found in
living cells, including amino acids, proteins (enzymes), nucleic
acids, and chlorophyll. 1.5% to 2% of plant dry matter is nitrogen
and approximately 16% of total plant protein. Thus, nitrogen
availability is a major limiting factor for crop plant growth and
production (Frink et al. (1999) Proc Natl Acad Sci USA 96(4):
1175-1180), and has as well a major impact on protein accumulation
and amino acid composition. Therefore, of great interest are crop
plants with an increased yield when grown under nitrogen-limiting
conditions.
[0153] Performance of the methods of the invention gives plants
grown under conditions of nutrient deficiency, particularly under
conditions of nitrogen deficiency, having enhanced yield-related
traits relative to control plants grown under comparable
conditions. Therefore, according to the present invention, there is
provided a method for enhancing yield-related traits in plants
grown under conditions of nutrient deficiency, which method
comprises modulating, preferably increasing, expression in the
roots of a plant, of a nucleic acid sequence encoding a 2-Cys PRX
polypeptide. Nutrient deficiency may result from a lack or excess
of nutrients such as nitrogen, phosphates and other
phosphorous-containing compounds, potassium, calcium, cadmium,
magnesium, manganese, iron and boron, amongst others.
[0154] The present invention encompasses plants or parts thereof
(including seeds) obtainable by the methods according to the
present invention. The plants or parts thereof comprise a nucleic
acid transgene encoding a 2-Cys PRX polypeptide as defined above,
operably linked to a root-specific promoter.
[0155] The invention also provides genetic constructs and vectors
to facilitate introduction and/or expression in plants of nucleic
acid sequences encoding 2-Cys PRX polypeptides. The gene constructs
may be inserted into vectors, which may be commercially available,
suitable for transforming into plants and suitable for expression
of the gene of interest in the transformed cells. The invention
also provides use of a gene construct as defined herein in the
methods of the invention.
[0156] More specifically, the present invention provides a
construct comprising: [0157] (a) a nucleic acid sequence encoding a
2-Cys PRX polypeptide as defined above; [0158] (b) one or more
control sequences capable of driving expression of the nucleic acid
sequence of (a); and optionally [0159] (c) a transcription
termination sequence.
[0160] Preferably, the nucleic acid sequence encoding a 2-Cys PRX
polypeptide is as defined above. The term "control sequence" and
"termination sequence" are as defined herein.
[0161] In one embodiment, one of the control sequences of a
construct is a organ-specific promoter, preferably a promoter for
expression in the roots of a plant. An example of a root-specific
promoter is a Rcc3 promoter, for example a rice Rcc3 promoter as
represented by SEQ ID NO: 80.
[0162] Plants are transformed with a vector comprising any of the
nucleic acid sequences described above. The skilled artisan is well
aware of the genetic elements that must be present on the vector in
order to successfully transform, select and propagate host cells
containing the sequence of interest. The sequence of interest is
operably linked to one or more control sequences (at least to a
promoter).
[0163] Advantageously, any type of promoter, whether natural or
synthetic, may be used to drive expression of the nucleic acid
sequence. According to a preferred feature of the invention, the
nucleic acid sequence encoding a 2-Cys PRX polypeptide is operably
linked to a root-specific promoter. The root-specific promoter is
preferably an RCc3 promoter (Plant Mol Biol. 1995 January;
27(2):237-48), more preferably the RCc3 promoter is from rice,
further preferably the RCc3 promoter is represented by a nucleic
acid sequence substantially similar to SEQ ID NO: 80, most
preferably the promoter is as represented by SEQ ID NO: 80.
Examples of other root-specific promoters which may also be used to
perform the methods of the invention are shown in Table 2b in the
"Definitions" section above.
[0164] It should be clear that the applicability of the present
invention is not restricted to the 2-Cys PRX polypeptide-encoding
nucleic acid sequence represented by SEQ ID NO: 1, nor is the
applicability of the invention restricted to expression of a 2-Cys
PRX polypeptide-encoding nucleic acid sequence when driven by a
root-specific promoter.
[0165] Other organ-specific promoters, for example for preferred
expression in leaves, stems, tubers, meristems, seeds (embryo
and/or endosperm), are useful in performing the methods of the
invention. See the "Definitions" section herein for definitions of
the various promoter types.
[0166] Optionally, one or more terminator sequences may be used in
the construct introduced into a plant. Additional regulatory
elements may include transcriptional as well as translational
enhancers. Those skilled in the art will be aware of terminator and
enhancer sequences that may be suitable for use in performing the
invention. An intron sequence may also be added to the 5'
untranslated region (UTR) or in the coding sequence to increase the
amount of the mature message that accumulates in the cytosol, as
described in the definitions section. Other control sequences
(besides promoter, enhancer, silencer, intron sequences, 3'UTR
and/or 5'UTR regions) may be protein and/or RNA stabilizing
elements. Such sequences would be known or may readily be obtained
by a person skilled in the art.
[0167] The genetic constructs of the invention may further include
an origin of replication sequence that is required for maintenance
and/or replication in a specific cell type. One example is when a
genetic construct is required to be maintained in a bacterial cell
as an episomal genetic element (e.g. plasmid or cosmid molecule).
Preferred origins of replication include, but are not limited to,
the f1-ori and colE1.
[0168] For the detection of the successful transfer of the nucleic
acid sequences as used in the methods of the invention and/or
selection of transgenic plants comprising these nucleic acid
sequences, it is advantageous to use marker genes (or reporter
genes). Therefore, the genetic construct may optionally comprise a
selectable marker gene. Selectable markers are described in more
detail in the "definitions" section herein.
[0169] It is known that upon stable or transient integration of
nucleic acid sequences into plant cells, only a minority of the
cells takes up the foreign DNA and, if desired, integrates it into
its genome, depending on the expression vector used and the
transfection technique used. To identify and select these
integrants, a gene coding for a selectable marker (such as the ones
described above) is usually introduced into the host cells together
with the gene of interest. These markers can for example be used in
mutants in which these genes are not functional by, for example,
deletion by conventional methods. Furthermore, nucleic acid
molecules encoding a selectable marker can be introduced into a
host cell on the same vector that comprises the sequence encoding
the polypeptides of the invention or used in the methods of the
invention, or else in a separate vector. Cells which have been
stably transfected with the introduced nucleic acid sequence can be
identified for example by selection (for example, cells which have
integrated the selectable marker survive whereas the other cells
die). The marker genes may be removed or excised from the
transgenic cell once they are no longer needed. Techniques for
marker gene removal are known in the art, useful techniques are
described above in the definitions section.
[0170] The invention also provides a method for the production of
transgenic plants having enhanced yield-related traits relative to
control plants, comprising introduction and expression in the roots
of a plant, of any nucleic acid sequence encoding a 2-Cys PRX
polypeptide as defined hereinabove.
[0171] More specifically, the present invention provides a method
for the production of transgenic plants having enhanced
yield-related traits, which method comprises: [0172] (i)
introducing and expressing in a plant, plant part, or plant cell a
nucleic acid sequence encoding 2-Cys PRX polypeptide, under the
control of a root-specific promoter; and [0173] (ii) cultivating
the plant cell under conditions promoting plant growth and
development.
[0174] The nucleic acid sequence of (i) may be any of the nucleic
acid sequences capable of encoding a 2-Cys PRX polypeptide as
defined herein.
[0175] The nucleic acid sequence may be introduced directly into a
plant cell or into the plant itself (including introduction into a
tissue, organ or any other part of a plant). According to a
preferred feature of the present invention, the nucleic acid
sequence is preferably introduced into a plant by transformation.
The term "transformation" is described in more detail in the
"definitions" section herein.
[0176] The genetically modified plant cells can be regenerated via
all methods with which the skilled worker is familiar. Suitable
methods can be found in the abovementioned publications by S. D.
Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
[0177] Generally after transformation, plant cells or cell
groupings are selected for the presence of one or more markers
which are encoded by plant-expressible genes co-transferred with
the gene of interest, following which the transformed material is
regenerated into a whole plant. To select transformed plants, the
plant material obtained in the transformation is, as a rule,
subjected to selective conditions so that transformed plants can be
distinguished from untransformed plants. For example, the seeds
obtained in the above-described manner can be planted and, after an
initial growing period, subjected to a suitable selection by
spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into
plants. Alternatively, the transformed plants are screened for the
presence of a selectable marker such as the ones described
above.
[0178] Following DNA transfer and regeneration, putatively
transformed plants may also be evaluated, for instance using
Southern analysis, for the presence of the gene of interest, copy
number and/or genomic organisation. Alternatively or additionally,
expression levels of the newly introduced DNA may be monitored
using Northern and/or Western analysis, both techniques being well
known to persons having ordinary skill in the art.
[0179] The generated transformed plants may be propagated by a
variety of means, such as by clonal propagation or classical
breeding techniques. For example, a first generation (or T1)
transformed plant may be selfed and homozygous second-generation
(or T2) transformants selected, and the T2 plants may then further
be propagated through classical breeding techniques. The generated
transformed organisms may take a variety of forms. For example,
they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain
the expression cassette); grafts of transformed and untransformed
tissues (e.g., in plants, a transformed rootstock grafted to an
untransformed scion).
[0180] The present invention clearly extends to any plant cell or
plant produced by any of the methods described herein, and to all
plant parts and propagules thereof. The present invention extends
further to encompass the progeny of a primary transformed or
transfected cell, tissue, organ or whole plant that has been
produced by any of the aforementioned methods, the only requirement
being that progeny exhibit the same genotypic and/or phenotypic
characteristic(s) as those produced by the parent in the methods
according to the invention.
[0181] The invention also includes host cells containing an
isolated nucleic acid sequence encoding a 2-Cys PRX polypeptide as
defined hereinabove. Preferred host cells according to the
invention are plant cells. Host plants for the nucleic acid
sequences or the vector used in the method according to the
invention, the expression cassette or construct or vector are, in
principle, advantageously all plants, which are capable of
synthesizing the polypeptides used in the inventive method.
[0182] The methods of the invention are advantageously applicable
to any plant. Plants that are particularly useful in the methods of
the invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous
plants including fodder or forage legumes, ornamental plants, food
crops, trees or shrubs. According to a preferred embodiment of the
present invention, the plant is a crop plant. Examples of crop
plants include soybean, sunflower, canola, alfalfa, rapeseed,
cotton, tomato, potato and tobacco. Further preferably, the plant
is a monocotyledonous plant. Examples of monocotyledonous plants
include sugarcane. More preferably the plant is a cereal. Examples
of cereals include rice, maize, wheat, barley, millet, rye,
triticale, sorghum and oats.
[0183] The invention also extends to harvestable parts of a plant
such as, but not limited to seeds, leaves, fruits, flowers, stems,
rhizomes, tubers and bulbs. The invention furthermore relates to
products derived, preferably directly derived, from a harvestable
part of such a plant, such as dry pellets or powders, oil, fat and
fatty acids, starch or proteins.
[0184] According to a preferred feature of the invention, the
modulated expression is increased expression. Methods for
increasing expression of nucleic acid sequences or genes, or gene
products, are well documented in the art and examples are provided
in the definitions section.
[0185] As mentioned above, a preferred method for modulating,
preferably increasing, expression of a nucleic acid sequence
encoding a 2-Cys PRX polypeptide is by introducing and expressing
in the roots of a plant, a nucleic acid sequence encoding a 2-Cys
PRX polypeptide; however the effects of performing the method, i.e.
enhancing yield-related traits may also be achieved using other
well known techniques, including but not limited to T-DNA
activation tagging, TILLING, homologous recombination. A
description of these techniques is provided in the definitions
section.
[0186] The present invention also encompasses use of nucleic acid
sequences encoding 2-Cys PRX polypeptides as described herein and
use of these 2-Cys PRX polypeptides in enhancing any of the
aforementioned yield-related traits in plants.
[0187] Nucleic acid sequences encoding 2-Cys PRX polypeptide
described herein, or the 2-Cys PRX polypeptides themselves, may
find use in breeding programmes in which a DNA marker is identified
which may be genetically linked to a 2-Cys PRX polypeptide-encoding
gene. The genes/nucleic acid sequences, or the 2-Cys PRX
polypeptides themselves may be used to define a molecular marker.
This DNA or protein marker may then be used in breeding programmes
to select plants having enhanced yield-related traits as defined
hereinabove in the methods of the invention.
[0188] Allelic variants of a 2-Cys PRX polypeptide-encoding
gene/nucleic acid sequence may also find use in marker-assisted
breeding programmes. Such breeding programmes sometimes require
introduction of allelic variation by mutagenic treatment of the
plants, using for example EMS mutagenesis; alternatively, the
programme may start with a collection of allelic variants of so
called "natural" origin caused unintentionally. Identification of
allelic variants then takes place, for example, by PCR. This is
followed by a step for selection of superior allelic variants of
the sequence in question and which enhance yield-related traits.
Selection is typically carried out by monitoring growth performance
of plants containing different allelic variants of the sequence in
question. Growth performance may be monitored in a greenhouse or in
the field. Further optional steps include crossing plants in which
the superior allelic variant was identified with another plant.
This could be used, for example, to make a combination of
interesting phenotypic features.
[0189] Nucleic acid sequences encoding 2-Cys PRX polypeptides may
also be used as probes for genetically and physically mapping the
genes that they are a part of, and as markers for traits linked to
those genes. Such information may be useful in plant breeding in
order to develop lines with desired phenotypes. Such use of 2-Cys
PRX polypeptide-encoding nucleic acid sequences requires only a
nucleic acid sequence of at least 15 nucleotides in length. The
2-Cys PRX polypeptide-encoding nucleic acid sequences may be used
as restriction fragment length polymorphism (RFLP) markers.
Southern blots (Sambrook J, Fritsch E F and Maniatis T (1989)
Molecular Cloning, A Laboratory Manual) of restriction-digested
plant genomic DNA may be probed with the 2-Cys PRX-encoding nucleic
acid sequences. The resulting banding patterns may then be
subjected to genetic analyses using computer programs such as
MapMaker (Lander et al. (1987) Genomics 1: 174-181) in order to
construct a genetic map. In addition, the nucleic acid sequences
may be used to probe Southern blots containing restriction
endonuclease-treated genomic DNAs of a set of individuals
representing parent and progeny of a defined genetic cross.
Segregation of the DNA polymorphisms is noted and used to calculate
the position of the 2-Cys PRX polypeptide-encoding nucleic acid
sequence in the genetic map previously obtained using this
population (Botstein et al. (1980) Am. J. Hum. Genet.
32:314-331).
[0190] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bernatzky and Tanksley (1986)
Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe
genetic mapping of specific cDNA clones using the methodology
outlined above or variations thereof. For example, F2 intercross
populations, backcross populations, randomly mated populations,
near isogenic lines, and other sets of individuals may be used for
mapping. Such methodologies are well known to those skilled in the
art.
[0191] The nucleic acid probes may also be used for physical
mapping (i.e., placement of sequences on physical maps; see
Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical
Guide, Academic press 1996, pp. 319-346, and references cited
therein).
[0192] In another embodiment, the nucleic acid probes may be used
in direct fluorescence in situ hybridisation (FISH) mapping (Trask
(1991) Trends Genet. 7:149-154). Although current methods of FISH
mapping favour use of large clones (several kb to several hundred
kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in
sensitivity may allow performance of FISH mapping using shorter
probes.
[0193] A variety of nucleic acid amplification-based methods for
genetic and physical mapping may be carried out using the nucleic
acid sequences. Examples include allele-specific amplification
(Kazazian (1989) J. Lab. Clin. Med 11:95-96), polymorphism of
PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics
16:325-332), allele-specific ligation (Landegren et al. (1988)
Science 241:1077-1080), nucleotide extension reactions (Sokolov
(1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter
et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook
(1989) Nucleic Acid Res. 17:6795-6807). For these methods, the
sequence of a nucleic acid is used to design and produce primer
pairs for use in the amplification reaction or in primer extension
reactions. The design of such primers is well known to those
skilled in the art. In methods employing PCR-based genetic mapping,
it may be necessary to identify DNA sequence differences between
the parents of the mapping cross in the region corresponding to the
instant nucleic acid sequence. This, however, is generally not
necessary for mapping methods.
[0194] The methods according to the present invention result in
plants having enhanced yield-related traits, as described
hereinbefore. These traits may also be combined with other
economically advantageous traits, such as further yield-enhancing
traits, tolerance to other abiotic and biotic stresses, traits
modifying various architectural features and/or biochemical and/or
physiological features.
2. Annexin-Like (ANN)
[0195] Surprisingly, it has now been found that modulating
expression in a plant of a nucleic acid encoding an annexin-like
(hereafter named ANN) polypeptide gives plants having enhanced
yield-related traits relative to control plants. According to a
first embodiment, the present invention provides a method for
enhancing yield-related traits in plants relative to control
plants, comprising modulating expression in a plant of a nucleic
acid encoding an ANN polypeptide.
[0196] A preferred method for modulating (preferably, increasing)
expression of a nucleic acid encoding an ANN polypeptide is by
introducing and expressing in a plant a nucleic acid encoding an
ANN polypeptide.
[0197] Any reference hereinafter to a "protein useful in the
methods of the invention" is taken to mean an ANN polypeptide as
defined herein. Any reference hereinafter to a "nucleic acid useful
in the methods of the invention" is taken to mean a nucleic acid
capable of encoding such an ANN polypeptide. The nucleic acid to be
introduced into a plant (and therefore useful in performing the
methods of the invention) is any nucleic acid encoding the type of
protein which will now be described, hereafter also named "ANN
nucleic acid" or "ANN gene".
[0198] An "ANN polypeptide" as defined herein refers to any
polypeptide comprising in its native form (i.e. the protein as it
is encoded in the genome) at least one, preferably two or more of
the following conserved signature sequences:
TABLE-US-00011 Signature sequence 1 (SEQ ID NO: 87)
(A/L/V)(M/V/L/I)(L/V/M/I/C)X(W/F)(I/V/M/T/A)
(L/P/Y/M/F)(D/E/S/H)(P/A)X(G/S/E/A)RDA
wherein X on position 4 may be any amino acid, preferably one of L,
S, I, V, Q, or M; and X on position 10 may be any amino acid,
preferably one of A, V, P, G, S, T or W.
TABLE-US-00012 Preferably, signature sequence 1 is
(A/L/V)(V/L/I)(L/V/M/I)X(W/F)(V/T/A)(L/Y/M/F) (D/E/S/H)PX(E/A)RDA
Signature sequence 2 (SEQ ID NO: 88):
A(F/I/V/C/G)XG(F/R/W/M)G(C/T/V)(D/N)(S/A/T/E)X
(T/A/V/L/M)(V/I/L)(I/T)X(I/V/T)L(T/A/G) (H/Q/K)(R/S)
wherein X in position 3 may be any amino acid, preferably one of K,
R, Q, S, E, A or M; X on position 10 may be any amino acid,
preferably one of T, S, K, N, G, D, A, E, Q, or R; X on position 14
may be any amino acid, preferably one of N, A, R, E, D, S, Q,
TABLE-US-00013 Preferably, signature sequence 2 is
A(F/I/V/C/G)XG(W/M)G(T/V)(D/N)EX(A/L/M)(I/L)(I/T)
X(I/V/T)L(A/G)(H/Q/K)(R/S) Signature sequence 3 (SEQ ID NO: 89):
(T/S)(D/N/E/T)(D/E/K)XXL(I/T/S/N)R(V/I/A/G)
(V/I/F)(V/T/C/S/A)(T/S)R(T/A)(E/D) (I/V/F/L/K/H)(D/S)
wherein X on position 4 may be any amino acid, preferably one of S,
T, D, E, G, W, N, K; X on position 5 may be any amino acid,
preferably one of T, A, S, M, H, D, G, W.
TABLE-US-00014 Preferably, signature sequence 3 is
(T/S)(D/E/T)(D/E/K)XXL(T/S/N)R(V/I/A/G)(V/I/F)
(V/T/C/S/A)(T/S)R(T/A)(E/D)(I/V/F/L/K/H)(D/S) Signature sequence 4
(SEQ ID NO: 90): (Y/H)(F/Y)(A/E/V/S)(K/E/D)(V/A/L/I)(L/V/I)
(R/H/D)X(S/A)(M/I/L) Preferably, signature sequence 4 is
(Y/H)(F/Y)(A/E/V/S)(K/E/D)(V/L/I)(L/V/I)(R/D) X(S/A)(I/L) Signature
sequence 5 (SEQ ID NO: 91):
(Y/G/K/S)(L/I/M)E(H/E)(D/H)(I/V/L)(G/A/E) Preferably, signature
sequence 5 is S(L/I/M)EE(D/H)(I/V/L)A Signature sequence 6 (SEQ ID
NO: 92): (F/L/V/I/T)(I/L/V)(R/Q/Y)(I/V)(F/L/V/I)(T/S/G/A) (E/D/T)RS
Preferably, signature sequence 6 is
(F/L/V/I/T)(I/L/V)(R/Y)(I/V)(L/V/I)(T/S/G/A)TRS Signature sequence
7 (SEQ ID NO: 93): Y(R/K/M/E/Q)X(F/T/L/M/I)(L/I)(L/I/V)(S/T/V/A)
L(V/I/L/A/M)(G/S)
wherein X on position 8 may be any amino acid, preferably one of K,
E, D, T, L, S, Q, R, N, or A. wherein X on position 3 may be any
amino acid, preferably one of T, D, N, K, S, R, A
[0199] Since the ANN polypeptide is related to annexins, the ANN
polypeptide useful in the methods of the invention preferably also
has one or more annexin domains (Pfam entry PF00191, SMART entry
SM00335, InterPro IPR001464, see also FIGS. 7 A and 7 B).
[0200] Preferably, the polypeptide sequence which when used in the
construction of a phylogenetic tree, such as the one depicted in
FIG. 8 (taken from Cantero et al., Plant Physiol. Biochem. 44,
13-24, 2006), tends to cluster with the group of ANN polypeptides
comprising the amino acid sequence represented by SEQ ID NO: 84 and
SEQ ID NO: 135, rather than with any other group.
[0201] The term "domain", "motif" and "signature" is defined in the
"definitions" section herein. Specialist databases exist for the
identification of domains, for example, SMART (Schultz et al.
(1998) Proc. Natl. Acad. Sci. USA 95, 5857-5864; Letunic et al.
(2002) Nucleic Acids Res 30, 242-244, InterPro (Mulder et al.,
(2003) Nucl. Acids. Res. 31, 315-318, Prosite (Bucher and Bairoch
(1994), A generalized profile syntax for biomolecular sequences
motifs and its function in automatic sequence interpretation. (In)
ISMB-94; Proceedings 2nd International Conference on Intelligent
Systems for Molecular Biology. Altman R., Brutlag D., Karp P.,
Lathrop R., Searls D., Eds., pp 53-61, AAAI Press, Menlo Park; Hulo
et al., Nucl. Acids. Res. 32:D134-D137, (2004), or Pfam (Bateman et
al., Nucleic Acids Research 30(1): 276-280 (2002). A set of tools
for in silico analysis of protein sequences is available on the
ExPASy proteomics server (Swiss Institute of Bioinformatics
(Gasteiger et al., ExPASy: the proteomics server for in-depth
protein knowledge and analysis, Nucleic Acids Res. 31:3784-3788
(2003)). Domains may also be identified using routine techniques,
such as by sequence alignment.
[0202] Methods for the alignment of sequences for comparison are
well known in the art, such methods include GAP, BESTFIT, BLAST,
FASTA and TFASTA. GAP uses the algorithm of Needleman and Wunsch
((1970) J Mol Biol 48: 443-453) to find the global (i.e. spanning
the complete sequences) alignment of two sequences that maximizes
the number of matches and minimizes the number of gaps. The BLAST
algorithm (Altschul et al. (1990) J Mol Biol 215: 403-10)
calculates percent sequence identity and performs a statistical
analysis of the similarity between the two sequences. The software
for performing BLAST analysis is publicly available through the
National Centre for Biotechnology Information (NCBI). Homologues
may readily be identified using, for example, the ClustalW multiple
sequence alignment algorithm (version 1.83), with the default
pairwise alignment parameters, and a scoring method in percentage.
Global percentages of similarity and identity may also be
determined using one of the methods available in the MatGAT
software package (Campanella et al., BMC Bioinformatics. 2003 Jul.
10; 4:29. MatGAT: an application that generates similarity/identity
matrices using protein or DNA sequences.). Minor manual editing may
be performed to optimise alignment between conserved motifs, as
would be apparent to a person skilled in the art. Furthermore,
instead of using full-length sequences for the identification of
homologues, specific domains may also be used. The sequence
identity values may be determined over the entire nucleic acid or
amino acid sequence or over selected domains or conserved motif(s),
using the programs mentioned above using the default
parameters.
[0203] Furthermore, ANN polypeptides (at least in their native
form) typically have calcium binding activity and capability of
binding to membranes. Tools and techniques for measuring membrane
binding activity are well known in the art, and include measurement
of effects on membrane surface hydrophobicity, vesicle leakage or
vesicle aggregation. In addition, ANN polypeptides may exhibit
enzymatic activity; for example, Annexin 1 from Arabidopsis
thaliana is reported to display peroxidase activity (Gorecka et
al., Biochem. Biophys. Res. Comm. 336, 868-875, 2005). Further
details are provided in Example 19.
[0204] The present invention is illustrated by transforming plants
with the nucleic acid sequence represented by SEQ ID NO: 83,
encoding respectively the polypeptide sequences of SEQ ID NO: 84.
However, performance of the invention is not restricted to these
sequences; the methods of the invention may advantageously be
performed using any ANN-encoding nucleic acid or ANN polypeptide as
defined herein.
[0205] Examples of nucleic acids encoding ANN polypeptides are
given in Table B1 of Example 14 herein. Such nucleic acids are
useful in performing the methods of the invention. The amino acid
sequences given in Table B1 of Example 14 are example sequences of
orthologues and paralogues of the ANN polypeptide represented by
SEQ ID NO: 84, the terms "orthologues" and "paralogues" being as
defined herein. Further orthologues and paralogues may readily be
identified by performing a so-called reciprocal blast search.
Typically, this involves a first BLAST involving BLASTing a query
sequence (for example using any of the sequences listed in Table B1
of Example 14) against any sequence database, such as the publicly
available NCBI database. BLASTN or TBLASTX (using standard default
values) are generally used when starting from a nucleotide
sequence, and BLASTP or TBLASTN (using standard default values)
when starting from a protein sequence. The BLAST results may
optionally be filtered. The full-length sequences of either the
filtered results or non-filtered results are then BLASTed back
(second BLAST) against sequences from the organism from which the
query sequence is derived (where the query sequence is SEQ ID NO:
83 or SEQ ID NO: 84, the second BLAST would therefore be against
Arabidopsis thaliana sequences). The results of the first and
second BLASTs are then compared. A paralogue is identified if a
high-ranking hit from the first blast is from the same species as
from which the query sequence is derived, a BLAST back then ideally
results in the query sequence amongst the highest hits; an
orthologue is identified if a high-ranking hit in the first BLAST
is not from the same species as from which the query sequence is
derived, and preferably results upon BLAST back in the query
sequence being among the highest hits.
[0206] High-ranking hits are those having a low E-value. The lower
the E-value, the more significant the score (or in other words the
lower the chance that the hit was found by chance). Computation of
the E-value is well known in the art. In addition to E-values,
comparisons are also scored by percentage identity. Percentage
identity refers to the number of identical nucleotides (or amino
acids) between the two compared nucleic acid (or polypeptide)
sequences over a particular length. In the case of large families,
ClustalW may be used, followed by a neighbour joining tree, to help
visualize clustering of related genes and to identify orthologues
and paralogues.
[0207] Nucleic acid variants may also be useful in practising the
methods of the invention. Examples of such variants include nucleic
acids encoding homologues and derivatives of any one of the amino
acid sequences given in Table B1 of Example 14, the terms
"homologue" and "derivative" being as defined herein. Also useful
in the methods of the invention are nucleic acids encoding
homologues and derivatives of orthologues or paralogues of any one
of the amino acid sequences given in Table B1 of Example 14.
Homologues and derivatives useful in the methods of the present
invention have substantially the same biological and functional
activity as the unmodified protein from which they are derived.
[0208] Further nucleic acid variants useful in practising the
methods of the invention include portions of nucleic acids encoding
ANN polypeptides, nucleic acids hybridising to nucleic acids
encoding ANN polypeptides, splice variants of nucleic acids
encoding ANN polypeptides, allelic variants of nucleic acids
encoding ANN polypeptides and variants of nucleic acids encoding
ANN polypeptides obtained by gene shuffling. The terms hybridising
sequence, splice variant, allelic variant and gene shuffling are as
described herein.
[0209] Nucleic acids encoding ANN polypeptides need not be
full-length nucleic acids, since performance of the methods of the
invention does not rely on the use of full-length nucleic acid
sequences. According to the present invention, there is provided a
method for enhancing yield-related traits in plants, comprising
introducing and expressing in a plant a portion of any one of the
nucleic acid sequences given in Table B1 of Example 14, or a
portion of a nucleic acid encoding an orthologue, paralogue or
homologue of any of the amino acid sequences given in Table B1 of
Example 14.
[0210] A portion of a nucleic acid may be prepared, for example, by
making one or more deletions to the nucleic acid. The portions may
be used in isolated form or they may be fused to other coding (or
non-coding) sequences in order to, for example, produce a protein
that combines several activities. When fused to other coding
sequences, the resultant polypeptide produced upon translation may
be bigger than that predicted for the protein portion.
[0211] Portions useful in the methods of the invention, encode an
ANN polypeptide as defined herein, and have substantially the same
biological activity as the amino acid sequences given in Table B1
of Example 14. Preferably, the portion is a portion of any one of
the nucleic acids given in Table B1 of Example 14, or is a portion
of a nucleic acid encoding an orthologue or paralogue of any one of
the amino acid sequences given in Table B1 of Example 14.
Preferably the portion is at least 400, 450, 500, 550, 600, 650,
700, consecutive nucleotides in length, the consecutive nucleotides
being of any one of the nucleic acid sequences given in Table B1 of
Example 14, or of a nucleic acid encoding an orthologue or
paralogue of any one of the amino acid sequences given in Table B1
of Example 14. Most preferably the portion is a portion of the
nucleic acid of SEQ ID NO: 83. Preferably, the portion encodes an
amino acid sequence which when used in the construction of a
phylogenetic tree, such as the one depicted in FIG. 8, tends to
cluster with the group of ANN comprising the amino acid sequence
represented by SEQ ID NO: 84 and SEQ ID NO: 135 rather than with
any other group.
[0212] Another nucleic acid variant useful in the methods of the
invention is a nucleic acid capable of hybridising, under reduced
stringency conditions, preferably under stringent conditions, with
a nucleic acid encoding an ANN polypeptide as defined herein, or
with a portion as defined herein.
[0213] According to the present invention, there is provided a
method for enhancing yield-related traits in plants, comprising
introducing and expressing in a plant a nucleic acid capable of
hybridizing to any one of the nucleic acids given in Table B1 of
Example 14, or comprising introducing and expressing in a plant a
nucleic acid capable of hybridising to a nucleic acid encoding an
orthologue, paralogue or homologue of any of the nucleic acid
sequences given in Table B1 of Example 14.
[0214] Hybridising sequences useful in the methods of the invention
encode an ANN polypeptide as defined herein, and have substantially
the same biological activity as the amino acid sequences given in
Table B1 of Example 14. Preferably, the hybridising sequence is
capable of hybridising to any one of the nucleic acids given in
Table B1 of Example 14, or to a portion of any of these sequences,
a portion being as defined above, or wherein the hybridising
sequence is capable of hybridising to a nucleic acid encoding an
orthologue or paralogue of any one of the amino acid sequences
given in Table B1 of Example 14. Most preferably, the hybridising
sequence is capable of hybridising to a nucleic acid as represented
by SEQ ID NO: 83 or to a portion thereof.
[0215] Preferably, the hybridising sequence encodes an amino acid
sequence which when used in the construction of a phylogenetic
tree, such as the one depicted in FIG. 8, tends to cluster with the
group of ANN polypeptides comprising the amino acid sequence
represented by SEQ ID NO: 84 and SEQ ID NO: 135, rather than with
any other group.
[0216] Another nucleic acid variant useful in the methods of the
invention is a splice variant encoding an ANN polypeptide as
defined hereinabove, a splice variant being as defined herein.
[0217] According to the present invention, there is provided a
method for enhancing yield-related traits in plants, comprising
introducing and expressing in a plant a splice variant of any one
of the nucleic acid sequences given in Table B1 of Example 14, or a
splice variant of a nucleic acid encoding an orthologue, paralogue
or homologue of any of the amino acid sequences given in Table B1
of Example 14.
[0218] Preferred splice variants are splice variants of a nucleic
acid represented by SEQ ID NO: 83, or a splice variant of a nucleic
acid encoding an orthologue or paralogue of SEQ ID NO: 84.
Preferably, the amino acid sequence encoded by the splice variant,
when used in the construction of a phylogenetic tree, such as the
one depicted in FIG. 8, tends to cluster with the group of ANN
polypeptides comprising the amino acid sequence represented by SEQ
ID NO: 84 and SEQ ID NO: 135 rather than with any other group.
[0219] Another nucleic acid variant useful in performing the
methods of the invention is an allelic variant of a nucleic acid
encoding an ANN polypeptide as defined hereinabove, an allelic
variant being as defined herein.
[0220] According to the present invention, there is provided a
method for enhancing yield-related traits in plants, comprising
introducing and expressing in a plant an allelic variant of any one
of the nucleic acids given in Table B1 of Example 14, or comprising
introducing and expressing in a plant an allelic variant of a
nucleic acid encoding an orthologue, paralogue or homologue of any
of the amino acid sequences given in Table B1 of Example 14.
[0221] The allelic variants useful in the methods of the present
invention have substantially the same biological activity as the
ANN polypeptide of SEQ ID NO: 84 and any of the amino acids
depicted in Table B1 of Example 14. Allelic variants exist in
nature, and encompassed within the methods of the present invention
is the use of these natural alleles. Preferably, the allelic
variant is an allelic variant of SEQ ID NO: 83 or an allelic
variant of a nucleic acid encoding an orthologue or paralogue of
SEQ ID NO: 84. Preferably, the amino acid sequence encoded by the
allelic variant, when used in the construction of a phylogenetic
tree, such as the one depicted in FIG. 8, tends to cluster with the
ANN polypeptides comprising the amino acid sequence represented by
SEQ ID NO: 84 and SEQ ID NO: 135, rather than with any other
group.
[0222] Gene shuffling or directed evolution may also be used to
generate variants of nucleic acids encoding ANN polypeptides as
defined above; the term "gene shuffling" being as defined
herein.
[0223] According to the present invention, there is provided a
method for enhancing yield-related traits in plants, comprising
introducing and expressing in a plant a variant of any one of the
nucleic acid sequences given in Table B1 of Example 14, or
comprising introducing and expressing in a plant a variant of a
nucleic acid encoding an orthologue, paralogue or homologue of any
of the amino acid sequences given in Table B1 of Example 14, which
variant nucleic acid is obtained by gene shuffling.
[0224] Preferably, the amino acid sequence encoded by the variant
nucleic acid obtained by gene shuffling, when used in the
construction of a phylogenetic tree such as the one depicted in
FIG. 8, tends to cluster with the group of ANN polypeptides
comprising the amino acid sequence represented by SEQ ID NO: 84 and
SEQ ID NO: 135, rather than with any other group.
[0225] Furthermore, nucleic acid variants may also be obtained by
site-directed mutagenesis. Several methods are available to achieve
site-directed mutagenesis, the most common being PCR based methods
(Current Protocols in Molecular Biology. Wiley Eds).
[0226] Nucleic acids encoding ANN polypeptides may be derived from
any natural or artificial source. The nucleic acid may be modified
from its native form in composition and/or genomic environment
through deliberate human manipulation. Preferably the ANN
polypeptide-encoding nucleic acid is from a plant, further
preferably from a dicotyledonous plant, more preferably from the
family Brassicaceae, most preferably the nucleic acid is from
Arabidopsis thaliana.
[0227] Performance of the methods of the invention gives plants
having enhanced yield-related traits. In particular performance of
the methods of the invention gives plants having increased yield,
especially increased seed yield relative to control plants. The
terms "yield" and "seed yield" are described in more detail in the
"definitions" section herein.
[0228] Reference herein to enhanced yield-related traits is taken
to mean an increase in biomass (weight) of one or more parts of a
plant, which may include aboveground (harvestable) parts and/or
(harvestable) parts below ground. In particular, such harvestable
parts are seeds, and performance of the methods of the invention
results in plants having increased seed yield relative to the seed
yield of control plants.
[0229] Taking corn as an example, a yield increase may be
manifested as one or more of the following: increase in the number
of plants established per hectare or acre, an increase in the
number of ears per plant, an increase in the number of rows, number
of kernels per row, kernel weight, thousand kernel weight, ear
length/diameter, increase in the seed filling rate (which is the
number of filled seeds divided by the total number of seeds and
multiplied by 100), among others. Taking rice as an example, a
yield increase may manifest itself as an increase in one or more of
the following: number of plants per hectare or acre, number of
panicles per plant, number of spikelets per panicle, number of
flowers (florets) per panicle (which is expressed as a ratio of the
number of filled seeds over the number of primary panicles),
increase in the seed filling rate (which is the number of filled
seeds divided by the total number of seeds and multiplied by 100),
increase in thousand kernel weight, among others.
[0230] The present invention provides a method for increasing
yield, especially seed yield of plants, relative to control plants,
which method comprises modulating expression, preferably increasing
expression, in a plant of a nucleic acid encoding an ANN
polypeptide as defined herein.
[0231] Since the transgenic plants according to the present
invention have increased yield, it is likely that these plants
exhibit an increased growth rate (during at least part of their
life cycle), relative to the growth rate of control plants at a
corresponding stage in their life cycle.
[0232] The increased growth rate may be specific to one or more
parts of a plant (including seeds), or may be throughout
substantially the whole plant. Plants having an increased growth
rate may have a shorter life cycle. The life cycle of a plant may
be taken to mean the time needed to grow from a dry mature seed up
to the stage where the plant has produced dry mature seeds, similar
to the starting material. This life cycle may be influenced by
factors such as early vigour, growth rate, greenness index,
flowering time and speed of seed maturation. The increase in growth
rate may take place at one or more stages in the life cycle of a
plant or during substantially the whole plant life cycle. Increased
growth rate during the early stages in the life cycle of a plant
may reflect enhanced vigour. The increase in growth rate may alter
the harvest cycle of a plant allowing plants to be sown later
and/or harvested sooner than would otherwise be possible (a similar
effect may be obtained with earlier flowering time). If the growth
rate is sufficiently increased, it may allow for the further sowing
of seeds of the same plant species (for example sowing and
harvesting of rice plants followed by sowing and harvesting of
further rice plants all within one conventional growing period).
Similarly, if the growth rate is sufficiently increased, it may
allow for the further sowing of seeds of different plants species
(for example the sowing and harvesting of corn plants followed by,
for example, the sowing and optional harvesting of soybean, potato
or any other suitable plant). Harvesting additional times from the
same rootstock in the case of some crop plants may also be
possible. Altering the harvest cycle of a plant may lead to an
increase in annual biomass production per acre (due to an increase
in the number of times (say in a year) that any particular plant
may be grown and harvested). An increase in growth rate may also
allow for the cultivation of transgenic plants in a wider
geographical area than their wild-type counterparts, since the
territorial limitations for growing a crop are often determined by
adverse environmental conditions either at the time of planting
(early season) or at the time of harvesting (late season). Such
adverse conditions may be avoided if the harvest cycle is
shortened. The growth rate may be determined by deriving various
parameters from growth curves, such parameters may be: T-Mid (the
time taken for plants to reach 50% of their maximal size) and T-90
(time taken for plants to reach 90% of their maximal size), amongst
others.
[0233] According to a preferred feature of the present invention,
performance of the methods of the invention gives plants having an
increased growth rate relative to control plants. Therefore,
according to the present invention, there is provided a method for
increasing the growth rate of plants, which method comprises
modulating expression, preferably increasing expression, in a plant
of a nucleic acid encoding an ANN polypeptide as defined
herein.
[0234] An increase in yield and/or growth rate occurs whether the
plant is under non-stress conditions or whether the plant is
exposed to various stresses compared to control plants. Plants
typically respond to exposure to stress by growing more slowly. In
conditions of severe stress, the plant may even stop growing
altogether. Mild stress on the other hand is defined herein as
being any stress to which a plant is exposed which does not result
in the plant ceasing to grow altogether without the capacity to
resume growth. Mild stress in the sense of the invention leads to a
reduction in the growth of the stressed plants of less than 40%,
35% or 30%, preferably less than 25%, 20% or 15%, more preferably
less than 14%, 13%, 12%, 11% or 10% or less in comparison to the
control plant under non-stress conditions. Due to advances in
agricultural practices (irrigation, fertilization, pesticide
treatments) severe stresses are not often encountered in cultivated
crop plants. As a consequence, the compromised growth induced by
mild stress is often an undesirable feature for agriculture. Mild
stresses are the everyday biotic and/or abiotic (environmental)
stresses to which a plant is exposed. Abiotic stresses may be due
to drought or excess water, anaerobic stress, salt stress, chemical
toxicity, oxidative stress and hot, cold or freezing temperatures.
The abiotic stress may be an osmotic stress caused by a water
stress (particularly due to drought), salt stress, oxidative stress
or an ionic stress. Biotic stresses are typically those stresses
caused by pathogens, such as bacteria, viruses, fungi and
insects.
[0235] In particular, the methods of the present invention may be
performed under non-stress conditions or under conditions of mild
drought to give plants having increased yield relative to control
plants. As reported in Wang et al. (Planta (2003) 218: 1-14),
abiotic stress leads to a series of morphological, physiological,
biochemical and molecular changes that adversely affect plant
growth and productivity. Drought, salinity, extreme temperatures
and oxidative stress are known to be interconnected and may induce
growth and cellular damage through similar mechanisms. Rabbani et
al. (Plant Physiol (2003) 133: 1755-1767) describes a particularly
high degree of "cross talk" between drought stress and
high-salinity stress. For example, drought and/or salinisation are
manifested primarily as osmotic stress, resulting in the disruption
of homeostasis and ion distribution in the cell. Oxidative stress,
which frequently accompanies high or low temperature, salinity or
drought stress, may cause denaturing of functional and structural
proteins. As a consequence, these diverse environmental stresses
often activate similar cell signalling pathways and cellular
responses, such as the production of stress proteins, up-regulation
of anti-oxidants, accumulation of compatible solutes and growth
arrest. The term "non-stress" conditions as used herein are those
environmental conditions that allow optimal growth of plants.
Persons skilled in the art are aware of normal soil conditions and
climatic conditions for a given location.
[0236] Performance of the methods of the invention gives plants
grown under non-stress conditions or under mild drought conditions
increased yield relative to control plants grown under comparable
conditions. Therefore, according to the present invention, there is
provided a method for increasing yield in plants grown under
non-stress conditions or under mild drought conditions, which
method comprises increasing expression in a plant of a nucleic acid
encoding an ANN polypeptide.
[0237] Performance of the methods of the invention gives plants
grown under conditions of nutrient deficiency, particularly under
conditions of nitrogen deficiency, increased yield relative to
control plants grown under comparable conditions. Therefore,
according to the present invention, there is provided a method for
increasing yield in plants grown under conditions of nutrient
deficiency, which method comprises increasing expression in a plant
of a nucleic acid encoding an ANN polypeptide. Nutrient deficiency
may result from a lack of nutrients such as nitrogen, phosphates
and other phosphorous-containing compounds, potassium, calcium,
cadmium, magnesium, manganese, iron and boron, amongst others.
[0238] The present invention encompasses plants or parts thereof
(including seeds) obtainable by the methods according to the
present invention. The plants or parts thereof comprise a nucleic
acid transgene encoding an ANN polypeptide as defined above.
[0239] The invention also provides genetic constructs and vectors
to facilitate introduction and/or expression in plants of nucleic
acids encoding ANN polypeptides. The gene constructs may be
inserted into vectors, which may be commercially available,
suitable for transforming into plants and suitable for expression
of the gene of interest in the transformed cells. The invention
also provides use of a gene construct as defined herein in the
methods of the invention.
[0240] More specifically, the present invention provides a
construct comprising: [0241] (a) a nucleic acid encoding an ANN
polypeptide as defined above; [0242] (b) one or more control
sequences capable of driving expression of the nucleic acid
sequence of (a); and optionally [0243] (c) a transcription
termination sequence.
[0244] Preferably, the nucleic acid encoding an ANN polypeptide is
as defined above. The term "control sequence" and "termination
sequence" are as defined herein.
[0245] Plants are transformed with a vector comprising any of the
nucleic acids described above. The skilled artisan is well aware of
the genetic elements that must be present on the vector in order to
successfully transform, select and propagate host cells containing
the sequence of interest. The sequence of interest is operably
linked to one or more control sequences (at least to a
promoter).
[0246] Advantageously, any type of promoter, whether natural or
synthetic, may be used to drive expression of the nucleic acid
sequence. A constitutive promoter is particularly useful in the
methods. See the "Definitions" section herein for definitions of
the various promoter types. Also useful in the methods of the
invention is a green tissue-specific promoter.
[0247] It should be clear that the applicability of the present
invention is not restricted to the ANN polypeptide-encoding nucleic
acid represented by SEQ ID NO: 83, nor is the applicability of the
invention restricted to expression of an ANN polypeptide-encoding
nucleic acid when driven by a constitutive promoter, or when driven
by a green tissue-specific promoter.
[0248] The constitutive promoter is preferably a GOS2 promoter,
preferably a GOS2 promoter from rice. Further preferably the
constitutive promoter is represented by a nucleic acid sequence
substantially similar to SEQ ID NO: 94, most preferably the
constitutive promoter is as represented by SEQ ID NO: 94. See Table
2a in the "Definitions" section herein for further examples of
constitutive promoters.
[0249] According to another preferred feature of the invention, the
nucleic acid encoding an ANN polypeptide is operably linked to a
green tissue-specific promoter. The green tissue specific promoter
is preferably an expansin promoter, further preferably an expansin
promoter from rice. Further preferably the green tissue-specific
promoter is represented by a nucleic acid sequence substantially
similar to SEQ ID NO: 95, most preferably the green tissue-specific
promoter is as represented by SEQ ID NO: 95. See Table 2g in the
"Definitions" section herein for further examples of green
tissue-specific promoters.
[0250] Optionally, one or more terminator sequences may be used in
the construct introduced into a plant. Additional regulatory
elements may include transcriptional as well as translational
enhancers. Those skilled in the art will be aware of terminator and
enhancer sequences that may be suitable for use in performing the
invention. An intron sequence may also be added to the 5'
untranslated region (UTR) or in the coding sequence to increase the
amount of the mature message that accumulates in the cytosol, as
described in the definitions section. Other control sequences
(besides promoter, enhancer, silencer, intron sequences, 3'UTR
and/or 5'UTR regions) may be protein and/or RNA stabilizing
elements. Such sequences would be known or may readily be obtained
by a person skilled in the art.
[0251] The genetic constructs of the invention may further include
an origin of replication sequence that is required for maintenance
and/or replication in a specific cell type. One example is when a
genetic construct is required to be maintained in a bacterial cell
as an episomal genetic element (e.g. plasmid or cosmid molecule).
Preferred origins of replication include, but are not limited to,
the f1-ori and colE1.
[0252] For the detection of the successful transfer of the nucleic
acid sequences as used in the methods of the invention and/or
selection of transgenic plants comprising these nucleic acids, it
is advantageous to use marker genes (or reporter genes). Therefore,
the genetic construct may optionally comprise a selectable marker
gene. Selectable markers are described in more detail in the
"definitions" section herein.
[0253] It is known that upon stable or transient integration of
nucleic acids into plant cells, only a minority of the cells takes
up the foreign DNA and, if desired, integrates it into its genome,
depending on the expression vector used and the transfection
technique used. To identify and select these integrants, a gene
coding for a selectable marker (such as the ones described above)
is usually introduced into the host cells together with the gene of
interest. These markers can for example be used in mutants in which
these genes are not functional by, for example, deletion by
conventional methods. Furthermore, nucleic acid molecules encoding
a selectable marker can be introduced into a host cell on the same
vector that comprises the sequence encoding the polypeptides of the
invention or used in the methods of the invention, or else in a
separate vector. Cells which have been stably transfected with the
introduced nucleic acid can be identified for example by selection
(for example, cells which have integrated the selectable marker
survive whereas the other cells die). The marker genes may be
removed or excised from the transgenic cell once they are no longer
needed. Techniques for marker gene removal are known in the art,
useful techniques are described above in the definitions
section.
[0254] The invention also provides a method for the production of
transgenic plants having enhanced yield-related traits relative to
control plants, comprising introduction and expression in a plant
of any nucleic acid encoding an ANN polypeptide as defined
hereinabove.
[0255] More specifically, the present invention provides a method
for the production of transgenic plants having increased enhanced
yield-related traits, particularly increased seed yield, which
method comprises: [0256] (i) introducing and expressing in a plant
or plant cell an ANN polypeptide-encoding nucleic acid; and [0257]
(ii) cultivating the plant cell under conditions promoting plant
growth and development.
[0258] The nucleic acid of (i) may be any of the nucleic acids
capable of encoding an ANN polypeptide as defined herein.
[0259] The nucleic acid may be introduced directly into a plant
cell or into the plant itself (including introduction into a
tissue, organ or any other part of a plant). According to a
preferred feature of the present invention, the nucleic acid is
preferably introduced into a plant by transformation. The term
"transformation" is described in more detail in the "definitions"
section herein.
[0260] The genetically modified plant cells can be regenerated via
all methods with which the skilled worker is familiar. Suitable
methods can be found in the abovementioned publications by S. D.
Kung and R. Wu, Potrykus or Hofgen and Willmitzer.
[0261] Generally after transformation, plant cells or cell
groupings are selected for the presence of one or more markers
which are encoded by plant-expressible genes co-transferred with
the gene of interest, following which the transformed material is
regenerated into a whole plant. To select transformed plants, the
plant material obtained in the transformation is, as a rule,
subjected to selective conditions so that transformed plants can be
distinguished from untransformed plants. For example, the seeds
obtained in the above-described manner can be planted and, after an
initial growing period, subjected to a suitable selection by
spraying. A further possibility consists in growing the seeds, if
appropriate after sterilization, on agar plates using a suitable
selection agent so that only the transformed seeds can grow into
plants. Alternatively, the transformed plants are screened for the
presence of a selectable marker such as the ones described
above.
[0262] Following DNA transfer and regeneration, putatively
transformed plants may also be evaluated, for instance using
Southern analysis, for the presence of the gene of interest, copy
number and/or genomic organisation. Alternatively or additionally,
expression levels of the newly introduced DNA may be monitored
using Northern and/or Western analysis, both techniques being well
known to persons having ordinary skill in the art.
[0263] The generated transformed plants may be propagated by a
variety of means, such as by clonal propagation or classical
breeding techniques. For example, a first generation (or T1)
transformed plant may be selfed and homozygous second-generation
(or T2) transformants selected, and the T2 plants may then further
be propagated through classical breeding techniques. The generated
transformed organisms may take a variety of forms. For example,
they may be chimeras of transformed cells and non-transformed
cells; clonal transformants (e.g., all cells transformed to contain
the expression cassette); grafts of transformed and untransformed
tissues (e.g., in plants, a transformed rootstock grafted to an
untransformed scion).
[0264] The present invention clearly extends to any plant cell or
plant produced by any of the methods described herein, and to all
plant parts and propagules thereof. The present invention extends
further to encompass the progeny of a primary transformed or
transfected cell, tissue, organ or whole plant that has been
produced by any of the aforementioned methods, the only requirement
being that progeny exhibit the same genotypic and/or phenotypic
characteristic(s) as those produced by the parent in the methods
according to the invention.
[0265] The invention also includes host cells containing an
isolated nucleic acid encoding an ANN polypeptide as defined
hereinabove. Preferred host cells according to the invention are
plant cells. Host plants for the nucleic acids or the vector used
in the method according to the invention, the expression cassette
or construct or vector are, in principle, advantageously all
plants, which are capable of synthesizing the polypeptides used in
the inventive method.
[0266] The methods of the invention are advantageously applicable
to any plant. Plants that are particularly useful in the methods of
the invention include all plants which belong to the superfamily
Viridiplantae, in particular monocotyledonous and dicotyledonous
plants including fodder or forage legumes, ornamental plants, food
crops, trees or shrubs. According to a preferred embodiment of the
present invention, the plant is a crop plant. Examples of crop
plants include soybean, sunflower, canola, alfalfa, rapeseed,
cotton, tomato, potato and tobacco. Further preferably, the plant
is a monocotyledonous plant. Examples of monocotyledonous plants
include sugarcane. More preferably the plant is a cereal. Examples
of cereals include rice, maize, wheat, barley, millet, rye,
triticale, sorghum and oats.
[0267] The invention also extends to harvestable parts of a plant
such as, but not limited to seeds, leaves, fruits, flowers, stems,
rhizomes, tubers and bulbs. The invention furthermore relates to
products derived, preferably directly derived, from a harvestable
part of such a plant, such as dry pellets or powders, oil, fat and
fatty acids, starch or proteins.
[0268] According to a preferred feature of the invention, the
modulated expression is increased expression. Methods for
increasing expression of nucleic acids or genes, or gene products,
are well documented in the art and examples are provided in the
definitions section.
[0269] As mentioned above, a preferred method for modulating
(preferably, increasing) expression of a nucleic acid encoding an
ANN polypeptide is by introducing and expressing in a plant a
nucleic acid encoding an ANN polypeptide; however the effects of
performing the method, i.e. enhancing yield-related traits may also
be achieved using other well known techniques, including but not
limited to T-DNA activation tagging, TILLING, homologous
recombination. A description of these techniques is provided in the
definitions section.
[0270] The present invention also encompasses use of nucleic acids
encoding ANN polypeptides as described herein and use of these ANN
polypeptides in enhancing any of the aforementioned yield-related
traits in plants.
[0271] Nucleic acids encoding ANN polypeptide described herein, or
the ANN polypeptides themselves, may find use in breeding
programmes in which a DNA marker is identified which may be
genetically linked to an ANN polypeptide-encoding gene. The nucleic
acids/genes, or the ANN polypeptides themselves may be used to
define a molecular marker. This DNA or protein marker may then be
used in breeding programmes to select plants having enhanced
yield-related traits as defined hereinabove in the methods of the
invention.
[0272] Allelic variants of an ANN polypeptide-encoding nucleic
acid/gene may also find use in marker-assisted breeding programmes.
Such breeding programmes sometimes require introduction of allelic
variation by mutagenic treatment of the plants, using for example
EMS mutagenesis; alternatively, the programme may start with a
collection of allelic variants of so called "natural" origin caused
unintentionally. Identification of allelic variants then takes
place, for example, by PCR. This is followed by a step for
selection of superior allelic variants of the sequence in question
and which give increased yield. Selection is typically carried out
by monitoring growth performance of plants containing different
allelic variants of the sequence in question. Growth performance
may be monitored in a greenhouse or in the field. Further optional
steps include crossing plants in which the superior allelic variant
was identified with another plant. This could be used, for example,
to make a combination of interesting phenotypic features.
[0273] Nucleic acids encoding ANN polypeptides may also be used as
probes for genetically and physically mapping the genes that they
are a part of, and as markers for traits linked to those genes.
Such information may be useful in plant breeding in order to
develop lines with desired phenotypes. Such use of ANN
polypeptide-encoding nucleic acids requires only a nucleic acid
sequence of at least 15 nucleotides in length. The ANN
polypeptide-encoding nucleic acids may be used as restriction
fragment length polymorphism (RFLP) markers. Southern blots
(Sambrook J, Fritsch E F and Maniatis T (1989) Molecular Cloning, A
Laboratory Manual) of restriction-digested plant genomic DNA may be
probed with the ANN-encoding nucleic acids. The resulting banding
patterns may then be subjected to genetic analyses using computer
programs such as MapMaker (Lander et al. (1987) Genomics 1:
174-181) in order to construct a genetic map. In addition, the
nucleic acids may be used to probe Southern blots containing
restriction endonuclease-treated genomic DNAs of a set of
individuals representing parent and progeny of a defined genetic
cross. Segregation of the DNA polymorphisms is noted and used to
calculate the position of the ANN polypeptide-encoding nucleic acid
in the genetic map previously obtained using this population
(Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
[0274] The production and use of plant gene-derived probes for use
in genetic mapping is described in Bernatzky and Tanksley (1986)
Plant Mol. Biol. Reporter 4: 37-41. Numerous publications describe
genetic mapping of specific cDNA clones using the methodology
outlined above or variations thereof. For example, F2 intercross
populations, backcross populations, randomly mated populations,
near isogenic lines, and other sets of individuals may be used for
mapping. Such methodologies are well known to those skilled in the
art.
[0275] The nucleic acid probes may also be used for physical
mapping (i.e., placement of sequences on physical maps; see
Hoheisel et al. In: Non-mammalian Genomic Analysis: A Practical
Guide, Academic press 1996, pp. 319-346, and references cited
therein).
[0276] In another embodiment, the nucleic acid probes may be used
in direct fluorescence in situ hybridisation (FISH) mapping (Trask
(1991) Trends Genet. 7:149-154). Although current methods of FISH
mapping favour use of large clones (several kb to several hundred
kb; see Laan et al. (1995) Genome Res. 5:13-20), improvements in
sensitivity may allow performance of FISH mapping using shorter
probes.
[0277] A variety of nucleic acid amplification-based methods for
genetic and physical mapping may be carried out using the nucleic
acids. Examples include allele-specific amplification (Kazazian
(1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplified
fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332),
allele-specific ligation (Landegren et al. (1988) Science
241:1077-1080), nucleotide extension reactions (Sokolov (1990)
Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al.
(1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989)
Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of
a nucleic acid is used to design and produce primer pairs for use
in the amplification reaction or in primer extension reactions. The
design of such primers is well known to those skilled in the art.
In methods employing PCR-based genetic mapping, it may be necessary
to identify DNA sequence differences between the parents of the
mapping cross in the region corresponding to the instant nucleic
acid sequence. This, however, is generally not necessary for
mapping methods.
[0278] The methods according to the present invention result in
plants having enhanced yield-related traits, as described
hereinbefore. These traits may also be combined with other
economically advantageous traits, such as further yield-enhancing
traits, tolerance to other abiotic and biotic stresses, traits
modifying various architectural features and/or biochemical and/or
physiological features.
Items
[0279] 1. A method for enhancing yield-related traits in plants
relative to control plants, comprising modulating, preferably
increasing, expression in the roots a plant, of a nucleic acid
sequence encoding 2-cysteine peroxiredoxin (2-Cys PRX), which 2-Cys
PRX polypeptide comprises from N-terminus to C-terminus: (1) a
plastidic transit peptide; and (2) a 2-Cys PRX domain, and
optionally selecting for plants having increased yield. [0280] 2.
Method according to item 1, wherein said 2-Cys PRX polypeptide
additionally comprise one or both of: (i) Motif 1 as represented by
SEQ ID NO: 77, or a motif having at least 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence
identity to SEQ ID NO: 77; or (ii) Motif 2 as represented by SEQ ID
NO: 78, or a motif having at least 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, 90%, 95%, 98%, 99% or more amino acid sequence identity
to SEQ ID NO: 78. [0281] 3. Method according to item 1 or 2,
wherein said 2-Cys PRX polypeptide, when used in the construction
of a phylogenetic tree, such as the one depicted in FIG. 3, tends
to cluster with the 2-Cys PRX clade of polypeptides comprising the
polypeptide sequence as represented by SEQ ID NO: 2, rather than
with any other PRX clade. [0282] 4. Method according to any
preceding item wherein said 2-Cys PRX polypeptide is a polypeptide
having in increasing order of preference at least 35%, 40%, 45%,
50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or more
sequence identity to the 2-Cys PRX polypeptide as represented by
SEQ ID NO: 2 or to any of the polypeptide sequences given in Table
A1 herein. [0283] 5. Method according to any preceding item,
wherein said nucleic acid sequence encoding a 2-Cys PRX polypeptide
is represented by any one of the nucleic acid sequences listed in
Table A1, or is a portion thereof, or is a sequence capable of
hybridising with any one of the nucleic acid sequences listed in
Table A1. [0284] 6. Method according to any preceding item, wherein
said nucleic acid sequence encodes an orthologue or paralogue of
any of the polypeptides listed in Table A1. [0285] 7. Method
according to any preceding item, wherein said modulated, preferably
increased, expression is effected by any one or more of: T-DNA
activation tagging, TILLING, or homologous recombination. [0286] 8.
Method according to any preceding item, wherein said modulated,
preferably increased, expression is effected by introducing and
expressing in the roots of a plant a nucleic acid sequence encoding
a 2-Cys PRX polypeptide. [0287] 9. Method according to any
preceding item, wherein said enhanced yield-related traits is one
or more of: (i) improved early vigour; (ii) increased aboveground
biomass; (iii) increased root biomass; (iv) increase number of
flowers per panicle; (v) increased seed fill rate; (vi) increased
total seed yield per plant; (vii) increased number of (filled)
seeds; (viii) increased harvest index; or (ix) increased thousand
kernel weight (TKW). [0288] 10. Method according to any preceding
item, wherein said enhanced yield-related traits are obtained under
abiotic stress. [0289] 11. Method according to item 10, wherein
said abiotic stress is osmotic stress, selected from one or more
of: water stress, salt stress, oxidative stress and ionic stress;
preferably wherein said water stress is drought stress and/or
reduced nutrient availability, preferably reduced nitrogen
availability. [0290] 12. Method according to item 10 or 11, wherein
said abiotic stress tolerance is manifested as enhanced
yield-related trait selected from one or more of: (i) improved
early vigour; (ii) increased aboveground biomass; (iii) increased
root (thick and thin) biomass; (iv) increase number of flowers per
panicle; (v) increased seed fill rate; (vi) increased total seed
yield per plant; (vii) increased number of (filled) seeds; (viii)
increased harvest index; or (ix) increased thousand kernel weight
(TKW), each relative to control plants. [0291] 13. Method according
to any of items 8 to 12, wherein said nucleic acid sequence is
operably linked to a root-specific promoter, preferably to an RCc3
promoter, further preferably to an RCc3 promoter substantially
similar to SEQ ID NO: 80, most preferably to a promoter as
represented by SEQ ID NO: 80. [0292] 14. Method according to any
preceding item, wherein said nucleic acid sequence encoding a 2-Cys
PRX polypeptide is of plant origin, preferably from a
dicotyledonous plant, more preferably from the family Brassicaceae,
most preferably from Brassica rapa. [0293] 15. Plant or part
thereof, including seeds, obtainable by a method according to any
preceding item, wherein said plant or part thereof comprises a
nucleic acid transgene encoding a 2-Cys PRX polypeptide, operably
linked to a root-specific promoter. [0294] 16. Construct
comprising: [0295] (a) a nucleic acid sequence encoding a 2-Cys PRX
polypeptide as defined in any one of items 1 to 6; [0296] (b) one
or more control sequences capable of driving expression of the
nucleic acid sequence of (a); and optionally [0297] (c) a
transcription termination sequence; wherein at least one of the
control sequences is a root-specific promoter, preferably an Rcc3
promoter. [0298] 17. Use of a construct according to item 16 in a
method for making plants having enhanced yield-related traits,
which enhanced yield-related traits is preferably one or more of:
(i) improved early vigour; (ii) increased aboveground biomass;
(iii) increased root (thick and thin) biomass; (iv) increase number
of flowers per panicle; (v) increased seed fill rate; (vi)
increased total seed yield per plant; (vii) increased number of
(filled) seeds; (viii) increased harvest index; or (ix) increased
thousand kernel weight (TKW), relative to control plants. [0299]
18. Plant, plant part or plant cell transformed with a construct
according to item 16. [0300] 19. Method for the production of a
transgenic plant having enhanced yield-related traits relative to
control plants, comprising: [0301] introducing and expressing in a
plant, plant part or plant cell a nucleic acid sequence encoding a
2-Cys PRX polypeptide as defined in any one of items 1 to 5, under
the control of a root-specific promoter; and [0302] (ii)
cultivating the plant cell under conditions promoting plant growth
and development. [0303] 20. Method according to item 19 wherein
said enhanced yield-related traits occur under increased abiotic
stress. [0304] 21. Transgenic plant having enhanced yield-related
traits relative to control plants, resulting from modulated,
preferably increased, expression in the roots, of a nucleic acid
sequence encoding a 2-Cys PRX polypeptide as defined in any one of
items 1 to 6, or a transgenic plant cell or plant part derived from
said transgenic plant. [0305] 22. Transgenic plant according to
item 15, 18 or 21, wherein said plant is a crop plant or a monocot
or a cereal, such as rice, maize, wheat, barley, millet, rye,
triticale, sorghum and oats, or a transgenic plant cell or plant
part derived from said transgenic plant. [0306] 23. Harvestable
parts of a plant comprising a nucleic acid sequence encoding a
2-Cys PRX polypeptide according to item 22, wherein said
harvestable parts are preferably seeds. [0307] 24. Products derived
from a plant according to item 22 and/or from harvestable parts of
a plant according to item 23. [0308] 25. Use of a nucleic acid
sequence encoding a 2-Cys PRX polypeptide as defined in any one of
items 1 to 6 in enhancing yield-related traits in plants,
preferably in increasing one or more of: (i) increased seed fill
rate; (ii) increased total seed yield per plant; (iii) increased
number of filled seeds; (iv) increased total number of seeds; (v)
increased thousand kernel weight (TKW) or (vi) increased harvest
index, relative to control plants. [0309] 26. Use according to item
25, wherein said enhanced yield-related traits occur under abiotic
stress. [0310] 27. A method for enhancing yield-related traits in
plants relative to control plants, comprising modulating expression
in a plant of a nucleic acid encoding an ANN polypeptide, wherein
said ANN polypeptide comprises one or more of the following motifs:
[0311] (i) Signature sequence 1 (SEQ ID NO: 87), [0312] (ii)
Signature sequence 2 (SEQ ID NO: 88), [0313] (iii) Signature
sequence 3 (SEQ ID NO: 89), [0314] (iv) Signature sequence 4 (SEQ
ID NO: 90), [0315] (v) Signature sequence 5 (SEQ ID NO: 91), [0316]
(vi) Signature sequence 6 (SEQ ID NO: 92), [0317] (vii) Signature
sequence 7 (SEQ ID NO: 93). [0318] 28. Method according to item 27,
wherein said ANN polypeptide comprises at least an annexin domain.
[0319] 29. Method according to item 27 or 28, wherein said
modulated expression is effected by introducing and expressing in a
plant a nucleic acid encoding an ANN polypeptide. [0320] 30. Method
according to any one of items 27 to 29, wherein said nucleic acid
encoding an ANN polypeptide encodes any one of the proteins listed
in Table B1 or is a portion of such a nucleic acid, or a nucleic
acid capable of hybridising with such a nucleic acid. [0321] 31.
Method according to any one of items 27 to 30, wherein said nucleic
acid sequence encodes an orthologue or paralogue of any of the
proteins given in Table B1. [0322] 32. Method according to any one
of items 27 to 31, wherein said enhanced yield-related traits
comprise increased yield, preferably increased seed yield relative
to control plants. [0323] 33. Method according to any one of items
27 to 32, wherein said enhanced yield-related traits are obtained
under non-stress conditions. [0324] 34. Method according to any one
of items 27 to 33, wherein said enhanced yield-related traits are
obtained under conditions of drought. [0325] 35. Method according
to any one of items 29 to 34, wherein said nucleic acid is operably
linked to a constitutive promoter, preferably to a GOS2 promoter,
most preferably to a GOS2 promoter from rice. [0326] 36. Method
according to any one of items 29 to 34, wherein said nucleic acid
is operably linked to a green-tissue specific promoter, preferably
to an expansin promoter, most preferably to an expansin promoter
from rice. [0327] 37. Method according to any preceding item,
wherein said nucleic acid encoding an ANN polypeptide is of plant
origin, preferably from a dicotyledonous plant, further preferably
from the family Brassicaceae, more preferably from the genus
Arabidopsis, most preferably from Arabidopsis thaliana. [0328] 38.
Plant or part thereof, including seeds, obtainable by a method
according to any preceding item, wherein said plant or part thereof
comprises a recombinant nucleic acid encoding an ANN polypeptide.
[0329] 39. Construct comprising: [0330] (i) nucleic acid encoding
an ANN polypeptide as defined in items 27 or 28; [0331] (ii) one or
more control sequences capable of driving expression of the nucleic
acid sequence of (i); and optionally [0332] (iii) a transcription
termination sequence. [0333] 40. Construct according to item 39,
wherein one of said control sequences is a constitutive promoter,
preferably a GOS2 promoter, most preferably a GOS2 promoter from
rice. [0334] 41. Construct according to item 39, wherein one of
said control sequences is a green-tissue specific promoter,
preferably an expansin promoter, most preferably an expansin
promoter from rice. [0335] 42. Use of a construct according to any
one of items 39 to 41 in a method for making plants having
increased yield, particularly increased biomass and/or increased
seed yield relative to control plants. [0336] 43. Plant, plant part
or plant cell transformed with a construct according to any one of
items 39 to 41. [0337] 44. Method for the production of a
transgenic plant having increased yield, particularly increased
seed yield relative to control plants, comprising: [0338] (i)
introducing and expressing in a plant a nucleic acid encoding an
ANN polypeptide as defined in item 27 or 28; and [0339] (ii)
cultivating the plant cell under conditions promoting plant growth
and development. [0340] 45. Transgenic plant having increased
yield, particularly increased seed yield, relative to control
plants, resulting from increased expression of a nucleic acid
encoding an ANN polypeptide as defined in item 27 or 28, or a
transgenic plant cell derived from said transgenic plant. [0341]
46. Transgenic plant according to item 38, 43, or 45, or a
transgenic plant cell derived thereof, wherein said plant is a crop
plant or a monocot or a cereal, such as rice, maize, wheat, barley,
millet, rye, triticale, sorghum and oats. [0342] 47. Harvestable
parts of a plant according to item 46, wherein said harvestable
parts are preferably seeds. [0343] 48. Products derived from a
plant according to item 46 and/or from harvestable parts of a plant
according to item 47. [0344] 49. Use of a nucleic acid encoding an
ANN polypeptide in increasing yield, particularly in increasing
seed yield in plants, relative to control plants.
DESCRIPTION OF FIGURES
[0345] The present invention will now be described with reference
to the following Figures in which:
[0346] FIG. 1 represents the catalytic and
inactivation/reactivation cycles of 2-Cys Prx enzymes, according to
Rhee et al., (2005) Free radical Biology and Medicine 38:
1543-1552.
[0347] FIG. 2 represents the output of a search (using default
values) of the Conserved Domains Database (CDD) at NCBI, using a
2-Cys PRX as represented by SEQ ID NO: 2. The top hit is entry
CD3015, PRX_Typ2cys.
[0348] FIG. 3 shows a phylogenetic tree built using the neighbour
joining clustering method, after a ClustalW (1.83) multiple
sequence alignment of 2-Cys PRX from eubacteria, plant algae,
animals, and 1-Cys PRX from plants. The 2-Cys PRX class is marked
with an accolade. The 2-Cys PRX as represented by SEQ ID NO: 2 is
boxed.
[0349] FIG. 4 shows a CLUSTAL W (1.83) multiple sequence alignment
of 2-Cys PRX from eubacteria, plant algae, animals, and 1-Cys PRX
from plants, using default values. Motif 1 as represented by SEQ ID
NO: 77 and Motif 2 as represented by SEQ ID NO: 78 are boxed.
Sequences shown are: gi|113596153|dbj|BAF20027.1| (SEQ ID NO: 162);
gi|113535696|dbj|BAF08079.1| (SEQ ID NO: 163);
gi|113533376|dbj|BAF05759.1| (SEQ ID NO: 164);
gi|115439131|ref|NP.sub.--001043845. (SEQ ID NO: 165);
gi|113532211|dbj|BAF04594.1| (SEQ ID NO: 166);
gi|115435844|ref|NP.sub.--001042680. (SEQ ID NO: 167);
gi|113611943|dbj|BAF22321.1| (SEQ ID NO: 168);
gi|113611944|dbj|BAF22322.1| (SEQ ID NO: 169);
gi|2499469|sp|Q61171|PRDX2_MOU (SEQ ID NO: 170);
gi|8394432|ref|NP.sub.--058865.1| (SEQ ID NO: 52);
gi|21553667|gb|AAM62760.1| (SEQ ID NO: 10);
gi|3265490|gb|AAG40040.2|AF32 (SEQ ID NO: 171);
gi|1119229|gb|AAG30570.1|AF31 (SEQ ID NO: 8);
gi|113564335|dbj|BAF14678.1| (SEQ ID NO: 172);
gi|15229806|ref|NP.sub.--187769.1| (SEQ ID NO: 173);
gi|21592588|gb|AAM64537.1| (SEQ ID NO: 6);
gi|3121825|sp|O24364|BAS1_SPIO (SEQ ID NO: 174);
gi|1498198|emb|CAA63909.1| (SEQ ID NO: 175);
gi|6002472|gb|AAF00001.1|AF052 (SEQ ID NO: 4);
gi|21912927|emb|CAC84143.2| (SEQ ID NO: 14);
gi|47027073|gb|AAT08751.1| (SEQ ID NO: 176);
gi|11558242|emb|CAC17803.1| (SEQ ID NO: 16);
gi|15131688|emb|CAC48323.1| (SEQ ID NO: 18);
gi|3328221|gb|AAC78473.1| (SEQ ID NO: 177);
gi|2499477|sp|Q96468|BAS1_HORV (SEQ ID NO: 178);
gi|2829687|sp|P80602|BAS1_WHEA (SEQ ID NO: 179);
gi|1076722|pir.lamda.S49173 (SEQ ID NO: 180);
gi|15446541|ref|NP.sub.--001047050. (SEQ ID NO: 20);
gi|13536581|dbj|BAF08964.1| (SEQ ID NO: 181);
gi|125539780|gb|EAY86175.1| (SEQ ID NO: 182);
gi|7339568|emb|CAB82860.1| (SEQ ID NO: 24);
gi|17232133|ref|NP.sub.--488681.1| (SEQ ID NO: 28);
gi|19509654|ref|ZP.sub.--01628800.1 (SEQ ID NO: 32);
gi|86609696|ref|YP.sub.--478458.11 (SEQ ID NO: 30);
gi|86605254|ref|YP.sub.--474017.1| (SEQ ID NO: 38);
gi|22298997|ref|NP.sub.--682244.1| (SEQ ID NO: 34);
gi|11465738|ref|NP.sub.--053882.1| (SEQ ID NO: 46);
gi|51209959|ref|YP.sub.--063623.1| (SEQ ID NO: 48);
gi|81301118|ref|YP.sub.--401326.1| (SEQ ID NO: 42);
gi|33865747|ref|NP.sub.--897306.1| (SEQ ID NO: 40);
gi|84518029|ref|ZP.sub.--01005378.1| (SEQ ID NO: 44);
gi|116059461|emb|CAL55168.1| (SEQ ID NO: 36);
gi|74272711|gb|ABA01151.1| (SEQ ID NO: 26);
gi|11995220|emb|CAC19677.1| (SEQ ID NO: 183);
gi|15455107|ref|NP.sub.--001051154. (SEQ ID NO: 184);
gi|15455105|ref|NP.sub.--001051153. (SEQ ID NO: 185);
gi|113595092|dbj|BAF18966.1| (SEQ ID NO: 186);
gi|115452325|ref|NP.sub.--001049763. (SEQ ID NO: 187); and
gi|113610859|dbj|BAF21237.1| (SEQ ID NO: 188).
[0350] FIG. 5 represents the binary vector for increased expression
in Oryza sativa of a 2-Cys PRX-encoding nucleic acid sequence under
the control of either a rice GOS2 promoter (pGOS2; SEQ ID NO: 79),
or a rice Rcc3 (pRcc3; SEQ ID NO: 80) promoter.
[0351] FIG. 6 details examples of sequences useful in performing
the methods according to the present invention.
[0352] FIG. 7 A represents SEQ ID NO: 84 with the annexin domains
as predicted by SMART indicated in bold underlined; FIG. 7 B shows
the annexin domains (predicted by SMART) in ANNEXIN 4 of
Arabidopsis thaliana (SEQ ID NO: 135).
[0353] FIG. 8 shows a phylogenetic tree (Cantero et al., Plant
Physiol. Biochem. 44, 13-24, 2006) of plant annexin proteins useful
in the methods of the present invention. The arrows indicate
annexin 1 (SEQ ID NO: 84) and annexin 4 (SEQ ID NO: 135) both from
Arabidopsis thaliana.
[0354] FIG. 9 represents a multiple alignment of various plant
annexin proteins. The identifiers refer to the database accessions;
NP.sub.--174810 corresponds to SEQ ID NO: 84. Conserved residues
are indicated by colons or dots. Sequences shown are:
NP.sub.--001063096 (SEQ ID NO: 141); NP.sub.--001061839 (SEQ ID NO:
145); ABE65753 (SEQ ID NO: 133); NP.sub.--181410 (SEQ ID NO: 131);
NP.sub.--001055408 (SEQ ID NO: 151); AAC33305 (SEQ ID NO: 97);
AAB71830 (SEQ ID NO: 99); CAB92956 (SEQ ID NO: 113); ABB55363 (SEQ
ID NO: 123); AAC97494 (SEQ ID NO: 115); 1DK5 (SEQ ID NO: 105);
CAA75213 (SEQ ID NO: 111); CAA75214 (SEQ ID NO: 121);
NP.sub.--174810 (SEQ ID NO: 84); AAD24540 (SEQ ID NO: 107);
AAC97493 (SEQ ID NO: 117); AAB67994 (SEQ ID NO: 103); AAR13288 (SEQ
ID NO: 109); AAZ41833 (SEQ ID NO: 101); NP.sub.--201307 (SEQ ID NO:
119); NP.sub.--196585 (SEQ ID NO: 125); AAZ67605 (SEQ ID NO: 129);
NP.sub.--196584 (SEQ ID NO: 137); CAA52903 (SEQ ID NO: 127);
NP.sub.--001048149 (SEQ ID NO: 143); NP.sub.--001057176 (SEQ ID NO:
147); NP.sub.--568271 (SEQ ID NO: 139); NP.sub.--181409 (SEQ ID NO:
135); NP.sub.--001063343 (SEQ ID NO: 149); NP.sub.--001061661 (SEQ
ID NO: 153); and NP.sub.--001051711 (SEQ ID NO: 155).
[0355] FIG. 10 represents the binary vector for increased
expression in Oryza sativa of an ANN-encoding nucleic acid under
the control of a rice GOS2 promoter (pGOS2).
[0356] FIG. 11 details examples of sequences useful in performing
the methods according to the present invention.
EXAMPLES
[0357] The present invention will now be described with reference
to the following examples, which are by way of illustration alone.
The following examples are not intended to completely define or
otherwise limit the scope of the invention.
[0358] DNA manipulation: unless otherwise stated, recombinant DNA
techniques are performed according to standard protocols described
in (Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd
Edition Cold Spring Harbor Laboratory Press, CSH, New York) or in
Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in
Molecular Biology, Current Protocols. Standard materials and
methods for plant molecular work are described in Plant Molecular
Biology Labfax (1993) by R. D. D. Croy, published by BIOS
Scientific Publications Ltd (UK) and Blackwell Scientific
Publications (UK).
Example 1
Identification of Sequences Related to the Nucleic Acid Sequence
Used in the Methods of the Invention
[0359] Sequences (full length cDNA, ESTs or genomic) related to the
nucleic acid sequence used in the methods of the present invention
were identified amongst those maintained in the Entrez Nucleotides
database at the National Center for Biotechnology Information
(NCBI) using database sequence search tools, such as the Basic
Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol.
215:403-410; and Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402). The program is used to find regions of local
similarity between sequences by comparing nucleic acid or
polypeptide sequences to sequence databases and by calculating the
statistical significance of matches. For example, the polypeptide
encoded by the nucleic acid sequence used in the present invention
was used for the TBLASTN algorithm, with default settings and the
filter to ignore low complexity sequences set off. The output of
the analysis was viewed by pairwise comparison, and ranked
according to the probability score (E-value), where the score
reflect the probability that a particular alignment occurs by
chance (the lower the E-value, the more significant the hit). In
addition to E-values, comparisons were also scored by percentage
identity. Percentage identity refers to the number of identical
nucleotides (or amino acids) between the two compared nucleic acid
(or polypeptide) sequences over a particular length. In some
instances, the default parameters may be adjusted to modify the
stringency of the search. For example the E-value may be increased
to show less stringent matches. This way, short nearly exact
matches may be identified.
[0360] Table A1 provides a list of nucleic acid sequences related
to the nucleic acid sequence used in the methods of the present
invention.
TABLE-US-00015 TABLE A1 Examples of 2-Cys PRX nucleic acid and
polypeptide sequences: Nucleic acid sequence NCBI SEQ Polypeptide
sequence SEQ Species accession ID NO NCBI accession ID NO Brassica
sp. SEQ ID NO: 1 1 SEQ ID NO: 2 2 Brassica rapa AF052202.1 3
AAF00001.1 4 Arabidopsis thaliana AY086974.1 5 AAM64537.1 6
Brassica napus AF311863.1 7 AAG30570.1 8 Arabidopsis thaliana
NM_120712.2 9 AAM62760.1 10 Spinacia oleracea X94219.1 11 O24364 12
Nicotiana tabacum AJ309009.2 13 CAC84143.2 14 Phaseolus vulgaris
AJ288895.1 15 CAC17803.1 16 Pisum sativum AJ315851.1| 17 CAC48323.1
18 Oryza sativa NM_001053585.1 19 NP_001047050.1 20 Os02g0537700
Secale cereale AF076920.1 21 AAC78473.1 22 Riccia fluitans
AJ005006.1 23 CAB82860.1 24 Chlamydomonas incerta DQ122920.1 25
ABA01151.1 26 Nostoc gi|17227497:5544705-5545316 27 NP_488681.1 28
Synechococcus sp gi|86607503:2357237-2357845 29 YP_478458.1 30
Nodularia spumigena gi|119509627:35267-35878 31 ZP_01628800.1 32
Thermosynechococcus elongatus gi|22297544:1516844-1517437 33
NP_682244.1 34 Ostreococcus tauri gi|118721427:14610-14671, 35
CAL55168.1 36 14874-14913, 15068-15658 Synechococcus
gi|86604733:552727-553335 37 YP_474017.1 38 Synechococcus
gi|33864539:1204065-1204667 39 NP_897306.1 40 Synechococcus
elongatus gi|81298811:2377107-2377703 41 YP_401326.1 42
Prochlorococcus marinus gi|84517401:555567-556163 43 ZP_01005378.1
44 Porphyra purpurea gi|11465652:76976-77575 45 NP_053882.1 46
Gracilaria tenuistipitata gi|51209843:100291-100971 47 YP_063623.1
48 Mus musculus X82067.1 49 Q61171 50 Rattus norvegicus NM_017169.1
51 NP_058865.1 52
[0361] In some instances, related sequences have tentatively been
assembled and publicly disclosed by research institutions, such as
The Institute for Genomic Research (TIGR). The Eukaryotic Gene
Orthologs (EGO) database may be used to identify such related
sequences, either by keyword search or by using the BLAST algorithm
with the nucleic acid sequence or polypeptide sequence of
interest.
Example 2
Alignment of 2-Cys PRX Polypeptide Sequences
[0362] Alignment of 2-Cys PRX polypeptide sequences from
eubacteria, plant algae, animals, and 1-Cys PRX polypeptide
sequences from plants (as outliers) was performed the Clustal
algorithm (1.83) of progressive alignment, using default values
(Thompson et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et
al. (2003). Nucleic Acids Res 31:3497-3500). Minor manual editing
may be done to further optimise the alignment. The 2-Cys PRX
polypeptides are aligned in FIG. 4. Motif 1 as represented by SEQ
ID NO: 77 and Motif 2 as represented by SEQ ID NO: 78 are
boxed.
[0363] A phylogenetic tree of 2-Cys PRX polypeptide sequences from
eubacteria, plant algae, animals, and 1-Cys PRX polypeptide
sequences from plants (as outliers) was constructed using a
neighbour-joining clustering algorithm, well known in the art (FIG.
3). The 2-Cys PRX class is marked with an accolade. The 2-Cys PRX
as represented by SEQ ID NO: 2 is boxed.
Example 3
Calculation of Global Percentage Identity Between Polypeptide
Sequences Useful in Performing the Methods of the Invention
[0364] Global percentages of similarity and identity between full
length polypeptide sequences useful in performing the methods of
the invention were determined using one of the methods available in
the art, the MatGAT (Matrix Global Alignment Tool) software (BMC
Bioinformatics. 2003 4:29. MatGAT: an application that generates
similarity/identity matrices using protein or DNA sequences.
Campanella J J, Bitincka L, Smalley J; software hosted by Ledion
Bitincka). MatGAT software generates similarity/identity matrices
for DNA or protein sequences without needing pre-alignment of the
data. The program performs a series of pair-wise alignments using
the Myers and Miller global alignment algorithm (with a gap opening
penalty of 12, and a gap extension penalty of 2), calculates
similarity and identity using for example Blosum 62 (for
polypeptides), and then places the results in a distance matrix.
Sequence similarity is shown in the bottom half of the dividing
line and sequence identity is shown in the top half of the diagonal
dividing line.
[0365] Parameters used in the comparison were:
TABLE-US-00016 Scoring matrix: Blosum62 First Gap: 12 Extending
gap: 2
[0366] Results of the software analysis are shown in Table A2 for
the global similarity and identity over the full length of the
polypeptide sequences. Percentage identity is given above the
diagonal in bold and percentage similarity is given below the
diagonal (normal face).
[0367] The percentage identity between the 2-Cys PRX polypeptide
sequences useful in performing the methods of the invention can be
as low as 35% amino acid identity compared to SEQ ID NO: 2.
TABLE-US-00017 TABLE A2 MatGAT results for global similarity and
identity over the full length of the polypeptide sequences. 1 2 3 4
5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 01. seqidno02 91 89 86 86
88 76 78 76 76 74 66 61 55 53 55 54 57 52 02. seqidno04 92 85 80 80
85 71 74 71 72 69 61 56 52 50 52 51 54 49 03. seqidno06 93 88 85 86
97 75 77 74 74 74 66 61 55 52 55 53 58 51 04. seqidno08 92 86 92 89
85 74 77 74 73 73 64 60 56 52 56 52 59 51 05. seqidno10 91 86 91 93
85 77 76 74 75 74 66 61 57 53 56 53 59 52 06. seqidno12 92 87 98 91
90 76 77 75 75 75 66 61 55 53 55 54 58 52 07. seqidno14 83 80 85 86
86 85 79 79 75 75 66 63 57 54 55 54 57 53 08. seqidno16 84 79 84 83
82 83 83 86 77 76 66 63 58 57 57 56 61 55 09. seqidno18 85 81 85 83
83 85 85 91 75 74 67 63 57 54 56 55 59 53 10. seqidno20 82 77 81 81
83 83 83 85 86 79 66 64 57 55 57 57 60 54 11. seqidno22 84 78 83 82
82 83 83 84 82 85 67 64 58 56 57 56 61 55 12. seqidno24 79 75 79 78
80 79 81 77 78 79 77 60 56 51 53 52 58 51 13. seqidno26 75 70 74 74
75 74 75 75 75 75 74 72 66 62 63 65 69 61 14. seqidno28 65 61 65 64
65 65 65 66 65 67 67 63 75 83 91 83 63 82 15. seqidno30 63 59 63 64
64 63 64 66 65 66 68 63 72 92 80 84 61 96 16. seqidno32 65 61 65 64
65 65 65 67 64 67 67 62 75 97 92 79 63 80 17. seqidno34 62 58 62 62
61 62 62 64 63 66 65 60 73 88 90 88 64 83 18. seqidno36 67 63 67 68
67 67 67 68 67 71 70 65 79 74 72 74 73 59 19. seqidno38 63 59 63 63
63 63 63 66 64 66 68 62 72 92 100 93 89 71 20. seqidno40 62 58 61
60 61 61 61 64 62 62 63 58 73 84 85 85 84 71 85 21. seqidno42 61 58
61 61 61 62 61 65 63 63 64 60 73 89 90 89 89 73 91 22. seqidno44 61
58 61 60 60 61 61 64 62 62 63 57 73 85 85 85 84 73 85 23. seqidno46
62 58 61 61 61 62 61 63 62 63 65 60 72 87 90 88 88 71 90 24.
seqidno48 64 61 65 64 65 65 64 67 66 64 67 62 73 74 73 74 71 71 72
25. seqidno50 58 54 58 57 58 58 56 58 57 58 59 55 64 73 73 73 73 64
72 26. seqidno52 57 54 57 56 56 57 55 57 56 58 58 55 63 72 72 72 72
64 72 27. seqidno54 41 39 40 40 40 40 40 42 40 39 41 40 39 45 45 46
46 40 45 28. seqidno56 50 48 50 50 48 49 49 50 50 52 53 49 53 55 52
55 52 49 52 29. seqidno58 36 36 35 34 35 36 36 36 37 36 37 36 40 43
43 44 41 38 42 30. seqidno60 41 41 41 40 40 40 40 40 37 42 41 39 43
28 28 28 27 39 25 31. seqidno62 41 41 43 40 41 43 41 40 40 42 41 40
46 42 39 42 40 43 39 32. seqidno64 27 30 27 27 27 31 27 29 30 33 28
30 35 38 38 37 40 35 40 33. seqidno66 36 37 33 35 35 34 38 33 33 37
34 36 33 30 29 30 27 30 29 34. seqidno68 39 40 39 39 41 39 39 41 38
41 40 35 42 39 34 39 34 40 34 35. seqidno70 34 38 34 34 34 33 32 36
32 35 34 31 33 26 31 26 25 33 26 36. seqidno72 34 33 35 34 34 35 32
36 33 34 36 34 33 27 25 26 25 28 25 37. seqidno74 33 33 32 36 32 33
33 34 32 35 33 33 34 27 29 29 28 31 31 38. seqidno76 30 28 29 30 31
29 30 34 29 32 30 30 35 36 35 38 36 36 35 20 21 22 23 24 25 26 27
28 29 30 31 32 33 34 35 36 37 38 01. seqidno02 53 53 51 50 47 47 46
20 46 19 24 25 16 18 23 18 19 17 17 02. seqidno04 49 51 48 47 46 45
44 21 45 19 22 26 16 19 21 20 18 18 18 03. seqidno06 52 52 50 50 48
46 46 19 46 18 24 25 16 16 22 18 21 18 16 04. seqidno08 52 53 52 50
50 47 45 20 46 19 23 24 15 17 21 17 21 16 16 05. seqidno10 53 53 51
51 49 47 46 20 47 18 24 24 15 18 22 18 20 16 17 06. seqidno12 52 52
51 50 48 48 47 21 46 19 25 25 16 17 22 18 21 19 16 07. seqidno14 53
55 52 51 50 46 45 22 45 20 23 24 15 18 22 18 20 17 16 08. seqidno16
55 57 53 53 51 48 48 21 48 19 21 23 17 17 22 20 20 18 17 09.
seqidno18 53 56 51 52 51 46 45 20 46 21 22 24 18 15 22 17 20 16 14
10. seqidno20 53 55 51 53 49 47 46 19 48 18 24 26 16 19 22 20 20 21
19 11. seqidno22 54 56 53 53 49 48 47 20 50 19 26 25 18 18 24 22 22
20 16 12. seqidno24 50 54 50 49 46 44 45 20 43 20 24 25 17 18 21 17
20 20 17 13. seqidno26 64 67 64 59 56 53 53 21 45 22 23 25 20 18 24
19 18 18 18 14. seqidno28 76 83 75 74 61 60 60 27 44 27 13 21 22 15
19 16 17 16 16 15. seqidno30 74 83 72 76 60 58 58 25 43 25 15 19 23
17 15 18 16 17 16 16. seqidno32 76 80 73 73 63 61 61 27 44 27 12 21
22 16 18 16 17 18 17 17. seqidno34 75 83 74 77 60 60 60 27 44 26 16
19 23 15 17 18 17 19 15 18. seqidno36 63 63 63 60 57 50 50 22 44 21
24 22 20 15 20 20 19 17 20 19. seqidno38 72 82 72 76 59 57 57 27 42
27 11 19 23 17 17 17 16 19 17 20. seqidno40 81 90 71 57 64 63 29 44
28 16 19 23 16 17 15 17 17 16 21. seqidno42 88 78 76 58 62 62 26 44
25 15 18 21 15 15 18 17 18 15 22. seqidno44 96 88 70 56 61 61 28 43
27 17 19 20 16 19 17 13 17 18 23. seqidno46 84 90 86 63 58 57 27 39
27 12 18 21 13 18 14 15 16 17 24. seqidno48 71 71 72 73 46 45 23 34
22 18 20 17 12 18 13 12 11 17 25. seqidno50 75 74 75 73 61 99 27 36
26 18 19 22 17 18 18 17 19 16 26. seqidno52 74 73 74 72 60 99 27 36
26 18 19 22 16 18 18 17 19 17 27. seqidno54 46 43 47 47 41 46 46 17
81 12 16 20 15 17 15 16 16 13 28. seqidno56 53 53 53 53 47 47 46 30
19 16 19 15 15 15 13 14 16 17 29. seqidno58 43 42 43 46 40 41 41 90
31 16 17 20 16 17 14 14 17 17 30. seqidno60 28 31 30 26 38 34 34 28
28 28 23 42 20 73 24 20 22 34 31. seqidno62 40 38 40 39 43 38 38 26
29 28 38 17 22 22 20 19 19 18 32. seqidno64 38 36 37 35 35 39 38 34
35 34 53 30 18 44 17 16 19 35 33. seqidno66 29 29 28 24 31 25 25 25
26 27 34 35 27 21 21 19 20 21 34. seqidno68 34 32 33 34 42 34 34 30
29 31 81 42 54 34 20 18 22 31 35. seqidno70 25 26 27 28 30 27 27 27
27 27 38 32 26 33 33 57 33 20 36. seqidno72 26 25 23 27 29 26 26 28
24 26 36 32 26 33 33 73 30 19 37. seqidno74 26 27 27 29 27 31 30 29
24 28 36 30 30 35 35 46 48 17 38. seqidno76 36 32 38 35 35 36 39 28
34 29 50 37 49 35 48 32 32 33
Example 4
Identification of Domains Comprised in Polypeptide Sequences Useful
in Performing the Methods of the Invention
[0368] The Conserved Domain Search service (CD-Search) is a
web-based tool for the detection of structural and functional
domains in protein sequences, hosted at NCBI. CD-Search uses
BLAST(R) to search a comprehensive collection of domain models.
Search results are displayed as domain architecture cartoons and
pairwise alignments between the query and domain-model consensus
sequences (Marchler-Bauer A, Bryant S H (2004), "CD-Search: protein
domain annotations on the fly", Nucleic Acids Res. 32 (W)327-331).
A search (using default values) performed using a 2-Cys PRX as
represented by SEQ ID NO: 2, gives as top hit is entry CD3015,
PRX_Typ2cys (FIG. 2, Table A3).
TABLE-US-00018 TABLE A3 CDD of the polypeptide sequence as
represented by SEQ ID NO: 2. Data- Accession Accession base number
name Description CDD CD3015 PRX_ Peroxiredoxin (PRX) family,
Typical Typ2cys 2-Cys PRX subfamily; PRXs are thiol- specific
antioxidant (TSA) proteins, which confer a protective role in cells
through its peroxidase activity by reducing hydrogen peroxide,
peroxynitrite, and organic hydroperoxides
Example 5
Subcellular Localisation Prediction of the Polypeptide Sequences
Useful in Performing the Methods of the Invention
[0369] TargetP 1.1 predicts the subcellular location of eukaryotic
proteins. The location assignment is based on the predicted
presence of any of the N-terminal pre-sequences: chloroplast
transit peptide (cTP), mitochondrial targeting peptide (mTP) or
secretory pathway signal peptide (SP). Scores on which the final
prediction is based are not really probabilities, and they do not
necessarily add to one. However, the location with the highest
score is the most likely according to TargetP, and the relationship
between the scores (the reliability class) may be an indication of
how certain the prediction is. The reliability class (RC) ranges
from 1 to 5, where 1 indicates the strongest prediction. TargetP is
maintained at the server of the Technical University of
Denmark.
[0370] For the sequences predicted to contain an N-terminal
presequence a potential cleavage site can also be predicted.
[0371] A number of parameters were selected, such as organism group
(non-plant or plant), cutoff sets (none, predefined set of cutoffs,
or user-specified set of cutoffs), and the calculation of
prediction of cleavage sites (yes or no).
[0372] The results of TargetP 1.1 analysis of the polypeptide
sequence as represented by SEQ ID NO: 2 are presented Table A4. The
"plant" organism group has been selected, no cutoffs defined, and
the predicted length of the transit peptide requested. The
predicted subcellular localization of the polypeptide sequence as
represented by SEQ ID NO: 2 is the plastidic compartment.
TABLE-US-00019 TABLE A4 TargetP 1.1 analysis of the polypeptide
sequence as represented by SEQ ID NO: 2 Length (AA) 268
Chloroplastic transit peptide 0.987 Mitochondrial transit peptide
0.014 Secretory pathway signal peptide 0.023 Other subcellular
targeting 0.018 Predicted Location Chloro Reliability class 1
Predicted transit peptide length 58
[0373] The predicted length according to TargetP1.1 is of 58 amino
acids (starting from the N-terminus), but this can only be verified
experimentally by sequencing the mature protein. Cheong et al
(1999) predict a 65 amino acid transit peptide for the polypeptide
as represented by SEQ ID NO: 2 (Plant Molec Biol 40: 825-834).
[0374] Many other algorithms can be used to perform such analyses,
including: [0375] ChloroP 1.1 hosted on the server of the Technical
University of Denmark; [0376] Protein Prowler Subcellular
Localisation Predictor version 1.2 hosted on the server of the
Institute for Molecular Bioscience, University of Queensland,
Brisbane, Australia; [0377] PENCE Proteome Analyst PA-GOSUB 2.5
hosted on the server of the University of Alberta, Edmonton,
Alberta, Canada; [0378] TMHMM, hosted on the server of the
Technical University of Denmark
Example 6
Functional Assay for a 2-Cys PRX Polypeptide
[0379] 2-Cys PRX polypeptides present peroxidase activity on
hydrogen peroxide, for example. Enzyme assays for 2-Cys PRX
proteins have been extensively described in the literature, and are
well-know to a person skilled in the art. Huang et al. (2007; Appl
Microbiol Biotechnol 74(1):84-92), Bernier-Villamor et al. (2004; J
Exp Bot 55(406):2191-9) and Caporaletti et al. (2007; Biochem
Biophys Res Commun 355(3):722-7) are recent publications describing
the enzymatic assay of 2-Cys PRX proteins.
Example 7
Cloning of the Nucleic Acid Sequence Used in the Methods of the
Invention
[0380] The nucleic acid sequence used in the methods of the
invention was amplified by PCR using as template a Brassica rapa
2-Cys PRX clone as described in Cheong et al., (1999; Plant Molec
Biol). PCR was performed using Hifi Taq DNA polymerase in standard
conditions, using 200 ng of template in a 50 .mu.l PCR mix. The
primers used were prm08756 (SEQ ID NO: 81; sense, start codon in
bold): 5'-ggggacaagtttgtacaaaaaagcaggcttaaacaatggcgt
ctgttgcttctt-3' and prm08757 (SEQ ID NO: 82; reverse,
complementary): 5'-gg
ggaccactttgtacaagaaagctgggttcgagctaaatagctgagaagag-3', which
include the AttB sites for Gateway recombination. The amplified PCR
fragment was purified also using standard methods. The first step
of the Gateway procedure, the BP reaction, was then performed,
during which the PCR fragment recombines in vivo with the pDONR201
plasmid to produce, according to the Gateway terminology, an "entry
clone", p2-Cys PRX. Plasmid pDONR201 was purchased from Invitrogen,
as part of the Gateway.RTM. technology.
[0381] The entry clone comprising SEQ ID NO: 1 was then used in an
LR reaction with two destination vectors used for Oryza sativa
transformation. The vectors contained as functional elements within
the T-DNA borders: a plant selectable marker; a screenable marker
expression cassette; and a Gateway cassette intended for LR in vivo
recombination with the nucleic acid sequence of interest already
cloned in the entry clone. The first destination vector comprised
upstream of this Gateway cassette the rice GOS2 promoter (SEQ ID
NO: 79) for strong constitutive expression, and the second
destination vector comprised the rice Rcc3 promoter for
root-specific expression (SEQ ID NO: 80).
[0382] After the LR recombination step, the resulting expression
vectors pGOS2::2-Cys PRX and pRcc3::2-Cys PRX (FIG. 5) were
separately transformed into Agrobacterium strain LBA4044 according
to methods well known in the art.
Example 8
Plant Transformation
Rice Transformation
[0383] The two Agrobacterium strains each containing one of the
expression vectors as described in Example 7, used to transform
Oryza sativa plants. Mature dry seeds of the rice japonica cultivar
Nipponbare were dehusked. Sterilization was carried out by
incubating for one minute in 70% ethanol, followed by 30 minutes in
0.2% HgCl.sub.2, followed by a 6 times 15 minutes wash with sterile
distilled water. The sterile seeds were then germinated on a medium
containing 2,4-D (callus induction medium). After incubation in the
dark for four weeks, embryogenic, scutellum-derived calli were
excised and propagated on the same medium. After two weeks, the
calli were multiplied or propagated by subculture on the same
medium for another 2 weeks. Embryogenic callus pieces were
sub-cultured on fresh medium 3 days before co-cultivation (to boost
cell division activity).
[0384] Agrobacterium strain LBA4404 containing the expression
vector was used for co-cultivation. Agrobacterium was inoculated on
AB medium with the appropriate antibiotics and cultured for 3 days
at 28.degree. C. The bacteria were then collected and suspended in
liquid co-cultivation medium to a density (OD.sub.600) of about 1.
The suspension was then transferred to a Petri dish and the calli
immersed in the suspension for 15 minutes. The callus tissues were
then blotted dry on a filter paper and transferred to solidified,
co-cultivation medium and incubated for 3 days in the dark at
25.degree. C. Co-cultivated calli were grown on 2,4-D-containing
medium for 4 weeks in the dark at 28.degree. C. in the presence of
a selection agent. During this period, rapidly growing resistant
callus islands developed. After transfer of this material to a
regeneration medium and incubation in the light, the embryogenic
potential was released and shoots developed in the next four to
five weeks. Shoots were excised from the calli and incubated for 2
to 3 weeks on an auxin-containing medium from which they were
transferred to soil. Hardened shoots were grown under high humidity
and short days in a greenhouse.
[0385] Approximately 35 independent T0 rice transformants were
generated for one construct. The primary transformants were
transferred from a tissue culture chamber to a greenhouse. After a
quantitative PCR analysis to verify copy number of the T-DNA
insert, only single copy transgenic plants that exhibit tolerance
to the selection agent were kept for harvest of T1 seed. Seeds were
then harvested three to five months after transplanting. The method
yielded single locus transformants at a rate of over 50% (Aldemita
and Hodges 1996, Chan et al. 1993, Hiei et al. 1994).
Example 9
Phenotypic Evaluation Procedure
9.1 Evaluation Setup
[0386] Approximately 35 independent T0 rice transformants were
generated. The primary transformants were transferred from a tissue
culture chamber to a greenhouse for growing and harvest of T1 seed.
Six events, of which the T1 progeny segregated 3:1 for
presence/absence of the transgene, were retained. For each of these
events, approximately 10 T1 seedlings containing the transgene
(hetero- and homo-zygotes) and approximately 10 T1 seedlings
lacking the transgene (nullizygotes) were selected by monitoring
visual marker expression. The transgenic plants and the
corresponding nullizygotes were grown side-by-side at random
positions. Greenhouse conditions were of shorts days (12 hours
light), 28.degree. C. in the light and 22.degree. C. in the dark,
and a relative humidity of 70%.
[0387] Four T1 events were further evaluated in the T2 generation
following the same evaluation procedure as for the T1 generation
but with more individuals per event. From the stage of sowing until
the stage of maturity the plants were passed several times through
a digital imaging cabinet. At each time point digital images
(2048.times.1536 pixels, 16 million colours) were taken of each
plant from at least 6 different angles.
Drought Screen
[0388] Plants from T2 seeds from 4 events were grown in potting
soil under normal conditions until they approached the heading
stage. They were then transferred to a "dry" section where
irrigation was withheld. Humidity probes were inserted in randomly
chosen pots to monitor the soil water content (SWC). When SWC went
below certain thresholds, the plants were automatically re-watered
continuously until a normal level was reached again. The plants
were then re-transferred again to normal conditions. The rest of
the cultivation (plant maturation, seed harvest) was the same as
for plants not grown under abiotic stress conditions. Growth and
yield parameters were recorded as detailed for growth under normal
conditions.
9.2 Statistical Analysis: F Test
[0389] A two factor ANOVA (analysis of variants) was used as a
statistical model for the overall evaluation of plant phenotypic
characteristics. An F test was carried out on all the parameters
measured of all the plants of all the events transformed with the
gene of the present invention. The F test was carried out to check
for an effect of the gene over all the transformation events and to
verify for an overall effect of the gene, also known as a global
gene effect. The threshold for significance for a true global gene
effect was set at a 5% probability level for the F test. A
significant F test value points to a gene effect, meaning that it
is not only the mere presence or position of the gene that is
causing the differences in phenotype.
[0390] Because two experiments with overlapping events were carried
out, a combined analysis was performed. This is useful to check
consistency of the effects over the two experiments, and if this is
the case, to accumulate evidence from both experiments in order to
increase confidence in the conclusion. The method used was a
mixed-model approach that takes into account the multilevel
structure of the data (i.e. experiment-event-segregants). P values
were obtained by comparing likelihood ratio test to chi square
distributions.
9.3 Parameters Measured
Biomass-Related Parameter Measurement
[0391] From the stage of sowing until the stage of maturity the
plants were passed several times through a digital imaging cabinet.
At each time 2-Cys PRXnt digital images (2048.times.1536 pixels, 16
million colours) were taken of each plant from at least 6 different
angles.
[0392] The early vigour is the plant (seedling) aboveground area
three weeks post-germination. Early vigour was determined by
counting the total number of pixels from aboveground plant parts
discriminated from the background. This value was averaged for the
pictures taken on the same time point from different angles and was
converted to a physical surface value expressed in square mm by
calibration. The results described below are for plants three weeks
post-germination.
[0393] The plant aboveground area (or leafy biomass) was determined
by counting the total number of pixels on the digital images from
aboveground plant parts discriminated from the background. This
value was averaged for the pictures taken on the same time point
from the different angles and was converted to a physical surface
value expressed in square mm by calibration. Experiments show that
the aboveground plant area measured this way correlates with the
biomass of plant parts above ground. The above ground area is the
area measured at the time point at which the plant had reached its
maximal leafy biomass.
[0394] To measure root-related parameters, plants were grown in
specially designed pots with transparent bottoms to allow
visualization of the roots. A digital camera recorded images
through the bottom of the pot during plant growth. Root features
such as total projected area (which can be correlated to total root
volume), average diameter and length of roots above a certain
thickness threshold (length of thick roots, or thick root length)
were deduced from the picture using of appropriate software.
Increase in root biomass is expressed as an increase in total root
biomass (measured as maximum biomass of roots observed during the
lifespan of a plant); or as an increase in the root/shoot index
(measured as the ratio between root mass and shoot mass in the
period of active growth of root and shoot).
Seed-Related Parameter Measurements
[0395] The mature primary panicles were harvested, counted, bagged,
barcode-labelled and then dried for three days in an oven at
37.degree. C. The panicles were then threshed and all the seeds
were collected and counted. The filled husks were separated from
the empty ones using an air-blowing device. The empty husks were
discarded and the remaining fraction was counted again. The filled
husks were weighed on an analytical balance. The number of filled
seeds was determined by counting the number of filled husks that
remained after the separation step. The total seed yield was
measured by weighing all filled husks harvested from a plant. Total
seed number per plant was measured by counting the number of husks
harvested from a plant. Thousand Kernel Weight (TKW) is
extrapolated from the number of filled seeds counted and their
total weight. The Harvest Index (HI) in the present invention is
defined as the ratio between the total seed yield and the above
ground area (mm.sup.2), multiplied by a factor 10.sup.6. The total
number of flowers per panicle as defined in the present invention
is the ratio between the total number of seeds and the number of
mature primary panicles. The seed fill rate as defined in the
present invention is the proportion (expressed as a %) of the
number of filled seeds over the total number of seeds (or
florets).
Example 10
Results of the Phenotypic Evaluation of the Transgenic Plants,
Grown Under Normal Growth Conditions
[0396] The results of the evaluation of transgenic rice plants
expressing a nucleic acid sequence encoding a 2-Cys PRX polypeptide
under the control of a root-specific promoter, and grown under
normal growth conditions, are presented below. Improved early
vigour was observed, as well as increased seed fill rate, increased
total seed yield per plant, increased number of filled seeds,
increased harvest index, and increased thousand kernel weight.
TABLE-US-00020 TABLE A5 Results of the evaluation of transgenic
rice plants expressing the nucleic acid sequence useful in
performing the methods of the invention, under the control of a
root-specific-promoter, grown under normal growth conditions. %
Increase for the % Increase for the three best events three best
events Trait in T1 generation in T2 generation Early vigour
(seedlings) 7% 5% Seed fill rate 1% 10% Total seed yield per plant
8% 10% Total numbers of filled seeds 6% 8% Harvest index 7% 9%
Thousand kernel weight 3% 3%
[0397] Transgenic rice plants expressing a nucleic acid sequence
encoding a 2-Cys PRX polypeptide under the control of a
constitutive promoter (a rice GOS2 promoter), and grown under
normal growth conditions, showed no difference in any of the traits
phenotypically examined as compared to control plants grown under
comparable normal growth conditions (data not shown).
Example 11
Results of the Phenotypic Evaluation of the Transgenic Plants,
Grown Under Stress Growth Conditions
[0398] The results of the evaluation of transgenic rice plants
expressing a nucleic acid sequence encoding a 2-Cys PRX polypeptide
under the control of a root-specific promoter, and grown under
drought-stress growth conditions, are presented below. Improved
early vigour was observed, as well as increased aboveground
biomass, increased root biomass, increased number of flowers per
panicle, increased seed fill rate, increased total seed yield per
plant, increased number of seeds, increased number of filled seeds,
and increased harvest index.
TABLE-US-00021 TABLE A6 Results of the evaluation of transgenic
rice plants expressing the nucleic acid sequence useful in
performing the methods of the invention, under the control of a
root-specific- promoter, grown under drought-stress growth
conditions. Trait % Increase in T2 generation (all events) Early
vigour (seedlings) 31% Aboveground biomass 11% Root biomass 6%
Flowers per panicle 8% Seed fill rate 8% Total seed yield per plant
18% Total number of seeds 10% Total numbers of filled seeds 17%
Harvest index 8%
Example 12
Examples of Transformation of Other Crops
Corn Transformation
[0399] Transformation of maize (Zea mays) is performed with a
modification of the method described by Ishida et al. (1996) Nature
Biotech 14(6): 745-50. Transformation is genotype-dependent in corn
and only specific genotypes are amenable to transformation and
regeneration. The inbred line A188 (University of Minnesota) or
hybrids with A188 as a parent are good sources of donor material
for transformation, but other genotypes can be used successfully as
well. Ears are harvested from corn plant approximately 11 days
after pollination (DAP) when the length of the immature embryo is
about 1 to 1.2 mm. Immature embryos are cocultivated with
Agrobacterium tumefaciens containing the expression vector, and
transgenic plants are recovered through organogenesis. Excised
embryos are grown on callus induction medium, then maize
regeneration medium, containing the selection agent (for example
imidazolinone but various selection markers can be used). The Petri
plates are incubated in the light at 25.degree. C. for 2-3 weeks,
or until shoots develop. The green shoots are transferred from each
embryo to maize rooting medium and incubated at 25.degree. C. for
2-3 weeks, until roots develop. The rooted shoots are transplanted
to soil in the greenhouse. T1 seeds are produced from plants that
exhibit tolerance to the selection agent and that contain a single
copy of the T-DNA insert.
Wheat Transformation
[0400] Transformation of wheat is performed with the method
described by Ishida et al. (1996) Nature Biotech 14(6): 745-50. The
cultivar Bobwhite (available from CIMMYT, Mexico) is commonly used
in transformation. Immature embryos are co-cultivated with
Agrobacterium tumefaciens containing the expression vector, and
transgenic plants are recovered through organogenesis. After
incubation with Agrobacterium, the embryos are grown in vitro on
callus induction medium, then regeneration medium, containing the
selection agent (for example imidazolinone but various selection
markers can be used). The Petri plates are incubated in the light
at 25.degree. C. for 2-3 weeks, or until shoots develop. The green
shoots are transferred from each embryo to rooting medium and
incubated at 25.degree. C. for 2-3 weeks, until roots develop. The
rooted shoots are transplanted to soil in the greenhouse. T1 seeds
are produced from plants that exhibit tolerance to the selection
agent and that contain a single copy of the T-DNA insert.
Soybean Transformation
[0401] Soybean is transformed according to a modification of the
method described in the Texas A&M U.S. Pat. No. 5,164,310.
Several commercial soybean varieties are amenable to transformation
by this method. The cultivar Jack (available from the Illinois Seed
foundation) is commonly used for transformation. Soybean seeds are
sterilised for in vitro sowing. The hypocotyl, the radicle and one
cotyledon are excised from seven-day old young seedlings. The
epicotyl and the remaining cotyledon are further grown to develop
axillary nodes. These axillary nodes are excised and incubated with
Agrobacterium tumefaciens containing the expression vector. After
the cocultivation treatment, the explants are washed and
transferred to selection media. Regenerated shoots are excised and
placed on a shoot elongation medium. Shoots no longer than 1 cm are
placed on rooting medium until roots develop. The rooted shoots are
transplanted to soil in the greenhouse. T1 seeds are produced from
plants that exhibit tolerance to the selection agent and that
contain a single copy of the T-DNA insert.
Rapeseed/Canola Transformation
[0402] Cotyledonary petioles and hypocotyls of 5-6 day old young
seedling are used as explants for tissue culture and transformed
according to Babic et al. (1998, Plant Cell Rep 17: 183-188). The
commercial cultivar Westar (Agriculture Canada) is the standard
variety used for transformation, but other varieties can also be
used. Canola seeds are surface-sterilized for in vitro sowing. The
cotyledon petiole explants with the cotyledon attached are excised
from the in vitro seedlings, and inoculated with Agrobacterium
(containing the expression vector) by dipping the cut end of the
petiole explant into the bacterial suspension. The explants are
then cultured for 2 days on MSBAP-3 medium containing 3 mg/l BAP,
3% sucrose, 0.7% Phytagar at 23.degree. C., 16 hr light. After two
days of co-cultivation with Agrobacterium, the petiole explants are
transferred to MSBAP-3 medium containing 3 mg/l BAP, cefotaxime,
carbenicillin, or timentin (300 mg/l) for 7 days, and then cultured
on MSBAP-3 medium with cefotaxime, carbenicillin, or timentin and
selection agent until shoot regeneration. When the shoots are 5-10
mm in length, they are cut and transferred to shoot elongation
medium (MSBAP-0.5, containing 0.5 mg/l BAP). Shoots of about 2 cm
in length are transferred to the rooting medium (MS0) for root
induction. The rooted shoots are transplanted to soil in the
greenhouse. T1 seeds are produced from plants that exhibit
tolerance to the selection agent and that contain a single copy of
the T-DNA insert.
Alfalfa Transformation
[0403] A regenerating clone of alfalfa (Medicago sativa) is
transformed using the method of (McKersie et al., 1999 Plant
Physiol 119: 839-847). Regeneration and transformation of alfalfa
is genotype dependent and therefore a regenerating plant is
required. Methods to obtain regenerating plants have been
described. For example, these can be selected from the cultivar
Rangelander (Agriculture Canada) or any other commercial alfalfa
variety as described by Brown D C W and A Atanassov (1985. Plant
Cell Tissue Organ Culture 4: 111-112). Alternatively, the RA3
variety (University of Wisconsin) has been selected for use in
tissue culture (Walker et al., 1978 Am J Bot 65:654-659). Petiole
explants are cocultivated with an overnight culture of
Agrobacterium tumefaciens C58C1 pMP90 (McKersie et al., 1999 Plant
Physiol 119: 839-847) or LBA4404 containing the expression vector.
The explants are cocultivated for 3 d in the dark on SH induction
medium containing 288 mg/L Pro, 53 mg/L thioproline, 4.35 g/L
K2SO4, and 100 .mu.m acetosyringinone. The explants are washed in
half-strength Murashige-Skoog medium (Murashige and Skoog, 1962)
and plated on the same SH induction medium without acetosyringinone
but with a suitable selection agent and suitable antibiotic to
inhibit Agrobacterium growth. After several weeks, somatic embryos
are transferred to BOi2Y development medium containing no growth
regulators, no antibiotics, and 50 g/L sucrose. Somatic embryos are
subsequently germinated on half-strength Murashige-Skoog medium.
Rooted seedlings were transplanted into pots and grown in a
greenhouse. T1 seeds are produced from plants that exhibit
tolerance to the selection agent and that contain a single copy of
the T-DNA insert.
Cotton Transformation
[0404] Cotton (Gossypium hirsutum L.) transformation is performed
using Agrobacterium tumefaciens, on hypocotyls explants. The
commercial cultivars such as Coker 130 or Coker 312 (SeedCo,
Lubbock, Tex.) are standard varieties used for transformation, but
other varieties can also be used. The seeds are surface sterilized
and germinated in the dark. Hypocotyl explants are cut from the
germinated seedlings to lengths of about 1-1.5 centimeter. The
hypotocyl explant is submersed in the Agrobacterium tumefaciens
inoculum containing the expression vector, for 5 minutes then
co-cultivated for about 48 hours on MS +1.8 mg/l KNO3+2% glucose at
24.degree. C., in the dark. The explants are transferred the same
medium containing appropriate bacterial and plant selectable
markers (renewed several times), until embryogenic calli is seen.
The calli are separated and subcultured until somatic embryos
appear. Plantlets derived from the somatic embryos are matured on
rooting medium until roots develop. The rooted shoots are
transplanted to potting soil in the greenhouse. T1 seeds are
produced from plants that exhibit tolerance to the selection agent
and that contain a single copy of the T-DNA insert.
Example 13
Examples of Other Stress Screens
Salt Stress Screen
[0405] Plants are grown on a substrate made of coco fibers and
argex (3 to 1 ratio). A normal nutrient solution is used during the
first two weeks after transplanting the plantlets in the
greenhouse. After the first two weeks, 25 mM of salt (NaCl) is
added to the nutrient solution, until the plants were
harvested.
Reduced Nutrient (Nitrogen) Availability Screen
[0406] Rice plants from T2 seeds are grown in potting soil under
normal conditions except for the nutrient solution. The pots are
watered from transplantation to maturation with a specific nutrient
solution containing reduced N nitrogen (N) content, usually between
7 to 8 times less. The rest of the cultivation (plant maturation,
seed harvest) is the same as for plants not grown under abiotic
stress. Growth and yield parameters are recorded as detailed for
growth under normal conditions.
Example 14
Identification of Sequences Related to the Nucleic Acid Sequence
Used in the Methods of the Invention
[0407] Sequences (full length cDNA, ESTs or genomic) related to the
nucleic acid sequence used in the methods of the present invention
were identified amongst those maintained in the Entrez Nucleotides
database at the National Center for Biotechnology Information
(NCBI) using database sequence search tools, such as the Basic
Local Alignment Tool (BLAST) (Altschul et al. (1990) J. Mol. Biol.
215:403-410; and Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402). The program is used to find regions of local
similarity between sequences by comparing nucleic acid or
polypeptide sequences to sequence databases and by calculating the
statistical significance of matches. For example, the polypeptide
encoded by the nucleic acid used in the present invention was used
for the TBLASTN algorithm, with default settings and the filter to
ignore low complexity sequences set off. The output of the analysis
was viewed by pairwise comparison, and ranked according to the
probability score (E-value), where the score reflect the
probability that a particular alignment occurs by chance (the lower
the E-value, the more significant the hit). In addition to
E-values, comparisons were also scored by percentage identity.
Percentage identity refers to the number of identical nucleotides
(or amino acids) between the two compared nucleic acid (or
polypeptide) sequences over a particular length. In some instances,
the default parameters may be adjusted to modify the stringency of
the search. For example the E-value may be increased to show less
stringent matches. This way, short nearly exact matches may be
identified.
[0408] Table B1 provides a list of nucleic acid sequences related
to the nucleic acid sequence useful in the methods of the present
invention.
TABLE-US-00022 TABLE B1 Examples of ANN polypeptides: Nucleic acid
Protein Plant Source SEQ ID NO: SEQ ID NO: Arabidopsis thaliana
ANNAT1 83 84 Gossypium hirsutum fiber annexin 96 97 Lavatera
thuringiaca annexin 98 99 Brassica rapa subsp. Pekinensis 80C09_22
100 101 Gossypium hirsutum annexin 102 103 Capsicum annuum Annexin
24 104 105 Nicotiana tabacum VCaB42 106 107 Gossypium hirsutum Anx1
108 109 Nicotiana tabacum annexin 110 111 Solanum tuberosum annexin
p34 112 113 Lycopersicon esculentum annexin p34 114 115
Lycopersicon esculentum annexin p35 116 117 Arabidopsis thaliana
ANNAT2 118 119 Nicotiana tabacum annexin 120 121 Solanum tuberosum
annexin p34-like 122 123 Arabidopsis thaliana ANN7 124 125 Medicago
sativa annexin 126 127 Brassica rapa subsp. pekinensis 80A08_20 128
129 Arabidopsis thaliana ANNAT3 130 131 Arabidopsis thaliana
annexin 5 132 133 Arabidopsis thaliana ANNAT4 134 135 Arabidopsis
thaliana ANN6 136 137 Arabidopsis thaliana ANN8 At5g12380 138 139
Oryza sativa (japonica cultivar-group) 140 141 Os09g0394900 Oryza
sativa (japonica cultivar-group) 142 143 Os02g0753800 Oryza sativa
(japonica cultivar-group) 144 145 Os08g0425700 Oryza sativa
(japonica cultivar-group) 146 147 Os06g0221200 Oryza sativa
(japonica cultivar-group) 148 149 Os09g0453300 Oryza sativa
(japonica cultivar-group) 150 151 Os05g0382900 Oryza sativa
(japonica cultivar-group) 152 153 Os08g0372900 Oryza sativa
(japonica cultivar-group) 154 155 Os03g0819300
[0409] In some instances, related sequences have tentatively been
assembled and publicly disclosed by research institutions, such as
The Institute for Genomic Research (TIGR). The Eukaryotic Gene
Orthologs (EGO) database may be used to identify such related
sequences, either by keyword search or by using the BLAST algorithm
with the nucleic acid or polypeptide sequence of interest.
Example 15
Alignment of ANN Polypeptide Sequences
[0410] Alignment of polypeptide sequences was performed using the
AlignX programme from the Vector NTI (Invitrogen) which is based on
the popular Clustal W algorithm of progressive alignment (Thompson
et al. (1997) Nucleic Acids Res 25:4876-4882; Chenna et al. (2003).
Nucleic Acids Res 31:3497-3500). Default values are for the gap
open penalty of 10, for the gap extension penalty of 0, 1 and the
selected weight matrix is Blosum 62 (if polypeptides are aligned).
Minor manual editing was done to further optimise the alignment.
Sequence conservation among ANN polypeptides is found throughout
the whole sequence. The ANN polypeptides are aligned in FIG. 9.
[0411] A phylogenetic tree of ANN polypeptides (such as the one
from FIG. 8) may be constructed using a neighbour-joining
clustering algorithm as provided in the AlignX programme from the
Vector NTI (Invitrogen).
Example 16
Calculation of Global Percentage Identity Between Polypeptide
Sequences Useful in Performing the Methods of the Invention
[0412] Global percentages of similarity and identity between full
length polypeptide sequences useful in performing the methods of
the invention were determined using one of the methods available in
the art, the MatGAT (Matrix Global Alignment Tool) software (BMC
Bioinformatics. 2003 4:29. MatGAT: an application that generates
similarity/identity matrices using protein or DNA sequences.
Campanella J J, Bitincka L, Smalley J; software hosted by Ledion
Bitincka). MatGAT software generates similarity/identity matrices
for DNA or protein sequences without needing pre-alignment of the
data. The program performs a series of pair-wise alignments using
the Myers and Miller global alignment algorithm (with a gap opening
penalty of 12, and a gap extension penalty of 2), calculates
similarity and identity using for example Blosum 62 (for
polypeptides), and then places the results in a distance matrix.
Sequence similarity is shown in the bottom half of the dividing
line and sequence identity is shown in the top half of the diagonal
dividing line.
[0413] Parameters used in the comparison were:
TABLE-US-00023 Scoring matrix: Blosum62 First Gap: 12 Extending
gap: 2
[0414] Results of the software analysis are shown in Table B2 for
the global similarity and identity over the full length of the
polypeptide sequences. Percentage identity is given above the
diagonal in bold and percentage similarity is given below the
diagonal (normal face).
[0415] The percentage identity between the ANN polypeptide
sequences useful in performing the methods of the invention can be
as low as about 30% amino acid identity compared to SEQ ID NO: 84
(NP.sub.--174810).
TABLE-US-00024 TABLE B2 MatGAT results for global similarity and
identity over the full length of the polypeptide sequences. 1 2 3 4
5 6 7 8 1. NP_174810 72.6 71.0 29.9 68.6 65.2 67.5 67.8 2. AAC33305
84.5 89.9 32.1 71.6 71.6 68.0 72.8 3. AAB71830 84.5 94.3 30.6 68.8
67.0 64.2 68.7 4. AAZ41833 36.4 37.4 36.8 32.6 29.4 33.4 33.9 5.
AAB67994 82.3 84.5 83.2 38.7 63.1 72.2 85.5 6. 1DK5 83.9 83.9 81.7
37.7 79.2 63.6 64.5 7. AAD24540 81.1 82.6 81.0 38.7 83.9 77.6 74.4
8. AAR13288 81.7 84.5 82.6 39.3 92.7 78.6 84.8 9. CAA75213 84.2
85.1 83.2 37.4 81.0 95.7 79.1 80.4 10. CAB92956 83.9 85.4 83.5 37.6
80.6 95.3 79.1 80.4 11. AAC97494 83.9 85.4 83.5 37.6 80.6 95.3 79.1
80.4 12. AAC97493 80.8 80.4 79.4 37.8 82.9 77.6 92.1 85.8 13.
NP_201307 79.8 81.4 80.1 44.5 85.5 79.2 83.9 84.5 14. CAA75214 83.6
84.2 82.3 37.1 80.0 94.7 78.2 79.7 15. ABB55363 83.3 84.8 82.9 37.3
79.7 94.7 78.5 79.7 16. NP_196585 80.4 80.4 79.1 41.2 84.2 78.9
82.0 82.6 17. CAA52903 80.8 82.0 81.6 38.6 83.8 79.2 82.3 83.2 18.
AAZ67605 79.2 79.1 79.1 41.1 82.6 78.0 81.6 82.0 19. NP_181410 59.2
58.6 58.6 27.8 61.4 58.4 61.7 63.2 20. ABE65753 58.4 56.3 54.7 27.0
58.5 54.0 56.6 59.5 21. NP_181409 56.4 55.2 53.6 25.3 53.9 54.7
54.5 54.2 22. NP_196584 77.4 78.9 78.6 41.2 81.8 78.0 81.1 81.8 23.
NP_568271 69.4 67.1 67.7 30.9 68.4 66.5 66.8 69.0 24. NP_001063096
55.8 55.7 53.8 27.4 59.0 54.7 57.0 58.5 25. NP_001048149 76.0 78.5
77.5 35.2 79.4 74.5 75.3 79.1 26. NP_001061839 55.1 54.5 52.6 27.1
57.6 53.7 57.0 56.1 27. NP_001057176 78.2 79.8 80.1 35.2 79.5 75.8
77.0 79.5 28. NP_001063343 53.0 53.6 52.4 26.1 53.6 50.3 52.0 52.0
29. NP_001055408 54.3 52.7 53.0 30.2 53.8 55.1 53.8 54.3 30.
NP_001061661 27.8 27.6 27.3 24.9 26.8 24.2 28.4 26.8 31.
NP_001051711 17.7 16.8 17.1 10.2 16.5 17.4 14.2 16.8 9 10 11 12 13
14 15 16 1. NP_174810 65.6 66.2 65.9 65.9 64.0 65.6 64.9 63.1 2.
AAC33305 72.8 72.8 73.4 65.5 67.5 71.8 71.4 66.5 3. AAB71830 69.0
68.7 69.0 63.3 64.4 68.0 67.3 63.3 4. AAZ41833 29.6 30.0 30.2 32.4
41.8 29.3 29.5 35.9 5. AAB67994 63.4 63.1 63.1 70.3 70.1 63.1 62.1
68.1 6. 1DK5 91.0 91.9 91.6 62.0 61.8 90.7 90.4 62.0 7. AAD24540
63.9 64.2 64.9 87.0 70.3 63.6 62.9 69.3 8. AAR13288 65.2 65.2 66.5
73.7 71.0 65.2 63.8 70.3 9. CAA75213 92.4 92.7 62.0 64.4 97.1 90.8
64.6 10. CAB92956 97.8 98.4 63.6 64.4 92.4 98.4 63.9 11. AAC97494
97.8 100.0 64.2 64.4 92.7 96.8 64.9 12. AAC97493 79.4 79.4 79.4
68.1 62.7 62.3 68.0 13. NP_201307 80.1 80.4 80.4 81.4 63.1 63.0
79.2 14. CAA75214 98.4 97.5 97.5 79.0 79.5 90.8 63.9 15. ABB55363
96.5 98.7 98.7 78.8 79.8 96.2 62.6 16. NP_196585 80.4 81.0 81.0
80.7 89.9 79.7 80.4 17. CAA52903 81.5 81.5 81.5 80.3 83.3 80.9 80.4
81.0 18. AAZ67605 79.4 79.7 79.7 80.1 89.9 78.8 79.1 95.6 19.
NP_181410 58.6 58.9 58.9 60.1 57.6 58.3 58.9 59.5 20. ABE65753 54.7
55.1 55.1 55.4 58.0 55.7 55.1 58.9 21. NP_181409 55.8 55.8 55.8
54.9 53.9 55.8 55.8 53.0 22. NP_196584 78.3 79.2 78.9 80.2 88.4
77.4 78.6 91.2 23. NP_568271 66.8 68.0 67.7 66.5 66.9 66.8 68.0
67.7 24. NP_001063096 54.9 54.6 54.6 57.5 59.0 55.2 54.4 59.2 25.
NP_001048149 77.1 77.1 77.1 74.9 76.0 76.1 75.9 76.6 26.
NP_001061839 55.1 53.9 53.9 56.1 58.3 54.8 53.6 57.9 27.
NP_001057176 76.7 77.0 77.0 74.8 76.3 75.7 76.7 74.8 28.
NP_001063343 50.8 51.1 51.1 51.4 52.4 50.8 50.8 53.0 29.
NP_001055408 53.0 53.2 53.2 53.5 53.8 53.2 53.2 53.8 30.
NP_001061661 28.1 27.3 27.3 29.1 29.4 28.1 29.4 28.1 31.
NP_001051711 17.2 17.5 17.8 16.8 16.4 16.2 17.1 15.2 17 18 19 20 21
22 23 24 1. NP_174810 65.0 62.8 38.5 34.3 32.3 60.8 49.4 35.5 2.
AAC33305 69.3 64.9 41.0 35.6 34.2 63.8 51.1 37.4 3. AAB71830 68.4
62.3 39.8 34.1 33.0 61.3 49.2 35.3 4. AAZ41833 33.1 35.3 19.9 17.5
16.8 34.8 22.7 19.2 5. AAB67994 70.5 67.5 39.6 34.9 35.1 64.9 51.3
36.8 6. 1DK5 63.6 61.1 40.0 33.5 34.2 59.8 48.6 35.2 7. AAD24540
69.6 69.3 42.5 36.9 33.7 67.6 51.4 37.2 8. AAR13288 69.3 68.7 43.5
36.6 35.3 67.3 51.7 37.5 9. CAA75213 64.2 62.7 41.9 35.6 35.0 61.9
48.6 36.5 10. CAB92956 65.2 62.7 41.3 35.3 34.1 61.6 49.2 35.5 11.
AAC97494 65.8 63.6 42.2 35.0 34.7 62.3 49.5 35.5 12. AAC97493 66.5
67.4 40.4 36.0 33.4 65.4 49.8 36.9 13. NP_201307 69.7 78.9 42.2
37.4 34.9 74.9 48.1 40.4 14. CAA75214 63.0 62.3 41.0 35.0 34.7 61.6
48.9 35.3 15. ABB55363 63.8 61.3 40.4 34.5 34.1 60.3 48.3 35.3 16.
NP_196585 69.0 89.9 41.9 38.2 34.7 82.4 50.5 38.7 17. CAA52903 68.4
40.8 35.1 33.8 67.0 49.1 36.3 18. AAZ67605 81.3 40.4 36.0 33.8 82.4
50.2 37.5 19. NP_181410 58.3 58.3 31.9 33.6 41.4 45.5 33.4 20.
ABE65753 54.7 58.5 51.7 27.4 36.1 34.3 53.2 21. NP_181409 51.4 52.0
54.2 47.6 32.6 34.7 30.1 22. NP_196584 80.8 91.5 58.6 58.5 51.7
49.5 37.9 23. NP_568271 68.0 68.4 63.2 56.6 55.8 66.0 36.5 24.
NP_001063096 57.1 58.9 55.1 72.2 46.1 59.1 58.9 25. NP_001048149
77.1 75.9 60.1 57.9 51.1 74.2 67.1 59.7 26. NP_001061839 55.5 57.0
54.5 69.2 46.4 58.6 57.3 84.4 27. NP_001057176 76.3 74.4 60.4 56.8
48.6 74.2 69.1 57.4 28. NP_001063343 48.3 52.0 48.6 48.9 50.2 52.4
50.2 50.2 29. NP_001055408 52.4 53.5 60.2 44.9 47.3 53.0 55.6 46.5
30. NP_001061661 25.8 27.3 26.3 26.0 28.9 27.3 27.6 26.5 31.
NP_001051711 19.2 16.8 16.8 17.1 17.2 16.7 19.0 19.0 25 26 27 28 29
30 31 1. NP_174810 59.6 36.8 60.2 28.4 37.2 12.4 11.3 2. AAC33305
63.3 38.8 64.5 30.2 38.9 14.1 10.1 3. AAB71830 59.8 36.0 61.9 29.1
37.8 15.1 9.2 4. AAZ41833 29.0 19.0 28.9 14.6 21.5 14.4 8.0 5.
AAB67994 63.1 39.0 61.8 30.6 39.0 12.5 10.1 6. 1DK5 60.8 33.9 59.5
29.0 41.3 12.2 10.5 7. AAD24540 61.1 40.7 62.6 28.3 39.4 14.9 9.5
8. AAR13288 63.6 38.8 63.5 30.2 40.8 14.3 7.9 9. CAA75213 62.0 35.6
60.7 30.5 39.1 13.0 10.5 10. CAB92956 62.0 34.8 61.0 29.5 39.1 12.5
9.9 11. AAC97494 63.0 34.8 61.9 29.5 39.7 12.5 8.6 12. AAC97493
59.5 39.8 59.4 28.0 38.3 13.7 11.6 13. NP_201307 59.6 39.6 60.8
27.9 40.1 12.9 10.7 14. CAA75214 60.8 35.3 59.4 29.8 38.9 13.0 9.9
15. ABB55363 61.0 34.0 60.6 29.2 38.4 13.2 10.2 16. NP_196585 61.4
38.8 58.2 28.3 39.4 12.9 11.4 17. CAA52903 60.3 36.0 59.6 27.5 37.6
13.0 9.4 18. AAZ67605 60.1 37.9 56.6 28.3 37.5 12.8 11.1 19.
NP_181410 41.1 30.3 42.4 25.5 41.4 15.0 11.1 20. ABE65753 37.0 50.2
35.5 27.0 28.8 14.7 12.3 21. NP_181409 33.0 28.2 31.0 25.4 30.2
14.1 10.3 22. NP_196584 59.1 37.5 58.1 29.0 37.6 12.8 11.3 23.
NP_568271 51.1 35.9 52.4 29.2 38.5 13.3 12.3 24. NP_001063096 37.8
68.5 38.6 30.4 29.3 14.0 12.1 25. NP_001048149 37.7 83.9 29.8 37.4
14.6 10.8 26. NP_001061839 57.3 38.4 31.8 30.9 16.8 15.5 27.
NP_001057176 90.2 58.3 28.1 39.8 15.4 11.4 28. NP_001063343 49.8
49.2 49.2 19.8 14.6 10.9 29. NP_001055408 51.6 46.5 54.8 38.4 15.5
12.1 30. NP_001061661 29.4 27.6 27.3 28.6 29.1 13.4 31.
NP_001051711 17.8 20.6 17.7 19.4 16.4 19.6
Example 17
Identification of Domains Comprised in Polypeptide Sequences Useful
in Performing the Methods of the Invention
[0416] The Integrated Resource of Protein Families, Domains and
Sites (InterPro) database is an integrated interface for the
commonly used signature databases for text- and sequence-based
searches. The InterPro database combines these databases, which use
different methodologies and varying degrees of biological
information about well-characterized proteins to derive protein
signatures. Collaborating databases include SWISS-PROT, PROSITE,
TrEMBL, PRINTS, Propom and Pfam, Smart and TIGRFAMs. Pfam is a
large collection of multiple sequence alignments and hidden Markov
models covering many common protein domains and families. Pfam is
hosted at the Sanger Institute server in the United Kingdom.
Interpro is hosted at the European Bioinformatics Institute in the
United Kingdom.
[0417] The results of the InterPro scan of the polypeptide sequence
as represented by SEQ ID NO: 84 are presented in Table B3.
TABLE-US-00025 TABLE B3 InterPro scan results (major accession
numbers) of the polypeptide sequence as represented by SEQ ID NO:
84. Amino acid coordinates on Database Accession number Accession
name SEQ ID NO 84 PRODOM PD000143 Annexin
[16-78]T-[88-153]T-[172-239]T- [246-313]T PRINTS PR00196 ANNEXIN
[25-47]T-[65-81]T-[92-113]T- [255-275]T-[299-312]T GENE3D
G3DSA:1.10.220.10 Annexin [13-82]T-[83-161]T-[166-238]T- [243-317]T
PANTHER PTHR10502 Annexin [9-312]T PFAM PF00191 Annexin
[15-80]T-[87-152]T-[170-236]T- [246-311]T SMART SM00335 ANX
[28-80]T-[100-152]T-[183- 232]T-[259-311]T PROFILE PS00223 ANNEXIN
[259-311]T SUPERFAMILY SSF47874 Annexin [1-316]T PRINTS PR01814
ANNEXINPLANT [119-133]T-[161-181]T-[227- 245]T PANTHER
PTHR10502:SF10 Annexin_like [9-312]T
Example 18
Topology Prediction of the Polypeptide Sequences Useful in
Performing the Methods of the Invention
[0418] TargetP 1.1 predicts the subcellular location of eukaryotic
proteins. The location assignment is based on the predicted
presence of any of the N-terminal pre-sequences: chloroplast
transit peptide (cTP), mitochondrial targeting peptide (mTP) or
secretory pathway signal peptide (SP). Scores on which the final
prediction is based are not really probabilities, and they do not
necessarily add to one. However, the location with the highest
score is the most likely according to TargetP, and the relationship
between the scores (the reliability class) may be an indication of
how certain the prediction is. The reliability class (RC) ranges
from 1 to 5, where 1 indicates the strongest prediction. TargetP is
maintained at the server of the Technical University of
Denmark.
[0419] For the sequences predicted to contain an N-terminal
presequence a potential cleavage site can also be predicted.
[0420] A number of parameters were selected, such as organism group
(non-plant or plant), cutoff sets (none, predefined set of cutoffs,
or user-specified set of cutoffs), and the calculation of
prediction of cleavage sites (yes or no).
[0421] The results of TargetP 1.1 analysis of the polypeptide
sequence as represented by SEQ ID NO: 84 are presented Table B4.
The "plant" organism group has been selected, no cutoffs defined,
and the predicted length of the transit peptide requested. The
subcellular localization of the polypeptide sequence as represented
by SEQ ID NO: 84 may be the cytoplasm or nucleus, no transit
peptide is predicted. This prediction is in agreement with earlier
reports that indicate that annexin proteins are associated to the
plasma membrane, vacuole and nuclear periphery (Clark & Roux,
Plant Physiol. 109, 1133-1139, 1995).
TABLE-US-00026 TABLE B4 TargetP 1.1 analysisof the polypeptide
sequence as represented by SEQ ID NO: 84 Length (AA) 317
Chloroplastic transit peptide 0.102 Mitochondrial transit peptide
0.126 Secretory pathway signal peptide 0.045 Other subcellular
targeting 0.905 Predicted Location / Reliability class 2 Predicted
transit peptide length /
[0422] Many other algorithms can be used to perform such analyses,
including: [0423] ChloroP 1.1 hosted on the server of the Technical
University of Denmark; [0424] Protein Prowler Subcellular
Localisation Predictor version 1.2 hosted on the server of the
Institute for Molecular Bioscience, University of Queensland,
Brisbane, Australia; [0425] PENCE Proteome Analyst PA-GOSUB 2.5
hosted on the server of the University of Alberta, Edmonton,
Alberta, Canada; [0426] TMHMM, hosted on the server of the
Technical University of Denmark
Example 19
Functional Assay for the ANN Polypeptide
[0427] Assay for annexin-membrane interactions (Dabitz et al.
Biochemistry 44, 16292-16300, 2005):
Membrane Binding Assay (Copelleting Assay).
[0428] Phospholipid vesicles are prepared following the protocol of
Reeves and Dowben (J. Cell. Physiol. 73, 49-60, 1969). To assess
the plant annexin-membrane binding behavior, a copelleting assay is
conducted (Hofmann and Huber, Methods Enzymol. 372, 186-216, 2003).
A total amount of 0.2 .mu.mol of phospholipids is used for each
individual sample (500 .mu.L), composed of 0.5 nmol of protein in
liposome buffer and varying amounts of calcium. As a control, a
sample of 0.1 nmol of protein in 100 .mu.L of 10% SDS is prepared
at this stage. All samples are centrifuged (23 000 rpm at 4.degree.
C. for 45 min), and the pellets are resuspended with 50 .mu.L of
10% SDS and then subjected to SDS-PAGE. Gels are stained with
Coomassie and analyzed densitometrically using ImageJ (Rasband, W.
ImageJ, version 1.30, National Institutes of Health, Bethesda, Md.,
2005). Each calcium concentration is assessed three times
independently. Curve fitting is performed with SigmaPlot using a
standard binding equation.
Phospholipid Vesicle Preparation.
[0429] For experiments assessing membrane surface hydrophobicity
and liposome leakage, the following protocols are used. Brain
phosphatidylserine (PS), egg phosphatidylcholine (PC), egg
phosphatidylethanolamine (PE), nitrobenzoxadiazole
phosphatidylethanolamine (NBD-PE), and lissamine rhodamine B
sulfonyl phosphatidylethanolamine (Rh-PE) are from a commercial
supplier. Multilamellar phospholipid vesicles (MLVs) are prepared
using the method of Bangham et al. (Preparation and use of
liposomes as models of biological membranes, in Methods in Membrane
Biology (Korn, E. D., Ed.) pp 1-68, Plenum Press, New York, 1974).
The MLVs are converted into large unilamellar vesicles (LUVs) by
five freeze-thaw cycles and subsequent extrusion (five times)
through 0.1 .mu.m Nucleopore filter membranes using an extruder
(Lipex Biomembranes, Vancouver, BC) at 30.degree. C. Phosphate
determination is performed according to the method of Chen et al.
(Anal. Chem. 28, 1756-1758, 1956).
Membrane Surface Hydrophobicity.
[0430] An increase in membrane surface hydrophobicity is the result
of dehydration of the phospholipid headgroups by either the binding
of protein to the membrane surface or the creation of water-free
interfaces between two vesicles, which occurs during aggregation.
Changing membrane surface hydrophobicity can be observed by
labeling vesicles with
N-[5-(dimethylamino)-naphthalene-2-sulfonyl)-1,2-dioleoylyl-sn-glycero-3--
PE (dansyl-PE), whose emission wavelength is proportional to the
dielectric constant of the probe environment. In this context, pure
PS, PS/PE (3:1), and PS/PC (1:1) LUVs containing 1 mol % dansyl-PE
are prepared and added to a 900 .mu.L buffer solution (final
phospholipid concentration of 45 .mu.M). The effect of annexin on
these vesicles is observed at different pH values by injecting 200
nM (0.18 nmol) protein into the calcium-free sample. The samples
are excited at 340 nm, and the fluorescence emission is recorded
from 400 to 600 nm (23). The calcium-dependent behaviour of surface
hydrophobicity is observed after monitoring the effect of protein
alone.
Liposome Leakage Assay.
[0431] Annexin-phospholipid interactions may cause the
destabilization of phospholipid vesicles which results in leakage
of the vesicle's interior. Vesicle leakage is monitored by the
fluorescence quenching of 8-aminonaphthalene-1,3,6-trisulfonic acid
(ANTS) in the presence of p-xylen-bis-pyridiniumbromid (DPX). The
watersoluble fluorophore ANTS and its quencher DPX are added to the
buffer solution while the vesicles are prepared. Excess ANTS/DPX
buffer solution is removed by gel filtration using a Sephadex G-50
column. In the undisturbed vesicles, the fluorophore and quencher
are spatially close so that DPX quenches the fluorescence of ANTS.
With an increasing level of vesicle leakage, ANTS and DPX are
diluted into the outer buffer solution, resulting in an increase in
the fluorescence of ANTS (Ellens et al., Biochemistry 24,
3099-3106, 1985).
Assay for Peroxidase Activity of Annexin 1 (Gorecka et al.
2005).
[0432] Peroxidase activity of recombinant annexin1 proteins
expressed in eukaryotic or prokaryotic systems may be tested with
two methods. The first method is based on the chemiluminescence of
oxidized luminal. Samples containing the proteins to be analyzed,
separated by non-denaturating electrophoresis, are transferred onto
a nitrocellulose membrane, covered with the developing solution
(ECL kit, Amersham) containing luminol, and exposed to X-ray
medical film for 1 hr according to the manufacturer's protocol.
Alternatively, peroxidase activity of recombinant annexin1 proteins
is determined using a fluorometric method with Amplex Red reagent
(Molecular Probes) on a Fluorolog 3 spectrofluorimeter (Jobin Yvon
Spex, Edison, N.J.) with 1-nm slits for both excitation and
emission. The assay medium (total volume of 100 .mu.l) contains 50
mM potassium phosphate buffer, pH 7.4, 2 mM H.sub.2O.sub.2, Amplex
Red reagent at a final concentration of 100 .mu.M. Measurements are
made in quartz cuvettes of optical path length of 10 mm (0.1 ml
volume). Fluorescence emission of the product of Amplex Red reagent
oxidation, resorufin, is recorded at .lamda..sub.em 590 nm
(.lamda..sub.exc 560 nm). For the determination of the effect of
protein phosphorylation on peroxidase activity AnnAt1 is prior to
measurements incubated with alkaline phosphatase (Sigma, 15 U/ml)
at 36.degree. C. for 10 min in a potassium phosphate buffer, pH
7.4. A sample without AnnAt1 is used as a control.
Example 20
Cloning of the Nucleic Acid Sequence Used in the Methods of the
Invention
[0433] The nucleic acid sequence used in the methods of the
invention was amplified by PCR using as template a custom-made
Arabidopsis thaliana seedlings cDNA library (in pCMV Sport 6.0;
Invitrogen, Paisley, UK). PCR was performed using Hifi Taq DNA
polymerase in standard conditions, using 200 ng of template in a 50
.mu.l PCR mix. The primers used were prm08727 (SEQ ID NO: 85;
sense, start codon in bold): 5'-ggggacaagtttgtacaaaaaagcaggctta
aacaatggcgactcttaaggtttct-3' and prm09025 (SEQ ID NO: 86; reverse,
complementary): 5'-ggggaccactttgtacaagaaagctgggtttaagcatcatcttcaccg
ag-3', which include the AttB sites for Gateway recombination. The
amplified PCR fragment was purified also using standard methods.
The first step of the Gateway procedure, the BP reaction, was then
performed, during which the PCR fragment recombines in vivo with
the pDONR201 plasmid to produce, according to the Gateway
terminology, an "entry clone", pANN. Plasmid pDONR201 was purchased
from Invitrogen, as part of the Gateway.RTM. technology.
[0434] The entry clone comprising SEQ ID NO: 83 was then used in an
LR reaction with a destination vector used for Oryza sativa
transformation. This vector contained as functional elements within
the T-DNA borders: a plant selectable marker; a screenable marker
expression cassette; and a Gateway cassette intended for LR in vivo
recombination with the nucleic acid sequence of interest already
cloned in the entry clone. A rice expansin promoter (SEQ ID NO: 95)
for green-tissue specific expression was located upstream of this
Gateway cassette.
[0435] In alternative embodiment, a destination vector comprising
the GOS2 promoter (SEQ ID NO: 94) was used resulting in the
expression vector pGOS2::ANN.
[0436] After the LR recombination step, the resulting expression
vector pEXP::ANN (FIG. 10) or pGOS2::ANN was transformed into
Agrobacterium strain LBA4044 according to methods well known in the
art.
Example 21
Plant Transformation
[0437] Transformation of rice plants was carried out according to
the procedure outlined in Example 8 herein.
Example 22
Phenotypic Evaluation Procedure
22.1 Evaluation Setup
[0438] Approximately 35 independent T0 rice transformants were
generated. The primary transformants were transferred from a tissue
culture chamber to a greenhouse for growing and harvest of T1 seed.
Six events, of which the T1 progeny segregated 3:1 for
presence/absence of the transgene, were retained. For each of these
events, approximately 10 T1 seedlings containing the transgene
(hetero- and homo-zygotes) and approximately 10 T1 seedlings
lacking the transgene (nullizygotes) were selected by monitoring
visual marker expression. The transgenic plants and the
corresponding nullizygotes were grown side-by-side at random
positions. Greenhouse conditions were of shorts days (12 hours
light), 28.degree. C. in the light and 22.degree. C. in the dark,
and a relative humidity of 70%.
[0439] Four T1 events were further evaluated in the T2 generation
following the same evaluation procedure as for the T1 generation
but with more individuals per event. From the stage of sowing until
the stage of maturity the plants were passed several times through
a digital imaging cabinet. At each time point digital images
(2048.times.1536 pixels, 16 million colours) were taken of each
plant from at least 6 different angles.
Drought Screen
[0440] Plants from T2 seeds were grown in potting soil under normal
conditions until they approached the heading stage. They were then
transferred to a "dry" section where irrigation was withheld.
Humidity probes were inserted in randomly chosen pots to monitor
the soil water content (SWC). When SWC went below certain
thresholds, the plants were automatically re-watered continuously
until a normal level was reached again. The plants were then
re-transferred again to normal conditions. The rest of the
cultivation (plant maturation, seed harvest) was the same as for
plants not grown under abiotic stress conditions. Growth and yield
parameters are recorded as detailed for growth under normal
conditions.
Nitrogen Use Efficiency Screen
[0441] Rice plants from T2 seeds are grown in potting soil under
normal conditions except for the nutrient solution. The pots are
watered from transplantation to maturation with a specific nutrient
solution containing reduced N nitrogen (N) content, usually between
7 to 8 times less. The rest of the cultivation (plant maturation,
seed harvest) is the same as for plants not grown under abiotic
stress. Growth and yield parameters are recorded as detailed for
growth under normal conditions.
22.2 Statistical Analysis: F Test
[0442] A two factor ANOVA (analysis of variants) was used as a
statistical model for the overall evaluation of plant phenotypic
characteristics. An F test was carried out on all the parameters
measured of all the plants of all the events transformed with the
gene of the present invention. The F test was carried out to check
for an effect of the gene over all the transformation events and to
verify for an overall effect of the gene, also known as a global
gene effect. The threshold for significance for a true global gene
effect was set at a 5% probability level for the F test. A
significant F test value points to a gene effect, meaning that it
is not only the mere presence or position of the gene that is
causing the differences in phenotype.
[0443] Because two experiments with overlapping events were carried
out, a combined analysis was performed. This is useful to check
consistency of the effects over the two experiments, and if this is
the case, to accumulate evidence from both experiments in order to
increase confidence in the conclusion. The method used was a
mixed-model approach that takes into account the multilevel
structure of the data (i.e. experiment-event-segregants). P values
were obtained by comparing likelihood ratio test to chi square
distributions.
22.3 Parameters Measured
Biomass-Related Parameter Measurement
[0444] From the stage of sowing until the stage of maturity the
plants were passed several times through a digital imaging cabinet.
At each time point digital images (2048.times.1536 pixels, 16
million colours) were taken of each plant from at least 6 different
angles.
[0445] The plant aboveground area (or leafy biomass) was determined
by counting the total number of pixels on the digital images from
aboveground plant parts discriminated from the background. This
value was averaged for the pictures taken on the same time point
from the different angles and was converted to a physical surface
value expressed in square mm by calibration. Experiments show that
the aboveground plant area measured this way correlates with the
biomass of plant parts above ground. The above ground area is the
area measured at the time point at which the plant had reached its
maximal leafy biomass.
Seed-Related Parameter Measurements
[0446] The mature primary panicles were harvested, counted, bagged,
barcode-labelled and then dried for three days in an oven at
37.degree. C. The panicles were then threshed and all the seeds
were collected and counted. The filled husks were separated from
the empty ones using an air-blowing device. The empty husks were
discarded and the remaining fraction was counted again. The filled
husks were weighed on an analytical balance. The number of filled
seeds was determined by counting the number of filled husks that
remained after the separation step. The total seed yield was
measured by weighing all filled husks harvested from a plant. Total
seed number per plant was measured by counting the number of husks
harvested from a plant. Thousand Kernel Weight (TKW) is
extrapolated from the number of filled seeds counted and their
total weight. The Harvest Index (HI) in the present invention is
defined as the ratio between the total seed yield and the above
ground area (mm.sup.2), multiplied by a factor 10.sup.6. The total
number of flowers per panicle as defined in the present invention
is the ratio between the total number of seeds and the number of
mature primary panicles. The seed fill rate as defined in the
present invention is the proportion (expressed as a %) of the
number of filled seeds over the total number of seeds (or
florets).
Example 23
Results of the Phenotypic Evaluation of the Transgenic Plants
[0447] Evaluation of transgenic rice plants expressing an ANN
nucleic acid operably linked to a constitutive promoter, grown
under non-stress conditions revealed an increase of more than 5%
was observed for total seed yield, number of filled seeds, fill
rate, harvest index and more than 3% for TKW. Under drought stress
conditions, an increase was observed for total seed yield, number
of filled seeds and fill rate.
[0448] Plants expressing an ANN nucleic acid operably linked to a
green-tissue specific promoter also exhibited an increased yield,
in particular increased TKW.
Example 24
Examples of Transformation of Other Crops
[0449] Transformation of other crops is described in Example 12
hereinabove.
Sequence CWU 1
1
1881848DNABrassica rapa 1ttgtacaaaa aagcaggctt aaacaatggc
gtctgttgct tcttcaacca ctctcatctc 60ctcctccgct agtgttctcc cagccaccaa
gtcttcgctt cttccatctc cctccctctc 120tttccttcca accctctcct
ctccttcccc atccgcttct ctccggctcc ctcgtccctc 180tcccctcacc
tcaatccgct cctcttctcg ccggagcttc gctgtcaagg cccaaaccga
240tgatcttcca ttggttggaa acaaggcgcc tgattttgag gcagaggctg
tgttcgatca 300ggagttcatc aaggtcaagc tctctgagta cattgggaag
aagtatgtga ttctcttttt 360ctaccccttg gacttcactt tcgtctgccc
aacagagatt actgccttca gtgaccgata 420tgcagaattt gagaagctga
acacagaagt gttaggtgtc tctgttgata gtgtgttctc 480ccaccttgct
tgggttcaaa ccgacagaaa atctggagga cttggtgatc tcaactatcc
540acttatatca gatgtcacta aatctatctc aaaatctttt ggagtgctca
tccatgatca 600gggaatagcg ttgagagggc ttttcataat agacaaggaa
ggagtgatcc aacattcaac 660catcaacaat cttggtattg gccgaagtgt
tgatgagaca atgagaaccc ttcaggcatt 720acagtacatc caggagaacc
ctgatgaagt ctgccctgca ggatggaaac caggggagaa 780gtcaatgaaa
cctgacccca agctcagcaa agagtacttc tcagctattt agctcgaacc 840cagctttc
8482268PRTBrassica rapa 2Met Ala Ser Val Ala Ser Ser Thr Thr Leu
Ile Ser Ser Ser Ala Ser 1 5 10 15 Val Leu Pro Ala Thr Lys Ser Ser
Leu Leu Pro Ser Pro Ser Leu Ser 20 25 30 Phe Leu Pro Thr Leu Ser
Ser Pro Ser Pro Ser Ala Ser Leu Arg Leu 35 40 45 Pro Arg Pro Ser
Pro Leu Thr Ser Ile Arg Ser Ser Ser Arg Arg Ser 50 55 60 Phe Ala
Val Lys Ala Gln Thr Asp Asp Leu Pro Leu Val Gly Asn Lys 65 70 75 80
Ala Pro Asp Phe Glu Ala Glu Ala Val Phe Asp Gln Glu Phe Ile Lys 85
90 95 Val Lys Leu Ser Glu Tyr Ile Gly Lys Lys Tyr Val Ile Leu Phe
Phe 100 105 110 Tyr Pro Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile
Thr Ala Phe 115 120 125 Ser Asp Arg Tyr Ala Glu Phe Glu Lys Leu Asn
Thr Glu Val Leu Gly 130 135 140 Val Ser Val Asp Ser Val Phe Ser His
Leu Ala Trp Val Gln Thr Asp 145 150 155 160 Arg Lys Ser Gly Gly Leu
Gly Asp Leu Asn Tyr Pro Leu Ile Ser Asp 165 170 175 Val Thr Lys Ser
Ile Ser Lys Ser Phe Gly Val Leu Ile His Asp Gln 180 185 190 Gly Ile
Ala Leu Arg Gly Leu Phe Ile Ile Asp Lys Glu Gly Val Ile 195 200 205
Gln His Ser Thr Ile Asn Asn Leu Gly Ile Gly Arg Ser Val Asp Glu 210
215 220 Thr Met Arg Thr Leu Gln Ala Leu Gln Tyr Ile Gln Glu Asn Pro
Asp 225 230 235 240 Glu Val Cys Pro Ala Gly Trp Lys Pro Gly Glu Lys
Ser Met Lys Pro 245 250 255 Asp Pro Lys Leu Ser Lys Glu Tyr Phe Ser
Ala Ile 260 265 3 841DNABrassica rapamisc_feature(662)..(662)n is
a, c, g, or t 3gccatatggc gtctgttgct tcttcaacca ctctcatctc
ctcctccgct agtgttctcc 60cagccaccaa gtcttcgctt cttccatctc cctccctctc
tttccttcca accctctcct 120ctccttcccc atccgcttct ctccggtccc
tcgtccctct cccctcacct caatccgctt 180cctcttctcg ccggagcttc
gctgtcaagg gccaaaccga tgatcttcca ttggttggaa 240acaaggcgcc
tgattttgag gcagagggtg tgttcgatca ggagttcatc aagttcatca
300aggtcaagct ctctgattac attgggaaaa agtatgtgat tctctttttt
ctaccccttg 360acttcacttt cgtctgccca acagaaatta ctgccttcag
tgaccgatat gcagaatttg 420agaagctgaa tacagaagtg ttaggtgttt
ctgttgatag tgtgagtgtg ttctcccacc 480ttgctggggt tcaaaccgac
agaaaatttg gaggacttgg tgatctcaac tatccactta 540tatcagatgt
cactaaatct atctcaaaat cttttggagt gctcatccat gatcagggaa
600tagcgttgag agggcttttc ataatagaca aggaaggagt gatccaacat
tcaaccatca 660anaatcttgg tattggccga agtgttgatg agacaatgag
aacccttcag gcattacagt 720acatccagga aggccctggt gaagtctgcc
ctgcaggatg gaaaccaggg gagaagtcaa 780tgaaacctga ccccaagctc
agcaaagagc tcttctcagc tatttagctc gaggctaagc 840c 8414273PRTBrassica
rapaUNSURE(219)..(219)Xaa can be any naturally occurring amino acid
4Met Ala Ser Val Ala Ser Ser Thr Thr Leu Ile Ser Ser Ser Ala Ser 1
5 10 15 Val Leu Pro Ala Thr Lys Ser Ser Leu Leu Pro Ser Pro Ser Leu
Ser 20 25 30 Phe Leu Pro Thr Leu Ser Ser Pro Ser Pro Ser Ala Ser
Leu Arg Ser 35 40 45 Leu Val Pro Leu Pro Ser Pro Gln Ser Ala Ser
Ser Ser Arg Arg Ser 50 55 60 Phe Ala Val Lys Gly Gln Thr Asp Asp
Leu Pro Leu Val Gly Asn Lys 65 70 75 80 Ala Pro Asp Phe Glu Ala Glu
Gly Val Phe Asp Gln Glu Phe Ile Lys 85 90 95 Phe Ile Lys Val Lys
Leu Ser Asp Tyr Ile Gly Lys Lys Tyr Val Ile 100 105 110 Leu Phe Phe
Leu Pro Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile 115 120 125 Thr
Ala Phe Ser Asp Arg Tyr Ala Glu Phe Glu Lys Leu Asn Thr Glu 130 135
140 Val Leu Gly Val Ser Val Asp Ser Val Ser Val Phe Ser His Leu Ala
145 150 155 160 Gly Val Gln Thr Asp Arg Lys Phe Gly Gly Leu Gly Asp
Leu Asn Tyr 165 170 175 Pro Leu Ile Ser Asp Val Thr Lys Ser Ile Ser
Lys Ser Phe Gly Val 180 185 190 Leu Ile His Asp Gln Gly Ile Ala Leu
Arg Gly Leu Phe Ile Ile Asp 195 200 205 Lys Glu Gly Val Ile Gln His
Ser Thr Ile Xaa Asn Leu Gly Ile Gly 210 215 220 Arg Ser Val Asp Glu
Thr Met Arg Thr Leu Gln Ala Leu Gln Tyr Ile 225 230 235 240 Gln Glu
Gly Pro Gly Glu Val Cys Pro Ala Gly Trp Lys Pro Gly Glu 245 250 255
Lys Ser Met Lys Pro Asp Pro Lys Leu Ser Lys Glu Leu Phe Ser Ala 260
265 270 Ile 51002DNAArabidopsis thaliana 5gtgattgaca gatagataag
agtgtttggt agctcagact cagagagtca caaagtgtgt 60gtagcagcaa tggcgtctgt
tgcttcttca actactctca tctcttctcc ctcttctagg 120gtttttccag
caaagtcttc actttcctct ccatctgttt ctttccttcg aaccctttct
180tctccttccg catctgcttc tctccgctcc ggatttgctc gacgctcttc
cctcagctcc 240acttctcgtc ggagctttgc tgtcaaagcc caggccgatg
atcttccact ggttggaaac 300aaggcgcctg attttgaggc agaggctgtg
tttgatcaag agttcatcaa ggttaagctc 360tctgattaca ttggaaagaa
gtatgtgatt ctctttttct acccattgga ctttactttc 420gtctgcccaa
cagagattac tgccttcagt gaccggcatt cagaatttga gaagttgaac
480accgaagtat taggtgtttc tgtcgatagt gtgttctctc accttgcatg
ggtccaaaca 540gacaggaaat ctggagggct tggtgatctg aactatcccc
ttatttcata tttcactaaa 600tcaatctcaa agtcgttcgg agtgctcatc
catgatcagg gaatagcact gagaggactt 660ttcataatcg acaaggaagg
agtgatccaa cattccacca tcaacaatct tggtattggc 720caaagcgttg
atgagacaat gagaaccctc caggcattac agtacatcca ggaaaacccg
780gatgaagtct gcccagcagg atggaagccg ggtgagaagt caatgaaacc
cgacccaaaa 840ctcagcaaag agtacttctc agctatttag aaactctact
atgatagcaa aaaggtacaa 900tctttgttat atgtgagcag agtttttttt
cttgtacgct aaaacaatcc tttgtttgat 960tctcactttg tccccaaaat
tataataaaa aactttttcc gc 10026266PRTArabidopsis thaliana 6Met Ala
Ser Val Ala Ser Ser Thr Thr Leu Ile Ser Ser Pro Ser Ser 1 5 10 15
Arg Val Phe Pro Ala Lys Ser Ser Leu Ser Ser Pro Ser Val Ser Phe 20
25 30 Leu Arg Thr Leu Ser Ser Pro Ser Ala Ser Ala Ser Leu Arg Ser
Gly 35 40 45 Phe Ala Arg Arg Ser Ser Leu Ser Ser Thr Ser Arg Arg
Ser Phe Ala 50 55 60 Val Lys Ala Gln Ala Asp Asp Leu Pro Leu Val
Gly Asn Lys Ala Pro 65 70 75 80 Asp Phe Glu Ala Glu Ala Val Phe Asp
Gln Glu Phe Ile Lys Val Lys 85 90 95 Leu Ser Asp Tyr Ile Gly Lys
Lys Tyr Val Ile Leu Phe Phe Tyr Pro 100 105 110 Leu Asp Phe Thr Phe
Val Cys Pro Thr Glu Ile Thr Ala Phe Ser Asp 115 120 125 Arg His Ser
Glu Phe Glu Lys Leu Asn Thr Glu Val Leu Gly Val Ser 130 135 140 Val
Asp Ser Val Phe Ser His Leu Ala Trp Val Gln Thr Asp Arg Lys 145 150
155 160 Ser Gly Gly Leu Gly Asp Leu Asn Tyr Pro Leu Ile Ser Tyr Phe
Thr 165 170 175 Lys Ser Ile Ser Lys Ser Phe Gly Val Leu Ile His Asp
Gln Gly Ile 180 185 190 Ala Leu Arg Gly Leu Phe Ile Ile Asp Lys Glu
Gly Val Ile Gln His 195 200 205 Ser Thr Ile Asn Asn Leu Gly Ile Gly
Gln Ser Val Asp Glu Thr Met 210 215 220 Arg Thr Leu Gln Ala Leu Gln
Tyr Ile Gln Glu Asn Pro Asp Glu Val 225 230 235 240 Cys Pro Ala Gly
Trp Lys Pro Gly Glu Lys Ser Met Lys Pro Asp Pro 245 250 255 Lys Leu
Ser Lys Glu Tyr Phe Ser Ala Ile 260 265 7988DNABrassica napus
7gtcgaccacg cgtccggaga gaaagagaga gagagcagag ttcttcagtc catggcttcc
60ttagcttcaa ccaccacact tatctcttca tctagcgttc ttcttccctc aaagccttct
120cctttttctc ccgccgcctc cttcctccga actcttcctt ctacctccgt
atctacctcc 180tcttctctcc gctcctgttt ctccagcatc agtcccctca
cctgcatccg ctcttcctct 240cgccctagct tcgccgtcaa ggcccaggct
gatgatttgc cactggttgg taacaaggcg 300cctgattttg aggcagaggc
tgtttttgac caagagttca tcaaggtgaa gctctcagag 360tacattggta
aaaagtatgt gattctgttt ctctaccctt tggacttcac ttttgtctgc
420cctacggaga ttactgcctt cagtgaccgt tatgaagaat ttgagaagct
aaacacggaa 480gtgttaggtg tctcagtcga cagtgtgttc tcgcatcttg
cgtgggtcca aacagagaga 540aagtcaggag ggctgggtga cctgaactac
ccacttgtct ctgatatcac taaatccatt 600tcaaaatctt ttggagtgct
catccctgat cagggcattg cactgagagg gcttttcatc 660atcgacaaaa
aaggagtcat acagcattcc acaatcaaca acctcggtat tggccgaagc
720gttgatgaga caatgagaac cctccaggca ttgcagtatg ttcaagaaaa
ccctgatgag 780gtttgccccg cgggatggaa gcctggggag aaatcgatga
agcctgaccc caagctgagc 840aaagagtatt tttcagctat ttaaaggctt
tttaaacaaa tgattggtga aaagcagagg 900catgttttgt cttcattgct
tatgtttctg ctatgtgtgt tttcctcaaa attgaataaa 960aataatggaa
tttaaaaaaa aaaaaaaa 9888270PRTBrassica napus 8Met Ala Ser Leu Ala
Ser Thr Thr Thr Leu Ile Ser Ser Ser Ser Val 1 5 10 15 Leu Leu Pro
Ser Lys Pro Ser Pro Phe Ser Pro Ala Ala Ser Phe Leu 20 25 30 Arg
Thr Leu Pro Ser Thr Ser Val Ser Thr Ser Ser Ser Leu Arg Ser 35 40
45 Cys Phe Ser Ser Ile Ser Pro Leu Thr Cys Ile Arg Ser Ser Ser Arg
50 55 60 Pro Ser Phe Ala Val Lys Ala Gln Ala Asp Asp Leu Pro Leu
Val Gly 65 70 75 80 Asn Lys Ala Pro Asp Phe Glu Ala Glu Ala Val Phe
Asp Gln Glu Phe 85 90 95 Ile Lys Val Lys Leu Ser Glu Tyr Ile Gly
Lys Lys Tyr Val Ile Leu 100 105 110 Phe Leu Tyr Pro Leu Asp Phe Thr
Phe Val Cys Pro Thr Glu Ile Thr 115 120 125 Ala Phe Ser Asp Arg Tyr
Glu Glu Phe Glu Lys Leu Asn Thr Glu Val 130 135 140 Leu Gly Val Ser
Val Asp Ser Val Phe Ser His Leu Ala Trp Val Gln 145 150 155 160 Thr
Glu Arg Lys Ser Gly Gly Leu Gly Asp Leu Asn Tyr Pro Leu Val 165 170
175 Ser Asp Ile Thr Lys Ser Ile Ser Lys Ser Phe Gly Val Leu Ile Pro
180 185 190 Asp Gln Gly Ile Ala Leu Arg Gly Leu Phe Ile Ile Asp Lys
Lys Gly 195 200 205 Val Ile Gln His Ser Thr Ile Asn Asn Leu Gly Ile
Gly Arg Ser Val 210 215 220 Asp Glu Thr Met Arg Thr Leu Gln Ala Leu
Gln Tyr Val Gln Glu Asn 225 230 235 240 Pro Asp Glu Val Cys Pro Ala
Gly Trp Lys Pro Gly Glu Lys Ser Met 245 250 255 Lys Pro Asp Pro Lys
Leu Ser Lys Glu Tyr Phe Ser Ala Ile 260 265 270 91146DNAArabidopsis
thaliana 9agctatttgg tttctctatc cgattcgtct ctctcacgcc ctcacgttta
tccacctcat 60cctcaaacca aaccacaaga cctctttttt aagtaaccaa tcacagagag
atagagagag 120agaacagagt caatgtcaat ggcgtctata gcttcttctt
cttccaccac cctactctct 180tcctctaggg ttcttcttcc ctccaagtct
tctcttttat ctcctaccgt ctctttcccc 240agaatcatac cctcttcctc
ggcatcatcc tcttctctct gttccgggtt ctccagtctc 300ggttccctca
ccaccaaccg ctccgcctca cgccggaact tcgccgtcaa ggctcaggct
360gatgatttac cactggtcgg taataaggcg cctgattttg aagctgaggc
agtttttgat 420caagagttca taaaggtgaa gctctctgag tacattggca
aaaagtatgt tattctattc 480ttctaccctt tggacttcac ttttgtctgc
cccactgaga ttactgcctt cagtgaccgt 540tatgaagaat ttgagaagct
aaacaccgaa gtattagggg tctctgtcga cagtgtgttc 600tcgcatcttg
cgtgggtcca aacagacaga aagtcgggag ggctcggtga tctgaattat
660cctcttgttt cggatatcac taaatccatt tcaaaatcgt ttggagtgct
catccctgat 720cagggcattg cactgagagg gcttttcatc atagacaagg
aaggagtcat tcagcattcc 780accatcaaca acctcggtat tggccgaagt
gttgatgaga caatgagaac cctccaggca 840ttacagtatg ttcaagaaaa
cccggatgaa gtgtgccctg cgggatggaa gccaggggag 900aaatcaatga
aacctgaccc caagctcagc aaagaatact tttcagctat ctagaggcta
960agattgaaca catgtttggt gaaaattagc aatcagagtt gttttattcg
tcttttcaaa 1020gttggagcag agttgttatt tttagccaaa gaacctttgt
atctatctca tctttctcct 1080gtttctgcta tgtgattctc cttaaattga
atcaaaaata aagaaatcct tcttttcttt 1140tgccaa 114610271PRTArabidopsis
thaliana 10Met Ala Ser Ile Ala Ser Ser Ser Ser Thr Thr Leu Leu Ser
Ser Ser 1 5 10 15 Arg Val Leu Leu Pro Ser Lys Ser Ser Leu Leu Ser
Pro Thr Val Ser 20 25 30 Val Pro Arg Thr Leu His Ser Ser Ser Ala
Ser Ser Ser Ser Leu Cys 35 40 45 Ser Gly Phe Ser Ser Leu Gly Ser
Leu Thr Thr Ser Arg Ser Ala Ser 50 55 60 Arg Arg Asn Phe Ala Val
Lys Ala Gln Ala Asp Asp Leu Pro Leu Val 65 70 75 80 Gly Asn Lys Ala
Pro Asp Phe Glu Ala Glu Ala Val Phe Asp Gln Glu 85 90 95 Phe Ile
Lys Val Lys Leu Ser Glu Tyr Ile Gly Lys Lys Tyr Val Ile 100 105 110
Leu Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile 115
120 125 Thr Ala Phe Ser Asp Arg Tyr Glu Glu Phe Glu Lys Leu Asn Thr
Glu 130 135 140 Val Leu Gly Val Ser Val Asp Ser Val Phe Ser His Leu
Ala Trp Val 145 150 155 160 Gln Thr Asp Arg Lys Ser Gly Gly Leu Gly
Asp Leu Asn Tyr Pro Leu 165 170 175 Val Ser Asp Ile Thr Lys Ser Ile
Ser Lys Ser Phe Gly Val Leu Ile 180 185 190 Pro Asp Gln Gly Ile Ala
Leu Arg Gly Leu Phe Ile Ile Asp Lys Glu 195 200 205 Gly Val Ile Gln
His Ser Thr Ile Asn Asn Leu Gly Ile Gly Arg Ser 210 215 220 Val Asp
Glu Thr Met Arg Thr Leu Gln Ala Leu Gln Tyr Val Gln Glu 225 230 235
240 Asn Pro Asp Glu Val Cys Pro Ala Gly Trp Lys Pro Gly Glu Lys Ser
245 250 255 Met Lys Pro Asp Pro Lys Leu Ser Lys Glu Tyr Phe Ser Ala
Ile 260 265 270 11867DNASpinacia oleracea 11gtgtgtagca gcaatggcgt
gtgttgcttc ttcaactact ctcatctctt ctccctcttc 60tagggttttt ccagcaaagt
cttcactttc ctctccatct gtttctttcc ttcgaaccct 120ttcttctcct
tccgcatctg cttctctccg ctccggattt gctcgacgct cttccctcag
180ctccacttct cgtcggagct ttgctgtcaa agcccaggcc gatgatcttc
cactggttgg 240aaacaaggcg cctgattttg aggcagaggc tgtgtttgat
caagagttca tcaaggttaa 300gctctctgat tacattggaa agaagtatgt
gattctgttt ttctacccat tggactttac 360tttcgtctgc ccaacagaga
ttactgcctt cagtgaccgg cattcagaat ttgagaagtt 420gaacaccgaa
gtattaggtg tttctgtcga tagtgtgttc tctcaccttg catgggtcca
480aacagacagg aaatctggag ggcttggtga tctgaactat ccccttattt
cagatgtcac 540taaatcaatc tcaaagtcgt tcggagtgct catccatgat
cagggaatag cactgagagg 600acttttcata atcgacaagg aaggagtgat
ccaacattcc accatcaaca atcttggtat 660tggccgaagc gttgatgaga
caatgagaac cctccaggca ttacagtaca caggaaaccc 720ggatgaagtc
tgcccagcag gatggaagcc gggtgagaag tcaatgaaac ccgacccaaa
780actcagcaag gagtacttct cagctattta gaactctact atgatagcaa
aggtacatct 840ttgttatatg tgagcagagt ttttctg
86712265PRTSpinacia oleracea 12Met Ala Cys Val Ala Ser Ser Thr Thr
Leu Ile Ser Ser Pro Ser Ser 1 5 10 15 Arg Val Phe Pro Ala Lys Ser
Ser Leu Ser Ser Pro Ser Val Ser Phe 20 25 30 Leu Arg Thr Leu Ser
Ser Pro Ser Ala Ser Ala Ser Leu Arg Ser Gly 35 40 45 Phe Ala Arg
Arg Ser Ser Leu Ser Ser Thr Ser Arg Arg Ser Phe Ala 50 55 60 Val
Lys Ala Gln Ala Asp Asp Leu Pro Leu Val Gly Asn Lys Ala Pro 65 70
75 80 Asp Phe Glu Ala Glu Ala Val Phe Asp Gln Glu Phe Ile Lys Val
Lys 85 90 95 Leu Ser Asp Tyr Ile Gly Lys Lys Tyr Val Ile Leu Phe
Phe Tyr Pro 100 105 110 Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile
Thr Ala Phe Ser Asp 115 120 125 Arg His Ser Glu Phe Glu Lys Leu Asn
Thr Glu Val Leu Gly Val Ser 130 135 140 Val Asp Ser Val Phe Ser His
Leu Ala Trp Val Gln Thr Asp Arg Lys 145 150 155 160 Ser Gly Gly Leu
Gly Asp Leu Asn Tyr Pro Leu Ile Ser Asp Val Thr 165 170 175 Lys Ser
Ile Ser Lys Ser Phe Gly Val Leu Ile His Asp Gln Gly Ile 180 185 190
Ala Leu Arg Gly Leu Phe Ile Ile Asp Lys Glu Gly Val Ile Gln His 195
200 205 Ser Thr Ile Asn Asn Leu Gly Ile Gly Arg Ser Val Asp Glu Thr
Met 210 215 220 Arg Thr Leu Gln Ala Leu Gln Tyr Thr Gly Asn Pro Asp
Glu Val Cys 225 230 235 240 Pro Ala Gly Trp Lys Pro Gly Glu Lys Ser
Met Lys Pro Asp Pro Lys 245 250 255 Leu Ser Lys Glu Tyr Phe Ser Ala
Ile 260 265 13990DNANicotiana tabacum 13ggcacgagct cctatccaat
ggcttgctct gcttcttcta cagcacttct ttcttccaac 60ccaaaagcag cttccatttc
ccccaaatcc tcctttcaag ctcccatttc tcaatgttta 120tctgtacctt
cctctttcaa tgggctccgt aattgcaagc cttttgtttc tcgtgtagcc
180cgttccctct ctactcgcgt tgctcaatcc caacgccgtc gtttcgttgt
tcgtgcctct 240agtgaacttc cacttgttgg aaatcaagcg ccagactttg
aggctgaagc tgtttttgat 300caagaattca tcaaggttaa actatctgag
tacattggga agaagtatgt cattctcttt 360ttctacccac tagactttac
atttgtttgc ccaacagaga tcactgcttt cagtgaccgt 420tatggagaat
ttgaaaagtt gaacacagaa atattgggtg tttccgtaga cagtgtgttc
480tcccaccttg cctgggttca aactgataga aagtctggtg gcctaggtga
tctgaactat 540ccattaattt ccgacgtgac caagtcaatt tcaaaatcat
acaatgttct gatccccgat 600cagggaattg cattgagagg acttttcatc
attgacaagg aaggagttat tcagcattca 660accattaaca atcttggaat
tggtcgtagt gttgatgaaa cattgagaac tcttcaggca 720ttgcaatacg
ttcaggataa cccggatgaa gtgtgcccag ctggatggaa gcctggggag
780aaatccatga agcctgaccc caagggtagc aaagaatact ttgcatccat
atgaggtgat 840gactgcaatt gctttatcta atttgttgtt taggaaggct
ggagacccta cttttctgtt 900acatttttct aatgtaccgg ctgagtttgg
tcatttttga gaatatatac acttgtacac 960ttttaaaaaa aaaaaaaaaa
aaaaaaaaaa 99014271PRTNicotiana tabacum 14Met Ala Cys Ser Ala Ser
Ser Thr Ala Leu Leu Ser Ser Asn Pro Lys 1 5 10 15 Ala Ala Ser Ile
Ser Pro Lys Ser Ser Phe Gln Ala Pro Ile Ser Gln 20 25 30 Cys Leu
Ser Val Pro Ser Ser Phe Asn Gly Leu Arg Asn Cys Lys Pro 35 40 45
Phe Val Ser Arg Val Ala Arg Ser Leu Ser Thr Arg Val Ala Gln Ser 50
55 60 Gln Arg Arg Arg Phe Val Val Arg Ala Ser Ser Glu Leu Pro Leu
Val 65 70 75 80 Gly Asn Gln Ala Pro Asp Phe Glu Ala Glu Ala Val Phe
Asp Gln Glu 85 90 95 Phe Ile Lys Val Lys Leu Ser Glu Tyr Ile Gly
Lys Lys Tyr Val Ile 100 105 110 Leu Phe Phe Tyr Pro Leu Asp Phe Thr
Phe Val Cys Pro Thr Glu Ile 115 120 125 Thr Ala Phe Ser Asp Arg Tyr
Gly Glu Phe Glu Lys Leu Asn Thr Glu 130 135 140 Ile Leu Gly Val Ser
Val Asp Ser Val Phe Ser His Leu Ala Trp Val 145 150 155 160 Gln Thr
Asp Arg Lys Ser Gly Gly Leu Gly Asp Leu Asn Tyr Pro Leu 165 170 175
Ile Ser Asp Val Thr Lys Ser Ile Ser Lys Ser Tyr Asn Val Leu Ile 180
185 190 Pro Asp Gln Gly Ile Ala Leu Arg Gly Leu Phe Ile Ile Asp Lys
Glu 195 200 205 Gly Val Ile Gln His Ser Thr Ile Asn Asn Leu Gly Ile
Gly Arg Ser 210 215 220 Val Asp Glu Thr Leu Arg Thr Leu Gln Ala Leu
Gln Tyr Val Gln Asp 225 230 235 240 Asn Pro Asp Glu Val Cys Pro Ala
Gly Trp Lys Pro Gly Glu Lys Ser 245 250 255 Met Lys Pro Asp Pro Lys
Gly Ser Lys Glu Tyr Phe Ala Ser Ile 260 265 270 15944DNAPhaseolus
vulgaris 15tctattctat ctacactcac tctctcactc tcccactctc ccatggcttc
ctcagctccc 60tgtgcttctc tcatatcctc aaaccctaac attctcttct ctcccaaatt
cccttcttct 120tccttttctt ccctctcctt ccccaattcc cccaactctc
ttttcaaacc tttacgcact 180tctctcaatc cttcatctcc ccctctcaga
accttcgttg ccagggcttc gagtgaactt 240ccattagttg ggaacacagc
accggatttt gaagcagagg ccgtttttga tcaggagttc 300atcaaggtca
aactatctga ttatattggg aaaaaatatg ttatcctctt tttctatcca
360ctggacttca cattcgtttg tccgacagaa atcactgcct tcagtgaccg
gtatgcagag 420tttgaggcac taaatacaga aattttgggt gtttcagttg
acagtgtttt ttcacacctt 480gcatgggttc aaactgatag aaagtcgggt
ggtcttggcg acttgaatta tccattgatt 540tctgatgtca ccaaatccat
ctcaaaatct tatgatgttc tcattcccga tcaggggatt 600gcattgagag
gattgttcat tattgacaag gaaggggtta ttcagcattc taccattaac
660aacctggcca ttggtagaag tgttgatgag acaaagagaa cgctccaggc
cttgcagtat 720gtgcaggaga acccagatga agtttgccca gctgggtgga
agcctggtga gaagtccatg 780aaaccagacc ctaaacttag caaagagtac
ttctctgcta tttagggagg ataatggttg 840aagagtagca attgctcata
tgtatcaatc aatgataatt tgtataatgc aacgcaagtt 900tataaagttt
tgattgagag ggtctcatga ttatacaaaa aaaa 94416260PRTPhaseolus vulgaris
16Met Ala Ser Ser Ala Pro Cys Ala Ser Leu Ile Ser Ser Asn Pro Asn 1
5 10 15 Ile Leu Phe Ser Pro Lys Phe Pro Ser Ser Ser Phe Ser Ser Leu
Ser 20 25 30 Phe Pro Asn Ser Pro Asn Ser Leu Phe Lys Pro Leu Arg
Thr Ser Leu 35 40 45 Asn Pro Ser Ser Pro Pro Leu Arg Thr Phe Val
Ala Arg Ala Ser Ser 50 55 60 Glu Leu Pro Leu Val Gly Asn Thr Ala
Pro Asp Phe Glu Ala Glu Ala 65 70 75 80 Val Phe Asp Gln Glu Phe Ile
Lys Val Lys Leu Ser Asp Tyr Ile Gly 85 90 95 Lys Lys Tyr Val Ile
Leu Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val 100 105 110 Cys Pro Thr
Glu Ile Thr Ala Phe Ser Asp Arg Tyr Ala Glu Phe Glu 115 120 125 Ala
Leu Asn Thr Glu Ile Leu Gly Val Ser Val Asp Ser Val Phe Ser 130 135
140 His Leu Ala Trp Val Gln Thr Asp Arg Lys Ser Gly Gly Leu Gly Asp
145 150 155 160 Leu Asn Tyr Pro Leu Ile Ser Asp Val Thr Lys Ser Ile
Ser Lys Ser 165 170 175 Tyr Asp Val Leu Ile Pro Asp Gln Gly Ile Ala
Leu Arg Gly Leu Phe 180 185 190 Ile Ile Asp Lys Glu Gly Val Ile Gln
His Ser Thr Ile Asn Asn Leu 195 200 205 Ala Ile Gly Arg Ser Val Asp
Glu Thr Lys Arg Thr Leu Gln Ala Leu 210 215 220 Gln Tyr Val Gln Glu
Asn Pro Asp Glu Val Cys Pro Ala Gly Trp Lys 225 230 235 240 Pro Gly
Glu Lys Ser Met Lys Pro Asp Pro Lys Leu Ser Lys Glu Tyr 245 250 255
Phe Ser Ala Ile 260 171044DNAPisum sativummisc_feature(968)..(968)n
is a, c, g, or t 17atggcttgct cagctccatt tgcttctctc ctatattcaa
accctaacac actcttctct 60cccaaattct cttctccgcg cctctcttct ctctcaatcc
ccaatgcacc caattctctc 120cccaaactac gcacttccct ccctctttcc
ctcaaccgct cctcttcctc tcgccgcact 180ttcgtcgtta gggcttctgg
tgaattacca ttagttggga actcagcgcc ggattttgaa 240gctgaagctg
ttttcgatca ggagtttatc aaggtcaaac tatctgaata tattgggaag
300aaatatgtta tcctcttttt ctacccattg gacttcacgt tcgtttgccc
aacagaaatc 360actgctttca gtgaccggca tgcagagttt gatgcaataa
atactgagat tttgggtgtt 420tcagttgaca gtgtgttctc gcaccttgca
tgggttcaat cagatagaaa gtcaggtggc 480cttggtgact tgaaatatcc
tctggtttct gatgtcacca aatccatatc ggaatcttac 540ggtgttctca
ttcccgatca gggaattgca ttgagaggat tgttcattat cgataaggaa
600ggggtgatcc aacattccac catcaacaac ctcggaattg gtagaagtgt
tgacgagaca 660aagagaacac tccaggcttt gcagtatgtg caggagaacc
cagatgaagt ttgccctgct 720gggtggaagc ctggtgagaa gtccatgaaa
ccagacccca aaggtagcaa agagtacttt 780gctgctgtgt agaatggcta
atagtaaatt gctatgagta ttaactactc atctgtatca 840tttgggatgt
aaaaggattt tgttttatgt aattctatcc attttgaatt atgaggccta
900tgggcttagc cataaaaata aaaagtatga ggtccaaaag tgtgtggtta
cagaagcatg 960cttgtgtncc ttgattttgg agtgaattat gaattgatgt
attatctgta aaaaaaaaaa 1020aaaaaaaaaa aaaaaaaaaa aaaa
104418263PRTPisum sativum 18Met Ala Cys Ser Ala Pro Phe Ala Ser Leu
Leu Tyr Ser Asn Pro Asn 1 5 10 15 Thr Leu Phe Ser Pro Lys Phe Ser
Ser Pro Arg Leu Ser Ser Leu Ser 20 25 30 Ile Pro Asn Ala Pro Asn
Ser Leu Pro Lys Leu Arg Thr Ser Leu Pro 35 40 45 Leu Ser Leu Asn
Arg Ser Ser Ser Ser Arg Arg Thr Phe Val Val Arg 50 55 60 Ala Ser
Gly Glu Leu Pro Leu Val Gly Asn Ser Ala Pro Asp Phe Glu 65 70 75 80
Ala Glu Ala Val Phe Asp Gln Glu Phe Ile Lys Val Lys Leu Ser Glu 85
90 95 Tyr Ile Gly Lys Lys Tyr Val Ile Leu Phe Phe Tyr Pro Leu Asp
Phe 100 105 110 Thr Phe Val Cys Pro Thr Glu Ile Thr Ala Phe Ser Asp
Arg His Ala 115 120 125 Glu Phe Asp Ala Ile Asn Thr Glu Ile Leu Gly
Val Ser Val Asp Ser 130 135 140 Val Phe Ser His Leu Ala Trp Val Gln
Ser Asp Arg Lys Ser Gly Gly 145 150 155 160 Leu Gly Asp Leu Lys Tyr
Pro Leu Val Ser Asp Val Thr Lys Ser Ile 165 170 175 Ser Glu Ser Tyr
Gly Val Leu Ile Pro Asp Gln Gly Ile Ala Leu Arg 180 185 190 Gly Leu
Phe Ile Ile Asp Lys Glu Gly Val Ile Gln His Ser Thr Ile 195 200 205
Asn Asn Leu Gly Ile Gly Arg Ser Val Asp Glu Thr Lys Arg Thr Leu 210
215 220 Gln Ala Leu Gln Tyr Val Gln Glu Asn Pro Asp Glu Val Cys Pro
Ala 225 230 235 240 Gly Trp Lys Pro Gly Glu Lys Ser Met Lys Pro Asp
Pro Lys Gly Ser 245 250 255 Lys Glu Tyr Phe Ala Ala Val 260
191078DNAOryza sativa 19acccaagctc ccaaacccct ctcgcaccca atccaaccca
atcccctcct catccactcc 60gctctgcggc catggccgcc tgctgctcct ccctcgccac
cgccgtctcc tcctcctccg 120ccaagcccct cgccggcatc ccccccgccg
cgccgcactc cctctccctc ccccgcgctc 180ccgccgccag gcccctccgc
ctctccgcct cctcatccag atccgcccgg gccagcagct 240tcgtcgcccg
cgccggcggt gtggacgatg cgccgctggt cgggaacaag gcgcccgact
300tcgatgcgga ggcagtcttc gaccaggagt tcatcaacgt gaagctgtcc
gactacatcg 360ggaagaagta cgtcattctc ttcttctacc cgttggactt
caccttcgtc tgcccgaccg 420agattaccgc tttcagtgac agatacgatg
agttcgagaa gttgaacact gagatcctcg 480gtgtttcaat tgacagtgtg
ttctcccatc ttgcatgggt gcagacagac aggaaatctg 540gtgggcttgg
tgatctgaaa tacccattga tttcagatgt tactaaatca atttcgaagt
600cctttggtgt cttgatccct gaccagggaa ttgctctgag aggacttttc
atcattgaca 660aggagggagt gattcagcac tctaccatta acaaccttgc
cattggacgc agtgtagatg 720agaccatgag gacccttcag gcgttgcagt
acgtccagga caacccggac gaggtgtgcc 780cggccggatg gaagcccggt
gacaagtcga tgaagcctga ccccaaggga agcaaggagt 840acttcgcggc
catctaagca cacatatgca tatgcctggt gatggatgta gggagttttt
900ttgctttcgc gagagccatt gcgtttcgtc tccaaagtgt agtaccgtgt
gctcgtctga 960tcggattttg ttacttgttc gccaccagct gttactttgt
tccctaacaa ataaggcttt 1020gttttggtcg tgttatacat gtatacatgt
tagtgcgttt caagatcgct tctgtttt 107820261PRTOryza sativa 20Met Ala
Ala Cys Cys Ser Ser Leu Ala Thr Ala Val Ser Ser Ser Ser 1 5 10 15
Ala Lys Pro Leu Ala Gly Ile Pro Pro Ala Ala Pro His Ser Leu Ser 20
25 30 Leu Pro Arg Ala Pro Ala Ala Arg Pro Leu Arg Leu Ser Ala Ser
Ser 35 40 45 Ser Arg Ser Ala Arg Ala Ser Ser Phe Val Ala Arg Ala
Gly Gly Val 50 55 60 Asp Asp Ala Pro Leu Val Gly Asn Lys Ala Pro
Asp Phe Asp Ala Glu 65 70 75 80 Ala Val Phe Asp Gln Glu Phe Ile Asn
Val Lys Leu Ser Asp Tyr Ile 85 90 95 Gly Lys Lys Tyr Val Ile Leu
Phe Phe Tyr Pro Leu Asp Phe Thr Phe 100 105 110 Val Cys Pro Thr Glu
Ile Thr Ala Phe Ser Asp Arg Tyr Asp Glu Phe 115 120 125 Glu Lys Leu
Asn Thr Glu Ile Leu Gly Val Ser Ile Asp Ser Val Phe 130 135 140 Ser
His Leu Ala Trp Val Gln Thr Asp Arg Lys Ser Gly Gly Leu Gly 145 150
155 160 Asp Leu Lys Tyr Pro Leu Ile Ser Asp Val Thr Lys Ser Ile Ser
Lys 165 170 175 Ser Phe Gly Val Leu Ile Pro Asp Gln Gly Ile Ala Leu
Arg Gly Leu 180 185 190 Phe Ile Ile Asp Lys Glu Gly Val Ile Gln His
Ser Thr Ile Asn Asn 195 200 205 Leu Ala Ile Gly Arg Ser Val Asp Glu
Thr Met Arg Thr Leu Gln Ala 210 215 220 Leu Gln Tyr Val Gln Asp Asn
Pro Asp Glu Val Cys Pro Ala Gly Trp 225 230 235 240 Lys Pro Gly Asp
Lys Ser Met Lys Pro Asp Pro Lys Gly Ser Lys Glu 245 250 255 Tyr Phe
Ala Ala Ile 260 211067DNASecale cereale 21tcttcatatt cgggaaccct
atctatctgg aggctaccgc ggccgccccc gggcactccc 60cgcctgacaa ccacggccat
ggcgtgcgcc ttctccgcct ccaccgtgtc cacggcggcc 120gcgctcgtcg
cgtccccgaa gccagccggg gcgccgagtg cctgtcgttt ccccgcgctt
180cgcaggggcc gcgcaggcct ccgctgcgcg cggctcgagg acgccagggc
ccgcagcttc 240gtcgcccgcg ccgcagccga gtacgacctg ccactggtgg
ggaacaaagc accggacttc 300gctgcggagg ccgtgttcga ccaggagttc
atcaacgtca agctatctga ttacattggg 360aagaagtatg tgattctttt
cttctaccct ctggacttca ccttcgtctg cccaactgag 420attacggctt
tcagcgacag acatgaggag ttcgagaaga taaacactga aattcttggt
480gtttcagttg atagtgtgtt ttcccatctt gcatgggtgc agacagagag
gaaatctggt 540ggacttggtg atcttaagta tcctctggtt tctgatgtca
ccaaatcaat ctcaaagtct 600tttggtgtat tgatccctga tcagggaatt
gctctgagag gattattcat gattgacaag 660gagggtgtga ttcagcattc
cactattaac aaccttggta ttggccgcag tgtggatgag 720accttgagaa
cccttcaggc tctgcaatac gtccaagaaa acccagacga ggtctgcccg
780gcaggatgga aacccgggga aaagtcgatg aagcctgacc ctaagggcag
caaggagtac 840ttcgctgcca tctagatgcg acctttgcgc tcacagtctg
agttttgtca tggccatttc 900tggttacttg tgttcttgtg acccgagttg
tagttatcac gcgtccaatt gcctctgtaa 960ttcctccaat aagggtttgt
ctgtgtgttg attttccctc ctccaatttg gaaagcccaa 1020tccaagattg
gaaataaaac cttctgccac ccaaaaaaaa aaaaaaa 106722258PRTSecale cereale
22Met Ala Cys Ala Phe Ser Ala Ser Thr Val Ser Thr Ala Ala Ala Leu 1
5 10 15 Val Ala Ser Pro Lys Pro Ala Gly Ala Pro Ser Ala Cys Arg Phe
Pro 20 25 30 Ala Leu Arg Arg Gly Arg Ala Gly Leu Arg Cys Ala Arg
Leu Glu Asp 35 40 45 Ala Arg Ala Arg Ser Phe Val Ala Arg Ala Ala
Ala Glu Tyr Asp Leu 50 55 60 Pro Leu Val Gly Asn Lys Ala Pro Asp
Phe Ala Ala Glu Ala Val Phe 65 70 75 80 Asp Gln Glu Phe Ile Asn Val
Lys Leu Ser Asp Tyr Ile Gly Lys Lys 85 90 95 Tyr Val Ile Leu Phe
Phe Tyr Pro Leu Asp Phe Thr Phe Val Cys Pro 100 105 110 Thr Glu Ile
Thr Ala Phe Ser Asp Arg His Glu Glu Phe Glu Lys Ile 115 120 125 Asn
Thr Glu Ile Leu Gly Val Ser Val Asp Ser Val Phe Ser His Leu 130
135 140 Ala Trp Val Gln Thr Glu Arg Lys Ser Gly Gly Leu Gly Asp Leu
Lys 145 150 155 160 Tyr Pro Leu Val Ser Asp Val Thr Lys Ser Ile Ser
Lys Ser Phe Gly 165 170 175 Val Leu Ile Pro Asp Gln Gly Ile Ala Leu
Arg Gly Leu Phe Met Ile 180 185 190 Asp Lys Glu Gly Val Ile Gln His
Ser Thr Ile Asn Asn Leu Gly Ile 195 200 205 Gly Arg Ser Val Asp Glu
Thr Leu Arg Thr Leu Gln Ala Leu Gln Tyr 210 215 220 Val Gln Glu Asn
Pro Asp Glu Val Cys Pro Ala Gly Trp Lys Pro Gly 225 230 235 240 Glu
Lys Ser Met Lys Pro Asp Pro Lys Gly Ser Lys Glu Tyr Phe Ala 245 250
255 Ala Ile 23876DNARiccia fluitans 23gttgggaaag gcagcaaata
tggcaaccgc ctgtgctgca gtgtctgcag tggctgttcc 60tgtggcctct gtagctaacc
acattgcgtc ttcatcatct gggaccccat cccttgccat 120tcccaggtct
tatgagggtt taaacaaatc cttcggcgct agaattgcac cccgatcaac
180ctccgctttt cgcaagcccg tcactggtgt ctccctcaag cagttctcga
agggaaaagt 240cgcttctgcg agatgtgcgt cacctcttgt tggaaatgtc
gccccggact tcgaggcgga 300ggccgttttt gaccaagagt tcgtgaagat
caagctctcg gagtacattg ggaagagata 360cgttgttctt ttcttctacc
ctcttgactt caccttcgtt tgcccaacag aaattaccgc 420atttagcgac
aaacacgaag agtttgagaa gttgaacacc gaagttattg gggtttctac
480tgacagtgtg ttttcccatc ttgcctggat tcaaactgac agaaaatctg
gaggacttgg 540tgacttgaag tacccacttg tgtccgactt gaccaagaag
atcgctgaag attttggagt 600attgatcccc gatcagggca ttgcattgcg
aggattgttc atcatcgaca aggagggcgt 660cattcagcac gcaaccatta
acaatttggc catcggcaga agtgtggagg agacgcttcg 720aactctgcag
gctgtacaat atgtgcagga gaacccagac gaggtctgcc ccgctggctg
780gaagccgggt gaaaagacca tgaagcctga cacaaagctc agcaaggagt
acttcgcaca 840agtataggcc gaaaatagct tcgtttggaa tacata
87624275PRTRiccia fluitans 24Met Ala Thr Ala Cys Ala Ala Val Ser
Ala Val Ala Val Pro Val Ala 1 5 10 15 Ser Val Ala Asn His Ile Ala
Ser Ser Ser Ser Gly Thr Pro Ser Leu 20 25 30 Ala Ile Pro Arg Ser
Tyr Glu Gly Leu Asn Lys Ser Phe Gly Ala Arg 35 40 45 Ile Ala Pro
Arg Ser Thr Ser Ala Phe Arg Lys Pro Val Thr Gly Val 50 55 60 Ser
Leu Lys Gln Phe Ser Lys Gly Lys Val Ala Ser Ala Arg Cys Ala 65 70
75 80 Ser Pro Leu Val Gly Asn Val Ala Pro Asp Phe Glu Ala Glu Ala
Val 85 90 95 Phe Asp Gln Glu Phe Val Lys Ile Lys Leu Ser Glu Tyr
Ile Gly Lys 100 105 110 Arg Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp
Phe Thr Phe Val Cys 115 120 125 Pro Thr Glu Ile Thr Ala Phe Ser Asp
Lys His Glu Glu Phe Glu Lys 130 135 140 Leu Asn Thr Glu Val Ile Gly
Val Ser Thr Asp Ser Val Phe Ser His 145 150 155 160 Leu Ala Trp Ile
Gln Thr Asp Arg Lys Ser Gly Gly Leu Gly Asp Leu 165 170 175 Lys Tyr
Pro Leu Val Ser Asp Leu Thr Lys Lys Ile Ala Glu Asp Phe 180 185 190
Gly Val Leu Ile Pro Asp Gln Gly Ile Ala Leu Arg Gly Leu Phe Ile 195
200 205 Ile Asp Lys Glu Gly Val Ile Gln His Ala Thr Ile Asn Asn Leu
Ala 210 215 220 Ile Gly Arg Ser Val Glu Glu Thr Leu Arg Thr Leu Gln
Ala Val Gln 225 230 235 240 Tyr Val Gln Glu Asn Pro Asp Glu Val Cys
Pro Ala Gly Trp Lys Pro 245 250 255 Gly Glu Lys Thr Met Lys Pro Asp
Thr Lys Leu Ser Lys Glu Tyr Phe 260 265 270 Ala Gln Val 275
251255DNAChlamydomonas incerta 25atggccgctc tgcagtccgc ttcccgctcc
tcggcggtgg ccttctcgcg ccaggcgcgc 60gtggccccgc gcgttgcctc cagcgttgct
cgccgcaacc tggtcgtgcg cgcttcccac 120gctgagaagc ctctggtcgg
ctccgtcgcc cctgacttca aggcccaggc cgtgttcgac 180caggagttcc
aggagattac cctgagcaag taccgcggca agtacgtggt gctgttcttc
240taccccctgg acttcacctt cgtgtgcccc accgagatca ccgccttctc
ggaccgctac 300aaggagttca aggacatcaa caccgaggtc ctgggcgtgt
ccgtggacag ccagttcacc 360cacctggcct ggattcagac cgaccgcaag
gagggtggcc tgggcgacct gaactacccc 420ctggtggctg acctgaagaa
ggagatctcc aaggcctacg gcgtcctgac cgaggacggc 480atctccctgc
gcggcctgtt catcatcgac aaggagggcg ttgtgcagca cgccaccatc
540aacaacctgg ctttcggccg ctcggtcgac gagaccaagc gtgtgctgca
ggccatccag 600tacgtgcagt ccaaccccga tgaggtctgc cccgccggct
ggaagcccgg tgacaagacc 660atgaagcccg accccaaggg ctccaaggag
tacttcgccg ccgtgtaaat tgacccttga 720ttgagagtca atgacacgcg
agggcgtcat cgcagtactc gggggcatgc tgcagatcag 780caggcatgcg
gacgagacca gtgcattggc aggctaggcg cacacgggag gcagagccag
840tgcggcggca gcggcgagcg gcggctgtgg aagcaggcgc tagcagcagc
ggcggccgcg 900gcggcgctgc tctccatggg tgcgcctgca agcagcatgt
gcatgtggac tcggtgcttc 960tcgttgatgg gtcagggcgg cgttgccggt
ggtgcggacc gggcggtaat cgcacgtagc 1020tcaattgttg cgtgcgggcg
ctgtgcgggc tggcgtgacg gcacgcaacc tgtgtggggc 1080ctgttggtac
gctcgcgata atgcagtgcg cggtccgagc ggagggacgc ggcggtgaat
1140agctgctgta gtttcaggca gggatttacc aggtgacggg tggttgcgcc
cacacccgaa 1200cggctgtgat cccaattttc catgagaggg cttgcagatg
gacggcgtgt gatcg 125526235PRTChlamydomonas incerta 26Met Ala Ala
Leu Gln Ser Ala Ser Arg Ser Ser Ala Val Ala Phe Ser 1 5 10 15 Arg
Gln Ala Arg Val Ala Pro Arg Val Ala Ser Ser Val Ala Arg Arg 20 25
30 Asn Leu Val Val Arg Ala Ser His Ala Glu Lys Pro Leu Val Gly Ser
35 40 45 Val Ala Pro Asp Phe Lys Ala Gln Ala Val Phe Asp Gln Glu
Phe Gln 50 55 60 Glu Ile Thr Leu Ser Lys Tyr Arg Gly Lys Tyr Val
Val Leu Phe Phe 65 70 75 80 Tyr Pro Leu Asp Phe Thr Phe Val Cys Pro
Thr Glu Ile Thr Ala Phe 85 90 95 Ser Asp Arg Tyr Lys Glu Phe Lys
Asp Ile Asn Thr Glu Val Leu Gly 100 105 110 Val Ser Val Asp Ser Gln
Phe Thr His Leu Ala Trp Ile Gln Thr Asp 115 120 125 Arg Lys Glu Gly
Gly Leu Gly Asp Leu Asn Tyr Pro Leu Val Ala Asp 130 135 140 Leu Lys
Lys Glu Ile Ser Lys Ala Tyr Gly Val Leu Thr Glu Asp Gly 145 150 155
160 Ile Ser Leu Arg Gly Leu Phe Ile Ile Asp Lys Glu Gly Val Val Gln
165 170 175 His Ala Thr Ile Asn Asn Leu Ala Phe Gly Arg Ser Val Asp
Glu Thr 180 185 190 Lys Arg Val Leu Gln Ala Ile Gln Tyr Val Gln Ser
Asn Pro Asp Glu 195 200 205 Val Cys Pro Ala Gly Trp Lys Pro Gly Asp
Lys Thr Met Lys Pro Asp 210 215 220 Pro Lys Gly Ser Lys Glu Tyr Phe
Ala Ala Val 225 230 235 27612DNANostoc sp. 27atgtccatca cctacggaac
acaagaaagc ctccgcgttg gtcaacaggc tcccgacttt 60acagcaacag ctgtagttga
tcaggaattc aagacaatta agctttccga ctatcgtggt 120aagtacgttg
tcttgttctt ctatccccta gactttacct ttgtttgccc cacggagatc
180acagcattta gcgatcgcta cgaagaattc aagaaactta acaccgaaat
tctcggtgtg 240tccgttgata gcgagttctc ccacctagct tggattcaaa
ctgatcgtaa gtctggtggt 300gttggcgacc taaattatcc cttagtttcc
gatattaaga aagaggttag cgacgcttac 360aacgtactag acccagcagc
aggtatcgct ttacgtggtc tgttcatcat cgataaagat 420ggtatcattc
agcacgctac cattaacaac ctagcttttg gtcgtagcgt tgatgaaacc
480ctacggacat tgcaagcaat ccagtatgtc cagtctcacc cagatgaagt
ttgccctgct 540ggttggcaac ctggggaaaa gaccatgact cccgaccctg
tgaagtccaa agtttacttc 600gctgctgtgt aa 61228203PRTNostoc sp. 28Met
Ser Ile Thr Tyr Gly Thr Gln Glu Ser Leu Arg Val Gly Gln Gln 1 5 10
15 Ala Pro Asp Phe Thr Ala Thr Ala Val Val Asp Gln Glu Phe Lys Thr
20 25 30 Ile Lys Leu Ser Asp Tyr Arg Gly Lys Tyr Val Val Leu Phe
Phe Tyr 35 40 45 Pro Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile
Thr Ala Phe Ser 50 55 60 Asp Arg Tyr Glu Glu Phe Lys Lys Leu Asn
Thr Glu Ile Leu Gly Val 65 70 75 80 Ser Val Asp Ser Glu Phe Ser His
Leu Ala Trp Ile Gln Thr Asp Arg 85 90 95 Lys Ser Gly Gly Val Gly
Asp Leu Asn Tyr Pro Leu Val Ser Asp Ile 100 105 110 Lys Lys Glu Val
Ser Asp Ala Tyr Asn Val Leu Asp Pro Ala Ala Gly 115 120 125 Ile Ala
Leu Arg Gly Leu Phe Ile Ile Asp Lys Asp Gly Ile Ile Gln 130 135 140
His Ala Thr Ile Asn Asn Leu Ala Phe Gly Arg Ser Val Asp Glu Thr 145
150 155 160 Leu Arg Thr Leu Gln Ala Ile Gln Tyr Val Gln Ser His Pro
Asp Glu 165 170 175 Val Cys Pro Ala Gly Trp Gln Pro Gly Glu Lys Thr
Met Thr Pro Asp 180 185 190 Pro Val Lys Ser Lys Val Tyr Phe Ala Ala
Val 195 200 29609DNASynechococcus sp. 29ctacttggca atcgccgcga
agaactcctt ggatttcact gggtcggggt gcatggtttt 60ctggccgggc tgccagttgg
ccgggcaaac ttcatcgggg tgagattgca cgtattggat 120agcttgcagg
gtgcgcaagg tttcatccac actgcggcca aaggccaggt tgttaatggt
180ggcgtgctgg atgatccctt ctttgtcgat gatgaacagg ccgcgcagcg
ccacaccggc 240ctccggatcc agaacattgt aggcagcgct gatctccttt
ttcaggtcag agaccagagg 300ataccttagc tcgcccaccc ctccggcttt
gcggtcggtc tggatccagg ccaagtgaga 360gtattcgctg tccaccgaga
cgcccaggat ctcggtatcc agcttggcaa agtcgtcata 420gcggtcgcta
aaggccgtga tctccgttgg gcagacaaag gtgaagtcca aggggtagaa
480gaacagcacc acatacttct taccccggta gtcggagagc ttcaccgtct
tgaattccat 540gtcatagacg gcggtggccg aaaaatcggg agcgggctgc
cccactcgca ggcatccttc 600ctgagacat 60930202PRTSynechococcus sp.
30Met Ser Gln Glu Gly Cys Leu Arg Val Gly Gln Pro Ala Pro Asp Phe 1
5 10 15 Ser Ala Thr Ala Val Tyr Asp Met Glu Phe Lys Thr Val Lys Leu
Ser 20 25 30 Asp Tyr Arg Gly Lys Lys Tyr Val Val Leu Phe Phe Tyr
Pro Leu Asp 35 40 45 Phe Thr Phe Val Cys Pro Thr Glu Ile Thr Ala
Phe Ser Asp Arg Tyr 50 55 60 Asp Asp Phe Ala Lys Leu Asp Thr Glu
Ile Leu Gly Val Ser Val Asp 65 70 75 80 Ser Glu Tyr Ser His Leu Ala
Trp Ile Gln Thr Asp Arg Lys Ala Gly 85 90 95 Gly Val Gly Glu Leu
Arg Tyr Pro Leu Val Ser Asp Leu Lys Lys Glu 100 105 110 Ile Ser Ala
Ala Tyr Asn Val Leu Asp Pro Glu Ala Gly Val Ala Leu 115 120 125 Arg
Gly Leu Phe Ile Ile Asp Lys Glu Gly Ile Ile Gln His Ala Thr 130 135
140 Ile Asn Asn Leu Ala Phe Gly Arg Ser Val Asp Glu Thr Leu Arg Thr
145 150 155 160 Leu Gln Ala Ile Gln Tyr Val Gln Ser His Pro Asp Glu
Val Cys Pro 165 170 175 Ala Asn Trp Gln Pro Gly Gln Lys Thr Met His
Pro Asp Pro Val Lys 180 185 190 Ser Lys Glu Phe Phe Ala Ala Ile Ala
Lys 195 200 31612DNANodularia spumigena 31atgtccctca cttacgcaac
agaaggatgc ctccgcgttg gtcaacaggc tcctgaattt 60acagccacag ctgtggtaga
tcaagaattt aagaccatta aactttccga ctatcgcggt 120aagtatgtgg
ttctgttttt ctacccctta gactttacct ttgtttgccc cactgagatc
180acagcattta gcgatcgcta cgaagaattt aagaaagtta acacagaagt
tctcggtgtt 240tccgttgata gcgaattctc tcacctagcc tggattcaaa
ctgaacgcaa gtctggtggt 300gtcggcgacc tcaattatcc cttagtttcg
gacatcaaaa aagagattag cgccacctac 360aatgtccttg acccagccgc
aggtattgct ttacgcggtt tgttcattat tgataaagat 420ggtatcatcc
agcattctac agtgaataac ctcgcctttg gtcgcagcgt tgatgaaacc
480ctgcggacat tgcaagccct tcagtatgtt cagtctcacc ccgatgaagt
ttgcccagcc 540ggttggcaac ctggtgatca aacaatggtt cctgaccctg
tgaagtcgaa agtctacttc 600tcggctgtct ag 61232203PRTNodularia
spumigena 32Met Ser Leu Thr Tyr Ala Thr Glu Gly Cys Leu Arg Val Gly
Gln Gln 1 5 10 15 Ala Pro Glu Phe Thr Ala Thr Ala Val Val Asp Gln
Glu Phe Lys Thr 20 25 30 Ile Lys Leu Ser Asp Tyr Arg Gly Lys Tyr
Val Val Leu Phe Phe Tyr 35 40 45 Pro Leu Asp Phe Thr Phe Val Cys
Pro Thr Glu Ile Thr Ala Phe Ser 50 55 60 Asp Arg Tyr Glu Glu Phe
Lys Lys Val Asn Thr Glu Val Leu Gly Val 65 70 75 80 Ser Val Asp Ser
Glu Phe Ser His Leu Ala Trp Ile Gln Thr Glu Arg 85 90 95 Lys Ser
Gly Gly Val Gly Asp Leu Asn Tyr Pro Leu Val Ser Asp Ile 100 105 110
Lys Lys Glu Ile Ser Ala Thr Tyr Asn Val Leu Asp Pro Ala Ala Gly 115
120 125 Ile Ala Leu Arg Gly Leu Phe Ile Ile Asp Lys Asp Gly Ile Ile
Gln 130 135 140 His Ser Thr Val Asn Asn Leu Ala Phe Gly Arg Ser Val
Asp Glu Thr 145 150 155 160 Leu Arg Thr Leu Gln Ala Leu Gln Tyr Val
Gln Ser His Pro Asp Glu 165 170 175 Val Cys Pro Ala Gly Trp Gln Pro
Gly Asp Gln Thr Met Val Pro Asp 180 185 190 Pro Val Lys Ser Lys Val
Tyr Phe Ser Ala Val 195 200 33594DNAThermosynechococcus elongatus
33ttagcccacg gcttcaaaat agactttgga tttgacaggg tcagggttca tcgtcttgtc
60accggggtgc cagcccgcgg ggcagacttc atcggggtga gtttgaacgt attgaatcgc
120ttggagtacc cgcagggtct catcaacact gcggccaaag gccaagttat
tgattgttgc 180gtgttggata atcccttctt tatcaatgat gaacagaccc
cgcagggcca cgccttcttc 240ggtcaggaca ttgtaggcag tgctgatgtc
ttttttcagg tcagacacca agggatattt 300aagatcgccg acaccaccag
ctttgcgatc agtttgtgtc caagccaagt gggagaactg 360gctatccaca
gacacgccca ggatttcggt gttcaatttg gcaaattcat cgtagcgatc
420gctaaaggca acaatttccg tggggcagac aaaggtaaag tccaagggat
agaaaaagag 480aacaacgtac ttaccgcgat agtccgagag cttgatggtt
ttgaactctt ggtcataaac 540agcaaccgct tcaaaatcgg gggcgggttg
accgacgcgc agacactcag acat 59434197PRTThermosynechococcus elongatus
34Met Ser Glu Cys Leu Arg Val Gly Gln Pro Ala Pro Asp Phe Glu Ala 1
5 10 15 Val Ala Val Tyr Asp Gln Glu Phe Lys Thr Ile Lys Leu Ser Asp
Tyr 20 25 30 Arg Gly Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp
Phe Thr Phe 35 40 45 Val Cys Pro Thr Glu Ile Val Ala Phe Ser Asp
Arg Tyr Asp Glu Phe 50 55 60 Ala Lys Leu Asn Thr Glu Ile Leu Gly
Val Ser Val Asp Ser Gln Phe 65 70 75 80 Ser His Leu Ala Trp Thr Gln
Thr Asp Arg Lys Ala Gly Gly Val Gly 85 90 95 Asp Leu Lys Tyr Pro
Leu Val Ser Asp Leu Lys Lys Asp Ile Ser Thr 100 105 110 Ala Tyr Asn
Val Leu Thr Glu Glu Gly Val Ala Leu Arg Gly Leu Phe 115 120 125 Ile
Ile Asp Lys Glu Gly Ile Ile Gln His Ala Thr Ile Asn Asn Leu 130 135
140 Ala Phe Gly Arg Ser Val Asp Glu Thr Leu Arg Val Leu Gln Ala Ile
145 150 155 160 Gln Tyr Val Gln Thr His Pro Asp Glu Val Cys Pro Ala
Gly Trp His 165 170 175 Pro Gly Asp Lys Thr Met Asn Pro Asp Pro Val
Lys Ser Lys Val Tyr 180 185 190 Phe Glu Ala Val Gly 195
35693DNAOstreococcus tauri 35atgttgtccg cgagtttgtc caagagcgcg
ttcacgccca gggcgtcggc gctccagaag 60agcgttaagg ggaagaactt ctcccgatcc
gccgtccgcg tggaagcgcg caagccgctc 120gtgggctacc cggcgccgga
gtttagcgcc gaggcggtgt tcgatcaaga gttccaagac 180atcaagctct
cggattaccg cggcaagtac gtcgtgctct tcttctaccc gctcgatttt
240acctttgtgt gcccgacgga aatcaccgcc ttctccgatc gctacgaaga
gttcgcgaag 300ctcaacaccg aagtcctcgg cgtgagcgtt gactccaagt
tctctcactt ggcgtggttg 360caaaccgacc gcaacgacgg cggcctcggc
gacttggcct acccgctcgt cagtgacctc 420aagcgcgaaa tctgcgaatc
gtacgatgtg ttgtacgaag acggcaccgc gctccgtggg 480ttgtacatca
tcgatcgtga gggcgtcatc cagcactaca catgcaacaa cgctccgttc
540ggccgcaacg tcgacgagtg cctgcgcgtg cttcaagcga
tccaatacgt tcaaaacaac 600ccagacgagg tgtgcccggc gggctggacc
ccgggtgcgg cgacgatgaa gccggatccg 660aagggctcga aggaatactt
caaggcgatc taa 69336230PRTOstreococcus tauri 36Met Leu Ser Ala Ser
Leu Ser Lys Ser Ala Phe Thr Pro Arg Ala Ser 1 5 10 15 Ala Leu Gln
Lys Ser Val Lys Gly Lys Asn Phe Ser Arg Ser Ala Val 20 25 30 Arg
Val Glu Ala Arg Lys Pro Leu Val Gly Tyr Pro Ala Pro Glu Phe 35 40
45 Ser Ala Glu Ala Val Phe Asp Gln Glu Phe Gln Asp Ile Lys Leu Ser
50 55 60 Asp Tyr Arg Gly Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu
Asp Phe 65 70 75 80 Thr Phe Val Cys Pro Thr Glu Ile Thr Ala Phe Ser
Asp Arg Tyr Glu 85 90 95 Glu Phe Ala Lys Leu Asn Thr Glu Val Leu
Gly Val Ser Val Asp Ser 100 105 110 Lys Phe Ser His Leu Ala Trp Leu
Gln Thr Asp Arg Asn Asp Gly Gly 115 120 125 Leu Gly Asp Leu Ala Tyr
Pro Leu Val Ser Asp Leu Lys Arg Glu Ile 130 135 140 Cys Glu Ser Tyr
Asp Val Leu Tyr Glu Asp Gly Thr Ala Leu Arg Gly 145 150 155 160 Leu
Tyr Ile Ile Asp Arg Glu Gly Val Ile Gln His Tyr Thr Cys Asn 165 170
175 Asn Ala Pro Phe Gly Arg Asn Val Asp Glu Cys Leu Arg Val Leu Gln
180 185 190 Ala Ile Gln Tyr Val Gln Asn Asn Pro Asp Glu Val Cys Pro
Ala Gly 195 200 205 Trp Thr Pro Gly Ala Ala Thr Met Lys Pro Asp Pro
Lys Gly Ser Lys 210 215 220 Glu Tyr Phe Lys Ala Ile 225 230
37609DNASynechococcus sp. 37ctacttggca accgccgcga agaactcctt
ggatttcacc gggtcggggt tcagggttct 60ctggccgggc tgccagttgg ccgggcaaac
ttcatcgggg tgagcttgca cgtactggat 120ggcctgcagg gtgcgcaggg
tttcatccac gctgcggcca aaggccaggt tgttgatggt 180ggcgtgctgg
atgatccctt ccttgtcgat gatgaacagg ccgcgcaaag ccacccctgc
240cgccggatcc aggacattgt aggcggcgct gatctccttt ttcaggtcgg
agaccagcgg 300atacctcagc tcgcccaccc ctccggcttt gcggtcggtc
tgaatccagg ccaggtgaga 360gtactcgcta tccaccgaga cgccgaggat
ctccgtgtcc agcttggcaa actcgtcgta 420gcgatcgctg aaagccgtga
tctccgtcgg gcagacgaag gtgaagtcca aggggtagaa 480gaacagcacc
acgtacttct tgccccggta gtcggagagc ctcaccgtct taaattccat
540gtcgtaaacg gcagtggccg aaaaatcggg agcgggctgc cccacccgca
gacatccttc 600ctgagacat 60938202PRTSynechococcus sp. 38Met Ser Gln
Glu Gly Cys Leu Arg Val Gly Gln Pro Ala Pro Asp Phe 1 5 10 15 Ser
Ala Thr Ala Val Tyr Asp Met Glu Phe Lys Thr Val Arg Leu Ser 20 25
30 Asp Tyr Arg Gly Lys Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp
35 40 45 Phe Thr Phe Val Cys Pro Thr Glu Ile Thr Ala Phe Ser Asp
Arg Tyr 50 55 60 Asp Glu Phe Ala Lys Leu Asp Thr Glu Ile Leu Gly
Val Ser Val Asp 65 70 75 80 Ser Glu Tyr Ser His Leu Ala Trp Ile Gln
Thr Asp Arg Lys Ala Gly 85 90 95 Gly Val Gly Glu Leu Arg Tyr Pro
Leu Val Ser Asp Leu Lys Lys Glu 100 105 110 Ile Ser Ala Ala Tyr Asn
Val Leu Asp Pro Ala Ala Gly Val Ala Leu 115 120 125 Arg Gly Leu Phe
Ile Ile Asp Lys Glu Gly Ile Ile Gln His Ala Thr 130 135 140 Ile Asn
Asn Leu Ala Phe Gly Arg Ser Val Asp Glu Thr Leu Arg Thr 145 150 155
160 Leu Gln Ala Ile Gln Tyr Val Gln Ala His Pro Asp Glu Val Cys Pro
165 170 175 Ala Asn Trp Gln Pro Gly Gln Arg Thr Leu Asn Pro Asp Pro
Val Lys 180 185 190 Ser Lys Glu Phe Phe Ala Ala Val Ala Lys 195 200
39603DNASynechococcus sp. 39tcaaccgatg gcggagaaat actccttgga
acctttcgga tcgggcttca tggtcttttc 60gccgggcgtc cagttggcgg ggcagacttc
atcggggttg gactgcacgt actggaaggc 120ctgaagcaca cgcagggttt
cgtccacatt ccggccaaca ggcaggttgt tgatcgtgga 180gtgcatgatc
acgccatcgg gatcgatgat gaacagtcca cgcaaagcaa cgccttcggc
240gtcgtccagc acgttgtatg cggtggcgat ttccttcttg aggtcagcga
ccaggggata 300gttgatgtcg cccagaccgc cctgattgcg gggagtctga
atccaggcca gatggctgaa 360ctggctgtca acggaaacgc cgaggacttc
ggtgttcttg ctggagaaat cggcgtagcg 420gtcgctgaag gccgtgattt
ctgtggggca gacgaaggtg aaatccaggg gatagaagaa 480gagcaccacg
tacttgccgc ggtactggga cagggagatt tccttgaatt cctggtccac
540cactgcagtg gcagtgaaat cgggggcctg ctggcccaca cgaaggcaac
cggtctcggt 600cat 60340200PRTSynechococcus sp. 40Met Thr Glu Thr
Gly Cys Leu Arg Val Gly Gln Gln Ala Pro Asp Phe 1 5 10 15 Thr Ala
Thr Ala Val Val Asp Gln Glu Phe Lys Glu Ile Ser Leu Ser 20 25 30
Gln Tyr Arg Gly Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp Phe 35
40 45 Thr Phe Val Cys Pro Thr Glu Ile Thr Ala Phe Ser Asp Arg Tyr
Ala 50 55 60 Asp Phe Ser Ser Lys Asn Thr Glu Val Leu Gly Val Ser
Val Asp Ser 65 70 75 80 Gln Phe Ser His Leu Ala Trp Ile Gln Thr Pro
Arg Asn Gln Gly Gly 85 90 95 Leu Gly Asp Ile Asn Tyr Pro Leu Val
Ala Asp Leu Lys Lys Glu Ile 100 105 110 Ala Thr Ala Tyr Asn Val Leu
Asp Asp Ala Glu Gly Val Ala Leu Arg 115 120 125 Gly Leu Phe Ile Ile
Asp Pro Asp Gly Val Ile Met His Ser Thr Ile 130 135 140 Asn Asn Leu
Pro Val Gly Arg Asn Val Asp Glu Thr Leu Arg Val Leu 145 150 155 160
Gln Ala Phe Gln Tyr Val Gln Ser Asn Pro Asp Glu Val Cys Pro Ala 165
170 175 Asn Trp Thr Pro Gly Glu Lys Thr Met Lys Pro Asp Pro Lys Gly
Ser 180 185 190 Lys Glu Tyr Phe Ser Ala Ile Gly 195 200
41597DNASynechococcus elongatus 41ctagactgca gcgaagaact ctttcgactt
aacagggtcg gggttcatcg tcgctgcacc 60cggttgccaa ttggcggggc aaacttcatc
ggggtgactt tggacgtact gaatggcttg 120cagcacccgc agggtttcat
caacgctgcg gccaaacgcc aggttgttga tggtggcgtg 180ctggatcaca
ccttctttgt cgatgatgaa cagaccgcgc agggcaatgc cttcagccgg
240atcaagcacg ttgtaggcag tgctgatttc tttcttgagg tcagcaacca
gcgggtaagc 300caagtcaccc aaaccacctt ctttacggct ggtttgaatc
caagccaagt ggctgaattg 360gctatcgacc gagacaccca agatttcggt
gttcagggct gaaaagtctg catagcgatc 420gctaaaagca gtaatttcgg
tcgggcaaac aaaggtgaag tcgaggggat agaagaacag 480aacgacgtat
ttgccccggt aattggatag cttgatcgtc tggaattcct gatcaacgac
540tgcagtcgct tcaaaatcgg gggccaattg gccgacgcgc agggctcctt cggtcat
59742198PRTSynechococcus elongatus 42Met Thr Glu Gly Ala Leu Arg
Val Gly Gln Leu Ala Pro Asp Phe Glu 1 5 10 15 Ala Thr Ala Val Val
Asp Gln Glu Phe Gln Thr Ile Lys Leu Ser Asn 20 25 30 Tyr Arg Gly
Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp Phe Thr 35 40 45 Phe
Val Cys Pro Thr Glu Ile Thr Ala Phe Ser Asp Arg Tyr Ala Asp 50 55
60 Phe Ser Ala Leu Asn Thr Glu Ile Leu Gly Val Ser Val Asp Ser Gln
65 70 75 80 Phe Ser His Leu Ala Trp Ile Gln Thr Ser Arg Lys Glu Gly
Gly Leu 85 90 95 Gly Asp Leu Ala Tyr Pro Leu Val Ala Asp Leu Lys
Lys Glu Ile Ser 100 105 110 Thr Ala Tyr Asn Val Leu Asp Pro Ala Glu
Gly Ile Ala Leu Arg Gly 115 120 125 Leu Phe Ile Ile Asp Lys Glu Gly
Val Ile Gln His Ala Thr Ile Asn 130 135 140 Asn Leu Ala Phe Gly Arg
Ser Val Asp Glu Thr Leu Arg Val Leu Gln 145 150 155 160 Ala Ile Gln
Tyr Val Gln Ser His Pro Asp Glu Val Cys Pro Ala Asn 165 170 175 Trp
Gln Pro Gly Ala Ala Thr Met Asn Pro Asp Pro Val Lys Ser Lys 180 185
190 Glu Phe Phe Ala Ala Val 195 43597DNAProchlorococcus marinus
43ttatagactt gagaaatact ccttgctccc ttctggatct ggcttcattg tcttttcccc
60tggagtccaa ttggcaggac atacctcgtc tgggttggct tgaacatatt gaaatgcttg
120aagaactctc aaggtctcat caacatttct tcctacaggt aggttgttaa
tagtagcgtg 180catgatcaca ccatctggat cgatgatata aagacctctt
aaagcaacac cctctgcatc 240gtcgagaacg ttataagcca atgaaatctc
tttctttaaa tcggcaacca agggataatt 300gatatcgcca atgcctccat
catttctttg agtttgaatc caggcaaggt ggctaaattg 360actgtctaca
gataccccta agacctcagt gttcttactt gaaaattcgg agtatctatc
420gctaaaagcg gtaatttcag ttggacatac aaaagtaaaa tctagagggt
aaaagaaaag 480cacaacatat ttacctctgt aatttgaaag tgatatttcc
ttgaattcct ggtctatcac 540tgcagtagca gtaaaatcag gagctttctg
gccaacacgg atacattcgt tcgtcat 59744198PRTProchlorococcus marinus
44Met Thr Asn Glu Cys Ile Arg Val Gly Gln Lys Ala Pro Asp Phe Thr 1
5 10 15 Ala Thr Ala Val Ile Asp Gln Glu Phe Lys Glu Ile Ser Leu Ser
Asn 20 25 30 Tyr Arg Gly Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu
Asp Phe Thr 35 40 45 Phe Val Cys Pro Thr Glu Ile Thr Ala Phe Ser
Asp Arg Tyr Ser Glu 50 55 60 Phe Ser Ser Lys Asn Thr Glu Val Leu
Gly Val Ser Val Asp Ser Gln 65 70 75 80 Phe Ser His Leu Ala Trp Ile
Gln Thr Gln Arg Asn Asp Gly Gly Ile 85 90 95 Gly Asp Ile Asn Tyr
Pro Leu Val Ala Asp Leu Lys Lys Glu Ile Ser 100 105 110 Leu Ala Tyr
Asn Val Leu Asp Asp Ala Glu Gly Val Ala Leu Arg Gly 115 120 125 Leu
Tyr Ile Ile Asp Pro Asp Gly Val Ile Met His Ala Thr Ile Asn 130 135
140 Asn Leu Pro Val Gly Arg Asn Val Asp Glu Thr Leu Arg Val Leu Gln
145 150 155 160 Ala Phe Gln Tyr Val Gln Ala Asn Pro Asp Glu Val Cys
Pro Ala Asn 165 170 175 Trp Thr Pro Gly Glu Lys Thr Met Lys Pro Asp
Pro Glu Gly Ser Lys 180 185 190 Glu Tyr Phe Ser Ser Leu 195
45600DNAPorphyra purpurea 45ttatgcagcc gcaaaataat ttttagattt
tataggatcc ggattcattg ttctatcacc 60aggtttccaa tttgctggac atacttcatc
tggatgggct tgaacatatt gaattgcttg 120cagaactctt aaagtttctt
caacgcttct tccaaactcc agattgttaa cggtagaata 180ttgaattata
cctttaggat ctataataaa taatcccctt agggctacac ccccactatt
240taatacatta taggcaatgc taatttcttt ttttagatct gatactaaag
gatactcaag 300atctcctaat ccaccagatt ctcgatctgt ttgcaaccaa
gctaagtgag aatattcgct 360atccacagaa acgcctaaga tttctgtgtt
aagttcagaa aaatcagaat acttatcact 420gaacgcggtt atttctgtag
ggcaaacaaa agtaaaatct aaagggtaaa aaaataagat 480gacatactta
tttttaaagt cagataattt tattgtttta aattcttggt cataaacagc
540tgtagctgaa aagtcaggcg cgatctggcc tacttgaaga caattgtgtc
ctgaaatcat 60046199PRTPorphyra purpurea 46Met Ile Ser Gly His Asn
Cys Leu Gln Val Gly Gln Ile Ala Pro Asp 1 5 10 15 Phe Ser Ala Thr
Ala Val Tyr Asp Gln Glu Phe Lys Thr Ile Lys Leu 20 25 30 Ser Asp
Phe Lys Asn Lys Tyr Val Ile Leu Phe Phe Tyr Pro Leu Asp 35 40 45
Phe Thr Phe Val Cys Pro Thr Glu Ile Thr Ala Phe Ser Asp Lys Tyr 50
55 60 Ser Asp Phe Ser Glu Leu Asn Thr Glu Ile Leu Gly Val Ser Val
Asp 65 70 75 80 Ser Glu Tyr Ser His Leu Ala Trp Leu Gln Thr Asp Arg
Glu Ser Gly 85 90 95 Gly Leu Gly Asp Leu Glu Tyr Pro Leu Val Ser
Asp Leu Lys Lys Glu 100 105 110 Ile Ser Ile Ala Tyr Asn Val Leu Asn
Ser Gly Gly Val Ala Leu Arg 115 120 125 Gly Leu Phe Ile Ile Asp Pro
Lys Gly Ile Ile Gln Tyr Ser Thr Val 130 135 140 Asn Asn Leu Glu Phe
Gly Arg Ser Val Glu Glu Thr Leu Arg Val Leu 145 150 155 160 Gln Ala
Ile Gln Tyr Val Gln Ala His Pro Asp Glu Val Cys Pro Ala 165 170 175
Asn Trp Lys Pro Gly Asp Arg Thr Met Asn Pro Asp Pro Ile Lys Ser 180
185 190 Lys Asn Tyr Phe Ala Ala Ala 195 47681DNAGracilaria
tenuistipitata 47atgttattat gttgttttat tactgttatt ttatataata
tagacaatac taaattttat 60aacaggaagt gtagtattaa aatgataaca aataataata
ttttgagagt tggtcaacaa 120gcccccaatt tttctgctat tgctgtatat
gatcaagagt ttaagaaaat aacactgtct 180gattacttgg gtaagtatgt
aatattactg ttttatcctt tagatttcac atttgtttgt 240ccaactgaga
tcactgcttt cagtgattca tataaagaga ttcaaagtct gaatacagaa
300gttttgggta tatctgttga cagtgaatat tcacatttag catggttgca
aatggaaaga 360gatattggag gcttaggaga tcttaattac ccgttagttt
ctgatttaac aaaacagatt 420agtgcttcat ataatgttct aacagaagaa
ggtaaagcat taagaggttt atttattgtt 480gatcagcaag gaattataca
atattcttta gttaataatt tagactttgg ccgtagtatt 540agtgaaacta
taagaacact taaagctatc caatatgtac aatctcaccc agatgaagtt
600tgtccagcaa attggcagcc aggaaaagct actataatta atagtcctca
aaaatcgaaa 660aattattttc aatctatata g 68148226PRTGracilaria
tenuistipitata 48Met Leu Leu Cys Cys Phe Ile Thr Val Ile Leu Tyr
Asn Ile Asp Asn 1 5 10 15 Thr Lys Phe Tyr Asn Arg Lys Cys Ser Ile
Lys Met Ile Thr Asn Asn 20 25 30 Asn Ile Leu Arg Val Gly Gln Gln
Ala Pro Asn Phe Ser Ala Ile Ala 35 40 45 Val Tyr Asp Gln Glu Phe
Lys Lys Ile Thr Leu Ser Asp Tyr Leu Gly 50 55 60 Lys Tyr Val Ile
Leu Leu Phe Tyr Pro Leu Asp Phe Thr Phe Val Cys 65 70 75 80 Pro Thr
Glu Ile Thr Ala Phe Ser Asp Ser Tyr Lys Glu Ile Gln Ser 85 90 95
Leu Asn Thr Glu Val Leu Gly Ile Ser Val Asp Ser Glu Tyr Ser His 100
105 110 Leu Ala Trp Leu Gln Met Glu Arg Asp Ile Gly Gly Leu Gly Asp
Leu 115 120 125 Asn Tyr Pro Leu Val Ser Asp Leu Thr Lys Gln Ile Ser
Ala Ser Tyr 130 135 140 Asn Val Leu Thr Glu Glu Gly Lys Ala Leu Arg
Gly Leu Phe Ile Val 145 150 155 160 Asp Gln Gln Gly Ile Ile Gln Tyr
Ser Leu Val Asn Asn Leu Asp Phe 165 170 175 Gly Arg Ser Ile Ser Glu
Thr Ile Arg Thr Leu Lys Ala Ile Gln Tyr 180 185 190 Val Gln Ser His
Pro Asp Glu Val Cys Pro Ala Asn Trp Gln Pro Gly 195 200 205 Lys Ala
Thr Ile Ile Asn Ser Pro Gln Lys Ser Lys Asn Tyr Phe Gln 210 215 220
Ser Ile 225 49924DNAMus musculus 49gcctagggct ctctcggttt cgagatctct
ttcctgtctc taaccgtgtc tggaagtcca 60tgtgtccggc tcttgttcac gcagtaatgg
cctccggcaa cgcgcaaatc ggaaagtcgg 120ctcctgactt cacggccaca
gcggtggtgg atggtgcctt caaggaaatc aagctttcgg 180actacagagg
gaagtacgtg gtcctctttt tctacccact ggacttcact tttgtttgcc
240ccacggagat catcgctttt agcgaccatg ctgaggactt ccgaaagcta
ggctgcgagg 300tgctgggagt gtctgtggac tctcagttca cccacctggc
gtggatcaat accccacgga 360aagagggagg cttgggcccc ctgaatatcc
ctctgcttgc tgacgtgact aaaagcttgt 420cccagaatta cggcgtgttg
aaaaatgatg agggcattgc ttacaggggt ctctttatca 480tcgatgccaa
gggtgtcctt cgccagatca cagtcaatga cctacctgtg ggccgctctg
540tagacgaggc tctccgccta gtccaggcct ttcagtatac agacgagcat
ggggaagtct 600gccctgctgg ctggaagccc ggcagtgaca ccatcaagcc
caatgtggat gacagcaagg 660aatacttctc caaacacaac tgagatgggt
aaacatcggt gagcctgaag cttggatttc 720acctgtgccc caacctggat
gtcctgtgct ggcccagaaa atgctagatt ttcctccact 780ctctgaaggg
gctggagtct aggctgaggc tttctcatta cccacctgga atctggtgaa
840tagtgatcct gccctgagca cacctagctg ggcccaggtc tataggaaac
caataaagta 900ttagggacag tgtaaaaaaa aaaa 92450198PRTMus musculus
50Met Ala Ser Gly Asn Ala Gln Ile Gly Lys Ser Ala Pro Asp Phe Thr 1
5 10 15 Ala Thr Ala Val Val Asp Gly Ala Phe Lys Glu Ile Lys Leu Ser
Asp 20 25 30 Tyr Arg Gly
Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp Phe Thr 35 40 45 Phe
Val Cys Pro Thr Glu Ile Ile Ala Phe Ser Asp His Ala Glu Asp 50 55
60 Phe Arg Lys Leu Gly Cys Glu Val Leu Gly Val Ser Val Asp Ser Gln
65 70 75 80 Phe Thr His Leu Ala Trp Ile Asn Thr Pro Arg Lys Glu Gly
Gly Leu 85 90 95 Gly Pro Leu Asn Ile Pro Leu Leu Ala Asp Val Thr
Lys Ser Leu Ser 100 105 110 Gln Asn Tyr Gly Val Leu Lys Asn Asp Glu
Gly Ile Ala Tyr Arg Gly 115 120 125 Leu Phe Ile Ile Asp Ala Lys Gly
Val Leu Arg Gln Ile Thr Val Asn 130 135 140 Asp Leu Pro Val Gly Arg
Ser Val Asp Glu Ala Leu Arg Leu Val Gln 145 150 155 160 Ala Phe Gln
Tyr Thr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp 165 170 175 Lys
Pro Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu 180 185
190 Tyr Phe Ser Lys His Asn 195 51877DNARattus norvegicus
51gaattcggca cgagggtcgt ccgcgtgtcc ggctcttgcc cacgcagtca tggcctccgg
60caacgcgcac atcggaaagc ctgcccctga cttcacgggc accgccgtgg tggatggtgc
120ctttaaggaa atcaagcttt cagactacag agggaagtac gtggtcctct
ttttctatcc 180actggacttc acttttgttt gccccacgga gatcatcgct
tttagcgacc acgctgagga 240cttccgaaag ctaggctgcg aggtgctggg
agtgtctgtg gactctcagt tcacccacct 300ggcctggatc aataccccac
ggaaggaggg aggcttgggc ccactgaata tccctctgct 360tgctgatgtg
actaaaagct tgtcccagaa ttacggcgtg ttgaaaaatg atgagggcat
420cgcttacagg ggcctcttta tcatcgatgc caagggtgtc cttcgccaga
tcacagtcaa 480cgacctacct gtgggacgct ctgtagatga ggctctccgc
ctcgtccagg cctttcagta 540tacagatgag catggggaag tctgtcctgc
tggctggaag cccggcagtg acaccatcaa 600acccaatgtg gatgacagca
aggaatactt ctccaaacac aactgagatg ggtaaacatc 660ggtgagcctg
aatcccggat ctcacctgcg cccttacctg gatgtcctgt gctggcccag
720aaaacgctag atcttcctct acattctaaa ggggctggag gctaggccga
ggctttctca 780ttacccacct ggaatctggt gaatagtgac cctgccctga
gcacacccag ctgggcccag 840gtctatagga aaccaataaa gtattaggga cagtgta
87752198PRTRattus norvegicus 52Met Ala Ser Gly Asn Ala His Ile Gly
Lys Pro Ala Pro Asp Phe Thr 1 5 10 15 Gly Thr Ala Val Val Asp Gly
Ala Phe Lys Glu Ile Lys Leu Ser Asp 20 25 30 Tyr Arg Gly Lys Tyr
Val Val Leu Phe Phe Tyr Pro Leu Asp Phe Thr 35 40 45 Phe Val Cys
Pro Thr Glu Ile Ile Ala Phe Ser Asp His Ala Glu Asp 50 55 60 Phe
Arg Lys Leu Gly Cys Glu Val Leu Gly Val Ser Val Asp Ser Gln 65 70
75 80 Phe Thr His Leu Ala Trp Ile Asn Thr Pro Arg Lys Glu Gly Gly
Leu 85 90 95 Gly Pro Leu Asn Ile Pro Leu Leu Ala Asp Val Thr Lys
Ser Leu Ser 100 105 110 Gln Asn Tyr Gly Val Leu Lys Asn Asp Glu Gly
Ile Ala Tyr Arg Gly 115 120 125 Leu Phe Ile Ile Asp Ala Lys Gly Val
Leu Arg Gln Ile Thr Val Asn 130 135 140 Asp Leu Pro Val Gly Arg Ser
Val Asp Glu Ala Leu Arg Leu Val Gln 145 150 155 160 Ala Phe Gln Tyr
Thr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp 165 170 175 Lys Pro
Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu 180 185 190
Tyr Phe Ser Lys His Asn 195 53 1089DNAOryza sativa 53gccttcgtct
cgacacgttt gcattgcagc agctcataag tttcttcttc gtttctgctc 60ccagtgctaa
ggcagcacag tcgttcgtcg ccatgccagg gctcaccatc ggcgacaccg
120tccccaacct ggagctggac tccacccacg gcaagatccg catccacgac
ttcgtcggcg 180acacctatgt catcctcttc tcccaccccg gcgacttcac
cccggtctgc accacggagc 240tggcagccat ggccggctac gccaaggagt
tcgacaagag gggcgtcaag ctgctcggca 300tctcctgcga cgacgtgcag
tctcacaagg actggatcaa ggacatcgag gcctacaagc 360ctgggaaccg
cgtgacgtac ccgatcatgg ccgatccgag ccgcgaggcc atcaagcagc
420tgaacatggt cgacccggac gagaaggatt ccaacggcgg ccacctcccg
tcccgcgcgc 480tgcacatcgt cggccccgac aagaaggtga agctgagctt
cctgtacccg gcgtgcgtgg 540ggcggaacat ggatgaggtg gtgcgtgcgg
tcgacgcgct gcagacggcg gcgaagcacg 600cggtggcgac gccggtgaac
tggaagcccg gcgagcgcgt cgtcatccct cccggcgtct 660ccgacgacga
ggcgaaggag aagttccccc aggggttcga caccgccgac ctgccgtccg
720gcaagggcta cctccgcttc accaaggtcg gctagatcat atcgatatcg
acctcgctct 780tcgtacatca tgtgcgccac gcgtgcgtga tagcgtgtgc
tggcgtgatg actatgcgag 840atgcatccct gtgtgtgttg gtgtggataa
tgccgctacg tttggaacag tagtgcattt 900actctgtgct actgtctgaa
ctttggctgt ttggcagact gtttatgtac ccgtatgttc 960gcccctgtac
taatagagtg ggtgttgtgg ttggcaagta ctctcctcgg acaacatttt
1020aactttgact actaataaca aacaaattaa aaagatcaat cagatgttac
tagacatctt 1080aattttatt 108954220PRTOryza sativa 54Met Pro Gly Leu
Thr Ile Gly Asp Thr Val Pro Asn Leu Glu Leu Asp 1 5 10 15 Ser Thr
His Gly Lys Ile Arg Ile His Asp Phe Val Gly Asp Thr Tyr 20 25 30
Val Ile Leu Phe Ser His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35
40 45 Glu Leu Ala Ala Met Ala Gly Tyr Ala Lys Glu Phe Asp Lys Arg
Gly 50 55 60 Val Lys Leu Leu Gly Ile Ser Cys Asp Asp Val Gln Ser
His Lys Asp 65 70 75 80 Trp Ile Lys Asp Ile Glu Ala Tyr Lys Pro Gly
Asn Arg Val Thr Tyr 85 90 95 Pro Ile Met Ala Asp Pro Ser Arg Glu
Ala Ile Lys Gln Leu Asn Met 100 105 110 Val Asp Pro Asp Glu Lys Asp
Ser Asn Gly Gly His Leu Pro Ser Arg 115 120 125 Ala Leu His Ile Val
Gly Pro Asp Lys Lys Val Lys Leu Ser Phe Leu 130 135 140 Tyr Pro Ala
Cys Val Gly Arg Asn Met Asp Glu Val Val Arg Ala Val 145 150 155 160
Asp Ala Leu Gln Thr Ala Ala Lys His Ala Val Ala Thr Pro Val Asn 165
170 175 Trp Lys Pro Gly Glu Arg Val Val Ile Pro Pro Gly Val Ser Asp
Asp 180 185 190 Glu Ala Lys Glu Lys Phe Pro Gln Gly Phe Asp Thr Ala
Asp Leu Pro 195 200 205 Ser Gly Lys Gly Tyr Leu Arg Phe Thr Lys Val
Gly 210 215 220 55465DNAOryza sativa 55atggcgtgcg ccttctccgt
ctcctctgcc gcggcgcctc tcgcctcccc gaagggggac 60ctgccgttgg tcgggaacaa
ggcgccggac ttcgaggcgg aggccatgtt cgaccagggg 120ttcatcaagt
ctaaatgcat gtttgtaagc tctgcagaga tcactgcttt cagcgacaga
180tatgaggagt ttgagaagat aaatactgaa gttctcggtg tttcgattga
cagtgtgggg 240attgctctga gaggattatt catcattgac aaggagggtg
tgattcagca ttctaccatt 300aacaaccttg ctattggccg tagcgtggat
gagacgctta ggacccttca ggccctacag 360tatgtccaag aaaacccgga
tgaggtttgc ccagctggat ggaaacctgg ggagaagtca 420atgaagcctg
accccaagga cagcaaggag gaacaagaat gctga 46556154PRTOryza sativa
56Met Ala Cys Ala Phe Ser Val Ser Ser Ala Ala Ala Pro Leu Ala Ser 1
5 10 15 Pro Lys Gly Asp Leu Pro Leu Val Gly Asn Lys Ala Pro Asp Phe
Glu 20 25 30 Ala Glu Ala Met Phe Asp Gln Gly Phe Ile Lys Ser Lys
Cys Met Phe 35 40 45 Val Ser Ser Ala Glu Ile Thr Ala Phe Ser Asp
Arg Tyr Glu Glu Phe 50 55 60 Glu Lys Ile Asn Thr Glu Val Leu Gly
Val Ser Ile Asp Ser Val Gly 65 70 75 80 Ile Ala Leu Arg Gly Leu Phe
Ile Ile Asp Lys Glu Gly Val Ile Gln 85 90 95 His Ser Thr Ile Asn
Asn Leu Ala Ile Gly Arg Ser Val Asp Glu Thr 100 105 110 Leu Arg Thr
Leu Gln Ala Leu Gln Tyr Val Gln Glu Asn Pro Asp Glu 115 120 125 Val
Cys Pro Ala Gly Trp Lys Pro Gly Glu Lys Ser Met Lys Pro Asp 130 135
140 Pro Lys Asp Ser Lys Glu Glu Gln Glu Cys 145 150 571085DNAOryza
sativa 57cattccatca cagacagttc gcagaatcgc agcagcttag cttaattact
tttttcacca 60actcaacttt cagttaattt ccggttaatc ctcgattcct catcatgcct
ggactcaccc 120tcggcgacgt cgtccccgac ctggagctcg acaccaccca
cggcaagatc cgcctccacg 180acttcgtcgg cgacgcctac gtcatcatct
tctcccaccc cgctgacttc acgccggtct 240gcacgacgga gctgtcggag
atggcgggct acgccggcga gttcgacaag aggggcgtca 300agctcctcgg
cttctcctgc gacgacgtcg agtcgcacaa ggactggatc aaggacatcg
360aggcctacaa gcctggccgc cgcgtcggct tcccgatcgt cgccgacccg
gacagggagg 420cgatcaggca gctcaacatg atcgacgccg acgagaagga
caccgccggc ggcgagctcc 480ccaaccgggc gctccacatc gtcgggccgg
acaagaaggt gaagctgagc ttcctgttcc 540cggcgtgcac ggggcggaac
atggcggagg tgctgcgcgc gacggacgcg ctgctgacgg 600cggcgaggca
ccgggtggcg acgccggtga actggaagcc cggcgagcgc gtcgtcatcc
660cccccggcgt ctccgacgag gaggccaagg cgaggttccc ggccgggttc
gagaccgccc 720agctgccctc caacaagtgc tacctccgct tcacccaggt
ggactgagag actgatggtg 780agggagggag ggagagatct gggccgtcgg
tttcgtgtgt aataaaccaa cgcacgacgt 840agatgcttcc acgtgtgtgt
ttcccgtgct gcttcgattg atcgatcgat cattcggtaa 900gtactctagt
tatgtgtaat ctgctgtttg ggtgtagtgg tgcatttgct gttctgttgc
960ctgaaagtga cgaacggatt atgtttgtca tttgtatgta aaatgtaacc
gtatgttttt 1020tatttatccc ttccgaaatt actgtggaat atagtgaagt
aatgctgtta ataaacagcc 1080cgttt 108558220PRTOryza sativa 58Met Pro
Gly Leu Thr Leu Gly Asp Val Val Pro Asp Leu Glu Leu Asp 1 5 10 15
Thr Thr His Gly Lys Ile Arg Leu His Asp Phe Val Gly Asp Ala Tyr 20
25 30 Val Ile Ile Phe Ser His Pro Ala Asp Phe Thr Pro Val Cys Thr
Thr 35 40 45 Glu Leu Ser Glu Met Ala Gly Tyr Ala Gly Glu Phe Asp
Lys Arg Gly 50 55 60 Val Lys Leu Leu Gly Phe Ser Cys Asp Asp Val
Glu Ser His Lys Asp 65 70 75 80 Trp Ile Lys Asp Ile Glu Ala Tyr Lys
Pro Gly Arg Arg Val Gly Phe 85 90 95 Pro Ile Val Ala Asp Pro Asp
Arg Glu Ala Ile Arg Gln Leu Asn Met 100 105 110 Ile Asp Ala Asp Glu
Lys Asp Thr Ala Gly Gly Glu Leu Pro Asn Arg 115 120 125 Ala Leu His
Ile Val Gly Pro Asp Lys Lys Val Lys Leu Ser Phe Leu 130 135 140 Phe
Pro Ala Cys Thr Gly Arg Asn Met Ala Glu Val Leu Arg Ala Thr 145 150
155 160 Asp Ala Leu Leu Thr Ala Ala Arg His Arg Val Ala Thr Pro Val
Asn 165 170 175 Trp Lys Pro Gly Glu Arg Val Val Ile Pro Pro Gly Val
Ser Asp Glu 180 185 190 Glu Ala Lys Ala Arg Phe Pro Ala Gly Phe Glu
Thr Ala Gln Leu Pro 195 200 205 Ser Asn Lys Cys Tyr Leu Arg Phe Thr
Gln Val Asp 210 215 220 591248DNAOryza sativa 59atcgattccc
ccaacatatt agggctcacg cctcccaaag tcaaaacagc ccagcccgaa 60caagcatttc
ctcgaacact tcgccctcca ccaccatggc cgccgccgcc tccaccctcg
120cctccctctc cgccaccgcg gccgcggccg ccggcaagcg cctcctcctc
tcctccccct 180cccgctccct ctccctctcc ctcgcctccc gcggccgcat
cgccgtcatg ccccacctcc 240gcgctggcat cctctccgcc gcaccgagga
gggcggtgtc ggcctcggcc ccggccgcgg 300ccaccatcgc ggtcggggac
aagctccccg acgcgacgct ctcctacttc gactcgcccg 360acggggagct
gaagacggtg accgtgcgcg acctcaccgc cgggaagaag gtggtcctct
420tcgcggtccc cggcgcgttc accccgacct gcacgcagaa gcacgtcccg
gggttcgtcg 480ccaaggccgg ggagctccgc gccaaggggg tcgacgccgt
ggcctgcgtc tccgtcaacg 540acgcgttcgt gatgcgggcg tggaaggaga
gcctcggcgt cggcgacgag gtgctcctcc 600tctccgacgg caacggcgag
ctcgcccgcg ccatgggcgt cgagctcgac ctctccgaca 660agcccgccgg
cctcggcgtg cggtcccgcc gctacgcgct cctggcggag gacggcgtcg
720tcaaggtgct caacctcgag gagggcggcg ccttcaccac cagcagcgcc
gaggagatgc 780tcaaggcgct ctgaagcgtg aacaactcaa gccatcctcc
acttttcatc tcaaatctcc 840atagctcggt tcgttgccta cttctctcaa
gtgttcgctt cttttcctga ataataaatc 900atggcaacaa tggtggaccg
tgcagagtag tgttgtcgtt ttgatgtgtg aagcttctat 960agcgaacata
gtgtgcaatt tttaggtaac atatatgagt cttggccttg cactgtttgt
1020caggtagtaa caacttggca cagctataga ctgtagtaac agagttcctt
tcatgttgaa 1080tggtgaggct gtgatgtgtt ctagagctga ataaacgtgc
tctggtaaat actgtcacca 1140gatcagacta tggagtagta gtaagatttt
gcttggttaa ttgggcaatg gctatttttc 1200aggatcgttc agttgagata
aacatgtttt gctgttcaga tgagttcg 124860232PRTOryza sativa 60Met Ala
Ala Ala Ala Ser Thr Leu Ala Ser Leu Ser Ala Thr Ala Ala 1 5 10 15
Ala Ala Ala Gly Lys Arg Leu Leu Leu Ser Ser Pro Ser Arg Ser Leu 20
25 30 Ser Leu Ser Leu Ala Ser Arg Gly Arg Ile Ala Val Met Pro His
Leu 35 40 45 Arg Ala Gly Ile Leu Ser Ala Ala Pro Arg Arg Ala Val
Ser Ala Ser 50 55 60 Ala Pro Ala Ala Ala Thr Ile Ala Val Gly Asp
Lys Leu Pro Asp Ala 65 70 75 80 Thr Leu Ser Tyr Phe Asp Ser Pro Asp
Gly Glu Leu Lys Thr Val Thr 85 90 95 Val Arg Asp Leu Thr Ala Gly
Lys Lys Val Val Leu Phe Ala Val Pro 100 105 110 Gly Ala Phe Thr Pro
Thr Cys Thr Gln Lys His Val Pro Gly Phe Val 115 120 125 Ala Lys Ala
Gly Glu Leu Arg Ala Lys Gly Val Asp Ala Val Ala Cys 130 135 140 Val
Ser Val Asn Asp Ala Phe Val Met Arg Ala Trp Lys Glu Ser Leu 145 150
155 160 Gly Val Gly Asp Glu Val Leu Leu Leu Ser Asp Gly Asn Gly Glu
Leu 165 170 175 Ala Arg Ala Met Gly Val Glu Leu Asp Leu Ser Asp Lys
Pro Ala Gly 180 185 190 Leu Gly Val Arg Ser Arg Arg Tyr Ala Leu Leu
Ala Glu Asp Gly Val 195 200 205 Val Lys Val Leu Asn Leu Glu Glu Gly
Gly Ala Phe Thr Thr Ser Ser 210 215 220 Ala Glu Glu Met Leu Lys Ala
Leu 225 230 61946DNAOryza sativa 61acacccaaac ccgacgaaca gccgcagctg
caggccacgc atcctcgccg tgaatctccc 60accgcgctcc ggcgatggca ttcgcggtct
ccaccgcctg caggccgtcc ctgctcctgc 120ccccgcgcca gcgctcgtcg
ccgccgcggc cgcggccgct cctctgcacg ccctccaccg 180ccgccttccg
ccgcggcgcc ctcagcgcga caacaacgcc aacgccggcg cgcgcagcac
240tgccgtcgac gacggggagg aacaggatcg tctgcggcaa ggtgagcaag
ggcagcgcgg 300cgcccaactt cacgctgagg gaccaggacg ggagggcggt
gtcgctgtcc aagttcaagg 360ggaggccggt ggtggtgtac ttctaccccg
ccgacgagac ccccggatgc accaagcagg 420cctgcgcctt ccgcgactcc
tacgagaagt tcaagaaggc cggcgccgag gtcatcggca 480tcagcggcga
cgacgccgcc tcccacaagg agttcaagaa gaagtacaag ctgccgttca
540cgctgctgag cgacgagggg aacaaggtga ggaaggagtg gggtgtgccg
gctgacctgt 600tcgggacgct gccgggaagg cagacgtacg tgctcgacaa
gaacggcgtc gtccagtaca 660tctacaacaa ccagttccag cccgagaagc
acattggcga gaccctcaag atcctccaga 720gcctctgatt ctcttcttct
tcttcctcct tttttaacta caatctctca tgtatgatcc 780atcacagtat
accgagaaat taatccatct gttaatctct tctcgatcgt ttttctccct
840cggcatgtgt atagctagtg tatctgtaac tctgtgagta tatatacagt
caaaatcggt 900gggctgctag ctctgaattt tgccgtaagg cactctgatt ttctct
94662217PRTOryza sativa 62Met Ala Phe Ala Val Ser Thr Ala Cys Arg
Pro Ser Leu Leu Leu Pro 1 5 10 15 Pro Arg Gln Arg Ser Ser Pro Pro
Arg Pro Arg Pro Leu Leu Cys Thr 20 25 30 Pro Ser Thr Ala Ala Phe
Arg Arg Gly Ala Leu Ser Ala Thr Thr Thr 35 40 45 Pro Thr Pro Ala
Arg Ala Ala Leu Pro Ser Thr Thr Gly Arg Asn Arg 50 55 60 Ile Val
Cys Gly Lys Val Ser Lys Gly Ser Ala Ala Pro Asn Phe Thr 65 70 75 80
Leu Arg Asp Gln Asp Gly Arg Ala Val Ser Leu Ser Lys Phe Lys Gly 85
90 95 Arg Pro Val Val Val Tyr Phe Tyr Pro Ala Asp Glu Thr Pro Gly
Cys 100 105 110 Thr Lys Gln Ala Cys Ala Phe Arg Asp Ser Tyr Glu Lys
Phe Lys Lys 115 120 125 Ala Gly Ala Glu Val Ile Gly Ile Ser Gly Asp
Asp Ala Ala Ser His 130 135 140 Lys Glu Phe Lys Lys Lys Tyr Lys Leu
Pro Phe Thr Leu Leu Ser Asp 145 150 155 160 Glu Gly Asn Lys Val Arg
Lys Glu Trp Gly Val Pro Ala Asp Leu Phe 165 170 175 Gly Thr Leu
Pro Gly Arg Gln Thr Tyr Val Leu Asp Lys Asn Gly Val 180 185 190 Val
Gln Tyr Ile Tyr Asn Asn Gln Phe Gln Pro Glu Lys His Ile Gly 195 200
205 Glu Thr Leu Lys Ile Leu Gln Ser Leu 210 215 63770DNAOryza
sativa 63atctcccttc cctgtcgatt accttctctc cttcctctgt tcctcctctc
ctccacacat 60ccaggcaggc aacacaagaa tcatccggga gagcgacatg gccccggttg
ccgtgggcga 120caccctcccc gacggccagc tggggtggtt cgacggggag
gacaagctgc agcaggtctc 180cgtccacggc ctcgccgccg gcaagaaggt
cgtcctcttc ggcgtccccg gtgccttcac 240cccgacctgc agcaatcagc
atgtgccagg attcataaat caggctgagc agctcaaagc 300caagggtgta
gacgacatct tgcttgtcag tgttaacgac ccctttgtca tgaaggcgtg
360ggcaaagtca taccctgaga ataagcatgt gaaattcctt gccgatggtt
tgggaacata 420caccaaggca cttggtcttg agcttgacct ttcggagaaa
gggcttggta ttcgttcgag 480acggtttgct ctccttgctg acaacctcaa
ggttactgtt gcaaacattg aggaaggtgg 540ccaattcaca atctctggtg
ctgaggagat cctcaaggca ctgtaagagc ttcagctctt 600aggaacggca
gcgatcactt ggacctatcg tgtcaatctt gtttaaattt gtctgcaaaa
660tacttgtgcg aataaaattg tcgatgagct gcctagttgt gaggacttta
tgataatgtt 720tgaatctgta tccactgttg aatcaagtag taatgttcag
tgctcatgtt 77064162PRTOryza sativa 64Met Ala Pro Val Ala Val Gly
Asp Thr Leu Pro Asp Gly Gln Leu Gly 1 5 10 15 Trp Phe Asp Gly Glu
Asp Lys Leu Gln Gln Val Ser Val His Gly Leu 20 25 30 Ala Ala Gly
Lys Lys Val Val Leu Phe Gly Val Pro Gly Ala Phe Thr 35 40 45 Pro
Thr Cys Ser Asn Gln His Val Pro Gly Phe Ile Asn Gln Ala Glu 50 55
60 Gln Leu Lys Ala Lys Gly Val Asp Asp Ile Leu Leu Val Ser Val Asn
65 70 75 80 Asp Pro Phe Val Met Lys Ala Trp Ala Lys Ser Tyr Pro Glu
Asn Lys 85 90 95 His Val Lys Phe Leu Ala Asp Gly Leu Gly Thr Tyr
Thr Lys Ala Leu 100 105 110 Gly Leu Glu Leu Asp Leu Ser Glu Lys Gly
Leu Gly Ile Arg Ser Arg 115 120 125 Arg Phe Ala Leu Leu Ala Asp Asn
Leu Lys Val Thr Val Ala Asn Ile 130 135 140 Glu Glu Gly Gly Gln Phe
Thr Ile Ser Gly Ala Glu Glu Ile Leu Lys 145 150 155 160 Ala Leu
651249DNAOryza sativa 65gagagccaac acggtgcatc tctcagccac acagccccac
ccgcgccatg tcactcgcca 60ccgccgccgc cggagcgcaa ccgttcgtcc gctcctcctc
ctccgccgcc gcggcgtcct 120cgtcgcggcc cctgctcgcc gtcgccgccg
cccgccaccg ccgcccgcat ggatctctcg 180ccgccgccgc cgccgcggca
aggcggcgtc gtcgtcgtcc gctcctccag gtgcgcgcgg 240caaggacgga
gtccacgggc gtctccgtcg ggttccgcgc gccccagttc gagctcccgg
300agccactgac ggggaagctc tggacattgg atgacttcga aggcaacccc
gcgctgctgg 360ttatgtttgt atgtaatcac tgtccattcg taaagcatct
caaaaaagat attgcgaagc 420tcacctcatt ctacatggag aaagggcttg
ctgctgttgc catatcctcg aactcaattg 480tgacacaccc acaggatggt
cctgattaca tagctgagga agcaaaattg tataaatact 540ctttcccgta
tctatatgat gagtctcaag aagttgctaa agcttttcga gccgtctgca
600cgccagagtt ttacttgttc aaaaaggatg gacgaaggcc atttgagctt
ttctaccatg 660ggcagtttga cgattcaaga ccgagtaaca acgtgccagt
taccggaagg gatttaagtc 720gtgcgattga ttgtgcactt agtggacaag
agctaccttt tgtgccaaaa cccagtgtcg 780ggtgcagcat caaatggcac
ccatgaagag cgtattgcat tgtcatgtgc tggaatatag 840atgtttttcc
cccttaaatt gaaggttgaa catggggatt gaggtgagcc atgctctcta
900ctactagaag tatggaagca cacacatagt agatttatga tagctaattt
cacatagtag 960atttatgata gctaatttat aatgtaattt ttaagggaaa
tagatgcagt tgaggccttg 1020tggagctgat tcttaacgtt gtgggggctg
ttcaacttga gagttgcaaa actagacatg 1080aatggcgtgg atagtgttat
gttgtgtgct ggtgtctcat cttggccgga aaaagaaaaa 1140ctgatggatg
taactggtat ttgtgcaaca atgggataat gcacacaagt acaataaccc
1200attattatgg ctaacacaac acccacgggt gaaaattaaa gatgagggc
124966252PRTOryza sativa 66Met Ser Leu Ala Thr Ala Ala Ala Gly Ala
Gln Pro Phe Val Arg Ser 1 5 10 15 Ser Ser Ser Ala Ala Ala Ala Ser
Ser Ser Arg Pro Leu Leu Ala Val 20 25 30 Ala Ala Ala Arg His Arg
Arg Pro His Gly Ser Leu Ala Ala Ala Ala 35 40 45 Ala Ala Ala Arg
Arg Arg Arg Arg Arg Pro Leu Leu Gln Val Arg Ala 50 55 60 Ala Arg
Thr Glu Ser Thr Gly Val Ser Val Gly Phe Arg Ala Pro Gln 65 70 75 80
Phe Glu Leu Pro Glu Pro Leu Thr Gly Lys Leu Trp Thr Leu Asp Asp 85
90 95 Phe Glu Gly Asn Pro Ala Leu Leu Val Met Phe Val Cys Asn His
Cys 100 105 110 Pro Phe Val Lys His Leu Lys Lys Asp Ile Ala Lys Leu
Thr Ser Phe 115 120 125 Tyr Met Glu Lys Gly Leu Ala Ala Val Ala Ile
Ser Ser Asn Ser Ile 130 135 140 Val Thr His Pro Gln Asp Gly Pro Asp
Tyr Ile Ala Glu Glu Ala Lys 145 150 155 160 Leu Tyr Lys Tyr Ser Phe
Pro Tyr Leu Tyr Asp Glu Ser Gln Glu Val 165 170 175 Ala Lys Ala Phe
Arg Ala Val Cys Thr Pro Glu Phe Tyr Leu Phe Lys 180 185 190 Lys Asp
Gly Arg Arg Pro Phe Glu Leu Phe Tyr His Gly Gln Phe Asp 195 200 205
Asp Ser Arg Pro Ser Asn Asn Val Pro Val Thr Gly Arg Asp Leu Ser 210
215 220 Arg Ala Ile Asp Cys Ala Leu Ser Gly Gln Glu Leu Pro Phe Val
Pro 225 230 235 240 Lys Pro Ser Val Gly Cys Ser Ile Lys Trp His Pro
245 250 67994DNAOryza sativa 67aaatcccaac agagaagcat ttcctcgaac
acatcgccgt cgccgccctc catggccgct 60cccaccgcag cagctctctc caccctctcc
accgccagcg tcacctccgg caagcgcttc 120atcacctcct ccttctccct
ctccttctcc tcccgccccc tcgcaacagg cgtccgcgcc 180gcgggggcga
gagcggcgcg gaggtcggcg gcgtcggcgt ccaccgtggt ggcgaccatc
240gccgtcggag acaagctccc cgacgcgacg ctgtcctact tcgacccggc
ggacggcgag 300ctgaagacgg tgacggtggc ggagctgacg gcgggcagga
aggcggtgct gttcgcggtg 360cccggcgcgt tcacgccgac gtgctcgcag
aagcacctcc cggggttcat cgagaaggcc 420ggggagctcc acgccaaggg
ggtggacgcc attgcctgcg tgtcggtgaa cgacgcgttc 480gtgatgcgcg
cgtggaagga gagcctgggc ctcggcgacg ccgacgtgct cctcctctcc
540gacggcaacc tggagctcac gcgcgcgctc ggcgtcgaga tggacctctc
cgacaagccc 600atggggctcg gcgtcaggtc gcgccgctac gcgctcctcg
ccgacgacgg cgtcgtcaag 660gtgctcaacc tcgaggaggg cggcgccttc
accaccagca gcgccgagga gatgctcaag 720gcgctctgaa gatggaatcc
gagctctcgt aggtggcaac aatggcagga tcagcccgtt 780gctctcgcgc
gttggtgagt agcgtcgtcg ttgtgaagag gaaattttgt gtgtgttttt
840tttcggttga atgttgcatg ccatgtgctt gacgaaatga cggaataaca
aaagaaaaaa 900aactactttt atttttttgt tgaaatttgc aaaccatgtg
tttgacgaaa tgtcgagata 960tgaaagctgt gaaatcgctt acgtcacgtg cacc
99468225PRTOryza sativa 68Met Ala Ala Pro Thr Ala Ala Ala Leu Ser
Thr Leu Ser Thr Ala Ser 1 5 10 15 Val Thr Ser Gly Lys Arg Phe Ile
Thr Ser Ser Phe Ser Leu Ser Phe 20 25 30 Ser Ser Arg Pro Leu Ala
Thr Gly Val Arg Ala Ala Gly Ala Arg Ala 35 40 45 Ala Arg Arg Ser
Ala Ala Ser Ala Ser Thr Val Val Ala Thr Ile Ala 50 55 60 Val Gly
Asp Lys Leu Pro Asp Ala Thr Leu Ser Tyr Phe Asp Pro Ala 65 70 75 80
Asp Gly Glu Leu Lys Thr Val Thr Val Ala Glu Leu Thr Ala Gly Arg 85
90 95 Lys Ala Val Leu Phe Ala Val Pro Gly Ala Phe Thr Pro Thr Cys
Ser 100 105 110 Gln Lys His Leu Pro Gly Phe Ile Glu Lys Ala Gly Glu
Leu His Ala 115 120 125 Lys Gly Val Asp Ala Ile Ala Cys Val Ser Val
Asn Asp Ala Phe Val 130 135 140 Met Arg Ala Trp Lys Glu Ser Leu Gly
Leu Gly Asp Ala Asp Val Leu 145 150 155 160 Leu Leu Ser Asp Gly Asn
Leu Glu Leu Thr Arg Ala Leu Gly Val Glu 165 170 175 Met Asp Leu Ser
Asp Lys Pro Met Gly Leu Gly Val Arg Ser Arg Arg 180 185 190 Tyr Ala
Leu Leu Ala Asp Asp Gly Val Val Lys Val Leu Asn Leu Glu 195 200 205
Glu Gly Gly Ala Phe Thr Thr Ser Ser Ala Glu Glu Met Leu Lys Ala 210
215 220 Leu 225 69959DNAOryza sativa 69tacctatgga gacggtcgcc
tcgctctcgc gcgccgcgct cgccggcgcg cccgccgcta 60cacgcgcgac agcgtcgccc
gtgaacaggg ccgtggtccc tgcggcgtcc cggccgcgcg 120ggggacgcct
ttgctgccga cgctcgctga cggccgtctc cgcggcggca ggggcttccc
180ctcccgtctc cccgtcgcct agccccgatg gcggctcccc cggcgtgtgg
gacgctctcg 240gcggcgtgtc cgtgctcgcc gccggcaccg gcgaagccgt
tcagctcagg gacctgtggg 300accccaccga gggggtggcc gtggtggcgc
tgctccggca cttcgggtgc ttctgctgct 360gggagctggc ctctgttctg
aaggaatcca tggcgaaatt cgacgctgcc ggggccaagc 420tgatcgccat
cggcgtcggg actcctgaca aagctcgcat tctcgccgat gggctgccgt
480tccctgttga tagcttgtac gctgaccccg agcgcaaggc ttacgacgta
ttggggcttt 540accatggtct gggtcggaca ttaatcagtc ctgcgaagat
gtactcgggg cttaattcca 600tcaagaaggt aaccaagaac tacacgctca
agggcacacc agcagacctg acgggtatct 660tgcagcaggg tggtatgctt
gtgttcagag ggaaagagtt gctgtactca tggaaagaca 720aaggcacggg
tgatcatgct cctctggatg atgtcctcaa cgcttgctgc aatcgaactt
780cttgaggtct ctagcagtcg gaagatgtgt atgtaaatat atgaaatgct
cagcatgcca 840aacagagagc aattagactc aacagtacta gatgttcgat
taattatgca ttgttggttt 900gcttatgtac ttagcatgat attggattag
ctacccaatg gacatgacac tacagtctg 95970259PRTOryza sativa 70Met Glu
Thr Val Ala Ser Leu Ser Arg Ala Ala Leu Ala Gly Ala Pro 1 5 10 15
Ala Ala Thr Arg Ala Thr Ala Ser Pro Val Asn Arg Ala Val Val Pro 20
25 30 Ala Ala Ser Arg Pro Arg Gly Gly Arg Leu Cys Cys Arg Arg Ser
Leu 35 40 45 Thr Ala Val Ser Ala Ala Ala Gly Ala Ser Pro Pro Val
Ser Pro Ser 50 55 60 Pro Ser Pro Asp Gly Gly Ser Pro Gly Val Trp
Asp Ala Leu Gly Gly 65 70 75 80 Val Ser Val Leu Ala Ala Gly Thr Gly
Glu Ala Val Gln Leu Arg Asp 85 90 95 Leu Trp Asp Pro Thr Glu Gly
Val Ala Val Val Ala Leu Leu Arg His 100 105 110 Phe Gly Cys Phe Cys
Cys Trp Glu Leu Ala Ser Val Leu Lys Glu Ser 115 120 125 Met Ala Lys
Phe Asp Ala Ala Gly Ala Lys Leu Ile Ala Ile Gly Val 130 135 140 Gly
Thr Pro Asp Lys Ala Arg Ile Leu Ala Asp Gly Leu Pro Phe Pro 145 150
155 160 Val Asp Ser Leu Tyr Ala Asp Pro Glu Arg Lys Ala Tyr Asp Val
Leu 165 170 175 Gly Leu Tyr His Gly Leu Gly Arg Thr Leu Ile Ser Pro
Ala Lys Met 180 185 190 Tyr Ser Gly Leu Asn Ser Ile Lys Lys Val Thr
Lys Asn Tyr Thr Leu 195 200 205 Lys Gly Thr Pro Ala Asp Leu Thr Gly
Ile Leu Gln Gln Gly Gly Met 210 215 220 Leu Val Phe Arg Gly Lys Glu
Leu Leu Tyr Ser Trp Lys Asp Lys Gly 225 230 235 240 Thr Gly Asp His
Ala Pro Leu Asp Asp Val Leu Asn Ala Cys Cys Asn 245 250 255 Arg Thr
Ser 711183DNAOryza sativa 71tccgcaacga cctccacacc gcaacggtcc
actcgctcgc ctccgtctcc ctcccgcgtc 60tagggtttcg ccacgtctca cgcagccatg
gccgcggcgg ccgcgtccac ctcgctcccc 120gtcccgcgcg tctccctccc
gccgtccgct cgcccagccg ccgctccccg gcacggtctc 180ctcatccccg
gtcgccgtgg gtgcttccgt ctccgcggct caccagcggc accggccgcc
240gccgcctcgg gctccccttc cgtgccttcc tcttccccgg aggctgggtc
gggcatcggg 300gatgccctcg gtggcgtcgc catctactcc gcggccaccg
gcgagcccgt gctgttcagg 360gacctgtggg accagaacga gggaatggct
gttgttgccc tgctaaggca ttttgggtgc 420ccttgctgtt gggagttggc
ctctgtgttg agggatacaa aagagagatt tgattcagct 480ggtgtcaagc
taatagccgt tggtgttggc actccagata aagcccgtat tcttgctgag
540cgtttaccat ttccattgga ctacctctac gcagatcctg agcgcaaggc
ctatgatctc 600ttgggtttgt attttggtat tggtcgcaca ttcttcaatc
cagccagtgc aagtgtgttt 660tcacgatttg actccctcaa ggaggcagtg
aagaactata caattgaagc caccccagat 720gatagggcta gtgttctaca
acagggtgga atgtttgtgt tcagagggaa agaattaata 780tatgcaagga
aagatgaggg cactggtgat catgcacctc tggatgatgt cctcaacatc
840tgttgtaaag cccctgcggc atgatattgt gtaatcaatg tcccatgaga
attttcatag 900cctggttctg ttcgtgtccc aaagttgtat gcagaaaagc
atctcttgat tttggaaggc 960tggcttctgc aaggatagta tctctttgtc
tgtacgtctg atctaccatg ctgttgatat 1020gtaatatatc agttgaaaac
ttgagggatg taggcagacc aaaggacttt ctcatgccat 1080aagctcagca
gttcttttcc ttttcctcat agaaatgtac taattataga agagaatctt
1140acactgtaca ataagtttgt gttaaagttg gcgaaatttt cct
118372258PRTOryza sativa 72Met Ala Ala Ala Ala Ala Ser Thr Ser Leu
Pro Val Pro Arg Val Ser 1 5 10 15 Leu Pro Pro Ser Ala Arg Pro Ala
Ala Ala Pro Arg His Gly Leu Leu 20 25 30 Ile Pro Gly Arg Arg Gly
Cys Phe Arg Leu Arg Gly Ser Pro Ala Ala 35 40 45 Pro Ala Ala Ala
Ala Ser Gly Ser Pro Ser Val Pro Ser Ser Ser Pro 50 55 60 Glu Ala
Gly Ser Gly Ile Gly Asp Ala Leu Gly Gly Val Ala Ile Tyr 65 70 75 80
Ser Ala Ala Thr Gly Glu Pro Val Leu Phe Arg Asp Leu Trp Asp Gln 85
90 95 Asn Glu Gly Met Ala Val Val Ala Leu Leu Arg His Phe Gly Cys
Pro 100 105 110 Cys Cys Trp Glu Leu Ala Ser Val Leu Arg Asp Thr Lys
Glu Arg Phe 115 120 125 Asp Ser Ala Gly Val Lys Leu Ile Ala Val Gly
Val Gly Thr Pro Asp 130 135 140 Lys Ala Arg Ile Leu Ala Glu Arg Leu
Pro Phe Pro Leu Asp Tyr Leu 145 150 155 160 Tyr Ala Asp Pro Glu Arg
Lys Ala Tyr Asp Leu Leu Gly Leu Tyr Phe 165 170 175 Gly Ile Gly Arg
Thr Phe Phe Asn Pro Ala Ser Ala Ser Val Phe Ser 180 185 190 Arg Phe
Asp Ser Leu Lys Glu Ala Val Lys Asn Tyr Thr Ile Glu Ala 195 200 205
Thr Pro Asp Asp Arg Ala Ser Val Leu Gln Gln Gly Gly Met Phe Val 210
215 220 Phe Arg Gly Lys Glu Leu Ile Tyr Ala Arg Lys Asp Glu Gly Thr
Gly 225 230 235 240 Asp His Ala Pro Leu Asp Asp Val Leu Asn Ile Cys
Cys Lys Ala Pro 245 250 255 Ala Ala 73917DNAOryza sativa
73ctcggcgcgg ccacagccgc agaaccacac ctaggccgct cgaagaccca cgtagcttcc
60atccaagctt accgccatgg ccgcgcgcgc gccgctcccc gtaccgcacg cggccgccac
120cagcccgcga ccggctgcgg cgtcgagcct cctccgcgcg aggggcccgt
gcgcctccct 180cctctacccg cgccgcctcc gcttctccgt tgcgccggtg
gccgccgcca agcccgaggc 240cgtcgggagg gccggggagg cagctgcggc
gccggtggaa gggctcgcga aatccctgca 300gggggtggag gtgttcgatc
tgagcggaaa ggcggtgccc gttgttgatc tgtggaagga 360caggaaggcc
atcgttgcgt tcgcccgcca ttttggatgc gtgctgtgcc gtaagagggc
420cgatcttctc gcggctaagc aggatgcaat ggaggctgca ggggttgctc
ttgttttaat 480cggaccaggt actgttgaac aggcaaaggc attttatgac
caaaccaaat tcaaaggaga 540agtatacgct gatccaagtc actcatcata
taatgccctt gaatttgcat ttgggctgtt 600ctcaacgttt actccatcgg
ccggtttgaa gattatacag ttgtacatgg aaggatacag 660gcaggattgg
gaactgtcgt tcgagaagac caccagaacg aaaggtggat ggtatcaagg
720gggcctactt gttgcaggac caggcatcga caatattttg tatatccaca
aggacaaaga 780agcaggagat gaccctgaca tggatgatgt cttgaaagct
tgctgttcct agatcactag 840tatcctatat catttctgtt aacctccaga
ccttgaagac acatgtaaat attttgccaa 900gttaaagtat gttatgt
91774251PRTOryza sativa 74Met Ala Ala Arg Ala Pro Leu Pro Val Pro
His Ala Ala Ala Thr Ser 1 5 10 15 Pro Arg Pro Ala Ala Ala Ser Ser
Leu Leu Arg Ala Arg Gly Pro Cys 20 25 30 Ala Ser Leu Leu Tyr Pro
Arg Arg Leu Arg Phe Ser Val Ala Pro Val 35 40 45 Ala Ala Ala Lys
Pro Glu Ala Val Gly Arg Ala Gly Glu Ala Ala Ala 50 55 60 Ala Pro
Val Glu Gly Leu Ala Lys Ser Leu Gln Gly Val Glu Val Phe 65 70 75 80
Asp Leu Ser Gly Lys Ala Val Pro Val Val Asp Leu Trp Lys Asp Arg 85
90 95 Lys Ala Ile Val Ala Phe Ala Arg His Phe Gly Cys Val Leu Cys
Arg 100 105 110 Lys Arg Ala Asp Leu Leu Ala Ala Lys Gln Asp Ala Met
Glu Ala Ala 115 120
125 Gly Val Ala Leu Val Leu Ile Gly Pro Gly Thr Val Glu Gln Ala Lys
130 135 140 Ala Phe Tyr Asp Gln Thr Lys Phe Lys Gly Glu Val Tyr Ala
Asp Pro 145 150 155 160 Ser His Ser Ser Tyr Asn Ala Leu Glu Phe Ala
Phe Gly Leu Phe Ser 165 170 175 Thr Phe Thr Pro Ser Ala Gly Leu Lys
Ile Ile Gln Leu Tyr Met Glu 180 185 190 Gly Tyr Arg Gln Asp Trp Glu
Leu Ser Phe Glu Lys Thr Thr Arg Thr 195 200 205 Lys Gly Gly Trp Tyr
Gln Gly Gly Leu Leu Val Ala Gly Pro Gly Ile 210 215 220 Asp Asn Ile
Leu Tyr Ile His Lys Asp Lys Glu Ala Gly Asp Asp Pro 225 230 235 240
Asp Met Asp Asp Val Leu Lys Ala Cys Cys Ser 245 250 75886DNAOryza
sativa 75acgcgtgagt tcgtgacgcg tcacgccccg cggccttccc ctcccaaaaa
gcggcaggac 60gcaacctgat ccccatcccc cgagcaagca aagcggagga acgcgatggc
gtcggcgctg 120ctgaggaagg cgacggtagg cggctccgcg gcggcggcgg
cggcgaggtg ggcttccagg 180gggctcgcgt cggtgggctc cggctccgac
atcgtctcgg cggcgcccgg cgtgtcgctg 240cagaaggccc gctcctggga
cgagggcgtc gccaccaact tctccaccac ccctctcaag 300gacatcttcc
atgggaagaa agtggtcatc ttcggcctgc ctggtgcata cacaggagtc
360tgttcacagg cacacgtccc tagttataaa aataacattg acaagttgaa
agcaaaaggg 420gttgactctg ttatctgtgt ctctgtgaat gacccttatg
ccctgaatgg atgggcagaa 480aagctacagg caaaagatgc tattgaattt
tatggtgatt ttgatgggag tttccacaaa 540agcttggatt tggaagtaga
cctctctgct gctttgcttg gccgccgttc ccacaggtgg 600tcagcctttg
ttgacgatgg gaagatcaag gctttcaatg ttgaggtagc tccttctgac
660ttcaaggttt ctggtgccga ggtgatcttg gaccaaatct gatccgagta
acgaaattct 720gtcgttgttt gttttctcat gcagcatgca tgcttttgct
gtagtaaata aacgaaaact 780cgactactcg agtatccatg taaagatgtt
tgtagtctgc cttgctacgc ccagaatatt 840tgttttcctg ttacaaatca
gcttgccggg caacatgttt gtcagc 88676198PRTOryza sativa 76Met Ala Ser
Ala Leu Leu Arg Lys Ala Thr Val Gly Gly Ser Ala Ala 1 5 10 15 Ala
Ala Ala Ala Arg Trp Ala Ser Arg Gly Leu Ala Ser Val Gly Ser 20 25
30 Gly Ser Asp Ile Val Ser Ala Ala Pro Gly Val Ser Leu Gln Lys Ala
35 40 45 Arg Ser Trp Asp Glu Gly Val Ala Thr Asn Phe Ser Thr Thr
Pro Leu 50 55 60 Lys Asp Ile Phe His Gly Lys Lys Val Val Ile Phe
Gly Leu Pro Gly 65 70 75 80 Ala Tyr Thr Gly Val Cys Ser Gln Ala His
Val Pro Ser Tyr Lys Asn 85 90 95 Asn Ile Asp Lys Leu Lys Ala Lys
Gly Val Asp Ser Val Ile Cys Val 100 105 110 Ser Val Asn Asp Pro Tyr
Ala Leu Asn Gly Trp Ala Glu Lys Leu Gln 115 120 125 Ala Lys Asp Ala
Ile Glu Phe Tyr Gly Asp Phe Asp Gly Ser Phe His 130 135 140 Lys Ser
Leu Asp Leu Glu Val Asp Leu Ser Ala Ala Leu Leu Gly Arg 145 150 155
160 Arg Ser His Arg Trp Ser Ala Phe Val Asp Asp Gly Lys Ile Lys Ala
165 170 175 Phe Asn Val Glu Val Ala Pro Ser Asp Phe Lys Val Ser Gly
Ala Glu 180 185 190 Val Ile Leu Asp Gln Ile 195 7722PRTArtificial
sequencemotif 1 77Pro Leu Val Gly Asn Xaa Ala Pro Asp Phe Xaa Ala
Glu Xaa Xaa Phe 1 5 10 15 Asp Gln Xaa Phe Xaa Xaa 20
7822PRTArtificial sequencemotif 2 78Tyr Pro Leu Xaa Ser Xaa Xaa Thr
Lys Xaa Ile Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Val Leu Ile Xaa Asp Gln
20 792194DNAOryza sativa 79aatccgaaaa gtttctgcac cgttttcacc
ccctaactaa caatataggg aacgtgtgct 60aaatataaaa tgagacctta tatatgtagc
gctgataact agaactatgc aagaaaaact 120catccaccta ctttagtggc
aatcgggcta aataaaaaag agtcgctaca ctagtttcgt 180tttccttagt
aattaagtgg gaaaatgaaa tcattattgc ttagaatata cgttcacatc
240tctgtcatga agttaaatta ttcgaggtag ccataattgt catcaaactc
ttcttgaata 300aaaaaatctt tctagctgaa ctcaatgggt aaagagagag
atttttttta aaaaaataga 360atgaagatat tctgaacgta ttggcaaaga
tttaaacata taattatata attttatagt 420ttgtgcattc gtcatatcgc
acatcattaa ggacatgtct tactccatcc caatttttat 480ttagtaatta
aagacaattg acttattttt attatttatc ttttttcgat tagatgcaag
540gtacttacgc acacactttg tgctcatgtg catgtgtgag tgcacctcct
caatacacgt 600tcaactagca acacatctct aatatcactc gcctatttaa
tacatttagg tagcaatatc 660tgaattcaag cactccacca tcaccagacc
acttttaata atatctaaaa tacaaaaaat 720aattttacag aatagcatga
aaagtatgaa acgaactatt taggtttttc acatacaaaa 780aaaaaaagaa
ttttgctcgt gcgcgagcgc caatctccca tattgggcac acaggcaaca
840acagagtggc tgcccacaga acaacccaca aaaaacgatg atctaacgga
ggacagcaag 900tccgcaacaa ccttttaaca gcaggctttg cggccaggag
agaggaggag aggcaaagaa 960aaccaagcat cctccttctc ccatctataa
attcctcccc ccttttcccc tctctatata 1020ggaggcatcc aagccaagaa
gagggagagc accaaggaca cgcgactagc agaagccgag 1080cgaccgcctt
ctcgatccat atcttccggt cgagttcttg gtcgatctct tccctcctcc
1140acctcctcct cacagggtat gtgcctccct tcggttgttc ttggatttat
tgttctaggt 1200tgtgtagtac gggcgttgat gttaggaaag gggatctgta
tctgtgatga ttcctgttct 1260tggatttggg atagaggggt tcttgatgtt
gcatgttatc ggttcggttt gattagtagt 1320atggttttca atcgtctgga
gagctctatg gaaatgaaat ggtttaggga tcggaatctt 1380gcgattttgt
gagtaccttt tgtttgaggt aaaatcagag caccggtgat tttgcttggt
1440gtaataaagt acggttgttt ggtcctcgat tctggtagtg atgcttctcg
atttgacgaa 1500gctatccttt gtttattccc tattgaacaa aaataatcca
actttgaaga cggtcccgtt 1560gatgagattg aatgattgat tcttaagcct
gtccaaaatt tcgcagctgg cttgtttaga 1620tacagtagtc cccatcacga
aattcatgga aacagttata atcctcagga acaggggatt 1680ccctgttctt
ccgatttgct ttagtcccag aatttttttt cccaaatatc ttaaaaagtc
1740actttctggt tcagttcaat gaattgattg ctacaaataa tgcttttata
gcgttatcct 1800agctgtagtt cagttaatag gtaatacccc tatagtttag
tcaggagaag aacttatccg 1860atttctgatc tccattttta attatatgaa
atgaactgta gcataagcag tattcatttg 1920gattattttt tttattagct
ctcacccctt cattattctg agctgaaagt ctggcatgaa 1980ctgtcctcaa
ttttgttttc aaattcacat cgattatcta tgcattatcc tcttgtatct
2040acctgtagaa gtttcttttt ggttattcct tgactgcttg attacagaaa
gaaatttatg 2100aagctgtaat cgggatagtt atactgcttg ttcttatgat
tcatttcctt tgtgcagttc 2160ttggtgtagc ttgccacttt caccagcaaa gttc
2194801264DNAOryza sativa 80tcgacgctac tcaagtggtg ggaggccacc
gcatgttcca acgaagcgcc aaagaaagcc 60ttgcagactc taatgctatt agtcgcctag
gatatttgga atgaaaggaa ccgcagagtt 120tttcagcacc aagagcttcc
ggtggctagt ctgatagcca aaattaagga ggatgccaaa 180acatgggtct
tggcgggcgc gaaacacctt gataggtggc ttacctttta acatgttcgg
240gccaaaggcc ttgagacggt aaagttttct atttgcgctt gcgcatgtac
aattttattc 300ctctattcaa tgaaattggt ggctcactgg ttcattaaaa
aaaaaagaat ctagcctgtt 360cgggaagaag aggattttgt tcgtgagaga
gagagagaga gagagagaga gagagagaga 420gaaggaggag gaggattttc
aggcttcgca ttgcccaacc tctgcttctg ttggcccaag 480aagaatccca
ggcgcccatg ggctggcagt ttaccacgga cctacctagc ctaccttagc
540tatctaagcg ggccgaccta gtagccacgt gcctagtgta gattaaagtt
gccgggccag 600caggaagcca cgctgcaatg gcatcttccc ctgtccttcg
cgtacgtgaa aacaaaccca 660ggtaagctta gaatcttctt gcccgttgga
ctgggacacc caccaatccc accatgcccc 720gatattcctc cggtctcggt
tcatgtgatg tcctctcttg tgtgatcacg gagcaagcat 780tcttaaacgg
caaaagaaaa tcaccaactt gctcacgcag tcacgctgca ccgcgcgaag
840cgacgcccga taggccaaga tcgcgagata aaataacaac caatgatcat
aaggaaacaa 900gcccgcgatg tgtcgtgtgc agcaatcttg gtcatttgcg
ggatcgagtg cttcacagct 960aaccaaatat tcggccgatg atttaacaca
ttatcagcgt agatgtacgt acgatttgtt 1020aattaatcta cgagccttgc
tagggcaggt gttctgccag ccaatccaga tcgccctcgt 1080atgcacgctc
acatgatggc agggcagggt tcacatgagc tctaacggtc gattaattaa
1140tcccggggct cgactataaa tacctcccta atcccatgat caaaaccatc
tcaagcagcc 1200taatcatctc cagctgatca agagctctta attagctagc
tagtgattag ctgcgcttgt 1260gatc 12648154DNAArtificial sequenceprimer
forward primer prm8756 81ggggacaagt ttgtacaaaa aagcaggctt
aaacaatggc gtctgttgct tctt 548252DNAArtificial sequenceprimer
reverse primer prm8757 82ggggaccact ttgtacaaga aagctgggtt
cgagctaaat agctgagaag ag 52831317DNAArabidopsis thaliana
83cggacaggcc acgtcgtgtc ctaaacctct tagcctttcc ctttataagt caatcttgtg
60tcggcttcga ctcccaacat acacaaaaca ctaaaagtag aagaaaaatg gcgactctta
120aggtttctga ttctgttcct gctccttctg atgatgctga gcaattgaga
accgcttttg 180aaggatgggg tacgaacgag gacttgatca tatcaatctt
ggctcacaga agtgctgaac 240agaggaaagt catcaggcaa gcataccacg
aaacctacgg cgaagacctt ctcaagactc 300ttgacaagga gctctctaac
gatttcgaga gagctatctt gttgtggact cttgaacccg 360gtgagcgtga
tgctttattg gctaatgaag ctacaaaaag atggacttca agcaaccaag
420ttcttatgga agttgcttgc acaaggacat caacgcagct gcttcacgct
aggcaagctt 480accatgctcg ctacaagaag tctcttgaag aggacgttgc
tcaccacact accggtgact 540tcagaaagct tttggtttct cttgttacct
catacaggta cgaaggagat gaagtgaaca 600tgacattggc taagcaagaa
gctaagctgg tccatgagaa aatcaaggac aagcactaca 660atgatgagga
tgttattaga atcttgtcca caagaagcaa agctcagatc aatgctactt
720ttaaccgtta ccaagatgat catggcgagg aaattctcaa gagtcttgag
gaaggagatg 780atgatgacaa gttccttgca cttttgaggt caaccattca
gtgcttgaca agaccagagc 840tttactttgt cgatgttctt cgttcagcaa
tcaacaaaac tggaactgat gaaggagcac 900tcactagaat tgtgaccaca
agagctgaga ttgacttgaa ggtcattgga gaggagtacc 960agcgcaggaa
cagcattcct ttggagaaag ctattaccaa agacactcgt ggagattacg
1020agaagatgct cgtcgcactt ctcggtgaag atgatgctta atcaatcaat
cctccacaga 1080gaaacataag ctgctctaca gcttctgtta tctcttatct
ccctctctct ctctttgatg 1140agtttcaaat cgtttgattt tgtttctaca
aaaaccttgt ttgtttctgt tgtgtgtttt 1200gagttcctaa ataatgcaaa
agagagagac agagagaacc agtgtggtct cttaagttat 1260atatatatga
agagcattgg cctaaaacac agactaacaa gtagttctgg ttttgac
131784317PRTArabidopsis thaliana 84Met Ala Thr Leu Lys Val Ser Asp
Ser Val Pro Ala Pro Ser Asp Asp 1 5 10 15 Ala Glu Gln Leu Arg Thr
Ala Phe Glu Gly Trp Gly Thr Asn Glu Asp 20 25 30 Leu Ile Ile Ser
Ile Leu Ala His Arg Ser Ala Glu Gln Arg Lys Val 35 40 45 Ile Arg
Gln Ala Tyr His Glu Thr Tyr Gly Glu Asp Leu Leu Lys Thr 50 55 60
Leu Asp Lys Glu Leu Ser Asn Asp Phe Glu Arg Ala Ile Leu Leu Trp 65
70 75 80 Thr Leu Glu Pro Gly Glu Arg Asp Ala Leu Leu Ala Asn Glu
Ala Thr 85 90 95 Lys Arg Trp Thr Ser Ser Asn Gln Val Leu Met Glu
Val Ala Cys Thr 100 105 110 Arg Thr Ser Thr Gln Leu Leu His Ala Arg
Gln Ala Tyr His Ala Arg 115 120 125 Tyr Lys Lys Ser Leu Glu Glu Asp
Val Ala His His Thr Thr Gly Asp 130 135 140 Phe Arg Lys Leu Leu Val
Ser Leu Val Thr Ser Tyr Arg Tyr Glu Gly 145 150 155 160 Asp Glu Val
Asn Met Thr Leu Ala Lys Gln Glu Ala Lys Leu Val His 165 170 175 Glu
Lys Ile Lys Asp Lys His Tyr Asn Asp Glu Asp Val Ile Arg Ile 180 185
190 Leu Ser Thr Arg Ser Lys Ala Gln Ile Asn Ala Thr Phe Asn Arg Tyr
195 200 205 Gln Asp Asp His Gly Glu Glu Ile Leu Lys Ser Leu Glu Glu
Gly Asp 210 215 220 Asp Asp Asp Lys Phe Leu Ala Leu Leu Arg Ser Thr
Ile Gln Cys Leu 225 230 235 240 Thr Arg Pro Glu Leu Tyr Phe Val Asp
Val Leu Arg Ser Ala Ile Asn 245 250 255 Lys Thr Gly Thr Asp Glu Gly
Ala Leu Thr Arg Ile Val Thr Thr Arg 260 265 270 Ala Glu Ile Asp Leu
Lys Val Ile Gly Glu Glu Tyr Gln Arg Arg Asn 275 280 285 Ser Ile Pro
Leu Glu Lys Ala Ile Thr Lys Asp Thr Arg Gly Asp Tyr 290 295 300 Glu
Lys Met Leu Val Ala Leu Leu Gly Glu Asp Asp Ala 305 310 315
8556DNAArtificial sequenceforward primer P08727 85ggggacaagt
ttgtacaaaa aagcaggctt aaacaatggc gactcttaag gtttct
568650DNAArtificial sequencereverse primer P09025 86ggggaccact
ttgtacaaga aagctgggtt taagcatcat cttcaccgag 508714PRTArtificial
sequencesignature sequence 1 87Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Arg Asp Ala 1 5 10 8819PRTArtificial sequencesignature
sequence 2 88Ala Xaa Xaa Gly Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa Xaa Leu 1 5 10 15 Xaa Xaa Xaa 8917PRTArtificial
sequencesignature sequence 3 89Xaa Xaa Xaa Xaa Xaa Leu Xaa Arg Xaa
Xaa Xaa Xaa Arg Xaa Xaa Xaa 1 5 10 15 Xaa 9010PRTArtificial
sequencesignature sequence 4 90Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
Xaa 1 5 10 917PRTArtificial sequencesignature sequence 5 91Xaa Xaa
Glu Xaa Xaa Xaa Xaa 1 5 929PRTArtificial sequencesignature sequence
6 92Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Ser 1 5 9310PRTArtificial
sequencesignature sequence 7 93Tyr Arg Xaa Phe Leu Leu Ser Leu Val
Gly 1 5 10 942194DNAOryza sativa 94aatccgaaaa gtttctgcac cgttttcacc
ccctaactaa caatataggg aacgtgtgct 60aaatataaaa tgagacctta tatatgtagc
gctgataact agaactatgc aagaaaaact 120catccaccta ctttagtggc
aatcgggcta aataaaaaag agtcgctaca ctagtttcgt 180tttccttagt
aattaagtgg gaaaatgaaa tcattattgc ttagaatata cgttcacatc
240tctgtcatga agttaaatta ttcgaggtag ccataattgt catcaaactc
ttcttgaata 300aaaaaatctt tctagctgaa ctcaatgggt aaagagagag
atttttttta aaaaaataga 360atgaagatat tctgaacgta ttggcaaaga
tttaaacata taattatata attttatagt 420ttgtgcattc gtcatatcgc
acatcattaa ggacatgtct tactccatcc caatttttat 480ttagtaatta
aagacaattg acttattttt attatttatc ttttttcgat tagatgcaag
540gtacttacgc acacactttg tgctcatgtg catgtgtgag tgcacctcct
caatacacgt 600tcaactagca acacatctct aatatcactc gcctatttaa
tacatttagg tagcaatatc 660tgaattcaag cactccacca tcaccagacc
acttttaata atatctaaaa tacaaaaaat 720aattttacag aatagcatga
aaagtatgaa acgaactatt taggtttttc acatacaaaa 780aaaaaaagaa
ttttgctcgt gcgcgagcgc caatctccca tattgggcac acaggcaaca
840acagagtggc tgcccacaga acaacccaca aaaaacgatg atctaacgga
ggacagcaag 900tccgcaacaa ccttttaaca gcaggctttg cggccaggag
agaggaggag aggcaaagaa 960aaccaagcat cctccttctc ccatctataa
attcctcccc ccttttcccc tctctatata 1020ggaggcatcc aagccaagaa
gagggagagc accaaggaca cgcgactagc agaagccgag 1080cgaccgcctt
ctcgatccat atcttccggt cgagttcttg gtcgatctct tccctcctcc
1140acctcctcct cacagggtat gtgcctccct tcggttgttc ttggatttat
tgttctaggt 1200tgtgtagtac gggcgttgat gttaggaaag gggatctgta
tctgtgatga ttcctgttct 1260tggatttggg atagaggggt tcttgatgtt
gcatgttatc ggttcggttt gattagtagt 1320atggttttca atcgtctgga
gagctctatg gaaatgaaat ggtttaggga tcggaatctt 1380gcgattttgt
gagtaccttt tgtttgaggt aaaatcagag caccggtgat tttgcttggt
1440gtaataaagt acggttgttt ggtcctcgat tctggtagtg atgcttctcg
atttgacgaa 1500gctatccttt gtttattccc tattgaacaa aaataatcca
actttgaaga cggtcccgtt 1560gatgagattg aatgattgat tcttaagcct
gtccaaaatt tcgcagctgg cttgtttaga 1620tacagtagtc cccatcacga
aattcatgga aacagttata atcctcagga acaggggatt 1680ccctgttctt
ccgatttgct ttagtcccag aatttttttt cccaaatatc ttaaaaagtc
1740actttctggt tcagttcaat gaattgattg ctacaaataa tgcttttata
gcgttatcct 1800agctgtagtt cagttaatag gtaatacccc tatagtttag
tcaggagaag aacttatccg 1860atttctgatc tccattttta attatatgaa
atgaactgta gcataagcag tattcatttg 1920gattattttt tttattagct
ctcacccctt cattattctg agctgaaagt ctggcatgaa 1980ctgtcctcaa
ttttgttttc aaattcacat cgattatcta tgcattatcc tcttgtatct
2040acctgtagaa gtttcttttt ggttattcct tgactgcttg attacagaaa
gaaatttatg 2100aagctgtaat cgggatagtt atactgcttg ttcttatgat
tcatttcctt tgtgcagttc 2160ttggtgtagc ttgccacttt caccagcaaa gttc
2194951244DNAOryza sativa 95aaaaccaccg agggacctga tctgcaccgg
ttttgatagt tgagggaccc gttgtgtctg 60gttttccgat cgagggacga aaatcggatt
cggtgtaaag ttaagggacc tcagatgaac 120ttattccgga gcatgattgg
gaagggagga cataaggccc atgtcgcatg tgtttggacg 180gtccagatct
ccagatcact cagcaggatc ggccgcgttc gcgtagcacc cgcggtttga
240ttcggcttcc cgcaaggcgg cggccggtgg ccgtgccgcc gtagcttccg
ccggaagcga 300gcacgccgcc gccgccgacc cggctctgcg tttgcaccgc
cttgcacgcg atacatcggg 360atagatagct actactctct ccgtttcaca
atgtaaatca ttctactatt ttccacattc 420atattgatgt taatgaatat
agacatatat atctatttag attcattaac atcaatatga 480atgtaggaaa
tgctagaatg acttacattg tgaattgtga aatggacgaa gtacctacga
540tggatggatg caggatcatg aaagaattaa tgcaagatcg tatctgccgc
atgcaaaatc 600ttactaattg cgctgcatat atgcatgaca gcctgcatgc
gggcgtgtaa gcgtgttcat 660ccattaggaa gtaaccttgt cattacttat
accagtacta catactatat agtattgatt 720tcatgagcaa atctacaaaa
ctggaaagca ataagaaata cgggactgga aaagactcaa 780cattaatcac
caaatatttc gccttctcca gcagaatata tatctctcca tcttgatcac
840tgtacacact gacagtgtac gcataaacgc agcagccagc ttaactgtcg
tctcaccgtc 900gcacactggc cttccatctc aggctagctt tctcagccac
ccatcgtaca tgtcaactcg 960gcgcgcgcac aggcacaaat tacgtacaaa
acgcatgacc aaatcaaaac caccggagaa 1020gaatcgctcc cgcgcgcggc
ggcgacgcgc acgtacgaac gcacgcacgc acgcccaacc 1080ccacgacacg
atcgcgcgcg acgccggcga caccggccgt ccacccgcgc cctcacctcg
1140ccgactataa atacgtaggc atctgcttga tcttgtcatc catctcacca
ccaaaaaaaa 1200aaggaaaaaa aaacaaaaca caccaagcca aataaaagcg acaa
1244961141DNAGossypium hirsutum 96cagattcaga aagaaataaa ggaagaagaa
gcaatggcca ctcttacagt gcccacgaca 60gttccttcgg tgtctgaaga ttgtgaacag
ctaagaaaag ccttttcagg atggggaact 120aatgagggct taatcataga
tatattgggt cacagaaatg ccgagcaacg aaacttgatt 180cgaaaaacct
acgctgaaac ctatggagag gatctcctca aggcactaga caaggagctc
240tcgaatgact ttgagaggct ggttttgctt tgggctcttg atcctgctga
acgtgatgcc 300cttttggcta atgaagccac caaaaggtgg acttcaagca
atcaggtcct tatggaaata 360gcttgcacaa ggtctgccaa ccaactgctt
cacgcaaggc aggcttatca tgctcgttat 420aagaagtcgc ttgaagagga
tgttgctcat cacacgactg gcgacttccg taagctcctc 480ctacctctag
tgagttcata cagatatgag ggagaggagg tgaacatgaa tctggcgaaa
540acagaggcga agttgcttca tgagaaaatt tcagacaaag cttacagtga
tgacgatgtc 600ataagggttt tggctacaag aagcaaggca cagatcaatg
caactctgaa tcactacaaa 660aatgaatatg gaaatgacat aaacaaggac
ttgaaggctg atcctaagga tgagttcctt 720gcactactaa ggtccacagt
gaagtgcttg gtctatccgg aaaagtattt tgagaaggtt 780cttcgcctag
caatcaatag acgaggaacg gatgaaggag ctcttactag agttgtttgc
840actagggctg aggttgatct aaagatcata gcagatgagt accagcgaag
gaacagtgtc 900ccactgactc gtgccattgt caaggacact catggagact
atgaaaaatt gctgctggta 960cttgcaggac atgtggagaa ttgaatctga
tatcatgaga caatttcctg gtgaataaat 1020gtttatgacc aaactataat
ggtctagtgt ggttattgat gttttcctgt ttttctatgt 1080agtattgcga
gttatatgct atccaagaat tcgaagtcta tttaaaaaaa aaaaaaaaaa 1140a
114197316PRTGossypium hirsutum 97Met Ala Thr Leu Thr Val Pro Thr
Thr Val Pro Ser Val Ser Glu Asp 1 5 10 15 Cys Glu Gln Leu Arg Lys
Ala Phe Ser Gly Trp Gly Thr Asn Glu Gly 20 25 30 Leu Ile Ile Asp
Ile Leu Gly His Arg Asn Ala Glu Gln Arg Asn Leu 35 40 45 Ile Arg
Lys Thr Tyr Ala Glu Thr Tyr Gly Glu Asp Leu Leu Lys Ala 50 55 60
Leu Asp Lys Glu Leu Ser Asn Asp Phe Glu Arg Leu Val Leu Leu Trp 65
70 75 80 Ala Leu Asp Pro Ala Glu Arg Asp Ala Leu Leu Ala Asn Glu
Ala Thr 85 90 95 Lys Arg Trp Thr Ser Ser Asn Gln Val Leu Met Glu
Ile Ala Cys Thr 100 105 110 Arg Ser Ala Asn Gln Leu Leu His Ala Arg
Gln Ala Tyr His Ala Arg 115 120 125 Tyr Lys Lys Ser Leu Glu Glu Asp
Val Ala His His Thr Thr Gly Asp 130 135 140 Phe Arg Lys Leu Leu Leu
Pro Leu Val Ser Ser Tyr Arg Tyr Glu Gly 145 150 155 160 Glu Glu Val
Asn Met Asn Leu Ala Lys Thr Glu Ala Lys Leu Leu His 165 170 175 Glu
Lys Ile Ser Asp Lys Ala Tyr Ser Asp Asp Asp Val Ile Arg Val 180 185
190 Leu Ala Thr Arg Ser Lys Ala Gln Ile Asn Ala Thr Leu Asn His Tyr
195 200 205 Lys Asn Glu Tyr Gly Asn Asp Ile Asn Lys Asp Leu Lys Ala
Asp Pro 210 215 220 Lys Asp Glu Phe Leu Ala Leu Leu Arg Ser Thr Val
Lys Cys Leu Val 225 230 235 240 Tyr Pro Glu Lys Tyr Phe Glu Lys Val
Leu Arg Leu Ala Ile Asn Arg 245 250 255 Arg Gly Thr Asp Glu Gly Ala
Leu Thr Arg Val Val Cys Thr Arg Ala 260 265 270 Glu Val Asp Leu Lys
Ile Ile Ala Asp Glu Tyr Gln Arg Arg Asn Ser 275 280 285 Val Pro Leu
Thr Arg Ala Ile Val Lys Asp Thr His Gly Asp Tyr Glu 290 295 300 Lys
Leu Leu Leu Val Leu Ala Gly His Val Glu Asn 305 310 315
981112DNALavatera thuringiaca 98aatggctact cttacagttc cctccacact
tccgtcagtg tctgaagatt gtgaacaact 60caggaaagcc ttctcaggat ggggaactaa
tgaggactta atcataaata tattgggtca 120ccgaaatgcg gacgaacgaa
actcgattcg aaaagcttat actgaaaccc atggagaaga 180tctcctcaag
gcactggaca aggaactctc aaatgacttt gagaggctgg ttctgctttg
240gactcttgat cctcctgaac gtgatgcact tttggcaaat gaagccacca
aaaggtggac 300ttcaagcaat caggtaatta tggaaatagc ctgcagaagt
tcttctgacc aactgcttcg 360cgcgaggcag gcttatcatg ttcgttataa
gaaatcgctt gaagaggatg ttgcccatca 420cacaactggc gacttccgta
agcttctcct acctcttgtg agttcataca gatacgaggg 480agatgaagtg
aacatgactc tggcgaaaac agaggccaag ttactccatg agaaaatctc
540aaacaaagct tacagtgatg acgatgtcat cagggttttg gctacgagaa
gcaagtcaca 600gatcaacgaa cgtcttaatc actacaaaaa tgaatacgca
actgatataa acaaggacct 660gaaggctgac cctaaggatg agttccttgc
actgctaagg tccacagtga agtgcttggt 720ctaccctgaa aagtatttcg
agaaggttct tcgtctagca atcaataaac gaggaacgga 780tgaaggagct
cttacgaggg ttgtttccac cagggctgag gttgatctaa agatcatagc
840agatgagtac cagcgaagga acagtgtccc actgactcgt gctattgtca
aggacactaa 900tggagactac gaaaaattgc tgctggtact tgctggagag
gtggaggctt gaaccggttt 960tcatgagatg attttgtgtt gaataaaaac
ttaatgaccg gaactctaat ggtctagtgt 1020tgctattatg ttatcctgtt
ttttttcttc tatggtactg tgagttttat gcaataaagg 1080cttgttattt
agaaaaaaaa aaaaaaaaaa aa 111299316PRTLavatera thuringiaca 99Met Ala
Thr Leu Thr Val Pro Ser Thr Leu Pro Ser Val Ser Glu Asp 1 5 10 15
Cys Glu Gln Leu Arg Lys Ala Phe Ser Gly Trp Gly Thr Asn Glu Asp 20
25 30 Leu Ile Ile Asn Ile Leu Gly His Arg Asn Ala Asp Glu Arg Asn
Ser 35 40 45 Ile Arg Lys Ala Tyr Thr Glu Thr His Gly Glu Asp Leu
Leu Lys Ala 50 55 60 Leu Asp Lys Glu Leu Ser Asn Asp Phe Glu Arg
Leu Val Leu Leu Trp 65 70 75 80 Thr Leu Asp Pro Pro Glu Arg Asp Ala
Leu Leu Ala Asn Glu Ala Thr 85 90 95 Lys Arg Trp Thr Ser Ser Asn
Gln Val Ile Met Glu Ile Ala Cys Arg 100 105 110 Ser Ser Ser Asp Gln
Leu Leu Arg Ala Arg Gln Ala Tyr His Val Arg 115 120 125 Tyr Lys Lys
Ser Leu Glu Glu Asp Val Ala His His Thr Thr Gly Asp 130 135 140 Phe
Arg Lys Leu Leu Leu Pro Leu Val Ser Ser Tyr Arg Tyr Glu Gly 145 150
155 160 Asp Glu Val Asn Met Thr Leu Ala Lys Thr Glu Ala Lys Leu Leu
His 165 170 175 Glu Lys Ile Ser Asn Lys Ala Tyr Ser Asp Asp Asp Val
Ile Arg Val 180 185 190 Leu Ala Thr Arg Ser Lys Ser Gln Ile Asn Glu
Arg Leu Asn His Tyr 195 200 205 Lys Asn Glu Tyr Ala Thr Asp Ile Asn
Lys Asp Leu Lys Ala Asp Pro 210 215 220 Lys Asp Glu Phe Leu Ala Leu
Leu Arg Ser Thr Val Lys Cys Leu Val 225 230 235 240 Tyr Pro Glu Lys
Tyr Phe Glu Lys Val Leu Arg Leu Ala Ile Asn Lys 245 250 255 Arg Gly
Thr Asp Glu Gly Ala Leu Thr Arg Val Val Ser Thr Arg Ala 260 265 270
Glu Val Asp Leu Lys Ile Ile Ala Asp Glu Tyr Gln Arg Arg Asn Ser 275
280 285 Val Pro Leu Thr Arg Ala Ile Val Lys Asp Thr Asn Gly Asp Tyr
Glu 290 295 300 Lys Leu Leu Leu Val Leu Ala Gly Glu Val Glu Ala 305
310 315 1002040DNABrassica rapa 100atggcgtctc tcaaagtccc taccaacgtt
cctcttcccg aggaagacgc cgagcaactc 60cacaaggctt ttgcaggatg gggtaccaac
gagaagctga tcatatcaat cctagctcac 120aggacctcag cacaacgcag
cttaatccgc agcgcttatg ccgctgctta caatgaggat 180ctcctcaagg
ccttagacaa agagctttct agtgactttg agcgagttgt catgttgtgg
240actcttgatc cagcggagag agatgctttc cttgctaaag aatctaccaa
aatgttcacc 300aagaacaatt gggttcttgt tgaaatcgca tgcactaggt
gtcctcttga tcttttcaag 360gtcaaacaag cttaccaagc acgttacaag
aaatctctag aggaagatgt tgcgcaacac 420acatctggtg accttcgtaa
gctcttgctt cctcttgtta gtacttttag gtacgaagga 480gatgaggtga
acatgaggct tgcaagatcc gaagctaagt tacttcacga gaaggtctca
540gagaaagcct ttagtgatga tgacttcatc agaatcttga caacaagaag
caaagcacag 600ctcggtgcaa ctcttaacca ctacaataat gagtacggaa
acgctattaa caagcacttg 660aaggaagatt cagatgatga gtacctgaag
ctactaagag ctgcgatcac gtgtttgaca 720taccctgaga agcattttga
gaaggttttg cgtctagcaa ttaacaaaat ggggactgat 780gagtgggcac
taacccgagt tgtgactaca cgaactgaag ttgatatgga acgtatcaaa
840gaggaatatc aacgaaggaa cagcattcct ttgcaccatg ccgtcgctaa
agacacttct 900ggtgattatg aggatatgct tgtttctctt ctcggacatg
gagatgaagg gactctcgac 960ggatcttttg ctggagataa gcgcctcggc
ttctcgtctt cttcacagtc ctcacgtcgt 1020ctcgccctat ctggtgggtc
gtctctccaa caacgaatcg agaggactct cttgccactg 1080ctgctctcgg
gttatcggag acaagctcca gtgaactccc ctctaactgc cccagacatg
1140tctcgatttt gcagatccga agaaccttct ggttctcttg gtctctcatc
atttggtggc 1200tccttcctca ccagagccgg gtctagcgtc aattcacctg
ctcctctctc atcaatttat 1260gttaatccgg cgaccgatgt aggtggaact
ccactccggc gaccagatct gttccttaaa 1320agtttgagaa gacaagcatc
ctccagcgac attcctcttc cccgctccat cctctccatc 1380ctccatgttt
ggcctcctcc acccttcaga ttgtgtaaac ttggtttaaa acgattgcat
1440gaagatccat accaccaacc actgcaaaca taccttctat cacagcggtt
tgcaaatttg 1500gcgtccgatg taggtgggaa ttcactccgg catacagttt
tgagccatat gtttatgaat 1560atgacgtctg atgtgagtgg gaatccactc
cggcctccag ctctgagcca tcaaaagcta 1620gtaaggccaa tttgtcggcg
cattattctc acctcttttt ctgttgtgga gatcacttta 1680ctaccatgtc
tcccttctat gaatggagaa aatttctcag attcttttcc gagcttcagt
1740tgcagtttac tcactggttt gttactttat ggagcggtcc gtacggggcc
tgaaggtgca 1800atcgagacta cttcggtttt tcttgttggt gaagactgtc
tttcaacgtc acttgtgact 1860atctctcaac tatccaactt tgccgtggaa
gctttattga cgcattcaaa cttgatattg 1920aattcgctgt caacttcata
tgaagattta ttatgcttgt ttctaattgc tattatagtt 1980catgaattgt
ccacaagagg atgtttagtt ctcttttggc tttgtagtcc ttgcatttga
2040101679PRTBrassica rapa 101Met Ala Ser Leu Lys Val Pro Thr Asn
Val Pro Leu Pro Glu Glu Asp 1 5 10 15 Ala Glu Gln Leu His Lys Ala
Phe Ala Gly Trp Gly Thr Asn Glu Lys 20 25 30 Leu Ile Ile Ser Ile
Leu Ala His Arg Thr Ser Ala Gln Arg Ser Leu 35 40 45 Ile Arg Ser
Ala Tyr Ala Ala Ala Tyr Asn Glu Asp Leu Leu Lys Ala 50 55 60 Leu
Asp Lys Glu Leu Ser Ser Asp Phe Glu Arg Val Val Met Leu Trp 65 70
75 80 Thr Leu Asp Pro Ala Glu Arg Asp Ala Phe Leu Ala Lys Glu Ser
Thr 85 90 95 Lys Met Phe Thr Lys Asn Asn Trp Val Leu Val Glu Ile
Ala Cys Thr 100 105 110 Arg Cys Pro Leu Asp Leu Phe Lys Val Lys Gln
Ala Tyr Gln Ala Arg 115 120 125 Tyr Lys Lys Ser Leu Glu Glu Asp Val
Ala Gln His Thr Ser Gly Asp 130 135 140 Leu Arg Lys Leu Leu Leu Pro
Leu Val Ser Thr Phe Arg Tyr Glu Gly 145 150 155 160 Asp Glu Val Asn
Met Arg Leu Ala Arg Ser Glu Ala Lys Leu Leu His 165 170 175 Glu Lys
Val Ser Glu Lys Ala Phe Ser Asp Asp Asp Phe Ile Arg Ile 180 185 190
Leu Thr Thr Arg Ser Lys Ala Gln Leu Gly Ala Thr Leu Asn His Tyr 195
200 205 Asn Asn Glu Tyr Gly Asn Ala Ile Asn Lys His Leu Lys Glu Asp
Ser 210 215 220 Asp Asp Glu Tyr Leu Lys Leu Leu Arg Ala Ala Ile Thr
Cys Leu Thr 225 230 235 240 Tyr Pro Glu Lys His Phe Glu Lys Val Leu
Arg Leu Ala Ile Asn Lys 245 250 255 Met Gly Thr Asp Glu Trp Ala Leu
Thr Arg Val Val Thr Thr Arg Thr 260 265 270 Glu Val Asp Met Glu Arg
Ile Lys Glu Glu Tyr Gln Arg Arg Asn Ser 275 280 285 Ile Pro Leu His
His Ala Val Ala Lys Asp Thr Ser Gly Asp Tyr Glu 290 295 300 Asp Met
Leu Val Ser Leu Leu Gly His Gly Asp Glu Gly Thr Leu Asp 305 310 315
320 Gly Ser Phe Ala Gly Asp Lys Arg Leu Gly Phe Ser Ser Ser Ser Gln
325 330 335 Ser Ser Arg Arg Leu Ala Leu Ser Gly Gly Ser Ser Leu Gln
Gln Arg 340 345 350 Ile Glu Arg Thr Leu Leu Pro Leu Leu Leu Ser Gly
Tyr Arg Arg Gln 355 360 365 Ala Pro Val Asn Ser Pro Leu Thr Ala Pro
Asp Met Ser Arg Phe Cys 370 375 380 Arg Ser Glu Glu Pro Ser Gly Ser
Leu Gly Leu Ser Ser Phe Gly Gly 385 390 395 400 Ser Phe Leu Thr Arg
Ala Gly Ser Ser Val Asn Ser Pro Ala Pro Leu 405 410 415 Ser Ser Ile
Tyr Val Asn Pro Ala Thr Asp Val Gly Gly Thr Pro Leu 420 425 430 Arg
Arg Pro Asp Leu Phe Leu Lys Ser Leu Arg Arg Gln Ala Ser Ser 435 440
445 Ser Asp Ile Pro Leu Pro Arg Ser Ile Leu Ser Ile Leu His Val Trp
450 455 460 Pro Pro Pro Pro Phe Arg Leu Cys Lys Leu Gly Leu Lys Arg
Leu His 465 470 475 480 Glu Asp Pro Tyr His Gln Pro Leu Gln Thr Tyr
Leu Leu Ser Gln Arg 485 490 495 Phe Ala Asn Leu Ala Ser Asp Val Gly
Gly Asn Ser Leu Arg His Thr 500 505 510 Val Leu Ser His Met Phe Met
Asn Met Thr Ser Asp Val Ser Gly Asn 515 520 525 Pro Leu Arg Pro Pro
Ala Leu Ser His Gln Lys Leu Val Arg Pro Ile 530 535 540 Cys Arg Arg
Ile Ile Leu Thr Ser Phe Ser Val Val Glu Ile Thr Leu 545 550 555 560
Leu Pro Cys Leu Pro Ser Met Asn Gly Glu Asn Phe Ser Asp Ser Phe 565
570 575 Pro Ser Phe Ser Cys Ser Leu Leu Thr Gly Leu Leu Leu Tyr Gly
Ala 580 585 590 Val Arg Thr Gly Pro Glu Gly Ala Ile Glu Thr Thr Ser
Val Phe Leu 595 600 605 Val Gly Glu Asp Cys Leu Ser Thr Ser Leu Val
Thr Ile Ser Gln Leu 610 615 620 Ser Asn Phe Ala Val Glu Ala Leu Leu
Thr His Ser Asn Leu Ile Leu 625 630 635 640 Asn Ser Leu Ser Thr Ser
Tyr Glu Asp Leu Leu Cys Leu Phe Leu Ile 645 650 655 Ala Ile Ile Val
His Glu Leu Ser Thr Arg Gly Cys Leu Val Leu Phe 660 665 670 Trp Leu
Cys Ser Pro Cys Ile 675 1021146DNAGossypium hirsutum 102caaccctcaa
agttccagtt cacgttcctt ctccttctga ggatgctgaa tggcaacttc 60ggaaagcttt
tgaaggctgg ggtacgaacg agcaattgat tatcgacata ttggctcaca
120ggaatgcagc acagcgcaat tcaattcgga aagtttatgg tgaagcttat
ggggaagatc 180ttctcaagtg tttggagaag gaacttacaa gtgatttcga
gcgggctgtg ctgcttttta 240cgttggaccc tgcagagcga gatgctcatc
tggctaatga agctacaaag aagttcacat 300caagcaattg gattctcatg
gagatagctt gcagtaggtc ttcgcatgaa ctactcaatg 360tgaaaaaggc
gtatcatgct cgttataaga aatcccttga agaagatgtt gctcaccaca
420ctaccggaga gtaccgcaag cttttggtcc ctcttgttag tgcattccga
tatgagggag 480aggaggtgaa catgacattg gcaaaatctg aggctaagat
acttcatgat aaaatttcgg 540acaagcatta taccgatgag gaggtgatta
ggattgtatc aacaaggagt aaggcacagc 600tcaatgcaac tctcaaccat
tacaatactt cattcggcaa tgctatcaac aaggatttga 660aggctgatcc
cagtgatgaa ttcctcaaat tactaagagc tgtgatcaag tgcttgacca
720ccccagagca atatttcgag aaggttttac gtcaagccat caataagttg
ggatccgatg 780aatgggctct tacccgagtc gtcacaactc gtgcagaggt
cgacatggta cgtattaagg 840aggcatatca acgaagaaac agcatccctc
tcgaacaagc aattgctaaa gatacttcgg 900gtgactatga gaagtttctt
cttgccttga tcggagctgg agatgcatga accgtcttcg 960gtattaagtt
cctctgtatg aatgtttagt ttgccttatc cgctatgact taataattta
1020tgcttggttt ttcatcgttt tcattatcta aagcattgct tgcttccatg
atagaacatt 1080caaaataaaa tgattgagtt cgtttaaaaa aaaaaaaaaa
aaaaaaaagg aaaaaaaaaa 1140aaaaaa 1146103315PRTGossypium hirsutum
103Thr Leu Lys Val Pro Val His Val Pro Ser Pro Ser Glu Asp Ala Glu
1 5 10 15 Trp Gln Leu Arg Lys Ala Phe Glu Gly Trp Gly Thr Asn Glu
Gln Leu 20 25 30 Ile Ile Asp Ile Leu Ala His Arg Asn Ala Ala Gln
Arg Asn Ser Ile 35 40 45 Arg Lys Val Tyr Gly Glu Ala Tyr Gly Glu
Asp Leu Leu Lys Cys Leu 50 55 60 Glu Lys Glu Leu Thr Ser Asp Phe
Glu Arg Ala Val Leu Leu Phe Thr 65
70 75 80 Leu Asp Pro Ala Glu Arg Asp Ala His Leu Ala Asn Glu Ala
Thr Lys 85 90 95 Lys Phe Thr Ser Ser Asn Trp Ile Leu Met Glu Ile
Ala Cys Ser Arg 100 105 110 Ser Ser His Glu Leu Leu Asn Val Lys Lys
Ala Tyr His Ala Arg Tyr 115 120 125 Lys Lys Ser Leu Glu Glu Asp Val
Ala His His Thr Thr Gly Glu Tyr 130 135 140 Arg Lys Leu Leu Val Pro
Leu Val Ser Ala Phe Arg Tyr Glu Gly Glu 145 150 155 160 Glu Val Asn
Met Thr Leu Ala Lys Ser Glu Ala Lys Ile Leu His Asp 165 170 175 Lys
Ile Ser Asp Lys His Tyr Thr Asp Glu Glu Val Ile Arg Ile Val 180 185
190 Ser Thr Arg Ser Lys Ala Gln Leu Asn Ala Thr Leu Asn His Tyr Asn
195 200 205 Thr Ser Phe Gly Asn Ala Ile Asn Lys Asp Leu Lys Ala Asp
Pro Ser 210 215 220 Asp Glu Phe Leu Lys Leu Leu Arg Ala Val Ile Lys
Cys Leu Thr Thr 225 230 235 240 Pro Glu Gln Tyr Phe Glu Lys Val Leu
Arg Gln Ala Ile Asn Lys Leu 245 250 255 Gly Ser Asp Glu Trp Ala Leu
Thr Arg Val Val Thr Thr Arg Ala Glu 260 265 270 Val Asp Met Val Arg
Ile Lys Glu Ala Tyr Gln Arg Arg Asn Ser Ile 275 280 285 Pro Leu Glu
Gln Ala Ile Ala Lys Asp Thr Ser Gly Asp Tyr Glu Lys 290 295 300 Phe
Leu Leu Ala Leu Ile Gly Ala Gly Asp Ala 305 310 315
1041180DNACapsicum annuum 104aaaaatggca agtctaaccg ttccagcaca
tgttccttcg gctgctgaag actgtgaaca 60actccgatct gccttcaaag gatggggaac
aaatgagaag ttgatcatat caattttggc 120tcatagaact gctgctcagc
gcaaattgat tcgtcaaact tatgctgaga ctttcggaga 180ggatctactt
aaagagttgg acagagaact tacccatgat tttgagaaat tggtgctagt
240gtggacgttg gatccttcag aacgtgatgc tcatttggct aaggaagcta
ctaagagatg 300gacaaaaagc aactttgttc ttgtggagct agcttgtacc
agatcgccta aagaactggt 360tttggctagg gaagcttatc atgcacgtta
caagaaatct cttgaagagg atgttgccta 420tcacactact ggggatcacc
gcaagctttt ggtacctctt gtgagctcct accgatatgg 480aggagaggag
gtggacttgc gccttgctaa agcagaatct aaaattctgc atgagaagat
540ctccgataag gcttacagtg atgatgaggt cattagaatt ttagccacaa
ggagcaaagc 600gcaactcaat gctactttga atcattacaa agatgaacat
ggtgaggata tcctaaagca 660attggaagat ggggatgagt ttgttgcact
attgagggcc accattaaag gtcttgtcta 720cccggagcac tattttgtgg
aggttcttcg tgatgcaatc aacaggagag ggacagagga 780agatcatctg
acaagagtta ttgctacaag ggctgaggtc gatctgaaga ttatcgctga
840tgagtaccag aagagggata gcattcccct gggtcgcgcc attgccaaag
atacaagagg 900agattatgag agtatgctgt tggctttgct tggacaagag
gaggactgag gaggatttgg 960ccacttatgt tttacaatga caagaataaa
tatgccatcc cctatatgag aattggcatc 1020cgttgtatgt ttgatgattg
agtgtggtct gtttatgagc ttttagtcct tttttcttct 1080cgtgagaaac
ttctaatatg caactttgtg ctgtctacat atgttttcta ataatatgca
1140tcgattagtt ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa
1180105322PRTCapsicum annuum 105Met Ala His His His His His His Met
Ala Ser Leu Thr Val Pro Ala 1 5 10 15 His Val Pro Ser Ala Ala Glu
Asp Cys Glu Gln Leu Arg Ser Ala Phe 20 25 30 Lys Gly Trp Gly Thr
Asn Glu Lys Leu Ile Ile Ser Ile Leu Ala His 35 40 45 Arg Thr Ala
Ala Gln Arg Lys Leu Ile Arg Gln Thr Tyr Ala Glu Thr 50 55 60 Phe
Gly Glu Asp Leu Leu Lys Glu Leu Asp Arg Glu Leu Thr His Asp 65 70
75 80 Phe Glu Lys Leu Val Leu Val Trp Thr Leu Asp Pro Ser Glu Arg
Asp 85 90 95 Ala His Leu Ala Lys Glu Ala Thr Lys Arg Trp Thr Lys
Ser Asn Phe 100 105 110 Val Leu Val Glu Leu Ala Cys Thr Arg Ser Pro
Lys Glu Leu Val Leu 115 120 125 Ala Arg Glu Ala Tyr His Ala Arg Tyr
Lys Lys Ser Leu Glu Glu Asp 130 135 140 Val Ala Tyr His Thr Thr Gly
Asp His Arg Lys Leu Leu Val Pro Leu 145 150 155 160 Val Ser Ser Tyr
Arg Tyr Gly Gly Glu Glu Val Asp Leu Arg Leu Ala 165 170 175 Lys Ala
Glu Ser Lys Ile Leu His Glu Lys Ile Ser Asp Lys Ala Tyr 180 185 190
Ser Asp Asp Glu Val Ile Arg Ile Leu Ala Thr Arg Ser Lys Ala Gln 195
200 205 Leu Asn Ala Thr Leu Asn His Tyr Lys Asp Glu His Gly Glu Asp
Ile 210 215 220 Leu Lys Gln Leu Glu Asp Gly Asp Glu Phe Val Ala Leu
Leu Arg Ala 225 230 235 240 Thr Ile Lys Gly Leu Val Tyr Pro Glu His
Tyr Phe Val Glu Val Leu 245 250 255 Arg Asp Ala Ile Asn Arg Arg Gly
Thr Glu Glu Asp His Leu Thr Arg 260 265 270 Val Ile Ala Thr Arg Ala
Glu Val Asp Leu Lys Ile Ile Ala Asp Glu 275 280 285 Tyr Gln Lys Arg
Asp Ser Ile Pro Leu Gly Arg Ala Ile Ala Lys Asp 290 295 300 Thr Arg
Gly Asp Tyr Glu Ser Met Leu Leu Ala Leu Leu Gly Gln Glu 305 310 315
320 Glu Asp 1061197DNANicotiana tabacum 106catccacaat tctccacagt
acaagaaaac aaaaaaatgg cgagtcttaa agttccaaca 60tctgttccag aaccttatga
agatgctgag caactcaaaa aagcttttgc tggatggggt 120acaaatgagg
cacttattat tcagattctg gcacatagaa atgcagcaca acgcaagtta
180atccgagaaa cttatgctgc agcttatgga gaggatcttc tcaaggactt
ggatgctgaa 240ctgacaagtg attttcagcg tgcagtgctt ctgtggactt
tgagtcctgc tgagcgcgac 300gcctacttgg ttaatgaagc taccaaacgt
ctgacttcta gcaattgggt tatcttggaa 360attgcttgta caaggtcttc
tgatgatctc tttaaggcga ggcaggccta ccatgctcga 420tacaagaaat
cacttgaaga agatgttgct tatcacacaa ctggggattt ccgtaagctt
480ttggttcctc ttttaactgc attcagatac gaaggagaag aggcgaacat
gacattggca 540agaaaggagg caaatatact acacgagaag atctctgaca
aggcttacaa tgatgaggag 600ctcatccgaa ttatttctac taggagtaaa
gcacagctga atgcaacatt caaccactac 660cttgaccaac atggcagtga
aatcaacaag gatctggaaa ctgattctga tgatgagtac 720ctgaaattac
tcagcgcagc aatagaatgc ttgaaaaccc cagagaaaca ctttgagaaa
780gttcttcgat tggctatcaa gggtacaggc acagacgaat gggaccttac
tagagttgtc 840actactcggg ctgaagttga catggaacgt atcaaagaag
agtaccataa gaggaacagt 900gttccattgg accgtgcaat tgctggagac
acttcaggag actatgaaag gatgcttctg 960gctttgattg ggcatggaga
tgcttgaatg gaatatgtgt tctaagattg gataagaaac 1020tatttcctaa
tgtctgaagt ttgaatttgt ttgatgatgt gtgcatgtat gcccagagtt
1080tggtttgcat tatatggatt taaataatcc aggtgttgtg ttttgttttt
ttcttcactt 1140gtcatagttt ggttctatat attcggactt cctcaaccag
tgatcttatt gtttatc 1197107316PRTNicotiana tabacum 107Met Ala Ser
Leu Lys Val Pro Thr Ser Val Pro Glu Pro Tyr Glu Asp 1 5 10 15 Ala
Glu Gln Leu Lys Lys Ala Phe Ala Gly Trp Gly Thr Asn Glu Ala 20 25
30 Leu Ile Ile Gln Ile Leu Ala His Arg Asn Ala Ala Gln Arg Lys Leu
35 40 45 Ile Arg Glu Thr Tyr Ala Ala Ala Tyr Gly Glu Asp Leu Leu
Lys Asp 50 55 60 Leu Asp Ala Glu Leu Thr Ser Asp Phe Gln Arg Ala
Val Leu Leu Trp 65 70 75 80 Thr Leu Ser Pro Ala Glu Arg Asp Ala Tyr
Leu Val Asn Glu Ala Thr 85 90 95 Lys Arg Leu Thr Ser Ser Asn Trp
Val Ile Leu Glu Ile Ala Cys Thr 100 105 110 Arg Ser Ser Asp Asp Leu
Phe Lys Ala Arg Gln Ala Tyr His Ala Arg 115 120 125 Tyr Lys Lys Ser
Leu Glu Glu Asp Val Ala Tyr His Thr Thr Gly Asp 130 135 140 Phe Arg
Lys Leu Leu Val Pro Leu Leu Thr Ala Phe Arg Tyr Glu Gly 145 150 155
160 Glu Glu Ala Asn Met Thr Leu Ala Arg Lys Glu Ala Asn Ile Leu His
165 170 175 Glu Lys Ile Ser Asp Lys Ala Tyr Asn Asp Glu Glu Leu Ile
Arg Ile 180 185 190 Ile Ser Thr Arg Ser Lys Ala Gln Leu Asn Ala Thr
Phe Asn His Tyr 195 200 205 Leu Asp Gln His Gly Ser Glu Ile Asn Lys
Asp Leu Glu Thr Asp Ser 210 215 220 Asp Asp Glu Tyr Leu Lys Leu Leu
Ser Ala Ala Ile Glu Cys Leu Lys 225 230 235 240 Thr Pro Glu Lys His
Phe Glu Lys Val Leu Arg Leu Ala Ile Lys Gly 245 250 255 Thr Gly Thr
Asp Glu Trp Asp Leu Thr Arg Val Val Thr Thr Arg Ala 260 265 270 Glu
Val Asp Met Glu Arg Ile Lys Glu Glu Tyr His Lys Arg Asn Ser 275 280
285 Val Pro Leu Asp Arg Ala Ile Ala Gly Asp Thr Ser Gly Asp Tyr Glu
290 295 300 Arg Met Leu Leu Ala Leu Ile Gly His Gly Asp Ala 305 310
315 1081086DNAGossypium hirsutum 108gggcaggttc ttcacaaaaa
agaaaagaaa aattacagtg aaaatggcaa cccttaaagt 60tccagctcat gtacctgccc
cttctgagga tgctgagcaa cttcgtaaag cttttgaagg 120atggggtaca
aatgagcaat tgattatcga cattttggct cacaggaatg cagctcagcg
180caatttgatt cgtaaaactt atcgtgaagc ttatggggaa gatctcctta
agtctttgga 240tgaggaactt tcaagtgact ttgagcgagc tgtggtgctg
tttactttgg accctgcaga 300gcgtgatgca tttctggctc atgaagctac
aaagaggttc acatcaagcc attgggttct 360catggaaatt gcttgcacta
ggtcttcaca tgaactgttc aatgtgagga aggcgtatca 420cgatctttac
aagaaatccc ttgaagaaga tgttgcgcac cataccaagg gagactaccg
480caagcttttg gtcccacttg ttagtgcatt ccgataccag ggagaggagg
tgaacatgac 540actggcaagg tcggaggcaa agatacttcg tgagaagata
tcagacaagc agtacagtga 600tgaggaggtc atcaggattg taacaacacg
gagtaaggca cagttaaatg ctactctgaa 660tcattacaat actgcatttg
ggaatgctat caacaaggat ttgaaggccg accctgaaga 720cgaattcctc
aaattgctga gagctgcaat caagtgcttg actgtccctg agaaatattt
780tgagaaggtg ctacgtcaag caatcaataa gctgggaaca gatgaatggg
ctcttactag 840agtggtcgcc actcgggcgg aggtagacat ggtgcgtatt
aaggaggaat atcagcgaag 900aaacagtgtg accctggaaa aagcgattgc
tggagatacc tctggagact atgagaaaat 960gctgcttgcg ttgattggag
ctggagacgt ctgagctgct ttcctatatt gagttgttgg 1020tatgaaaatt
tagtttgcaa tttgaggtgt gagttatgtt tgtttggttg agggtgtgcc 1080aatcgc
1086109316PRTGossypium hirsutum 109Met Ala Thr Leu Lys Val Pro Ala
His Val Pro Ala Pro Ser Glu Asp 1 5 10 15 Ala Glu Gln Leu Arg Lys
Ala Phe Glu Gly Trp Gly Thr Asn Glu Gln 20 25 30 Leu Ile Ile Asp
Ile Leu Ala His Arg Asn Ala Ala Gln Arg Asn Leu 35 40 45 Ile Arg
Lys Thr Tyr Arg Glu Ala Tyr Gly Glu Asp Leu Leu Lys Ser 50 55 60
Leu Asp Glu Glu Leu Ser Ser Asp Phe Glu Arg Ala Val Val Leu Phe 65
70 75 80 Thr Leu Asp Pro Ala Glu Arg Asp Ala Phe Leu Ala His Glu
Ala Thr 85 90 95 Lys Arg Phe Thr Ser Ser His Trp Val Leu Met Glu
Ile Ala Cys Thr 100 105 110 Arg Ser Ser His Glu Leu Phe Asn Val Arg
Lys Ala Tyr His Asp Leu 115 120 125 Tyr Lys Lys Ser Leu Glu Glu Asp
Val Ala His His Thr Lys Gly Asp 130 135 140 Tyr Arg Lys Leu Leu Val
Pro Leu Val Ser Ala Phe Arg Tyr Gln Gly 145 150 155 160 Glu Glu Val
Asn Met Thr Leu Ala Arg Ser Glu Ala Lys Ile Leu Arg 165 170 175 Glu
Lys Ile Ser Asp Lys Gln Tyr Ser Asp Glu Glu Val Ile Arg Ile 180 185
190 Val Thr Thr Arg Ser Lys Ala Gln Leu Asn Ala Thr Leu Asn His Tyr
195 200 205 Asn Thr Ala Phe Gly Asn Ala Ile Asn Lys Asp Leu Lys Ala
Asp Pro 210 215 220 Glu Asp Glu Phe Leu Lys Leu Leu Arg Ala Ala Ile
Lys Cys Leu Thr 225 230 235 240 Val Pro Glu Lys Tyr Phe Glu Lys Val
Leu Arg Gln Ala Ile Asn Lys 245 250 255 Leu Gly Thr Asp Glu Trp Ala
Leu Thr Arg Val Val Ala Thr Arg Ala 260 265 270 Glu Val Asp Met Val
Arg Ile Lys Glu Glu Tyr Gln Arg Arg Asn Ser 275 280 285 Val Thr Leu
Glu Lys Ala Ile Ala Gly Asp Thr Ser Gly Asp Tyr Glu 290 295 300 Lys
Met Leu Leu Ala Leu Ile Gly Ala Gly Asp Val 305 310 315
1101196DNANicotiana tabacum 110gcatttacga gttttgacaa tcatatcttc
cacagacaga aaaaaaatgg ctagtcttac 60tgttccggca gaagttcctt cagttgctga
agactgtgaa caactccgat ctgccttcaa 120aggatgggga acaaatgaga
agttgatcat atcaattttg gctcatagaa atgctgctca 180acgcaagttg
attcaacaga cttatgctga gacttttggt gaagatctcc ttaaagagtt
240ggacagagaa cttaccaatg attttgagaa attggtggta gtgtggacat
tggatccttc 300agaacgcgat gcctatttgg ctaaggaagc tactaagaga
tggacaaaaa gcaattttgt 360tcttgtggag attgcttgta ccagatctcc
taaagaattg gttttggcaa gggaagctta 420tcatgctcgt ttcaagaaat
ctcttgaaga ggacgttgct tatcacacta ctggggaaca 480cccccagctt
ttggtacctc ttgtgagctc ctaccgatat ggaggggacg aggtggactt
540gcgccttgct aaagcagaag ctaaaatact gcacgagaag atctccgata
aggcttacag 600tgacgatgag gtcatcagaa ttctagccac aaggagcaaa
gcacagatca atgctactct 660gaaccattac aaagatgaat atgaagaaga
tatcctgaag caattggaag agggggatga 720gtttgttgga ctattgaggg
caaccataaa aggtcttgtc taccccgagc actacttcgt 780ggaggttctt
cgagatgcaa ttaacaggag aggaacagat gaagatcatc tgaccagagt
840tatcgctaca agggctgagg ttgatatgaa gattatcgct gatgagtacc
agaagaggga 900tagcatccct ctgggtcggg ccatcgccaa agatacaaga
ggagattatg agagtatgtt 960gttggctctg cttggacaag aggaggacta
agaaggtttt gcttctgttt cataatgacc 1020agaataaaca tgctatcccc
tatatttgag agttggcatc cgttgtatgc ttgatgatta 1080agcgtggtct
gtttaacgtg agcttttagt ccttttcttc ttgtgataaa ctttgaatgt
1140acaactttat gctatctaag aatgtttttc taaaaaaaaa aaaaaaaaaa aaaaaa
1196111314PRTNicotiana tabacum 111Met Ala Ser Leu Thr Val Pro Ala
Glu Val Pro Ser Val Ala Glu Asp 1 5 10 15 Cys Glu Gln Leu Arg Ser
Ala Phe Lys Gly Trp Gly Thr Asn Glu Lys 20 25 30 Leu Ile Ile Ser
Ile Leu Ala His Arg Asn Ala Ala Gln Arg Lys Leu 35 40 45 Ile Gln
Gln Thr Tyr Ala Glu Thr Phe Gly Glu Asp Leu Leu Lys Glu 50 55 60
Leu Asp Arg Glu Leu Thr Asn Asp Phe Glu Lys Leu Val Val Val Trp 65
70 75 80 Thr Leu Asp Pro Ser Glu Arg Asp Ala Tyr Leu Ala Lys Glu
Ala Thr 85 90 95 Lys Arg Trp Thr Lys Ser Asn Phe Val Leu Val Glu
Ile Ala Cys Thr 100 105 110 Arg Ser Pro Lys Glu Leu Val Leu Ala Arg
Glu Ala Tyr His Ala Arg 115 120 125 Phe Lys Lys Ser Leu Glu Glu Asp
Val Ala Tyr His Thr Thr Gly Glu 130 135 140 His Pro Gln Leu Leu Val
Pro Leu Val Ser Ser Tyr Arg Tyr Gly Gly 145 150 155 160 Asp Glu Val
Asp Leu Arg Leu Ala Lys Ala Glu Ala Lys Ile Leu His 165 170 175 Glu
Lys Ile Ser Asp Lys Ala Tyr Ser Asp Asp Glu Val Ile Arg Ile 180 185
190 Leu Ala Thr Arg Ser Lys Ala Gln Ile Asn Ala Thr Leu Asn His Tyr
195 200 205 Lys Asp Glu Tyr Glu Glu Asp Ile Leu Lys Gln Leu Glu Glu
Gly Asp 210 215 220 Glu Phe Val Gly Leu Leu Arg Ala Thr Ile Lys Gly
Leu Val Tyr Pro 225 230 235 240 Glu His Tyr Phe Val Glu Val Leu Arg
Asp Ala Ile Asn Arg Arg Gly 245 250 255 Thr Asp Glu Asp His Leu Thr
Arg Val Ile Ala Thr Arg Ala Glu Val 260 265 270 Asp Met Lys Ile Ile
Ala Asp Glu Tyr Gln Lys Arg Asp Ser Ile Pro 275 280 285 Leu Gly Arg
Ala Ile Ala Lys Asp Thr Arg Gly Asp Tyr Glu Ser Met 290 295 300 Leu
Leu Ala Leu Leu Gly Gln Glu Glu Asp 305 310 112945DNASolanum
tuberosum 112atggcaagtc ttacagttcc ggcagaagtt ccttccgtag ctgaagactg
tgaacaactc 60cgatctgcct tcaaaggatg gggaacgaac gagaagttga ttatatcaat
tttggctcat 120agaaatgctg ctcagcgcaa attgattcga cagacttatg
ctgaaacttt tggggaagat 180ctacttaaag agttggacag agaacttacc
catgattttg agaaattggt gctaatatgg 240acactggatc cgtcagaacg
tgatgcctat ttggctaagg aagctactaa gagatggaca 300aaaagcaact
ttgttcttgt ggagatagct tgtactagat ctcctaaaga actggttttg
360gcaagggaag cttatcatgc tcgtaacaag aaatctcttg aagaggacgt
tgcttatcac 420actactgggg atcaccgcaa gcttttggta cctcttgtga
gctcctaccg atatggagga 480gacgaggtgg acttgcgcct tgctaaagca
gaatctaaag tactgcatga gaagatctcc 540gataaggctt acagtgacga
tgaggtcatt agaattttag ccacaaggag caaagcgcaa 600ctcaatgcta
ctttgaatca ttacaaagat gaatatggtg aggatatcct aaagcaattg
660gaagatgagg atgagtttgt tgcactattg agggccacca taaaaggtct
tgtctaccct 720gagcactatt tcgtggaggt tcttcgtgat gcaattaaca
ggagaggaac agaggaagat 780catctgagcc gagttatcgc tacaagggct
gaggtggatc tgaagactat cgctaacgag 840taccagaaga gggatagcat
tcctctgggt cgcgccattg ccaaagatac aggaggagat 900tatgagaata
tgctggtggc tttacttgga caagaggagg aatga 945113314PRTSolanum
tuberosum 113Met Ala Ser Leu Thr Val Pro Ala Glu Val Pro Ser Val
Ala Glu Asp 1 5 10 15 Cys Glu Gln Leu Arg Ser Ala Phe Lys Gly Trp
Gly Thr Asn Glu Lys 20 25 30 Leu Ile Ile Ser Ile Leu Ala His Arg
Asn Ala Ala Gln Arg Lys Leu 35 40 45 Ile Arg Gln Thr Tyr Ala Glu
Thr Phe Gly Glu Asp Leu Leu Lys Glu 50 55 60 Leu Asp Arg Glu Leu
Thr His Asp Phe Glu Lys Leu Val Leu Ile Trp 65 70 75 80 Thr Leu Asp
Pro Ser Glu Arg Asp Ala Tyr Leu Ala Lys Glu Ala Thr 85 90 95 Lys
Arg Trp Thr Lys Ser Asn Phe Val Leu Val Glu Ile Ala Cys Thr 100 105
110 Arg Ser Pro Lys Glu Leu Val Leu Ala Arg Glu Ala Tyr His Ala Arg
115 120 125 Asn Lys Lys Ser Leu Glu Glu Asp Val Ala Tyr His Thr Thr
Gly Asp 130 135 140 His Arg Lys Leu Leu Val Pro Leu Val Ser Ser Tyr
Arg Tyr Gly Gly 145 150 155 160 Asp Glu Val Asp Leu Arg Leu Ala Lys
Ala Glu Ser Lys Val Leu His 165 170 175 Glu Lys Ile Ser Asp Lys Ala
Tyr Ser Asp Asp Glu Val Ile Arg Ile 180 185 190 Leu Ala Thr Arg Ser
Lys Ala Gln Leu Asn Ala Thr Leu Asn His Tyr 195 200 205 Lys Asp Glu
Tyr Gly Glu Asp Ile Leu Lys Gln Leu Glu Asp Glu Asp 210 215 220 Glu
Phe Val Ala Leu Leu Arg Ala Thr Ile Lys Gly Leu Val Tyr Pro 225 230
235 240 Glu His Tyr Phe Val Glu Val Leu Arg Asp Ala Ile Asn Arg Arg
Gly 245 250 255 Thr Glu Glu Asp His Leu Ser Arg Val Ile Ala Thr Arg
Ala Glu Val 260 265 270 Asp Leu Lys Thr Ile Ala Asn Glu Tyr Gln Lys
Arg Asp Ser Ile Pro 275 280 285 Leu Gly Arg Ala Ile Ala Lys Asp Thr
Gly Gly Asp Tyr Glu Asn Met 290 295 300 Leu Val Ala Leu Leu Gly Gln
Glu Glu Glu 305 310 1141160DNALycopersicon esculentum 114atggcaagtc
ttacagttcc ggcagaagtt ccttcagtcg ctgaagactg tgaacaactc 60cgatctgcct
tcaaaggatg gggaacgaat gagaagttga ttatatcaat tttggctcat
120agaaatgcgg ctcaacgcaa attgattcga cagacttatg ctgagacttt
tggggaagat 180ctgcttaaag agttggacag agaacttact catgattttg
agaaattggt ggtagtatgg 240acactggatc ctgcagaacg tgatgcctat
ttggctaagg aagctactaa gagatggaca 300aaaagcaact ttgttcttgt
ggagatagct tgtaccagat ctcctaaaga actggttttg 360gcaagagaag
cttatcatgc tcgtaacaag aaatctctcg aagaggacgt tgcttatcac
420actactgggg atcaccgcaa gcttttggta cctcttgtga gctcctaccg
atatggggga 480gatgaggtgg acttgcgact tgctaaagca gaatctaaag
tgctgcatga gaagatctcc 540gataaggctt acagtgacga tgaggtcatt
agaattttag ccacaaggag caaagcgcaa 600ctcaatgcta ctttgaatca
ttacaaagat gaatatggtg aggatatcct aaagcaatta 660gaagatgagg
atgagtttgt tgcactgtta agggccacca taaaaggtct tgtctacccc
720gagcactatt tcgtggaggt tcttcgtgat gcaattaaca ggagaggaac
agaggaagat 780catctaaccc gagttatcgc tacaagggct gaggtcgatc
tgaagactat cgctaacgag 840taccagaaga gggatagcgt tcctctgggt
cgcgccattg ccaaagatac aggaggagat 900tatgagaata tgctggtggc
tttacttgga caagaggagg aataagaagc ggattggctc 960acttctgttt
ataatgacca gataatatgc cattctccat atatttcaga gttggcatgt
1020gtttgatgat tgagagtggt ctgttcacat gagctttagt ccttttcttc
ttgtgagaaa 1080ctttgaatat gaatctttgt gctgtctaaa aatgttctct
aatgatttgc atccactaaa 1140aaaaaaaaaa aaaaaaaaaa
1160115314PRTLycopersicon esculentum 115Met Ala Ser Leu Thr Val Pro
Ala Glu Val Pro Ser Val Ala Glu Asp 1 5 10 15 Cys Glu Gln Leu Arg
Ser Ala Phe Lys Gly Trp Gly Thr Asn Glu Lys 20 25 30 Leu Ile Ile
Ser Ile Leu Ala His Arg Asn Ala Ala Gln Arg Lys Leu 35 40 45 Ile
Arg Gln Thr Tyr Ala Glu Thr Phe Gly Glu Asp Leu Leu Lys Glu 50 55
60 Leu Asp Arg Glu Leu Thr His Asp Phe Glu Lys Leu Val Val Val Trp
65 70 75 80 Thr Leu Asp Pro Ala Glu Arg Asp Ala Tyr Leu Ala Lys Glu
Ala Thr 85 90 95 Lys Arg Trp Thr Lys Ser Asn Phe Val Leu Val Glu
Ile Ala Cys Thr 100 105 110 Arg Ser Pro Lys Glu Leu Val Leu Ala Arg
Glu Ala Tyr His Ala Arg 115 120 125 Asn Lys Lys Ser Leu Glu Glu Asp
Val Ala Tyr His Thr Thr Gly Asp 130 135 140 His Arg Lys Leu Leu Val
Pro Leu Val Ser Ser Tyr Arg Tyr Gly Gly 145 150 155 160 Asp Glu Val
Asp Leu Arg Leu Ala Lys Ala Glu Ser Lys Val Leu His 165 170 175 Glu
Lys Ile Ser Asp Lys Ala Tyr Ser Asp Asp Glu Val Ile Arg Ile 180 185
190 Leu Ala Thr Arg Ser Lys Ala Gln Leu Asn Ala Thr Leu Asn His Tyr
195 200 205 Lys Asp Glu Tyr Gly Glu Asp Ile Leu Lys Gln Leu Glu Asp
Glu Asp 210 215 220 Glu Phe Val Ala Leu Leu Arg Ala Thr Ile Lys Gly
Leu Val Tyr Pro 225 230 235 240 Glu His Tyr Phe Val Glu Val Leu Arg
Asp Ala Ile Asn Arg Arg Gly 245 250 255 Thr Glu Glu Asp His Leu Thr
Arg Val Ile Ala Thr Arg Ala Glu Val 260 265 270 Asp Leu Lys Thr Ile
Ala Asn Glu Tyr Gln Lys Arg Asp Ser Val Pro 275 280 285 Leu Gly Arg
Ala Ile Ala Lys Asp Thr Gly Gly Asp Tyr Glu Asn Met 290 295 300 Leu
Val Ala Leu Leu Gly Gln Glu Glu Glu 305 310 1161242DNALycopersicon
esculentum 116atgtctagtc ttaaagttcc agcatcagtt ccagatcctt
atgaagatgc tgagcaactc 60aaaaaagctt ttaaaggatg gggcacaaat gaggaactta
ttattcagat tctggctcat 120aggaatgcca gacaacgcaa gttaatccga
gattcttatg ctgctgctta tggagaggat 180cttctcaagg acttggattc
tgaactgaca agtgattttc agcgtgtggt gcttctctgg 240actttgagtc
ctgctgagcg cgacgcctac ttggttaatg aggctaccaa acgtctgact
300gctagcaatt ggggtatcat ggaaattgct tgtaccaggt cttctgatga
tctttttaag 360gcgaggcagg cctaccatgc tccatacaag aaatcacttg
aagaagatgt tgcttatcat 420acagtggggg atttccgtaa gcttttggtt
cctcttataa ctgcattcag atatgaagga 480gatgaggtga acatgacatt
agcaagaaag ggaagcaaat atctgcatga gaagatctct 540gacaaggctt
accatgacga ggagatcatc cgaatcattt ctactaggag taaagcacag
600ctgagtgcta cgttcaacca ctaccatgat caccatggcc atgaaatcat
caaggatctg 660gaagctgatg atgacgatga gtacctgaaa ctactcagag
cagcaataga atgcttgaaa 720cccagagaac actttgagaa agttcttcga
ttggctatca agaagctggg tacagacgaa 780tgggatctta ctagagttgt
tgccactcgg gctgaagttg acatggagcg tatcaaagaa 840gagtaccata
ggaggaacag tgttacattg gaccgtgcaa ttgctggaga cacttcagga
900gactatgaaa aaatgcttct ggctctgatt gggcacggag atgcttgaat
tacatgtgct 960gaaaccttaa gataataaaa aactctactt attttctgaa
ctttcatttg cttttatgat 1020ctatggtgtg tactctcaga gtttggttct
gtgtttatat gaactaaaaa cactcgggag 1080ttgagttgtg ttttgttttc
gccttcactt ttcatttcgg acttctactg gttttgcctg 1140ctaaataagc
atagcttcaa ctttggcttg aacggatctt gtttctttat aactcagaaa
1200tagattatgt atcttggttc gtaaaaaaaa aaaaaaaaaa aa
1242117315PRTLycopersicon esculentum 117Met Ser Ser Leu Lys Val Pro
Ala Ser Val Pro Asp Pro Tyr Glu Asp 1 5 10 15 Ala Glu Gln Leu Lys
Lys Ala Phe Lys Gly Trp Gly Thr Asn Glu Glu 20 25 30 Leu Ile Ile
Gln Ile Leu Ala His Arg Asn Ala Arg Gln Arg Lys Leu 35 40 45 Ile
Arg Asp Ser Tyr Ala Ala Ala Tyr Gly Glu Asp Leu Leu Lys Asp 50 55
60 Leu Asp Ser Glu Leu Thr Ser Asp Phe Gln Arg Val Val Leu Leu Trp
65 70 75 80 Thr Leu Ser Pro Ala Glu Arg Asp Ala Tyr Leu Val Asn Glu
Ala Thr 85 90 95 Lys Arg Leu Thr Ala Ser Asn Trp Gly Ile Met Glu
Ile Ala Cys Thr 100 105 110 Arg Ser Ser Asp Asp Leu Phe Lys Ala Arg
Gln Ala Tyr His Ala Pro 115 120 125 Tyr Lys Lys Ser Leu Glu Glu Asp
Val Ala Tyr His Thr Val Gly Asp 130 135 140 Phe Arg Lys Leu Leu Val
Pro Leu Ile Thr Ala Phe Arg Tyr Glu Gly 145 150 155 160 Asp Glu Val
Asn Met Thr Leu Ala Arg Lys Gly Ser Lys Tyr Leu His 165 170 175 Glu
Lys Ile Ser Asp Lys Ala Tyr His Asp Glu Glu Ile Ile Arg Ile 180 185
190 Ile Ser Thr Arg Ser Lys Ala Gln Leu Ser Ala Thr Phe Asn His Tyr
195 200 205 His Asp His His Gly His Glu Ile Ile Lys Asp Leu Glu Ala
Asp Asp 210 215 220 Asp Asp Glu Tyr Leu Lys Leu Leu Arg Ala Ala Ile
Glu Cys Leu Lys 225 230 235 240 Pro Arg Glu His Phe Glu Lys Val Leu
Arg Leu Ala Ile Lys Lys Leu 245 250 255 Gly Thr Asp Glu Trp Asp Leu
Thr Arg Val Val Ala Thr Arg Ala Glu 260 265 270 Val Asp Met Glu Arg
Ile Lys Glu Glu Tyr His Arg Arg Asn Ser Val 275 280 285 Thr Leu Asp
Arg Ala Ile Ala Gly Asp Thr Ser Gly Asp Tyr Glu Lys 290 295 300 Met
Leu Leu Ala Leu Ile Gly His Gly Asp Ala 305 310 315
1181226DNAArabidopsis thaliana 118ttacttaagt aggacgacgt gcgtctgctt
cgtctcatta caaagcagaa gaaacacaaa 60cagaggcaga gatcttaaga gttaaagact
aatcccaaca atggcgtctc tcaaagtccc 120aagcaatgtt cctcttcccg
aagatgacgc cgagcaactc cacaaggctt tttcaggatg 180gggtaccaac
gagaagctga tcatatcaat actagctcac aggaacgcag cacaacgcag
240cttgatccgc agcgtttatg cagctaccta caatgaggat cttctcaaag
cattagacaa 300agagctttct agcgactttg agagagctgt gatgttgtgg
actcttgatc caccagagag 360agatgcttat ttggctaaag aatccaccaa
gatgttcacc aagaacaatt gggttcttgt 420tgaaatcgct tgcacaaggc
ctgctcttga gcttatcaag gtcaagcaag cttaccaagc 480tcgatacaag
aaatcaatcg aggaagatgt cgcgcaacac acatctggtg accttcgtaa
540gctcttgctt cctcttgtga gcactttcag gtatgaagga gatgatgtga
acatgatgct 600tgcaagatct gaagctaaga tacttcacga gaaggtctca
gagaaatctt acagtgacga 660tgacttcatc agaatcttga caacaagaag
caaagcacag ctcggtgcaa cactcaacca 720ctacaacaac gagtatggaa
acgccattaa caagaacttg aaggaagagt cggacgacaa 780tgactacatg
aaactactaa gagctgtaat cacatgtttg acataccctg agaagcattt
840tgagaaggtt cttcgtctat caatcaacaa aatgggaaca gacgaatggg
gactaacccg 900agtcgtgact acacgaactg aagttgacat ggaacgcatc
aaagaggaat atcagcgaag 960aaacagcatt cctttggacc gtgctatcgc
caaagacact tctggtgact atgaggacat 1020gcttgttgct cttctcggac
atggcgatgc ttgaaactgt ttcaactttc gagttcctcc 1080tttctcttac
tgcatggttt gttttaaata aaagagttgt gaaactggtt ctgcaactat
1140ttatcaatga tcgtttgagt ttgttaaatt tgaatcaaaa tctgtttttc
tttcttttaa 1200atacaatcta aagcacaaac taaagc
1226119317PRTArabidopsis thaliana 119Met Ala Ser Leu Lys Val Pro
Ser Asn Val Pro Leu Pro Glu Asp Asp 1 5 10 15 Ala Glu Gln Leu His
Lys Ala Phe Ser Gly Trp Gly Thr Asn Glu Lys 20 25 30 Leu Ile Ile
Ser Ile Leu Ala His Arg Asn Ala Ala Gln Arg Ser Leu 35 40 45 Ile
Arg Ser Val Tyr Ala Ala Thr Tyr Asn Glu Asp Leu Leu Lys Ala 50 55
60 Leu Asp Lys Glu Leu Ser Ser Asp Phe Glu Arg Ala Val Met Leu Trp
65 70 75 80 Thr Leu Asp Pro Pro Glu Arg Asp Ala Tyr Leu Ala Lys Glu
Ser Thr 85 90 95 Lys Met Phe Thr Lys Asn Asn Trp Val Leu Val Glu
Ile Ala Cys Thr 100 105 110 Arg Pro Ala Leu Glu Leu Ile Lys Val Lys
Gln Ala Tyr Gln Ala Arg 115 120 125 Tyr Lys Lys Ser Ile Glu Glu Asp
Val Ala Gln His Thr Ser Gly Asp 130 135 140 Leu Arg Lys Leu Leu Leu
Pro Leu Val Ser Thr Phe Arg Tyr Glu Gly 145 150 155 160 Asp Asp Val
Asn Met Met Leu Ala Arg Ser Glu Ala Lys Ile Leu His 165 170 175 Glu
Lys Val Ser Glu Lys Ser Tyr Ser Asp Asp Asp Phe Ile Arg Ile 180 185
190 Leu Thr Thr Arg Ser Lys Ala Gln Leu Gly Ala Thr Leu Asn His Tyr
195 200 205 Asn Asn Glu Tyr Gly Asn Ala Ile Asn Lys Asn Leu Lys Glu
Glu Ser 210 215 220 Asp Asp Asn Asp Tyr Met Lys Leu Leu Arg Ala Val
Ile Thr Cys Leu 225 230 235 240 Thr Tyr Pro Glu Lys His Phe Glu Lys
Val Leu Arg Leu Ser Ile Asn 245 250 255 Lys Met Gly Thr Asp Glu Trp
Gly Leu Thr Arg Val Val Thr Thr Arg 260 265 270 Thr Glu Val Asp Met
Glu Arg Ile Lys Glu Glu Tyr Gln Arg Arg Asn 275 280 285 Ser Ile Pro
Leu Asp Arg Ala Ile Ala Lys Asp Thr Ser Gly Asp Tyr 290 295 300 Glu
Asp Met Leu Val Ala Leu Leu Gly His Gly Asp Ala 305 310 315
1201172DNANicotiana tabacum 120ctgcttgcat tttcgagtct tgacaatcat
aaaaatggct agtcttactg ttccggcaga 60agttccttca gtagctgaag actgtgaaca
actccgatct gccttcaaag ggtggggaac 120aaacgagaag ttgatcatat
caattttggc tcatagaaat gctgctcaac gcaagttgat 180tcaacagact
tatgctgaga cttttggtga agatctcctt aaagagttgg acagagaact
240taccaatgat tttgagaaat tggtggtagt gtggacattg gatccttcag
aacgcgatgc 300ctatttggct aaggaagcta ctaagagatg gacaaaaagc
aattttgttc ttgtggaaat 360agcttgtacc agatctccta aagaattggt
tttggcacgg gaagcttatc atgctcgtta 420caagaaatct cttgaagagg
acgttgctta tcacactact ggggaacacc gcaagctttt 480ggtagctctt
gtgagctcct atcgatatgg aggagacgag gtggacttgc gtcttgctaa
540agctgaagct aaaatactgc atgagaagat ctccgataag gcttacagtg
acaatgaggt 600catcagaatt ctagccacaa ggagtaaagc acagatcaat
gctactctga atcattacaa 660agatgaatat gaagaggata tcctaaagca
attggaagag ggggatgagt ttgttggact 720attgagggca accataaaag
gtcttgtcta caccgagcac tacttcgtgg aggttcttcg 780agatgcaatt
aacaggagag gaacagagga agatcatctg accagagtta tcgctacaag
840ggctgaggtt gatatgaaga ctatcgctga tgagtaccag aagagggata
gcatccatct 900gggtcgcgcc attgccaaag atacaagagg agattatgag
agtatgttgt tggctctgct 960tggacaagag gaggactaag aaggatttgc
tttataatga ccggaataaa tatgatatcc 1020cctatatttg agagttggca
tccgctgtat gtttgatgat tgagcgtggt ctgtttaacg 1080tgagcgttga
gtccttttct tctcactttg aatatgcaac tttatgctat ctaagaatat
1140ttttttataa aaaaaaaaaa aaaaaaaaaa aa 1172121314PRTNicotiana
tabacum 121Met Ala Ser Leu Thr Val Pro Ala Glu Val Pro Ser Val Ala
Glu Asp 1 5 10 15 Cys Glu Gln Leu Arg Ser Ala Phe Lys Gly Trp Gly
Thr Asn Glu Lys 20 25 30 Leu Ile Ile Ser Ile Leu Ala His Arg Asn
Ala Ala Gln Arg Lys Leu 35 40 45 Ile Gln Gln Thr Tyr Ala Glu Thr
Phe Gly Glu Asp Leu Leu Lys Glu 50 55 60 Leu Asp Arg Glu Leu Thr
Asn Asp Phe Glu Lys Leu Val Val Val Trp 65 70 75 80 Thr Leu Asp Pro
Ser Glu Arg Asp Ala Tyr Leu Ala Lys Glu Ala Thr 85 90 95 Lys Arg
Trp Thr Lys Ser Asn Phe Val Leu Val Glu Ile Ala Cys Thr 100 105 110
Arg Ser Pro Lys Glu Leu Val Leu Ala Arg Glu Ala Tyr His Ala Arg 115
120 125 Tyr Lys Lys Ser Leu Glu Glu Asp Val Ala Tyr His
Thr Thr Gly Glu 130 135 140 His Arg Lys Leu Leu Val Ala Leu Val Ser
Ser Tyr Arg Tyr Gly Gly 145 150 155 160 Asp Glu Val Asp Leu Arg Leu
Ala Lys Ala Glu Ala Lys Ile Leu His 165 170 175 Glu Lys Ile Ser Asp
Lys Ala Tyr Ser Asp Asn Glu Val Ile Arg Ile 180 185 190 Leu Ala Thr
Arg Ser Lys Ala Gln Ile Asn Ala Thr Leu Asn His Tyr 195 200 205 Lys
Asp Glu Tyr Glu Glu Asp Ile Leu Lys Gln Leu Glu Glu Gly Asp 210 215
220 Glu Phe Val Gly Leu Leu Arg Ala Thr Ile Lys Gly Leu Val Tyr Thr
225 230 235 240 Glu His Tyr Phe Val Glu Val Leu Arg Asp Ala Ile Asn
Arg Arg Gly 245 250 255 Thr Glu Glu Asp His Leu Thr Arg Val Ile Ala
Thr Arg Ala Glu Val 260 265 270 Asp Met Lys Thr Ile Ala Asp Glu Tyr
Gln Lys Arg Asp Ser Ile His 275 280 285 Leu Gly Arg Ala Ile Ala Lys
Asp Thr Arg Gly Asp Tyr Glu Ser Met 290 295 300 Leu Leu Ala Leu Leu
Gly Gln Glu Glu Asp 305 310 1221215DNASolanum tuberosum
122atcactctgc attttcgagt cttcacaatc atatcctcct aaccacacac
agaagaaaaa 60aaaatggcaa gtcttacagt tccggcagaa gttccttccg tcgctgaaga
ctgtgaacaa 120ctccgatctg ccttcaaagg atggggaacg aacgagaagt
tgattatatc aattttggct 180catagaaatg ctgctcagcg caaattgatt
cgacagactt atgctgaaac ttttggggaa 240gatctactta aagagattgg
gacaggaaga aacttaaccc atgattttga gaaattggtg 300ctaatatgga
cactggatcc gtcagaacgt gatgcctatt tggctaagga agctactaag
360agatggacaa aaagcaactt tgttcttgtg gagatagctt gtactagatc
tcctaaagaa 420ctggttttgg caagggaagc ttatcatgct cgtaacaaga
aatctctcga agaggacgtt 480gcttatcaca ctactgggga tcaccgcaag
cttttggtac ctcttgtgag ctcctaccga 540tatggaggag acgaggtgga
cttgcgcctt gctaaagcag aatctaaagt actgcatgag 600aagatctccg
ataaggctta cagtgacgat gaggtcatta gaattttagc cacaaggagc
660aaagcgcaac tcaatgctac tttgaatcat tacaaagatg aatatggtga
ggatatccta 720aagcaattgg aagatgagga tgagtttgtt gcactattga
gggccaccat aaaaggtctt 780gtctaccctg agcactattt cgtggaggtt
cttcgtgatg caattaacag gagaggaaca 840gaggaagatc atctgagccg
agttattgct acaagggctg aggtcgatct gaagactatc 900gctaacgagt
accagaagag ggatagcatt cctctgggtc gcgccattgc caaagataca
960ggaggagatt atgagaatat gctggtggct ttacttggac aagaggagga
atgaggagga 1020ttggctcact tctgtgttat aatgaccaga ataaatatgc
catctcccat atatttcaga 1080gttggcatct gtttgatgat tgagtgtggt
ctgttttcac atgagctttt agtccttttc 1140ttcgtgtgag aaactttgaa
tatgcatctt tgtgctgtct aaaaatattt tctaaaaaaa 1200aaaaaaaaaa aaaaa
1215123316PRTSolanum tuberosum 123Met Ala Ser Leu Thr Val Pro Ala
Glu Val Pro Ser Val Ala Glu Asp 1 5 10 15 Cys Glu Gln Leu Arg Ser
Ala Phe Lys Gly Trp Gly Thr Asn Glu Lys 20 25 30 Leu Ile Ile Ser
Ile Leu Ala His Arg Asn Ala Ala Gln Arg Lys Leu 35 40 45 Ile Arg
Gln Thr Tyr Ala Glu Thr Phe Gly Glu Asp Leu Leu Lys Glu 50 55 60
Ile Gly Thr Gly Arg Asn Leu Thr His Asp Phe Glu Lys Leu Val Leu 65
70 75 80 Ile Trp Thr Leu Asp Pro Ser Glu Arg Asp Ala Tyr Leu Ala
Lys Glu 85 90 95 Ala Thr Lys Arg Trp Thr Lys Ser Asn Phe Val Leu
Val Glu Ile Ala 100 105 110 Cys Thr Arg Ser Pro Lys Glu Leu Val Leu
Ala Arg Glu Ala Tyr His 115 120 125 Ala Arg Asn Lys Lys Ser Leu Glu
Glu Asp Val Ala Tyr His Thr Thr 130 135 140 Gly Asp His Arg Lys Leu
Leu Val Pro Leu Val Ser Ser Tyr Arg Tyr 145 150 155 160 Gly Gly Asp
Glu Val Asp Leu Arg Leu Ala Lys Ala Glu Ser Lys Val 165 170 175 Leu
His Glu Lys Ile Ser Asp Lys Ala Tyr Ser Asp Asp Glu Val Ile 180 185
190 Arg Ile Leu Ala Thr Arg Ser Lys Ala Gln Leu Asn Ala Thr Leu Asn
195 200 205 His Tyr Lys Asp Glu Tyr Gly Glu Asp Ile Leu Lys Gln Leu
Glu Asp 210 215 220 Glu Asp Glu Phe Val Ala Leu Leu Arg Ala Thr Ile
Lys Gly Leu Val 225 230 235 240 Tyr Pro Glu His Tyr Phe Val Glu Val
Leu Arg Asp Ala Ile Asn Arg 245 250 255 Arg Gly Thr Glu Glu Asp His
Leu Ser Arg Val Ile Ala Thr Arg Ala 260 265 270 Glu Val Asp Leu Lys
Thr Ile Ala Asn Glu Tyr Gln Lys Arg Asp Ser 275 280 285 Ile Pro Leu
Gly Arg Ala Ile Ala Lys Asp Thr Gly Gly Asp Tyr Glu 290 295 300 Asn
Met Leu Val Ala Leu Leu Gly Gln Glu Glu Glu 305 310 315
1241176DNAArabidopsis thaliana 124catacagaaa tttcacttgt tcgaaaaatg
gcttctctca aagttcccgc cactgttcct 60cttcccgaag aagacgctga gcaactctac
aaagccttta aaggatgggg aaccaatgag 120aggatgatca tatcaatctt
ggctcacaga aatgcaacgc aacgtagttt cattcgtgcc 180gtttatgctg
ctaactacaa taaggatctt ctcaaggaat tagacagaga gctttccggt
240gactttgagc gagctgtgat gttgtggact tttgaaccag cggagagaga
tgcttatttg 300gcaaaagaat ctaccaaaat gttcaccaaa aacaattggg
ttcttgtcga aatcgcttgt 360actagatctg ctcttgaact ctttaatgcc
aagcaagcat accaagcccg ctacaagacc 420tccctcgagg aagacgtcgc
ataccacaca tctggagaca ttcgaaagct cttggtacct 480cttgtgagca
cttttaggta cgatggagat gaagtgaaca tgacgttagc taggtccgag
540gctaagatac ttcacgagaa gatcaaggaa aaggcttatg ctgatgatga
tctcataaga 600atcttgacaa ccaggagcaa agcacaaatc agcgcaactc
tcaatcacta caaaaacaat 660ttcggaactt ccatgagcaa atacctaaag
gaggattcgg aaaacgaata cattcaattg 720ctcaaagccg tgatcaaatg
cttgacatat ccagagaagt attttgagaa agttctacgt 780caagccatca
acaaattggg aacagatgag tggggactaa cgagagtggt cactacacga
840gcagagtttg acatggaacg gatcaaagag gaatatatac gtagaaacag
tgttcctctt 900gatcgagcca ttgctaaaga cactcatggt gactatgagg
atatacttct cgctcttctc 960ggacatgacc atgcttgaaa taacatttgc
aagttttgtt taagaaaaaa aactaaattt 1020tatcgctttg tgtttaataa
aacagttgtg gttggacttg caacttggtc atgttaagaa 1080tttagtgtct
tcagtttcat ttgtcgtcga tgttttcagt tatttttttt tttaaatcta
1140aaaattataa aaccatatca aaaattatta ttgatc
1176125316PRTArabidopsis thaliana 125Met Ala Ser Leu Lys Val Pro
Ala Thr Val Pro Leu Pro Glu Glu Asp 1 5 10 15 Ala Glu Gln Leu Tyr
Lys Ala Phe Lys Gly Trp Gly Thr Asn Glu Arg 20 25 30 Met Ile Ile
Ser Ile Leu Ala His Arg Asn Ala Thr Gln Arg Ser Phe 35 40 45 Ile
Arg Ala Val Tyr Ala Ala Asn Tyr Asn Lys Asp Leu Leu Lys Glu 50 55
60 Leu Asp Arg Glu Leu Ser Gly Asp Phe Glu Arg Ala Val Met Leu Trp
65 70 75 80 Thr Phe Glu Pro Ala Glu Arg Asp Ala Tyr Leu Ala Lys Glu
Ser Thr 85 90 95 Lys Met Phe Thr Lys Asn Asn Trp Val Leu Val Glu
Ile Ala Cys Thr 100 105 110 Arg Ser Ala Leu Glu Leu Phe Asn Ala Lys
Gln Ala Tyr Gln Ala Arg 115 120 125 Tyr Lys Thr Ser Leu Glu Glu Asp
Val Ala Tyr His Thr Ser Gly Asp 130 135 140 Ile Arg Lys Leu Leu Val
Pro Leu Val Ser Thr Phe Arg Tyr Asp Gly 145 150 155 160 Asp Glu Val
Asn Met Thr Leu Ala Arg Ser Glu Ala Lys Ile Leu His 165 170 175 Glu
Lys Ile Lys Glu Lys Ala Tyr Ala Asp Asp Asp Leu Ile Arg Ile 180 185
190 Leu Thr Thr Arg Ser Lys Ala Gln Ile Ser Ala Thr Leu Asn His Tyr
195 200 205 Lys Asn Asn Phe Gly Thr Ser Met Ser Lys Tyr Leu Lys Glu
Asp Ser 210 215 220 Glu Asn Glu Tyr Ile Gln Leu Leu Lys Ala Val Ile
Lys Cys Leu Thr 225 230 235 240 Tyr Pro Glu Lys Tyr Phe Glu Lys Val
Leu Arg Gln Ala Ile Asn Lys 245 250 255 Leu Gly Thr Asp Glu Trp Gly
Leu Thr Arg Val Val Thr Thr Arg Ala 260 265 270 Glu Phe Asp Met Glu
Arg Ile Lys Glu Glu Tyr Ile Arg Arg Asn Ser 275 280 285 Val Pro Leu
Asp Arg Ala Ile Ala Lys Asp Thr His Gly Asp Tyr Glu 290 295 300 Asp
Ile Leu Leu Ala Leu Leu Gly His Asp His Ala 305 310 315
1261182DNAMedicago sativa 126ctctcacgtt ccgtctccat cagaagacag
tgaacaattg cgcggtgctt ttcaaggatg 60gggaacgaat gaaggcttga taatatcgat
cctggctcat agaaatgcag ctcagcgtaa 120gtcgatccgc gaaacttaca
cgcagaccca tggagaagat cttcttaaag atcttgacaa 180agaactttca
agtgattttg agaaagctgt gctgctgtgg acattggatc cggccgagcg
240tgatgcattt ttagccaatc aagcaactaa aatgttgact tcaaacaatt
cgatcatcgt 300ggaaattgct tccacaagat ctccacttga acttcttaag
gcaaagcaag catatcaagt 360ccgtttcaaa aagtcccttg aagaagatgt
tgcctatcat acttctggtg acatccgcaa 420gcttttggtt cctcttgtgg
gcatacaccg ttatgaggga gatgaggtga acatgacatt 480ggcaaaatct
gaagctaaat tgcttcatga gaagattgcg gataaggctt acaatcatga
540tgacctgatc aggattgtaa caacaaggag taaagcgcaa ttaaatgcaa
ctttgaatca 600ctataacaat gagtttggga atgtaataga caaggatttg
gaaactgatt cggatgatga 660atatctgaaa ttattgaggg cagcaattaa
gggcttgacc taccctgaga aatattttga 720ggaactcctt aggctggcta
taaacaagat gggaaccgat gaaaatgctc ttactagagt 780ggtgacaact
agagctgagg ttgatttgca gcgaattgcg gaggaatacc agagaagaaa
840cagtgttcct ctggaccgtg caattgacaa agacacttct ggagactatc
agaaaattct 900ccttgcactg atgggacatg atgagtaagt tcttaatctg
tccagtagtc atggagtggc 960tgtttggact atctgttttc ccttcatcat
cagcgtgatt ttgctgcgga tctcttgata 1020gtatacagaa ttcggtgact
tgctgtggta actatgcttg tgatatgtat gaactattgt 1080ggttttaaat
aatatgtttt gaatatggac tgaaattcaa aacagaactt tgccttctta
1140aataatgaaa cagctatcat atttctctcc ttaaaaaaaa aa
1182127308PRTMedicago sativa 127Ser His Val Pro Ser Pro Ser Glu Asp
Ser Glu Gln Leu Arg Gly Ala 1 5 10 15 Phe Gln Gly Trp Gly Thr Asn
Glu Gly Leu Ile Ile Ser Ile Leu Ala 20 25 30 His Arg Asn Ala Ala
Gln Arg Lys Ser Ile Arg Glu Thr Tyr Thr Gln 35 40 45 Thr His Gly
Glu Asp Leu Leu Lys Asp Leu Asp Lys Glu Leu Ser Ser 50 55 60 Asp
Phe Glu Lys Ala Val Leu Leu Trp Thr Leu Asp Pro Ala Glu Arg 65 70
75 80 Asp Ala Phe Leu Ala Asn Gln Ala Thr Lys Met Leu Thr Ser Asn
Asn 85 90 95 Ser Ile Ile Val Glu Ile Ala Ser Thr Arg Ser Pro Leu
Glu Leu Leu 100 105 110 Lys Ala Lys Gln Ala Tyr Gln Val Arg Phe Lys
Lys Ser Leu Glu Glu 115 120 125 Asp Val Ala Tyr His Thr Ser Gly Asp
Ile Arg Lys Leu Leu Val Pro 130 135 140 Leu Val Gly Ile His Arg Tyr
Glu Gly Asp Glu Val Asn Met Thr Leu 145 150 155 160 Ala Lys Ser Glu
Ala Lys Leu Leu His Glu Lys Ile Ala Asp Lys Ala 165 170 175 Tyr Asn
His Asp Asp Leu Ile Arg Ile Val Thr Thr Arg Ser Lys Ala 180 185 190
Gln Leu Asn Ala Thr Leu Asn His Tyr Asn Asn Glu Phe Gly Asn Val 195
200 205 Ile Asp Lys Asp Leu Glu Thr Asp Ser Asp Asp Glu Tyr Leu Lys
Leu 210 215 220 Leu Arg Ala Ala Ile Lys Gly Leu Thr Tyr Pro Glu Lys
Tyr Phe Glu 225 230 235 240 Glu Leu Leu Arg Leu Ala Ile Asn Lys Met
Gly Thr Asp Glu Asn Ala 245 250 255 Leu Thr Arg Val Val Thr Thr Arg
Ala Glu Val Asp Leu Gln Arg Ile 260 265 270 Ala Glu Glu Tyr Gln Arg
Arg Asn Ser Val Pro Leu Asp Arg Ala Ile 275 280 285 Asp Lys Asp Thr
Ser Gly Asp Tyr Gln Lys Ile Leu Leu Ala Leu Met 290 295 300 Gly His
Asp Glu 305 128951DNABrassica rapa 128atggcgtctc tcaaagttcc
tgctagtgtt cctcttccag aagaagatgc cgagcagctc 60cagaaggcct ttaaaggatg
gggaaccaac gagaggatga tcatatcaat cttggctcac 120agaaatgccg
agcaacgcag cttcatccgt gctgtttatg ctgctaacta caataaggat
180cttctcaagg aattagacaa agagctatcc ggtgacttcg agcgagctgt
gatgttgtgg 240acacttgaac cagcggagag agatgcgtat ttggctaagg
aatcaacaaa aatgttcact 300aaagacaatt gggttctagt tgaaatcgct
tgtactagat cttcccttga gtttttcaag 360gccaagcaag cataccaagt
tcgctacaag acatctattg aggaagatgt cgcctaccac 420acatctggag
atgtccgaaa gctcttggtt cctcttgtga gtacctttag gtacgatgga
480gatgaagtaa acatgatgat tgctaagtct gaggctaaga tacttcacga
gaagatggag 540gcgaaggatt acaatgatgg agatctcatt agaatcctga
caacaagaag caaagctcaa 600atcagtgcaa cactcaacca cttcaaaaat
aagttcggaa cttccattac aaaatacctt 660aaagaggatt ccgacaacga
atatgttcag ctacttaaag ccgtgatcaa atgcttgact 720tatccagaga
aatattttga gaaagttctt cgtcaagcca tcaacaaaat gggaactgac
780gagtggggac ttactagagt ggtcaccaca cgagctgagc tcgacatgga
acggatcaaa 840gaggaatact tgcgcaggaa cagtgtccca cttgaccgag
ccattgccaa agacactcat 900ggtgactatg aggatattct tctagctctt
atcggacatg gccatgcttg a 951129316PRTBrassica rapa 129Met Ala Ser
Leu Lys Val Pro Ala Ser Val Pro Leu Pro Glu Glu Asp 1 5 10 15 Ala
Glu Gln Leu Gln Lys Ala Phe Lys Gly Trp Gly Thr Asn Glu Arg 20 25
30 Met Ile Ile Ser Ile Leu Ala His Arg Asn Ala Glu Gln Arg Ser Phe
35 40 45 Ile Arg Ala Val Tyr Ala Ala Asn Tyr Asn Lys Asp Leu Leu
Lys Glu 50 55 60 Leu Asp Lys Glu Leu Ser Gly Asp Phe Glu Arg Ala
Val Met Leu Trp 65 70 75 80 Thr Leu Glu Pro Ala Glu Arg Asp Ala Tyr
Leu Ala Lys Glu Ser Thr 85 90 95 Lys Met Phe Thr Lys Asp Asn Trp
Val Leu Val Glu Ile Ala Cys Thr 100 105 110 Arg Ser Ser Leu Glu Phe
Phe Lys Ala Lys Gln Ala Tyr Gln Val Arg 115 120 125 Tyr Lys Thr Ser
Ile Glu Glu Asp Val Ala Tyr His Thr Ser Gly Asp 130 135 140 Val Arg
Lys Leu Leu Val Pro Leu Val Ser Thr Phe Arg Tyr Asp Gly 145 150 155
160 Asp Glu Val Asn Met Met Ile Ala Lys Ser Glu Ala Lys Ile Leu His
165 170 175 Glu Lys Met Glu Ala Lys Asp Tyr Asn Asp Gly Asp Leu Ile
Arg Ile 180 185 190 Leu Thr Thr Arg Ser Lys Ala Gln Ile Ser Ala Thr
Leu Asn His Phe 195 200 205 Lys Asn Lys Phe Gly Thr Ser Ile Thr Lys
Tyr Leu Lys Glu Asp Ser 210 215 220 Asp Asn Glu Tyr Val Gln Leu Leu
Lys Ala Val Ile Lys Cys Leu Thr 225 230 235 240 Tyr Pro Glu Lys Tyr
Phe Glu Lys Val Leu Arg Gln Ala Ile Asn Lys 245 250 255 Met Gly Thr
Asp Glu Trp Gly Leu Thr Arg Val Val Thr Thr Arg Ala 260 265 270 Glu
Leu Asp Met Glu Arg Ile Lys Glu Glu Tyr Leu Arg Arg Asn Ser 275 280
285 Val Pro Leu Asp Arg Ala Ile Ala Lys Asp Thr His Gly Asp Tyr Glu
290 295 300 Asp Ile Leu Leu Ala Leu Ile Gly His Gly His Ala 305 310
315 1301235DNAArabidopsis thaliana 130ccttgaggaa aaaaaactaa
aacggctcta aattgaaaac aaaagaagag agagagagag 60agagacgaag aaacagagag
attctctcga aaatggccac cattagagta ccaaacgaag 120ttccttctcc
agctcaggat tctgaaactc tcaaacaagc tattcgcgga tggggaacag
180atgagaaggc gattatacga gttttagggc aaagagacca gagccagaga
aggaagatta 240gagaaagttt tagagagatt tatggcaaag atcttatcga
tgttctatcc tccgaactgt 300ctggtgattt catgaaagct gtggtttcgt
ggacgtatga tccagcagag agagacgcaa 360ggcttgtgaa caagattttg
aacaaggaga agaagaagaa aagcttagag aatttgaagg 420ttatagtaga
gatctcttgc acgacttccc caaaccattt gattgctgtg aggaaagctt
480attgttcact ctttgactct tctcttgaag aacacattgc ttcttctctg
ccttttcctc 540ttgcaaagtt actggtgaca ttggcaagta cattcagata
tgacaaagat aggactgatg 600cagaagtagc tactattgag gcggctatgc
tacgtgaagc catagagaag aaacaattag 660atcatgacca tgtcctgtac
atattaggaa cgcgtagtat ctatcagctc agagaaactt 720ttgttgctta
caagaagaat tatggggtca caattgataa ggatgttgat ggatgtccag
780gagatgctga tctgagaagt ctattgaagg
tggcaatctt ttgtattgat actcctgaga 840aacactttgc aaaggtggta
agagattcga ttgagggttt tggaacagat gaggattcgt 900tgacgagggc
gattgtgacg cgtgcagaga tcgatttgat gaaagtaaga ggagagtatt
960tcaacatgta taatacaagc atggacaatg ctattactgg tgatatttct
ggagactaca 1020aggacttcat tatcacctta cttggatcca aaatctgatc
gttctttcgt ttctttgtca 1080gttgttatat tcttggcttt gcttgtgact
tgtataatca atcaatacat tgtattccaa 1140ctccagtttg aattgtttaa
aaaataatca aatttctctt gattcttgca tttttgaatc 1200aaagcaaatc
tatgtttaat tttgttttca aaatt 1235131321PRTArabidopsis thaliana
131Met Ala Thr Ile Arg Val Pro Asn Glu Val Pro Ser Pro Ala Gln Asp
1 5 10 15 Ser Glu Thr Leu Lys Gln Ala Ile Arg Gly Trp Gly Thr Asp
Glu Lys 20 25 30 Ala Ile Ile Arg Val Leu Gly Gln Arg Asp Gln Ser
Gln Arg Arg Lys 35 40 45 Ile Arg Glu Ser Phe Arg Glu Ile Tyr Gly
Lys Asp Leu Ile Asp Val 50 55 60 Leu Ser Ser Glu Leu Ser Gly Asp
Phe Met Lys Ala Val Val Ser Trp 65 70 75 80 Thr Tyr Asp Pro Ala Glu
Arg Asp Ala Arg Leu Val Asn Lys Ile Leu 85 90 95 Asn Lys Glu Lys
Lys Lys Lys Ser Leu Glu Asn Leu Lys Val Ile Val 100 105 110 Glu Ile
Ser Cys Thr Thr Ser Pro Asn His Leu Ile Ala Val Arg Lys 115 120 125
Ala Tyr Cys Ser Leu Phe Asp Ser Ser Leu Glu Glu His Ile Ala Ser 130
135 140 Ser Leu Pro Phe Pro Leu Ala Lys Leu Leu Val Thr Leu Ala Ser
Thr 145 150 155 160 Phe Arg Tyr Asp Lys Asp Arg Thr Asp Ala Glu Val
Ala Thr Ile Glu 165 170 175 Ala Ala Met Leu Arg Glu Ala Ile Glu Lys
Lys Gln Leu Asp His Asp 180 185 190 His Val Leu Tyr Ile Leu Gly Thr
Arg Ser Ile Tyr Gln Leu Arg Glu 195 200 205 Thr Phe Val Ala Tyr Lys
Lys Asn Tyr Gly Val Thr Ile Asp Lys Asp 210 215 220 Val Asp Gly Cys
Pro Gly Asp Ala Asp Leu Arg Ser Leu Leu Lys Val 225 230 235 240 Ala
Ile Phe Cys Ile Asp Thr Pro Glu Lys His Phe Ala Lys Val Val 245 250
255 Arg Asp Ser Ile Glu Gly Phe Gly Thr Asp Glu Asp Ser Leu Thr Arg
260 265 270 Ala Ile Val Thr Arg Ala Glu Ile Asp Leu Met Lys Val Arg
Gly Glu 275 280 285 Tyr Phe Asn Met Tyr Asn Thr Ser Met Asp Asn Ala
Ile Thr Gly Asp 290 295 300 Ile Ser Gly Asp Tyr Lys Asp Phe Ile Ile
Thr Leu Leu Gly Ser Lys 305 310 315 320 Ile 132951DNAArabidopsis
thaliana 132atggcaacaa tgaaaatacc aatgacggta ccttctcctc gagtcgatgc
tgaccaactc 60tttaaggcct tcaaaggaag aggctgcgat acttcggtga tcatcaacat
cttagctcat 120cgcaatgcaa cacaacgagc tctcatcgaa caagaatacg
aaaccaaatt ctcggatgac 180ctccgaaaac gtctccactc tgagcttcat
ggtcatctca agaaagccgt tcttttgtgg 240atgcctgaag cagtggagcg
agacgcttca atactgaaac gctccttaag aggagccgtg 300actgatcata
aagcgattgc tgagattata tgcacacgat ctggctctca gcttcgtcag
360atcaaacagg tctactcaaa cactttcggt gtgaaacttg aagaggacat
cgaatccgaa 420gcttctggca atcacaaaag agttttgctc gcgtatttga
acactacgcg atatgaagga 480ccagagatcg ataatgcgag tgtagagaac
gatgctagga ctctcaagag cgcggttgca 540aggaagcata aatctgatga
ccagacgttg attcagatat tcactgaccg aagcaggact 600catttggtcg
ctgtaagatc tacttaccgt tccatgtacg gcaaagaact tggaaaggcc
660ataagagatg agactcgcgg gaacttcgag catgtccttc taacaatttt
acaatgtgct 720gaaaactctt gtttctattt cgcaaaggca ttgaggaaat
caatgaaagg attaggaaca 780gatgacacgg cgttgataag aatcgtggtg
acgagagcag aggtggatat gcagttcatc 840atcacagaat accgtaagag
atacaagaag actttgtaca atgctgttca ttctgataca 900actagtcatt
acaggacttt tctcctctct cttttaggcc ccaacgtttg a
951133316PRTArabidopsis thaliana 133Met Ala Thr Met Lys Ile Pro Met
Thr Val Pro Ser Pro Arg Val Asp 1 5 10 15 Ala Asp Gln Leu Phe Lys
Ala Phe Lys Gly Arg Gly Cys Asp Thr Ser 20 25 30 Val Ile Ile Asn
Ile Leu Ala His Arg Asn Ala Thr Gln Arg Ala Leu 35 40 45 Ile Glu
Gln Glu Tyr Glu Thr Lys Phe Ser Asp Asp Leu Arg Lys Arg 50 55 60
Leu His Ser Glu Leu His Gly His Leu Lys Lys Ala Val Leu Leu Trp 65
70 75 80 Met Pro Glu Ala Val Glu Arg Asp Ala Ser Ile Leu Lys Arg
Ser Leu 85 90 95 Arg Gly Ala Val Thr Asp His Lys Ala Ile Ala Glu
Ile Ile Cys Thr 100 105 110 Arg Ser Gly Ser Gln Leu Arg Gln Ile Lys
Gln Val Tyr Ser Asn Thr 115 120 125 Phe Gly Val Lys Leu Glu Glu Asp
Ile Glu Ser Glu Ala Ser Gly Asn 130 135 140 His Lys Arg Val Leu Leu
Ala Tyr Leu Asn Thr Thr Arg Tyr Glu Gly 145 150 155 160 Pro Glu Ile
Asp Asn Ala Ser Val Glu Asn Asp Ala Arg Thr Leu Lys 165 170 175 Ser
Ala Val Ala Arg Lys His Lys Ser Asp Asp Gln Thr Leu Ile Gln 180 185
190 Ile Phe Thr Asp Arg Ser Arg Thr His Leu Val Ala Val Arg Ser Thr
195 200 205 Tyr Arg Ser Met Tyr Gly Lys Glu Leu Gly Lys Ala Ile Arg
Asp Glu 210 215 220 Thr Arg Gly Asn Phe Glu His Val Leu Leu Thr Ile
Leu Gln Cys Ala 225 230 235 240 Glu Asn Ser Cys Phe Tyr Phe Ala Lys
Ala Leu Arg Lys Ser Met Lys 245 250 255 Gly Leu Gly Thr Asp Asp Thr
Ala Leu Ile Arg Ile Val Val Thr Arg 260 265 270 Ala Glu Val Asp Met
Gln Phe Ile Ile Thr Glu Tyr Arg Lys Arg Tyr 275 280 285 Lys Lys Thr
Leu Tyr Asn Ala Val His Ser Asp Thr Thr Ser His Tyr 290 295 300 Arg
Thr Phe Leu Leu Ser Leu Leu Gly Pro Asn Val 305 310 315
1341337DNAArabidopsis thaliana 134acagaaacca aaccaagagc cggaaatcaa
aaacagtaat aaaagatcaa ctgcaagaaa 60atggctcttc ctctcgagct cgaaagcctc
actgaagcca tctcagctgg gatgggaatg 120ggagttgatg agaatgcatt
gataagcaca ctggggaaat cgcaaaagga acatagaaaa 180ttgtttagga
aagcaagcaa aagtttcttt gttgaagatg aggaaagagc ttttgagaaa
240tgtcatgatc acttcgtcag acacctcaag cttgagttct cccgcttcaa
tactgcggtg 300gtgatgtggg caatgcatcc atgggagaga gatgcaaggt
tggtgaagaa agctttgaag 360aaaggagaag aagcttacaa cctcatcgtt
gaggtctcat gcacacgctc tgctgaggat 420ctcctcggtg cacgtaaagc
ttaccactct ctcttcgacc aatcaatgga agaagacatt 480gcctctcacg
tccacggtcc tcagcgcaag ttgcttgtgg ggctcgtgag tgcttataga
540tacgaaggaa ataaggtgaa ggatgattct gccaaatccg atgctaagat
tctagccgaa 600gcagtggctt cttcaggcga agaagccgtg gagaaggatg
aggttgttag gattttgacc 660acaagaagca aacttcatct ccaacatctc
tacaaacact ttaacgaaat caaaggctct 720gatcttcttg ggggtgtatc
taagtcttct cttctcaatg aagcattgat ttgtttgctc 780aaaccggctc
tgtatttcag caagattttg gatgcgtctc tgaacaaaga cgcagacaag
840actaccaaga aatggttgac aagagtgttc gttacaagag cagatcatag
tgatgagatg 900aatgagatca aagaagagta caataacctt tatggtgaga
ctttggctca aagaatccaa 960gagaagataa aagggaacta cagagatttc
ttgctcacac ttctctccaa atccgattga 1020tttcgtgttg agaaacctat
taccaatact tttggttatt gaagatttat gatttccctt 1080tttatggttt
tatgtttcta attcctaaat ttgcgttttc tctaccgttt ggtaataaag
1140acatgaaaat ttgatgaact cggtgaatcg agagtaagag ttttgcgatt
gtgacaatga 1200gtgattaata caaggattaa gctccaataa aaaaatgttg
cataaatcag aaatgaaact 1260tgtaactctt cttttcttta tgtgaaactt
gtaactctat ttgaaagatt ctatgtgacc 1320actaaaccga attacgg
1337135319PRTArabidopsis thaliana 135Met Ala Leu Pro Leu Glu Leu
Glu Ser Leu Thr Glu Ala Ile Ser Ala 1 5 10 15 Gly Met Gly Met Gly
Val Asp Glu Asn Ala Leu Ile Ser Thr Leu Gly 20 25 30 Lys Ser Gln
Lys Glu His Arg Lys Leu Phe Arg Lys Ala Ser Lys Ser 35 40 45 Phe
Phe Val Glu Asp Glu Glu Arg Ala Phe Glu Lys Cys His Asp His 50 55
60 Phe Val Arg His Leu Lys Leu Glu Phe Ser Arg Phe Asn Thr Ala Val
65 70 75 80 Val Met Trp Ala Met His Pro Trp Glu Arg Asp Ala Arg Leu
Val Lys 85 90 95 Lys Ala Leu Lys Lys Gly Glu Glu Ala Tyr Asn Leu
Ile Val Glu Val 100 105 110 Ser Cys Thr Arg Ser Ala Glu Asp Leu Leu
Gly Ala Arg Lys Ala Tyr 115 120 125 His Ser Leu Phe Asp Gln Ser Met
Glu Glu Asp Ile Ala Ser His Val 130 135 140 His Gly Pro Gln Arg Lys
Leu Leu Val Gly Leu Val Ser Ala Tyr Arg 145 150 155 160 Tyr Glu Gly
Asn Lys Val Lys Asp Asp Ser Ala Lys Ser Asp Ala Lys 165 170 175 Ile
Leu Ala Glu Ala Val Ala Ser Ser Gly Glu Glu Ala Val Glu Lys 180 185
190 Asp Glu Val Val Arg Ile Leu Thr Thr Arg Ser Lys Leu His Leu Gln
195 200 205 His Leu Tyr Lys His Phe Asn Glu Ile Lys Gly Ser Asp Leu
Leu Gly 210 215 220 Gly Val Ser Lys Ser Ser Leu Leu Asn Glu Ala Leu
Ile Cys Leu Leu 225 230 235 240 Lys Pro Ala Leu Tyr Phe Ser Lys Ile
Leu Asp Ala Ser Leu Asn Lys 245 250 255 Asp Ala Asp Lys Thr Thr Lys
Lys Trp Leu Thr Arg Val Phe Val Thr 260 265 270 Arg Ala Asp His Ser
Asp Glu Met Asn Glu Ile Lys Glu Glu Tyr Asn 275 280 285 Asn Leu Tyr
Gly Glu Thr Leu Ala Gln Arg Ile Gln Glu Lys Ile Lys 290 295 300 Gly
Asn Tyr Arg Asp Phe Leu Leu Thr Leu Leu Ser Lys Ser Asp 305 310 315
1361110DNAArabidopsis thaliana 136gtctcatcta gagagctaga gaaatattca
gtggtcggag aatggcgtct ctcaaaattc 60cagcaaatat tcctcttccc gaagaagact
ccgagcagct ccacaaggca ttcaaaggat 120ggggaactaa tgaagggatg
atcatatcaa ttttggctca tagaaacgca acgcaacgca 180gtttcattcg
tgccgtttat gctgctaact acaataagga tcttctcaag gaattagacg
240gagagctttc tggtgacttt gagagagttg tgatgttgtg gactcttgat
ccaacggaga 300gagatgcgta tttggccaat gaatctacca aattgttcac
caaaaacatt tgggtcctag 360ttgaaatcgc ttgtactaga ccttctcttg
agtttttcaa gaccaagcaa gcataccatg 420ttcgctacaa gacctctctc
gaggaagatg ttgcatacca tacatctgga aatatccgaa 480agctattggt
tcctcttgtg agcaccttca ggtacgatgg aaatgctgat gaggtcaacg
540tgaagctggc tagatccgaa gctaagacac ttcacaagaa gatcactgag
aaggcttaca 600ctgatgaaga tctcatcaga atcttgacaa caaggagcaa
agcacagatc aatgcaacac 660tcaatcactt caaggacaag tttggaagtt
ccattaacaa gtttctcaaa gaagattcga 720acgatgatta tgttcaatta
ctcaaaaccg cgatcaaatg cttgacatat ccagagaagt 780actttgagaa
ggttctacgt cgagccatca acaggatggg aacagacgag tgggcactta
840ctagagtggt cactacaaga gcagaggtcg acctggagcg gatcaaagaa
gaatacttac 900gcaggaacag tgttcctctt gatcgagcca ttgctaatga
cacttctggt gactacaagg 960atatgcttct cgcccttctt ggacatgacc
atgcttgaaa caacatcatc gtttcatagt 1020cttttataag acagttgtta
tttgtttttc attttctttg aactttggtc cttagttttt 1080acattttact
gcaacaactt attctggttt 1110137318PRTArabidopsis thaliana 137Met Ala
Ser Leu Lys Ile Pro Ala Asn Ile Pro Leu Pro Glu Glu Asp 1 5 10 15
Ser Glu Gln Leu His Lys Ala Phe Lys Gly Trp Gly Thr Asn Glu Gly 20
25 30 Met Ile Ile Ser Ile Leu Ala His Arg Asn Ala Thr Gln Arg Ser
Phe 35 40 45 Ile Arg Ala Val Tyr Ala Ala Asn Tyr Asn Lys Asp Leu
Leu Lys Glu 50 55 60 Leu Asp Gly Glu Leu Ser Gly Asp Phe Glu Arg
Val Val Met Leu Trp 65 70 75 80 Thr Leu Asp Pro Thr Glu Arg Asp Ala
Tyr Leu Ala Asn Glu Ser Thr 85 90 95 Lys Leu Phe Thr Lys Asn Ile
Trp Val Leu Val Glu Ile Ala Cys Thr 100 105 110 Arg Pro Ser Leu Glu
Phe Phe Lys Thr Lys Gln Ala Tyr His Val Arg 115 120 125 Tyr Lys Thr
Ser Leu Glu Glu Asp Val Ala Tyr His Thr Ser Gly Asn 130 135 140 Ile
Arg Lys Leu Leu Val Pro Leu Val Ser Thr Phe Arg Tyr Asp Gly 145 150
155 160 Asn Ala Asp Glu Val Asn Val Lys Leu Ala Arg Ser Glu Ala Lys
Thr 165 170 175 Leu His Lys Lys Ile Thr Glu Lys Ala Tyr Thr Asp Glu
Asp Leu Ile 180 185 190 Arg Ile Leu Thr Thr Arg Ser Lys Ala Gln Ile
Asn Ala Thr Leu Asn 195 200 205 His Phe Lys Asp Lys Phe Gly Ser Ser
Ile Asn Lys Phe Leu Lys Glu 210 215 220 Asp Ser Asn Asp Asp Tyr Val
Gln Leu Leu Lys Thr Ala Ile Lys Cys 225 230 235 240 Leu Thr Tyr Pro
Glu Lys Tyr Phe Glu Lys Val Leu Arg Arg Ala Ile 245 250 255 Asn Arg
Met Gly Thr Asp Glu Trp Ala Leu Thr Arg Val Val Thr Thr 260 265 270
Arg Ala Glu Val Asp Leu Glu Arg Ile Lys Glu Glu Tyr Leu Arg Arg 275
280 285 Asn Ser Val Pro Leu Asp Arg Ala Ile Ala Asn Asp Thr Ser Gly
Asp 290 295 300 Tyr Lys Asp Met Leu Leu Ala Leu Leu Gly His Asp His
Ala 305 310 315 138951DNAArabidopsis thaliana 138atggccacca
ttgtttctcc tccacatttc tcccctgtcg aagacgctga aaacatcaag 60gcggcttgtc
aaggatgggg aaccaatgaa aatgccatca tctcgatctt aggacaccgg
120aatttgttcc agaggaagct cataagacaa gcttaccagg agatttacca
tgaggatctc 180attcaccagc tcaaatctga gctctctggc aattttgaga
gagctatttg cttgtgggtc 240ttggatcctc cagagagaga tgctctcttg
gctaacttgg ctcttcaaaa gcctattcct 300gactacaagg ttcttgtcga
aattgcctgc atgagatccc ctgaagatat gttagctgct 360agacgtgctt
accgttgcct ctacaagcat tctcttgagg aagacttggc ctcccgtact
420attggcgaca tcaggagact cttggttgca atggtgtctg cttataaata
tgatggagaa 480gaaattgatg agatgctggc gcaatcagag gctgcgattc
ttcatgatga aatccttggt 540aaggctgttg atcacgaaga aacgatcagg
gtgttaagta caaggagcag catgcagctt 600agcgcaatct tcaaccgcta
caaggatata tatggcacat cgatcactaa ggatctcctc 660aatcacccta
caaatgagta cctgagtgca ctacgtgcag ccatcaggtg catcaaaaac
720cctacccggt attatgcaaa ggttttgcgc aattcaatca acacggtggg
gactgatgaa 780gatgctctga accgtgtgat tgtcacacga gcagaaaagg
acctgacgaa tataactggg 840ctgtacttta agaggaacaa tgtgagtctc
gatcaagcta tagcaaaaga gacatcaggg 900gactacaagg cctttcttct
agctttgcta ggacatggaa aacaacttta g 951139316PRTArabidopsis thaliana
139Met Ala Thr Ile Val Ser Pro Pro His Phe Ser Pro Val Glu Asp Ala
1 5 10 15 Glu Asn Ile Lys Ala Ala Cys Gln Gly Trp Gly Thr Asn Glu
Asn Ala 20 25 30 Ile Ile Ser Ile Leu Gly His Arg Asn Leu Phe Gln
Arg Lys Leu Ile 35 40 45 Arg Gln Ala Tyr Gln Glu Ile Tyr His Glu
Asp Leu Ile His Gln Leu 50 55 60 Lys Ser Glu Leu Ser Gly Asn Phe
Glu Arg Ala Ile Cys Leu Trp Val 65 70 75 80 Leu Asp Pro Pro Glu Arg
Asp Ala Leu Leu Ala Asn Leu Ala Leu Gln 85 90 95 Lys Pro Ile Pro
Asp Tyr Lys Val Leu Val Glu Ile Ala Cys Met Arg 100 105 110 Ser Pro
Glu Asp Met Leu Ala Ala Arg Arg Ala Tyr Arg Cys Leu Tyr 115 120 125
Lys His Ser Leu Glu Glu Asp Leu Ala Ser Arg Thr Ile Gly Asp Ile 130
135 140 Arg Arg Leu Leu Val Ala Met Val Ser Ala Tyr Lys Tyr Asp Gly
Glu 145 150 155 160 Glu Ile Asp Glu Met Leu Ala Gln Ser Glu Ala Ala
Ile Leu His Asp 165 170 175 Glu Ile Leu Gly Lys Ala Val Asp His Glu
Glu Thr Ile Arg Val Leu 180 185 190 Ser Thr Arg Ser Ser Met Gln Leu
Ser Ala Ile Phe Asn Arg Tyr Lys 195 200 205 Asp Ile Tyr Gly Thr Ser
Ile Thr Lys Asp Leu Leu Asn His Pro Thr 210 215 220 Asn Glu Tyr Leu
Ser Ala Leu Arg Ala Ala Ile Arg Cys Ile Lys Asn 225 230
235 240 Pro Thr Arg Tyr Tyr Ala Lys Val Leu Arg Asn Ser Ile Asn Thr
Val 245 250 255 Gly Thr Asp Glu Asp Ala Leu Asn Arg Val Ile Val Thr
Arg Ala Glu 260 265 270 Lys Asp Leu Thr Asn Ile Thr Gly Leu Tyr Phe
Lys Arg Asn Asn Val 275 280 285 Ser Leu Asp Gln Ala Ile Ala Lys Glu
Thr Ser Gly Asp Tyr Lys Ala 290 295 300 Phe Leu Leu Ala Leu Leu Gly
His Gly Lys Gln Leu 305 310 315 1401298DNAOryza sativa
140gttgcagatt actaccacca cctccccaaa atcccaatcg aatcgaaatc
gaatcgagtc 60gagtcgccgc cggagccgga gacggaggcg gcagcggcgc agcggtaatg
gcgagcctca 120ccctgccgcc ggcgcccacc aaccctcgcc aggacgccat
cgacctccac aaggccttca 180aagggtttgg ctgtgatagt acaacagtta
taaatatact tactcatcgt gactcgatgc 240aacgcgcgct cattcaacag
gaatacagga ctatgtattc tgaggatctc tctcgccgta 300tatcatctga
actcagtgga caccacaaga aagcaatgct gctatggatt cttgatcctg
360ctggacgaga tgcaactgtt ttgagagaag ctctgagtgg tgatactatt
gacctgagag 420cagccactga gataatatgt tccaggacac catcgcagct
gcaaataatg aaacagactt 480atcatgcaaa atttggtact tatcttgagc
acgacattgg tcagcgcaca tcaggcgacc 540atcagaagct cttgcttgct
tatgtgggga ttccacgcta tgaaggtcct gaggttgatc 600ctactatagt
gacacacgat gcaaaggacc tctataaagc tggtgagaaa aggctgggca
660ctgatgagaa gaccttcatc cgcattttca ctgaacgcag ctgggcacac
atggcatctg 720ttgcctctgc ttaccatcat atgtatgatc ggtcactgga
gaaggttgtg aagagcgaaa 780catctggaaa ctttgaactt gctctgctaa
ctatcctcag atgcgctgag aatccagcca 840agtattttgc aaaggtcttg
cggaagtcca tgaaaggtat gggcactgat gatagtacac 900ttataagggt
tgtagtaaca aggactgaga tcgacatgca atatatcaag gctgagtact
960acaagaaata caaaaaatca ttagctgaag ctatccattc cgagacctca
ggaaattatc 1020gaacattcct cctttctcta gttggtagcc attaggctac
atttcgtcga ccctgtggca 1080cttgacgttc catgactatc ctaaatgcag
tggttctacc tggaaactgt aaaatttcgc 1140catcattgtg ctctctattc
gtgtgtgctt gcttaaaaat gtgtgtatat atataacctg 1200ggcattaaat
agttggtgct taatatggtt tggtggttcc atctgacaag tcactcgtta
1260ctcggtgcat ttattcgaat aagtgatggt atttggtc 1298141315PRTOryza
sativa 141Met Ala Ser Leu Thr Leu Pro Pro Ala Pro Thr Asn Pro Arg
Gln Asp 1 5 10 15 Ala Ile Asp Leu His Lys Ala Phe Lys Gly Phe Gly
Cys Asp Ser Thr 20 25 30 Thr Val Ile Asn Ile Leu Thr His Arg Asp
Ser Met Gln Arg Ala Leu 35 40 45 Ile Gln Gln Glu Tyr Arg Thr Met
Tyr Ser Glu Asp Leu Ser Arg Arg 50 55 60 Ile Ser Ser Glu Leu Ser
Gly His His Lys Lys Ala Met Leu Leu Trp 65 70 75 80 Ile Leu Asp Pro
Ala Gly Arg Asp Ala Thr Val Leu Arg Glu Ala Leu 85 90 95 Ser Gly
Asp Thr Ile Asp Leu Arg Ala Ala Thr Glu Ile Ile Cys Ser 100 105 110
Arg Thr Pro Ser Gln Leu Gln Ile Met Lys Gln Thr Tyr His Ala Lys 115
120 125 Phe Gly Thr Tyr Leu Glu His Asp Ile Gly Gln Arg Thr Ser Gly
Asp 130 135 140 His Gln Lys Leu Leu Leu Ala Tyr Val Gly Ile Pro Arg
Tyr Glu Gly 145 150 155 160 Pro Glu Val Asp Pro Thr Ile Val Thr His
Asp Ala Lys Asp Leu Tyr 165 170 175 Lys Ala Gly Glu Lys Arg Leu Gly
Thr Asp Glu Lys Thr Phe Ile Arg 180 185 190 Ile Phe Thr Glu Arg Ser
Trp Ala His Met Ala Ser Val Ala Ser Ala 195 200 205 Tyr His His Met
Tyr Asp Arg Ser Leu Glu Lys Val Val Lys Ser Glu 210 215 220 Thr Ser
Gly Asn Phe Glu Leu Ala Leu Leu Thr Ile Leu Arg Cys Ala 225 230 235
240 Glu Asn Pro Ala Lys Tyr Phe Ala Lys Val Leu Arg Lys Ser Met Lys
245 250 255 Gly Met Gly Thr Asp Asp Ser Thr Leu Ile Arg Val Val Val
Thr Arg 260 265 270 Thr Glu Ile Asp Met Gln Tyr Ile Lys Ala Glu Tyr
Tyr Lys Lys Tyr 275 280 285 Lys Lys Ser Leu Ala Glu Ala Ile His Ser
Glu Thr Ser Gly Asn Tyr 290 295 300 Arg Thr Phe Leu Leu Ser Leu Val
Gly Ser His 305 310 315 1421222DNAOryza sativa 142ctccccgccg
cataaatccc cttcgcctcc ccgccgcgcc ccgcggcgtc gcacgatctc 60actgaggcat
aaagtgagag accgtgattg gatcgatcac cggagcgacg atcaatggcg
120acgctcaccg tgcccgccgc cgtgccgccc gtcgccgagg actgcgagca
gctgcgcaag 180gcgttcaaag ggtggggcac gaacgagaag ctcatcatct
ccatcctcgc ccaccgcgac 240gcggcgcagc gccgggcgat ccgccgcgcc
tacgccgagg cgtacggcga ggagctgctc 300cgcgccctca acgacgagat
ccacggcaaa ttcgagaggg cggtgatcca gtggacgctg 360gacccggcgg
agcgggacgc ggtgctggcg aacgaggagg cgaggaagtg gcacccgggg
420ggccgcgcgc tcgtcgagat cgcgtgcacg cgcactccat cgcagctctt
cgctgcgaag 480caggcgtacc acgagcgctt caagaggtcg ctcgaggagg
acgtcgcggc gcacatcacc 540ggcgactacc gtaagctttt ggtgccactt
gtgactgtat atcgctatga tgggccagag 600gtgaacacat cgttggcaca
ttctgaagcc aaaatactcc atgagaagat ccatgacaag 660gcttacagtg
acgatgaaat catcaggatt ctcaccacaa ggagcaaagc acagttacta
720gcaacattca atagttacaa tgatcagttc ggccatccaa tcactaagga
tcttaaagct 780gatcctaagg acgagttcct tggtacacta agggcgatca
taagatgctt cacttgccct 840gacagatact ttgagaaagt cattcgattg
gctctaggag gaatgggcac agacgagaac 900tctcttacaa ggatcataac
aactcgtgcc gaggtagacc tgaagctgat aaaggaggcc 960taccagaaga
gaaacagtgt cccattggag cgagctgttg ctaaagatac aaccagagac
1020tacgaggata tactccttgc cctccttgga gcagagtgag gtgtatatct
gctccatctc 1080gtctgtctga tcctccttgt ttgatcggaa aataagatct
gcatagaact gtgttctatt 1140ttgttgtttc tgaatgatac aagtgagcta
gtctgcatag cagtgctcat ataataaaat 1200ctgtcctgca tactggtttg tc
1222143314PRTOryza sativa 143Met Ala Thr Leu Thr Val Pro Ala Ala
Val Pro Pro Val Ala Glu Asp 1 5 10 15 Cys Glu Gln Leu Arg Lys Ala
Phe Lys Gly Trp Gly Thr Asn Glu Lys 20 25 30 Leu Ile Ile Ser Ile
Leu Ala His Arg Asp Ala Ala Gln Arg Arg Ala 35 40 45 Ile Arg Arg
Ala Tyr Ala Glu Ala Tyr Gly Glu Glu Leu Leu Arg Ala 50 55 60 Leu
Asn Asp Glu Ile His Gly Lys Phe Glu Arg Ala Val Ile Gln Trp 65 70
75 80 Thr Leu Asp Pro Ala Glu Arg Asp Ala Val Leu Ala Asn Glu Glu
Ala 85 90 95 Arg Lys Trp His Pro Gly Gly Arg Ala Leu Val Glu Ile
Ala Cys Thr 100 105 110 Arg Thr Pro Ser Gln Leu Phe Ala Ala Lys Gln
Ala Tyr His Glu Arg 115 120 125 Phe Lys Arg Ser Leu Glu Glu Asp Val
Ala Ala His Ile Thr Gly Asp 130 135 140 Tyr Arg Lys Leu Leu Val Pro
Leu Val Thr Val Tyr Arg Tyr Asp Gly 145 150 155 160 Pro Glu Val Asn
Thr Ser Leu Ala His Ser Glu Ala Lys Ile Leu His 165 170 175 Glu Lys
Ile His Asp Lys Ala Tyr Ser Asp Asp Glu Ile Ile Arg Ile 180 185 190
Leu Thr Thr Arg Ser Lys Ala Gln Leu Leu Ala Thr Phe Asn Ser Tyr 195
200 205 Asn Asp Gln Phe Gly His Pro Ile Thr Lys Asp Leu Lys Ala Asp
Pro 210 215 220 Lys Asp Glu Phe Leu Gly Thr Leu Arg Ala Ile Ile Arg
Cys Phe Thr 225 230 235 240 Cys Pro Asp Arg Tyr Phe Glu Lys Val Ile
Arg Leu Ala Leu Gly Gly 245 250 255 Met Gly Thr Asp Glu Asn Ser Leu
Thr Arg Ile Ile Thr Thr Arg Ala 260 265 270 Glu Val Asp Leu Lys Leu
Ile Lys Glu Ala Tyr Gln Lys Arg Asn Ser 275 280 285 Val Pro Leu Glu
Arg Ala Val Ala Lys Asp Thr Thr Arg Asp Tyr Glu 290 295 300 Asp Ile
Leu Leu Ala Leu Leu Gly Ala Glu 305 310 1441152DNAOryza sativa
144agcacagcac agcacacatc tcgtccagtc catccatggc gagcctgagc
gtgccgccgg 60tgccgacgga cccgcggcgc gacgcgatcg acctccacag ggcgttcaag
gggttcggct 120gcgacgccac ggcggtgacc gccatcctcg cccaccgcga
cgcctcccag cgcgccctaa 180tccggcgcca ctacgcggcg gtctaccacc
aggacctcct ccaccgcctc gccgccgagc 240tctcgggcca ccacaagcgc
gccgtcctgc tctgggtgct cgacccggcg tcccgcgacg 300ccgccgtcct
ccaccaggcg ctcaacggcg acgtcaccga catgagggcg gccaccgagg
360tggtgtgctc caggacgccg tcgcagctgc tcgtggtgag gcaggcctac
ctcgccaggt 420tcggcggcgg cggcggcggc ggcctcgagc acgacgtcgc
cgtcagggcg tccggcgacc 480accagaggct gcttctggcg tacctgcgct
cgccgcggta cgaggggccc gaggtggtcg 540acatggcggc ggcggcgcgc
gacgccaggg agctgtacag ggccggcgag aggcggctcg 600gcaccgacga
gaggacgttc atccgcgtct tctccgagcg cagcgccgcc cacatggcgg
660ccgtcgccgc cgcgtaccac cacatgtacg accgctccct cgagaaggct
gtgaagagtg 720aaacttcagg gaactttggg tttggcctgc tgacaatcct
caggtgcgcc gagagcccgg 780ccaagtactt cgccaaggtg ctccacgagg
cgatgaaggg gctgggcacc aacgacacga 840cgctgatcag ggtggtgacg
acgagggcgg aggtggacat gcagtacatc aaggcggagt 900accaccggag
ctacaagcgc tcgctcgccg acgccgtcca ctccgagacc tccggcaact
960accgcacctt cctcctctcc ctcatcggcc gcgaccgcta acgtcgattg
gtttcggtct 1020ctttgagcgt gtgttaaggg acgcatttgt tccatagcgc
acaaacatgg caattattta 1080tgtgcgtgtg tagtggtgtg ttcgaacgtt
cgtttttcgt gtaataaaaa aaattgagtt 1140tgctgtcttg tg
1152145321PRTOryza sativa 145Met Ala Ser Leu Ser Val Pro Pro Val
Pro Thr Asp Pro Arg Arg Asp 1 5 10 15 Ala Ile Asp Leu His Arg Ala
Phe Lys Gly Phe Gly Cys Asp Ala Thr 20 25 30 Ala Val Thr Ala Ile
Leu Ala His Arg Asp Ala Ser Gln Arg Ala Leu 35 40 45 Ile Arg Arg
His Tyr Ala Ala Val Tyr His Gln Asp Leu Leu His Arg 50 55 60 Leu
Ala Ala Glu Leu Ser Gly His His Lys Arg Ala Val Leu Leu Trp 65 70
75 80 Val Leu Asp Pro Ala Ser Arg Asp Ala Ala Val Leu His Gln Ala
Leu 85 90 95 Asn Gly Asp Val Thr Asp Met Arg Ala Ala Thr Glu Val
Val Cys Ser 100 105 110 Arg Thr Pro Ser Gln Leu Leu Val Val Arg Gln
Ala Tyr Leu Ala Arg 115 120 125 Phe Gly Gly Gly Gly Gly Gly Gly Leu
Glu His Asp Val Ala Val Arg 130 135 140 Ala Ser Gly Asp His Gln Arg
Leu Leu Leu Ala Tyr Leu Arg Ser Pro 145 150 155 160 Arg Tyr Glu Gly
Pro Glu Val Val Asp Met Ala Ala Ala Ala Arg Asp 165 170 175 Ala Arg
Glu Leu Tyr Arg Ala Gly Glu Arg Arg Leu Gly Thr Asp Glu 180 185 190
Arg Thr Phe Ile Arg Val Phe Ser Glu Arg Ser Ala Ala His Met Ala 195
200 205 Ala Val Ala Ala Ala Tyr His His Met Tyr Asp Arg Ser Leu Glu
Lys 210 215 220 Ala Val Lys Ser Glu Thr Ser Gly Asn Phe Gly Phe Gly
Leu Leu Thr 225 230 235 240 Ile Leu Arg Cys Ala Glu Ser Pro Ala Lys
Tyr Phe Ala Lys Val Leu 245 250 255 His Glu Ala Met Lys Gly Leu Gly
Thr Asn Asp Thr Thr Leu Ile Arg 260 265 270 Val Val Thr Thr Arg Ala
Glu Val Asp Met Gln Tyr Ile Lys Ala Glu 275 280 285 Tyr His Arg Ser
Tyr Lys Arg Ser Leu Ala Asp Ala Val His Ser Glu 290 295 300 Thr Ser
Gly Asn Tyr Arg Thr Phe Leu Leu Ser Leu Ile Gly Arg Asp 305 310 315
320 Arg 1461272DNAOryza sativa 146cgggtctcct ctcctccccc gccgacgcgc
actcgatccc ccccgcctcc gcctccgcct 60ccgcctccgc gtcgcccatc tcgagatccc
ccgcatggcg acgctcaccg tcccctccgc 120cgtcccgccc gtcgccgacg
actgcgacca gctccgcaag gccttccaag ggtggggcac 180gaacgaggcg
ctcatcatct ccatcctggc ccaccgcgac gcggcgcagc ggcgcgccat
240ccgccgcgcc tacgccgaca cctacggcga ggagctcctc cgcagcatca
ccgacgagat 300ctccggcgac ttcgagaggg ccgtgatcct gtggacgctg
gacccggcgg agcgcgacgc 360ggtgctcgcc aacgaggtcg cgaggaagtg
gtacccaggg agcgggagcc gcgtgctggt 420cgagatcgcg tgcgcgcgcg
gccccgcgca gctgttcgcg gtcaggcagg cctaccacga 480gcgcttcaag
cgctcgctcg aggaggacgt cgcggcgcac gccactggtg acttccgcaa
540gctcttggtg ccacttataa gtgcttaccg ctatgagggg ccggaagtca
acacaaagtt 600ggcacattca gaagccaaaa ttctgcatga gaagatccag
cataaggcat atggtgatga 660tgagatcatc agaattctca ctactaggag
caaggctcag ttgattgcga cattcaatcg 720ttacaatgat gaatatggtc
acccaatcaa caaggatctc aaggctgatc ccaaggacga 780gttcctttcc
acgctgcgtg caatcatccg ctgcttctgt tgccctgaca ggtacttcga
840gaaagtcatc aggttggcca tcgcaggcat gggaacagac gagaactccc
tcactaggat 900cattaccact cgtgccgagg tggatctgaa gctgatcacg
gaggcgtacc agaagaggaa 960cagtgtcccg ctggagcgtg cggtcgcagg
ggacacctcc ggggactacg agaggatgct 1020tcttgctctt ctgggtcagg
agcagtgagc catgcctatc ttgcccagtc acacacttca 1080tgtgatcatg
tcatatcaga gaataaacct gttatgcagg ggacacagcc gtggtgatta
1140tgatgttgtt tttccagtgt acggtactgt ttgctgcagc ttgcataaca
gtgacgatga 1200aataaatcat agtggaatgc gttggctcat gggacctcac
ttattttgca actttttgac 1260aggtcttatt tc 1272147317PRTOryza sativa
147Met Ala Thr Leu Thr Val Pro Ser Ala Val Pro Pro Val Ala Asp Asp
1 5 10 15 Cys Asp Gln Leu Arg Lys Ala Phe Gln Gly Trp Gly Thr Asn
Glu Ala 20 25 30 Leu Ile Ile Ser Ile Leu Ala His Arg Asp Ala Ala
Gln Arg Arg Ala 35 40 45 Ile Arg Arg Ala Tyr Ala Asp Thr Tyr Gly
Glu Glu Leu Leu Arg Ser 50 55 60 Ile Thr Asp Glu Ile Ser Gly Asp
Phe Glu Arg Ala Val Ile Leu Trp 65 70 75 80 Thr Leu Asp Pro Ala Glu
Arg Asp Ala Val Leu Ala Asn Glu Val Ala 85 90 95 Arg Lys Trp Tyr
Pro Gly Ser Gly Ser Arg Val Leu Val Glu Ile Ala 100 105 110 Cys Ala
Arg Gly Pro Ala Gln Leu Phe Ala Val Arg Gln Ala Tyr His 115 120 125
Glu Arg Phe Lys Arg Ser Leu Glu Glu Asp Val Ala Ala His Ala Thr 130
135 140 Gly Asp Phe Arg Lys Leu Leu Val Pro Leu Ile Ser Ala Tyr Arg
Tyr 145 150 155 160 Glu Gly Pro Glu Val Asn Thr Lys Leu Ala His Ser
Glu Ala Lys Ile 165 170 175 Leu His Glu Lys Ile Gln His Lys Ala Tyr
Gly Asp Asp Glu Ile Ile 180 185 190 Arg Ile Leu Thr Thr Arg Ser Lys
Ala Gln Leu Ile Ala Thr Phe Asn 195 200 205 Arg Tyr Asn Asp Glu Tyr
Gly His Pro Ile Asn Lys Asp Leu Lys Ala 210 215 220 Asp Pro Lys Asp
Glu Phe Leu Ser Thr Leu Arg Ala Ile Ile Arg Cys 225 230 235 240 Phe
Cys Cys Pro Asp Arg Tyr Phe Glu Lys Val Ile Arg Leu Ala Ile 245 250
255 Ala Gly Met Gly Thr Asp Glu Asn Ser Leu Thr Arg Ile Ile Thr Thr
260 265 270 Arg Ala Glu Val Asp Leu Lys Leu Ile Thr Glu Ala Tyr Gln
Lys Arg 275 280 285 Asn Ser Val Pro Leu Glu Arg Ala Val Ala Gly Asp
Thr Ser Gly Asp 290 295 300 Tyr Glu Arg Met Leu Leu Ala Leu Leu Gly
Gln Glu Gln 305 310 315 1481491DNAOryza sativa 148agtaatacgc
aaggaatacc tggatcatac gatacgaata tctagagaca aaacatgatt 60tgagtgattg
atgatcgaga aagaagctca aaaggtcttg aaaagtcgaa acgtccatca
120ctgaaattcg gtttcatgcc ctcagatccc attgctgagt aggacgcaga
ttttcttcct 180tcctaccatt tcctttctct tgcttccttt ttggtcattt
gagatagctt tatccatcct 240ttagcaaaaa ggaaaccaat agctagcaat
acttgccatc atatatacct gggcaatggc 300cggccaagca aactcagttc
tctacattga acacttcagg tttgagtaga catggcctct 360cggtgtcttg
ttaccacagg ttttgaggat gagtgcagag agatccatga tgcgtgcaac
420cagccacgcc gtttgagcgt tctcttggct catcggagcc catcggagag
gcagaaaatc 480aaggcgactt accgtacagt gttcggcgaa gatctcgccg
gagaagtgca gaaaatcctc 540atggtcaacc aggaagatga gctctgcaag
ctgctctacc tgtgggtgct cgacccgtcg 600gagcgcgacg cgatcatggc
tcgggacgcc gtcgagaatg gcggcgccac ggattaccgg 660gtcctggtgg
agatcttcac acgccggaag cagaaccagc tcttcttcac caatcaggca
720taccttgcca ggttcaagaa gaacctggag caggacatgg tcacagagcc
gtctcatcct 780taccagaggc tattggtagc acttgcaacc tcccacaagt
cgcaccacga tgaacttagt 840cggcacattg caaaatgtga cgccaggagg
ctctatgatg cgaagaacag cggcatggga 900tcggtcgacg aggctgtcat
tcttgagatg ttcagcaaga ggagcatccc acagctcagg 960ctagcattct
gcagttacaa gcacatatat gggcatgact acaccaaggc actgaagaaa
1020aatggcttcg gtgagtttga acaatctttg agggttgttg tgaagtgcat
ctacaatcct 1080tccatgtatt tctccaagct gctgcataga agtctgcaat
gctcagcgac caataaaagg 1140ttggttacaa gggctatttt gggcagtgac
gatgtcgata tggacaagat caagtcagtg 1200ttcaaaagta gttatggaaa
ggaccttgag gatttcatcc ttgaaagctt gcctgagaat 1260gattacagag
actttctttt aggtgcggcc aaggggtcaa gggcctcatg aagtctgtgg
1320agagagatcc ttgaattatc tagggaaagt aaagggtgca tatactgctt
tgcatgtaag 1380agcaaattga ccatcaaaaa cagcagtttt atgttatctg
agaataggat ttaggtgaga 1440acatcatgct cattttgttt attttgggtg
aaaaaagtta tcagttcaac t 1491149319PRTOryza sativa 149Met Ala Ser
Arg Cys Leu Val Thr Thr Gly Phe Glu Asp Glu Cys Arg 1 5 10 15 Glu
Ile His Asp Ala Cys Asn Gln Pro Arg Arg Leu Ser Val Leu Leu 20 25
30 Ala His Arg Ser Pro Ser Glu Arg Gln Lys Ile Lys Ala Thr Tyr Arg
35 40 45 Thr Val Phe Gly Glu Asp Leu Ala Gly Glu Val Gln Lys Ile
Leu Met 50 55 60 Val Asn Gln Glu Asp Glu Leu Cys Lys Leu Leu Tyr
Leu Trp Val Leu 65 70 75 80 Asp Pro Ser Glu Arg Asp Ala Ile Met Ala
Arg Asp Ala Val Glu Asn 85 90 95 Gly Gly Ala Thr Asp Tyr Arg Val
Leu Val Glu Ile Phe Thr Arg Arg 100 105 110 Lys Gln Asn Gln Leu Phe
Phe Thr Asn Gln Ala Tyr Leu Ala Arg Phe 115 120 125 Lys Lys Asn Leu
Glu Gln Asp Met Val Thr Glu Pro Ser His Pro Tyr 130 135 140 Gln Arg
Leu Leu Val Ala Leu Ala Thr Ser His Lys Ser His His Asp 145 150 155
160 Glu Leu Ser Arg His Ile Ala Lys Cys Asp Ala Arg Arg Leu Tyr Asp
165 170 175 Ala Lys Asn Ser Gly Met Gly Ser Val Asp Glu Ala Val Ile
Leu Glu 180 185 190 Met Phe Ser Lys Arg Ser Ile Pro Gln Leu Arg Leu
Ala Phe Cys Ser 195 200 205 Tyr Lys His Ile Tyr Gly His Asp Tyr Thr
Lys Ala Leu Lys Lys Asn 210 215 220 Gly Phe Gly Glu Phe Glu Gln Ser
Leu Arg Val Val Val Lys Cys Ile 225 230 235 240 Tyr Asn Pro Ser Met
Tyr Phe Ser Lys Leu Leu His Arg Ser Leu Gln 245 250 255 Cys Ser Ala
Thr Asn Lys Arg Leu Val Thr Arg Ala Ile Leu Gly Ser 260 265 270 Asp
Asp Val Asp Met Asp Lys Ile Lys Ser Val Phe Lys Ser Ser Tyr 275 280
285 Gly Lys Asp Leu Glu Asp Phe Ile Leu Glu Ser Leu Pro Glu Asn Asp
290 295 300 Tyr Arg Asp Phe Leu Leu Gly Ala Ala Lys Gly Ser Arg Ala
Ser 305 310 315 1501393DNAOryza sativa 150atggaaaaat accatgaaat
acaaatggaa aaaatagggt agagcccaga gcattggatg 60tgcagattaa agctatacta
ctagcattca agtttttttc aactctgctg cgtaggaggc 120gtgtgtgtgc
catgtgttgc tggtgctgct gcctggactg catccataac atacctccac
180tcaatctcct cttcctccat ttctcccccc attctctctc ctcctcagct
gcttctgcag 240gtggaggaga agcagcagca gcagcagctg ttgctcccat
ggcttccatc tctgtcccaa 300acccagctcc ttcccctaca gaggatgcag
agagcataag aaaggcagtg caaggatggg 360gaacggacga gaatgcgctg
atcgagatcc tcggccaccg gacggcggcg cagcgggcgg 420agatcgccgt
cgcctacgag ggcctctacg acgagaccct cctcgacagg ctccactccg
480agctctccgg cgacttccgt agcgcgttga tgctgtggac gatggacccg
gcggcgcggg 540acgccaagct ggccaacgag gccctgaaga agaagaagaa
gggcgagctc cgccacatct 600gggtgctcgt cgaggtcgcc tgcgcgtcgt
cgccggacca cctcgtcgcc gtcaggaagg 660cctaccgcgc cgcctacgcc
tcgtcgctgg aggaggacgt ggcgtcgtgc tcgctgttcg 720gggacccgct
caggcggttc ctggtgcgcc tcgtgagctc ctaccggtac ggcggcggtg
780gcgtcgacgg cgagctggcg atcgccgagg cggcggagct gcacgacgcg
gtggtgggca 840gggggcaggc gctgcacggc gacgacgtcg tccgcatcgt
cggcacgagg agcaaggcgc 900agctcgcggt gacgctggag cggtacaggc
aggagcacgg caagggcatc gacgaggtcc 960tcgacggccg ccgcggcgac
cagctcgcgg cggtgctcaa ggccgcgctc tggtgcctca 1020cctcgccgga
gaagcatttc gctgaggtga tccggacatc gattctaggg cttggcaccg
1080acgaggagat gctgacgaga gggatcgtgt cgcgggcgga ggtggacatg
gagaaggtga 1140aggaggagta caaggtcagg tacaacacca cggtcaccgc
cgacgtccgc ggcgacacgt 1200cggggtacta catgaacacg cttctcaccc
tcgtcggccc tgagaagtag ccatgtagca 1260gcttggacat tttattgctt
gctcatttga tttgaacaaa atacaccgtg tgatgttgca 1320gttattagta
aaatgcgagt aggatcgatg ttgttttcgt tgggtggatt aataatggag
1380catgttttat cgc 1393151372PRTOryza sativa 151Met Cys Cys Trp Cys
Cys Cys Leu Asp Cys Ile His Asn Ile Pro Pro 1 5 10 15 Leu Asn Leu
Leu Phe Leu His Phe Ser Pro His Ser Leu Ser Ser Ser 20 25 30 Ala
Ala Ser Ala Gly Gly Gly Glu Ala Ala Ala Ala Ala Ala Val Ala 35 40
45 Pro Met Ala Ser Ile Ser Val Pro Asn Pro Ala Pro Ser Pro Thr Glu
50 55 60 Asp Ala Glu Ser Ile Arg Lys Ala Val Gln Gly Trp Gly Thr
Asp Glu 65 70 75 80 Asn Ala Leu Ile Glu Ile Leu Gly His Arg Thr Ala
Ala Gln Arg Ala 85 90 95 Glu Ile Ala Val Ala Tyr Glu Gly Leu Tyr
Asp Glu Thr Leu Leu Asp 100 105 110 Arg Leu His Ser Glu Leu Ser Gly
Asp Phe Arg Ser Ala Leu Met Leu 115 120 125 Trp Thr Met Asp Pro Ala
Ala Arg Asp Ala Lys Leu Ala Asn Glu Ala 130 135 140 Leu Lys Lys Lys
Lys Lys Gly Glu Leu Arg His Ile Trp Val Leu Val 145 150 155 160 Glu
Val Ala Cys Ala Ser Ser Pro Asp His Leu Val Ala Val Arg Lys 165 170
175 Ala Tyr Arg Ala Ala Tyr Ala Ser Ser Leu Glu Glu Asp Val Ala Ser
180 185 190 Cys Ser Leu Phe Gly Asp Pro Leu Arg Arg Phe Leu Val Arg
Leu Val 195 200 205 Ser Ser Tyr Arg Tyr Gly Gly Gly Gly Val Asp Gly
Glu Leu Ala Ile 210 215 220 Ala Glu Ala Ala Glu Leu His Asp Ala Val
Val Gly Arg Gly Gln Ala 225 230 235 240 Leu His Gly Asp Asp Val Val
Arg Ile Val Gly Thr Arg Ser Lys Ala 245 250 255 Gln Leu Ala Val Thr
Leu Glu Arg Tyr Arg Gln Glu His Gly Lys Gly 260 265 270 Ile Asp Glu
Val Leu Asp Gly Arg Arg Gly Asp Gln Leu Ala Ala Val 275 280 285 Leu
Lys Ala Ala Leu Trp Cys Leu Thr Ser Pro Glu Lys His Phe Ala 290 295
300 Glu Val Ile Arg Thr Ser Ile Leu Gly Leu Gly Thr Asp Glu Glu Met
305 310 315 320 Leu Thr Arg Gly Ile Val Ser Arg Ala Glu Val Asp Met
Glu Lys Val 325 330 335 Lys Glu Glu Tyr Lys Val Arg Tyr Asn Thr Thr
Val Thr Ala Asp Val 340 345 350 Arg Gly Asp Thr Ser Gly Tyr Tyr Met
Asn Thr Leu Leu Thr Leu Val 355 360 365 Gly Pro Glu Lys 370
1521341DNAOryza sativa 152ggtcttttcc ggccgctccc ggcgcccgcc
ggccatgtca acaacaagct cgtcaaaaac 60cgccacgtgc cctctctgcc acgccgacgt
gctgctgcca cggcggcggt cagccggctc 120ctcgacgcac agaagccacg
acctcgacga cggccccgct ccgccgtcgc cggagtccag 180ctgccgcagc
aacgccgcgg tgtgcgggtg cgcccgctgc cggcgccctg atctgatcag
240tcccggaacc ggcatgcgga tgaggaacag attcggcgat ggccccaaca
ggatatcagc 300ccagagtgga gcatggctgt gccgagagcg cacggcggag
acttgccgga gcacaagtag 360ccggtccggc cgctaccggc gcctgcagcc
tgcagcggtc gccggaacac gacgcgccga 420actcataaag cggctgcaag
agctctgcca cccagcaaat aaccttccaa attgttctgc 480ttcttggcaa
ccacagcgac agcggcagaa aacaattgat cgaattccag atagcctaga
540ttgtggggtg acaatggaaa ggggcaagaa caagcgtgat ggaagtgaca
atgggctcat 600cttctctaac ctaatgcacg gtgttgctgc cggcatctat
gggtatcctc ctcaccaggg 660atacactcag gctcagagct acctactgct
gccggaagca tatccacctc ctccgtggac 720ataccctctt tctagtgctt
accctcctca acctgttggt tacccttcag gtggctaccc 780tcctgcagtc
tactctgact cgtatctgca ccaaggtagc agagttgcgc gggagcaatg
840ccctctatca tattccaata atgctgtcac ttgcagggag gatgggcaaa
tgaactgtga 900aaatggaaca gtaaatatgg agaaaagtgc aatgtcctca
aataagatgg ctactagtct 960actaaagagt tgcggcaatg tgatgccatg
cagaaatatg gagagaagtg gcccagccat 1020gtataaggtg gacatgcgcg
gcagtacgaa gcaattctct atgggcagca agatgatgat 1080gtgtctgatt
gtgtttggat gtctgatagc tgccttggat atgtttagaa atgttgcaca
1140aaaacagatg ttttctgtcg ttagtttact ttcttttgta gtcgcgacct
atgtctgcta 1200ggagtctcta catgtaccgt aaaattgctc tttgtgtaat
gtgtacttct tcatcctgta 1260aaaatagaat cccaatcaaa ctatatatgg
tttgtctgtc gggctttcaa tacaatctga 1320gtgtcctctc tttacctttg t
1341153388PRTOryza sativa 153Met Ser Thr Thr Ser Ser Ser Lys Thr
Ala Thr Cys Pro Leu Cys His 1 5 10 15 Ala Asp Val Leu Leu Pro Arg
Arg Arg Ser Ala Gly Ser Ser Thr His 20 25 30 Arg Ser His Asp Leu
Asp Asp Gly Pro Ala Pro Pro Ser Pro Glu Ser 35 40 45 Ser Cys Arg
Ser Asn Ala Ala Val Cys Gly Cys Ala Arg Cys Arg Arg 50 55 60 Pro
Asp Leu Ile Ser Pro Gly Thr Gly Met Arg Met Arg Asn Arg Phe 65 70
75 80 Gly Asp Gly Pro Asn Arg Ile Ser Ala Gln Ser Gly Ala Trp Leu
Cys 85 90 95 Arg Glu Arg Thr Ala Glu Thr Cys Arg Ser Thr Ser Ser
Arg Ser Gly 100 105 110 Arg Tyr Arg Arg Leu Gln Pro Ala Ala Val Ala
Gly Thr Arg Arg Ala 115 120 125 Glu Leu Ile Lys Arg Leu Gln Glu Leu
Cys His Pro Ala Asn Asn Leu 130 135 140 Pro Asn Cys Ser Ala Ser Trp
Gln Pro Gln Arg Gln Arg Gln Lys Thr 145 150 155 160 Ile Asp Arg Ile
Pro Asp Ser Leu Asp Cys Gly Val Thr Met Glu Arg 165 170 175 Gly Lys
Asn Lys Arg Asp Gly Ser Asp Asn Gly Leu Ile Phe Ser Asn 180 185 190
Leu Met His Gly Val Ala Ala Gly Ile Tyr Gly Tyr Pro Pro His Gln 195
200 205 Gly Tyr Thr Gln Ala Gln Ser Tyr Leu Leu Leu Pro Glu Ala Tyr
Pro 210 215 220 Pro Pro Pro Trp Thr Tyr Pro Leu Ser Ser Ala Tyr Pro
Pro Gln Pro 225 230 235 240 Val Gly Tyr Pro Ser Gly Gly Tyr Pro Pro
Ala Val Tyr Ser Asp Ser 245 250 255 Tyr Leu His Gln Gly Ser Arg Val
Ala Arg Glu Gln Cys Pro Leu Ser 260 265 270 Tyr Ser Asn Asn Ala Val
Thr Cys Arg Glu Asp Gly Gln Met Asn Cys 275 280 285 Glu Asn Gly Thr
Val Asn Met Glu Lys Ser Ala Met Ser Ser Asn Lys 290 295 300 Met Ala
Thr Ser Leu Leu Lys Ser Cys Gly Asn Val Met Pro Cys Arg 305 310 315
320 Asn Met Glu Arg Ser Gly Pro Ala Met Tyr Lys Val Asp Met Arg Gly
325 330 335 Ser Thr Lys Gln Phe Ser Met Gly Ser Lys Met Met Met Cys
Leu Ile 340 345 350 Val Phe Gly Cys Leu Ile Ala Ala Leu Asp Met Phe
Arg Asn Val Ala 355 360 365 Gln Lys Gln Met Phe Ser Val Val Ser Leu
Leu Ser Phe Val Val Ala 370 375 380 Thr Tyr Val Cys 385
1541276DNAOryza sativa 154ataggtcaac ttgaatcttg ttcaaaaatt
cttttcaagt cttaggtagg tcactgttgg 60tgccaccttc ttgagaagtt tatgatctgc
atgttcacct aagatatcca gtcaagtgct 120aaaaaggtca caaaaatggg
aggcagaaag gacaatcatg actcctcaaa tgccgacaaa 180gggttccatg
gagcgtatcc aagcggttac cctggtgcat atcccctaat gcaaggatac
240cctaattcac ctggacaata tccgactccc ggtggatacc ctagtgcacc
accgggacaa 300tacccaccag ccggtgggta ccctggtgca caatatccac
caagcggtta ccctccatca 360caaggtgggt accctccagg agcgtatcca
ccatcaggat atccacaaca accaggctac 420ccgccagctg gttacccagg
tcatggccat ggtccaccca tgcaaggagg tgggcatggt 480gcaggcgcat
ctggctatgg agcgctgctc gccggaggcg ccgcggtggc ggctgctgcg
540gtgggagctc acatggtacg acccggcggc ggtggcggcc acgggatgtt
cggccaccat 600ggtggcaaat tcaagaaagg aaagttcaag catggcaagt
acggcaagca caagaagttt 660gggcgcaagt ggaagtgata agcaaactaa
attgcactgc agtttgcctc cggtttcttg 720tttggttgaa gtgctatagc
atgatgggat taagtgtcca tttaccatat atatatatat 780atattcatcg
aaatataacc atacgagatc ttattttaaa gataattgta ataaatataa
840tggtgtaatg gatcacaaat tagaaaaaag gtttaggaga aaaaacattt
tgagcttgct 900aacagaaaat taaacccacc ataagcaatc agaataggcc
acatgtgaat ggctatatcc 960ggttaaatcc gccgaatcca atcatcgaac
tcttatttcc cacaaaccaa actcatccag 1020cctcatgcac ctaactaatc
acatccatcc ttagacgatg catgcgtgtg gacattcttg 1080ccaaaaccaa
caattagccg tgagagccca aacaactgag tactccagcc tacgattgtc
1140aggatatttt ctcatctaac ttattctagt attaggagtt atgaagatat
gaaaagccat 1200tttagttcag aaaattgtac gataaatcat gtaacctgtt
tctgaaatgg aaataaaata 1260tgagaaaaag atacta 1276155180PRTOryza
sativa 155Met Gly Gly Arg Lys Asp Asn His Asp Ser Ser Asn Ala Asp
Lys Gly 1 5 10 15 Phe His Gly Ala Tyr Pro Ser Gly Tyr Pro Gly Ala
Tyr Pro Leu Met 20 25 30 Gln Gly Tyr Pro Asn Ser Pro Gly Gln Tyr
Pro Thr Pro Gly Gly Tyr 35 40 45 Pro Ser Ala Pro Pro Gly Gln Tyr
Pro Pro Ala Gly Gly Tyr Pro Gly 50 55 60 Ala Gln Tyr Pro Pro Ser
Gly Tyr Pro Pro Ser Gln Gly Gly Tyr Pro 65 70 75 80 Pro Gly Ala Tyr
Pro Pro Ser Gly Tyr Pro Gln Gln Pro Gly Tyr Pro 85 90 95 Pro Ala
Gly Tyr Pro Gly His Gly His Gly Pro Pro Met Gln Gly Gly 100 105 110
Gly His Gly Ala Gly Ala Ser Gly Tyr Gly Ala Leu Leu Ala Gly Gly 115
120 125 Ala Ala Val Ala Ala Ala Ala Val Gly Ala His Met Val Arg Pro
Gly 130 135 140 Gly Gly Gly Gly His Gly Met Phe Gly His His Gly Gly
Lys Phe Lys 145 150 155 160 Lys Gly Lys Phe Lys His Gly Lys Tyr Gly
Lys His Lys Lys Phe Gly 165 170 175 Arg Lys Trp Lys 180
15632PRTChlamydomonas sp. 156Met Ala Met Ala Met Arg Ser Thr Phe
Ala Ala Arg Val Gly Ala Lys 1 5 10 15 Pro Ala Val Arg Gly Ala Arg
Pro Ala Ser Arg Met Ser Cys Met Ala 20 25 30 15732PRTChlamydomonas
sp. 157Met Gln Val Thr Met Lys Ser Ser Ala Val Ser Gly Gln Arg Val
Gly 1 5 10 15 Gly Ala Arg Val Ala Thr Arg Ser Val Arg Arg Ala Gln
Leu Gln Val 20 25 30 15852PRTArabidopsis thaliana 158Met Ala Ser
Leu Met Leu Ser Leu Gly Ser Thr Ser Leu Leu Pro Arg 1 5 10 15 Glu
Ile Asn Lys Asp Lys Leu Lys Leu Gly Thr Ser Ala Ser Asn Pro 20 25
30 Phe Leu Lys Ala Lys Ser Phe Ser Arg Val Thr Met Thr Val Ala Val
35 40 45 Lys Pro Ser Arg 50 15954PRTArabidopsis thaliana 159Met Ala
Thr Gln Phe Ser Ala Ser Val Ser Leu Gln Thr Ser Cys Leu 1 5 10 15
Ala Thr Thr Arg Ile Ser Phe Gln Lys Pro Ala Leu Ile Ser Asn His 20
25 30 Gly Lys Thr Asn Leu Ser Phe Asn Leu Arg Arg Ser Ile Pro Ser
Arg 35 40 45 Arg Leu Ser Val Ser Cys 50 16070PRTArabidopsis
thaliana 160Met Ala Ser Ile Ala Ala Ser Ala Ser Ile Ser Leu Gln Ala
Arg Pro 1 5 10 15 Arg Gln Leu Ala Ile Ala Ala Ser Gln Val Lys Ser
Phe Ser Asn Gly 20 25 30 Arg Arg Ser Ser Leu Ser Phe Asn Leu Arg
Gln Leu Pro Thr Arg Leu 35 40 45 Thr Val Ser Cys Ala Ala Lys Pro
Glu Thr Val Asp Lys Val Cys Ala 50 55 60 Val Val Arg Lys Gln Leu 65
70 16174PRTArabidopsis thaliana 161Met Ala Ser Ile Ala Thr Ser Ala
Ser Thr Ser Leu Gln Ala Arg Pro 1 5 10 15 Arg Gln Leu Val Ile Gly
Ala Lys Gln Val Lys Ser Phe Ser Tyr Gly 20 25 30 Ser Arg Ser Asn
Leu Ser Phe Asn Leu Arg Gln Leu Pro Thr Arg Leu 35
40 45 Thr Val Tyr Cys Ala Ala Lys Pro Glu Thr Val Asp Lys Val Cys
Ala 50 55 60 Val Val Arg Lys Gln Leu Ser Leu Lys Glu 65 70
162232PRTUnknownPRX protein 162Met Ala Ala Ala Ala Ser Thr Leu Ala
Ser Leu Ser Ala Thr Ala Ala 1 5 10 15 Ala Ala Ala Gly Lys Arg Leu
Leu Leu Ser Ser Pro Ser Arg Ser Leu 20 25 30 Ser Leu Ser Leu Ala
Ser Arg Gly Arg Ile Ala Val Met Pro His Leu 35 40 45 Arg Ala Gly
Ile Leu Ser Ala Ala Pro Arg Arg Ala Val Ser Ala Ser 50 55 60 Ala
Pro Ala Ala Ala Thr Ile Ala Val Gly Asp Lys Leu Pro Asp Ala 65 70
75 80 Thr Leu Ser Tyr Phe Asp Ser Pro Asp Gly Glu Leu Lys Thr Val
Thr 85 90 95 Val Arg Asp Leu Thr Ala Gly Lys Lys Val Val Leu Phe
Ala Val Pro 100 105 110 Gly Ala Phe Thr Pro Thr Cys Thr Gln Lys His
Val Pro Gly Phe Val 115 120 125 Ala Lys Ala Gly Glu Leu Arg Ala Lys
Gly Val Asp Ala Val Ala Cys 130 135 140 Val Ser Val Asn Asp Ala Phe
Val Met Arg Ala Trp Lys Glu Ser Leu 145 150 155 160 Gly Val Gly Asp
Glu Val Leu Leu Leu Ser Asp Gly Asn Gly Glu Leu 165 170 175 Ala Arg
Ala Met Gly Val Glu Leu Asp Leu Ser Asp Lys Pro Ala Gly 180 185 190
Leu Gly Val Arg Ser Arg Arg Tyr Ala Leu Leu Ala Glu Asp Gly Val 195
200 205 Val Lys Val Leu Asn Leu Glu Glu Gly Gly Ala Phe Thr Thr Ser
Ser 210 215 220 Ala Glu Glu Met Leu Lys Ala Leu 225 230
163225PRTUnknownPRX protein 163Met Ala Ala Pro Thr Ala Ala Ala Leu
Ser Thr Leu Ser Thr Ala Ser 1 5 10 15 Val Thr Ser Gly Lys Arg Phe
Ile Thr Ser Ser Phe Ser Leu Ser Phe 20 25 30 Ser Ser Arg Pro Leu
Ala Thr Gly Val Arg Ala Ala Gly Ala Arg Ala 35 40 45 Ala Arg Arg
Ser Ala Ala Ser Ala Ser Thr Val Val Ala Thr Ile Ala 50 55 60 Val
Gly Asp Lys Leu Pro Asp Ala Thr Leu Ser Tyr Phe Asp Pro Ala 65 70
75 80 Asp Gly Glu Leu Lys Thr Val Thr Val Ala Glu Leu Thr Ala Gly
Arg 85 90 95 Lys Ala Val Leu Phe Ala Val Pro Gly Ala Phe Thr Pro
Thr Cys Ser 100 105 110 Gln Lys His Leu Pro Gly Phe Ile Glu Lys Ala
Gly Glu Leu His Ala 115 120 125 Lys Gly Val Asp Ala Ile Ala Cys Val
Ser Val Asn Asp Ala Phe Val 130 135 140 Met Arg Ala Trp Lys Glu Ser
Leu Gly Leu Gly Asp Ala Asp Val Leu 145 150 155 160 Leu Leu Ser Asp
Gly Asn Leu Glu Leu Thr Arg Ala Leu Gly Val Glu 165 170 175 Met Asp
Leu Ser Asp Lys Pro Met Gly Leu Gly Val Arg Ser Arg Arg 180 185 190
Tyr Ala Leu Leu Ala Asp Asp Gly Val Val Lys Val Leu Asn Leu Glu 195
200 205 Glu Gly Gly Ala Phe Thr Thr Ser Ser Ala Glu Glu Met Leu Lys
Ala 210 215 220 Leu 225 164162PRTUnknownPRX protein 164Met Ala Pro
Val Ala Val Gly Asp Thr Leu Pro Asp Gly Gln Leu Gly 1 5 10 15 Trp
Phe Asp Gly Glu Asp Lys Leu Gln Gln Val Ser Val His Gly Leu 20 25
30 Ala Ala Gly Lys Lys Val Val Leu Phe Gly Val Pro Gly Ala Phe Thr
35 40 45 Pro Thr Cys Ser Asn Gln His Val Pro Gly Phe Ile Asn Gln
Ala Glu 50 55 60 Gln Leu Lys Ala Lys Gly Val Asp Asp Ile Leu Leu
Val Ser Val Asn 65 70 75 80 Asp Pro Phe Val Met Lys Ala Trp Ala Lys
Ser Tyr Pro Glu Asn Lys 85 90 95 His Val Lys Phe Leu Ala Asp Gly
Leu Gly Thr Tyr Thr Lys Ala Leu 100 105 110 Gly Leu Glu Leu Asp Leu
Ser Glu Lys Gly Leu Gly Ile Arg Ser Arg 115 120 125 Arg Phe Ala Leu
Leu Ala Asp Asn Leu Lys Val Thr Val Ala Asn Ile 130 135 140 Glu Glu
Gly Gly Gln Phe Thr Ile Ser Gly Ala Glu Glu Ile Leu Lys 145 150 155
160 Ala Leu 165162PRTUnknownPRX protein 165Met Ala Pro Val Ala Val
Gly Asp Thr Leu Pro Asp Gly Gln Leu Gly 1 5 10 15 Trp Phe Asp Gly
Glu Asp Lys Leu Gln Gln Val Ser Val His Gly Leu 20 25 30 Ala Ala
Gly Lys Lys Val Val Leu Phe Gly Val Pro Gly Ala Phe Thr 35 40 45
Pro Thr Cys Ser Asn Gln His Val Pro Gly Phe Ile Asn Gln Ala Glu 50
55 60 Gln Leu Lys Ala Lys Gly Val Asp Asp Ile Leu Leu Val Ser Val
Asn 65 70 75 80 Asp Pro Phe Val Met Lys Ala Trp Ala Lys Ser Tyr Pro
Glu Asn Lys 85 90 95 His Val Lys Phe Leu Ala Asp Gly Leu Gly Thr
Tyr Thr Lys Ala Leu 100 105 110 Gly Leu Glu Leu Asp Leu Ser Glu Lys
Gly Leu Gly Ile Arg Ser Arg 115 120 125 Arg Phe Ala Leu Leu Ala Asp
Asn Leu Lys Val Thr Val Ala Asn Ile 130 135 140 Glu Glu Gly Gly Gln
Phe Thr Ile Ser Gly Ala Glu Glu Ile Leu Lys 145 150 155 160 Ala Leu
166198PRTUnknownPRX protein 166Met Ala Ser Ala Leu Leu Arg Lys Ala
Thr Val Gly Gly Ser Ala Ala 1 5 10 15 Ala Ala Ala Ala Arg Trp Ala
Ser Arg Gly Leu Ala Ser Val Gly Ser 20 25 30 Gly Ser Asp Ile Val
Ser Ala Ala Pro Gly Val Ser Leu Gln Lys Ala 35 40 45 Arg Ser Trp
Asp Glu Gly Val Ala Thr Asn Phe Ser Thr Thr Pro Leu 50 55 60 Lys
Asp Ile Phe His Gly Lys Lys Val Val Ile Phe Gly Leu Pro Gly 65 70
75 80 Ala Tyr Thr Gly Val Cys Ser Gln Ala His Val Pro Ser Tyr Lys
Asn 85 90 95 Asn Ile Asp Lys Leu Lys Ala Lys Gly Val Asp Ser Val
Ile Cys Val 100 105 110 Ser Val Asn Asp Pro Tyr Ala Leu Asn Gly Trp
Ala Glu Lys Leu Gln 115 120 125 Ala Lys Asp Ala Ile Glu Phe Tyr Gly
Asp Phe Asp Gly Ser Phe His 130 135 140 Lys Ser Leu Asp Leu Glu Val
Asp Leu Ser Ala Ala Leu Leu Gly Arg 145 150 155 160 Arg Ser His Arg
Trp Ser Ala Phe Val Asp Asp Gly Lys Ile Lys Ala 165 170 175 Phe Asn
Val Glu Val Ala Pro Ser Asp Phe Lys Val Ser Gly Ala Glu 180 185 190
Val Ile Leu Asp Gln Ile 195 167198 PRTUnknownPRX protein 167Met Ala
Ser Ala Leu Leu Arg Lys Ala Thr Val Gly Gly Ser Ala Ala 1 5 10 15
Ala Ala Ala Ala Arg Trp Ala Ser Arg Gly Leu Ala Ser Val Gly Ser 20
25 30 Gly Ser Asp Ile Val Ser Ala Ala Pro Gly Val Ser Leu Gln Lys
Ala 35 40 45 Arg Ser Trp Asp Glu Gly Val Ala Thr Asn Phe Ser Thr
Thr Pro Leu 50 55 60 Lys Asp Ile Phe His Gly Lys Lys Val Val Ile
Phe Gly Leu Pro Gly 65 70 75 80 Ala Tyr Thr Gly Val Cys Ser Gln Ala
His Val Pro Ser Tyr Lys Asn 85 90 95 Asn Ile Asp Lys Leu Lys Ala
Lys Gly Val Asp Ser Val Ile Cys Val 100 105 110 Ser Val Asn Asp Pro
Tyr Ala Leu Asn Gly Trp Ala Glu Lys Leu Gln 115 120 125 Ala Lys Asp
Ala Ile Glu Phe Tyr Gly Asp Phe Asp Gly Ser Phe His 130 135 140 Lys
Ser Leu Asp Leu Glu Val Asp Leu Ser Ala Ala Leu Leu Gly Arg 145 150
155 160 Arg Ser His Arg Trp Ser Ala Phe Val Asp Asp Gly Lys Ile Lys
Ala 165 170 175 Phe Asn Val Glu Val Ala Pro Ser Asp Phe Lys Val Ser
Gly Ala Glu 180 185 190 Val Ile Leu Asp Gln Ile 195
168220PRTUnknownPRX protein 168Met Pro Gly Leu Thr Ile Gly Asp Thr
Val Pro Asn Leu Glu Leu Asp 1 5 10 15 Ser Thr His Gly Lys Ile Arg
Ile His Asp Phe Val Gly Asp Thr Tyr 20 25 30 Val Ile Leu Phe Ser
His Pro Gly Asp Phe Thr Pro Val Cys Thr Thr 35 40 45 Glu Leu Ala
Ala Met Ala Gly Tyr Ala Lys Glu Phe Asp Lys Arg Gly 50 55 60 Val
Lys Leu Leu Gly Ile Ser Cys Asp Asp Val Gln Ser His Lys Asp 65 70
75 80 Trp Ile Lys Asp Ile Glu Ala Tyr Lys Pro Gly Asn Arg Val Thr
Tyr 85 90 95 Pro Ile Met Ala Asp Pro Ser Arg Glu Ala Ile Lys Gln
Leu Asn Met 100 105 110 Val Asp Pro Asp Glu Lys Asp Ser Asn Gly Gly
His Leu Pro Ser Arg 115 120 125 Ala Leu His Ile Val Gly Pro Asp Lys
Lys Val Lys Leu Ser Phe Leu 130 135 140 Tyr Pro Ala Cys Val Gly Arg
Asn Met Asp Glu Val Val Arg Ala Val 145 150 155 160 Asp Ala Leu Gln
Thr Ala Ala Lys His Ala Val Ala Thr Pro Val Asn 165 170 175 Trp Lys
Pro Gly Glu Arg Val Val Ile Pro Pro Gly Val Ser Asp Asp 180 185 190
Glu Ala Lys Glu Lys Phe Pro Gln Gly Phe Asp Thr Ala Asp Leu Pro 195
200 205 Ser Gly Lys Gly Tyr Leu Arg Phe Thr Lys Val Gly 210 215 220
169220PRTUnknownPRX protein 169Met Pro Gly Leu Thr Leu Gly Asp Val
Val Pro Asp Leu Glu Leu Asp 1 5 10 15 Thr Thr His Gly Lys Ile Arg
Leu His Asp Phe Val Gly Asp Ala Tyr 20 25 30 Val Ile Ile Phe Ser
His Pro Ala Asp Phe Thr Pro Val Cys Thr Thr 35 40 45 Glu Leu Ser
Glu Met Ala Gly Tyr Ala Gly Glu Phe Asp Lys Arg Gly 50 55 60 Val
Lys Leu Leu Gly Phe Ser Cys Asp Asp Val Glu Ser His Lys Asp 65 70
75 80 Trp Ile Lys Asp Ile Glu Ala Tyr Lys Pro Gly Arg Arg Val Gly
Phe 85 90 95 Pro Ile Val Ala Asp Pro Asp Arg Glu Ala Ile Arg Gln
Leu Asn Met 100 105 110 Ile Asp Ala Asp Glu Lys Asp Thr Ala Gly Gly
Glu Leu Pro Asn Arg 115 120 125 Ala Leu His Ile Val Gly Pro Asp Lys
Lys Val Lys Leu Ser Phe Leu 130 135 140 Phe Pro Ala Cys Thr Gly Arg
Asn Met Ala Glu Val Leu Arg Ala Thr 145 150 155 160 Asp Ala Leu Leu
Thr Ala Ala Arg His Arg Val Ala Thr Pro Val Asn 165 170 175 Trp Lys
Pro Gly Glu Arg Val Val Ile Pro Pro Gly Val Ser Asp Glu 180 185 190
Glu Ala Lys Ala Arg Phe Pro Ala Gly Phe Glu Thr Ala Gln Leu Pro 195
200 205 Ser Asn Lys Cys Tyr Leu Arg Phe Thr Gln Val Asp 210 215 220
170198PRTUnknownPRX protein 170Met Ala Ser Gly Asn Ala Gln Ile Gly
Lys Ser Ala Pro Asp Phe Thr 1 5 10 15 Ala Thr Ala Val Val Asp Gly
Ala Phe Lys Glu Ile Lys Leu Ser Asp 20 25 30 Tyr Arg Gly Lys Tyr
Val Val Leu Phe Phe Tyr Pro Leu Asp Phe Thr 35 40 45 Phe Val Cys
Pro Thr Glu Ile Ile Ala Phe Ser Asp His Ala Glu Asp 50 55 60 Phe
Arg Lys Leu Gly Cys Glu Val Leu Gly Val Ser Val Asp Ser Gln 65 70
75 80 Phe Thr His Leu Ala Trp Ile Asn Thr Pro Arg Lys Glu Gly Gly
Leu 85 90 95 Gly Pro Leu Asn Ile Pro Leu Leu Ala Asp Val Thr Lys
Ser Leu Ser 100 105 110 Gln Asn Tyr Gly Val Leu Lys Asn Asp Glu Gly
Ile Ala Tyr Arg Gly 115 120 125 Leu Phe Ile Ile Asp Ala Lys Gly Val
Leu Arg Gln Ile Thr Val Asn 130 135 140 Asp Leu Pro Val Gly Arg Ser
Val Asp Glu Ala Leu Arg Leu Val Gln 145 150 155 160 Ala Phe Gln Tyr
Thr Asp Glu His Gly Glu Val Cys Pro Ala Gly Trp 165 170 175 Lys Pro
Gly Ser Asp Thr Ile Lys Pro Asn Val Asp Asp Ser Lys Glu 180 185 190
Tyr Phe Ser Lys His Asn 195 171271PRTUnknownPRX protein 171Met Ala
Ser Ile Ala Ser Ser Ser Ser Thr Thr Leu Leu Ser Ser Ser 1 5 10 15
Arg Val Leu Leu Pro Ser Lys Ser Ser Leu Leu Ser Pro Thr Val Ser 20
25 30 Phe Pro Arg Ile Ile Pro Ser Ser Ser Ala Ser Ser Ser Ser Leu
Cys 35 40 45 Ser Gly Phe Ser Ser Leu Gly Ser Leu Thr Thr Asn Arg
Ser Ala Ser 50 55 60 Arg Arg Asn Phe Ala Val Lys Ala Gln Ala Asp
Asp Leu Pro Leu Val 65 70 75 80 Gly Asn Lys Ala Pro Asp Phe Glu Ala
Glu Ala Val Phe Asp Gln Glu 85 90 95 Phe Ile Lys Val Lys Leu Ser
Glu Tyr Ile Gly Lys Lys Tyr Val Ile 100 105 110 Leu Phe Phe Tyr Pro
Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile 115 120 125 Thr Ala Phe
Ser Asp Arg Tyr Glu Glu Phe Glu Lys Leu Asn Thr Glu 130 135 140 Val
Leu Gly Val Ser Val Asp Ser Val Phe Ser His Leu Ala Trp Val 145 150
155 160 Gln Thr Asp Arg Lys Ser Gly Gly Leu Gly Asp Leu Asn Tyr Pro
Leu 165 170 175 Val Ser Asp Ile Thr Lys Ser Ile Ser Lys Ser Phe Gly
Val Leu Ile 180 185 190 Pro Asp Gln Gly Ile Ala Leu Arg Gly Leu Phe
Ile Ile Asp Lys Glu 195 200 205 Gly Val Ile Gln His Ser Pro Ile Asn
Asn Leu Gly Ile Gly Arg Ser 210 215 220 Val Asp Glu Thr Met Arg Thr
Leu Gln Ala Leu Gln Tyr Val Gln Glu 225 230 235 240 Asn Pro Asp Glu
Val Cys Pro Ala Gly Trp Lys Pro Gly Glu Lys Ser 245 250 255 Met Lys
Pro Asp Pro Lys Leu Ser Lys Glu Tyr Phe Ser Ala Ile 260 265 270
172154PRTUnknownPRX protein 172Met Ala Cys Ala Phe Ser Val Ser Ser
Ala Ala Ala Pro Leu Ala Ser 1 5 10 15 Pro Lys Gly Asp Leu Pro Leu
Val Gly Asn Lys Ala Pro Asp Phe Glu 20 25 30 Ala Glu Ala Met Phe
Asp Gln Gly Phe Ile Lys Ser Lys Cys Met Phe 35 40 45 Val Ser Ser
Ala Glu Ile Thr Ala Phe Ser Asp Arg Tyr Glu Glu Phe 50 55 60 Glu
Lys Ile Asn Thr Glu Val Leu Gly Val Ser Ile Asp Ser Val Gly 65 70
75 80 Ile Ala Leu Arg Gly Leu Phe Ile Ile Asp Lys Glu Gly Val Ile
Gln 85 90 95 His Ser Thr Ile Asn Asn Leu Ala Ile Gly Arg Ser Val
Asp Glu Thr 100 105 110 Leu Arg Thr Leu Gln Ala Leu Gln Tyr Val Gln
Glu Asn Pro Asp Glu 115 120 125 Val Cys Pro Ala Gly Trp Lys Pro Gly
Glu Lys Ser Met Lys Pro Asp 130 135 140
Pro Lys Asp Ser Lys Glu Glu Gln Glu Cys 145 150 173266PRTUnknownPRX
protein 173Met Ala Ser Val Ala Ser Ser Thr Thr Leu Ile Ser Ser Pro
Ser Ser 1 5 10 15 Arg Val Phe Pro Ala Lys Ser Ser Leu Ser Ser Pro
Ser Val Ser Phe 20 25 30 Leu Arg Thr Leu Ser Ser Pro Ser Ala Ser
Ala Ser Leu Arg Ser Gly 35 40 45 Phe Ala Arg Arg Ser Ser Leu Ser
Ser Thr Ser Arg Arg Ser Phe Ala 50 55 60 Val Lys Ala Gln Ala Asp
Asp Leu Pro Leu Val Gly Asn Lys Ala Pro 65 70 75 80 Asp Phe Glu Ala
Glu Ala Val Phe Asp Gln Glu Phe Ile Lys Val Lys 85 90 95 Leu Ser
Asp Tyr Ile Gly Lys Lys Tyr Val Ile Leu Phe Phe Tyr Pro 100 105 110
Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile Thr Ala Phe Ser Asp 115
120 125 Arg His Ser Glu Phe Glu Lys Leu Asn Thr Glu Val Leu Gly Val
Ser 130 135 140 Val Asp Ser Val Phe Ser His Leu Ala Trp Val Gln Thr
Asp Arg Lys 145 150 155 160 Ser Gly Gly Leu Gly Asp Leu Asn Tyr Pro
Leu Ile Ser Asp Val Thr 165 170 175 Lys Ser Ile Ser Lys Ser Phe Gly
Val Leu Ile His Asp Gln Gly Ile 180 185 190 Ala Leu Arg Gly Leu Phe
Ile Ile Asp Lys Glu Gly Val Ile Gln His 195 200 205 Ser Thr Ile Asn
Asn Leu Gly Ile Gly Arg Ser Val Asp Glu Thr Met 210 215 220 Arg Thr
Leu Gln Ala Leu Gln Tyr Ile Gln Glu Asn Pro Asp Glu Val 225 230 235
240 Cys Pro Ala Gly Trp Lys Pro Gly Glu Lys Ser Met Lys Pro Asp Pro
245 250 255 Lys Leu Ser Lys Glu Tyr Phe Ser Ala Ile 260 265
174265PRTUnknownPRX protein 174Met Ala Cys Val Ala Ser Ser Thr Thr
Leu Ile Ser Ser Pro Ser Ser 1 5 10 15 Arg Val Phe Pro Ala Lys Ser
Ser Leu Ser Ser Pro Ser Val Ser Phe 20 25 30 Leu Arg Thr Leu Ser
Ser Pro Ser Ala Ser Ala Ser Leu Arg Ser Gly 35 40 45 Phe Ala Arg
Arg Ser Ser Leu Ser Ser Thr Ser Arg Arg Ser Phe Ala 50 55 60 Val
Lys Ala Gln Ala Asp Asp Leu Pro Leu Val Gly Asn Lys Ala Pro 65 70
75 80 Asp Phe Glu Ala Glu Ala Val Phe Asp Gln Glu Phe Ile Lys Val
Lys 85 90 95 Leu Ser Asp Tyr Ile Gly Lys Lys Tyr Val Ile Leu Phe
Phe Tyr Pro 100 105 110 Leu Asp Phe Thr Phe Val Cys Pro Thr Glu Ile
Thr Ala Phe Ser Asp 115 120 125 Arg His Ser Glu Phe Glu Lys Leu Asn
Thr Glu Val Leu Gly Val Ser 130 135 140 Val Asp Ser Val Phe Ser His
Leu Ala Trp Val Gln Thr Asp Arg Lys 145 150 155 160 Ser Gly Gly Leu
Gly Asp Leu Asn Tyr Pro Leu Ile Ser Asp Val Thr 165 170 175 Lys Ser
Ile Ser Lys Ser Phe Gly Val Leu Ile His Asp Gln Gly Ile 180 185 190
Ala Leu Arg Gly Leu Phe Ile Ile Asp Lys Glu Gly Val Ile Gln His 195
200 205 Ser Thr Ile Asn Asn Leu Gly Ile Gly Arg Ser Val Asp Glu Thr
Met 210 215 220 Arg Thr Leu Gln Ala Leu Gln Tyr Thr Gly Asn Pro Asp
Glu Val Cys 225 230 235 240 Pro Ala Gly Trp Lys Pro Gly Glu Lys Ser
Met Lys Pro Asp Pro Lys 245 250 255 Leu Ser Lys Glu Tyr Phe Ser Ala
Ile 260 265 175265PRTUnknownPRX protein 175Met Ala Ser Val Ala Ser
Ser Thr Thr Leu Ile Ser Ser Pro Ser Ser 1 5 10 15 Arg Val Phe Pro
Ala Lys Ser Ser Leu Ser Ser Pro Ser Val Ser Phe 20 25 30 Leu Arg
Thr Leu Ser Ser Pro Ser Ala Ser Ala Ser Leu Arg Ser Gly 35 40 45
Phe Ala Arg Arg Ser Ser Leu Ser Ser Thr Ser Arg Arg Ser Phe Ala 50
55 60 Val Lys Ala Gln Ala Asp Asp Leu Pro Leu Val Gly Asn Lys Ala
Pro 65 70 75 80 Asp Phe Lys Ala Glu Ala Val Phe Asp Gln Glu Phe Ile
Lys Val Lys 85 90 95 Leu Ser Asp Tyr Asn Gly Lys Lys Tyr Val Ile
Leu Phe Phe Tyr Pro 100 105 110 Leu Asp Phe Thr Phe Val Cys Pro Thr
Glu Ile Thr Ala Phe Ser Asp 115 120 125 Arg His Ser Glu Phe Glu Lys
Leu Asn Thr Glu Val Leu Gly Val Ser 130 135 140 Val Asp Ser Val Phe
Ser His Leu Ala Trp Val Gln Thr Asp Arg Lys 145 150 155 160 Ser Gly
Gly Leu Gly Asp Leu Asn Tyr Pro Leu Ile Ser Asp Val Thr 165 170 175
Lys Ser Ile Ser Lys Ser Phe Gly Val Leu Ile His Asp Gln Gly Ile 180
185 190 Ala Leu Arg Gly Leu Phe Ile Ile Asp Lys Glu Gly Val Ile Gln
His 195 200 205 Ser Thr Ile Asn Asn Leu Gly Ile Gly Arg Ser Val Asp
Glu Thr Met 210 215 220 Arg Thr Leu Gln Ala Leu Gln Tyr Thr Gly Asn
Pro Asp Glu Val Cys 225 230 235 240 Pro Ala Gly Trp Lys Ser Gly Glu
Lys Ser Met Lys Pro Asp Pro Lys 245 250 255 Leu Ser Lys Glu Tyr Phe
Ser Ala Ile 260 265 176196PRTUnknownPRX protein 176Glu Leu Pro Leu
Val Gly Asn Ser Ala Pro Gly Phe Glu Ala Glu Ala 1 5 10 15 Val Phe
Asp Gln Glu Phe Ile Lys Val Lys Leu Ser Glu Tyr Ile Gly 20 25 30
Lys Lys Tyr Val Ile Leu Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val 35
40 45 Cys Pro Thr Glu Ile Thr Ala Phe Ser Asp Arg Tyr Ser Glu Phe
Glu 50 55 60 Lys Val Asn Thr Glu Val Leu Gly Val Ser Val Asp Ser
Val Phe Ser 65 70 75 80 His Leu Ala Trp Val Gln Thr Asp Arg Lys Ser
Gly Gly Leu Gly Asp 85 90 95 Leu Asn Tyr Pro Leu Val Ser Asp Val
Thr Lys Ser Ile Ser Lys Ser 100 105 110 Tyr Gly Val Leu Ile Pro Asp
Gln Gly Ile Ala Leu Arg Gly Leu Phe 115 120 125 Ile Ile Asp Lys Glu
Gly Val Ile Gln His Ser Thr Ile Asn Asn Leu 130 135 140 Gly Ile Gly
Arg Ser Val Asp Glu Thr Met Arg Thr Leu Gln Ala Leu 145 150 155 160
Gln Tyr Val Gln Glu Asn Pro Asp Glu Val Cys Pro Ala Gly Trp Lys 165
170 175 Pro Gly Glu Lys Ser Met Lys Pro Asp Pro Lys Arg Ser Lys Glu
Tyr 180 185 190 Phe Ala Ser Ile 195 177258PRTUnknownPRX protein
177Met Ala Cys Ala Phe Ser Ala Ser Thr Val Ser Thr Ala Ala Ala Leu
1 5 10 15 Val Ala Ser Pro Lys Pro Ala Gly Ala Pro Ser Ala Cys Arg
Phe Pro 20 25 30 Ala Leu Arg Arg Gly Arg Ala Gly Leu Arg Cys Ala
Arg Leu Glu Asp 35 40 45 Ala Arg Ala Arg Ser Phe Val Ala Arg Ala
Ala Ala Glu Tyr Asp Leu 50 55 60 Pro Leu Val Gly Asn Lys Ala Pro
Asp Phe Ala Ala Glu Ala Val Phe 65 70 75 80 Asp Gln Glu Phe Ile Asn
Val Lys Leu Ser Asp Tyr Ile Gly Lys Lys 85 90 95 Tyr Val Ile Leu
Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val Cys Pro 100 105 110 Thr Glu
Ile Thr Ala Phe Ser Asp Arg His Glu Glu Phe Glu Lys Ile 115 120 125
Asn Thr Glu Ile Leu Gly Val Ser Val Asp Ser Val Phe Ser His Leu 130
135 140 Ala Trp Val Gln Thr Glu Arg Lys Ser Gly Gly Leu Gly Asp Leu
Lys 145 150 155 160 Tyr Pro Leu Val Ser Asp Val Thr Lys Ser Ile Ser
Lys Ser Phe Gly 165 170 175 Val Leu Ile Pro Asp Gln Gly Ile Ala Leu
Arg Gly Leu Phe Met Ile 180 185 190 Asp Lys Glu Gly Val Ile Gln His
Ser Thr Ile Asn Asn Leu Gly Ile 195 200 205 Gly Arg Ser Val Asp Glu
Thr Leu Arg Thr Leu Gln Ala Leu Gln Tyr 210 215 220 Val Gln Glu Asn
Pro Asp Glu Val Cys Pro Ala Gly Trp Lys Pro Gly 225 230 235 240 Glu
Lys Ser Met Lys Pro Asp Pro Lys Gly Ser Lys Glu Tyr Phe Ala 245 250
255 Ala Ile 178210PRTUnknownPRX protein 178Asp Ala Arg Ala Arg Ser
Phe Val Ala Arg Ala Ala Ala Glu Tyr Asp 1 5 10 15 Leu Pro Leu Val
Gly Asn Lys Ala Pro Asp Phe Ala Ala Glu Ala Val 20 25 30 Phe Asp
Gln Glu Phe Ile Asn Val Lys Leu Ser Asp Tyr Ile Gly Lys 35 40 45
Lys Tyr Val Ile Leu Phe Phe Tyr Pro Leu Asp Phe Thr Phe Val Cys 50
55 60 Pro Thr Glu Ile Thr Ala Phe Ser Asp Arg His Glu Glu Phe Glu
Lys 65 70 75 80 Ile Asn Thr Glu Ile Leu Gly Val Ser Val Asp Ser Val
Phe Ser His 85 90 95 Leu Ala Trp Val Gln Thr Glu Arg Lys Ser Gly
Gly Leu Gly Asp Leu 100 105 110 Lys Tyr Pro Leu Val Ser Asp Val Thr
Lys Ser Ile Ser Lys Ser Phe 115 120 125 Gly Val Leu Ile Pro Asp Gln
Gly Ile Ala Leu Arg Gly Leu Phe Ile 130 135 140 Ile Asp Lys Glu Gly
Val Ile Gln His Ser Thr Ile Asn Asn Leu Gly 145 150 155 160 Ile Gly
Arg Ser Val Asp Glu Thr Leu Arg Thr Leu Gln Ala Leu Gln 165 170 175
Tyr Val Lys Lys Pro Asp Glu Val Cys Pro Ala Gly Trp Lys Pro Gly 180
185 190 Glu Lys Ser Met Lys Pro Asp Pro Lys Gly Ser Lys Glu Tyr Phe
Ala 195 200 205 Ala Ile 210 179210PRTUnknownPRX protein 179Asp Ala
Arg Ala Arg Ser Phe Val Ala Arg Ala Ala Ala Glu Tyr Asp 1 5 10 15
Leu Pro Leu Val Gly Asn Lys Ala Pro Asp Phe Ala Ala Glu Ala Val 20
25 30 Phe Asp Gln Glu Phe Ile Asn Val Lys Leu Ser Asp Tyr Ile Gly
Lys 35 40 45 Lys Tyr Val Ile Leu Phe Phe Tyr Pro Leu Asp Phe Thr
Phe Val Cys 50 55 60 Pro Thr Glu Ile Thr Ala Phe Ser Asp Arg His
Glu Glu Phe Glu Lys 65 70 75 80 Ile Asn Thr Glu Ile Leu Gly Val Ser
Val Asp Ser Val Phe Ser His 85 90 95 Leu Ala Trp Val Gln Thr Glu
Arg Lys Ser Gly Gly Leu Gly Asp Leu 100 105 110 Lys Tyr Pro Leu Val
Ser Asp Val Thr Lys Ser Ile Ser Lys Ser Phe 115 120 125 Gly Val Leu
Ile Pro Asp Gln Gly Ile Ala Leu Arg Gly Leu Phe Ile 130 135 140 Ile
Asp Lys Glu Gly Val Ile Gln His Ser Thr Ile Asn Asn Leu Gly 145 150
155 160 Ile Gly Arg Ser Val Asp Glu Thr Leu Arg Thr Leu Arg Ala Leu
Gln 165 170 175 Tyr Val Lys Lys Pro Asp Glu Val Cys Pro Ala Gly Trp
Lys Pro Gly 180 185 190 Glu Lys Ser Met Lys Pro Asp Pro Lys Gly Ser
Lys Glu Tyr Phe Ala 195 200 205 Ala Ile 210 180242PRTUnknownPRX
protein 180Asp Ala Arg Ala Arg Ser Phe Val Ala Arg Ala Ala Ala Glu
Tyr Asp 1 5 10 15 Leu Pro Leu Val Gly Asn Lys Ala Pro Asp Phe Ala
Ala Glu Ala Val 20 25 30 Phe Asp Gln Glu Phe Ile Asn Val Lys Leu
Ser Asp Tyr Ile Gly Lys 35 40 45 Lys Tyr Val Ile Leu Phe Phe Tyr
Pro Leu Asp Phe Thr Phe Val Cys 50 55 60 Pro Thr Glu Ile Thr Ala
Phe Ser Asp Arg His Glu Glu Phe Glu Lys 65 70 75 80 Ile Asn Thr Glu
Ile Leu Gly Val Ser Val Asp Ser Val Phe Ser His 85 90 95 Leu Ala
Trp Val Gln Thr Glu Arg Lys Ser Gly Gly Leu Gly Asp Leu 100 105 110
Lys Tyr Pro Leu Val Ser Asp Val Thr Lys Ser Ile Ser Lys Ser Phe 115
120 125 Gly Val Leu Ile Pro Asp Gln Gly Ile Ala Leu Arg Gly Leu Phe
Ile 130 135 140 Ile Asp Lys Glu Gly Val Ile Gln His Ser Thr Ile Asn
Asn Leu Gly 145 150 155 160 Ile Gly Arg Ser Val Asp Glu Thr Leu Arg
Thr Leu Gln Ala Leu Gln 165 170 175 Tyr Val Lys Lys Pro Asp Glu Val
Cys Pro Ala Gly Trp Lys Pro Gly 180 185 190 Glu Lys Ser Met Lys Pro
Asp Leu Gly Pro Lys Arg Ser Thr Arg Cys 195 200 205 Tyr Leu Glu Arg
Thr Phe Ala Leu Ser Cys Gly Val Leu Ser Trp Pro 210 215 220 Phe Leu
Gly Tyr Met Cys Phe Cys Asp Pro Ser Cys Ser Tyr His Ala 225 230 235
240 Tyr Asn 181261PRTUnknownPRX protein 181Met Ala Ala Cys Cys Ser
Ser Leu Ala Thr Ala Val Ser Ser Ser Ser 1 5 10 15 Ala Lys Pro Leu
Ala Gly Ile Pro Pro Ala Ala Pro His Ser Leu Ser 20 25 30 Leu Pro
Arg Ala Pro Ala Ala Arg Pro Leu Arg Leu Ser Ala Ser Ser 35 40 45
Ser Arg Ser Ala Arg Ala Ser Ser Phe Val Ala Arg Ala Gly Gly Val 50
55 60 Asp Asp Ala Pro Leu Val Gly Asn Lys Ala Pro Asp Phe Asp Ala
Glu 65 70 75 80 Ala Val Phe Asp Gln Glu Phe Ile Asn Val Lys Leu Ser
Asp Tyr Ile 85 90 95 Gly Lys Lys Tyr Val Ile Leu Phe Phe Tyr Pro
Leu Asp Phe Thr Phe 100 105 110 Val Cys Pro Thr Glu Ile Thr Ala Phe
Ser Asp Arg Tyr Asp Glu Phe 115 120 125 Glu Lys Leu Asn Thr Glu Ile
Leu Gly Val Ser Ile Asp Ser Val Phe 130 135 140 Ser His Leu Ala Trp
Val Gln Thr Asp Arg Lys Ser Gly Gly Leu Gly 145 150 155 160 Asp Leu
Lys Tyr Pro Leu Ile Ser Asp Val Thr Lys Ser Ile Ser Lys 165 170 175
Ser Phe Gly Val Leu Ile Pro Asp Gln Gly Ile Ala Leu Arg Gly Leu 180
185 190 Phe Ile Ile Asp Lys Glu Gly Val Ile Gln His Ser Thr Ile Asn
Asn 195 200 205 Leu Ala Ile Gly Arg Ser Val Asp Glu Thr Met Arg Thr
Leu Gln Ala 210 215 220 Leu Gln Tyr Val Gln Asp Asn Pro Asp Glu Val
Cys Pro Ala Gly Trp 225 230 235 240 Lys Pro Gly Asp Lys Ser Met Lys
Pro Asp Pro Lys Gly Ser Lys Glu 245 250 255 Tyr Phe Ala Ala Ile 260
182276PRTUnknownPRX protein 182Met Ala Ala Cys Cys Ser Ser Leu Ala
Thr Ala Val Ser Ser Ser Ser 1 5 10 15 Ala Lys Pro Leu Ala Gly Ile
Pro Pro Ala Ser Pro His Ser Leu Ser 20 25 30 Leu Pro Arg Ser Pro
Ala Ala Ala Ala Arg Pro Leu Arg Leu Ser Ala 35 40 45 Ser Ser Ser
Arg Ser Ala Arg Ala Ser Ser Phe Val Ala Arg Ala Gly 50 55 60 Gly
Val Asp Asp Ala Pro Leu Val Gly Asn Lys Ala Pro Asp Phe Asp 65 70
75 80 Ala Glu Ala Val Phe Asp Gln Glu Phe
Ile Asn Val Lys Leu Ser Asp 85 90 95 Tyr Ile Gly Lys Lys Tyr Val
Ile Leu Phe Phe Tyr Pro Leu Asp Phe 100 105 110 Thr Phe Val Cys Pro
Thr Glu Ile Thr Ala Phe Ser Asp Arg Tyr Asp 115 120 125 Glu Phe Glu
Lys Leu Asn Thr Glu Ile Leu Gly Val Ser Ile Asp Ser 130 135 140 Val
Phe Ser His Leu Ala Trp Val Gln Thr Asp Arg Lys Ser Gly Gly 145 150
155 160 Leu Gly Asp Leu Lys Tyr Pro Leu Ile Ser Asp Val Thr Lys Ser
Ile 165 170 175 Ser Lys Ser Phe Gly Val Leu Ile Pro Asp Gln Gly Ile
Ala Leu Arg 180 185 190 Gly Leu Phe Ile Ile Asp Lys Glu Gly Val Ile
Gln His Ser Thr Ile 195 200 205 Asn Asn Leu Ala Ile Gly Arg Ser Val
Asp Glu Thr Met Arg Thr Leu 210 215 220 Gln Ala Ser Ser Leu Glu Tyr
Thr Leu Leu Ser Ala His Thr Ala Leu 225 230 235 240 Gln Tyr Val Gln
Asp Asn Pro Asp Glu Val Cys Pro Ala Gly Trp Lys 245 250 255 Pro Gly
Asp Lys Ser Met Lys Pro Asp Pro Lys Gly Ser Lys Glu Tyr 260 265 270
Phe Ala Ala Ile 275 183199PRTUnknownPRX protein 183Arg Ala Ser His
Ala Glu Lys Pro Leu Val Gly Ser Val Ala Pro Asp 1 5 10 15 Phe Lys
Ala Gln Ala Val Phe Asp Gln Glu Phe Gln Glu Ile Thr Leu 20 25 30
Ser Lys Tyr Arg Gly Lys Tyr Val Val Leu Phe Phe Tyr Pro Leu Asp 35
40 45 Phe Thr Phe Val Cys Pro Thr Glu Ile Thr Ala Phe Ser Asp Arg
Tyr 50 55 60 Lys Glu Phe Lys Asp Ile Asn Thr Glu Val Leu Gly Val
Ser Val Asp 65 70 75 80 Ser Gln Phe Thr His Leu Ala Trp Ile Gln Thr
Asp Arg Lys Glu Gly 85 90 95 Gly Leu Gly Asp Leu Ala Tyr Pro Leu
Val Ala Asp Leu Lys Lys Glu 100 105 110 Ile Ser Lys Ala Tyr Gly Val
Leu Thr Glu Asp Gly Ile Ser Leu Arg 115 120 125 Gly Leu Phe Ile Ile
Asp Lys Glu Gly Val Val Gln His Ala Thr Ile 130 135 140 Asn Asn Leu
Ala Phe Gly Arg Ser Val Asp Glu Thr Lys Arg Val Leu 145 150 155 160
Gln Ala Ile Gln Tyr Val Gln Ser Asn Pro Asp Glu Val Cys Pro Ala 165
170 175 Gly Trp Lys Pro Gly Asp Lys Thr Met Lys Pro Asp Pro Lys Gly
Ser 180 185 190 Lys Glu Tyr Phe Ser Ala Val 195 184259PRTUnknownPRX
protein 184Met Glu Thr Val Ala Ser Leu Ser Arg Ala Ala Leu Ala Gly
Ala Pro 1 5 10 15 Ala Ala Thr Arg Ala Thr Ala Ser Pro Val Asn Arg
Ala Val Val Pro 20 25 30 Ala Ala Ser Arg Pro Arg Gly Gly Arg Leu
Cys Cys Arg Arg Ser Leu 35 40 45 Thr Ala Val Ser Ala Ala Ala Gly
Ala Ser Pro Pro Val Ser Pro Ser 50 55 60 Pro Ser Pro Asp Gly Gly
Ser Pro Gly Val Trp Asp Ala Leu Gly Gly 65 70 75 80 Val Ser Val Leu
Ala Ala Gly Thr Gly Glu Ala Val Gln Leu Arg Asp 85 90 95 Leu Trp
Asp Pro Thr Glu Gly Val Ala Val Val Ala Leu Leu Arg His 100 105 110
Phe Gly Cys Phe Cys Cys Trp Glu Leu Ala Ser Val Leu Lys Glu Ser 115
120 125 Met Ala Lys Phe Asp Ala Ala Gly Ala Lys Leu Ile Ala Ile Gly
Val 130 135 140 Gly Thr Pro Asp Lys Ala Arg Ile Leu Ala Asp Gly Leu
Pro Phe Pro 145 150 155 160 Val Asp Ser Leu Tyr Ala Asp Pro Glu Arg
Lys Ala Tyr Asp Val Leu 165 170 175 Gly Leu Tyr His Gly Leu Gly Arg
Thr Leu Ile Ser Pro Ala Lys Met 180 185 190 Tyr Ser Gly Leu Asn Ser
Ile Lys Lys Val Thr Lys Asn Tyr Thr Leu 195 200 205 Lys Gly Thr Pro
Ala Asp Leu Thr Gly Ile Leu Gln Gln Gly Gly Met 210 215 220 Leu Val
Phe Arg Gly Lys Glu Leu Leu Tyr Ser Trp Lys Asp Lys Gly 225 230 235
240 Thr Gly Asp His Ala Pro Leu Asp Asp Val Leu Asn Ala Cys Cys Asn
245 250 255 Arg Thr Ser 185258PRTUnknownPRX protein 185Met Ala Ala
Ala Ala Ala Ser Thr Ser Leu Pro Val Pro Arg Val Ser 1 5 10 15 Leu
Pro Pro Ser Ala Arg Pro Ala Ala Ala Pro Arg His Gly Leu Leu 20 25
30 Ile Pro Gly Arg Arg Gly Cys Phe Arg Leu Arg Gly Ser Pro Ala Ala
35 40 45 Pro Ala Ala Ala Ala Ser Gly Ser Pro Ser Val Pro Ser Ser
Ser Pro 50 55 60 Glu Ala Gly Ser Gly Ile Gly Asp Ala Leu Gly Gly
Val Ala Ile Tyr 65 70 75 80 Ser Ala Ala Thr Gly Glu Pro Val Leu Phe
Arg Asp Leu Trp Asp Gln 85 90 95 Asn Glu Gly Met Ala Val Val Ala
Leu Leu Arg His Phe Gly Cys Pro 100 105 110 Cys Cys Trp Glu Leu Ala
Ser Val Leu Arg Asp Thr Lys Glu Arg Phe 115 120 125 Asp Ser Ala Gly
Val Lys Leu Ile Ala Val Gly Val Gly Thr Pro Asp 130 135 140 Lys Ala
Arg Ile Leu Ala Glu Arg Leu Pro Phe Pro Leu Asp Tyr Leu 145 150 155
160 Tyr Ala Asp Pro Glu Arg Lys Ala Tyr Asp Leu Leu Gly Leu Tyr Phe
165 170 175 Gly Ile Gly Arg Thr Phe Phe Asn Pro Ala Ser Ala Ser Val
Phe Ser 180 185 190 Arg Phe Asp Ser Leu Lys Glu Ala Val Lys Asn Tyr
Thr Ile Glu Ala 195 200 205 Thr Pro Asp Asp Arg Ala Ser Val Leu Gln
Gln Gly Gly Met Phe Val 210 215 220 Phe Arg Gly Lys Glu Leu Ile Tyr
Ala Arg Lys Asp Glu Gly Thr Gly 225 230 235 240 Asp His Ala Pro Leu
Asp Asp Val Leu Asn Ile Cys Cys Lys Ala Pro 245 250 255 Ala Ala
186217PRTUnknownPRX protein 186Met Ala Phe Ala Val Ser Thr Ala Cys
Arg Pro Ser Leu Leu Leu Pro 1 5 10 15 Pro Arg Gln Arg Ser Ser Pro
Pro Arg Pro Arg Pro Leu Leu Cys Thr 20 25 30 Pro Ser Thr Ala Ala
Phe Arg Arg Gly Ala Leu Ser Ala Thr Thr Thr 35 40 45 Pro Thr Pro
Ala Arg Ala Ala Leu Pro Ser Thr Thr Gly Arg Asn Arg 50 55 60 Ile
Val Cys Gly Lys Val Ser Lys Gly Ser Ala Ala Pro Asn Phe Thr 65 70
75 80 Leu Arg Asp Gln Asp Gly Arg Ala Val Ser Leu Ser Lys Phe Lys
Gly 85 90 95 Arg Pro Val Val Val Tyr Phe Tyr Pro Ala Asp Glu Thr
Pro Gly Cys 100 105 110 Thr Lys Gln Ala Cys Ala Phe Arg Asp Ser Tyr
Glu Lys Phe Lys Lys 115 120 125 Ala Gly Ala Glu Val Ile Gly Ile Ser
Gly Asp Asp Ala Ala Ser His 130 135 140 Lys Glu Phe Lys Lys Lys Tyr
Lys Leu Pro Phe Thr Leu Leu Ser Asp 145 150 155 160 Glu Gly Asn Lys
Val Arg Lys Glu Trp Gly Val Pro Ala Asp Leu Phe 165 170 175 Gly Thr
Leu Pro Gly Arg Gln Thr Tyr Val Leu Asp Lys Asn Gly Val 180 185 190
Val Gln Tyr Ile Tyr Asn Asn Gln Phe Gln Pro Glu Lys His Ile Gly 195
200 205 Glu Thr Leu Lys Ile Leu Gln Ser Leu 210 215
187251PRTUnknownPRX protein 187Met Ala Ala Arg Ala Pro Leu Pro Val
Pro His Ala Ala Ala Thr Ser 1 5 10 15 Pro Arg Pro Ala Ala Ala Ser
Ser Leu Leu Arg Ala Arg Gly Pro Cys 20 25 30 Ala Ser Leu Leu Tyr
Pro Arg Arg Leu Arg Phe Ser Val Ala Pro Val 35 40 45 Ala Ala Ala
Lys Pro Glu Ala Val Gly Arg Ala Gly Glu Ala Ala Ala 50 55 60 Ala
Pro Val Glu Gly Leu Ala Lys Ser Leu Gln Gly Val Glu Val Phe 65 70
75 80 Asp Leu Ser Gly Lys Ala Val Pro Val Val Asp Leu Trp Lys Asp
Arg 85 90 95 Lys Ala Ile Val Ala Phe Ala Arg His Phe Gly Cys Val
Leu Cys Arg 100 105 110 Lys Arg Ala Asp Leu Leu Ala Ala Lys Gln Asp
Ala Met Glu Ala Ala 115 120 125 Gly Val Ala Leu Val Leu Ile Gly Pro
Gly Thr Val Glu Gln Ala Lys 130 135 140 Ala Phe Tyr Asp Gln Thr Lys
Phe Lys Gly Glu Val Tyr Ala Asp Pro 145 150 155 160 Ser His Ser Ser
Tyr Asn Ala Leu Glu Phe Ala Phe Gly Leu Phe Ser 165 170 175 Thr Phe
Thr Pro Ser Ala Gly Leu Lys Ile Ile Gln Leu Tyr Met Glu 180 185 190
Gly Tyr Arg Gln Asp Trp Glu Leu Ser Phe Glu Lys Thr Thr Arg Thr 195
200 205 Lys Gly Gly Trp Tyr Gln Gly Gly Leu Leu Val Ala Gly Pro Gly
Ile 210 215 220 Asp Asn Ile Leu Tyr Ile His Lys Asp Lys Glu Ala Gly
Asp Asp Pro 225 230 235 240 Asp Met Asp Asp Val Leu Lys Ala Cys Cys
Ser 245 250 188252PRTUnknownPRX protein 188Met Ser Leu Ala Thr Ala
Ala Ala Gly Ala Gln Pro Phe Val Arg Ser 1 5 10 15 Ser Ser Ser Ala
Ala Ala Ala Ser Ser Ser Arg Pro Leu Leu Ala Val 20 25 30 Ala Ala
Ala Arg His Arg Arg Pro His Gly Ser Leu Ala Ala Ala Ala 35 40 45
Ala Ala Ala Arg Arg Arg Arg Arg Arg Pro Leu Leu Gln Val Arg Ala 50
55 60 Ala Arg Thr Glu Ser Thr Gly Val Ser Val Gly Phe Arg Ala Pro
Gln 65 70 75 80 Phe Glu Leu Pro Glu Pro Leu Thr Gly Lys Leu Trp Thr
Leu Asp Asp 85 90 95 Phe Glu Gly Asn Pro Ala Leu Leu Val Met Phe
Val Cys Asn His Cys 100 105 110 Pro Phe Val Lys His Leu Lys Lys Asp
Ile Ala Lys Leu Thr Ser Phe 115 120 125 Tyr Met Glu Lys Gly Leu Ala
Ala Val Ala Ile Ser Ser Asn Ser Ile 130 135 140 Val Thr His Pro Gln
Asp Gly Pro Asp Tyr Ile Ala Glu Glu Ala Lys 145 150 155 160 Leu Tyr
Lys Tyr Ser Phe Pro Tyr Leu Tyr Asp Glu Ser Gln Glu Val 165 170 175
Ala Lys Ala Phe Arg Ala Val Cys Thr Pro Glu Phe Tyr Leu Phe Lys 180
185 190 Lys Asp Gly Arg Arg Pro Phe Glu Leu Phe Tyr His Gly Gln Phe
Asp 195 200 205 Asp Ser Arg Pro Ser Asn Asn Val Pro Val Thr Gly Arg
Asp Leu Ser 210 215 220 Arg Ala Ile Asp Cys Ala Leu Ser Gly Gln Glu
Leu Pro Phe Val Pro 225 230 235 240 Lys Pro Ser Val Gly Cys Ser Ile
Lys Trp His Pro 245 250
* * * * *