U.S. patent application number 10/112887 was filed with the patent office on 2003-10-02 for genes for modifying plant traits xi.
Invention is credited to Heard, Jacqueline.
Application Number | 20030188330 10/112887 |
Document ID | / |
Family ID | 28453448 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030188330 |
Kind Code |
A1 |
Heard, Jacqueline |
October 2, 2003 |
Genes for modifying plant traits xi
Abstract
The invention relates to plant transcription factor
polypeptides, polynucleotides that encode them, homologs and
orthologs from a variety of plant species, and methods of using the
polynucleotides and polypeptides to produce transgenic plants
having advantageous properties compared to a reference plant.
Sequence information related to these polynucleotides and
polypeptides can also be used in bioinformatic search methods and
is also disclosed.
Inventors: |
Heard, Jacqueline; (San
Mateo, CA) |
Correspondence
Address: |
MATTHEW KASER
Mendel Biotechnology, Inc.
21375 Cabot Blvd.
Hayward
CA
94545
US
|
Family ID: |
28453448 |
Appl. No.: |
10/112887 |
Filed: |
March 18, 2002 |
Current U.S.
Class: |
800/278 ;
530/370; 536/23.6 |
Current CPC
Class: |
C12N 15/8267 20130101;
C12N 15/8214 20130101; C12N 15/8247 20130101; C12N 15/8282
20130101; C07K 14/415 20130101; C12N 15/8273 20130101; C12N 15/8261
20130101; Y02A 40/146 20180101; C12N 15/8251 20130101; C12N 15/8271
20130101; C12N 15/8216 20130101 |
Class at
Publication: |
800/278 ;
536/23.6; 530/370 |
International
Class: |
A01H 001/00; C12N
015/82; C07H 021/04; C07K 014/415 |
Claims
What is claimed is:
1. A transgenic plant comprising a recombinant polynucleotide
having a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence encoding a polypeptide comprising a
sequence selected from those of SEQ ID NOs:2, 4, or a complementary
nucleotide sequence thereof; (b) a nucleotide sequence encoding a
polypeptide comprising a conservatively substituted variant of a
polypeptide of (a); (c) one of SEQ ID NOs: 1, 3, or a complementary
nucleotide sequence thereof; (d) a nucleotide sequence comprising
silent substitutions in a nucleotide sequence of (c); (e) a
nucleotide sequence which hybridizes under stringent conditions to
a nucleotide sequence of one or more of: (a), (b), (c), or (d); (f)
a nucleotide sequence comprising at least 15 consecutive
nucleotides outside of a conserved domain of any of (a)-(e); (g) a
nucleotide sequence comprising a subsequence or fragment of any of
(a)-(f), which subsequence or fragment encodes a polypeptide that
modifies a plant's trait; (h) a nucleotide sequence having at least
31% sequence identity to a nucleotide sequence of any of (a)-(g);
(i) a nucleotide sequence having at least 60% sequence identity to
a nucleotide sequence of any of (a)-(g); (j) a nucleotide sequence
having at least 95% sequence identity to a nucleotide sequence of
any of (a)-(g); (k) a nucleotide sequence which encodes a
polypeptide having at least 31% sequence identity outside of a
conserved domain of a polypeptide of SEQ ID NOs:2 or 4; (l) a
nucleotide sequence which encodes a polypeptide having at least 60%
sequence identity outside of a conserved domain of a polypeptide of
SEQ ID NOs:2 or 4; (m) a nucleotide sequence which encodes a
polypeptide having at least 75% sequence identity outside of a
conserved domain of a polypeptide of SEQ ID NOs:2 or 4; (n) a
nucleotide sequence which encodes a polypeptide having at least 95%
sequence identity outside of a conserved domain of a polypeptide of
SEQ ID NOs:2 or 4; (o) a nucleotide sequence encoding a polypeptide
having an amino acid domain with at least 86% sequence identity to
a conserved domain of a polypeptide of SEQ ID NOs:2 or 4; (p) a
nucleotide sequence encoding a polypeptide having an amino acid
domain with at least about 90% sequence identity to a conserved
domain of a polypeptide of SEQ ID NOs:2 or 4; (q) a nucleotide
sequence encoding a polypeptide having an amino acid domain with at
least about 95% sequence identity to a conserved domain of a
polypeptide of SEQ ID NOs:2 or 4; (r) a nucleotide sequence
encoding a polypeptide having an amino acid domain with at least
about 98% sequence identity to a conserved domain of a polypeptide
of SEQ ID NOs:2 or 4; (s) a nucleotide sequence which encodes a
polypeptide having at least 31% sequence identity over the entire
length of a polypeptide of SEQ ID NOs:2 or 4; (t) a nucleotide
sequence which encodes a polypeptide having at least 60% sequence
identity over the entire length of a polypeptide of SEQ ID NOs:2 or
4; (u) a nucleotide sequence which encodes a polypeptide having at
least 75% sequence identity over the entire length of a polypeptide
of SEQ ID NOs:2 or 4; and (v) a nucleotide sequence which encodes a
polypeptide having at least 95% sequence identity over the entire
length of a polypeptide of SEQ ID NOs:2 or 4; wherein the plant
possesses an altered trait as compared to a wild type plant, or the
plant exhibits an altered phenotype as compared to a wild type
plant, or the plant expresses an altered level of one or more genes
associated with a plant trait as compared to a wild type plant.
2. The transgenic plant of claim 1, further comprising a
constitutive, inducible, or tissue-specific promoter operably
linked to said nucleotide sequence.
3. The transgenic plant of claim 1, wherein the plant is selected
from the group consisting of: soybean, wheat, corn, potato, cotton,
rice, oilseed rape, sunflower, alfalfa, sugarcane, turf, banana,
blackberry, blueberry, strawberry, raspberry, cantaloupe, carrot,
cauliflower, coffee, cucumber, eggplant, grapes, honeydew, lettuce,
mango, melon, onion, papaya, peas, peppers, pineapple, pumpkin,
spinach, squash, sweet corn, tobacco, tomato, watermelon, mint and
other labiates, rosaceous fruits, and vegetable brassicas.
4. An isolated or recombinant polynucleotide comprising a
nucleotide sequence selected from the group consisting of: (a) a
nucleotide sequence encoding a polypeptide comprising a sequence
selected from those of SEQ ID NOs:2, 4, or a complementary
nucleotide sequence thereof; (b) a nucleotide sequence encoding a
polypeptide comprising a conservatively substituted variant of a
polypeptide of (a); (c) one of SEQ ID NOs: 1, 3, or a complementary
nucleotide sequence thereof; (d) a nucleotide sequence comprising
silent substitutions in a nucleotide sequence of (c); (e) a
nucleotide sequence which hybridizes under stringent conditions to
a nucleotide sequence of one or more of: (a), (b), (c), or (d); (f)
a nucleotide sequence comprising at least 15 consecutive
nucleotides outside of a conserved domain of any of (a)-(e); (g) a
nucleotide sequence comprising a subsequence or fragment of any of
(a)-(f), which subsequence or fragment encodes a polypeptide that
modifies a plant's trait; (h) a nucleotide sequence having at least
31% sequence identity to a nucleotide sequence of any of (a)-(g);
(i) a nucleotide sequence having at least 60% sequence identity to
a nucleotide sequence of any of (a)-(g); (j) a nucleotide sequence
having at least 95% sequence identity to a nucleotide sequence of
any of (a)-(g); (k) a nucleotide sequence which encodes a
polypeptide having at least 31% sequence identity outside of a
conserved domain of a polypeptide of SEQ ID NOs:2 or 4; (l) a
nucleotide sequence which encodes a polypeptide having at least 60%
sequence identity outside of a conserved domain of a polypeptide of
SEQ ID NOs:2 or 4; (m) a nucleotide sequence which encodes a
polypeptide having at least 75% sequence identity outside of a
conserved domain of a polypeptide of SEQ ID NOs:2 or 4; (n) a
nucleotide sequence which encodes a polypeptide having at least 95%
sequence identity outside of a conserved domain of a polypeptide of
SEQ ID NOs:2 or 4; (o) a nucleotide sequence encoding a polypeptide
having an amino acid domain with at least 86% sequence identity to
a conserved domain of a polypeptide of SEQ ID NOs:2 or 4; (p) a
nucleotide sequence encoding a polypeptide having an amino acid
domain with at least about 90% sequence identity to a conserved
domain of a polypeptide of SEQ ID NOs:2 or 4; (q) a nucleotide
sequence encoding a polypeptide having an amino acid domain with at
least about 95% sequence identity to a conserved domain of a
polypeptide of SEQ ID NOs:2 or 4; (r) a nucleotide sequence
encoding a polypeptide having an amino acid domain with at least
about 98% sequence identity to a conserved domain of a polypeptide
of SEQ ID NOs:2 or 4; (s) a nucleotide sequence which encodes a
polypeptide having at least 31% sequence identity over the entire
length of a polypeptide of SEQ ID NOs:2 or 4; (t) a nucleotide
sequence which encodes a polypeptide having at least 60% sequence
identity over the entire length of a polypeptide of SEQ ID NOs:2 or
4; (u) a nucleotide sequence which encodes a polypeptide having at
least 75% sequence identity over the entire length of a polypeptide
of SEQ ID NOs:2 or 4; and (v) a nucleotide sequence which encodes a
polypeptide having at least 95% sequence identity over the entire
length of a polypeptide of SEQ ID NOs:2 or 4.
5. The isolated or recombinant polynucleotide of claim 4, further
comprising a constitutive, inducible, or tissue-specific promoter
operably linked to the nucleotide sequence.
6. An isolated or recombinant polypeptide comprising a subsequence
of at least about 15 contiguous amino acids encoded by the
recombinant or isolated polynucleotide of claim 4.
7. A method of using the isolated or recombinant polynucleotide of
claim 4 for producing a plant having a modified trait, the method
comprising selecting a polynucleotide that encodes a polypeptide,
inserting the polynucleotide into an expression vector, introducing
the vector into a plant or a cell of a plant to overexpress the
polypeptide, thereby producing a modified plant, and selecting for
a modified trait.
8. The transgenic plant of claim 1, wherein the trait is selected
from the group consisting of: enhanced tolerance to freezing,
chilling, heat, drought, water saturation, radiation and ozone;
improved tolerance to microbial, fungal or viral diseases; improved
tolerance to pest infestations, decreased herbicide sensitivity;
improved tolerance of heavy metals or enhanced ability to take up
heavy metals; improved growth under poor photoconditions.
9. The transgenic plant of claim 1, wherein the trait is an
alteration in the level of one or more of the compounds selected
from the group consisting of: taxol, tocopherol, tocotrienol,
sterols, phytosterols, vitamins, wax monomers, anti-oxidants, amino
acids, lignins, cellulose, tannins, prenyllipids, glucosinolates,
and terpenoids.
10. The transgenic plant of claim 1, wherein the trait is an
alteration in one or more physical characteristics selected from
the group consisting of: the number of trichomes, fruit and seed
size and number, yield of stems, leaves, or roots, stability of
seeds during storage, susceptibility of the seed to shattering,
root hair length and quantity, internode distances, or the quality
of seed coat.
11. The transgenic plant of claim 1, wherein the trait is an
alteration in a plant growth characteristic selected from the group
consisting of: growth rate, germination rate of seeds, vigor of
plants and seedlings, leaf and flower senescence, male sterility,
apomixis, flowering time, flower abscission, rate of nitrogen
uptake, biomass or transpiration characteristics, apical dominance,
branching patterns, number of organs, organ identity, and organ
shape or size.
12. The transgenic plant of claim 1, wherein the trait is an
alteration in one or more characteristics selected from the group
consisting of protein or oil production, seed protein or oil
production, insoluble sugar level, soluble sugar level, and starch
composition.
13. The method of claim 7, wherein the trait is selected from the
group consisting of: enhanced tolerance to freezing, chilling,
heat, drought, water saturation, radiation and ozone; improved
tolerance to microbial, fungal or viral diseases; improved
tolerance to pest infestations, decreased herbicide sensitivity;
improved tolerance of heavy metals or enhanced ability to take up
heavy metals, improved growth under poor photoconditions.
14. The method of claim 7, wherein the trait is an alteration in
the level of one or more of the compounds selected from the group
consisting of: taxol, tocopherol, tocotrienol, sterols,
phytosterols, vitamins, wax monomers, anti-oxidants, amino acids,
lignins, cellulose, tannins, prenyllipids, glucosinolates, and
terpenoids.
15. The method of claim 7, wherein the trait is an alteration in
one or more physical characteristics selected from the group
consisting of: the number of trichomes, fruit and seed size and
number, yield of stems, leaves, or roots, stability of seeds during
storage, susceptibility of the seed to shattering, root hair length
and quantity, internode distances, or the quality of seed coat.
16. The method of claim 7, wherein the trait is an alteration in a
plant growth characteristic selected from the group consisting of:
growth rate, germination rate of seeds, vigor of plants and
seedlings, leaf and flower senescence, male sterility, apomixis,
flowering time, flower abscission, rate of nitrogen uptake, biomass
or transpiration characteristics, apical dominance, branching
patterns, number of organs, organ identity, and organ shape or
size.
17. The method of claim 7, wherein the trait is an alteration in
one or more characteristics selected from the group consisting of
protein or oil production, seed protein or oil production,
insoluble sugar level, soluble sugar level, and starch
composition.
18. A plant produced by the method of claim 13.
19. A plant produced by the method of claim 14.
20. A plant produced by the method of claim 15.
21. A plant produced by the method of claim 16.
22. A plant produced by the method of claim 17.
23. A method of using the isolated or recombinant polynucleotide of
claim 4 for producing a plant having a modified trait, the method
comprising selecting a polynucleotide that when expressed produces
an antisense nucleic acid sequence, inserting the polynucleotide
into an expression vector, introducing the vector into a plant or a
cell of a plant to express the antisense nucleic acid, thereby
producing a modified plant, and selecting for a modified trait.
24. The method of claim 23, wherein the trait is selected from the
group consisting of: enhanced tolerance to freezing, chilling,
heat, drought, water saturation, radiation and ozone; improved
tolerance to microbial, fungal or viral diseases; improved
tolerance to pest infestations, decreased herbicide sensitivity;
improved tolerance of heavy metals or enhanced ability to take up
heavy metals, improved growth under poor photoconditions.
25. The method of claim 23, wherein the trait is an alteration in
the level of one or more of the compounds selected from the group
consisting of: taxol, tocopherol, tocotrienol, sterols,
phytosterols, vitamins, wax monomers, anti-oxidants, amino acids,
lignins, cellulose, tannins, prenyllipids, glucosinolates, and
terpenoids.
26. The method of claim 23, wherein the trait is an alteration in
one or more physical characteristics selected from the group
consisting of: the number of trichomes, fruit and seed size and
number, yield of stems, leaves, or roots, stability of seeds during
storage, susceptibility of the seed to shattering, root hair length
and quantity, internode distances, or the quality of seed coat.
27. The method of claim 23, wherein the trait is an alteration in a
plant growth characteristic selected from the group consisting of:
growth rate, germination rate of seeds, vigor of plants and
seedlings, leaf and flower senescence, male sterility, apomixis,
flowering time, flower abscission, rate of nitrogen uptake, biomass
or transpiration characteristics, apical dominance, branching
patterns, number of organs, organ identity, and organ shape or
size.
28. The method of claim 23, wherein the trait is an alteration in
one or more characteristics selected from the group consisting of
protein or oil production, seed protein or oil production,
insoluble sugar level, soluble sugar level, and starch
composition.
29. A plant produced by the method of claim 24.
30. A plant produced by the method of claim 25.
31. A plant produced by the method of claim 26.
32. A plant produced by the method of claim 27.
33. A plant produced by the method of claim 28.
34. An isolated or recombinant polypeptide comprising a subsequence
of at least about 10 contiguous amino acids encoded by the
recombinant or isolated polynucleotide of claim 4, wherein the
contiguous amino acids are outside of a conserved domain.
35. An isolated or recombinant polypeptide comprising a subsequence
of at least about 20 contiguous amino acids encoded by the
recombinant or isolated polynucleotide of claim 4, wherein the
contiguous amino acids are outside of a conserved domain.
36. An isolated or recombinant polypeptide comprising a subsequence
of at least about 30 contiguous amino acids encoded by the
recombinant or isolated polynucleotide of claim 4, wherein the
contiguous amino acids are outside of a conserved domain.
37. An isolated or recombinant polypeptide comprising a subsequence
of at least about 10 contiguous amino acids encoded by the
recombinant or isolated polynucleotide of claim 4, wherein the
contiguous amino acids are within a conserved domain.
38. An isolated or recombinant polypeptide comprising a subsequence
of at least about 20 contiguous amino acids encoded by the
recombinant or isolated polynucleotide of claim 4, wherein the
contiguous amino acids are within a conserved domain.
39. An isolated or recombinant polypeptide comprising a subsequence
of at least about 30 contiguous amino acids encoded by the
recombinant or isolated polynucleotide of claim 4, wherein the
contiguous amino acids are within a conserved domain.
40. A polypeptide having at least 31% sequence identity over the
entire length of a polypeptide of SEQ ID NOs:2 or 4, or the length
of the polypeptide itself.
41. A polypeptide having at least 60% sequence identity over the
entire length of a polypeptide of SEQ ID NOs:2 or 4, or the length
of the polypeptide itself.
42. A polypeptide having at least 75% sequence identity over the
entire length of a polypeptide of SEQ ID NOs:2 or 4, or the length
of the polypeptide itself.
43. A polypeptide having at least 95% sequence identity over the
entire length of a polypeptide of SEQ ID NOs:2 or 4, or the length
of the polypeptide itself.
Description
FIELD OF THE INVENTION AND INTRODUCTION
[0001] This invention relates to the field of plant biology. More
particularly, the present invention pertains to compositions and
methods for phenotypically modifying a plant.
[0002] A plant's traits, such as its biochemical, developmental, or
phenotypic characteristics, can be controlled through a number of
cellular processes. One important way to manipulate that control is
through transcription factors--proteins that influence the
expression of a particular gene or sets of genes. Transgenic plants
that comprise cells having altered levels of at least one selected
transcription factor, for example, possess advantageous or
desirable traits. Strategies for manipulating traits by altering a
plant cell's transcription factor content can therefore result in
plants and crops with commercially valuable properties. Applicants
have identified polynulceotides encoding transcription factors,
developed numerous transgenic plants using these polynucleotides,
and have analyzed the plants for a variety of important traits. In
so doing, applicants have identified important polynucleotide and
polypeptide sequences for producing commercially valuable plants
and crops as well as the methods for making them and using them.
Other aspects and embodiments of the invention are described below
and can be derived from the teachings of this disclosure as a
whole.
BACKGROUND OF THE INVENTION
[0003] Transcription factors can modulate gene expression, either
increasing or decreasing (inducing or repressing) the rate of
transcription. This modulation results in differential levels of
gene expression at various developmental stages, in different
tissues and cell types, and in response to different exogenous
(e.g., environmental) and endogenous stimuli throughout the life
cycle of the organism.
[0004] Because transcription factors are key controlling elements
of biological pathways, altering the expression levels of one or
more transcription factors can change entire biological pathways in
an organism. For example, manipulation of the levels of selected
transcription factors may result in increased expression of
economically useful proteins or metabolic chemicals in plants or to
improve other agriculturally relevant characteristics. Conversely,
blocked or reduced expression of a transcription factor may reduce
biosynthesis of unwanted compounds or remove an undesirable trait.
Therefore, manipulating transcription factor levels in a plant
offers tremendous potential in agricultural biotechnology for
modifying a plant's traits.
[0005] The present invention provides novel transcription factors
useful for modifying a plant's phenotype in desirable ways.
SUMMARY OF THE INVENTION
[0006] In a first aspect, the invention relates to a recombinant
polynucleotide comprising a nucleotide sequence selected from the
group consisting of: (a) a nucleotide sequence encoding a
polypeptide comprising a polypeptide sequence selected from those
of the Sequence Listing, SEQ ID NOs:2 and 4, or a complementary
nucleotide sequence thereof; (b) a nucleotide sequence encoding a
polypeptide comprising a variant of a polypeptide of (a) having one
or more, or between 1 and about 5, or between 1 and about 10, or
between 1 and about 30, conservative amino acid substitutions; (c)
a nucleotide sequence comprising a sequence selected from those of
SEQ ID NOs:1 and 3, or a complementary nucleotide sequence thereof;
(d) a nucleotide sequence comprising silent substitutions in a
nucleotide sequence of (c); (e) a nucleotide sequence which
hybridizes under stringent conditions over substantially the entire
length of a nucleotide sequence of one or more of: (a), (b), (c),
or (d); (f) a nucleotide sequence comprising at least 10 or 15, or
at least about 20, or at least about 30 consecutive nucleotides of
a sequence of any of (a)-(e), or at least 10 or 15, or at least
about 20, or at least about 30 consecutive nucleotides outside of a
region encoding a conserved domain of any of (a)-(e); (g) a
nucleotide sequence comprising a subsequence or fragment of any of
(a)-(f), which subsequence or fragment encodes a polypeptide having
a biological activity that modifies a plant's characteristic,
functions as a transcription factor, or alters the level of
transcription of a gene or transgene in a cell; (h) a nucleotide
sequence having at least 31% sequence identity to a nucleotide
sequence of any of (a)-(g); (i) a nucleotide sequence having at
least 60%, or at least 70%, or at least 80%, or at least 90 or at
least 95% sequence identity to a nucleotide sequence of any of
(a)-(g) or a 10 or 15 nucleotide, or at least about 20, or at least
about 30 nucleotide region of a sequence of (a)-(g) that is outside
of a region encoding a conserved domain; (j) a nucleotide sequence
that encodes a polypeptide having at least 31% sequence identity to
a polypeptide listed in the Sequence Listing or in Tables 6 and 7;
(k) a nucleotide sequence which encodes a polypeptide having at
least 60%, or at least 70%, or at least 80%, or at least 90%, or at
least 95% sequence identity to a polypeptide listed in the Sequence
Listing or in Tables 6 and 7; and (1) a nucleotide sequence that
encodes a conserved domain of a polypeptide having at least 85%, or
at least 90%, or at least 95%, or at least 98% sequence identity to
a conserved domain of a polypeptide listed in the Sequence Listing
or in Tables 6 and 7. The recombinant polynucleotide may further
comprise a constitutive, inducible, or tissue-specific promoter
operably linked to the nucleotide sequence. The invention also
relates to compositions comprising at least two of the
above-described polynucleotides.
[0007] In a second aspect, the invention comprises an isolated or
recombinant polypeptide comprising a subsequence of at least about
10, or at least about 15, or at least about 20, or at least about
30 contiguous amino acids encoded by the recombinant or isolated
polynucleotide described above, or comprising a subsequence of at
least about 8, or at least about 12, or at least about 15, or at
least about 20, or at least about 30 contiguous amino acids outside
a conserved domain.
[0008] In a third aspect, the invention comprises an isolated or
recombinant polynucleotide which encodes a polypeptide which is a
paralog of the isolated polypeptide described in paragraph 6 above.
In one aspect, the invention is an paralog which, when expressed in
Arabidopsis, modifies a trait of the Arabidopsis plant.
[0009] In a fourth aspect, the invention comprises an isolated or
recombinant polynucleotide which encodes a polypeptide which is an
ortholog of the isolated polypeptide described in paragraph 6
above. In one aspect, the invention is an ortholog which, when
expressed in Arabidopsis, modifies a trait of the Arabidopsis
plant.
[0010] In a fifth aspect, the invention comprises an isolated or
recombinant polynucleotide which encodes a polypeptide which is a
paralog of the isolated polypeptide described in paragraph 6 above.
In one aspect, the invention is an paralog which, when expressed in
Arabidopsis, modifies a trait of the Arabidopsis plant.
[0011] In another aspect, the invention comprises an isolated
polypeptide which is an ortholog of the isolated polypeptide
described in paragraph 6 above. In one aspect, the invention is an
ortholog which, when expressed in Arabidopsis, modifies a trait of
the Arabidopsis plant.
[0012] In yet another aspect, the invention comprises an isolated
synthetic polypeptide which is a homolog of the isolated
polypeptide described in paragraph 6 above. In one aspect, the
invention is a synthetic polypeptide which, when expressed in
Arabidopsis, modifies a trait of the Arabidopsis plant.
[0013] In another aspect, the invention is a transgenic plant
comprising one or more of the above-described recombinant
polynucleotides. In yet another aspect, the invention is a plant
with altered expression levels of a polynucleotide described above
or a plant with altered expression or activity levels of an
above-described polypeptide. Further, the invention is a plant
lacking a nucleotide sequence encoding a polypeptide described
above or substantially lacking a polypeptide described above. The
plant may be any plant, including, but not limited to, Arabidopsis,
mustard, soybean, wheat, corn, potato, cotton, rice, oilseed rape,
sunflower, alfalfa, sugarcane, turf, banana, blackberry, blueberry,
strawberry, raspberry, cantaloupe, carrot, cauliflower, coffee,
cucumber, eggplant, grapes, honeydew, lettuce, mango, melon, onion,
papaya, peas, peppers, pineapple, pumpkin, spinach, squash, sweet
corn, tobacco, tomato, watermelon, rosaceous fruits, vegetable
brassicas, and mint or other labiates. In yet another aspect, the
inventions is an isolated plant material of a plant, including, but
not limited to, plant tissue, fruit, seed, plant cell, embryo,
protoplast, pollen, and the like. In yet another aspect, the
invention is a transgenic plant tissue culture of regenerable
cells, including, but not limited to, embryos, meristematic cells,
microspores, protoplast, pollen, and the like.
[0014] In a further aspect the invention provides a method of using
the polynucleotide composition to breed progeny from a parent plant
including crossing plants, producing seeds from transgenic plants,
and methods of breeding using transgenic plants.
[0015] In a further aspect, the invention provides a progeny plant
derived from a parental plant wherein said progeny plant exhibits
at least three fold greater messenger RNA levels than said parental
plant, wherein the messenger RNA encodes a DNA-binding protein
which is capable of binding to a DNA regulatory sequence and
inducing expression of a plant trait gene, wherein the progeny
plant is characterized by a change in the plant trait compared to
said parental plant. In yet a further aspect, the progeny plant
exhibits at least ten fold greater messenger RNA levels compared to
said parental plant. In yet a further aspect, the progeny plant
exhibits at least fifty fold greater messenger RNA levels compared
to said parental plant.
[0016] In a further aspect, the invention relates to a cloning or
expression vector comprising the isolated or recombinant
polynucleotide described above or cells comprising the cloning or
expression vector.
[0017] In yet a further aspect, the invention relates to a
composition produced by incubating a polynucleotide of the
invention with a nuclease, a restriction enzyme, a polymerase; a
polymerase and a primer; a cloning vector, or with a cell.
[0018] Furthermore, the invention relates to a method for producing
a plant having a modified trait. The method comprises altering the
expression of an isolated or recombinant polynucleotide of the
invention or altering the expression or activity of a polypeptide
of the invention in a plant to produce a modified plant, and
selecting the modified plant for a modified trait. In one aspect,
the plant is a monocot plant. In another aspect, the plant is a
dicot plant. In another aspect the recombinant polynucleotide is
from a dicot plant and the plant is a monocot plant. In yet another
aspect the recombinant polynucleotide is from a monocot plant and
the plant is a dicot plant. In yet another aspect the recombinant
polynucleotide is from a monocot plant and the plant is a monocot
plant. In yet another aspect the recombinant polynucleotide is from
a dicot plant and the plant is a dicot plant.
[0019] In another aspect, the invention relates to a method of
identifying a factor that is modulated by or interacts with a
polypeptide encoded by a polynucleotide of the invention. The
method comprises expressing a polypeptide encoded by the
polynucleotide in a plant; and identifying at least one factor that
is modulated by or interacts with the polypeptide. In one
embodiment the method for identifying modulating or interacting
factors is by detecting binding by the polypeptide to a promoter
sequence, or by detecting interactions between an additional
protein and the polypeptide in a yeast two hybrid system, or by
detecting expression of a factor by hybridization to a microarray,
subtractive hybridization, or differential display.
[0020] In yet another aspect, the invention is a method of
identifying a molecule that modulates activity or expression of a
polynucleotide or polypeptide of interest. The method comprises
placing the molecule in contact with a plant comprising the
polynucleotide or polypeptide encoded by the polynucleotide of the
invention and monitoring one or more of the expression level of the
polynucleotide in the plant, the expression level of the
polypeptide in the plant, and modulation of an activity of the
polypeptide in the plant. In a farther aspect, the invention is a
method of using a molecule that modulates activity or expression of
a polynucleotide or polypeptide of interest. The polynucleotide may
be selected from the group comprising SEQ ID NOs:1 and 3, a variant
or ortholog thereof. In the alternative, the polypeptide may be
selected from the group comprising SEQ ID NOs:2 and 4, a variant or
ortholog thereof. The method comprises placing the molecule in
contact with a plant comprising the polynucleotide or polypeptide
encoded by the polynucleotide of the invention and monitoring one
or more of the expression level of the polynucleotide in the plant,
the expression level of the polypeptide in the plant, and
modulation of an activity of the polypeptide in the plant.
[0021] In yet another aspect, the invention relates to an
integrated system, computer or computer readable medium comprising
one or more character strings corresponding to a polynucleotide of
the invention, or to a polypeptide encoded by the polynucleotide.
The integrated system, computer or computer readable medium may
comprise a link between one or more sequence strings to a modified
plant trait.
[0022] In yet another aspect, the invention is a method for
identifying a sequence similar or homologous to one or more
polynucleotides of the invention, or one or more polypeptides
encoded by the polynucleotides. The method comprises providing a
sequence database, and querying the sequence database with one or
more target sequences corresponding to the one or more
polynucleotides or to the one or more polypeptides to identify one
or more sequence members of the database that display sequence
similarity or homology to one or more of the one or more target
sequences.
[0023] The method may further comprise of linking the one or more
of the polynucleotides of the invention, or encoded polypeptides,
to a modified plant phenotype.
[0024] Brief Description of the Sequence Listing and Tables
[0025] The Sequence Listing provides exemplary polynucleotide and
polypeptide sequences of the invention. The traits associated with
the use of the sequences are included in the "Examples" section of
the invention disclosure.
[0026] Tables 1 through 5 are shown and described in the invention
disclosure.
[0027] Table 6 lists a summary of orthologous and homologous
sequences of the polynucleotide sequences and polypeptide sequences
of the invention (SEQ ID NOs:1-4) identified using BLAST (TBLASTX
program). The first column shows the polynucleotide sequence
identifier (SEQ ID NO), the second column shows the transcription
factor CDNA identifier (Gene ID), the third column shows the
orthologous or homologous polynucleotide GenBank Accession Number
(Test Sequence ID), the fourth column shows the orthologous or
homologous polynucleotide sequence identifier (Test Sequence SEQ ID
NO), the fifth column shows the calculated probability value that
the sequence identity is due to chance (Smallest Sum Probability),
and the sixth column shows the orthologous or homologous GenBank
annotation (Test Sequence GenBank Annotation).
[0028] Table 7 lists orthologous and homologous sequences of the
polynucleotide sequences and polypeptide sequences of the invention
(SEQ ID NOs:1-4) identified using BLAST (TBLASTX program). The
first column shows the polynucleotide sequence identifier (SEQ ID
NO), the second column shows the transcription factor cDNA
identifier (Gene ID), the third column shows the orthologous or
homologous polynucleotide GenBank Accession Number (Test Sequence
ID), the fourth column shows the orthologous or homologous
polynucleotide sequence GenBank annotation (Test Sequence GenBank
Annotation) identifier (Test Sequence SEQ ID NO), the fifth column
shows the reading frame of the Test sequence which encodes the
orthologous or homologous sequence (Reading Frame), the sixth
column shows the calculated score value of the aligned sequences
(High Score), the seventh column shows the calculated probability
value that the sequence identity is due to chance (Smallest Sum
Probability), and the eighth column shows the number of regions of
the orthologous or homologous Test Sequences which aligned with the
sequence encoded by the transcription factor cDNA sequence GenBank
annotation (N).
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0029] In an important aspect, the present invention relates to
polynucleotides and polypeptides, e.g. for modifying phenotypes of
plants. Throughout this disclosure, various information sources are
referred to and/or are specifically incorporated. The information
sources include scientific journal articles, patent documents,
textbooks, and world wide web browser-inactive page addresses, for
example. While the reference to these information sources clearly
indicates that they can be used by one of skill in the art,
applicants specifically incorporate each and every one of the
information sources cited herein, in their entirety, whether or not
a specific mention of "incorporation by reference" is noted. The
contents and teachings of each and every one of the information
sources can be relied on and used to make and use embodiments of
the invention.
[0030] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a plant" includes a plurality of such
plants, and a reference to "a stress" is a reference to one or more
stresses and equivalents thereof known to those skilled in the art,
and so forth.
[0031] The polynucleotides of the invention encode plant
transcription factors. As one of ordinary skill in the art
recognizes, transcription factors can be identified by the presence
of a region or domain of structural similarity or identity to a
specific consensus sequence or the presence of a specific consensus
DNA-binding site or DNA-binding site motif (see, for example,
Riechmann et al., Science 290: 2105-2110 (2000)). The plant
transcription factors may belong to one of the following
transcription factor families: the AP2 (APETALA2) domain
transcription factor family (Riechmann and Meyerowitz (1998) Biol.
Chem. 379:633-646); the MYB transcription factor family (Martin and
Paz-Ares, (1997) Trends Genet. 13:67-73); the MADS domain
transcription factor family (Riechmann and Meyerowitz (1997) Biol.
Chem. 378:1079-1101); the WRKY protein family (Ishiguro and
Nakamura (1994) Mol. Gen. Genet. 244:563-571); the ankyrin-repeat
protein family (Zhang et al. (1992) Plant Cell 4:1575-1588); the
zinc finger protein (Z) family (Klug and Schwabe (1995) FASEB J. 9:
597-604); the homeobox (HB) protein family (Duboule (1994)
Guidebook to the Homeobox Genes, Oxford University Press); the
CAAT-element binding proteins (Forsburg and Guarente (1989) Genes
Dev. 3:1166-1178); the squamosa promoter binding proteins (SPB)
(Klein et al. (1996) Mol. Gen. Genet. 1996 250:7-16); the NAM
protein family (Souer et al. (1996) Cell 85:159-170); the IAA/AUX
proteins (Rouse et al. (1998) Science 279:1371-1373); the HLH/MYC
protein family (Littlewood et al. (1994) Prot. Profile 1:639-709);
the DNA-binding protein (DBP) family (Tucker et al. (1994) EMBO J.
13:2994-3002); the bZIP family of transcription factors (Foster et
al. (1994) FASEB J. 8:192-200); the Box P-binding protein (the
BPF-1) family (da Costa e Silva et al. (1993) Plant J. 4:125-135);
the high mobility group (HMG) family (Bustin and Reeves (1996)
Prog. Nucl. Acids Res. Mol. Biol. 54:35-100); the scarecrow (SCR)
family (Di Laurenzio et al. (1996) Cell 86:423-433); the GF14
family (Wu et al. (1997) Plant Physiol. 114:1421-1431); the
polycomb (PCOMB) family (Kennison (1995) Annu. Rev. Genet.
29:289-303); the teosinte branched (TEO) family (Luo et al. (1996)
Nature 383:794-799; the ABI3 family (Giraudat et al. (1992) Plant
Cell 4:1251-1261); the triple helix (TH) family (Dehesh et al.
(1990) Science 250:1397-1399); the EIL family (Chao et al. (1997)
Cell 89:1133-44); the AT-HOOK family (Reeves and Nissen (1990)
Journal of Biological Chemistry 265:8573-8582); the SIFA family
(Zhou et al. (1995) Nucleic Acids Res. 23:1165-1169); the bZIPT2
family (Lu and Ferl (1995) Plant Physiol. 109:723); the YABBY
family (Bowman et al. (1999) Development 126:2387-96); the PAZ
family (Bohmert et al. (1998) EMBO J. 17:170-80); miscellaneous
(MISC) transcription factors including the DPBF family (Kim et al.
(1997) Plant J. 11: 1237-1251) and the SPF1 family (Ishiguro and
Nakamura (1994) Mol. Gen. Genet. 244:563-571); the golden (GLD)
family (Hall et al. (1998) Plant Cell 10:925-936); the TUBBY family
(Boggin et al, (1999) Science 286:2119-2125); the heat shock family
(Wu C (1995) Annu Rev Cell Dev Biol 11:441-469); the ENBP family
(Christiansen et al (1996) Plant Mol Biol 32:809-821); the
RING-zinc family (Jensen et al. (1998) FEBS letters 436:283-287);
the PDBP family (Janik et al Virology. (1989)168:320-329); the PCF
family (Cubas P, et al. Plant J. (1999)18:215-22); the SRS
(SHI-related) family (Fridborg et al Plant Cell (1999)
11:1019-1032); the CPP (cysteine-rich polycomb-like) family
(Cvitanich et al Proc. Natl. Acad. Sci. U S A. (2000)
97:8163-8168); the ARF (auxin response factor) family (Ulmasov, et
al. (1999) Proc. Natl. Acad. Sci. USA 96: 5844-5849); the SWI/SNF
family (Collingwood et al J. Mol. End. 23:255-275); the ACBF family
(Seguin et al Plant Mol Biol. (1997) 35:281-291); PCGL (CG-1 like)
family (Plant Mol Biol. (1994) 25:921-924); the ARID family
(Vazquez et al Development. (1999) 126: 733-42); the Jumonji family
(Balciunas et al Trends Biochem Sci. (2000) 25: 274-276); the
bZIP-NIN family (Schauser et al Nature. (1999) 402: 191-195); the
E2F family Kaelin et al (1992) Cell 70: 351-364); and the GRF-like
family (Knaap et al (2000) Plant Physiol. 122: 695-704. As
indicated by any part of the list above and as known in the art,
transcription factors have been sometimes categorized by class,
family, and sub-family according to their structural content and
consensus DNA-binding site motif, for example. Many of the classes
and many of the families and sub-families are listed here. However,
the inclusion of one sub-family and not another, or the inclusion
of one family and not another, does not mean that the invention
does not encompass polynucleotides or polypeptides of a certain
family or sub-family. The list provided here is merely an example
of the types of transcription factors and the knowledge available
concerning the consensus sequences and consensus DNA-binding site
motifs that help define them as known to those of skill in the art
(each of the references noted above are specifically incorporated
herein by reference). A transcription factor may include, but is
not limited to, any polypeptide that can activate or repress
transcription of a single gene or a number of genes. This
polypeptide group includes, but is not limited to, DNA-binding
proteins, DNA-binding protein binding proteins, protein kinases,
protein phosphatases, GTP-binding proteins, and receptors, and the
like.
[0032] In addition to methods for modifying a plant phenotype by
employing one or more polynucleotides and polypeptides of the
invention described herein, the polynucleotides and polypeptides of
the invention have a variety of additional uses. These uses include
their use in the recombinant production (i.e, expression) of
proteins; as regulators of plant gene expression, as diagnostic
probes for the presence of complementary or partially complementary
nucleic acids (including for detection of natural coding nucleic
acids); as substrates for further reactions, e.g., mutation
reactions, PCR reactions, or the like; as substrates for cloning
e.g., including digestion or ligation reactions; and for
identifying exogenous or endogenous modulators of the transcription
factors.
[0033] A "polynucleotide" is a nucleic acid sequence comprising a
plurality of polymerized nucleotides, e.g., at least about 15
consecutive polymerized nucleotide, optionally at least about 30
consecutive nucleotides, at least about 50 consecutive nucleotides.
In many instances, a polynucleotide comprises a nucleotide sequence
encoding a polypeptide (or protein) or a domain or fragment
thereof. Additionally, the polynucleotide may comprise a promoter,
an intron, an enhancer region, a polyadenylation site, a
translation initiation site, 5' or 3' untranslated regions, a
reporter gene, a selectable marker, or the like. The polynucleotide
can be single stranded or double stranded DNA or RNA. The
polynucleotide optionally comprises modified bases or a modified
backbone. The polynucleotide can be, e.g., genomic DNA or RNA, a
transcript (such as an MRNA), a cDNA, a PCR product, a cloned DNA,
a synthetic DNA or RNA, or the like. The polynucleotide can
comprise a sequence in either sense or antisense orientations.
[0034] A "recombinant polynucleotide" is a polynucleotide that is
not in its native state, e.g., the polynucleotide comprises a
nucleotide sequence not found in nature, or the polynucleotide is
in a context other than that in which it is naturally found, e.g.,
separated from nucleotide sequences with which it typically is in
proximity in nature, or adjacent (or contiguous with) nucleotide
sequences with which it typically is not in proximity. For example,
the sequence at issue can be cloned into a vector, or otherwise
recombined with one or more additional nucleic acid.
[0035] A "synthetic polynucleotide" is a polynucleotide not fund in
nature and encodes a polypeptide not found in nature. The encoded
polypeptide comprises at least four consecutive amino acid residues
of a polypeptide found in nature.
[0036] An "isolated polynucleotide" is a polynucleotide whether
naturally occurring or recombinant, that is present outside the
cell in which it is typically found in nature, whether purified or
not. Optionally, an isolated polynucleotide is subject to one or
more enrichment or purification procedures, e.g., cell lysis,
extraction, centrifugation, precipitation, or the like.
[0037] A "polypeptide" is an amino acid sequence comprising a
plurality of consecutive polymerized amino acid residues e.g., at
least about 15 consecutive polymerized amino acid residues,
optionally at least about 30 consecutive polymerized amino acid
residues, at least about 50 consecutive polymerized amino acid
residues. In many instances, a polypeptide comprises a polymerized
amino acid residue sequence which is a transcription factor or a
domain or portion or fragment thereof. Additionally, the
polypeptide may comprise a localization domain, 2) an activation
domain, 3) a repression domain, 4) an oligomerization domain or 5)
a DNA-binding domain, or the like. The polypeptide optionally
comprises modified amino acid residues, naturally occurring amino
acid residues not encoded by a codon, non-naturally occurring amino
acid residues.
[0038] A "recombinant polypeptide" is a polypeptide produced by
translation of a recombinant polynucleotide. A "synthetic
polypeptide" is a polypeptide created by consecutive polymerization
of isolated amino acid residues using methods well known in the
art. An "isolated polypeptide," whether a naturally occurring or a
recombinant polypeptide, is more enriched in (or out of) a cell
than the polypeptide in its natural state in a wild type cell,
e.g., more than about 5% enriched, more than about 10% enriched, or
more than about 20%, or more than about 50%, or more, enriched,
i.e., alternatively denoted: 105%, 110%, 120%, 150% or more,
enriched relative to wild type standardized at 100%. Such an
enrichment is not the result of a natural response of a wild type
plant. Alternatively, or additionally, the isolated polypeptide is
separated from other cellular components with which it is typically
associated, e.g., by any of the various protein purification
methods herein.
[0039] A "synthetic polypeptide" is a polypeptide not fund in
nature and has activity of a polypeptide found in nature. The
polypeptide comprises at least four consecutive amino acid residues
of a polypeptide found in nature.
[0040] "Altered" nucleic acid sequences encoding polypeptide
include those sequences with deletions, insertions, or
substitutions of different nucleotides, resulting in a
polynucleotide encoding a polypeptide with at least one functional
characteristic of the polypeptide. Included within this definition
are polymorphisms which may or may not be readily detectable using
a particular oligonucleotide probe of the polynucleotide encoding
polypeptide, and improper or unexpected hybridization to allelic
variants, with a locus other than the normal chromosomal locus for
the polynucleotide sequence encoding polypeptide. The encoded
polypeptide protein may also be "altered", and may contain
deletions, insertions, or substitutions of amino acid residues
which produce a silent change and result in a functionally
equivalent polypeptide. Deliberate amino acid substitutions may be
made on the basis of similarity in residue side chain chemistry,
including, but not limited to, polarity, charge, solubility,
hydrophobicity, hydrophilicity, and/or the amphipathic nature of
the residues, as long as the biological activity of polypeptide is
retained. For example, negatively charged amino acids may include
aspartic acid and glutamic acid, positively charged amino acids may
include lysine and arginine, and amino acids with uncharged polar
head groups having similar hydrophilicity values may include
leucine, isoleucine, and valine; glycine and alanine; asparagine
and glutamine; serine and threonine; and phenylalanine and
tyrosine. Alignments between different polypeptide sequences may be
used to calculate "percentage sequence similarity".
[0041] The term "plant" includes whole plants, shoot vegetative
organs/structures (e.g., leaves, stems and tubers), roots, flowers
and floral organs/structures (e.g., bracts, sepals, petals,
stamens, carpels, anthers and ovules), seed (including embryo,
endosperm, and seed coat) and fruit (the mature ovary), plant
tissue (e.g., vascular tissue, ground tissue, and the like) and
cells (e.g., guard cells, egg cells, and the like), and progeny of
same. The class of plants that can be used in the method of the
invention is generally as broad as the class of higher and lower
plants amenable to transformation techniques, including angiosperms
(monocotyledonous and dicotyledonous plants), gymnosperms, ferns,
horsetails, psilophytes, lycophytes, bryophytes, and multicellular
algae. (See for example, Daly et al. 2001 Plant Physiology
127:1328-1333; and also Tudge, C., The Variety of Life, Oxford
University Press, New York, 2000, pp. 547-606.)
[0042] A "transgenic plant" refers to a plant that contains genetic
material not found in a wild type plant of the same species,
variety or cultivar. The genetic material may include a transgene,
an insertional mutagenesis event (such as by transposon or T-DNA
insertional mutagenesis), an activation tagging sequence, a mutated
sequence, a homologous recombination event or a sequence modified
by chimeraplasty. Typically, the foreign genetic material has been
introduced into the plant by human manipulation, but any method can
be used as one of skill in the art recognizes.
[0043] A transgenic plant may contain an expression vector or
cassette. The expression cassette typically comprises a
polypeptide-encoding sequence operably linked (i.e., under
regulatory control of) to appropriate inducible or constitutive
regulatory sequences that allow for the expression of polypeptide.
The expression cassette can be introduced into a plant by
transformation or by breeding after transformation of a parent
plant. A plant refers to a whole plant as well as to a plant part,
such as seed, fruit, leaf, or root, plant tissue, plant cells or
any other plant material, e.g., a plant explant, as well as to
progeny thereof, and to in vitro systems that mimic biochemical or
cellular components or processes in a cell.
[0044] "Ectopic expression or altered expression" in reference to a
polynucleotide indicates that the pattern of expression in, e.g., a
transgenic plant or plant tissue, is different from the expression
pattern in a wild type plant or a reference plant of the same
species. The pattern of expression may also be compared with a
reference expression pattern in a wild type plant of the same
species. For example, the polynucleotide or polypeptide is
expressed in a cell or tissue type other than a cell or tissue type
in which the sequence is expressed in the wild type plant, or by
expression at a time other than at the time the sequence is
expressed in the wild type plant, or by a response to different
inducible agents, such as hormones or environmental signals, or at
different expression levels (either higher or lower) compared with
those found in a wild type plant. The term also refers to altered
expression patterns that are produced by lowering the levels of
expression to below the detection level or completely abolishing
expression. The resulting expression pattern can be transient or
stable, constitutive or inducible. In reference to a polypeptide,
the term "ectopic expression or altered expression" further may
relate to altered activity levels resulting from the interactions
of the polypeptides with exogenous or endogenous modulators or from
interactions with factors or as a result of the chemical
modification of the polypeptides.
[0045] A "fragment" or "domain," with respect to a polypeptide,
refers to a subsequence of the polypeptide. In some cases, the
fragment or domain, is a subsequence of the polypeptide which
performs at least one biological finction of the intact polypeptide
in substantially the same manner, or to a similar extent, as does
the intact polypeptide. For example, a polypeptide fragment can
comprise a recognizable structural motif or finctional domain such
as a DNA-binding site or domain that binds to a DNA promoter
region, an activation domain, or a domain for protein-protein
interactions. Fragments can vary in size from as few as 6 amino
acids to the full length of the intact polypeptide, but are
preferably at least about 30 amino acids in length and more
preferably at least about 60 amino acids in length. In reference to
a polynucleotide sequence, "a fragment" refers to any subsequence
of a polynucleotide, typically, of at least about 15 consecutive
nucleotides, preferably at least about 30 nucleotides, more
preferably at least about 50 nucleotides, of any of the sequences
provided herein. [00451 A "conserved domain", with respect to a
polypeptide, refers to a domain within a transcription factor
family which exhibits a higher degree of sequence homology, such as
at least 65% sequence identity including conservative
substitutions, and preferably at least 80% sequence identity, and
more preferably at least 85%, or at least about 86%, or at least
about 87%, or at least about 88%, or at least about 90%, or at
least about 95%, or at least about 98% amino acid residue sequence
identity of a polypeptide of consecutive amino acid residues. A
fragment or domain can be referred to as outside a consensus
sequence or outside a consensus DNA-binding site that is known to
exist or that exists for a particular transcription factor class,
family, or sub-family. In this case, the fragment or domain will
not include the exact amino acids of a consensus sequence or
consensus DNA-binding site of a transcription factor class, family
or sub-family, or the exact amino acids of a particular
transcription factor consensus sequence or consensus DNA-binding
site. Furthermore, a particular fragment, region, or domain of a
polypeptide, or a polynucleotide encoding a polypeptide, can be
"outside a conserved domain" if all the amino acids of the
fragment, region, or domain fall outside of a defined conserved
domain(s) for a polypeptide or protein. The conserved domains for
each of polypeptides of SEQ ID NOs:2 and 4 are listed in Table 4 as
described in Example VII. A comparison of the regions of the
polypeptides in SEQ ID NOs:2 and 4 allows one of skill in the art
to identify conserved domain(s) for any of the polypeptides listed
or referred to in this disclosure, including those in Tables 6 and
7.
[0046] A "trait" refers to a physiological, morphological,
biochemical, or physical characteristic of a plant or particular
plant material or cell. In some instances, this characteristic is
visible to the human eye, such as seed or plant size, or can be
measured by biochemical techniques, such as detecting the protein,
starch, or oil content of seed or leaves, or by the observation of
the expression level of a gene or genes, e.g., by employing
Northern analysis, RT-PCR, microarray gene expression assays, or
reporter gene expression systems, or by agricultural observations
such as stress tolerance, yield, or pathogen tolerance. Any
technique can be used to measure the amount of, comparative level
of, or difference in any selected chemical compound or
macromolecule in the transgenic plants, however.
[0047] "Trait modification" refers to a detectable difference in a
characteristic in a plant ectopically expressing a polynucleotide
or polypeptide of the present invention relative to a plant not
doing so, such as a wild type plant. In some cases, the trait
modification can be evaluated quantitatively. For example, the
trait modification can entail at least about a 2% increase or
decrease in an observed trait (difference), at least a 5%
difference, at least about a 10% difference, at least about a 20%
difference, at least about a 30%, at least about a 50%, at least
about a 70%, or at least about a 100%, or an even greater
difference compared with a wild type plant. It is known that there
can be a natural variation in the modified trait. Therefore, the
trait modification observed entails a change of the normal
distribution of the trait in the plants compared with the
distribution observed in wild type plant.
[0048] Trait modifications of particular interest include those to
seed (such as embryo or endosperm), fruit, root, flower, leaf,
stem, shoot, seedling or the like, including: enhanced tolerance to
environmental conditions including freezing, chilling, heat,
drought, water saturation, radiation and ozone; improved tolerance
to microbial, fungal or viral diseases; improved tolerance to pest
infestations, including nematodes, mollicutes, parasitic higher
plants or the like; decreased herbicide sensitivity; improved
tolerance of heavy metals or enhanced ability to take up heavy
metals; improved growth under poor photoconditions (e.g., low light
and/or short day length), or changes in expression levels of genes
of interest. Other phenotype that can be modified relate to the
production of plant metabolites, such as variations in the
production of taxol, tocopherol, tocotrienol, sterols,
phytosterols, vitamins, wax monomers, anti-oxidants, amino acids,
lignins, cellulose, tannins, prenyllipids (such as chlorophylls and
carotenoids), glucosinolates, and terpenoids, enhanced or
compositionally altered protein or oil production (especially in
seeds), or modified sugar (insoluble or soluble) and/or starch
composition. Physical plant characteristics that can be modified
include cell development (such as the number of trichomes), fruit
and seed size and number, yields of plant parts such as stems,
leaves and roots, the stability of the seeds during storage,
characteristics of the seed pod (e.g., susceptibility to
shattering), root hair length and quantity, internode distances, or
the quality of seed coat. Plant growth characteristics that can be
modified include growth rate, germination rate of seeds, vigor of
plants and seedlings, leaf and flower senescence, male sterility,
apomixis, flowering time, flower abscission, rate of nitrogen
uptake, biomass or transpiration characteristics, as well as plant
architecture characteristics such as apical dominance, branching
patterns, number of organs, organ identity, organ shape or
size.
[0049] Examples of plant trait modifications and how to measure and
determine those plant traits or characteristics are provided in the
invention disclosure and the "Examples" section, Table 5. The
disclosures are intended to illustrate but not limit which plant
trait or characteristic may be modified by the invention.
[0050] Polypeptides and Polynucleotides of the Invention
[0051] The present invention provides, among other things,
transcription factors (TFs), and transcription factor homologue
polypeptides, and isolated or recombinant polynucleotides encoding
the polypeptides, or novel variant polypeptides or polynucleotides
encoding novel variants of transcription factors derived from the
specific sequences provided here. These polypeptides and
polynucleotides may be employed to modify a plant's
characteristic.
[0052] Exemplary polynucleotides encoding the polypeptides of the
invention were identified in the Arabidopsis thaliana GenBank
database using publicly available sequence analysis programs and
parameters. Sequences initially identified were then further
characterized to identify sequences comprising specified sequence
strings corresponding to sequence motifs present in families of
known transcription factors. In addition, farther exemplary
polynucleotides encoding the polypeptides of the invention were
identified in the plant GenBank database using publicly available
sequence analysis programs and parameters. Sequences initially
identified were then further characterized to identify sequences
comprising specified sequence strings corresponding to sequence
motifs present in families of known transcription factors.
Polynucleotide sequences meeting such criteria were confirmed as
transcription factors.
[0053] Additional polynucleotides of the invention were identified
by screening Arabidopsis thaliana and/or other plant cDNA libraries
with probes corresponding to known transcription factors under low
stringency hybridization conditions. Additional sequences,
including fall length coding sequences were subsequently recovered
by the rapid amplification of cDNA ends (RACE) procedure, using a
conunercially available kit according to the manufacturer's
instructions. Where necessary, multiple rounds of RACE are
performed to isolate 5' and 3' ends. The fall length cDNA was then
recovered by a routine end-to-end polymerase chain reaction (PCR)
using primers specific to the isolated 5' and 3' ends. Exemplary
sequences are provided in the Sequence Listing.
[0054] The polynucleotides of the invention can be or were
ectopically expressed in overexpressor or knockout plants and the
changes in the characteristic(s) or trait(s) of the plants
observed. Therefore, the polynucleotides and polypeptides can be
employed to improve the characteristics of plants.
[0055] Producing Polypeptides
[0056] The polynucleotides of the invention include sequences that
encode transcription factors and transcription factor homologue
polypeptides and sequences complementary thereto, as well as unique
fragments of coding sequence, or sequence complementary thereto.
Such polynucleotides can be, e.g., DNA or RNA, e.g., mRNA, cRNA,
synthetic RNA, genomic DNA, cDNA synthetic DNA, oligonucleotides,
etc. The polynucleotides are either double-stranded or
single-stranded, and include either, or both sense (i.e., coding)
sequences and antisense (i.e., non-coding, complementary)
sequences. The polynucleotides include the coding sequence of a
transcription factor, or transcription factor homologue
polypeptide, in isolation, in combination with additional coding
sequences (e.g., a purification tag, a localization signal, as a
fusion-protein, as a pre-protein, or the like), in combination with
non-coding sequences (e.g., introns or inteins, regulatory elements
such as promoters, enhancers, terminators, and the like), and/or in
a vector or host environment in which the polynucleotide encoding a
transcription factor or transcription factor homologue polypeptide
is an endogenous or exogenous gene.
[0057] A variety of methods exist for producing the polynucleotides
of the invention. Procedures for identifying and isolating DNA
clones are well known to those of skill in the art, and are
described in, e.g., Berger and Kimmel, Guide to Molecular Cloning
Techniques, Methods in Enzymology volume 152 Academic Press, Inc.,
San Diego, Calif. ("Berger"); Sambrook et al., Molecular Cloning--A
Laboratory Manual (2nd Ed.), Vol. 1-3, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y., 1989 ("Sambrook") and Current
Protocols in Molecular Biology, F. M. Ausubel et al., eds., Current
Protocols, a joint venture between Greene Publishing Associates,
Inc. and John Wiley & Sons, Inc., (supplemented through 2000)
("Ausubel").
[0058] Alternatively, polynucleotides of the invention, can be
produced by a variety of in vitro amplification methods adapted to
the present invention by appropriate selection of specific or
degenerate primers. Examples of protocols sufficient to direct
persons of skill through in vitro amplification methods, including
the polymerase chain reaction (PCR) the ligase chain reaction
(LCR), Qbeta-replicase amplification and other RNA polymerase
mediated techniques (e.g., NASBA), e.g., for the production of the
homologous nucleic acids of the invention are found in Berger,
Sambrook, and Ausubel (all supra), as well as Mullis et al., (1987)
PCR Protocols A Guide to Methods and Applications (Innis et al.
eds) Academic Press Inc. San Diego, Calif. (1990) (Innis). Improved
methods for cloning in vitro amplified nucleic acids are described
in Wallace et al., U.S. Pat. No. 5,426,039. Improved methods for
amplifying large nucleic acids by PCR are summarized in Cheng et
al. (1994) Nature 369: 684-685 and the references cited therein, in
which PCR amplicons of up to 40 kb are generated. One of skill will
appreciate that essentially any RNA can be converted into a double
stranded DNA suitable for restriction digestion, PCR expansion and
sequencing using reverse transcriptase and a polymerase. See, e.g.,
Ausubel, Sambrook and Berger, all supra.
[0059] Alternatively, polynucleotides and oligonucleotides of the
invention can be assembled from fragments produced by solid-phase
synthesis methods. Typically, fragments of up to approximately 100
bases are individually synthesized and then enzymatically or
chemically ligated to produce a desired sequence, e.g., a
polynucletotide encoding all or part of a transcription factor. For
example, chemical synthesis using the phosphoramidite method is
described, e.g., by Beaucage et al. (1981) Tetrahedron Letters
22:1859-69; and Matthes et al. (1984) EMBO J. 3:801-5. According to
such methods, oligonucleotides are synthesized, purified, annealed
to their complementary strand, ligated and then optionally cloned
into suitable vectors. And if so desired, the polynucleotides and
polypeptides of the invention can be custom ordered from any of a
number of commercial suppliers.
[0060] Homologous Sequences
[0061] Sequences homologous, i.e., that share significant sequence
identity or similarity, to those provided in the Sequence Listing,
derived from Arabidopsis thaliana or from other plants of choice
are also an aspect of the invention. Homologous sequences can be
derived from any plant including monocots and dicots and in
particular agriculturally important plant species, including but
not limited to, crops such as soybean, wheat, corn, potato, cotton,
rice, oilseed rape (including canola), sunflower, alfalfa,
sugarcane and turf; or fruits and vegetables, such as banana,
blackberry, blueberry, strawberry, and raspberry, cantaloupe,
carrot, cauliflower, coffee, cucumber, eggplant, grapes, honeydew,
lettuce, mango, melon, onion, papaya, peas, peppers, pineapple,
pumpkin, spinach, squash, sweet corn, tobacco, tomato, watermelon,
rosaceous fruits (such as apple, peach, pear, cherry and plum) and
vegetable brassicas (such as broccoli, cabbage, cauliflower,
brussel sprouts and kohlrabi). Other crops, fruits and vegetables
whose phenotype can be changed include barley, rye, millet,
sorghum, currant, avocado, citrus fruits such as oranges, lemons,
grapefruit and tangerines, artichoke, cherries, nuts such as the
walnut and peanut, endive, leek, roots, such as arrowroot, beet,
cassava, turnip, radish, yam, and sweet potato, and beans. The
homologous sequences may also be derived from woody species, such
pine, poplar and eucalyptus, or mint or other labiates.
[0062] Transcription factors that are homologous to the listed
sequences will typically share at least about 30% amino acid
sequence identity, or at least about 30% amino acid sequence
identity outside of a known consensus sequence or consensus
DNA-binding site. More closely related transcription factors can
share at least about 50%, about 60%, about 65%, about 70%, about
75% or about 80% or about 90% or about 95% or about 98% or more
sequence identity with the listed sequences, or with the listed
sequences but excluding or outside a known consensus sequence or
consensus DNA-binding site, or with the listed sequences excluding
one or all conserved domain. Factors that are most closely related
to the listed sequences share, e.g., at least about 85%, about 90%
or about 95% or more % sequence identity to the listed sequences,
or to the listed sequences but excluding or outside a known
consensus sequence or consensus DNA-binding site or outside one or
all conserved domain. At the nucleotide level, the sequences will
typically share at least about 40% nucleotide sequence identity,
preferably at least about 50%, about 60%, about 70% or about 80%
sequence identity, and more preferably about 85%, about 90%, about
95% or about 97% or more sequence identity to one or more of the
listed sequences, or to a listed sequence but excluding or outside
a known consensus sequence or consensus DNA-binding site, or
outside one or all conserved domain. The degeneracy of the genetic
code enables major variations in the nucleotide sequence of a
polynucleotide while maintaining the amino acid sequence of the
encoded protein. Conserved domains within a transcription factor
family may exhibit a higher degree of sequence homology, such as at
least 65% sequence identity including conservative substitutions,
and preferably at least 80% sequence identity, and more preferably
at least 85%, or at least about 86%, or at least about 87%, or at
least about 88%, or at least about 90%, or at least about 95%, or
at least about 98% sequence identity. Transcription factors that
are homologous to the listed sequences should share at least 30%,
or at least about 60%, or at least about 75%, or at least about
80%, or at least about 90%, or at least about 95% amino acid
sequence identity over the entire length of the polypeptide or the
homolog. In addition, transcription factors that are homologous to
the listed sequences should share at least 30%, or at least about
60%, or at least about 75%, or at least about 80%, or at least
about 90%, or at least about 95% amino acid sequence similarity
over the entire length of the polypeptide or the homolog.
[0063] Identifying Orthologs And Paralogs
[0064] Several different methods are known by those of skill in the
art for identifying and defining these functionally homologous
sequences. Three general methods for defining paralogs and
orthologs are described; a paralog or ortholog may be identified by
only one or more of the methods described below.
[0065] Orthologs and paralogs are evolutionarily related genes that
have similar sequence and similar functions. Paralogs are related
genes within a single species and are most likely a result of gene
duplication, whereas orthologs are related genes in different
species derived from a common ancestral molecule prior to
speciation.
[0066] Within a single plant species, gene duplication may causes
two copies of a particular gene, giving rise to two or more genes
with similar sequence and similar function known as paralogs. A
paralog is therefore a similar gene with a similar function within
the same species. Paralogs typically cluster together or in the
same lade (a group of similar genes) when a gene family phylogeny
is analyzed using programs such as CLUSTAL (Thompson et al. (1994)
Nucleic Acids Res. 22:4673-4680; Higgins et al. (1996) Methods
Enzymol. 266 383-402). Groups of similar genes can also be
identified using by pair-wise BLAST analysis (Feng and Doolittle
(1987) J. Mol. Evol. 25:351-360). For example, a lade of very
similar MADS domain transcription factors from Arabidopsis all
share a common function in flowering time (Ratcliffe et al. (2001)
Plant Physiol. 126:122-132), and a group of very similar AP2 domain
transcription factors from Arabidopsis are involved in tolerance of
plants to freezing (Gilmour et al. (1998) Plant J. 16:433-442).
Analysis of groups of similar genes with similar function that fall
within one lade can yield sub-sequences that are particular to the
lade. These sub-sequences, known as consensus sequences, can not
only be used to define the sequences within each lade, but define
the functions of these genes, since genes within each lade
typically share the same function. (See also, for example, Mount,
D. W. (2001) Bioinformatics: Sequence and Genome Analysis Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. page
543.)
[0067] Speciation, the production of new species from a parental
species, can also give rise to two or more genes with similar
sequence and similar function. These genes, termed orthologs, often
have an identical function within their host plants and are often
interchangeable between species without losing function. Because
plants have common ancestors, many genes in any plant species will
have a corresponding orthologous gene in another plant species.
Once a phylogenic tree for a gene family of one species has been
constructed using a program such as CLUSTAL (Thompson et al. (1994)
Nucleic Acids Res. 22:4673-4680; Higgins et al. (1996) Methods
Enzymol. 266:383-402), potential orthologous sequences can placed
into the phylogenetic tree and its relationship to genes from the
species of interest can be determined. Once the ortholog pair has
been identified, the function of the test ortholog can be
determined by determining the function of the reference
ortholog.
[0068] Orthologs can also be identified by pair-wise BLAST analysis
by aligning a set of reference sequences against a set of test
sequences. Test sequences with the closest match to a particular
reference sequence, as determined by the P-value of the BLAST
analysis, can be taken and individually aligned against the
reference set of sequences. The individual test sequence will
either best match the particular reference sequence, in which case
it is likely to be an ortholog, or not, in which case it may not be
an ortholog.
[0069] A further way of identifying an ortholog is by identifying a
consensus sequence within the candidate ortholog. Using pair-wise
BLAST analysis, or programs such as CLUSTAL alignment program, sets
of similar genes, or clades, can be identified. The particular
sub-sequences which defining within a particular clade have in
common to differentiate themselves can be derived from an alignment
of those sequences. Orthologs would have the consensus sequence, or
a sequence similar to the consensus sequence. Orthologs might also
have a consensus sequence outside a conserved domain, which could
be particular to that family of orthologous sequences.
[0070] Corresponding orthologs may bridge the monocot/dicot
division of the plant kingdom and orthologous pairs of genes can be
identified in rice and Arabidopsis, corn and Arabidopsis and
Antirhinnum and corn. For example Peng et al showed that a mutant
of the Arabidonsis gene termed Gibberellin Insensitive (GAI; mutant
termed gai) encoded a transcription factor and which conferred a
reduction in gibberellin responsiveness in the native plant (Peng
et al. 1997 Genes and Development 11:3194-3205). In addition, Peng
et al. subsequently showed that the Arabidopsis GAI protein has 62
% amino acid residue identity with the wheat Rht-D 1 a protein and
62% amino acid residue identity with the maize d8. Peng et al.
showed that transgenic rice plants containing a mutant GAI allele
give reduced response to gibberellin and are dwarfed, mimicking the
dwarfed wheat variety from which the mutant Rht-Dla gene was
isolated. Peng et al. taught that Arabidopsis GAI protein is an
ortholog of the wheat Rht-Dla and maize d8 proteins. (Peng et al.
1999 Nature 400:256-261.)
[0071] In addition Fu et al. (2001 Plant Cell 13:1791-1802), Nandi
et al. (2000 Curr. Biol. 10:215-218), Coupland (1995 Nature
377:482-483), and Weigel and Nilsson (1995 Nature 377:482-500) show
that an Arabidopsis transcription factor expressed in an exogenous
plant species elicits the same or very similar phenotypic response.
Furthermore, Kater et al. (1998 Plant Cell 10:171-182), Mandel et
al. (1992 Cell 71-133-143), and Suzuki et al. (2001 Plant J.
28:409-418) showed that a transcription factor expressed in another
plant species elicits the same or very similar phenotypic response
of the endogenous sequence, as often predicted in earlier studies
of Arabidopsis transcription factors in Arabidopsis.
[0072] Identifying Polynucleotides Or Nucleic Acids By
Hybridization
[0073] Polynucleotides homologous to the sequences illustrated in
the Sequence Listing can be identified, e.g., by hybridization to
each other under stringent or under highly stringent conditions.
Single stranded polynucleotides hybridize when they associate based
on a variety of well characterized physical-chemical forces, such
as hydrogen bonding, solvent exclusion, base stacking and the like.
The stringency of a hybridization reflects the degree of sequence
identity of the nucleic acids involved, such that the higher the
stringency, the more similar are the two polynucleotide strands.
Stringency is influenced by a variety of factors, including
temperature, salt concentration and composition, organic and
non-organic additives, solvents, etc. present in both the
hybridization and wash solutions and incubations (and number), as
described in more detail in the references cited above.
[0074] An example of stringent hybridization conditions for
hybridization of complementary nucleic acids which have more than
100 complementary residues on a filter in a Southern or northern
blot is about 5.degree. C. to 20.degree. C. lower than the thermal
melting point (T.sub.m) for the specific sequence at a defined
ionic strength and pH. The T.sub.m is the temperature (under
defined ionic strength and pH) at which 50% of the target sequence
hybridizes to a perfectly matched probe. Nucleic acid molecules
that hybridize under stringent conditions will typically hybridize
to a probe based on either the entire cDNA or selected portions,
e.g., to a unique subsequence, of the cDNA under wash conditions of
0.2.times. SSC to 2.0.times. SSC, 0.1% SDS at 50-65.degree. C. For
example, high stringency is about 0.2.times. SSC, 0.1% SDS at
65.degree. C. Ultra-high stringency will be the same conditions
except the wash temperature is raised about 3 or about 5.degree.
C., and ultra-ultra-high stringency will be the same conditions
except the wash temperature is raised about 6 or about 9.degree. C.
For identification of less closely related homologues washes can be
performed at a lower temperature, e.g., 500 C. In general,
stringency is increased by raising the wash temperature and/or
decreasing the concentration of SSC, as known in the art.
[0075] As another example, stringent conditions can be selected
such that an oligonucleotide that is perfectly complementary to the
coding oligonucleotide hybridizes to the coding oligonucleotide
with at least about a 5-10.times.higher signal to noise ratio than
the ratio for hybridization of the perfectly complementary
oligonucleotide to a nucleic acid encoding a transcription factor
known as of the filing date of the application. Conditions can be
selected such that a higher signal to noise ratio is observed in
the particular assay which is used, e.g., about 15.times.,
25.times., 35.times., 50.times. or more. Accordingly, the subject
nucleic acid hybridizes to the unique coding oligonucleotide with
at least a 2.times. higher signal to noise ratio as compared to
hybridization of the coding oligonucleotide to a nucleic acid
encoding known polypeptide. Again, higher signal to noise ratios
can be selected, e.g., about 5.times., 10.times., 25.times.,
35.times., 50.times. or more. The particular signal will depend on
the label used in the relevant assay, e.g., a fluorescent label, a
colorimetric label, a radioactive label, or the like.
[0076] Alternatively, transcription factor homolog polypeptides can
be obtained by screening an expression library using antibodies
specific for one or more transcription factors. With the provision
herein of the disclosed transcription factor, and transcription
factor homologue nucleic acid sequences, the encoded polypeptide(s)
can be expressed and purified in a heterologous expression system
(e.g., E. coli) and used to raise antibodies (monoclonal or
polyclonal) specific for the polypeptide(s) in question. Antibodies
can also be raised against synthetic peptides derived from
transcription factor, or transcription factor homologue, amino acid
sequences. Methods of raising antibodies are well known in the art
and are described in Harlow and Lane (1988) Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York. Such
antibodies can then be used to screen an expression library
produced from the plant from which it is desired to clone
additional transcription factor homologues, using the methods
described above. The selected cDNAs can be confirmed by sequencing
and enzymatic activity.
[0077] Sequence Variations
[0078] It will readily be appreciated by those of skill in the art,
that any of a variety of polynucleotide sequences are capable of
encoding the transcription factors and transcription factor
homologue polypeptides of the invention. Due to the degeneracy of
the genetic code, many different polynucleotides can encode
identical and/or substantially similar polypeptides in addition to
those sequences illustrated in the Sequence Listing.
[0079] For example, Table 1 illustrates, e.g., that the codons AGC,
AGT, TCA, TCC, TCG, and TCT all encode the same amino acid: serine.
Accordingly, at each position in the sequence where there is a
codon encoding serine, any of the above trinucleotide sequences can
be used without altering the encoded polypeptide.
1TABLE 1 Amino acid Possible Codons Alanine Ala A GCA GCC GCG GCU
Cysteine Cys C TGC TGT Aspartic acid Asp D GAC GAT Glutamic acid
Glu E GAA GAG Phenylalanine Phe F TTC TTT Glycine Gly G GGA GGC GGG
GGT Histidine His H CAC CAT Isoleucine Ile I ATA ATC ATT Lysine Lys
K AAA AAG Leucine Leu L TTA TTG CTA CTC CTG CTT Methionine Met M
ATG Asparagine Asn N AAC AAT Proline Pro P CCA CCC CCG CCT
Glutamine Gln Q CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGT
Serine Ser S AGC AGT TCA TCC TCG TCT Threonine Thr T ACA ACC ACG
ACT Valine Val V GTA GTC GTG GTT Tryptophan Trp W TGG Tyrosine Tyr
Y TAC TAT
[0080] Sequence alterations that do not change the amino acid
sequence encoded by the polynucleotide are termed "silent"
variations. With the exception of the codons ATG and TGG, encoding
methionine and tryptophan, respectively, any of the possible codons
for the same amino acid can be substituted by a variety of
techniques, e.g., site-directed mutagenesis, available in the art.
Accordingly, any and all such variations of a sequence selected
from the above table are a feature of the invention.
[0081] In addition to silent variations, other conservative
variations that alter one, or a few amino acids in the encoded
polypeptide, can be made without altering the function of the
polypeptide, these conservative variants are, likewise, a feature
of the invention.
[0082] For example, substitutions, deletions and insertions
introduced into the sequences provided in the Sequence Listing are
also envisioned by the invention. Such sequence modifications can
be engineered into a sequence by site-directed mutagenesis (Wu
(ed.) Meth. Enzymol. (1993) vol. 217, Academic Press) or the other
methods noted below. Amino acid substitutions are typically of
single residues; insertions usually will be on the order of about
from 1 to 10 amino acid residues; and deletions will range about
from 1 to 30 residues. In preferred embodiments, deletions or
insertions are made in adjacent pairs, e.g., a deletion of two
residues or insertion of two residues. Substitutions, deletions,
insertions or any combination thereof can be combined to arrive at
a sequence. The mutations that are made in the polynucleotide
encoding the transcription factor should not place the sequence out
of reading frame and should not create complementary regions that
could produce secondary mRNA structure. Preferably, the polypeptide
encoded by the DNA performs the desired function.
[0083] Conservative substitutions are those in which at least one
residue in the amino acid sequence has been removed and a different
residue inserted in its place. Such substitutions generally are
made in accordance with the Table 2 when it is desired to maintain
the activity of the protein. Table 2 shows amino acids which can be
substituted for an amino acid in a protein and which are typically
regarded as conservative substitutions.
2 TABLE 2 Conservative Residue 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
[0084] Similar substitutions are those in which at least one
residue in the amino acid sequence has been removed and a different
residue inserted in its place. Such substitutions generally are
made in accordance with the Table 3 when it is desired to maintain
the activity of the protein. Table 3 shows amino acids which can be
substituted for an amino acid in a protein and which are typically
regarded as structural and functional substitutions. For example, a
residue in column 1 of Table 3 may be substituted with residue in
column 2; in addition, a residue in column 2 of Table 3 may be
substituted with the residue of column 1.
3 TABLE 3 Residue Similar Substitutions Ala Ser; Thr; Gly; Val;
Leu; Ile Arg Lys; His; Gly Asn Gln; His; Gly; Ser; Thr Asp Glu,
Ser; Thr Gln Asn; Ala Cys Ser; Gly Glu Asp Gly Pro; Arg His Asn;
Gln; Tyr; Phe; Lys; Arg Ile Ala; Leu; Val; Gly; Met Leu Ala; Ile;
Val; Gly; Met Lys Arg; His; Gln; Gly; Pro Met Leu; Ile; Phe Phe
Met; Leu; Tyr; Trp; His; Val; Ala Ser Thr; Gly; Asp; Ala; Val; Ile;
His Thr Ser; Val; Ala; Gly Trp Tyr; Phe; His Tyr Trp; Phe; His Val
Ala; Ile; Leu; Gly; Thr; Ser; Glu
[0085] Substitutions that are less conservative than those in Table
2 or Table 3 can be selected by picking residues that differ more
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. The substitutions which in general are expected
to produce the greatest changes in protein properties will be those
in which (a) a hydrophilic residue, e.g., seryl or threonyl, is
substituted for (or by) a hydrophobic residue, e.g., leucyl,
isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline
is substituted for (or by) any other residue; (c) a residue having
an electropositive side chain, e.g., lysyl, arginyl, or histidyl,
is substituted for (or by) an electronegative residue, e.g.,
glutamyl or aspartyl; or (d) a residue having a bulky side chain,
e.g., phenylalanine, is substituted for (or by) one not having a
side chain, e.g., glycine.
[0086]
[0087] Further Modifying Sequences of the
Invention--Mutation/Forced Evolution
[0088] In addition to generating silent or conservative
substitutions as noted, above, the present invention optionally
includes methods of modifying the sequences of the Sequence
Listing. In the methods, nucleic acid or protein modification
methods are used to alter the given sequences to produce new
sequences and/or to chemically or enzymatically modify given
sequences to change the properties of the nucleic acids or
proteins. These sequences may be synthetic polynuceotides and
synthetic polypetides.
[0089] Thus, in one embodiment, given nucleic acid sequences are
modified, e.g., according to standard mutagenesis or artificial
evolution methods to produce modified sequences. For example,
Ausubel, supra, provides additional details on mutagenesis methods.
Artificial forced evolution methods are described, e.g., by Stemmer
(1994) Nature 370:389-391, and Stemmer (1994) Proc. Natl. Acad.
Sci. USA 91:10747-10751, and U.S. Pat. Nos. 5,811,238, 5,837,500,
and 6,242,568. Many other mutation and evolution methods are also
available and expected to be within the skill of the practitioner.
A synthetic polypeptide encoded by a synthetic polynucleotide may
have as little as 20% amino acid residue sequence identity to the
polypeptide encoded by the claimed polynucleotides and still modify
a plant's trait or characteristic.
[0090] Similarly, chemical or enzymatic alteration of expressed
nucleic acids and polypeptides can be performed by standard
methods. For example, sequence can be modified by addition of
lipids, sugars, peptides, organic or inorganic compounds, by the
inclusion of modified nucleotides or amino acids, or the like. For
example, protein modification techniques are illustrated in
Ausubel, supra. Further details on chemical and enzymatic
modifications can be found herein. These modification methods can
be used to modify any given sequence, or to modify any sequence
produced by the various mutation and artificial evolution
modification methods noted herein.
[0091] Accordingly, the invention provides for modification of any
given nucleic acid by mutation, evolution, chemical or enzymatic
modification, or other available methods, as well as for the
products produced by practicing such methods, e.g., using the
sequences herein as a starting substrate for the various
modification approaches.
[0092] For example, optimized coding sequence containing codons
preferred by a particular prokaryotic or eukaryotic host can be
used e.g., to increase the rate of translation or to produce
recombinant RNA transcripts having desirable properties, such as a
longer half-life, as compared with transcripts produced using a
non-optimized sequence. Translation stop codons can also be
modified to reflect host preference. For example, preferred stop
codons for S. cerevisiae and mammals are TAA and TGA, respectively.
The preferred stop codon for monocotyledonous plants is TGA,
whereas insects and E. coli prefer to use TAA as the stop
codon.
[0093] The polynucleotide sequences of the present invention can
also be engineered in order to alter a coding sequence for a
variety of reasons, including but not limited to, alterations which
modify the sequence to facilitate cloning, processing and/or
expression of the gene product. For example, alterations are
optionally introduced using techniques which are well known in the
art, e.g., site-directed mutagenesis, to insert new restriction
sites, to alter glycosylation patterns, to change codon preference,
to introduce splice sites, etc.
[0094] Furthermore, a fragment or domain derived from any of the
polypeptides of the invention can be combined with domains derived
from other transcription factors or synthetic domains to modify the
biological activity of a transcription factor. For instance, a
DNA-binding domain derived from a transcription factor of the
invention can be combined with the activation domain of another
transcription factor or with a synthetic activation domain. A
transcription activation domain assists in initiating transcription
from a DNA-binding site. Examples include the transcription
activation region of VP16 or GAL4 (Moore et al. (1998) Proc. Natl.
Acad. Sci. USA 95: 376-381; and Aoyama et al. (1995) Plant Cell
7:1773-1785), peptides derived from bacterial sequences (Ma and
Ptashne (1987) Cell 51; 113-119) and synthetic peptides (Giniger
and Ptashne, (1987) Nature 330:670-672).
[0095] Expression and Modification of Polypeptides
[0096] Typically, polynucleotide sequences of the invention are
incorporated into recombinant DNA (or RNA) molecules that direct
expression of polypeptides of the invention in appropriate host
cells, transgenic plants, in vitro translation systems, or the
like. Due to the inherent degeneracy of the genetic code, nucleic
acid sequences which encode substantially the same or a
functionally equivalent amino acid sequence can be substituted for
any listed sequence to provide for cloning and expressing the
relevant homologue.
[0097] Vectors, Promoters, and Expression Systems
[0098] The present invention includes recombinant constructs
comprising one or more of the nucleic acid sequences herein. The
constructs typically comprise a vector, such as a plasmid, a
cosmid, a phage, a virus (e.g., a plant virus), a bacterial
artificial chromosome (BAC), a yeast artificial chromosome (YAC),
or the like, into which a nucleic acid sequence of the invention
has been inserted, in a forward or reverse orientation. In a
preferred aspect of this embodiment, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and are
commercially available.
[0099] General texts which describe molecular biological techniques
useful herein, including the use and production of vectors,
promoters and many other relevant topics, include Berger, Sambrook
and Ausubel, supra. Any of the identified sequences can be
incorporated into a cassette or vector, e.g., for expression in
plants. A number of expression vectors suitable for stable
transformation of plant cells or for the establishment of
transgenic plants have been described including those described in
Weissbach and Weissbach, (1989) Methods for Plant Molecular
Biology, Academic Press, and Gelvin et al., (1990) Plant Molecular
Biology Manual, Kluwer Academic Publishers. Specific examples
include those derived from a Ti plasmid of Agrobacterium
tumefaciens, as well as those disclosed by Herrera-Estrella et al.
(1983) Nature 303: 209, Bevan (1984) Nucl Acid Res. 12: 8711-8721,
Klee (1985) Bio/Technology 3: 637-642, for dicotyledonous
plants.
[0100] Alternatively, non-Ti vectors can be used to transfer the
DNA into monocotyledonous plants and cells by using free DNA
delivery techniques. Such methods can involve, for example, the use
of liposomes, electroporation, microprojectile bombardment, silicon
carbide whiskers, and viruses. By using these methods transgenic
plants such as wheat, rice (Christou (1991) Bio/Technology 9:
957-962) and corn (Gordon-Kamm (1990) Plant Cell 2: 603-618) can be
produced. An immature embryo can also be a good target tissue for
monocots for direct DNA delivery techniques by using the particle
gun (Weeks et al. (1993) Plant Physiol 102: 1077-1084; Vasil (1993)
Bio/Technology 10: 667-674; Wan and Lemeaux (1994) Plant Physiol
104: 37-48, and for Agrobacterium-mediated DNA transfer (Ishida et
al. (1996) Nature Biotech 14: 745-750).
[0101] Typically, plant transformation vectors include one or more
cloned plant coding sequence (genomic or cDNA) under the
transcriptional control of 5' and 3' regulatory sequences and a
dominant selectable marker. Such plant transformation vectors
typically also contain a promoter (e.g., a regulatory region
controlling inducible or constitutive, environmentally-or
developmentally-regulated, or cell- or tissue-specific expression),
a transcription initiation start site, an RNA processing signal
(such as intron splice sites), a transcription termination site,
and/or a polyadenylation signal.
[0102] Examples of constitutive plant promoters which can be useful
for expressing the TF sequence include: the cauliflower mosaic
virus (CaMV) 35S promoter, which confers constitutive, high-level
expression in most plant tissues (see, e.g., Odel et al. (1985)
Nature 313:810); the nopaline synthase promoter (An et al. (1988)
Plant Physiol 88:547); and the octopine synthase promoter (Fromm et
al. (1989) Plant Cell 1: 977).
[0103] A variety of plant gene promoters that regulate gene
expression in response to environmental, hormonal, chemical,
developmental signals, and in a tissue-active manner can be used
for expression of a TF sequence in plants. Choice of a promoter is
based largely on the phenotype of interest and is determined by
such factors as tissue (e.g., seed, fruit, root, pollen, vascular
tissue, flower, carpel, etc.), inducibility (e.g., in response to
wounding, heat, cold, drought, light, pathogens, etc.), timing,
developmental stage, and the like. Numerous known promoters have
been characterized and can favorable be employed to promote
expression of a polynucleotide of the invention in a transgenic
plant or cell of interest. For example, tissue specific promoters
include: seed-specific promoters (such as the napin, phaseolin or
DC3 promoter described in U.S. Pat. No. 5,773,697), fruit-specific
promoters that are active during fruit ripening (such as the dru 1
promoter (U.S. Pat. No. 5,783,393), or the 2A11 promoter (U.S. Pat.
No. 4,943,674) and the tomato polygalacturonase promoter (Bird et
al. (1988) Plant Mol Biol 11:651), root-specific promoters, such as
those disclosed in U.S. Pat. Nos. 5,618,988, 5,837,848 and
5,905,186, pollen-active promoters such as PTA29, PTA26 and PTA13
(U.S. Pat. No. 5,792,929), promoters active in vascular tissue
(Ringli and Keller (1998) Plant Mol Biol 37:977-988),
flower-specific (Kaiser et al, (1995) Plant Mol Biol 28:231-243),
pollen (Baerson et al. (1994) Plant Mol Biol 26:1947-1959), carpels
(Ohl et al. (1990) Plant Cell 2:837-848), pollen and ovules
(Baerson et al. (1993) Plant Mol Biol 22:255-267), auxin-inducible
promoters (such as that described in van der Kop et al. (1999)
Plant Mol Biol 39:979-990 or Baumann et al. (1999) Plant Cell
11:323-334), cytokinin-inducible promoter (Guevara-Garcia (1998)
Plant Mol Biol 38:743-753), promoters responsive to gibberellin
(Shi et al. (1998) Plant Mol Biol 38:1053-1060, Willmott et al.
(1998) 38:817-825) and the like. Additional promoters are those
that elicit expression in response to heat (Ainley et al. (1993)
Plant Mol Biol 22: 13-23), light (e.g., the pea rbcS-3A promoter,
Kuhlemeier et al. (1989) Plant Cell 1:471, and the maize rbcS
promoter, Schaffner and Sheen (1991) Plant Cell 3: 997); wounding
(e.g., wunI, Siebertz et al. (1989) Plant Cell 1: 961); pathogens
(such as the PR-1 promoter described in Buchel et al. (1999) Plant
Mol. Biol. 40:387-396, and the PDF1.2 promoter described in Manners
et al. (1998) Plant Mol. Biol. 38:1071-80), and chemicals such as
methyl jasmonate or salicylic acid (Gatz et al. (1997) Annu Rev
Plant Physiol Plant Mol Biol 48: 89-108). In addition, the timing
of the expression can be controlled by using promoters such as
those acting at senescence (Gan and Amasino (1995) Science 270:
1986-1988); or late seed development (Odell et al. (1994) Plant
Physiol 106:447-458).
[0104] Plant expression vectors can also include RNA processing
signals that can be positioned within, upstream or downstream of
the coding sequence. In addition, the expression vectors can
include additional regulatory sequences from the 3'-untranslated
region of plant genes, e.g., a 3' terminator region to increase
mRNA stability of the mRNA, such as the PI-II terminator region of
potato or the octopine or nopaline synthase 3' terminator
regions.
[0105] Additional Expression Elements
[0106] Specific initiation signals can aid in efficient translation
of coding sequences. These signals can include, e.g., the ATG
initiation codon and adjacent sequences. In cases where a coding
sequence, its initiation codon and upstream sequences are inserted
into the appropriate expression vector, no additional translational
control signals may be needed. However, in cases where only coding
sequence (e.g., a mature protein coding sequence), or a portion
thereof, is inserted, exogenous transcriptional control signals
including the ATG initiation codon can be separately provided. The
initiation codon is provided in the correct reading frame to
facilitate transcription. Exogenous transcriptional elements and
initiation codons can be of various origins, both natural and
synthetic. The efficiency of expression can be enhanced by the
inclusion of enhancers appropriate to the cell system in use.
[0107] Expression Hosts
[0108] The present invention also relates to host cells which are
transduced with vectors of the invention, and the production of
polypeptides of the invention (including fragments thereof) by
recombinant techniques. Host cells are genetically engineered (i.e,
nucleic acids are introduced, e.g., transduced, transformed or
transfected) with the vectors of this invention, which may be, for
example, a cloning vector or an expression vector comprising the
relevant nucleic acids herein. The vector is optionally a plasmid,
a viral particle, a phage, a naked nucleic acid, etc. The
engineered host cells can be cultured in conventional nutrient
media modified as appropriate for activating promoters, selecting
transformants, or amplifying the relevant gene. The culture
conditions, such as temperature, pH and the like, are those
previously used with the host cell selected for expression, and
will be apparent to those skilled in the art and in the references
cited herein, including, Sambrook and Ausubel.
[0109] The host cell can be a eukaryotic cell, such as a yeast
cell, or a plant cell, or the host cell can be a prokaryotic cell,
such as a bacterial cell. Plant protoplasts are also suitable for
some applications. For example, the DNA fragments are introduced
into plant tissues, cultured plant cells or plant protoplasts by
standard methods including electroporation (Fromm et al., (1985)
Proc. Natl. Acad. Sci. USA 82, 5824, infection by viral vectors
such as cauliflower mosaic virus (CaMV) (Hohn et al., (1982)
Molecular Biology of Plant Tumors, (Academic Press, New York) pp.
549-560; U.S. Pat. No. 4,407,956), high velocity ballistic
penetration by small particles with the nucleic acid either within
the matrix of small beads or particles, or on the surface (Klein et
al., (1987) Nature 327, 70-73), use of pollen as vector (WO
85/01856), or use of Agrobacterium tumefaciens or A. rhizogenes
carrying a T-DNA plasmid in which DNA fragments are cloned. The
T-DNA plasmid is transmitted to plant cells upon infection by
Agrobacterium tumefaciens, and a portion is stably integrated into
the plant genome (Horsch et al. (1984) Science 233:496-498; Fraley
et al. (1983) Proc. Natl. Acad. Sci. USA 80, 4803).
[0110] The cell can include a nucleic acid of the invention which
encodes a polypeptide, wherein the cells expresses a polypeptide of
the invention. The cell can also include vector sequences, or the
like. Furthermnore, cells and transgenic plants which include any
polypeptide or nucleic acid above or throughout this specification,
e.g., produced by transduction of a vector of the invention, are an
additional feature of the invention.
[0111] For long-term, high-yield production of recombinant
proteins, stable expression can be used. Host cells transformed
with a nucleotide sequence encoding a polypeptide of the invention
are optionally cultured under conditions suitable for the
expression and recovery of the encoded protein from cell culture.
The protein or fragment thereof produced by a recombinant cell may
be secreted, membrane-bound, or contained intracellularly,
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides encoding mature proteins of the
invention can be designed with signal sequences which direct
secretion of the mature polypeptides through a prokaryotic or
eukaryotic cell membrane.
[0112] Modified Amino Acids
[0113] Polypeptides of the invention may contain one or more
modified amino acids. The presence of modified amino acids may be
advantageous in, for example, increasing polypeptide half-life,
reducing polypeptide antigenicity or toxicity, increasing
polypeptide storage stability, or the like. Amino acid(s) are
modified, for example, co-translationally or post-translationally
during recombinant production or modified by synthetic or chemical
means.
[0114] Non-limiting examples of a modified amino acid include
incorporation or other use of acetylated amino acids, glycosylated
amino acids, sulfated amino acids, prenylated (e.g., famesylated,
geranylgeranylated) amino acids, PEG modified (e.g., "PEGylated")
amino acids, biotinylated amino acids, carboxylated amino acids,
phosphorylated amino acids, etc. References adequate to guide one
of skill in the modification of amino acids are replete throughout
the literature.
[0115] The modified amino acids may prevent or increase affinity of
the polypeptide for another molecule, including, but not limited
to, polynucleotide, proteins, carbohydrates, lipids and lipid
derivatives, and other organic or synthetic compounds.
[0116] Identification of Additional Factors
[0117] A transcription factor provided by the present invention can
also be used to identify additional endogenous or exogenous
molecules that can affect a phentoype or trait of interest. On the
one hand, such molecules include organic (small or large molecules)
and/or inorganic compounds that affect expression of (i.e.,
regulate) a particular transcription factor. Alternatively, such
molecules include endogenous molecules that are acted upon either
at a transcriptional level by a transcription factor of the
invention to modify a phenotype as desired. For example, the
transcription factors can be employed to identify one or more
downstream gene with which is subject to a regulatory effect of the
transcription factor. In one approach, a transcription factor or
transcription factor homologue of the invention is expressed in a
host cell, e.g, a transgenic plant cell, tissue or explant, and
expression products, either RNA or protein, of likely or random
targets are monitored, e.g., by hybridization to a microarray of
nucleic acid probes corresponding to genes expressed in a tissue or
cell type of interest, by two-dimensional gel electrophoresis of
protein products, or by any other method known in the art for
assessing expression of gene products at the level of RNA or
protein. Alternatively, a transcription factor of the invention can
be used to identify promoter sequences (i.e., binding sites)
involved in the regulation of a downstream target. After
identifying a promoter sequence, interactions between the
transcription factor and the promoter sequence can be modified by
changing specific nucleotides in the promoter sequence or specific
amino acids in the transcription factor that interact with the
promoter sequence to alter a plant trait. Typically, transcription
factor DNA-binding sites are identified by gel shift assays. After
identifying the promoter regions, the promoter region sequences can
be employed in double-stranded DNA arrays to identify molecules
that affect the interactions of the transcription factors with
their promoters (Bulyk et al. (1999) Nature Biotechnology
17:573-577). A test promoter region element of a transcription
factor gene may also be screened using a phage-display analysis and
a phage library which comprises polynucleotides encoding any
transcription factor to identify a transcription factor so encoded
which binds to the test promoter region element. Such phage-display
methods are well known in the art.
[0118] The identified transcription factors are also useful to
identify proteins that modify the activity of the transcription
factor. Such modification can occur by covalent modification, such
as by phosphorylation, or by protein-protein (homo
or-heteropolymer) interactions. Any method suitable for detecting
protein-protein interactions can be employed. Among the methods
that can be employed are co-immunoprecipitation, cross-linking and
co-purification through gradients or chromatographic columns, and
the two-hybrid yeast system.
[0119] The two-hybrid system detects protein interactions in vivo
and is described in Chien, et al., (1991), Proc. Natl. Acad. Sci.
USA 88, 9578-9582 and is commercially available from Clontech (Palo
Alto, Calif.). In such a system, plasmids are constructed that
encode two hybrid proteins: one consists of the DNA-binding domain
of a transcription activator protein fused to the TF polypeptide
and the other consists of the transcription activator protein's
activation domain fused to an unknown protein that is encoded by a
cDNA that has been recombined into the plasmid as part of a cDNA
library. The DNA-binding domain fusion plasmid and the cDNA library
are transformed into a strain of the yeast Saccharomyces cerevisiae
that contains a reporter gene (e.g., lacZ) whose regulatory region
contains the transcription activator's binding site. Either hybrid
protein alone cannot activate transcription of the reporter gene.
Interaction of the two hybrid proteins reconstitutes the functional
activator protein and results in expression of the reporter gene,
which is detected by an assay for the reporter gene product. Then,
the library plasmids responsible for reporter gene expression are
isolated and sequenced to identify the proteins encoded by the
library plasmids. After identifying proteins that interact with the
transcription factors, assays for compounds that interfere with the
TF protein-protein interactions can be preformed.
[0120] Identification of Modulators
[0121] In addition to the intracellular molecules described above,
extracellular molecules that alter activity or expression of a
transcription factor, either directly or indirectly, can be
identified. For example, the methods can entail first placing a
candidate molecule in contact with a plant or plant cell. The
molecule can be introduced by topical administration, such as
spraying or soaking of a plant, and then the molecule's effect on
the expression or activity of the TF polypeptide or the expression
of the polynucleotide monitored. Changes in the expression of the
TF polypeptide can be monitored by use of polyclonal or monoclonal
antibodies, gel electrophoresis or the like. Changes in the
expression of the corresponding polynucleotide sequence can be
detected by use of microarrays, Northerns, quantitative PCR, or any
other technique for monitoring changes in mRNA expression. These
techniques are exemplified in Ausubel et al. (eds) Current
Protocols in Molecular Biology, John Wiley & Sons (1998, and
supplements through 2001). Such changes in the expression levels
can be correlated with modified plant traits and thus identified
molecules can be useful for soaking or spraying on fruit, vegetable
and grain crops to modify traits in plants.
[0122] Essentially any available composition can be tested for
modulatory activity of expression or activity of any nucleic acid
or polypeptide herein. Thus, available libraries of compounds such
as chemicals, polypeptides, nucleic acids and the like can be
tested for modulatory activity. Often, potential modulator
compounds can be dissolved in aqueous or organic (e.g., DMSO-based)
solutions for easy delivery to the cell or plant of interest in
which the activity of the modulator is to be tested. Optionally,
the assays are designed to screen large modulator composition
libraries by automating the assay steps and providing compounds
from any convenient source to assays, which are typically run in
parallel (e.g., in microtitre formats on microtitre plates in
robotic assays).
[0123] In one embodiment, high throughput screening methods involve
providing a combinatorial library containing a large number of
potential compounds (potential modulator compounds). Such
"combinatorial chemical libraries" are then screened in one or more
assays, as described herein, to identify those library members
(particular chemical species or subclasses) that display a desired
characteristic activity. The compounds thus identified can serve as
target compounds.
[0124] A combinatorial chemical library can be, e.g., a collection
of diverse chemical compounds generated by chemical synthesis or
biological synthesis. For example, a combinatorial chemical library
such as a polypeptide library is formed by combining a set of
chemical building blocks (e.g., in one example, amino acids) in
every possible way for a given compound length (i.e., the number of
amino acids in a polypeptide compound of a set length). Exemplary
libraries include peptide libraries, nucleic acid libraries,
antibody libraries (see, e.g., Vaughn et al. (1996) Nature
Biotechnology, 14(3):309-314 and PCT/US96/10287), carbohydrate
libraries (see, e.g., Liang et al. Science (1996) 274:1520-1522 and
U.S. Pat. No. 5,593,853), peptide nucleic acid libraries (see,
e.g., U.S. Pat. No. 5,539,083), and small organic molecule
libraries (see, e.g., benzodiazepines, Baum C&EN January 18,
page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;
thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;
pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholino
compounds, U.S. Pat. No. 5,506,337) and the like.
[0125] Preparation and screening of combinatorial or other
libraries is well known to those of skill in the art. Such
combinatorial chemical libraries include, but are not limited to,
peptide libraries (see, e.g., U.S. Pat. No. 5,010,175, Furka, Int.
J. Pept. Prot. Res. 37:487-493 (1991) and Houghton et al. Nature
354:84-88 (1991)). Other chemistries for generating chemical
diversity libraries can also be used.
[0126] In addition, as noted, compound screening equipment for
high-throughput screening is generally available, e.g., using any
of a number of well known robotic systems that have also been
developed for solution phase chemistries useful in assay systems.
These systems include automated workstations including an automated
synthesis apparatus and robotic systems utilizing robotic arms. Any
of the above devices are suitable for use with the present
invention, e.g., for high-throughput screening of potential
modulators. The nature and implementation of modifications to these
devices (if any) so that they can operate as discussed herein will
be apparent to persons skilled in the relevant art.
[0127] Indeed, entire high throughput screening systems are
commercially available. These systems typically automate entire
procedures including all sample and reagent pipetting, liquid
dispensing, timed incubations, and final readings of the microplate
in detector(s) appropriate for the assay. These configurable
systems provide high throughput and rapid start up as well as a
high degree of flexibility and customization. Similarly,
microfluidic implementations of screening are also commercially
available.
[0128] The manufacturers of such systems provide detailed protocols
the various high throughput. Thus, for example, Zymark Corp.
provides technical bulletins describing screening systems for
detecting the modulation of gene transcription, ligand binding, and
the like. The integrated systems herein, in addition to providing
for sequence alignment and, optionally, synthesis of relevant
nucleic acids, can include such screening apparatus to identify
modulators that have an effect on one or more polynucleotides or
polypeptides according to the present invention.
[0129] In some assays it is desirable to have positive controls to
ensure that the components of the assays are working properly. At
least two types of positive controls are appropriate. That is,
known transcriptional activators or inhibitors can be incubated
with cells/plants/etc. in one sample of the assay, and the
resulting increase/decrease in transcription can be detected by
measuring the resulting increase in RNA/protein expression, etc.,
according to the methods herein. It will be appreciated that
modulators can also be combined with transcriptional activators or
inhibitors to find modulators which inhibit transcriptional
activation or transcriptional repression. Either expression of the
nucleic acids and proteins herein or any additional nucleic acids
or proteins activated by the nucleic acids or proteins herein, or
both, can be monitored.
[0130] In an embodiment, the invention provides a method for
identifying compositions that modulate the activity or expression
of a polynucleotide or polypeptide of the invention. For example, a
test compound, whether a small or large molecule, is placed in
contact with a cell, plant (or plant tissue or explant), or
composition comprising the polynucleotide or polypeptide of
interest and a resulting effect on the cell, plant, (or tissue or
explant) or composition is evaluated by monitoring, either directly
or indirectly, one or more of: expression level of the
polynucleotide or polypeptide, activity (or modulation of the
activity) of the polynucleotide or polypeptide. In some cases, an
alteration in a plant phenotype can be detected following contact
of a plant (or plant cell, or tissue or explant) with the putative
modulator, e.g., by modulation of expression or activity of a
polynucleotide or polypeptide of the invention. Modulation of
expression or activity of a polynucleotide or polypeptide of the
invention may also be caused by molecular elements in a signal
transduction second messenger pathway and such modulation can
affect similar elements in the same or another signal transduction
second messenger pathway.
[0131] Subsequences
[0132] Also contemplated are uses of polynucleotides, also referred
to herein as oligonucleotides, typically having at least 12 bases,
preferably at least 15, more preferably at least 20, 30, or 50
bases, which hybridize under at least highly stringent (or
ultra-high stringent or ultra-ultra- high stringent conditions)
conditions to a polynucleotide sequence described above. The
polynucleotides may be used as probes, primers, sense and antisense
agents, and the like, according to methods as noted supra.
[0133] Subsequences of the polynucleotides of the invention,
including polynucleotide fragments and oligonucleotides are useful
as nucleic acid probes and primers. An oligonucleotide suitable for
use as a probe or primer is at least about 15 nucleotides in
length, more often at least about 18 nucleotides, often at least
about 21 nucleotides, frequently at least about 30 nucleotides, or
about 40 nucleotides, or more in length. A nucleic acid probe is
useful in hybridization protocols, e.g., to identify additional
polypeptide homologues of the invention, including protocols for
microarray experiments. Primers can be annealed to a complementary
target DNA strand by nucleic acid hybridization to form a hybrid
between the primer and the target DNA strand, and then extended
along the target DNA strand by a DNA polymerase enzyme. Primer
pairs can be used for amplification of a nucleic acid sequence,
e.g., by the polymerase chain reaction (PCR) or other nucleic-acid
amplification methods. See Sambrook and Ausubel, supra.
[0134] In addition, the invention includes an isolated or
recombinant polypeptide including a subsequence of at least about
15 contiguous amino acids encoded by the recombinant or isolated
polynucleotides of the invention. For example, such polypeptides,
or domains or fragments thereof, can be used as immunogens, e.g.,
to produce antibodies specific for the polypeptide sequence, or as
probes for detecting a sequence of interest. A subsequence can
range in size from about 15 amino acids in length up to and
including the full length of the polypeptide.
[0135] Production of Transgenic Plants
[0136] Modification of Traits
[0137] The polynucleotides of the invention are favorably employed
to produce transgenic plants with various traits, or
characteristics, that have been modified in a desirable manner,
e.g., to improve the seed characteristics of a plant. For example,
alteration of expression levels or patterns (e.g., spatial or
temporal expression patterns) of one or more of the transcription
factors (or transcription factor homologues) of the invention, as
compared with the levels of the same protein found in a wild type
plant, can be used to modify a plant's traits. An illustrative
example of trait modification, improved characteristics, by
altering expression levels of a particular transcription factor is
described fuirther in the Examples and the Sequence Listing.
[0138] Antisense and Cosuppression Approaches
[0139] In addition to expression of the nucleic acids of the
invention as gene replacement or plant phenotype modification
nucleic acids, the nucleic acids are also useful for sense and
anti-sense suppression of expression, e.g., to down-regulate
expression of a nucleic acid of the invention, e.g., as a further
mechanism for modulating plant phenotype. That is, the nucleic
acids of the invention, or subsequences or anti-sense sequences
thereof, can be used to block expression of naturally occurring
homologous nucleic acids. A variety of sense and anti-sense
technologies are known in the art, e.g., as set forth in
Lichtenstein and Nellen (1997) Antisense Technology: A Practical
Approach IRL Press at Oxford University, Oxford, England. In
general, sense or anti-sense sequences are introduced into a cell,
where they are optionally amplified, e.g., by transcription. Such
sequences include both simple oligonucleotide sequences and
catalytic sequences such as ribozymes.
[0140] For example, a reduction or elimination of expression (i.e.,
a "knock-out") of a transcription factor or transcription factor
homologue polypeptide in a transgenic plant, e.g., to modify a
plant trait, can be obtained by introducing an antisense construct
corresponding to the polypeptide of interest as a cDNA. For
antisense suppression, the transcription factor or homologue cDNA
is arranged in reverse orientation (with respect to the coding
sequence) relative to the promoter sequence in the expression
vector. The introduced sequence need not be the full length cDNA or
gene, and need not be identical to the cDNA or gene found in the
plant type to be transformed. Typically, the antisense sequence
need only be capable of hybridizing to the target gene or RNA of
interest. Thus, where the introduced sequence is of shorter length,
a higher degree of homology to the endogenous transcription factor
sequence will be needed for effective antisense suppression. While
antisense sequences of various lengths can be utilized, preferably,
the introduced antisense sequence in the vector will be at least 30
nucleotides in length, and improved antisense suppression will
typically be observed as the length of the antisense sequence
increases. Preferably, the length of the antisense sequence in the
vector will be greater than 100 nucleotides. Transcription of an
antisense construct as described results in the production of RNA
molecules that are the reverse complement of mRNA molecules
transcribed from the endogenous transcription factor gene in the
plant cell.
[0141] Suppression of endogenous transcription factor gene
expression can also be achieved using a ribozyme. Ribozymes are RNA
molecules that possess highly specific endoribonuclease activity.
The production and use of ribozymes are disclosed in U.S. Pat. No.
4,987,071 and U.S. Pat. No. 5,543,508. Synthetic ribozyme sequences
including antisense RNAs can be used to confer RNA cleaving
activity on the antisense RNA, such that endogenous MRNA molecules
that hybridize to the antisense RNA are cleaved, which in turn
leads to an enhanced antisense inhibition of endogenous gene
expression.
[0142] Vectors in which RNA encoded by a transcription factor or
transcription factor homologue cDNA is over-expressed can also be
used to obtain co-suppression of a corresponding endogenous gene,
e.g., in the manner described in U.S. Pat. No. 5,231,020 to
Jorgensen. Such co-suppression (also termed sense suppression) does
not require that the entire transcription factor cDNA be introduced
into the plant cells, nor does it require that the introduced
sequence be exactly identical to the endogenous transcription
factor gene of interest. However, as with antisense suppression,
the suppressive efficiency will be enhanced as specificity of
hybridization is increased, e.g., as the introduced sequence is
lengthened, and/or as the sequence similarity between the
introduced sequence and the endogenous transcription factor gene is
increased.
[0143] Vectors expressing an untranslatable form of the
transcription factor mRNA, e.g., sequences comprising one or more
stop codon, or nonsense mutation) can also be used to suppress
expression of an endogenous transcription factor, thereby reducing
or eliminating it's activity and modifying one or more traits.
Methods for producing such constructs are described in U.S. Pat.
No. 5,583,021. Preferably, such constructs are made by introducing
a premature stop codon into the transcription factor gene.
Alternatively, a plant trait can be modified by gene silencing
using double-strand RNA (Sharp (1999) Genes and Development 13:
139-141).
[0144] Another method for abolishing the expression of a gene is by
insertion mutagenesis using the T-DNA of Agrobacterium tumefaciens.
After generating the insertion mutants, the mutants can be screened
to identify those containing the insertion in a transcription
factor or transcription factor homologue gene. Plants containing a
single transgene insertion event at the desired gene can be crossed
to generate homozygous plants for the mutation (Koncz et al. (1992)
Methods in Arabidopsis Research. World Scientific).
[0145] Alternatively, a plant phenotype can be altered by
eliminating an endogenous gene, such as a transcription factor or
transcription factor homologue, e.g., by homologous recombination
(Kempin et al. (1997) Nature 389:802).
[0146] A plant trait can also be modified by using the Cre-lox
system (for example, as described in U.S. Pat. No. 5,658,772). A
plant genome can be modified to include first and second lox sites
that are then contacted with a Cre recombinase. If the lox sites
are in the same orientation, the intervening DNA sequence between
the two sites is excised. If the lox sites are in the opposite
orientation, the intervening sequence is inverted.
[0147] The polynucleotides and polypeptides of this invention can
also be expressed in a plant in the absence of an expression
cassette by manipulating the activity or expression level of the
endogenous gene by other means. For example, by ectopically
expressing a gene by T-DNA activation tagging (Ichikawa et al.
(1997) Nature 390 698-701; Kakimoto et al. (1996) Science 274:
982-985). This method entails transforming a plant with a gene tag
containing multiple transcriptional enhancers and once the tag has
inserted into the genome, expression of a flanking gene coding
sequence becomes deregulated. In another example, the
transcriptional machinery in a plant can be modified so as to
increase transcription levels of a polynucleotide of the invention
(See, e.g., PCT Publications WO 96/06166 and WO 98/53057 which
describe the modification of the DNA-binding specificity of zinc
finger proteins by changing particular amino acids in the
DNA-binding motif).
[0148] The transgenic plant can also include the machinery
necessary for expressing or altering the activity of a polypeptide
encoded by an endogenous gene, for example by altering the
phosphorylation state of the polypeptide to maintain it in an
activated state.
[0149] Transgenic plants (or plant cells, or plant explants, or
plant tissues) incorporating the polynucleotides of the invention
and/or expressing the polypeptides of the invention can be produced
by a variety of well established techniques as described above.
Following construction of a vector, most typically an expression
cassette, including a polynucleotide, e.g., encoding a
transcription factor or transcription factor homologue, of the
invention, standard techniques can be used to introduce the
polynucleotide into a plant, a plant cell, a plant explant or a
plant tissue of interest. Optionally, the plant cell, explant or
tissue can be regenerated to produce a transgenic plant.
[0150] The plant can be any higher plant, including gymnosperms,
monocotyledonous and dicotyledenous plants. Suitable protocols are
available for Leguminosae (alfalfa, soybean, clover, etc.),
Umbelliferae (carrot, celery, parsnip), Cruciferae (cabbage,
radish, rapeseed, broccoli, etc.), Curcurbitaceae (melons and
cucumber), Gramineae (wheat, corn, rice, barley, millet, etc.),
Solanaceae (potato, tomato, tobacco, peppers, etc.), and various
other crops. See protocols described in Ammirato et al. (1984)
Handbook of Plant Cell Culture--Crop Species, Macmillan Publ. Co.
Shimamoto et al. (1989) Nature 338:274-276; Fronim et al. (1990)
Bio/Technolog 8:833-839; and Vasil et al. (1990) Bio/Technology
8:429-434.
[0151] Transformation and regeneration of both monocotyledonous and
dicotyledonous plant cells is now routine, and the selection of the
most appropriate transformation technique will be determined by the
practitioner. The choice of method will vary with the type of plant
to be transformed; those skilled in the art will recognize the
suitability of particular methods for given plant types. Suitable
methods can include, but are not limited to: electroporation of
plant protoplasts; liposome-mediated transformation; polyethylene
glycol (PEG) mediated transformation; transformation using viruses;
micro-injection of plant cells; micro-projectile bombardment of
plant cells; vacuum infiltration; and Agrobacterium tumeficiens
mediated transformation. Transformation means introducing a
nucleotide sequence into a plant in a manner to cause stable or
transient expression of the sequence.
[0152] Successful examples of the modification of plant
characteristics by transformation with cloned sequences which serve
to illustrate the current knowledge in this field of technology,
and which are herein incorporated by reference, include: U.S. Pat.
Nos. 5,571,706; 5,677,175; 5,510,471; 5,750,386; 5,597,945;
5,589,615; 5,750,871; 5,268,526; 5,780,708; 5,538,880; 5,773,269;
5,736,369 and 5,610,042.
[0153] Following transformation, plants are preferably selected
using a dominant selectable marker incorporated into the
transformation vector. Typically, such a marker will confer
antibiotic or herbicide resistance on the transformed plants, and
selection of transformants can be accomplished by exposing the
plants to appropriate concentrations of the antibiotic or
herbicide.
[0154] After transformed plants are selected and grown to maturity,
those plants showing a modified trait are identified. The modified
trait can be any of those traits described above. Additionally, to
confirm that the modified trait is due to changes in expression
levels or activity of the polypeptide or polynucleotide of the
invention can be determined by analyzing mRNA expression using
Northern blots, RT-PCR or microarrays, or protein expression using
immunoblots or Western blots or gel shift assays.
[0155] Integrated Systems--Sequence Identity
[0156] Additionally, the present invention may be an integrated
system, computer or computer readable medium that comprises an
instruction set for determining the identity of one or more
sequences in a database. In addition, the instruction set can be
used to generate or identify sequences that meet any specified
criteria. Furthermore, the instruction set may be used to associate
or link certain functional benefits, such improved characteristics,
with one or more identified sequence.
[0157] For example, the instruction set can include, e.g., a
sequence comparison or other alignment program, e.g., an available
program such as, for example, the Wisconsin Package Version 10.0,
such as BLAST, FASTA, PILEUP, FINDPATTERNS or the like (GCG,
Madision, Wis.). Public sequence databases such as GenBank, EMBL,
Swiss-Prot and PIR or private sequence databases such as PhytoSeq
(Incyte Pharmaceuticals, Palo Alto, Calif.) can be searched.
[0158] Alignment of sequences for comparison can be conducted by
the local homology algorithm of Smith and Waterman (1981) Adv.
AMol. Math. 2:482, by the homology alignment algorithm of Needleman
and Wunsch (1970) J. Mol. Biol. 48:443, by the search for
similarity method of Pearson and Lipman (1988) Proc. Natl. Acad.
Sci. U.S.A. 85: 2444, by computerized implementations of these
algorithms. After alignment, sequence comparisons between two (or
more) polynucleotides or polypeptides are typically performed by
comparing sequences of the two sequences over a comparison window
to identify and compare local regions of sequence similarity. The
comparison window can be a segment of at least about 20 contiguous
positions, usually about 50 to about 200, more usually about 100 to
about 150 contiguous positions. A description of the method is
provided in Ausubel et al., supra.
[0159] A variety of methods for determining sequence relationships
can be used, including manual alignment and computer assisted
sequence alignment and analysis. This later approach is a preferred
approach in the present invention, due to the increased throughput
afforded by computer assisted methods. As noted above, a variety of
computer programs for performing sequence alignment are available,
or can be produced by one of skill.
[0160] One example algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al. J. Mol. Biol
215:403-410 (1990). Software for performing BLAST analyses is
publicly available, e.g., through the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.go- v/). This
algorithm involves first identifying high scoring sequence pairs
(HSPs) by identifying short words of length W in the query
sequence, which either match or satisfy some positive-valued
threshold score T when aligned with a word of the same length in a
database sequence. T is referred to as the neighborhood word score
threshold (Altschul et al., supra). These initial neighborhood word
hits act as seeds for initiating searches to find longer HSPs
containing them. The word hits are then extended in both directions
along each sequence for as far as the cumulative alignment score
can be increased. Cumulative scores are calculated using, for
nucleotide sequences, the parameters M (reward score for a pair of
matching residues; always>0) and N (penalty score for
mismatching residues; always<0). For amino acid sequences, a
scoring matrix is used to calculate the cumulative score. Extension
of the word hits in each direction are halted when: the cumulative
alignment score falls off by the quantity X from its maximum
achieved value; the cumulative score goes to zero or below, due to
the accumulation of one or more negative-scoring residue
alignments; or the end of either sequence is reached. The BLAST
algorithm parameters W, T, and X determine the sensitivity and
speed of the alignment. The BLASTN program (for nucleotide
sequences) uses as defaults a wordlength (W) of 11, an expectation
(E) of 10, a cutoff of 100, M=5, N=-4, and a comparison of both
strands. For amino acid sequences, the BLASTP program uses as
defaults a wordlength (W) of 3, an expectation (E) of 10, and the
BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc.
Natl. Acad. Sci. USA 89:10915). Unless otherwise indicated,
"sequence identity" here refers to the % sequence identity
generated from a tblastx using the NCBI version of the algorithm at
the default settings using gapped alignments with the filter "off"
(http://www.ncbi.nlm.nih.gov/). Additionally, BLASTX and TBLASTX
programs may be used.
[0161] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul
(1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence (and, therefore, in this context,
homologous) if the smallest sum probability in a comparison of the
test nucleic acid to the reference nucleic acid is less than about
0.1, or less than about 0.01, and or even less than about 0.001. An
additional example of a useful sequence alignment algorithm is
PILEUP. PILEUP creates a multiple sequence alignment from a group
of related sequences using progressive, pairwise alignments. The
program can align, e.g., up to 300 sequences of a maximum length of
5,000 letters.
[0162] The integrated system, or computer typically includes a user
input interface allowing a user to selectively view one or more
sequence records corresponding to the one or more character
strings, as well as an instruction set which aligns the one or more
character strings with each other or with an additional character
string to identify one or more region of sequence similarity. The
system may include a link of one or more character strings with a
particular phenotype or gene function. Typically, the system
includes a user readable output element which displays an alignment
produced by the alignment instruction set.
[0163] The methods of this invention can be implemented in a
localized or distributed computing environment. In a distributed
environment, the methods may implemented on a single computer
comprising multiple processors or on a multiplicity of computers.
The computers can be linked, e.g. through a common bus, but more
preferably the computer(s) are nodes on a network. The network can
be a generalized or a dedicated local or wide-area network and, in
certain preferred embodiments, the computers may be components of
an intra-net or an internet.
[0164] Thus, the invention provides methods for identifying a
sequence similar or homologous to one or more polynucleotides as
noted herein, or one or more target polypeptides encoded by the
polynucleotides, or otherwise noted herein and may include linking
or associating a given plant phenotype or gene function with a
sequence. In the methods, a sequence database is provided (locally
or across an inter or intra net) and a query is made against the
sequence database using the relevant sequences herein and
associated plant phenotypes or gene functions.
[0165] Any sequence herein can be entered into the database, before
or after querying the database. This provides for both expansion of
the database and, if done before the querying step, for insertion
of control sequences into the database. The control sequences can
be detected by the query to ensure the general integrity of both
the database and the query. As noted, the query can be performed
using a web browser based interface. For example, the database can
be a centralized public database such as those noted herein, and
the querying can be done from a remote terminal or computer across
an internet or intranet.
EXAMPLES
[0166] The following examples are intended to illustrate but not
limit the present invention.
Example I
Full Length Gene Identification and Cloning
[0167] Putative transcription factor sequences (genomic or ESTs)
related to known transcription factors were identified in the
Arabidopsis thaliana GenBank database using the tblastn sequence
analysis program using default parameters and a P-value cutoff
threshold of -4 or -5 or lower, depending on the length of the
query sequence. Putative transcription factor sequence hits were
then screened to identify those containing particular sequence
strings. If the sequence hits contained such sequence strings, the
sequences were confirmed as transcription factors.
[0168] Alternatively, Arabidopsis thaliana cDNA libraries derived
from different tissues or treatments, or genomic libraries were
screened to identify novel members of a transcription family using
a low stringency hybridization approach. Probes were synthesized
using gene specific primers in a standard PCR reaction (annealing
temperature 60.degree. C.) and labeled with .sup.32P dCTP using the
High Prime DNA Labeling Kit (Boehringer Mannheim). Purified
radiolabelled probes were added to filters immersed in Church
hybridization medium (0.5 M NaPO.sub.4 pH 7.0, 7% SDS, 1% w/v
bovine serum albumin) and hybridized overnight at 60.degree. C.
with shaking. Filters were washed two times for 45 to 60 minutes
with 1.times.SCC, 1% SDS at 60.degree. C.
[0169] To identify additional sequence 5' or 3' of a partial cDNA
sequence in a cDNA library, 5' and 3' rapid amplification of cDNA
ends (RACE) was performed using the Marathon.TM. cDNA amplification
kit (Clontech, Palo Alto, Calif.). Generally, the method entailed
first isolating poly(A) mRNA, performing first and second strand
cDNA synthesis to generate double stranded cDNA, blunting cDNA
ends, followed by ligation of the Marathon.TM. Adaptor to the cDNA
to form a library of adaptor-ligated ds cDNA.
[0170] Gene-specific primers were designed to be used along with
adaptor specific primers for both 5' and 3' RACE reactions. Nested
primers, rather than single primers, were used to increase PCR
specificity. Using 5' and 3' RACE reactions, 5' and 3' RACE
fragments were obtained, sequenced and cloned. The process can be
repeated until 5' and 3' ends of the full-length gene were
identified. Then the full-length cDNA was generated by PCR using
primers specific to 5' and 3' ends of the gene by end-to-end
PCR.
Example II
Construction of Expression Vectors
[0171] The sequence was amplified from a genomic or cDNA library
using primers specific to sequences upstream and downstream of the
coding region. The expression vector was pMEN20 or pMEN65, which
are both derived from pMON316 (Sanders et al, (1987) Nucleic Acids
Research 15:1543-58) and contain the CaMV 35S promoter to express
transgenes. To clone the sequence into the vector, both pMEN20 and
the amplified DNA fragment were digested separately with SalI and
NotI restriction enzymes at 37.degree. C. for 2 hours. The
digestion products were subject to electrophoresis in a 0.8%
agarose gel and visualized by ethidium bromide staining. The DNA
fragments containing the sequence and the linearized plasmid were
excised and purified by using a Qiaquick gel extraction kit
(Qiagen, Calif.). The fragments of interest were ligated at a ratio
of 3:1 (vector to insert). Ligation reactions using T4 DNA ligase
(New England Biolabs, MA) were carried out at 16.degree. C. for 16
hours. The ligated DNAs were transformed into competent cells of
the E. coli strain DH5alpha by using the heat shock method. The
transformations were plated on LB plates containing 50 mg/l
kanamycin (Sigma, Mo.).
[0172] Individual colonies were grown overnight in five milliliters
of LB broth containing 50 mg/l kanamycin at 37.degree. C. Plasmid
DNA was purified by using Qiaquick Mini Prep kits (Qiagen,
Calif.).
Example III
Transformation of Agrobacterium with the Expression Vector
[0173] After the plasmid vector containing the gene was
constructed, the vector was used to transform Agrobacterium
tumefaciens cells expressing the gene products. The stock of
Agrobacterium tumefaciens cells for transformation were made as
described by Nagel et al. (1990) FEMS Microbiol Letts. 67: 325-328.
Agrobacterium strain ABI was grown in 250 ml LB medium (Sigma)
overnight at 28.degree. C. with shaking until an absorbance
(A.sub.600) of 0.5-1.0 was reached. Cells were harvested by
centrifugation at 4,000.times.g for 15 min at 4.degree. C. Cells
were then resuspended in 250 .mu.l chilled buffer (1 mM HEPES, pH
adjusted to 7.0 with KOH). Cells were centrifuged again as
described above and resuspended in 125 .mu.l chilled buffer. Cells
were then centrifuged and resuspended two more times in the same
HEPES buffer as described above at a volume of 100 .mu.l and 750
.mu.l, respectively. Resuspended cells were then distributed into
40 .mu.l aliquots, quickly frozen in liquid nitrogen, and stored at
-80.degree. C.
[0174] Agrobacterium cells were transformed with plasmids prepared
as described above following the protocol described by Nagel et al.
For each DNA construct to be transformed, 50-100 ng DNA (generally
resuspended in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0) was mixed with 40
.mu.l of Agrobacterium cells. The DNA/cell mixture was then
transferred to a chilled cuvette with a 2 mm electrode gap and
subject to a 2.5 kV charge dissipated at 25 .mu.F and 200 .mu.F
using a Gene Pulser II apparatus (Bio-Rad). After electroporation,
cells were immediately resuspended in 1.0 ml LB and allowed to
recover without antibiotic selection for 2-4 hours at 28.degree. C.
in a shaking incubator. After recovery, cells were plated onto
selective medium of LB broth containing 100 .mu.g/ml spectinomycin
(Sigma) and incubated for 24-48 hours at 28.degree. C. Single
colonies were then picked and inoculated in fresh medium. The
presence of the plasmid construct was verified by PCR amplification
and sequence analysis.
Example IV
Transformation of Arabidopsis Plants With Agrobacterium tumefaciens
With Expression Vector
[0175] After transformation of Agrobacterium tumefaciens with
plasmid vectors containing the gene, single Agrobacterium colonies
were identified, propagated, and used to transform Arabidopsis
plants. Briefly, 500 ml cultures of LB medium containing 50 mg/l
kanamycin were inoculated with the colonies and grown at 28.degree.
C. with shaking for 2 days until an absorbance (A.sub.600)
of>2.0 is reached. Cells were then harvested by centrifugation
at 4,000.times.g for 10 min, and resuspended in infiltration medium
(1/2.times.Murashige and Skoog salts (Sigma), 1.times.Gamborg's B-5
vitamins (Sigma), 5.0% (w/v) sucrose (Sigma), 0.044 .mu.M
benzylamino purine (Sigma), 200 .mu.l/1 Silwet L-77 (Lehle Seeds)
until an absorbance (A.sub.600) of 0.8 was reached.
[0176] Prior to transformation, Arabidopsis thaliana seeds (ecotype
Columbia) were sown at a density of .about.10 plants per 4" pot
onto Pro-Mix BX potting medium (Hummert International) covered with
fiberglass mesh (18 mm.times.16 mm). Plants were grown under
continuous illumination (50-75 .mu.E/m.sup.2/sec) at 22-23.degree.
C. with 65-70% relative humidity. After about 4 weeks, primary
inflorescence stems (bolts) are cut off to encourage growth of
multiple secondary bolts. After flowering of the mature secondary
bolts, plants were prepared for transformation by removal of all
siliques and opened flowers.
[0177] The pots were then immersed upside down in the mixture of
Agrobacterium infiltration medium as described above for 30 sec,
and placed on their sides to allow draining into a 1'.times.2' flat
surface covered with plastic wrap. After 24 h, the plastic wrap was
removed and pots are turned upright. The immersion procedure was
repeated one week later, for a total of two immersions per pot.
Seeds were then collected from each transformation pot and analyzed
following the protocol described below.
Example V
Identification of Arabidopsis Primary Transformants
[0178] Seeds collected from the transformation pots were sterilized
essentially as follows. Seeds were dispersed into in a solution
containing 0.1% (v/v) Triton X-100 (Sigma) and sterile H.sub.2O and
washed by shaking the suspension for 20 min. The wash solution was
then drained and replaced with fresh wash solution to wash the
seeds for 20 min with shaking. After removal of the second wash
solution, a solution containing 0.1% (v/v) Triton X-100 and 70%
ethanol (Equistar) was added to the seeds and the suspension was
shaken for 5 min. After removal of the ethanol/detergent solution,
a solution containing 0.1% (v/v) Triton X-100 and 30% (v/v) bleach
(Clorox) was added to the seeds, and the suspension was shaken for
10 min. After removal of the bleach/detergent solution, seeds were
then washed five times in sterile distilled H.sub.2O. The seeds
were stored in the last wash water at 4.degree. C. for 2 days in
the dark before being plated onto antibiotic selection medium
(1.times.Murashige and Skoog salts (pH adjusted to 5.7 with 1M
KOH), 1.times.Gamborg's B-5 vitamins, 0.9% phytagar (Life
Technologies), and 50 mg/l kanamycin). Seeds were germinated under
continuous illumination (50-75 .mu.E/m.sup.2/sec) at 22-23.degree.
C. After 7-10 days of growth under these conditions, kanamycin
resistant primary transformants (T.sub.1 generation) were visible
and obtained. These seedlings were transferred first to fresh
selection plates where the seedlings continued to grow for 3-5 more
days, and then to soil (Pro-Mix BX potting medium).
[0179] Primary transformants were crossed and progeny seeds
(T.sub.2) collected; kanamycin resistant seedlings were selected
and analyzed. The expression levels of the recombinant
polynucleotides in the transformants varies from about a 5%
expression level increase to a least a 100% expression level
increase. Similar observations are made with respect to polypeptide
level expression.
Example VI
Identification of Arabidopsis Plants with Transcription Factor Gene
Knockouts
[0180] The screening of insertion mutagenized Arabidopsis
collections for null mutants in a known target gene was essentially
as described in Krysan et al (1999) Plant Cell 11:2283-2290.
Briefly, gene-specific primers, nested by 5-250 base pairs to each
other, were designed from the 5' and 3' regions of a known target
gene. Similarly, nested sets of primers were also created specific
to each of the T-DNA or transposon ends (the "right" and "left"
borders). All possible combinations of gene specific and
T-DNA/transposon primers were used to detect by PCR an insertion
event within or close to the target gene. The amplified DNA
fragments were then sequenced which allows the precise
determination of the T-DNA/transposon insertion point relative to
the target gene. Insertion events within the coding or intervening
sequence of the genes were deconvoluted from a pool comprising a
plurality of insertion events to a single unique mutant plant for
functional characterization. The method is described in more detail
in Yu and Adam, U.S. application Ser. No. 09/177,733 filed Oct. 23,
1998.
Example VII
Identification of Modified Phenotypes in Overexpression or Gene
Knockout Plants
[0181] Experiments were performed to identify those transformants
or knockouts that exhibited modified biochemical characteristics.
Among the biochemicals that were assayed were insoluble sugars,
such as arabinose, fucose, galactose, mannose, rhamnose or xylose
or the like; prenyl lipids, such as lutein, beta-carotene,
xanthophyll-1, xanthophyll-2, chlorophylls A or B, or alpha-,
delta- or gamma-tocopherol or the like; fatty acids, such as 16:0
(palmitic acid), 16:1 (palmitoleic acid), 18:0 (stearic acid), 18:1
(oleic acid), 18:2 (linoleic acid), 20:0, 18:3 (linolenic acid),
20:1 (eicosenoic acid), 20:2, 22: 1 (erucic acid) or the like;
waxes, such as by altering the levels of C29, C31, or C33 alkanes;
sterols, such as brassicasterol, campesterol, stigmasterol,
sitosterol or stigmastanol or the like, glucosinolates, protein or
oil levels.
[0182] Fatty acids were measured using two methods depending on
whether the tissue was from leaves or seeds. For leaves, lipids
were extracted and esterified with hot methanolic H2SO4 and
partitioned into hexane from methanolic brine. For seed fatty
acids, seeds were pulverized and extracted in
methanol:heptane:toluene:2,2-dimethoxypropane:H2SO4 (39:34:20:5:2)
for 90 minutes at 80.degree. C. After cooling to room temperature
the upper phase, containing the seed fatty acid esters, was
subjected to GC analysis. Fatty acid esters from both seed and leaf
tissues were analyzed with a Supelco SP-2330 column.
[0183] Glucosinolates were purified from seeds or leaves by first
heating the tissue at 95.degree. C. for 10 minutes. Preheated
ethanol:water (50:50) is and after heating at 95.degree. C. for a
further 10 minutes, the extraction solvent is applied to a DEAE
Sephadex column which had been previously equilibrated with 0.5 M
pyridine acetate. Desulfoglucosinolates were eluted with 300 ul
water and analyzed by reverse phase HPLC monitoring at 226 nm.
[0184] For wax alkanes, samples were extracted using an identical
method as fatty acids and extracts were analyzed on a HP 5890 GC
coupled with a 5973 MSD. Samples were chromatographed on a J&W
DB35 mass spectrometer (J&W Scientific).
[0185] To measure prenyl lipids levels, seeds or leaves were
pulverized with 1 to 2% pyrogallol as an antioxidant. For seeds,
extracted samples were filtered and a portion removed for
tocopherol and carotenoid/chlorophyll analysis by HPLC. The
remaining material was saponified for sterol determination. For
leaves, an aliquot was removed and diluted with methanol and
chlorophyll A, chlorophyll B, and total carotenoids measured by
spectrophotometry by determining absorbance at 665.2 nm, 652.5 nm,
and 470 nm. An aliquot was removed for tocopherol and
carotenoid/chlorophyll composition by HPLC using a Waters uBondapak
C18 column (4.6 mm.times.150 mm). The remaining methanolic solution
was saponified with 10% KOH at 80.degree. C. for one hour. The
samples were cooled and diluted with a mixture of methanol and
water. A solution of 2% methylene chloride in hexane was mixed in
and the samples were centrifuged. The aqueous methanol phase was
again re-extracted 2% methylene chloride in hexane and, after
centrifugation, the two upper phases were combined and evaporated.
2% methylene chloride in hexane was added to the tubes and the
samples were then extracted with one ml of water. The upper phase
was removed, dried, and resuspended in 400 ul of 2% methylene
chloride in hexane and analyzed by gas chromatography using a 50 m
DB-5 ms (0.25 mm ID, 0.25 um phase, J&W Scientific).
[0186] Insoluble sugar levels were measured by the method
essentially described by Reiter et al., Plant Journal 12:335-345.
This method analyzes the neutral sugar composition of cell wall
polymers found in Arabidopsis leaves. Soluble sugars were separated
from sugar polymers by extracting leaves with hot 70% ethanol. The
remaining residue containing the insoluble polysaccharides was then
acid hydrolyzed with allose added as an internal standard. Sugar
monomers generated by the hydrolysis were then reduced to the
corresponding alditols by treatment with NaBH4, then were
acetylated to generate the volatile alditol acetates which were
then analyzed by GC-FID. Identity of the peaks was determined by
comparing the retention times of known sugars converted to the
corresponding alditol acetates with the retention times of peaks
from wild-type plant extracts. Alditol acetates were analyzed on a
Supelco SP-2330 capillary column (30 m.times.250 um.times.0.2 um)
using a temperature program beginning at 180.degree. C. for 2
minutes followed by an increase to 220.degree. C. in 4 minutes.
After holding at 220.degree. C. for 10 minutes, the oven
temperature is increased to 240.degree. C. in 2 minutes and held at
this temperature for 10 minutes and brought back to room
temperature.
[0187] To identify plants with alterations in total seed oil or
protein content, 150 mg of seeds from T2 progeny plants were
subjected to analysis by Near Infrared Reflectance (NIR) using a
Foss NirSystems Model 6500 with a spinning cup transport
system.
[0188] Experiments were performed to identify those transformants
or knockouts that exhibited an improved pathogen tolerance. For
such studies, the transformants were exposed to biotropic fungal
pathogens, such as Erisyphe orontii, and necrotropic flimgal
pathogens, such as Fusarium oxysporum. Fusarium oxysporum isolates
cause vascular wilts and damping off of various annual vegetables,
perennials and weeds (Mauch-Mani and Slusarenko (1994) Molecular
Plant-Microbe Interactions 7: 378-383). For Fusarium oxysporum
experiments, plants grown on petri dishes were sprayed with a fresh
spore suspension of F. oxysporum. The spore suspension was prepared
as follows: A plug of fungal hyphae from a plate culture was placed
on a fresh potato dextrose agar plate and allowed to spread for one
week. 5 ml sterile water was then added to the plate, swirled, and
pipetted into 50 ml Armstrong Fusarium medium. Spores were grown
overnight in Fusarium medium and then sprayed onto plants using a
Preval paint sprayer. Plant tissue was harvested and frozen in
liquid nitrogen 48 hours post infection.
[0189] Erysiphe orontii is a causal agent of powdery mildew. For
Erysiphe orontii experiments, plants were grown approximately 4
weeks in a greenhouse under 12 hour light (20.degree. C.,.about.30%
relative humidity (rh)). Individual leaves were infected with E.
orontii spores from infected plants using a camel's hair brush, and
the plants were transferred to a Percival growth chamber
(20.degree. C., 80% rh.). Plant tissue was harvested and frozen in
liquid nitrogen 7 days post infection.
[0190] Botrytis cinerea is a necrotrophic pathogen. Botrytis
cinerea was grown on potato dextrose agar in the light. A spore
culture was made by spreading 10 ml of sterile water on the ftingus
plate, swirling and transferring spores to 10 ml of sterile water.
The spore inoculum (approx. 105 spores/ml) was used to spray 10
day-old seedlings grown under sterile conditions on MS (minus
sucrose) media. Symptoms were evaluated every day up to
approximately 1 week.
[0191] Infection with bacterial pathogens Pseudomonas syringae pv
maculicola strain 4326 and pv maculicola strain 4326 was performed
by hand inoculation at two doses. Two inoculation doses allows the
differentiation between plants with enhanced susceptibility and
plants with enhanced resistance to the pathogen. Plants were grown
for 3 weeks in the greenhouse, then transferred to the growth
chamber for the remainder of their growth. Psm ES4326 was hand
inoculated with 1 ml syringe on 3 fully-expanded leaves per plant
(41/2 wk old), using at least 9 plants per overexpressing line at
two inoculation doses, OD=0.005 and OD=0.0005. Disease scoring
occured at day 3 post-inoculation with pictures of the plants and
leaves taken in parallel.
[0192] In some instances, expression patterns of the
pathogen-induced genes (such as defense genes) was monitored by
microarray experiments. cDNAs were generated by PCR and resuspended
at a final concentration of.about.100 ng/ul in 3.times.SSC or 150
mM Na-phosphate (Eisen and Brown (1999) Meth. in Enzymol.
303:179-205). The cDNAs were spotted on microscope glass slides
coated with polylysine. The prepared cDNAs were aliquoted into 384
well plates and spotted on the slides using an x-y-z gantry
(OnmiGrid) purchased from GeneMachines (Menlo Park, Calif.)
outfitted with quill type pins purchased from Telechem
International (Sunnyvale, Calif.). After spotting, the arrays were
cured for a minimum of one week at room temperature, rehydrated and
blocked following the protocol recommended by Eisen and Brown
(1999). ]Sample total RNA (10 ug) samples were labeled using
fluorescent Cy3 and Cy5 dyes. Labeled samples were resuspended in
4.times.SSC/0.03% SDS/4 ug salmon sperm DNA/2 ug tRNA/50 mM
Na-pyrophosphate, heated for 95.degree. C. for 2.5 minutes, spun
down and placed on the array. The array was then covered with a
glass coverslip and placed in a sealed chamber. The chamber was
then kept in a water bath at 62.degree. C. overnight. The arrays
were washed as described in Eisen and Brown (1999) and scanned on a
General Scanning 3000 laser scanner. The resulting files are
subsequently quantified using Imagene a software purchased from
BioDiscovery (Los Angeles, Calif.).
[0193] Experiments were performed to identify those transformants
or knockouts that exhibited an improved environmental stress
tolerance. For such studies, the transformants were exposed to a
variety of environmental stresses. Plants were exposed to chilling
stress (6 hour exposure to 4-8.degree. C.), heat stress (6 hour
exposure to 32-37.degree. C.), high salt stress (6 hour exposure to
200 mM NaCl), drought stress (168 hours after removing water from
trays), osmotic stress (6 hour exposure to 3 M mannitol), or
nutrient limitation (nitrogen, phosphate, and potassium) (Nitrogen:
all components of MS medium remained constant except N was reduced
to 20 mg/l of NH 4 NO3, or Phosphate: All components of MS medium
except KH 2 PO 4, which was replaced by K2SO4, Potassium: All
components of MS medium except removal of KNO3 and KH2PO4, which
were replaced by NaH4PO4).
[0194] Experiments were performed to identify those transformants
or knockouts that exhibited a modified structure and development
characteristics. For such studies, the transformants were observed
by eye to identify novel structural or developmental
characteristics associated with the ectopic expression of the
polynucleotides or polypeptides of the invention.
[0195] Experiments were performed to identify those transformants
or knockouts that exhibited modified sugar-sensing. For such
studies, seeds from transformants were germinated on media
containing 5% glucose or 9.4% sucrose which normally partially
restrict hypocotyl elongation. Plants with altered sugar sensing
may have either longer or shorter hypocotyls than normal plants
when grown on this media. Additionally, other plant traits may be
varied such as root mass.
[0196] Flowering time was measured by the number of rosette leaves
present when a visible inflorescence of approximately 3 cm is
apparent Rosette and total leaf number on the progeny stem are
tightly correlated with the timing of flowering (Koomneef et al
(1991) Mol. Gen. Genet 229:57-66. The vernalization response was
measured. For vernalization treatments, seeds were sown to MS agar
plates, sealed with micropore tape, and placed in a 4.degree. C.
cold room with low light levels for 6-8 weeks. The plates were then
transferred to the growth rooms alongside plates containing freshly
sown non-vernalized controls. Rosette leaves were counted when a
visible inflorescence of approximately 3 cm was apparent.
[0197] Modified phenotypes observed for particular overexpressor or
knockout plants are provided in Table 4 and Table 5. For a
particular overexpressor that shows a less beneficial
characteristic, it may be more useful to select a plant with a
decreased expression of the particular transcription factor. For a
particular knockout that shows a less beneficial characteristic, it
may be more useful to select a plant with an increased expression
of the particular transcription factor.
[0198] The sequences of the Sequence Listing or those discloses
here can be used to prepare transgenic plants and plants with
altered traits. The specific transgenic plants listed below are
produced from the sequences of the Sequence Listing, as noted.
Table 4 and Table 5 provide exemplary polynucleotide and
polypeptide sequences of the invention. Table 4 includes, from left
to right for each sequence: the first column shows the
polynucleotide SEQ ID NO; the second column shows the polypeptide
SEQ ID NO encoded by the polynucleotide; the third column shows the
Mendel Gene ID No., GID; the fourth column, CDS, shows the start
and stop nucleotide positions of the encoded polypeptide,
respectively, with respect to the polynucleotide co-ordinates; the
fifth column shows the amino acid residue positions of the
conserved domain in amino acid (AA) co-ordinates; and the sixth
column shows if the polynucleotide was knocked out (KO) or
overexpressed (OE) in a transgenic plant. Table 5 includes, from
left to right for each sequence: the first column shows the
nucleotide SEQ ID NO.; the second column shows the polypeptide SEQ
ID NO encoded by the polynucleotide; the third column shows the
Mendel Gene ID No., GID; the fourth column shows if the
polynucleotide was knocked out (KO) or overexpressed (OE) in a
transgenic plant; and the fifth column shows the trait resulting
from the knock out or overexpression of the polynucleotide in the
transgenic plant.
4TABLE 4 SEQ ID NO SEQ ID NO Conserved domain in AA Knockout or
(polynucleotide) (polypeptide) GID CDS coordinates overexpressor 1
2 G481 103 . . . 528 20-109 OE 3 4 G1466 16 . . . 1278 154-420
OE
[0199]
5TABLE 5 SEQ ID SEQ ID NO (poly- NO nucle- (poly- Observed
phenotype Overexpressor otide) peptide) GID or trait or knockout 1
2 G481 Germination assay: OE High sucrose, osmotic stress 3 4 G1466
Seed composition assay: OE High seed oil and protein content
[0200] G481: Better Germination on High Sucrose
[0201] Seed of plants overexpressing sequence G481 (SEQ ID NOs: 1
and 2) showed slightly better germination when grown on high
sucrose medium (5% glucose or 9.4% sucrose). The plants showed
longer radicle and more cotyledon expansion. In one line (line 8)
analysis of leaf insoluble sugars showed a slight increase in
decrease in Rhamnose levels (16.9% vs. approximately 11.4% in
wild-type plants) and a decrease in Arabinose and Xylose levels
(15.3% and 12.8% vs. approximately 19.0% and 21% in wild-type
plants, respectively).
[0202] In wild-type plants, G481 was predominantly expressed in
flower and silique, and to a lesser extent, in rosette, embryo, and
germinating seed.
[0203] The potential utility of G481 includes a possible role in
sugar sensing, a plant mechanism that has been shown to be involved
in the following: 1) altering storage compound accumulation (oil
and/or protein) in seeds which could impact yield and seed quality,
and 2) altering photosynthetic rate which could also impact yield
in vegetative tissues as well as seed. Sugars are key regulatory
molecules that affect diverse processes in higher plants including
germination, growth, flowering, senescence, sugar metabolism and
photosynthesis. Sucrose is the major transport form of
photosynthate and its flux through cells has been shown to affect
gene expression and alter storage compound accumulation in seeds
(source-sink relationships).
[0204] The enhanced germination phenotype of transgenic plants
overexpressing G481 under a condition of osmotic stress (such as
high concentrations of sucrose) suggests the gene could also be
used to improve plant tolerance to water deficit related conditions
such as drought stress, salt stress, and freezing stress. Thus G481
could be used to engineer plants with enhanced stress tolerance
that could ultimately impact survivability and yield.
[0205] G1466: Increased Seed Oil; Decreased Protein Content
[0206] Seed of plants overexpressing sequences G1466 (SEQ ID NOs:3
and 4) was subjected to NIR analysis and an increase in seed oil
content compared with seed from wild-type plants was identified
(39% vs. approximately 35% in wild-type plants). In addition, a
slight decrease in seed protein content compared with seed from
wild-type plants was identified (20.8% vs. approximately 23% in
wild-type plants).
[0207] In wild-type plants, G1466 was expressed in all tissues
examined.
[0208] Therefore, G1466 could be used to modify high value seed
quality traits such protein, oil and carbohydrates content and
composition in any plant in which the expression of this gene is
altered. Altering the amount of seed oil, protein or carbohydrate
could effectively increase the yield and quality. Altering the
composition of seeds would improve feed quality by altering
availability of energy and phosphorus, and to improve the amino
acid balance of grain meal or to improve oil quality for human food
and industrial uses. Altering seed composition with G1466 could
also improved grain quality for the wet milling industry as well as
provide a means for the production of novel polymers and
chemicals.
Example VIII
Identification of Homologous Sequences
[0209] Homologous sequences from Arabidopsis and plant species
other than Arabidopsis were identified using database sequence
search tools, such as the Basic Local Alignment Search Tool (BLAST)
(Altschul et al. (1990) J. Mol. Biol. 215:403-410; and Altschul et
al. (1997) Nucl. Acid Res. 25: 3389-3402). The tblastx sequence
analysis programs were employed using the BLOSUM-62 scoring matrix
(Henikoff, S. and Henikoff, J. G. (1992) Proc. Natl. Acad. Sci. 89:
10915-10919).
[0210] Identified orthologs and homologs of Arabidopsis sequences
are provided in Tables 6 and 7. The percent sequence identity among
these sequences can be as low as 47%, or even lower sequence
identity. Additionally, the entire NCBI GenBank database was
filtered for sequences from all plants except Arabidopsis thaliana
by selecting all entries in the NCBI GenBank database associated
with NCBI taxonomic ID 33090 (Viridiplantae; all plants) and
excluding entries associated with taxonomic ID 3701 (Arabidopsis
thaliana). These sequences are compared to sequences representing
genes of SEQ IDs NOs:2 and 4 using the Washington University
TBLASTX algorithm (version 2.0a19MP) at the default settings using
gapped alignments with the filter "off". For each gene of SEQ IDs
NOs:2 and 4 individual comparisons were ordered by probability
score (P-value), where the score reflects the probability that a
particular alignment occurred by chance. For example, a score of
3.6e-40 is 3.6.times.10.sup.-40. In addition to P-values,
comparisons were also scored by percentage identity. Percentage
identity reflects the degree to which two segments of DNA or
protein are identical over a particular length. The identified
homologous polynucleotide and polypeptide sequences and homologues
of the Arabidopsis polynucleotides and polypeptides may be
orthologs of the Arabidopsis polynucleotides and polypeptides.
[0211] As shown in Table 6, polynucleotide and polypeptide
sequences which were identified as orthologous and homologous of
SEQ ID NOs: 1 and 2 were found in Gossypium arboreum, Glycine max,
Zea mays, Gossypium hirsutum, Medicago truncatula, Lycopersicon
esculentum, Solanum tuberosum, Triticum aestivum, Hordeum vulgare,
Triticum monococcum, Oryza sativa, Vemonia galamensis, Argemone
mexicana, and Triticum aestivum.
[0212] As shown in Table 6, polynucleotide and polypeptide
sequences which were identified as orthologous and homologous of
SEQ ID NOs:3 and 4 were found in Brassica oleracea, Lycopersicon
esculentum, Lycopersicon pennellii, Lotus japonicus, Oryza sativa,
Solanum tuberosum, Medicago truncatula, Glycine max, Euphorbia
esula, Gossypium arboreum, Antirrhinum hispanicum, Oryza sativa
(japonica cultivar-group), and Triticum aestivum. Additional
orthologous and homologous polynucleotides and polypeptide
sequences from other plant species are shown in Table 7.
[0213] All references, publications, patent documents, web pages,
and other documents cited or mentioned herein are hereby
incorporated by reference in their entirety for all purposes.
Although the invention has been described with reference to
specific embodiments and examples, it should be understood that one
of ordinary skill can make various modifications without departing
from the spirit of the invention. The scope of the invention is not
limited to the specific embodiments and examples provided.
6TABLE 6 Smallest Test Sum SEQ ID NO Gene ID Sequence ID
Probability Test Sequence Species Test Sequence GenBank Annotation
1 G481 BG440251 9.00E-42 [Gossypium arboreum] GA_Ea0006K20f
Gossypium arboreum 7-10 d 1 G481 BM887558 3.90E-41 [Glycine max]
sam40c09.y1 Gm-c1068 Glycine max cDNA clone SOY 1 G481 ZMNFYB
1.70E-40 [Zea mays] Z. mays mRNA for CAAT-box DNA binding protein
subun 1 G481 AI728916 2.40E-40 [Gossypium hirsutum] BNLGHi12022
Six-day Cotton fiber Gossypi 1 G481 AW775623 3.80E-40 [Medicago
truncatula] EST334688 DSIL Medicago truncatula cDNA 1 G481 AW738727
9.80E-40 [Lycopersicon esculentum] EST340154 tomato flower buds,
anthe 1 G481 BG599785 1.70E-38 [Solanum tuberosum] EST504680 cSTS
Solanum tuberosum cDNA clo 1 G481 BE413647 2.50E-38 [Triticum
aestivum] SCU001.E10.R990714 ITEC SCU Wheat Endospe 1 G481 BF065056
5.80E-38 [Hordeum vulgare] HV_CEb0022M01f Hordeum vulgare seedling
gre 1 G481 BG314203 1.00E-37 [Triticum monococcum]
WHE2460_E10_I20ZS Triticum monococcum i 1 G481 gi22380 1.10E-45
[Zea mays] CAAT-box DNA binding protein subunit B (NF-YB). 1 G481
gi15408794 2.60E-30 [Oryza sativa] putative CCAAT-binding
transcription factor 1 G481 gi16902054 1.00E-28 [Vernonia
galamensis] CCAAT-box binding factor HAP3 B domai 1 G481 gi16902050
2.70E-28 [Glycine max] CCAAT-box binding factor HAP3 B domain. 1
G481 gi16902056 4.30E-28 [Argemone mexicana] CCAAT-box binding
factor HAP3 B domain. 1 G481 gi16902058 1.50E-23 [Triticum
aestivum] CCAAT-box binding factor HAP3 B domain. 3 G1466 BH596941
1.40E-65 [Brassica oleracea] BOHFG41TR BOHF Brassica oleracea
genomic 3 G1466 BE462774 0.00014 [Lycopersicon esculentum]
EST325108 tomato flower buds 0-3 mm 3 G1466 AW399721 0.00049
[Lycopersicon pennellii] EST310221 L. pennellii trichome, Cor 3
G1466 AV413010 0.00054 [Lotus japonicus] AV413010 Lotus japonicus
young plants (two- 3 G1466 OSJN00182 0.0029 [Oryza sativa]
chromosome 4 clone OSJNBa0086O06, *** SEQUENC 3 G1466 BG599234
0.0047 [Solanum tuberosum] EST504134 cSTS Solanum tuberosum cDNA
clo 3 G1466 BG580068 0.0095 [Medicago truncatula] EST481790 GVN
Medicago truncatula cDNA 3 G1466 BI316339 0.026 [Glycine max]
saf02a08.y1 Gm-c1065 Glycine max cDNA clone GEN 3 G1466 BG409415
0.026 [Euphorbia esula] 00787 leafy spurge Lambda HybriZAP 2.1 two-
3 G1466 BF278686 0.11 [Gossypium arboreum] GA_Eb0035E21f Gossypium
arboreum 7-10 d 3 G1466 gi8096405 2.30E-12 [Oryza sativa] Similar
to Arabidopsis thaliana chromosome 1 3 G1466 gi13161526 3.40E-08
[Antirrhinum hispanicum] S locus F-box (SLF)-S2 protein. 3 G1466
gi18844804 0.083 [Oryza sativa (japonica cultivar-group)] contains
ESTs D151 3 G1466 gi9858770 0.83 [Lycopersicon esculentum] BAC19.2.
3 G1466 gi6505722 1 [Triticum aestivum] ribosomal RNA apurinic site
specificly
[0214]
7TABLE 7 SEQ ID Test Reading High Smallest Sum NO Gene ID Sequence
ID Test Sequence GenBank Annotation Frame Score Probability N 1
G481 BG440251 BG440251 GA_Ea0006K20f Gossypium arboreum 7 . . . 3
263 9.00E-42 2 1 G481 BM887558 BM887558 sam40c09.y1 Gm-c1068
Glycine max cD . . . 3 268 3.90E-41 2 1 G481 BG362898 BG362898
sac13e07.y1 Gm-c1040 Glycine max cD . . . 3 263 4.90E-41 2 1 G481
AW395227 AW395227 sh45e04.y1 Gm-c1017 Glycine max cDN . . . 1 264
9.10E-41 2 1 G481 BM525962 BM525962 sak74b11.y1 Gm-c1036 Glycine
max cD . . . 3 264 9.20E-41 2 1 G481 BI972318 BI972318 sag90a01.y1
Gm-c1084 Glycine max cD . . . 3 264 9.70E-41 2 1 G481 BG363233
BG363233 sac11h11.y1 Gm-c1040 Glycine max cD . . . 3 264 9.90E-41 2
1 G481 BE021941 BE021941 sm64d05.y1 Gm-c1028 Glycine max cDN . . .
1 264 1.00E-40 2 1 G481 BI316766 BI316766 saf73a12.y1 Gm-c1078
Glycine max cD . . . 3 264 1.10E-40 2 1 G481 BE803572 BE803572
sr60e11.y1 Gm-c1052 Glycine max cDN . . . 2 264 1.10E-40 2 1 G481
ZMNFYB X59714 Z. mays mRNA for CAAT-box DNA binding . . . 2 262
1.70E-40 2 1 G481 BF071234 BF071234 st06h05.y1 Gm-c1065 Glycine max
cDN . . . 3 258 2.00E-40 2 1 G481 AI728916 AI728916 BNLGHi12022
Six-day Cotton fiber Go . . . 3 261 2.40E-40 2 1 G481 BF597252
BF597252 su96c06.y1 Gm-c1056 Glycine max cDN . . . 2 264 2.90E-40 2
1 G481 AW597630 AW597630 sj96g06.y1 Gm-c1023 Glycine max cDN . . .
2 259 3.40E-40 2 1 G481 AW775623 AW775623 EST334688 DSIL Medicago
truncatula . . . 2 259 3.80E-40 2 1 G481 AW733618 AW733618
sk75h06.y1 Gm-c1016 Glycine max cDN . . . 3 257 6.00E-40 2 1 G481
AW738727 AW738727 EST340154 tomato flower buds, anthe . . . 1 249
9.80E-40 2 1 G481 BG642751 BG642751 EST510945 tomato shoot/meristem
Lyc . . . 1 249 9.90E-40 2 1 G481 BE441135 BE441135 EST408405
tomato developing/immatur . . . 1 249 9.90E-40 2 1 G481 AW621652
AW621652 EST312450 tomato root during/after . . . 1 249 1.10E-39 2
1 G481 AI900024 AI900024 sb97g11.y1 Gm-c1012 Glycine max cDN . . .
1 264 3.20E-39 2 1 G481 BG445358 BG445358 GA_Ea0027N18f Gossypium
arboreum 7 . . . 2 243 1.40E-38 2 1 G481 BG599785 BG599785
EST504680 cSTS Solanum tuberosum cD . . . 2 254 1.70E-38 2 1 G481
BG350430 BG350430 091D09 Mature tuber lambda ZAP Sola . . . 3 254
1.70E-38 2 1 G481 BE413647 BE413647 SCU001.E10.R990714 ITEC SCU
Wheat E . . . 3 248 2.50E-38 2 1 G481 BE516510 BE516510
WHE611_D10_H19ZA Wheat ABA-treated . . . 2 248 3.00E-38 2 1 G481
BF065056 BF065056 HV_CEb0022M01f Hordeum vulgare seed . . . 3 247
5.80E-38 2 1 G481 BG314203 BG314203 WHE2460_E10_I20ZS Triticum
monococc . . . 1 243 1.00E-37 2 1 G481 AI725612 AI725612
BNLGHi12445 Six-day Cotton fiber Go . . . 2 247 1.20E-37 2 1 G481
AL387357 AL387357 MtBC42A04F1 MtBC Medicago truncatul . . . 3 231
2.00E-37 2 1 G481 AW907348 AW907348 EST343471 potato stolon,
Cornell Un . . . 2 246 2.40E-37 2 1 G481 BG274786 BG274786
WHE2234_C03_E06ZS Aegilops speltoid . . . 1 248 2.50E-37 2 1 G481
AW459387 AW459387 sh23f03.y1 Gm-c1016 Glycine max cDN . . . 2 233
3.40E-37 2 1 G481 BE804236 BE804236 sr77b04.y1 Gm-c1052 Glycine max
cDN . . . 2 254 4.30E-37 2 1 G481 BE210041 BE210041 so38b01.y1
Gm-c1039 Glycine max cDN . . . 3 231 5.70E-37 2 1 G481 AW980494
AW980494 EST391647 GVN Medicago truncatula c . . . 1 229 6.30E-37 2
1 G481 BG263362 BG263362 WHE2341_B02_C03ZS Wheat pre-anthesi . . .
1 238 7.10E-37 2 1 G481 BH532457 BH532457 BOGLV08TR BOGL Brassica
oleracea ge . . . 2 225 8.00E-37 2 1 G481 BG847452 BG847452
1024017D03.y1 C. reinhardtii CC-169 . . . 2 236 8.20E-37 2 1 G481
BG857007 BG857007 1024049D01.y1 C. reinhardtii CC-169 . . . 3 236
9.30E-37 2 1 G481 BG858372 BG858372 1024057C11.y1 C. reinhardtii
CC-169 . . . 3 236 9.50E-37 2 1 G481 BG850689 BG850689
1024029A11.y2 C. reinhardtii CC-169 . . . 2 236 9.50E-37 2 1 G481
BI718232 BI718232 1031024F10.y1 C. reinhardtii CC-169 . . . 3 236
9.70E-37 2 1 G481 BI719728 BI719728 1031045D08.y1 C. reinhardtii
CC-169 . . . 3 236 9.80E-37 2 1 G481 BI875221 BI875221 963122G10.y1
C. reinhardtii CC-1690 . . . 3 236 9.90E-37 2 1 G481 BE496857
BE496857 WHE0761_D09_H17ZS Wheat heat-stress . . . 1 238 1.00E-36 2
1 G481 BF651151 BF651151 NF101H10EC1F1090 Elicited cell cult . . .
3 227 1.10E-36 2 1 G481 BE441739 BE441739 925009A11.x1 C.
reinhardtii CC-2290 . . . 2 236 1.30E-36 2 1 G481 BG846124 BG846124
1024012C11.y1 C. reinhardtii CC-169 . . . 1 234 1.50E-36 2 1 G481
AX288144 AX288144 Sequence 15 from Patent WO0177311. . . . 2 231
2.90E-36 2 1 G481 AW570530 AW570530 sj63c01.y1 Gm-c1033 Glycine max
cDN . . . 2 263 2.90E-36 2 1 G481 AX180950 AX180950 Sequence 1 from
Patent WO0145493. 8 . . . 2 231 3.30E-36 2 1 G481 BF585526 BF585526
FM1_23_E09.g1_A003 Floral-Induced M . . . 1 224 3.50E-36 2 1 G481
BI271802 BI271802 NF013D06FL1F1057 Developing flower . . . 1 226
5.40E-36 2 1 G481 BF270944 BF270944 GA_Eb0010B11f Gossypium
arboreum 7 . . . 1 240 5.60E-36 2 1 G481 AI731275 AI731275
BNLGHi9078 Six-day Cotton fiber Gos . . . 2 221 1.30E-35 2 1 G481
BI967397 BI967397 GM830001B20E03 Gm-r1083 Glycine max . . . -3 226
1.40E-35 2 1 G481 BF585616 BF585616 FM1_23_E09.b1_A003
Floral-Induced M . . . 1 265 1.80E-35 2 1 G481 BF263449 BF263449
HV_CEa0006M10f Hordeum vulgare seed . . . 1 230 2.70E-35 2 1 G481
BF263455 BF263455 HV_CEa0006M16f Hordeum vulgare seed . . . 1 230
2.70E-35 2 1 G481 BF459554 BF459554 061A04 Mature tuber lambda ZAP
Sola . . . 2 227 2.90E-35 2 1 G481 BF460267 BF460267 073E08 Mature
tuber lambda ZAP Sola . . . 2 209 3.00E-35 3 1 G481 BG594268
BG594268 EST492946 cSTS Solanum tuberosum cD . . . 3 236 5.10E-35 2
1 G481 BI469382 BI469382 sai11b10.y1 Gm-c1053 Glycine max cD . . .
1 225 5.50E-35 2 1 G481 AI731250 AI731250 BNLGHi9010 Six-day Cotton
fiber Gos . . . 3 236 6.40E-35 2 1 G481 BG850688 BG850688
1024029A11.y1 C. reinhardtii CC-169 . . . 2 236 7.50E-35 2 1 G481
AW132359 AW132359 se03b02.y1 Gm-c1013 Glycine max cDN . . . 1 264
8.10E-35 2 1 G481 BM269434 BM269434 MEST409-G11.univ ISUM5-RN Zea
mays . . . -2 216 8.70E-35 2 1 G481 AW035570 AW035570 EST281308
tomato callus, TAMU Lycop . . . 3 249 9.30E-35 2 1 G481 BE418716
BE418716 SCL074.B01R990724 ITEC SCL Wheat Le . . . 1 227 1.20E-34 2
1 G481 AW648378 AW648378 EST326832 tomato germinating seedli . . .
1 223 1.20E-34 2 1 G481 AV424305 AV424305 AV424305 Lotus japonicus
young plan . . . -3 219 1.50E-34 2 1 G481 BF715909 BF715909
saa11e08.y1 Gm-c1058 Glycine max cD . . . 3 222 2.20E-34 2 1 G481
BI423967 BI423967 sah64c11.y1 Gm-c1049 Glycine max cD . . . 1 215
2.30E-34 2 1 G481 BG890447 BG890447 EST516298 cSTD Solanum
tuberosum cD . . . 2 220 3.00E-34 2 1 G481 AI495007 AI495007
sa89f03.y1 Gm-c1004 Glycine max cDN . . . 3 220 5.70E-34 2 1 G481
AW760103 AW760103 sl58b03.y1 Gm-c1027 Glycine max cDN . . . 3 225
7.40E-34 2 1 G481 BF517889 BF517889 NXSI_029_D01_F NXSI (Nsf Xylem
Side . . . 3 222 1.70E-33 2 1 G481 BE060015 BE060015 sn39h06.y1
Gm-c1027 Glycine max cDN . . . 2 219 1.90E-33 2 1 G481 AW625817
AW625817 EST319724 tomato radicle, 5 d post- . . . 3 211 2.90E-33 2
1 G481 AW397727 AW397727 sg83f04.y1 Gm-c1026 Glycine max cDN . . .
3 210 3.10E-33 2 1 G481 BI207873 BI207873 EST525913 cTOS
Lycopersicon esculen . . . 2 227 3.20E-33 2 1 G481 AV632945
AV632945 AV632945 Chlamydomonas reinhardtii . . . 3 236 3.40E-33 2
1 G481 AW931634 AW931634 EST357477 tomato fruit mature green . . .
1 223 4.90E-33 2 1 G481 BM888735 BM888735 952068E04.y1 952 --BMS
tissue from . . . 2 214 5.10E-33 2 1 G481 AI486503 AI486503
EST244824 tomato ovary, TAMU Lycope . . . 3 223 5.40E-33 2 1 G481
BE641101 BE641101 Cri2_2_E11_SP6 Ceratopteris Spore L . . . 3 224
8.60E-33 2 1 G481 BI953657 BI953657 HVSMEm0013M03f Hordeum vulgare
gree . . . 1 230 9.80E-33 2 1 G481 BM341536 BM341536 MEST336-C11.T3
ISUM5-RN Zea mays cD . . . -1 214 1.80E-32 2 1 G481 AX180957
AX180957 Sequence 8 from Patent WO0145493. 8 . . . 2 231 2.70E-32 2
1 G481 AW201996 AW201996 sf09g11.y1 Gm-c1027 Glycine max cDN . . .
1 207 2.90E-32 2 1 G481 BG135204 BG135204 EST468096 tomato crown
gall Lycoper . . . 3 230 3.20E-32 2 1 G481 BI406257 BI406257 158C12
Mature tuber lambda ZAP Sola . . . 2 201 1.20E-31 2 1 G481 BM341107
BM341107 MEST330-D11.T3 ISUM5-RN Zea mays cD . . . -1 214 1.30E-31
2 1 G481 AI782351 AI782351 EST263230 tomato susceptible, Corne . .
. 2 208 2.50E-31 2 1 G481 BM331836 BM331836 MEST171-B11.T3 ISUM5-RN
Zea mays cD . . . -3 214 3.30E-31 2 1 G481 BM268414 BM268414
MEST395-C12.univ ISUM5-RN Zea mays . . . -2 214 3.40E-31 2 1 G481
BM337630 BM337630 MEST215-B12.T3 ISUM5-RN Zea mays cD . . . -2 214
3.40E-31 2 1 G481 BM349646 BM349646 MEST253-D11.T3 ISUM5-RN Zea
mays cD . . . -2 214 3.40E-31 2 1 G481 BE356560 BE356560
DG1_126_D05.b1_A002 Dark Grown 1 (D . . . 3 265 3.70E-31 2 1 G481
AV632044 AV632044 AV632044 Chlamydomonas reinhardtii . . . 2 236
4.50E-31 2 1 G481 BE054369 BE054369 GA_Ea0002A05f Gossypium
arboreum 7 . . . 1 263 5.80E-31 2 1 G481 AC108500 AC108500 Oryza
sativa chromosome 5 clone OJ1 . . . -3 230 6.20E-31 2 1 G481
BI129814 BI129814 G095P88Y Populus cambium cDNA libra . . . 1 266
6.90E-31 2 1 G481 BG318871 BG318871 NXPV_020_H08_F NXPV (Nsf Xylem
Plan . . . 1 216 7.60E-31 2 1 G481 AV420653 AV420653 AV420653 Lotus
japonicus young plan . . . 1 256 1.70E-30 2 1 G481 AW348165
AW348165 GM210001A21D7 Gm-r1021 Glycine max . . . -3 196 4.60E-30 2
1 G481 AI442376 AI442376 sa26b07.y1 Gm-c1004 Glycine max cDN . . .
2 197 5.10E-30 2 1 G481 AI442765 AI442765 sa26b07.x1 Gm-c1004
Glycine max cDN . . . -1 192 1.70E-29 2 1 G481 BE604847 BE604847
WHE1713-1716_D19_D19ZS Wheat heat s . . . 3 241 1.90E-29 2 1 G481
AW043377 AW043377 ST32F09 Pine TriplEx shoot tip libr . . . 1 233
3.90E-29 2 1 G481 AP003271 AP003271 Oryza sativa genomic DNA,
chromosom . . . -2 240 7.60E-29 2 1 G481 BG832836 BG832836
NXPV_081_C10_F NXPV (Nsf Xylem Plan . . . 1 222 9.10E-29 2 1 G481
AP004366 AP004366 Oryza sativa chromosome 1 clone P04 . . . -3 240
9.30E-29 2 1 G481 BE603222 BE603222 HVSMEh0102J16f Hordeum vulgare
5-45 . . . 2 204 3.50E-28 2 1 G481 AW754604 AW754604 PC04B12 Pine
TriplEx pollen cone li . . . 2 197 3.70E-28 2 1 G481 AW756413
AW756413 sl21a12.y1 Gm-c1036 Glycine max cDN . . . 1 201 4.00E-28 2
1 G481 AW432980 AW432980 si03a01.y1 Gm-c1029 Glycine max cDN . . .
3 201 4.40E-28 2 1 G481 BG551755 BG551755 sad42f11.y1 Gm-c1075
Glycine max cD . . . 3 201 4.80E-28 2 1 G481 BF595304 BF595304
su76f03.y1 Gm-c1055 Glycine max cDN . . . 2 201 5.10E-28 2 1 G481
BM308208 BM308208 sak43a12.y1 Gm-c1036 Glycine max cD . . . 2 200
5.20E-28 2 1 G481 AC104284 AC104284 Oryza sativa chromosome 5 clone
OJ1 . . . -3 224 5.70E-28 2 1 G481 BM528842 BM528842 sak69b03.y1
Gm-c1036 Glycine max cD . . . 2 200 5.70E-28 2 1 G481 BI268123
BI268123 NF116D11IN1F1094 Insect herbivory M . . . 3 224 9.10E-28 2
1 G481 AW981720 AW981720 PC15H07 Pine TriplEx pollen cone li . . .
3 204 9.10E-28 2 1 G481 AA660543 AA660543 00429 MtRHE Medicago
truncatula cDN . . . 3 187 1.30E-27 2 1 G481 BE726750 BE726750
894093C12.y3 C. reinhardtii CC-1690 . . . 2 236 1.90E-27 2 1 G481
BI309186 BI309186 EST530596 GPOD Medicago truncatula . . . 2 224
4.00E-27 2 1 G481 AW931376 AW931376 EST357219 tomato fruit mature
green . . . 2 244 7.10E-27 2 1 G481 BF636140 BF636140
NF060H09DT1F1079 Drought Medicago t . . . 3 224 1.70E-26 2 1 G481
BG526135 BG526135 57-6 Stevia field grown leaf cDNA S . . . -1 228
2.50E-26 2 1 G481 BI311277 BI311277 EST5313027 GESD Medicago
truncatula . . . 2 192 2.60E-26 2 1 G481 BI721770 BI721770
1031057H04.y1 C. reinhardtii CC-169 . . . 2 212 2.90E-26 2 1 G481
AW200790 AW200790 se93e11.y1 Gm-c1027 Glycine max cDN . . . 2 225
3.50E-26 2 1 G481 AY058919 AY058919 Vernonia galamensis CCAAT-box
bindi . . . 1 187 3.80E-26 2 1 G481 BM109471 BM109471 EST557007
potato roots Solanum tube . . . 3 220 4.20E-26 2 1 G481 AY058917
AY058917 Glycine max clone se2.11d12 CCAAT-b . . . 1 188 1.00E-25 2
1 G481 AY058920 AY058920 Argemone mexicana CCAAT-box binding . . .
1 189 1.30E-25 2 1 G481 AU088581 AU088581 AU088581 Rice callus
Oryza sativa c . . . 2 178 1.40E-25 2 1 G481 AW720671 AW720671
LjNEST6a3rc Lotus japonicus nodule . . . 2 192 2.10E-25 2 1 G481
BI419749 BI419749 LjNEST14e12r Lotus japonicus nodule . . . 1 192
2.10E-25 2 1 G481 AW719547 AW719547 LjNEST6a3r Lotus japonicus
nodule I . . . 2 192 2.20E-25 2 1 G481 BM134935 BM134935
WHE0460_A02_A03ZS Wheat Fusarium gr . . . 2 211 2.60E-25 2 1 G481
AI965590 AI965590 sc74b05.y1 Gm-c1018 Glycine max cDN . . . 2 208
4.00E-25 2 1 G481 BI875522 BI875522 963125B06.y1 C. reinhardtii
CC-1690 . . . 3 212 5.70E-25 2 1 G481 BI531782 BI531782
1024116E03.y1 C. reinhardtii CC-169 . . . 3 212 5.90E-25 2 1 G481
AW688588 AW688588 NF009C11ST1F1000 Developing stem Me . . . 2 218
8.00E-25 2 1 G481 BG368375 BG368375 HVSMEi0018C01f Hordeum vulgare
20 D . . . 1 214 8.20E-25 2 1 G481 BF270164 BF270164 GA_Eb0007A21f
Gossypium arboreum 7 . . . 2 206 1.70E-24 2 1 G481 BH472297
BH472297 BOGJF90TF BOGJ Brassica oleracea ge . . . 1 187 1.70E-24 2
1 G481 AY058918 AY058918 Glycine max clone ses2w.pk0015.a4 C . . .
1 178 1.10E-23 2 1 G481 BF291752 BF291752 WHE2205_F04_K07ZS
Aegilops speltoid . . . 3 205 1.10E-23 2 1 G481 BF169598 BF169598
NXCI_125_B04_F NXCI (Nsf Xylem Comp . . . 2 210 8.30E-23 2 1 G481
BH470962 BH470962 BOGNF35TF BOGN Brassica oleracea ge . . . 3 185
1.00E-22 2 1 G481 BI952722 BI952722 HVSMEm0007l19f Hordeum vulgare
gree . . . 1 230 2.20E-22 2 1 G481 AL506199 AL506199 AL506199
Hordeum vulgare Barke deve . . . 1 179 2.30E-22 2 1 G481 BH659234
BH659234 BOMDK68TR BO_2_3_KB Brassica olerac . . . 3 179 4.00E-22 2
1 G481 BE802539 BE802539 sr32f02.y1 Gm-c1050 Glycine max cDN . . .
2 221 1.40E-21 2 1 G481 BE196056 BE196056 HVSMEh0091D23f Hordeum
vulgare 5-45 . . . 3 211 1.60E-21 2 1 G481 AL509098 AL509098
AL509098 Hordeum vulgare Barke deve . . . 3 179 2.70E-21 2 1 G481
AP003266 AP003266 Oryza sativa genomic DNA, chromosom . . . -3 194
3.00E-21 2 1 G481 AF410176 AF410176 Zea mays leafy cotyledon1
(Lec1) mR . . . 3 179 3.30E-21 2 1 G481 AX365282 AX365282 Sequence
18 from Patent WO0206499. . . . 3 179 3.30E-21 2 1 G481 AY058921
AY058921 Triticum aestivum CCAAT-box binding . . . 1 175 5.70E-21 2
1 G481 BG662094 BG662094 Ljirnpest38-110-g8 Ljirnp Lambda Hy . . .
1 266 8.90E-20 1 1 G481 BH645253 BH645253 BOMFL56TR BO_2_3_KB
Brassica olerac . . . 3 166 2.70E-19 2 1 G481 AP004179 AP004179
Oryza sativa chromosome 2 clone OJ1 . . . 3 180 4.60E-19 2 1 G481
BM500534 BM500534 PAC000000000627 Pioneer AF-1 array . . . 2 179
8.20E-19 2 1 G481 BH701005 BH701005 BOMMD16TR BO_2_3_KB Brassica
olerac . . . 2 248 5.60E-18 1 1 G481 BH678940 BH678940 BOMIF09TF
BO_2_3_KB Brassica olerac . . . -1 244 1.10E-17 1 1 G481 AP004791
AP004791 Oryza sativa (japonica cultivar-gro . . . -1 183 9.20E-17
2 1 G481 BE121888 BE121888 894015G05.y1 C. reinhardtii CC-1690 . .
. 1 236 1.30E-16 1 1 G481 AX288136 AX288136 Sequence 7 from Patent
WO0177311. 1 . . . 3 231 3.90E-16 1 1 G481 AV411210 AV411210
AV411210 Lotus japonicus young plan . . . 2 228 1.10E-15 1 1 G481
AV425835 AV425835 AV425835 Lotus japonicus young plan . . . 2 228
1.10E-15 1 1 G481 BI206716 BI206716 EST524756 cTOS Lycopersicon
esculen . . . 3 227 1.10E-15 1 1 G481 BF645376 BF645376
NF040B05EC1F1044 Elicited cell cult . . . 2 224 1.90E-15 1 1 G481
BM347760 BM347760 MEST281-G09.T3 lSUM5-RN Zea mays cD . . . -3 219
6.10E-15 1 1 G481 AW648379 AW648379 EST326833 tomato germinating
seedli . . . 1 218 1.40E-14 1 1 G481 BI176409 BI176409 EST521199 P.
infestans-challenged l . . . 2 142 1.90E-14 2 1 G481 BM109406
BM109406 EST556942 potato roots Solanum tube . . . 1 214 1.90E-14 1
1 G481 BM348480 BM348480 MEST291-E08.T3 lSUM5-RN Zea mays cD . . .
-3 214 2.30E-14 1 1 G481 BI531808 BI531808 1024116G03.y1 C.
reinhardtii CC-169 . . . 3 122 2.70E-14 2 1 G481 BM158109 BM158109
NXLV_029_E11_F NXLV (Nsf Xylem Late . . . 3 212 4.40E-14 1 1 G481
AI966550 AI966550 sc51h01.y1 Gm-c1015 Glycine max cDN . . . 2 212
5.90E-14 1 1 G481 BM341073 BM341073 MEST329-H08.T3 ISUM5-RN Zea
mays cD . . . -2 210 6.00E-14 1 1 G481 BM335521 BM335521
MEST162-H08.T3 ISUM5-RN Zea mays cD . . . -2 210 6.60E-14 1 1 G481
AV422691 AV422691 AV422691 Lotus japonicus young plan . . . 2 138
2.20E-13 2 1 G481 BG039303 BG039303 NXSI_097_E11_F NXSI (Nsf Xylem
Side . . . 3 205 2.70E-13 1 1 G481 BF068031 BF068031 st86h12.y1
Gm-c1054 Glycine max cDN . . . 1 205 3.50E-13 1 1 G481 BF777951
BF777951 NXSI_079_003_F NXSI (Nsf Xylem Side . . . 2 205 4.80E-13 1
1 G481 BI720257 BI720257 1031048F06.y1 C. reinhardtii CC-169 . . .
3 204
4.90E-13 1 1 G481 BM441686 BM441686 EBed07_SQ001_E05_R IGF Barley
EBed0 . . . 2 115 1.70E-12 2 1 G481 AX365284 AX365284 Sequence 20
from Patent WO0206499. . . . 3 124 4.10E-12 2 1 G481 BH153040
BH153040 Gm_ISb001_083_P16R ISU Soybean BAC . . . -1 185 2.90E-11 1
1 G481 BG589029 BG589029 EST490838 MHRP-Medicago truncatula . . . 2
187 3.30E-11 1 1 G481 BG350792 BG350792 098C07 Mature tuber lambda
ZAP Sola . . . 1 185 1.90E-10 1 1 G481 BM094268 BM094268
sah27d01.y1 Gm-c1036 Glycine max cD . . . 3 167 3.20E-09 1 1 G481
BM271333 BM271333 sak08b06.y1 Gm-c1075 Glycine max cD . . . 1 156
5.10E-08 1 1 G481 AW693654 AW693654 NF066H08ST1F1000 Developing
stem Me . . . 3 161 6.30E-08 1 1 G481 C19290 C19290 C19290 Rice
panicle at ripening stage . . . 1 153 1.40E-07 1 1 G481 AL388746
AL388746 MtBC50E04F1 MtBC Medicago truncatul . . . -2 145 8.90E-07
1 1 G481 BI068503 BI068503 C022P78U Populus strain T89 leaves . . .
1 142 2.70E-06 1 1 G481 BH591716 BH591716 BOGTY51TR BOGT Brassica
oleracea ge . . . -2 139 3.50E-06 1 1 G481 BM333505 BM333505
MEST156-F03.T3 ISUM5-RN Zea mays cD . . . -2 138 4.40E-06 1 1 G481
BH645120 BH645120 BOHZM09TF BO_2_3_KB Brassica olerac . . . 3 149
5.50E-06 1 1 G481 BM112643 BM112643 EST560179 potato roots Solanum
tube . . . 2 143 9.40E-06 1 1 G481 BE356637 BE356637
DG1_126_D05.g1_A002 Dark Grown 1 (D . . . 1 133 1.80E-05 1 1 G481
BH580896 BH580896 BOGLY95TR BOGL Brassica oleracea ge . . . -2 146
2.20E-05 1 1 G481 BE400220 BE400220 AWB001.E09F000328 ITEC AWB
Wheat Me . . . 3 132 2.20E-05 1 1 G481 AW981721 AW981721 PC15H08
Pine TriplEx pollen cone li . . . 2 129 4.40E-05 1 1 G481 BI325183
BI325183 baa05b02.x1 Cassava EYC library1 Ma . . . -1 124 0.00029 1
1 G481 BE822946 BE822946 GM700019A20C12 Gm-r1070 Glycine max . . .
-3 130 0.00055 1 1 G481 BI271659 BI271659 NF026A03FL1F1020
Developing flower . . . 1 121 0.00076 1 1 G481 AMTAM4 X59057 A.
majus transposable element Tam4 DNA . . . 2 134 0.0019 1 1 G481
BG653330 BG653330 sad87a07.y1 Gm-c1055 Glycine max cD . . . 3 114
0.0023 1 1 G481 BG644353 BG644353 EST505972 KV3 Medicago truncatula
c . . . 2 126 0.004 1 1 G481 BG650027 BG650027 sad90h01.y1 Gm-c1055
Glycine max cD . . . 1 110 0.0056 1 1 G481 AW734322 AW734322
sk81f11.y1 Gm-c1016 Glycine max cDN . . . 3 110 0.0057 1 1 G481
BG046421 BG046421 saa63e06.y1 Gm-c1060 Glycine max cD . . . 2 110
0.0059 1 1 G481 BG726061 BG726061 sae06d09.y1 Gm-c1055 Glycine max
CD . . . 3 110 0.0062 1 1 G481 BF070629 BF070629 st23b12.y1
Gm-c1065 Glycine max cDN . . . 1 110 0.0062 1 1 G481 AI443631
AI443631 sa42e05.y1 Gm-c1004 Glycine max cDN . . . 2 110 0.0065 1 1
G481 BF009637 BF009637 ss81h09.y1 Gm-c1064 Glycine max cDN . . . 1
109 0.0076 1 1 G481 BF627138 BF627138 HVSMEb0004A20f Hordeum
vulgare seed . . . 1 125 0.008 1 1 G481 AV420244 AV420244 AV420244
Lotus japonicus young plan . . . 3 109 0.0085 1 1 G481 BH637694
BH637694 1008018B12.2EL_x1 1008 --RescueMu G . . . -2 111 0.0097 1
1 G481 AI967494 AI967494 Ljirnpest03-196-b3 Ljirnp Lambda Hy . . .
2 109 0.01 1 1 G481 AV416827 AV416827 AV416827 Lotus japonicus
young plan . . . 1 109 0.011 1 1 G481 BG131195 BG131195 EST464087
tomato crown gall Lycoper . . . 3 107 0.012 1 1 G481 BF052943
BF052943 EST438173 potato leaves and petiole . . . 1 107 0.012 1 1
G481 AV409183 AV409183 AV409183 Lotus japonicus young plan . . . 3
108 0.015 1 1 G481 AW776198 AW776198 EST335263 DSIL Medicago
truncatula . . . 1 105 0.022 1 1 G481 AL368819 AL368819 MtBA27A03F1
MtBA Medicago truncatul . . . 3 105 0.026 1 1 G481 C99407 C99407
C99407 Rice panicle at ripening stage . . . -3 105 0.026 1 1 G481
BH448006 BH448006 BOGBH37TR BOGB Brassica oleracea ge . . . -1 113
0.049 1 1 G481 AW185273 AW185273 se89c10.y1 Gm-c1023 Glycine max
cDN . . . 3 110 0.061 1 1 G481 AW423569 AW423569 sh68f08.y1
Gm-c1015 Glycine max cDN . . . 3 110 0.065 1 1 G481 BG507868
BG507868 sac82d02.y1 Gm-c1072 Glycine max cD . . . 3 110 0.083 1 1
G481 C19737 C19737 C19737 Rice panicle at ripening stage . . . 3
100 0.089 1 1 G481 AW719575 AW719575 LjNEST6a11r Lotus japonicus
nodule . . . 1 109 0.095 1 1 G481 AW706867 AW706867 sk07d05.y1
Gm-c1023 Glycine max cDN . . . 3 110 0.099 1 1 G481 AI460665
AI460665 sa71g05.y1 Gm-c1004 Glycine max cDN . . . 2 110 0.11 1 1
G481 AW759521 AW759521 sl44d11.y1 Gm-c1027 Glycine max cDN . . . 2
110 0.11 1 1 G481 BF595796 BF595796 sv04f12.y1 Gm-c1056 Glycine max
cDN . . . 2 110 0.11 1 1 G481 BM178052 BM178052 saj68e01.y1
Gm-c1072 Glycine max cD . . . 3 109 0.16 1 1 G481 BI424397 BI424397
saf34c08.y4 Gm-c1077 Glycine max cD . . . 1 110 0.16 1 1 G481
BG044257 BG044257 saa25h07.y1 Gm-c1059 Glycine max cD . . . 2 110
0.17 1 1 G481 AW234956 AW234956 sf21b08.y1 Gm-c1028 Glycine max cDN
. . . 1 110 0.17 1 1 G481 AW759820 AW759820 sl54e01.y1 Gm-c1027
Glycine max cDN . . . 3 110 0.18 1 1 G481 AI959799 AI959799
sc94d03.y1 Gm-c1019 Glycine max cDN . . . 1 110 0.18 1 1 G481
AW201988 AW201988 sf09f10.y1 Gm-c1027 Glycine max cDN . . . 3 109
0.2 1 1 G481 AW164642 AW164642 se74f06.y1 Gm-c1023 Glycine max cDN
. . . 3 110 0.2 1 1 G481 AT002114 AT002114 AT002114 Flower bud cDNA
Brassica r . . . 2 98 0.23 1 1 G481 BF113032 BF113032 EST440542
tomato breaker fruit Lyco . . . 1 107 0.25 1 1 G481 BE659989
BE659989 1010 GmaxSC Glycine max cDNA, mRNA . . . 2 110 0.26 1 1
G481 OSA300218 AJ300218 Oryza sativa nf-yb1 gene and nf-YB1 . . . 2
111 0.27 1 1 G481 BG597547 BG597547 EST496225 cSTS Solanum
tuberosum cD . . . 1 107 0.32 1 1 G481 BF644204 BF644204
NF060B12EC1F1101 Elicited cell cult . . . 2 105 0.33 1 1 G481
AF464906 AF464906 Glycine max repressor protein (Dr1) . . . 1 110
0.34 1 1 G481 BE436801 BE436801 EST407919 tomato breaker fruit, TlG
. . . 2 107 0.36 1 1 G481 BE659987 BE659987 7-F12 GmaxSC Glycine
max cDNA, mRNA . . . 1 109 0.36 1 1 G481 BG451060 BG451060
NF098C04DT1F1024 Drought Medicago t . . . 3 105 0.42 1 1 G481
BM436739 BM436739 WA009B06_53061 An expressed sequen . . . 3 107
0.45 1 1 G481 BF273545 BF273545 GA_Eb0018J12f Gossypium arboreum 7
. . . 1 104 0.5 1 1 G481 BM411335 BM411335 EST585662 tomato breaker
fruit Lyco . . . 3 107 0.5 1 1 G481 AW830697 AW830697 sm06h11.y1
Gm-c1027 Glycine max cDN . . . 2 91 0.54 1 1 G481 BE202566 BE202566
EST392975 KV1 Medicago truncatula c . . . 2 105 0.55 1 1 G481
BG240158 BG240158 OV1_18_F02.b1_A002 Ovary 1 (OV1) So . . . 3 101
0.56 1 1 G481 BM817060 BM817060 HC01C02_T3.ab1 HC Hordeum vulgare c
. . . 1 103 0.58 1 1 G481 BE998485 BE998485 EST430208 GVSN Medicago
truncatula . . . 3 105 0.58 1 1 G481 BE022456 BE022456 sm74b08.y1
Gm-c1015 Glycine max cDN . . . 2 103 0.6 1 1 G481 BF647976 BF647976
NF013H04EC1F1042 Elicited cell cult . . . 1 105 0.63 1 1 G481
BG102166 BG102166 RHIZ2_21_F06.b1_A003 Rhizome2 (RHIZ . . . 3 101
0.63 1 1 G481 AU084707 AU084707 AU084707 Cryptomeria japonica inner
. . . 1 101 0.68 1 1 G481 BG648823 BG648823 EST510442 HOGA Medicago
truncatula . . . 1 105 0.7 1 1 G481 BG648909 BG648909 EST510528
HOGA Medicago truncatula . . . 2 105 0.72 1 1 G481 BM380524
BM380524 MEST521-B07.univ ISUM6 Zea mays cDN . . . -3 103 0.75 1 1
G481 BG593107 BG593107 EST491785 cSTS Solanum tuberosum cD . . . 3
103 0.81 1 1 G481 BG052069 BG052069 RHIZ2_5_F11.b1_A003 Rhizome2
(RHIZ2 . . . 3 101 0.9 1 1 G481 BE497740 BE497740 WHE0956_G06_M12ZS
Wheat pre-anthesi . . . 2 100 0.92 1 1 G481 BG873649 BG873649
MEST8-E10.T7-1 ISUM3-TL Zea mays cD . . . 3 101 0.94 1 1 G481
AW432997 AW432997 si03b08.y1 Gm-c1029 Glycine max cDN . . . 2 88
0.96 1 1 G481 AW064635 AW064635 ST33H06 Pine TriplEx shoot tip libr
. . . 2 100 0.96 1 1 G481 AX365283 AX365283 Sequence 19 from Patent
WO0206499. . . . 1 101 0.97 1 1 G481 BG648613 BG648613 EST510232
HOGA Medicago truncatula . . . 3 99 0.993 1 1 G481 BE640725
BE640725 Cri2_1_E07_SP6 Ceratopteris Spore L . . . 2 100 0.995 1 1
G481 AF464902 AF464902 Oryza sativa repressor protein (Dr1 . . . 2
101 0.996 1 1 G481 AF464903 AF464903 Triticum aestivum repressor
protein . . . 2 100 0.999 1 1 G481 BE449790 BE449790 EST361228
tomato root, plants pre-a . . . 3 97 0.9991 1 1 G481 BI206380
BI206380 EST524420 cTOS Lycopersicon esculen . . . 1 97 0.9996 1 1
G481 GI-22380 CAAT-box DNA binding protein subunit B (N . . . 1 262
1.10E-45 3 1 G481 GI-115840 CBFA_MAIZE CCAAT-BINDING 1 262 1.10E-45
3 TRANSCRIPTION FAC . . . 1 G481 GI-7443522 S22820 transcription
factor NF-Y, CCAAT-bi . . . 1 262 1.10E-45 3 1 G481 GI-15408794
putative CCAAT-binding transcription facto . . . 1 194 2.60E-30 2 1
G481 GI-16902054 CCAAT-box binding factor HAP3 B domain [Ve . . . 1
187 1.00E-28 2 1 G481 GI-16902050 CCAAT-box binding factor HAP3 B
domain [Gl . . . 1 188 2.70E-28 2 1 G481 GI-16902056 CCAAT-box
binding factor HAP3 B domain [Ar . . . 1 189 4.30E-28 2 1 G481
GI-15321716 AF410176_1 leafy cotyledon1 [Zea mays] 1 179 1.20E-26 3
1 G481 GI-16902052 CCAAT-box binding factor HAP3 B domain [Gl . . .
1 178 2.90E-26 2 1 G481 GI-15408793 hypothetical
protein.about.similar to CCAAT-bind . . . 1 180 2.70E-24 2 1 G481
GI-16902058 CCAAT-box binding factor HAP3 B domain [Tr . . . 1 175
1.50E-23 2 1 G481 GI-18481628 AF464906_1 repressor protein [Glycine
max ] 1 110 2.10E-09 2 1 G481 GI-13928060 NF-YB1 protein [Oryza
sativa ] 1 111 0.00069 1 1 G481 GI-18481620 AF464902_1 repressor
protein [Oryza sativa] 1 101 0.037 1 1 G481 GI-18481622 AF464903_1
repressor protein [Triticum aes . . . 1 100 0.052 1 3 G1466
BH596941 BH596941 BOHFG41TR BOHF Brassica oleracea ge . . . 1 557
1.40E-65 2 3 G1466 BH497171 BH497171 BOHKV94TF BOHK Brassica
oleracea ge . . . -1 531 7.70E-49 1 3 G1466 BH515525 BH515525
BOGZE84TR BOGZ Brassica oleracea ge . . . -1 518 1.60E-47 1 3 G1466
BH685857 BH685857 BOHWL93TF BO_2_3_KB Brassica olerac . . . 1 409
6.90E-36 1 3 G1466 BH556937 BH556937 BOHCT42TF BOHC Brassica
oleracea ge . . . 3 374 3.20E-32 1 3 G1466 BH716964 BH716964
BOMNM26TR BO_2_3_KB Brassica olerac . . . -2 371 6.40E-32 1 3 G1466
BH556945 BH556945 BOHCT42TR BOHC Brassica oleracea ge . . . -3 205
3.40E-24 2 3 G1466 BH586673 BH586673 BOHPK25TF BOHP Brassica
oleracea ge . . . -1 293 1.80E-23 1 3 G1466 BH479514 BH479514
BOHEU46TF BOHE Brassica oleracea ge . . . -3 293 2.50E-23 1 3 G1466
BH442040 BH442040 BOHJW94TR BOHJ Brassica oleracea ge . . . 2 284
2.00E-22 1 3 G1466 BH468201 BH468201 BOHAW05TF BOHA Brassica
oleracea ge . . . 3 263 6.80E-20 1 3 G1466 BH554599 BH554599
BOHES28TF BOHE Brassica oleracea ge . . . -2 126 4.40E-17 3 3 G1466
BH443975 BH443975 BOGYY14TR BOGY Brassica oleracea ge . . . -1 220
7.20E-15 1 3 G1466 BH478506 BH478506 BOGJO28TR BOGJ Brassica
oleracea ge . . . 2 184 3.10E-11 1 3 G1466 BH248066 BH248066
BOGAU25TR BOGA Brassica oleracea ge . . . 2 164 7.00E-08 1 3 G1466
BH455609 BH455609 BOHPV23TF BOHP Brassica oleracea ge . . . -1 160
6.60E-07 1 3 G1466 BH453095 BH453095 BOGVR55TF BOGV Brassica
oleracea ge . . . 2 141 8.30E-05 1 3 G1466 BH536461 BH536461
BOHMJ41TR BOHM Brassica oleracea ge . . . 2 139 8.90E-05 1 3 G1466
BE462774 BE462774 EST325108 tomato flower buds 0-3 mm . . . 1 129
0.00014 1 3 G1466 AW399721 AW399721 EST310221 L. pennellii
trichome, Co . . . 3 124 0.00049 1 3 G1466 AV413010 AV413010
AV413010 Lotus japonicus young plan . . . 3 119 0.00054 1 3 G1466
AW032605 AW032605 EST276164 tomato callus, TAMU Lycop . . . 3 114
0.0021 1 3 G1466 BI927221 BI927221 EST547110 tomato flower, 3-8 mm
b . . . 2 121 0.0023 1 3 G1466 OSJN00182 AL662981 Oryza sativa
chromosome 4 clone OSJ . . . 3 132 0.0029 1 3 G1466 BG662160
BG662160 Ljirnpest39-183-g3 Ljirnp Lambda Hy . . . 2 111 0.0037 1 3
G1466 BG599234 BG599234 EST504134 cSTS Solanum tuberosum cD . . . 3
125 0.0047 1 3 G1466 BG133465 BG133465 EST466357 tomato crown gall
Lycoper . . . 3 110 0.0059 1 3 G1466 BG596415 BG596415 EST495093
cSTS Solanum tuberosum cD . . . 3 109 0.0061 1 3 G1466 BI922297
BI922297 EST542201 tomato callus Lycopersico . . . 3 114 0.0065 1 3
G1466 BG097828 BG097828 EST462347 potato leaves and petiole . . . 1
122 0.0081 1 3 G1466 BI178485 BI178485 EST519430 cSTE Solanum
tuberosum cD . . . 1 120 0.0095 1 3 G1466 BG580068 BG580068
EST481790 GVN Medicago truncatula c . . . 3 114 0.0095 1 3 G1466
AW932502 AW932502 EST358345 tomato fruit mature green . . . 3 118
0.011 1 3 G1466 BH700517 BH700517 BOMEG26TR BO_2_3_KB Brassica
olerac . . . 1 106 0.013 1 3 G1466 AV414925 AV414925 AV414925 Lotus
japonicus young plan . . . 1 106 0.014 1 3 G1466 BG887252 BG887252
EST513103 cSTD Solanum tuberosum cD . . . 3 121 0.015 1 3 G1466
BH649717 BH649717 BOHTZ83TF BO_2_3_KB Brassica olerac . . . -2 121
0.016 1 3 G1466 AW691672 AW691672 NF047G10ST1F1000 Developing stem
Me . . . 3 105 0.02 1 3 G1466 BI316339 BI316339 saf02a08.y1
Gm-c1065 Glycine max cD . . . 1 115 0.026 1 3 G1466 BG409415
BG409415 00787 leafy spurge Lambda HybriZAP . . . 2 115 0.026 1 3
G1466 AP003837 AP003837 Oryza sativa chromosome 7 clone OJ1 . . .
-2 123 0.027 1 3 G1466 AW928976 AW928976 EST337860 tomato flower
buds 8 mm t . . . 2 115 0.03 1 3 G1466 OSJN00167 AL662965 Oryza
sativa chromosome 4 clone OSJ . . . -2 122 0.034 1 3 G1466 BH660321
BH660321 BOMGE76TR BO_2_3_KB Brassica olerac . . . -3 116 0.036 1 3
G1466 AW395660 AW395660 sg73f12.y1 Gm-c1007 Glycine max cDN . . . 3
102 0.037 1 3 G1466 BE459691 BE459691 EST414983 tomato
developing/immatur . . . 2 102 0.038 1 3 G1466 BH558685 BH558685
BOGEH17TR BOGE Brassica oleracea ge . . . 1 116 0.05 1 3 G1466
BH486360 BH486360 BOHLY92TF BOHL Brassica oleracea ge . . . 3 116
0.056 1 3 G1466 AW218077 AW218077 EST296792 tomato flower buds,
anthe . . . 1 114 0.078 1 3 G1466 AW650052 AW650052 EST328506
tomato germinating seedli . . . 1 112 0.08 1 3 G1466 BH698615
BH698615 BOHUQ33TR BO_2_3_KB Brassica olerac . . . 3 113 0.081 1 3
G1466 BG596140 BG596140 EST494818 cSTS Solanum tuberosum cD . . . 1
114 0.093 1 3 G1466 BE205043 BE205043 EST397719 KV0 Medicago
truncatula c . . . 2 112 0.097 1 3 G1466 BE919377 BE919377
EST423230 potato leaves and petiole . . . 3 111 0.097 1 3 G1466
BH437084 BH437084 BOHKZ27TF BOHK Brassica oleracea ge . . . -3 99
0.1 1 3 G1466 BF278686 BF278686 GA_Eb0035E21f Gossypium arboreum 7
. . . 3 111 0.11 1 3 G1466 BI933949 BI933949 EST553838 tomato
flower, anthesis L . . . 3 113 0.12 1 3 G1466 BF096307 BF096307
EST360356 tomato nutrient deficient . . . 2 109 0.13 1 3 G1466
BH737844 BH737844 BOMAX76TF BO_2_3_KB Brassica olerac . . . -1 109
0.14 1 3 G1466 BI929823 BI929823 EST549712 tomato flower, 3-8 mm b
. . . 3 104 0.17 1 3 G1466 BH513097 BH513097 BOGEX21TR BOGE
Brassica oleracea ge . . . -1 112 0.17 1 3 G1466 BE203214 BE203214
EST403236 KV1 Medicago truncatula c . . . 1 110 0.18 1 3 G1466
BE202448 BE202448 EST392897 KV1 Medicago truncatula c . . . 2 104
0.19 1 3 G1466 BF644564 BF644564 NF016D06EC1F1057 Elicited cell
cult . . . 1 110 0.19 1 3 G1466 BI420528 BI420528 LjNEST58c10r
Lotus japonicus nodule . . . 3 96 0.2 1 3 G1466 BF010496 BE010496
NXCI_084_G01_F NXCI (Nsf Xylem Comp . . . 2 95 0.2 1 3 G1466
BI308763 BI308763 EST530173 GPOD Medicago truncatula . . . 3 107
0.21 1 3 G1466 BH469525 BH469525 BOGSA70TF BOGS Brassica oleracea
ge . . . 2 111 0.22 1 3 G1466 BE442810 BE442810 WHE1106_C02_E04ZS
Wheat etiolated s . . . 1 110 0.23 1 3 G1466 BH569775 BH569775
BOGMV91TF BOGM Brassica oleracea ge . . . -3 109 0.23 1 3 G1466
BH723965 BH723965 BOMHF56TF BO_2_3_KB Brassica olerac . . . -2 110
0.25 1 3 G1466 BH475440 BH475440 BOHNA92TF BOHN Brassica oleracea
ge . . . -2 109 0.26 1 3 G1466 BH730453 BH730453 BOHVD24TR
BO_2_3_KB Brassica olerac . . . 1 110 0.27 1 3 G1466 AC082644
AC082644 Oryza sativa chromosome 3 BAC OSJNB . . . -2 113 0.28 1 3
G1466 AZ124264 AZ124264 T223049b Medicago truncatula BAC li . . .
-3 93 0.31 1 3 G1466 BI922762 BI922762 EST542666 tomato callus
Lycopersico . . . 3 104 0.34 1 3 G1466 AI443156 AI443156 sa50f01.y1
Gm-c1004 Glycine max cDN . . . 3 105 0.34 1 3 G1466 BE249557
BE249557 NF022C06LF1F1049 Developing leaf Me . . . 1 94 0.34 1 3
G1466 AL379504 AL379504 MtBB45G03F1 MtBB Medicago truncatul . . . 1
102 0.35 1 3 G1466 BF646352 BF646352 NF068G05EC1F1039 Elicited cell
cult . . . 1 106 0.47 1 3 G1466 BH498652 BH498652 BOGZR84TR BOGZ
Brassica oleracea ge . . . -2 107 0.48 1 3 G1466 BM442042 BM442042
EBan01_SQ002_A06_R IGF Barley EBan0 . . . 2 96 0.49 1 3 G1466
BG447666 BG447666 NF019E03ST1F1000 Developing stem Me . . . 1 106
0.49 1 3 G1466 AV427033 AV427033 AV427033 Lotus japonicus young
plan . . . 1 98 0.5 1 3 G1466 AW216996 AW216996 EST295710 tomato
callus, TAMU Lycop . . . 2 92 0.53 1 3 G1466 AW648500 AW648500
EST326954 tomato germinating seedli . . . 3 102 0.55 1 3 G1466
AW649521 AW649521 EST327975
tomato germinating seedli . . . 1 102 0.59 1 3 G1466 BF187212
BF187212 EST443499 potato stolon, Cornell Un . . . 3 102 0.62 1 3
G1466 BE202443 BE202443 EST392892 KV1 Medicago truncatula c . . . 3
104 0.65 1 3 G1466 BE344294 BE344294 EST409456 potato stolon,
Cornell Un . . . 3 102 0.66 1 3 G1466 C72509 C72509 C72509 Rice
panicle at flowering stag . . . 2 89 0.67 1 3 G1466 BH743977
BH743977 gt29g06.b1 BoBuds01 Brassica olerac . . . 2 101 0.68 1 3
G1466 AI967453 AI967453 Ljirnpest02-129-d2 Ljirnp Lambda Hy . . . 1
99 0.7 1 3 G1466 BI419994 BI419994 LjNEST42f9r Lotus japonicus
nodule . . . 2 102 0.7 1 3 G1466 BG646874 BG646874 EST508493 HOGA
Medicago truncatula . . . 2 104 0.7 1 3 G1466 AW694435 AW694435
NF076B06ST1F1048 Developing stem Me . . . 1 102 0.75 1 3 G1466
AP004055 AP004055 Oryza sativa chromosome 2 clone OJ1 . . . 1 107
0.77 1 3 G1466 AP004144 AP004144 Oryza sativa chromosome 2 clone
OJ1 . . . -3 107 0.77 1 3 G1466 OSJN00115 AL606997 Oryza sativa
chromosome 4 clone OSJ . . . -1 107 0.77 1 3 G1466 AP002071
AP002071 Oryza sativa genomic DNA, chromosom . . . -1 107 0.77 1 3
G1466 BE095245 BE095245 00306 leafy spurge Lambda HybriZAP . . . 1
88 0.79 1 3 G1466 BM526605 BM526605 sal43e01.y1 Gm-c1059 Glycine
max cD . . . 2 100 0.8 1 3 G1466 BI933721 BI933721 EST553610 tomato
flower, anthesis L . . . 3 102 0.86 1 3 G1466 BI272408 BI272408
NF020H09FL1F1079 Developing flower . . . 3 102 0.87 1 3 G1466
BI933639 BI933639 EST553528 tomato flower, anthesis L . . . 3 102
0.89 1 3 G1466 BM108916 BM108916 EST556452 potato roots Solanum
tube . . . 1 102 0.89 1 3 G1466 BH581102 BH581102 BOHKD50TF BOHK
Brassica oleracea ge . . . 3 95 0.9 1 3 G1466 BH692394 BH692394
BOMKE64TF BO_2_3_KB Brassica olerac . . . 3 99 0.92 1 3 G1466
BG131783 BG131783 EST464675 tomato crown gall Lycoper . . . 3 97
0.93 1 3 G1466 AI896339 AI896339 EST265782 tomato callus, TAMU
Lycop . . . 2 94 0.93 1 3 G1466 BG404995 BG404995 sac46g02.y1
Gm-c1062 Glycine max cD . . . 1 97 0.95 1 3 G1466 AP004042 AP004042
Oryza sativa chromosome 8 clone OJ1 . . . 1 104 0.95 1 3 G1466
AP004708 AP004708 Oryza sativa chromosome 8 clone P07 . . . -3 104
0.95 1 3 G1466 AP004274 AP004274 Oryza sativa chromosome 7 clone
P04 . . . -1 104 0.95 1 3 G1466 BE919578 BE919578 EST423347 potato
leaves and petiole . . . 1 100 0.95 1 3 G1466 BF634570 BF634570
NF061B09DT1F1076 Drought Medicago t . . . 3 100 0.96 1 3 G1466
BI420155 BI420155 LjNEST53f7r Lotus japonicus nodule . . . 3 96
0.97 1 3 G1466 AQ917137 AQ917137 T233170b Medicago truncatula BAC
li . . . -1 99 0.97 1 3 G1466 AW720029 AW720029 LjNEST15c9r Lotus
japonicus nodule . . . 2 96 0.98 1 3 G1466 AW568479 AW568479
si59c09.y1 Gm-r1030 Glycine max cDN . . . 3 97 0.98 1 3 G1466
BI420626 BI420626 LjNEST59e5r Lotus japonicus nodule . . . 3 96
0.99 1 3 G1466 BE433506 BE433506 EST400035 tomato breaker fruit,
TIG . . . 2 97 0.99 1 3 G1466 BI785473 BI785473 sai41d06.y1
Gm-c1065 Glycine max cD . . . 3 97 0.991 1 3 G1466 BH737087
BH737087 BOMGM48TR BO_2_3_KB Brassica olerac . . . 3 98 0.991 1 3
G1466 BM324672 BM324672 PIC1_34_B01.b1_A002 Pathogen-infect . . . 3
98 0.992 1 3 G1466 BH425892 BH425892 BOGDN33TF BOGD Brassica
oleracea ge . . . -3 98 0.994 1 3 G1466 BH645340 BH645340 BOHWO42TF
BO_2_3_KB Brassica olerac . . . -3 97 0.995 1 3 G1466 AW687534
AW687534 NF010F07RT1F1062 Developing root Me . . . 3 96 0.997 1 3
G1466 BF176934 BF176934 EM1_4_E11.b1_A002 Embryo 1 (EM1) So . . . 3
96 0.997 1 3 G1466 BI419241 BI419241 LjNEST44a5r Lotus japonicus
nodule . . . 3 96 0.998 1 3 G1466 AL380183 AL380183 MtBB50H09F1
MtBB Medicago truncatul . . . 1 94 0.998 1 3 G1466 AC079128
AC079128 Oryza sativa chromosome 10 clone OS . . . 3 101 0.998 1 3
G1466 BF070938 BF070938 st85e05.y1 Gm-c1054 Glycine max cDN . . . 3
95 0.999 1 3 G1466 AW928966 AW928966 EST337850 tomato flower buds 8
mm t . . . 3 86 0.9995 1 3 G1466 BH429646 BH429646 BOGSF37TF BOGS
Brassica oleracea ge . . . -3 97 0.9997 1 3 G1466 BH560749 BH560749
BOGPC01TR BOGP Brassica oleracea ge . . . -1 97 0.9997 1 3 G1466
AP004150 AP004150 Oryza sativa chromosome 2 clone OJ1 . . . -1 100
0.9998 1 3 G1466 AP004077 AP004077 Oryza sativa chromosome 2 clone
OJ1 . . . 1 100 0.9998 1 3 G1466 BG130763 BG130763 EST463655 tomato
crown gall Lycoper . . . 3 96 0.9998 1 3 G1466 BH484808 BH484808
BOGQC69TR BOGQ Brassica oleracea ge . . . 1 97 0.9998 1 3 G1466
BF651138 BF651138 NF101G05EC1F1038 Elicited cell cult . . . 1 96
0.9999 1 3 G1466 BF650226 BF650226 NF090F10EC1F1089 Elicited cell
cult . . . 3 96 0.9999 1 3 G1466 BF647598 BF647598 NF012A06EC1F1039
Elicited cell cult . . . 3 96 0.9999 1 3 G1466 BG447988 BG447988
NF103H10EC1F1090 Elicited cell cult . . . 3 96 0.9999 1 3 G1466
BH480188 BH480188 BOGZW54TR BOGZ Brassica oleracea ge . . . 3 97
0.99991 1 3 G1466 BF519054 BF519054 EST456514 DSIL Medicago
truncatula . . . 3 96 0.99994 1 3 G1466 GI-8096405 Similar to
Arabdopsis thaliana chromosome . . . -3 137 2.30E-12 2 3 G1466
GI-8096413 hypothetical protein [Oryza sativa] -3 135 2.20E-11 2 3
G1466 GI-8096416 hypothetical protein [Oryza sativa] -3 111
9.40E-09 3 3 G1466 GI-13161526 S locus F-box (SLF)-S2 protein
[Antirrhinu . . . -3 92 3.40E-08 3 3 G1466 GI-13161540 SLF-S2
protein [Antirrhinum hispanicum] -3 92 3.40E-08 3 3 G1466
GI-13161528 S locus F-box (SLF)-S2-like protein [Antir . . . -1 87
1.20E-07 3 3 G1466 GI-8096415 hypothetical protein [Oryza sativa]
-1 102 3.60E-07 2 3 G1466 GI-18854994 AC087599_5 unknown protein
[Oryza sativa] -1 90 1.10E-05 3 3 G1466 GI-14028986 AC079128_10
Unknown protein [Oryza sativa] -1 101 4.60E-05 2 3 G1466
GI-18854992 AC087599_3 putative transposase [Oryza sat . . . -3 93
0.0014 3 3 G1466 GI-12039340 AC082644_9 hypothetical protein [Oryza
sat . . . -1 113 0.0029 1 3 G1466 GI-14018043 AC079936_2
Hypothetical protein [Oryza sat . . . -1 87 0.012 2 3 G1466
GI-15451623 AC091734_4 Hypothetical protein [Oryza sat . . . -1 87
0.012 2 3 G1466 GI-15528755 hypothetical protein [Oryza sativa] -1
96 0.012 2 3 G1466 GI-8096406 hypothetical protein [Oryza sativa]
-3 98 0.013 2 3 G1466 GI-8096410 hypothetical protein [Oryza
sativa] -1 103 0.022 1 3 G1466 GI-18844804 contains ESTs
D15126(C0122),C97919(C0122).about.. . . -1 99 0.083 1 3 G1466
GI-18449949 AC099733_6 Unknown protein [Oryza sativa] -1 81 0.099 2
3 G1466 GI-18464016 AC090873_9 Hypothetical protein [Oryza sat . .
. -3 69 0.23 2 3 G1466 GI-19224986 AC077693_1 putative transposase
protein, 5 . . . -3 93 0.24 2 3 G1466 GI-12039332 AC082644_1
hypothetical protein [Oryza sat . . . -1 91 0.5 1 3 G1466
GI-18461280 similar to Oryza sativa chromosome 1, P045 . . . -3 72
0.61 2 3 G1466 GI-9858770 AF273333_2 BAC19.2 [Lycopersicon
esculentum] -1 70 0.83 2 3 G1466 GI-12039337 AC082644_6
hypothetical protein [Oryza sat . . . -1 87 0.85 1 3 G1466
GI-18642684 AC074283_6 Hypothetical protein [Oryza sat . . . -1 79
0.9 2 3 G1466 GI-18642688 AC074283_7 Unknown protein [Oryza sativa]
-1 75 0.97 1 3 G1466 GI-18087878 AC087182_15 hypothetical protein
[Oryza sa . . . -1 87 0.98 1 3 G1466 GI-18873858 AC079874_27
hypothetical protein [Oryza sa . . . -3 81 0.994 1 3 G1466
GI-6505722 ribosomal RNA apurinic site specific lyase -1 81 0.99993
1 3 G1466 GI-6513849 ribosomal RNA apurinic site specific lyase -1
81 0.99993 1
[0215]
Sequence CWU 1
1
4 1 832 DNA Arabidopsis thaliana CDS (103)...(528) G481 1
gagcgtttcg tagaaaaatt cgatttctct aaagccctaa aactaaaacg actatcccca
60 attccaagtt ctagggtttc catcttcccc aatctagtat aa atg gcg gat acg
114 Met Ala Asp Thr 1 cct tcg agc cca gct gga gat ggc gga gaa agc
ggc ggt tcc gtt agg 162 Pro Ser Ser Pro Ala Gly Asp Gly Gly Glu Ser
Gly Gly Ser Val Arg 5 10 15 20 gag cag gat cga tac ctt cct ata gct
aat atc agc agg atc atg aag 210 Glu Gln Asp Arg Tyr Leu Pro Ile Ala
Asn Ile Ser Arg Ile Met Lys 25 30 35 aaa gcg ttg cct cct aat ggt
aag att gga aaa gat gct aag gat aca 258 Lys Ala Leu Pro Pro Asn Gly
Lys Ile Gly Lys Asp Ala Lys Asp Thr 40 45 50 gtt cag gaa tgc gtc
tct gag ttc atc agc ttc atc act agc gag gcc 306 Val Gln Glu Cys Val
Ser Glu Phe Ile Ser Phe Ile Thr Ser Glu Ala 55 60 65 agt gat aag
tgt caa aaa gag aaa agg aaa act gtg aat ggt gat gat 354 Ser Asp Lys
Cys Gln Lys Glu Lys Arg Lys Thr Val Asn Gly Asp Asp 70 75 80 ttg
ttg tgg gca atg gca aca tta gga ttt gag gat tac ctg gaa cct 402 Leu
Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Asp Tyr Leu Glu Pro 85 90
95 100 cta aag ata tac cta gcg agg tac agg gag ttg gag ggt gat aat
aag 450 Leu Lys Ile Tyr Leu Ala Arg Tyr Arg Glu Leu Glu Gly Asp Asn
Lys 105 110 115 gga tca gga aag agt gga gat gga tca aat aga gat gct
ggt ggc ggt 498 Gly Ser Gly Lys Ser Gly Asp Gly Ser Asn Arg Asp Ala
Gly Gly Gly 120 125 130 gtt tct ggt gaa gaa atg ccg agc tgg taa
aagaagttgc aagtagtgat 548 Val Ser Gly Glu Glu Met Pro Ser Trp * 135
140 taagaacaat cgccaaatga tcaagggaaa ttagagatca gtgagttgtt
tatagttgag 608 ctgatcgaca actatttcgg gtttactctc aatttcggtt
atgttagttt gaacgtttgg 668 tttattgttt ccggtttagt tggttgtatt
taaagatttc tctgttagat gttgagaaca 728 cttgaatgaa ggaaaaattt
gtccacatcc tgttgttatt ttcgattcac tttcggaatt 788 tcatagctaa
tttattctca tttaatacca aatccttaaa ttaa 832 2 141 PRT Arabidopsis
thaliana 2 Met Ala Asp Thr Pro Ser Ser Pro Ala Gly Asp Gly Gly Glu
Ser Gly 1 5 10 15 Gly Ser Val Arg Glu Gln Asp Arg Tyr Leu Pro Ile
Ala Asn Ile Ser 20 25 30 Arg Ile Met Lys Lys Ala Leu Pro Pro Asn
Gly Lys Ile Gly Lys Asp 35 40 45 Ala Lys Asp Thr Val Gln Glu Cys
Val Ser Glu Phe Ile Ser Phe Ile 50 55 60 Thr Ser Glu Ala Ser Asp
Lys Cys Gln Lys Glu Lys Arg Lys Thr Val 65 70 75 80 Asn Gly Asp Asp
Leu Leu Trp Ala Met Ala Thr Leu Gly Phe Glu Asp 85 90 95 Tyr Leu
Glu Pro Leu Lys Ile Tyr Leu Ala Arg Tyr Arg Glu Leu Glu 100 105 110
Gly Asp Asn Lys Gly Ser Gly Lys Ser Gly Asp Gly Ser Asn Arg Asp 115
120 125 Ala Gly Gly Gly Val Ser Gly Glu Glu Met Pro Ser Trp 130 135
140 3 1342 DNA Arabidopsis thaliana CDS (16)...(1278) G1466 3
caacaagctt caaaa atg ggg gaa gaa gaa gag aac cct aat tcg atc gac 51
Met Gly Glu Glu Glu Glu Asn Pro Asn Ser Ile Asp 1 5 10 att ctt ccc
gag cta ctt gaa gaa gtt ctc ctt aga ttg ccc acg aaa 99 Ile Leu Pro
Glu Leu Leu Glu Glu Val Leu Leu Arg Leu Pro Thr Lys 15 20 25 tcg
atc ctc aaa tgc aga atc gtc tca aaa caa tgg agg tca ctc ctg 147 Ser
Ile Leu Lys Cys Arg Ile Val Ser Lys Gln Trp Arg Ser Leu Leu 30 35
40 gaa tcg agt agg ttc gcg gag agg cat atg agt ctt caa aac agc cgc
195 Glu Ser Ser Arg Phe Ala Glu Arg His Met Ser Leu Gln Asn Ser Arg
45 50 55 60 cgg aga atc tta gct gct tac aac tgc gac tgc ggc gga cgg
agg aag 243 Arg Arg Ile Leu Ala Ala Tyr Asn Cys Asp Cys Gly Gly Arg
Arg Lys 65 70 75 ctc cta ccc gag tca cgg ttt gaa ggg gac gaa gag
att gtc tat ctg 291 Leu Leu Pro Glu Ser Arg Phe Glu Gly Asp Glu Glu
Ile Val Tyr Leu 80 85 90 cac tgc gac gcc tca cga ccc tcg atg act
tgc caa ggt gtg atc tgc 339 His Cys Asp Ala Ser Arg Pro Ser Met Thr
Cys Gln Gly Val Ile Cys 95 100 105 ttc ccc gag caa gat tgg atc atc
gtt ttg aac cca tcg act agc caa 387 Phe Pro Glu Gln Asp Trp Ile Ile
Val Leu Asn Pro Ser Thr Ser Gln 110 115 120 ctt cgg cga ttc cct tcc
ggc ttg aac cat aac tgc aga ttt aga att 435 Leu Arg Arg Phe Pro Ser
Gly Leu Asn His Asn Cys Arg Phe Arg Ile 125 130 135 140 gga tta tgg
aag act ttc tct ccg gga aac tgg gta atg ggg ttt ggt 483 Gly Leu Trp
Lys Thr Phe Ser Pro Gly Asn Trp Val Met Gly Phe Gly 145 150 155 aga
gac aaa gtg aat ggg agg tat aaa gtg gtg agg atg tct ttt gct 531 Arg
Asp Lys Val Asn Gly Arg Tyr Lys Val Val Arg Met Ser Phe Ala 160 165
170 ttc tgg aga gtt agg caa gag gag cct gtg gtg gaa tgt ggt gtt ctt
579 Phe Trp Arg Val Arg Gln Glu Glu Pro Val Val Glu Cys Gly Val Leu
175 180 185 gat gtt gat act ggt gaa tgg cgg aag ctg agt cca cct cct
tat gtg 627 Asp Val Asp Thr Gly Glu Trp Arg Lys Leu Ser Pro Pro Pro
Tyr Val 190 195 200 gtc aat gtg gga agc aaa tcg gta tgc gtg aat gga
tct atc tac tgg 675 Val Asn Val Gly Ser Lys Ser Val Cys Val Asn Gly
Ser Ile Tyr Trp 205 210 215 220 tta cac att cag acg gtt tac aga ata
cta gcc ttg gat ctt cac aaa 723 Leu His Ile Gln Thr Val Tyr Arg Ile
Leu Ala Leu Asp Leu His Lys 225 230 235 caa gag ttt cat aaa gtc cca
gtg ccg cct acg cag atc act gtg gac 771 Gln Glu Phe His Lys Val Pro
Val Pro Pro Thr Gln Ile Thr Val Asp 240 245 250 act cag atg gtg aac
ctt gag gac cgt ctc gta ctt gct ata acc aga 819 Thr Gln Met Val Asn
Leu Glu Asp Arg Leu Val Leu Ala Ile Thr Arg 255 260 265 gtt agc cct
gaa tgg ata cta gag gta tgg ggc atg gat aca tac aaa 867 Val Ser Pro
Glu Trp Ile Leu Glu Val Trp Gly Met Asp Thr Tyr Lys 270 275 280 gaa
aaa tgg agc aag act tac tcc ata agt ttg gat cat aga gtt gtt 915 Glu
Lys Trp Ser Lys Thr Tyr Ser Ile Ser Leu Asp His Arg Val Val 285 290
295 300 tcc tgg cga agg cag aaa agg tgg ttc acg ccc gtg gca gtt tct
aag 963 Ser Trp Arg Arg Gln Lys Arg Trp Phe Thr Pro Val Ala Val Ser
Lys 305 310 315 caa gcg aat ctt gtc ttc tat gac aat aag aag agg cta
ttc aaa tat 1011 Gln Ala Asn Leu Val Phe Tyr Asp Asn Lys Lys Arg
Leu Phe Lys Tyr 320 325 330 tat cca gtg aaa gat gag att cgt tgt ctc
tcc tta gac att tgt gtt 1059 Tyr Pro Val Lys Asp Glu Ile Arg Cys
Leu Ser Leu Asp Ile Cys Val 335 340 345 ctg tct cct tac gtg gaa aac
ttg gtc cct ctt ccg tta aag cca agc 1107 Leu Ser Pro Tyr Val Glu
Asn Leu Val Pro Leu Pro Leu Lys Pro Ser 350 355 360 cat ccg cat cct
act ccg aaa aat tca gat ttt gaa atg agg ata tca 1155 His Pro His
Pro Thr Pro Lys Asn Ser Asp Phe Glu Met Arg Ile Ser 365 370 375 380
aga tgc cgc ttg ttt tcg acg cca ggt tct tgg ata tcc aaa att ttg
1203 Arg Cys Arg Leu Phe Ser Thr Pro Gly Ser Trp Ile Ser Lys Ile
Leu 385 390 395 aaa tgg aat gtt atg act cta gag att ttg ttt acc tct
cta gca ata 1251 Lys Trp Asn Val Met Thr Leu Glu Ile Leu Phe Thr
Ser Leu Ala Ile 400 405 410 gtt ggt tat ata tgc tta cct ctc tag
gagttattta tcttgtttca 1298 Val Gly Tyr Ile Cys Leu Pro Leu * 415
420 aaatattagt tggttatata tgctttcgag atctttctga taaa 1342 4 420 PRT
Arabidopsis thaliana 4 Met Gly Glu Glu Glu Glu Asn Pro Asn Ser Ile
Asp Ile Leu Pro Glu 1 5 10 15 Leu Leu Glu Glu Val Leu Leu Arg Leu
Pro Thr Lys Ser Ile Leu Lys 20 25 30 Cys Arg Ile Val Ser Lys Gln
Trp Arg Ser Leu Leu Glu Ser Ser Arg 35 40 45 Phe Ala Glu Arg His
Met Ser Leu Gln Asn Ser Arg Arg Arg Ile Leu 50 55 60 Ala Ala Tyr
Asn Cys Asp Cys Gly Gly Arg Arg Lys Leu Leu Pro Glu 65 70 75 80 Ser
Arg Phe Glu Gly Asp Glu Glu Ile Val Tyr Leu His Cys Asp Ala 85 90
95 Ser Arg Pro Ser Met Thr Cys Gln Gly Val Ile Cys Phe Pro Glu Gln
100 105 110 Asp Trp Ile Ile Val Leu Asn Pro Ser Thr Ser Gln Leu Arg
Arg Phe 115 120 125 Pro Ser Gly Leu Asn His Asn Cys Arg Phe Arg Ile
Gly Leu Trp Lys 130 135 140 Thr Phe Ser Pro Gly Asn Trp Val Met Gly
Phe Gly Arg Asp Lys Val 145 150 155 160 Asn Gly Arg Tyr Lys Val Val
Arg Met Ser Phe Ala Phe Trp Arg Val 165 170 175 Arg Gln Glu Glu Pro
Val Val Glu Cys Gly Val Leu Asp Val Asp Thr 180 185 190 Gly Glu Trp
Arg Lys Leu Ser Pro Pro Pro Tyr Val Val Asn Val Gly 195 200 205 Ser
Lys Ser Val Cys Val Asn Gly Ser Ile Tyr Trp Leu His Ile Gln 210 215
220 Thr Val Tyr Arg Ile Leu Ala Leu Asp Leu His Lys Gln Glu Phe His
225 230 235 240 Lys Val Pro Val Pro Pro Thr Gln Ile Thr Val Asp Thr
Gln Met Val 245 250 255 Asn Leu Glu Asp Arg Leu Val Leu Ala Ile Thr
Arg Val Ser Pro Glu 260 265 270 Trp Ile Leu Glu Val Trp Gly Met Asp
Thr Tyr Lys Glu Lys Trp Ser 275 280 285 Lys Thr Tyr Ser Ile Ser Leu
Asp His Arg Val Val Ser Trp Arg Arg 290 295 300 Gln Lys Arg Trp Phe
Thr Pro Val Ala Val Ser Lys Gln Ala Asn Leu 305 310 315 320 Val Phe
Tyr Asp Asn Lys Lys Arg Leu Phe Lys Tyr Tyr Pro Val Lys 325 330 335
Asp Glu Ile Arg Cys Leu Ser Leu Asp Ile Cys Val Leu Ser Pro Tyr 340
345 350 Val Glu Asn Leu Val Pro Leu Pro Leu Lys Pro Ser His Pro His
Pro 355 360 365 Thr Pro Lys Asn Ser Asp Phe Glu Met Arg Ile Ser Arg
Cys Arg Leu 370 375 380 Phe Ser Thr Pro Gly Ser Trp Ile Ser Lys Ile
Leu Lys Trp Asn Val 385 390 395 400 Met Thr Leu Glu Ile Leu Phe Thr
Ser Leu Ala Ile Val Gly Tyr Ile 405 410 415 Cys Leu Pro Leu 420
* * * * *
References