U.S. patent application number 13/678170 was filed with the patent office on 2013-07-25 for transgenic plants.
This patent application is currently assigned to MONSANTO TECHNOLOGY LLC. The applicant listed for this patent is MONSANTO TECHNOLOGY LLC. Invention is credited to Stanton B. Dotson, Changlin Fu, Linda Lutfiyya, Jindong Sun, Jingrui Wu, Kimberly Zobrist.
Application Number | 20130191942 13/678170 |
Document ID | / |
Family ID | 34555488 |
Filed Date | 2013-07-25 |
United States Patent
Application |
20130191942 |
Kind Code |
A1 |
Sun; Jindong ; et
al. |
July 25, 2013 |
Transgenic Plants
Abstract
Disclosed herein are transgenic plants having recombinant DNA
which expresses a G1073 transcription factor which provides
enhanced resistance and/or tolerance to water deficit. More
specifically the DNA constructs comprise a polynucleotide which
encodes at least a functional part of a G1073 transcription factor
or a homologous transcription factor.
Inventors: |
Sun; Jindong; (St. Charles,
MO) ; Zobrist; Kimberly; (Greenville, IL) ;
Wu; Jingrui; (Chesterfield, MO) ; Fu; Changlin;
(Chesterfield, MO) ; Dotson; Stanton B.;
(Chesterfield, MO) ; Lutfiyya; Linda; (St. Louis,
MO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
MONSANTO TECHNOLOGY LLC; |
St. Louis |
MO |
US |
|
|
Assignee: |
MONSANTO TECHNOLOGY LLC
St. Louis
MO
|
Family ID: |
34555488 |
Appl. No.: |
13/678170 |
Filed: |
November 15, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12972782 |
Dec 20, 2010 |
8324482 |
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13678170 |
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10783710 |
Feb 21, 2004 |
7888557 |
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12972782 |
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60449054 |
Feb 22, 2003 |
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Current U.S.
Class: |
800/278 |
Current CPC
Class: |
C07K 14/4702 20130101;
C12N 15/8273 20130101; C07K 14/415 20130101; Y02A 40/146 20180101;
C12N 15/8247 20130101; C12N 15/8261 20130101 |
Class at
Publication: |
800/278 |
International
Class: |
C07K 14/415 20060101
C07K014/415 |
Claims
1.-6. (canceled)
7. A method for imparting water deficit tolerance to a crop plant
variety as compared to said crop variety lacking recombinant DNA
expressing a G1073 transcription factor when said crop varieties
are grown in a water deficient environment, said method comprising
inserting into the genome of said variety recombinant DNA which
expresses a transcription factor comprising an amino acid sequence
with at least about 80% sequence identity to the amino acid
sequence of SEQ ID NO: 11 and screening for identification of the
water deficit tolerance trait having consensus amino acid sequence
of SEQ ID NO:11.
8. A method of imparting water deficit tolerance to a crop plant by
crossing a first transgenic crop with a second crop plant wherein
pollen from said first transgenic crop contains recombinant DNA
which expresses a transcription factor comprising an amino acid
sequence with at least about 80% sequence identity to the consensus
amino acid sequence of SEQ ID NO:11, and wherein said method
further comprises a screening process for identification of the
water deficit tolerance trait.
9. The method of claim 8 wherein one of said crops comprises
recombinant DNA which expresses a protein that confers at least one
of an herbicide resistance trait or a pest resistance trait.
10.-13. (canceled)
14. The method of claim 7, wherein said transcription factor
comprises an amino acid sequence with at least about 50% sequence
identity to the amino acid sequence of SEQ ID NO:1.
15. The method of claim 7, wherein said transcription factor
comprises an amino acid sequence of SEQ ID NO:11.
16. The method of claim 8, wherein said transcription factor
comprises an amino acid sequence with at least about 50% sequence
identity to the amino acid sequence of SEQ ID NO:1.
17. The method of claim 8, wherein said transcription factor
comprises an amino acid sequence of SEQ ID NO:11.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 10/783,710, filed Feb. 21, 2004 and incorporated herein by
reference in its entirety, which claims priority to U.S.
Provisional Application 60/449,054 filed Feb. 22, 2003,
incorporated herein by reference in its entirety.
JOINT RESEARCH STATEMENT
[0002] The claimed invention, in the field of functional genomics
and the characterization of plant genes for the improvement of
plants, was made by or on behalf of Mendel Biotechnology, Inc. and
Monsanto Company as a result of activities undertaken within the
scope of a joint research agreement in effect on or before the date
the claimed invention was made.
INCORPORATION OF SEQUENCE LISTING
[0003] The sequences in the enclosed Sequence Listing are identical
to the sequences in the Sequence Listing and computer readable form
of prior U.S. Provisional Application 60/449,054 filed Feb. 22,
2003, which contain the file named "G1073FINAL.ST25.txt" which is
21 kb and was created on 21 Feb. 2003 and which is incorporated
herein by reference.
FIELD OF THE INVENTION
[0004] Disclosed herein are DNA useful for producing transgenic
plants and seeds and methods of making and using such transgenic
plants and seed.
BACKGROUND OF THE INVENTION
[0005] Water deficit can have adverse effects on plants such as
yield reductions, increased susceptibility to disease and pests,
reduced plant growth and reproductive failure. An object of this
invention is to provide plants which can express genes to
ameliorate the adverse effects of water deficit. Useful genes for
expression especially during water deficit are genes which promote
aspects of plant growth or fertility, genes which impart disease
resistance, genes which impart pest resistance, and the like.
[0006] Considering the complexity of water use in land plants,
especially during conditions that produce water deficit, relatively
few genes specifically associated with this aspect of physiology
have been identified. It would be of benefit to the art to increase
the number and variety of genes involved in regulating water use in
plants, more particularly, in corn plants, and even more
particularly in corn plants experiencing water deficit. It would be
especially advantageous to identify transcription factors which can
be used in directing the production of proteins which are
beneficial to the plant when produced during water deficit.
[0007] Transcription factors are investigated for improving plant
properties and traits in transgenic plants. Reference is made to WO
02079403 of Mendel Biotechnology, Inc. which claims priority from
U.S. application Ser. No. 09/823,676 (incorporated herein by
reference) for a disclosure of a variety of Arabidopsis thaliana
transcription factors including one identified as G1073 which are
alleged to be useful for modifying plant biomass, for methods of
building DNA constructs which express transcript factors, and for
methods of producing transformed plants with DNA constructs which
express transcription factors.
[0008] One of the goals of plant genetic engineering is to produce
plants with agronomically, horticulturally or economically
important traits including tolerance to any of a variety of
environmental stresses such as water deficit. Many transgenic crop
plants have recombinant DNA that confers herbicide and/or pest
resistance traits. Incorporation of additional recombinant DNA for
conferring crop improvement traits in crop plants presents a
challenge of using DNA constructs of increased complexity.
SUMMARY OF THE INVENTION
[0009] We have discovered that transcription factors G1073 and
homologs (G1073 transcription factors) are useful for imparting
enhanced resistance and/or tolerance to water deficit in transgenic
plants. The present invention is directed to DNA which encode at
least a functional part of a G1073 transcription factor which is
useful in transgenic plants for enhancing yield when the plants are
subjected to water deficit. One aspect of this invention provides
methods for providing transgenic plants with an enhanced resistance
and/or tolerance to water deficit. More particularly the method
comprises transforming plants with recombinant DNA construct
comprising DNA which encodes at least a functional part of a G1073
transcription factor, e.g. which imparts resistance to and/or
tolerance to water deficit. Another aspect of the invention
provides transgenic seed for growing a plant which is resistant to
water deficit as compared to wild type wherein the genome of said
seed comprises recombinant DNA which expresses at least a
functional part of such a G1073 transcription factor, e.g. having
an amino acid sequence comprising at least 50 contiguous amino
acids of a G1073 transcription factor. In another aspect of the
invention such transgenic seed has in its genome recombinant DNA
which expresses a transcription factor polypeptide having an amino
acid sequences which is at least 50% identical (and preferably of
higher identity) with a synthetic consensus amino acid sequence
from a conserved region of a G1073 transcription factor. In one
aspect of the invention the recombinant DNA is exogenous DNA. In
another aspect of the invention the DNA expressing a G1073
transcription factor is DNA from the Arabidopsis thaliana
transcription factor G1073. In yet another aspect of the invention
the DNA expressing a G1073 transcription factor is not derived from
the Arabidopsis thaliana transcription factor G1073, but rather is
derived from DNA expressing a homologous G1073 transcription factor
from another species.
[0010] This invention also provides plants grown from such
transgenic seed with recombinant DNA expressing a G1073
transcription factor. Transformed plants with tolerance and/or
resistance to water deficit should inherently provide enhanced
yield as compared to wild type plants which are stunted by or
succumb to water deficit. One aspect of the invention provides
transgenic plants with stacked engineered traits, e.g. a crop
improvement trait provided by recombinant DNA expressing a G1073
transcription factor in combination with herbicide and/or pest
resistance traits.
[0011] Another aspect of the invention provides hybrid corn with
stacked engineered traits. One embodiment of such hybrid corn is
the progeny of a transgenic ancestor corn plant having in its
genome a recombinant DNA which expresses a G1073 transcription
factor in combination with an herbicide and/or pest resistance
trait. Embodiments of such hybrid corn have a transgenic male
ancestor corn plant which has in its genome recombinant DNA which
confers herbicide resistance and/or pest resistance.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 is an amino acid sequence alignment of: [0013] (a)
all of SEQ ID NO:7 representing conserved amino acid sequence in an
Arabidopsis transcription factor denoted "G1067" which is disclosed
in application Ser. No. 09/934,455; [0014] (b) all of SEQ ID NO:8
representing conserved amino acid sequence in an Arabidopsis
transcription factor denoted "G1073" which has the full amino acid
sequence of SEQ ID NO:1; [0015] (c) residues 1-59 and 67-106 of SEQ
ID NO:9 representing conserved amino acid sequence in a cotton
transcription factor which has the full amino acid sequence of SEQ
ID NO:3; [0016] (d) residues 1-59 and 69-108 of SEQ ID NO:10
representing conserved amino acid sequence in a rice transcription
factor which has the full amino acid sequence of SEQ ID NO:2; and
[0017] (e) all of SEQ ID NO: 11 which is a consensus representation
of those conserved amino acid sequences.
DETAILED DESCRIPTION OF THE INVENTION
[0018] As used herein a "G1073 transcription factor" means a
protein which is expressed by DNA of SEQ ID NO:4-6 and a protein
having the amino acid sequence of SEQ ID NO:1-3 and a protein
having the conserved amino acid sequence of SEQ ID NO:7-10 and a
protein having the consensus amino acid sequence of SEQ ID NO:11
and a homologue protein from another species and parts of such
proteins that function to provide the water-deficit-tolerance trait
exhibited in Arabidopsis thaliana, e.g. in the assay illustrated in
the example below.
[0019] As used herein "water deficit" is a plant condition
characterized by water potential in a plant tissue of less than
-0.7 megapascals (MPa), e.g. -0.8 Mpa. Water potential in maize is
conveniently measured by clamping a leaf segment in a pressurizable
container so that a cut cross section of leaf is open to
atmospheric pressure. Gauge pressure (above atmospheric pressure)
on the contained leaf section is increased until water begins to
exude from the atmospheric-pressure-exposed cross section; the
gauge pressure at incipient water exudation is reported as negative
water potential in the plant tissue, e.g. 7 bars of gauge pressure
is reported as -0.7 MPa water potential. Water deficit can be
induced by withholding water from plants for sufficient time that
wild type plants are deleteriously affected, e.g. as manifested by
reduced yield, stunted growth, retarded development, death or the
like. The plants of this invention show a remarkable risibility
after periods of water deficit that are adverse to wild type
plants.
[0020] As used herein "yield" of a crop plant means the production
of a crop, e.g. shelled corn kernels or soybean or cotton fiber,
per unit of production area, e.g. in bushels per acre or metric
tons per hectare, often reported on a moisture adjusted basis, e.g.
corn is typically reported at 15.5% moisture. Moreover a bushel of
corn is defined by law in the State of Iowa as 56 pounds by weight,
a useful conversion factor for corn yield is: 100 bushels per acre
is equivalent to 6.272 metric tons per hectare. Other measurements
for yield are in common practice. As used herein a "transgenic"
organism, e.g. plant or seed, is one whose genome has been altered
by the incorporation of exogenous genetic material or additional
copies of native genetic material, e.g.
[0021] by transformation or recombination of the organism or an
ancestor organism. Transgenic plants include progeny plants of an
original plant derived from a transformation process including
progeny of breeding transgenic plants with wild type plants or
other transgenic plants. Crop plants of interest in the present
invention include, but are not limited to soy, cotton, canola,
maize, wheat, sunflower, sorghum, alfalfa, barley, millet, rice,
tobacco, fruit and vegetable crops, and turfgrass.
[0022] As used herein an "herbicide resistance" trait is a
characteristic of a transgenic plant that is resistant to dosages
of an herbicide that is typically lethal to a progenitor plant.
Such herbicide resistance can arise from a natural mutation or more
typically from incorporation of recombinant DNA that confers
herbicide resistance. Herbicides for which resistance is useful in
a plant include glyphosate herbicides, phosphinothricin herbicides,
oxynil herbicides, imidazolinone herbicides, dinitroaniline
herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos
herbicides, sulfonamide herbicides and gluphosinate herbicides. To
illustrate that the production of transgenic plants with herbicide
resistance is a capability of those of ordinary skill in the art
reference is made to U.S. patent application publications
2003/0106096A1 and 2002/0112260A1 and U.S. Pat. Nos. 5,034,322;
6,107,549 and 6,376,754, all of which are incorporated herein by
reference.
[0023] As used herein an "pest resistance" trait is a
characteristic of a transgenic plant is resistant to attack from a
plant pest such as a virus, a nematode, a larval insect or an adult
insect that typically is capable of inflicting crop yield loss in a
progenitor plant. Such pest resistance can arise from a natural
mutation or more typically from incorporation of recombinant DNA
that confers pest resistance. For insect resistance, such
recombinant DNA can, for example, encode an insect lethal protein
such as a delta endotoxin of Bacillus thuringiensis bacteria or be
transcribed to a dsRNA targeted for suppression of an essential
gene in the insect. To illustrate that the production of transgenic
plants with pest resistance is a capability of those of ordinary
skill in the art reference is made to U.S. Pat. Nos. 5,250,515 and
5,880,275 which disclose plants expressing an endotoxin of Bacillus
thuringiensis bacteria, to U.S. Pat. No. 6,506,599 which discloses
control of invertebrates which feed on transgenic plants which
express dsRNA for suppressing a target gene in the invertebrate, to
U.S. Pat. No. 5,986,175 which discloses the control of viral pests
by transgenic plants which express viral replicase, and to U.S.
Patent Application Publication 2003/0150017 A1 which discloses
control of pests by a transgenic plant which express a dsRNA
targeted to suppressing a gene in the pest, all of which are
incorporated herein by reference.
[0024] SEQ ID NO: 1 provides the amino acid sequence of Arabidopsis
thaliana transcription factor G1073, which are disclosed in U.S.
application Ser. No. 09/823,676, filed Mar. 26, 2001, incorporated
herein by reference.
[0025] SEQ ID NO:2 provides the amino acid sequence of part of the
rice (Oryza sativa) polypeptide which is a homolog of the
Arabidopsis thaliana G1073 transcription factor.
[0026] SEQ ID NO:3 provides the amino acid sequence of part of the
cotton (Gossypium hirsutum) polypeptide which is a homolog of the
Arabidopsis thaliana G1073 transcription factor.
[0027] SEQ ID NO:4 provides DNA from the gene encoding an
Arabidopsis thaliana G1073 transcription factor of SEQ ID NO:1.
[0028] SEQ ID NO:5 provides DNA from the gene encoding a rice
transcription factor of SEQ ID NO:2.
[0029] SEQ ID NO:6 provides DNA from the gene encoding a cotton
transcription factor of SEQ ID NO:3.
[0030] SEQ ID NO:7 provides a conserved region of the amino acid
sequence of the Arabidopsis thaliana transcription factor G1067
which is disclosed in U.S. application Ser. No. 09/934,455,
incorporated herein by reference.
[0031] SEQ ID NO: 8 provides a conserved region of the amino acid
sequence of the Arabidopsis thaliana transcription factor G1073
(SEQ ID NO:1).
[0032] SEQ ID NO:9 provides a conserved region of the amino acid
sequence of the cotton transcription factor (SEQ ID NO:3).
[0033] SEQ ID NO:10 provides a conserved region of the amino acid
sequence of the rice transcription factor (SEQ ID NO:2).
[0034] SEQ ID NO:11 is an synthetic consensus amino acid sequence
developed from alignment of the conserved region of SEQ ID NO: 7
through 10. The alignment is illustrated in FIG. 1.
[0035] SEQ ID NO: 12 provides DNA from the gene encoding an
Arabidopsis thaliana G1067 transcription factor, a conserved region
of which is SEQ ID NO:7.
[0036] Polynucleotides of the present invention are DNA that is
used to impart the desired agronomic trait, e.g. such biological
properties by providing for enhanced protein activity in a
transgenic plants by overexpression of the polynucleotide, e.g.
with a constitutive promoter or a promoter which is active during
water deficit.
[0037] Protein and Polypeptide Molecules--Proteins of the present
invention are whole proteins or at least a sufficient portion of
the protein to impart the relevant biological activity of the
protein, e.g. resistance and/or tolerance to water deficit in
transgenic plants as compared to wild type, as provided by
constitutive expression of the Arabidopsis thaliana G1073
transcription factor or a functionally homologous transcription
factor. The term "protein" also includes molecules consisting of
one or more polypeptide chains. Thus, a polypeptide useful in the
present invention may constitute an entire gene product or one or
more functional portion of a natural protein which provides the
agronomic trait of this invention, i.e. enhanced yield despite
exposure to water deficit.
[0038] Homologs of the polypeptides of the present invention may be
identified by comparison of the amino acid sequence of the
polypeptide to amino acid sequences of polypeptides from the same
or different plant sources, e.g. manually or by using known
homology-based search algorithms such as those commonly known and
referred to as BLAST, FASTA, and Smith-Waterman.
[0039] A further aspect of the invention comprises functional
homolog proteins which differ in one or more amino acids from those
of a polypeptide provided herein as the result of one or more of
the well-known conservative amino acid substitutions, e.g. valine
is a conservative substitute for alanine and threonine is a
conservative substitute for serine. Conservative substitutions for
an amino acid within the native polypeptide sequence can be
selected from other members of a class to which the naturally
occurring amino acid belongs. Representative amino acids within
these various classes include, but are not limited to: (1) acidic
(negatively charged) amino acids such as aspartic acid and glutamic
acid; (2) basic (positively charged) amino acids such as arginine,
histidine, and lysine; (3) neutral polar amino acids such as
glycine, serine, threonine, cysteine, tyrosine, asparagine, and
glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such
as alanine, leucine, isoleucine, valine, proline, phenylalanine,
tryptophan, and methionine. Conserved substitutes for an amino acid
within a native amino acid sequence can be selected from other
members of the group to which the naturally occurring amino acid
belongs. For example, a group of amino acids having aliphatic side
chains is glycine, alanine, valine, leucine, and isoleucine; a
group of amino acids having aliphatic-hydroxyl side chains is
serine and threonine; a group of amino acids having
amide-containing side chains is asparagine and glutamine; a group
of amino acids having aromatic side chains is phenylalanine,
tyrosine, and tryptophan; a group of amino acids having basic side
chains is lysine, arginine, and histidine; and a group of amino
acids having sulfur-containing side chains is cysteine and
methionine. Naturally conservative amino acids substitution groups
are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine,
lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and
asparagine-glutamine. A further aspect of the invention comprises
polypeptides which differ in one or more amino acids from those of
a described protein sequence as the result of deletion or insertion
of one or more amino acids in a native sequence.
[0040] Polypeptides of the present invention that are variants of
the polypeptides provided herein will generally demonstrate
significant identity with the polypeptides provided herein. Of
particular interest are polypeptides having at least 50% sequence
identity, more preferably at least about 70% sequence identity or
higher, e.g. at least about 80% sequence identity with (a) an
synthetic consensus amino acid sequence of SEQ ID NO:11 or (b) a
conserved amino acid region of SEQ ID NO: 7 through 10 or (c) an
amino acid sequence of SEQ ID NO:1 through 3, or (d) other
functional homologs of any polypeptide identified in (a) through
(c). Of course useful polypeptides also include those with higher
identity to such a polypeptide sequence, e.g. 90%, to 100%
identity. Other polypeptides of interest have at least 50 or more,
e.g. at least 60 or 70 of the amino acids of a conserved segment of
the transcription factors proteins as defined by SEQ ID NO:7
through 10 and the synthetic consensus amino acid sequence of SEQ
ID NO:11. Of course useful polypeptides also include those with
higher percentage of the amino acids in an protein segment of SEQ
ID NO:7 thorough 11.
[0041] Recombinant DNA Constructs--The present invention
contemplates the use of polynucleotides which encode a protein
effective for imparting resistance and/or tolerance to water
deficit in plants. Such polynucleotides are assembled in
recombinant DNA constructs using methods known to those of ordinary
skill in the art. A useful technology for building DNA constructs
and vectors for transformation is the GATEWAY.TM. cloning
technology (available from Invitrogen Life Technologies, Carlsbad,
Calif.) uses the site specific recombinase LR cloning reaction of
the Integrase/att system from bacterophage lambda vector
construction, instead of restriction endonucleases and ligases. The
LR cloning reaction is disclosed in U.S. Pat. Nos. 5,888,732 and
6,277,608, U.S. Patent Application Publications 2001283529,
2001282319 and 20020007051, all of which are incorporated herein by
reference. The GATEWAY.TM. Cloning Technology Instruction Manual
which is also supplied by Invitrogen also provides concise
directions for routine cloning of any desired RNA into a vector
comprising operable plant expression elements.
[0042] Transgenic DNA constructs used for transforming plant cells
will comprise the heterologous DNA which one desires to introduced
into and a promoter to express the heterologous DNA in the host
maize cells. As is well known in the art such constructs typically
also comprise a promoter and other regulatory elements, 3'
untranslated regions (such as polyadenylation sites), transit or
signal peptides and marker genes elements as desired. For instance,
see U.S. Pat. Nos. 5,858,642 and 5,322,938 which disclose versions
of the constitutive promoter derived from cauliflower mosaic virus
(CaMV35S), U.S. Pat. No. 6,437,217 which discloses a maize RS81
promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin
promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324
promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1
promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3
promoter, U.S. Pat. No. 6,177,611 which discloses constitutive
maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3
oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice
actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which
discloses a root specific promoter, U.S. Pat. No. 6,084,089 which
discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which
discloses light inducible promoters, U.S. Pat. No. 6,140,078 which
discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which
discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060
which discloses phosphorus deficiency inducible promoters, U.S.
Patent Application Publication 2002/0192813A1 which discloses 5',
3' and intron elements useful in the design of effective plant
expression vectors, U.S. patent application Ser. No. 09/078,972
which discloses a coixin promoter, U.S. patent application Ser. No.
09/757,089 which discloses a maize chloroplast aldolase promoter,
all of which are incorporated herein by reference.
[0043] In many aspects of the invention it is preferred that the
promoter element in the DNA construct should be capable of causing
sufficient expression to result in the production of an effective
amount of the transcription factor in water deficit conditions.
Such promoters can be identified and isolated from the regulatory
region of plant genes which are over expressed in water deficit
conditions. Specific water-deficit-inducible promoters for use in
this invention are derived from the 5' regulatory region of genes
identified as a heat shock protein 17.5 gene (HSP17.5), an HVA22
gene (HVA22), and a cinnamic acid 4-hydroxylase (CA4H) gene (CA4H)
of Zea maize. Such water-deficit-inducible promoters are disclosed
in U.S. provisional application Ser. No. 60/435,987, filed Dec. 20,
2002, incorporated herein by reference.
[0044] In general it is preferred to introduce heterologous DNA
randomly, i.e. at a non-specific location, in the plant genome. In
special cases it may be useful to target heterologous DNA insertion
in order to achieve site specific integration, e.g. to replace an
existing gene in the genome. In some other cases it may be useful
to target a heterologous DNA integration into the genome at a
predetermined site from which it is known that gene expression
occurs. Several site specific recombination systems exist which are
known to function implants include cre-lox as disclosed in U.S.
Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No.
5,527,695, both incorporated herein by reference.
[0045] Constructs and vectors may also include a transit peptide
for targeting of a gene target to a plant organelle, particularly
to a chloroplast, leucoplast or other plastid organelle. For a
description of the use of a chloroplast transit peptide see U.S.
Pat. No. 5,188,642, incorporated herein by reference.
[0046] In practice DNA is introduced into only a small percentage
of target cells in any one experiment. Marker genes are used to
provide an efficient system for identification of those cells that
are stably transformed by receiving and integrating a transgenic
DNA construct into their genomes. Preferred marker genes provide
selective markers which confer resistance to a selective agent,
such as an antibiotic or herbicide. Potentially transformed cells
are exposed to the selective agent. In the population of surviving
cells will be those cells where, generally, the
resistance-conferring gene has been integrated and expressed at
sufficient levels to permit cell survival. Cells may be tested
further to confirm stable integration of the exogenous DNA. Useful
selective marker genes include those conferring resistance to
antibiotics such as kanamycin (nptII), hygromycin B (aph IV) and
gentamycin (aac3 and aacC4) or resistance to herbicides such as
glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such
selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435;
5,780,708 and 6,118,047, all of which are incorporated herein by
reference. Screenable markers which provide an ability to visually
identify transformants can also be employed, e.g., a gene
expressing a colored or fluorescent protein such as a luciferase or
green fluorescent protein (GFP) or a gene expressing a
beta-glucuronidase or uidA gene (GUS) for which various chromogenic
substrates are known.
[0047] Transformation Methods and Transgenic Plants--Methods and
compositions for transforming plants by introducing a transgenic
DNA construct into a plant genome in the practice of this invention
can include any of the well-known and demonstrated methods.
Preferred methods of plant transformation are microprojectile
bombardment as illustrated in U.S. Pat. Nos. 5,015,580; 5,550,318;
5,538,880; 6,160,208; 6,399,861 and 6,403,865 and
Agrobacterium-mediated transformation as illustrated in U.S. Pat.
Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840 and 6,384,301, all
of which are incorporated herein by reference. See also U.S.
application Ser. No. 09/823,676, incorporated herein by reference,
for a description of vectors, transformation methods, and
production of transformed Arabidopsis thaliana plants where
transcription factors such as G1073 are constitutively expressed by
a CaMV35S promoter.
[0048] Transformation methods of this invention to provide plants
with enhanced environmental stress tolerance are preferably
practiced in tissue culture on media and in a controlled
environment. "Media" refers to the numerous nutrient mixtures that
are used to grow cells in vitro, that is, outside of the intact
living organism. Recipient cell targets include, but are not
limited to, meristem cells, callus, immature embryos and gametic
cells such as microspores, pollen, sperm and egg cells. It is
contemplated that any cell from which a fertile plant may be
regenerated is useful as a recipient cell. Callus may be initiated
from tissue sources including, but not limited to, immature
embryos, seedling apical meristems, microspores and the like. Those
cells which are capable of proliferating as callus also are
recipient cells for genetic transformation. Practical
transformation methods and materials for making transgenic plants
of this invention, e.g. various media and recipient target cells,
transformation of immature embryos and subsequent regeneration of
fertile transgenic plants are disclosed in U.S. Pat. No. 6,194,636
and U.S. patent application Ser. No. 09/757,089, which are
incorporated herein by reference.
[0049] The seeds of this invention can be harvested from fertile
transgenic plants and be used to grow progeny generations of
transformed plants of this invention including hybrid plants line
comprising the DNA construct expressing a transcription factor
which provides the benefits of resistance and/or tolerance to water
deficit.
Breeding of Transgenic Plants
[0050] In addition to direct transformation of a plant with a
recombinant DNA construct, transgenic plants can be prepared by
crossing a first plant having a recombinant DNA construct with a
second plant lacking the construct. For example, recombinant DNA
can be introduced into a plant line that is amenable to
transformation to produce a transgenic plant which can be crossed
with a second plant line to introgress the recombinant DNA into the
second plant line.
[0051] In one aspect of the invention a transgenic plant with
recombinant DNA conferring a crop improvement trait is crossed with
a transgenic plant having recombinant DNA conferring herbicide
and/or pest resistance to produce progeny plants having recombinant
DNA that confers both the crop improvement trait and the herbicide
and/or pest resistance trait. Preferably, in such breeding for
combining traits the transgenic plant donating the crop improvement
trait is a female line and the transgenic plant donating the
herbicide and/or pest resistance trait is a male line. The progeny
of this cross will segregate such that some of the plant will carry
the DNA for both parental traits and some will carry DNA for one
parental trait; such plants can be identified by markers associated
with parental recombinant DNA Progeny plants carrying DNA for both
parental traits can be crossed back into the female parent line
multiple times, e.g. usually 6 to 8 generations, to produce a
progeny plant with substantially the same genotype as one original
transgenic parental line but for the recombinant DNA of the other
transgenic parental line.
[0052] In yet another aspect of the invention hybrid transgenic
seed, e.g. a hybrid transgenic corn seed, is produced by crossing a
female transgenic corn line containing recombinant DNA conferring a
crop improvement trait with a male transgenic corn line containing
recombinant DNA conferring herbicide and/or pest resistance. In a
preferred aspect of this invention hybrid transgenic corn seed is
produced by crossing a female transgenic corn line with recombinant
DNA conferring both a crop improvement trait and herbicide
resistance with a male transgenic corn line with recombinant DNA
conferring both herbicide resistance and pest resistance.
[0053] Having now generally described the invention, the same will
be more readily understood through reference to the following
example which is provided by way of illustration, and is not
intended to be limiting of the present invention, unless
specified.
EXAMPLES
[0054] These examples illustrates the use of a polynucleotide
encoding transcription factor G1073 to provide a transgenic plant
exhibiting enhanced tolerance for and/or resistance to growing
conditions of water deficit.
[0055] Transgenic Arabidopsis thaliana was prepared with an
exogenous DNA construct comprising a constitutive promoter of CaMV
35S operably linked to a polynucleotide of SEQ ID NO: 12 encoding
Arabidopsis thaliana transcription factor G1073 of SEQ ID NO:1.
Transgenic and wild type plants were potted in garden soil in a
controlled environmental growth chamber in a 12hour light/dark
cycle. When the plants were at the early flowering stage, they were
screened for water-deficit tolerance. Water was withheld until the
wild type plants began wilting. Carbon dioxide assimilation rates
were measured at growth and saturated conditions. Growth conditions
were light at 200 .mu.mol m.sup.-2 s.sup.-1 and 350 ppm CO.sub.2.
Saturating conditions were light at 1000 .mu.mol m.sup.-2 s.sup.-1
and 1000 ppm CO.sub.2. Wild type plants had smaller stomata
conductance and lower CO.sub.2 assimilation rates than did the
transgenic plants.
[0056] Transgenic soybean was prepared with an exogenous DNA
construct comprising a constitutive promoter CaMV35S operably
linked to a polynucleotide of SEQ ID NO: 12 encoding Arabidopsis
thaliana transcription factor G1073 of SEQ ID NO:1. When grown in
water-deficit assay conditions the transgenic soybean showed
enhanced resistance and/or tolerance to water deficit as compared
to wild type.
[0057] Transgenic corn was prepared with an exogenous DNA construct
comprising a constitutive promoter of the rice actin 1 gene
operably linked to a polynucleotide of SEQ ID NO: 12 encoding
Arabidopsis thaliana transcription factor G1073 of SEQ ID NO:1.
Transgenic corn exhibited various enhance traits, e.g. increased
biomass, increased seed oil, increased yield and the ability to
utilize high levels of nitrogen.
Sequence CWU 1
1
121270PRTArabidopsis thaliana 1Met Glu Leu Asn Arg Ser Glu Ala Asp
Glu Ala Lys Ala Glu Thr Thr 1 5 10 15 Pro Thr Gly Gly Ala Thr Ser
Ser Ala Thr Ala Ser Gly Ser Ser Ser 20 25 30 Gly Arg Arg Pro Arg
Gly Arg Pro Ala Gly Ser Lys Asn Lys Pro Lys 35 40 45 Pro Pro Thr
Ile Ile Thr Arg Asp Ser Pro Asn Val Leu Arg Ser His 50 55 60 Val
Leu Glu Val Thr Ser Gly Ser Asp Ile Ser Glu Ala Val Ser Thr 65 70
75 80 Tyr Ala Thr Arg Arg Gly Cys Gly Val Cys Ile Ile Ser Gly Thr
Gly 85 90 95 Ala Val Thr Asn Val Thr Ile Arg Gln Pro Ala Ala Pro
Ala Gly Gly 100 105 110 Gly Val Ile Thr Leu His Gly Arg Phe Asp Ile
Leu Ser Leu Thr Gly 115 120 125 Thr Ala Leu Pro Pro Pro Ala Pro Pro
Gly Ala Gly Gly Leu Thr Val 130 135 140 Tyr Leu Ala Gly Gly Gln Gly
Gln Val Val Gly Gly Asn Val Ala Gly 145 150 155 160 Ser Leu Ile Ala
Ser Gly Pro Val Val Leu Met Ala Ala Ser Phe Ala 165 170 175 Asn Ala
Val Tyr Asp Arg Leu Pro Ile Glu Glu Glu Glu Thr Pro Pro 180 185 190
Pro Arg Thr Thr Gly Val Gln Gln Gln Gln Pro Glu Ala Ser Gln Ser 195
200 205 Ser Glu Val Thr Gly Ser Gly Ala Gln Ala Cys Glu Ser Asn Leu
Gln 210 215 220 Gly Gly Asn Gly Gly Gly Gly Val Ala Phe Tyr Asn Leu
Gly Met Asn 225 230 235 240 Met Asn Asn Phe Gln Phe Ser Gly Gly Asp
Ile Tyr Gly Met Ser Gly 245 250 255 Gly Ser Gly Gly Gly Gly Gly Gly
Ala Thr Arg Pro Ala Phe 260 265 270 2295PRTOryza sativa 2Met Glu
His Ser Lys Met Ser Pro Asp Lys Ser Pro Val Gly Glu Gly 1 5 10 15
Asp His Ala Gly Gly Ser Gly Ser Gly Gly Val Gly Gly Asp His Gln 20
25 30 Pro Ser Ser Ser Ala Met Val Pro Val Glu Gly Gly Ser Gly Ser
Ala 35 40 45 Gly Gly Ser Gly Ser Gly Gly Pro Thr Arg Arg Pro Arg
Gly Arg Pro 50 55 60 Pro Gly Ser Lys Asn Lys Pro Lys Pro Pro Ile
Ile Val Thr Arg Asp 65 70 75 80 Ser Pro Asn Ala Leu His Ser His Val
Leu Glu Val Ala Gly Gly Ala 85 90 95 Asp Val Val Asp Cys Val Ala
Glu Tyr Ala Arg Arg Arg Gly Arg Gly 100 105 110 Val Cys Val Leu Ser
Gly Gly Gly Ala Val Val Asn Val Ala Leu Arg 115 120 125 Gln Pro Gly
Ala Ser Pro Pro Gly Ser Met Val Ala Thr Leu Arg Gly 130 135 140 Arg
Phe Glu Ile Leu Ser Leu Thr Gly Thr Val Leu Pro Pro Pro Ala 145 150
155 160 Pro Pro Gly Ala Ser Gly Leu Thr Val Phe Leu Ser Gly Gly Gln
Gly 165 170 175 Gln Val Ile Gly Gly Ser Val Val Gly Pro Leu Val Ala
Ala Gly Pro 180 185 190 Val Val Leu Met Ala Ala Ser Phe Ala Asn Ala
Val Tyr Glu Arg Leu 195 200 205 Pro Leu Glu Gly Glu Glu Glu Glu Val
Ala Ala Pro Ala Ala Gly Gly 210 215 220 Glu Ala Gln Asp Gln Val Ala
Gln Ser Ala Gly Pro Pro Gly Gln Gln 225 230 235 240 Pro Ala Ala Ser
Gln Ser Ser Gly Val Thr Gly Gly Asp Gly Thr Gly 245 250 255 Gly Ala
Gly Gly Met Ser Leu Tyr Asn Leu Ala Gly Asn Val Gly Gly 260 265 270
Tyr Gln Leu Pro Gly Asp Asn Phe Gly Gly Trp Ser Gly Ala Gly Ala 275
280 285 Gly Gly Val Arg Pro Pro Phe 290 295 3230PRTGossypium
hirsutum 3Ala Phe Gly Ser His Tyr Lys Leu Trp Arg Arg Ser Thr Thr
Ser Gly 1 5 10 15 Lys Lys Pro Arg Gly Arg Pro Ala Gly Ser Lys Asn
Lys Pro Lys Ser 20 25 30 Pro Ile Ile Val Ala Arg Asp Ser Pro Asn
Ser Leu Arg Ser His Val 35 40 45 Leu Glu Ile Ser Ser Gly Ser Asp
Ile Val Asp Ser Val Trp Gly Tyr 50 55 60 Ala Arg Arg Arg Gly Arg
Gly Val Cys Val Leu Ser Gly Thr Gly Ala 65 70 75 80 Val Thr Asn Val
Thr Leu Arg Gln Pro Ala Ala Pro Pro Gly Ser Val 85 90 95 Val Thr
Leu His Gly Arg Phe Glu Ile Leu Ser Leu Thr Gly Thr Ser 100 105 110
Leu Pro Pro Pro Ala Pro Pro Gly Ala Gly Gly Leu Thr Val Tyr Leu 115
120 125 Ala Gly Val Gln Gly Gln Val Val Gly Gly Ser Val Val Gly Pro
Leu 130 135 140 Met Ala Ser Gly Pro Val Val Leu Met Ala Ala Ser Phe
Ala Asn Ala 145 150 155 160 Val Tyr Asp Arg Leu Pro Leu Glu Glu Glu
Asp Pro Pro Thr Val His 165 170 175 Glu Gln Gln Pro Ala Ala Ser Gln
Ser Ser Gly Leu Thr Gly Ser Gly 180 185 190 Gly Gly Asn Asn Asn Asn
Cys Gly Thr Thr Gly Thr Gly Val Gly Gly 195 200 205 Gly Gly Gly Gly
Val Pro Phe Tyr Asn Leu Gly Pro Asn Met Gly Thr 210 215 220 Tyr Pro
Phe Pro Gly Leu 225 230 4974DNAArabidopsis thaliana 4ccccccgacc
tgcctctaca gagacctgaa gattccagaa ccccacctga tcaaaaataa 60catggaactt
aacagatctg aagcagacga agcaaaggcc gagaccactc ccaccggtgg
120agccaccagc tcagccacag cctctggctc ttcctccgga cgtcgtccac
gtggtcgtcc 180tgcaggttcc aaaaacaaac ccaaacctcc gacgattata
actagagata gtcctaacgt 240ccttagatca cacgttcttg aagtcacctc
cggttcggac atatccgagg cagtctccac 300ctacgccact cgtcgcggct
gcggcgtttg cattataagc ggcacgggtg cggtcactaa 360cgtcacgata
cggcaacctg cggctccggc tggtggaggt gtgattaccc tgcatggtcg
420gtttgacatt ttgtctttga ccggtactgc gcttccaccg cctgcaccac
cgggagcagg 480aggtttgacg gtgtatctag ccggaggtca aggacaagtt
gtaggaggga atgtggctgg 540ttcgttaatt gcttcgggac cggtagtgtt
gatggctgct tcttttgcaa acgcagttta 600tgataggtta ccgattgaag
aggaagaaac cccaccgccg agaaccaccg gggtgcagca 660gcagcagccg
gaggcgtctc agtcgtcgga ggttacgggg agtggggccc aggcgtgtga
720gtcaaacctc caaggtggaa atggtggagg aggtgttgct ttctacaatc
ttggaatgaa 780tatgaacaat tttcaattct ccgggggaga tatttacggt
atgagcggcg gtagcggagg 840aggtggtggc ggtgcgacta gacccgcgtt
ttagagtttt agcgttttgg tgacaccttt 900tgttgcgttt gcgtgtttga
cctcaaacta ctaggctact agctatagcg gttgcgaaat 960gcgaatatta ggtt
97451071DNAOryza sativa 5atggccggga tggaccctgg cgggggcggc
gccggcgccg gcagctcacg gtacttccac 60catctgctcc gaccgcagca gccgtcgccg
ctgtcaccgc tgtcgccgac atcccatgtc 120aagatggagc actccaagat
gtcacccgac aagagccccg tgggcgaggg agatcacgcg 180ggagggagtg
gaagcggcgg cgtcggcggt gaccaccagc cgtcgtcgtc ggccatggtg
240cccgtcgagg gtggcagcgg cagcgccggc ggtagtggct cgggtgggcc
gacgcggcgc 300ccgcgcgggc gcccgcccgg gtccaagaac aagccgaagc
cgcccatcat cgtgacgcgc 360gacagcccga acgcgctgca ctcgcacgtg
ctcgaggtcg ccggcggcgc cgacgtcgtc 420gactgcgtgg ccgagtacgc
ccgccgccga gggcgcggcg tgtgcgtgct gagcggcggc 480ggcgccgtcg
tcaacgtggc gctgcggcag ccgggcgcgt cgccgccggg cagcatggtg
540gccacgctgc ggggccggtt cgagatccta tctctcacgg gcacggtcct
gccgcctccc 600gcgccacccg gcgcgagcgg cctcaccgtg ttcctctccg
gcggccaggg ccaggtgatc 660ggcggcagcg tggtgggccc gctggtcgcc
gcggggcccg tcgtcctgat ggcggcctca 720ttcgcgaacg ccgtgtacga
gcggctgccg ctggagggcg aggaagagga ggtcgccgcg 780cccgccgccg
gaggcgaagc acaagatcaa gtggcacaat cagctggacc cccagggcag
840caaccggcgg cgtcacagtc ctccggcgtg acaggaggcg acggcaccgg
cggcgccggt 900ggcatgtcgc tctacaacct cgccgggaat gtgggaggct
atcagctccc cggagacaac 960ttcggaggtt ggagcggcgc cggcgccggc
ggagtcaggc caccgttctg acccatgtct 1020tagcatccag ttcaaaaatt
ctccaaatta agaattgcgc agtgcagaag c 10716693DNAGossypium hirsutum
6gcgttcggca gccactacaa gctctggagg aggagtacca cgtcgggaaa aaaacctaga
60ggacgtccag cgggatccaa gaacaagccg aaatcaccca taatcgttgc tcgcgacagt
120ccgaactcgt tgagatccca cgtgctcgaa atctcttccg gttcagacat
agttgactcg 180gtgtggggct acgcacggcg gcgcggccgt ggcgtttgtg
tactcagcgg gaccggtgcc 240gtcacgaatg tcacgttaag gcaaccggct
gctccacctg gaagtgtcgt aacactacac 300ggtcggttcg agattttatc
tttaaccggg acttctctcc caccgccagc accgcctgga 360gctggtggat
tgacggttta tctcgccggc gttcaaggtc aagtagtcgg aggaagcgtg
420gtgggaccgt taatggcttc aggtccagtc gtattaatgg ctgcatcgtt
cgccaatgca 480gtttacgata ggttacctct cgaagaagaa gacccaccaa
ccgttcacga acaacaacca 540gcagcttcac aatcatccgg attaaccggc
agtggcggcg gaaacaacaa caactgtgga 600acaaccggaa ccggcgtagg
cggcggcggc ggcggggttc ctttctataa tttgggacca 660aacatgggaa
cttatccatt tccaggatta tga 693799PRTArabidopsis thaliana 7Ala Lys
Pro Pro Ile Ile Val Thr Arg Asp Ser Pro Asn Ala Leu Arg 1 5 10 15
Ser His Val Leu Glu Val Ser Pro Gly Ala Asp Ile Val Glu Ser Val 20
25 30 Ser Thr Tyr Ala Arg Arg Arg Gly Arg Gly Val Ser Val Leu Gly
Gly 35 40 45 Asn Gly Thr Val Ser Asn Val Thr Leu Arg Gln Val Val
Thr Leu His 50 55 60 Gly Arg Phe Glu Ile Leu Ser Leu Thr Gly Thr
Val Leu Pro Pro Pro 65 70 75 80 Ala Pro Pro Gly Ala Gly Gly Leu Ser
Ile Phe Leu Ala Gly Gly Gln 85 90 95 Gly Gln Val 899PRTArabidopsis
thaliana 8Pro Lys Pro Pro Thr Ile Ile Thr Arg Asp Ser Pro Asn Val
Leu Arg 1 5 10 15 Ser His Val Leu Glu Val Thr Ser Gly Ser Asp Ile
Ser Glu Ala Val 20 25 30 Ser Thr Tyr Ala Thr Arg Arg Gly Cys Gly
Val Cys Ile Ile Ser Gly 35 40 45 Thr Gly Ala Val Thr Asn Val Thr
Ile Arg Gln Val Ile Thr Leu His 50 55 60 Gly Arg Phe Asp Ile Leu
Ser Leu Thr Gly Thr Ala Leu Pro Pro Pro 65 70 75 80 Ala Pro Pro Gly
Ala Gly Gly Leu Thr Val Tyr Leu Ala Gly Gly Gln 85 90 95 Gly Gln
Val 9107PRTGossypium hirsutum 9Pro Lys Ser Pro Ile Ile Val Ala Arg
Asp Ser Pro Asn Ser Leu Arg 1 5 10 15 Ser His Val Leu Glu Ile Ser
Ser Gly Ser Asp Ile Val Asp Ser Val 20 25 30 Trp Gly Tyr Ala Arg
Arg Arg Gly Arg Gly Val Cys Val Leu Ser Gly 35 40 45 Thr Gly Ala
Val Thr Asn Val Thr Leu Arg Gln Pro Ala Ala Pro Pro 50 55 60 Gly
Ser Val Val Thr Leu His Gly Arg Phe Glu Ile Leu Ser Leu Thr 65 70
75 80 Gly Thr Ser Leu Pro Pro Pro Ala Pro Pro Gly Ala Gly Gly Leu
Thr 85 90 95 Val Tyr Leu Ala Gly Val Gln Gly Gln Val Val 100 105
10109PRTOryza sativa 10Pro Lys Pro Pro Ile Ile Val Thr Arg Asp Ser
Pro Asn Ala Leu His 1 5 10 15 Ser His Val Leu Glu Val Ala Gly Gly
Ala Asp Val Val Asp Cys Val 20 25 30 Ala Glu Tyr Ala Arg Arg Arg
Gly Arg Gly Val Cys Val Leu Ser Gly 35 40 45 Gly Gly Ala Val Val
Asn Val Ala Leu Arg Gln Pro Gly Ala Ser Pro 50 55 60 Pro Gly Ser
Met Val Ala Thr Leu Arg Gly Arg Phe Glu Ile Leu Ser 65 70 75 80 Leu
Thr Gly Thr Val Leu Pro Pro Pro Ala Pro Pro Gly Ala Ser Gly 85 90
95 Leu Thr Val Phe Leu Ser Gly Gly Gln Gly Gln Val Ile 100 105
11108PRTArtificial sequenceSynthetic construct 11Xaa Lys Xaa Pro
Xaa Ile Xaa Xaa Arg Asp Ser Pro Asn Xaa Leu Xaa 1 5 10 15 Ser His
Val Leu Glu Xaa Xaa Xaa Gly Xaa Asp Xaa Xaa Xaa Xaa Val 20 25 30
Xaa Xaa Tyr Ala Xaa Arg Arg Gly Xaa Gly Val Xaa Xaa Xaa Xaa Gly 35
40 45 Xaa Gly Xaa Val Xaa Asn Val Xaa Xaa Arg Gln Xaa Xaa Xaa Xaa
Xaa 50 55 60 Xaa Xaa Xaa Xaa Val Xaa Thr Leu Xaa Gly Arg Phe Xaa
Ile Leu Ser 65 70 75 80 Leu Thr Gly Thr Xaa Leu Pro Pro Pro Ala Pro
Pro Gly Ala Xaa Gly 85 90 95 Leu Xaa Xaa Xaa Leu Ala Gly Xaa Gln
Gly Gln Val 100 105 121473DNAArabidopsis thaliana 12tctcaagctt
ctctctcctt tttttcccat agcacatcag aatcgctaaa tacgactcct 60atgcaaagaa
gaagctactt ctttctcttg ccctaattaa tctacctaac tagggtttcc
120tcttaccttt catgagagag atcatttaac ataagtcacc ttttttatat
cttttgcttc 180gtctttaatt tagttctgtt cttggtctgt ttctatattt
tgtcggcttg cgtaaccgat 240cacaccttaa tgctttagct attgtttcct
caaaatcatg agttttgact tctcgatctg 300agttttcttt ttctctcttt
acgctcttct tcacctagct accaatatat gaacgagcag 360gatcaagaat
cgagaaattg atttgagctg gcgaataagc agtggtggga tagggaatta
420gtagatgcgg cggcgatgga aggcggttac gagcaaggcg gtggagcttc
tagatacttc 480cataacctct ttagaccgga gattcaccac caacagcttc
aaccgcaggg cgggatcaat 540cttatcgacc agcatcatca tcagcaccag
caacatcaac aacaacaaca accgtcggat 600gattcaagag aatctgacca
ttcaaacaaa gatcatcatc aacagggtcg acccgattca 660gacccgaata
catcaagctc agcaccggga aaacgtccac gtggacgtcc accaggatct
720aagaacaaag ccaagccacc gatcatagta actcgtgata gccccaacgc
gcttagatct 780cacgttcttg aagtatctcc tggagctgac atagttgaga
gtgtttccac gtacgctagg 840aggagaggga gaggcgtctc cgttttagga
ggaaacggca ccgtatctaa cgtcactctc 900cgtcagccag tcactcctgg
aaatggcggt ggtgtgtccg gaggaggagg agttgtgact 960ttacatggaa
ggtttgagat tctttcgcta acggggactg ttttgccacc tcctgcaccg
1020cctggtgccg gtggtttgtc tatattttta gccggagggc aaggtcaggt
ggtcggagga 1080agcgttgtgg ctccccttat tgcatcagct ccggttatac
taatggcggc ttcgttctca 1140aatgcggttt tcgagagact accgattgag
gaggaggaag aagaaggtgg tggtggcgga 1200ggaggaggag gaggagggcc
accgcagatg caacaagctc catcagcatc tccgccgtct 1260ggagtgaccg
gtcagggaca gttaggaggt aatgtgggtg gttatgggtt ttctggtgat
1320cctcatttgc ttggatgggg agctggaaca ccttcaagac caccttttta
attgaatttt 1380aatgtccgga aatttatgtg tttttatcat cttgaggagt
cgtctttcct ttgggatatt 1440tggtgtttaa tgtttagttg atatgcatat ttt
1473
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