U.S. patent application number 13/159353 was filed with the patent office on 2012-01-19 for tubulin regulatory elements for use in plants.
Invention is credited to Gregory R. Heck, Marianne Malven, James D. Masucci, Jinsong You.
Application Number | 20120015810 13/159353 |
Document ID | / |
Family ID | 34272575 |
Filed Date | 2012-01-19 |
United States Patent
Application |
20120015810 |
Kind Code |
A1 |
Heck; Gregory R. ; et
al. |
January 19, 2012 |
TUBULIN REGULATORY ELEMENTS FOR USE IN PLANTS
Abstract
The present invention provides polynucleotide molecules isolated
from Zea mays and Oryza sativa and useful for expressing transgenes
in plants. The present invention also provides expression
constructs containing the polynucleotide molecules useful for
expressing transgenes in plants. The present invention also
provides transgenic plants and seeds containing the polynucleotide
molecules useful for expressing transgenes in plants.
Inventors: |
Heck; Gregory R.; (Crystal
Lake Park, MO) ; Malven; Marianne; (Ellisville,
MO) ; Masucci; James D.; (Ballwin, MO) ; You;
Jinsong; (Manchester, MO) |
Family ID: |
34272575 |
Appl. No.: |
13/159353 |
Filed: |
June 13, 2011 |
Related U.S. Patent Documents
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Patent Number |
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12912072 |
Oct 26, 2010 |
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13159353 |
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12389314 |
Feb 19, 2009 |
7838654 |
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12912072 |
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10925392 |
Aug 25, 2004 |
7511130 |
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12389314 |
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60497523 |
Aug 25, 2003 |
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Current U.S.
Class: |
504/206 ;
435/320.1; 536/24.1; 800/298; 800/306; 800/312; 800/314; 800/317.2;
800/317.3; 800/317.4; 800/320; 800/320.1; 800/320.2; 800/320.3;
800/322 |
Current CPC
Class: |
C12N 15/823 20130101;
C12N 15/8223 20130101 |
Class at
Publication: |
504/206 ;
536/24.1; 435/320.1; 800/298; 800/320.3; 800/320.1; 800/320;
800/320.2; 800/317.3; 800/317.4; 800/317.2; 800/312; 800/314;
800/306; 800/322 |
International
Class: |
A01H 5/00 20060101
A01H005/00; A01P 13/00 20060101 A01P013/00; A01H 5/10 20060101
A01H005/10; A01N 57/20 20060101 A01N057/20; C12N 15/113 20100101
C12N015/113; C12N 15/63 20060101 C12N015/63 |
Claims
1. An isolated polynucleotide molecule having gene regulatory
activity and comprising at least 95 contiguous bases of a
polynucleotide sequence selected from the group consisting of SEQ
ID NO: 1-4.
2. An isolated polynucleotide molecule having gene regulatory
activity and comprising a polynucleotide sequence selected from the
group consisting of SEQ ID NO: 1-5.
3. The isolated polynucleotide molecule according to claim 2,
wherein said isolated polynucleotide molecule comprises a
polynucleotide sequence which exhibits a substantial percent
sequence identity of about 98% identity with the polynucleotide
sequence of SEQ ID NO: 1-5.
4. A DNA construct comprising an isolated polynucleotide molecule
having gene regulatory activity and comprising a polynucleotide
sequence selected from the group consisting of SEQ ID NO: 1-5,
wherein said isolated polynucleotide molecule is operably linked to
a transcribable polynucleotide molecule.
5. The DNA construct of claim 4, wherein said transcribable
polynucleotide molecule is a marker gene.
6. The DNA construct of claim 4, wherein said transcribable
polynucleotide molecule is a gene of agronomic interest.
7. The DNA construct of claim 6, wherein said gene of agronomic
interest is a herbicide tolerance gene selected from the group
consisting of genes that encode for phosphinothricin
acetyltransferase, glyphosate resistant
5-enolpyruvylshikimate-3-phosphate synthase, hydroxyphenyl pyruvate
dehydrogenase, dalapon dehalogenase, bromoxynil resistant
nitrilase, anthranilate synthase, glyphosate oxidoreductase and
glyphosate-N-acetyl transferase.
8. A transgenic plant stably transformed with the DNA construct of
claim 4.
9. The transgenic plant of claim 8, wherein said plant is a
monocotyledonous selected from the group consisting of wheat,
maize, rye, rice, oat, barley, turfgrass, sorghum, millet and
sugarcane.
10. The transgenic plant of claim 8, wherein said plant is a
dicotyledonous plant selected from the group consisting of tobacco,
tomato, potato, soybean, cotton, canola, sunflower and alfalfa.
11. A seed of said transgenic plant of claim 9.
12. A seed of said transgenic plant of claim 10.
13. A method of inhibiting weed growth in a field of transgenic
glyphosate tolerant crop plants comprising: i) planting the
transgenic plants transformed with an expression cassette
comprising (a) an isolated polynucleotide molecule having gene
regulatory activity and comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO: 1-5 and operably
linked to a DNA molecule encoding a glyphosate tolerance gene and
ii) applying glyphosate to the field at an application rate that
inhibits the growth of weeds, wherein the growth and yield of the
transgenic crop plant is not substantially affected by the
glyphosate application.
14. In the method of claim 13, wherein said glyphosate tolerance
gene is selected from the group consisting of a gene encoding for a
glyphosate resistant 5-enolpyruvylshikimate-3-phosphate synthase, a
glyphosate oxidoreductase, and a
glyphosate-N-acetyltransferase.
15. In the method of claim 13, wherein the transgenic plants are
capable of tolerating an application rate up to 256
ounces/acre.
16. In the method of claim 13, wherein the transgenic plants are
capable of tolerating an application rate ranging from 8
ounces/acre to 128 ounces/acre.
17. In the method of claim 13, wherein the transgenic plants are
capable of tolerating an application rate ranging from 32
ounces/acre to 96 ounces/acre.
18. In the method of claim 13, wherein the application of
glyphosate is at least once during the growth of the crop.
Description
INCORPORATION OF SEQUENCE LISTING
[0001] Two copies of the sequence listing (Seq. Listing Copy 1 and
Seq. Listing Copy 2) and a computer-readable form of the sequence
listing, all on CD-ROMs, each containing the file named
53419-B.txt, which is 10,240 bytes (measured in MS-DOS) and was
created on Aug. 24, 2004, are hereby incorporated by reference.
FIELD OF THE INVENTION
[0002] The invention relates to the field of plant molecular
biology and plant genetic engineering and polynucleotide molecules
useful for the expression of transgenes in plants.
BACKGROUND
[0003] One of the goals of plant genetic engineering is to produce
plants with agronomically desirable characteristics or traits. The
proper expression of a desirable transgene in a transgenic plant is
one way to achieve this goal. Regulatory elements such as
promoters, leaders, and introns are non-coding polynucleotide
molecules which play an integral part in the overall expression of
genes in living cells. Isolated regulatory elements that function
in plants are therefore useful for modifying plant phenotypes
through the methods of genetic engineering.
[0004] Many regulatory elements are available and are useful for
providing good overall expression of a transgene. For example,
constitutive promoters such as P-FMV, the promoter from the 35S
transcript of the Figwort mosaic virus (U.S. Pat. No. 6,051,753);
P-CaMV 35S, the promoter from the 35S RNA transcript of the
Cauliflower mosaic virus (U.S. Pat. No. 5,530,196); P-Rice Actin 1,
the promoter from the actin 1 gene of Oryza sativa (U.S. Pat. No.
5,641,876); and P-NOS, the promoter from the nopaline synthase gene
of Agrobacterium tumefaciens are known to provide some level of
gene expression in most or all of the tissues of a plant during
most or all of the plant's lifespan. While previous work has
provided a number of regulatory elements useful to affect gene
expression in transgenic plants, there is still a great need for
novel regulatory elements with beneficial expression
characteristics. Many previously identified regulatory elements
fail to provide the patterns or levels of expression required to
fully realize the benefits of expression of selected genes in
transgenic crop plants.
[0005] Spatial organization within the eukaryotic cell and directed
movements of the cell contents are mediated by the cytoskeleton, a
network of filamentous protein polymers that permeates the cytosol.
Tubulin is one of the three major families of proteins making up
the cytoskeleton. Members of this multi-gene family have been
reported in almost all eukaryotic species including yeast, humans,
mouse, Drosophila, tobacco, maize, rice, soybean, potato and
Arbabidopsis. There are two types of tubulin proteins in higher
eukaryotes, .alpha.- and .beta.-tubulin. Plant .alpha.- and
.beta.-tubulins are encoded by two gene families, each constituted
by a number of different isotypes.
[0006] We hypothesized that a promoter from an .alpha.-tubulin gene
might have a constitutive expression pattern and that the promoter
and regulatory elements could be useful to direct expression of a
transgene such as a glyphosate resistant
5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) transgene to
produce a glyphosate tolerant plant. The efficient production of
glyphosate tolerant plants requires the use of a promoter and
regulatory elements capable of directing transgene expression in
all tissues including the most sensitive reproductive organs such
as anthers and meristem tissues. The present invention thus
provides such promoters and regulatory elements isolated from an
.alpha.-tubulin gene of Oryza sativa.
SUMMARY
[0007] In one embodiment the invention provides polynucleotide
molecules isolated from Oryza sativa useful for modulating
transgene expression in plants. In another embodiment the invention
provides expression constructs containing the polynucleotide
molecules useful for modulating transgene expression in plants. In
another embodiment the invention provides transgenic plants and
seeds containing the polynucleotide molecules useful for modulating
transgene expression in plants.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] FIG. 1: pMON77978 plasmid map. Genetic elements and their
relative position are shown. P=promoter; I=Intron; L=5' UTR;
CR=coding region; T=3' region plus downstream sequence;
nptII=kanamycin resistance gene, for plant and microbial
selection.
[0009] FIG. 2: pMON70453 plasmid map. Genetic elements and their
relative position are shown. P=promoter; I=Intron; L=5' UTR;
TS=transit peptide sequence; CR=coding region; T=3' region plus
downstream sequence; CP4=glyphosate resistance gene for plant
selection; SPC/STR=aad for microbial selection; left and right
T-DNA borders are shown.
DETAILED DESCRIPTION
[0010] The following definitions and methods are provided to better
define the present invention and to guide those of ordinary skill
in the art in the practice of the present invention. Unless
otherwise noted, terms are to be understood according to
conventional usage by those of ordinary skill in the relevant
art.
[0011] The invention disclosed herein provides polynucleotide
molecules having gene regulatory activity from Oryza sativa. The
design, construction, and use of these polynucleotide molecules are
one object of this invention. The polynucleotide sequences of these
polynucleotide molecules are provided as SEQ ID NO: 1-5. These
polynucleotide molecules are capable of affecting the transcription
of operably linked transcribable polynucleotide molecules in both
vegetative and reproductive tissues of plants and therefore can
selectively regulate expression of transgenes in these tissues.
[0012] As used herein, the term "polynucleotide molecule" refers to
the single- or double-stranded DNA or RNA of genomic or synthetic
origin, i.e., a polymer of deoxyribonucleotide or ribonucleotide
bases, respectively, read from the 5' (upstream) end to the 3'
(downstream) end.
[0013] As used herein, the term "polynucleotide sequence" refers to
the sequence of a polynucleotide molecule. The nomenclature for DNA
bases as set forth at 37 CFR .sctn.1.822 is used.
[0014] As used herein, the term "gene regulatory activity" refers
to a polynucleotide molecule capable of affecting transcription or
translation of an operably linked transcribable polynucleotide
molecule. An isolated polynucleotide molecule having gene
regulatory activity may provide temporal or spatial expression or
modulate levels and rates of expression of the operably linked
transcribable polynucleotide molecule. An isolated polynucleotide
molecule having gene regulatory activity may comprise a promoter,
intron, leader, or 3' transcriptional termination region.
[0015] As used herein, the term "promoter" refers to a
polynucleotide molecule that is involved in recognition and binding
of RNA polymerase II and other proteins (trans-acting transcription
factors) to initiate transcription. A "plant promoter" is a native
or non-native promoter that is functional in plant cells. A plant
promoter can be used as a 5' regulatory element for modulating
expression of an operably linked gene or genes. Plant promoters may
be defined by their temporal, spatial, or developmental expression
pattern.
[0016] A promoter comprises subfragments that have promoter
activity. Subfragments may comprise enhancer domains and may be
useful for constructing chimeric promoters. Subfragments of SEQ ID
NO: 1 comprise at least about 75, 85, 90, 95, 110, 125, 250, 400,
750, 1000, 1300, 1500, 1800, and 2000 contiguous nucleotides of the
polynucleotide sequence of SEQ ID NO: 1, up to the full 2190
nucleotides of SEQ ID NO: 1. Subfragments of SEQ ID NO: 2 comprise
at least about 95, 110, 125, 250, 400, 750, 1000, 1300, 1500, and
1800 contiguous nucleotides of the polynucleotide sequence of SEQ
ID NO: 2, up to the full 1260 nucleotides of SEQ ID NO: 2.
[0017] As used herein, the term "enhancer domain" refers to a
cis-acting transcriptional regulatory element, a.k.a. cis-element,
which confers an aspect of the overall control of gene expression.
An enhancer domain may function to bind transcription factors,
trans-acting protein factors that regulate transcription. Some
enhancer domains bind more than one transcription factor, and
transcription factors may interact with different affinities with
more than one enhancer domain. Enhancer domains can be identified
by a number of techniques, including deletion analysis, i.e.,
deleting one or more nucleotides from the 5' end or internal to a
promoter; DNA binding protein analysis using DNase I footprinting,
methylation interference, electrophoresis mobility-shift assays, in
vivo genomic footprinting by ligation-mediated PCR, and other
conventional assays; or by DNA sequence similarity analysis with
known cis-element motifs by conventional DNA sequence comparison
methods. The fine structure of an enhancer domain can be further
studied by mutagenesis (or substitution) of one or more nucleotides
or by other conventional methods. Enhancer domains can be obtained
by chemical synthesis or by isolation from promoters that include
such elements, and they can be synthesized with additional flanking
nucleotides that contain useful restriction enzyme sites to
facilitate subsequence manipulation. Thus, the design,
construction, and use of enhancer domains according to the methods
disclosed herein for modulating the expression of operably linked
polynucleotide molecules are encompassed by the present
invention.
[0018] As used herein, the term "chimeric" refers to the product of
the fusion of portions of two or more different polynucleotide
molecules. As used herein, the term "chimeric promoter" refers to a
promoter produced through the manipulation of known promoters or
other polynucleotide molecules. Such chimeric promoters may combine
enhancer domains that can confer or modulate gene expression from
one or more promoters, for example, by fusing a heterologous
enhancer domain from a first promoter to a second promoter with its
own partial or complete regulatory elements. Thus, the design,
construction, and use of chimeric promoters according to the
methods disclosed herein for modulating the expression of operably
linked polynucleotide molecules are encompassed by the present
invention.
[0019] As used herein, the term "percent sequence identity" refers
to the percentage of identical nucleotides in a linear
polynucleotide sequence of a reference polynucleotide molecule (or
its complementary strand) as compared to a test polynucleotide
molecule (or its complementary strand) when the two sequences are
optimally aligned (with appropriate nucleotide insertions,
deletions, or gaps totaling less than 20 percent of the reference
sequence over the window of comparison). Optimal alignment of
sequences for aligning a comparison window are well known to those
skilled in the art and may be conducted by tools such as the local
homology algorithm of Smith and Waterman, the homology alignment
algorithm of Needleman and Wunsch, the search for similarity method
of Pearson and Lipman, and preferably by computerized
implementations of these algorithms such as GAP, BESTFIT, FASTA,
and TFASTA available as part of the GCG.RTM. Wisconsin Package.RTM.
(Accelrys Inc., Burlington, Mass.). An "identity fraction" for
aligned segments of a test sequence and a reference sequence is the
number of identical components which are shared by the two aligned
sequences divided by the total number of components in the
reference sequence segment, i.e., the entire reference sequence or
a smaller defined part of the reference sequence. Percent sequence
identity is represented as the identity fraction multiplied by 100.
The comparison of one or more polynucleotide sequences may be to a
full-length polynucleotide sequence or a portion thereof, or to a
longer polynucleotide sequence.
[0020] As used herein, the term "substantial percent sequence
identity" refers to a percent sequence identity of at least about
70% sequence identity, at least about 80% sequence identity, at
least about 90% sequence identity, or even greater sequence
identity, such as about 98% or about 99% sequence identity. Thus,
one embodiment of the invention is a polynucleotide molecule that
has at least about 70% sequence identity, at least about 80%
sequence identity, at least about 90% sequence identity, or even
greater sequence identity, such as about 98% or about 99% sequence
identity with a polynucleotide sequence described herein.
Polynucleotide molecules that are capable of regulating
transcription of operably linked transcribable polynucleotide
molecules and have a substantial percent sequence identity to the
polynucleotide sequences of the polynucleotide molecules provided
herein are encompassed within the scope of this invention.
Promoter Isolation and Modification Methods
[0021] Any number of methods well known to those skilled in the art
can be used to isolate fragments of a promoter disclosed herein.
For example, PCR (polymerase chain reaction) technology can be used
to amplify flanking regions from a genomic library of a plant using
publicly available sequence information. A number of methods are
known to those of skill in the art to amplify unknown
polynucleotide molecules adjacent to a core region of known
polynucleotide sequence. Methods include but are not limited to
inverse PCR (IPCR), vectorette PCR, Y-shaped PCR, and genome
walking approaches. Polynucleotide fragments can also be obtained
by other techniques such as by directly synthesizing the fragment
by chemical means, as is commonly practiced by using an automated
oligonucleotide synthesizer. For the present invention, the
polynucleotide molecules were isolated from genomic DNA by
designing PCR primers based on available sequence information.
[0022] Novel chimeric promoters can be designed or engineered by a
number of methods. For example, a chimeric promoter may be produced
by fusing an enhancer domain from a first promoter to a second
promoter. The resultant chimeric promoter may have novel expression
properties relative to the first or second promoters. Novel
chimeric promoters can be constructed such that the enhancer domain
from a first promoter is fused at the 5' end, at the 3' end, or at
any position internal to the second promoter. The location of the
enhancer domain fusion relative to the second promoter may cause
the resultant chimeric promoter to have novel expression properties
relative to a fusion made at a different location.
[0023] Those of skill in the art are familiar with the standard
resource materials that describe specific conditions and procedures
for the construction, manipulation, and isolation of macromolecules
(e.g., polynucleotide molecules, plasmids, etc.), as well as the
generation of recombinant organisms and the screening and isolation
of polynucleotide molecules.
Constructs
[0024] As used herein, the term "construct" refers to any
recombinant polynucleotide molecule such as a plasmid, cosmid,
virus, autonomously replicating polynucleotide molecule, phage, or
linear or circular single-stranded or double-stranded DNA or RNA
polynucleotide molecule, derived from any source, capable of
genomic integration or autonomous replication, comprising a
polynucleotide molecule where one or more polynucleotide molecule
has been operably linked.
[0025] As used herein, the term "operably linked" refers to a first
polynucleotide molecule, such as a promoter, connected with a
second transcribable polynucleotide molecule, such as a gene of
interest, where the polynucleotide molecules are so arranged that
the first polynucleotide molecule affects the function of the
second polynucleotide molecule. The two polynucleotide molecules
may be part of a single contiguous polynucleotide molecule and may
be adjacent. For example, a promoter is operably linked to a gene
of interest if the promoter regulates or mediates transcription of
the gene of interest in a cell.
[0026] As used herein, the term "transcribable polynucleotide
molecule" refers to any polynucleotide molecule capable of being
transcribed into a RNA molecule. Methods are known for introducing
constructs into a cell in such a manner that the transcribable
polynucleotide molecule is transcribed into a functional mRNA
molecule that is translated and therefore expressed as a protein
product. Constructs may also be constructed to be capable of
expressing antisense RNA molecules, in order to inhibit translation
of a specific RNA molecule of interest. For the practice of the
present invention, conventional compositions and methods for
preparing and using constructs and host cells are well known to one
skilled in the art, see for example, Molecular Cloning: A
Laboratory Manual, 3.sup.rd edition Volumes 1, 2, and 3 (2000) J.
F. Sambrook, D. W. Russell, and N. Irwin, Cold Spring Harbor
Laboratory Press.
[0027] Constructs of the present invention would typically contain
a promoter operably linked to a transcribable polynucleotide
molecule operably linked to a 3' transcription termination
polynucleotide molecule. In addition, constructs may include but
are not limited to additional regulatory polynucleotide molecules
from the 3'-untranslated region (3' UTR) of plant genes (e.g., a 3'
UTR to increase mRNA stability of the mRNA, such as the PI-II
termination region of potato or the octopine or nopaline synthase
3' termination regions). Constructs may include but are not limited
to the 5' untranslated regions (5' UTR) of an mRNA polynucleotide
molecule which can play an important role in translation initiation
and can also be a genetic component in a plant expression
construct. For example, non-translated 5' leader polynucleotide
molecules derived from heat shock protein genes have been
demonstrated to enhance gene expression in plants (see for example,
U.S. Pat. No. 5,659,122 and U.S. Pat. No. 5,362,865, all of which
are hereby incorporated by reference). These additional upstream
and downstream regulatory polynucleotide molecules may be derived
from a source that is native or heterologous with respect to the
other elements present on the promoter construct.
[0028] Thus, one embodiment of the invention is a promoter such as
provided in SEQ ID NO: 1-2, operably linked to a transcribable
polynucleotide molecule so as to direct transcription of said
transcribable polynucleotide molecule at a desired level or in a
desired tissue or developmental pattern upon introduction of said
construct into a plant cell. In some cases, the transcribable
polynucleotide molecule comprises a protein-coding region of a
gene, and the promoter provides for transcription of a functional
mRNA molecule that is translated and expressed as a protein
product. Constructs may also be constructed for transcription of
antisense RNA molecules or other similar inhibitory RNA in order to
inhibit expression of a specific RNA molecule of interest in a
target host cell.
[0029] Exemplary transcribable polynucleotide molecules for
incorporation into constructs of the present invention include, for
example, polynucleotide molecules or genes from a species other
than the target gene species, or even genes that originate with or
are present in the same species, but are incorporated into
recipient cells by genetic engineering methods rather than
classical reproduction or breeding techniques. Exogenous gene or
genetic element is intended to refer to any gene or polynucleotide
molecule that is introduced into a recipient cell. The type of
polynucleotide molecule included in the exogenous polynucleotide
molecule can include a polynucleotide molecule that is already
present in the plant cell, a polynucleotide molecule from another
plant, a polynucleotide molecule from a different organism, or a
polynucleotide molecule generated externally, such as a
polynucleotide molecule containing an antisense message of a gene,
or a polynucleotide molecule encoding an artificial or modified
version of a gene.
[0030] The promoters of the present invention can be incorporated
into a construct using marker genes as described and tested in
transient analyses that provide an indication of gene expression in
stable plant systems. As used herein the term "marker gene" refers
to any transcribable polynucleotide molecule whose expression can
be screened for or scored in some way. Methods of testing for
marker gene expression in transient assays are known to those of
skill in the art. Transient expression of marker genes has been
reported using a variety of plants, tissues, and DNA delivery
systems. For example, types of transient analyses can include but
are not limited to direct gene delivery via electroporation or
particle bombardment of tissues in any transient plant assay using
any plant species of interest. Such transient systems would include
but are not limited to electroporation of protoplasts from a
variety of tissue sources or particle bombardment of specific
tissues of interest. The present invention encompasses the use of
any transient expression system to evaluate promoters or promoter
fragments operably linked to any transcribable polynucleotide
molecules, including but not limited to selected reporter genes,
marker genes, or genes of agronomic interest. Examples of plant
tissues envisioned to test in transients via an appropriate
delivery system would include but are not limited to leaf base
tissues, callus, cotyledons, roots, endosperm, embryos, floral
tissue, pollen, and epidermal tissue.
[0031] Any scorable or screenable marker gene can be used in a
transient assay. Exemplary marker genes for transient analyses of
the promoters or promoter fragments of the present invention
include a GUS gene (U.S. Pat. No. 5,599,670, hereby incorporated by
reference) or a GFP gene (U.S. Pat. No. 5,491,084 and U.S. Pat. No.
6,146,826, both of which are hereby incorporated by reference). The
constructs containing the promoters or promoter fragments operably
linked to a marker gene are delivered to the tissues and the
tissues are analyzed by the appropriate mechanism, depending on the
marker. The quantitative or qualitative analyses are used as a tool
to evaluate the potential expression profile of the promoters or
promoter fragments when operatively linked to genes of agronomic
interest in stable plants.
[0032] Thus, in one preferred embodiment, a polynucleotide molecule
of the present invention as shown in SEQ ID NO: 1-5 is incorporated
into a construct such that a promoter of the present invention is
operably linked to a transcribable polynucleotide molecule that
provides for a selectable, screenable, or scorable marker. Markers
for use in the practice of the present invention include, but are
not limited to transcribable polynucleotide molecules encoding
.beta.-glucuronidase (GUS), green fluorescent protein (GFP),
luciferase (LUC), proteins that confer antibiotic resistance, or
proteins that confer herbicide tolerance. Useful antibiotic
resistance markers, including those genes encoding proteins
conferring resistance to kanamycin (nptII), hygromycin B (aph IV),
streptomycin or spectinomycin (aad, spec/strep) and gentamycin
(aac3 and aacC4) are known in the art. Herbicides for which
transgenic plant tolerance has been demonstrated and the method of
the present invention can be applied, include but are not limited
to: glyphosate, glufosinate, sulfonylureas, imidazolinones,
bromoxynil, delapon, cyclohezanedione, protoporphyrionogen oxidase
inhibitors, and isoxasflutole herbicides. Polynucleotide molecules
encoding proteins involved in herbicide tolerance are known in the
art, and include, but are not limited to a polynucleotide molecule
encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)
described in U.S. Pat. No. 5,627,061, U.S. Pat. No. 5,633,435, and
U.S. Pat. No. 6,040,497 and aroA described in U.S. Pat. No.
5,094,945 for glyphosate tolerance, all of which are hereby
incorporated by reference; a polynucleotide molecule encoding
bromoxynil nitrilase (Bxn) described in U.S. Pat. No. 4,810,648 for
Bromoxynil tolerance, which is hereby incorporated by reference; a
polynucleotide molecule encoding phytoene desaturase (crtI)
described in Misawa et al, (1993) Plant Journal 4:833-840 and
Misawa et al, (1994) Plant Journal 6:481-489 for norflurazon
tolerance; a polynucleotide molecule encoding acetohydroxyacid
synthase (AHAS, aka ALS) described in Sathasiivan et al. (1990)
Polynucleotides Research 18:2188-2193 for tolerance to sulfonylurea
herbicides; and the bar gene described in DeBlock, et al. (1987)
EMBO Journal 6:2513-2519 for glufosinate and bialaphos
tolerance.
[0033] In one embodiment of the invention, a polynucleotide
molecule as shown in SEQ ID NO: 1-5 is incorporated into a
construct such that a polynucleotide molecule of the present
invention is operably linked to a transcribable polynucleotide
molecule that is a gene of agronomic interest. As used herein, the
term "gene of agronomic interest" refers to a transcribable
polynucleotide molecule that includes but is not limited to a gene
that provides a desirable characteristic associated with plant
morphology, physiology, growth and development, yield, nutritional
enhancement, disease or pest resistance, or environmental or
chemical tolerance. The expression of a gene of agronomic interest
is desirable in order to confer an agronomically important trait. A
gene of agronomic interest that provides a beneficial agronomic
trait to crop plants may be, for example, including, but not
limited to genetic elements comprising herbicide resistance (U.S.
Pat. No. 5,633,435 and U.S. Pat. No. 5,463,175), increased yield
(U.S. Pat. No. 5,716,837), insect control (U.S. Pat. No. 6,063,597;
U.S. Pat. No. 6,063,756; U.S. Pat. No. 6,093,695; U.S. Pat. No.
5,942,664; and U.S. Pat. No. 6,110,464), fungal disease resistance
(U.S. Pat. No. 5,516,671; U.S. Pat. No. 5,773,696; U.S. Pat. No.
6,121,436; U.S. Pat. No. 6,316,407, and U.S. Pat. No. 6,506,962),
virus resistance (U.S. Pat. No. 5,304,730 and U.S. Pat. No.
6,013,864), nematode resistance (U.S. Pat. No. 6,228,992),
bacterial disease resistance (U.S. Pat. No. 5,516,671), starch
production (U.S. Pat. No. 5,750,876 and U.S. Pat. No. 6,476,295),
modified oils production (U.S. Pat. No. 6,444,876), high oil
production (U.S. Pat. No. 5,608,149 and U.S. Pat. No. 6,476,295),
modified fatty acid content (U.S. Pat. No. 6,537,750), high protein
production (U.S. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No.
5,512,466), enhanced animal and human nutrition (U.S. Pat. No.
5,985,605 and U.S. Pat. No. 6,171,640), biopolymers (U.S. Pat. No.
5,958,745 and U.S. Patent Publication No. US20030028917),
environmental stress resistance (U.S. Pat. No. 6,072,103),
pharmaceutical peptides (U.S. Pat. No. 6,080,560), improved
processing traits (U.S. Pat. No. 6,476,295), improved digestibility
(U.S. Pat. No. 6,531,648) low raffinose (U.S. Pat. No. 6,166,292),
industrial enzyme production (U.S. Pat. No. 5,543,576), improved
flavor (U.S. Pat. No. 6,011,199), nitrogen fixation (U.S. Pat. No.
5,229,114), hybrid seed production (U.S. Pat. No. 5,689,041), and
biofuel production (U.S. Pat. No. 5,998,700). The genetic elements,
methods, and transgenes described in the patents listed above are
hereby incorporated by reference.
[0034] Alternatively, a transcribable polynucleotide molecule can
effect the above mentioned phenotypes by encoding a RNA molecule
that causes the targeted inhibition of expression of an endogenous
gene, for example via antisense, inhibitory RNA (RNAi), or
cosuppression-mediated mechanisms. The RNA could also be a
catalytic RNA molecule (i.e., a ribozyme) engineered to cleave a
desired endogenous mRNA product. Thus, any polynucleotide molecule
that encodes a protein or mRNA that expresses a phenotype or
morphology change of interest may be useful for the practice of the
present invention.
[0035] The constructs of the present invention are generally double
Ti plasmid border DNA constructs that have the right border (RB or
AGRtu.RB) and left border (LB or AGRtu.LB) regions of the Ti
plasmid isolated from Agrobacterium tumefaciens comprising a T-DNA,
that along with transfer molecules provided by the Agrobacterium
cells, permits the integration of the T-DNA into the genome of a
plant cell. The constructs also contain the plasmid backbone DNA
segments that provide replication function and antibiotic selection
in bacterial cells, for example, an Escherichia coli origin of
replication such as ori322, a broad host range origin of
replication such as oriV or oriRi, and a coding region for a
selectable marker such as Spec/Strp that encodes for Tn7
aminoglycoside adenyltransferase (aadA) conferring resistance to
spectinomycin or streptomycin, or a gentamicin (Gm, Gent)
selectable marker gene. For plant transformation, the host
bacterial strain is often Agrobacterium tumefaciens ABI, C58, or
LBA4404, however, other strains known to those skilled in the art
of plant transformation can function in the present invention.
Transformed Plants And Plant Cells
[0036] As used herein, the term "transformed" refers to a cell,
tissue, organ, or organism into which has been introduced a foreign
polynucleotide molecule, such as a construct. The introduced
polynucleotide molecule may be integrated into the genomic DNA of
the recipient cell, tissue, organ, or organism such that the
introduced polynucleotide molecule is inherited by subsequent
progeny. A "transgenic" or "transformed" cell or organism also
includes progeny of the cell or organism and progeny produced from
a breeding program employing such a transgenic plant as a parent in
a cross and exhibiting an altered phenotype resulting from the
presence of a foreign polynucleotide molecule. A plant
transformation construct containing a promoter of the present
invention may be introduced into plants by any plant transformation
method. Methods and materials for transforming plants by
introducing a plant expression construct into a plant genome in the
practice of this invention can include any of the well-known and
demonstrated methods including electroporation as illustrated in
U.S. Pat. No. 5,384,253; microprojectile bombardment as illustrated
in U.S. Pat. Nos. 5,015,580; U.S. Pat. No. 5,550,318; U.S. Pat. No.
5,538,880; U.S. Pat. No. 6,160,208; U.S. Pat. No. 6,399,861; and
U.S. Pat. No. 6,403,865; Agrobacterium-mediated transformation as
illustrated in U.S. Pat. No. 5,824,877; U.S. Pat. No. 5,591,616;
U.S. Pat. No. 5,981,840; and U.S. Pat. No. 6,384,301; and
protoplast transformation as illustrated in U.S. Pat. No.
5,508,184, all of which are hereby incorporated by reference.
[0037] Methods for specifically transforming dicots are well known
to those skilled in the art. Transformation and plant regeneration
using these methods have been described for a number of crops
including, but not limited to, cotton (Gossypium hirsutum), soybean
(Glycine max), peanut (Arachis hypogaea), and members of the genus
Brassica.
[0038] Methods for transforming monocots are well known to those
skilled in the art. Transformation and plant regeneration using
these methods have been described for a number of crops including,
but not limited to, barley (Hordeum vulgarae); maize (Zea mays);
oats (Avena sativa); orchard grass (Dactylis glomerata); rice
(Oryza sativa, including indica and japonica varieties); sorghum
(Sorghum bicolor); sugar cane (Saccharum sp); tall fescue (Festuca
arundinacea); turfgrass species (e.g. species: Agrostis
stolonifera, Poa pratensis, Stenotaphrum secundatum); wheat
(Triticum aestivum), and alfalfa (Medicago sativa). It is apparent
to those of skill in the art that a number of transformation
methodologies can be used and modified for production of stable
transgenic plants from any number of target crops of interest.
[0039] The transformed plants are analyzed for the presence of the
genes of interest and the expression level and/or profile conferred
by the promoters of the present invention. Those of skill in the
art are aware of the numerous methods available for the analysis of
transformed plants. For example, methods for plant analysis
include, but are not limited to Southern blots or northern blots,
PCR-based approaches, biochemical analyses, phenotypic screening
methods, field evaluations, and immunodiagnostic assays.
[0040] 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 plant lines
comprising the construct of this invention and expressing a gene of
agronomic interest.
[0041] The present invention also provides for parts of the plants
of the present invention. Plant parts, without limitation, include
seed, endosperm, ovule and pollen. In a particularly preferred
embodiment of the present invention, the plant part is a seed.
[0042] Still yet another aspect of the invention is a method of
inhibiting weed growth in a field of transgenic crop plants
comprising first planting the transgenic plants transformed with an
expression cassette comprising an isolated polynucleotide molecule
having gene regulatory activity and comprising a polynucleotide
sequence selected from the group consisting of SEQ ID NO: 1-5 and
operably linked to a DNA molecule encoding a glyphosate tolerance
gene and then applying glyphosate to the field at an application
rate that inhibits the growth of weeds, wherein the growth and
yield of the transgenic crop plant is not substantially affected by
the glyphosate application. The glyphosate application rate is the
effective rate necessary to control weeds in a particular
glyphosate tolerant crop; these rates may range from 8 ounces/acre
to 256 ounces/acre, preferably 16 ounces/acre to 128 ounces/acre,
and more preferably 32 ounces/acre to 96 ounces/acre. The
glyphosate is applied at least once during the growth of the
glyphosate tolerant crop and may be applied 2, 3, or 4 times during
the growth of the crop or more as necessary to control weeds in the
field.
[0043] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples that
follow represent techniques discovered by the inventors to function
well in the practice of the invention. However, those of skill in
the art should, in light of the present disclosure, appreciate that
many changes can be made in the specific embodiments that are
disclosed and still obtain a like or similar result without
departing from the spirit and scope of the invention, therefore all
matter set forth or shown in the accompanying drawings is to be
interpreted as illustrative and not in a limiting sense.
EXAMPLES
Example 1
Constitutive Gene Identification
[0044] Rice genes having a constitutive expression pattern were
identified as the first step in isolating heterologous elements for
use in the construction of transgene cassettes. Genes involved in
basic cellular functions such as cytoskeleton formation are often
constitutively expressed throughout the plant. These genes often
exist in gene families, however, and while the overall expression
of the family members may be constitutive throughout the plant,
individual members may have more restricted, temporal,
developmental, or organ/tissue/cell type specific expression
patterns. With this understanding, specific gene families were
focused on for the selection of individual candidate genes using
data from gene expression studies.
[0045] The 5' untranslated region (UTR) sequences of members of
selected multi-gene families (including tubulin, actin, histone,
etc.) were identified using rice genomic sequence. Primers were
designed to anneal to the 5' UTR sequences and PCR was performed
using standard methods. The subsequent polynucleotide molecule
products were arrayed on nylon filters for mRNA expression
analysis. The filters were then probed with cDNA molecules derived
from rice mRNA pools from root, leaf, grain, anther, ovary, or
glume tissue. The experiment was repeated twice and results were
used to analyze the expression of the selected genes. From
analyzing the expression data, twenty genes were chosen for further
analysis using real time PCR with 3' UTR specific primers. The
primers were used in conjunction with the SYBR Green kit (Perkin
Elmer Inc., Wellesley, Mass.) using a Taqman machine (Applied
Biosystems, Foster City, Calif.) and standard protocols supplied by
the manufacturer to amplify sequences out of leaf, root, anther,
gynoecium, or apex cDNA. Results were then compared to the level of
expression for the rice actin 1 gene, whose promoter and first
intron are known to provide high glyphosate tolerance when operably
linked to a glyphosate resistant EPSPS gene. An .alpha.-tubulin
gene, designated Os-TubA-3, was shown to be expressed in rice in
all tissues at levels higher than the ract1 gene.
[0046] The generally constitutive expression pattern of the
Os-TubA-3 gene was further confirmed when a BLASTN analysis was
performed using its 3' UTR sequence as a query of rice EST
libraries prepared from various rice organs at various
developmental stages (see Table 1). Results were again compared to
the level of expression for the rice actin 1 gene. A particularly
desirable expression feature of the TubA-3 gene was EST
representation in developing floral structures, including anthers.
A BLASTN search using the Os-TubA-3 3' UTR sequence was done to
identify a rice BAC which contained a complete genomic copy of the
Os-TubA-3 gene including the promoter region.
TABLE-US-00001 TABLE 1 Occurrence of respective 3' UTR sequences in
rice EST libraries. Total number Rice actin 1 Os-TubA-3 of 5' and
occur- occur- Library 3' reads rences rences panicle, cracking-3/4
open floret 20,227 7 >16 developing panicle 7,909 5 >16 late
anther 5,956 14 4 developing seed 7,453 1 8 dry seed 9,362 0 0
germ. seed 9,743 0 1 vegetative apex 7,672 2 >16 leaf, 3-5 leaf
10,040 0 1 leaf, 3-4 tiller 9,209 1 0 leaf, elong-boot 7,897 1 3
root, 3-5 leaf 10,524 2 4 root, 3-4 tiller 10,624 1 9 root, third
tiller-milk 7,481 1 3
Example 2
Constructs
[0047] The Os-TubA-3 promoter was isolated from the upstream
genomic region of the Os-TubA-3 gene for incorporation into an
expression cassette and subsequent characterization in transgenic
plants. Oligonucleotides OsTUBA136-1 and OsTUBA136-2 (provided as
SEQ ID NO: 6 and SEQ ID NO: 7, respectively) were designed to
amplify the 5' region (promoter and 5' UTR). The upstream promoter
region of the Os-TubA-3 gene was amplified starting immediately
upstream of the translation initiation codon and ending
approximately 1.2 kb immediately upstream of the start of
transcription initiation site (inferred by looking at the longest
5'UTR present for the Os-TubA-3 gene in the EST collection). The
promoter sequence is provided as SEQ ID NO: 2. The leader sequence
is provided as SEQ ID NO: 5. The first intron of the Os-TubA-3 gene
(which lies downstream of the start of translation) was amplified
with oligonucleotides OsTUBA136-3 and OsTubA136-4 (provided as SEQ
ID NO: 8 and SEQ ID NO: 9, respectively). The intron sequence is
provided as SEQ ID NO: 4. The intron was then removed from its
native context and placed within the 5'UTR immediately upstream of
the translation initiation codon to produce a chimeric Os-TubA-3
promoter. The chimeric promoter sequence is provided as SEQ ID NO:
1.
[0048] Constructs for in planta Os-TubA-3 promoter characterization
for expression of an operably linked transgene were then created.
The promoter was ligated into a plant expression construct such
that the promoter was operably linked to the transgene of
interest.
[0049] The Os-TubA-3 3' region (500-600 bp of sequence immediately
downstream of the translation termination codon which includes the
3' UTR plus adjacent downstream sequence) was also amplified using
oligonucleotides OsTUBA136-5 and OsTUBA136-6 (provided as SEQ ID
NO: 10 and SEQ ID NO: 11). The 3' region sequence is provided as
SEQ ID NO: 3. The 3' region was used in constructing the plant
transformation vector in order to provide further element diversity
over existing constructs and to capture any regulatory capacity
(transcriptional or at the level of mRNA stability) present within
the sequence. The Os-TubA-3 3' region was ligated onto the 3' end
of the transgene of interest. For GUS activity characterization the
plant transformation vector pMON77978 as shown in FIG. 1 contains
the GUS reporter gene as the transgene of interest. For glyphosate
tolerance characterization the plant transformation vector
pMON70453 as shown in FIG. 2 contains the CTP2/CP4 EPSPS gene as
the transgene of interest.
Example 3
Promoter Characterization in Transient Systems
[0050] The plant expression vector pMON77978 was used to transform
maize callus using particle bombardment for in planta promoter
characterization. The GUS activity was then analyzed
quantitatively. Results are shown in Table 2. The Os-TubA-3
promoter was shown to have a desirable level of expression in this
transient system.
TABLE-US-00002 TABLE 2 Quantitative Analysis of GUS activity in
maize callus. Construct GUS activity (pmoles/.mu.g protein/hour)
Os-TubA-3 (pMON77978) 17.51 .+-. 2.16 E35S (pMON77952) 33.53 .+-.
11.28 Blank Control 1.67 .+-. 0.502 promoterless GUS vector
(pMON77951)
Example 4
Promoter Characterization in Transgenic Plants
[0051] The plant expression vector pMON70453 was used to transform
corn using an Agrobacterium mediated transformation method. For
example, a disarmed Agrobacterium strain C58 harboring a binary DNA
construct of the present invention is used. The DNA construct is
transferred into Agrobacterium by a triparental mating method
(Ditta et al., Proc. Natl. Acad. Sci. 77:7347-7351, 1980). Liquid
cultures of Agrobacterium are initiated from glycerol stocks or
from a freshly streaked plate and grown overnight at 26.degree.
C.-28.degree. C. with shaking (approximately 150 rpm) to mid-log
growth phase in liquid LB medium, pH 7.0 containing the appropriate
antibiotics. The Agrobacterium cells are resuspended in the
inoculation medium (liquid CM4C) and the density is adjusted to
OD.sub.660 of 1. Freshly isolated Type II immature HiIIxLH198 and
HiII corn embryos are inoculated with Agrobacterium containing a
construct and co-cultured several days in the dark at 23.degree. C.
The embryos are then transferred to delay media and incubated at
28.degree. C. for several or more days. All subsequent cultures are
kept at this temperature. The embryos are transferred to a first
selection medium containing carbenicillin 500/0.5 mM glyphosate.
Two weeks later, surviving tissue are transferred to a second
selection medium containing carbenicillin 500/1.0 mM glyphosate.
Subculture surviving callus every 2 weeks until events can be
identified. This may take about 3 subcultures on 1.0 mM glyphosate.
Once events are identified, bulk up the tissue to regenerate. The
plantlets (events) are transferred to MSOD media in culture vessel
and kept for two weeks. The transformation efficiency is determined
by dividing the number of events produced by the number of embryos
inoculated. Then the plants with roots are transferred into soil.
Those skilled in the art of monocot transformation methods can
modify this method to provide substantially identical transgenic
monocot plants containing the DNA compositions of the present
invention, or use other methods, such as particle gun, that are
known to provide transgenic monocot plants.
[0052] Approximately 25 events per construct were generated. Events
were selected on glyphosate containing medium, transferred to soil,
and then moved to the greenhouse. In the greenhouse, plants were
sprayed with glyphosate (0.84 kg glyphosate acid
equivalentsha.sup.-1) using the Roundup Ultra formulation at
approximately V4 leaf stage. Plants that survived without injury
(<10% chlorosis and malformation) were kept and transferred to
large pots. At approximately V8 stage, a second glyphosate
application was made as before. This second spray was to evaluate
male reproductive tolerance. Events from the new constructs were
scored for male fertility upon maturation of the tassels. The Male
Fertility Rating (MFR) is scored on a range where MFR=1 is
completely sterility (for tassels lacking developed florets) and
MFR=5 is full pollen shed (for fully developed anthers with pollen
shed); MFR=4-5 is considered commercially viable. A combination of
Taqman and Southern analysis were used to evaluate the transgene
copy number in the events going to the greenhouse. Southern
analysis using the new elements also showed that these heterologous
sequences do not exhibit cross hybridization to endogenous maize
sequences--a significant quality for event characterization. These
early evaluations are part of a process to select cassettes
equivalent to P-Os.Act1/CP4. Important criteria for a successful
construct include good transformation efficiency (number of events
produced/# explants inoculated) and the ability to reproducibly
provide vegetatively and reproductively tolerant transformants
carrying a single copy of the transgene. A summary of the early
transformation and greenhouse evaluations is shown in Table 3.
Single copy events that passed greenhouse evaluations were advanced
to field evaluations.
TABLE-US-00003 TABLE 3 Early transformation and greenhouse
evaluations. Single copy and Single copy, vegetatively vegetatively
Transformation Number tolerant tolerant and Construct Frequency of
Events Single Copy in R0 MFR = 4-5 in R0 P-Os.Act1 5.1% 24 33% 21%
21 pMON30167 P-Os.TubA-3 5.2% 49 53% 37% 33 pMON70453
[0053] Field evaluations were done in Puerto Rico with F2
generation corn plants. Plants were treated with two applications
of 3.36 kg glyphosate acid equivalentsha.sup.-1 in Roundup
UltraMax.TM. formulation (4.times. above current field use rate).
One treatment was done at V4 stage and a second treatment was done
at V8 stage. The vegetative ratings for chlorosis (<10%
chlorosis) and malformation (<10% malformation) were taken 10
days after treatment. Male fertility ratings (MFR) were taken at
tassel maturity. For comparison, commercial event NK603 (pMON25496
containing P-Os.Act1/CP4 EPSPS::P-e35S/CP4 EPSPS) was evaluated.
Results are shown in Table 4.
TABLE-US-00004 TABLE 4 Field Evaluation. Events that Events that
Events that passed passed V8 malfor- passed V8 vegetative ratings
Construct Number of Events mation rating chlorosis rating and MFR =
4-5 P-Os.Act1 + E35S 1 1 1 1 NK603 P-Os.TubA-3 7 6 6 6
pMON70453
[0054] CP4 EPSPS protein accumulation (.mu.g/mg of total protein
shown as mean.+-.standard error) in various corn tissues was then
measured for single copy events in hemi-zygous F1 plants that
showed good field efficacy for glyphosate tolerance. For
comparison, commercial event NK603 has approximately 21 ppm or
approximately 1.4 .mu.g CP4 EPSPS/mg total protein in V4 leaf
lamina stage when grown under similar conditions. Results are shown
in Table 5.
TABLE-US-00005 TABLE 5 CP4 EPSPS accumulation in various corn
tissues. V4 leaf V9-10 leaf Number of lamina at V4 lamina at
Immature Construct single copy events stage V9-10 stage Tassel
(5-10 cm) Root tip (~1 cm) P-Os.TubA-3 10 0.041 .+-. 0.005 0.070
.+-. 0.006 0.144 .+-. 0.014 0.095 .+-. 0.016 pMON70453
[0055] Having illustrated and described the principles of the
present invention, it should be apparent to persons skilled in the
art that the invention can be modified in arrangement and detail
without departing from such principles. We claim all modifications
that are within the spirit and scope of the appended claims. All
publications and published patent documents cited in this
specification are incorporated herein by reference to the same
extent as if each individual publication or patent application is
specifically and individually indicated to be incorporated by
reference.
Sequence CWU 1
1
1112190DNAOryza sativa 1gcctcgagac aacaacatgc ttctcatcaa catggaggga
agagggaggg agaaagtgtc 60gcctggtcac ctccattgtc acactagcca ctggccagct
ctcccacacc accaatgcca 120ggggcgagct ttagcacagc caccgcttca
cctccaccac cgcactaccc tagcttcgcc 180caacagccac cgtcaacgcc
tcctctccgt caacataaga gagagagaga agaggagagt 240agccatgtgg
ggaggaggaa tagtacatgg ggcctaccgt ttggcaagtt attttgggtt
300gccaagttag gccaataagg ggagggattt ggccatccgg ttggaaaggt
tattggggta 360gtatcttttt actagaattg tcaaaaaaaa atagtttgag
agccatttgg agaggatgtt 420gcctgttaga ggtgctctta ggacatcaaa
ttccataaaa acatcagaaa aattctctcg 480atgaagattt ataaccacta
aaactgccct caattcgaag ggagttcaaa acaattaaaa 540tcatgttcga
attgagtttc aatttcactt taaccccttt gaaatctcaa tggtaaaaca
600tcaacccgtc aggtagcatg gttcttttta ttcctttcaa aaagagttaa
ttacaaacag 660aatcaaaact aacagttagg cccaaggccc atccgagcaa
acaatagatc atgggccagg 720cctgccacca ccctccccct cctggctccc
gctcttgaat ttcaaaatcc aaaaatatcg 780gcacgactgg ccgccgacgg
agcgggcgga aaatgacgga acaacccctc gaattctacc 840ccaactacgc
ccaccaaccc acacgccact gacaatccgg tcccaccctt gtgggcccac
900ctacaagcga gacgtcagtc gctcgcagca accagtgggc ccacctccca
gtgagcggcg 960ggtagatctg gactcttacc cacccacact aaacaaaacg
gcatgaatat tttgcactaa 1020aaccctcaga aaaattccga tattccaaac
cagtacagtt cctgaccgtt ggaggagcca 1080aagtggagcg gagtgtaaaa
ttgggaaact taatcgaggg ggttaaacgc aaaaacgccg 1140aggcgcctcc
cgctctatag aaaggggagg agtgggaggt ggaaacccta ccacaccgca
1200gagaaaggcg tcttcgtact cgcctctctc cgcgccctcc tccgccgccg
ctcgccgccg 1260ttcgtctccg ccgccaccgg ctagccatcc aggtaaaaca
aacaaaaacg gatctgatgc 1320ttccattcct ccgtttctcg tagtagcgcg
cttcgatctg tgggtggatc tgggtgatcc 1380tggggtgtgg ttcgttctgt
ttgatagatc tgtcggtgga tctggccttc tgtggttgtc 1440gatgtccgga
tctgcgtttt gatcagtggt agttcgtgga tctggcgaaa tgttttggat
1500ctggcagtga gacgctaaga atcgggaaat gatgcaatat taggggggtt
tcggatgggg 1560atccactgaa ttagtctgtc tccctgctga taatctgttc
ctttttggta gatctggtta 1620gtgtatgttt gtttcggata gatctgatca
atgcttgttt gttttttcaa attttctacc 1680taggttgtat aggaatggca
tgcggatctg gttggattgc catgatccgt gctgaaatgc 1740ccctttggtt
gatggatctt gatattttac tgctgttcac ctagatttgt actcccgttt
1800atacttaatt tgttgcttat tatgaataga tctgtaactt aggcacatgt
atggacggag 1860tatgtggatc tgtagtatgt acattgctgc gagctaagaa
ctatttcaga gcaagcacag 1920aaaaaaatat ttagacagat tgggcaacta
tttgatggtc tttggtatca tgctttgtag 1980tgctcgtttc tgcgtagtaa
tcttttgatc tgatctgaag ataggtgcta ttatattctt 2040aaaggtcatt
agaacgctat ctgaaaggct gtattatgtg gattggttca cctgtgactc
2100cctgttcgtc ttgtcttgat aaatcctgtg ataaaaaaaa ttcttaaggc
gtaatttgtt 2160gaaatcttgt tttgtcctat gcagcctgat 219021206DNAOryza
sativa 2gcctcgagac aacaacatgc ttctcatcaa catggaggga agagggaggg
agaaagtgtc 60gcctggtcac ctccattgtc acactagcca ctggccagct ctcccacacc
accaatgcca 120ggggcgagct ttagcacagc caccgcttca cctccaccac
cgcactaccc tagcttcgcc 180caacagccac cgtcaacgcc tcctctccgt
caacataaga gagagagaga agaggagagt 240agccatgtgg ggaggaggaa
tagtacatgg ggcctaccgt ttggcaagtt attttgggtt 300gccaagttag
gccaataagg ggagggattt ggccatccgg ttggaaaggt tattggggta
360gtatcttttt actagaattg tcaaaaaaaa atagtttgag agccatttgg
agaggatgtt 420gcctgttaga ggtgctctta ggacatcaaa ttccataaaa
acatcagaaa aattctctcg 480atgaagattt ataaccacta aaactgccct
caattcgaag ggagttcaaa acaattaaaa 540tcatgttcga attgagtttc
aatttcactt taaccccttt gaaatctcaa tggtaaaaca 600tcaacccgtc
aggtagcatg gttcttttta ttcctttcaa aaagagttaa ttacaaacag
660aatcaaaact aacagttagg cccaaggccc atccgagcaa acaatagatc
atgggccagg 720cctgccacca ccctccccct cctggctccc gctcttgaat
ttcaaaatcc aaaaatatcg 780gcacgactgg ccgccgacgg agcgggcgga
aaatgacgga acaacccctc gaattctacc 840ccaactacgc ccaccaaccc
acacgccact gacaatccgg tcccaccctt gtgggcccac 900ctacaagcga
gacgtcagtc gctcgcagca accagtgggc ccacctccca gtgagcggcg
960ggtagatctg gactcttacc cacccacact aaacaaaacg gcatgaatat
tttgcactaa 1020aaccctcaga aaaattccga tattccaaac cagtacagtt
cctgaccgtt ggaggagcca 1080aagtggagcg gagtgtaaaa ttgggaaact
taatcgaggg ggttaaacgc aaaaacgccg 1140aggcgcctcc cgctctatag
aaaggggagg agtgggaggt ggaaacccta ccacaccgca 1200gagaaa
12063582DNAOryza sativa 3cagggttctt gcctggtgcc ttggcaatgc
ttgattactg ctgctatcct atgatctgtc 60cgtgtgggct tctatctatc agtttgtgtg
tctggttttg aaaaacattt gcttttcgat 120tatgtagggt ttgcttgtag
ctttcgctgc tgtgacctgt gttgtttatg tgaaccttct 180ttgtggcatc
tttaatatcc aagttcgtgg tttgtcgtaa aacgaagcct ctacttcgta
240aagttgtgtc tatagcattg aaatcgtttt tttgctcgag aataattgtg
acctttagtt 300ggcgtgaaac tagttttgga tatctgattc tctggttcgc
aatcttgaga tcgtcgctgc 360ttaggtgagc taagtgatgt tcctaagtaa
atgctcctca ccagaatacg tagctgtgtg 420aaaagagaac gcgtgaatac
gtagctgtgt aaagattgtg tcccaagtaa acctcagtga 480tttttgtttg
gatttttaat ttagaaacat tcgactggga gcggctagag ccacacccaa
540gttcctaact atgataaagt tgctctgtaa cagaaaacac ca 5824892DNAOryza
sativa 4gtaaaacaaa caaaaacgga tctgatgctt ccattcctcc gtttctcgta
gtagcgcgct 60tcgatctgtg ggtggatctg ggtgatcctg gggtgtggtt cgttctgttt
gatagatctg 120tcggtggatc tggccttctg tggttgtcga tgtccggatc
tgcgttttga tcagtggtag 180ttcgtggatc tggcgaaatg ttttggatct
ggcagtgaga cgctaagaat cgggaaatga 240tgcaatatta ggggggtttc
ggatggggat ccactgaatt agtctgtctc cctgctgata 300atctgttcct
ttttggtaga tctggttagt gtatgtttgt ttcggataga tctgatcaat
360gcttgtttgt tttttcaaat tttctaccta ggttgtatag gaatggcatg
cggatctggt 420tggattgcca tgatccgtgc tgaaatgccc ctttggttga
tggatcttga tattttactg 480ctgttcacct agatttgtac tcccgtttat
acttaatttg ttgcttatta tgaatagatc 540tgtaacttag gcacatgtat
ggacggagta tgtggatctg tagtatgtac attgctgcga 600gctaagaact
atttcagagc aagcacagaa aaaaatattt agacagattg ggcaactatt
660tgatggtctt tggtatcatg ctttgtagtg ctcgtttctg cgtagtaatc
ttttgatctg 720atctgaagat aggtgctatt atattcttaa aggtcattag
aacgctatct gaaaggctgt 780attatgtgga ttggttcacc tgtgactccc
tgttcgtctt gtcttgataa atcctgtgat 840aaaaaaaatt cttaaggcgt
aatttgttga aatcttgttt tgtcctatgc ag 892586DNAOryza sativa
5ggcgtcttcg tactcgcctc tctccgcgcc ctcctccgcc gccgctcgcc gccgttcgtc
60tccgccgcca ccggctagcc atccag 86632DNAArtificial SequenceSynthetic
oligonucleotide primer 6gacaagcttg cctcgagaca acaacatgct tc
32738DNAArtificial SequenceSynthetic oligonucleotide primer
7attccatggc ggctagccgg tggcggcgga gacgaacg 38839DNAArtificial
SequenceSynthetic oligonucleotide primer 8cgagctagcc atccaggtaa
aacaaacaaa aacggatct 39934DNAArtificial SequenceSynthetic
oligonucleotide primer 9attccatgga tcaggctgca taggacaaaa caag
341033DNAArtificial SequenceSynthetic oligonucleotide primer
10tagagagctc cagggttctt gcctggtgcc ttg 331133DNAArtificial
SequenceSynthetic oligonucleotide primer 11acttctagat ggtgttttct
gttacagagc aac 33
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