U.S. patent application number 12/472260 was filed with the patent office on 2009-11-12 for generation of plants with altered oil content.
This patent application is currently assigned to AGRINOMICS LLC. Invention is credited to JOHN DAVIES, HEIN TSOENG NG, SANDRA PETERS.
Application Number | 20090282581 12/472260 |
Document ID | / |
Family ID | 35432020 |
Filed Date | 2009-11-12 |
United States Patent
Application |
20090282581 |
Kind Code |
A1 |
DAVIES; JOHN ; et
al. |
November 12, 2009 |
GENERATION OF PLANTS WITH ALTERED OIL CONTENT
Abstract
The present disclosure is directed to methods of generating
plants with an altered oil content phenotype.
Inventors: |
DAVIES; JOHN; (PORTLAND,
OR) ; PETERS; SANDRA; (PORTLAND, OR) ; NG;
HEIN TSOENG; (BEAVERTON, OR) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET, SUITE 1600
PORTLAND
OR
97204
US
|
Assignee: |
AGRINOMICS LLC
|
Family ID: |
35432020 |
Appl. No.: |
12/472260 |
Filed: |
May 26, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11596833 |
Nov 17, 2006 |
7554009 |
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PCT/US2005/018919 |
May 26, 2005 |
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12472260 |
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60575202 |
May 28, 2004 |
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Current U.S.
Class: |
800/281 |
Current CPC
Class: |
C12N 15/8247 20130101;
C07K 14/415 20130101; A23K 10/30 20160501 |
Class at
Publication: |
800/281 |
International
Class: |
A01H 3/00 20060101
A01H003/00 |
Claims
1. A method of generating a plant having a high oil phenotype
comprising identifying a plant that has an allele in its HIO1002
gene that results in increased oil content compared to plants
lacking the allele and generating progeny of said identified plant,
wherein the generated progeny inherit the allele and have the high
oil phenotype.
2. The method of claim 1, wherein identifying a plant comprises
employing candidate gene/quantitative trait locus (QTL)
methodology.
3. The method of claim 1, wherein identifying a plant comprises
employing targeting induced local lesion in genome (TILLING)
methodology.
4. The method of claim 3, wherein TILLING methodology comprises
inducing lesions in a plant part.
5. The method of claim 4, wherein the plant part is a seed.
6. The method of claim 5, wherein inducing lesions in a plant part
comprises ethyl methyl sulfonate (EMS) treatment of the seed.
7. The method of claim 5, further comprising planting the seed in
conditions that allow the seed to grow into a plant.
8. The method of claim 7, further comprising isolating DNA from the
resulting plant and identifying a plant that has an allele in its
HIO1002 gene by polymerase chain reaction (PCR).
9. The method of claim 8, wherein amplification of a nucleic acid
sequence with 95% sequence identity to SEQ ID NOs: 1, 3 or 5
indicates a plant has an allele in its HIO1002 gene that results in
increased oil content.
10. The method of claim 8, wherein amplification of a nucleic acid
sequence with the sequence as set forth in SEQ ID NOs: 1, 3 or 5
indicates a plant has an allele in its HIO1002 gene that results in
increased oil content.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional of U.S. National Stage application Ser.
No. 11/596,833, filed Nov. 17, 2006, which is the .sctn.371 U.S.
National Stage of International Application No. PCT/US2005/018919,
filed May 26, 2005, which was published in English under PCT
Article 21(2), which in turn claims the benefit of U.S. Provisional
Application No. 60/575,202, filed May 28, 2004, the contents of
said applications are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] The ability to manipulate the composition of crop seeds,
particularly the content and composition of seed oils, has
important applications in the agricultural industries, relating
both to processed food oils and to oils for animal feeding. Seeds
of agricultural crops contain a variety of valuable constituents,
including oil, protein and starch. Industrial processing can
separate some or all of these constituents for individual sale in
specific applications. For instance, nearly 60% of the U.S. soybean
crop is crushed by the soy processing industry. Soy processing
yields purified oil, which is sold at high value, while the
remainder is sold principally for lower value livestock feed (U.S.
Soybean Board, 2001 Soy Stats). Canola seed is crushed to produce
oil and the co-product canola meal (Canola Council of Canada).
Nearly 20% of the 1999/2000 U.S. corn crop was industrially
refined, primarily for production of starch, ethanol and oil (Corn
Refiners Association). Thus, it is often desirable to maximize oil
content of seeds. For instance, for processed oilseeds such as soy
and canola, increasing the absolute oil content of the seed will
increase the value of such grains. For processed corn it may be
desired to either increase or decrease oil content, depending on
utilization of other major constituents. Decreasing oil may improve
the quality of isolated starch by reducing undesired flavors
associated with oil oxidation. Alternatively, in ethanol
production, where flavor is unimportant, increasing oil content may
increase overall value. In many fed grains, such as corn and wheat,
it is desirable to increase seed oil content, because oil has
higher energy content than other seed constituents such as
carbohydrate. Oilseed processing, like most grain processing
businesses, is a capital-intensive business; thus small shifts in
the distribution of products from the low valued components to the
high value oil component can have substantial economic impacts for
grain processors.
[0003] Biotechnological manipulation of oils can provide
compositional alteration and improvement of oil yield.
Compositional alterations include high oleic soybean and corn oil
(U.S. Pat. Nos. 6,229,033 and 6,248,939), and laurate-containing
seeds (U.S. Pat. No. 5,639,790), among others. Work in
compositional alteration has predominantly focused on processed
oilseeds but has been readily extendable to non-oilseed crops,
including corn. While there is considerable interest in increasing
oil content, the only currently practiced biotechnology in this
area is High-Oil Corn (HOC) technology (DuPont, U.S. Pat. No.
5,704,160). HOC employs high oil pollinators developed by classical
selection breeding along with elite (male-sterile) hybrid females
in a production system referred to as TopCross. The TopCross High
Oil system raises harvested grain oil content in maize from about
3.5% to about 7%, improving the energy content of the grain.
[0004] While it has been fruitful, the HOC production system has
inherent limitations. First, the system of having a low percentage
of pollinators responsible for an entire field's seed set contains
inherent risks, particularly in drought years. Second, oil contents
in current HOC fields have plateaued at about 9% oil. Finally,
high-oil corn is not primarily a biochemical change, but rather an
anatomical mutant (increased embryo size) that has the indirect
result of increasing oil content. For these reasons, an alternative
high oil strategy, particularly one that derives from an altered
biochemical output, would be especially valuable.
[0005] The most obvious target crops for the processed oil market
are soy and rapeseed, and a large body of commercial work (e.g.,
U.S. Pat. No. 5,952,544; PCT application WO9411516) demonstrates
that Arabidopsis is an excellent model for oil metabolism in these
crops. Biochemical screens of seed oil composition have identified
Arabidopsis genes for many critical biosynthetic enzymes and have
led to identification of agronomically important gene orthologs.
For instance, screens using chemically mutagenized populations have
identified lipid mutants whose seeds display altered fatty acid
composition (Lemieux et al., 1990; James and Dooner, 1990). T-DNA
mutagenesis screens (Feldmann et al., 1989) that detected altered
fatty acid composition identified the omega 3 desaturase (FAD3) and
delta-12 desaturase (FAD2) genes (U.S. Pat. No. 5,952,544; Yadav et
al., 1993; Okuley et al., 1994). A screen which focused on oil
content rather than oil quality, analyzed chemically-induced
mutants for wrinkled seeds or altered seed density, from which
altered seed oil content was inferred (Focks and Benning, 1998).
Another screen, designed to identify enzymes involved in production
of very long chain fatty acids, identified a mutation in the gene
encoding a diacylglycerol acyltransferase (DGAT) as being
responsible for reduced triacyl glycerol accumulation in seeds
(Katavic V et al., 1995). It was further shown that seed-specific
over-expression of the DGAT cDNA was associated with increased seed
oil content (Jako et al., 2001).
[0006] Activation tagging in plants refers to a method of
generating random mutations by insertion of a heterologous nucleic
acid construct comprising regulatory sequences (e.g., an enhancer)
into a plant genome. The regulatory sequences can act to enhance
transcription of one or more native plant genes; accordingly,
activation tagging is a fruitful method for generating
gain-of-function, generally dominant mutants (see, e.g., Hayashi et
al., 1992; Weigel D et al. 2000). The inserted construct provides a
molecular tag for rapid identification of the native plant whose
mis-expression causes the mutant phenotype. Activation tagging may
also cause loss-of-function phenotypes. The insertion may result in
disruption of a native plant gene, in which case the phenotype is
generally recessive.
[0007] Activation tagging has been used in various species,
including tobacco and Arabidopsis, to identify many different kinds
of mutant phenotypes and the genes associated with these phenotypes
(Wilson et al., 1996, Schaffer et al., 1998, Fridborg et al., 1999;
Kardailsky et al., 1999; Christensen S et al., 1998).
SUMMARY OF THE INVENTION
[0008] The invention provides a transgenic plant having a high oil
phenotype. The transgenic plant comprises a transformation vector
comprising a nucleotide sequence that encodes or is complementary
to a sequence that encodes a HIO1002 polypeptide. In preferred
embodiments, the transgenic plant is selected from the group
consisting of rapeseed, soy, corn, sunflower, cotton, cocoa,
safflower, oil palm, coconut palm, flax, castor and peanut. The
invention further provides a method of producing oil comprising
growing the transgenic plant and recovering oil from said
plant.
[0009] The invention also provides a transgenic plant cell having a
high oil phenotype. The transgenic plant cell comprises a
transformation vector comprising a nucleotide sequence that encodes
or is complementary to a sequence that encodes a High Oil
(hereinafter "HIO1002") polypeptide. In preferred embodiments, the
transgenic plant cell is selected from the group consisting of
rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm,
coconut palm, flax, castor and peanut. In other embodiments, the
plant cell is a seed, pollen, propagule, or embryo cell. The
invention further provides feed, meal, grain, food, or seed
comprising a nucleic acid sequence that encodes a HIO1002
polypeptide. The invention also provides feed, meal, grain, food,
or seed comprising the HIO1002 polypeptide, or an ortholog
thereof.
[0010] The transgenic plant of the invention is produced by a
method that comprises introducing into progenitor cells of the
plant a plant transformation vector comprising a nucleotide
sequence that encodes or is complementary to a sequence that
encodes a HIO1002 polypeptide, and growing the transformed
progenitor cells to produce a transgenic plant, wherein the HIO1002
polynucleotide sequence is expressed causing the high oil
phenotype. The invention further provides plant cells obtained from
said transgenic plant.
[0011] The present invention also provides a container of over
about 10,000, more preferably about 20,000, and even more
preferably about 40,000 seeds where over about 10%, more preferably
about 25%, more preferably about 50%, and even more preferably
about 75% or more preferably about 90% of the seeds are seeds
derived from a plant of the present invention.
[0012] The present invention also provides a container of over
about 10 kg, more preferably about 25 kg, and even more preferably
about 50 kg seeds where over about 10%, more preferably about 25%,
more preferably about 50%, and even more preferably about 75% or
more preferably about 90% of the seeds are seeds derived from a
plant of the present invention.
[0013] Any of the plants or parts thereof of the present invention
may be processed to produce a feed, meal, or oil preparation. A
particularly preferred plant part for this purpose is a seed. In a
preferred embodiment the feed, meal, or oil preparation is designed
for ruminant animals. Methods to produce feed, meal, and oil
preparations are known in the art. See, for example, U.S. Pat. Nos.
4,957,748; 5,100,679; 5,219,596; 5,936,069; 6,005,076; 6,146,669;
and 6,156,227. The meal of the present invention may be blended
with other meals. In a preferred embodiment, the meal produced from
plants of the present invention or generated by a method of the
present invention constitutes greater than about 0.5%, about 1%,
about 5%, about 10%, about 25%, about 50%, about 75%, or about 90%
by volume or weight of the meal component of any product. In
another embodiment, the meal preparation may be blended and can
constitute greater than about 10%, about 25%, about 35%, about 50%,
or about 75% of the blend by volume.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0014] Unless otherwise indicated, all technical and scientific
terms used herein have the same meaning as they would to one
skilled in the art of the present invention. Practitioners are
particularly directed to Sambrook et al., 1989, and Ausubel F M et
al., 1993, for definitions and terms of the art. It is to be
understood that this invention is not limited to the particular
methodology, protocols, and reagents described, as these may
vary.
[0015] As used herein, the term "vector" refers to a nucleic acid
construct designed for transfer between different host cells. An
"expression vector" refers to a vector that has the ability to
incorporate and express heterologous DNA fragments in a foreign
cell. Many prokaryotic and eukaryotic expression vectors are
commercially available. Selection of appropriate expression vectors
is within the knowledge of those having skill in the art.
[0016] A "heterologous" nucleic acid construct or sequence has a
portion of the sequence that is not native to the plant cell in
which it is expressed. Heterologous, with respect to a control
sequence refers to a control sequence (i.e. promoter or enhancer)
that does not function in nature to regulate the same gene the
expression of which it is currently regulating. Generally,
heterologous nucleic acid sequences are not endogenous to the cell
or part of the genome in which they are present, and have been
added to the cell, by infection, transfection, microinjection,
electroporation, or the like. A "heterologous" nucleic acid
construct may contain a control sequence/DNA coding sequence
combination that is the same as, or different from a control
sequence/DNA coding sequence combination found in the native
plant.
[0017] As used herein, the term "gene" means the segment of DNA
involved in producing a polypeptide chain, which may or may not
include regions preceding and following the coding region, e.g. 5'
untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer"
sequences, as well as intervening sequences (introns) between
individual coding segments (exons) and non-transcribed regulatory
sequence.
[0018] As used herein, "recombinant" includes reference to a cell
or vector, that has been modified by the introduction of a
heterologous nucleic acid sequence or that the cell is derived from
a cell so modified. Thus, for example, recombinant cells express
genes that are not found in identical form within the native
(non-recombinant) form of the cell or express native genes that are
otherwise abnormally expressed, under expressed or not expressed at
all as a result of deliberate human intervention.
[0019] As used herein, the term "gene expression" refers to the
process by which a polypeptide is produced based on the nucleic
acid sequence of a gene. The process includes both transcription
and translation; accordingly, "expression" may refer to either a
polynucleotide or polypeptide sequence, or both. Sometimes,
expression of a polynucleotide sequence will not lead to protein
translation. "Over-expression" refers to increased expression of a
polynucleotide and/or polypeptide sequence relative to its
expression in a wild-type (or other reference [e.g.,
non-transgenic]) plant and may relate to a naturally-occurring or
non-naturally occurring sequence.
[0020] "Ectopic expression" refers to expression at a time, place,
and/or increased level that does not naturally occur in the
non-altered or wild-type plant. "Under-expression" refers to
decreased expression of a polynucleotide and/or polypeptide
sequence, generally of an endogenous gene, relative to its
expression in a wild-type plant. The terms "mis-expression" and
"altered expression" encompass over-expression, under-expression,
and ectopic expression.
[0021] The term "introduced" in the context of inserting a nucleic
acid sequence into a cell, means "transfection", or
"transformation" or "transduction" and includes reference to the
incorporation of a nucleic acid sequence into a eukaryotic or
prokaryotic cell where the nucleic acid sequence may be
incorporated into the genome of the cell (for example, chromosome,
plasmid, plastid, or mitochondrial DNA), converted into an
autonomous replicon, or transiently expressed (for example,
transfected mRNA).
[0022] As used herein, a "plant cell" refers to any cell derived
from a plant, including cells from undifferentiated tissue (e.g.,
callus) as well as plant seeds, pollen, propagules and embryos.
[0023] As used herein, the terms "native" and "wild-type" relative
to a given plant trait or phenotype refers to the form in which
that trait or phenotype is found in the same variety of plant in
nature.
[0024] As used herein, the term "modified" regarding a plant trait,
refers to a change in the phenotype of a transgenic plant relative
to the similar non-transgenic plant. An "interesting phenotype
(trait)" with reference to a transgenic plant refers to an
observable or measurable phenotype demonstrated by a T1 and/or
subsequent generation plant, which is not displayed by the
corresponding non-transgenic (i.e., a genotypically similar plant
that has been raised or assayed under similar conditions). An
interesting phenotype may represent an improvement in the plant or
may provide a means to produce improvements in other plants. An
"improvement" is a feature that may enhance the utility of a plant
species or variety by providing the plant with a unique and/or
novel quality. An "altered oil content phenotype" refers to
measurable phenotype of a genetically modified plant, where the
plant displays a statistically significant increase or decrease in
overall oil content (i.e., the percentage of seed mass that is
oil), as compared to the similar, but non-modified plant. A high
oil phenotype refers to an increase in overall oil content.
As used herein, a "mutant" polynucleotide sequence or gene differs
from the corresponding wild type polynucleotide sequence or gene
either in terms of sequence or expression, where the difference
contributes to a modified plant phenotype or trait. Relative to a
plant or plant line, the term "mutant" refers to a plant or plant
line which has a modified plant phenotype or trait, where the
modified phenotype or trait is associated with the modified
expression of a wild type polynucleotide sequence or gene.
[0025] As used herein, the term "T1" refers to the generation of
plants from the seed of T0 plants. The T1 generation is the first
set of transformed plants that can be selected by application of a
selection agent, e.g., an antibiotic or herbicide, for which the
transgenic plant contains the corresponding resistance gene. The
term "T2" refers to the generation of plants by self-fertilization
of the flowers of T1 plants, previously selected as being
transgenic. T3 plants are generated from T2 plants, etc. As used
herein, the "direct progeny" of a given plant derives from the seed
(or, sometimes, other tissue) of that plant and is in the
immediately subsequent generation; for instance, for a given
lineage, a T2 plant is the direct progeny of a T1 plant. The
"indirect progeny" of a given plant derives from the seed (or other
tissue) of the direct progeny of that plant, or from the seed (or
other tissue) of subsequent generations in that lineage; for
instance, a T3 plant is the indirect progeny of a T1 plant.
[0026] As used herein, the term "plant part" includes any plant
organ or tissue, including, without limitation, seeds, embryos,
meristematic regions, callus tissue, leaves, roots, shoots,
gametophytes, sporophytes, pollen, and microspores. Plant cells can
be obtained from any plant organ or tissue and cultures prepared
therefrom. The class of plants which can be used in the methods of
the present invention is generally as broad as the class of higher
plants amenable to transformation techniques, including both
monocotyledenous and dicotyledenous plants.
[0027] As used herein, "transgenic plant" includes a plant that
comprises within its genome a heterologous polynucleotide. The
heterologous polynucleotide can be either stably integrated into
the genome, or can be extra-chromosomal. Preferably, the
polynucleotide of the present invention is stably integrated into
the genome such that the polynucleotide is passed on to successive
generations. A plant cell, tissue, organ, or plant into which the
heterologous polynucleotides have been introduced is considered
"transformed", "transfected", or "transgenic". Direct and indirect
progeny of transformed plants or plant cells that also contain the
heterologous polynucleotide are also considered transgenic.
[0028] Various methods for the introduction of a desired
polynucleotide sequence encoding the desired protein into plant
cells are available and known to those of skill in the art and
include, but are not limited to: (1) physical methods such as
microinjection, electroporation, and microprojectile mediated
delivery (biolistics or gene gun technology); (2) virus mediated
delivery methods; and (3) Agrobacterium-mediated transformation
methods.
[0029] The most commonly used methods for transformation of plant
cells are the Agrobacterium-mediated DNA transfer process and the
biolistics or microprojectile bombardment mediated process (i.e.,
the gene gun). Typically, nuclear transformation is desired but
where it is desirable to specifically transform plastids, such as
chloroplasts or amyloplasts, plant plastids may be transformed
utilizing a microprojectile-mediated delivery of the desired
polynucleotide.
[0030] Agrobacterium-mediated transformation is achieved through
the use of a genetically engineered soil bacterium belonging to the
genus Agrobacterium. A number of wild-type and disarmed strains of
Agrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti
or Ri plasmids can be used for gene transfer into plants. Gene
transfer is done via the transfer of a specific DNA known as
"T-DNA" that can be genetically engineered to carry any desired
piece of DNA into many plant species.
[0031] Agrobacterium-mediated genetic transformation of plants
involves several steps. The first step, in which the virulent
Agrobacterium and plant cells are first brought into contact with
each other, is generally called "inoculation". Following the
inoculation, the Agrobacterium and plant cells/tissues are
permitted to be grown together for a period of several hours to
several days or more under conditions suitable for growth and T-DNA
transfer. This step is termed "co-culture". Following co-culture
and T-DNA delivery, the plant cells are treated with bactericidal
or bacteriostatic agents to kill the Agrobacterium remaining in
contact with the explant and/or in the vessel containing the
explant. If this is done in the absence of any selective agents to
promote preferential growth of transgenic versus non-transgenic
plant cells, then this is typically referred to as the "delay"
step. If done in the presence of selective pressure favoring
transgenic plant cells, then it is referred to as a "selection"
step. When a "delay" is used, it is typically followed by one or
more "selection" steps.
[0032] With respect to microprojectile bombardment (U.S. Pat. No.
5,550,318 (Adams et al.); U.S. Pat. No. 5,538,880 (Lundquist et.
al.), U.S. Pat. No. 5,610,042 (Chang et al.); and PCT Publication
WO 95/06128 (Adams et al.); each of which is specifically
incorporated herein by reference in its entirety), particles are
coated with nucleic acids and delivered into cells by a propelling
force. Exemplary particles include those comprised of tungsten,
platinum, and preferably, gold.
[0033] An illustrative embodiment of a method for delivering DNA
into plant cells by acceleration is the Biolistics Particle
Delivery System (BioRad, Hercules, Calif.), which can be used to
propel particles coated with DNA or cells through a screen, such as
a stainless steel or Nytex screen, onto a filter surface covered
with monocot plant cells cultured in suspension.
[0034] Microprojectile bombardment techniques are widely
applicable, and may be used to transform virtually any plant
species. Examples of species that have been transformed by
microprojectile bombardment include monocot species such as maize
(International Publication No. WO 95/06128 (Adams et al.)), barley,
wheat (U.S. Pat. No. 5,563,055 (Townsend et al.) incorporated
herein by reference in its entirety), rice, oat, rye, sugarcane,
and sorghum; as well as a number of dicots including tobacco,
soybean (U.S. Pat. No. 5,322,783 (Tomes et al.), incorporated
herein by reference in its entirety), sunflower, peanut, cotton,
tomato, and legumes in general (U.S. Pat. No. 5,563,055 (Townsend
et al.) incorporated herein by reference in its entirety).
[0035] To select or score for transformed plant cells regardless of
transformation methodology, the DNA introduced into the cell
contains a gene that functions in a regenerable plant tissue to
produce a compound that confers upon the plant tissue resistance to
an otherwise toxic compound. Genes of interest for use as a
selectable, screenable, or scorable marker would include but are
not limited to GUS, green fluorescent protein (GFP), luciferase
(LUX), antibiotic or herbicide tolerance genes. Examples of
antibiotic resistance genes include the penicillins, kanamycin (and
neomycin, G418, bleomycin); methotrexate (and trimethoprim);
chloramphenicol; kanamycin and tetracycline. 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 (Barry, et
al.), U.S. Pat. No. 5,633,435 (Barry, et al.), and U.S. Pat. No.
6,040,497 (Spencer, et al.) and aroA described in U.S. Pat. No.
5,094,945 (Comai) for glyphosate tolerance; a polynucleotide
molecule encoding bromoxynil nitrilase (Bxn) described in U.S. Pat.
No. 4,810,648 (Duerrschnabel, et al.) for Bromoxynil tolerance; a
polynucleotide molecule encoding phytoene desaturase (crtl)
described in Misawa et al., (1993) Plant J. 4:833-840 and Misawa et
al., (1994) Plant J. 6:481-489 for norflurazon tolerance; a
polynucleotide molecule encoding acetohydroxyacid synthase (AHAS,
aka ALS) described in Sathasiivan et al. (1990) Nucl. Acids Res.
18:2188-2193 for tolerance to sulfonylurea herbicides; and the bar
gene described in DeBlock, et al. (1987) EMBO J. 6:2513-2519 for
glufosinate and bialaphos tolerance.
[0036] The regeneration, development, and cultivation of plants
from various transformed explants are well documented in the art.
This regeneration and growth process typically includes the steps
of selecting transformed cells and culturing those individualized
cells through the usual stages of embryonic development through the
rooted plantlet stage. Transgenic embryos and seeds are similarly
regenerated. The resulting transgenic rooted shoots are thereafter
planted in an appropriate plant growth medium such as soil. Cells
that survive the exposure to the selective agent, or cells that
have been scored positive in a screening assay, may be cultured in
media that supports regeneration of plants. Developing plantlets
are transferred to soil less plant growth mix, and hardened off,
prior to transfer to a greenhouse or growth chamber for
maturation.
[0037] The present invention can be used with any transformable
cell or tissue. By transformable as used herein is meant a cell or
tissue that is capable of further propagation to give rise to a
plant. Those of skill in the art recognize that a number of plant
cells or tissues are transformable in which after insertion of
exogenous DNA and appropriate culture conditions the plant cells or
tissues can form into a differentiated plant. Tissue suitable for
these purposes can include but is not limited to immature embryos,
scutellar tissue, suspension cell cultures, immature inflorescence,
shoot meristem, nodal explants, callus tissue, hypocotyl tissue,
cotyledons, roots, and leaves.
[0038] Any suitable plant culture medium can be used. Examples of
suitable media would include but are not limited to MS-based media
(Murashige and Skoog, Physiol. Plant, 15:473-497, 1962) or N6-based
media (Chu et al., Scientia Sinica 18:659, 1975) supplemented with
additional plant growth regulators including but not limited to
auxins, cytokinins, ABA, and gibberellins. Those of skill in the
art are familiar with the variety of tissue culture media, which
when supplemented appropriately, support plant tissue growth and
development and are suitable for plant transformation and
regeneration. These tissue culture media can either be purchased as
a commercial preparation, or custom prepared and modified. Those of
skill in the art are aware that media and media supplements such as
nutrients and growth regulators for use in transformation and
regeneration and other culture conditions such as light intensity
during incubation, pH, and incubation temperatures that can be
optimized for the particular variety of interest.
[0039] One of ordinary skill will appreciate that, after an
expression cassette is stably incorporated in transgenic plants and
confirmed to be operable, it can be introduced into other plants by
sexual crossing. Any of a number of standard breeding techniques
can be used, depending upon the species to be crossed.
Identification of Plants with an Altered Oil Content Phenotype
[0040] We used an Arabidopsis activation tagging screen to identify
the association between the gene we have identified and designated
"HIO1002," (At1g73650, GI#30698954) encoding a protein
(GI#18410409), and an altered oil content phenotype (specifically,
a high oil phenotype). Splice variants and their corresponding
GenBank entries are noted below. Briefly, and as further described
in the Examples, a large number of Arabidopsis plants were mutated
with the pSKI015 vector, which comprises a T-DNA from the Ti
plasmid of Agrobacterium tumifaciens, a viral enhancer element, and
a selectable marker gene (Weigel et al., 2000). When the T-DNA
inserts into the genome of transformed plants, the enhancer element
can cause up-regulation genes in the vicinity, generally within
about 10 kilobase (kb) of the insertion. T1 plants were exposed to
the selective agent in order to specifically recover transformed
plants that expressed the selectable marker and therefore harbored
T-DNA insertions. Samples of approximately 15-20 T2 seeds were
collected from transformed T1 plants, and lipids were extracted
from whole seeds. Gas chromatography (GC) analysis was performed to
determine fatty acid content and composition of seed samples.
[0041] An Arabidopsis line that showed a high-oil phenotype was
identified. The association of the HIO1002 gene with the high oil
phenotype was discovered by analysis of the genomic DNA sequence
flanking the T-DNA insertion in the identified line. Accordingly,
HIO1002 genes and/or polypeptides may be employed in the
development of genetically modified plants having a modified oil
content phenotype ("a HIO1002 phenotype"). HIO1002 genes may be
used in the generation of oilseed crops that provide improved oil
yield from oilseed processing and in the generation of feed grain
crops that provide increased energy for animal feeding. HIO1002
genes may further be used to increase the oil content of specialty
oil crops, in order to augment yield of desired unusual fatty
acids. Transgenic plants that have been genetically modified to
express HIO1002 can be used in the production of oil, wherein the
transgenic plants are grown, and oil is obtained from plant parts
(e.g. seed) using standard methods.
HIO1002 Nucleic Acids and Polypeptides
[0042] Arabidopsis HIO1002 nucleic acid sequence is provided in SEQ
ID NO:1 and in Genbank entry GI#30698954. The corresponding protein
sequence is provided in SEQ ID NO:2 and in GI#18410409. Two
putative splice variants were also identified which encode proteins
that differ from SEQ ID NO:2 at the COOH terminus: GI#30698952 (SEQ
ID NO:3) with a corresponding protein sequence as in GI#30698953
(SEQ ID NO:4) that is 100% identical with SEQ ID NO:2 up to E288,
and ending with LG at amino acid positions 289 and 290,
respectively; and GI#42572098 (SEQ ID NO:5) with a corresponding
protein sequence as in GI#42572099 (SEQ ID NO:6) that is 100%
identical with SEQ ID NO:2 up to E288, and ending with
GRLQNSKEKEVKDD at amino acids 289-302, respectively. Nucleic acids
and/or proteins that are orthologs or paralogs of Arabidopsis
HIO1002, are described in Example 3 below.
[0043] As used herein, the term "HIO1002 polypeptide" refers to a
full-length HIO1002 protein or a fragment, derivative (variant), or
ortholog thereof that is "functionally active," meaning that the
protein fragment, derivative, or ortholog exhibits one or more or
the functional activities associated with the polypeptide of SEQ ID
NO:2, 4, or 6. In one preferred embodiment, a functionally active
HIO1002 polypeptide causes an altered oil content phenotype when
mis-expressed in a plant. In a further preferred embodiment,
mis-expression of the HIO1002 polypeptide causes a high oil
phenotype in a plant. In another embodiment, a functionally active
HIO1002 polypeptide is capable of rescuing defective (including
deficient) endogenous HIO1002 activity when expressed in a plant or
in plant cells; the rescuing polypeptide may be from the same or
from a different species as that with defective activity. In
another embodiment, a functionally active fragment of a full length
HIO1002 polypeptide (i.e., a native polypeptide having the sequence
of SEQ ID NO:2, 4, or 6, or a naturally occurring ortholog thereof)
retains one of more of the biological properties associated with
the full-length HIO1002 polypeptide, such as signaling activity,
binding activity, catalytic activity, or cellular or extra-cellular
localizing activity. A HIO1002 fragment preferably comprises a
HIO1002 domain, such as a C- or N-terminal or catalytic domain,
among others, and preferably comprises at least 10, preferably at
least 20, more preferably at least 25, and most preferably at least
50 contiguous amino acids of a HIO1002 protein. Functional domains
can be identified using the PFAM program (Bateman A et al., 1999
Nucleic Acids Res 27:260-262). A preferred HIO1002 fragment
comprises of one or more transmembrane domains.
[0044] Functionally active variants of full-length HIO1002
polypeptides or fragments thereof include polypeptides with amino
acid insertions, deletions, or substitutions that retain one of
more of the biological properties associated with the full-length
HIO1002 polypeptide. In some cases, variants are generated that
change the post-translational processing of a HIO1002 polypeptide.
For instance, variants may have altered protein transport or
protein localization characteristics or altered protein half-life
compared to the native polypeptide.
[0045] As used herein, the term "HI01002 nucleic acid" encompasses
nucleic acids with the sequence provided in or complementary to the
sequence provided in SEQ ID NO:1, 3, or 5, as well as functionally
active fragments, derivatives, or orthologs thereof. A HIO1002
nucleic acid of this invention may be DNA, derived from genomic DNA
or cDNA, or RNA.
[0046] In one embodiment, a functionally active HIO1002 nucleic
acid encodes or is complementary to a nucleic acid that encodes a
functionally active HIO1002 polypeptide. Included within this
definition is genomic DNA that serves as a template for a primary
RNA transcript (i.e., an mRNA precursor) that requires processing,
such as splicing, before encoding the functionally active HIO1002
polypeptide. A HIO1002 nucleic acid can include other non-coding
sequences, which may or may not be transcribed; such sequences
include 5' and 3' UTRs, polyadenylation signals and regulatory
sequences that control gene expression, among others, as are known
in the art. Some polypeptides require processing events, such as
proteolytic cleavage, covalent modification, etc., in order to
become fully active. Accordingly, functionally active nucleic acids
may encode the mature or the pre-processed HIO1002 polypeptide, or
an intermediate form. A HIO1002 polynucleotide can also include
heterologous coding sequences, for example, sequences that encode a
marker included to facilitate the purification of the fused
polypeptide, or a transformation marker.
[0047] In another embodiment, a functionally active HIO1002 nucleic
acid is capable of being used in the generation of loss-of-function
HIO1002 phenotypes, for instance, via antisense suppression,
co-suppression, etc.
[0048] In one preferred embodiment, a HIO1002 nucleic acid used in
the methods of this invention comprises a nucleic acid sequence
that encodes or is complementary to a sequence that encodes a
HIO1002 polypeptide having at least 50%, 60%, 70%, 75%, 80%, 85%,
90%, 95% or more sequence identity to the polypeptide sequence
presented in SEQ ID NO:2, 4 or 6.
[0049] In another embodiment a HIO1002 polypeptide of the invention
comprises a polypeptide sequence with at least 50% or 60% identity
to the HIO1002 polypeptide sequence of SEQ ID NO:2, 4 or 6, and may
have at least 70%, 80%, 85%, 90% or 95% or more sequence identity
to the HIO1002 polypeptide sequence of SEQ ID NO:2, 4, or 6, such
as one or more transmembrane domains. In another embodiment, a
HIO1002 polypeptide comprises a polypeptide sequence with at least
50%, 60%, 70%, 80%, 85%, 90% or 95% or more sequence identity to a
functionally active fragment of the polypeptide presented in SEQ ID
NO:2, 4, or 6. In yet another embodiment, a HIO1002 polypeptide
comprises a polypeptide sequence with at least 50%, 60%, 70%, 80%,
or 90% identity to the polypeptide sequence of SEQ ID NO:2, 4 or 6
over its entire length and comprises of one or more transmembrane
domains.
[0050] In another aspect, a HIO1002 polynucleotide sequence is at
least 50% to 60% identical over its entire length to the HIO1002
nucleic acid sequence presented as SEQ ID NO:1, 3, or 5, or nucleic
acid sequences that are complementary to such a HIO1002 sequence,
and may comprise at least 70%, 80%, 85%, 90% or 95% or more
sequence identity to the HIO1002 sequence presented as SEQ ID NO:1,
3, or 5, or a functionally active fragment thereof, or
complementary sequences.
[0051] As used herein, "percent (%) sequence identity" with respect
to a specified subject sequence, or a specified portion thereof, is
defined as the percentage of nucleotides or amino acids in the
candidate derivative sequence identical with the nucleotides or
amino acids in the subject sequence (or specified portion thereof),
after aligning the sequences and introducing gaps, if necessary to
achieve the maximum percent sequence identity, as generated by the
program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997)
215:403-410) with search parameters set to default values. The HSP
S and HSP S2 parameters are dynamic values and are established by
the program itself depending upon the composition of the particular
sequence and composition of the particular database against which
the sequence of interest is being searched. A "% identity value" is
determined by the number of matching identical nucleotides or amino
acids divided by the sequence length for which the percent identity
is being reported. "Percent (%) amino acid sequence similarity" is
determined by doing the same calculation as for determining % amino
acid sequence identity, but including conservative amino acid
substitutions in addition to identical amino acids in the
computation. A conservative amino acid substitution is one in which
an amino acid is substituted for another amino acid having similar
properties such that the folding or activity of the protein is not
significantly affected. Aromatic amino acids that can be
substituted for each other are phenylalanine, tryptophan, and
tyrosine; interchangeable hydrophobic amino acids are leucine,
isoleucine, methionine, and valine; interchangeable polar amino
acids are glutamine and asparagine; interchangeable basic amino
acids are arginine, lysine and histidine; interchangeable acidic
amino acids are aspartic acid and glutamic acid; and
interchangeable small amino acids are alanine, serine, threonine,
cysteine and glycine.
[0052] Derivative nucleic acid molecules of the subject nucleic
acid molecules include sequences that selectively hybridize to the
nucleic acid sequence of SEQ ID NO:1. The stringency of
hybridization can be controlled by temperature, ionic strength, pH,
and the presence of denaturing agents such as formamide during
hybridization and washing. Conditions routinely used are well known
(see, e.g., Current Protocol in Molecular Biology, Vol. 1, Chap.
2.10, John Wiley & Sons, Publishers (1994); Sambrook et al.,
Molecular Cloning, Cold Spring Harbor (1989)). In some embodiments,
a nucleic acid molecule of the invention is capable of hybridizing
to a nucleic acid molecule containing the nucleotide sequence of
SEQ ID NO:1 under stringent hybridization conditions that are:
prehybridization of filters containing nucleic acid for 8 hours to
overnight at 65.degree. C. in a solution comprising 6.times. single
strength citrate (SSC) (1.times.SSC is 0.15 M NaCl, 0.015 M Na
citrate; pH 7.0), 5.times.Denhardt's solution, 0.05% sodium
pyrophosphate and 100 .mu.g/ml herring sperm DNA; hybridization for
18-20 hours at 65.degree. C. in a solution containing 6.times.SSC,
1.times.Denhardt's solution, 100 .mu.g/ml yeast tRNA and 0.05%
sodium pyrophosphate; and washing of filters at 65.degree. C. for 1
h in a solution containing 0.1.times.SSC and 0.1% SDS (sodium
dodecyl sulfate). In other embodiments, moderately stringent
hybridization conditions are used that are: pretreatment of filters
containing nucleic acid for 6 h at 40.degree. C. in a solution
containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH 7.5), 5
mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 .mu.g/ml denatured
salmon sperm DNA; hybridization for 18-20 h at 40.degree. C. in a
solution containing 35% formamide, 5.times.SSC, 50 mM Tris-HCl (pH
7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 .mu.g/ml
salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by
washing twice for 1 hour at 55.degree. C. in a solution containing
2.times.SSC and 0.1% SDS. Alternatively, low stringency conditions
can be used that comprise: incubation for 8 hours to overnight at
37.degree. C. in a solution comprising 20% formamide, 5.times.SSC,
50 mM sodium phosphate (pH 7.6), 5.times.Denhardt's solution, 10%
dextran sulfate, and 20 .mu.g/ml denatured sheared salmon sperm
DNA; hybridization in the same buffer for 18 to 20 hours; and
washing of filters in 1.times.SSC at about 37.degree. C. for 1
hour.
[0053] As a result of the degeneracy of the genetic code, a number
of polynucleotide sequences encoding a HIO1002 polypeptide can be
produced. For example, codons may be selected to increase the rate
at which expression of the polypeptide occurs in a particular host
species, in accordance with the optimum codon usage dictated by the
particular host organism (see, e.g., Nakamura et al., 1999). Such
sequence variants may be used in the methods of this invention.
[0054] The methods of the invention may use orthologs of the
Arabidopsis HIO1002. Methods of identifying the orthologs in other
plant species are known in the art. Normally, orthologs in
different species retain the same function, due to presence of one
or more protein motifs and/or 3-dimensional structures. In
evolution, when a gene duplication event follows speciation, a
single gene in one species, such as Arabidopsis, may correspond to
multiple genes (paralogs) in another. As used herein, the term
"orthologs" encompasses paralogs. When sequence data is available
for a particular plant species, orthologs are generally identified
by sequence homology analysis, such as BLAST analysis, usually
using protein bait sequences. Sequences are assigned as a potential
ortholog if the best hit sequence from the forward BLAST result
retrieves the original query sequence in the reverse BLAST (Huynen
M A and Bork P, Proc Natl Acad Sci (1998) 95:5849-5856; Huynen M A
et al., Genome Research (2000) 10:1204-1210).
[0055] Programs for multiple sequence alignment, such as CLUSTAL
(Thompson J D et al., 1994, Nucleic Acids Res 22:4673-4680) may be
used to highlight conserved regions and/or residues of orthologous
proteins and to generate phylogenetic trees. In a phylogenetic tree
representing multiple homologous sequences from diverse species
(e.g., retrieved through BLAST analysis), orthologous sequences
from two species generally appear closest on the tree with respect
to all other sequences from these two species. Structural threading
or other analysis of protein folding (e.g., using software by
ProCeryon, Biosciences, Salzburg, Austria) may also identify
potential orthologs. Nucleic acid hybridization methods may also be
used to find orthologous genes and are preferred when sequence data
are not available. Degenerate PCR and screening of cDNA or genomic
DNA libraries are common methods for finding related gene sequences
and are well known in the art (see, e.g., Sambrook, 1989;
Dieffenbach and Dveksler, 1995). For instance, methods for
generating a cDNA library from the plant species of interest and
probing the library with partially homologous gene probes are
described in Sambrook et al. A highly conserved portion of the
Arabidopsis HIO1002 coding sequence may be used as a probe. HIO1002
ortholog nucleic acids may hybridize to the nucleic acid of SEQ ID
NO:1 under high, moderate, or low stringency conditions. After
amplification or isolation of a segment of a putative ortholog,
that segment may be cloned and sequenced by standard techniques and
utilized as a probe to isolate a complete cDNA or genomic clone.
Alternatively, it is possible to initiate an EST project to
generate a database of sequence information for the plant species
of interest. In another approach, antibodies that specifically bind
known HIO1002 polypeptides are used for ortholog isolation (see,
e.g., Harlow and Lane, 1988, 1999). Western blot analysis can
determine that a HIO1002 ortholog (i.e., an orthologous protein) is
present in a crude extract of a particular plant species. When
reactivity is observed, the sequence encoding the candidate
ortholog may be isolated by screening expression libraries
representing the particular plant species. Expression libraries can
be constructed in a variety of commercially available vectors,
including lambda gt11, as described in Sambrook, et al., 1989. Once
the candidate ortholog(s) are identified by any of these means,
candidate orthologous sequence are used as bait (the "query") for
the reverse BLAST against sequences from Arabidopsis or other
species in which HIO1002 nucleic acid and/or polypeptide sequences
have been identified.
[0056] HIO1002 nucleic acids and polypeptides may be obtained using
any available method. For instance, techniques for isolating cDNA
or genomic DNA sequences of interest by screening DNA libraries or
by using polymerase chain reaction (PCR), as previously described,
are well known in the art. Alternatively, nucleic acid sequence may
be synthesized. Any known method, such as site directed mutagenesis
(Kunkel T A et al., 1991), may be used to introduce desired changes
into a cloned nucleic acid.
[0057] In general, the methods of the invention involve
incorporating the desired form of the HIO1002 nucleic acid into a
plant expression vector for transformation of in plant cells, and
the HIO1002 polypeptide is expressed in the host plant.
[0058] An isolated HIO1002 nucleic acid molecule is other than in
the form or setting in which it is found in nature and is
identified and separated from least one contaminant nucleic acid
molecule with which it is ordinarily associated in the natural
source of the HIO1002 nucleic acid. However, an isolated HIO1002
nucleic acid molecule includes HIO1002 nucleic acid molecules
contained in cells that ordinarily express HIO1002 where, for
example, the nucleic acid molecule is in a chromosomal location
different from that of natural cells.
Generation of Genetically Modified Plants with an Altered Oil
Content Phenotype
[0059] HIO1002 nucleic acids and polypeptides may be used in the
generation of genetically modified plants having a modified oil
content phenotype. As used herein, a "modified oil content
phenotype" may refer to modified oil content in any part of the
plant; the modified oil content is often observed in seeds. In a
preferred embodiment, altered expression of the HIO1002 gene in a
plant is used to generate plants with a high oil phenotype.
[0060] The methods described herein are generally applicable to all
plants. Although activation tagging and gene identification is
carried out in Arabidopsis, the HIO1002 gene (or an ortholog,
variant or fragment thereof) may be expressed in any type of plant.
In a preferred embodiment, the invention is directed to
oil-producing plants, which produce and store triacylglycerol in
specific organs, primarily in seeds. Such species include soybean
(Glycine max), rapeseed and canola (including Brassica napus, B.
campestris), sunflower (Helianthus annus), cotton (Gossypium
hirsutum), corn (Zea mays), cocoa (Theobroma cacao), safflower
(Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm
(Cocos nucifera), flax (Linum usitatissimum), castor (Ricinus
communis) and peanut (Arachis hypogaea). The invention may also be
directed to fruit- and vegetable-bearing plants, grain-producing
plants, nut-producing plants, rapid cycling Brassica species,
alfalfa (Medicago sativa), tobacco (Nicotiana), turfgrass (Poaceae
family), other forage crops, and wild species that may be a source
of unique fatty acids.
[0061] The skilled artisan will recognize that a wide variety of
transformation techniques exist in the art, and new techniques are
continually becoming available. Any technique that is suitable for
the target host plant can be employed within the scope of the
present invention. For example, the constructs can be introduced in
a variety of forms including, but not limited to as a strand of
DNA, in a plasmid, or in an artificial chromosome. The introduction
of the constructs into the target plant cells can be accomplished
by a variety of techniques, including, but not limited to
Agrobacterium-mediated transformation, electroporation,
microinjection, microprojectile bombardment calcium-phosphate-DNA
co-precipitation or liposome-mediated transformation of a
heterologous nucleic acid. The transformation of the plant is
preferably permanent, i.e. by integration of the introduced
expression constructs into the host plant genome, so that the
introduced constructs are passed onto successive plant generations.
Depending upon the intended use, a heterologous nucleic acid
construct comprising an HIO1002 polynucleotide may encode the
entire protein or a biologically active portion thereof.
[0062] In one embodiment, binary Ti-based vector systems may be
used to transfer polynucleotides. Standard Agrobacterium binary
vectors are known to those of skill in the art, and many are
commercially available (e.g., pBI121 Clontech Laboratories, Palo
Alto, Calif.). A construct or vector may include a plant promoter
to express the nucleic acid molecule of choice. In a preferred
embodiment, the promoter is a plant promoter.
[0063] The optimal procedure for transformation of plants with
Agrobacterium vectors will vary with the type of plant being
transformed. Exemplary methods for Agrobacterium-mediated
transformation include transformation of explants of hypocotyl,
shoot tip, stem or leaf tissue, derived from sterile seedlings
and/or plantlets. Such transformed plants may be reproduced
sexually, or by cell or tissue culture. Agrobacterium
transformation has been previously described for a large number of
different types of plants and methods for such transformation may
be found in the scientific literature. Of particular relevance are
methods to transform commercially important crops, such as rapeseed
(De Block et al., 1989), sunflower (Everett et al., 1987), and
soybean (Christou et al., 1989; Kline et al., 1987).
[0064] Expression (including transcription and translation) of
HIO1002 may be regulated with respect to the level of expression,
the tissue type(s) where expression takes place and/or
developmental stage of expression. A number of heterologous
regulatory sequences (e.g., promoters and enhancers) are available
for controlling the expression of a HIO1002 nucleic acid. These
include constitutive, inducible and regulatable promoters, as well
as promoters and enhancers that control expression in a tissue- or
temporal-specific manner. Exemplary constitutive promoters include
the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and 5,783,394),
the nopaline synthase (NOS) promoter (Ebert et al., Proc. Natl.
Acad. Sci. (U.S.A.) 84:5745-5749, 1987), the octopine synthase
(OCS) promoter (which is carried on tumor-inducing plasmids of
Agrobacterium tumefaciens), the caulimovirus promoters such as the
cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant
Mol. Biol. 9:315-324, 1987) and the CaMV 35S promoter (Odell et
al., Nature 313:810-812, 1985 and Jones J D et al, 1992), the melon
actin promoter (published PCT application WO0056863), the figwort
mosaic virus 35S-promoter (U.S. Pat. No. 5,378,619), the
light-inducible promoter from the small subunit of
ribulose-1,5-bis-phosphate carboxylase (ssRUBISCO), the Adh
promoter (Walker et al., Proc. Natl. Acad. Sci. (U.S.A.)
84:6624-6628, 1987), the sucrose synthase promoter (Yang et al.,
Proc. Natl. Acad. Sci. (U.S.A.) 87:4144-4148, 1990), the R gene
complex promoter (Chandler et al., The Plant Cell 1:1175-1183,
1989), the chlorophyll a/b binding protein gene promoter the CsVMV
promoter (Verdaguer B et al., 1998); these promoters have been used
to create DNA constructs that have been expressed in plants, e.g.,
PCT publication WO 84/02913. Exemplary tissue-specific promoters
include the tomato E4 and E8 promoters (U.S. Pat. No. 5,859,330)
and the tomato 2AII gene promoter (Van Haaren M J J et al.,
1993).
[0065] In one preferred embodiment, HIO1002 expression is under
control of regulatory sequences from genes whose expression is
associated with early seed and/or embryo development. Indeed, in a
preferred embodiment, the promoter used is a seed-enhanced
promoter. Examples of such promoters include the 5' regulatory
regions from such genes as napin (Kridl et al., Seed Sci. Res.
1:209:219, 1991), globulin (Belanger and Kriz, Genet., 129:
863-872, 1991, GenBank Accession No. L22295), gamma zein Z 27
(Lopes et al., Mol Gen Genet., 247:603-613, 1995), L3 oleosin
promoter (U.S. Pat. No. 6,433,252), phaseolin (Bustos et al., Plant
Cell, 1(9):839-853, 1989), arcelin5 (US 2003/0046727), a soybean 7S
promoter, a 7S.alpha. promoter (US 2003/0093828), the soybean
7S.alpha.' beta conglycinin promoter, a 7S .alpha.' promoter
(Beachy et al., EMBO J., 4:3047, 1985; Schuler et al., Nucleic Acid
Res., 10(24):8225-8244, 1982), soybean trypsin inhibitor (Riggs et
al., Plant Cell 1(6):609-621, 1989), ACP (Baerson et al., Plant
Mol. Biol., 22(2):255-267, 1993), stearoyl-ACP desaturase (Slocombe
et al., Plant Physiol. 104(4):167-176, 1994), soybean a' subunit of
.beta.-conglycinin (Chen et al., Proc. Natl. Acad. Sci.
83:8560-8564, 1986), Vicia faba USP(P-Vf.Usp, SEQ ID NO: 1, 2, and
3 in (US 2003/229918) and Zea mays L3 oleosin promoter (Hong et
al., Plant Mol. Biol., 34(3):549-555, 1997). Also included are the
zeins, which are a group of storage proteins found in corn
endosperm. Genomic clones for zein genes have been isolated
(Pedersen et al., Cell 29:1015-1026, 1982; and Russell et al.,
Transgenic Res. 6(2):157-168) and the promoters from these clones,
including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and genes, could
also be used. Other promoters known to function, for example, in
corn include the promoters for the following genes: waxy, Brittle,
Shrunken 2, Branching enzymes I and II, starch synthases,
debranching enzymes, oleosins, glutelins and sucrose synthases.
Legume genes whose promoters are associated with early seed and
embryo development include V. faba legumin (Baumlein et al., 1991,
Mol Gen Genet. 225:121-8; Baumlein et al., 1992, Plant J 2:233-9),
V. faba usp (Fiedler et al., 1993, Plant Mol Biol 22:669-79), pea
convicilin (Bown et al., 1988, Biochem J 251:717-26), pea lectin
(dePater et al., 1993, Plant Cell 5:877-86), P. vulgaris beta
phaseolin (Bustos et al., 1991, EMBO J. 10:1469-79), P. vulgaris
DLEC2 and PHS [beta] (Bobb et al., 1997, Nucleic Acids Res
25:641-7), and soybean beta-Conglycinin, 7S storage protein
(Chamberland et al., 1992, Plant Mol Biol 19:937-49).
[0066] Cereal genes whose promoters are associated with early seed
and embryo development include rice glutelin ("GluA-3," Yoshihara
and Takaiwa, 1996, Plant Cell Physiol 37:107-11; "GluB-1," Takaiwa
et al., 1996, Plant Mol Biol 30:1207-21; Washida et al., 1999,
Plant Mol Biol 40:1-12; "Gt3," Leisy et al., 1990, Plant Mol Biol
14:41-50), rice prolamin (Zhou & Fan, 1993, Transgenic Res
2:141-6), wheat prolamin (Hammond-Kosack et al., 1993, EMBO J.
12:545-54), maize zein (Z4, Matzke et al., 1990, Plant Mol Biol
14:323-32), and barley B-hordeins (Entwistle et al., 1991, Plant
Mol Biol 17:1217-31).
[0067] Other genes whose promoters are associated with early seed
and embryo development include oil palm GLO7A (7S globulin,
Morcillo et al., 2001, Physiol Plant 112:233-243), Brassica napus
napin, 2S storage protein, and napA gene (Josefsson et al., 1987, J
Biol Chem 262:12196-201; Stalberg et al., 1993, Plant Mol Biol 1993
23:671-83; Ellerstrom et al., 1996, Plant Mol Biol 32:1019-27),
Brassica napus oleosin (Keddie et al., 1994, Plant Mol Biol
24:327-40), Arabidopsis oleosin (Plant et al., 1994, Plant Mol Biol
25:193-205), Arabidopsis FAE1 (Rossak et al., 2001, Plant Mol Biol
46:717-25), Canavalia gladiata conA (Yamamoto et al., 1995, Plant
Mol Biol 27:729-41), and Catharanthus roseus strictosidine synthase
(Str, Ouwerkerk and Memelink, 1999, Mol Gen Genet 261:635-43). In
another preferred embodiment, regulatory sequences from genes
expressed during oil biosynthesis are used (see, e.g., U.S. Pat.
No. 5,952,544). Alternative promoters are from plant storage
protein genes (Bevan et al., 1993, Philos Trans R Soc Lond B Biol
Sci 342:209-15). Additional promoters that may be utilized are
described, for example, in U.S. Pat. Nos. 5,378,619; 5,391,725;
5,428,147; 5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441;
5,633,435; and 4,633,436.
[0068] In yet another aspect, in some cases it may be desirable to
inhibit the expression of endogenous HIO1002 in a host cell.
Exemplary methods for practicing this aspect of the invention
include, but are not limited to antisense suppression (Smith, et
al., 1988; van der Krol et al., 1988); co-suppression (Napoli, et
al., 1990); ribozymes (PCT Publication WO 97/10328); and
combinations of sense and antisense (Waterhouse, et al., 1998).
Methods for the suppression of endogenous sequences in a host cell
typically employ the transcription or transcription and translation
of at least a portion of the sequence to be suppressed. Such
sequences may be homologous to coding as well as non-coding regions
of the endogenous sequence. Antisense inhibition may use the entire
cDNA sequence (Sheehy et al., 1988), a partial cDNA sequence
including fragments of 5' coding sequence, (Cannon et al., 1990),
or 3' non-coding sequences (Ch'ng et al., 1989). Cosuppression
techniques may use the entire cDNA sequence (Napoli et al., 1990;
van der Krol et al., 1990), or a partial cDNA sequence (Smith et
al., 1990).
[0069] Standard molecular and genetic tests may be performed to
further analyze the association between a gene and an observed
phenotype. Exemplary techniques are described below.
[0070] 1. DNA/RNA Analysis
[0071] The stage- and tissue-specific gene expression patterns in
mutant versus wild-type lines may be determined, for instance, by
in situ hybridization. Analysis of the methylation status of the
gene, especially flanking regulatory regions, may be performed.
Other suitable techniques include overexpression, ectopic
expression, expression in other plant species and gene knock-out
(reverse genetics, targeted knock-out, viral induced gene silencing
[VIGS, see Baulcombe D, 1999]).
[0072] In a preferred application expression profiling, generally
by microarray analysis, is used to simultaneously measure
differences or induced changes in the expression of many different
genes. Techniques for microarray analysis are well known in the art
(Schena M et al., Science (1995) 270:467-470; Baldwin D et al.,
1999; Dangond F, Physiol Genomics (2000) 2:53-58; van Hal N L et
al., J Biotechnol (2000) 78:271-280; Richmond T and Somerville S,
Curr Opin Plant Biol (2000) 3:108-116). Expression profiling of
individual tagged lines may be performed. Such analysis can
identify other genes that are coordinately regulated as a
consequence of the overexpression of the gene of interest, which
may help to place an unknown gene in a particular pathway.
[0073] 2. Gene Product Analysis
[0074] Analysis of gene products may include recombinant protein
expression, antisera production, immunolocalization, biochemical
assays for catalytic or other activity, analysis of phosphorylation
status, and analysis of interaction with other proteins via yeast
two-hybrid assays.
[0075] 3. Pathway Analysis
[0076] Pathway analysis may include placing a gene or gene product
within a particular biochemical, metabolic or signaling pathway
based on its mis-expression phenotype or by sequence homology with
related genes. Alternatively, analysis may comprise genetic crosses
with wild-type lines and other mutant lines (creating double
mutants) to order the gene in a pathway, or determining the effect
of a mutation on expression of downstream "reporter" genes in a
pathway.
Generation of Mutated Plants with an Altered Oil Content
Phenotype
[0077] The invention further provides a method of identifying
plants that have mutations in endogenous HIO1002 that confer
altered oil content, and generating altered oil content progeny of
these plants that are not genetically modified. In one method,
called "TILLING" (for targeting induced local lesions in genomes),
mutations are induced in the seed of a plant of interest, for
example, using EMS treatment. The resulting plants are grown and
self-fertilized, and the progeny are used to prepare DNA samples.
HIO1002-specific PCR is used to identify whether a mutated plant
has a HIO1002 mutation. Plants having HIO1002 mutations may then be
tested for altered oil content, or alternatively, plants may be
tested for altered oil content, and then HIO1002-specific PCR is
used to determine whether a plant having altered oil content has a
mutated HIO1002 gene. TILLING can identify mutations that may alter
the expression of specific genes or the activity of proteins
encoded by these genes (see Colbert et al (2001) Plant Physiol
126:480-484; McCallum et al (2000) Nature Biotechnology
18:455-457).
[0078] In another method, a candidate gene/Quantitative Trait Locus
(QTLs) approach can be used in a marker-assisted breeding program
to identify alleles of or mutations in the HIO1002 gene or
orthologs of HIO1002 that may confer altered oil content (see Bert
et al., Theor Appl Genet. 2003 June; 107(1):181-9; and Lionneton et
al., Genome. 2002 December; 45(6):1203-15). Thus, in a further
aspect of the invention, a HIO1002 nucleic acid is used to identify
whether a plant having altered oil content has a mutation in
endogenous HIO1002 or has a particular allele that causes altered
oil content.
[0079] While the invention has been described with reference to
specific methods and embodiments, it will be appreciated that
various modifications and changes may be made without departing
from the invention. All publications cited herein are expressly
incorporated herein by reference for the purpose of describing and
disclosing compositions and methodologies that might be used in
connection with the invention. All cited patents, patent
applications, and sequence information in referenced websites and
public databases are also incorporated by reference.
EXAMPLES
Example 1
Generation of Plants with a HIO1002 Phenotype by Transformation
with an Activation Tagging Construct
[0080] Mutants were generated using the activation tagging "ACTTAG"
vector, pSKI015 (GI#6537289; Weigel D et al., 2000). Standard
methods were used for the generation of Arabidopsis transgenic
plants, and were essentially as described in published application
PCT WO0183697. Briefly, T0 Arabidopsis (Col-0) plants were
transformed with Agrobacterium carrying the pSKI015 vector, which
comprises T-DNA derived from the Agrobacterium Ti plasmid, an
herbicide resistance selectable marker gene, and the 4.times.CaMV
35S enhancer element. Transgenic plants were selected at the T1
generation based on herbicide resistance.
[0081] T3 seed was analyzed by Near Infrared Spectroscopy (NIR) at
the time of harvest. NIR infrared spectra were captured using a
Bruker 22 N/F. Bruker Software was used to estimate total seed oil
and total seed protein content using data from NIR analysis and
reference methods according to the manufacturers instructions. Oil
contents predicted by our calibration (ren oil 1473 1d+sline.q2,
Predicts Hexane Extracted Oil), which followed the general method
of AOCS Procedure AM1-92, Official Methods and Recommended
Practices of the American Oil Chemists Society, 5th Ed., AOCS,
Champaign, Ill., were compared for 38,090 individual ACTTAG lines.
Subsequent to seed compositional analysis, the position of the
ACTTAG element in the genome of in each line was determined by
inverse PCR and sequencing. 38,090 lines with recovered flanking
sequences were considered in this analysis.
[0082] Since the 38,090 lines were planted and grown over a
12-month period, the seed oil content values were normalized to
minimize the effect of environmental differences which may alter
seed oil content. The average seed oil content and its standard
deviation, for each day lines were planted, were calculated. The
seed oil content was expressed as a "relative standard deviation
distance" (SD distance) which was calculated by subtracting the
average seed oil content for the planting day from seed oil content
for each line and dividing the difference by the standard deviation
for that day. This normalization allows comparison of seed oil
content in seed from plants grown throughout the year.
[0083] Genes that cause a high seed oil phenotype when
over-expressed were identified by evaluating all of the genes
affected by ACTTAG elements in the 38,090 lines. This was
accomplished by the following procedure; first, the genes likely to
be activated by the ACTTAG element in each line were identified and
the seed oil content of the line was assigned to these genes;
second, the seed oil content when a particular gene is
over-expressed was determined by averaging the individual seed oil
values for each gene. Since 38,090 lines were evaluated and each
element affects an average of 2.5 genes, each gene will have an
average of 4 seed oil values. The genes with the highest average SD
distance were determined to be those that cause a high seed oil
phenotype when over-expressed.
[0084] Seed from plants over-expressing At1g73650 have an oil
content of 128% of the planting day average, as is shown in the
following Table 1.
TABLE-US-00001 TABLE 1 Average Standard n (# of oil deviation
ACTTAG content of lines of the oil relative planted for Seed the
content standard the date Oil planting for the deviation plant
Planting with NIR content date planting Tair distance count
Description Line ID date measurement) (%) (%) date At1g73650
2.960951 1 expressed W000203274 Oct. 7, 2002 1184 32.016 25.025
2.361 protein
Example 2
Characterization of the T-DNA Insertion in Plants Exhibiting the
Altered Oil Content Phenotype
[0085] We performed standard molecular analyses, essentially as
described in patent application PCT WO0183697, to determine the
site of the T-DNA insertion associated with the altered oil content
phenotype. Briefly, genomic DNA was extracted from plants
exhibiting the altered oil content phenotype. PCR, using primers
specific to the pSKI015 vector, confirmed the presence of the 35S
enhancer in plants from the HIO1002 oil line, and Southern blot
analysis verified the genomic integration of the ACTTAG T-DNA and
showed the presence of the T-DNA insertions in each of the
transgenic lines.
[0086] Inverse PCR was used to recover genomic DNA flanking the
T-DNA insertion, which was then subjected to sequence analysis
using a basic BLASTN search and/or a search of the Arabidopsis
Information Resource (TAIR) database (available at the
arabidopsis.org website).
Example 3
Recapitulation of HIO1002 Phenotype
[0087] To test whether over-expression of At1g73650 causes a high
seed oil phenotype, oil content in seeds from transgenic plants
over-expressing this gene was compared with oil content in seeds
from non-transgenic control plants. To do this, At1g73650 was
cloned into a plant transformation vector behind the seed specific
CsVMV promoter and transformed into Arabidopsis plants using the
floral dip method. The plant transformation vector contains the
nptII gene driven by the RE4 promoter, to provide resistance to
kanamyacin, and serve as a selectable marker. Seed from the
transformed plants were plated on agar medium containing kanamycin.
After 7 days, transgenic plants were identified as healthy green
plants and transplanted to soil. Non-transgenic control plants were
germinated on agar medium, allowed to grow for 7 days and then
transplanted to soil. Twenty-two transgenic seedlings and 10
non-transgenic control plants were transplanted to random positions
in the same 32 cell flat. The plants were grown to maturity,
allowed to self-fertilize and set seed. Seed was harvested from
each plant and its oil content estimated by Near Infrared (NIR)
Spectroscopy using methods previously described. The percent oil in
the seed harvested from each plant as determined by NIR
spectroscopy is presented in Table 3. The Relative Oil value is
determined by dividing the predicted oil value by the average oil
value in control seed (i.e. seed from plants without the
trangene).
[0088] The effect of over-expression of At1g73650 on seed oil has
been tested in two experiments. In both experiments, the plants
over-expressing At1g73650 had higher seed oil content than the
control plants grown in the same flat. Across the experiments, the
average seed oil content of plants over-expressing At1g73650 was
4.5% greater than the untransformed controls. The seed oil content
in plants over-expressing At1g73650 was significantly greater than
non-transgenic control plants (two-way ANOVA; P=0.0160).
TABLE-US-00002 TABLE 2 Percent Relative Experiment Plant Transgene
Oil Oil 1 DX07122001 CsVMV:At1g73650 29.89 110.48 1 DX07122002
CsVMV:At1g73650 26.7 98.68 1 DX07122003 CsVMV:At1g73650 27.33
101.03 1 DX07122004 CsVMV:At1g73650 26.13 96.59 1 DX07122005
CsVMV:At1g73650 24.09 89.04 1 DX07122006 CsVMV:At1g73650 27.38
101.2 1 DX07122007 CsVMV:At1g73650 30.58 113.05 1 DX07122009
CsVMV:At1g73650 28.05 103.68 1 DX07122010 CsVMV:At1g73650 28.15
104.06 1 DX07122011 CsVMV:At1g73650 26 96.11 1 DX07122012
CsVMV:At1g73650 25.16 93 1 DX07122013 CsVMV:At1g73650 25.64 94.78 1
DX07122014 CsVMV:At1g73650 27.04 99.97 1 DX07122015 CsVMV:At1g73650
25.86 95.6 1 DX07122016 CsVMV:At1g73650 26.26 97.08 1 DX07122017
CsVMV:At1g73650 30.09 111.23 1 DX07122018 CsVMV:At1g73650 28.19
104.22 1 DX07122019 CsVMV:At1g73650 29.95 110.7 1 DX07122020
CsVMV:At1g73650 29 107.18 1 DX07122021 CsVMV:At1g73650 28.88 106.77
1 DX07122022 CsVMV:At1g73650 27.29 100.88 1 DX07140001 None 26.33
97.33 1 DX07140002 None 26.54 98.12 1 DX07140003 None 28.19 104.21
1 DX07140004 None 26.17 96.72 1 DX07140005 None 26.05 96.29 1
DX07140006 None 25.39 93.86 1 DX07140007 None 25.87 95.64 1
DX07140008 None 31.93 118.04 1 DX07140009 None 28.13 103.99 1
DX07140010 None 25.92 95.79 2 DX07167001 CsVMV:At1g73650 31.77
106.66 2 DX07167002 CsVMV:At1g73650 32.21 108.14 2 DX07167003
CsVMV:At1g73650 31.63 106.18 2 DX07167004 CsVMV:At1g73650 30.2
101.37 2 DX07167005 CsVMV:At1g73650 30.5 102.4 2 DX07167006
CsVMV:At1g73650 31.62 106.17 2 DX07167007 CsVMV:At1g73650 30.55
102.57 2 DX07167008 CsVMV:At1g73650 36.57 122.77 2 DX07167009
CsVMV:At1g73650 32.7 109.78 2 DX07167010 CsVMV:At1g73650 29.27
98.25 2 DX07167011 CsVMV:At1g73650 30 100.71 2 DX07167012
CsVMV:At1g73650 29.58 99.29 2 DX07167013 CsVMV:At1g73650 33.75
113.31 2 DX07167014 CsVMV:At1g73650 30.63 102.84 2 DX07167015
CsVMV:At1g73650 35.07 117.74 2 DX07167016 CsVMV:At1g73650 34.49
115.79 2 DX07167017 CsVMV:At1g73650 32.99 110.75 2 DX07167018
CsVMV:At1g73650 33.06 110.99 2 DX07167019 CsVMV:At1g73650 31.52
105.8 2 DX07167020 CsVMV:At1g73650 32.8 110.13 2 DX07167022
CsVMV:At1g73650 30.37 101.94 2 DX07185001 None 29.9 100.39 2
DX07185002 None 28.47 95.59 2 DX07185003 None 28.83 96.79 2
DX07185004 None 29.03 97.45 2 DX07185005 None 29.05 97.51 2
DX07185007 None 27.27 91.56 2 DX07185008 None 31.06 104.26 2
DX07185009 None 32.22 108.18 2 DX07185010 None 32.25 108.28
Example 4
Analysis of Arabidopsis HIO1002 Sequence
[0089] Sequence analyses were performed with BLAST (Altschul et
al., 1990, J. Mol. Biol. 215:403-410), PFAM (Bateman et al., 1999,
Nucleic Acids Res 27:260-262), PSORT (Nakai K, and Horton P, 1999,
Trends Biochem Sci 24:34-6), and/or CLUSTAL (Thompson J D et al.,
1994, Nucleic Acids Res 22:4673-4680).
TBLASTN Against ESTs:
[0090] The candidate gene At1g73650 is supported by the full-length
cDNAs GI:15809973 and GI:27363285. There are many ESTs from diverse
plant species showing similarity to At1g73650. Where possible, ESTs
contigs of each species were made. The top hit for each of the
following species are listed below and included in the orthologue
Table 2: Triticum aestivum, Glycine max, Mentha.times.piperita,
Populus tremula, Oryza sativa, Lycopersicon esculentum, Solanum
tuberosum, and Beta vulgaris.
1. Sugar Beet ESTs with the Following GenBank IDS: gil26121630 2.
Potato ESTs with the Following GenBank IDS: gil12588047 gil15259347
3. Tomato ESTs with the Following GenBank IDS: gil5896833
gil5896833 gil5896833 gil12628275 gil12628275 gil12628275
gil16242572 4. Rice ESTs with the Following GenBank IDS: gil5456487
gil13420908 gil700187 5. Poplar ESTs with the Following GenBank
IDS: gil3857688 gil23959842 gil23960044 gil23963690 gil23997967
gil23998879 gil24062476 gil24065210 gil28609230 6. Soybean ESTs
with the Following GenBank IDS: gil9205433 gil12488109 gil12773915
gil15286042 gil18731515 7. Corn ESTs with the Following GenBank
IDS: gil5739680 gil21208932 gil407242 gil407243 8. Cotton ESTs with
the Following GenBank IDS: gil3326250 9. Wheat ESTs with the
Following GenBank IDS: gil9742370 gil20309688 gil25194283
gil25235692 gil25237006 gil25243036 gil25261519 gil25280706
gil25431065 gil25547029 gil25560875 gil32546080 gil32670437
BLASTP Against Amino Acids:
[0091] The protein At1g73650 has homology to proteins from other
organisms. The top seven BLAST results for At1g73650 are listed
below and are included in the Orthologue Table 3 below.
[0092] 1. One EST contig from Beta vulgaris
[0093] exl13978852lexsl13978851
[0094] 2. One EST contig from Solanum tubersom
[0095] exl11208765lexsl11208764
[0096] 3. One EST contig from Lycopersicon esculentum
[0097] exl5891894lexsl5891893
[0098] 4. One EST contig from Oryza sativa
[0099] exl7258057lexsl7258056
[0100] 5. One EST contig from Populus tremula
[0101] exl10187045lexsl10187044
[0102] 6. One EST contig from Glycine max
[0103] exl9156811lexsl9156810
[0104] 7. One EST contig from Zea mays
[0105] exl7766057lexsl7766056
[0106] 8. One EST contig from Gossypium hirsutum
[0107] exl9283238lexsl9283237
[0108] 9. One EST contig from Triticum aestivum
[0109] exl11024927lexsl11024926
TABLE-US-00003 TABLE 3 Ortholog Gene % ID to Score(s) (BLAST, Name
Species GI # HIO1002 Clustal, etc.) One Solanum gi|12588047 Length:
585 TBLASTN EST tubersom gi|15259347 Identities: Score: 691 contig
consensus: 0.653 Probability: 2.300000e-68 from SEQ ID NO: 7
Positives: 0.774 potato Frames: 3 One Beta gi|26121630 Length: 504
TBLASTN EST vulgaris Identities: Score: 577 contig 0.667
Probability: 1.300000e-56 from Positives: 0.758 sugar Frames: 1
beet One Glycine gi|9205433 Length: 605 TBLASTN EST max gi|12488109
Identities: Score: 592 contig gi|12773915 0.614 Probability:
1.500000e-57 from gi|15286042 Positives: 0.733 soybean gi|18731515
Frames: 2 consensus: SEQ ID NO: 11 One Triticum gi|9742370 Length:
1569 TBLASTN EST aestivum gi|20309688 Identities: Score: 992 contig
gi|25194283 0.638 Probability: 4.000000e-100 from gi|25235692
Positives: 0.745 wheat gi|25237006 Frames: 1 gi|25243036
gi|25261519 gi|25280706 gi|25431065 gi|25547029 gi|25560875
gi|32546080 gi|32670437 consensus: SEQ ID NO: 13 One Oryza
gi|5456487 Length: 463 TBLASTN EST sativa gi|13420908 Identities:
Score: 469 contig gi|700187 0.766 Probability: 9.800000e-45 from
consensus: Positives: 0.901 rice SEQ ID NO: 9 Frames: 1 One Populus
gi|3857688 Length: 1262 TBLASTN EST tremula gi|23959842 Identities:
Score: 1040 contig gi|23960044 0.674 Probability: 9.900000e-106
from gi|23963690 Positives: 0.774 poplar gi|23997967 Frames: 2
gi|23998879 gi|24062476 gi|24065210 gi|28609230 consensus: SEQ ID
NO: 10 One Lycopersicon gi|5896833 Length: 927 TBLASTN EST
esculentum gi|5896833 Identities: Score: 949 contig gi|5896833
0.652 Probability: 3.600000e-106 from gi|12628275 Positives: 0.769
tomato gi|12628275 Frames: 2 gi|12628275 gi|16242572 consensus: SEQ
ID NO: 8 One Zea mays gi|5739680 Length: 864 TBLASTN EST
gi|21208932 Identities: Score: 450 contig gi|407242 0.626
Probability: 3.000000e-50 from gi|407243 Positives: 0.772 corn
consensus: Frames: 3 SEQ ID NO: 12 One Gossypium gi|3326250 Length:
681 TBLASTN EST hirsutum Identities: Score: 415 contig 0.629
Probability: 8.600000e-40 from Positives: 0.750 cotton Frames:
1
Closest Plant Homologs:
TABLE-US-00004 [0110] At1g73650 Arabidopsis gi|18410409 Identities:
BLASTP thaliana 0.893 Score: 1351 Positives: 0.893 P =
3.700000e-137 Frames: N At1g18180 Arabidopsis gi|15221003
Identities: BLASTP thaliana 0.733 Score: 1122 Positives: 0.806 P =
6.900000e-113 Frames: N gi|13937298 Oryza gi|13937298 Identities:
BLASTP sativa 0.641 Score: 1004 Positives: 0.760 P = 2.200000e-100
Frames: N gi|31213708 Anopheles gi|31213708 Identities: BLASTP
gambiae gi|21299548 0.415 Score: 592 Positives: 0.588 P =
1.000000e-56 Frames: N gi|21355723 Drosophila gi|21355723
Identities: BLASTP melanogaster gi|28574404 0.394 Score: 555
gi|16648114 Positives: 0.564 P = 8.300000e-53 gi|23093921 Frames: N
gi|28380570 gi|38105747 Magnaporthe gi|38105747 Identities: BLASTP
grisea 0.416 Score: 509 70-15 Positives: 0.549 P = 6.300000e-48
Frames: N gi|43807390 environmental gi|43807390 Identities: BLASTP
sequence 0.336 Score: 418 Positives: 0.529 P = 2.700000e-38 Frames:
N
[0111] This NCBI entry for At1g73650 (NP.sub.--849882.1) is a
predicted trans-membrane protein. There are six predicted
trans-membrane domains (predicted by TMHMM; amino acid residues
9-31; 64-86; 99-118; 138-155; 185-204; 209-231). The first
trans-membrane domain overlaps with the signal anchor sequence
(predicted by SignalP; probability=0.990; amino acid residues
1-28).
[0112] Psort2 predicts that At1g73650 may be localized to the
endoplasmic reticulum, plasma membrane or the mitochondria (32%
endoplasmic reticulum, 28% plasma membrane, 24% mitochondrial, 4%
nuclear, 4% golgi apparatus, 4% vacuolar, 4% vesicles of secretory
system by Psort2). However, At1g73650 is unlikely to be targeted to
the mitochondria based on TargetP prediction.
[0113] Pfam analysis predicts that At1g73650 has one domain of
unknown function (PF06966, amino acid residues 24-252):
TABLE-US-00005 hmm- Model Domain seq-f* seq-t f hmm-t score E-value
PF06966 1/1 24 252.. 1 266 [ ] 453.0 4.9e-133 *Seq-f refers to
"sequence-from" and seq-t refers to "sequence-to." The two periods
following the seq-t number indicate that the matching region was
within the sequence and did not extend to either end. The two
brackets indicate that the match spanned the entire length of the
profile HMM. hmm-f and hmm-t refer to the beginning and ending
coordinates of the matching portion of the profile HMM.
Example 5
[0114] Transformed explants of rapeseed, soy, corn, sunflower,
cotton, cocoa, safflower, oil palm, coconut palm, flax, castor and
peanut are obtained through Agrobacterium tumefaciens-mediated
transformation or microparticle bombardment. Plants are regenerated
from transformed tissue. The greenhouse grown plants are then
analyzed for the gene of interest expression levels as well as oil
levels.
Example 6
[0115] This example provides analytical procedures to determine oil
and protein content, mass differences, amino acid composition, free
amino acid levels, and micronutrient content of transgenic maize
plants.
[0116] Oil levels (on a mass basis and as a percent of tissue
weight) of first generation single corn kernels and dissected germ
and endosperm are determined by low-resolution .sup.1H nuclear
magnetic resonance (NMR) (Tiwari et al., JAOCS, 51:104-109 (1974);
or Rubel, JAOCS, 71:1057-1062 (1994)), whereby NMR relaxation times
of single kernel samples are measured, and oil levels are
calculated based on regression analysis using a standard curve
generated from analysis of corn kernels with varying oil levels as
determined gravimetrically following accelerated solvent
extraction. One-way analysis of variance and the Student's T-test
(JMP, version 4.04, SAS Institute Inc., Cary, N.C., USA) are
performed to identify significant differences between transgenic
and non-transgenic kernels as determined by transgene-specific
PCR.
[0117] Oil levels and protein levels in second generation seed are
determined by NIT spectroscopy, whereby NIT spectra of pooled seed
samples harvested from individual plants are measured, and oil and
protein levels are calculated based on regression analysis using a
standard curve generated from analysis of corn kernels with varying
oil or protein levels, as determined gravimetrically following
accelerated solvent extraction or elemental (% N) analysis,
respectively. One-way analysis of variance and the Student's T-test
are performed to identify significant differences in oil (% kernel
weight) and protein (% kernel weight) between seed from marker
positive and marker negative plants.
[0118] The levels of free amino acids are analyzed from each of the
transgenic events using the following procedure. Seeds from each of
the transgenic plants are crushed individually into a fine powder
and approximately 50 mg of the resulting powder is transferred to a
pre-weighed centrifuge tube. The exact sample weight is recorded
and 1.0 ml of 5% trichloroacetic acid is added to each sample tube.
The samples are mixed at room temperature by vortex and then
centrifuged for 15 minutes at 14,000 rpm on an Eppendorf
microcentrifuge (Model 5415C, Brinkmann Instrument, Westbury,
N.Y.). An aliquot of the supernatant is removed and analyzed by
HPLC (Agilent 1100) using the procedure set forth in Agilent
Technical Publication "Amino Acid Analysis Using the Zorbax
Eclipse-AAA Columns and the Agilent 1100 HPLC," Mar. 17, 2000.
[0119] Quantitative determination of total amino acids from corn is
performed by the following method. Kernels are ground and
approximately 60 mg of the resulting meal is acid-hydrolyzed using
6 N HCl under reflux at 100.degree. C. for 24 hrs. Samples are
dried and reconstituted in 0.1 N HCl followed by precolumn
derivatization with .alpha.-phthalaldehyde (OPA0 for HPLC analysis.
The amino acids are separated by a reverse-phase Zorbax Eclipse
XDB-C18 HPLC column on an Agilent 1100 HPLC (Agilent, Palo Alto,
Calif.). The amino acids are detected by fluorescence. Cysteine,
proline, asparagine, glutamine, and tryptophan are not included in
this amino acid screen (Henderson et al., "Rapid, Accurate,
Sensitive and Reproducible HPLC Analysis of Amino acids, Amino Acid
Analysis Using Zorbax Eclipse-AAA Columns and the Agilent 1100
HPLC," Agilent Publication (2000); see, also, "Measurement of
Acid-Stable Amino Acids," AACC Method 07-01 (American Association
of Cereal Chemists, Approved Methods, 9th edition (LCCC#
95-75308)). Total tryptophan is measured in corn kernels using an
alkaline hydrolysis method as described (Approved Methods of the
American Association of Cereal Chemists--10.sup.th edition, AACC
ed, (2000) 07-20 Measurement of Tryptophan--Alakline
Hydrolysis).
[0120] Tocopherol and tocotrienol levels in seeds are assayed by
methods well-known in the art. Briefly, 10 mg of seed tissue are
added to 1 g of microbeads (Biospec Product Inc, Barlesville,
Okla.) in a sterile microfuge tube to which 500 .mu.l 1% pyrogallol
(Sigma Chemical Co., St. Louis, Mo.)/ethanol have been added. The
mixture is shaken for 3 minutes in a mini Beadbeater (Biospec) on
"fast" speed, then filtered through a 0.2 .mu.m filter into an
autosampler tube. The filtered extracts are analyzed by HPLC using
a Zorbax silica HPLC column (4.6 mm.times.250 mm) with a
fluorescent detection, an excitation at 290 nm, an emission at 336
nm, and bandpass and slits. Solvent composition and running
conditions are as listed below with solvent A as hexane and solvent
B as methyl-t-butyl ether. The injection volume is 20 .mu.l, the
flow rate is 1.5 ml/minute and the run time is 12 minutes at
40.degree. C. The solvent gradient is 90% solvent A, 10% solvent B
for 10 minutes; 25% solvent A, 75% solvent B for 11 minutes; and
90% solvent A, 10% solvent B for 12 minutes. Tocopherol standards
in 1% pyrogallol/ethanol are run for comparison
(.alpha.-tocopherol, .gamma.-tocopherol, .beta.-tocopherol,
.delta.-tocopherol, and tocopherol (tocol)). Standard curves for
alpha, beta, delta, and gamma tocopherol are calculated using
Chemstation software (Hewlett Packard). Tocotrienol standards in 1%
pyrogallol/ethanol are run for comparison (.alpha.-tocotrienol,
.gamma.-tocotrienol, .beta.-tocotrienol, .delta.-tocotrienol).
Standard curves for .alpha.-, .beta.-, .delta.-, and
.gamma.-tocotrienol are calculated using Chemstation software
(Hewlett Packard).
[0121] Carotenoid levels within transgenic corn kernels are
determined by a standard protocol (Craft, Meth. Enzymol.,
213:185-205 (1992)). Plastiquinols and phylloquinones are
determined by standard protocols (Threlfall et al., Methods in
Enzymology, XVIII, part C, 369-396 (1971); and Ramadan et al., Eur.
Food Res. Technol., 214(6):521-527 (2002)).
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Harlow E and Lane D, Antibodies: A Laboratory Manual, Cold Spring
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1:285-297 1992. [0144] Kardailsky I et al., Science 286: 1962-1965,
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et al., Methods Enzymol. 204:125-39, 1991. [0148] Lemieux B., et
al., 1990, Theor Appl Genet. 80, 234-240. [0149] Nakamura Y et al.,
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2:279-289, 1990. [0151] Okuley et al., Plant Cell 6(1):147-158,
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Manual (Second Edition), Cold Spring Harbor Press, Plainview, N.Y.,
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Sheehy et al., Proc. Natl. Acad. Sci. USA (1988) 85:8805-8809.
[0155] Smith, et al., Nature 334:724-726, 1988. [0156] Smith et
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al., Nucleic Acids Res 22:4673-4680, 1994. [0158] Van der Krol et
al., Biotechniques (1988) 6:958-976. [0159] Van der Krol et al.,
The Plant Cell (1990) 2:291-299. [0160] Van Haaren M J J et al.,
Plant Mol Bio 21:625-640, 1993. [0161] Verdaguer B et al., Plant
Mol Biol 37:1055-1067, 1998. [0162] Waterhouse, et al., Proc. Natl.
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8: 659-671, 1996. [0165] Yadav N S et al., (1993) Plant Physiol
103, 467-476.
Sequence CWU 1
1
1311272DNAArabidopsis thaliana 1ttaaagtaat gacttttttt tatatgatat
attctcaatt ttgttactcg gaaacaatag 60aaaggataac gaatcgtttc tcttcgatcg
aaagaatata tatttggaag ttttctctgc 120ttttttgttt tcgtttgata
gaatgggaac agttctcgat tcgcattttc tggctctcac 180tgctattgtc
actgtgattt accagttcat attcttcgta atcacggctc ttttcaaatt
240tgatcaagtc actgactttg ctggtagcac aaacttcgtt atacttgctg
tgttaacact 300tgttctcaaa gcctcttggc attttcgaca gatagtattg
actttgctag ttgtggtatg 360gggtcttcgc ttggggattt tccttctaat
gaggatcttg caatgggggg aagatcgtcg 420ctttgatgaa cagcgtggaa
atatagtgag actaatcatt ttctggactc ttcaggctgt 480gtgggtttgg
acggttagct tacctttaac acttgttaat gcaagtgatg gtggtggatc
540tcttaaaccc gcagatgtta tcggttggac tatgtgggtt ttcggtttct
tgattgaagc 600tgcagctgat caacagaagc tatcattcaa aaactctcct
gaaaacagag gaaaatggtg 660tgatgttgga gtctggaagt attcaagaca
tccaaactac ttcggtgaga tgttactgtg 720gtggggaatc tttgtggctg
catcgcctgt gcttgaaggt gcagagtatc ttgtcatatt 780cggaccactc
tttctcactt tgctacttct attcgtcagc ggcataccat tactcgaggc
840atcggctgac aaaaaacatg gaaactcagg agcttacaga tcctacaaga
agacaacaag 900tcctctgatt ctgttcccaa gaggagtgta tgggaactta
ccaggatggt tcaagacggt 960ctttctcttc gagtttccat tttacagccg
aaatctccct caagaggtgg ctgtttagta 1020actgcttatt ctgtttcagt
ttcatctttt atatcttact ttagtgtttg atgagtttgg 1080cttctctttg
ttgtatgtgt gcaagaaagt tagggtagac tccaaaacag caaagagaaa
1140gaagtaaaag atgactgatt tgtgatactc atatttgagt tattgttctt
cacaacactc 1200tttgataaaa tttgtgtaaa aaattgcccg taagaaactg
aatttgtttg aaatattgga 1260cagtcttgac ag 12722291PRTArabidopsis
thaliana 2Met Gly Thr Val Leu Asp Ser His Phe Leu Ala Leu Thr Ala
Ile Val1 5 10 15Thr Val Ile Tyr Gln Phe Ile Phe Phe Val Ile Thr Ala
Leu Phe Lys 20 25 30Phe Asp Gln Val Thr Asp Phe Ala Gly Ser Thr Asn
Phe Val Ile Leu 35 40 45Ala Val Leu Thr Leu Val Leu Lys Ala Ser Trp
His Phe Arg Gln Ile 50 55 60Val Leu Thr Leu Leu Val Val Val Trp Gly
Leu Arg Leu Gly Ile Phe65 70 75 80Leu Leu Met Arg Ile Leu Gln Trp
Gly Glu Asp Arg Arg Phe Asp Glu 85 90 95Gln Arg Gly Asn Ile Val Arg
Leu Ile Ile Phe Trp Thr Leu Gln Ala 100 105 110Val Trp Val Trp Thr
Val Ser Leu Pro Leu Thr Leu Val Asn Ala Ser 115 120 125Asp Gly Gly
Gly Ser Leu Lys Pro Ala Asp Val Ile Gly Trp Thr Met 130 135 140Trp
Val Phe Gly Phe Leu Ile Glu Ala Ala Ala Asp Gln Gln Lys Leu145 150
155 160Ser Phe Lys Asn Ser Pro Glu Asn Arg Gly Lys Trp Cys Asp Val
Gly 165 170 175Val Trp Lys Tyr Ser Arg His Pro Asn Tyr Phe Gly Glu
Met Leu Leu 180 185 190Trp Trp Gly Ile Phe Val Ala Ala Ser Pro Val
Leu Glu Gly Ala Glu 195 200 205Tyr Leu Val Ile Phe Gly Pro Leu Phe
Leu Thr Leu Leu Leu Leu Phe 210 215 220Val Ser Gly Ile Pro Leu Leu
Glu Ala Ser Ala Asp Lys Lys His Gly225 230 235 240Asn Ser Gly Ala
Tyr Arg Ser Tyr Lys Lys Thr Thr Ser Pro Leu Ile 245 250 255Leu Phe
Pro Arg Gly Val Tyr Gly Asn Leu Pro Gly Trp Phe Lys Thr 260 265
270Val Phe Leu Phe Glu Phe Pro Phe Tyr Ser Arg Asn Leu Pro Gln Glu
275 280 285Val Ala Val 29031192DNAArabidopsis thaliana 3ttaaagtaat
gacttttttt tatatgatat attctcaatt ttgttactcg gaaacaatag 60aaaggataac
gaatcgtttc tcttcgatcg aaagaatata tatttggaag ttttctctgc
120ttttttgttt tcgtttgata gaatgggaac agttctcgat tcgcattttc
tggctctcac 180tgctattgtc actgtgattt accagttcat attcttcgta
atcacggctc ttttcaaatt 240tgatcaagtc actgactttg ctggtagcac
aaacttcgtt atacttgctg tgttaacact 300tgttctcaaa gcctcttggc
attttcgaca gatagtattg actttgctag ttgtggtatg 360gggtcttcgc
ttggggattt tccttctaat gaggatcttg caatgggggg aagatcgtcg
420ctttgatgaa cagcgtggaa atatagtgag actaatcatt ttctggactc
ttcaggctgt 480gtgggtttgg acggttagct tacctttaac acttgttaat
gcaagtgatg gtggtggatc 540tcttaaaccc gcagatgtta tcggttggac
tatgtgggtt ttcggtttct tgattgaagc 600tgcagctgat caacagaagc
tatcattcaa aaactctcct gaaaacagag gaaaatggtg 660tgatgttgga
gtctggaagt attcaagaca tccaaactac ttcggtgaga tgttactgtg
720gtggggaatc tttgtggctg catcgcctgt gcttgaaggt gcagagtatc
ttgtcatatt 780cggaccactc tttctcactt tgctacttct attcgtcagc
ggcataccat tactcgaggc 840atcggctgac aaaaaacatg gaaactcagg
agcttacaga tcctacaaga agacaacaag 900tcctctgatt ctgttcccaa
gaggagtgta tgggaactta ccaggatggt tcaagacggt 960ctttctcttc
gagtttccat tttacagccg aaatctccct caagagttag ggtagactcc
1020aaaacagcaa agagaaagaa gtaaaagatg actgatttgt gatactcata
tttgagttat 1080tgttcttcac aacactcttt gataaaattt gtgtaaaaaa
ttgcccgtaa gaaactgaat 1140ttgtttgaaa tattggacag tcttgacaga
ttatctcagt tgtatttggt tc 11924290PRTArabidopsis thaliana 4Met Gly
Thr Val Leu Asp Ser His Phe Leu Ala Leu Thr Ala Ile Val1 5 10 15Thr
Val Ile Tyr Gln Phe Ile Phe Phe Val Ile Thr Ala Leu Phe Lys 20 25
30Phe Asp Gln Val Thr Asp Phe Ala Gly Ser Thr Asn Phe Val Ile Leu
35 40 45Ala Val Leu Thr Leu Val Leu Lys Ala Ser Trp His Phe Arg Gln
Ile 50 55 60Val Leu Thr Leu Leu Val Val Val Trp Gly Leu Arg Leu Gly
Ile Phe65 70 75 80Leu Leu Met Arg Ile Leu Gln Trp Gly Glu Asp Arg
Arg Phe Asp Glu 85 90 95Gln Arg Gly Asn Ile Val Arg Leu Ile Ile Phe
Trp Thr Leu Gln Ala 100 105 110Val Trp Val Trp Thr Val Ser Leu Pro
Leu Thr Leu Val Asn Ala Ser 115 120 125Asp Gly Gly Gly Ser Leu Lys
Pro Ala Asp Val Ile Gly Trp Thr Met 130 135 140Trp Val Phe Gly Phe
Leu Ile Glu Ala Ala Ala Asp Gln Gln Lys Leu145 150 155 160Ser Phe
Lys Asn Ser Pro Glu Asn Arg Gly Lys Trp Cys Asp Val Gly 165 170
175Val Trp Lys Tyr Ser Arg His Pro Asn Tyr Phe Gly Glu Met Leu Leu
180 185 190Trp Trp Gly Ile Phe Val Ala Ala Ser Pro Val Leu Glu Gly
Ala Glu 195 200 205Tyr Leu Val Ile Phe Gly Pro Leu Phe Leu Thr Leu
Leu Leu Leu Phe 210 215 220Val Ser Gly Ile Pro Leu Leu Glu Ala Ser
Ala Asp Lys Lys His Gly225 230 235 240Asn Ser Gly Ala Tyr Arg Ser
Tyr Lys Lys Thr Thr Ser Pro Leu Ile 245 250 255Leu Phe Pro Arg Gly
Val Tyr Gly Asn Leu Pro Gly Trp Phe Lys Thr 260 265 270Val Phe Leu
Phe Glu Phe Pro Phe Tyr Ser Arg Asn Leu Pro Gln Glu 275 280 285Leu
Gly 29051337DNAArabidopsis thaliana 5ttaaagtaat gacttttttt
tatatgatat attctcaatt ttgttactcg gaaacaatag 60aaaggataac gaatcgtttc
tcttcgatcg aaagaatata tatttggaag ttttctctgc 120ttttttgttt
tcgtttgata gaatgggaac agttctcgat tcgcattttc tggctctcac
180tgctattgtc actgtgattt accagttcat attcttcgta atcacggctc
ttttcaaatt 240tgatcaagtc actgactttg ctggtagcac aaacttcgtt
atacttgctg tgttaacact 300tgttctcaaa gcctcttggc attttcgaca
gatagtattg actttgctag ttgtggtatg 360gggtcttcgc ttggggattt
tccttctaat gaggatcttg caatgggggg aagatcgtcg 420ctttgatgaa
cagcgtggaa atatagtgag actaatcatt ttctggactc ttcaggctgt
480gtgggtttgg acggttagct tacctttaac acttgttaat gcaagtgatg
gtggtggatc 540tcttaaaccc gcagatgtta tcggttggac tatgtgggtt
ttcggtttct tgattgaagc 600tgcagctgat caacagaagc tatcattcaa
aaactctcct gaaaacagag gaaaatggtg 660tgatgttgga gtctggaagt
attcaagaca tccaaactac ttcggtgaga tgttactgtg 720gtggggaatc
tttgtggctg catcgcctgt gcttgaaggt gcagagtatc ttgtcatatt
780cggaccactc tttctcactt tgctacttct attcgtcagc ggcataccat
tactcgaggc 840atcggctgac aaaaaacatg gaaactcagg agcttacaga
tcctacaaga agacaacaag 900tcctctgatt ctgttcccaa gaggagtgta
tgggaactta ccaggatggt tcaagacggt 960ctttctcttc gagtttccat
tttacagccg aaatctccct caagagggta gactccaaaa 1020cagcaaagag
aaagaagtaa aagatgactg atttgtgata ctcatatttg agttattgtt
1080cttcacaaca ctctttgata aaatttgtgt aaaaaattgc ccgtaagaaa
ctgaatttgt 1140ttgaaatatt ggacagtctt gacagattat ctcagttgta
tttggttctt ttttttatgt 1200gattttgttt tttggttaaa aaaagttccc
attccaagaa tcgaacccgg gtctcctggg 1260tgaaagccaa atatcctaac
cgctggacga catcggattt gattatttga ttatttaatt 1320actttatatg ttgattt
13376302PRTArabidopsis thaliana 6Met Gly Thr Val Leu Asp Ser His
Phe Leu Ala Leu Thr Ala Ile Val1 5 10 15Thr Val Ile Tyr Gln Phe Ile
Phe Phe Val Ile Thr Ala Leu Phe Lys 20 25 30Phe Asp Gln Val Thr Asp
Phe Ala Gly Ser Thr Asn Phe Val Ile Leu 35 40 45Ala Val Leu Thr Leu
Val Leu Lys Ala Ser Trp His Phe Arg Gln Ile 50 55 60Val Leu Thr Leu
Leu Val Val Val Trp Gly Leu Arg Leu Gly Ile Phe65 70 75 80Leu Leu
Met Arg Ile Leu Gln Trp Gly Glu Asp Arg Arg Phe Asp Glu 85 90 95Gln
Arg Gly Asn Ile Val Arg Leu Ile Ile Phe Trp Thr Leu Gln Ala 100 105
110Val Trp Val Trp Thr Val Ser Leu Pro Leu Thr Leu Val Asn Ala Ser
115 120 125Asp Gly Gly Gly Ser Leu Lys Pro Ala Asp Val Ile Gly Trp
Thr Met 130 135 140Trp Val Phe Gly Phe Leu Ile Glu Ala Ala Ala Asp
Gln Gln Lys Leu145 150 155 160Ser Phe Lys Asn Ser Pro Glu Asn Arg
Gly Lys Trp Cys Asp Val Gly 165 170 175Val Trp Lys Tyr Ser Arg His
Pro Asn Tyr Phe Gly Glu Met Leu Leu 180 185 190Trp Trp Gly Ile Phe
Val Ala Ala Ser Pro Val Leu Glu Gly Ala Glu 195 200 205Tyr Leu Val
Ile Phe Gly Pro Leu Phe Leu Thr Leu Leu Leu Leu Phe 210 215 220Val
Ser Gly Ile Pro Leu Leu Glu Ala Ser Ala Asp Lys Lys His Gly225 230
235 240Asn Ser Gly Ala Tyr Arg Ser Tyr Lys Lys Thr Thr Ser Pro Leu
Ile 245 250 255Leu Phe Pro Arg Gly Val Tyr Gly Asn Leu Pro Gly Trp
Phe Lys Thr 260 265 270Val Phe Leu Phe Glu Phe Pro Phe Tyr Ser Arg
Asn Leu Pro Gln Glu 275 280 285Gly Arg Leu Gln Asn Ser Lys Glu Lys
Glu Val Lys Asp Asp 290 295 3007585DNAArtificial sequencesynthetic
construct 7gcaaagaccg aacagggaaa gttatggatt cccatttctt gggcctcact
gccattgtta 60ccttgggtta tcagttaaca tttttcatta tcacagcact cttcaggttt
gataaagtta 120ctgactttgc gggaggtacg aacttcatta tacttgctat
tttgaccttg gtgctaaagg 180gttcatggca ctttcgacag gtggttttgt
ctctatttgt agttatatgg ggccttcggt 240tggggctttt cttattaatg
aggatattac agtgggggga ggacagacgt tttgatgaca 300aacgtgacaa
tcttggaaaa ctggcaatat tttggatact tcaggcaatc tgggtatgga
360ctgttagttt accagtgaca gttgttaatg caagtgacaa gcagccttct
gttcaggctc 420aagacatcat tggttggatt atgtggatta ttgggatttt
agttgagatt acagctgacc 480aacaaaaatt ggcgttcaaa aactcctcgg
agaacagagg aaagtggtgc agtgttggcc 540tttggaaata ttctcgtcat
ccaaactatt ttggtgagat tttac 5858927DNAArtificial sequencesynthetic
construct 8ttctttgtga atggggacag ttatagactc ccatttgttg ggcctaaccg
ccattgttac 60agtggtttac caattaatct tttttataat cacagcactc ctcaaatttg
ataaagtcac 120agatttctca ggaggtacaa atttcattgt gcttgcagaa
ttaactttga tactaaaagg 180ttcatggaat tttcgacagg tggttttgtc
tgtgtttgtc gtgatatggg gccttcgact 240ggggctattt ctattaatga
ggatattaca atggggcgag gatagacgtt ttgatgataa 300gcgtgataat
ctggggaaac tggcaatatt ttggctattt caggcaatct gggtgtggac
360tgtgagttta ccagtgacag ttgttaatgc aagtgaccac caaccatctg
ttcagacggt 420agacatcatt ggttggatta tgtggtctat cggaatatca
gttgagatta cagctgacca 480acaaaaattg gcattcaaaa actccccgga
aaacagagga aagtggtgca gtgatggcct 540ttggaaatat tctcgccatc
caaactattt tggtgagatt ttactttggt ggggaatttt 600cctggcttcc
acaccggtgc tagagggtgc tgaatggctg gttgtgtttg gaccagtctt
660cataaccttg ctgctccttt ttgttagtgg catacccttg cttgaggcat
caggagacaa 720gaagtttgga aatgttggtg catacagatc atataagaga
aaaactagcc ctcttattct 780gctgcctcct ggtgtgtatg gggagtcttc
ctcaatggtt caaaaccata ctcctttttg 840agtttccttt ttacagtcgc
aatcttccgc agaaaagagt aagctggaaa tagacaaccc 900agccaggaag
cagtagaaca gacccga 9279465DNAArtificial sequencesynthetic construct
9gtgagaggag gaggataaac ggaggaggcc gcgatgggaa ccgtgctgga ctcccacttc
60ctggcgctca cggccatcgt cactgtggga tatcagctgg tgttcttcat catcaccgcg
120ctcctgcggt tcgataaagt cactgatttc gcaggaagta cgaatttcat
catacttgca 180atcctcacgc tagctttgaa gggagcatgg catttccgtc
aggttgtatt gactgtgctc 240gttgtgattt ggggtcttcg cttaggacta
ttcttattaa tgaggattct tcaatgggga 300gaggacaaac ggtttgatga
aatgcgcgat aacttgggaa aactagcagt tttctggatt 360ttccaggctg
tttggggtat ggactggtcc agcttgcctg gttacaattg tgnacgcaag
420tggccagcga tccctccaat tgaagcccgt gatatnattg gttgg
465101262DNAArtificial sequencesynthetic construct 10aagagaaaat
aaaagaagag acaccccata aggccataag cataaaagga gctgacaaat 60gaacacaaca
aactgaaaaa atcagtcttt atagatcgaa caaaactata gtgaagtaca
120acgtaatccc ctcaaaatgg gaaccgtttt agactcccat tttctagcac
tcactgccat 180cgtcactgtg ggataccaac tgctgttttt cgttatcact
gcccttctga agtttgataa 240agtcactgac tttgccggaa gcacaaattt
tattatacta gctgtgttga ctctggtttt 300gaaagggaca tggcatttca
gacaggtagt cttaagtttc cttgtagtat cttggggtct 360tcgcctggga
ctgttcctgt tactgaggat tctgcagtgg ggggaggata gacgttttga
420tgaaatgcgt agtaacttgg ggaaattagc tgttttctgg atatttcagg
ctgtctgggt 480gtggactgtg agtttacctg taacagtggt taatggaatt
gacagggatc cttccgttca 540ggctgcagac ataattggct ggattatgtg
gtctgttggt gtttcagttg aagctacagc 600tgatcaacaa aaactgacat
tcaagaatgc tccacaaaac agagggaaat ggggcaatgt 660tggattatgg
aatatatctc gtcacccaaa ctattttggc gaaattctcc tttggtgggg
720tatttttgtg gcttctgcac ctgtactgga aggtgctgaa tggctggtga
tccttgggcc 780aatctttctc acattgttgc ttctttttgt cagtggcata
ccattgcttg agcaatctgc 840tgacaagaaa tttggcaacg tggctgcata
cagggcatat aaaaggacaa ccagccctct 900aattccactg ccccaagcag
tgtacaggag cttaccttca tggttcaaaa ctgttttcct 960ctttgaattt
cctctctaca gtcgtaattt tccagaagag gggtcaacat gaacaagaca
1020aggtggaggc agcgatggat cgaagattgg ctagaagttc aacaggggaa
tctgccctgt 1080tttggtaact ttaatgtact aggaatagtt ttagctgtta
agctacatag acgccatgtt 1140ctttctatta ctagctgtcg gttgattttc
tttctcctgc ctgaaacatg tctccacctc 1200tactgttctg tccactgagt
ttccttccca cacaatcaat gctgatttct tatatttaag 1260tt
126211605DNAArtificial sequencesynthetic construct 11gtttatccaa
cgaggtctgt tttattattg caagagttaa gaactcgact caagaagaca 60gcaaaagtac
tgagccatgg gtaccgtcat cgagtcccat ttcttagcct tcactgccct
120tgtcactatt gcctatcagt ttctgttttt cgtcgtcact gcgctcctca
aattcgacaa 180agtcactgat tttgctggaa gtacgaattt catcataatt
gctgcattga ccctggtcat 240caaagcctcc tggtattttc gacagataat
cctcactttg tttgttggga catggggact 300tcgcctctgc cttttcctcc
tattcagaat tttgcagtgg ggagaggacc ggcgcttcga 360tgaaatgcgt
agtaatttgg gtagactggc catattttgg atatttcagg ctgtctgggt
420gtgggttgtg agtttacctg ttacgttggt taatgcaagc gacagaaatc
ccttcctaca 480ggttgttgat atagtagggt ggattttgtg ggctgtgggt
tttattgtgg aaggtacagc 540agatcaacaa aagctgcatt tcaaaagatc
ttcagaaaat agagggaagt ggtgtaatgt 600tggac 60512864DNAArtificial
sequencesynthetic construct 12gcacgagcag aaacccctcg attgaagcca
gggatataat cggttggata atatggttgg 60tcgggatatg tgtagaagca acagctgatc
agcagaagct tgtgttcaaa aattctccaa 120gtaacagggg aaagtggtgc
aacgccggtc tttggaagta ctctcgccac ccgaactact 180ttggggagat
attcctttgg tggggagtat ttgtagcatc gaccccagtt atctcagatg
240ccgaatggct tgtgattctt ggacctatat tcctgactct tctgcttctt
ttcgttagtg 300gaatcccact cctcgagtca tctgctgata agcgctatgg
acgattggag gaataccgtg 360catacaagaa cactacaagc cctcttatcc
catgccacct gctgtgtatg gttccctgcc 420tgcgtggttc aaggttgcct
tcctccttga gctcccgctc tacaatcctg ggccgggagg 480tgatccaatc
aaaccagaat gagcccattg atgggtcagc ctgatgttta tgttgctatt
540gtgtgcgtgg cagttattcc accttcgatt ccaaggggtt acagtggagt
ttggcccctg 600atttatcgct cgcgaggctc aaacatgtat cttgttggtt
gaaagttaga tttcttttgt 660tgccctccct gcctccatgt gcagatgaat
agatgcttag aaacctgaac gtggaacgtg 720ttcaccttcc tcgctccaaa
aaagtgggaa cggaacaatc ttgtccatat agtgttaaag 780tatttttttt
taagtacggt accacgttcc tgtttctggt tcccatcaat ccgagaactg
840attagatgat caaaaaactc gtgc 864131569DNAArtificial
sequencesynthetic construct 13gcacgaggag gcgatcgcac cacggtcccg
tctcgcacag ccgcgcattc gcaccaccag 60ggtcgcatcg cacgcattcg cccgcctccc
tgtccagcct ccagccacaa gtgacaactg 120acaagtcctc ctgacctcct
ccccctccca acgcgctccg ccgggtcgca tcgccgccgc 180cgccgccgtc
gaccgaaccc agtccaaccc ccgttaaaag cccggccggc gcgcgtgctc
240gcttctcact tcattttaat tctcaaagaa aggaaaggga gaaggataaa
gaaacagacc 300aacaaatcca ctgacggccg gcggggaaga aaaggcgaga
agatgggaac agtgctggac 360tcccacttcc tggcgctcac cgccctcgtc
accgtcgggt
accagctggt gttcttcatc 420atcacagccc tcctccgctt cgacaaggtc
acggatttcg caggcagtac aaattttgtc 480ataatcgccg tcctggtagc
agctttgaag ggaacatggc acttccgtca gatcgtgttg 540acagtgcttg
ttataatctg gggacttcgt ctggcagtgt ttttactaat gaggattttg
600caatggggag aggacaaacg gtttgatgag atgcgcagta acttgggaaa
attagctgtc 660ttctggactt ttcaggctgt ctgggtttgg actgtcagct
tgcctgttac aattgtgaac 720gcaagtagca gaaacccttc tattgaagct
cgggatatca ttggttggat aatgtgggtc 780atcgggctat ctgtggaagc
tatagctgat cagcaaaagc ttaaattcaa gaactctcca 840agcaataggg
gaaagtggtg taatgtgggc ctttggagtt atactcggca cccaaattac
900tttggggaga tgttcctttg gtggggggtg tttgtagcat caaccccggt
tctctcagga 960gctgaatggc ttgtaatctt ggggcccatc ttcctgacgc
tcttgcttct tttcgttagt 1020gggatcccac ttcttgagtc atctgctgat
aagcgctttg gtcggtctga ggaataccgc 1080acatacaaga aaaccacaag
ccctcttatc ccattgccgc cggtcgtgta tggagccctg 1140cccgattggt
tcaaggtggc attcctcctg gagctgcccc tctacaaccc cggaccggaa
1200cgcgaccccg tcagctgagt gatgaatgtg cattgttctt ctgatcacaa
gtgattatac 1260atgtaatgta ctgcttttat tctggatggg tgattctttt
gcccattgat ctcaagggtt 1320tacggtgaag tttgtccata tgtatctatc
tccacgggat gttcaaagca tagattgtgt 1380acttgtcttt aatagggata
ttgtgtactt gtgttaatag tggaaaagtt tgattttatg 1440ttgtacccaa
agtctgaatt tttctctagt ttctatggga attttactct ttgtcacgaa
1500ctccatttaa aatttctatt tctaaacggg cttcgatggt ttgtgaagct
tgcatgagtt 1560catatttaa 1569
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