U.S. patent application number 12/329472 was filed with the patent office on 2009-04-16 for generation of plants with altered oil content.
This patent application is currently assigned to Agrinomics LLC. Invention is credited to Stephanie K. Clendennen, Jeremy E. Coate, Nancy Anne Federspiel, Jonathan Lightner, Debra K. Schuster.
Application Number | 20090099378 12/329472 |
Document ID | / |
Family ID | 28454749 |
Filed Date | 2009-04-16 |
United States Patent
Application |
20090099378 |
Kind Code |
A1 |
Lightner; Jonathan ; et
al. |
April 16, 2009 |
GENERATION OF PLANTS WITH ALTERED OIL CONTENT
Abstract
The present invention is directed to plants that display an
altered oil content phenotype due to altered expression of ICL
nucleic acid. The invention is further directed to methods of
generating plants with an altered oil content phenotype.
Inventors: |
Lightner; Jonathan; (Des
Moines, IA) ; Coate; Jeremy E.; (Ithaca, NY) ;
Clendennen; Stephanie K.; (Kingsport, TN) ;
Federspiel; Nancy Anne; (Menlo Park, CA) ; Schuster;
Debra K.; (Portland, OR) |
Correspondence
Address: |
KLARQUIST SPARKMAN, LLP
121 S.W. SALMON STREET, SUITE 1600
PORTLAND
OR
97204
US
|
Assignee: |
Agrinomics LLC
|
Family ID: |
28454749 |
Appl. No.: |
12/329472 |
Filed: |
December 5, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10508442 |
May 18, 2005 |
7476779 |
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PCT/US03/08739 |
Mar 19, 2003 |
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12329472 |
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60366108 |
Mar 20, 2002 |
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Current U.S.
Class: |
554/9 ; 800/281;
800/306; 800/312; 800/314; 800/320.1; 800/322 |
Current CPC
Class: |
A23D 9/00 20130101; C12N
9/88 20130101; C12N 15/8247 20130101 |
Class at
Publication: |
554/9 ; 800/281;
800/320.1; 800/306; 800/314; 800/322; 800/312 |
International
Class: |
C11B 1/00 20060101
C11B001/00; A01H 1/00 20060101 A01H001/00; A01H 5/00 20060101
A01H005/00 |
Claims
1. A method of producing an altered oil content phenotype in a
plant, said method comprising: a) 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 an ICL polypeptide, and b) growing the transformed
progenitor cells to produce a transgenic plant, wherein said
polynucleotide sequence is expressed, and said transgenic plant
exhibits an altered oil content phenotype.
2. The method of claim 1 wherein the ICL polypeptide has at least
50% sequence identity to the amino acid sequence presented as SEQ
ID NO:2, and wherein the sequence comprises an isocitrate lyase
domain.
3. The method of claim 1 wherein the ICL polypeptide has at least
80% sequence identity to the amino acid sequence presented as SEQ
ID NO:2.
4. The method of claim 1 wherein the ICL polypeptide has at least
90% sequence identity to the amino acid sequence presented as SEQ
ID NO:2.
5. The method of claim 1 wherein the ICL polypeptide has the amino
acid sequence presented as SEQ ID NO:2.
6. The method of claim 1 wherein an ICL polypeptide is
over-expressed in the transgenic plant, and wherein the altered oil
content phenotype is a high oil phenotype.
7. A plant obtained by a method of claim 1.
8. The plant of claim 7, which is selected from the group
consisting of rapeseed, soy, corn, sunflower, cotton, cocoa,
safflower, oil palm, coconut palm, flax, castor and peanut.
9. A plant part obtained from a plant according to claim 7.
10. The plant part of claim 9, which is a seed.
11. Oil obtained from a plant of claim 7.
12. A transgenic plant comprising a chimeric DNA construct
comprising a plant specific promoter and a DNA encoding an ICL,
whereby the transgenic plant has an increased level of ICL relative
to a non-transgenic plant and wherein oil from the plant have
increased content compared to a plant lacking the chimeric DNA
construct.
13. A method of improving the oil content produced from a plant,
said method comprising: a) introducing into a plant a chimeric DNA
construct comprising a plant specific transcription initiation
region and a DNA encoding an ICL which, when introduced into cells
of said plant increases ICL activity in an amount sufficient to
increase the quantity of oil produced by the plant compared to a
plant lacking the chimeric DNA construct.
14. The method according to claim 13, wherein the DNA encoding ICL
is from a plant selected from the group consisting of rapeseed,
soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut
palm, flax, castor and peanut.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional patent
application 60/366,108 filed on Mar. 20, 2002. The content of the
prior application is hereby incorporated in its entirety.
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.
[0003] Industrial processing can separate some or all of these
constituents for individual sale in specific applications. For
instance, nearly 60% of the US 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 (US 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 US
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.
[0004] 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.
[0005] 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.
[0006] 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).
[0007] 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 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.
[0008] 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).
[0009] We used activation tagging techniques to identify the
association between Arabidopsis isocitrate lyase (ICL) and an
altered oil content phenotype. Isocitrate lyase (EC: 4.1.3.1) is an
enzyme that catalyzes the conversion of isocitrate to succinate and
glyoxylate. This is the first step in the glyoxylate bypass, a
specialized metabolic pathway that serves as an alternative to the
tricarboxylic acid cycle in bacteria, fungi and plants. Arabidopsis
ICL mutants have been isolated that are deficient in the glyoxylate
cycle, which plays a central role in the use of stored oil in
oilseeds (Eastmond and Graham, 2000, Trends Plant Sci 6:72-8;
Eastmond et al. 2001, Proc Natl Acad Sci USA 97:5669-74).
SUMMARY OF THE INVENTION
[0010] The present invention provides a method of producing an
altered oil content phenotype in a plant. The method comprises
introducing into plant progenitor cells a vector comprising a
nucleotide sequence that encodes or is complementary to a sequence
encoding an ICL polypeptide and growing a transgenic plant that
expresses the nucleotide sequence. In one embodiment, the ICL
polypeptide has at least 50% sequence identity to the amino acid
sequence presented in SEQ ID NO:2 and comprises an isocitrate lyase
domain. In other embodiments, the ICL polypeptide has at least 80%
or 90% sequence identity to or has the amino acid sequence
presented in SEQ ID NO:2.
[0011] In one preferred embodiment of the invention, the altered
oil content phenotype is a high oil phenotype.
[0012] The invention further provides plants, plant parts, and oils
obtained by the methods described herein. Preferred plants include
rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm,
coconut palm, flax, castor and peanut. Preferred plant parts
include seeds.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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. "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.
[0019] 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).
[0020] 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, progagules and embryos.
[0021] 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.
[0022] 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 an 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] As used herein, "transgenic plant" includes reference to 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.
Identification of Plants with an Altered Oil Content Phenotype
[0027] We used an Arabidopsis activation tagging screen to identify
the association between the gene encoding an isocitrate lyase
(ICL), and an altered oil content phenotype (specifically, a high
oil phenotype). 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.
[0028] An Arabidopsis line that showed a high-oil phenotype, was
identified, wherein oils (i.e., fatty acids) constituted
approximately 37% of seed mass. The association of the ICL 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, ICL genes and/or polypeptides may be employed in
the development of genetically modified plants having a modified
oil content phenotype. ICL 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. ICL genes may further be used
to increase the oil content of specialty oil crops, in order to
augment yield of desired unusual fatty acids.
ICL Nucleic Acids and Polypeptides
[0029] Arabidopsis ICL nucleic acid (cds) sequence is provided in
SEQ ID NO:1 and in Genbank entry GI 4589440, complement of
nucleotides 12755-12726, 12609-12203, 11579-10977, 10700-10108,
9768-9671. The corresponding protein sequence is provided in SEQ ID
NO:2 and in GI 11994639.
[0030] As used herein, the term "ICL polypeptide" refers to a
full-length ICL 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. In one preferred embodiment, a functionally active ICL
polypeptide causes an altered oil content phenotype when
mis-expressed in a plant. In a further preferred embodiment,
mis-expression of the ICL polypeptide causes a high oil phenotype
in a plant. In another embodiment, a functionally active ICL
polypeptide is capable of rescuing defective (including deficient)
endogenous ICL 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 ICL
polypeptide (i.e., a native polypeptide having the sequence of SEQ
ID NO:2 or a naturally occurring ortholog thereof) retains one of
more of the biological properties associated with the full-length
ICL polypeptide, such as signaling activity, binding activity,
catalytic activity, or cellular or extra-cellular localizing
activity. Preferred ICL polypeptides display enzymatic (isocitrate
lyase) activity. An ICL fragment preferably comprises an ICL
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 an ICL protein. Functional domains can be
identified using the PFAM program (Bateman A et al., 1999 Nucleic
Acids Res 27:260-262; website at pfam.wustl.edu). A preferred ICL
fragment comprises an ICL domain (PF00463). The ICL domain of SEQ
ID NO:2 is found at approximately amino acid residues 26-551.
Functionally active variants of full-length ICL 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 ICL
polypeptide. In some cases, variants are generated that change the
post-translational processing of an ICL polypeptide. For instance,
variants may have altered protein transport or protein localization
characteristics or altered protein half-life compared to the native
polypeptide.
[0031] As used herein, the term "ICL nucleic acid" encompasses
nucleic acids with the sequence provided in or complementary to the
sequence provided in SEQ ID NO:1, as well as functionally active
fragments, derivatives, or orthologs thereof. An ICL nucleic acid
of this invention may be DNA, derived from genomic DNA or cDNA, or
RNA.
[0032] In one embodiment, a functionally active ICL nucleic acid
encodes or is complementary to a nucleic acid that encodes a
functionally active ICL 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 ICL
polypeptide. An ICL 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 ICL polypeptide, or an
intermediate form. An ICL 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.
[0033] In another embodiment, a functionally active ICL nucleic
acid is capable of being used in the generation of loss-of-function
ICL phenotypes, for instance, via antisense suppression,
co-suppression, etc.
[0034] In one preferred embodiment, an ICL nucleic acid used in the
methods of this invention comprises a nucleic acid sequence that
encodes or is complementary to a sequence that encodes an ICL
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.
[0035] In another embodiment an ICL polypeptide of the invention
comprises a polypeptide sequence with at least 50% or 60% identity
to the ICL polypeptide sequence of SEQ ID NO:2, and may have at
least 70%, 80%, 85%, 90% or 95% or more sequence identity to the
ICL polypeptide sequence of SEQ ID NO:2. In another embodiment, an
ICL 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, such as an ICL domain. In yet another embodiment, an ICL
polypeptide comprises a polypeptide sequence with at least 50%,
60%, 70%, 80%, or 90% identity to the polypeptide sequence of SEQ
ID NO:2 over its entire length and comprises an ICL domain.
[0036] In another aspect, an ICL polynucleotide sequence is at
least 50% to 60% identical over its entire length to the ICL
nucleic acid sequence presented as SEQ ID NO:1, or nucleic acid
sequences that are complementary to such an ICL sequence, and may
comprise at least 70%, 80%, 85%, 90% or 95% or more sequence
identity to the ICL sequence presented as SEQ ID NO:1 or a
functionally active fragment thereof, or complementary
sequences.
[0037] 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. (1990)
215:403-410; website at blast.wustl.edu/blast/README.html) 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.
[0038] Derivative nucleic acid molecules of the subject nucleic
acid molecules include sequences that 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 comprise:
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 0.2.times.SSC and 0.1% SDS (sodium
dodecyl sulfate). In other embodiments, moderately stringent
hybridization conditions are used that comprise: 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.
[0039] As a result of the degeneracy of the genetic code, a number
of polynucleotide sequences encoding an ICL 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.
[0040] The methods of the invention may use orthologs of the
Arabidopsis ICL. 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). 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, 1989). 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 ICL coding
sequence may be used as a probe. ICL 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 ICL polypeptides are used
for ortholog isolation (see, e.g., Harlow and Lane, 1988, 1999).
Western blot analysis can determine that an ICL 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 ICL nucleic acid and/or polypeptide
sequences have been identified.
[0041] ICL 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.
[0042] In general, the methods of the invention involve
incorporating the desired form of the ICL nucleic acid into a plant
expression vector for transformation of in plant cells, and the ICL
polypeptide is expressed in the host plant.
[0043] An isolated ICL 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 ICL
nucleic acid. However, an isolated ICL nucleic acid molecule
includes ICL nucleic acid molecules contained in cells that
ordinarily express ICL 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
[0044] ICL 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 ICL gene in a plant
is used to generate plants with a high oil phenotype.
[0045] The methods described herein are generally applicable to all
plants. Although activation tagging and gene identification is
carried out in Arabidopsis, the ICL 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 (Gossypiun 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.
[0046] 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 ICL polynucleotide may encode the entire
protein or a biologically active portion thereof.
[0047] 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.).
[0048] 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).
[0049] Expression (including transcription and translation) of ICL
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 an ICL 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 35S CaMV
(Jones J D et al, 1992), the CsVMV promoter (Verdaguer B et al.,
1998) and the melon actin promoter (published PCT application
WO0056863). 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).
[0050] In one preferred embodiment, ICL expression is under control
of regulatory sequences from genes whose expression is associated
with early seed and/or embryo development. 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). 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).
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).
[0051] In yet another aspect, in some cases it may be desirable to
inhibit the expression of endogenous ICL 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).
[0052] 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.
[0053] 1. DNA/RNA Analysis
[0054] 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]).
[0055] 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.
[0056] 2. Gene Product Analysis
[0057] 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.
[0058] 3. Pathway Analysis
[0059] 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.
[0060] 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 High Oil Phenotype by Transformation
with an Activation Tagging Construct
[0061] Mutants were generated using the activation tagging "ACTTAG"
vector, pSKI015 (GI 6537289; Weigel 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. T2 seed was collected
from T1 plants and stored in an indexed collection, and a portion
of the T2 seed was accessed for the screen.
[0062] Quantitative determination of seed fatty acid content was
performed using the follows methods. An aliquot of 15 to 20 T2
seeds from each line tested, which generally contained homozygous
insertion, homozygous wild-type, and heterozygous genotypes in a
standard 1:1:2 ratio, was massed on UMT-2 ultra-microbalance
(Mettler-Toledo Co., Ohio, USA) and then transferred to a glass
extraction vial. Whole seeds were trans-esterified in 500 ul 2.5%
H.sub.2SO.sub.4 in MeOH for 3 hours at 80.degree. C., following the
method of Browse et al. (Biochem J 235:25-31, 1986) with
modifications. A known amount of heptadecanoic acid was included in
the reaction as an internal standard. 750 ul of water and 400 ul of
hexane were added to each vial, which was then shaken vigorously
and allowed to phase separate. Reaction vials were loaded directly
onto GC for analysis and the upper hexane phase was sampled by the
autosampler. Gas chromatography with Flame Ionization detection was
used to separate and quantify the fatty acid methyl esters. Agilent
6890 Plus GC's were used for separation with Agilent Innowax
columns (30 m.times.0.25 mm ID, 250 um film thickness). The carrier
gas was hydrogen at a constant flow of 2.5 ml/minute. 1 ul of
sample was injected in splitless mode (inlet temperature
220.quadrature.C, Purge flow 15 ml/min at 1 minute). The oven was
programmed for an initial temperature of 105.degree. C., initial
time 0.5 minutes, followed by a ramp of 60.degree. C. per minute to
175.degree. C., a 40.degree. C./minute ramp to 260.degree. C. with
a final hold time of 2 minutes. Detection was by Flame Ionization
(Temperature 275.degree. C., Fuel flow 30.0 ml/min, Oxidizer 400.0
ml/min). Instrument control and data collection and analysis was
using the Millennium Chromatography Management System (Version 3.2,
Waters Corporation, Milford, Mass.). Integration and quantification
were performed automatically, but all analyses were subsequently
examined manually to verify correct peak identification and
acceptable signal to noise ratio before inclusion of the derived
results in the study.
[0063] The ACTTAG line designated WO00063887 was identified as
having a high oil phenotype. Specifically, fatty acids constituted
37% of seed mass, compared to 30% or less in wild type.
Example 2
Characterization of the T-DNA Insertion in Plants Exhibiting the
Altered Oil Content Phenotype
[0064] 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 line WO00063887, and Southern blot analysis
verified the genomic integration of the ACTTAG T-DNA. There
appeared to be a complex T-DNA insertion, in which several T-DNAs
inserted as both inverted and tandem repeats, and which included
fragments of the pSKI015 backbone. Right border sequences flanked
the insertion at both upstream and downstream ends. Approximately
3/4 (73/101) of the T2 plants displayed the dominant herbicide
resistance phenotype, which was strong evidence that the T-DNA
insertions were at a single locus.
[0065] Plasmid rescue was used to recover genomic DNA flanking the
T-DNA insertion, which was then subjected to sequence analysis.
[0066] The sequence flanking the downstream right T-DNA border was
subjected to a basic BLASTN search and/or a search of the
Arabidopsis Information Resource (TAIR) database (available at the
arabidopsis.org website), which revealed sequence identity to P1
cloneMSD21 (GI#4589440), mapped to chromosome 3. The downstream
right border boundary was at nucleotide 2879 of P1 clone MSD21.
Sequence analysis revealed that the T-DNA had inserted in the
vicinity (i.e., within about 10 kb) of the gene whose nucleotide
sequence is presented as SEQ ID NO: 1 and GI 4589440, complement of
nucleotides 12755-12726, 12609-12203, 11579-10977, 10700-10108,
9768-9671, and which we designated ICL. Specifically, the
downstream right border of the T-DNA was approximately 9.7 kb 3' to
the start codon of SEQ ID NO:1.
[0067] The insertion was predicted to be dominant or semi-dominant
based on T3 data, as shown in Table 1. Higher than normal oil
content was observed in more than half of the T3 pools ("families")
from individual T2 plants that had shown a high oil phenotype and
were either homozygous or heterozygous for the ACTTAG T-DNA
insertion. We used a cut-off of 32% as the threshold for "high-oil"
in scoring the T3 pools (oil content in wild-type plants is <30%
of seed mass). A score of "1" in the "High oil" column in Table 1
indicates that the particular pool was scored as high oil. Eleven
of 17 lines produced high oil seed, indicating dominant or
semi-dominant inheritance.
TABLE-US-00001 TABLE 1 Oil Content in T3 pools from individual T2
plants. High oil Family # Mean Std Error n= (>32%) 1 23.5% 1.86%
2 2 30.9% 1.04% 6 3 35.9% 0.62% 3 1 4 33.7% 1.09% 7 1 5 30.1% 1.00%
3 6 33.3% 0.65% 3 1 7 32.7% 1.04% 6 1 8 33.5% 0.24% 3 1 9 35.1%
0.68% 3 1 10 33.9% 0.42% 3 1 11 32.7% 0.67% 3 1 12 34.9% 0.31% 3 1
13 30.1% 1.19% 6 14 30.8% 0.17% 3 15 30.8% 0.22% 3 16 33.8% 0.29% 3
1 17 32.3% 0.69% 3 1
Example 3
Analysis of Arabidopsis ICL Sequence
[0068] The amino acid sequence predicted from the ICL nucleic acid
sequence is presented in SEQ ID NO:2 and GI 11994639.
[0069] Sequence analyses were performed with BLAST (Altschul et
al., 1990, J. Mol. Biol. 215:403-410) and PFAM (Bateman et al.,
1999, Nucleic Acids Res 27:260-262), among others. BLASTP analysis
indicated that the Arabidopsis contains a single ICL gene. (A
variant predicted protein derived from the same nucleotide
sequence, which differs only in the first several amino acids of
the amino-terminal end, is presented in GI 15233130; the
discrepancy may be based on an incorrect gene prediction.) We
identified ICL (ICL) orthologs in a variety of plant species, as
presented in Table 2. When the same sequences are provided in
multiple Genbank entries, more than one GI number may be
provided.
TABLE-US-00002 TABLE 2 % Identity to SEQ ID Species (common name)
GI number(s) NO: 2 Brassica napus (canola) 113026, 167144 95
Brassica napus (canola) 2143227 94 Gossypium hirsutum 113029, 18486
84 (cotton) Cucurbita maxima (winter 8134299, 1695645 84 squash)
Ricinus communis (castor 113032, 169707, 84 bean) 68210 Cucumis
sativus 1351840, 1052578 84 (cucumber) Lycopersicon esculentum
1351841, 624211 81 (tomato) Ipomoea batatas (sweet 12005499 80
potato) Glycine max (soybean) 1168290, 349329 76 Glycine max
(soybean) 1168289 76 Dendrobium crumenatum 11131348 77 (orchid)
Pinus taeda (pine) 3831487, 1353642 75 Zea mays (corn) 1562544
(partial 67* sequence) Oryza sativa (rice) 18201655 (partial 78*
sequence) Solanum tuberosum translation of 84* (potato) 9250075
(EST) *For Zea mays, Oryza sativa and Solanum tuberosum sequences,
percent identity calculations were performed only over the partial
sequence.
[0070] While ICL mutants have been identified (Eastmond and Graham,
2000, supra), the association between ICL/ICL and an altered oil
content phenotype has not previously been reported.
Example 4
Application to Molecular Breeding
[0071] The disclosed ICL gene sequences may be used as molecular
probes to monitor occurrence and segregation of the ICL gene in
commercial oilseed germplasm. For instance, radiolabelled ICL
fragments may be used as RFLP markers (Helentjaris et al. TAG
(1986) 72:761-769). The utility of RFLP markers in plant breeding
is well established (Tanksley et al., Bio/Technology (1989)
7:257-264. Other sequence-based markers may be generated using the
disclosed ICL sequences or closely related sequences. These include
Single Nucleotide Polymorphisms (Jander et al. Plant Physiology
(2002) 129: 440-450), and cleavage amplified polymorphic DNAs
(Glazebrook et al., 1998 pp 173-182, in Arabidopsis Protocols,
Humana Press Totowa, N.J.).
[0072] PCR probes designed to amplify ICL sequences may be used to
quantify the expression level of ICL genes in commercial germplasm
of oilseed crops. Detection of altered expression of ICL sequences
can allow selection of germplasm for increased oil content.
Example 5
Constitutive Overexpression of ICL Gene Sequences in Transgenic
Plants to Produce Increased Seed Oil Content
[0073] Plasmids containing the Arabidopsis ICL gene sequence under
the control of the CsVMV promoter (Verdauger et al. 1996, Plant Mol
Biology 31(6): 1129-1139) were constructed (designated construct
pNT-4506), and used to transform wild type Arabidopsis.
[0074] Oligonucleotide primers were designed to amplify the ICL
gene, TAIR gene name At3g21720, from Arabidopsis genomic DNA. A 5'
EcoRI restriction site and 3' SpeI restriction site were engineered
into the primers to facilitate subcloning. The 5' primer was
designed upstream of the ICL start codon and the 3' primer was
designed downstream of the stop.
[0075] The ICL gene was subcloned into a cloning vector using the
pCR-Script Amp Cloning Kit from Stratagene (LaJolla, Calif.). The
amplified gene was verified by complete sequencing of both strands
and comparing the sequence to the published Genbank sequence
acc#AB025634. The 3.2 kb ICL gene was isolated from the pCR-Script
Amp vector by digestion with EcoRI and SpeI and cloned into a plant
expression vector (pAG-4217) containing the CsVMV promoter. The
resulting plasmid, pNT-4506 (CsVMV) was verified by PCR,
restriction digestion and sequencing across the junctions.
[0076] 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 pNT-4506 vector,
which comprises T-DNA derived from the Agrobacterium Ti plasmid, an
antibiotic resistance selectable marker gene, and the ICL coding
sequence under the control of the CsVMV promoter. Individual
transgenic plants were selected at the T1 generation based on
antibiotic resistance and transferred to individual 2'' pots to
grow to maturity. An equal number of wild type (Col-0) plants were
subjected to the same growth process and transplantation without
selection. At flowering individual plants were fitted with an
ARACONS.TM. (Lehle Seeds, Round Rock Tex.) seed harvesting device.
T2 seed was collected from individual T1 plants and wild type
controls and stored in barcoded tubes for analysis. To allow
non-destructive determination of oil content, Near Infrared
Reflectance (NIR) spectroscopy was employed essentially as
described in AOCS Procedure Am1-92 (Official Methods of the AOCS,
Fifth Edition, AOCS, Champaign, Ill.). Briefly, an IFS 28/N NIR
Spectrophotometer (Bruker Optics, Billerica, Mass.) was used to
determine oil content as measured by AOAC Method 920.39 (Fat
(Crude) or Ether Extract in Animal Feed, AOAC International,
Official Methods of Analysis, 17.sup.th Edition, AOAC
International, Gaithersburg Md.). Thirty-nine reference samples
having oil contents (determined by the reference method) between 23
and 41% were subjected to NIR analysis using the IFS28/.N and used
to construct a calibration curve using the manufacturer-supplied
software (OPUS, Quant2, Bruker Optics, Billerica, Mass.). The
correlation coefficient of the calibration was 0.9853.
[0077] Three independent spectra were taken of each T2 and controls
seed pool and the resulting oil determinations for each sample were
averaged. Independent transgenic events often behave differently in
terms of gene expression due to a variety of factors. In practice
the skilled artisan recognizes that many transgenic events produce
little effect and that selections can be made from those events
displaying the best performance. Despite the expectation that many
of the events may have little effect as a first approximation we
compared all CsVMV events with all the controls to determine if
there was a significant overall difference. Table 4 shows the
results of a t-test comparing means oil content of all control
lines with mean oil content of all CsVMV lines. It can be seen in
Table 3 that the mean oil content of the ICL events is higher than
the mean oil content of the controls. This difference in mean oil
content is highly statistically significant (P<0.001) for both
one and two tailed tests. Comparing the top 10 pNT4506
transformants to the wild type controls indicated that seed oil
content could be increased as much as 5% relative to the wild
type.
TABLE-US-00003 TABLE 3 Comparison of Mean oil content between
transgenic and control Control CsVMV Mean 37.76506977 38.60838462
Variance 1.266189066 1.521660202 Observations 43 52 Hypothesized
Mean Difference 0 Df 92 t Stat -3.480467455 P(T <= t) one-tail
0.00038362 t Critical one-tail 1.661585429 P(T <= t) two-tail
0.000767239 t Critical two-tail 1.986086318 t-Test: Two-Sample
Assuming Unequal Variances
Example 6
Seed Specific Expression of ICL Gene Sequences in Transgenic Plants
to Produce Altered Seed Oil Content
[0078] Plasmids were constructed containing the Arabidopsis ICL
gene sequence under the control of a promoter isolated from the
putative cherry (Prunus avium) ortholog of the almond (Prunus
amygdalus) prunin gene, which we have designated the PRU promoter
(see U.S. provisional patent application No. 60/400,170), and used
to transform wild type Arabidopsis.
[0079] Oligonucleotide primers were designed to amplify the ICL
gene, TAIR gene name At3g21720, from Arabidopsis genomic DNA. A 5'
EcoRI restriction site and 3' SpeI restriction site were engineered
into the primers to facilitate subcloning. The 5' primer was
designed upstream of the ICL start codon and the 3' primer was
designed downstream of the stop.
[0080] The ICL gene was subcloned into a cloning vector using the
pCR-Script Amp Cloning Kit from Stratagene (LaJolla, Calif.). The
amplified gene was verified by sequencing both strands and
comparing the sequence to the published Genbank sequence
acc#AB025634. The gene was also isolated from the pCR-Script Amp
vector by digestion with SmaI and SacII and cloned into a plant
expression vector (pNT-4269+MCS) containing the Pru promoter, which
was digested with EcoRV and SacII. The resulting plasmid, pNT-4706
(Pru) was verified by PCR, restriction digestion and sequencing
across the junctions. Verification PCR primers were chosen such
that the forward primer was located in the gene and the reverse
primer was located in the terminator.
[0081] Comparing the PRU::ICL events with all the controls reveals
an overall decrease in oil content in the transgenics that is
statistically significant (P T<=t=0.03). Thus, depending on the
tissue and temporal specificity of the promoter directing
expression of ICL genes different and opposite effects in oil
content may be created.
TABLE-US-00004 TABLE 4 T-test of All PRU events vs. all Controls
PRU:ICL Control Mean 36.90017 37.76507 Variance 4.34653 1.266189
Observations 38 43 Hypothesized Mean Difference 0 Df 55 t Stat
-2.28058 P(T <= t) one-tail 0.013236 t Critical one-tail
1.673034 P(T <= t) two-tail 0.026471 t Critical two-tail
2.004044 t-Test: Two-Sample Assuming Unequal Variances
Example 7
Constitutive Overexpression of a Glycine max ICL Gene Sequence in
Transgenic Plants to Produce Increased Seed Oil Content
[0082] Plasmids containing a Glycine max ICL gene sequence under
the control of the CsVMV promoter (Verdauger et al. 1996, Plant Mol
Biology 31(6)1129-1139) were constructed (designated construct
pNT-4508), and used to transform wild type Arabidopsis. RNA was
extracted from soybean cotyledons 3 days post inhibition using Tri
Reagent (Product number T9424, Molecular Research Center, Inc.).
The RNA was reverse transcribed using a mixture of oligonucleotide
GmICLR1 and oligo(dT)13 with Promega MMLV-RT (Product number
M1701). The GmICL gene was amplified from the cDNA using the Expand
High Fidelity PCR System from Roche and the GmICL1_F and GmICL1_R
primer set.
TABLE-US-00005 (SEQ ID NO: 3) GmICL1_F
5'-CCATGGCTGCATCATTATTTATG-3' (SEQ ID NO: 4) GmICL1_R
5'-CACTTTCACATTCTGGCCTTAG-3' (SEQ ID NO: 5) GmICLR1
5'-TCACATTCTGGCCTTAGCAACCACAATACTGC-3'
[0083] The GmICL PCR product was cloned using the pCR2.1 TA Cloning
Kit from Invitrogen (Product number K2000-01). The amplified gene
was verified by sequencing both the upper and lower strands and
comparing the sequence to cDNA contigs that were constructed from
ESTs and partial cDNA sequences. The gene was determined to be
GmICL2. The 1.7 kb GmICL2 gene was isolated from the pCR2.1 vector
by digestion with EcoRI. The gene was cloned into a plant
expression vector containing the CsVMV promoter (pAG-4217) which
was also digested with EcoRI and dephosphorylated with Shrimp
Alkaline Phosphatase (Roche, Product number 1758250).
[0084] Standard methods were used for the generation of Arabidopsis
transgenic plants, and were essentially as described in Example 5.
Oil content was determined by NIR essentially as described in
Example 5.
[0085] Three independent spectra are taken of each T2 and controls
seed pool and the resulting oil determinations for each sample are
averaged. Oil Contents for CsVMV transgenics and Col-0 controls can
then be compared and high oil events selected for further
propagation.
[0086] Expression of a Glycine max and Arabidopsis ICL Gene
Sequences in Transgenic Plants Under the Control of Tissue Specific
Promoters
[0087] Plasmids containing the Arabidopsis ICL gene sequence or
Glycine max ICL (GmICL) sequence under the control of the PRU
promoter (the associated promoter sequence recovered from the
putative cherry (Prunus avium) orthologs of the almond (Prunus
amygdalus) prunin gene, designated as the PRU promoter), or other
tissue specific promoters of choice are created using standard
methods. For instance, the GmICL2 gene is isolated from the pCR2.1
vector by digestion with BamHI and EcoRV and cloned into the
Gateway entry vector pENTR1A also digested with BamHI and EcoRV.
The GmICL2 gene is then cloned into a plant expression vector
(pNT-4287) containing the Pru promoter using the Gateway System
(Invitrogen) according to the manufacturers instructions. pNT-4287
is converted into a Gateway destination vector by the addition of a
Gateway Reading Frame. The Arabidopsis ICL gene is isolated from
the pCR-Script Amp vector by digestion with SmaI and SacII and
cloned into a plant expression vector (pNT-4269+MCS) containing the
Pru promoter which was digested with EcoRV and SacII. Both SmaI and
EcoRV are blunt cutters. The resulting plasmids, pNT-4708
(PRU-GmICL) and pNT-4706 (Pru-Hio1.4 Arabidopsis thaliana) are
verified by PCR, restriction digestion and sequencing across the
junctions. Verification PCR primers were chosen such that the
forward primer was located in the gene and the reverse primer was
located in the terminator.
[0088] Standard methods are used for the generation of Arabidopsis
transgenic plants, and are essentially as described in Example 5.
Oil content is determined by NIR essentially as described in
Example 5.
[0089] Three independent spectra are taken of each T2 and controls
seed pool and the resulting oil determinations for each sample are
averaged. Oil Contents for CsVMV transgenics and Col-0 controls can
then be compared and high oil transgenic events selected for
further propagation.
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Sequence CWU 1
1
511731DNAArabidopsis thaliana 1atggctgcat ctttctctgt cccctctatg
ataatggaag aagaagggag attcgaagcg 60gaggttgcgg aagtgcagac ttggtggagc
tcagagaggt tcaagctaac aaggcgccct 120tacactgccc gtgacgtggt
ggctctacgt ggccatctca agcaaggcta tgcttcgaac 180gagatggcta
agaagctgtg gagaacgctc aaaagccatc aagccaacgg tacggcctct
240cgcaccttcg gagcgttgga ccctgttcag gtgaccatga tggctaaaca
tttggacacc 300atctatgtct ctggttggca gtgctcgtcc actcacacat
ccactaatga gcctggtcct 360gatcttgctg attatccgta cgacaccgtt
cctaacaagg ttgaacacct cttcttcgct 420cagcagtacc atgacagaaa
gcagagggag gcaagaatga gcatgagcag agaagagagg 480acaaaaactc
cgttcgtgga ctacctaaag cccatcatcg ccgacggaga caccggcttt
540ggcggcacca ccgccaccgt caaactctgc aagcttttcg ttgaaagagg
cgccgctggg 600gtccacatcg aggaccagtc ctccgtcacc aagaagtgtg
gccacatggc cggaaaggtc 660ctcgtggcag tcagcgaaca catcaaccgc
cttgtcgcgg ctcggctcca gttcgacgtg 720atgggtacag agaccgtcct
tgttgctaga acagatgcgg tcgcagctac tctgatccag 780tcgaacattg
acgcgaggga ccaccagttc atcctcggtg ccactaaccc gagccttaga
840ggcaagagtt tgtcctcgct tctggctgag ggaatgactg taggcaagaa
tggtccggcg 900ttgcaatcca ttgaagatca gtggcttggc tcggccggtc
ttatgacttt ctcggaagct 960gtcgtgcagg ccatcaagcg catgaacctc
aacgagaacg agaagaatca gagactgagc 1020gagtggttaa cccatgcaag
gtatgagaac tgcctgtcaa atgagcaagg ccgagtgtta 1080gcagcaaaac
ttggtgtgac agatcttttc tgggactggg acttgccgag aaccagagaa
1140ggattctacc ggttccaagg ctcggtcgca gcggccgtgg tccgtggctg
ggcctttgca 1200cagatcgcag acatcatctg gatggaaacg gcaagccctg
atctcaatga atgcacccaa 1260ttcgccgaag gtatcaagtc caagacaccg
gaggtcatgc tcgcctacaa tctctcgccg 1320tccttcaact gggacgcttc
cggtatgacg gatcagcaga tggttgagtt cattccgcgg 1380attgctaggc
tcggatattg ttggcagttc ataacgcttg cgggtttcca tgcggatgct
1440cttgtggttg atacatttgc aaaggattac gctaggcgcg ggatgttggc
ttatgtggag 1500aggatacaaa gagaagagag gacccatggg gttgacactt
tggctcacca gaaatggtcc 1560ggtgctaatt actatgatcg ttatcttaag
accgtccaag gtggaatctc ctccactgca 1620gccatgggaa aaggtgtcac
tgaagaacag ttcaaggaga gttggacaag gccgggagct 1680gatggaatgg
gtgaagggac tagccttgtg gtcgccaagt caagaatgta a
17312576PRTArabidopsis thaliana 2Met Ala Ala Ser Phe Ser Val Pro
Ser Met Ile Met Glu Glu Glu Gly1 5 10 15Arg Phe Glu Ala Glu Val Ala
Glu Val Gln Thr Trp Trp Ser Ser Glu20 25 30Arg Phe Lys Leu Thr Arg
Arg Pro Tyr Thr Ala Arg Asp Val Val Ala35 40 45Leu Arg Gly His Leu
Lys Gln Gly Tyr Ala Ser Asn Glu Met Ala Lys50 55 60Lys Leu Trp Arg
Thr Leu Lys Ser His Gln Ala Asn Gly Thr Ala Ser65 70 75 80Arg Thr
Phe Gly Ala Leu Asp Pro Val Gln Val Thr Met Met Ala Lys85 90 95His
Leu Asp Thr Ile Tyr Val Ser Gly Trp Gln Cys Ser Ser Thr His100 105
110Thr Ser Thr Asn Glu Pro Gly Pro Asp Leu Ala Asp Tyr Pro Tyr
Asp115 120 125Thr Val Pro Asn Lys Val Glu His Leu Phe Phe Ala Gln
Gln Tyr His130 135 140Asp Arg Lys Gln Arg Glu Ala Arg Met Ser Met
Ser Arg Glu Glu Arg145 150 155 160Thr Lys Thr Pro Phe Val Asp Tyr
Leu Lys Pro Ile Ile Ala Asp Gly165 170 175Asp Thr Gly Phe Gly Gly
Thr Thr Ala Thr Val Lys Leu Cys Lys Leu180 185 190Phe Val Glu Arg
Gly Ala Ala Gly Val His Ile Glu Asp Gln Ser Ser195 200 205Val Thr
Lys Lys Cys Gly His Met Ala Gly Lys Val Leu Val Ala Val210 215
220Ser Glu His Ile Asn Arg Leu Val Ala Ala Arg Leu Gln Phe Asp
Val225 230 235 240Met Gly Thr Glu Thr Val Leu Val Ala Arg Thr Asp
Ala Val Ala Ala245 250 255Thr Leu Ile Gln Ser Asn Ile Asp Ala Arg
Asp His Gln Phe Ile Leu260 265 270Gly Ala Thr Asn Pro Ser Leu Arg
Gly Lys Ser Leu Ser Ser Leu Leu275 280 285Ala Glu Gly Met Thr Val
Gly Lys Asn Gly Pro Ala Leu Gln Ser Ile290 295 300Glu Asp Gln Trp
Leu Gly Ser Ala Gly Leu Met Thr Phe Ser Glu Ala305 310 315 320Val
Val Gln Ala Ile Lys Arg Met Asn Leu Asn Glu Asn Glu Lys Asn325 330
335Gln Arg Leu Ser Glu Trp Leu Thr His Ala Arg Tyr Glu Asn Cys
Leu340 345 350Ser Asn Glu Gln Gly Arg Val Leu Ala Ala Lys Leu Gly
Val Thr Asp355 360 365Leu Phe Trp Asp Trp Asp Leu Pro Arg Thr Arg
Glu Gly Phe Tyr Arg370 375 380Phe Gln Gly Ser Val Ala Ala Ala Val
Val Arg Gly Trp Ala Phe Ala385 390 395 400Gln Ile Ala Asp Ile Ile
Trp Met Glu Thr Ala Ser Pro Asp Leu Asn405 410 415Glu Cys Thr Gln
Phe Ala Glu Gly Ile Lys Ser Lys Thr Pro Glu Val420 425 430Met Leu
Ala Tyr Asn Leu Ser Pro Ser Phe Asn Trp Asp Ala Ser Gly435 440
445Met Thr Asp Gln Gln Met Val Glu Phe Ile Pro Arg Ile Ala Arg
Leu450 455 460Gly Tyr Cys Trp Gln Phe Ile Thr Leu Ala Gly Phe His
Ala Asp Ala465 470 475 480Leu Val Val Asp Thr Phe Ala Lys Asp Tyr
Ala Arg Arg Gly Met Leu485 490 495Ala Tyr Val Glu Arg Ile Gln Arg
Glu Glu Arg Thr His Gly Val Asp500 505 510Thr Leu Ala His Gln Lys
Trp Ser Gly Ala Asn Tyr Tyr Asp Arg Tyr515 520 525Leu Lys Thr Val
Gln Gly Gly Ile Ser Ser Thr Ala Ala Met Gly Lys530 535 540Gly Val
Thr Glu Glu Gln Phe Lys Glu Ser Trp Thr Arg Pro Gly Ala545 550 555
560Asp Gly Met Gly Glu Gly Thr Ser Leu Val Val Ala Lys Ser Arg
Met565 570 575323DNAArtificialOligonucleotide 3ccatggctgc
atcattattt atg 23422DNAArtificialOligonucleotide 4cactttcaca
ttctggcctt ag 22532DNAArtificialOligonucleotide 5tcacattctg
gccttagcaa ccacaatact gc 32
* * * * *