WO2006047159A1 - Generation of plants with altered oil content - Google Patents

Generation of plants with altered oil content Download PDF

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Publication number
WO2006047159A1
WO2006047159A1 PCT/US2005/037528 US2005037528W WO2006047159A1 WO 2006047159 A1 WO2006047159 A1 WO 2006047159A1 US 2005037528 W US2005037528 W US 2005037528W WO 2006047159 A1 WO2006047159 A1 WO 2006047159A1
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plant
oil
sequence
plants
seed
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PCT/US2005/037528
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French (fr)
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Jonathan Lightner
Hein Tsoeng Ng (Medard)
John P. Davies
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Agrinomics Llc
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Publication of WO2006047159A1 publication Critical patent/WO2006047159A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants

Definitions

  • 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).
  • oil Corn Refiners Association
  • 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.
  • ethanol production where flavor is unimportant, increasing oil content may increase overall value.
  • 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.
  • compositional alteration 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 High-Oil Corn
  • 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. 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.
  • T-DNA mutagenesis screens (Feldmann et ah, 1989) that detected altered fatty acid composition identified the omega 3 desaturase (FAD 3) and delta- 12 desaturase (FAD2) genes (U.S. Pat No 5952544; 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).
  • DGAT diacylglycerol acyltransferase
  • seed-specific over-expression of the DGAT cDNA was associated with increased seed oil content (Jako et al, 2001).
  • 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.
  • Activation tagging has been used in various species, including tobacco and
  • 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 HIO 128.4 polypeptide, hi 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.
  • 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 "HIO 128.4") polypeptide.
  • 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.
  • 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 HIO 128.4 polypeptide.
  • the invention also provides feed, meal, grain, food, or seed comprising the HIO 128.4 polypeptide, or an ortholog thereof.
  • 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 HIO128.4 polypeptide, and growing the transformed progenitor cells to produce a transgenic plant, wherein the HIO128.4 polynucleotide sequence is expressed causing the high oil phenotype.
  • the invention provides a plant that is the direct progeny or the indirect progeny of a plant grown from said progenitor cells.
  • the invention further provides plant cells obtained from said transgenic plant.
  • the invention also provides plant cells from a plant that is the direct progeny or the indirect progeny of a plant grown from said progenitor cells.
  • 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.
  • 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.
  • Any of the plants or parts thereof of the present invention may be processed to produce a feed, food, meal, or oil preparation.
  • a particularly preferred plant part for this purpose is a seed.
  • Li a preferred embodiment the feed, food, meal, or oil preparation is designed for ruminant animals.
  • Methods to produce feed, food, meal, and oil preparations are known in the art. See, for example, U.S. Patents 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, hi 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.
  • 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.
  • vector refers to a nucleic acid construct designed for transfer between different host cells.
  • 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.
  • 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.
  • control sequence i.e. promoter or enhancer
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mi-expression and altered expression encompass over-expression, under- expression, and ectopic expression.
  • 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) .
  • 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.
  • mutant and wild-type refers to the form in which that trait or phenotype is found in the same variety of plant in nature.
  • 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 Tl 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.
  • 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.
  • 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.
  • 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.
  • Tl refers to the generation of plants from the seed of TO plants.
  • the Tl 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.
  • T2 refers to the generation of plants by self-fertilization of the flowers of Tl plants, previously selected as being transgenic.
  • T3 plants are generated from T2 plants, etc.
  • 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 Tl 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 Tl plant.
  • 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.
  • 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.
  • 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.
  • Various methods for the introduction of a desired polynucleotide sequence encoding the desired protein into plant cells 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.
  • Agrobacterium-mediated DNA transfer process and the biolistics or microprojectile bombardment mediated process (i.e., the gene gun).
  • the gene gun i.e., the gene gun
  • 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.
  • Agrobacterium-mediated transformation is achieved through the use of a genetically engineered soil bacterium belonging to the genus Agrob ⁇ cterium.
  • a number of wild-type and disarmed strains of Agrob ⁇ cterium tumef ⁇ ciens and Agrob ⁇ cterium 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.
  • Agrobacterium-mediated genetic transformation of plants involves several steps.
  • the first step in which the virulent Agrob ⁇ cterium and plant cells are first brought into contact with each other, is generally called “inoculation”.
  • the Agrob ⁇ cterium 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”.
  • the plant cells are treated with bactericidal or bacteriostatic agents to kill the Agrob ⁇ cterium 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.
  • 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.
  • An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System (BioRad, Hercules, CA), 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.
  • Microprojectile bombardment techniques are widely applicable, and may be used to transform virtually any plant species.
  • 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. Patent 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. Patent No. 5,322,783 (Tomes et al), incorporated herein by reference in its entirety), sunflower, peanut, cotton, tomato, and legumes in general (U.S. Patent No.
  • 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 include, but are not limited to a polynucleotide molecule encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) described in U.S. Patent No. 5,627,061 (Barry, et al.), U.S. Patent No 5,633,435 (Barry, et al), and U.S. Patent No 6,040,497 (Spencer, et al.) and aroA described in U.S. Patent No.
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • 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.
  • 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.
  • Any suitable plant culture medium can be used.
  • suitable media would include but are not limited to MS-based media (Murashige and Skoog,
  • 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.
  • 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.
  • 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.
  • HIO128.4 Arabidopsis activation tagging
  • 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).
  • the enhancer element can cause up-regulation of genes in the vicinity, generally within about 10 kilobase (kb) of the enhancer. Tl plants were exposed to the selective agent in order to specifically recover transformed plants.
  • NIR Near Infrared Spectroscopy
  • HIO128.4 genes and/or polypeptides maybe employed in the development of genetically modified plants having a modified oil content phenotype ("a HIO128.4 phenotype").
  • HIO128.4 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. HIO128.4 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 HIO128.4 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.
  • Arabidopsis HIO 128.4 nucleic acid (genomic DNA) sequence is provided in SEQ ID NO: 1 and in Genbank entry GI#30693148.
  • the corresponding protein sequence is provided in SEQ ID NO:2 and in GI#15240340. Nucleic acids and/or proteins that are orthologs or paralogs of Arabidopsis HIO 128.4, are described in Example 3 below.
  • HIO128.4 polypeptide refers to a full-length HIO 128.4 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.
  • a functionally active HIO128.4 polypeptide causes an altered oil content phenotype when mis-expressed in a plant.
  • mis-expression of the HIO128.4 polypeptide causes a high oil phenotype in a plant.
  • a functionally active HIO128.4 polypeptide is capable of rescuing defective (including deficient) endogenous HIO128.4 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.
  • a functionally active fragment of a full length HIO128.4 polypeptide i.e., a native polypeptide having the sequence of SEQ ID NO:2 or a naturally occurring ortholog thereof
  • a HIO 128.4 fragment preferably comprises a HIO 128.4 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 HIO128.4 protein.
  • Functional domains can be identified using the PFAM program (Bateman A et at, 1999 Nucleic Acids Res 27:260-262).
  • a preferred HIO128.4 fragment comprises of one or more calmodulin-related protein 2, touch-induced (TCH2) proteins.
  • variants of full-length HIO128.4 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 HIO128.4 polypeptide.
  • variants are generated that change the post-translational processing of a HIO 128.4 polypeptide. For instance, variants may have altered protein transport or protein localization characteristics or altered protein half-life compared to the native polypeptide.
  • HIO128.4 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.
  • a HIO128.4 nucleic acid of this invention may be DNA, derived from genomic DNA or cDNA, or RNA.
  • a functionally active HIO 128.4 nucleic acid encodes or is complementary to a nucleic acid that encodes a functionally active HIO128.4 polypeptide.
  • 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 HIO128.4 polypeptide.
  • a HIO 128.4 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.
  • functionally active nucleic acids may encode the mature or the pre-processed HIO128.4 polypeptide, or an intermediate form.
  • a HIO128.4 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.
  • a functionally active HIO 128.4 nucleic acid is capable of being used in the generation of loss-of-function HIO128.4 phenotypes, for instance, via antisense suppression, co-suppression, etc.
  • a HIO 128.4 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 HIO128.4 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.
  • a HIO128.4 polypeptide of the invention comprises a polypeptide sequence with at least 50% or 60% identity to the HIO128.4 polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the HIO 128.4 polypeptide sequence of SEQ ID NO:2, such as one or more calmodulin-related protein 2, touch-induced (TCH2) proteins.
  • a HIO128.4 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.
  • a HIO128.4 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 of one or more calmodulin-related protein 2, touch-induced (TCH2) proteins.
  • a HIO128.4 polynucleotide sequence is at least 50% to 60% identical over its entire length to the HIO 128.4 nucleic acid sequence presented as SEQ ID NO:1, or nucleic acid sequences that are complementary to such a HIO128.4 sequence, and may comprise at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the HIO 128.4 sequence presented as SEQ ID NO: 1 or a functionally active fragment thereof, or complementary sequences.
  • 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-
  • 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.
  • 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.
  • 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)).
  • 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° C in a solution comprising 6X single strength citrate (SSC) (IX SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 ⁇ g/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, IX Denhardt's solution, 100 ⁇ g/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for 1 h in a solution containing 0.1X SSC and 0.1% SDS (sodium dodecyl sulfate).
  • SSC single strength citrate
  • moderately stringent hybridization conditions are used that are: pretreatment of filters containing nucleic acid for 6 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 ⁇ g/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 ⁇ g/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in a solution containing 2X SSC and 0.1% SDS.
  • low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37° C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour.
  • a number of polynucleotide sequences encoding a HIO128.4 polypeptide can be produced.
  • 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.
  • the methods of the invention may use orthologs of the Arabidopsis HIO 128.4. 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.
  • JD 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.
  • 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
  • Nucleic acid hybridization methods may also be used to find orthologous genes and are preferred when sequence data are not available.
  • HIO128.4 ortholog nucleic acids may hybridize to the nucleic acid of SEQ ID NO: 1 under high, moderate, or low stringency conditions.
  • HIO128.4 ortholog i.e., an orthologous protein
  • 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 gtl 1, 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 HIO128.4 nucleic acid and/or polypeptide sequences have been identified.
  • HIO 128.4 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 TA et ah, 1991), maybe used to introduce desired changes into a cloned nucleic acid.
  • the methods of the invention involve incorporating the desired form of the HIO 128.4 nucleic acid into a plant expression vector for transformation of plant cells, and the HIO128.4 polypeptide is expressed in the host plant.
  • An isolated HIO 128.4 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 HIO128.4 nucleic acid.
  • an isolated HIO128.4 nucleic acid molecule includes HIO 128.4 nucleic acid molecules contained in cells that ordinarily express HIO 128.4 where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • HIO128.4 nucleic acids and polypeptides may be used in the generation of genetically modified plants having a modified oil content phenotype.
  • 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.
  • altered expression of the HIO128.4 gene in a plant is used to generate plants with a high oil phenotype.
  • the methods described herein are generally applicable to all plants. Although activation tagging and gene identification is carried out in Arabidopsis, the HIO128.4 gene (or an ortholog, variant or fragment thereof) may be expressed in any type of plant, hi 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 ⁇ Tlieobroma cacao), saffiower ⁇ 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.
  • 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.
  • 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.
  • a heterologous nucleic acid construct comprising a HIO 128.4 polynucleotide may encode the entire protein or a biologically active portion thereof.
  • binary Ti-based vector systems may be used to transfer polynucleotides.
  • Standard Agrob ⁇ cterium binary vectors are known to those of skill in the art, and many are commercially available (e.g., pBI121 Clontech Laboratories, Palo Alto, CA).
  • a construct or vector may include a plant promoter to express the nucleic acid molecule of choice.
  • the promoter is a plant promoter.
  • the optimal procedure for transformation of plants with Agrob ⁇ cterium vectors will vary with the type of plant being transformed.
  • Exemplary methods for Agrob ⁇ cterium-medi&ted 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.
  • HIO128.4 may be regulated with respect to the level of expression, the tissue type(s) where expression takes place and/or developmental stage of expression.
  • heterologous regulatory sequences are available for controlling the expression of a HIO128.4 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. Patent Nos. 5,783,393 and 5,783,394), the nopaline synthase (NOS) promoter (Ebert et al, Proc. Natl. Acad.
  • OCS octopine synthase
  • CaMV cauliflower mosaic virus
  • tissue-specific promoters include the tomato E4 and E8 promoters (U.S. Patent No. 5,859,330) and the tomato 2AII gene promoter (Van Haaren MJJ et al, 1993).
  • HIO 128.4 expression is under control of regulatory sequences from genes whose expression is associated with early seed and/or embryo development.
  • the promoter used is a seed-enhanced promoter. Examples of such promoters include the 5 1 regulatory regions from such genes as napin (Kridl et al, Seed ScL Res. 1:209:219, 1991), globulin (Belanger and Kriz, Genet., 129: 863-872, 1991, GenBank Accession No.
  • zeins are a group of storage proteins found in corn endosperm. Genomic clones for zein genes have been isolated (Pedersen et ah, Cell 29:1015-1026, 1982; and Russell et ah, Transgenic
  • 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.
  • V.faba legumin (Baumlein et ah, 1991, MoI Gen Genet 225:121-8; Baumlein et ah, 1992, Plant J 2:233-9), V.faba usp (Fiedler et ah, 1993, Plant MoI Biol 22:669-79), pea convicilin (Bown et ah, 1988, Biochem J 251:717-26), pea lectin (dePater et ah, 1993, Plant Cell 5:877-86), P. vulgaris beta phaseolin (Bustos et ah, 1991, EMBO J 10:1469- 79), P.
  • Cereal genes whose promoters are associated with early seed and embryo development include rice glutelin ("GluA-3,” Yoshihara and Takaiwa, 1996, Plant Cell Physiol 37:124-11; "GIuB-I,” Takaiwa et ah, 1996, Plant MoI Biol 30:1207-21; Washida et ah, 1999, Plant MoI Biol 40:1-12; "Gt3,” Leisy et ah, 1990, Plant MoI Biol 14:41-50), ⁇ ceprolamin (Zhou & Fan, 1993, Transgenic Res 2:141-6), wheat prolamin (Hammond-Kosack et ah, 1993, EMBO J 12:545-54), maize zein (Z4, Matzke et ah, 1990, Plant MoI Biol 14:323-32), and barley B-hordeins (Entwistle et ah, 1991, Plant MoI Biol 17:1217-31).
  • genes whose promoters are associated with early seed and embryo development include oil palm GL07A (7S globulin, Morcillo et ah, 2001, Physiol Plant 112:233-243), Brassica napus napin, 2S storage protein, and napA gene (Josefsson et ah, 1987, J Biol Chem 262:12196-201; Stalberg et ah, 1993, Plant MoI Biol 1993 23:671-83; Ellerstrom et ah, 1996, Plant MoI Biol 32:1019-27),
  • Brassica napus oleosin (Keddie et ah, 1994, Plant MoI Biol 24:327-40), Arabidopsis oleosin (Plant et ah, 1994, Plant MoI Biol 25:193-205), Arabidopsis FAEl (Rossak et ah, 2001, Plant MoI Biol 46:717-25), Canavalia gladiata conA (Yamamoto et ah, 1995, Plant MoI Biol 27:729-41), and Catharanthus roseus strictosidine synthase (Str, Ouwerkerk and Memelink, 1999, MoI Gen Genet 261:635-43).
  • regulatory sequences from genes expressed during oil biosynthesis are used (see, e.g., US 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. Patents 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.
  • exemplary methods for practicing this aspect of the invention include, but are not limited to antisense suppression (Smith, et «/.,1988; van der Krol et ah, 1988); co-suppression (Napoli, et ah, 1990); ribozymes (PCT Publication WO 97/10328); and combinations of sense and antisense (Waterhouse, et ah, 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 ah, 1988), a partial cDNA sequence including fragments of 5' coding sequence, (Cannon et ah, 1990), or 3' non-coding sequences (Ch'ng et ah, 1989).
  • Cosuppression techniques may use the entire cDNA sequence (Napoli et ah, 1990; van der Krol et ah, 1990), or a partial cDNA sequence (Smith et ah, 1990). 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.
  • 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]).
  • microarray analysis 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 NL et al, J
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention further provides a method of identifying plants that have mutations in, or an allele of, endogenous HIO128.4 that confer a HIO128.4 phenotype, and generating progeny of these plants that also have the HIO128.4 phenotype and are not genetically modified.
  • 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.
  • HIO 128.4- specific PCR is used to identify whether a mutated plant has a HIO128.4 mutation.
  • Plants having HIO128.4 mutations may then be tested for the HIO128.4 phenotype, or alternatively, plants may be tested for the HIO 128.4 phenotype, and then HIO128.4-specific PCR is used to determine whether a plant having the HIO128.4 phenotype has a mutated HIO128.4 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).
  • 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 HIO 128.4 gene or orthologs of HIO 128.4 that may confer the HIO128.4 phenotype (see Foolad et al, Theor Appl Genet. (2002) 104(6-7):945- 958; Rothan et al, Theor Appl Genet (2002) 105(l):145-159); Dekkers and Hospital, Nat Rev Genet. (2002) Jan;3(l):22-32).
  • QTLs Quality of Traitative Trait Locus
  • a HIO128.4 nucleic acid is used to identify whether a plant having a HIO 128.4 phenotype has a mutation in endogenous HIO128.4 or has a particular allele that causes the HIO128.4 phenotype compared to plants lacking the mutation or allele, and generating progeny of the identified plant that have inherited the HIO128.4 mutation or allele and have the HIO128.4 phenotype.
  • Activation Tagging Construct 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 WOOl 83697. Briefly, TO Arabidopsis (CoI-O) 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 4X CaMV 35S enhancer element. Transgenic plants were selected at the Tl generation based on herbicide resistance.
  • NIR Near Infrared Spectroscopy
  • Line W000041352 (IN009897) had a NIR determined oil content of 39.2% relative to a flat average oil content of 35.2% (111% of Flat Average).
  • Line W000210144 (TN088075) had a NIR determined oil content of 30.2% relative to a flat average oil content of 24.2% (124% of Flat Average).
  • Line W000041352 (IN009897) had a confirmed ACTTAG insertion on Chromosome 5 at bp 15023687.
  • Line W000210144 (IN088075) had a confirmed ACTTAG insertion on Chromosome 5 at bp 15020099.
  • 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 publicly available Arabidopsis Information Resource (TAIR) database.
  • TAIR Arabidopsis Information Resource
  • ACTTAG line IN009897 there was sequence identity to nucleotides 31217 - 8456 on Arabidopsis BAC clone K22F20 on chromosome 5 (GI#3449314), placing the left border junction upstream from nucleotide 8456 (GI#3449314).
  • Left border of IN009897 T-DNA was about 6897 bp 5' of the translation start site.
  • ACTTAG line IN088075 there was sequence identity to nucleotides 4490 - 4828 on Arabidopsis BAC clone K22F20 on chromosome 5 (GI#3449314), placing the left border junction upstream from nucleotide 4828 (GI#3449314).
  • Left border IN088075 T-DNA was about 3309 bp 5' of the translation start site.
  • the candidate gene At5g37770 is supported by the full-length cDNA (Tair Accession: Sequence 3708394) and by the NCBI entry GI#30693148.
  • the contigged sequence is presented as SEQ ID NO: 4.
  • the contigged sequence is presented as SEQ ID NO: 6.
  • the contigged sequence is presented as SEQ ID NO: 8.
  • the contigged sequence is presented as SEQ ID NO: 9.
  • the contigged sequence is presented as SEQ ID NO: 10.
  • the contigged sequence is presented as SEQ ID NO: 11.
  • the protein At5g37770 (TCH2) has homology to large numbers of EF-hand- containing proteins from plants, fungi and animals.
  • the Arabidopsis genome encodes more than 200 such proteins.
  • the top 3 BLAST results and the BLAST results for Atlg73630, At2g36180, At5gl7470, At3g03400, At3g03410, At2gl5680 are listed below. This is because these genes form a subclade among the EF-hand-containing proteins in Arabidopsis.
  • At2g36180 Arabidopsis > ⁇ i
  • 152275931 Length 144 BLASTP thaliana > ⁇ il20197940
  • At3g03400 Arabidopsis >qi
  • At2g15680 Arabidopsis > ⁇ il 15226592
  • Length 187 .
  • BLASTP thaliana > ⁇ il25411654
  • At5g37770 is a non-secretory protein that lacks transmembrane domain (predicted by TMHMM (Krogh et al, J. MoI. Biol. 2001 305:567-580)) and signal peptide (predicted by SignalP).
  • TMHMM transmembrane domain
  • SignalP signal peptide
  • At5g37770 has 4 EF-hand domains (PF00036, SM00054).
  • Many Ca2+-binding proteins belong to the same evolutionary family and share a type of Ca2+-binding domain known as the EF-hand. This type of domain consists of a twelve-residue loop flanked on both sides by a twelve-residue- helical domain.
  • the Ca2+ ion is coordinated in a pentagonal bipyramidal configuration.
  • the six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, -Y, -X and -Z.
  • the invariant GIu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand).
  • Ca2+ is an important messenger mediating plant responses to hormones, developmental cues and external stimuli. Changes in intracellular concentration of Ca2+ is implicated in regulating diverse cellular process such as cytoplasmic streaming, thigmotropism, gravitropism, cell division, cell elongation, cell differentiation, cell polarity, photomorphogenesis and plant defense and stress responses. Transient increase in the concentration of Ca2+ in the cytoplasm is sensed by Ca2+ binding proteins. The conformation of these protein changes when they bind Ca2+, resulting in modulation of its activity or its ability to interact with other proteins or nucleic acids and modulate their function or activity.
  • calmodulin which contains EF-hand motifs.
  • At5g37770 is one of two proteins that is induced by touch, rain, wind, wounding, darkness, increase in extracellular Ca2+ concentration and heat shock. At5g37770 is related to the calmodulins and belongs to a subclade within the group IV family of EF-hand containing proteins. The blastP alignments between At5g37770 and other proteins in the subclade were described above. The three- dimensional structure of At5g37770 (TCH2) has been solved and shown to resemble the structure of calmodulin.
  • Calmodulins are highly conserved small molecular weight acidic proteins that have 4 EF-hands and bind four molecules of Ca2+.
  • At5g37770 is involved in signal transduction in Arabidopsis. Binding of Ca2+ to At5g37770 is likely to result in conformational change which then allows At5g37770 to interact with target proteins and to modulate their activity or function.
  • *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.
  • At5g37770 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.
  • Arabidopsis plants of the CoI-O ecotype were transformed by Agrobacterium mediated transformation using the floral dip method, with a construct containing the coding sequences of the HIO128.4 gene (At5g37770) behind the CsVMV promoter and in front of the nos terminator or a control gene unrelated to pathogen resistance. Both of these constructs contain the nptll gene directed by the RE4 promoter to confer kanamycin resistance in plants and serve as a selectable marker.
  • Tl seed was harvested from the transformed plants and transformants selected by germinating seed on agar medium containing kanamycin. Kanamycin resistant transformants, identified as healthy green plants, were transplanted to soil after 7 days. Control plants were germinated on agar medium without kanamycin, transplanted to soil after 7 days. Twenty-two plants containing the CsVMV:: HIO 128.4 transgene along with 10 CoI-O control plants were grown in the same flat in the growth room. All plants were allowed to self-fertilize and set seed.
  • NIR Near Infrared Spectroscopy
  • 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 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.
  • OiI 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 1 H nuclear magnetic resonance (NMR) (Tiwari et ah, 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.
  • NMR nuclear magnetic resonance
  • JMP version 4.04, SAS Institute Inc., Cary, NC, USA
  • 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.
  • 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.
  • 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, NY).
  • the amino acids are separated by a reverse-phase Zorbax Eclipse XDB-C 18 HPLC column on an Agilent 1100 HPLC (Agilent, Palo Alto, CA).
  • the amino acids are detected by fluorescence. Cysteine, proline, asparagine, glutamine, and tryptophan are not included in this amino acid screen (Henderson et ah, "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 th edition, AACC ed, (2000) 07-20 Measurement of Tryptophan - Alakline Hydrolysis).
  • 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, OK) in a sterile microfuge tube to which 500 ⁇ 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 ⁇ m filter into an autosampler tube.
  • the filtered extracts are analyzed by HPLC using a Zorbax silica HPLC column (4.6 mmx250 mm) with a fluorescent detection, an excitation at 290 ran, an- emission at 336 ran, 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 ⁇ l, the flow rate is 1.5 ml/minute and the run time is 12 minutes at 4O 0 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 ( ⁇ - tocopherol, ⁇ -tocopherol, ⁇ -tocopherol, ⁇ -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 ( ⁇ -tocotrienol, ⁇ - tocotrienol, ⁇ - tocotrienol, ⁇ - tocotrienol).
  • Standard curves for ⁇ -, ⁇ -, ⁇ -, and ⁇ -tocotrienol are calculated using Chemstation software (Hewlett Packard).
  • 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 ah, Methods in Enzymology, XVIII, part C, 369-396 (1971); and Ramadan et ah, Eur. Food Res. Technoh, 214(6):521-527 (2002)).

Abstract

The present invention is directed to plants that display an altered oil content phenotype due to altered expression of a HIO128.4 nucleic acid. The invention is further directed to methods of generating plants with an altered oil content phenotype.

Description

GENERATION OF PLANTS WITH ALTERED OIL CONTENT
REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application Number 60/621 , 107, filed October 22, 2004, the contents of which are hereby incorporated by reference.
BACKGROUND OF THE INVENTION
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 feed 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.
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. 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.
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 ah, 1990; James and Dooner, 1990). T-DNA mutagenesis screens (Feldmann et ah, 1989) that detected altered fatty acid composition identified the omega 3 desaturase (FAD 3) and delta- 12 desaturase (FAD2) genes (U.S. Pat No 5952544; 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). 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. 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
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 HIO 128.4 polypeptide, hi 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.
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 "HIO 128.4") 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 HIO 128.4 polypeptide. The invention also provides feed, meal, grain, food, or seed comprising the HIO 128.4 polypeptide, or an ortholog thereof.
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 HIO128.4 polypeptide, and growing the transformed progenitor cells to produce a transgenic plant, wherein the HIO128.4 polynucleotide sequence is expressed causing the high oil phenotype. In other embodiments, the invention provides a plant that is the direct progeny or the indirect progeny of a plant grown from said progenitor cells. The invention further provides plant cells obtained from said transgenic plant. The invention also provides plant cells from a plant that is the direct progeny or the indirect progeny of a plant grown from said progenitor cells. 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.
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.
Any of the plants or parts thereof of the present invention may be processed to produce a feed, food, meal, or oil preparation. A particularly preferred plant part for this purpose is a seed. Li a preferred embodiment the feed, food, meal, or oil preparation is designed for ruminant animals. Methods to produce feed, food, meal, and oil preparations are known in the art. See, for example, U.S. Patents 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, hi 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
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 FM 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. 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.
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. 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. 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.
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.
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) .
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.
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.
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 Tl 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.
As used herein, the term "Tl" refers to the generation of plants from the seed of TO plants. The Tl 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 Tl 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 Tl 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 Tl plant.
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.
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.
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.
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. Agrobacterium-mediated transformation is achieved through the use of a genetically engineered soil bacterium belonging to the genus Agrobαcterium. A number of wild-type and disarmed strains of Agrobαcterium tumefαciens and Agrobαcterium 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.
Agrobacterium-mediated genetic transformation of plants involves several steps. The first step, in which the virulent Agrobαcterium and plant cells are first brought into contact with each other, is generally called "inoculation". Following the inoculation, the Agrobαcterium 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 Agrobαcterium 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.
With respect to microprojectile bombardment (U.S. Patent No. 5,550,318 (Adams et al); U.S. Patent No. 5,538,880 (Lundquist et. al), U.S. Patent 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. An illustrative embodiment of a method for delivering DNA into plant cells by acceleration is the Biolistics Particle Delivery System (BioRad, Hercules, CA), 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. 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. Patent 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. Patent No. 5,322,783 (Tomes et al), incorporated herein by reference in its entirety), sunflower, peanut, cotton, tomato, and legumes in general (U.S. Patent No. 5,563,055 (Townsend et al.) incorporated herein by reference in its entirety). 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. Patent No. 5,627,061 (Barry, et al.), U.S. Patent No 5,633,435 (Barry, et al), and U.S. Patent No 6,040,497 (Spencer, et al.) and aroA described in U.S. Patent No. 5,094,945 (Comai) for glyphosate tolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) described in U.S. Patent 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.
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.
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.
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.
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 Phenotvpe
We used an Arabidopsis activation tagging (ACTTAG) screen to identify the association between the gene we have designated "HIO128.4," (At5g37770; GI#30693148) encoding one or more calmodulin-related protein 2, touch-induced (TCH2) proteins (synonym: T31G3.4; GI#15240340), 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 of genes in the vicinity, generally within about 10 kilobase (kb) of the enhancer. Tl plants were exposed to the selective agent in order to specifically recover transformed plants. To amplify the seed stocks, about eighteen seeds from the Tl plants were sown in soil and, after germination, exposed to the selective agent to recover transformed T2 plants. T3 seed from these plants was harvested and pooled. Oil content of the seed was estimated using Near Infrared Spectroscopy (NIR) as described in Example 1.
An Arabidopsis line that showed a high-oil phenotype was identified wherein oil content (i.e., fatty acids) constituted about 39.2% compared to a flat average oil content of 35.2% for seed from other plants planted on that day (a 11% increase in oil). The association of the HIO128.4 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, HIO128.4 genes and/or polypeptides maybe employed in the development of genetically modified plants having a modified oil content phenotype ("a HIO128.4 phenotype"). HIO128.4 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. HIO128.4 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 HIO128.4 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.
HIO128.4 Nucleic Acids and Polypeptides
Arabidopsis HIO 128.4 nucleic acid (genomic DNA) sequence is provided in SEQ ID NO: 1 and in Genbank entry GI#30693148. The corresponding protein sequence is provided in SEQ ID NO:2 and in GI#15240340. Nucleic acids and/or proteins that are orthologs or paralogs of Arabidopsis HIO 128.4, are described in Example 3 below.
As used herein, the term "HIO128.4 polypeptide" refers to a full-length HIO 128.4 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 HIO128.4 polypeptide causes an altered oil content phenotype when mis-expressed in a plant. In a further preferred embodiment, mis-expression of the HIO128.4 polypeptide causes a high oil phenotype in a plant. Li another embodiment, a functionally active HIO128.4 polypeptide is capable of rescuing defective (including deficient) endogenous HIO128.4 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. Li another embodiment, a functionally active fragment of a full length HIO128.4 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 HIO 128.4 polypeptide, such as signaling activity, binding activity, catalytic activity, or cellular or extra-cellular localizing activity. A HIO 128.4 fragment preferably comprises a HIO 128.4 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 HIO128.4 protein. Functional domains can be identified using the PFAM program (Bateman A et at, 1999 Nucleic Acids Res 27:260-262). A preferred HIO128.4 fragment comprises of one or more calmodulin-related protein 2, touch-induced (TCH2) proteins. Functionally active variants of full-length HIO128.4 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 HIO128.4 polypeptide. In some cases, variants are generated that change the post-translational processing of a HIO 128.4 polypeptide. For instance, variants may have altered protein transport or protein localization characteristics or altered protein half-life compared to the native polypeptide.
As used herein, the term "HIO128.4 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. A HIO128.4 nucleic acid of this invention may be DNA, derived from genomic DNA or cDNA, or RNA. In one embodiment, a functionally active HIO 128.4 nucleic acid encodes or is complementary to a nucleic acid that encodes a functionally active HIO128.4 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 HIO128.4 polypeptide. A HIO 128.4 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 HIO128.4 polypeptide, or an intermediate form. A HIO128.4 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.
In another embodiment, a functionally active HIO 128.4 nucleic acid is capable of being used in the generation of loss-of-function HIO128.4 phenotypes, for instance, via antisense suppression, co-suppression, etc.
In one preferred embodiment, a HIO 128.4 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 HIO128.4 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.
In another embodiment a HIO128.4 polypeptide of the invention comprises a polypeptide sequence with at least 50% or 60% identity to the HIO128.4 polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the HIO 128.4 polypeptide sequence of SEQ ID NO:2, such as one or more calmodulin-related protein 2, touch-induced (TCH2) proteins. In another embodiment, a HIO128.4 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. In yet another embodiment, a HIO128.4 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 of one or more calmodulin-related protein 2, touch-induced (TCH2) proteins.
In another aspect, a HIO128.4 polynucleotide sequence is at least 50% to 60% identical over its entire length to the HIO 128.4 nucleic acid sequence presented as SEQ ID NO:1, or nucleic acid sequences that are complementary to such a HIO128.4 sequence, and may comprise at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the HIO 128.4 sequence presented as SEQ ID NO: 1 or a functionally active fragment thereof, or complementary sequences. 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-
2.0al9 (Altschul et al, J. MoI. Biol. (1990) 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.
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° C in a solution comprising 6X single strength citrate (SSC) (IX SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5X Denhardt's solution, 0.05% sodium pyrophosphate and 100 μg/ml herring sperm DNA; hybridization for 18-20 hours at 65° C in a solution containing 6X SSC, IX Denhardt's solution, 100 μg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65° C for 1 h in a solution containing 0.1X 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° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40° C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55° C in a solution containing 2X SSC and 0.1% SDS. Alternatively, low stringency conditions can be used that comprise: incubation for 8 hours to overnight at 37° C in a solution comprising 20% formamide, 5 x SSC, 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1 x SSC at about 37° C for 1 hour. As a result of the degeneracy of the genetic code, a number of polynucleotide sequences encoding a HIO128.4 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.
The methods of the invention may use orthologs of the Arabidopsis HIO 128.4. 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 MA and Bork P, Proc Natl Acad Sci (1998) 95:5849- 5856; Huynen MA et al, Genome Research (2000) 10:1204-1210). Programs for multiple sequence alignment, such as CLUSTAL (Thompson
JD 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 a A highly conserved portion of the Arabidopsis HIO128.4 coding sequence may be used as a probe. HIO128.4 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 HIO128.4 polypeptides are used for ortholog isolation (see, e.g., Harlow and Lane, 1988, 1999). Western blot analysis can determine that a HIO128.4 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 gtl 1, 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 HIO128.4 nucleic acid and/or polypeptide sequences have been identified.
HIO 128.4 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 TA et ah, 1991), maybe used to introduce desired changes into a cloned nucleic acid.
In general, the methods of the invention involve incorporating the desired form of the HIO 128.4 nucleic acid into a plant expression vector for transformation of plant cells, and the HIO128.4 polypeptide is expressed in the host plant. An isolated HIO 128.4 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 HIO128.4 nucleic acid. However, an isolated HIO128.4 nucleic acid molecule includes HIO 128.4 nucleic acid molecules contained in cells that ordinarily express HIO 128.4 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
HIO128.4 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 HIO128.4 gene in a plant is used to generate plants with a high oil phenotype.
The methods described herein are generally applicable to all plants. Although activation tagging and gene identification is carried out in Arabidopsis, the HIO128.4 gene (or an ortholog, variant or fragment thereof) may be expressed in any type of plant, hi 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 {Tlieobroma cacao), saffiower {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. 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 a HIO 128.4 polynucleotide may encode the entire protein or a biologically active portion thereof. In one embodiment, binary Ti-based vector systems may be used to transfer polynucleotides. Standard Agrobαcterium binary vectors are known to those of skill in the art, and many are commercially available (e.g., pBI121 Clontech Laboratories, Palo Alto, CA). 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. The optimal procedure for transformation of plants with Agrobαcterium vectors will vary with the type of plant being transformed. Exemplary methods for Agrobαcterium-medi&ted 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. Agrobαcterium 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 αl, 1989), sunflower (Everett et αl., 1987), and soybean (Christou et αl., 1989; Kline et αl., 1987). Expression (including transcription and translation) of HIO128.4 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 HIO128.4 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. Patent Nos. 5,783,393 and 5,783,394), the nopaline synthase (NOS) promoter (Ebert et al, Proc. Natl. Acad. ScL (U.S.A.) 84:5145- 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 MoI. Biol. P:315-324, 1987) and the CaMV 35S promoter (Odell et al, Nature 313:810- 812, 1985 and Jones JD et al, 1992), the melon actin promoter (published PCT application WO0056863), the figwort mosaic virus 35S-promoter (U.S. Patent No. 5,378,619), the light-inducible promoter from the small subunit of ribulose-l,5-bis- phosphate carboxylase (ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl. Acad. ScI (U.S.A.) 84:6624-662%, 1987), the sucrose synthase promoter (Yang et al, Proc. Natl. Acad. ScL (U.S.A.) 57:4144-4148, 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. Patent No. 5,859,330) and the tomato 2AII gene promoter (Van Haaren MJJ et al, 1993).
In one preferred embodiment, HIO 128.4 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 51 regulatory regions from such genes as napin (Kridl et al, Seed ScL 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, MoI Gen Genet., 247:603-613, 1995), L3 oleosin promoter (US Patent 6,433,252), phaseolin (Bustos et al, Plant Cell, l(9):839-853, 1989), arcelin5 (US 2003/0046727), a soybean 7S promoter, a 7Sα promoter (US 2003/0093828), the soybean 7Sα' beta conglycinin promoter, a 7S α' promoter (Beachy et ah, EMBOJ., 4:3047, 1985; Schuler et ah, Nucleic Acid Res., 10(24): 8225-8244, 1982), soybean trypsin inhibitor (Riggs et ah, Plant Cell l(6):609-621, 1989), ACP (Baerson et ah, Plant MoI. Biol, 22(2):255-267, 1993), stearoyl-ACP desaturase (Slocombe etal, Plant Physiol. 104(4): 167- 176, 1994), soybean a' subunit of β-conglycinin (Chen et ah, Proc. Natl. Acad. ScL 83:8560- 8564, 1986), Viciafaba USP (P-Vf.Usp, SEQ ID NO: 1, 2, and 3 in (US 2003/229918) and Zea mays L3 oleosin promoter (Hong et ah, Plant MoI. 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 ah, Cell 29:1015-1026, 1982; and Russell et ah, Transgenic
Res. <5(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 ah, 1991, MoI Gen Genet 225:121-8; Baumlein et ah, 1992, Plant J 2:233-9), V.faba usp (Fiedler et ah, 1993, Plant MoI Biol 22:669-79), pea convicilin (Bown et ah, 1988, Biochem J 251:717-26), pea lectin (dePater et ah, 1993, Plant Cell 5:877-86), P. vulgaris beta phaseolin (Bustos et ah, 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 ah, 1992, Plant MoI 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 37:124-11; "GIuB-I," Takaiwa et ah, 1996, Plant MoI Biol 30:1207-21; Washida et ah, 1999, Plant MoI Biol 40:1-12; "Gt3," Leisy et ah, 1990, Plant MoI Biol 14:41-50), ήceprolamin (Zhou & Fan, 1993, Transgenic Res 2:141-6), wheat prolamin (Hammond-Kosack et ah, 1993, EMBO J 12:545-54), maize zein (Z4, Matzke et ah, 1990, Plant MoI Biol 14:323-32), and barley B-hordeins (Entwistle et ah, 1991, Plant MoI Biol 17:1217-31). Other genes whose promoters are associated with early seed and embryo development include oil palm GL07A (7S globulin, Morcillo et ah, 2001, Physiol Plant 112:233-243), Brassica napus napin, 2S storage protein, and napA gene (Josefsson et ah, 1987, J Biol Chem 262:12196-201; Stalberg et ah, 1993, Plant MoI Biol 1993 23:671-83; Ellerstrom et ah, 1996, Plant MoI Biol 32:1019-27),
Brassica napus oleosin (Keddie et ah, 1994, Plant MoI Biol 24:327-40), Arabidopsis oleosin (Plant et ah, 1994, Plant MoI Biol 25:193-205), Arabidopsis FAEl (Rossak et ah, 2001, Plant MoI Biol 46:717-25), Canavalia gladiata conA (Yamamoto et ah, 1995, Plant MoI Biol 27:729-41), and Catharanthus roseus strictosidine synthase (Str, Ouwerkerk and Memelink, 1999, MoI Gen Genet 261:635-43). In another preferred embodiment, regulatory sequences from genes expressed during oil biosynthesis are used (see, e.g., US 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. Patents 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.
In yet another aspect, in some cases it may be desirable to inhibit the expression of endogenous HIO128.4 in a host cell. Exemplary methods for practicing this aspect of the invention include, but are not limited to antisense suppression (Smith, et «/.,1988; van der Krol et ah, 1988); co-suppression (Napoli, et ah, 1990); ribozymes (PCT Publication WO 97/10328); and combinations of sense and antisense (Waterhouse, et ah, 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 ah, 1988), a partial cDNA sequence including fragments of 5' coding sequence, (Cannon et ah, 1990), or 3' non-coding sequences (Ch'ng et ah, 1989). Cosuppression techniques may use the entire cDNA sequence (Napoli et ah, 1990; van der Krol et ah, 1990), or a partial cDNA sequence (Smith et ah, 1990). 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.
1. DNA/RNA analysis 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]).
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 NL 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.
2. Gene Product Analysis
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.
3. Pathway Analysis
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 a HIOl 28.4 Phenotvpe
The invention further provides a method of identifying plants that have mutations in, or an allele of, endogenous HIO128.4 that confer a HIO128.4 phenotype, and generating progeny of these plants that also have the HIO128.4 phenotype and 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. HIO 128.4- specific PCR is used to identify whether a mutated plant has a HIO128.4 mutation. Plants having HIO128.4 mutations may then be tested for the HIO128.4 phenotype, or alternatively, plants may be tested for the HIO 128.4 phenotype, and then HIO128.4-specific PCR is used to determine whether a plant having the HIO128.4 phenotype has a mutated HIO128.4 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).
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 HIO 128.4 gene or orthologs of HIO 128.4 that may confer the HIO128.4 phenotype (see Foolad et al, Theor Appl Genet. (2002) 104(6-7):945- 958; Rothan et al, Theor Appl Genet (2002) 105(l):145-159); Dekkers and Hospital, Nat Rev Genet. (2002) Jan;3(l):22-32).
Thus, in a further aspect of the invention, a HIO128.4 nucleic acid is used to identify whether a plant having a HIO 128.4 phenotype has a mutation in endogenous HIO128.4 or has a particular allele that causes the HIO128.4 phenotype compared to plants lacking the mutation or allele, and generating progeny of the identified plant that have inherited the HIO128.4 mutation or allele and have the HIO128.4 phenotype.
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 public databases are also incorporated by reference.
EXAMPLES
EXAMPLE 1
Generation of Plants with a HIO128.4 Phenotype by Transformation with an
Activation Tagging Construct 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 WOOl 83697. Briefly, TO Arabidopsis (CoI-O) 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 4X CaMV 35S enhancer element. Transgenic plants were selected at the Tl generation based on herbicide resistance. T3 seed pools were analyzed by Near Infrared Spectroscopy (NIR) intact at time of harvest. NIR infrared spectra were captured using a Bruker 22 N/F near infrared spectrometer. 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 content predicting calibrations were developed following the general method of AOCS Procedure Am 1-92, Official Methods and Recommended Practices of the American Oil Chemists Society, 5th Ed., AOCS, Champaign 111). Calibrations allowing NIR predictions of Crude Oil (Ether Extract) (PDXOU3, AOAC Method 920.39 (Fat(Crude) or Ether Extract in Animal Feed, AOAC International, Official Methods of Analysis, 17th Edition, AOAC International , Gaithersburg Maryland) and Crude Oil ASE (Ren Oil, Accelerated Solvent Extraction) NIR predicted oil contents were determined for over 81 ,307 individual ACTTAG lines. To identify high oil lines the NIR oil result was compared to the mean oil result for all ACTTAG lines planted in the same flat (Relative oil content). Oil Content distance (SD_Distance) was determined by taking the NIR predicted oil content of the individual, subtracting the mean oil content for the flat from which the individual came, then dividing the result by the flat standard deviation. This creates a real number measure of distance, in standard deviations, that each samples result lies from the flat average. From 81,307 oil determinations high oil results were selected by eliminating all lines with an ASE predicted oil content SD_ DISTANCE of <2. ACTTAG lines that had ASE oil predictions with an SD_Distance >=2 were compared to identify lines sharing a flanking sequence tag placement location that occurred in the same 10kb region in two distinct lines. The genome coordinates in this subset of high oil ACTTAG lines was evaluated and lines having two or more independent ACTTAG insertions, also displaying high oil, were identified and the flanking sequence confirmed by PCR and sequencing.
Line W000041352 (IN009897) had a NIR determined oil content of 39.2% relative to a flat average oil content of 35.2% (111% of Flat Average). Line W000210144 (TN088075) had a NIR determined oil content of 30.2% relative to a flat average oil content of 24.2% (124% of Flat Average).
Line W000041352 (IN009897) had a confirmed ACTTAG insertion on Chromosome 5 at bp 15023687. Line W000210144 (IN088075) had a confirmed ACTTAG insertion on Chromosome 5 at bp 15020099.
EXAMPLE 2 Characterization of the T-DNA Insertion in Plants Exhibiting the Altered Oil
Content Phenotvpe.
We performed standard molecular analyses, essentially as described in patent application PCT WOOl 83697, 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 lines IN009897 and IN088075, 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. 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 publicly available Arabidopsis Information Resource (TAIR) database. For ACTTAG line IN009897, there was sequence identity to nucleotides 31217 - 8456 on Arabidopsis BAC clone K22F20 on chromosome 5 (GI#3449314), placing the left border junction upstream from nucleotide 8456 (GI#3449314). Left border of IN009897 T-DNA was about 6897 bp 5' of the translation start site.
For ACTTAG line IN088075, there was sequence identity to nucleotides 4490 - 4828 on Arabidopsis BAC clone K22F20 on chromosome 5 (GI#3449314), placing the left border junction upstream from nucleotide 4828 (GI#3449314). Left border IN088075 T-DNA was about 3309 bp 5' of the translation start site.
EXAMPLE 3 Analysis of Arabidopsis HIO128.4 Sequence
Sequence analyses were performed with BLAST (Altschul et al., 1990, J. MoI. 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 JD et al, 1994, Nucleic Acids Res 22:4673-4680).
TBLASTN against est others results:
The candidate gene At5g37770 is supported by the full-length cDNA (Tair Accession: Sequence 3708394) and by the NCBI entry GI#30693148.
There are many ESTs from diverse plant species showing similarity to At5g37770. If 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" (Table 1): Triticum aestivum, Gossypium hirsutum, Zea mays, Glycine max, Mentha x piperita, Populus tremula, Oryza sativa, Lycopersicon esculentum, Solanum tuberosum, and Beta vulgaris.
1. One EST contig from wheat
The contigged sequence is presented as SEQ ID NO: 3. 2. One EST contig from cotton
The contigged sequence is presented as SEQ ID NO: 4.
3. One EST contig from maize The contigged sequence is presented as SEQ ID NO: 5.
4. One EST contig from soybean
The contigged sequence is presented as SEQ ID NO: 6.
5. One EST from mint gi|7244443 MLl 81 peppermint glandular trichome Mentha x piperita cDNA, MRNA sequence
6. One EST contig from poplars The contigged sequence is presented as SEQ ID NO: 7.
7. One EST contig from rice
The contigged sequence is presented as SEQ ID NO: 8.
8. One EST from tomato
The contigged sequence is presented as SEQ ID NO: 9.
9. One EST contig from potato
The contigged sequence is presented as SEQ ID NO: 10.
10. One EST contig from sugar beet
The contigged sequence is presented as SEQ ID NO: 11.
BLASTP against a protein database based on a translation of the Arabidopsis genome sequences from the Arabidopsis genome initiative (ATHl aa):
The protein At5g37770 (TCH2) has homology to large numbers of EF-hand- containing proteins from plants, fungi and animals. The Arabidopsis genome encodes more than 200 such proteins. In order to identify genes that are most likely to carryout similar function as At5g37770 (TCH2), the top 3 BLAST results and the BLAST results for Atlg73630, At2g36180, At5gl7470, At3g03400, At3g03410, At2gl5680 are listed below. This is because these genes form a subclade among the EF-hand-containing proteins in Arabidopsis.
1. At5g37770 itself (several redundant entries)
>gi[15240340|ref|NP 198593.11 calmodulin-related protein 2, touch-induced (TCH2) [Arabidopsis thaliana] >gi|3123295lsplP25070jTCH2 ARATH Calmodulin- related protein 2, touch-induced >gi|2583169lgb|AAB82713.1| calmodulin-related protein [Arabidopsis thaliana] >gill0177164|dbi|BAB10353.1| calmodulin-related protein 2, touch-induced [Arabidopsis thaliana] >gi|21554396lgb|AAM63501.1| touch-induced calmodulin-related protein TCH2 [Arabidopsis thaliana] Score = 718 (257.8 bits), Expect = 2.5e-70, P = 2.5e-70
2. Atlg66400 from Arabidopsis (5 redundant entries)
>gi|21618074|gb|AAM67124.1[ calmodulin-related protein [Arabidopsis thaliana] >gi|18408502|ref]NP 564874.1| calmodulin-related protein, putative [Arabidopsis thaliana] >gij25295746|pir||D96689 calmodulin-related protein, 72976-72503 [imported] - Arabidopsis thaliana >gi[123243991gb|AAG52166.1|AC020665 11 calmodulin-related protein; 72976-72503 [Arabidopsis thaliana] >gill7380758[gb|AAL36209.11 putative calmodulin-related protein [Arabidopsis thaliana] >gil21436399lgb|AAM51400.1| putative calmodulin-related protein [Arabidopsis thaliana] Score = 565 (203.9 bits), Expect = 4.1e-54, P = 4.1e-54
3. Atlgl8210 from Arabidopsis (7 redundant entries)
>gi|15221030|ref1NP 173259.1| calcium-binding protein, putative [Arabidopsis thaliana] >gi|30685754|ref]NP 849686.1| calcium-binding protein, putative [Arabidopsis thaliana] >gi|25295745|pir||A86317 protein T10O22.19 [imported] - Arabidopsis thaliana >gi|8671778lgblAAF78384.1|AC069551 17 T10O22.19 [Arabidopsis thaliana] >gil9719735|gblAAF97837.11AC034107 20 Strong similarity to calcium-binding protein (PCA23) from Olea europaea gblAF078680 and contains multiple EF-hand PF|00036 domains. ESTs gb|T21585. gb[T21589, gb|T41586. gb[Z37721, gb|Z29218, gb| All 00607, gb|AI997012. gblAV540453. gb|AV544989. gb|AV544493, gb|AV554674 come from this gene. [Arabidopsis thaliana] >gi|15027897|gb|AAK76479.1| putative calcium-binding protein [Arabidopsis thaliana] >gi|19310789lgb]AAL85125.11 putative calcium-binding protein
[Arabidopsis thaliana]
Score = 309 (113.8 bits), Expect = 5.5e-27, P = 5.5e-27
4. At5gl7470 from Arabidopsis (several redundant entries)
>gi| 15237969[ref|NP 197249.1| calmodulin-related protein, putative [Arabidopsis thaliana] >gi| 11276988 |pir||T51473 calmodulin-like protein - Arabidopsis thaliana >gi|9755771|emblCAC01891.11 calmodulin-like protein [Arabidopsis thaliana]
Score = 307 (113.1 bits), Expect = 9.0e-27, P = 9.0e-27
5. Atlg73630 from Arabidopsis (several redundant entries)
>gi| 15219514|ref|NP 177504.1| calcium-binding protein, putative [Arabidopsis thaliana] >gi]25295748|pir||C96763 protein calmodulin F25P22.4 [imported] - Arabidopsis thaliana >gi[ 12324222]gb|AAG52088.1 |AC01267? 26 putative calmodulin; 12692-13183 [Arabidopsis thaliana]
Score = 299 (110.3 bits), Expect = 6.3e-26, P = 6.3e-26
6. At2g36180 from Arabidopsis (several redundant entries)
>gi|l 5227593 [ref]NP 181160.11 calmodulin-related protein, putative [Arabidopsis thaliana] >gi|20197940|gb[AAD21447.2] putative touch-induced calmodulin [Arabidopsis thaliana] >gil20198113lgblAAM15404.1| putative touch-induced calmodulin [Arabidopsis thaliana] Score = 288 (106.4 bits), Expect = 9.2e-25, P = 9.2e-25 7. At3g03400 from Arabidopsis (2 redundant entries)
>gi]15228545|ref|NP 186990.11 calmodulin-related protein, putative [Arabidopsis thaliana] >gil6017122lgblAAP01605.11AC009895 26 calmodulin-like protein [Arabidopsis thaliana] Score = 286 (105.7 bits), Expect = 1.5e-24, P = 1.5e-24
8. At3g03410 from Arabidopsis (2 redundant entries)
>gi|15228547|reflNP 186991.1| calmodulin-related protein, putative [Arabidopsis thaliana] >gi|6017121|gb|AAF01604.1|AC009895 25 calmodulin-like protein [Arabidopsis thaliana] Score = 249 (92.7 bits), Expect = 1.3e-20, P = 1.3e-20
9. At2gl5680 from Arabidopsis (several redundant entries)
>gi[15226592|ref1NP 179170.1] calmodulin-related protein, putative [Arabidopsis thaliana] >gi|25411654|pir||A84532 probable calmodulin-like protein [imported] - Arabidopsis thaliana >gi[4335734|gb|AAD17412.11 putative calmodulin-like protein [Arabidopsis thaliana] >gi|22135992lgb|AAM91578,ll putative calmodulin-like protein [Arabidopsis thaliana] >gi[301029221gb|AAP21379.1| At2gl5680 [Arabidopsis thaliana] Score = 297 (109.6 bits), Expect = 1.0e-25, P = 1.0e-25
Table 1.
Figure imgf000034_0001
"One EST contig Zea mays αil5268949 Length = 861 TBLASTN from maize αil14204033 Identities = 31/55 (56%), Score = 179 (68.1 bits),
Positives = 43/55 (78%) , Expect = 1.9e-26, Sum
I P(2) = 1.9e-26
"One EST From Glycine max GΪ:634Ϊ742 ~ ' Length =1231 ' TBLASTN soybean Gl:6665878 Identities = 74/137 (54%), Score = 375 (137.1 bits),
Gl:7589322 Positives = 101/137 (73%) Expect = 7.4e-35, P =
GI:10255162 7.4e-35
Gl:23728227
GI:23050798
GI:26059028
Gl:24135637
Gl:31561226
One EST from ~~" Mentha x gi|7244443"" Length = 630 \ TBLASTN
Mint piperita Identities = 56/136 (41%), Score = 262 (97.3 bits),
Positives = 80/136 (58%) ' Expect = 2.7e-24, P =
2.7e-24
One EST contig " Populus ~ GI:J8007875 Length = 846 TBLASTN from poplars tremula GM24062599 Identities = 77/138 (55%), Score = 392 (143.0 bits),
GI:24063571 Positives = 99/138 (71%) Expect = 7.1e-37, P =
GI:24103197 7.1e-37
Gl:24102671
Gl:23999523
Gl:23968457
Gl:23969174
GI:24005672
GI:24002461
GI:24055344
One EST contig Oryza sativa GM5852938 Length = 923 TBLASTN from rice Gl:16578533 Identities = 56/124 (45%), Score = 272 (100.8 bits),
Gl:25777884 Positives = 75/124 (60%) Expect = 3.7e-27, Sum
Gl:25797682 P(2) = 3.7e-27
One EST from Lycopersicon GI:5893030 Length = 1663 TBLASTN tomato esculentum Gl:5892792 Identities = 61/113 (53%), Score = 314 (115.6 bits),
Gl:5892166 Positives = 80/113 (70%) Expect = 1.2e-35, Sum
G 1:5604769 P(2) = 1.2Θ-35
GI:5600642
Gl:5279946
Gl:5269676
Gl:5269645
Gl:4387818
Gl:4385435
Gl: 16217807
Gl:16218153
GI:6528098
GI:6528056
Gl:6528144
Gl:9432869
Figure imgf000036_0001
Table 2. Closest Arabidopsis homologs • •
At1g66400 Arabidopsis >qi|21618074 Length = 157, Identities = ' BLASTP thaliana >αil18408502 111/148 (75%), Positives Score = 565 (203.9 bits),
>αil25295746 = 125/148 (84%) Expect = 4.1e-54, P =
>αi|12324399 4.1e-54
>qil 17380758
>αi|21436399
AtI g18210 Arabidopsis >αil15221030 Length = 170, Identities = BLASTP thaliana >gi|30685754| 62/139 (44%), Positives = Score = 309 (113.8 bits),
>qil25295745| 91/139 (65%) Expect = 5.5e-27, P =
>qil8671778 5.5e-27
>αi|9719735
>αil 15027897
>αil 19310789
At5g17470 Arabidopsis ">qϊl15237969 Length = 146" " BLASTP thaliana >αi| 11276988 Identities = 60/733 (45%), Score = 307 (113.1 bits),
>αil9755771 Positives = 87/133 (65%) Expect = 8.9e-27, P =
8.9e-27
At1g73630 Arabidopsis ">αil15219514 Length = 163 BLASTP thaliana >αil25295748 Identities = 61/143 (42%), Score = 299 (110.3 bits),
>αi 112324222 Positives = 90/143 (62%) Expect = 6.3e-26, P =
6.3e-26
At2g36180 Arabidopsis >αi| 152275931 Length = 144 BLASTP thaliana >αil20197940| Identities = 60/131 (45%), Score = 288 (106.4 bits),
>αil20198113| Positives = 80/131 (61%) Expect = 9.2e-25, P =
9.2e-25
At3g03400 Arabidopsis >qi|15228545J Length = 137 BLASTP thaliana >αil6017122l Identities = 56/132 (42%), Score = 286 (105.7 bits),
Positives = 83/132 (62%) Expect = 1.5e-24, P =
1.5e-24 At3gO341O Arabidopsis >αil15228547| Length = 131 BLASTP thaliana >αil6017121| Identities = 52/132 (39%), • Score = 249 (92.7 bits),
Positives = 79/132 (59%) \ Expect = 1.3e-20, P =
1.3e-20
At2g15680 Arabidopsis >αil 15226592| Length = 187 . BLASTP thaliana >αil25411654| Identities = 59/137 (43%), \ Score = 297 (109.6 bits),
>αil4335734| Positives = 90/137 (55%) Expect = 1. Oe-25, P =
>qi|22135992| I 1.0e-25
>qi|30102922|
At5g37770 is a non-secretory protein that lacks transmembrane domain (predicted by TMHMM (Krogh et al, J. MoI. Biol. 2001 305:567-580)) and signal peptide (predicted by SignalP). Psort2 analysis suggests that At5g37770 is a cytoplasmic or a nuclear protein (32% nuclear, 28% cytoplasmic, 24% mitochondrial, 8% cytoskeletal by Psort2).
Pfam analysis showed that At5g37770 has 4 EF-hand domains (PF00036, SM00054). Many Ca2+-binding proteins belong to the same evolutionary family and share a type of Ca2+-binding domain known as the EF-hand. This type of domain consists of a twelve-residue loop flanked on both sides by a twelve-residue- helical domain. In an EF-hand loop the Ca2+ ion is coordinated in a pentagonal bipyramidal configuration. The six residues involved in the binding are in positions 1, 3, 5, 7, 9 and 12; these residues are denoted by X, Y, Z, -Y, -X and -Z. The invariant GIu or Asp at position 12 provides two oxygens for liganding Ca (bidentate ligand).
Ca2+ is an important messenger mediating plant responses to hormones, developmental cues and external stimuli. Changes in intracellular concentration of Ca2+ is implicated in regulating diverse cellular process such as cytoplasmic streaming, thigmotropism, gravitropism, cell division, cell elongation, cell differentiation, cell polarity, photomorphogenesis and plant defense and stress responses. Transient increase in the concentration of Ca2+ in the cytoplasm is sensed by Ca2+ binding proteins. The conformation of these protein changes when they bind Ca2+, resulting in modulation of its activity or its ability to interact with other proteins or nucleic acids and modulate their function or activity. One such class of proteins is calmodulin, which contains EF-hand motifs. At5g37770 (TCH2) is one of two proteins that is induced by touch, rain, wind, wounding, darkness, increase in extracellular Ca2+ concentration and heat shock. At5g37770 is related to the calmodulins and belongs to a subclade within the group IV family of EF-hand containing proteins. The blastP alignments between At5g37770 and other proteins in the subclade were described above. The three- dimensional structure of At5g37770 (TCH2) has been solved and shown to resemble the structure of calmodulin.
Calmodulins are highly conserved small molecular weight acidic proteins that have 4 EF-hands and bind four molecules of Ca2+.
In this context, it seems likely that At5g37770 is involved in signal transduction in Arabidopsis. Binding of Ca2+ to At5g37770 is likely to result in conformational change which then allows At5g37770 to interact with target proteins and to modulate their activity or function.
Model Domain seq-f seq-t hmm-f hmm-t score E-value
SM00054 1/4 17 45 .. 1 29 [] 40.1 7.1e-09
PF00036 1/4 17 45 .. 1 29 [] 42.7 l.le-09
SM00054 2/4 53 81 .. 1 29 [] 25.5 0.00017
PF00036 2/4 53 81 .. 1 29 [] 30.3 6.3e-06
PF00036 3/4 94 122 .. 1 29 [] 37.9 3.2e-08
SM00054 3/4 94 122 .. 1 29 [] 39.0 1.4e-08
PF00036 4/4 130 158 .. 1 29 [] 38.0 3e-08
SM00054 4/4 130 158 .. 1 29 [] 34.9 2.6e-07
*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 4
Recapitulation of the High Oil Phenotype
To test whether over-expression of At5g37770 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, Arabidopsis plants of the CoI-O ecotype were transformed by Agrobacterium mediated transformation using the floral dip method, with a construct containing the coding sequences of the HIO128.4 gene (At5g37770) behind the CsVMV promoter and in front of the nos terminator or a control gene unrelated to pathogen resistance. Both of these constructs contain the nptll gene directed by the RE4 promoter to confer kanamycin resistance in plants and serve as a selectable marker. Tl seed was harvested from the transformed plants and transformants selected by germinating seed on agar medium containing kanamycin. Kanamycin resistant transformants, identified as healthy green plants, were transplanted to soil after 7 days. Control plants were germinated on agar medium without kanamycin, transplanted to soil after 7 days. Twenty-two plants containing the CsVMV:: HIO 128.4 transgene along with 10 CoI-O control plants were grown in the same flat in the growth room. All plants were allowed to self-fertilize and set seed.
To evaluate the high seed oil phenotype T2 seed pools were analyzed by Near Infrared Spectroscopy (NIR) at time of harvest. NIR infrared spectra were captured using a Bruker 22 N/F spectrometer. 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 Id + sline.q2, Predicts Hexane Extracted Oil), followed the general method of AOCS Procedure AM 1-92, Official Methods and Recommended Practices of the American Oil Chemists Society, 5th Ed., AOCS, Champaign, IU. Seed from transgenic plants and control plants grown in the same flat were compared and the results are presented in Table 3.
To compare the effect of over-expression of HIO128.4 on seed oil accumulation, four experiments were performed. In three out of four experiments, the plants over-expressing HIO 128.4 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 HIO128.4 was 4.0% greater than the untransformed controls; the seed oil content in plants over-expressing HIO 128.4 was significantly greater than non-transgenic control plants (two-way ANOVA (analysis of variance); P = 0.0013).
Table 3.
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
EXAMPLE 5
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 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. OiI 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 1H nuclear magnetic resonance (NMR) (Tiwari et ah, 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, NC, USA) are performed to identify significant differences between transgenic and non-transgenic kernels as determined by transgene-specific PCR.
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.
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, NY). 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," March 17, 2000. Quantitative deteraiination 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°C for 24 hrs. Samples are dried and reconstituted in 0.1 N HCl followed by precolumn derivatization with α-phthalaldehyde (OPAO for HPLC analysis. The amino acids are separated by a reverse-phase Zorbax Eclipse XDB-C 18 HPLC column on an Agilent 1100 HPLC (Agilent, Palo Alto, CA). The amino acids are detected by fluorescence. Cysteine, proline, asparagine, glutamine, and tryptophan are not included in this amino acid screen (Henderson et ah, "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 -10th edition, AACC ed, (2000) 07-20 Measurement of Tryptophan - Alakline Hydrolysis).
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, OK) in a sterile microfuge tube to which 500 μl 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 μm filter into an autosampler tube. The filtered extracts are analyzed by HPLC using a Zorbax silica HPLC column (4.6 mmx250 mm) with a fluorescent detection, an excitation at 290 ran, an- emission at 336 ran, 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 μl, the flow rate is 1.5 ml/minute and the run time is 12 minutes at 4O0C. 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 (α- tocopherol, γ-tocopherol, β-tocopherol, δ-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 (α-tocotrienol, γ- tocotrienol, β- tocotrienol, δ- tocotrienol). Standard curves for α-, β-, δ-, and γ-tocotrienol are calculated using Chemstation software (Hewlett Packard).
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 ah, Methods in Enzymology, XVIII, part C, 369-396 (1971); and Ramadan et ah, Eur. Food Res. Technoh, 214(6):521-527 (2002)).
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Claims

IT IS CLAIMED:
1. A transgenic plant comprising a plant transformation vector comprising a nucleotide sequence that encodes or is complementary to a sequence that encodes a HIO128.4 polypeptide, comprising the amino acid sequence of SEQ E) NO:2, or an ortholog thereof, whereby the transgenic plant has a high oil phenotype relative to control plants.
2. The transgenic plant of Claim 1, which is selected from the group consisting of rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor and peanut.
3. A plant part obtained from the plant according to Claim 1.
4. The plant part of Claim 3, which is a seed.
5. Meal, feed, or food produced from the seed of claim 4.
6. A method of producing oil comprising growing the transgenic plant of Claim 1 and recovering oil from said plant.
7. The method of claim 6, wherein the oil is recovered from a seed of the plant.
8. A method of producing a high oil 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 a HIO128.4 polypeptide comprising the amino acid sequence of SEQ E) NO:2, or an ortholog thereof, 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 relative to control plants.
9. A plant obtained by a method of claim 8.
10. The plant of Claim 9, which is selected from the group consisting of rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor and peanut.
11. The plant of claim 9, wherein the plant is selected from the group consisting of a plant grown from said progenitor cells, a plant that is the direct progeny of a plant grown from said progenitor cells, and a plant that is the indirect progeny of a plant grown from said progenitor cells.
12. A method of generating a plant having a high oil phenotype comprising identifying a plant that has an allele or mutation in its HIO 128.4 gene that results in the high oil phenotype compared to plants lacking the allele or mutation and generating progeny of said identified plant, wherein the generated progeny inherit the allele and have the high oil phenotype.
13. The method of claim 12 that employs candidate gene/QTL methodology.
14. The method of claim 12 that employs TILLING methodology.
15. A feed, meal, grain, food, or seed comprising a polypeptide encoded by the nucleic acid sequence as set forth in SEQ ID NO: 1.
16. A feed, meal, grain, food, or seed comprising a polypeptide comprising an amino acid sequence as set forth in SEQ ID NO:2, or an ortholog thereof.
PCT/US2005/037528 2004-10-22 2005-10-20 Generation of plants with altered oil content WO2006047159A1 (en)

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