WO2006076596A2 - Production de plantes a teneur en huile modifiee - Google Patents

Production de plantes a teneur en huile modifiee Download PDF

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Publication number
WO2006076596A2
WO2006076596A2 PCT/US2006/001280 US2006001280W WO2006076596A2 WO 2006076596 A2 WO2006076596 A2 WO 2006076596A2 US 2006001280 W US2006001280 W US 2006001280W WO 2006076596 A2 WO2006076596 A2 WO 2006076596A2
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Prior art keywords
plant
plants
seed
oil
hio
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WO2006076596A3 (fr
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John P. Davies
Hein Tsoeng Ng (Medard)
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Agrinomics LLC
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Agrinomics LLC
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Priority to MX2007008435A priority Critical patent/MX2007008435A/es
Priority to BRPI0606643-7A priority patent/BRPI0606643A2/pt
Priority to US11/813,858 priority patent/US7663020B2/en
Priority to CN2006800022466A priority patent/CN101103116B/zh
Publication of WO2006076596A2 publication Critical patent/WO2006076596A2/fr
Publication of WO2006076596A3 publication Critical patent/WO2006076596A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically 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
    • C12N15/8247—Phenotypically 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

Definitions

  • the present disclosure is related to transgenic plants and plant cells with altered oil content, as well as methods of making plants having altered oil content and producing oil from such plants.
  • 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 animal feeds.
  • 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 remaining seed meal is sold for livestock feed (U.S. Soybean Board, 2001 Soy Stats). Canola seed is also 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 acid soybean and corn oil (U.S. Patent Nos. 6,229,033 and 6,248,939), and laurate- containing seeds (U.S. Patent 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. Patent 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.
  • HOC production system 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 content in current HOC fields has 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 al, 1989, Science 243: 1351-1354) that detected altered fatty acid composition identified the omega 3 desaturase (FAD3) and delta- 12 desaturase (FAD2) genes (U.S. Patent No. 5952544; Yadav et al, 1993, Plant Physiol. 103, 467-476; Okuley et ⁇ /., 1994, Plant Cell 6(1):147-158).
  • 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, Plant Physiol. 118:91-101).
  • 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, Plant Physiol.108(l):399-409). It was further shown that seed-specific over-expression of the DGAT cDNA was associated with increased seed oil content (Jako et al, 2001, Plant Physiol.126(2):861-74). Arabidopsis is also a model for understanding the accumulation of seed components that affect meal quality. For example, Arabidopsis contains albumin and globulin seed storage proteins found in many dicotyledonous plants including canola and soybean (Shewry 1995, Plant Cell
  • 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, Science 258: 1350-1353; Weigel D et al, 2000, Plant Physiology, 122:1003-1013).
  • 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 ah, 1996, Plant Cell 8: 659-671; Schaffer et ah, 1998, Cell 93: 1219-1229; Fridborg et ah, 1999, Plant Cell 11: 1019- 1032; Kardailsky et ah, 1999, Science 286: 1962-1965; and Christensen S et ah, 1998, 9 th International Conference on Arabidopsis Research, Univ. of Wisconsin- Madison, June 24-28, Abstract 165).
  • transgenic plants having a high oil having a high oil (hereinafter "HIO") phenotype.
  • HIO high oil
  • Transgenic plants with a HIO phenotype have an altered or increased oil content in any part of the plant, for example the seeds, relative to control, non- transgenic, or wild-type plants.
  • meal, feed, or food generated from any part of a transgenic plant having a HIO phenotype comprises a transformation vector comprising a nucleotide sequence that encodes or is complementary to a sequence that encodes a HIO polypeptide.
  • expression of a HIO polypeptide in a transgenic plant causes an altered or increased oil content in the transgenic plant.
  • 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 disclosure further provides a method of producing oil comprising growing the transgenic plant and recovering oil from said plant.
  • the disclosure further provides feed, meal, grain, or seed comprising a nucleic acid sequence that encodes a HIO polypeptide.
  • the disclosure also provides feed, meal, grain, or seed comprising the HIO polypeptide or an ortholog thereof.
  • the disclosed transgenic plants are 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 HIO polypeptide, and growing the transformed progenitor cells.to produce a transgenic plant, wherein the HIO polynucleotide sequence is expressed causing the high oil phenotype in the transgenic plant.
  • the disclosed transgenic plant is the direct progeny or the indirect progeny of a plant grown from said progenitor cells.
  • the method produces transgenic plants wherein expression of the HIO polypeptide causes a HIO phenotype in the transgenic plant, relative to control, non-transgenic, or wild-type plants.
  • Additional methods are disclosed herein of generating a plant having a HIO phenotype, wherein a plant is identified that has an allele in its HIO nucleic acid sequence that results in a HIO phenotype, compared to plants lacking the allele.
  • the plant can generate progeny, wherein the progeny inherit the allele and have a HIO phenotype.
  • the method employs candidate gene/QTL methodology or TILLING methodology.
  • transgenic plant cell having a HIO phenotype.
  • the transgenic plant cell comprises a transformation vector comprising a HIO nucleotide sequence that encodes or is complementary to a sequence that encodes a HIO polypeptide.
  • the transgenic plant cell is selected from the group consisting of canola, 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 plant cells are obtained from the disclosed transgenic plant.
  • the disclosure also provides plant cells from a plant that is the direct progeny or the indirect progeny of a plant grown from said progenitor cells, or plant cells from a plant that is the direct progeny or the indirect progeny of a plant grown from said progenitor cells.
  • the present disclosure 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 disclosure.
  • the present disclosure 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 disclosure.
  • Any of the plants or parts thereof of the present disclosure may be processed to produce a feed, food, meal, or oil preparation.
  • a particularly preferred plant part for this purpose is a seed.
  • the feed, food, meal, or oil preparation is designed for 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 disclosure may be blended with other meals, hi a preferred embodiment, the meal produced from plants of the present disclosure or generated by a method of the present disclosure 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.
  • HIO high oil
  • phenotype refers to plants, or any part of a plant (for example, seeds), with an altered oil content (phenotype).
  • altered oil content includes an increased oil content in plants or seeds, compared to a control, non-transgenic, or wildtype plant.
  • the term “content” refers to the type and relative amount of, for instance, a seed or seed meal component.
  • the term “meal” refers to seed components remaining following the extraction of oil from the seed. Examples of components of meal include protein and fiber.
  • fiber refers to non-digestible components of the plant seed including cellular components such as cellulose, hemicellulose, pectin, lignin, and phenolics.
  • 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.
  • 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.
  • a heterologous nucleic acid sequence include a HIO nucleic acid sequence, or a fragment, derivative (variant), or ortholog thereof.
  • 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 1 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 sequences.
  • 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.
  • gene expression refers to the process by which a polypeptide is produced based on the nucleic acid sequence of a gene.
  • expression may refer to either a polynucleotide or polypeptide sequence, or both, i 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.
  • Eutopic 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 includes “transfection,” “transformation,” and “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 any type of cell that is found in a seed, a pollen grain, a propagule, or an embryo, or a structure associated therewith.
  • undifferentiated tissue e.g., callus
  • a wild-type plant is also a control plant.
  • a wild-type plant is a non-transgenic plant.
  • the term "modified” regarding a plant trait refers to a change in the phenotype of a transgenic plant (for example, a transgenic plant with an altered oil content) in any part of the transgenic plant, for example the seeds, relative to a similar non-transgenic plant.
  • the term “altered” refers to either an increase or a decrease of a plant trait or phenotype (for example, oil content) in a transgenic plant, relative to a similar non-transgenic plant.
  • a transgenic plant with a modified trait includes a plant with an increased oil content, or HIO content, relative to a 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 plant ⁇ 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 (for example, increased oil content or HIO content in seeds of 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 phenotype or quality.
  • Such transgenic plants may have an improved phenotype, such as an HIO phenotype.
  • altered oil content phenotype refers to a measurable phenotype of a genetically modified (transgenic) 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 (non-transgenic) plant.
  • a high oil (HIO) 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 or altered plant phenotype or trait.
  • the term “mutant” refers to a plant or plant line which has a modified or altered plant phenotype or trait, where the modified or altered phenotype or trait is associated with the modified or altered 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.
  • a selection agent e.g., an antibiotic or herbicide
  • 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.
  • a transgenic plant cell having a HIO phenotype is provided herein.
  • the transgenic plant cell comprises a transformation vector comprising a HIO nucleotide sequence that encodes or is complementary to a sequence that encodes a HIO polypeptide
  • the transgenic plant cell is of a plant selected from the group consisting of canola, 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, including any type of cell that is found in a seed, a pollen grain, a propagule, or an embryo, or a structure associated therewith.
  • the disclosure 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 class of plants which can be used in the methods of the present disclosure is generally as broad as - li ⁇
  • 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 disclosure 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.
  • Transgenic plants with a HIO phenotype may include an improved oil quantity or an altered oil content in any part of the transgenic plant, for example in the seeds. Also provided is oil derived from the seeds of transgenic plants, wherein the seeds have altered oil content. Further provided herein is meal, feed, or food produced from any part of the transgenic plant with a HIO phenotype.
  • the disclosed transgenic plants comprise a transformation vector comprising a HIO nucleotide sequence that encodes or is complementary to a sequence that encodes a "HIO" polypeptide.
  • expression of a HIO polypeptide in a transgenic plant causes an altered oil content in the transgenic plant
  • the transgenic plant is selected from the group consisting of canola, rapeseed, soy, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut palm, flax, castor, and peanut.
  • a method of producing oil or seed meal comprising growing the transgenic plant and recovering oil and/or seed meal from said plant.
  • the disclosure further provides feed, meal, grain, or seed comprising a nucleic acid sequence that encodes a HIO polypeptide.
  • the disclosure also provides feed, meal, grain, or seed comprising the HIO polypeptide, or an ortholog thereof.
  • 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 Agrobacterium.
  • a number of wild-type and disarmed strains of Agrobacterium tumefaciens and Agrobacterium rhizogenes harboring Ti or Ri plasmids can be used for gene transfer into plants. Gene transfer is done via the transfer of a specific DNA known as "T- DNA” that can be genetically engineered to carry any desired piece of DNA into many plant species.
  • Agrobacterium-mediated genetic transformation of plants involves several steps.
  • the first step in which the virulent Agrobacterium and plant cells are first brought into contact with each other, is generally called “inoculation.”
  • the Agrobacterium and plant cells/tissues are permitted to be grown together for a period of several hours to several days or more under conditions suitable for growth and T-DNA transfer.
  • This step is termed "co-culture.”
  • the plant cells are treated with bactericidal or bacteriostatic agents to kill the Agrobacterium remaining in contact with the explant and/or in the vessel containing the explant.
  • 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.
  • BioRad Hercules, CA
  • 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 (PCT Publication No. WO 95/06128), barley, wheat (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, 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, U.S. Patent No 5,633,435, and U.S. Patent No 6,040,497 and aroA described in U.S. Patent No. 5,094,945 for glyphosate tolerance; a polynucleotide molecule encoding bromoxynil nitrilase (Bxn) described in U.S. Patent No.
  • EPSPS 5-enolpyruvylshikimate-3-phosphate synthase
  • Bxn bromoxynil nitrilase
  • 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, 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.
  • auxins cytokinins
  • ABA cytokinins
  • gibberellins gibberellins.
  • 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.
  • An Arabidopsis activation tagging (ACTTAG) screen was used to identify the association between the genes identified and designated HIO# (listed in column 1 of Table 1 below) and altered oil content phenotypes (specifically, high oil phenotypes). 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).
  • the enhancer element can cause up-regulation 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 T2 seed from each Tl plant 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 was estimated using one of two methods; measurement of fatty acid content and composition in T2 seeds using Gas Chromatography (GC) for HIO30.1 or estimation of total lipid content of T3 seeds using NIR infrared Spectroscopy (NIR) for HIOlOlB.
  • GC Gas Chromatography
  • NIR NIR infrared Spectroscopy
  • HIO nucleic acids and/or polypeptides may be employed in the development of genetically modified plants having a modified oil content phenotype ("a HIO phenotype").
  • HIO nucleic acids 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.
  • HIO nucleic acids 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 HIO polypeptides 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.
  • HIO Nucleic Acids and Polypeptides The HIO nucleic acids discovered in the activation tagging screen are listed in column 1 of Table 1.
  • the Arabidopsis Information Resource (TAIR) identification numbers are provided in column 2.
  • Columns 3-4 provide Genbank identifier numbers (GI#s) for the nucleotide and polypeptide sequences, respectively.
  • Column 5 lists the putative biochemical function and/or protein name.
  • Column 6 lists conserved protein domains.
  • Column 7 lists the relative seed oil content of plants over-expressing the HIO nucleic acid.
  • Column 8 provides GI#s for nucleic acid and/or polypeptide sequences of orthologous genes from other plant species.
  • Arabidopsis HIO30.1 nucleic acid (genomic DNA) sequence is provided in SEQ ID NO: 1 and in GenBank entry GI#30694055.
  • the corresponding protein sequence is provided in SEQ ID NO: 2 and in GI#15232503.
  • Arabidopsis HIOlOlB nucleic acid is provided in SEQ ID NO: 3 and in GenBank entry GI#30680675.
  • the corresponding protein sequence is provided in SEQ ID NO: 4 and in GI#30680676.
  • HIO polypeptide refers to any polypeptide that when expressed in a plant causes a HIO phenotype in any part of the plant, for example the seeds.
  • the present disclosure 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 disclosure.
  • the present disclosure 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 disclosure.
  • HIO polypeptide refers to a full-length HIO protein or a fragment, derivative (variant), or ortholog thereof that is “functionally active,” such that the protein fragment, derivative, or ortholog exhibits one or more or the functional activities associated with the full-length HIO polypeptide, hi one preferred embodiment, a functionally active HIO polypeptide causes a HIO phenotype in a transgenic plant. In one preferred embodiment, a functionally active HIO polypeptide causes an altered oil content phenotype when mis-expressed in a plant.
  • mis-expression of the HIO polypeptide causes a high oil phenotype in a plant
  • a functionally active HIO polypeptide is capable of rescuing defective (including deficient) endogenous HIO 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 HIO polypeptide retains one of more of the biological properties associated with the full-length HIO polypeptide, such as signaling activity, binding activity, catalytic activity, or cellular or extra-cellular localizing activity.
  • a HIO fragment preferably comprises a HIO 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 HIO protein.
  • Functional domains of HIO genes are listed in column 6 of Table 1 and can be identified using the PFAM program (Bateman A et ah, 1999 Nucleic Acids Res. 27:260-262) or INTERPRO (Mulder et ah, 2003, Nucleic Acids Res. 31, 315-318) program.
  • variants of full-length HIO 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 HIO polypeptide.
  • variants are generated that change the post-translational processing of a HIO polypeptide. For instance, variants may have altered protein transport or protein localization characteristics, or altered protein half-life, compared to the native polypeptide.
  • HIO nucleic acid encompasses nucleic acids with the sequence provided in or complementary to the sequence of the GenBank entry referenced in column 3 of Table 1, as well as functionally active fragments, derivatives, or orthologs thereof.
  • a HIO nucleic acid of this disclosure may be DNA, derived from genomic DNA or cDNA, or RNA.
  • a functionally active HIO nucleic acid encodes or is complementary to a nucleic acid that encodes a functionally active HIO 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 HIO 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 HIO polypeptide.
  • a HIO 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.
  • HIO 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 HIO polypeptide, or an intermediate form.
  • a HIO 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 nucleic acid is capable of being used in the generation of loss-of-function HIO phenotypes, for instance, via antisense suppression, co-suppression, etc.
  • a HIO nucleic acid used in the methods of this disclosure comprises a nucleic acid sequence that encodes or is complementary to a sequence that encodes a HIO polypeptide having at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity to the disclosed HIO polypeptide sequence of the GenBank entry referenced in column 4 of Table 1 (for example the amino acid sequence set forth as SEQ ID NO: 2 or SEQ ID NO: 4).
  • a HIO polypeptide of the disclosure comprises a polypeptide sequence with at least 50% or 60% identity to the HIO polypeptide sequence as set forth as SEQ ID NO: 2 or SEQ ID NO: 4, and may have at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to the disclosed HIO polypeptide sequence, and may include a conserved protein domain of the disclosed HIO polypeptide, such as the protein domain(s) listed in column 6 of Table 1.
  • a HIO polypeptide comprises a polypeptide sequence with at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to a functionally active fragment of the polypeptide of the GenBank entry referenced in column 4 of Table 1, for example the amino acid sequence as set forth as SEQ ID NO: 2 or SEQ ID NO: 4.
  • a HIO polypeptide comprises a polypeptide sequence with at least 50%, 60 %, 70%, 80%, or 90% identity to the polypeptide sequence of the GenBank entry referenced in column 4 of Table 1, for example the amino acid sequence as set forth as SEQ ID NO: 2 or SEQ ID NO: 4, over its entire length and comprises a conserved protein domain(s) listed in column 6 of Table 1.
  • a HIO polynucleotide sequence is at least 50% to 60% identical over its entire length to a disclosed HIO nucleic acid sequence, such as the nucleic acid sequence set forth as SEQ ID NO: 1 or SEQ ID NO: 3, or nucleic acid sequences that are complementary to such a HIO sequence, and may comprise at least 70%, 80%, 85%, 90% or 95% or more sequence identity over its entire length to the disclosed HIO nucleic acid sequence (for example, SEQ ID NO: 1 or SEQ ID NO: 3, or the GenBank entry referenced in column 3 of Table 1) or a functionally active fragment thereof, or nucleic acid sequences that are complementary to such a HIO sequence
  • a disclosed HIO nucleic acid comprises a nucleic acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 3, or nucleic acid sequences that are complementary to such a HIO sequence, and nucleic acid sequences that have substantial sequence homology to a such HIO sequences.
  • substantially sequence homology refers to those nucleic acid sequences that have slight or inconsequential sequence variations from such HIO sequences, i.e., the sequences function in substantially the same manner and encode a HIO polypeptide.
  • 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 disclosed nucleic acid sequence, for example the nucleic acid sequence as set forth as SEQ ID NO: 1 or SEQ ID NO: 3.
  • 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 disclosure is capable of hybridizing to a nucleic acid molecule , for example a nucleic acid molecule with a nucleic acid sequence as set forth as SEQ ID NO: 1 or SEQ ID NO: 3, 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 (s
  • 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 raM 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 HIO 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 disclosure.
  • the methods of the disclosure may use orthologs of the Arabidopsis HIO.
  • Putative orthologs of each of the Arabidopsis HIO genes identified in Table 1 ,below, are identified in column 8 of Table 1.
  • Methods of identifying these and orthologs of HIO genes from 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.
  • a gene duplication event follows speciation, a single gene in one species, such as Arabidopsis, may correspond to multiple genes (paralogs) in another.
  • 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 Sd (1998) 95:5849-5856; Huynen MA et al, Genome Research (2000) 10:1204-1210).
  • Programs for multiple sequence alignment may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees, hi 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, Molecular Cloning: A
  • HIO ortholog nucleic acids may hybridize to the nucleic acid of the GenBank entry referenced in column 3 of Table 1 under high, moderate, or low stringency conditions.
  • HIO ortholog ⁇ i.e., a protein orthologous to a disclosed HIO polypeptide n
  • 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 HIO nucleic acid and/or polypeptide sequences have been identified. HIO nucleic acids and polypeptides may be obtained using any available method.
  • nucleic acid sequence may be synthesized. Any known method, such as site directed mutagenesis (Kunkel TA et al., 1991), may be used to introduce desired changes into a cloned nucleic acid.
  • site directed mutagenesis Kelkel TA et al., 1991
  • the methods of the disclosure involve incorporating the desired form of the HIO nucleic acid into a plant expression vector for transformation of in plant cells, and the HIO polypeptide is expressed in the host plant.
  • an "isolated" HIO 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 HIO nucleic acid.
  • an isolated HIO nucleic acid molecule includes HIO nucleic acid molecules contained in cells that ordinarily express HIO where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • the disclosed HIO nucleic acids and polypeptides may be used in the generation of transgenic plants having a modified or altered oil content phenotype.
  • an "altered oil content (phenotype)" may refer to altered oil content in any part of the plant.
  • altered expression of the HIO gene in a plant is used to generate plants with a high oil content (phenotype).
  • the altered oil content is often observed in seeds.
  • Examples of a transgenic plant include plants comprising a plant transformation vector with a nucleotide sequence that encodes or is complementary to a sequence that encodes a HIO polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2 or SEQ ID NO: 4, or an ortholog thereof.
  • Any of the plants or parts thereof of the present disclosure may be processed to produce a feed, food, meal, or oil preparation.
  • Transgenic plants such as corn, soybean and canola containing the disclosed nucleic acid sequences, can be used in the production of vegetable oil and meal.
  • a particularly preferred plant part for this purpose is a seed.
  • Vegetable oil is used in a variety of food products, while meal from seed is used as an animal feed, hi 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.
  • the seed is cleaned to remove plant stalks and other material and then flaked in roller mills to break the hulls.
  • the crushed seed is heated to 75- 100° C to denature hydrolytic enzymes, lyse the unbroken oil containing cells, and allow small oil droplets to coalesce. Most of the oil is then removed (and can be recovered) by pressing the seed material in a screw press. The remaining oil is removed from the presscake by extraction with and organic solvents, such as hexane.
  • the solvent is removed from the meal by heating it to approximately 100° C. After drying, the meal is then granulated to a consistent form.
  • the meal, containing the protein, digestible carbohydrate, and fiber of the seed may be mixed with other materials prior to being used as an animal feed.
  • the meal of the present disclosure may be blended with other meals.
  • the meal produced from plants of the present disclosure or generated by a method of the present disclosure 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.
  • transgenic plants are generally applicable to all plants.
  • activation tagging and gene identification is carried out in Arabidopsis
  • the HIO nucleic acid sequence (or an ortholog, variant or fragment thereof) may be expressed in any type of plant.
  • oil-producing plants produce and store triacylglycerol in specific organs, primarily in seeds.
  • Such species include soybean (Glycine max), rapeseed and canola (including Brassica napus, B.
  • campestris sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn (Zea mays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linum usitatissimum), castor (Ricinus communis), and peanut (Arachis hypogaea).
  • 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 may also be a source of unique fatty acids.
  • 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, Agrobacte ⁇ um-mediated transformation, electroporation, microinjection, microprojectile bombardment, calcium-phosphate-DNA co-precipitation, or liposome-mediated transformation of a heterologous nucleic acid.
  • the transfo ⁇ nation 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 polynucleotide may encode the entire protein or a biologically active portion thereof.
  • binary Ti-based vector systems may be used to transfer polynucleotides.
  • Standard Agrobacterium binary vectors are known to those of skill in the art, and many are commercially available (e.g., pBI121 Clontech Laboratories, Palo Alto, CA).
  • a construct or vector may include a plant promoter to express the nucleic acid molecule of choice.
  • the promoter is a plant promoter.
  • Agrobacterium vectors The optimal procedure for transformation of plants with Agrobacterium vectors will vary with the type of plant being transformed.
  • Exemplary methods for Agrobacterium-mediated transformation include transformation of explants of hypocotyl, shoot tip, stem or leaf tissue, derived from sterile seedlings and/or plantlets. Such transformed plants may be reproduced sexually, or by cell or tissue culture.
  • Agrobacterium transformation has been previously described for a large number of different types of plants and methods for such transformation may be found in the scientific literature.
  • HIO nucleic acid sequence 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 are available for controlling the expression of an HIO 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.
  • octopine synthase (OCS) promoter which is carried on tumor-inducing plasmids of Agrobacterium tumefaciens
  • OCS octopine synthase
  • the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al, Plant MoI. Biol. 9:315-324, 1987) and the CaMV 35S promoter (Odell et al, Nature 373:810-812, 1985 and Jones JD et al, 1992, Transgenic Res., 1:285-297
  • CaMV cauliflower mosaic virus
  • figwort mosaic virus 35S-promoter U.S. Patent No.
  • 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, Plant MoI Bio., 21:625-640).
  • expression of the HIO nucleic acid sequence 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.
  • promoters include the 5' 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 (U.S. Patent No.
  • soybean a' subunit of ⁇ -conglycinin (Chen et al, Proc. Natl Acad. Sci. 83:8560- 8564, 1986), Viciafaba USP (P-Vf.Usp, SEQ ID NO: 1, 2, and 3 in (U.S. Application No. 2003/229918) and Zea mays L3 oleosin promoter (Hong et al, Plant MoI. Biol, 34(3):549-555, 1997).
  • zeins which are a group of storage proteins found in corn endosperm.
  • Genomic clones for zein genes have been isolated (Pedersen et al, Cell, 29:1015-1026, 1982; and Russell et al, Transgenic Res. 6(2): 157-168) and the promoters from these clones, including the 15 kD, 16 kD, 19 kD, 22 kD, 27 kD and genes, could also be used.
  • Other promoters known to function, for example, in corn include the promoters for the following genes: waxy, Brittle, Shrunken 2, Branching enzymes I and II, starch synthases, debranching enzymes, oleosins, glutelins and sucrose synthases.
  • Legume genes whose promoters are associated with early seed and embryo development include V.
  • Cereal genes whose promoters are associated with early seed and embryo development include rice glutelin ("GluA-3,” Yoshihara and Takaiwa, 1996, Plant Cell Physiol. 37:107-11; "GIuB-I,” Takaiwa et al, 1996, Plant MoI Biol. 30:1207- 21; Washida et al, 1999, Plant MoI Biol. 40:1-12; "Gt3,” Leisy et al, 1990, Plant MoI Biol. 14:41-50), ⁇ ceprolamin (Zhou & Fan, 1993, Transgenic Res. 2:141-6), wheat prolamin (Hammond-Kosack et al, 1993, EMBO J.
  • genes whose promoters are associated with early seed and embryo development include oil palm GLO7A (7S globulin, Morcillo et al, 2001, Physiol. Plant 112:233-243), Brassica napus napin, 2S storage protein, and napA gene (Josefsson et al, 1987, J. Biol. Chem. 262:12196-201; Stalberg et ⁇ /., 1993, Plant MoI. Biol. 1993 23:671-83; Ellerstrom et al, 1996, Plant MoI. Biol. 32:1019-27), Brassica napus oleosin (Keddie et al, 1994, Plant MoI. Biol.
  • exemplary methods for practicing this aspect of the disclosure include, but are not limited to antisense suppression (Smith, et al, 1988, Nature, 334:724-726; van der Krol et al, 1988, BioTechniques, 6:958-976); co-suppression (Napoli, et al, 1990, Plant Cell, 2:279-289); ribozymes (PCT Publication WO 97/10328); and combinations of sense and antisense (Waterhouse, et al, 1998, Proc. Natl. Acad. ScL USA, 95:13959- 13964).
  • Methods for the suppression of endogenous sequences in a host cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be suppressed. Such sequences may be homologous to coding as well as non-coding regions of the endogenous sequence. Antisense inhibition may use the entire cDNA sequence (Sheehy et al, 1988, Proc. Natl. Acad. Sd. USA, 85:8805-8809), a partial cDNA sequence including fragments of 5 1 coding sequence, (Cannon et al, 1990, Plant MoI. Biol, 15:39-47), or 3' non-coding sequences (Ch'ng et al, 1989, Proc. Natl. Acad. Sd.
  • Cosuppression techniques may use the entire cDNA sequence (Napoli et al, 1990, Plant Cell, 2:279-289; van der Krol et al, 1990, Plant Cell, 2:291-299), or a partial cDNA sequence (Smith et al, 1990, MoI Gen. Genetics, 224:477-481). Standard molecular and genetic tests may be performed to further analyze the association between a nucleic acid sequence 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 over-expression, ectopic expression, expression in other plant species and gene knock-out (reverse genetics, targeted knock-out, viral induced gene silencing (VIGS; see, Baulcombe D 5 1999, Arch. Virol. Suppl. 15:189-201).
  • expression profiling 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, Cur. Opin. Plant Biol. 2(2):96-103; Dangond F, Physiol Genomics (2000) 2:53-58; van Hal NL et al, JBiotechnol. (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 over-expression 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.
  • Additional methods are disclosed herein of identifying plants that have mutations in endogenous HIO polypeptides that confer altered oil content, and generating a plant having a HIO phenotype, wherein a plant is identified that has an allele in its HIO nucleic acid sequence that results in a HIO phenotype, compared to plants lacking the allele.
  • the plant can generate progeny, wherein the progeny inherit the allele and have a HIO phenotype.
  • provided herein is a method of identifying plants that have mutations in the endogenous HIO nucleic acid sequence that confer a HIO phenotype and generating progeny of these plants with a HIO phenotype that 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 (ethylmethane sulfonate) treatment.
  • EMS ethylmethane sulfonate
  • the resulting plants are grown and self- fertilized, and the progeny are used to prepare DNA samples.
  • HIO-specific PCR is used to identify whether a mutated plant has a mutation in the HIO nucleic acid sequence. Plants having HIO mutations may then be tested for altered oil content, or alternatively, plants may be tested for altered oil content, and then PCR amplification and sequencing of the HIO nucleic acid sequence is used to determine whether a plant having altered oil content has a mutated HIO nucleic acid sequence.
  • 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 nucleic acid sequence or orthologs of the HIO nucleic acid sequence that may confer altered oil content (see Bert et al, Theor Appl Genet, 2003 Jun;107(l):181-9; and Lionneton et al, Genome, 2002 Dec;45(6):1203-15).
  • a HIO nucleic acid is used to identify whether a plant having altered oil content has a mutation an endogenous HIO nucleic acid sequence or has a particular allele that causes altered oil content.
  • Quantitative determination of fatty acid content in T2 seeds was performed using the following methods for HIO30.1.
  • the seed sample was massed on UMT-2 ultra-microbalance (Mettler-Toledo Co., Ohio, USA) and then transferred to a glass extraction vial. Lipids were extracted from the seeds and trans-esterified in 500 ⁇ l 2.5% H 2 SO 4 in MeOH for 3 hours at 80 0 C, following the method of Browse et al. (Biochem. J., 235:25-31, 1986) with modifications.
  • a known amount of heptadecanoic acid was included in the reaction as an internal standard. 750 ⁇ l of water and 400 ⁇ l of hexane were added to each vial, which was then shaken vigorously and allowed to phase separate. Reaction vials were loaded directly onto GC for analysis and the upper hexane phase was sampled by the autosampler. Gas chromatography with Flame Ionization detection was used to separate and quantify the fatty acid methyl esters. Agilent 6890 Plus GCs were used for separation with Agilent Innowax columns (30m x 0.25mm ID, 250 ⁇ m film thickness).
  • the carrier gas was Hydrogen at a constant flow of 2.5 ml/minute, l ⁇ l of sample was injected in splitless mode (inlet temperature 220 0 C, Purge flow 15ml/min at 1 minute).
  • the oven was programmed for an initial temperature of 105 0 C, initial time 0.5 minutes, followed by a ramp of 6O 0 C per minute to 175°C, a 4O 0 C /minute ramp to 26O 0 C with a final hold time of 2 minutes.
  • Detection was by Flame Ionization (Temperature 275°C, Fuel flow 30.0 ml/min, Oxidizer 400.0 ml/min).
  • the ACTTAG line W000086431 was identified as having a high oil phenotype and designated as HIO30.1. Specifically, oil constituted 34.8% of seed mass (w/w) in HIO30.1 compared to an average oil content of 28.7% of other ACTTAG lines grown and analyzed in the same conditions (i.e. reference lines). Reanalysis of the same seed was performed in triplicate. Oil constituted 32.1% of seed mass, confirming an increase in oil content relative to the reference lines.
  • T2 seed stocks of the ACTTAG lines about eighteen T2 seed 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. T3 seed pools were analyzed for oil content by Near Infrared Spectroscopy
  • NIR 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 manufacturer's instructions. Oil content predicting calibrations were developed following the general method of AOCS Procedure Ami -92, Official Methods and Recommended Practices of the American Oil Chemists Society, 5th Ed., AOCS, Champaign 111).
  • Inverse PCR was used to recover genomic DNA flanking the T-DNA insertion, which was then subjected to sequence analysis using a basic BLASTN search and/or a search of the Arabidopsis Information Resource (TAIR) database.
  • TAIR Arabidopsis Information Resource
  • the ACTTAG elements in approximately 40% of the lines analyzed were placed on the genome. At the time of the analysis, 478 high oil lines (as defined above) had successful placements of the ACTTAG element on the genome. Seed from both IN022173 and IN023577 had high oil (107%) and ACTTAG inserts within 10kb of one another.
  • the gene Atlg08520 (HIOlOlB) lies between the insertion sites of the ACTTAG elements in these lines.
  • Plasmid rescue and inverse PCR were used to recover genomic DNA flanking the T-DNA insertion, which was then subjected to sequence analysis using a basic BLASTN search of the Arabidopsis Genomic DNA TAIR database
  • the W000086431 line (HIO30) has T-DNA inserted at three distinct loci.
  • T2 plants were grown to maturity and seed harvested from these plants was used to determine that high oil phenotype.
  • the seed oil content from these was determined by GC analysis as described in Example 1.
  • the genotype of the T2 seed was inferred by analyzing the T3 seed for the presence or absence of the T-DNA at the site of the insertion by PCR using primers that are specific to the corresponding genomic region and the T-DNA. The results show that the loci 2 and 3 were tightly linked. Furthermore, the average oil content of T3 seed containing the T-DNA insert at loci 2 and 3 was higher than those families lacking the insert at these loci.
  • T2 individuals homozygous for loci 2 and 3 produced seed with an oil content of 115.4% of the reference and T2 individuals hemizygous for these loci produced seed with an oil content of 118.4% of the reference while T2 individuals lacking the T-DNA at these loci had an average oil content of 105% of a reference sample of seed from wild-type CoI-O plants. Because the homozygotes and hemizygotes for the high oil loci display a similar increase in oil content, we conclude that loci 2 and 3 are linked with the high oil phenotype and the phenotype is caused by a dominant mutation. By contrast, the average oil content of T3 families containing the T-DNA insert at locus 1 was lower than or about the same as those lacking the insert at the corresponding locus. It is concluded that locus 1 is not linked to the high oil phenotype.
  • At3g54400 HIO30.1
  • oil content in seeds from transgenic plants over-expressing this gene was compared with oil content in seeds from non-transgenic control plants.
  • At3g54400 was cloned into a plant transformation vector behind the seed specific PRU promoter and transformed into Arabidopsis plants using the floral dip method.
  • the plant transformation vector contains the nptll gene, which provides resistance to kanamyacin, and serves as a selectable marker. Seed from the transformed plants were plated on agar medium containing kanamycm. After seven days, transgenic plants were identified as healthy green plants and transplanted to soil.
  • Non-transgenic control plants were germinated on agar medium, allowed to grow for seven days and then transplanted to soil. Twenty-two transgenic seedlings and ten non-transgenic control plants were transplanted to random positions in the same 32 cell flat. The plants were grown to maturity, allowed to self-fertilize and set seed. Seed was harvested from each plant and its oil content estimated by Near Infrared (MR) Spectroscopy using methods previously described.
  • MR Near Infrared
  • Atlg08520 HIOlOlB
  • oil content in seeds from transgenic plants over-expressing this gene was compared with oil content in seeds from non-transgenic control plants.
  • Atlg08520 was cloned into a plant transformation vector behind the seed specific PRU promoter and transformed into Arabidopsis plants using the floral dip method.
  • the plant transformation vector contains the nptll gene, which provides resistance to kanamyacin, and serves as a selectable marker. Seed from the transformed plants were plated on agar medium containing kanamycin. After 7 days, transgenic plants were identified as healthy green plants and transplanted to soil.
  • Non-transgenic control plants were germinated on agar medium, allowed to grow for 7 days and then transplanted to soil. Twenty-two transgenic seedlings and 10 non-transgenic control plants were transplanted to random positions in the same 32 cell flat. The plants were grown to maturity, allowed to self-fertilize and set seed. Seed was harvested from each plant and its oil content estimated by Near Infrared (NIR) Spectroscopy using methods previously described.
  • NIR Near Infrared
  • seed oil content from the progeny of a transgenic line displaying a high oil phenotype was compared with oil content in seeds from non-transgenic control plants.
  • T2 seed from DX06813012 was plated on agar medium containing kanamycin to identify plants containing the transgene. After seven days, transgenic plants were identified as healthy green plants and transplanted to soil. Non-transgenic control plants were germinated on agar medium, allowed to grow for seven days and then transplanted to soil. Twenty- two transgenic seedlings and ten non-transgenic control plants were transplanted to random positions in the same 32 cell flat. The plants were grown to maturity, allowed to self-fertilize and set seed. Seed was harvested from each plant and its oil content estimated by Near Infrared (NTR) Spectroscopy using methods previously described.
  • NTR Near Infrared
  • the seed oil content in the progeny of DX06813012 was higher than the seed oil content of control plants grown in the same tray, see Table 4.
  • seed oil content from the progeny of 4 transgenic lines displaying high oil phenotypes was compared with oil content in seeds from non-transgenic control plants.
  • T2 seed from Z003907005, Z003907008, Z003907013 and Z003907018 were plated on agar medium containing kanamycin to identify plants containing the transgene. After seven days, transgenic plants were identified as healthy green plants and transplanted to soil.
  • Non-transgenic control plants were germinated on agar medium, allowed to grow for seven days and then transplanted to soil. Twenty-two transgenic seedlings from each line and ten non-transgenic control plants were transplanted to random positions in four 32 cell flats. The plants were grown to maturity, allowed to self-fertilize and set seed. Seed was harvested from each plant and its oil content estimated by Near Infrared (NIR) Spectroscopy using methods previously described.
  • NIR Near Infrared
  • the seed oil content in the progeny of the transgenic lines was higher than the seed oil content of control plants grown in the same tray, see Table 5.

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Abstract

La présente invention concerne des plantes et des cellules végétales présentant un phénotype à teneur en huile modifiée en raison d'une expression modifiée d'un acide nucléique à haute teneur en huile. L'invention concerne également des procédés de production de plantes présentant un phénotype à teneur en huile modifiée.
PCT/US2006/001280 2005-01-12 2006-01-11 Production de plantes a teneur en huile modifiee Ceased WO2006076596A2 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
MX2007008435A MX2007008435A (es) 2005-01-12 2006-01-11 Generacion de plantas con contenido alterado de aceite.
BRPI0606643-7A BRPI0606643A2 (pt) 2005-01-12 2006-01-11 geração de plantas com teor alterado de óleo
US11/813,858 US7663020B2 (en) 2006-01-11 2006-01-11 Generation of plants with altered oil content
CN2006800022466A CN101103116B (zh) 2005-01-12 2006-01-11 生产改变含油量的植物

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US60/643,674 2005-01-12

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WO2006076596A3 WO2006076596A3 (fr) 2007-01-04

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110527738A (zh) * 2019-08-28 2019-12-03 中国农业科学院油料作物研究所 甘蓝型油菜种子油酸含量的主效qtl位点、snp分子标记及其应用

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EP1033405A3 (fr) * 1999-02-25 2001-08-01 Ceres Incorporated Fragments d'ADN avec des séquences déterminées et polypeptides encodées par lesdits fragments
AU2003304464A1 (en) * 2002-03-15 2005-03-29 Monsanto Technology Llc Nucleic acid molecules associated with oil in plants
WO2004093532A2 (fr) * 2003-04-22 2004-11-04 Agrinomics Llc Generation de plantes avec un contenu en huile modifie
WO2004093528A2 (fr) * 2003-04-22 2004-11-04 Agrinomics Llc Génération de plantes à teneur modifiée en huile

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110527738A (zh) * 2019-08-28 2019-12-03 中国农业科学院油料作物研究所 甘蓝型油菜种子油酸含量的主效qtl位点、snp分子标记及其应用

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AR059287A1 (es) 2008-03-26
WO2006076596A3 (fr) 2007-01-04
CN101103116B (zh) 2012-09-05
ZA200705263B (en) 2009-02-25
BRPI0606643A2 (pt) 2009-07-14
MX2007008435A (es) 2007-09-06
PY0600557A (es) 2010-06-01

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