WO2005006846A2 - Production de vegetaux a teneur en huile modifiee - Google Patents

Production de vegetaux a teneur en huile modifiee Download PDF

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WO2005006846A2
WO2005006846A2 PCT/US2004/022578 US2004022578W WO2005006846A2 WO 2005006846 A2 WO2005006846 A2 WO 2005006846A2 US 2004022578 W US2004022578 W US 2004022578W WO 2005006846 A2 WO2005006846 A2 WO 2005006846A2
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plant
hiol
sequence
oil
phenotype
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WO2005006846A3 (fr
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Jonathan Lightner
Hein Tsoeng Ng
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Agrinomics LLC
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Agrinomics LLC
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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

  • 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 com oil (US Pat Nos 6,229,033 and 6,248,939), and laurate-containing seeds (US Pat No 5,639,790), among others.
  • HOC High-Oil Com
  • DuPont US 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.
  • Biochemical screens of seed oil composition have identified Arabidopsis genes for many critical biosynthetic enzymes and have led to identification of agronomically important gene orthologs. For instance, screens using chemically mutagenized populations have identified lipid mutants whose seeds display altered fatty acid composition (Lemieux et al, 1990; James and Dooner, 1990). T-DNA mutagenesis screens (Feldmann et al, 1989) that detected altered fatty acid composition identified the omega 3 desaturase (FAD3) and delta- 12 desaturase (FAD2) genes (US Pat No 5952544; Yadav et al, 1993; Okuley et al, 1994).
  • FAD3 omega 3 desaturase
  • FAD2 delta- 12 desaturase
  • 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
  • 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).
  • 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 HIOl 10.3 polypeptide.
  • the transgenic plant is selected from the group consisting of rapeseed, soy, com, 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 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 HIOl 10.3 polypeptide, and growing the transformed progenitor cells to produce a transgenic plant, wherein the HIOl 10.3 polynucleotide sequence is expressed causing the high oil phenotype.
  • 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.
  • 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.
  • 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.
  • “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.
  • transfection 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.
  • tissue e.g., callus
  • 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.
  • 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.
  • 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 insertion.
  • Tl plants were exposed to the selective agent in order to specifically recover transformed plants that expressed the selectable marker and therefore harbored T-DNA insertions.
  • HIOl 10.3 genes and/or polypeptides may be employed in the development of genetically modified plants having a modified oil content phenotype ("a HIOl 10.3 phenotype").
  • HIOl 10.3 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.
  • HIOl 10.3 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 HIOl 10.3 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.
  • HIOl 10.3 Nucleie Acids and Polypeptides Arabidopsis HIOl 10.3 nucleic acid (genomic DNA) sequence is provided in SEQ ID NO:
  • HIOl 10.3 polypeptide refers to a full-length HIOl 10.3 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 HIOl 10.3 polypeptide causes an altered oil content phenotype when mis-expressed in a plant.
  • mis- expression of the HIOl 10.3 polypeptide causes a high oil phenotype in a plant.
  • a functionally active HIO110.3 polypeptide is capable of rescuing defective (including deficient) endogenous HIOl 10.3 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 HIOl 10.3 polypeptide retains one of more of the biological properties associated with the full-length HIOl 10.3 polypeptide, such as signaling activity, binding activity, catalytic activity, or cellular or extra-cellular localizing activity.
  • a HIOl 10.3 fragment preferably comprises a HIOl 10.3 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 HIOl 10.3 protein.
  • HIOl 10.3 fragment comprises of one or more non-secretory proteins (synonym: T2E6.18).
  • Functionally active variants of full-length HIOl 10.3 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 HIOl 10.3 polypeptide. In some cases, variants are generated that change the post-translational processing of a HIOl 10.3 polypeptide.
  • HIOl 10.3 nucleic acid encompasses nucleic acids with the sequence provided in or complementary to the sequence provided in SEQ ID NO:l, as well as functionally active fragments, derivatives, or orthologs thereof.
  • a HIOl 10.3 nucleic acid of this invention may be DNA, derived from genomic DNA or cDNA, or RNA.
  • a functionally active HIOl 10.3 nucleic acid encodes or is complementary to a nucleic acid that encodes a functionally active HIOl 10.3 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 HIOl 10.3 polypeptide.
  • a HIOl 10.3 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 HIOl 10.3 polypeptide, or an intermediate form.
  • a HIOl 10.3 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 HIOl 10.3 nucleic acid is capable of being used in the generation of loss-of-function HIOl 10.3 phenotypes, for instance, via antisense suppression, co-suppression, etc.
  • a HIOl 10.3 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 HIOl 10.3 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 HIOl 10.3 polypeptide of the invention comprises a polypeptide sequence with at least 50% or 60% identity to the HIOl 10.3 polypeptide sequence of SEQ ID NO:2, and may have at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the HIOl 10.3 polypeptide sequence of SEQ ID NO:2, such as one or ⁇ more non-secretory proteins (synonym: T2E6.18).
  • a HIOl 10.3 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 HIOl 10.3 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 non-secretory proteins (synonym: T2E6.18).
  • a HIOl 10.3 polynucleotide sequence is at least 50% to 60% identical over its entire length to the HIO110.3 nucleic acid sequence presented as SEQ JD NO:l, or nucleic acid sequences that are complementary to such a HIOl 10.3 sequence, and may comprise at least 70%, 80%, 85%, 90% or 95% or more sequence identity to the HIO110.3 sequence presented as SEQ ID NO:l 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-2.0al9 (Altschul et al, J. Mol. Biol. (1997) 215:403-410; website at blast.wustl.edu/blast/README.html) with search parameters set to default values.
  • the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched.
  • a "% identity value” is determined by the number of matching identical nucleotides or amino acids divided by the sequence length for which the percent identity is being reported.
  • Percent (%) amino acid sequence similarity is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation.
  • a conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected.
  • Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, cysteine and glycine.
  • Derivative nucleic acid molecules of the subject nucleic acid molecules include sequences that selectively hybridize to the nucleic acid sequence of SEQ ID NO:l.
  • 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:l 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 HIOl 10.3 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).
  • sequence variants may be used in the methods of this invention.
  • the methods of the invention may use orthologs of the Arabidopsis HIOl 10.3. 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.
  • orthologs encompasses paralogs.
  • 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 may be used to highlight conserved regions and/or residues of orthologous proteins and to generate phylogenetic trees.
  • CLUSTAL Thimpson JD et al, 1994, Nucleic Acids Res 22:4673-4680
  • 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 may also identify potential orthologs.
  • Nucleic acid hybridization methods may also be used to find orthologous genes and are preferred when sequence data are not available.
  • Degenerate PCR and screening of cDNA or genomic DNA libraries are common methods for finding related gene sequences and are well known in the art (see, e.g., Sambrook, 1989; Dieffenbach and Dveksler, 1995). For instance, methods for generating a cDNA library from the plant species of interest and probing the library with partially homologous gene probes are described in Sambrook et al. A highly conserved portion of the Arabidopsis HIOl 10.3 coding sequence may be used as a probe.
  • HIOl 10.3 ortholog nucleic acids may hybridize to the nucleic acid of SEQ ID NO:l 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 HIOl 10.3 polypeptides are used for ortholog isolation (see, e.g., Harlow and Lane, 1988, 1999).
  • Western blot analysis can determine that a HIOl 10.3 ortholog (i.e., an orthologous protein) is present in a crude extract of a particular plant species.
  • 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 gtll, 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 HIOl 10.3 nucleic acid and/or polypeptide sequences have been identified.
  • HIOl 10.3 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 al, 1991), may be used to introduce desired changes into a cloned nucleic acid. In general, the methods of the invention involve incorporating the desired form of the HIOl 10.3 nucleic acid into a plant expression vector for transformation of in plant cells, and the HIOl 10.3 polypeptide is expressed in the host plant.
  • PCR polymerase chain reaction
  • An isolated HIOl 10.3 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 HIOl 10.3 nucleic acid.
  • an isolated HIOl 10.3 nucleic acid molecule includes HIOl 10.3 nucleic acid molecules contained in cells that ordinarily express HIOl 10.3 where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
  • HIOl 10.3 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 HIOl 10.3 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 HIOl 10.3 gene (or an ortholog, variant or fragment thereof) may be expressed in any type of plant.
  • 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), com (Zea mays), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconut palm (Cocos nucifera), flax (Linum usitatissimum), castor (Ricinus communis) and peanut (Arachis hypogaea).
  • the invention may also be directed to fruit- and vegetable-bearing plants, grain-producing plants, nut-producing plants, rapid cycling Brassica species, alfalfa (Medicago sativa), tobacco (Nicotiana), turfgrass (Poaceae family), other forage crops, and wild species that may be a source of unique fatty acids.
  • 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 Agr ⁇ b ⁇ cte ⁇ 'wm-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 an HIOl 10.3 polynucleotide may encode the entire protein or a biologically active portion thereof.
  • binary Ti-based vector systems may be used to transfer polynucleo tides.
  • 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).
  • the optimal procedure for transformation of plants with Agrobacterium vectors will vary with the type of plant being transformed.
  • Exemplary methods for Agrobacterium- ⁇ aa wX& ⁇ transformation include transformation of explants of hypocotyl, shoot tip, stem or leaf tissue, derived from sterile seedlings and or plantlets. Such transformed plants may be reproduced sexually, or by cell or tissue culture.
  • Agrobacterium transformation has been previously described for a large number of different types of plants and methods for such transformation may be found in the scientific literature. Of particular relevance are methods to transform commercially important crops, such as rapeseed (De Block et al, 1989), sunflower (Everett et al, 1987), and soybean (Christou et al, 1989; Kline et al, 1987).
  • Expression (including transcription and translation) of HIO110.3 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 HIOl 10.3 nucleic acid.
  • constitutive promoters include the raspberry E4 promoter (U.S. Patent Nos. 5,783,393 and 5,783,394), the 35S CaMV (Jones JD et al, 1992), the CsVMV promoter (Verdaguer B et al, 1998) and the melon actin promoter (published PCT application WO0056863).
  • tissue-specific promoters include the tomato E4 and E8 promoters (U.S. Patent No.
  • HIO110.3expression is under control of regulatory sequences from genes whose expression is associated with early seed and or embryo development.
  • Legume genes whose promoters are associated with early seed and embryo development include V.faba legumm (Baumlein et al, 1991, Mol Gen Genet 225:121-8; Baumlein et al, 1992, Plant J 2:233-9), 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 37:110-11; "GluB-1,” Takaiwa et al, 1996, Plant Mol Biol 30:1207-21; Washida et al, 1999, Plant Mol Biol 40:1-12; "Gt3,” Leisy et al, 1990, Plant Mol Biol 14:41-50), rice prolamin (Zhou & Fan, 1993, Transgenic Res 2:141-6), wheat prolamin (Hammond-Kosack et al, 1993, EMBO J 12:545-54), maize zein (Z4, Matzke et al, 1990, Plant Mol Biol 14:323-32), and barley B-hordeins (Entwistle et al, 1991, Plant Mol Biol 17:1217-31).
  • genes whose promoters are associated with early seed and embryo development include oil palm GL07A (7S globulin, Morcillo et al, 2001, Physiol Plant 112:233-243), Brassica napus napin, 2S storage protein, and napA gene (Josefsson et al, 1987, J Biol Chem 262:12196-201; Stalberg et al, 1993, Plant Mol Biol 1993 23:671-83; Ellerstrom et al, 1996, Plant Mol Biol 32:1019-27), Brassica napus oleosin (Keddie et al, 1994, Plant Mol Biol 24:327-40), Arabidopsis oleosin (Plant et al, 1994, Plant Mol Biol 25:193-205), Arabidopsis FAEl (Rossak et al, 2001, Plant Mol Biol 46:717-25), Canavalia gladiata conA (Yamamoto et al, 1995, Plant Mol Biol
  • 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).
  • Exemplary methods for practicing this aspect of the invention include, but are not limited to antisense suppression (Smith, et ⁇ -.,1988; van der Krol et al, 1988); co-suppression (Napoli, et al, 1990); ribozymes (PCT Publication WO 97/10328); and combinations of sense and antisense (Waterhouse, et al, 1998).
  • Methods for the suppression of endogenous sequences in a host cell typically employ the transcription or transcription and translation of at least a portion of the sequence to be suppressed. Such sequences may be homologous to coding as well as non-coding regions of the endogenous sequence.
  • Antisense inhibition may use the entire cDNA sequence (Sheehy et al, 1988), a partial cDNA sequence including fragments of 5' coding sequence, (Cannon et al, 1990), or 3' non-coding sequences (Ch'ng et al, 1989).
  • Cosuppression techniques may use the entire cDNA sequence (Napoli et al, 1990; van der Krol et al, 1990), or a partial cDNA sequence (Smith et al, 1990). 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 perfo ⁇ ned.
  • 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).
  • expression profiling generally by microarray analysis, is used to simultaneously measure differences or induced changes in the expression of many different genes.
  • 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.
  • 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.
  • 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 (NTR) intact at time of harvest. NIR infrared spectra were captured using a Bruker 22 N/F. Bruker Software was used to estimate total seed oil and total seed protein content using data from NIR analysis and reference methods according to the manufacturers instructions. Oil contents predicted by our calibration (PDX Oil 3, Predicts Hexane Extracted Oil) were compared for 40,000 individual ACTTAG lines. To identify high oil lines the NIR oil result was compared to the mean oil result for all ACTTAG lines planted on the same day (Relative oil content).
  • NTR Near Infrared Spectroscopy
  • Line W000097868 (IN033255) had a NIR determined oil content of 39.8% relative to a planting day average oil content of 35.7% (111% of PDA).
  • Line W000142648 had a NIR determined oil content of 39.8% relative to a planting day average oil content of 35.7% (111% of PDA).
  • Line W000097868 (LN033255) had a confirmed ACTTAG insertion on Chromosome 1 at bp 17164913.
  • Line W000142648 (IN044809) had a confirmed ACTTAG insertion on
  • Chromosome 1 at bp 17165460 Chromosome 1 at bp 17165460.
  • ACTTAG line IN033255 For ACTTAG line IN033255, there was sequence identity to nucleotides 64283- 64817 on Arabidopsis BAC clone T2E6 chromosome 1 (GI#9743359), placing the left border junction upstream from nucleotide 64817 (GI#9743359). The opposite flank (predicted right border junction) of this insert was not determined. Left border of IN033255 T-DNA was about 8448 bp 5' of the translation start site.
  • ACTTAG line E 044809 there was sequence identity to nucleotides 63685- 64270 on Arabidopsis BAC clone T2E6 chromosome 1 (GI#9743359), placing the left border junction downstream from nucleotide 63685 (GI#9743359). The opposite flank (predicted right border junction) of this insert was not determined. Left border IN044809 T-DNA is about 9578 bp 5' of the translation start site.
  • Atlg47750 has homology to a number of proteins from plants.
  • the top 10 BLAST results for Atlg47750 are listed below and are included in the Ortholog Table. 1. Itself (several redundant entries) >gi
  • AI995247 come from this gene >gi
  • the cellular localization of Atlg47750 is predicted by Psort2 (32.0 %: cytoplasmic, 32%: nuclear, 12%: mitochondrial, 8%: vesicles of secretory system, 8%: cytoskeletal).
  • Psort2 3.0 %: cytoplasmic, 32%: nuclear, 12%: mitochondrial, 8%: vesicles of secretory system, 8%: cytoskeletal).
  • Atlg47750 is likely to be a transmembrane protein.
  • Pfam analysis showed that Atlg47750 is a member of the peroxisomal biogenesis factor 11 (PEX11) family of proteins (PF05648).
  • PEXll peroxisomal biogenesis factor 11
  • yeast, mouse and human overexpression of PEXll promotes peroxisome division.
  • loss of PEXll results in reduced peroxisome abundance (see Li and Gould 2003; Li et al 2002; Carlo et al, 2000).
  • peroxisome is required for fatty acid metabolism (e.g. beta oxidation of fatty acids).
  • Atlg47750 can lead to change in seed oil content in Arabidopsis.
  • the invention further provides a method of identifying plants that have mutations in, or an allele of, endogenous HIOl 10.3 that confer a HIOl 10.3 phenotype, and generating progeny of these plants that also have the HIOl 10.3 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 trea ⁇ nent.
  • the resulting plants are grown and self-fertilized, and the progeny are used to prepare DNA samples.
  • HIOl 10.3-specific PCR is used to identify whether a mutated plant has a HIOl 10.3 mutation. Plants having HIOl 10.3 mutations may then be tested for the HIOl 10.3 phenotype, or alternatively, plants may be tested for the HIOl 10.3 phenotype, and then HIOl 10.3-specific PCR is used to determine whether a plant having the HIO110.3phenotype has a mutated HIO110.3gene.
  • TR-LD G 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 HIOl 10.3 gene or orthologs of HIOl 10.3 that may confer the HIOl 10.3 phenotype (see Foolad et al, Theor Appl Genet. (2002) 104(6-7):945-958; Rothan et al, Theor Appl
  • a HIOl 10.3 nucleic acid is used to identify whether a plant having a HIOl 10.3 phenotype has a mutation in endogenous HIOl 10.3 or has a particular allele that causes the HIOl 10.3 phenotype compared to plants lacking the mutation or allele, and generating progeny of the identified plant that have inherited the HIOl 10.3 mutation or allele and have the HIOl 10.3 phenotype.
  • EXAMPLE 5 Recapitulation of the High Oil Phenotype To confirm that over-expression of Atlg47750 causes the high seed oil phenotype in HIOl 10.3, Arabidopsis plants of the Col-0 ecotype were transformed by Agrobacterium mediated transformation with a construct containing the coding sequences of the HIOl 10.3 gene (Atlg47750) 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 npiS. gene to confer kanamycin resistance in plants. Tl seed was harvested from the transformed plants and transformants selected by germinating seed on agar medium containing kanamycin. Kanamycin resistant transformants were transplanted to soil after 7 days.
  • Control plants were germinated on agar medium without kanamycin, transplanted to soil after 7 days. Twenty plants containing the CsVMV::HIO110.3 transgene along with 10 Col-0 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
  • NIR Infrared Spectroscopy
  • 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 AMI -92, Official Methods and Recommended Practices of the American Oil Chemists Society, 5th Ed., AOCS, Champaign, HI. Seed from transgenic plants and control plants grown in the same flat were compared. T2 seed from plants containing the CsVMV::HI0110.3 transgene contained an average of 3% more oil than the seed from the control plants (Table 2). This increase was determined to be statistically significant (P ⁇ 0.05) by a T-test.

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Abstract

L'invention concerne des végétaux présentant un phénotype à teneur en huile modifiée en raison de l'expression modifiée d'un acide nucléique HIO110.3. L'invention concerne également des procédés de production de végétaux à phénotype à teneur en huile modifiée.
PCT/US2004/022578 2003-07-14 2004-07-13 Production de vegetaux a teneur en huile modifiee Ceased WO2005006846A2 (fr)

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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENEMBL [Online] 15 August 2000 CHAO ET AL., XP002983139 Database accession no. (AC012463) *
DATABASE GENEMBL [Online] 27 March 2003 HAAS ET AL., XP002983140 Database accession no. (AY074249) *

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