CN1893817A - Generation of plants with altered oil content - Google Patents

Abstract

The present invention is directed to plants that display an altered oil content phenotype due to altered expression of a HIO30.5 nucleic acid. The invention is further directed to methods of generating plants with an altered oil content phenotype.

Description

Produce the plant that changes oil content

Related application

The application requires the priority of the U.S. Provisional Patent Application 60/530,828 of submission on December 17th, 2003, and its content all is incorporated herein by reference.

Background of invention

The content of composition, the especially seed oils of manipulation crop seeds and the ability of composition have important use and are worth in the agro-industry that relates to processed food oils and animal feeding oils.The seed of crops contains multiple valuable component and comprises oils, albumen and starch.Industrial processes can separate some or all these components are used for selling separately of specialized application.For example, almost 60% U.S. soybean crops squeeze by soybean processing industry.Soybean processing produces the refined oil of selling goods at a high figure, and residue is sold (US Soybean Board, 2001 Soy Stats) mainly as the feed of low value.Canada's rape (canola) seed is squeezed produces oils and by product Canada coarse colza meal (Canadian rape association).Almost the american corn crops in 20% 1999/2000 year are mainly used in generation starch, ethanol and oils (corn refining dealer association) through industrial refining.Therefore, usually need to make the oil content maximization of seed.For example, for for the processing oilseed of soybean and Canadian rape, the absolute oil content that improves seed will improve the value of this cereal.For the corn of processing, may need to improve or reduce oil content according to the application of other key component.Reducing oils can be by reducing the quality that the undesirable taste relevant with the oils oxidation improves the starch that separates.Perhaps, in the unessential ethanol of taste produces, improve oil content and can improve total value.In many grain troughs, such as corn and wheat, may wish to improve oil content in plants, because oils has higher energy content than other seed components such as carbohydrate.Oilseed processing is just as most of cereal processing industries are intensive capital industries; Therefore the less change of the product distribution of product from the low value component to the high value oil component may have the remarkable economical influence for the cereal processor.

The biotechnology of oils is handled can provide the change of composition and the improvement of oil yield.The change of forming especially comprises the soybean and the corn oil (United States Patent (USP) 6,229,033 and 6,248,939) of high oleic acid, and the seed (United States Patent (USP) 5,639,790) etc. that contains laurate.The work that composition changes mainly concentrates on the oilseed of processing but can extend to non--oilseed crops at an easy rate comprises corn.Though have suitable interest to improving oil content, unique feasible biotechnology is high oils corn (HOC) technology (Du Pont, United States Patent (USP) 5,704,160) at present in this field.HOC utilizes by the selection breeding of standard hybridizes the female high oils pollina who obtains together with the improved seeds that are called topcross (TopCross) in the production system (infertile male).The cereal oil content that the high oils of topcross system makes harvest corn from～3.5% bring up to～7%, improved the energy content of cereal.

Though the HOC production system is fruitful, it still has intrinsic limitation.At first, the system of undertaking low pollina's percentage that the seed in whole soils plants contains inherent risk, especially in the year of drought.The second, current HOC field oil content is steadily in about 9% oils.At last, high oils corn master changes if it were not for biochemistry, but for the mutant on the anatomy that improves oil content generation indirect consequence (improving the plumule size).For this reason, selectable high oils strategy, it will be very important especially deriving from the high oils strategy that changes biochemical output.

The most tangible target crops in process oil market are soybean and rape seed, and a large amount of commercial pursuit (for example United States Patent (USP) 5,952, and 544; PCT applies for WO9411516) prove that arabidopsis is the good model of oils metabolism in these crops.The biochemistry screening of seed oil compositions has been identified the many crucial biosynthetic enzyme genes of arabidopsis and has been caused agricultural to go up directly being identified to homologue of important gene.For example, the screening that utilizes mutagenesis to handle the group has identified that its seed shows the lipid mutant (Lemieux B etc. 1990, James and Dooner, 1990) that the fatty acid composition changes.Detect the T-DNA mutagenesis screening (Feldmann etc. 1989) of fatty acid composition change and identified ω 3 dehydrases (FAD3) and δ-12 dehydrase (FAD2) gene (United States Patent (USP) 5952544; Yadav etc., 1993; Okuley etc., 1994).The mutant that wrinkled seed that concentrates on oil content but not induce on the screening analytical chemistry of oils quality or seed density change is inferred the oil content in plants (Focks and Benning, 1998) of change in view of the above.Be used for identifying the another kind of Screening and Identification that participates in the enzyme that unusual long-chain fatty acid produces coding be responsible for reducing the sudden change (Katavic V etc., 1995) of the gene of the diacylglycerol acyltransferase (DGAT) that triacylglycerol is assembled in the seed.In addition, the seed specific that also shows DGAT cDNA is crossed the oil content in plants relevant (Jako etc., 2001) of expressing with raising.

Activation labeling acts in the plant refer to a kind of by insertion comprise regulate sequence (for example, enhancer) the heterologous nucleic acids construct in Plant Genome and produce the method for random mutation.Regulate sequence and can play the effect that the one or more natural plant genes of enhancing are transcribed; Therefore, activate labeling acts and be and a kind ofly be used to produce function and obtain (gain-of-function), normally the effective ways of dominant mutant (referring to, for example, Hayashi etc., 1992; Weigel D etc., 2000).The construct that inserts provides a kind of molecular label, and it is used for Rapid identification because its wrong natural plants of expressing the mutagenesis phenotype.Activate the phenotype that labeling acts can cause afunction simultaneously.Insertion can cause the destruction of natural plant gene, and phenotype is normally recessive in this case.

Activate labeling acts and be used for multiple species, comprise tobacco and arabidopsis, with identify various mutant phenotypes and with the gene of these phenotypic correlations (Wilson, etc., 1996, Schaffer etc., 1998, Fridborg etc., 1999; Kardailsky etc., 1999; Christensen S etc., 1998).

Summary of the invention

The invention provides and have the phenotypic genetically modified plants of high oils content.Genetically modified plants comprise and contain coding or be complementary to the conversion carrier of nucleotide sequence that coding comprises the sequence of the HIO30.5 polypeptide of amino acid sequence of SEQ ID NO:2 or its lineal homologue.In preferred embodiments, genetically modified plants are selected from rape, soybean, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut, flax, castor-oil plant and peanut.The present invention also provides the method that produces oils, comprises cultivating genetically modified plants and reclaiming oils from described plant.The present invention further provides to produce and had the method for high oily phenotype plant, described method is tested and appraised the allelic plant with HIO30.5 gene and carries out, the allelomorph of described HIO30.5 gene causes the increase of comparing oil content with the plant of filial generation that lacks this equipotential gene and produce the plant of described evaluation, the filial generation that wherein produces this equipotential gene of heredity and be high oil meter type.Genetically modified plants of the present invention import the plant conversion carrier of nucleotide sequence that contains coding or be complementary to the sequence of coding HIO30.5 polypeptide by comprising to the CFU-GM of plant, and cultivate the method generation that the CFU-GM that transforms produces genetically modified plants, the expression of HIO30.5 polynucleotide sequence wherein produces high oils content phenotype.

Detailed Description Of The Invention

Definition

Unless otherwise stated, whole technology used herein and scientific terminology have the identical implication of knowing with those skilled in the art.The professional is especially with reference to Sambrook etc., and 1989, and Ausubel FM etc., 1993 definition and term.Should be appreciated that to the invention is not restricted to described ad hoc approach that flow process and reagent are because these can change.

Term described herein " carrier " is meant and is designed for the nucleic acid construct that shifts between different host cell." expression vector " is meant and can integrates the also carrier of expressing heterologous dna fragmentation in the allos cell.Many protokaryons and eucaryote expression vector can commercially available acquisitions.It is known to select suitable expression vector to be that personnel are stated in this area.

But " allos " nucleic acid construct or sequence have the wild-type sequence that a part of sequence is not a plant cell express therein.The control sequence of allos is meant the control sequence (being promotor or enhancer) of the homologous genes expression that not natural adjusting is being regulated at present.Usually, heterologous nucleic acid sequence be not they existing in the endogenous generation of cell or genomic a part of institute, and by infection, transfection, microinjection, electroporations etc. add in the cell." allos " nucleic acid construct can contain control sequence/dna encoding combined sequence, and it is identical or different with control sequence/dna encoding combined sequence of finding in the wild-type plant.

Term described herein " gene " is meant and participates in the dna fragmentation that polypeptide chain produces, it may comprise or not comprise before the code area and zone afterwards, and for example 5 ' non-translational region (5 ' UTR) or " leading " sequence and 3 ' UTR or " afterbody " sequence and independent encode fragment (exon) and the intron sequences (intron) between non--transcriptional regulatory sequences.

" recombinant " used herein comprises cell or the carrier of having modified by the nucleotide sequence that imports allos, and perhaps described cell derives from the cell of modification like this.Therefore, for example, do not find the gene of same form in the cell of form that recombinant cell is expressed in natural (non--recombinant) or be expressed under the human intervention natural gene of the unconventionality expression of expressing fully or not expressing fully.

Term described herein " gene expression " is meant the method based on the polypeptide of the nucleotide sequence generation of gene.Described method comprises to be transcribed and translates; Therefore " expression " can be at polynucleotides or peptide sequence or both.Sometimes, the expression of polynucleotide sequence can not cause protein translation." cross express " is meant with respect to its expression in wild type (perhaps other reference [for example, non--transgenosis]) plant, polynucleotides and/or peptide sequence are expressed to be increased and may relate to that nature exists or non--and the sequence of existence naturally." ectopic expression " be meant not-change or wild-type plant in not abiogenous, at a time, somewhere and/or the expression of improving the standard." downward modulation is expressed " is meant polynucleotides and/or peptide sequence, and normally endogenous gene is expressed with respect to its reduction of expressing in wild-type plant.Term " false demonstration " and " change and express " comprised expression, and downward modulation is expressed and ectopic expression.

Nucleotide sequence is being inserted in the notion of cell, term " introducing " is meant " transfection ", perhaps " conversion " or " transduction ", and comprise that nucleotide sequence is incorporated in eucaryon or the prokaryotic, wherein nucleotide sequence can be integrated into genome (chromosome for example, the plasmid of cell, plastid or mitochondrial DNA), be converted into self-replicating, perhaps moment is expressed (for example, the mRNA of transfection).

" plant cell " used herein is meant the arbitrary cell that derives from plant, comprises from undifferentiated tissue (for example callus) and plant seed, pollen, the cell of brood body (progagules) and embryo.

Term described herein " natural " and " wild type " are meant to have feature or the phenotypic form of finding the plant of identical type itself with respect to given plant trait or phenotype.

Term described herein is meant the phenotypic change of genetically modified plants with respect to similarly non--genetically modified plants about " modification " of plant characteristic." purpose phenotype (proterties) " for genetically modified plants, refer to observable or measurable phenotype by T1 and/or progeny plants proof, corresponding non-transgenic plant does not show this phenotype (the similar plant of genotype of cultivating or analyzing yet promptly) under condition of similarity.The purpose phenotype can be represented the improvement to plant, and a kind of method that produces improvement in other plant maybe can be provided." improvement " is a kind of by unique and/or new quality are provided to plant, and strengthens the feature of the practicality of plant species or kind." change oil content phenotype " refers to the phenotype measured of genetically modified plant, wherein with similarly, but be that the plant of non-modification is compared, this plant shows the overall oil content (that is, oils accounts for the percentage of seed quality) that statistics goes up significantly to be increased or reduce.High oil meter type refers to that overall oil content increases.

" mutant " polynucleotide sequence used herein or gene and corresponding wild type polynucleotide sequence or gene are in sequence or express differently, and difference wherein causes phenotype or the proterties of the plant of modifying.With respect to plant or plant strain system, term " mutant " is meant that plant or strain cording have the plant phenotype or the proterties of modification, and wherein phenotype of Xiu Shiing or proterties are expressed relevant with the modification of wild type polynucleotide sequence or gene.

Term described herein " T1 " is meant the plant filial generation from the seed of T0 plant.The T1 filial generation is can be by the first serial plant transformed of application choice reagent screening, and described selective reagent is for example antibiotic or weed killer herbicide, and genetically modified plants contain corresponding resistant gene thus.Term " T2 " is meant that the flower by the T1 plant carries out the plant filial generation that self-fertilization produces, and is screened as genetically modified plants before the described T1 plant.The T3 plant is by generations such as T2 plants." the lineal filial generation " of specified plant described here derives from the seed (perhaps, sometimes from other tissue) of described plant and is the filial generation that follows closely; For example, for specific family, the T2 plant is the lineal filial generation of T1 plant." indirectly filial generation " of specified plant derives from the seed (or other tissue) of the lineal filial generation of described plant, or from the seed (or other tissue) of this family filial generation subsequently; For example, the T3 plant is the indirect filial generation of T1 plant.

Term described herein " plant part " comprises plant organ or tissue arbitrarily, includes, but are not limited to seed, embryo, meristematic tissue zone, callus, leaf, root, bud, gametophyte, sporophyte, pollen and microspore.Plant cell can be available from plant organ or tissue and culture prepared therefrom arbitrarily.The floristics that can be used for the inventive method normally widely, it can be the higher plant that can be used for transformation technology, comprises monocotyledonous and dicots plant.

Described here " genetically modified plants " are included in the plant that its genome contains heterologous polynucleotide.The polynucleotides of allos can stably be incorporated in the genome, maybe can be inserted into outside the chromosome.Preferably, thus polynucleotides of the present invention are incorporated into with being stabilized and make polynucleotides by continuous passage in the genome.Plant cell, tissue, organ or the plant that has been imported into heterologous polynucleotide are considered to " conversion ", " transfection " or " genetically modified " plant.Also containing the conversion plant of this heterologous polynucleotide or the direct line and the indirect filial generation of plant cell also is considered to be genetically modified.

The evaluation of plant with oil content phenotype of change

We use arabidopsis to activate label (ACTTAG) screening, identify our called after " HIO30.5 " (At3g52280; Association between the oil content phenotype of gene GI#18409525:1-1110) and change (high oil meter type particularly).Briefly, and as further describing among the embodiment, with the pSKI015 carrier many arabidopsis thalianas are suddenlyd change, described carrier comprises the T-DNA from the Agrobacterium tumefaciems Ti-plasmids, virus enhancer element and selectable marker gene (Weigel etc., 2000).When T-DNA was inserted in the genome that transforms plant, near the rise of gene enhancer element can cause was usually in about 10 kilobase (kb) of enhancer.In order to identify genetically modified plants, the T1 plant is exposed to selective agent, express selected marker so that reclaim specifically, and have the conversion plant of T-DNA.Collect the T2 seed from these plants.Extract lipid from about 15-20T2 seed.Carry out gas-chromatography (GC) analysis, to measure the content of fatty acid and the composition of seed specimen.

The arabidopsis system that shows high oil meter type is identified, wherein oil content (fatty acid also promptly) and about 35% (oil increases by 22%) of comparing the formation seed quality in identical time cultivation with the average oil content about 28.7% of the seed of other plant of analyzing.Be tested and appraised site that T-DNA inserts fragment and found related between HIO30.5 gene and the high oil meter type, and as shown in the Examples, the heredity that has confirmed the existence of high oily seed phenotypes and T-DNA be divided into from.Therefore, HIO30.5 gene and/or polypeptide can be used for developing the plant of the genetic modification of the oil content phenotype (" HIO30.5 phenotype ") with modification.The HIO30.5 gene can be used for producing the oilseed crop, and it provides the oil production of raising from oilseed processing, and is used to produce the feed grain crop that can provide energy to increase and is used for animal feeding.The HIO30.5 gene can be further used for increasing the oil content of special oil crop, so that increase the output of required rare fatty acid.Carried out genetic modification and can be used for producing oil, wherein utilized standard method to cultivate genetically modified plants, and obtain oil from plant part (for example, seed) with the genetically modified plants of expressing HIO30.5.

HIO30.5 nucleic acid and polypeptide

Arabidopsis HIO30.5 nucleic acid (genomic DNA) sequence is provided among SEQ ID NO:1 and the Genbank accession number GI#18409525:1-1110.Corresponding protein sequence is provided among SEQ ID NO:2 and the GI#15231174.The lineal homologue of arabidopsis HIO30.5 or the nucleic acid of collateral line homologue and/or albumen are described in the following examples 3.

The fragment that refers to total length HIO30.5 albumen or its " functional activity " as term used herein " HIO30.5 polypeptide ", derivative (variant) or lineal homologue, refer to this protein fragments, derivative or lineal homologue show one or more functional activities relevant with the polypeptide of SEQ ID NO:2.In a preferred embodiment, functional activity HIO30.5 polypeptide when wrong expression the in plant, causes the oil content phenotype that changes.In embodiment preferred further, the mistake of HIO30.5 polypeptide in plant expressed and caused high oil meter type.In another embodiment, functional activity HIO30.5 polypeptide can be saved (comprising disappearance) endogenous HIO30.5 activity of defective when expressing in plant or plant cell; This rescue polypeptide can be from identical with the plant with defective activity or different species.In another embodiment, the functional activity fragment of total length HIO30.5 polypeptide (also is, natural polypeptides or its naturally occurring lineal homologue with sequence of SEQ ID NO:2) keep one or more biological propertyes relevant with total length HIO30.5 polypeptide, signal activity for example, in conjunction with active, catalytic activity or cell or extracellular disposition activity.The HIO30.5 fragment preferably includes the β subunit of the transcription initiation factor IIF domain of HIO30.5 domain, for example C-or N-end or catalyst structure domain, especially, preferably include at least 10 of HIO30.5 albumen, preferably at least 20, more preferably at least 25, at least 50 continuous amino acid most preferably.Functional domain can use the PFAM program to identify (Bateman A etc., 1999 Nucleic Acids Res 27:260-262).Preferred HIO30.5 fragment comprises bromine domain (bromodomain) (Pfam00439).The functional activity variant of total length HIO30.5 polypeptide or its fragment comprise having aminoacid insertion, and disappearance is perhaps replaced, but keep the polypeptide of one or more biological propertyes relevant with total length HIO30.5 polypeptide.Sometimes, the variant of generation is processed after changing the translation of HIO30.5 polypeptide.For example, compare with natural polypeptides, variant can have the protein transport or the albumen locating features of change, the perhaps half life period of the albumen of Gai Bianing.

Term used herein " HIO30.5 nucleic acid " comprises and has the sequence that SEQ ID NO:1 provides, the perhaps nucleic acid of the sequence of the sequence complementation that provides with SEQ ID NO:1, and functional activity fragment, derivative or lineal homologue.HIO30.5 nucleic acid of the present invention can be the DNA that derives from genomic DNA or cDNA, perhaps RNA.

In one embodiment, the HIO30.5 nucleic acid coding of functional activity or be complementary to the nucleic acid of the HIO30.5 polypeptide of encoding function activity.Genomic DNA is included in this definition, its as original rna transcription this () template also promptly, the mRNA precursor, it needed processing, for example montage before the active HIO30.5 polypeptide of encoding function.HIO30.5 nucleic acid can comprise other non-coding sequence, and it can be transcribed or not transcribed; This sequence comprises 5 ' and 3 ' UTRs, the adjusting sequence that polyadenylation signal and controlling gene especially known in the art are expressed.Some polypeptide need be processed incident, and for example proteolysis is sheared, covalent modification, or the like, so that activation fully.Therefore, HIO30.5 polypeptide functional activity nucleic acid codified maturation or that process in advance, perhaps intermediate forms.The HIO30.5 polynucleotides can also comprise allogeneic coding sequence, for example, and the mark of the promotion fused polypeptide purifying that coding comprises or the sequence of transformation marker.

In another embodiment, functional activity HIO30.5 nucleic acid for example can be used for, and by Antisense Suppression, suppresses to wait the HIO30.5 phenotype that produces afunction altogether.

In a preferred embodiment, the HIO30.5 nucleic acid that is used for the inventive method, comprise the nucleotide sequence of encoding or being complementary to the sequence of coding HIO30.5 polypeptide, the peptide sequence that this HIO30.5 polypeptide and SEQ ID NO:2 provide has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or above sequence homogeneity.

In another embodiment, HIO30.5 polypeptide of the present invention comprises the peptide sequence that the HIO30.5 peptide sequence with SEQ ID NO:2 has at least 50% or 60% homogeneity, and can have at least 70% with the HIO30.5 peptide sequence of SEQ ID NO:2,80%, 85%,, 90% or 95% or above sequence homogeneity.In another embodiment, the HIO30.5 polypeptide comprises that the functional activity fragment with the polypeptide shown in the SEQ ID NO:2 has at least 50%, 60%, 70%, 80%, 85%, 90% or 95% or the peptide sequence of above sequence homogeneity.In another embodiment, the HIO30.5 polypeptide comprises that the total length with the peptide sequence of SEQ ID NO:2 has at least 50%, 60%, and the peptide sequence of 70%, 80% or 90% sequence homogeneity also contains the bromine domain.

In yet another aspect, the total length of the HIO30.5 nucleotide sequence shown in HIO30.5 polynucleotide sequence and the SEQ ID NO:1 or have at least 50% to 60% homogeneity with the nucleotide sequence of this HIO30.5 sequence complementation, and can comprise and the HIO30.5 sequence shown in the SEQ ID NO:1 or its functional activity fragment, or complementary series has at least 70%, 80%, 85%, 90% or 95% or above sequence homogeneity.

Used herein, for specific target sequence or its specific part, " percentage (%) sequence homogeneity " is defined as if necessary obtaining maximum percentage sequence homogeneity, as passing through WU-BLAST-2.0al9 program (Altschul etc., J.Mol.Biol. (1990) 215:403-410 produced, wherein search parameter is made as default value, after carrying out sequence alignment and introducing breach, nucleotide or amino acid and middle nucleotide of target sequence (or its specific part) or the identical percentage of amino acid in candidate's the derived sequence.HSP S and HSP S2 parameter are mobility values, and are to depend on the composition of particular sequence and the composition of the certain database of therein interested sequence being searched for and definite by program itself." % homogeneity value " is by determining the identical nucleotide of coupling or amino acid whose number divided by the sequence length of the sequence of the percentage homogeneity of report." percentage (%) amino acid sequence similarity " determined by carrying out the calculating identical with definite percentage amino acid sequence identity, but comprised also except identical amino acid that in calculating conserved amino acid replaces.Conserved amino acid replaces, and promptly wherein another amino acid of amino acid with similar characteristic replaces, and the folding or activity of albumen is not had remarkable influence.The aromatic amino acid that can replace mutually is a phenyl alanine, tryptophan and tyrosine; Interchangeable hydrophobic amino acid is a leucine, isoleucine, methionine and valine; Interchangeable polar amino acid is glutamine and asparagine; Interchangeable basic amino acid is an arginine, lysine and histidine; Interchangeable acidic amino acid is aspartic acid and glutamic acid; Interchangeable p1 amino acid is alanine, serine, threonine, cysteine and glycine.

The derivative nucleic acids molecule of target nucleic acid molecules comprises can be optionally and the sequence of the nucleic acid array hybridizing of SEQ ID NO:1.The rigorous degree of hybridization can pass through temperature, ion strength, and pH, and the existence of denaturant such as formamide is controlled during hybridization and the washing.Normally used condition be called optical imaging (referring to, for example, Current Protocol in MolecularBiology, Vol.1, Chap.2.10, John Wiley ﹠amp; Sons, Publishers (1994); Sambrook etc., Molecular Cloning, Cold Spring Harbor (1989)).In some embodiment, nucleic acid molecules of the present invention can be under following rigorous hybridization conditions and the making nucleic acid molecular hybridization that comprises the nucleotide sequence of SEQ ID NO:1: the filter paper that contains nucleic acid, (1X SSC is 0.15M NaCl, the 0.015M sodium citrate at the citrate that contains single times of concentration of 6X (SSC); PH 7.0), 5X Denhardt solution, in the solution of the herring sperm dna of 0.05% sodium pyrophosphate and 100 μ g/ml, 65 ℃ of prehybridizations 8 hours are to spending the night; Containing 6X SSC, 1XDenhardt solution in the solution of 100 μ g/ml yeast tRNA and 0.05% sodium pyrophosphate, was hybridized 18-20 hour for 65 ℃; In the solution that contains 0.1X SSC and 0.1%SDS (lauryl sodium sulfate), 65 ℃ were washed filter paper 1 hour.In other embodiment, use following medium rigorous hybridization conditions: the filter paper that comprises nucleic acid, containing 35% formamide, 5X SSC, 50mMTris-HCl (pH7.5), 5mM EDTA, 0.1%PVP, 1%Ficoll, in the solution of 1%BSA and 500 μ g/ml denatured salmon sperm dnas, 40 ℃ of preliminary treatment 6 hours; Containing 35% formamide, 5X SSC, 50mM Tris-HCl (pH7.5), 5mM EDTA, 0.02%PVP, 0.02%Ficoll, 0.2%BSA in the solution of 100 μ g/ml salmon sperm dnas and 10% (wt/vol) dextran sulfate, was hybridized 18-20 hour for 40 ℃; Then, in the solution that contains 2X SSC and 0.1%SDS, 55 ℃ of washed twice 1 hour.Perhaps, can use following low rigorous condition: containing 20% formamide, 5xSSC, 50mM sodium phosphate (pH7.6), 5XDenhardt solution in the solution of 10% dextran sulfate and 20 μ g/ml denatured sheared salmon sperm dnas, is hatched 8 hours to spending the night for 37 ℃; Hybridization is 18 to 20 hours in identical buffer solution; In 1xSSC, about 37 ℃ of washing filter paper 1 hour.

Owing to the degeneracy of genetic code, can produce the polynucleotide sequence of many coding HIO30.5 polypeptide.For example, use according to the biological best codon that shows of specific host, can select codon with increase the expression of polypeptide in specific host species (referring to, for example, Nakamura etc., 1999).This sequence variants can be used in the method for the present invention.

Method of the present invention can be used the lineal homologue of arabidopsis HIO30.5.The method of identifying lineal homologue in the other plant kind is known in the art.In general, the lineal homologue in the different plant species keeps identical functions, owing to have one or more albumen motifs and/or three-dimensional structure.During evolution, when species form back producer repeated events, species, for example the individual gene in the arabidopsis may be corresponding to a plurality of genes in another species (collateral line homologue).Used herein, term " lineal homologue " comprises the collateral line homologue.When sequence data can be used for specific plant species, can pass through sequence homology analysis usually, for example BLAST analyzes, and generally uses albumen bait sequence, identifies lineal homologue.If to original search sequence among the reverse BLAST, this sequence then is possible lineal homologue (Huynen MA and Bork P, Proc Natl AcadSci (1998) 95:5849-5856 from forward BLAST result's optimum sequence retrieval; Huynen MA etc., Genome Research (2000) 10:1204-1210).The program that is used for a plurality of sequence alignments, for example CLUSTAL (Thompson JD etc., 1994, Nucleic Acids Res 22:4673-4680) can be used for the conservative region and/or the residue of outstanding lineal homologous protein, and produces phylogenetic tree.In the phylogenetic tree of expression from a plurality of homologous sequences (for example, retrieving by the BLAST analysis) of different plant species, from the lineal homologous sequence of 2 species, with respect to the every other sequence from 2 species, it is the most close to seem on phylogenetic tree usually.Get lines crossed other analyses (for example, using ProCeryon, Biosciences, Salzburg, the software of Austria) of (threading) or protein folding of structure also can identified possible lineal homologue.Nucleic acid hybridization also can be used for seeking orthologous gene, and when can not to obtain sequence data be preferred.The screening of degenerate pcr and cDNA or genome dna library is a common methods of seeking the related gene sequence, and be well known in the art (referring to, for example, Sambrook, 1989; Dieffenbach and Dveksler, 1995).For example, described among the Sambrook etc. and be used for producing the cDNA library and with the method in homeologous gene probe detection library from interested plant species.The high conservative of arabidopsis HIO30.5 coded sequence partly can be used as probe.The lineal homologous nucleic acid of HIO30.5 can be at height, under moderate or the low rigorous condition with the nucleic acid hybridization of SEQ ID NO:1.Amplification or separate after the part of lineal homologue of supposition can be cloned this part and checks order, and be separated complete cDNA or genomic clone as probe by standard technique.Perhaps, can start the sequence information data storehouse that the EST scheme produces interested plant species.In another approach, can specificity in conjunction with the antibody of known HIO30.5 polypeptide, be used for lineal homologue separate (referring to, for example, Harlow andLane, 1988,1999).The Western engram analysis can determine that the lineal homologue of HIO30.5 (lineal homologous protein also promptly) is present in the crude extract of specified plant species.When observing response, can show the expression library of specified plant species by screening, the sequence of separating the lineal homologue of coding candidate.Described in Sambrook etc. 1989, can be in the obtainable carrier of various commerce the construction expression library, comprise λ gt11.In case identified candidate's lineal homologue by any of these method, candidate's lineal homologous sequence can be used as bait (" inquiry "), is used for from the arabidopsis or other the species that have wherein identified HIO30.5 nucleic acid and/or peptide sequence sequence being carried out reverse BLAST.

Can use any available method to obtain HIO30.5 nucleic acid and polypeptide.For example, by foregoing screening DNA library or by using polymerase chain reaction (PCR) (PCR), the technology of separating interested cDNA or genomic dna sequence is well known in the art.Perhaps, but synthetic nucleic acid sequence.Any known method, for example site-directed mutagenesis (Kunkel TA etc., 1991) can be used for required change is incorporated in clone's the nucleic acid.

Usually, method of the present invention comprises the HIO30.5 nucleic acid of desired form is incorporated into the plant expression vector that is used for transformed plant cells, and express the HIO30.5 polypeptide in host plant.

The HIO30.5 nucleic acid molecules that separates is to be different from the form of natural discovery or the nucleic acid molecules of setting, and identifies from least one impurity nucleic acid molecules and separate, and this impurity nucleic acid molecules is combined in the natural origin of HIO30.5 nucleic acid usually.Yet the HIO30.5 nucleic acid molecules of separation comprises the HIO30.5 nucleic acid molecules in the cell that is included in common expression HIO30.5, and for example, this nucleic acid molecules is arranged in the chromosome position with the nucleic acid molecule differ of n cell.

Generation has the plant of genetic modification of the oil content phenotype of change

HIO30.5 nucleic acid and polypeptide can be used for producing the plant of the genetic modification of the oil content phenotype with modification.Used herein, " the oil content phenotype of modification " can refer to the oil content of modifying in the plant any part; In the seed of being everlasting, observe the oil content of modification.In a preferred embodiment, the change of HIO30.5 gene expression can be used for producing the plant with high oil meter type in the plant.

Method described here generally is applicable to all plants.Although in arabidopsis, carried out activation labeling acts and identified for genes, the HIO30.5 gene (or its lineal homologue, variant or fragment) can in any floristics, express.In a preferred embodiment, the present invention relates to oil-produced vegetable, it mainly is to produce and store triacylglycerol in seed at the specificity organ.These species comprise soybean (Glycine max), rape seed and canola (comprise cabbage type rape (Brassica napus), B.campestris), sunflower (Helianthus annus), cotton (upland cotton), corn (Zea maya), cocoa (Theobroma cacao), safflower (Carthamustinctorius), oil palm (Elaeis guineensis), coconut (Cocos nucifera), flax (Linumusitatissimum), castor-oil plant (Ricinus communis) and peanut (Arachis hypogaea).The invention still further relates to the plant that produces fruit and vegetables, produce the plant of cereal, produce the plant of nut, the rape kind of fast cycle, alfalfa (Medicago sativa), tobacco (Nicotiana), turfgrass (Poaceae family), other forage crop and may be the wildlife species of rare fatty acid source.

Those skilled in the art should understand the transformation technology miscellaneous that this area exists, and new technology constantly becomes available.Any technology that is suitable for the target host plant can be used in the scope of the present invention.For example, can introduce various forms of constructs, include but not limited to the DNA chain, to plasmid, or in the artificial chromosome.Can construct be incorporated in the target plant cell by various technology, include but not limited to conversion, electroporation, microinjection, microparticle bombardment, calcium phosphate-DNA coprecipitation or the liposome-mediated conversion of the soil Agrobacterium mediation of heterologous nucleic acids.Plant Transformation is preferably permanent, also promptly is incorporated in the host plant gene group by the expression construct that makes importing, so that the construct of this importing passes to continuous plant from generation to generation.Planned purposes comprises complete albumen or its biologic activity part of heterologous nucleic acids construct codified of HIO30.5 polynucleotides.

In one embodiment, double base can be used for shifting polynucleotides based on the carrier system of Ti.The soil Agrobacterium binary vector of standard is well known by persons skilled in the art, and many be can commercial obtain (for example, pBI121 Clontech Laboratories, Palo Alto, CA).

The best approach that transforms plant with the soil Agrobacterium carrier changes with floristics to be transformed.The illustrative methods that is used for the conversion of soil Agrobacterium mediation comprises being converted and comes from the plumular axis of aseptic seedling and/or plantlet, bud, the explant of stem or leaf texture.But this plant transformed sexual propagation, or by cell or tissue cultivation breeding.Before describe the soil Agrobacterium conversion and be used for many dissimilar plants, and can in scientific literature, find this method for transformation.Wherein relevant especially is to transform commercially important crop, rape seed (De Block etc., 1989) for example, sunflower (Everett etc., 1987) and soybean (Christou etc., 1989; Kline etc., 1987) method.

According to expression, the types of organization of expression and/or the developmental stage of expression take place, can regulate the expression (comprise and transcribe and translate) of HIO30.5.Many allos are regulated sequence (for example, promotor and enhancer) and be can be used for controlling the HIO30.5 expression of nucleic acids.These comprise composing type, induction type and adjustable promotor, and can be with the promotor and the enhancer of tissue or temporal-specific mode regulating and expressing.Exemplary constitutive promoter comprises raspberry E4 promotor (United States Patent (USP) 5,783,393 and 5,783,394), 35S CaMV (Jones JD etc., 1992), CsVMV promotor (Verdaguer B etc., 1998) and muskmelon actin promoter (disclosed PCT application WO0056863).Exemplary tissue-specific promoter comprises tomato E4 and E8 promotor (United States Patent (USP) 5,859,330) and tomato 2AII gene promoter (Van Haaren MJJ etc., 1993).

In a preferred embodiment, HIO30.5 is expressed in from it and expresses under the regulation and control with the adjusting sequence of the gene that seed and/or embryonic development are relevant in early days.The leguminous plant gene that its promotor is relevant with embryonic development with early stage seed comprises V.faba legumin (Baumlein etc., 1991, Mol Gen Genet 225:121-8; Baumlein etc., 1992, Plant J 2:233-9), V.faba usp (Fiedler etc., 1993, Plant Mol Biol 22:669-79), pea convicilin (Bown etc., 1988, Biochem J 251:717-26), pisum sativum agglutinin (dePater etc., 1993, Plant Cell 5:877-86), P.vulgaris β Kidney bean albumen (Bustos etc., 1991, EMBO J 10:1469-79), P.vulgaris DLEC2 and PHS[β] (Bobb etc., 1997, Nucleic Acids Res 25:641-7) and soybean β-conglycinin, 7S storage protein (Chamberland etc., 1992, Plant Mol Biol 19:937-49).The cereal gene that its promotor is relevant with embryonic development with early stage seed comprises paddy rice glutelin (" GluA-3, " Yoshiharaand Takaiwa, 1996, Plant Cell Physiol 37:107-11; " GluB-1, " Takaiwa etc., 1996, Plant Mol Biol 30:1207-21; Washida etc., 1999, Plant Mol Biol 40:1-12; " Gt3, " Leisy etc., 1990, Plant Mol Biol 14:41-50), paddy rice prolamin (Zhou ﹠amp; Fan, 1993, Transgenic Res 2:141-6), wheat prolamin (Hammond-Kosack etc., 1993, EMBO J 12:545-54), zeins (Z4, Matzke etc., 1990, Plant Mol Biol 14:323-32) and barley B-hordein (Entwistle etc., 1991, Plant Mol Biol 17:1217-31).Other its promotor and the early stage seed gene relevant with embryonic development, comprise oil palm GL07A (7S globulin, Morcillo etc., 2001, Physiol Plant 112:233-243), cabbage type rape napin, 2S storage protein and napA gene (Josefsson etc., 1987, J Biol Chem 262:12196-201; Stalberg etc., 1993, Plant Mol Biol 1993 23:671-83; Ellerstrom etc., 1996, Plant Mol Biol 32:1019-27), cabbage type rape oleosin (Keddie etc., 1994, Plant Mol Biol 24:327-40), arabidopsis oleosin (Plant etc., 1994, Plant Mol Biol 25:193-205), arabidopsis FAEl (Rossak etc., 2001, Plant Mol Biol 46:717-25), sword bean conA (Yamamoto etc., 1995, Plant Mol Biol 27:729-41) and the different lima bean glycosides of catharanthus roseus synzyme (Str, Ouwerkerk and Memelink, 1999, Mol Gen Genet 261:635-43).In another preferred embodiment, the adjusting sequence of the gene of expressing during the next comfortable oily biosynthesis of use (referring to, for example, United States Patent (USP) 5,952,544).Selectable promotor comes from plant storage protein gene (Bevan etc., 1993, Philos Trans R Soc Lond B Biol Sci342:209-15).

In yet another aspect, may need to suppress the expression of endogenous HIO30.5 in the host cell sometimes.The illustrative methods that is used to implement this aspect of the present invention includes, but are not limited to Antisense Suppression (Smith etc., 1988; Van der Krol etc., 1988); Suppress altogether (Napoli, etc., 1990); Ribozyme (the open WO 97/10328 of PCT); With justice and the combination of antisense (Waterhouse, etc., 1998).The method that suppresses endogenous sequence in the host cell is generally used transcribing or transcribe and translating of at least a portion of waiting to suppress sequence.This sequence can with the coding and the noncoding region homology of endogenous sequence.Antisense Suppression can be used complete cDNA sequence (Sheehy etc., 1988), and Partial cDNA Sequence comprises the fragment of 5 ' coded sequence (Cannon etc., 1990) or 3 ' non-coding sequence (Ch ' ng etc., 1989).The technology that suppresses altogether can be used complete cDNA sequence (Napoli etc., 1990; Van der Krol etc., 1990) or Partial cDNA Sequence (Smith etc., 1990).

Can carry out the molecule and the genetic test of standard, related with between analyzing gene further and the observed phenotype.Example technique is described below.

1.DNA/RNA analyze

For example, by in situ hybridization, can by the contrast of mutant and wild type system determine the stage-with the tissue-specific gene expression pattern.Can carry out gene, especially the analysis of flank regulatory region methylation status.Other applicable technology comprised expression, ectopic expression, expression in the other plant species and gene knockout (reverse genetics, target knocks out, the gene silencing of virus induction [VIGS is referring to Baulcombe D, 1999]).

In a preferred application, the expression and distribution by microarray analysis is used to measure simultaneously difference in many different genes expression or the change of inducing.The technology that is used for microarray analysis is (Schena M etc., Science (1995) 270:467-470 well known in the art; Baldwin D etc., 1999; Dangond F, Physiol Genomics (2000) 2:53-58; Van Hal NL etc., JBiotechnol (2000) 78:271-280; Richmond T and Somerville S, Curr OpinPlant Biol (2000) 3:108-116).Can carry out the expression and distribution analysis of the strain system of independent label.This analysis can identify that it can help unknown gene is placed specific approach owing to crossing of gene of interest expressed other gene of coordinating adjusting.

2. the analysis of gene outcome

The gene outcome analysis can comprise expression of recombinant proteins, and antiserum produces, immunolocalization, the biochemical analysis of catalysis or other activity, the phosphorylation status analysis and by the yeast two-hybrid analysis carry out and other albumen between transactional analysis.

3. path analysis

Path analysis can comprise, based on its mistake express phenotype or by with the sequence homology of related gene, gene or gene outcome are placed specific biochemistry, in metabolism or the signal pathway.Perhaps, analysis can comprise that the heredity with wild type system and other mutantion line intersects (generation double-mutant), so that gene is specified in the approach, or determines to suddenly change to the effect of downstream " report " gene expression in approach.

Generation has the mutant plant of the phenotype that changes oil content

The present invention further provides the method for identifying the non-transgenic plant that has the sudden change that can give these plants and offspring HIO30.5 phenotype thereof in endogenous HIO30.5 or its allelomorph.。In the method for a kind of being called " TILLING " (be used for induce local patholoic change), for example, use EMS to handle induced mutation in the seed of plant interested at the genome target.The plant that cultivation obtains, and self-fertilization, the offspring is used to prepare the DNA sample.The HIO30.5-specific PCR is used to identify whether the plant of sudden change has the HIO30.5 sudden change.Test has the change of the oil content in plants of HIO30.5 sudden change then, and perhaps, the change of the oil content of test plants uses the HIO30.5-specific PCR to determine to have the HIO30.5 the gene whether plant that changes oil content has sudden change then.TILLING can identify the expression that can change specific gene or by the sudden change of the activity of the albumen of these gene codes (referring to (2001) PlantPhysiol 126:480-484 such as Colbert; McCallum etc. (2000) Nature Biotechnology 18:455-457).

In another approach, candidate gene/quantitative trait locus (QTL) method can be used for the auxiliary breeding project of mark, to identify allelomorph or the sudden change in the lineal homologue of HIO30.5 gene or HIO30.5, its change that can give oil content is (referring to Bert etc., Theor ApplGenet.2003 Jun; 107 (1): 181-9; And Lionneton etc., Genome.2002 Dec; 45 (6): 1203-15).Therefore, aspect the present invention further in, HIO30.5 nucleic acid is used to identify whether the plant of the oil content with change has sudden change in endogenous HIO30.5, or does not have the specific allelomorph that can cause oil content to change.

Though described the present invention with reference to specific method and embodiment, only be to be understood that otherwise deviate from the present invention and can carry out various modifications and change.Here all publications of quoting are all clearly here as with reference to introducing, and are used to describe and the composition that openly can use with the present invention and the purpose of method.The patent of all references, the sequence information in the public database of patent application and reference also is incorporated herein by reference.

Embodiment

Embodiment 1

Utilize activation label construct to transform and produce plant with HIO30.5 phenotype

Use and activate label " ACTTAG " carrier, pSKI015 produces mutant (GI#6537289; Weigel D etc., 2000).Use standard method to produce the arabidopsis genetically modified plants, basically described in disclosed application PCT WO0183697.Briefly, T0 arabidopsis (Col-0) plant transforms with the soil Agrobacterium that carries the pSKI015 carrier, and this carrier comprises the T-DNA that derives from the soil Agrobacterium Ti-plasmids, Herbicid resistant selected marker and 4X CaMV35S enhancer element.According to Herbicid resistant, select genetically modified plants from generation to generation at T1.Select the T2 seed and carry out preservation by the T1 plant, and the part of evaluation T2 seed is used for screening with the set that indexes.

Utilize following method to carry out the quantitative assay of seed content of fatty acid.Usually contain the insert that isozygotys with 1: 1: 2 ratio of standard, 15 to 20 T2 seed samples of each strain of the test of homozygous wildtype and heterozygous genes type are at UMT-2 ultramicrobalance (Mettler-Toledo Co., Ohio weighs on USA) and changes the glass extraction flask then over to.Extract lipid from seed, and according to the amending method of people such as Browse (Biochem J 235:25-31,1986) method with all seeds at 80 ℃ of 2.5%H at 500ul ₂SO ₄MeOH solution transfer esterification 3 hours.The Heptadecanoic acide of known quantity adds in the reaction as internal standard compound.The hexane of the water of adding 750 μ l and 400 μ l firmly shakes then and is separated in each bottle.Reaction bulb directly is loaded into to be analyzed on the GC and gets upper strata hexane phase by Autosampler.Gas chromatography with flame ionization detection is used for the separation and the mensuration of fatty acid methyl ester.Agilent 6890Plus GC ' s is used for Agilent Innowax post (30m * 0.25mm ID, 250um film thickness) and separates.Carrier gas is the hydrogen with 2.5ml/ minute constant flow.Inject the sample (entering 220 ℃ of temperature, purge flow 15ml/min 1 minute) of 1 μ l in the mode of not shunting.Baking oven is set to 105 ℃ initial temperature, zero-time 0.5 minute, and the gradient with 60 ℃ of per minutes rises to 175 ℃ then, and 40 ℃/minute rise to 260 ℃, final 2 minutes retention times.Detect (275 ℃ of temperature, fuel oil stream 30.0ml/ minute, oxidant 400.0ml/min) by flame ionization.Utilize control of Millennium chromatogram management system monitors instrument and data collection and analyze (version 3 .2, WatersCorporation, Milford, MA).Automatically integrate and quantitative analysis, but all analyses are all carried out desk checking subsequently confirm that correct peak differentiates and acceptable signal-to-interference ratio before gathering with the result who obtains in being obtained studying.

The ACTTAG strain of called after WO00086431 is accredited as high oil meter type.Particularly, with the ACTTAG strain of other growth and average 28.7% the average oil content of analyzing (being datum line) under the same conditions compare, oil constitutes 34.8% (w/w) of seed quality.Carry out reanalysing of same seed in triplicate.Oil constitutes 32.1% of seed quality, confirms the increase with respect to the benchmark oil content.

Embodiment 2

The sign of T-DNA insetion sequence in the plant of the oil content phenotype of display change

We have carried out the standard molecule analysis as shown in patent application PCT WO0183697 basically, to determine inserting the site with the T-DNA of the oil content phenotypic correlation that changes.In brief, from the plant extract genomic DNA of the oil content phenotype of display change.The PCR that the Auele Specific Primer of use pSKI015 carrier carries out has confirmed to have the 35S enhancer in the plant from WO00086431 system, and the Southern engram analysis has confirmed the genome conformity of ACTTAG T-DNA.

Plasmid rescue and inverse PCR are used to reclaim the genomic DNA of T-DNA insetion sequence flank, use the BLASTN search arabidopsis thaliana genomic dna TAIR database (can obtain in the arabidopsis information resources website that the public can enter) on basis to carry out sequence analysis then.WO00086431 ties up to the T-DNA that three different sites have insertion.

In order to determine which insetion sequence causes high seed oil phenotype, check high seed oil phenotype and T-DNA existence be divided into from.18 T2 plants are cultivated maturation and determine the seed oil phenotype by the seed that these plants are collected.As determining the seed oil content of these plants as described in the embodiment 1.By using PCR to corresponding genome area and the specific primer of T-DNA, analyze in the T3 seed T-DNA in the existence of inserting the site or do not exist, infer the genotype of T2 seed.The result shows that site 2 and 3 closely connects.In addition, 2 and 3 to comprise the average oil content of T3 seed of T-DNA insetion sequence higher than the average oil content of those families that lack insetion sequences in these sites in the site.Site 2 produces the seed of comparing 115.4% oil content with reference with the individual homozygote of 3 T2, T2 individual semizygote in these sites produces the seed of comparing 118.4% oil content with reference, and the T2 individuality that lacks T-DNA in these sites has 105% the average oil content of reference sample of the seed of wild type Col-0 plant.Because the homozygote of high oil level point and semizygote show that oil content similarly increases, we infer that site 2 and phenotype relevant with high oil meter type with 3 caused by dominant mutation.On the contrary, the 1 average oil content that comprises the T3 family of T-DNA insetion sequence is compared lower or much at one than the situation that lacks insetion sequence in corresponding site in the site.We infer that site 1 is uncorrelated with high oil meter type.

Sequence analysis discloses the initiation codon of the nucleotide sequence be shown as SEQ NO:1, our called after HIO30.5, be site 3 about 1kb 5 ' the upstream boundary of T-DNA.

Embodiment 3

The analysis of arabidopsis HIO32.2 sequence

Utilize BLAST (Altschul etc., 1997, J.Mol.Biol.215:403-410), PFAM (Bateman etc., 1999, Nucleic Acids Res 27:260-262), PSORT (Nakai K and Horton P, 1999, Trends Biochem Sci 24:34-6) and/or CLUSTAL (Thompson JD etc., 1994, Nucleic Acids Res 22:4673-4680) carries out sequence analysis.

The BLASTN of SEQ ID NO:1 and GenBank sequence has recovered the arabidopsis paralogous gene of candidate gene itself (adding its genomic DNA accession number) and supposition, At2g34900 (2 accession number).The At3g52280 candidate gene is not expressed as the cDNA clone and exists without any the arabidopsis ESTs relevant with this gene yet:

Produce the sequence that height-fragment of keeping the score is right: the P that keeps the score (N) N

gi|18409525|ref|NM_115088.1|Arabidopsjs thaliana chromos...5550 5.6e-244

gi|6434227|emb|AL132972.1|ATT25B15 Arabidopsis thaliana D...1155 1.0e-175

gi|18403687|ref|NM_129043.1|Arabidopsis thaliana chromos...609 9.0e-191

gi|20197115|gb|Ac004238.3|Arabidopsis thaliana chromosom...341 1.7e-052

Reappeared similar result at the amino acid whose BLASTP among the GenBank, and identified other 3 by gene A t2g34900At5g10550 ﹠amp; The arabidopsis homologue of the deduction of At5g65630 coding has 39%, 13% and 14% sequence homogeneity respectively with SEQ ID NO:2.Identified albumen (GI#7523514) equally, had 15% sequence homogeneity with SEQ ID NO:2 from the prediction of paddy rice.Best 10 the results are shown in following and comprise people that the coding controlling gene expression relevant with development models is relevant and the gene of fly.

Produce the sequence that height-fragment of keeping the score is right: the p that keeps the score (N) N

gi|15231174|ref|NP_190796.1|unknown protein；protein id：...1570 1.1e-160

gi|15226857|ref|NP_181036.1|putative RING3 protein；prot...606 1.5e-581

gi|15238195|ref|NP_196617.1|bromodomain protein-like；...239 1.0e-262

gi|7523514|dbj|BAA94242.1|Similar to Arabidopsis thalian...278 3.4e-231

gi|15239091|ref|NP_201366.1|putative protein；protein id...212 1.2e-212

gi|7657218|ref|NP_055114.1|bromodomain-containing protei...215 7.9e-212

gi|3184498|gb|AAC27978.1|R31546_1[Homo sapiens] 215 8.2e-212

gi|120558|sp|P13709|FSH_DROME FEMALE STERILE HOMEOTIC PRO...216 1.0e-202

gi|24640482|ref|NP_511078.2|CG2252-PB [Drosophila melano...216 1.0e-202

gi|1588281|prf||2208296A RING3 protein 255 1.2e-201

The BLASTN of SEQ ID NO:1 and GenBank sequence has identified following arabidopsis thaliana sequence, is presented in the bracket with the % homogeneity of SEQ ID NO:1: At2g34900GI#15226857 (39%); At5g10550 GI#15238195 (13%) and At5g65630GI#15239091 (14%).

The following ESTs from non--arabidopsis thaliana kind that reappears at the BLASTN of ESTs is presented in the bracket with the % homogeneity of SEQ ID NO:2: potato (potato) GI#s13608255 (58%) and 21921887 (30%); Cotton (Gossypium) GI#5050723 (61%); Soybean (green soy bean) GI#9835128 (30%) turnip GI#1019482 (51%); And cereal (corn) GI#24765979 (34%).

SEQ ID NOs 3 and 4 is respectively from the sequence of tomato and wheat, and its representative is compiled to the ESTs of reproduction of the contig of lowest number.Wherein contig is represented the part code area, can be that the technical staff determines full cDNA sequence by technical field of molecular biology.

SEQ ID NO:3 is from tomato (Lycoprsicon esculentum), and is the contig (contig) of following sequence: GI#s 13778307,10804738, and 9504093,6985312,4381395.Has 58% homogeneity with SEQ ID NO:1.

SEQ ID NO:4 is from wheat (Triticum aestivum), and is GI#s21837181,19950212,20123686,20547632,19957908 and 20299669 contig.Has 22% homogeneity with SEQ ID NO:1.

The PFam analyzing and testing arrives the bromine domain (PF00439) from the residue 87 to 200 of SEQ ID NO:2.With prediction play a part transcribe the factor and conform to, it is (60%) that nuclear is originated that PSORT2 analyzes prophesy At3g52280 gene outcome.This gene may be played the part of the role who regulates the gene expression that participates in fatty acid metabolism or relevant approach.

Embodiment 4

Confirm phenotype/genotypic association

RT-PCR the analysis showed that the expression excessively in from the plant that shows the HIO30.5 phenotype of HIO30.5 gene.Particularly, really extract RNA from cluster leaf and/or the angle of the plant of the demonstration HIO30.5 phenotype of collecting in multiple developmental stage and storehouse.Utilization is specific to sequence shown in the SEQ ID NO:1, and the primer that is specific to the predicted gene of contiguous other of T-DNA insert and is specific to the actin gene (positive control) of constitutive expression carries out RT-PCR.The result shows that the phenotypic plant of demonstration HIO30.5 crosses the mRNA that expresses the HIO30.5 gene, shows that the enhancing of HIO30.5 gene is expressed relevant with the HIO30.5 phenotype.

The phenotypic dominant inheritance pattern of HIO30.5 confirms by genetic analysis.Usually, genetic analysis relates to generation and the analysis of first filial generation F1.Usually, carry out F1 hybridization by collected pollen by the T2 plant, described T2 plant is used for wild-type plant is pollinated.Such hybridization is by taking from 4 flowers of the bion of each selection and utilizing the T2 flower to carry out as female as the flower of male pollen donor and wild-type plant.4-5 hybridization is used for independent purpose.To assemble by the seed that same individual hybridization forms, plant and cultivate ripe first filial generation F1.

Embodiment 5

The recapitulaion of high oil meter type

In order to confirm whether cross expressing of At3g52280 causes high oilseed phenotype, will compare from the oil content of the seed of crossing the genetically modified plants of expressing this gene and oil content from the seed of non-transgenic check plant.In order to carry out this comparison, At3g52280 to be cloned in the plant conversion carrier, At3g52280 is positioned at seed specific PRU promotor back, and utilizes floral dip method to be transformed in the arabidopsis thaliana.The nptII gene that can provide kalamycin resistance is provided this conversion carrier, as selected marker.To in containing the agar medium of kanamycin, cultivate from the seed that transforms plant.After 7 days, identify the healthy green plants of genetically modified plants and be transplanted to soil.The non-transgenic check plant germinates on agar medium, grows and transfers on the soil then in 7 days.22 genetically modified seedling and 10 not genetically modified check plants are transferred to the random site on 32 identical lattice level lands.Plant growing is to ripe and selfing and knot.From each plant results seed, by method as described below oil content by Near Infrared (NIR) Wave Spectrum assessment seed.

Use Bruker 22N/F near infrared spectroscopy to catch the NIR infrared spectrum.Use is from the NIR data of sample and reference method, according to the specification of manufacturer Bruker software is used to assess total seed oil and total seed protein content.Developed oil content prediction calibration according to the conventional method of AOCS program Aml-92, AOCS program Aml-92 is the formal method and API Rp (the Ofiicial Methods andRecommended Practices of the American Oil Chemists Society of American Oil Chemists Society, the 5th edition, AOCS, Champaign I11).Developed the calibration that is used for NIR prediction crude oil ASE (Ren Oil, Accelerated Solvent Extraction).

In two experiments, checked the influence of expressing seed oil of crossing of At3g52280.In two experiments, cross the plant of expressing At3g52280 and have the seed oil content higher than the check plant that is grown in identical level land.In the whole experiment, the average seed oil content of crossing the plant of expressing At3g52280 is higher by 4% than unconverted contrast.The seed oil content of crossing the plant of expressing At3g52280 is significantly higher than non--transgenosis check plant (two-way variance analysis; P=0.0208), referring to table l.

Table 1

Experiment	Plant ID	Transgenosis	The mean value of prediction	Relative value mean value
					1	DX04624001	PRU::HIO30.5	34.7317	105.1313
1	DX04624002	PRU::HIO30.5	35.3328	106.9509
					1	DX04624003	PRU::HIO30.5	32.2034	97.4782
1	DX04624004	PRU::HIO30.5	35.2082	106.5738
					1	DX04624005	PRU::HIO30.5	36.3858	110.1381
1	DX04624006	PRU::HIO30.5	35.3761	107.0819
					1	DX04624007	PRU::HIO30.5	34.4489	104.2752
1	DX04624008	PRU::HIO30.5	35.1352	106.3526
					1	DX04624009	PRU::HIO30.5	31.4731	95.2678
1	DX04624010	PRU::HIO30.5	34.1334	103.3205
					1	DX04624011	PRU::HIO30.5	33.6363	101.8156
1	DX04624012	PRU::HIO30.5	32.135	97.2713
					1	DX04624013	PRU::HIO30.5	33.0094	99.918
1	DX04624014	PRU::HIO30.5	32.8502	99.4363
					1	DX04624015	PRU::HIO30.5	35.2295	106.6383

1	DX04624016	PRU::HIO30.5	32.6427	98.808
					1	DX04624017	PRU::HIO30.5	33.6761	101.936
1	DX04624018	PRU::HIO30.5	32.826	99.3629
					1	DX04624019	PRU::HIO30.5	31.7287	96.0416
1	DX04624020	PRU::HIO30.5	30.4119	92.0554
					1	DX04624021	PRU::HIO30.5	32.6628	98.8688
1	DX04624022	PRU::HIO30.5	32.7279	99.066
					1	DX04642001	None	31.8656	96.4559
1	DX04642002	None	36.3384	109.9948
					1	DX04642003	None	31.6187	95.7083
1	DX04642004	None	32.7062	99.0003
					1	DX04642005	None	35.1348	106.3515
1	DX04642006	None	34.2222	103.5891
					1	DX04642007	None	34.5412	104.5548
1	DX04642008	None	32.7261	99.0604
					1	DX04642009	None	28.0916	85.0321
1	DX04642010	None	33.12	100.2529
					2	DX06812001	PRU::HIO30.5	29.234	104.5474
2	DX06812002	PRU::HIO30.5	28.1495	100.6689
					2	DX06812004	PRU::HIO30.5	28.9208	103.4271
2	DX06812005	PRU::HIO30.5	32.4947	116.2083
					2	DX06812006	PRU::HIO30.5	27.9377	99.9113
2	DX06812007	PRU::HIO30.5	28.9667	103.5916
					2	DX06812008	PRU::HIO30.5	25.6801	91.8377
2	DX06812009	PRU::HIO30.5	27.4305	98.0976
					2	DX06812010	PRU::HIO30.5	28.2639	101.0781
2	DX06812011	PRU::HIO30.5	30.168	107.8874
					2	DX06812012	PRU::HIO30.5	32.4399	116.0125
2	DX06812013	PRU::HIO30.5	28.9043	103.3685
					2	DX06812015	PRU::HIO30.5	32.1043	114.8121
2	DX06812016	PRU::HIO30.5	31.3237	112.0206
					2	DX06812017	PRU::HIO30.5	30.9057	110.5259
2	DX06812018	PRU::HIO30.5	32.561	116.4454
					2	DX06812019	PRU::HIO30.5	30.4487	108.8914
2	DX06812020	PRU::HIO30.5	31.5006	112.6532
					2	DX06812021	PRU::HIO30.5	28.7262	102.7315
2	DX06812022	PRU::HIO30.5	30.3064	108.3826
					2	DX06794001	None	27.007	96.5832
2	DX06794002	None	25.7445	92.0681
					2	DX06794003	None	25.7293	92.0138
2	DX06794004	None	27.9487	99.951
					2	DX06794005	None	28.8503	103.1751
2	DX06794006	None	27.7921	99.391
					2	DX06794007	None	30.0356	107.414
2	DX06794008	None	29.9573	107.1342
					2	DX06794009	None	27.1836	97.2146
2	DX06794010	None	29.376	105.0551

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Sequence table

＜110〉Agrinomics LLC (Agrinomics, LLC)

＜120〉produce the plant (GENERATION OF PLANTS WITH ALTERED OIL CONTENT) that changes oil content

<130>SCT062603-66

<150>60/530,828

<151>2003-12-17

<160>4

<170>PatentIn version 3.2

<210>1

<211>1110

<212>DNA

<213>Arabidopsis thaliana

<400>1

atggctgact cagtgccagg tcatgtcgcc ggcggaggtt tacagggttt ctctgtcgac 60

gccgagtgca tcaagcagcg tgtcgacgag gttttacagt gggttgattc gcttgagcat 120

aaactgaaag aagtggagga gttttactcg agcattggtg tctcaaactc tggctctatt 180

ggtaaagata cagaaaaagg gagacatgtt gtggggatta ggaagattca acaagaggct 240

gcgcgtagag aagctgttgc tgctaaaaga atgcaggacc ttatgcgtca atttggaaca 300

atctttcgtc agataactca gcataagtgc gcttggccgt ttatgcatcc tgtcaatgtt 360

gaaggtcttg gtttgcatga ctactttgag gttattgaca agcctatgga cttcagcacg 420

ataaaaaatc aaatggaggc taaggatggt accgggtaca aacatgtcat gcaaatttat 480

gctgatatgc ggctagtgtt tgagaacgca atgaattata atgaagaaac aagtgatgtt 540

tactctatgg cgaaaaagtt actggaaaag tttgaggaga agtgggcaca ctttcttcca 600

aaggttcaag aagaggagaa aatacgagag gaagaggaga agcaagcagc aaaggaggcg 660

ctgctagcaa aagaagcatc tcatatcaaa acaactagag aattggggaa tgagatttgc 720

catgctaatg acgagctgga gaagctaatg cgtaaagttg tggaaagatg caggaaaatc 780

acaattgagg agaagcgtaa tattggattg gcattgttaa aactgtcacc ggatgatcta 840

cagaaagtgt tgggtatagt tgctcaggct aacccgagct ttcaacctag agcagaggaa 900

gttagtattg agatggacat actggacgaa ccaacactat ggaggctaaa gttctttgtg 960

aaggatgcgt tggataatgc aatgaagaag aagaaagaag aggagacaaa gactagagaa 1020

ttgagtggag cacaaaagaa agaggtttcg aagaaacgaa atgcgactac aaagttagct 1080

gagaggaaaa cgaagcggtc acgcatatga 1110

<210>2

<211>369

<212>PRT

<213>Arabidopsis thaliana

<400>2

Met Ala Asp Ser Val Pro Gly His Val Ala Gly Gly Gly Leu Gln Gly

1 5 10 15

Phe Ser Val Asp Ala Glu Cys Ile Lys Gln Arg Val Asp Glu Val Leu

20 25 30

Gln Trp Val Asp Ser Leu Glu His Lys Leu Lys Glu Val Glu Glu Phe

35 40 45

Tyr Ser Ser Ile Gly Val Ser Asn Ser Gly Ser Ile Gly Lys Asp Thr

50 55 60

Glu Lys Gly Arg His Val Val Gly Ile Arg Lys Ile Gln Gln Glu Ala

65 70 75 80

Ala Arg Arg Glu Ala Val Ala Ala Lys Arg Met Gln Asp Leu Met Arg

85 90 95

Gln Phe Gly Thr Ile Phe Arg Gln Ile Thr Gln His Lys Cys Ala Trp

100 105 110

Pro Phe Met His Pro Val Asn Val Glu Gly Leu Gly Leu His Asp Tyr

115 120 125

Phe Glu Val Ile Asp Lys Pro Met Asp Phe Ser Thr Ile Lys Asn Gln

130 135 140

Met Glu Ala Lys Asp Gly Thr Gly Tyr Lys His Val Met Gln Ile Tyr

145 150 155 160

Ala Asp Met Arg Leu Val Phe Glu Asn Ala Met Asn Tyr Asn Glu Glu

165 170 175

Thr Ser Asp Val Tyr Ser Met Ala Lys Lys Leu Leu Glu Lys Phe Glu

180 185 190

Glu Lys Trp Ala His Phe Leu Pro Lys Val Gln Glu Glu Glu Lys Ile

195 200 205

Arg Glu Glu Glu Glu Lys Gln Ala Ala Lys Glu Ala Leu Leu Ala Lys

210 215 220

Glu Ala Ser His Ile Lys Thr Thr Arg Glu Leu Gly Asn Glu Ile Cys

225 230 235 240

His Ala Asn Asp Glu Leu Glu Lys Leu Met Arg Lys Val Val Glu Arg

245 250 255

Cys Arg Lys Ile Thr Ile Glu Glu Lys Arg Asn Ile Gly Leu Ala Leu

260 265 270

Leu Lys Leu Ser Pro Asp Asp Leu Gln Lys Val Leu Gly Ile Val Ala

275 280 285

Gln Ala Asn Pro Ser Phe Gln Pro Arg Ala Glu Glu Val Ser Ile Glu

290 295 300

Met Asp Ile Leu Asp Glu Pro Thr Leu Trp Arg Leu Lys Phe Phe Val

305 310 315 320

Lys Asp Ala Leu Asp Asn Ala Met Lys Lys Lys Lys Glu Glu Glu Thr

325 330 335

Lys Thr Arg Glu Leu Ser Gly Ala Gln Lys Lys Glu Val Ser Lys Lys

340 345 350

Arg Asn Ala Thr Thr Lys Leu Ala Glu Arg Lys Thr Lys Arg Ser Arg

355 360 365

Ile

<210>3

<211>965

<212>DNA

<213>Arabidopsis thaliana

<400>3

gccttcatcg gcggcacaac ctgttgctta tctccgatct actccggcga acggcgaaat 60

tcgagctatg gagaatctaa acggcttagt tcctgacatc cctatacaag agcagaaaga 120

caatgctact gcttctgaaa atttcggtcg aagtgttgat gaaatggttg ggaaggttga 180

tcagattgag cagagactga atgaggttga gcatttctat tccaatacta gtaaaaaaca 240

atcaaacacg ccaagaggtg gctctattct gaaagacaaa gagaaacaga tgtctagctt 300

tagaaggcgg cagcaggatg catcacgtag agaagcagct ggttccagga gaatgcaaga 360

acttatgcgc caatttggta ctatattacg tcagatcact cagcacaagt gggcagaacc 420

ttttatggaa cctgtggatg tcaagggtct tggattacac gattattttg aggttattga 480

gaagcctatg gacttcagta ctatcaaaaa caagatggaa gcaaaggatg gttctggcta 540

taagcatgtc cgagagatat gtgctgacgt gaggctaata tttaagaatg cgatgaaata 600

caacgaggaa agggatgacg ttcatgtaat ggcgaaaacc ttgttgggaa aattcgagga 660

gaaatggttg cagcttttgc ccaaagttga tgaagaggaa aaacggcgaa aggaagagga 720

agcagaagcc caacaggata tgcagctcgc tcaagaggct gctcatgcaa aaatggcaaa 780

agatttaact attgagcttg atgaggtgga catgcaactg gaagaactca gggacttggt 840

gcttcagaaa tgcagaaaga tttcaacgga agacaggaaa caacttggga atgcgctcac 900

taagctgtct cctgacgatc ttaataaagc actattgatt gtggcacaga atgatcctac 960

ttttc 965

<210>4

<211>1290

<212>DNA

<213>Arabidopsis thaliana

<400>4

cgaggggcgg caatggaggg agcgtggccc catccggcgc cgatggagcc gaccaccgcc 60

gcggatgggc ctcaagtgtc ggaggtgaac tcgttccggc gccaggtcga cgacctcctc 120

tccaagaccg acgtgctgga gaagagggtg aacgaggtgg tagggttcta caattccaag 180

aagcacagca gtggaggccg caaggctggc ggcaggtacg cggaaaacgg tgccagggac 240

agccacggca atgggatgcc cgacctcatg cgccagtttg ccggtatcat tcgccagatt 300

acatctcatg aatgggcaca gccgtttttg caaccggtag acgtcgtagg tcttcaactt 360

gatgactatc accagattat aacaaaacct atggatttct cgaccatccg aaacaaaatg 420

gaagggaagg aaagtaccac atataacagt gtccgagaaa tatattctga tgttagatta 480

gtttttacca atgcaatgaa gtacaatgtt gaaggtcacc ctgttaacat aatggccaag 540

ttcctactcg agagatttga ggagaaatgg cttcaccttc tccctgaagt tgagaacgag 600

gaaagggaac gggaggaacc aaatgatgct ccaaccataa gcatttctcc ggaagctgca 660

attgcaaaat tagctgaaga tactggtaat gagctgaatg agattaataa gcagctggag 720

gagctccaga aaatggtggt tcagagatgc aggaaaatga cgacggacga gaagagaaaa 780

ctcggcgcag gtctttgcca attgtctcca gaagatctta acaaggcgct agagttggtc 840

gcgcaagaca atcctagctt ccaaactaca gctgaagaag tggaccttga catggatgct 900

cagagcgaga cgaccctctg gaggctgaag ttctttgtga gggaagcatt ggaacaacag 960

gcgaatgtag gcgtagtggc ctgtggcaag atcgatgaaa acacgaagag gagccgcgac 1020

atgtacaatg ctctagccaa gaccgtttcg aaacggatta agagataacc ttagcggctc 1080

ggcctttctg atggttcatg gtatttctgt agaaaatagg gccatgttct cccttgtgtg 1140

aagacatgtt gtatatatac gaagatgaat ctgcactaag atatgcttgg tcgcacccgg 1200

tctcatgtgg catgggtagc atgctattgg tagctatgta aaataaagag caatgttgaa 1260

cacctctgag aaacaagaat tattccttga 1290

Claims (12)

1. the genetically modified plants that comprise plant conversion carrier, this plant conversion carrier contains coding or is complementary to the nucleotide sequence that coding comprises the sequence of the HIO30.5 polypeptide of amino acid sequence of SEQ ID NO:2 or its lineal homologue, therefore with respect to check plant, these genetically modified plants have high oil meter type.

2. the genetically modified plants of claim 1, it is selected from rape seed, soybean, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut, flax, castor-oil plant and peanut.

3. accessory rights requires the plant part that 1 plant obtains.

4. the plant part of claim 3, it is a seed.

5. method that produces oil, it comprises the genetically modified plants of cultivating claim 1, and from described plant recovered oil.

6. produce the method for high oily phenotype plant, described method comprises:

A) plant conversion carrier is imported in the CFU-GM of plant, this plant conversion carrier contain coding or be complementary to coding comprise the HIO30.5 polypeptide of amino acid sequence of SEQ ID NO:2 or its lineal homologue sequence nucleotide sequence and

B) CFU-GM of cultivating this conversion is wherein expressed described polynucleotide sequence to produce genetically modified plants, and with respect to check plant, the oil content phenotype of described genetically modified plants display change.

7. pass through the plant of the method acquisition of claim 6.

8. the plant of claim 7, it is selected from rape seed, soybean, corn, sunflower, cotton, cocoa, safflower, oil palm, coconut, flax, castor-oil plant and peanut.

9. the plant of claim 7, plant wherein are selected from the plant that comes from described group of cell growth, the indirect offspring's of the direct offspring's of the plant that comes from described group of cell growth plant and the plant that comes from described group of cell growth plant.

10. produce and have the method for high oily phenotype plant, comprise and identify allelic plant with HIO30.5 gene, the allelomorph of described HIO30.5 gene causes the increase of comparing oil content with the plant of filial generation that lacks this equipotential gene and produce the plant of described evaluation, the filial generation that wherein produces this equipotential gene of heredity and be high oil meter type.

11. the method for claim 10, it uses candidate gene/QTL method.

12. the method for claim 10, it uses the TILLING method.