CN101103116A

CN101103116A - Generation of plants with altered oil content

Info

Publication number: CN101103116A
Application number: CNA2006800022466A
Authority: CN
Inventors: 约翰·P·戴维斯; 海因·措恩格·额
Original assignee: Agrinomics LLC
Current assignee: Agrinomics LLC
Priority date: 2005-01-12
Filing date: 2006-01-11
Publication date: 2008-01-09
Anticipated expiration: 2026-01-11
Also published as: ZA200705263B; AR059287A1; WO2006076596A2; MX2007008435A; BRPI0606643A2; WO2006076596A3; CN101103116B

Abstract

The present disclosure is directed to plants and plant cells that display an altered oil content phenotype due to altered expression of a HIO nucleic acid. The disclosure is further directed to methods of generating plants with an altered oil content phenotype.

Description

Produce the plant that changes oleaginousness

Related application

The application requires the right of priority of the U.S. Provisional Patent Application 60/643,674 of submission on January 12nd, 2005, and its content all is incorporated herein by reference.

Technical field

The application's disclosure relates to and has transgenic plant and the vegetable cell that changes oleaginousness, and produces and have the plant that changes oleaginousness and from the method for this plant production oil.

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 rearing oils.The seed of farm crop contains multiple valuable component, 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 farm crop squeeze by soyabean processing industry.Soyabean processing produces the refining oil of selling goods at a high figure, and resistates is sold (USSoybean Board, 2001 Soy Stats) mainly as feed.Canada's rape (canola) seed is squeezed produces oils and byproduct Canada coarse colza meal (Canadian rape association).Almost the american corn farm crop 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 oleaginousness maximization of seed.For example, for for the processing oleaginous seed of soybean and Canadian rape, the absolute oleaginousness that improves seed will improve the value of this cereal.For the corn of processing, may need to improve or reduce oleaginousness according to the application of other main ingredient.Reducing oils can be by reducing the quality that the undesirable taste relevant with the oils oxidation improves isolating starch.Perhaps, in the unimportant ethanol of taste produces, improve oleaginousness 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 intrinsic energy than other seed components such as carbohydrate.Oleaginous seed processing is just as most of cereal processing industries are fund-intensive industries; Therefore the less change of the distribution of the product from the low value component to the high value oil component may just have the remarkable economical influence for the cereal processor.

The biotechnology of oils is handled can provide the change of composition and the raising of oil yield.The change of forming especially comprises high oleic soybean and Semen Maydis oil (United States Patent (USP) 6,229,033 and 6,248,939), and the seed (United States Patent (USP) 5,639,790) etc. that contains laurate.The work that composition changes mainly concentrates on the oleaginous seed of processing but can extend to non--oleaginous seed farm crop at an easy rate comprises corn.Though have suitable interest to improving oleaginousness, 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 oleaginousness that the high oils of topcross system makes harvest corn from～3.5% bring up to～7%, improved the intrinsic energy of cereal.

Though the HOC production system is fruitful, it still has the inherent limitation.At first, the system of undertaking low pollina's per-cent that the seed in whole soils plants contains inherent risk, especially in the year of drought.The second, current HOC field oleaginousness is steadily in about 9% oils.At last, high oils corn master changes if it were not for biological chemistry, but to the mutant on the anatomy that improves oleaginousness 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 farm crop in process oil market are soybean and Semen Brassicae campestris, and a large amount of commercial pursuit (for example United States Patent (USP) 5,952, and 544; PCT applies for WO9411516) prove that Arabidopis thaliana is the metabolic good model of oils in these farm crop.The lineal homologue that the biological chemistry screening of seed oil compositions has been identified the many crucial biosynthetic enzyme genes of Arabidopis thaliana and caused agricultural to go up important gene is identified out.For example, the screening that utilizes chemomorphosis to handle the group has been identified the lipid mutant that its seed shows that lipid acid composition changes (Lemieux etc. 1990, Thero.Appl.Genet.80,234-240; James and Dooner, 1990, Thero.Appl.Genet.80,241-245).Detect the T-DNA mutagenesis screening (Feldmann etc. 1989, Science 243:1351-1354) of lipid acid composition change and identified ω 3 desaturases (FAD3) and δ-12 desaturase (FAD2) gene (United States Patent (USP) 5952544; Yadav etc., 1993, Plant Physiol.103,467-476; Okuley et al., 1994, Plant Cell 6 (1): 147-158).Concentrate on oleaginousness but not inductive wrinkled seed or seed density change on the screening analytical chemistry of oils quality mutant, infer in view of the above change oil content in plants (Focks and Benning, 1998, PlantPhysiol.118:91-101).

Be used for identifying the another kind of Screening and Identification that participates in the enzyme that unusual longer chain fatty acid produces coding be responsible for reducing sudden change (the Katavic V etc. of the gene of triacylglycerol accumulative diacylglycerol acyltransferase (DGAT) in the seed; 1995, Plant Physiol.108 (1): 399-409).In addition, the seed-specific that also shows DGAT cDNA is crossed oil content in plants relevant (Jako etc., 2001, the Plant Physiol.126 (2): 861-74) that expresses with raising.Arabidopis thaliana also is the cumulative model that is used to understand the seed components that influences the meal quality.For example, Arabidopis thaliana contains in a lot of dicotyledonss, comprises albumin and the sphaeroprotein seed storage protein (Shewry1995, Plant Cell 7:945-956) found in Canadian rape and the soybean.The component of synthon, biochemical route such as Mierocrystalline cellulose and xylogen is guarded in these vascular plants, and the arabidopsis mutant body that influences these components also separated (, summarizing among the Current Opinion in PlantBiology 1:179-185) at Chapel and Carpita 1998.

Activation labeling acts in the plant is a kind of by comprising the method that the heterologous nucleic acids construct of regulating sequence (for example, enhanser) inserts generation random mutation in the Plant Genome.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, Science 258:1350-1353; WeigelD et al., 2000, Plant Physiology, 122:1003-1013).The construct that inserts provides a kind of molecular label, and it is used for Rapid identification because its wrong natural phant 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 to multiple species, comprise tobacco and Arabidopis thaliana, identify various mutant phenotypes and with the gene of these phenotypic correlations (Wilson, etc., 1996, Plant Cell 8:659-671; Schaffer et al., 1998, Cell 93:1219-1229; Fridborg et al., 1999, Plant Cell 11:1019-1032; Kardailsky et al., 1999, Science 286:1962-1965; And Christensen S et al., 1998,9 ^ThInternational Conference on ArabidopsisResearch, Univ.of Wisconsin-Madison, June 24-28, Abstract 165).

Summary of the invention

The invention provides and have high oils the transgenic plant of (below be called " HIO ") phenotype.Have the arbitrary part of the transgenic plant of HIO phenotype plant, for example seed is with respect to contrast, and not genetically modified or wild-type plant has oleaginousness change or that increase.The present invention also provides the oil of the seed that derives from transgenic plant, and seed wherein has oleaginousness change or that improve.The present invention also provides meal, feed or the food that produces from arbitrary part of the transgenic plant with HIO phenotype.

In certain embodiments, these transgenic plant comprise the conversion carrier that contains coding or be complementary to the nucleotide sequence of coding HIO polypeptide.In specific embodiment, the expression of HIO polypeptide in transgenic plant causes the oleaginousness that transgenic plant change or improve.In preferred embodiments, transgenic plant are selected from Semen Brassicae campestris, soybean, corn, Sunflower Receptacle, cotton, cocoa, safflower, oil palm, coconut, flax, castor-oil plant and peanut.The present invention also provides the method that produces oils, comprises cultivating transgenic plant and reclaiming oils from described plant.Feed, meal, cereal or the seed of the nucleotide sequence that contains coding HIO polypeptide have been the present invention further provides.The present invention also provides the feed that contains HIO polypeptide or its lineal homologue, meal, cereal or seed.

Transgenic plant disclosed by the invention produce by the method that comprises the steps: plant conversion carrier is imported in the progenitor cell of plant, this plant conversion carrier contains coding or is complementary to the nucleotide sequence of the sequence of coding HIO polypeptide, and the progenitor cell of cultivating this conversion is to produce transgenic plant, wherein express described HIO polynucleotide sequence, cause the high oil meter type of transgenic plant.In other embodiments, transgenic plant disclosed by the invention are from described group of cell growth and direct offspring or the indirect offspring of next plant.In concrete non-limiting example, the HIO polypeptide expression is with respect to contrast in the transgenic plant that described method produces, and non-transgenic or wild-type plant cause the HIO phenotype of transgenic plant.

The invention also discloses and produce the additive method with HIO phenotype plant, wherein plant is accredited as in its HIO nucleotide sequence and has allelotrope, and lacks the allelotrope that this allelic plant compares in the described HIO nucleotide sequence and causes the HIO phenotype.This plant can produce filial generation, filial generation this equipotential gene of heredity that wherein produces and be the HIO phenotype.In some embodiments of this method, this method is used candidate gene/QTL method or is used the TILLING method.

The present invention also provides the transgenic plant cells with HIO phenotype.Described transgenic plant cells comprises the conversion carrier of HIO nucleotide sequence that contains coding or be complementary to the sequence of coding HIO polypeptide.In preferred embodiments, this transgenic plant cells is from being selected from Canadian rape, Semen Brassicae campestris, soybean, corn, Sunflower Receptacle, cotton, cocoa, safflower, oil palm, coconut, flax, the plant of castor-oil plant and peanut.In other embodiments, described vegetable cell is seed, pollen, propagulum or embryo cell.In some embodiments, this vegetable cell is available from transgenic plant disclosed by the invention.The present invention also provides from as direct offspring of the plant that comes from the growth of described group of cell or the vegetable cell of offspring's plant indirectly, perhaps from as direct offspring of the plant that comes from described group of cell growth or the vegetable cell of offspring's plant indirectly.

The present invention also provide surpass about 10,000, more preferably from about 20,000, even the container of preferred about 40,000 seeds, wherein surpass about 10%, more preferably from about 25%, more preferably from about 50%, even more preferably from about 75% or more preferably from about 90% seed is the seed that derives from plant of the present invention.

The present invention also provides and has surpassed about 10kg, 25kg more preferably from about, even the container of 50kg seed more preferably from about, wherein surpass about 10%, more preferably from about 25%, more preferably from about 50%, even more preferably from about 75% or more preferably from about 90% seed is the seed that derives from plant of the present invention.

Arbitrary plant of the present invention or its part can processed generation feed, food, meal or oil formulations.The especially preferred plant part that is used for this purpose is a seed.In preferred embodiments, feed, meal or oil formulation are designed to animal.Produce feed, food, the method for meal and oil formulation is well known in the art.For example, referring to United States Patent (USP) 4,957,748; 5,100,679; 5,219,596; 5,936,069; 6,005,076; 6,146,669; And 6,156,227.Meal of the present invention can mix with other meal.In preferred embodiments, originate from plant of the present invention or constitute by the meal that method of the present invention produces arbitrary product the meal component surpass about 0.5%, about 1%, about 5%, about 10%, about 25%, about 50%, about 75% or about 90% volume or weight.In another embodiment, can mix meal preparation and can constitute mixture and surpass about 10%, about 25%, about 35%, about 50% or about 75% volume.

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 " high oil (HIO) phenotype " is meant the plant with change oleaginousness (phenotype), perhaps arbitrary part of plant (for example, seed).As provided by the invention, change oleaginousness and comprise than contrast, non-transgenic or wild-type plant, oleaginousness increases in plant or the seed.

Term described herein " content " is meant the type and the relative content of seed for example or seed meal component.

Term described herein " meal " is meant in remaining seed components after Extraction oil of seed.The example of meal component comprises albumen and fiber.

Term described herein " fiber " is meant the indigestible component of plant seed, comprises such as Mierocrystalline cellulose hemicellulose, pectin, the cellular component of xylogen and phenolic compound.

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 eukaryote expression vector can commercially available acquisitions.It is known to select suitable expression vector to be that personnel are stated in this area.

But a part of sequence that " allogenic " nucleic acid construct or sequence have is not the wild-type sequence of vegetable cell expresses therein.Allogenic control sequence is meant the control sequence (being promotor or enhanser) 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, but by infection, transfection, microinjection, electroporations etc. add in the cell." allogenic " 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.The concrete limiting examples of heterologous nucleic acid sequence comprises the HIO nucleotide sequence, or its fragment, derivative (variant) or lineal homologue.

Term described herein " gene " is meant and participates in the dna fragmentation that polypeptide chain produces, it may comprise or not comprise before the coding region 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 chou " used herein comprises that by importing cell or the carrier that allogenic nucleotide sequence is modified, 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 reconstitution cell is expressed in natural (non--recombinant chou) or be expressed in artificial unconventionality expression down, the natural gene that downward modulation is expressed or do not expressed fully of deliberately intervening.

Term described herein " genetic expression " is meant the process based on the polypeptide of the nucleotide sequence generation of gene.Described process comprises to be transcribed and translates; Therefore " expression " can be at polynucleotide 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, polynucleotide 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 polynucleotide 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 ", " conversion " and " transduction ", and comprise that nucleotide sequence is incorporated in eucaryon or the prokaryotic cell prokaryocyte, wherein nucleotide sequence can be integrated into genome (karyomit(e) 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).

" vegetable cell " used herein is meant the arbitrary cell that derives from plant, comprise from undifferentiated tissue (for example callus) and at seed, pollen granule, the cell of the arbitrary type that exists in propagulum (progagules) or embryo or its dependency structure.

Term described herein " natural " and " wild-type " are meant to have the feature found the plant of identical type itself or the form of phenotype with respect to given plant trait or phenotype.In one embodiment, wild-type plant also is a control plant.In another embodiment, wild-type plant is a non-transgenic plant.

Term about plant characteristic described herein " modification " is meant the arbitrary part of transgenic plant (for example, having the transgenic plant that change oleaginousness) transgenic plant, for example in the seed with respect to the change of the phenotypes of similarly non--transgenic plant.Term described herein " change " is meant raising or the decline with respect to similarly non--transgenic plant of the plant traits of transgenic plant or phenotype (for example, oleaginousness).In a concrete limiting examples, the transgenic plant with improved proterties comprise with respect to similarly non--transgenic plant having the raising oleaginousness, perhaps the plant of HIO content.

" purpose phenotype (proterties) " for transgenic plant, 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 simulated condition.The purpose phenotype can be represented the improvement (for example, oleaginousness that improves or HIO content) to plant in the seed of plant, a kind of method that produces improvement in other plant maybe can be provided." improvement " is a kind of by unique and/or new phenotype or quality are provided to plant, and strengthens the feature of the practicality of plant species or kind.Such transgenic plant can have improved phenotype, such as the HIO phenotype.

Term " change oleaginousness phenotype " refers to the phenotype measured of genetic modification (transgenosis) plant, wherein with similarly, but the plant that is non-modification (non-transgenic) is compared, and this plant shows the overall oleaginousness (that is, oils accounts for the per-cent of seed quality) that statistics goes up significantly to be increased or reduce.High oil (HIO) phenotype refers to that overall oleaginousness increases.

" mutant " polynucleotide sequence used herein or gene are different in sequence or expression with corresponding wild type polynucleotide sequence or gene, and difference wherein causes phenotype or the proterties of plant that modify or change.With respect to plant or plant strain system, term " mutant " is meant that plant or plant strain cording have plant phenotype or proterties modification or that change, and phenotype of wherein modifying or change or proterties are expressed relevant with the modification or the change 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 selection, and described selective reagent is for example microbiotic or weedicide, and transgenic plant contain corresponding resistant gene thus.Term " T2 " is meant that the flower by the T1 plant carries out the plant filial generation that selfing produces, and is selected as transgenic plant before the described T1 plant.The T3 plant is by generations such as T2 plants." directly 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 direct filial generation of T1 plant." indirectly filial generation " of specified plant derives from the seed (or other tissue) of the direct 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 sporule.Vegetable cell can be available from plant organ or tissue and culture prepared therefrom arbitrarily.The invention provides transgenic plant cells with HIO phenotype.Described transgenic plant cells comprises the conversion carrier of HIO nucleotide sequence that contains coding or be complementary to the sequence of coding HIO polypeptide.In preferred embodiments, this transgenic plant cells is from being selected from Canadian rape, Semen Brassicae campestris, soybean, corn, Sunflower Receptacle, cotton, cocoa, safflower, oil palm, coconut, flax, the plant of castor-oil plant and peanut.In other embodiments, described vegetable cell is a seed, pollen, and propagulum or embryo cell are included in seed, pollen granule, the cell of the arbitrary type that exists in propagulum (progagules) or embryo or its dependency structure.The present invention also provides from as direct offspring of the plant that comes from described growth of progenitor cells or the vegetable cell of offspring's plant indirectly.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 " transgenic plant " are included in the plant that its genome contains heterologous polynucleotide.Allogenic polynucleotide can stably be incorporated in the genome, maybe can be inserted into outside the karyomit(e).Preferably, thus polynucleotide of the present invention are incorporated into with being stabilized and make polynucleotide by continuous passage in the genome.Vegetable 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 and indirect filial generation of vegetable cell also is considered to be genetically modified.

The invention provides transgenic plant with HIO phenotype.Transgenic plant with HIO phenotype can for example contain the oleaginousness of improved oil mass or change in arbitrary part of transgenic plant in the seed.The present invention also provides the oil of the seed that derives from transgenic plant, and seed wherein has the oleaginousness of change.The present invention also provides meal, feed or the food that produces from arbitrary part of the transgenic plant with HIO phenotype.

In certain embodiments, these transgenic plant comprise the conversion carrier of the HIO nucleotide sequence of the sequence that contains the coding or be complementary to coding " HIO " polypeptide.In specific embodiment, the expression of HIO polypeptide in transgenic plant causes the oleaginousness that transgenic plant change.In preferred embodiments, transgenic plant are selected from Canadian rape, Semen Brassicae campestris, soybean, corn, Sunflower Receptacle, cotton, cocoa, safflower, oil palm, coconut, flax, castor-oil plant and peanut.The present invention also provides the method that produces oils or seed meal, comprises cultivating transgenic plant and reclaiming oils and/or seed meal from described plant.Feed, meal, cereal or the seed of the nucleotide sequence that contains coding HIO polypeptide have been the present invention further provides.The present invention also provides the feed that contains HIO polypeptide or its lineal homologue, meal, cereal or seed.

Can utilize the several different methods that the polynucleotide sequence of required coding desirable proteins is imported vegetable cell, and these methods also are known to those skilled in the art, include but not limited to: (1) such as microinjection, the physical method of sending (particle gun (biolistic) or gene gun technology) of electroporation and particulate mediation; (2) virus-mediated delivery technique; And (3) agriculture bacillus mediated method for transformation.

The method of the transformed plant cells of normal use is the agriculture bacillus mediated DNA method for transformation and the method (that is particle gun) of particle gun or microparticle bombardment mediation.Usually, consideration conveyization is desirable, but transforms plasmid when being used for specificity, during such as chloroplast(id) or amyloplast, can utilize the required polynucleotide transforming plant plastides grain of particulate mediated delivery.

Agriculture bacillus mediated conversion realizes by using the genetically engineered soil bacteria that belongs to Agrobacterium.Have a large amount of wild-types of the agrobacterium tumefaciens (Agrobacterium tumefaciens) of Ti or Ri plasmid and Agrobacterium rhizogenes (Agrobacterium Rhizogenes) and unload first (disarmed) strain and can be used for carrying out gene transformation to plant.Carry out gene transformation by being transformed in the plant of a lot of kinds by the genetically engineered specific DNA that is called " T-DNA " that carries arbitrary required dna fragmentation.

The genetic transformation of agriculture bacillus mediated plant comprises some steps.The first step at first contacts with each other toxic Agrobacterium and vegetable cell, and this step is called " inoculation " usually.After the inoculation, time or longer time that Agrobacterium and vegetable cell/organize together were cultivated several hours to several days, the culture condition conversion with T-DNA that is suitable for growing.This step is called " cultivating altogether ".Cultivate altogether and after T-DNA sends, kill the Agrobacterium that contacts and/or be retained in explant in the container that contains explant with killing bacterium or bacteriostatic agent processing vegetable cell.If promote transgenic plant cells to compare under the condition of preferred growth of non-transgenic plant cell to carry out that this step is called " delay " step usually lacking arbitrary selective agent.If carry out existing selectivity to press well under the condition of transgenic plant cells, this step is called " selection " step.When using " delay " step, and then one or more " selection step " usually.

For microparticle bombardment (United States Patent (USP) 5,550,318; United States Patent (USP) 5,538,880, United States Patent (USP) 5,610,042; And the open WO95/06128 of PCT; Each all specifically is incorporated herein by reference herein), particle coating has nucleic acid and is delivered in the cell by propulsive force.Exemplary particle comprises tungsten, platinum, and preferred gold.

Is particle gun particle delivery system (Biolistics Particle Delivery System) (BioRad by acceleration with the exemplary embodiment that DNA is delivered to the method in the vegetable cell, Hercule, CA), the particle that this system can be used to apply DNA or cell advances past sieve, on the filter surfaces that covers to the monocot plant cell with suspension culture such as stainless steel or Nytex sieve.

The microparticle bombardment technology is the technology of widespread use, and can almost be used to transform arbitrary plant variety.Example by microparticle bombardment plant transformed kind comprises the monocotyledons kind, such as corn (international open WO 95/06128), barley, wheat (United States Patent (USP) 5,563,055, be incorporated herein by reference in full herein), rice, oat, rye (rye), sugarcane and Chinese sorghum; And comprise tobacco in a large number, the dicotyledons of soybean (United States Patent (USP) 5,322,783 is incorporated herein by reference herein in full), Sunflower Receptacle, peanut, cotton, tomato and beans (United States Patent (USP) 5,563,055 is incorporated herein by reference herein in full).

In order not consider that method for transformation selects the plant transformed cell or keep the score, the DNA in the transfered cell contains in reproducible plant tissue performance and produces and give gene to the compound of other toxic chemical resistances to plant tissue.As selectable, the goal gene of the mark that can screen or can keep the score includes but not limited to GUS, green fluorescent protein (GFP), luciferase (LUX), microbiotic or herbicide tolerant gene.The example of antibiotics resistance gene comprises penicillin, kantlex (and Xin Meisu, G418, bleomycin); Methotrexate (and Trimethoprim BP); Paraxin and tsiklomitsin.The proteic polynucleotide molecule that coding participates in herbicide tolerant is well known in the art, include but not limited to United States Patent (USP) 5,627,061, United States Patent (USP) 5,633,435 and the polynucleotide molecule and the United States Patent (USP) 5 of the coding 5-enol acetonyl thick grass hydrochlorate-3-phosphoric acid salt synthetic enzyme (EPSPS) of United States Patent (USP) 6,040,497 record, 094,945 aroA that is used to tolerate glyphosate that describes; Be described in United States Patent (USP) 4,810,648 are used to tolerate coding bromoxynil lytic enzyme (Nitrilase) polynucleotide molecule (Bxn) of bromoxynil (bromoxynil); Be described in Misawa etc., (Plant J.4:833-840,1993) and Misawa et al, (Plant J.6:481-489,1994) are used to tolerate the polynucleotide molecule of the coding phytoene dehydrogenase (crtI) of norflurazon (norfiurazon); Be described in the polynucleotide molecule that (Nucl.Acids Res.18:2188-2193,1990) such as Sathasiivan are used to tolerate the coding acetohydroxy acid synthetic enzyme (AHAS is also referred to as ALS) of sulfonylurea herbicide; And be described in the bar gene that DeBlock etc. (EMBO J.6:2513-2519,1987) is used to tolerate careless ammonium phosphine (Glufosinate) and bialaphos (bialaphos).

From the explant regeneration of multiple conversion, it is well known in the art growing and cultivating plants.This regeneration and process of growth generally include to be selected cell transformed and grows the stage that seedling etap of taking root cultivates these independent cells by the plumule of routine.Regeneration of transgenic plumule and seed similarly.The seedling that the transgenosis that is produced is taken root is planted in suitable plant growth medium then, in soil.The cell that exposes selective reagent but survive, the positive cell of perhaps keeping the score in screening is analyzed can be cultivated in supporting plant materials regenerated substratum.Developmental seedling is transferred in the less plant-growth mixture of soil, and transferred to the greenhouse or be used for making before the sophisticated growth room (seedling) to catch a cold and become cold-resistant.

The present invention can or organize use with cell transformed." transformable " used herein is meant and can further breeds cell or the tissue that produces plant materials.Those skilled in the art recognize that vegetable cell or tissue behind a large amount of insertion foreign DNAs can transform, and under suitable culture condition, this vegetable cell or tissue can form the plant of differentiation.The tissue that is suitable for these purposes can include but not limited to immature plumule, small org, suspended cell culture, immature inflorescence, stem meristematic tissue, knot explant, callus, hypocotyl tissue, cotyledon, root and leaf.

Can use arbitrary suitable medium.The example of suitable medium includes but not limited to substratum (Murashige and the Skoog based on MS-, Physiol.Plant, 15:473-497,1962) or be filled with and include but not limited to plant hormone, phytokinin, the substratum based on N6-of the other plant growth regulator of ABA and Plant hormones regulators,gibberellins (Chu etc., Scientia Sinica 18:659,1975).Those skilled in the art know multiple tissue culture medium (TCM), and it can be supported plant tissue growth and growth and be suitable for Plant Transformation and regeneration when carrying out suitably replenishing.These tissue culture medium (TCM)s can be used as the commercial preparation, and perhaps user's preparation and improved reagent are sold.Those skilled in the art will recognize that the substratum and culture medium supplemented agent and other culture condition that are used to transform with regenerated such as nutrition agent and growth regulator, such as the optical density(OD) between incubation period, pH and culture temperature can be optimized for the culture temperature of specific multiple purpose.

Those skilled in the art will recognize that the expression cassette stable integration and can import in other the plant materials by sexual hybridization to transgenic plant and after confirming to operate.Depend on the kind of being hybridized, can use the breeding technology of a large amount of standards.

The evaluation of plant with oleaginousness phenotype of change

We use Arabidopis thaliana activate label (ACTTAG) screening identify we identified and the gene 15232503 of called after HIO# (being set forth in 1 hurdle in the following table 1) and the oleaginousness phenotype (high oil meter type particularly) that changes between association.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 tumefaciens 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 enhanser.For specificity reclaims plant transformed, the T1 plant is exposed to selective agent.For the seed original seed that increases, will be in soil from about 18 T2 planting seeds of every kind of T1 plant, and after rudiment, be exposed to the T2 plant that selective agent reclaims conversion.Results are from the T3 seeds of these plants and compile.Utilize one of two kinds of following methods to estimate oil-contg; Utilization is used for the gas-chromatography (GC) of HIO30.1 to be analyzed, and fatty acid content in the mensuration T2 seed and composition or utilization are used for total lipid content of near infrared spectroscopy (NIR) the estimation T3 seed of HIO101B.

Insert segmental genomic dna sequence and found related between HIO nucleic acid and the high oil meter type by analyzing side joint T-DNA in the strain identified.Therefore, HIO nucleic acid and/or polypeptide can be used for developing the plant of the genetic modification of the oleaginousness phenotype (" HIO phenotype ") with modification.HIO nucleic acid can be used for producing the oleaginous seed crop, and it provides the oil offtake of raising from oleaginous seed processing, and is used to produce the feed grains crop that can provide energy to increase and is used for animal rearing.HIO nucleic acid can be further used for increasing the oleaginousness of special oil crop, so that increase the output of required rare lipid acid.The transgenic plant that carried out genetic modification expression HIO polypeptide can be used for producing oil, and wherein utilize standard method to cultivate transgenic plant, and obtain oil from plant part (for example, seed).

HIO nucleic acid and polypeptide

The HIO nucleic acid of finding in activating label filtration is listed in the 1st hurdle of table 1.Arabidopis thaliana information source (TAIR) is identified and number to be provided at the 2nd hurdle.The 3-4 hurdle provides the Genbank of Nucleotide and peptide sequence to identify number (GI#) respectively.Possible biochemical function and/or protein name have been enumerated in the 5th hurdle.Conservative protein structure domain has been enumerated on the 6th hurdle.The relative seed oil content of the plant of overexpression HIO nucleic acid has been enumerated on the 7th hurdle.The 8th hurdle provides from the nucleic acid of the lineal homologue gene of other plant kind and/or the GI# of peptide sequence.

Arabidopis thaliana HIO30.1 nucleic acid (genomic dna) sequence is provided in SEQ ID NO:1 and Genbank accession number GI#30694055.Corresponding protein sequence is provided among SEQ ID NO:2 and the GI#15232503.Arabidopis thaliana HIO101B nucleic acid is provided among SEQ ID NO:3 and the Genbank accession number GI#30680675.The corresponding proteins sequence is provided among SEQ IDNO:4 and the GI#30680 76.

Refer to when in plant, expressing, cause arbitrary part of plant, for example arbitrary polypeptide of seed HIO phenotype as term used herein " HIO polypeptide ".The present invention also provide surpass about 10,000, more preferably from about 20,000, even the container of preferred about 40,000 seeds, wherein surpass about 10%, more preferably from about 25%, more preferably from about 50%, even more preferably from about 75% or more preferably from about 90% seed is the seed that derives from plant of the present invention.

The fragment that refers to total length HIO albumen or its " functionally active " as term used herein " HIO polypeptide ", derivative (variant) or lineal homologue, as this protein fragments, derivative or lineal homologue show one or more functionally activies relevant with total length HIO polypeptide.In a preferred embodiment, functionally active HIO polypeptide causes the HIO phenotype in transgenic plant.In embodiment preferred further, the mistake of HIO polypeptide in plant expressed and caused high oil meter type.In another embodiment, functionally active HIO polypeptide can be saved (comprising disappearance) endogenous HIO activity of defective when expressing in plant or vegetable cell; This rescue polypeptide can from have the identical or different species of the active plant of defective.In another embodiment, the functionally active fragment of total length HIO polypeptide keeps one or more biological characteristicses relevant with total length HIO polypeptide, and signal activity for example is in conjunction with activity, catalytic activity or cell or extracellular disposition activity.

The HIO fragment preferably includes the HIO structural domain, and for example C-or N-end or catalyst structure domain especially, preferably include proteic at least 10 of HIO, and preferably at least 20, more preferably at least 25, at least 50 continuous amino acids most preferably.The functional domain of HIO gene is set forth in the 6th hurdle of table 1, and can use PFAM program (Bateman A etc., 1999 Nucleic AcidsRes 27:260-262) or INTERPRO (Mulder etc., 2003, Nucleic Acid Res.31,315-318) program is identified.Total length HIO polypeptide or its segmental functionally active variant comprise having the aminoacid insertion sequence, and disappearance is perhaps replaced, but keep the polypeptide of one or more biological characteristicses relevant with total length HIO polypeptide.Sometimes, the variant of generation can change the translation post-treatment of HIO polypeptide.For example, compare with natural polypeptides, variant can have the protein transport or the albumen locating features of change, the perhaps proteic transformation period of Gai Bianing.

Term used herein " HIO nucleic acid " comprises the sequence that the GenBank login sequence on the 3rd hurdle with table 1 provides, perhaps with the nucleic acid of its complementary sequence, and functionally active fragment, derivative or lineal homologue.HIO nucleic acid of the present invention can be the DNA that derives from genomic dna or cDNA, perhaps RNA.

In one embodiment, the HIO nucleic acid encoding of functionally active or be complementary to the nucleic acid of the active HIO polypeptide of encoding function.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 HIO polypeptide of encoding function.HIO 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, functionally active nucleic acid codified HIO polypeptide maturation or preprocess, perhaps intermediate forms.The HIO polynucleotide can also comprise allogeneic coding sequence, for example, and the mark of the promotion fusion polypeptide purifying that coding comprises or the sequence of transformation marker.In another embodiment, functionally active HIO nucleic acid for example can be used for, and by Antisense Suppression, suppresses to wait the HIO phenotype that produces afunction altogether.The present invention also provides feed, meal, cereal, food or the seed of the nucleotide sequence that contains coding HIO polypeptide.

In a preferred embodiment, be used for the HIO nucleic acid of the inventive method, comprise the nucleotide sequence of encoding or being complementary to the sequence of coding HIO polypeptide, the HIO peptide sequence that the GenBank login sequence on the 4th hurdle of this HIO polypeptide and table 1 provides (for example, SEQ ID NO:2 or 4 list aminoacid sequence) has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

In another embodiment, HIO polypeptide of the present invention comprises the peptide sequence that has at least 50% or 60% identity with the HIO peptide sequence of SEQ ID NO:2 or 4, and can have at least 70%, 80%, 85% with disclosed HIO peptide sequence, 90%, 95%, 97%, 98% or 99% sequence identity, and the conservative protein structural domain that can comprise disclosed HIO polypeptide is such as the cited protein structure domain in the 6th hurdle of table 1.In another embodiment, the HIO polypeptide comprises the functionally active fragment of the peptide sequence that the GenBank login sequence with the 4th hurdle of table 1 provides, for example SEQ ID NO:2 or 4 listed aminoacid sequences have at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, the peptide sequence of 97%, 98% or 99% sequence identity.In another embodiment, the HIO polypeptide comprises the full-length polypeptide sequence that the GenBank login sequence with the 4th hurdle of table 1 provides, for example SEQ ID NO:2 or 4 listed aminoacid sequences, have at least 50%, 60%, the peptide sequence of 70%, 80% or 90% sequence identity, and comprise the cited conservative protein structural domain in the 6th hurdle of table 1.

In yet another aspect, the total length of HIO polynucleotide sequence and disclosed HIO nucleotide sequence, the total length of the HIO nucleotide sequence shown in SEQ ID NO:1 or 3 or have at least 50% to 60% identity with this HIO sequence complementary nucleotide sequence, and can comprise with disclosed HIO nucleotide sequence (for example, SEQ ID NO:1 or 3, the perhaps sequence that provides of the GenBank login sequence on the 3rd hurdle of table 1) or its functionally active fragment, or have at least 70% with this HIO sequence complementary nucleotide sequence, 80%, 85%, 90% or 95% or above sequence identity.In another embodiment, disclosed HIO nucleic acid comprises the nucleotide sequence shown in SEQ IDNO:1 or the SEQ ID NO:3, perhaps with such HIO sequence complementary nucleotide sequence, and has the nucleotide sequence of significant sequence homology with such HIO sequence.Phrase used herein " significant sequence homology " be meant with such as the HIO sequence, have those nucleotide sequences of small or unessential sequence variations, promptly bring into play the sequence of function and coding HIO polypeptide in substantially the same mode.

Used herein, " per-cent (%) the sequence identity " of specific target sequence or its specific part is defined as obtaining if desired maximum per-cent sequence identity, 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 per-cent 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." % identity value " is by determining the identical Nucleotide of coupling or amino acid whose number divided by the sequence length of the sequence of the per-cent identity of report." per-cent (%) amino acid sequence similarity " determined by carrying out the calculating identical with definite per-cent 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 characteristics replaces, and proteic folding or activity is not had remarkable influence.The die aromatischen Aminosaeuren that can replace mutually is a phenylalanine, tryptophane and tyrosine; Interchangeable hydrophobic amino acid is a leucine, Isoleucine, methionine(Met) and Xie Ansuan; Interchangeable polare Aminosaeren is glutamine and asparagine; Interchangeable basic aminoacids is an arginine, Methionin and Histidine; Interchangeable acidic amino acid is aspartic acid and L-glutamic acid; Interchangeable p1 amino acid is L-Ala, Serine, Threonine, halfcystine and glycine.

The derivative nucleic acids molecule of target nucleic acid molecules comprises can be optionally and disclosed nucleotide sequence, for example the sequence of SEQ ID NO:1 or 3 nucleic acid array hybridizing.The rigorous degree of hybridization can pass through temperature, ionic strength, and pH, and the existence of denaturing agent such as methane amide is controlled during hybridization and the washing.Normally used condition be called optical imaging (referring to, for example, CurrentProtocol in Molecular Biology, Vol.1, Chap.2.10, John Wiley﹠amp; Sons, Publishers (1994); Sambrook etc., Molecular Cloning, Cold Spring Harbor (1989)).

In some embodiment, nucleic acid molecule of the present invention can be under following rigorous hybridization conditions and nucleic acid molecule, the making nucleic acid molecular hybridization that for example has the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3: the filter paper that contains nucleic acid, (1X SSC is 0.15M NaCl, the 0.015M Trisodium Citrate at the Citrate trianion that contains single times of concentration of 6X (SSC); PH 7.0), 5XDenhardt solution, in the solution of the herring sperm dna of 0.05% trisodium phosphate and 100 μ g/ml, 65 ℃ of prehybridizations 8 hours are to spending the night; Containing 6X SSC, 1X Denhardt solution in the solution of 100 μ g/ml yeast tRNA and 0.05% trisodium phosphate, was hybridized 18-20 hour for 65 ℃; In the solution that contains 0.1X SSC and 0.1%SDS (sodium lauryl sulphate), 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% methane amide, 5X SSC, 50mM Tris-HCl (pH 7.5), 5mMEDTA, 0.1%PVP, 1%Ficoll, in the solution of 1%BSA and 500 μ g/ml denatured salmon sperm dnas, 40 ℃ of pre-treatment 6 hours; Containing 35% methane amide, 5X SSC, 50mMTris-HCl (pH 7.5), 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% methane amide, 5 x SSC, 50 mM sodium phosphates (pH 7.6), 5X Denhardt 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 damping fluid; In 1 x SSC, about 37 ℃ of washing filter paper 1 hour.

Owing to the degeneracy of genetic code, can produce the polynucleotide sequence of many coding HIO 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 Arabidopis thaliana HIO.The possible lineal homologue of each Arabidopis thaliana HIO gene of identifying in following table 1 is identified the 8th hurdle at table 1.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 Arabidopis thaliana 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 primary search sequence among the reverse BLAST, this sequence then is possible lineal homologue (Huynen MA and Bork P, Proc NatlAcad Sci (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 genealogical tree.In the genealogical 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 genealogical 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 is preferred in the time can not obtaining sequence data.Degenerate pcr and cDNA or genome dna library screening are the common methods of seeking the genes involved sequence, and be well known in the art (referring to, for example, Sambrook, 1989, Molecular Cloning:A Laboratory Manual (second edition), Cold Spring Harbor Press, Plainview, N.Y.; Dieffenbach and Dveksler, 1995, PCR Primer:A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY).For example, described among the Sambrook etc. and be used for producing the cDNA library and with the method in portion homologous gene probe detection library from interested plant species.The high conservative of Arabidopis thaliana HIO encoding sequence partly can be used as probe.The lineal homologous nucleic acid of HIO can be at height, under moderate or the low rigorous condition with the nucleic acid hybridization shown in the GenBank accession number on table 1 the 3rd hurdle.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, specificity is in conjunction with the antibody of known HIO polypeptide, be used for lineal homologue separate (referring to, for example, Harlow and Lane, 1988,1999, Antibodies:ALaboratory Manual, Cold Spring Harbor Laboratory Press, New York).The Western engram analysis can determine that the lineal homologue of HIO (the lineal homologous protein of disclosed HIO polypeptide 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 Arabidopis thaliana or other the species that have wherein identified HIO nucleic acid and/or peptide sequence sequence being carried out reverse BLAST.

Can use any available method to obtain HIO nucleic acid and polypeptide.For example, by foregoing screening DNA library or by using polymerase chain reaction (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 HIO nucleic acid of desired form is incorporated into the plant expression vector that is used for transformed plant cells, and express the HIO polypeptide in host plant.

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

Table 1

1.HIO#	2.Tair	3. nucleic acid seq. GI#	4. polypeptide seq.GI#	5. possible biochemical function/protein name	6. Bao Shou protein structure domain	7. relative seed oil content (%)	8. lineal homologous gene: nucleic acid/polypeptide seq.GI#
							8. lineal homologous gene: nucleic acid/polypeptide seq.GI#			Nucleic acid GI#	Polypeptide GI#	Species
							HIO30-2F (HIO30.1)	At3g54400	gi\|30694055 SEQ ID NO：1	Nucleic acid GI#	Polypeptide GI#	Species	gi\|15232503 SEQ ID NO：2	The aspartyl protease family protein	IPR001461 PEPA A 1, stomach en-	112％	gi\|30681717 SEQ ID NO：5	gi\|15240680	Arabidopis thaliana
gi\|50939760 SEQ ID NO：6	gi\|50939761	Paddy rice (japonica cultivar-group)															gi\|30681717 SEQ ID NO：5	gi\|15240680	Arabidopis thaliana
gi\|50939760 SEQ ID NO：6	gi\|50939761	Paddy rice (japonica cultivar-group)	gi\|50904526 SEQ ID NO：7	gi\|50904527	Paddy rice (japonica cultivar-group)
HIO101B	At1g08520	gi\|30680675 SEQ ID NO：3	gi\|50904526 SEQ ID NO：7	gi\|50904527	Paddy rice (japonica cultivar-group)	gi\|30680676 SEQ ID NO：4				Magnesium-chelatase subunit chlD, chloroplast(id), possible /Mg-protoporphyrin IX chelatase, possible (CHLD)	The IPR000523 magnesium chelatase, the ChlI subunit	106％					gi\|2239150 SEQ ID NO：8	gi\|2239151	Tobacco
			gi\|2318116 SEQ ID NO：9	gi\|2318117	Pea												gi\|2239150 SEQ ID NO：8	gi\|2239151	Tobacco
			gi\|2318116 SEQ ID NO：9	gi\|2318117	Pea		Gi\|41386844; Residue 13673-2771 (in the other direction) is included among the gi\|32982486 SEQ ID NO:10	gi\|50540744	Paddy rice (japonica cultivar-group)

Generation has the plant of genetic modification of the oleaginousness phenotype of change

HIO nucleic acid disclosed in this invention and polypeptide can be used for producing the plant of the genetic modification of the oleaginousness phenotype with modification.Used herein, " oleaginousness of modification (phenotype) " can refer to the oleaginousness of modifying in the plant any part.In preferred embodiments, the change of HIO gene in plant expressed and is used for producing the plant with high oil content (phenotype).Usually in seed, observe the oleaginousness of change.The example of transgenic plant comprises containing to have coding or be complementary to the plant conversion carrier of nucleotide sequence of sequence that coding has the HIO polypeptide of the aminoacid sequence shown in SEQ ID NO:2 or the SEQ ID NO:4.

Arbitrary plant of the present invention or its part can processed generation feed, meal or oil formulations.Such as the corn that contains nucleotide sequence disclosed in this invention, the transgenic plant of soybean and Canadian rape can be used for producing vegetables oil and meal.The especially preferred plant part that is used for this purpose is a seed.Vegetables oil is used in the multiple foodstuff products, and is used as animal-feed from the meal of seed.In preferred embodiments, feed, food, meal or oil formulation are designed to ruminating animal.Produce feed, food, the method for meal and oil formulation is well known in the art.For example, referring to United States Patent (USP) 4,957,748; 5,100,679; 5,219,596; 5,936,069; 6,005,076; 6,146,669; And 6,156,227.Concrete but in the limiting examples, behind transgenic plant results seed, clean seed and remove stem, bar, handle and other materials, thereby in roller mill, roll over broken broken kind of subshell then of meal preparation.The broken seed of rolling is heated to 75-100 ℃ makes the lytic enzyme sex change, dissolving contains the uncracked cell of oil, and little oil droplet is assembled.Extract (and can reclaim) most oil by squeezing seed in expeller.By using organic solvent, extract remaining oil from press cake as hexane extraction.Remove solvent by meal being heated to about 100 ℃ from meal.After the drying, make meal become granulation to unified shape.The albumen that contains seed, the meal of degradable carbohydrate and fiber can as before the animal-feed with other material mixing.

Meal of the present invention can mix with other meal.In preferred embodiments, originate from plant of the present invention or constitute by the meal that method of the present invention produces arbitrary product the meal component surpass about 0.5%, about 1%, about 5%, about 10%, about 25%, about 50%, about 75% or about 90% volume or weight.In another embodiment, can mix meal preparation and can constitute mixture and surpass about 10%, about 25%, about 35%, about 50% or about 75% volume.

Method described here generally is applicable to all plants.Although in Arabidopis thaliana, carried out activation labeling acts and gene identification, the HIO nucleotide sequence (or its lineal homologue, variant or fragment) can in any floristics, express.In a preferred embodiment, oil-produced vegetable at the specificity organ, mainly is to produce and store triacylglycerol in seed.These species comprise soybean (Glycine max), Semen Brassicae campestris and Canadian rape (comprise swede type rape (Brassicanapus), B.campestris)), Sunflower Receptacle (Helianthus annus), cotton (upland cotton), corn (Zea maya), cocoa (Theobroma cacao), safflower (Carthamus tinctorius), oil palm (Elaeis guineensis), coconut (Cocos nucifera), flax (Linum usitatissimum), 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 (Poaceaefamily), other fodder crop and may be the wildlife species of rare fatty acid source.

Those skilled in the art should understand this area and have transformation technology miscellaneous, 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 vegetable 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 the successive plant from generation to generation.Planned purposes comprises complete albumen or its biologic activity part of heterologous nucleic acids construct codified of HIO polynucleotide.

In one embodiment, the carrier system based on Ti of double base can be used for shifting polynucleotide.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).Construct or carrier can comprise that plant promoter expresses selected nucleic acid molecule.In preferred embodiments, promotor is a plant promoter.

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, and for example the rape species comprise Canadian rape and Semen Brassicae campestris (De Block etc., 1989, Plant Physiol., 91:694-701), Sunflower Receptacle (Everett etc., 1987, Bio/Technology, 5:1201) and soybean (Christou etc., 1989, Proc.Natl.Acad.Sci USA, 86:7500-7504; Kline et al., 1987, Nature, method 327:70).

According to expression level, the types of organization of expression and/or the etap of expression take place, can regulate the expression (comprise and transcribe and translate) of HIO nucleotide sequence.Many allos are regulated sequence (for example, promotor and enhanser) and be can be used for controlling the HIO expression of nucleic acids.These comprise composing type, induction type and adjustable promotor, and can be with the promotor and the enhanser 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), rouge alkali synthetase (NOS) promotor (Ebert etc., Proc.Natl.Acad.Sci (U.S.A) 84:5745-5749,1987), octopine synthetic enzyme (OCS) promotor (its plasmid by the induced tumor of agrobacterium tumefaciens carries), such as cauliflower mosaic virus (CaMV) 19S promotor (Lawton etc., Plant Mol.Biol, 9:315-324,1987) and CaMV35S promotor (Odell et al., Nature 313:810-812,1985 and JonesJD et al, 1992) cauliflower disease virus promoter, radix scrophulariae mosaic virus 35 S-promotor (United States Patent (USP) 5,378,619), photoinduced from 1, the promotor (ssRUBISCO) of 5-diphosphoribulose carboxylase small subunit, Adh promotor (Walker etc., Proc.Natl.Acad.Sci. (U.S.A.) 84:6624-6628,1987), sucrose synthase promotor (Yang etc., Proc.Natl.Acad.Sci. (U.S.A.) 87:4144-4148,1990), R gene combined promoter (Chandler etc., The PlantCell 1:1175-1183,1989), the chlorophyll a/b binding protein gene promotor, CsVMV promotor (Verdaguer B et al., 1998, Plant Mol Biol., 37:1055-1067), muskmelon actin promoter (disclosed PCT application WO0056863), and seed-specific PRU promotor (U.S. Patent Application Publication U.S.20040064854, Clendennen etc.) 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 et al., 1993, Plant Mol Bio., 21:625-640).

In a preferred embodiment, HIO is expressed in from it and expresses under the regulation and control with the adjusting sequence of the gene that seed and/or fetal development are relevant in early days.In fact, in preferred embodiments, employed promotor is a seed enhanced promotor.The example of this promotor comprises from such as napin (Kridl etc., Seed Sci.Res.1:209:219,1991), sphaeroprotein (Belanger and Kriz, Genet., 129:863-872,1991, GenBank Accession No.L22295), γ zein Z 27 (Lopes etc., Mol Gen Genet., 247:603-613,1995), L3 oleosin promotor (United States Patent (USP) 6,433,252), Kidney bean albumen (Bustos etc., Plant Cell, 1 (9): 839-853,1989), arcelin5 (US application 2003/0046727), soybean 7S promotor, 7S α promotor (US application 2003/0093828), soybean 7S α ' β soya bean protein promotor, 7S α ' promotor (Beachy etc., EMBO J., 4:3047,1985; Schuler etc., Nucleic Acid Res., 10 (24): 8225-8244,1982), Trypsin inhibitor SBTI (Riggs etc., Plant Cell 1 (6): 609-621,1989), ACP (Baerson etc., Plant Mol.Biol., 22 (2): 255-267,1993), stearyl-ACP desaturase (Slocombe etc., PlantPhysiol.104 (4): 167-176,1994), soybean a ' subunit (Chen etc., the Proc.Natl.Acad.Sci.83:8560-8564 of β soya bean protein, 1986), broad bean USP (P-Vf.Usp, SEQ ID NO:1,2, with 3 (US application 2003/229918) and corn L3 oleosin promotor (Hong etc., Plant Mol.Biol., 34 (3): 549-555,1997) isogenic 5 ' regulation domain.Also comprising zein, is one group of storage protein finding in corn embryosperm.The genomic clone of zein spirit-soluble gene separated (Pedersen etc., Cell 29:1015-1026,1982; And Russell etc., Transgenic Res.6 (2): 157-168) and from these clones' promotor, comprise 15kD, 16kD, 19kD, 22kD, 27kD and these genes also can be utilized.Known other promotors of for example bringing into play function in corn comprise the promotor of following gene: waxy, Brittle, and Shrunken 2, q enzyme I and II, amylosynthease, debranching enzyme enzyme, oleosin, gluten and sucrose synthase.The leguminous plants gene that its promotor is relevant with fetal development with early stage seed comprises V.faba legumin (Baumlein etc., 1991, MolGen 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 Cell5:877-86), P.vulgaris β Kidney bean albumen (Bustos etc., 1991, EMBO J 10:1469-79), P.vulgaris DLEC2 and PHS[β] (Bobb etc., 1997, NucleicAcids 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 fetal development with early stage seed comprises paddy rice gluten (" GluA-3, " Yoshihara and 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 prolamine (Zhou﹠amp; Fan, 1993, Transgenic Res 2:141-6), wheat prolamine (Hammond-Kosack etc., 1993, EMBO J 12:545-54), zein (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 fetal development, comprise oil palm GL07A (7S sphaeroprotein, Morcillo etc., 2001, Physiol Plant 112:233-243), swede type rape napin, 2S storage protein and napA gene (Josefsson etc., 1987, J BiolChem 262:12196-201; Stalberg etc., 1993, Plant Mol Biol 1993 23:671-83; Ellerstrom etc., 1996, Plant Mol Biol 32:1019-27), swede type rape oleosin (Keddie etc., 1994, Plant Mol Biol 24:327-40), Arabidopis thaliana oleosin (Plant etc., 1994, Plant Mol Biol 25:193-205), Arabidopis thaliana 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 Vinca synthetic enzyme (Str, Ouwerkerk andMemelink, 1999, Mol Gen Genet 261:635-43).In another preferred embodiment, the adjusting sequence of the gene of expressing during the next comfortable oily biosynthesizing 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 Sci 342:209-15).Other spendable promotors for example are described in United States Patent (USP) 5,378,619; 5,391,725; 5,428,147; 5,447,858; 5,608,144; 5,608,144; 5,614,399; 5,633,441; 5,633,435 and 4,633,436.

In yet another aspect, may need to suppress the expression of endogenous HIO 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, Nature, 334:724-726; Van der Krol et al., 1988, BioTechniques, 6:958-976); Suppress altogether (Napoli, etc., 1990, Plant Cell, 2:279-289); Ribozyme (the open WO 97/10328 of PCT); With justice and the combination of antisense (Waterhouse, etc., 1998, Proc.Natl.Acad.Sci.USA, 95:13959-13964).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 non-coding region homology of endogenous sequence.Antisense Suppression can be used complete cDNA sequence (Sheehy etc., 1988, Sheehy et al., 1988, Proc.Natl.Acad.Sci.USA, 85:8805-8809), Partial cDNA Sequence comprises 5 ' encoding sequence (Cannon etc., 1990, Plant Mol.Biol., 15:39-47) or 3 ' non-coding sequence (Ch ' ng etc., 1989, Proc.Natl.Acad.Sci.USA, fragment 86:10006-10010).The technology that suppresses altogether can be used complete cDNA sequence (Napoli etc., 1990, Plant Cell, 2:279-289; Van der Krol et al., 1990, Plant Cell, 2:291-299) or Partial cDNA Sequence (Smith etc., 1990, Mol.Gen.Genetics, 224:477-481).

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 Appropriate technology comprised expression, ectopic expression, expression in the other plant species and gene knockout (reverse genetics, target knocks out, and [VIGS is referring to Baulcombe D for the gene silencing of virus induction, 1999, Arch.Virol.Suppl.15:189-201]).

In a preferred application, the expression and distribution by microarray analysis is used to measure simultaneously difference or the inductive of many different genes in expressing and changes.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, Cur.Opin.Plant Biol.2 (2): 96-103; Dangond F, Physiol Genomics (2000) 2:53-58; Van Hal NL et al., J Biotechnol. (2000) 78:271-280; Richmond T and Somerville S, Curr.Opin.Plant Biol.2000 3:108-116).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 product

The gene product analysis can comprise expression of recombinant proteins, and antiserum(antisera) produces, immunolocalization, catalysis or other active biochemical analysis, 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 genes involved, gene or gene product are placed specific biological chemistry, 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 " genetic expression in approach.

Generation has the mutant plant of the phenotype that changes oleaginousness

The present invention further provides and has identified in giving the endogenous HIO polypeptide that changes oleaginousness to have the plant of sudden change and the plant that generation has the HIO phenotype, wherein this plant is accredited as in its HIO nucleotide sequence and has allelotrope, compares with this allelic plant of shortage and produces the HIO phenotype.This plant can produce the offspring, wherein descendant inheritting allelotrope and have the HIO phenotype.For example, the invention provides evaluation and in endogenous HIO nucleotide sequence, have sudden change and the generation that can give the HIO phenotype and have the HIO phenotype, but these not genetically modified plant offsprings method.

In the method for a kind of being called " TILLING ", (be used for), for example, use EMS (ethyl methane sulfonate) to handle induced mutation in the seed of plant interested in genome target inductive local patholoic change.The plant that cultivation obtains, and autogamy, the offspring is used to prepare the DNA sample.The HIO-specific PCR is used to identify whether the plant of sudden change has sudden change in the HIO nucleotide sequence.Test the oleaginousness of the change of the plant with HIO sudden change then, perhaps the oleaginousness of test plants change uses the order-checking of pcr amplification and HIO nucleotide sequence to determine to have the HIO the nucleotide sequence whether plant that changes oleaginousness has sudden change then.TILLING can identify the expression that can change specific gene or by the proteic active sudden change of these genes encodings (referring to (2001) Plant Physiol 126:480-484 such as Colbert; McCallum etc. (2000) NatureBiotechnology 18:455-457).

In another approach, candidate gene/quantitative trait locus (QTL) method can be used for the auxiliary breeding project of mark, with the allelotrope of the oleaginousness that can give change in the lineal homologue of identifying HIO nucleotide sequence or HIO nucleotide sequence or sudden change (referring to Bert et al., TheorAppl Genet., 2003 Jun; 107 (1): 181-9; And Lionneton et al., Genome, 2002Dec; 45 (6): 1203-15.Therefore, aspect the present invention further in, HIO nucleic acid is used to identify whether the plant of the oleaginousness with change has sudden change in endogenous HIO nucleotide sequence, or has the specific allelotrope of the oleaginousness that causes 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 website of patent application and reference and the sequence information in the public database also are incorporated herein by reference.

Embodiment

Embodiment 1

Utilize activation label construct to transform and produce plant with HIO phenotype

This embodiment has described to produce has the transgenic plant that change oil-contg.

Use and activate label " ACTTAG " carrier, pSKI015 produces mutant (GI#6537289; Weigel D etc., 2000, Plant Physiology, 122:1003-1013).Use standard method to produce the Arabidopis thaliana transgenic plant, basically described in disclosed application PCT WO0183697.Briefly, T0 Arabidopis thaliana (Co1-0) 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 CaMV 35S enhancer element.According to Herbicid resistant, select transgenic plant from generation to generation and gather in the crops the T2 seed at T1.

Utilize the method for the following HIO30.1 of being used for to carry out the quantitative assay of T2 seed fatty acid content.Strain from each detection is the sample of 15 to 20 T2 seeds.The standard proportional that this sample contains usually with 1: 1: 2 has homology insertion fragment, does not insert fragment and hemizygote and inserts segmental plant.(Mettler-Toledo Co., Ohio weigh on USA) and transfer to then in the glass extraction flask this seed sample at the UMT-2 ultramicrobalance.From seed extraction lipid and at 80 ℃, 500 μ l 2.5%H ₂SO ₄Methanol solution transfer-esterification 3 hours, carry out the method for (Biochem J., 235:25-31,1986) such as Browse then.The margaric acid of known quantity is included in the reaction as interior mark.Add the water of 750 μ l and the hexane of 400 μ l in each bottle, thermal agitation and making is separated then.Reaction flask is directly gone up sample to GC, be used for analyzing, and get upper strata hexane phase by self-actuated sampler.Vapor-phase chromatography with flame ionization detection is used for the separation and the mensuration of fatty acid methyl ester.Agilent 6890 Plus GC ' s are used for Agilent Innowax post (30m * 0.25mm ID, 250um film thickness) and separate.Carrier gas is the hydrogen with 2.5ml/ minute constant flow.Inject the sample (220 ℃ of temperature ins, purge flow 15ml/min 1 minute) of 1 μ l in the mode of not shunting.Baking oven is set to 105 ℃ starting temperature, time of origin 0.5 minute, and the gradient with 60 ℃ of per minutes rises to 175 ℃ then, and 40 ℃/minute rise to 260 ℃, final 2 minutes hold-times.Detect (275 ℃ of temperature, fuel oil stream 30.0ml/ minute, oxygenant 400.0ml/min) by flame ionization.Utilize instrument control of Millennium chromatogram management system monitors and data gathering and analysis (version 3 .2, Waters Corporation, Milford, MA).Automatically integrate and quantitative analysis, but all analyses are all carried out manual checking subsequently confirm correct peak identification and acceptable signal-to-interference ratio before gathering with the result who obtains in being obtained studying.

ACTTAG strain W000086431 is accredited as high oil meter type and called after HIO30.1.Particularly, with the ACTTAG strain of other growth and average 28.7% the average oleaginousness of analyzing (being reference line) under the same conditions compare, oil constitutes 34.8% (w/w) of HIO30.1 seed quality.Carry out reanalysing of same seed in triplicate.Oil constitutes 32.1% increase that confirms with respect to the reference line oleaginousness of seed quality.

For the seed original seed of the ACTTAG strain that increases, about 18 T2 planting seeds in soil, and after rudiment, are exposed to the T2 plant that selective agent reclaims conversion.Results are from the T3 seeds of these plants and compile.

When results, analyze the set of T3 seed, and analyze the insertion site of the ACTTAG element of T3 seed set by inverse PCR and dna sequencing by the near infrared spectroscopy (NIR) that is used for HIO101B.Utilize Bruker 22 N/F near infrared spectrometers to catch the NIR infrared spectra.Utilize data that NIR analyzes and content and the total seed protein content of estimating total seed oil with reference to manufacturer's explanation by Bruker software.The method of AOCS official and recommended practice (Official and Recommended Practices of American Oil ChemistsSociety), the 5th version, AOCS, the general method of the AOCS method Am 1-92 of Champaign III, the oil-contg of exploitation calibrated predicted.Allow to carry out thick oil (ether extract) (PDXOil3, AOAC method 920.39 (ether extract (the AOAC world of fat (crude product) or animal-feed kind, official's analytical procedure (AOAC International, Official Methodsof Analysis), the 17th edition, AOAC International, Gaithersburg Maryland) and the calibration value of thick oily ASE (Ren Oil, accelerated solvent extracts) NIR prediction.Our oil-contg (PDX Oil3, the oil of prediction hexane extraction) of calibrated predicted is compared with 29,746 independent T3 ACTTAG seeds set.The oil-contg of average N IR prediction is 35.9%.Higher preceding 15% (prediction oil 〉=38%) sample of oil-contg is considered further to analyze.Limited by this, the set of 3,870 T3 seeds has high oil content.Inverse PCR is used to reclaim side joint T-DNA and inserts segmental genomic dna, uses the BLASTN search and/or Arabidopis thaliana information source (TAIR) database on basis to carry out sequential analysis then.ACTTAG element in about 40% the strain of the being analyzed system is placed on the genome.When analyzing, 478 high oily strain systems (as defined above) have successfully carried out the placement of ACTTAG element in genome.Seed from IN022173 and IN023577 has high oil (107%) and have ACTTAG insertion fragment in 10kb each other.Gene A tlg08520 (HIO101B) is in these strains system between the insertion site at the ACTTAG element.

Embodiment 2

The sign of T-DNA insertion sequence in the plant of the oleaginousness of display change (HIO) phenotype

We have carried out the standard molecule analysis as shown in patent application PCT WO0183697 basically, determine that the T-DNA among the W000086431 (HIO30.1) inserts the site.In brief, from the plant extract genomic dna of the oleaginousness phenotype of display change.The PCR that the Auele Specific Primer of use pSKI015 carrier carries out has confirmed to have the 35S enhanser in the plant from W000086431 system, and the Southern engram analysis has confirmed the genome conformity of ACTTAG T-DNA.

Plasmid rescue and anti-phase PCR are used to reclaim the genomic dna of T-DNA insertion sequence flank, use the BLASTN search arabidopsis thaliana genomic dna TAIR database (can obtain in the Arabidopis thaliana information resources website that the public can enter) on basis to carry out sequential analysis then.W000086431 system (HIO30) has the T-DNA of insertion in three different sites.

In order to determine which insertion sequence causes high seed oil phenotype, check high seed oil phenotype and T-DNA existence be divided into from.Determine the seed oil phenotype with 18 T2 plant culturing maturations and by the seed that these plants are collected.As described in embodiment 1, analyze the seed oil content of determining these plants by GC.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 site 2 and 3 close linkages.In addition, in the site the 2 and 3 average oleaginousness that comprise the T3 seed of T-DNA insertion sequence lack the average oleaginousness of those families of this insertion sequence than in these sites higher.The individual homozygote of site 2 and 3 T2 produces the seed of contrast 115.4% oleaginousness and the individual hemizygote of T2 in these sites produces the seed of contrast 118.4% oleaginousness, and the individuality that lacks T-DNA in these sites has 105% average oleaginousness from the seed control sample of wild-type Col-0 plant.Because the homozygote of high oil level point and hemizygote show that oleaginousness similarly increases, we infer that site 2 and phenotype relevant with high oil meter type with 3 caused by dominant mutation.By contrast, the 1 average oleaginousness that contains the T3 family of T-DNA insertion sequence is lower than or approximates the average oleaginousness that lacks those plants of insertion sequence in corresponding site greatly in the site.Reach a conclusion.Site 1 is not relevant with high oil meter type.

About 8kb place of 5 ' in the downstream, border of the initiator codon of Nucleotide that sequential analysis discloses called after HIO30.1 T-DNA insertion sequence of 3 in the site.

Embodiment 3

The analysis of Arabidopis thaliana HIO sequence

With BLAST (Altschul etc., 1990, 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), InterPro (Mulder etc., 2003, Nucleic Acids Res., 31,315-318) and/or CLUSTAL (Thompson JD etc., 1994, Nucleic Acids Res 22:4673-4680) carries out sequential analysis.

Embodiment 4

The repetition of high oil (HIO) phenotype

In order to check cross expressing of At3g54400 (HIO30.1) whether to cause high seed oil phenotype, will from the oil-contg the seed of crossing the transgenic plant of expressing this gene with compare from the oil-contg in non--transgenosis control plant seed.In order so to do, to be cloned into after the seed-specific PRU promotor At3g54400 in the plant conversion carrier and to utilize the flower pickling process to be transformed in the arabidopsis thaliana.This plant conversion carrier contains the nptII gene, provides at the resistance of kantlex and as selected marker.To be seeded on the nutrient agar that contains kantlex from the seed of plant transformed.After 7 days, transgenic plant are accredited as healthy green plants and are transplanted on the soil.Non--rudiment on nutrient agar of transgenosis control plant, grow and be transplanted on the soil then in 7 days.22 genetically modified seedling and 10 not genetically modified control plants are transferred to the random site on 32 identical lattice level lands.Plant-growth is to ripe and selfing and knot.From each plant results seed, by the oleaginousness of aforesaid method by near infrared (NIR) Wave Spectrum assessment seed.

In 2 experiments, checked the influence of expressing seed oil of crossing of At3g54400.In 2 experiments, cross the plant of expressing At3g54400 and have the seed oil content higher than the control plant that is grown in identical level land.In the whole experiment, the average seed oil content of crossing the plant of expressing At3g54400 is higher by 3% than unconverted contrast.The seed oil content of crossing the plant of expressing At3g54400 is significantly higher than (the two-way variance analysis (ANOVA) of non--transgenosis control plant; P=0.0477).

Table 2

Experiment	Plant ID	Transgenosis	The mean value of prediction	Relative value mean value
Experiment	Plant ID	Transgenosis	The mean value of prediction	Relative value mean value	1	G002735001	Pru::HIO30.1	33.6166	103.5698
1	G002735002	Pru::HIO30.1	32.4107	99.8547	1	G002735001	Pru::HIO30.1	33.6166	103.5698
1	G002735002	Pru::HIO30.1	32.4107	99.8547	1	G002735003	Pru::HIO30.1	32.5525	100.2913
1	G002735004	Pru::HIO30.1	33.0253	101.7482	1	G002735003	Pru::HIO30.1	32.5525	100.2913
1	G002735004	Pru::HIO30.1	33.0253	101.7482	1	G002735005	Pru::HIO30.1	34.7112	106.9422
1	G002735008	Pru::HIO30.1	30.3566	93.5262	1	G002735005	Pru::HIO30.1	34.7112	106.9422
1	G002735008	Pru::HIO30.1	30.3566	93.5262	1	G002735010	Pru::HIO30.1	33.7207	103.8905
1	G002735011	Pru::HIO30.1	33.7407	103.9523	1	G002735010	Pru::HIO30.1	33.7207	103.8905
1	G002735011	Pru::HIO30.1	33.7407	103.9523	1	G002735012	Pru::HIO30.1	35.5092	109.4009
1	G002735013	Pru::HIO30.1	32.5828	100.3848	1	G002735012	Pru::HIO30.1	35.5092	109.4009
1	G002735013	Pru::HIO30.1	32.5828	100.3848	1	G002735014	Pru::HIO30.1	30.9291	95.29
1	G002735015	Pru::HIO30.1	33.2569	102.4616	1	G002735014	Pru::HIO30.1	30.9291	95.29
1	G002735015	Pru::HIO30.1	33.2569	102.4616	1	G002735016	Pru::HIO30.1	29.9704	92.3363
1	G002735017	Pru::HIO30.1	28.7109	88.4558	1	G002735016	Pru::HIO30.1	29.9704	92.3363
1	G002735017	Pru::HIO30.1	28.7109	88.4558	1	G002735018	Pru::HIO30.1	32.977	101.5994
1	G002735019	Pru::HIO30.1	32.9081	101.3871	1	G002735018	Pru::HIO30.1	32.977	101.5994

1	G002735020	Pru::HIO30.1	32.0975	98.8895
1	G002735020	Pru::HIO30.1	32.0975	98.8895	1	G002735021	Pru::HIO30.1	32.5745	100.3592
1	G002735022	Pru::HIO30.1	32.4342	99.927	1	G002735021	Pru::HIO30.1	32.5745	100.3592
1	G002735022	Pru::HIO30.1	32.4342	99.927	1	G002736001	Do not have	31.1896	96.0925
1	G002736002	Do not have	31.7842	97.9243	1	G002736001	Do not have	31.1896	96.0925
1	G002736002	Do not have	31.7842	97.9243	1	G002736003	Do not have	33.9313	104.5394
1	G002736005	Do not have	32.7659	100.9489	1	G002736003	Do not have	33.9313	104.5394
1	G002736005	Do not have	32.7659	100.9489	1	G002736006	Do not have	32.6891	100.7124
1	G002736007	Do not have	31.9202	98.3435	1	G002736006	Do not have	32.6891	100.7124
1	G002736007	Do not have	31.9202	98.3435	1	G002736009	Do not have	30.7591	94.7662
1	G002736010	Do not have	34.6237	106.6728	1	G002736009	Do not have	30.7591	94.7662
1	G002736010	Do not have	34.6237	106.6728	2	DX06813001	Pru::HIO30.1	26.9581	99.2933
2	DX06813002	Pru::HIO30.1	31.5024	116.0309	2	DX06813001	Pru::HIO30.1	26.9581	99.2933
2	DX06813002	Pru::HIO30.1	31.5024	116.0309	2	DX06813003	Pru::HIO30.1	27.7407	102.1759
2	DX06813005	Pru::HIO30.1	27.8096	102.4296	2	DX06813003	Pru::HIO30.1	27.7407	102.1759
2	DX06813005	Pru::HIO30.1	27.8096	102.4296	2	DX06813006	Pru::HIO30.1	27.6955	102.0092
2	DX06813007	Pru::HIO30.1	29.4047	108.3048	2	DX06813006	Pru::HIO30.1	27.6955	102.0092
2	DX06813007	Pru::HIO30.1	29.4047	108.3048	2	DX06813008	Pru::HIO30.1	31.3112	115.3268
2	DX06813009	Pru::HIO30.1	28.395	104.5858	2	DX06813008	Pru::HIO30.1	31.3112	115.3268
2	DX06813009	Pru::HIO30.1	28.395	104.5858	2	DX06813010	Pru::HIO30.1	27.2328	100.305
2	DX06813011	Pru::HIO30.1	27.8172	102.4574	2	DX06813010	Pru::HIO30.1	27.2328	100.305
2	DX06813011	Pru::HIO30.1	27.8172	102.4574	2	DX06813012	Pru::HIO30.1	31.7126	116.8053
2	DX06813013	Pru::HIO30.1	27.7825	102.3297	2	DX06813012	Pru::HIO30.1	31.7126	116.8053
2	DX06813013	Pru::HIO30.1	27.7825	102.3297	2	DX06813016	Pru::HIO30.1	30.1171	110.9288
2	DX06813021	Pru::HIO30.1	29.7314	109.508	2	DX06813016	Pru::HIO30.1	30.1171	110.9288
2	DX06813021	Pru::HIO30.1	29.7314	109.508	2	DX06795001	Do not have	26.4647	97.4759
2	DX06795002	Do not have	27.6689	101.9113	2	DX06795001	Do not have	26.4647	97.4759
2	DX06795002	Do not have	27.6689	101.9113	2	DX06795003	Do not have	27.9777	103.0487
2	DX06795004	Do not have	28.3052	104.2549	2	DX06795003	Do not have	27.9777	103.0487

2	DX06795005	Do not have	26.8591	98.9286
2	DX06795005	Do not have	26.8591	98.9286	2	DX06795006	Do not have	26.8493	98.8927
2	DX06795007	Do not have	24.7616	91.2032	2	DX06795006	Do not have	26.8493	98.8927
2	DX06795007	Do not have	24.7616	91.2032	2	DX06795008	Do not have	26.9898	99.4099
2	DX06795009	Do not have	29.5455	108.8232	2	DX06795008	Do not have	26.9898	99.4099
2	DX06795009	Do not have	29.5455	108.8232	2	DX06795010	Do not have	26.078	96.0517

In order to check cross expressing of Atlg08520 (HIO101B) whether to cause high seed oil phenotype, will from the oil-contg the seed of crossing the transgenic plant of expressing this gene with compare from the oil-contg in non--transgenosis control plant seed.In order so to do, to be cloned into after the seed-specific PRU promotor Atlg08520 in the plant conversion carrier and to utilize the flower pickling process to be transformed in the arabidopsis thaliana.This plant conversion carrier contains the nptII gene, provides at the resistance of kantlex and as selected marker.To be seeded on the nutrient agar that contains kantlex from the seed of plant transformed.After 7 days, transgenic plant are accredited as healthy green plants and are transplanted on the soil.Non--rudiment on nutrient agar of transgenosis control plant, grow and be transplanted on the soil then in 7 days.22 genetically modified seedling and 10 not genetically modified control plants are transferred to the random site on 32 identical lattice level lands.Plant-growth is to ripe and selfing and knot.From each plant results seed, by the oleaginousness of aforesaid method by near infrared (NIR) Wave Spectrum assessment seed.

Referring to table 3, in 3 experiments, checked the influence of expressing seed oil of crossing of Atlg08520.In 3 experiments, cross the plant of expressing Atlg08520 and have the seed oil content higher than the control plant that is grown in identical level land.In the whole experiment, the average seed oil content of crossing the plant of expressing Atlg08520 is higher by 5% than unconverted contrast.The seed oil content of crossing the plant of expressing Atlg08520 is significantly higher than (the two-way variance analysis (ANOVA) of non--transgenosis control plant; P=0.0030).

Table 3

Experiment	Plant ID	Transgenosis	The oil of prediction	Oil is worth relatively
Experiment	Plant ID	Transgenosis	The oil of prediction	Oil is worth relatively	1	Z003907002	Pru::HIO101B	34.5351	109.0118
1	Z003907003	Pru::HIO101B	35.7637	112.8898	1	Z003907002	Pru::HIO101B	34.5351	109.0118
1	Z003907003	Pru::HIO101B	35.7637	112.8898	1	Z003907004	Pru::HIO101B	36.1101	113.9832
1	Z003907005	Pru::HIO101B	35.0344	110.5877	1	Z003907004	Pru::HIO101B	36.1101	113.9832
1	Z003907005	Pru::HIO101B	35.0344	110.5877	1	Z003907006	Pru::HIO101B	33.6465	106.2067
1	Z003907008	Pru::HIO101B	31.572	99.6584	1	Z003907006	Pru::HIO101B	33.6465	106.2067
1	Z003907008	Pru::HIO101B	31.572	99.6584	1	Z003907009	Pru::HIO101B	34.8969	110.1536
1	Z003907011	Pru::HIO101B	33.2621	104.9933	1	Z003907009	Pru::HIO101B	34.8969	110.1536
1	Z003907011	Pru::HIO101B	33.2621	104.9933	1	Z003907012	Pru::HIO101B	33.7056	106.3933
1	Z003907013	Pru::HIO101B	34.043	107.4582	1	Z003907012	Pru::HIO101B	33.7056	106.3933
1	Z003907013	Pru::HIO101B	34.043	107.4582	1	Z003907017	Pru::HIO101B	30.5754	96.5128
1	Z003907019	Pru::HIO101B	30.5735	96.5067	1	Z003907017	Pru::HIO101B	30.5754	96.5128
1	Z003907019	Pru::HIO101B	30.5735	96.5067	1	Z003907021	Pru::HIO101B	35.6997	112.6879
1	Z003910001	Do not have	32.4184	102.3301	1	Z003907021	Pru::HIO101B	35.6997	112.6879
1	Z003910001	Do not have	32.4184	102.3301	1	Z003910002	Do not have	31.8453	100.5212
1	Z003910003	Do not have	33.7541	106.5466	1	Z003910002	Do not have	31.8453	100.5212
1	Z003910003	Do not have	33.7541	106.5466	1	Z003910004	Do not have	32.409	102.3006
1	Z003910005	Do not have	31.2691	98.7024	1	Z003910004	Do not have	32.409	102.3006
1	Z003910005	Do not have	31.2691	98.7024	1	Z003910006	Do not have	30.9758	97.7767
1	Z003910007	Do not have	30.6926	96.8827	1	Z003910006	Do not have	30.9758	97.7767
1	Z003910007	Do not have	30.6926	96.8827	1	Z003910008	Do not have	31.7123	100.1015
1	Z003910009	Do not have	30.4135	96.0018	1	Z003910008	Do not have	31.7123	100.1015
1	Z003910009	Do not have	30.4135	96.0018	1	Z003910010	Do not have	31.3115	98.8364
2	Z003985002	Pru::HIO101B	29.2672	94.4525	1	Z003910010	Do not have	31.3115	98.8364
2	Z003985002	Pru::HIO101B	29.2672	94.4525	2	Z003985003	Pru::HIO101B	34.2542	110.5467
2	Z003985006	Pru::HIO101B	34.5119	111.3785	2	Z003985003	Pru::HIO101B	34.2542	110.5467
2	Z003985006	Pru::HIO101B	34.5119	111.3785	2	Z003985008	Pru::HIO101B	33.6445	108.579

2	Z003985010	Pru::HIO101B	29.9842	96.7665
2	Z003985010	Pru::HIO101B	29.9842	96.7665	2	Z003985011	Pru::HIO101B	31.1537	100.5408
2	Z003985013	Pru::HIO101B	36.1835	116.7732	2	Z003985011	Pru::HIO101B	31.1537	100.5408
2	Z003985013	Pru::HIO101B	36.1835	116.7732	2	Z003985018	Pru::HIO101B	35.0497	113.1142
2	Z003985019	Pru::HIO101B	35.7475	115.3661	2	Z003985018	Pru::HIO101B	35.0497	113.1142
2	Z003985019	Pru::HIO101B	35.7475	115.3661	2	Z004001001	Do not have	34.5363	111.4571
2	Z004001002	Do not have	31.3602	101.207	2	Z004001001	Do not have	34.5363	111.4571
2	Z004001002	Do not have	31.3602	101.207	2	Z004001003	Do not have	25.9146	83.6327
2	Z004001004	Do not have	28.7292	92.7163	2	Z004001003	Do not have	25.9146	83.6327
2	Z004001004	Do not have	28.7292	92.7163	2	Z004001005	Do not have	27.8834	89.9866
2	Z004001006	Do not have	30.4942	98.4123	2	Z004001005	Do not have	27.8834	89.9866
2	Z004001006	Do not have	30.4942	98.4123	2	Z004001007	Do not have	31.7341	102.4136
2	Z004001008	Do not have	27.5432	88.8887	2	Z004001007	Do not have	31.7341	102.4136
2	Z004001008	Do not have	27.5432	88.8887	2	Z004001009	Do not have	35.3457	114.0693
2	Z004001010	Do not have	36.3209	117.2164	2	Z004001009	Do not have	35.3457	114.0693
2	Z004001010	Do not have	36.3209	117.2164	3	DX06630001	Pru::HIO101B	29.8335	97.0689
3	DX06630002	Pru::HIO101B	29.1628	94.8866	3	DX06630001	Pru::HIO101B	29.8335	97.0689
3	DX06630002	Pru::HIO101B	29.1628	94.8866	3	DX06630003	Pru::HIO101B	29.5618	96.185
3	DX06630004	Pru::HIO101B	34.1125	110.9915	3	DX06630003	Pru::HIO101B	29.5618	96.185
3	DX06630004	Pru::HIO101B	34.1125	110.9915	3	DX06630005	Pru::HIO101B	29.9095	97.3163
3	DX06630009	Pru::HIO101B	31.2482	101.6719	3	DX06630005	Pru::HIO101B	29.9095	97.3163
3	DX06630009	Pru::HIO101B	31.2482	101.6719	3	DX06630010	Pru::HIO101B	31.3747	102.0834
3	DX06630011	Pru::HIO101B	29.3162	95.3858	3	DX06630010	Pru::HIO101B	31.3747	102.0834
3	DX06630011	Pru::HIO101B	29.3162	95.3858	3	DX06630012	Pru::HIO101B	33.8001	109.975
3	DX06630013	Pru::HIO101B	32.1773	104.6949	3	DX06630012	Pru::HIO101B	33.8001	109.975
3	DX06630013	Pru::HIO101B	32.1773	104.6949	3	DX06630014	Pru::HIO101B	33.8088	110.0032
3	DX06630015	Pru::HIO101B	30.963	100.7439	3	DX06630014	Pru::HIO101B	33.8088	110.0032
3	DX06630015	Pru::HIO101B	30.963	100.7439	3	DX06612001	Do not have	30.3733	98.8253
3	DX06612002	Do not have	30.1554	98.1162	3	DX06612001	Do not have	30.3733	98.8253

3	DX06612003	Do not have	30.7265	99.9744
3	DX06612003	Do not have	30.7265	99.9744	3	DX06612004	Do not have	31.8674	103.6867
3	DX06612005	Do not have	30.4838	99.1849	3	DX06612004	Do not have	31.8674	103.6867
3	DX06612005	Do not have	30.4838	99.1849	3	DX06612006	Do not have	29.9795	97.5438
3	DX06612007	Do not have	30.9704	100.7679	3	DX06612006	Do not have	29.9795	97.5438
3	DX06612007	Do not have	30.9704	100.7679	3	DX06612008	Do not have	31.4789	102.4224
3	DX06612009	Do not have	31.0681	101.0858	3	DX06612008	Do not have	31.4789	102.4224
3	DX06612009	Do not have	31.0681	101.0858	3	DX06612010	Do not have	30.2404	98.3927

Embodiment 5

In order to check in the plant of expressing HIO30.1 the high seed oil phenotype can heredity, will from the offspring's of the transgenic line that shows high oil meter type (DX06813012) seed oil content with compare from the oil-contg non--transgenosis control plant seed.In order so to do, will to be seeded in from the T2 seed of DX06813012 and to identify on the nutrient agar that contains kantlex and contain genetically modified plant.After 7 days, transgenic plant are accredited as healthy green plants and are transplanted on the soil.Non--rudiment on nutrient agar of transgenosis control plant, grow and be transplanted on the soil then in 7 days.22 genetically modified seedling and 10 not genetically modified control plants are transferred to the random site on 32 identical lattice level lands.Plant-growth is to ripe and selfing and knot.From each plant results seed, by the oleaginousness of aforesaid method by near infrared (NIR) Wave Spectrum assessment seed.

DX06813012 offspring's seed oil content is higher than the seed oil content of the control plant that is grown in identical level land, referring to table 4.DX06813012 offspring's average seed oil content is higher by 4.6% than the seed oil content of unconverted contrast.Being determined to increase by the T check is significant (P value=0.0015).

Table 4

Experiment numbers	Plant ID	Seed generation	The parent	Transgenosis	The mean value of prediction	Relative value mean value
Experiment numbers	Plant ID	Seed generation	The parent	Transgenosis	The mean value of prediction	Relative value mean value	1	DX08262001	T3	DX06813012	Pru::HIO30.1	35.32	105.46
1	DX08262002	T3	DX06813012	Pru::HIO30.1	34.89	104.18	1	DX08262001	T3	DX06813012	Pru::HIO30.1	35.32	105.46
1	DX08262002	T3	DX06813012	Pru::HIO30.1	34.89	104.18	1	DX08262003	T3	DX06813012	Pru::HIO30.1	32.89	98.22
1	DX08262004	T3	DX06813012	Pru::HIO30.1	34.31	102.47	1	DX08262003	T3	DX06813012	Pru::HIO30.1	32.89	98.22
1	DX08262004	T3	DX06813012	Pru::HIO30.1	34.31	102.47	1	DX08262005	T3	DX06813012	Pru::HIO30.1	33.8	100.93
1	DX08262006	T3	DX06813012	Pru::HIO30.1	36.18	108.04	1	DX08262005	T3	DX06813012	Pru::HIO30.1	33.8	100.93
1	DX08262006	T3	DX06813012	Pru::HIO30.1	36.18	108.04	1	DX08262007	T3	DX06813012	Pru::HIO30.1	33.26	99.32
1	DX08262008	T3	DX06813012	Pru::HIO30.1	32.13	95.94	1	DX08262007	T3	DX06813012	Pru::HIO30.1	33.26	99.32
1	DX08262008	T3	DX06813012	Pru::HIO30.1	32.13	95.94	1	DX08262009	T3	DX06813012	Pru::HIO30.1	34.82	103.98
1	DX08262010	T3	DX06813012	Pru::HIO30.1	35.42	105.77	1	DX08262009	T3	DX06813012	Pru::HIO30.1	34.82	103.98
1	DX08262010	T3	DX06813012	Pru::HIO30.1	35.42	105.77	1	DX08262011	T3	DX06813012	Pru::HIO30.1	33.97	101.44
1	DX08262012	T3	DX06813012	Pru::HIO30.1	34.34	102.56	1	DX08262011	T3	DX06813012	Pru::HIO30.1	33.97	101.44
1	DX08262012	T3	DX06813012	Pru::HIO30.1	34.34	102.56	1	DX08262013	T3	DX06813012	Pru::HIO30.1	36.79	109.85
1	DX08262014	T3	DX06813012	Pru::HIO30.1	33.43	99.83	1	DX08262013	T3	DX06813012	Pru::HIO30.1	36.79	109.85
1	DX08262014	T3	DX06813012	Pru::HIO30.1	33.43	99.83	1	DX08262015	T3	DX06813012	Pru::HIO30.1	35.89	107.18
1	DX08262016	T3	DX06813012	Pru::HIO30.1	33.79	100.9	1	DX08262015	T3	DX06813012	Pru::HIO30.1	35.89	107.18
1	DX08262016	T3	DX06813012	Pru::HIO30.1	33.79	100.9	1	DX08262017	T3	DX06813012	Pru::HIO30.1	36.4	108.69
1	DX08262018	T3	DX06813012	Pru::HIO30.1	37.13	110.88	1	DX08262017	T3	DX06813012	Pru::HIO30.1	36.4	108.69
1	DX08262018	T3	DX06813012	Pru::HIO30.1	37.13	110.88	1	DX08262019	T3	DX06813012	Pru::HIO30.1	34.5	103.01
1	DX08262020	T3	DX068 13012	Pru::HIO30.1	35.8	106.92	1	DX08262019	T3	DX06813012	Pru::HIO30.1	34.5	103.01
1	DX08262020	T3	DX068 13012	Pru::HIO30.1	35.8	106.92	1	DX08262021	T3	DX06813012	Pru::HIO30.1	37.71	112.62
1	DX08262022	T3	DX06813012	Pru::HIO30.1	37.92	113.23	1	DX08262021	T3	DX06813012	Pru::HIO30.1	37.71	112.62
1	DX08262022	T3	DX06813012	Pru::HIO30.1	37.92	113.23	1	DX08278001	T3	COL-0	Do not have	34.14	101.94
1	DX08278002	T3	COL-0	Do not have	33.98	101.47	1	DX08278001	T3	COL-0	Do not have	34.14	101.94
1	DX08278002	T3	COL-0	Do not have	33.98	101.47	1	DX08278003	T3	COL-0	Do not have	31.94	95.39
1	DX08278004	T3	COL-0	Do not have	33.86	101.1	1	DX08278003	T3	COL-0	Do not have	31.94	95.39
1	DX08278004	T3	COL-0	Do not have	33.86	101.1	1	DX08278005	T3	COL-0	Do not have	32.46	96.93
1	DX08278006	T3	COL-0	Do not have	32.91	98.27	1	DX08278005	T3	COL-0	Do not have	32.46	96.93
1	DX08278006	T3	COL-0	Do not have	32.91	98.27	1	DX08278007	T3	COL-0	Do not have	33.53	100.14
1	DX08278008	T3	COL-0	Do not have	33.69	100.62	1	DX08278007	T3	COL-0	Do not have	33.53	100.14
1	DX08278008	T3	COL-0	Do not have	33.69	100.62	1	DX08278009	T3	COL-0	Do not have	33.26	99.32
1	DX08278010	T3	COL-0	Do not have	35.1	104.82	1	DX08278009	T3	COL-0	Do not have	33.26	99.32

High seed oil phenotype can be hereditary in the plant of expressing HIO101B in order to check, will be from showing high oil meter type (Z003907005, Z003907008, Z003907013 and Z003907018) 4 kinds of transgenic lines the offspring seed oil content with compare from the oil-contg in non--transgenosis control plant seed.In order so to do, will be from Z003907005, Z003907008, the T2 seed of Z003907013 and Z003907018 are seeded in and identify on the nutrient agar that contains kantlex and contain genetically modified plant.After 7 days, transgenic plant are accredited as healthy green plants and are transplanted on the soil.Non--rudiment on nutrient agar of transgenosis control plant, grow and be transplanted on the soil then in 7 days.To transfer to the random site on 32 identical lattice level lands from 22 genetically modified seedling of each strain system and 10 not genetically modified control plants.Plant-growth is to ripe and selfing and knot.From each plant results seed, by the oleaginousness of aforesaid method by near infrared (NIR) Wave Spectrum assessment seed.

These transgenic lines offspring's seed oil content is higher than the seed oil content of the control plant that is grown in identical level land, referring to table 5.Z003907005, Z003907008, Z003907013 and Z003907018 offspring's the average seed oil content seed oil content than the identical unconverted contrast in level land respectively is high by 6.3,10.1, and 3.0 and 3.3%.Being determined to increase by the T check is significant (P value difference=0.0015,0.003,0.026 and 0.013).

Table 5

Experiment numbers	Plant	Seed generation	The parent	Transgenosis	The mean value of prediction	Relative value mean value
Experiment numbers	Plant	Seed generation	The parent	Transgenosis	The mean value of prediction	Relative value mean value	1	DX06905001	T3	Z003907005	Pru::HIO101B	34.34	114.9
1	DX06905002	T3	Z003907005	Pru::HIO101B	34.42	115.2	1	DX06905001	T3	Z003907005	Pru::HIO101B	34.34	114.9
1	DX06905002	T3	Z003907005	Pru::HIO101B	34.42	115.2	1	DX06905003	T3	Z003907005	Pru::HIO101B	32.17	107.7
1	DX06905004	T3	Z003907005	Pru::HIO101B	32.2	107.8	1	DX06905003	T3	Z003907005	Pru::HIO101B	32.17	107.7
1	DX06905004	T3	Z003907005	Pru::HIO101B	32.2	107.8	1	DX06905005	T3	Z003907005	Pru::HIO101B	34.04	113.9
1	DX06905006	T3	Z003907005	Pru::HIO101B	31.8	106.5	1	DX06905005	T3	Z003907005	Pru::HIO101B	34.04	113.9
1	DX06905006	T3	Z003907005	Pru::HIO101B	31.8	106.5	1	DX06905007	T3	Z003907005	Pru::HIO101B	32.13	107.6
1	DX06905008	T3	Z003907005	Pru::HIO101B	33.73	112.9	1	DX06905007	T3	Z003907005	Pru::HIO101B	32.13	107.6
1	DX06905008	T3	Z003907005	Pru::HIO101B	33.73	112.9	1	DX06905009	T3	Z003907005	Pru::HIO101B	31.78	106.4
1	DX06905010	T3	Z003907005	Pru::HIO101B	33.19	111.1	1	DX06905009	T3	Z003907005	Pru::HIO101B	31.78	106.4
1	DX06905010	T3	Z003907005	Pru::HIO101B	33.19	111.1	1	DX06905011	T3	Z003907005	Pru::HIO101B	29.43	98.5
1	DX06905012	T3	Z003907005	Pru::HIO101B	33.73	112.9	1	DX06905011	T3	Z003907005	Pru::HIO101B	29.43	98.5
1	DX06905012	T3	Z003907005	Pru::HIO101B	33.73	112.9	1	DX06905013	T3	Z003907005	Pru::HIO101B	30.89	103.4
1	DX06905014	T3	Z003907005	Pru::HIO101B	25.1	84.0	1	DX06905013	T3	Z003907005	Pru::HIO101B	30.89	103.4
1	DX06905014	T3	Z003907005	Pru::HIO101B	25.1	84.0	1	DX06905015	T3	Z003907005	Pru::HIO101B	29.56	99.0
1	DX06905016	T3	Z003907005	Pru::HIO101B	29.69	99.4	1	DX06905015	T3	Z003907005	Pru::HIO101B	29.56	99.0
1	DX06905016	T3	Z003907005	Pru::HIO101B	29.69	99.4	1	DX06905017	T3	Z003907005	Pru::HIO101B	31.21	104.5
1	DX06905018	T3	Z003907005	Pru::HIO101B	32.73	109.6	1	DX06905017	T3	Z003907005	Pru::HIO101B	31.21	104.5
1	DX06905018	T3	Z003907005	Pru::HIO101B	32.73	109.6	1	DX06905019	T3	Z003907005	Pru::HIO101B	30.23	101.2
1	DX06905020	T3	Z003907005	Pru::HIO101B	32.88	110.1	1	DX06905019	T3	Z003907005	Pru::HIO101B	30.23	101.2
1	DX06905020	T3	Z003907005	Pru::HIO101B	32.88	110.1	1	DX06905021	T3	Z003907005	Pru::HIO101B	32.27	108.0

1	DX06905022	T3	Z003907005	Pru::HIO101B	30.9	103.4
1	DX06905022	T3	Z003907005	Pru::HIO101B	30.9	103.4	1	DX06919001	T3	COL-0	Do not have	29.03	97.2
1	DX06919002	T3	COL-0	Do not have	30.87	103.3	1	DX06919001	T3	COL-0	Do not have	29.03	97.2
1	DX06919002	T3	COL-0	Do not have	30.87	103.3	1	DX06919003	T3	COL-0	Do not have	32.64	109.3
1	DX06919004	T3	COL-0	Do not have	31.55	105.6	1	DX06919003	T3	COL-0	Do not have	32.64	109.3
1	DX06919004	T3	COL-0	Do not have	31.55	105.6	1	DX06919005	T3	COL-0	Do not have	31.18	104.4
1	DX06919006	T3	COL-0	Do not have	28.45	95.2	1	DX06919005	T3	COL-0	Do not have	31.18	104.4
1	DX06919006	T3	COL-0	Do not have	28.45	95.2	1	DX06919007	T3	COL-0	Do not have	28.96	97.0
1	DX06919008	T3	COL-0	Do not have	28.62	95.8	1	DX06919007	T3	COL-0	Do not have	28.96	97.0
1	DX06919008	T3	COL-0	Do not have	28.62	95.8	1	DX06919009	T3	COL-0	Do not have	30.38	101.7
1	DX06919010	T3	COL-0	Do not have	27.04	90.5	1	DX06919009	T3	COL-0	Do not have	30.38	101.7
1	DX06919010	T3	COL-0	Do not have	27.04	90.5	2	DX07000001	T3	Z003907008	Pru::HIO101B	30.72	103.6
2	DX07000003	T3	Z003907008	Pru::HIO101B	30.12	101.6	2	DX07000001	T3	Z003907008	Pru::HIO101B	30.72	103.6
2	DX07000003	T3	Z003907008	Pru::HIO101B	30.12	101.6	2	DX07000004	T3	Z003907008	Pru::HIO101B	32.09	108.3
2	DX07000005	T3	Z003 907008	Pru::HIO101B	34.17	115.3	2	DX07000004	T3	Z003907008	Pru::HIO101B	32.09	108.3
2	DX07000005	T3	Z003 907008	Pru::HIO101B	34.17	115.3	2	DX07000007	T3	Z003907008	Pru::HIO101B	33.99	114.6
2	DX07000008	T3	Z003907008	Pru::HIO101B	33.14	111.8	2	DX07000007	T3	Z003907008	Pru::HIO101B	33.99	114.6
2	DX07000008	T3	Z003907008	Pru::HIO101B	33.14	111.8	2	DX07000009	T3	Z003907008	Pru::HIO101B	32.91	111.0
2	DX07000010	T3	Z003907008	Pru::HIO101B	33.94	114.5	2	DX07000009	T3	Z003907008	Pru::HIO101B	32.91	111.0
2	DX07000010	T3	Z003907008	Pru::HIO101B	33.94	114.5	2	DX07008001	T3	COL-0	Do not have	30.17	101.8
2	DX07008002	T3	COL-0	Do not have	27.75	93.6	2	DX07008001	T3	COL-0	Do not have	30.17	101.8
2	DX07008002	T3	COL-0	Do not have	27.75	93.6	2	DX07008003	T3	COL-0	Do not have	27.48	92.7
2	DX07008004	T3	COL-0	Do not have	32.74	110.4	2	DX07008003	T3	COL-0	Do not have	27.48	92.7
2	DX07008004	T3	COL-0	Do not have	32.74	110.4	2	DX07008005	T3	COL-0	Do not have	29.53	99.6
2	DX07008006	T3	COL-0	Do not have	27.46	92.6	2	DX07008005	T3	COL-0	Do not have	29.53	99.6
2	DX07008006	T3	COL-0	Do not have	27.46	92.6	2	DX07008007	T3	COL-0	Do not have	32.13	108.4
2	DX07008008	T3	COL-0	Do not have	30.97	104.5	2	DX07008007	T3	COL-0	Do not have	32.13	108.4
2	DX07008008	T3	COL-0	Do not have	30.97	104.5	2	DX07008010	T3	COL-0	Do not have	28.55	96.3
3	DX06908001	T3	Z003907013	Pru::HIO101B	32.84	105.0	2	DX07008010	T3	COL-0	Do not have	28.55	96.3
3	DX06908001	T3	Z003907013	Pru::HIO101B	32.84	105.0	3	DX06908002	T3	Z003907013	Pru::HIO101B	33.59	107.4
3	DX06908003	T3	Z003907013	Pru::HIO101B	33.03	105.7	3	DX06908002	T3	Z003907013	Pru::HIO101B	33.59	107.4
3	DX06908003	T3	Z003907013	Pru::HIO101B	33.03	105.7	3	DX06908004	T3	Z003907013	Pru::HIO101B	32.38	103.6
3	DX06908005	T3	Z003907013	Pru::HIO101B	32.79	104.9	3	DX06908004	T3	Z003907013	Pru::HIO101B	32.38	103.6
3	DX06908005	T3	Z003907013	Pru::HIO101B	32.79	104.9	3	DX06908006	T3	Z003907013	Pru::HIO101B	32.36	1 03.5
3	DX06908007	T3	Z003907013	Pru::HIO101B	34.42	110.1	3	DX06908006	T3	Z003907013	Pru::HIO101B	32.36	1 03.5
3	DX06908007	T3	Z003907013	Pru::HIO101B	34.42	110.1	3	DX06908008	T3	Z003907013	Pru::HIO101B	34.02	108.8
3	DX06908009	T3	Z003907013	Pru::HIO101B	31.19	99.8	3	DX06908008	T3	Z003907013	Pru::HIO101B	34.02	108.8
3	DX06908009	T3	Z003907013	Pru::HIO101B	31.19	99.8	3	DX06908010	T3	Z003907013	Pru::HIO101B	29.75	95.2
3	DX06908011	T3	Z003907013	Pru::HIO101B	32.78	104.9	3	DX06908010	T3	Z003907013	Pru::HIO101B	29.75	95.2
3	DX06908011	T3	Z003907013	Pru::HIO101B	32.78	104.9	3	DX06908012	T3	Z003907013	Pru::HIO101B	32.25	103.2
3	DX06908013	T3	Z003907013	Pru::HIO101B	32.1	102.7	3	DX06908012	T3	Z003907013	Pru::HIO101B	32.25	103.2
3	DX06908013	T3	Z003907013	Pru::HIO101B	32.1	102.7	3	DX06908014	T3	Z003907013	Pru::HIO101B	31.23	99.9
3	DX06908015	T3	Z003907013	Pru::HIO101B	32.26	103.2	3	DX06908014	T3	Z003907013	Pru::HIO101B	31.23	99.9
3	DX06908015	T3	Z003907013	Pru::HIO101B	32.26	103.2	3	DX06908016	T3	Z003907013	Pru::HIO101B	32.25	103.2
3	DX06908017	T3	Z003907013	Pru::HIO101B	32.13	102.8	3	DX06908016	T3	Z003907013	Pru::HIO101B	32.25	103.2

3	DX06908018	T3	Z003907013	Pru::HIO101B	32.1	102.7
3	DX06908018	T3	Z003907013	Pru::HIO101B	32.1	102.7	3	DX06908019	T3	Z003907013	Pru::HIO101B	31.15	99.6
3	DX06908020	T3	Z003907013	Pru::HIO101B	30.27	96.8	3	DX06908019	T3	Z003907013	Pru::HIO101B	31.15	99.6
3	DX06908020	T3	Z003907013	Pru::HIO101B	30.27	96.8	3	DX06908021	T3	Z003907013	Pru::HIO101B	32.89	105.2
3	DX06908022	T3	Z003907013	Pru::HIO101B	30.98	99.1	3	DX06908021	T3	Z003907013	Pru::HIO101B	32.89	105.2
3	DX06908022	T3	Z003907013	Pru::HIO101B	30.98	99.1	3	DX06922001	T3	COL-0	Do not have	30.65	98.0
3	DX06922002	T3	COL-0	Do not have	32.52	104.0	3	DX06922001	T3	COL-0	Do not have	30.65	98.0
3	DX06922002	T3	COL-0	Do not have	32.52	104.0	3	DX06922004	T3	COL-0	Do not have	32.1	102.7
3	DX06922005	T3	COL-0	Do not have	29.57	94.6	3	DX06922004	T3	COL-0	Do not have	32.1	102.7
3	DX06922005	T3	COL-0	Do not have	29.57	94.6	3	DX06922006	T3	COL-0	Do not have	31.56	101.0
3	DX06922007	T3	COL-0	Do not have	31.24	99.9	3	DX06922006	T3	COL-0	Do not have	31.56	101.0
3	DX06922007	T3	COL-0	Do not have	31.24	99.9	3	DX06922008	T3	COL-0	Do not have	31.85	101.9
3	DX06922009	T3	COL-0	Do not have	30.34	97.0	3	DX06922008	T3	COL-0	Do not have	31.85	101.9
3	DX06922009	T3	COL-0	Do not have	30.34	97.0	3	DX06922010	T3	COL-0	Do not have	31.54	100.9
4	DX06911001	T3	Z003985018	Pru::HIO101B	28.83	102.3	3	DX06922010	T3	COL-0	Do not have	31.54	100.9
4	DX06911001	T3	Z003985018	Pru::HIO101B	28.83	102.3	4	DX06911003	T3	Z003985018	Pru::HIO101B	27.82	98.7
4	DX06911004	T3	Z003985018	Pru::HIO101B	27.08	96.1	4	DX06911003	T3	Z003985018	Pru::HIO101B	27.82	98.7
4	DX06911004	T3	Z003985018	Pru::HIO101B	27.08	96.1	4	DX06911005	T3	Z003985018	Pru::HIO101B	28.37	100.6
4	DX06911006	T3	Z003985018	Pru::HIO101B	28.65	101.6	4	DX06911005	T3	Z003985018	Pru::HIO101B	28.37	100.6
4	DX06911006	T3	Z003985018	Pru::HIO101B	28.65	101.6	4	DX06911007	T3	Z003985018	Pru::HIO101B	29.94	106.2
4	DX06911008	T3	Z003985018	Pru::HIO101B	30.08	106.7	4	DX06911007	T3	Z003985018	Pru::HIO101B	29.94	106.2
4	DX06911008	T3	Z003985018	Pru::HIO101B	30.08	106.7	4	DX06911009	T3	Z003985018	Pru::HIO101B	28.09	99.6
4	DX06911010	T3	Z003985018	Pru::HIO101B	28.84	102.3	4	DX06911009	T3	Z003985018	Pru::HIO101B	28.09	99.6
4	DX06911010	T3	Z003985018	Pru::HIO101B	28.84	102.3	4	DX06911011	T3	Z003985018	Pru::HIO101B	28.68	101.7
4	DX06911012	T3	Z003985018	Pru::HIO101B	28.1	99.7	4	DX06911011	T3	Z003985018	Pru::HIO101B	28.68	101.7
4	DX06911012	T3	Z003985018	Pru::HIO101B	28.1	99.7	4	DX06911013	T3	Z003985018	Pru::HIO101B	31.92	113.2
4	DX06911014	T3	Z003985018	Pru::HIO101B	31.01	110.0	4	DX06911013	T3	Z003985018	Pru::HIO101B	31.92	113.2
4	DX06911014	T3	Z003985018	Pru::HIO101B	31.01	110.0	4	DX06911015	T3	Z003985018	Pru::HIO101B	29.71	105.4
4	DX06911016	T3	Z003985018	Pru::HIO101B	27.73	98.4	4	DX06911015	T3	Z003985018	Pru::HIO101B	29.71	105.4
4	DX06911016	T3	Z003985018	Pru::HIO101B	27.73	98.4	4	DX06911017	T3	Z003985018	Pru::HIO101B	28.75	102.0
4	DX06911018	T3	Z003985018	Pru::HIO101B	29.01	102.9	4	DX06911017	T3	Z003985018	Pru::HIO101B	28.75	102.0
4	DX06911018	T3	Z003985018	Pru::HIO101B	29.01	102.9	4	DX06911019	T3	Z003985018	Pru::HIO101B	31.19	110.6
4	DX06911020	T3	Z003985018	Pru::HIO101B	28.7	101.8	4	DX06911019	T3	Z003985018	Pru::HIO101B	31.19	110.6
4	DX06911020	T3	Z003985018	Pru::HIO101B	28.7	101.8	4	DX06911021	T3	Z003985018	Pru::HIO101B	29.4	104.3
4	DX06911022	T3	Z003985018	Pru::HIO101B	29.96	106.3	4	DX06911021	T3	Z003985018	Pru::HIO101B	29.4	104.3
4	DX06911022	T3	Z003985018	Pru::HIO101B	29.96	106.3	4	DX06925001	T3	COL-0	Do not have	27.25	96.7
4	DX06925002	T3	COL-0	Do not have	28.38	100.7	4	DX06925001	T3	COL-0	Do not have	27.25	96.7
4	DX06925002	T3	COL-0	Do not have	28.38	100.7	4	DX06925003	T3	COL-0	Do not have	29	102.9
4	DX06925004	T3	COL-0	Do not have	27.52	97.6	4	DX06925003	T3	COL-0	Do not have	29	102.9
4	DX06925004	T3	COL-0	Do not have	27.52	97.6	4	DX06925005	T3	COL-0	Do not have	27.07	96.0
4	DX06925006	T3	COL-0	Do not have	29.36	104.1	4	DX06925005	T3	COL-0	Do not have	27.07	96.0
4	DX06925006	T3	COL-0	Do not have	29.36	104.1	4	DX06925007	T3	COL-0	Do not have	27.98	99.2
4	DX06925008	T3	COL-0	Do not have	28.83	102.3	4	DX06925007	T3	COL-0	Do not have	27.98	99.2
4	DX06925008	T3	COL-0	Do not have	28.83	102.3	4	DX06925009	T3	COL-0	Do not have	28.24	100.2
4	DX06925010	T3	COL-0	Do not have	28.31	100.4	4	DX06925009	T3	COL-0	Do not have	28.24	100.2

Content description disclosed by the invention because the Zhi Wu ﹠amp of the oleaginousness phenotype of the expression display change of the change of HIO nucleic acid; The discovery of vegetable cell, and the methods involving and the composition that are used to utilize this discovery.Clearly, the fine detail of the method that the present invention describes can be changed or modify, only otherwise deviate from spirit of the present invention.We require this modification and change in the scope and spirit of specification sheets of the present invention and claim.

＜110〉Agrinomics LLC (Agrinomics LLC)

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

<130>SCT073199-66

<150>US 60/643,674

<151>2005-01-12

<160>10

<170>PatentIn version 3.3

<210>1

<211>1554

<212>DNA

<213>Arabidopsis thaliana

<400>1

acaaacacta gaaaccacaa agacaaaatc aaggtcttaa acaatgagat cccatctctt 60

gattttgtta atctctctct taatcttgaa atctgaatcc ataaactgca atgaaaagag 120

ccattcctca gatctaagag tgttccacat taacagtcta tgttctccat tcaaaacttc 180

tgtttcatgg gcagatacac ttcttcaaga taaggctcgt ttcctatact tgtcaagcct 240

cgctggcgtt aggaaatcat cagttccaat cgcctctggt cgggccatcg ttcagagccc 300

gacttacatc gtgagggcta acatcgggac accggctcag cccatgctcg tggctcttga 360

cactagcaat gacgctgctt ggattccttg ttctggctgc gttggctgtt cttcctctgt 420

tctctttgac ccttccaagt caagctcctc tcgtactctt caatgcgaag ctcctcagtg 480

taaacaggct ccaaatccaa gttgcacagt aagcaaatca tgtggtttca acatgaccta 540

cggtggttca accatcgaag catatctgac acaagacaca ctaacattgg ccagtgacgt 600

catcccaaac tacacctttg ggtgcatcaa caaagctagt ggaacatcgt tgccagcgca 660

aggactcatg ggcttaggtc gtggtccatt gtctttaatc tcacagtcac aaaatcttta 720

tcagtctaca ttctcgtatt gcttgcctaa tagtaagtcc agcaatttct ccggatcact 780

aagattggga cctaagaacc aaccgatccg gatcaagacc actccattgt taaagaaccc 840

tagaagatca tcgctttact atgttaactt ggttgggatt cgtgtcggaa acaaaattgt 900

ggatattcct acaagtgcac tcgcctttga tccggccacc ggagccggca ccatctttga 960

ctcggggacg gtctacacaa ggctagtcga accagcttac gtggcggtga gaaacgagtt 1020

caggagacgt gttaagaacg caaacgcaac ttcactagga ggtttcgaca catgctactc 1080

cggctccgtc gtgttcccgt cggtgacgtt tatgttcgcc ggaatgaacg tgactctgcc 1140

tccagacaac cttctcatcc acagctccgc aggtaacctc agctgcctcg ccatggctgc 1200

agctccggtc aacgttaact ctgtccttaa cgtcatcgct agtatgcagc aacagaacca 1260

ccgagttctc atcgacgttc caaattccag gctcggaatt tcccgtgaaa cttgcaccta 1320

agttttatcg atttgtattt ttgttttcgg tcgatttcgt aatgcgtttt gaacttttga 1380

attttggaaa ctatataagt taatgatttt tgttaattct caaacgattg taaaatcttt 1440

cgtatgattt tctttcgatg ttctctgttc aaaaaggatt gtactcatga atttgcggat 1500

gcaataagcc tggaacagag gacctttatt attttaaata tatatatgtt ccaa 1554

<210>2

<211>425

<212>PRT

<213>Arabidopsis thaliana

<400>2

Met Arg Ser His Leu Leu Ile Leu Leu Ile Ser Leu Leu Ile Leu Lys

1 5 10 15

Ser Glu Ser Ile Asn Cys Asn Glu Lys Ser His Ser Ser Asp Leu Arg

20 25 30

Val Phe His Ile Asn Ser Leu Cys Ser Pro Phe Lys Thr Ser Val Ser

35 40 45

Trp Ala Asp Thr Leu Leu Gln Asp Lys Ala Arg Phe Leu Tyr Leu Ser

50 55 60

Ser Leu Ala Gly Val Arg Lys Ser Ser Val Pro Ile Ala Ser Gly Arg

65 70 75 80

Ala Ile Val Gln Ser Pro Thr Tyr Ile Val Arg Ala Asn Ile Gly Thr

85 90 95

Pro Ala Gln Pro Met Leu Val Ala Leu Asp Thr Ser Asn Asp Ala Ala

100 105 110

Trp Ile Pro Cys Ser Gly Cys Val Gly Cys Ser Ser Ser Val Leu Phe

115 120 125

Asp Pro Ser Lys Ser Ser Ser Ser Arg Thr Leu Gln Cys Glu Ala Pro

130 135 140

Gln Cys Lys Gln Ala Pro Asn Pro Ser Cys Thr Val Ser Lys Ser Cys

145 150 155 160

Gly Phe Asn Met Thr Tyr Gly Gly Ser Thr Ile Glu Ala Tyr Leu Thr

165 170 175

Gln Asp Thr Leu Thr Leu Ala Ser Asp Val Ile Pro Asn Tyr Thr Phe

180 185 190

Gly Cys Ile Asn Lys Ala Ser Gly Thr Ser Leu Pro Ala Gln Gly Leu

195 200 205

Met Gly Leu Gly Arg Gly Pro Leu Ser Leu Ile Ser Gln Ser Gln Asn

210 215 220

Leu Tyr Gln Ser Thr Phe Ser Tyr Cys Leu Pro Asn Ser Lys Ser Ser

225 230 235 240

Asn Phe Ser Gly Ser Leu Arg Leu Gly Pro Lys Asn Gln Pro Ile Arg

245 250 255

Ile Lys Thr Thr Pro Leu Leu Lys Asn Pro Arg Arg Ser Ser Leu Tyr

260 265 270

Tyr Val Asn Leu Val Gly Ile Arg Val Gly Asn Lys Ile Val Asp Ile

275 280 285

Pro Thr Ser Ala Leu Ala Phe Asp Pro Ala Thr Gly Ala Gly Thr Ile

290 295 300

Phe Asp Ser Gly Thr Val Tyr Thr Arg Leu Val Glu Pro Ala Tyr Val

305 310 315 320

Ala Val Arg Asn Glu Phe Arg Arg Arg Val Lys Asn Ala Asn Ala Thr

325 330 335

Ser Leu Gly Gly Phe Asp Thr Cys Tyr Ser Gly Ser Val Val Phe Pro

340 345 350

Ser Val Thr Phe Met Phe Ala Gly Met Asn Val Thr Leu Pro Pro Asp

355 360 365

Asn Leu Leu Ile His Ser Ser Ala Gly Asn Leu Ser Cys Leu Ala Met

370 375 380

Ala Ala Ala Pro Val Asn Val Asn Ser Val Leu Asn Val Ile Ala Ser

385 390 395 400

Met Gln Gln Gln Asn His Arg Val Leu Ile Asp Val Pro Asn Ser Arg

405 410 415

Leu Gly Ile Ser Arg Glu Thr Cys Thr

420 425

<210>3

<211>2548

<212>DNA

<213>Arabidopsis thaliana

<400>3

gcaatcagga aaggatgacg agacaaaaga tagagaagca aaagtaagct gataaggttt 60

gatacagtag aaaatactat ctcttaactt cttcttcttc ttcttcttct tctcctatct 120

ttgaaaatgg cgatgactcc ggtcgcgtca tcatctccag tttcaacctg cagactcttt 180

cgctgcaatc tcctccctga tctcttacct aagcctctgt ttctctccct ccccaaacga 240

aacagaattg cctcgtgccg cttcactgta cgtgcctccg cgaatgctac cgtcgaatcc 300

cctaacggtg tccctgcctc cacatcagat acggatacgg agacggatac cacctcctat 360

ggccgacagt ttttcccttt ggccgcagtt gttggccagg aaggcataaa aactgctctt 420

ttacttggcg cggttgatcg tgaaatcgga gggattgcca tttcaggtcg tagaggcact 480

gcaaaaacag tcatggcgcg agggcttcat gaaatcctcc ctcctattga agttgttgta 540

ggctcaatat caaatgctga cccagcttgt ccagatgagt gggaagatga cttagatgag 600

cgcatagagt acaatgctga caataccatt aagactgaga ttgtcaaatc tcctttcatt 660

cagattccac taggagttac agaagacaga ctcattgggt ctgttgatgt tgaggagtct 720

gtgaaaaggg ggacaactgt tttccaacct ggtcttttgg ctgaagccca tagaggagtg 780

ttgtatgttg atgaaataaa tctcttagat gagggaatta gtaatttgct tctcaatgta 840

ttgacggatg gtgttaatat agttgaaaga gaaggaatca gctttaggca cccgtgcaaa 900

ccacttttaa ttgcaaccta taaccctgaa gaaggtgctg ttcgagagca cttgctagac 960

cgtgttgcca ttaatttaag tgcagaccta cctatgagtt ttgaagatcg tgtcgcagca 1020

gttggaattg ccacacagtt tcaggaacgc tgtaatgagg tttttagaat ggtaaatgaa 1080

gagacagaaa cagcaaagac gcagattata ttggctagag aatatttgaa agatgtcaag 1140

ataagtagag agcaattgaa gtatctggtt ttggaagctg tccgaggtgg tgtccaggga 1200

caccgcgccg aattgtatgc agctcgtgtg gcgaagtgtt tagctgcaat tgaaggacga 1260

gaaaaagtca caatcgatga cctcagaaag gccgttgagc tggtcattct tcctcgttca 1320

tcactagatg agactccacc tgaacaacaa aaccaaccac cacctcctcc acctcctcca 1380

caaaatagcg aatctggaga agaagaaaat gaagaagaac aagaagaaga agaagaggat 1440

gaaagcaatg aagaaaatga aaatgagcag caacaggacc aaatacctga agagtttata 1500

tttgacgctg agggcggtct ggtggatgag aaactcctct tctttgctca acaagcccag 1560

aaacgtcggg ggaaagctgg cagggcgaag aatgtcatat tctcagaaga tagaggacgc 1620

tacataaagc caatgcttcc aaagggtcca gtaaaaagat tagctgtgga tgcaaccctt 1680

agagcagctg caccatacca gaaattgcgc agagagaagg atatctcagg aactaggaaa 1740

gtctttgttg agaagacaga tatgagggcc aaaagaatgg caaggaaagc tggagccctg 1800

gttatctttg tggttgatgc aagtggcagt atggcattga atcgtatgca aaacgccaaa 1860

ggtgctgcac tcaaactact ggcagagagc tatactagca gggatcaggt ttcgattatt 1920

cctttccgag gggatgctgc ggaagtgctc ttgccccctt ctagatcaat agcaatggca 1980

aggaatcgtc ttgagagact tccttgtggt ggtggttctc ctcttgccca tggtttaaca 2040

acggctgtaa gagtaggact taacgcagag aagagtggtg atgtcgggcg cataatgatt 2100

gttgcgataa ccgatggtcg agccaacatt acactgaaaa gatcaactga tccggagtct 2160

attgccccag atgctcctag acccacgtcc aaagaattga aggatgagat tctggaagtt 2220

gctgggaaga tatacaaggc agggatgtct cttctagtga ttgataccga gaacaagttt 2280

gtttcaactg gttttgcaaa ggagatcgca agagttgctc aaggaaaata ttattacttg 2340

ccaaatgctt cggatgctgt aatctcggcc accactaggg atgcactatc tgatctgaag 2400

aattcttgac ctcattttgt ctgcaatacc ttgattctgg atcaaagtgt atattctaat 2460

tgtgcaacac atcagtcact aaacatgaaa ttataagaaa tgaaattttg ttttaacata 2520

ctcttaaaat gaatttccga ttttattc 2548

<210>4

<211>760

<212>PRT

<213>Arabidopsis thaliana

<400>4

Met Ala Met Thr Pro Val Ala Ser Ser Ser Pro Val Ser Thr Cys Arg

1 5 10 15

Leu Phe Arg Cys Asn Leu Leu Pro Asp Leu Leu Pro Lys Pro Leu Phe

20 25 30

Leu Ser Leu Pro Lys Arg Asn Arg Ile Ala Ser Cys Arg Phe Thr Val

35 40 45

Arg Ala Ser Ala Asn Ala Thr Val Glu Ser Pro Asn Gly Val Pro Ala

50 55 60

Ser Thr Ser Asp Thr Asp Thr Glu Thr Asp Thr Thr Ser Tyr Gly Arg

6 70 75 80

Gln Phe Phe Pro Leu Ala Ala Val Val Gly Gln Glu Gly Ile Lys Thr

85 90 95

Ala Leu Leu Leu Gly Ala Val Asp Arg Glu Ile Gly Gly Ile Ala Ile

100 105 110

Ser Gly Arg Arg Gly Thr Ala Lys Thr Val Met Ala Arg Gly Leu His

115 120 125

Glu Ile Leu Pro Pro Ile Glu Val Val Val Gly Ser Ile Ser Asn Ala

130 135 140

Asp Pro Ala Cys Pro Asp Glu Trp Glu Asp Asp Leu Asp Glu Arg Ile

145 150 155 160

Glu Tyr Asn Ala Asp Asn Thr Ile Lys Thr Glu Ile Val Lys Ser Pro

165 170 175

Phe Ile Gln Ile Pro Leu Gly Val Thr Glu Asp Arg Leu Ile Gly Ser

180 185 190

Val Asp Val Glu Glu Ser Val Lys Arg Gly Thr Thr Val Phe Gln Pro

195 200 205

Gly Leu Leu Ala Glu Ala His Arg Gly Val Leu Tyr Val Asp Glu Ile

210 215 220

Asn Leu Leu Asp Glu Gly Ile Ser Asn Leu Leu Leu Asn Val Leu Thr

225 230 235 240

Asp Gly Val Asn Ile Val Glu Arg Glu Gly Ile Ser Phe Arg His Pro

245 250 255

Cys Lys Pro Leu Leu Ile Ala Thr Tyr Asn Pro Glu Glu Gly Ala Val

260 265 270

Arg Glu His Leu Leu Asp Arg Val Ala Ile Asn Leu Ser Ala Asp Leu

275 280 285

Pro Met Ser Phe Glu Asp Arg Val Ala Ala Val Gly Ile Ala Thr Gln

290 295 300

Phe Gln Glu Arg Cys Asn Glu Val Phe Arg Met Val Asn Glu Glu Thr

305 310 315 320

Glu Thr Ala Lys Thr Gln Ile Ile Leu Ala Arg Glu Tyr Leu Lys Asp

325 330 335

Val Lys Ile Ser Arg Glu Gln Leu Lys Tyr Leu Val Leu Glu Ala Val

340 345 350

Arg Gly Gly Val Gln Gly His Arg Ala Glu Leu Tyr Ala Ala Arg Val

355 360 365

Ala Lys Cys Leu Ala Ala Ile Glu Gly Arg Glu Lys Val Thr Ile Asp

370 375 380

Asp Leu Arg Lys Ala Val Glu Leu Val Ile Leu Pro Arg Ser Ser Leu

385 390 395 400

Asp Glu Thr Pro Pro Glu Gln Gln Asn Gln Pro Pro Pro Pro Pro Pro

405 410 415

Pro Pro Gln Asn Ser Glu Ser Gly Glu Glu Glu Asn Glu Glu Glu Gln

420 425 430

Glu Glu Glu Glu Glu Asp Glu Ser Asn Glu Glu Asn Glu Asn Glu Gln

435 440 445

Gln Gln Asp Gln Ile Pro Glu Glu Phe Ile Phe Asp Ala Glu Gly Gly

450 455 460

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

465 470 475 480

Arg Gly Lys Ala Gly Arg Ala Lys Asn Val Ile Phe Ser Glu Asp Arg

485 490 495

Gly Arg Tyr Ile Lys Pro Met Leu Pro Lys Gly Pro Val Lys Arg Leu

500 505 510

Ala Val Asp Ala Thr Leu Arg Ala Ala Ala Pro Tyr Gln Lys Leu Arg

515 520 525

Arg Glu Lys Asp Ile Ser Gly Thr Arg Lys Val Phe Val Glu Lys Thr

530 535 540

Asp Met Arg Ala Lys Arg Met Ala Arg Lys Ala Gly Ala Leu Val Ile

545 550 555 560

Phe Val Val Asp Ala Ser Gly Ser Met Ala Leu Asn Arg Met Gln Asn

565 570 575

Ala Lys Gly Ala Ala Leu Lys Leu Leu Ala Glu Ser Tyr Thr Ser Arg

580 585 590

Asp Gln Val Ser Ile Ile Pro Phe Arg Gly Asp Ala Ala Glu Val Leu

595 600 605

Leu Pro Pro Ser Arg Ser Ile Ala Met Ala Arg Asn Arg Leu Glu Arg

6l0 615 620

Leu Pro Cys Gly Gly Gly Ser Pro Leu Ala His Gly Leu Thr Thr Ala

625 630 635 640

Val Arg Val Gly Leu Asn Ala Glu Lys Ser Gly Asp Val Gly Arg Ile

645 650 655

Met Ile Val Ala Ile Thr Asp Gly Arg Ala Asn Ile Thr Leu Lys Arg

660 665 670

Ser Thr Asp Pro Glu Ser Ile Ala Pro Asp Ala Pro Arg Pro Thr Ser

675 680 685

Lys Glu Leu Lys Asp Glu Ile Leu Glu Val Ala Gly Lys Ile Tyr Lys

690 695 700

Ala Gly Met Ser Leu Leu Val Ile Asp Thr Glu Asn Lys Phe Val Ser

705 710 715 720

Thr Gly Phe Ala Lys Glu Ile Ala Arg Val Ala Gln Gly Lys Tyr Tyr

725 730 735

Tyr Leu Pro Asn Ala Ser Asp Ala Val Ile Ser Ala Thr Thr Arg Asp

740 745 750

Ala Leu Ser Asp Leu Lys Asn Ser

755 760

<210>5

<211>1592

<212>DNA

<213>Arabidopsis thaliana

<400>5

ataacccatc caacccaaaa gcatacaata caatgtctac tcttgtttta ttcctccaac 60

ttttttccat cctaccatta gcacttgggt tgaaccaccc aaattgtgac ctgaccaaga 120

ctcaagacca aggctctacc ctaagaatct tccacataga cagcccttgc tcccccttca 180

aatcatcatc cccactctca tgggaagcgc gtgtgctcca gacgctggct caagaccagg 240

cccgtctcca gtacctgagc agcctcgtcg ccgggagatc tgtagtcccc attgcctcag 300

ggcgtcaaat gttgcaaagc actacataca ttgtcaaggc tttgatcggt actccggctc 360

agccgctgct cctagccatg gacacgagca gtgacgtggc ttggattcct tgctccggct 420

gcgttggctg tccttctaac acggcctttt cccctgctaa gtctacatcc ttcaagaacg 480

ttagctgcag cgctcctcag tgtaagcagg taccaaaccc cacgtgtgga gcacgcgcgt 540

gctctttcaa cctcacctat ggaagctcct ccattgcggc taacctctct caagacacga 600

tacgtcttgc cgccgatccg atcaaagcct ttacattcgg atgcgtcaac aaagtcgccg 660

gtggaggaac catcccacca cctcaaggct tgttgggctt gggacgaggc ccactttcac 720

tcatgtcaca ggctcagtct atttacaagt ccactttctc ttactgtttg ccaagtttca 780

ggtctctgac cttctctggt tctctcagac tcggccctac ctcccagccc cagcgcgtga 840

agtacactca acttctcaga aaccctagaa ggtcttctct gtactacgtc aacctcgttg 900

cgatccgcgt tggtcggaaa gtcgtcgatt taccgccggc agctattgct tttaatcctt 960

caaccggcgc tggaaccatc ttcgactccg gaactgtgta cacgcggctg gctaaaccgg 1020

tttacgaggc agtgaggaac gagttcagga agcgcgtgaa gccaaccaca gccgtggtga 1080

cgtcactcgg gggtttcgac acgtgctact cagggcaagt caaggtgccg acgataacat 1140

tcatgttcaa gggagtgaac atgacgatgc cggctgataa cctgatgtta cacagtaccg 1200

ccggaagcac gtcgtgcctc gccatggctg cagcgccgga aaacgtaaac tctgtcgtca 1260

atgtgatcgc aagcatgcag cagcagaacc accgtgtcct catcgacgtc cccaatggac 1320

ggctcggttt ggcacgtgaa cgatgctctt agaaagatga atgagacaat aagagcctac 1380

tcatatttat gcagagactt gtttgtttgt gttatttgtg tgtttcattt ttgtcttgtc 1440

ttgcgtcgtt tgcttcacca gtcttagagg gcggaggtgt gtagcatttg tgctatctag 1500

taatttatgt ttttaattgt tcttcaatta tctagagtgt tggatctaag ctaatcaaat 1560

aatgaaacta tattatattt ccttacggta ta 1592

<210>6

<211>1350

<212>DNA

<213>Oryza sativa

<400>6

atggcgctga gaatgagcat cgcggcgatg tcggtgttgg cggtggcggc ggtgctcgtt 60

gtggccggca cggcggcggc ggcggcggcg tcgtgcccgg cgacgccgcc ggacgcgggg 120

gcgacgctgc aggtgtcgca cgcgttcggg ccgtgctcgc cgctgggggc ggagtcggcg 180

gcgccgtcgt gggcggggtt cctggcggac caggcggcgc gcgacgcgtc gaggctgctg 240

tacctcgact cgctggcggt gaaggggcgc gcgtacgcgc cgatcgcgtc ggggaggcag 300

ctgctgcaga cgccgacgta cgtggtgcgc gcccgcctcg gcacgcccgc gcagcagctg 360

ctcctcgccg tcgacaccag caacgacgcc gcctggatcc cctgctccgg ctgcgcgggc 420

tgccccacct cgtcgccgtt caacccggcc gcctccgcct cctaccgccc ggtgccgtgc 480

ggctcgccgc agtgcgtgct ggcgcccaac ccgtcgtgct cccccaacgc caagtcctgc 540

ggcttcagcc tctcctacgc cgactcctcg ctccaggccg cgctgtcgca ggacaccctc 600

gccgtcgccg gcgacgtcgt gaaggcctac accttcggct gcttgcagag ggccaccggc 660

acggcggcgc cgccgcaggg cctcctcggg ctcggccgtg gcccgctctc cttcctgtct 720

cagaccaagg acatgtacgg ggccacgttc tcctactgcc tcccgagctt caagtcgctc 780

aacttctccg gcacgctccg gctcggccgg aacgggcagc cgcggcggat caagacgacg 840

ccgctgctcg ccaacccgca ccgctcgtcg ctctactacg tcaacatgac cggcatccgc 900

gtggggaaaa aggtggtgtc gatcccggcg tccgcgctgg cgttcgaccc ggcgacgggc 960

gccggcacgg tgctcgactc ggggacgatg ttcacgcgcc tggtggcgcc ggtgtacctg 1020

gcgctccgcg acgaggtccg ccgccgcgtc ggcgccggcg ccgccgccgt gtcctccctc 1080

ggcgggttcg acacgtgcta caacaccacg gtggcgtggc cgccggtgac gctgctgttc 1140

gacggcatgc aggtgacgct gccggaggag aacgtggtga tccacaccac gtacggcacc 1200

accagctgcc tggccatggc ggcggcgccc gacggcgtca acacggtgct caacgtcatc 1260

gccagcatgc agcagcagaa ccaccgcgtc ctcttcgacg tgcccaacgg ccgcgtcggc 1320

ttcgcccgcg agagctgcac cgccgcctag 1350

<210>7

<211>1338

<212>DNA

<213>Oryza sativa

<400>7

atgtcactcc ttgcattacc actcctagcc ttcttgtcca tattcttgac tcctacaacg 60

gcagtgagca gctcaacgct gcagctggca cgttctcact cggtgacgcc caatgccggg 120

gcgccgctca gcgcgtgggc cgcgtccgta gccgcccagt cggcggcgga cacggcgcgc 180

atcgtcagca tgctcacgtc gggggcgggg cctctgacga ccagagccaa gcccaaaccc 240

aagaacaggg caaacccacc cgtgccgatc gcgccggggc gtcagatcct cagcatcccg 300

aactacatcg cccgcgcggg cctcggcacg ccggcgcaga cgctcctcgt ggccatcgac 360

cccagcaacg acgcggcgtg ggtgccatgc agcgcctgcg ccggctgcgc cgcctcgtcg 420

ccgtccttct ccccgacgca gtcgtccacg taccgcaccg tgccctgcgg ctcgccgcag 480

tgcgcgcagg tgcccagccc gtcctgcccc gcgggcgtcg gctcctcctg cgggttcaac 540

ctcacctacg cggcgtccac gttccaggcc gtgctcgggc aggactcgct ggccctcgag 600

aacaacgtcg tcgtgtcgta caccttcggg tgcctccgtg tcgtcagcgg caactccgtg 660

ccgccgcagg ggctcatcgg cttcggccgc gggccgctct ccttcttgtc gcagaccaag 720

gacacgtacg gctccgtgtt ctcctactgc ctcccgaact acaggtcgtc taacttctcc 780

ggcacactaa agctcggccc catcgggcaa cccaagagga tcaagacgac gccgttgctc 840

tacaaccctc atcgcccttc actctactac gtcaacatga tcgggatccg cgtcggcagc 900

aaggtcgtgc aagtcccgca atccgcgctc gcgttcaacc cggtcacggg cagcggcacc 960

atcatcgacg ccgggacaat gttcacgcgg cttgccgccc cggtgtacgc cgccgtgcgc 1020

gacgcgttcc ggggcagggt gcgcacgccg gtggcgccgc cgctgggcgg gttcgacacg 1080

tgctacaacg tgaccgtctc ggtgcctacc gtcacgttca tgttcgccgg ggcggtcgcc 1140

gtgacgctgc cggaggagaa cgtgatgatc cacagctcgt cgggcggcgt cgcgtgcctg 1200

gcgatggccg cggggccctc ggacggcgtg aacgcggcgc tcaatgtgct ggccagcatg 1260

cagcagcaga accaacgcgt gctgttcgac gtcgccaacg gccgcgtcgg cttctcccgt 1320

gagctttgca cggcctga 1338

<210>8

<211>2495

<212>DNA

<213>Nicotiana tabacum

<400>8

catctaaaat cctaaatcaa aaacttcgat gctataaaaa tggggttttg ttcaacttca 60

accctcccac aaacatcact atccaattct caatcttcaa cattcttcac atacttaaaa 120

ccatgcccaa ttctatcctc cacatattta aggccgaaac ggctaaaatt tcgcctcaga 180

ataagtgcca ctgcaactat tgattcacct aatggcgctg ttgcagtagt ggaacctgaa 240

aaacaacctg agaaaatttc ctttggtaga cagtattttc ctctagctgc tgttattgga 300

caggatgcta ttaaaactgc tcttttactt ggggccattg accgtgagat aggaggaatt 360

gcaatatgtg ggaagcgtgg aacagcgaaa acgttaatgg cacgtggatt gcatgctatt 420

ctgccaccaa ttgaagtagt tgttggctca atggcaaatg ctgatccgaa ctgtcccgat 480

gagtgggaag acgggctagc tgacagagca gaatatgggt ctgatggtaa tatcaagacc 540

cagatagtta aatccccatt tgttcagatt ccccttggtg tcacagaaga tagattgatt 600

ggctctgttg atgtcgagga gtccgtgaaa tctggaacca ctgtctttca accaggcctc 660

ctcgcagaag ctcatcgagg agttctatat gttgatgaga ttaatctatt agatgaaggt 720

ataagtaacc tacttctgaa tgtattgact gagggagtca atattgtaga aagagaggga 780

atcagctttc gacatccatg caaaccacta ctaattgcta cctataaccc tgaagagggt 840

gcggttcgtg agcatctgct agaccgtatt gcgattaatt taagtgcaga tcttccaatg 900

agttttgacg atcgtgttgc agctgttgac atagcaacac gttttcagga gtgtagcaat 960

gaggttttta aaatggtgga tgaagaaaca gacagtgcaa aaacccagat aatattggca 1020

agggagtatt taaaggatgt cacaatcagt agagatcaac taaaatactt ggtcatggaa 1080

gcaattcgtg gtggctgcca ggggcaccga gctgaacttt atgctgctcg tgtagccaaa 1140

tgcttagctg ccatcgatgg acgtgaaaaa gttggtgttg atgagctgaa aaaagctgta 1200

gagcttgtca tcctcccacg ttcaactata gttgaaaacc caccagacca gcaaaaccag 1260

cagccacctc ctccccctcc ccctccccaa aatcaagatt cttcagaaga gcagaatgaa 1320

gaagaagaaa aagaagaaga agatcaagag gatgagaaag atagagaaaa tgaacagcaa 1380

cagccacaag tccctgatga gtttattttt gatgcggaag gtggtttagt ggatgaaaaa 1440

cttctcttct ttgcacaaca agcacaaaga cgcaaaggaa aagctggacg agcaaagaag 1500

gtcatctttt ccgaagatag aggtcgatat ataaagccaa tgcttccaaa gggtccagtg 1560

aagagattgg cagttgatgc aactctaaga gcagcggcac catatcagaa gttacgaaga 1620

gcaaaggaca tccaaaaaac tcgcaaggtt tatgtagaga aaactgacat gagagccaaa 1680

agaatggcac gcaaagccgg agctctggtg atattcgtag ttgacgctag tgggagtatg 1740

gcactgaata gaatgcagaa tgccaaagga gcagcactta aactacttgc agagagttat 1800

acaagcagag atcaggtctg tatcattccc ttccgcggag atgctgctga agttttgttg 1860

ccaccttcta ggtcaatatc gatggcaaga aatcgtcttg agagacttcc ctgtggaggg 1920

ggttctcccc ttgctcatgg gcttacgacg gcagttagag ttggaatgaa tgcagaaaag 1980

agtggtgatg ttggacgtat catgattgtt gcaattactg atggtagagc taacatctct 2040

cttaaaagat ccacagaccc tgaagctgaa gcttctgatg cacccagacc ttcttcccaa 2100

gagctgaagg atgagattct cgaggtggct ggtaaaatat acaaaacagg aatgtctctc 2160

ctcgtcatag atacagaaaa taagtttgtt tctactggtt ttgcgaaaga aatcgcgaga 2220

gtagctcaag ggaagtacta ttatttacca aatgcttcag atgctgtgat atctgcagca 2280

acaaaggatg cattatctgc attaaaggaa tcttgaccta aactcgatcg aattaattgt 2340

aaatgttgtt ttgagtatag attattggga ggatataaga gcttgcttga taattcttat 2400

cttttgttgt actaattgaa cttatttctc aattatgcaa tcagggtaat gaagattctt 2460

ttcatttcaa aaaaaaaaaa aaaaaaggaa ttcga 2495

<210>9

<211>2607

<212>DNA

<213>Pisum sativum

<400>9

gtggcaccgt gatcgttggt tctaaattca atcaaatggg tttcagtttg acacacacac 60

ctcacaccac tgcttccccc aatcttcaac tccgatttca ctctcttctt cctccttcat 120

tcacatcaca accgtttctc tctttgcatt ccacatttcc accaaaacgc accgttccaa 180

aacttcgcgc tcaatccgaa aatggagctg ttctgcaagc ttctgaggag aagctcgatg 240

cttccaatta cggaagacag tacttccctc tcgctgctgt tataggccaa gatgctatta 300

aaactgctct tttacttggg gctactgacc ctaggattgg agggattgct atatcaggaa 360

ggcggggaac tgctaaaaca ataatggcgc gtggaatgca tgcaattctt ccgcctattg 420

aagttgtaca aggttccatt gccaatgcag atccctcgtg ccctgaagag tgggaagatg 480

gtctttacaa acgcgtggaa tatgattctg atggaaatgt taaaactcat atcatcaagt 540

ctccttttgt tcagattcct cttggagtca cagaggacag actcattgga tcagttgatg 600

ttgaggagtc tgtgaagaca ggcacaactg ttttccaacc aggcctactc gctgaagctc 660

atagaggtgt tttatatgtt gatgaaatta atcttttgga cgagggtatc agtaatttgc 720

tccttaatgt actgactgaa ggagtaaata ttgttgaaag agagggaatc agctttaggc 780

acccatgcag gccccttctg attgctacct ataaccctga cgaaggttct gttcgtgaac 840

atctgctaga ccgcattgca attaatttga gtgcagatct tccaatgagt tttgaaaacc 900

gtgttgaagc tgttggaatt gcaacagaat ttcaggataa ctgtggccaa gtatttaaaa 960

tggttgatga ggatacagac aatgcaaaga cacagatcat cttggctaga gagtatctca 1020

aggatgttac tattagcaaa gaacaattaa aatacctggt tatcgaggct ttacgaggtg 1080

gtgtccaggg acacagagct gagctgtatg ctgctcgtgt tgctaagtgc ttagctgctc 1140

tggagggacg tgaaaaggtt tatgtggatg accttaaaaa agccgtagaa ttggtcattc 1200

ttccccggtc aatcattacc gatactccac ctgagcaaca aaatcaacct cctccgccac 1260

caccgcctcc acaaaaccaa gaatctaatg aagaacagaa tgaagaggaa gaacaagaag 1320

aagaggaaga ggatgacaat gatgaagaga atgaacaaca gcaagaccaa ttacctgaag 1380

aatttatctt tgatgctgaa gggggtttgg tggatgaaaa acttctcttc tttgcccaac 1440

aagcacagag acgccgtggg aaggctggaa gggcaaaaaa tgtcatattt tcagaggaca 1500

gaggccgata catcaagcca atgcttccaa agggtcctgt aaagagatta gcagttgatg 1560

caacccttag agctgctgca ccttatcaaa agttgcgaag ggaaaaagac accgaaaacc 1620

gtagaaaagt atatgttgaa aaaactgaca tgagggcaaa gagaatggcg cgtaaagcag 1680

gagcattggt catatttgtg gttgatgcta gtggaagcat ggcattgaac agaatgcaga 1740

atgcaaaagg tgcggcactt aagcttctgg cagaaagtta tacaagcagg gatcaggtat 1800

ctataattcc attccgtgga gattctgcag aagttctcct accaccttct agatcaattg 1860

caatggcaag gaaacgtctt gaaagactgc catgtggtgg agggtcgccc cttgcacatg 1920

gtcttaccac agctgttagg gttggattaa atgcagagaa aagtggtgat gttggacgta 1980

taatgattgt tgcaatcact gatggtcgtg ccaacatatc attgaaaagg tcaaatgacc 2040

ctgaagctgc tgccgctagt gatgccccta aacctacatc gcaagaatta aaggatgaaa 2100

ttattgaggt cgctgcgaag atatataaaa caggaatgtc tctccttgtc atcgacactg 2160

aaaacaagtt tgtgtcaact ggtttcgcta aagagattgc tagagttgct caagggaagt 2220

attattattt gccaaatgct tctgacgcag ttgtctcgtt ggcaacaagg gaagctttag 2280

cagctctgaa gagttcatga aactgaatga gaagcaacca atcttgacac ccctcccaat 2340

ttttgttaca taatgttatt gtaaattgcg caactataaa tgtctggtgg gaagaaagca 2400

tactcttata gcaagttcca ttatttccta ttttcgatga gattttgttt ttagtttctg 2460

tgtggattgt tgtaagtata aatttctctt actagtagac tcttcgaagt cagtcactaa 2520

ctgttaggag ggtggactcg gctggacaat taattttcac acaactttat aaataaatta 2580

gaatcttttg taaaagaaaa aaaaaaa 2607

<210>10

<211>2571

<212>DNA

<213>Oryza sativa

<400>10

gaccgcgctc tctccctccc ctcccatggc gatggccacc accgcgctct ccgcctccct 60

cccgcgccta ctcccgcctc gccgccgccg cttcccgacg ccctcctcct cctccccctc 120

cgccgcatcc acctccacct cccgcgtcgt ccgcctgcgg gccgccgcgg cctcggcgcc 180

atccgaggtc ctcgactcca ccaacggggc catcccctcg gggaagggcg gcggcgggca 240

gcagtacggg agggagtatt tccccttggc cgctgtcgtc gggcaggatg caattaaaac 300

tgctctgctg cttggggcaa ttgaccgtga aattggaggc atcgccatct cagggaagcg 360

tgggacagca aagacagtga tggctcgtgg cttgcacgct atgcttccac ctattgaagt 420

ggttgtaggc tcgattgcaa atgctgaccc taactaccca gaagaatggg aggagggttt 480

ggctaaccaa gttcaatatg atgctgatgg taacttgaag accgagatta tcaaaacacc 540

ttttgtgcag atcccgcttg gtatcactga ggataggtta atcggatcag tcgatgttga 600

agcatctgtg aaatcaggaa ctactgtgtt tcaacctggc cttcttgctg aagctcacag 660

aggcgttctt tatgttgatg agataaatct attggatgag ggcgtaagca atctacttct 720

gaatgtcttg actgagggag tcaatattgt ggaaagagag ggcattagct ttcgtcatcc 780

atgcaaacca cttctaattg ctacttacaa cccagaggaa ggatctgtac gtgaacactt 840

acttgatcgt attgcaatta atttaagcgc tgatctgcca atgagttttg atgatcgtgt 900

ggcagctgtg gatattgcaa cacaatttca agagtccagc aaagaggttt ttaaaatggt 960

ggaagaagaa actgaggttg caaaaaccca gataattttg gcaagagaat atctgaaaga 1020

tgttgcaatc agcacagagc agctcaaata tcttgtcatg gaagctatac gcggtggctg 1080

tcaggggcac cgggctgagc tgtatgctgc tcgagtggca aaatgtcttg ctgctatgga 1140

agggcgtgaa aaagtatatg tggatgacct taagaaagct gtagagctag ttattctacc 1200

tcgatcaatc ctatctgata acccacagga gcagcaagac caacaacctc ctccaccccc 1260

accgccaccc cctccacaag atcaagattc tcaagaagat caagatgaag acgaggaaga 1320

ggaccaagag gacgatgatg aagaaaatga acagcaggac cagcagatac ctgaggagtt 1380

catttttgat gctgaaggtg gtatagtaga tgagaagctc cttttctttg ctcagcaagc 1440

tcaaagacgg cgagggaaag ctggacgagc aaagaatctc atattctcat ctgatagggg 1500

acgatacata ggttctatgc ttcccaaggg tccaataagg aggttagctg ttgatgccac 1560

acttcgagca gctgcaccat accagaaact gaggagagag aaagatcgtg acaagacaag 1620

aaaggttttt gttgaaaaaa ctgacatgag agccaaaaga atggctcgaa aagcaggcgc 1680

actggtcata tttgttgtgg atgctagcgg tagcatggct ctgaatcgca tgcagaatgc 1740

gaaaggtgca gcattaaagt tgcttgcaga aagctacaca agcagagatc aggtttcaat 1800

cattccattt cgtggagatt ttgctgaggt tcttcttcca ccttcaagat ccatagcaat 1860

ggcccgcaat cgtcttgaga agttaccatg tggtggcggt tctcctttag ctcacggcct 1920

tagcacagct gtcagagtgg gtttgaatgc tgaaaagagc ggtgatgttg gacgtatcat 1980

gattgttgca atcaccgatg gaagagctaa tgtgtcactg aagaaatcga ctgacccaga 2040

agccacttca gatgctccaa gaccttcttc tcaagaatta aaggatgaga tacttgaggt 2100

ggctggcaaa atatacaagg ctggaatttc acttcttgtt attgataccg agaacaagtt 2160

tgtatccaca ggatttgcca aggaaattgc aagggtcgcc caaggtaaat actattacct 2220

gccgaatgct tcagacgctg ttatttccgc cgccaccaag actgcactct cggacctgaa 2280

gagttcgtga tcctggagag cgttttacct tcagataatg agtggttttt accttttacc 2340

ttgtttggtg cagcagtgtc catgtttcgt gtaactttgg gacgtttcgg ctgtgataac 2400

caattttggc ataggatttt taccgtgaga gttggaattc gggcgtagca ccgtgtaaag 2460

aatcatataa tccctcttct gtctaaataa ttggccatgt aaatatggtg ttattgcgta 2520

cagttctaag taataataac attcataatt tatgtgaaga aagaaattgc c 2571

Claims

1. the transgenic plant 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 HIO polypeptide of aminoacid sequence of SEQ ID NO:2 or 4 or its lineal homologue, with respect to control plant, these transgenic plant have high oil meter type thus.

2. the transgenic plant of claim 1, it is selected from Semen Brassicae campestris, soybean, corn, Sunflower Receptacle, cotton, cocoa, safflower, oil palm, coconut, flax, castor-oil plant and peanut.

3. Accessory Right requires the plant part that 1 plant obtains.

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

5. Accessory Right requires meal, feed or the food that 4 seed is produced.

6. a method that produces oil comprises the transgenic plant of cultivating claim 1, and from described plant refiltered oil.

7. the method for claim 6, recovery of oil wherein is from the seed of described plant.

8. produce the method for high oil meter type plant, described method comprises:

A) plant conversion carrier is imported in the progenitor cell of plant, this plant conversion carrier contains coding or is complementary to the nucleotide sequence that coding comprises the sequence of the HIO polypeptide of aminoacid sequence of SEQ ID NO:2 or 4 or its lineal homologue, and

B) progenitor cell of cultivating this conversion is wherein expressed described polynucleotide sequence to produce transgenic plant, and with respect to control plant, the oleaginousness phenotype of described transgenic plant display change.

9. pass through the plant of the method acquisition of claim 8.

10. the plant of claim 9, it is selected from Semen Brassicae campestris, soybean, corn, Sunflower Receptacle, cotton, cocoa, safflower, oil palm, coconut, flax, castor-oil plant and peanut.

11. the plant of claim 9, plant wherein are selected from the plant that comes from the growth of described group of cell, 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.

Has the method for high oil meter type plant 12. produce, comprise that evaluation has the plant of allelotrope or sudden change in its HIO103.1 or HIO101B nucleotide sequence, and the filial generation that produces the plant of described evaluation, with lack allelotrope or the sudden change that this allelic plant compares in described HIO103.1 or the HIO101B nucleotide sequence and cause high oil meter type, filial generation this equipotential gene of heredity that wherein produces and be high oil meter type.

13. the method for claim 12, it uses candidate gene/QTL method.

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

15. feed, meal, grain, food or seed comprise the polypeptide by the nucleic acid sequence encoding of SEQ ID NO:1 or 3.

16. feed, meal, grain, food or seed comprise the aminoacid sequence that contains SEQ ID NO:2 or 4 or the polypeptide of its lineal homologue.