CN103602691B - Relate to the enzyme of triterpene synthesis - Google Patents

Abstract

The invention discloses the enzyme relating to triterpene synthesis.Particularly, relate to the polynucleotide of separation, the enzyme that described polynucleotide encoding is made up of carboxypeptidase albumen, methyltransgerase and Transglucosylase, described enzyme relates to the biosynthesizing of the triterpene that the beta-amyrin in Plants and Seeds derives.The present invention also relates to the structure of recombinant dna construct, described construct comprises all or part of separation polynucleotide of the present invention, and described polynucleotide may be operably coupled to a few regulating and controlling sequence with sense or antisense direction.

Description

Relate to the enzyme of triterpene synthesis

The divisional application of the Chinese patent application 200780053491.4 " relating to the enzyme of triterpene synthesis " in the application to be the applying date be on June 25th, 2007.

Technical field

The present invention relates to field of plant molecular biology.More particularly, the present invention relates to the polynucleotide of the enzyme related in the triterpene biosynthesizing that beta-amyrin in coded plant and seed derives.The present invention also comprises transgenic plant, and wherein the expression level of polynucleotide of the present invention changes level or the structural modification of the triterpene (comprising saponin(e) causing beta-amyrin derivative.

Background technology

Terpenoid is also referred to as isoprenoid, and they constitute maximum natural product family, and such product is existing more than 22, and 000 kind of individually oriented compound is described.Triterpene or terpenoid (hemiterpene, monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene, Polyprenol etc.) play various different function, as hormone, Photosynthesis Pigment, electron carrier, polysaccharide fraction conditioning agent and membrane structure component in plant.Plant terpene compound is mainly present in resin, latex, wax and oil.

Triterpenoid is relevant to various plants characteristic, comprises animal food preference and the resistance to pathogenic agent and predator.Triterpene major storage is at plant root, and storage form is their glycoside, saponin(e (see people such as PriceK.R, 1987, CRCCrit.Rev.FoodSci.Nutr.26:27-133).Therefore, such as, the mutant of diploid oat, black oat, it lacks main avenacin, avenacin A-1 (so-called saponin(e lack or " Sad " mutant), has proved to lack the disease resistance (people such as PapadopoulouK., 1999, Proc.Natl.Acad.Sci.U.S.A.96:12923-12928).These mutant are easier to the infringement being subject to many different root fungal infections, comprise gaeumannomyces graminis, and it is concerning normally nonpathogenic oat.Genetic analysis shows that disease susceptibility improves and the reduction of avenacin level has cause-effect relationship.In addition, when inoculating gaeumannomyces graminis, the disease symptoms that the sad mutant (being about 15% of wild-type levels) that avenacin level reduces is only limited compared with other mutant lacking avenacin completely, avenacin level and disease resistance connect by further.

The saponin(e in diet may be useful (see the people such as people (2004) J.Med.Food7:67-78 and Vis, E.H. (2005) Nutr.Cancer51:37-44 such as such as Shi, J.A.) to have the data of accumulation to show.Same, prove that soybean dietary saponin(e is to prevention rat hypercholesterolemia and atherosclerosis useful people (1984) such as (, Nutr.Rep.Int.29:1039-1046) Oakenfull.Because saponin(e only has slightly damaged in the process from soybean to soybean isolate, saponin(e level in soybean improves and the saponin(e level caused in isolate improved (people (2002) Phytochem.Anal.13:343-348 such as Berhow, M.A.; HuJ. people (2002) is waited, J.Agric.FoodChem.50:2587-2594).Therefore the saponin(e level improved in soybean will be the effective ways improving saponin(e amount in human diet.In addition, the raising of saponin(e level can be the compound used in drug development and provides source.

Triterpene and sterol synthesize via Isoprenoid pathway.In this approach, bimolecular farnesylpyrophosphate head-head is connected to form squalene, and this is a kind of triterpene.Then squalene is converted into 2,3-epoxy squalene.Multiple oxidosqualene cyclase catalysis 2, the cyclisation of 3-epoxy squalene forms multiple many rings skeleton, comprises one or more cycloartenols, lanosterol, Lupeol, different composite precision oleyl alcohol (isomultiflorenol) (a kind of triterpenoid), beta-amyrin, alpha-amyrin and Arabidopis thaliana alcohol (thalianol) (a kind of triterpenoid).Branching-point should be formed between sterol and triterpenoid saponin biosynthetic pathway by the cyclisation event of oxidosqualene cyclase catalysis.Multiple oxidosqualene cyclase is evolved relevant people (1998) such as (, Eur.J.Biochem.256:238-244) Kushiro, T. and produces multiple three rings, Fourth Ring and five ring structures, and this structure can be modified further.

Triterpenoid saponin(e, via Isoprenoid pathway, forms pentacyclic triterpene based compound by cyclisation 2,3-epoxy squalene, mainly volatile oil (beta-amyrin) or dammarane skeleton synthesis.Triterpenoid main chain stands multiple modification (oxidation, replacement and glycosylation) subsequently, and described modification is mediated by Cytochrome P450 dependent form monooxygenase, glycosyltransferase (GT) and other enzyme.Usually about the enzyme related in saponin(e biosynthesizing and bio-chemical pathway understand few.Although there is considerable commercial benefits in the natural product that this type of is important, produce this important genetic mechanism needed for Secondary Metabolite Production in Plants family and remain unknown to a great extent.This may be that part is due to the complicacy of molecule and lack for the approach intermediate of biochemical research.But, understand first committed step in saponin(e biosynthesizing at present, it is undertaken by oxidosqualene cyclase beta-amyrin synthase (product of Sad1 gene), clone this gene recently and described its feature (people such as HaralampidisK., 2001, Proc.Natl.Acad.Sci.U.S.A.98:13431-13436).Another committed step mediates (by Sad2 genes encoding) by cytochrome P 450 enzymes AsCYP51H10, has been subject to research people such as (, 2006, Proc.Natl.Acad.Sci.U.S.A103:18848-18853) QiX. recently.AsCYP51H10 (SAD2) is required for avenacin synthesis.The accurate biological chemical functional of AsCYP51H10 is unknown.But Sad2 mutant accumulation beta-amyrin, therefore AsCYP51H10 is oxidized beta-amyrin (or derivatives thereof) necessary (Fig. 1) at one or more site (C12, C13, C16, C21 and/or C30).

The enzyme of other class of structure comparison (Fig. 1) prediction except Cytochrome P450 also changes into beta-amyrin required for avenacin A-1.These enzymes comprise glycosyltransferase (GT), acyltransferase and methyltransgerase (MT).Glycosyltransferase belongs to a large enzyme family, the enzyme of this family by sugar unit from activated donor molecular transfer to wide spectrum potential receptor molecule.Potential acceptor comprises protein, lipid, polysaccharide and small molecules, and they can relate to various different cell processes as Cell wall synthesis and signal transduction (people such as CoutinhoPM, 2003, J.Mol.Biol., 328:307-317).In 77 GT families of representative leap all zones, GT family 1 is wherein maximum (CoutinhoPM and HenrissatB, family 1 by the reversion catalyst mechanism that via sugar shift operated GT forms 1999:Carbohydrateactiveenzymeswebsitehttp: //afmb.cnrs-mrs.ff/CAZY/), usually lower molecular weight acceptor molecule (VogtT and JonesP is acted on, 2000, TrendsPlantSci.5: 380-386; LimE-K and BowlesDJ, 2004, EMBOJ23:2915-2922).By analogy micromolecular with other glycosylation, the collateralization sugar chain of prediction avenacin A-1 synthesizes by being added in aglycone component by sugared unit continuously, most probablely passes through that three different glycosyltransferases (GT) are active to be synthesized.Glycosylated first step relates to and is added to by L-arabinose on the C3 hydroxyl of aglycone, transferase mediated by cytosine arabinoside.After this two D-Glucose molecules are added, a C2 site at pectinose, another is in C4 site, mediates (the people such as TownsendB by (or may a two) Transglucosylase, 2006, PhytochemistryRevs.5:109-114).

Acyl group is the custom composition of the natural product of plant origin, and can change their chemistry and physical property.Therefore may affect the biological effect of natural product in ecological interaction, and affect other critical process as ubcellular exchange and sequestering action (such as taking in or resident mark by acting on vacuole).Have been found that the Plant acyl transferring enzyme that a class is new recently.These enzymes-serine carboxypeptidase sample acyltransferase-there is homology with peptase, but shortage peptidase activity, on the contrary can acidylate natural product (MilkowskiC & StrackD, (2003), Phytochemistry65:517; The people such as FraserCM (2005) PlantPhysiology138:1136).Although other Plant acyl transferring enzyme generally uses coenzyme thioesters as acry radical donor, these SCPL use acyl glucose donor.The member of feature description the best of SCPL acyltransferase family is tomato enzyme GAC-Lp, the formation of this enzyme catalysis glucose polyester, described glucose polyester contributes to the insect-resistant (LiAX & SteffensJC, 2000, PNAS97:6902) in wild-type tomato; Arabidopis thaliana enzyme SNG1, it is synthesis of phenyl phenoxy propionic acid mustard seed acyl malate (a kind of UV protective material) necessary (people such as LandryLG, 1995, PlantPhysiology109:1159; The people such as LehfeldtC, 2000, PlantCell12:1295); Second Arabidopis thaliana enzyme SNG2, it relates to mustard seed phatidylcholine people such as (, 2001, PlantJ28:83) ShirleyAM in synthesis seed; With swede type rape acyltransferase BnSCT, the formation of its catalysis mustard seed acid esters, this ester and bitter taste, convergency and seed oil extract problem relevant (people such as MilkowskiC, 2004, PlantJ.38:80; The people such as BaumertA, 2005, Phytochemistry66:1334).Although the enzyme related in these are modified and gene still fail to understand its feature, how the natural product of other important plant origins many is prepared by glucose-ester-dependent acyltransferase reaction by biochemical analysis is known.Example is included in the antioxidant chlorogenic acid in sweet potato (Ipomoeabatatas), the cyanin in wild Radix Dauci Sativae (Daucuscarota), Nutgalls Weibull in Oak Tree (Quercusrobur) and at rape (MilkowskiC & StrackD (2003) Phytochemistry65:517; The people such as FraserCM (2005) PlantPhysiology138:1136) in mustard seed acyl-and the glucosinolate of benzoyl-ester.There is the avenacin [14] that four kinds of different structures are relevant.Avenacin A-1 (being present in the main avenacin of oat root) and B-1 N-methyl anthranilic acid carry out acidylate; and avenacin A-2 and B-2 phenylformic acid carry out acidylate (HostettmannK and MarstonA; 1995; Saponins; CambridgeUniversityPress; Cambridge, UK).

The O-that SAMe-dependent methyltransgerase relates to many phytochemicals production methylates (people such as FrickS., 2001, Phytochemistry56:1-4).Play an important role in the compou nd synthesis of these enzymes needed for xylogen precursor and other plant defense (people such as GangDR, 2002, PlantCell14:505-519.

Summary of the invention

The present invention relates to the separation polynucleotide of the enzyme related in the synthesis of coding triterpene.Specifically, the present invention relates to the new serine carboxypeptidase sample acyltransferase of coding, new methyltransgerase is separated polynucleotide with new glucosylation enzyme family 1.These separation are called AsSCPL1 (serine carboxypeptidase sample acyltransferase), AsMT1 (methyltransgerase) and AsGT2 (Transglucosylase) from the new enzyme of black oat.

The gene identification that coding is responsible for the enzyme of triterpene synthesis in various types of grain will allow their manipulation.The change that will cause triterpenoid saponin level or structure is handled in triterpene synthesis.The raising of saponin(e output will cause the raising of plants against pests ability.The food deriving from the plant with high triterpene level is considered to have the effect reducing cholesterol, but triterpene level reduces and it is believed that food can be made to have better local flavor.Therefore, the transgenic plant with the triterpene level of change can produce resistance to insect, and the food of the Spawn preparation changed by triterpene level or structure will have nutritive value or the better local flavor of raising.

In another embodiment, the present invention relates to the polynucleotide of separation, described polynucleotide comprise the nucleotide sequence of encoding serine carboxypeptidase sample isopenicillin-n acyltransferase polypeptides, the aminoacid sequence of described polypeptide, when comparing with SEQIDNO:2, has the sequence iden of at least 95% based on ClustalV comparison method; Or the nucleotide sequence of coding methyltransferase polypeptides, the aminoacid sequence of described polypeptide, when comparing with SEQIDNO:4, has the sequence iden of at least 95% based on ClustalV comparison method; Or the nucleotide sequence of coding Transglucosylase, the aminoacid sequence of described enzyme, when comparing with SEQIDNO:6, has the sequence iden of at least 95% based on ClustalV comparison method; Or comprise the nucleotide sequence of total length complementary sequence of (a), (b) or (c).

In another embodiment, the present invention relates to and comprise the carrier that the present invention is separated polynucleotide.

In another embodiment, the present invention relates to recombinant dna construct, this construct comprises the polynucleotide of the present invention of the first enzyme of coding triterpene approach at least partially, and described portion being operable ground connects at least one regulating and controlling sequence.

In another embodiment, the present invention relates to recombinant dna construct, this construct comprises the polynucleotide of the present invention of the first enzyme of coding triterpene approach at least partially, described portion being operable ground connects at least one regulating and controlling sequence, also comprise at least the second polynucleotide at least partially, described second polynucleotide encoding regulates the polypeptide of the triterpene approach at least expression of the second enzyme.

In another embodiment, the present invention relates to the separation host cell comprising recombinant dna construct of the present invention.This host cell can be yeast cell, bacterial cell or vegetable cell.

The present invention also comprises the composition of plant and plant part, and described composition comprises isolated polypeptide of the present invention or polynucleotide.The present invention also comprises transformed plant, and described plant origin is in the transformed host cell of higher plant and seed or cereal, and they derive from this type of transformed plant.These type of transgenic plant comprise the triterpene that those beta-amyrins with level change derive or the plant with modification triterpene.

In another embodiment, the present invention relates to the transgenic plant comprising recombinant chou of the present invention, wherein regulate sequence to be allogeneic promoter, wherein transgenic plant have the triterpene level of change when compared with the triterpene level of wild-type plant.

The present invention also relates to the method changing polypeptide expression level in vegetable cell, described method comprises: with the nucleic acid fragment transformed plant tissue from separation polynucleotide of the present invention at least partially, wherein said polynucleotide can change native serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase; Described plant tissue is regenerated in transgenic plant; And assessment is when comparing with the plant of the wild type expression level with corresponding native serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase, the expression level change of the serine carboxypeptidase sample acyltransferase of described transgenic plant, methyltransgerase or Transglucosylase.

The present invention also relates to the method producing plant at least one fungi to resistance, described method comprises: the recombinant dna construct transformed plant cells of the first enzyme of triterpene approach of encoding with at least one the present invention; Under the condition promoting transgenic plant regeneration, the transformed plant cells from step (a) is grown; And the transgenic plant of appraisal procedure (b) are not when with the comparing by the plant that described recombinant dna construct is transformed of same species, situation is improved to the resistance of at least one fungi.Recombinant precursor also can comprise at least one second polynucleotide, described polynucleotide encoding regulates the polypeptide of at least one the second expression of enzymes of triterpene approach, expect that these polynucleotide include but not limited to the nucleotide sequence of the enzyme of the first two step in polynucleotide of the present invention and coding pass, described enzyme is the oxidosqualene cyclase beta-amyrin synthase (product of Sad1 gene; HaralampidisK. wait people, 2001, Proc.Natl.Acad.Sci.U.S.A.98:13431-13436) and/or cytochrome P 450 enzymes CYP51H10 (by Sad2 genes encoding; QiX. people is waited, 2006, Proc.Natl.Acad.Sci.U.S.A.103:18848-18853).

The present invention also relates to produce the method for the plant of serine carboxypeptidase sample acyltransferase, methyltransgerase or the Transglucosylase with change level, described method comprises: by the recombinant dna construct transformed plant cells of the first enzyme of at least one coding triterpene according to claim 5 approach; Under the condition promoting transgenic plant regeneration, the transformed plant cells from step (a) is grown; And assessment is when comparing with the amount of serine carboxypeptidase sample acyltransferase, methyltransgerase or the Transglucosylase in not transformed by the described recombinant dna construct plant of same species, the expression level change of the serine carboxypeptidase sample acyltransferase of the transgenic plant of step (b), methyltransgerase or Transglucosylase.

The present invention also relates to the method for the plant for the production of the triterpenoid saponin level with change, described method comprises: the recombinant dna construct transformed plant cells of the first enzyme of triterpene approach of encoding with at least one the present invention; Under the condition promoting transgenic plant regeneration, the transformed plant cells from step (a) is grown; And the triterpenoid saponin level of the transgenic plant of appraisal procedure (b) when comparing with the triterpenoid saponin amount in not transformed by the described recombinant dna construct plant of same species changes.Recombinant precursor also can comprise at least one second polynucleotide at least partially; described polynucleotide encoding regulates the polypeptide of at least one the second expression of enzymes in triterpene approach, expects that these polynucleotide include but not limited to polynucleotide of the present invention (acyltransferase, methyltransgerase and Transglucosylase), Sad1 and Sad2.

The present invention also relates to the method for the plant for the production of the triterpenoid saponin level with raising, described method comprises: by the recombinant dna construct transformed plant cells of the first enzyme of coding triterpene approach of at least one claim 5; Under the condition promoting transgenic plant regeneration, the transformed plant cells from step (a) is grown; And the triterpenoid saponin level of the transgenic plant of appraisal procedure (b) when comparing with the triterpenoid saponin amount in not transformed by the described recombinant dna construct plant of same species improves.Recombinant precursor also can comprise at least one second polynucleotide; described polynucleotide encoding regulates the polypeptide of at least one the second expression of enzymes in triterpene approach, expects that these polynucleotide include but not limited to polynucleotide of the present invention (acyltransferase, methyltransgerase and Transglucosylase), Sad1 and Sad2.

The present invention also relates to the method for the plant for the production of the triterpenoid saponin level with reduction, described method comprises: the recombinant dna construct transformed plant cells of the first enzyme of triterpene approach of encoding with at least one the present invention; Under the condition promoting transgenic plant regeneration, the transformed plant cells from step (a) is grown; And the triterpenoid saponin level of the transgenic plant of appraisal procedure (b) when comparing with the triterpenoid saponin amount in not transformed by the described recombinant dna construct plant of same species reduces.Recombinant precursor also can comprise at least one second polynucleotide at least partially; described polynucleotide encoding regulates the polypeptide of at least one the second expression of enzymes in triterpene approach, expects that these polynucleotide include but not limited to polynucleotide of the present invention (acyltransferase, methyltransgerase and Transglucosylase), Sad1 and Sad2.

The present invention also comprises the cereal from transgenic plant of the present invention.

Accompanying drawing explanation

According to the following detailed description and accompanying drawing and sequence list, can comprehend the present invention, the following detailed description and accompanying drawing and sequence list form a application's part.

Fig. 1 illustrates the structure of beta-amyrin and avenacin A-1, and explanation must there occurs multiple modification to derive the latter from the former.

Fig. 2 illustrates the genomic dna sequence of a 317kb, and this sequence comprises five genes from the biosynthetic enzyme of the prediction of black oat (beta-amyrin synthase (Sad1), Cytochrome P450 CYP51H10 (Sad2), t, serine carboxypeptidase sample albumin A sSCPL1, methyltransgerase AsMT1 and Transglucosylase AsGT2.

Fig. 3 is to the Northern engram analysis (beta-amyrin synthase (SAD1), Cytochrome P450 CYP51H10 (SAD2), serine carboxypeptidase sample albumin A sSCPL1, methyltransgerase AsMT1, Transglucosylase AsGT2 and GAPDH contrast) of the biosynthetic enzyme of five predictions, and described enzyme is arranged in radical bud, the leaf of oat and spends tissue.

Sequence description schematically illustrates accompanying sequence list.Nucleotide is represented with single letter in this sequence list, with 3 independent letter codes for amino acid, defined in the IUPAC-IUB standard described by NucleicAcidsResearch13:3021-3030 (1985) and BiochemicalJournal219 (2): 345-373 (1984).

SEQIDNO:1 is the cDNA nucleotide sequence of coding from the serine carboxypeptidase sample polypeptide of black oat (AsSCPL1).

SEQIDNO:2 is the AsSCPL1 aminoacid sequence derived from the genomic fragment shown in the cDNA fragment shown in SEQIDNO:1 or SEQIDNO:7.

SEQIDNO:3 is the cDNA nucleotide sequence of coding from the sulfonylmethyl transferring enzyme of black oat (AsMT1).

SEQIDNO:4 is the AsMT1 aminoacid sequence derived from the genomic fragment shown in the cDNA fragment shown in SEQIDNO:3 or SEQIDNO:8.

SEQIDNO:5 is the cDNA nucleotide sequence of coding from the Transglucosylase of black oat (AsGT2).

SEQIDNO:6 is the AsGT2 aminoacid sequence derived from the genomic fragment shown in the cDNA fragment shown in SEQIDNO:5 or SEQIDNO:9.

SEQIDNO:7 is the nucleotide sequence of the genomic fragment of coding AsSCPL1 polypeptide.

SEQIDNO:8 is the nucleotide sequence of the genomic fragment of coding AsMT1.

SEQIDNO:9 is the nucleotide sequence of the genomic fragment of coding AsGT2.

Embodiment

The disclosure of every section of listed herein reference is incorporated herein by reference all in full.

Singulative " one " as used herein and in the appended claims and " described " comprise plural references, unless clearly separately indicated in context.Therefore, such as, the connotation of " a strain plant " comprises this type of plant of many strains, and the connotation of " cell " comprises one or more cell and equivalent known to those skilled in the art thereof, etc.

Disclosed hereinly context of many terms and abbreviation.Give as given a definition.

" open reading frame " is abbreviated as ORF.

" polymerase chain reaction " is abbreviated as PCR.

Pathways metabolism or biosynthetic pathway can be thought to betide in cell by enzymatic series of chemical on Biochemical Significance, in order to realize treating that cell uses or to treat the formation of meta-bolites of storage of cells, or start another pathways metabolism (being thus called flow generating step).This classpath a lot of is all very meticulous, and relates to the product progressively modifying to make it to be formed the exact chemical structures with expectation to initial substance.

" nucleic acid " used herein means polynucleotide, and comprises deoxyribonucleotide or the ribonucleotide bases polymkeric substance of strand or double-strand.Nucleic acid also can comprise fragment and modified Nucleotide.Therefore, term " polynucleotide ", " nucleotide sequence ", " nucleotide sequence " or " nucleic acid fragment " are used interchangeably, and are RNA or the DNA polymkeric substance as strand or double-strand, optional containing nucleotide base that is synthesis, non-natural or that change.Nucleotide (usually with their 5 '-monophosphate form exist) can refer to by their single-letter title following: " A " represents adenylic acid (AMP) or deoxyadenylic acid (respectively for RNA or DNA), " C " represents cytidylic acid or deoxycytidylic acid(dCMP), " G " represents guanylic acid or dGMP, " U " represents uridylic acid, " T " represents deoxythymidylic acid, " R " represents purine (A or G), " Y " represents pyrimidine (C or T), " K " represents G or T, " H " represents A or C or T, " I " represents inosine, and " N " represents any Nucleotide.

Term " separation " polynucleotide be a kind of by substantially separately or from the biology of wherein natural generation polynucleotide by other polynucleotide of conventional nucleic acid purification method purifying, that is, other karyomit(e) and extrachromosomal DNA and RNA.The polynucleotide of recombination of polynucleotide and chemosynthesis also contained in this term.Separation polynucleotide of the present invention can comprise all or part of separation polynucleotide, such as, comprise the polynucleotide being selected from following nucleotide sequence: the total length complementary sequence of SEQIDNO:1, SEQIDNO:3, SEQIDNO:5, SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9 or this type of nucleotide sequence.

Triterpenoid saponin(e, via Isoprenoid pathway, forms pentacyclic triterpene based compound by cyclisation 2,3-epoxy squalene, mainly volatile oil (beta-amyrin) or dammarane skeleton synthesis.Triterpenoid main chain stands multiple modification subsequently; comprise oxidation, acylations, methylate, glycosylation and other replace, described modification is mediated by Cytochrome P450 dependent form monooxygenase, glycosyltransferase, acyltransferase, methyltransgerase and other enzyme.

Triterpene, also referred to as triterpenoid, includes but not limited to saponin(e and sterol.

" saponin(e " refers to the glycoside conjugate of the cyclisation triterpene of natural accumulation in plant.Cyclisation triterpene includes but not limited to lanosterol, cycloartenol, beta-amyrin, alpha-amyrin, Lupeol, isomultiflorenol and thalianol." triterpenoid saponin " finger ring triterpene join glycoconjugate, the triterpene except those derive from lanosterol or cycloartenol." steroidal saponins " refers to derive from lanosterol or cycloartenol glycoside conjugate.Sapogenol is the triterpenoid saponin obtained via external acid hydrolysis, provides a relative value to their amount therefrom extracting the triterpenoid saponin existed in the tissue of saponin(e that is measured as.

The level of triterpenoid saponin causes sapogenol to measure by measuring.The observed value of sapogenol is directly related to the level of triterpenoid saponin.Sapogenol is the triterpenoid saponin obtained via external acid hydrolysis, and provide a relative value to their amount therefrom extracting the triterpenoid saponin existed in the tissue of saponin(e that is measured as, this value is directly related to the amount of triterpenoid saponin.

Triterpenoid saponin level can use technology known in the art to measure.Such as, HPLC-MS or HPLC with light scattering detector can be used to carry out measuring (see people such as such as Rupasinghe, HP., (2003), J.Agri.FoodChem.51:5888-5894).Alternatively, the HPLC with UV detector can be used to carry out measuring (people such as HubertJ, (2005), J.Agric.FoodChem.53:3923-3930).Other method comprises use GC-FAB.(see people such as such as Gee, (1993), JSciFoodAgric.63:201-209).Other method relates to use tlc (TLC) and is separated saponin(e (see such as OleszekWA. (2002) J.Chromatogr.A967:147-162. in conjunction with densitometry; GurfinkelDM, and RaoAV (2002) J.Agric.FoodChem.50:426-430.

It is also possible for using other method to measure triterpenoid saponin.Such as, panimmunity measuring method (such as radioimmunoassay or ELISA) is used also to be suitable (WangCC, PrasainJK, with BarnesS. (2002) J.Chromatogr.BAnalyt.Technol.Biomed.LifeSci.777:3-28, the people such as AhamedA, (2003), Biochem.Biophys.Res.Commun.302:587-592).

" the triterpenoid saponin level of raising, " refers to that triterpenoid saponin level is present in the triterpenoid saponin level in the non-transformed plant of same species higher than those for the present invention, and it is owing to having transformed nucleic acid fragment of the present invention that level improves.Such as, " the triterpenoid saponin level of raising, " can refer to that triterpenoid saponin level is present in the triterpenoid saponin level in same species plant higher than those, and this plant does not have the recombinant DNA molecules comprising the polynucleotide of coding oxidosqualene cyclase of the present invention." the triterpenoid saponin level of raising " can be improve at least 100ppm, 250ppm, 500ppm, 750ppm, 1000ppm, 1250ppm, 1500ppm, 3000ppm, 6000, or their integer any.

" level of change " or " expression of change " refers to that the amount of the gene product produced in genetically modified organism or ratio are different from normal bio or non-transformed biology.

" the triterpenoid saponin level of change, " refers to that the amount of triterpenoid saponin level or ratio are different from those triterpenoid saponin levels do not transformed in the non-transformed plant of nucleic acid fragment of the present invention being present in same species for the present invention.

" the triterpenoid saponin level of reduction, " refers to that triterpenoid saponin level is present in the triterpenoid saponin level do not transformed in the non-transformed plant of nucleic acid fragment of the present invention of same species lower than those for the present invention.

Term " has the subfragment of suitable function " and " functionally suitable subfragment " is used interchangeably in this article.These terms refer to a part or the subsequence of the nucleic acid fragment of separation, and wherein no matter whether described fragment or subfragment encode activated enzyme, and it all maintains the ability changing genetic expression or cause certain phenotype.Such as, described fragment or subfragment can be used to design mosaic gene, to produce desired phenotype in plant in post-conversion.Can by nucleic acid fragment or subfragment be connected with affiliated promoter sequence with the direction relative to plant promoter sequences sense or antisense, thus the mosaic gene designed for suppressing, no matter whether wherein said nucleic acid fragment or subfragment encode activated enzyme.

At one group of amino acid that specific position is conservative in the aligned sequences of protein relevant in term " conserved domain " or " motif " fingering.Although can change at the amino acid of other position between homologous protein, be essential amino acid structure, stability or activity that the amino acid of specific position high conservative represents protein.Because they are identified by their high conservatives in the aligned sequences of protein homology thing family, thus they can be used as protein that identification marking or " signature " determine to have new mensuration sequence whether belong to before the protein families of qualification.

Term " homology ", " homology ", " similar substantially " and " corresponding substantially " are used interchangeably in this article.They refer to the nucleic acid fragment of ability that the change of wherein one or more nucleotide bases can not affect nucleic acid fragment mediated gene and expresses or produce certain phenotype.These terms also refer to the modification (such as lack or insert one or more Nucleotide) of nucleic acid fragment of the present invention, relative to initial not modified nucleic acid fragment, substantially can not change the functional performance of gained nucleic acid fragment.Therefore, as would be understood by one skilled in the art, these concrete exemplary sequence are not only contained in the present invention.

In addition, technician recognizes, the nucleotide sequence similar substantially contained of the present invention also by them at medium stringency condition (as 0.5XSSC, 0.1%SDS, 60 DEG C) under, with the ability of the sequence hybridization exemplified by this paper, or hybridization limited to any part of nucleotide sequence disclosed herein and hybridization to the ability of the sequence suitable with any nucleotide sequence function disclosed herein.Stringency can be regulated to screen the similar fragment of moderate such as from the homologous sequence of edge biology far away, to screening highly similar fragment such as from the gene of nearly edge bioautography functional enzyme.Stringency is determined in washing after hybridization.

Term " selective cross " comprises and referring under stringent hybridization condition, nucleotide sequence can detection level (such as at least 2 times to background) be hybridized with higher than the hybridization of itself and non-target nucleic acid sequences with specific nucleic acid target sequence, and refers to substantially eliminate non-target nucleic acid.The sequence of selective cross has the sequence iden of about at least 80% or the sequence iden of 90% usually each other, at most and comprise 100% sequence iden (i.e. complete complementary).

Term " stringent condition " or " stringent hybridization condition " comprise and refer to probe by selective cross to the condition of its target sequence.Stringent condition is sequence dependent and by different because of different environment.By controlling the severity of hybridization and/or wash conditions, can identify and the target sequence of probe 100% complementation (homology detects).Alternatively, adjustable stringent condition to allow some mispairing in sequence, the similarity (allos detection) of more low degree to be detected.Usually, the length of probe is less than about 1000 Nucleotide, and optional length is less than 500 Nucleotide.

Usually, stringent condition will be those conditions following: pH7.0 to 8.3 time salt concn lower than about 1.5M sodium ion, usually about 0.01 to 1.0M Na ion concentration (or other salt), and 30 DEG C are at least about for short probe (such as 10 to 50 Nucleotide) temperature, and 60 DEG C are at least about for long probe (such as more than 50 Nucleotide) temperature.Stringent condition also can be realized by the destabilizing agent adding such as methane amide and so on.Exemplary low stringency condition is included in 37 DEG C and hybridizes in the damping fluid containing 30 to 35% methane amides, 1MNaCl, 1%SDS (sodium lauryl sulphate), and washs with 1X to 2XSSC (20XSSC=3.0MNaCl/0.3M trisodium citrate) at 50 to 55 DEG C.Exemplary medium stringency condition is included in 37 DEG C and hybridizes in 40 to 45% methane amides, 1MNaCl, 1%SDS, and washs with 0.5X to 1XSSC at 55 to 60 DEG C.Exemplary high stringent condition is included in 37 DEG C and hybridizes in 50% methane amide, 1MNaCl, 1%SDS, and washs with 0.1XSSC at 60 to 65 DEG C.

Specificity depends on post-hybridization washing usually, and the ionic strength of last washing soln and temperature are key factors.For DNA-DNA crossbred, T _mcan with people such as Meinkoth, Anal.Biochem.138:267-284 (1984): T _m=81.5 DEG C+16.6 (logM)+0.41 (%GC)-0.61 (%form)-500/L; Wherein M is the molarity of univalent cation, and %GC is the per-cent of guanosine and cytidylic acid(CMP) in DNA, and %form is the per-cent of methane amide in hybridization solution, and L is the length of the crossbred represented with base pair.T _mthe complementary target sequence of (under the ionic strength determined and pH) 50% and the temperature during probe hybridization mated completely.Often there is the mispairing of 1%, T _mreduce about 1 DEG C; Therefore, adjustable T _mhybridization and/or wash conditions with there is the sequence hybridization expecting identity.Such as, if will seek to have>=the sequence of 90% identity, then can by T _mreduce by 10 DEG C.Usually, under the ionic strength determined and pH, stringent condition is chosen as the melting temperature (Tm) (T than particular sequence and complementary sequence thereof _m) low about 5 DEG C.But pole stringent condition can adopt than melting temperature (Tm) (T _m) hybridization and/or washing at the temperature of low 1,2,3 or 4 DEG C; Medium stringency condition can adopt than melting temperature (Tm) (T _m) hybridization and/or washing at the temperature of low 6,7,8,9 or 10 DEG C; Low stringency condition can adopt than melting temperature (Tm) (T _m) hybridization and/or washing at low 11,12,13,14,15 or 20 DEG C of temperature.Utilize described equation, hybridization and cleaning composition and desirable T _m, it will be understood to those of skill in the art that the change of the severity of hybridization and/or washing soln is described in itself.If required extent of mismatch causes T _mlower than 45 DEG C (aqueous solution) or 32 DEG C (formamide soln), then preferably increase the concentration of SSC higher temperature can be used.Can find in the following documents the detailed guidance of nucleic acid hybridization: Tijssen, LaboratoryTechniquesinBiochemistryandMolecularBiology--H ybridizationwithNucleicAcidProbes, part i, 2nd chapter " Overviewofprinciplesofhybridizationandthestrategyofnucle icacidprobeassays ", Elsevier, NewYork (1993); And CurrentProtocolsinMolecularBiology, the 2nd chapter, the people such as Ausubel edit, GreenePublishingandWiley-Interscience, NewYork (1995).Hybridization and/or wash conditions can carry out at least 10,30,60,90,120 or 240 minutes.

" sequence iden " or " identity " in nucleic acid or peptide sequence context refers to that nucleic acid base in two sequences or amino-acid residue are when being identical when specifying comparison maximum match in comparison window.

Therefore, " percentage of sequence identity " refers to the value determined by comparing two sections of sequences maximizing coupling on a comparison window, polynucleotide wherein in described comparison window or the part of peptide sequence can comprise additional or lack in (i.e. gap), to obtain the maximum match between two sections of sequences compared with the reference sequences of (do not comprise additional or lack).The method of calculation of described per-cent are, the quantity of adding up the site that identical nucleic acid base or amino-acid residue occur in two sections of sequences is simultaneously counted to obtain match bit, this match bit is counted divided by the total number of sites in comparison window, again result is multiplied by 100, thus obtains percentage of sequence identity.The available example of percentage of sequence identity includes but not limited to 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% and 95%, or from 50% to 100% arbitrary integer per-cent.Above-mentioned consistence can utilize random procedure as herein described to determine.

Alignment and percentage identities or similarity can be determined with being designed for the multiple comparative approach detecting homologous sequence, these methods include, but is not limited to LASERGENE bioinformation and calculate bag (DNASTARInc., Madison, WI) MegAlig ^tMprogram.When should be appreciated that in the context of present application for patent use sequence analysis software is analyzed, except as otherwise noted, otherwise analytical results is by " default value " based on referenced program.Any value or parameter set that software loads at first when initializers is first referred at this " default value " used.

" ClustalV comparison method " is corresponding to being called that ClustalV is (in Higgins and Sharp, CABIOS, 5:151-153 (1989); The people such as Higgins, D.G. (1992) Comput.Appl.Biosci.8:189-191), and be present in the MegAlign of LASERGENE information biology computation software package (DNASTARInc., Madison, WI) ^tMin program.For multiple ratio pair, default value corresponds to Gap Penalty (GAPPENALTY)=10 and Gap Length Penalty (GAPLENGTHPENALTY)=10.The default parameters carrying out the percentage identities calculating of paired comparison and protein sequence by Clustal method is KTUPLE=1, gap penalty=3, window (WINDOW)=5 and DIAGONALSSAVED=5.And for nucleic acid, these parameters are KTUPLE=2, gap penalty=5, window=4 and DIAGONALSSAVED=4.After ClustalV program aligned sequences, by checking that " sequence distance " table in same program obtains " percentage identities ".

" BLASTN comparison method " is the algorithm in order to adopt default parameters to compare nucleotide sequence provided by NCBI (NationalCenterforBiotechnologyInformation, NCBI).

Those skilled in the art is perfectly clear, and the sequence iden of multiple degree can be used for identifying polypeptide from other species, and wherein this kind of polypeptide has same or analogous function or activity.The available example of identity per-cent includes but not limited to any integer percent between 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or 50% to 100%.In fact, any integer amino acid identities between 50% to 100% can be used for describing the present invention, such as 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.Any total length of this nucleotide fragment isolation or the sequence of partial complementarity are also useful.

" Codon degeneracy " refers to the divergent degree of the genetic code changed when allowing nucleotides sequence to be listed in the aminoacid sequence not affecting coded polypeptide.Therefore, the present invention relates to any nucleic acid fragment comprising the nucleotide sequence of the aminoacid sequence of encode all or major portion, AsSCPL1, AsMT1 and AsGT2 albumen of described amino acid sequence encode as shown in SEQIDNO:2, SEQIDNO:4 and SEQIDNO:6.Technician knows " codon bias " that use the given amino acid whose nucleotide codon of appointment to show by particular host cell.Therefore, when synthetic polyribonucleotides is expressed for the specific gene improved in host cell, the frequency designing the preferred codon that these polynucleotide make its codon usage frequency use close to host cell is wished.

As used herein, " nucleic acid fragment " can be assembled by using the oligonucleotide component of method known to those skilled in the art chemosynthesis.These components are connected and carry out annealing to form larger nucleic acid fragment, then its enzyme catalysis can be carried out assembling building complete needed for nucleic acid fragment." chemosynthesis " relevant with nucleic acid fragment refers to assemble component nucleotide in vitro.Can adopt improve set up method to complete nucleic acid fragment Manual chemical synthesis, or can use many kinds can business obtain machine in one to complete robotics synthesis.Therefore, based on optimization nucleotide sequence to reflect the codon bias of host cell, nucleic acid fragment can be customized in order to optimization genetic expression.If codon uses those codons being partial to host's preference, then technician can understand the possibility of successful genetic expression.The determination of preferred codon can based on to the detection of gene (wherein sequence information can obtain) deriving from host cell.

" gene " refers to the nucleic acid fragment can expressing specified protein, and it comprises the regulating and controlling sequence (3 ' non-coding sequence) after the regulating and controlling sequence before encoding sequence (5 ' non-coding sequence) and encoding sequence." natural gene " refers to the naturally occurring gene with its oneself regulating and controlling sequence." mosaic gene " refers to it is not any gene of natural gene, is not the adjustment sequence existed together and encoding sequence under being included in its natural environment.Therefore, mosaic gene can comprise the regulating and controlling sequence and encoding sequence that come from different sources, or comprises and come from same source but to be different from the regulating and controlling sequence and encoding sequence that naturally occurring mode arranges." alien gene " refers to the gene be not present under normal circumstances in host living beings, and it imports host living beings by transgenosis.Alien gene can comprise the natural gene be inserted in non-native organism, or mosaic gene." transgenosis " is by the gene in method for transformation quiding gene group.

When being applied to vegetable cell, term " genome " is not only included in the chromosomal DNA found in nucleus, and is included in the organelle DNA found in the subcellular component (such as plastosome, plastid) of cell.

" through the optimized gene of codon " is that its codon usage frequency is through the gene of design in order to imitate the preferred codon usage frequency of host cell.

" allelotrope " karyomit(e) occupies the one in several selective alternative form of the gene of locating point.When all allelotrope that site given on karyomit(e) exists are identical, plant is homozygote in this site.If the allelotrope that on karyomit(e), given site exists is different, plant is heterozygote in this site.

" encoding sequence " refers to the DNA sequence dna of encoding particular amino acid sequence." regulating and controlling sequence " refers to be positioned at the upstream (5 ' non-coding sequence) of encoding sequence, middle or downstream (3 ' non-coding sequence), and affects the nucleotide sequence of the transcribing of related coding sequences, RNA processing or stability or translation.Regulating and controlling sequence can include, but are not limited to: promotor, translation leader sequence, intron, polyadenylation recognition sequence, RNA Processing position, effector binding site and loop-stem structure.

" promotor " refers to can control coding sequence or the functional r NA DNA sequence dna of expressing.Promoter sequence by nearside and comparatively distal upstream elements form, the latter often refers to enhanser.Therefore, " enhanser " is the DNA sequence dna that can stimulate promoter activity, and it can be the innate element of promotor, or inserts to strengthen promotor level or tissue-specific aheterologous element.Promotor wholely can come from natural gene, or is made up of the different elements coming from different naturally occurring promotors, or even comprises the DNA fragmentation of synthesis.Those skilled in the art should be appreciated that different promotors can in different tissues or cell type, or in the different etap, or respond the expression of different envrionment conditionss and guiding gene.It will also be appreciated that the exact range owing in most of the cases can't determine regulating and controlling sequence completely, the DNA fragmentation of some modification may have identical promoter activity.The promotor causing gene to express in most cells type is as a rule commonly referred to " constitutive promoter ".The continuous dissimilar new promotor finding to can be used in vegetable cell at present; Multi-instance can be found in the following documents: the BiochemistryofPlants15:1-82 (1989) that Okamuro, J.K.andGoldberg, R.B. edit.

" translation leader sequence " refers to the polynucleotide sequence between gene promoter sequence and encoding sequence.Translation leader sequence is present in the mRNA upstream after processing completely of translation initiation sequence.Translation leader sequence can affect the primary transcription process of mRNA, mRNA stability or translation efficiency.Describe the example (Turner, R. and Foster, G.D., Mol.Biotechnol.3:225-236 (1995)) of translation leader sequence.

" 3 ' non-coding sequence ", " transcription terminator " or " terminator sequence " refers to be positioned at encoding sequence downstream and to comprise can affect mRNA processing or the polyadenylation recognition sequence of genetic expression and the DNA sequence dna of other sequence encoding conditioning signals.Polyadenylation signal is characterized by usually affects the 3 ' end that polyadenylic acid sheet adds mRNA precursor to.The people such as Ingelbrecht, I.L. are exemplified with the purposes PlantCell1:671-680 (1989) of different 3 ' non-coding sequence.

" rna transcription thing " refers to transcribe produced product by the DNA sequence dna of RNA polymerase catalysis.When rna transcription thing and DNA sequence dna copy complete complementary, it refers to primary transcript.When rna transcription thing is the RNA sequence deriving from the primary transcript of transcribing post-treatment, it refers to ripe RNA." messenger RNA(mRNA) or " mRNA " refer to intronless and can be become the RNA of protein by cell translation." cDNA " refers to complementary with mRNA template and uses ThermoScript II from the DNA of mRNA templated synthesis.CDNA can be strand, or uses the Klenow fragment of DNA polymerase i to change into double-strand.The RNA that " has justice " refer to comprise mRNA and can in cell or In Vitro Translation become the rna transcription thing of protein.

" sense-rna " refers to and all or part of target primary transcript or mRNA complementation, and stops the RNA (United States Patent (USP) 5,107,065) of expression of target gene.Sense-rna can with any part of specific gene transcript, namely 5 ' non-coding sequence, 3 ' non-coding sequence, intron or encoding sequence are complementary." functional r NA " refer to sense-rna, ribozyme rna or other can not translate but cell processes be had to the RNA of influence.Term " complementation " and " reverse complemental " are used interchangeably concerning mRNA transcribes, and in order to define courier's sense-rna.

Term " is operably connected " association of the nucleotide sequence referred on single core acid fragment, makes the function of one of them nucleotide sequence be subject to the regulation and control of another nucleotide sequence.Such as, when promotor can regulate the expression of encoding sequence (that is, what this encoding sequence was subject to this promotor transcribes control), then this promotor is operably connected with this encoding sequence.Encoding sequence can may be operably coupled to regulating and controlling sequence with the orientation of sense or antisense.In another example, complementary RNA regions of the present invention, no matter directly or indirectly connect, 5 ' end and target mRNA or 3 ' hold and to be connected with target mRNA or to be positioned at that target mRNA or the first complementary region are held with 5 ' and its complement is held with 3 ' and is connected with target mRNA, all can form available connection.

Standard recombinant dna used herein and molecule clone technology are known in the art and have in such as Publication about Document and more fully describe: Sambrook, J., Fritsch, E.F.andManiatis, T., MolecularCloning:ALaboratoryManual; ColdSpringHarborLaboratory:ColdSpringHarbor, NY (1989).Method for transformation known by those skilled in the art, and is described in hereafter.

" PCR " or " polymerase chain reaction " is the technology for the synthesis of a large amount of specific DNA fragments, and it comprises the circulation (PerkinElmerCetusInstruments, Norwalk, CT) of a series of repetition.Typically, double-stranded DNA, by thermally denature, is annealed with it at a lower temperature with the primer of 3 ' end regions complementation of target fragment, is then extended at intermediate temperatures for two kinds.Above-mentioned three continuous print steps of one group are called as one " circulation ".

Term " restructuring " refers to such as by chemosynthesis or the artificial combination by handling the sequence fragments that the nucleic acid fragment realize two that is separated is separated originally with genetic engineering technique.

Term " plasmid ", " carrier " and " box " refer to the extra-chromosomal element of the gene usually carrying the part not belonging to cell centre metabolism, and are usually the forms of circular double stranded DNA fragment.This class component can be the nucleotide sequence (linear or ring-type) being derived from the autonomously replicating sequence in any source, genome integration sequence, phage or strand or double-stranded DNA or RNA, wherein multiple nucleotide sequence has connected or has recombinated and entered in a kind of unique design body, and the promoter fragment of selected gene product can be introduced in cell together with corresponding 3 ' end non-translated sequence with DNA sequence dna by this unique design body." conversion box " refers to containing alien gene and also containing the specific support of element being conducive to transforming particular host cell and transforming except this alien gene." expression cassette " refers to and comprises alien gene, and has the specific support (namely referring to nucleotide sequence or fragment to be moved into discontinuous nucleic acid fragment wherein) of the element beyond the described gene that can make described gene Enhanced expressing in host.

Term " recombinant precursor ", " expression construct ", " chimeric constructs ", " construct " and " recombinant dna construct " are used interchangeably herein.Recombinant precursor comprises artificial nucleic acid fragment combination, the regulating and controlling sequence such as, existed not together under natural condition and encoding sequence.Such as inserted structure can comprise the regulating and controlling sequence and encoding sequence that come from different sources, or comprises and come from same source but to be different from the regulating and controlling sequence and encoding sequence that naturally occurring mode arranges.This type of construct can use alone or uses with carrier combinations.If use carrier, then the method in order to transformed host cell is depended in the selection of carrier, and this host cell is known by those skilled in the art.Such as plasmid vector can be used.Technician knows to successfully transform, screening and breed the host cell comprising any nucleic acid fragment be separated of the present invention, must be present in the genetic elements on carrier.Technician also will recognize, different separate transformation events will cause expression (people such as Jones, EMBOJ., the 4:2411-2418 (1985) of different levels and pattern; The people such as DeAlmeida, Mol.Gen.Genetics218:78-86 (1989)), therefore, multiple events must be screened to obtain the strain that expression level and pattern are expected in display.Such screening can be completed by the immunoblotting assay of the Northern engram analysis of the Southern engram analysis of DNA, mrna expression, protein expression or phenotype analytical etc.

Term used herein " expression " refers to functional end product (such as, mRNA or protein (both can be precursor also can be mature form)) production.

" importing refers to and nucleic acid (such as expression construct) or albumen is provided in cell term.Importing comprise refer to nucleic acid integration is entered in eucaryon or prokaryotic cell prokaryocyte, can be integrated in the genome of cell in this cell amplifying nucleic acid, and comprise refer to nucleic acid or albumen are temporarily supplied to cell.Import to comprise and refer to stable or temporary method for transformation and sexual hybridization.Therefore, nucleic acid fragment (such as recombinant dna construct/expression construct) " importing " of inserting in intracellular situation is meant " transfection " or " conversion " or " transduction ", and comprise and refer to nucleic acid fragment to be integrated in eucaryon or prokaryotic cell prokaryocyte, can be integrated in the genome (as karyomit(e), plasmid, plastid or Mitochondrial DNA) of cell in this cell amplifying nucleic acid fragment, be transformed into autonomous replicon or transient expression (mRNA of such as transfection).

" maturation " protein refers to the polypeptide (namely eliminated and be present in any propetide in initial translation product or the former polypeptide of peptide) through post translational processing." precursor " protein refers to the primary translation product of mRNA, i.e. propetide and peptide is former still exists.Propetide and peptide former can be (but being not limited to) intracellular localization signals.

" signal peptide " is a kind of aminoacid sequence (Chrispeels, M. (1991) Ann.Rev.PlantPhys.PlantMol.Biol.42:21-53) of the secretory system that leads with albumen collaborative translation and by albumen.If by described albumen guiding vacuole, vacuole target signal (the same) can be added in addition, if or by described albumen guiding endoplasmic reticulum, endoplasmic reticulum retention signal (the same) can be added.If albumen to be led nucleus, will the signal peptide of any existence be removed and substitute (Raikhel, N. (1992) PlantPhys.100:1627-1632) in order to nuclear localization signal." chloroplast transit peptides " is the chloroplast(id) or prepare other plastid type existed in the cell of albumen wherein of leading with albumen collaborative translation and by albumen." chloroplast transit sequence " refers to the nucleotide sequence of encoding chloroplast transit peptide.

" stable conversion " refers to that genome nucleic acid fragment being proceeded to host living beings is to produce stable hereditary property, and this genome comprises Matrix attachment region and organelle gene group.On the contrary " instantaneous conversion " refer to nucleic acid fragment to proceed in the core of host living beings or comprise in the organoid of DNA, when without integrating or causing genetic expression without when stable hereditary property.Host living beings containing transformed nucleic acid fragments is called as " transgenosis " organism.

" transgenosis " refers to the plant or cell that contain heterologous polynucleotide in its genome as used herein.Preferably, heterologous polynucleotide is stably integrated in genome, makes these polynucleotide can be passed to continuous print from generation to generation.Heterologous polynucleotide can be entered in genome by the thin consolidation individually or as expression construct.Transgenosis used herein comprises because heterologous nucleic acids exists and has changed genotypic any cell, clone, callus, tissue, plant part or plant, comprises those transgenosiss of initially carrying out this type of transgenosis changed and being produced by sexual hybridization or vegetative propagation from initial transgenosis." transgenosis " is not contained by conventional plant breeding method or genome (chromogene group or the karyomit(e) alia gene group) change that caused by the naturally-occurring event of such as random cross fertilization, non-recombinant virus infections, non-recombinant Bacterial Transformation, non-recombinant swivel base or spontaneous mutation and so on as the term is employed herein.

Term " suppression " refers to when comparing with the detectable enzyme activity level of natural enzyme in plant, the reduction of detectable enzyme activity level in transgenic plant.In plant, the enzyme activity level of natural enzyme is called that " wild-type " is active herein.Term " suppression " comprises reduction, reduces, lowers, weakens, suppresses, eliminates and stop.This reduction may be the level translating into natural enzyme owing to reducing natural mRNA.Also may be because n DNA is transcribed into the amount reduction of mRNA and/or the degraded quickening of natural mRNA.Term " natural enzyme " refers to the enzyme of natural generation in required cell.

Enzyme level in plant completes by multiple method known in the art, comprises following methods." co-suppression " refer to produce can suppress identical or fully similar natural gene to be expressed have adopted rna transcription (United States Patent (USP) 5,231,020).Before this, by being conceived to have devised co-suppression construct in plant (see people such as Vaucheret with the nucleotide sequence (it causes all RNA with the sequence of process LAN with homology to reduce) that sense orientation process LAN and endogenous mRNA have a homology, 1998, PlantJ., 16:651-659; And Gura, 2000, Nature404:804-808)." Antisense Suppression " refers to produce the sense-rna transcript that target protein can be suppressed to express.Plant virus sequence can be used for guiding the suppression to near-end mRNA encoding sequence (disclosed in 20 days Augusts in 1998 the open WO98/36083 of PCT patent).Describe " hair clip " structure and integrate all or part of of mRNA encoding sequence with complementary direction, cause the RNA expressed to form potential " stem-ring " structure (disclosed in the 21 days October in 1999 the open WO99/53050 of PCT patent).In this case, the polynucleotide of the genes involved that stem is inserted with sense or antisense direction relative to promotor by correspondence are formed, and ring is formed by the polynucleotide of some genes involveds, and in construct, these polynucleotide do not have complementary sequence.Which increase the co-suppression in the transgenic plant of acquisition or reticent frequency.About the summary that hair clip suppresses, see people such as Wesley, S.V., 2003, MethodsinMolecularBiology, PlantFunctionalGenomics:MethodsandProtocols236:273-286.Wherein stem by least 30 constructs that ring is formed by arbitrary nucleotide sequence from treating the Nucleotide of suppressor gene to be formed also effectively for suppressing (disclosed in the 2 days December in 1999 WO99/61632).Poly-T and the poly-A sequence stem produced in stem-ring structure is used to have described (disclosed in the 3 days January in 2002 WO02/00894).But another kind of modification relates to the tumor-necrosis factor glycoproteins of use synthesis to promote the formation of the stem in stem-ring structure.The level of the polynucleotide that the transgenic organism display produced with this recombinant dna fragment is encoded by the nucleotide fragments forming loop-stem structure reduces, as PCT patent disclosed in 3 days January in 2002 discloses described in WO02/00904.Describe the purposes producing dsRNA construct.The gene specific sense and antisense RNA that common promotor guides induced gene to suppress in these constructs transcribes (see people (2000) RNA6:1069-1076 such as such as Shi, H.; The people such as Bastin, P. (2000) J.CellSci.113:3321-3328; The people such as Giordano, E. (2002) Genetics160:637-648; LaCount, D.J. and Donelson, J.E. U.S. Patent application 20020182223, is published on December 5th, 2002; The people such as Tran, N. (2003) BMCBiotechnol.3:21; With the U.S. Provisional Application 60/578,404 of applicant, be filed on June 9th, 2004).

Other method for enzyme level includes but not limited to use and can form the polynucleotide (U.S. Patent Publication 4 that catalysis RNA maybe can have ribozyme activity, 987,071, be published on January 22nd, 1991), and tiny RNA (also referred to as miRNA) interferes people (2003) Nature425:257-263 such as () Javier.

For the polynucleotide passage sequence that suppresses and need the polynucleotide passage sequence existed in repressed gene to be not 100% identical.Such as, the polynucleotide deriving from the Gene Partial of coding for alpha subunit (U.S. Patent Publication 6,362,399) are used successfully to inhibit the subunit of all soybean seed storage proteins β-soybean companion sphaeroprotein.β-soybean companion sphaeroprotein is a kind of heterogeneous glycoprotein, is made up of the variation of three high negative electricity subunits (being accredited as α, α ' and β).The polynucleotide sequence of coding for alpha and α ' subunit is about 85% identical each other, but the polynucleotide sequence of coding β subunit and α with α ' subunit are respectively about 75% identical to about 80%.Therefore, shown suppressing required target effective with the target polynucleotide region that need suppress at least about 75% identical polynucleotide.Described polynucleotide can be identical at least about 80% with required target sequence, identical at least about 90%, identical at least about 95%, or about 100% is identical.

Natural gene " can suppress express part " refers to a part or the subfragment of the nucleic acid fragment be separated, wherein no matter whether described fragment or subfragment can be translated into activated enzyme, and it all maintains the ability changing genetic expression or cause certain phenotype.Such as, described fragment or subfragment can be used to design mosaic gene or recombinant dna fragment, to produce desired phenotype in plant in post-conversion.Can by nucleic acid fragment or subfragment be connected with affiliated promoter sequence with the direction relative to plant promoter sequences sense or antisense, thus the mosaic gene designed for suppressing, no matter whether wherein said nucleic acid fragment or subfragment translate into activated enzyme.Recombinant dna fragment can be designed to comprise the nucleic acid fragment that can promote that stem-ring structure is formed.In stem-ring structure, ring or stem comprise a part needs repressed gene.Nucleic acid fragment should have the extension at least about 20 adjacent Nucleotide, and this extension is identical with needing repressed gene.The extension of adjacent Nucleotide can be any number, from least about 20, or about 32, to the size needing repressed whole gene.

Term " plant " comprises whole plant, plant organ, plant tissue, seed and vegetable cell, the filial generation of seed and same plant.Vegetable cell includes but not limited to the cell deriving from following material: seed, suspension culture, embryo, meristematic region, callus, leaf, root, bud, gametophyte, sporophyte, pollen and sporule.

" filial generation " comprises any subsequent generation of plant.

" at least one fungi is had to the plant of resistance " and refers to comprise the plant of recombinant dna construct of the present invention, when with this plant of fungi infestation, relative to the plant without recombinant dna construct of the present invention, its energy is anti-infective or standing infection to a greater extent, causes infringement minimizing, health degree increases and output is lost less or do not lose.Fungi is pathogenic agent normally." pathogenic agent " or " fungal pathogens " refers to will cause the fungi of plant disease under the condition not comprising recombinant dna construct of the present invention.The transgenic plant comprising recombinant dna construct of the present invention normally have more the plant of resistance at least one fungi than the same species plant without recombinant dna construct of the present invention.

plant expression system, expression cassette and carrier and conversion

Promotor guides vegetable cell mechanism to produce the DNA sequence dna of RNA from the adjacent encoding sequence downstream (3 ') of promotor.The etap when speed that the rna transcription thing that described promoter region affects gene synthesizes, synthesis and cell type.Described rna transcription thing is processed into mRNA, and the latter is as the template of the aminoacid sequence for RNA sequence being translated as coded polypeptide.5 ' end untranslated leader is region mRNA being positioned at upstream, protein-coding region, and it may play a role to the initial sum of mRNA translation.3 ' Transcription Termination/polyadenylation signal is the untranslated region being positioned at downstream, protein-coding region, and its effect in vegetable cell causes the termination of rna transcription and polyadenylic acid to the interpolation of RNA3 ' end.

The source of the promotor expressed for driving encoding sequence is unimportant, as long as it has enough transcriptional activities to pass through at reasonable time, the interpretable mRNA expressing desired nucleic acid fragment in desired host tissue, thus reaches object of the present invention.(namely endogenous) promotor of xenogenesis or non-xenogenesis all can be used for implementing the present invention.Such as, suitable promotor includes but not limited to: the α of β soybean companion globin promoter causes subunit, Ku Nizi (Kunitz) trypsin inhibitor 3 promotor, annexin promotor, glycinin Gy1 promotor, the β soybean companion β subunit of globin promoter, P34/GlyBdm30K promotor, albumin promoter, LegA1 promotor and LegA2 promotor.

Above-mentioned annexin or P34 promotor are described in PCT patent disclosure WO04/071178 (being published on August 26th, 2004).The activity level of above-mentioned annexin promotor and any known strong promoter are suitable, and described strong promoter is as: (1) CaMV35S promotor (people such as Atanassova, PlantMol.Biol.37:275-285 (1998); BattrawandHall, PlantMol.Biol.15:527-538 (1990); The people such as Holtorf, PlantMol.Biol.29:637-646 (1995); The people such as Jefferson, EMBOJ.6:3901-3907 (1987); The people such as Wilmink, PlantMol.Biol.28:949-955 (1995)); (2) Arabidopis thaliana oleosin (oleosin) promotor (people such as Plant, PlantMol.Biol.25:193-205 (1994); Li, TexasA & MUniversityPh.D.dissertation, pp.107-128 (1997)); (3) Arabidopis thaliana ubiquitin extension protein promotor (people such as Callis, JBiol.Chem.265 (21): 12486-93 (1990)); (4) tomato ubiquitin gene promotor (people such as Rollfinke, Gene.211 (2): 267-76 (1998)); (5) Soybean heat shock protein promoter (people such as Schoffl, MolGenGenet.217 (2-3): 246-53 (1989)); And (6) corn H3 histone gene promotor (people such as Atanassova, PlantMolBiol.37 (2): 275-85 (1989)).

Another useful property of above-mentioned annexin promotor is that it is growing the express spectra in seed.The most active in seed in the growth of above-mentioned annexin promotor (within pollinating latter 10 days) in early days, and substantially keep silent in period subsequently.The express spectra of above-mentioned annexin promotor and all different of many seed specific promoters, the latter is as usually shown the most highly active seed storage protein promoter (people such as Chen, Dev.Genet.10:112-122 (1989) at development later stage; The people such as Ellerstrom, PlantMol.Biol.32:1019-1027 (1996); The people such as Keddie, PlantMol.Biol.24:327-340 (1994); The people such as Plant, (the same); Li, (the same)).Above-mentioned annexin promotor has more conventional express spectra, but still known from other seed specific promoters is different.Therefore, when hope in early days etap process LAN or suppress a gene time, above-mentioned annexin promotor is a very attractive selection.Such as, may wish that process LAN one the regulation and control gene of early embryonic development or one participated in the gene of metabolism before seed maturity.

After identifying the suitable promotor being applicable to expressing encoding sequence, be utilized as the traditional method known by those skilled in the art, described promotor is operably connected with sense orientation.

The recombinant DNA of standard used herein and molecule clone technology are known in the art, and the people such as Sambrook, J. are at MolecularCloning:ALaboratoryManual; The second edition; ColdSpringHarborLaboratoryPress:ColdSpringHarbor, NewYork, 1989 (hereinafter referred to as " people such as Sambrook; 1989 ") or Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A.andStruhl, K., editor; At CurrentProtocolsinMolecularBiology; JohnWileyandSons:NewYork, has more detailed description in 1990 (hereinafter referred to as " people such as Ausubel, 1990 ").

After constructing recombinant precursor, by the method (such as transfection, conversion and electroporation) be well known for ordinary skill in the art, vegetable cell can be imported.

Also recombinant precursor of the present invention can be imported a kind of vegetable cell; Or also each recombinant precursor can be directed into different vegetable cells.

As previously mentioned, the phraseology in vegetable cell can be temporary also can be stable.

Plant part comprises differentiation and undifferentiated tissue, includes but not limited to following part: the cell of root, stem, bud, leaf, pollen, seed, tumor tissues and various ways and culture (such as individual cells, protoplastis, embryo and callus).Plant tissue can be present in plant or plant organ, tissue or cell culture.

Term " plant organ " refers to the plant tissue of the morphology and function different piece forming plant or organizes group.Term " genome " refers to: (1) be present in organism each cell or virus or organoid in a complete set of genetic material (gene and non-coding sequence); And/or (2) are as a complete set of karyomit(e) of (monoploid) unit from parent's heredity.

Obtain the method for transgenic plant for transform dicotyledonous plants (mainly utilizing agrobacterium tumefaciens), be published in following documents together with additive method: cotton (United States Patent (USP) 5,004,863; United States Patent (USP) 5,159,135); Soybean (United States Patent (USP) 5,569,834; United States Patent (USP) 5,416,011); For Brassica plants (Brassica) those (United States Patent (USP) 5,463,174); Peanut (people such as Cheng, PlantCellRep.15:653-657 (1996); The people PlantCellRep.14:699-703 (1995) such as McKently); Papaya (the people Bio/technology9:752-758 (1991) such as Ling, K.); And pea (the people PlantCellRep.15:254-258 (1995) such as Grant).To the summary of other common methods of Plant Transformation see Newell, C.A. (Mol.Biotechnol.16:53-65 (2000)).One of these method for transformation make use of rhizobiaceae (Tepfler, M.andCasse-Delbart, F.Microbiol.Sci.4:24-28 (1987)).The transformation of soybean that the utilization of having announced directly carries DNA to carry out, utilizes PEG to merge (PCT patent disclosure WO92/17598), utilize electroporation (people such as Chowrira, G.M., Mol.Biotechnol.3:17-23 (1995); The people such as Christou, P., Proc.Natl.Acad.Sci.84:3962-3966 (1987)), utilize microinjection and utilize biolistic bombardment (people such as McCabe, D.E, Bio/Technology6:923 (1988); The people such as Christou, PlantPhysiol.87:671-674 (1988)) carry out.

Exist multiple for the method from plant tissue aftergrowth.The concrete grammar of regeneration will depend on starting plant tissue and concrete plant species to be regenerated.From single plant protoplast transformants or from multiple explant regeneration through transforming, to grow and cultivate plants be (Weissbach and Weissbach (editor) known in the art, be loaded in: MethodsforPlantMolecularBiology, (Eds.), Academic:SanDiego, CA (1988)).This regeneration and growth method generally include following steps: select the cell of conversion and cultivate these individually oriented cells by the usual stage of embryonic development and by taking root the plantlet stage.Transgenosis embryo and seed regenerate in a similar fashion.Subsequently the genetically modified seedling of taking root of gained is planted in the Suitable plant growth substratum of such as soil and so on.Preferably, the plant of regeneration is carried out self-pollination to produce the transgenic plant of isozygotying.Or, the seed bearing plant of product deriving from strain important on the pollen of aftergrowth and agronomy is hybridized.On the contrary, the plant from these important strains is used for pollinate regenerated plants.Method well-known to those skilled in the art is utilized to cultivate the transgenic plant of the present invention containing required polypeptide.

Except program discussed above, professional is also familiar with describing the standard source data of the specified conditions for following purpose and program: macromolecular structure, operation and be separated (such as DNA molecular, plasmid etc.); The structure of recombinant dna fragment and recombinant expressed body; And clone screening be separated.For example, see: the people such as Sambrook, MolecularCloning:ALaboratoryManual, ColdSpringHarbor:NY (1989); The people such as Maliga, MethodsinPlantMolecularBiology, ColdSpringHarbor:NY (1995); The people such as Birren, GenomeAnalysis:DetectingGenes, the 1st volume, ColdSpringHarbor:NY (1998); The people such as Birren, GenomeAnalysis:AnalyzingDNA, the 2nd volume, ColdSpringHarbor:NY (1998); PlantMolecularBiology:ALaboratoryManual, editor, Clark, Springer:NY (1997).

The present invention relates to the separation polynucleotide of encoding serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase.As used herein; " polynucleotide " refer to the polynucleotide of coding novel serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase, and described enzyme relates to the triterpene that beta-amyrin in plant derives or the biosynthesizing with the triterpene changing modification.These separation are called AsSCPL1 (serine carboxypeptidase sample acyltransferase), AsMT1 (methyltransgerase) and AsGT2 (Transglucosylase) from the enzyme of black oat.

Sad7 mutant can not produce avenacin; triterpene glycoside (the people such as QiX that n-methyl oaminobenzoate (avenacin A-1 and B-1) or benzoate (avenacin A-2 and B-2) acyl group site have hydroxyl can not be accumulated in; 2004, Proc.Natl.Acad.Sci.U.S.A.101:8233).Therefore, there is not triterpene acidylate in them.Identify the mutant (table 1) that four have Sad7 phenotype.Each in them has and has infringement to polynucleotide of the present invention, by the function mRNA causing polynucleotide can not express encode functional protein.These data show (ASCPL1) serine carboxypeptidase sample acyltransferase of the nonmutationed polynucleotide encoding of the present invention from black oat together with biochemical data provided herein; this enzyme is responsible for the acidylate of triterpene main chain, and this acylation process does not occur in Sad7 mutant.Disclose the cDNA genomic fragment (SEQIDNO:1 and SEQIDNO:7) of coding AsSCPL1.

table 1: characterize Sad7 mutant.The base positions (subscript the 2nd row) changed of display based on comparing of mutant DNA sequence dna and SEQIDNO:1.

The methyltransgerase related in Sad9 gene product and the biosynthesizing of phenylpropionic acid class has homology, be included in blade and respond pathogenic agent and to attack and UV stimulates the barley methyltransgerase (people such as ChristensenA induced, 1998, PlantMol.Biol.36:219).Because this methyltransgerase is needed for avenacin synthesis, its effect may be the anthranilate part (Fig. 1) of avenacin A-1 and B-1 of methylating.Identify the mutant (table 2) that four have Sad9 phenotype.Each in them has and has infringement to polynucleotide of the present invention, by the function mRNA causing polynucleotide can not express encode functional protein.These data show the nonmutationed polynucleotide encoding of the present invention (AsMT1) methyltransgerase from black oat together with biochemical data provided herein, and this enzyme is responsible for methylating the anthranilate part of avenacin A-1 and B-1.Disclose the cDNA genomic fragment (SEQIDNO:3 and SEQIDNO:8) of coding AsMT1.

table 2:Sad9 mutant characterizes.The base positions (subscript the 2nd row) changed of display based on comparing of mutant DNA sequence dna and SEQIDNO:3.

The enzyme of Sad10 Transglucosylase and saccharification Whitfield's ointment and other benzoic acid derivative has homology.Disclose coding from the cDNA of the glucosylation of black oat (AsGT2) and genomic fragment (SEQIDNO:5 and SEQIDNO:9).

The gene identification that coding is responsible for the enzyme of triterpene synthesis in various types of grain will allow their manipulation.The change that will cause triterpenoid saponin level or structure is handled in triterpene synthesis.

Nucleic acid fragment of the present invention can be used for being separated cDNA and encoding from the gene of the homologous protein of identical or other microbial species.It is well known in the art for using sequence dependent code to be separated homologous gene.The example of sequence dependent form code includes but not limited to the various application (such as, polymerase chain reaction, ligase chain reaction) of nucleic acid hybridization and DNA and RNA amplification method such as nucleic acid amplification technologies.

Such as; to encode the gene of other acyltransferase (specifically those serine carboxypeptidase sample acyltransferases), Transglucosylase or methyltransgerase; directly can be separated with the form of cDNA or genomic dna; described separation uses method well known to those skilled in the art, makes DNA hybridization probe with the library of screening from any required plant by using all or part of nucleic acid fragment of the present invention.Can design and pass through methods known in the art, based on of the present invention or the sequent synthesis specific oligonucleotide probe (people such as Sambrook, MolecularCloning:ALaboratoryManual, ColdSpringHarbor:NY (1989)).Such as, and whole sequence can be directly used in by the known method of those of skill in the art, and random primer DNA marker, nick translation or end-labelling, carry out synthesized dna probe, or use obtainable in-vitro transcription system to synthesize rna probe.In addition, can Auele Specific Primer be designed and use it for amplification part or the sequence of the present invention of total length.The amplified production of gained can directly mark or mark after amplified reaction in amplified reaction process, and is used as cDNA or genomic fragment that probe is separated total length under suitable stringent condition.

In addition, two of a nucleic acid fragment of the present invention short-movie section can be used in polymerase chain reaction code the longer nucleic acid fragment from DNA or RNA amplification encoding homologous genes.Polymerase chain reaction also can carry out with the library of the nucleic acid fragment of clone, and the sequence source of one of them primer is from nucleic acid fragment of the present invention, and the sequence of another primer utilizes the existence of the 3 ' polyadenylic acid sheet held of the mRNA precursor of coded plant gene.Alternatively, second primer sequence can based on the sequence deriving from cloning vector.Such as, technician can according to RACE code (people such as Frohman, 1988, Proc.Natl.Acad.Sci.U.S.A., 85:8998-9002), cDNA is produced by the copy in the region between holding with pcr amplification Single locus and 3 ' or 5 ' in transcript.With 3 ' and 5 ' primer of direction orientation can use sequences Design of the present invention.Use can 3 ' RACE or the 5 ' RACE system (BRL) that obtains of business, can be separated specific 3 ' or 5 ' cDNA fragment (people such as Ohara, 1989, Proc.Natl.Acad.Sci.U.S.A86:5673-5677; The people such as Loh, 1989, Science243:217-220).Capable of being combined use by 3 ' and 5 ' product of RACE Program Generating to generate full-length cDNA (FrohmanandMartin, 1989, Techniques1:165).

The operability of nucleotide sequence of the present invention and its derived amino acid sequence is conducive to immunoscreening cDNA expression library.The synthetic peptide can synthesizing aminoacid sequence of the present invention represents part.These peptide immune animals can be used to produce polyclone or monoclonal antibody, and described antibody has the specificity to the peptide or albumen comprising described aminoacid sequence.Then these antibody can be used for screening cDNA expression library to be separated concerned full length cDNA clone (Lerner, 1984, Adv.Immunol.36:1-34; Sambrook).

The plasmid vector comprising separation polynucleotide of the present invention can be built.The selection of plasmid vector is depended on and will be used for the method for transformed host cell.Technician knows to successfully transform, screening and breed the host cell comprising mosaic gene, must be present in the genetic elements on plasmid vector.Technician also will recognize, different separate transformation events will cause expression (people such as Jones, 1985, EMBOJ., 4:2411-2418 of different levels and pattern; The people such as DeAlmeida, 1989, Mol.Gen.Genetics218:78-86), therefore, multiple events may must be screened to obtain the strain that expression level and pattern are expected in display.Such screening can be analyzed by the Western of the Northern engram analysis of the Southern engram analysis of DNA, mrna expression, protein expression or phenotype analytical completes.

Polypeptide of the present invention imported different cellular compartments with regard to some application or promote that they may be useful from the secretion cell.Therefore recombinant dna construct of the present invention can be supplemented by imagination further, this is undertaken by the encoding sequence changing target signal in the suitable cell of coding, as encoding transport signals (Keegstra, 1989, Cell56:247-253), there is or not have the signal sequence (Chrispeels of endoplasmic reticulum retention signal, 1991, Ann.Rev.PlantPhys.PlantMol.Biol.42:21-53) or remove or do not remove the nuclear localization signal (Raikhel of existing target signal, N., 1992, PlantPhys.100:1627-1632).When the reference of quoting provides the example of each signal in these signals, described list is incomplete, may find more target signal in the future.

The such as expression of chimeric serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase causes respectively compared with the level produced in unconverted host cell, serine carboxypeptidase sample acyltransferase, methyltransgerase or the Transglucosylase protein level change coded by producing in transformed host cell.The transgenic plant or the plant part that comprise polynucleotide of the present invention also cause plant to have the triterpene level of change under the control of allogeneic promoter, described polynucleotide such as SEQIDNO:1, SEQIDNO:3 and SEQIDNO:5, SEQIDNO:7, SEQIDNO:8 and SEQIDNO:9.Plant can be selected from monocotyledons and dicotyledons.Monocotyledons includes but not limited to corn, oat, rice, wheat, barley, palm etc.Dicotyledons includes but not limited to Arabidopis thaliana, soybean, oleaginous seed rape, peanut, Sunflower Receptacle, safflower, cotton, tobacco, tomato, potato, cocoa beans etc.Plant part includes but not limited to such as seed and cereal.Therefore, separation polynucleotide of the present invention can be attached to and can import and in the recombinant precursor copied in host cell.

Therefore, the invention still further relates to the method for transformant, described method comprises with recombinant precursor transformant of the present invention and selects those with the present invention's cell that wherein a kind of recombinant precursor is transformed.Also pay close attention to the method for the production of conversion of plant, described method comprises by any polynucleotide transformed plant cells of the present invention and from transformed Plant cell regeneration plant.

Nucleic acid fragment of the present invention can be used to produce transgenic plant; in transgenic plant, the level of acyltransferase of the present invention, Transglucosylase or methyltransgerase is higher than normal plants, or they do not produce the cell type of these enzymes under normal circumstances or exist in the etap.This effect that triterpene in those cells that change is produced.It is believed that the overexpression of the polynucleotide of the present invention that the polynucleotide being optionally responsible for the enzyme of other step in saponin(e biosynthetic pathway with coding combine improves the resistance at least one fungi.

The overexpression of serine carboxypeptidase sample acyltransferase of the present invention, Transglucosylase or methyltransgerase completes by following steps: first build recombinant dna construct; wherein coding region is operably connected to a promotor, and this promotor can guide the first enzyme of triterpene approach (as serine carboxypeptidase sample acyltransferase of the present invention, Transglucosylase or methyltransgerase) in required tissue and the expression in the required etap.Recombinant dna construct can comprise the promoter sequence and translation leader sequence that derive from homologous genes.3 ' non-coding sequence of encoding transcription termination signal also can be provided.Above-mentioned recombinant dna construct also can comprise the intron that one or more promote genetic expression.

In order to improve the flow of triterpene approach, the above-mentioned recombinant dna construct of serine carboxypeptidase sample acyltransferase, methyltransgerase or glucosylation expression of enzymes that can guide can be attached in transgenic plant.

In addition, the recombinant precursor comprising gene of the present invention can be built, wherein except may be operably coupled to the coding region of the promotor that can guide the first expression of enzymes of triterpene approach, recombinant precursor also comprises at least one other coding region, and this coding region may be operably coupled to the promotor that can guide at least one the second expression of enzymes of the present invention.Expect to be separated and include but not limited to from the example of the assortment of genes of the present invention of black oat: AsSCPL1+AsMT1; AsSCPL1+AsGT2; AsGT2+AsMT1 and AsSCPL1+AsMT1+AsGT2.

Also the construct by being combined in the enzyme of the first two step in above-mentioned recombinant precursor in transgenic plant and coding pass obtains the approach flux improved, and in described approach, the enzyme of the first two step is the oxidosqualene cyclase beta-amyrin synthase (product of Sad1 gene; HaralampidisK. wait people, 2001, Proc.Natl.Acad.Sci.U.S.A.98:13431-13436) and/or cytochrome P 450 enzymes CYP51H10 (by Sad2 genes encoding; QiX. people is waited, 2006, Proc.Natl.Acad.Sci.U.S.A.103:18848-18853).Have previously been described construct (people such as Osbourn, on March 6th, 2007, US, 7,186,884B2 for these two genes of overexpression; The people such as Osbourn, US2006-0112448A1).

In addition, the recombinant precursor comprising gene of the present invention can be built, wherein except may be operably coupled to the coding region of the promotor that can guide the first expression of enzymes of triterpene approach, recombinant precursor also comprises at least one other coding region, and this coding region may be operably coupled to the promotor that can guide at least one the second expression of enzymes of triterpene approach.Expect to be separated from the gene of the present invention of black oat and to include but not limited to from the example combinations of other gene of triterpene approach:

AsSCPL1+Sad1；AsMT1+Sad1；AsGT2+Sad1；AsSCPL1+Sad2；AsMT1+Sad2；AsGT2+Sad2；AsSCPL1+AsMT1；AsSCPL1+AsGT2；AsGT2+AsMT1；AsSCPL1+AsMT1+AsGT2；AsSCPL1+AsMT1+AsGT2+Sad1；AsSCPL1+AsMT1+AsGT2+Sad2；AsSCPL1+Sad1+Sad2；AsMT1+Sad1+Sad2；AsGT2+Sad1+Sad2；AsSCPL1+AsMT1+Sad1+Sad2；AsSCPL1+AsGT2+Sad1+Sad2；AsMT1+AsGT2+Sad1+Sad2；AsSCPL1+AsMT1+AsGT2+Sad1+Sad2；

AsSCPL1+AsMT1+Sad1; AsSCPL1+AsGT2+Sad1; AsGT2+AsMT1+Sad1; AsSCPL1+AsMT1+Sad2; AsSCPL1+AsGT2+Sad2; And AsGT2+AsMT1+Sad2;

Also the expression can expecting to reduce or eliminate serine carboxypeptidase sample acyltransferase of the present invention, methyltransgerase or Transglucosylase in plant is applied for some.Suppress polynucleotide of the present invention that plant can be caused to produce less saponin(e, this can improve local flavor then.In order to reach this object; can build the recombinant dna construct being designed for this fermentoid of co-suppression, this is constructed by and is connected on plant promoter sequences by the polynucleotide of coding serine carboxypeptidase sample acyltransferase of the present invention, methyltransgerase or Transglucosylase.Alternatively, can build the mosaic gene being designed for the sense-rna of expressing all or part of nucleic acid fragment of the present invention, this is constructed by and gene or gene fragment is reversely connected on plant promoter sequences.Via conversion, co-suppression or antisense mosaic gene can be imported in plant, wherein reduce or eliminate the expression of corresponding native gene.Also can prepare the construct of chimeric nucleic acid fragment causing forming stem-ring structure, wherein polynucleotide part of the present invention is stem or ring or structure.Use the nucleotide sequence of coding serine carboxypeptidase sample acyltransferase of the present invention, methyltransgerase or Transglucosylase to prepare the construct suppressing it to express with RNAi form at least partially be also possible.Can by any recombinant dna construct transfered cell mentioned above to eliminate the expression of serine carboxypeptidase sample acyltransferase of the present invention, methyltransgerase or Transglucosylase in plant.In addition, this type of builds physical efficiency and comprises oxidosqualene cyclase, the recombinant precursor combination (product of Sad1 gene of beta-amyrin synthase fragment; (people such as HaralampidisK., 2001, Proc.Natl.Acad.Sci.U.S.A.98:13431-13436) and/or cytochrome P 450 enzymes CYP51H10 are (by Sad2 genes encoding; QiX. people is waited, 2006, Proc.Natl.Acad.Sci.U.S.A.103:18848-18853) with the institute in suppression approach in steps.Expect that the example combinations being separated the gene of the present invention from black oat and other gene from triterpene approach (can carry out the level combining to change or reduce triterpenoid saponin) includes but not limited to:

AsSCPL1+Sad1；AsMT1+Sad1；AsGT2+Sad1；AsSCPL1+Sad2；AsMT1+Sad2；AsGT2+Sad2；AsSCPL1+AsMT1；AsSCPL1+AsGT2；AsGT2+AsMT1；AsSCPL1+AsMT1+AsGT2；AsSCPL1+AsMT1+AsGT2+Sad1；AsSCPL1+AsMT1+AsGT2+Sad2；AsSCPL1+Sad1+Sad2；AsMT1+Sad1+Sad2；AsGT2+Sad1+Sad2；AsSCPL1+AsMT1+Sad1+Sad2；AsSCPL1+AsGT2+Sad1+Sad2；AsMT1+AsGT2+Sad1+Sad2；AsSCPL1+AsMT1+AsGT2+Sad1+Sad2；

AsSCPL1+AsMT1+Sad1; AsSCPL1+AsGT2+Sad1; AsGT2+AsMT1+Sad1; AsSCPL1+AsMT1+Sad2; AsSCPL1+AsGT2+Sad2; And AsGT2+AsMT1+Sad2;

In certain embodiments, the nucleotide sequence of the present embodiment can with any combination stacked of concerned polynucleotide sequence to produce the plant with desired phenotype, and described sequence can be genetically modified or not genetically modified.Such as, the polynucleotide of described embodiment can carry out stacking with other polynucleotide any of this embodiment or other gene.The combination generated also can comprise any one in multiple copies of concerned polynucleotide.The polynucleotide of described embodiment also can occur stacking to produce the plant with multiple desired characteristic with other gene any or the assortment of genes, include but not limited to that characteristic needed for animal-feed is if high oil base is because of (such as U.S. Patent Publication 6,232,529); Amino acid (such as thionin (U.S. Patent Publication 5,990,389; 5,885,801; 5,885,802; With 5,703,409); Barley high-lysine (people such as Williamson, (1987), Eur.J.Biochem.165:99-106; And WO98/20122); With homomethionine albumen (people such as Pedersen, (1986), J.Biol.Chem.261:6279; The people such as Kirihara, (1988), Gene71:359; With people such as Musumura, (1989), PlantMol.Biol.12:123)); Digestibility (the reserve protein (U.S. Patent Application Serial Number 10/053,410 is filed in November 7 calendar year 2001) such as modified improved; With Trx (U.S. Patent Application Serial Number 10/005,429 is filed in December 3 calendar year 2001)), to be above-mentionedly openly incorporated herein by reference.The polynucleotide of described embodiment also can (such as B. thuringiensis Toxin protein (U.S. Patent Publication 5,366,892 stacking with the characteristic desired by anti-insect, disease or weedicide; 5,747,450; 5,737,514; 5723,756; 5,593,881; The people such as Geiser (1986) Gene48:109); Lectin (people (1994) PlantMol.Biol.24:825 such as VanDamme); Fumonisins detoxification (fumoBisindetoxification) gene (U.S. Patent Publication 5,792,931); Nontoxicity and disease-resistant gene (people (1994) Science266:789 such as Jones; The people such as Martin (1993) Science262:1432; The people such as Mindrinos (1994) Cell78:1089); Cause acetolactate synthase (ALS) mutant of Herbicid resistant as S4 and/or the Hra mutant (people such as Lee, (1988) EMBOJ.7 (5): 1241-1248), anti-glutamine synthase inhibitor is as glufosinates or basta (such as bar gene; The people such as DeBlock (1987) EMBOJ.6:2513-2518); Give the HPPD gene that HPPD suppresses Herbicid resistant, described weedicide is as mesotrione or different azoles humulone (people such as Matringe, (2005), PestManagementScience61:269-276; The people such as Dufourmantel, (2007) PlantBiotech.J.5:118-133; Also see WO1997049816), give the gene (Li and Nicholl, (2005) PestManagmentScience61:277-285) that PPO suppresses Herbicid resistant; Synthetic auxin resistant gene (the people such as U.S. Patent application 2005/014737 and Herman, (2005), J.Biol.Chem.280:24759-24767), and glyphosate resistance (epsps gene, gat gene are disclosed in the gene in U.S. Patent Publication Shen Qing Publication US2004/0082770, WO02/36782 and WO03/092360 as those)); With the characteristic desired by process or process product as high oil (such as U.S. Patent Publication 6,232,529); Modified oil (such as fatty acid desaturase gene (U.S. Patent Publication 5,952,544; WO94/11516)); Treated starch (such as ADPG pyrophosphorylase (AGPase), starch synthase (SS), starch collateralization enzyme (SBE) and starch remove collateralization enzyme (SDBE)); And polymer biological plastics (such as U.S. Patent Publication 5.602,321; β-ketothiolase, poly(hydrobutyl ester) synthase and acetoacetyl-CoA reductase (people (1988) J.Bacteriol.170:5837-5847 such as Schubert), be conducive to expressing polyhydroxyalkanoatefrom (PHAs)), it is openly incorporated herein by reference.Also can combine the polynucleotide of described embodiment and the polynucleotide of agronomic characteristics are provided, described agronomic characteristics is if male sterile (for example, see U.S. Patent Publication 5.583,210), stalk intensity, flowering time, output increased or transformation technology characteristic are as Cycle Regulation or gene target (such as WO99/61619; WO00/17364; WO99/25821), it is openly incorporated herein by reference.

Produce these stacking combinations by any method, include but not limited to by any ordinary method or method or gene transformation cross breeding plants.If described characteristic is stacking by genetic transformation plant, then concerned polynucleotide sequence can combine with any order at any time.Such as, the transgenic plant comprising one or more desired characteristic can be used as target, import more characteristics by follow-up conversion.Characteristic and concerned polynucleotide can be imported by cotransformation code, described polynucleotide are provided by any combination transforming box simultaneously.Such as, add importing two sequences, two sequences can be included in the conversion box of separation (trans) or to be included in (cis) in same conversion box.Promote that sequence is expressed by identical promoters or different promoters.In some instances, may expect to import the conversion box by suppressing concerned polynucleotide to be expressed.This can suppress any combination of box or overexpression box to combinationally use with other to combine to generate desired characteristic in plant.Recognize further and Site-specific recombinase system can be used at the stacking polynucleotide sequence of required genomic locus.See such as WO99/25821, WO99/25854, WO99/25840, WO99/25855 and WO99/25853, they are all incorporated herein by reference.

Embodiment of the present invention can effective anti-various plants fungal pathogens.The above-mentioned plant causing the recombinant precursor producing the horizontal saponin(e of higher triterpene to produce anti-at least one fungi can be used.Some specificity fungal pathogens of staple crop include but not limited to following bacterial classification: soybean: Kidney bean charcoal rot bacterium, dry thread Pyrenomycetes, Sclerotinia sclerotiorum, Fusarium oxysporum, beanpod carving maize ear rot and Phoma asparagi Sacc (Phomopsissojae), soybean north stem canker, white thin,tough silk bacterium, Cercospora kikuchii, Germ To Soybean Frogeye Leaf Spot, C. dematium (Colletotichumtruncatum), paragutta rod spore mould fallen leaves germ, Septoria Glycines, Phyllosticta hydrangea, alternaric bacteria, soybean Powdery Mildew, Fusarium semitectum, brown stem rot bacterium, black fruit bacterium glycine, soybean rest fungus, lanthanum element is to sickle-like bacteria, canola: black spot of cabbage bacterium, low sauerkraut seed oil black shank bacterium, dry thread Pyrenomycetes, Sclerotinia sclerotiorum, rape ring spot bacterium, Fusarlum roseum, alternaric bacteria, clover: Herba Medicaginis stem point mould, clover tail spore, the false cup fungi of clover, clover cilium bacterium L, Fusarium oxysporum, verticillium albo atrum reinke & berthold, stemphylium bacterium, clover stemphylium bacterium, clover anthrax bacteria, the little bare hull bacterium of clover, rust of alfalfa bacterium, Scleroitinia trifoliorum in alfalfa bacterium, clover shell many spores tikka bacterium, clover stemphylium botryosum, common leaf spot of alfalfa bacterium, wheat: wheat bar ustilago, black embryo of wheat germ rod method, the mould germ of Wheat Black, wheat sickle-like bacteria, fusarium culmorum, wheat loose smut bacterium, wheat shell two spore, wheat class Population of Xanthomonas Oryzae Pv, the raw anthrax-bacilus of standing grain, wheat powdery mildew, puccinia graminis bacterium, Puccinia recondita, strip rust bacteria, wheat brown patch germ, glume blight bacterium, leaf spoting bacteria, oat septoria musiva, wheat Phyllostachys pubescens, dry thread Pyrenomycetes, Rhizoctonia cerealis, gaeumannomyces graminis, Cochliobolus sativus, Claviceps purpurea, ryegrass LPC, bunt of wheat bacterium, wheat loose smut bacterium, tilletia indica mitra, dry thread Pyrenomycetes, sunflower Receptacle: Plasmopara Halstedll, Sclerotinia sclerotiorum, Septoria helianthi, Sunflower Receptacle brown rot germ, Sunflower Receptacle alternaria, Alternaria zinniae, Botrytis cinerea germ, Sunflower Receptacle Phoma sp black stem bacterium, Kidney bean shell ball spore bacterium, two spore powdery mildews, Rhizopus oryzae, rhizopus arrhizus, rhizopus stolonifer, Puccinia helianthi, verticillium dahliae, cephalosporium sp, corn: fine strain of millet anthrax bacteria (Glomerellagraminicola), Diplodia zea bacterium (Diplodiamaydis), corn top rot fusarium moniliforme, endogenous colyliform reaping hook is mould, Fusarium graminearum (Gibberellazeae), flavus, southern corn leaf blight, T (southern corn leaf blight), D. carbonum I, II & III (Cochlioboluscarbonum), Exserohilum turcicum I, II & III, obstruct compacted shape bacterium, rice brown patch germ, maize leaf spoting bacteria, corn eye spot bacterium, jowar tail spore, Ustilago maydis (D C.) Corola., corn handle rest fungus, Puccinia polysora, Kidney bean shell ball spore bacterium, penicillium oxalicum, rice Alternaria, standing grain black bud branch is mould, lunata, the curved spore such as not, Maize Curvularia, viride, the tough and tensile ustilago of corn, corn dry rot germ, maize crazy top bacterium, high grain woods smut-fungi, corn rust bacterium, wheat class Population of Xanthomonas Oryzae Pv, cephalosporium acremonium, chinese sorghum: Exserohilum Turcicum, sorghum leaf purple spot bacterium, Chinese sorghum cercospora glue bacterium, Chinese sorghum thick spot shell two spore, jowar rest fungus, Kidney bean shell ball spore bacterium, the mould pine root fungus of the black chopped spring onion of Chinese sorghum, fusarium moniliforme, alternaric bacteria, for chinese sorghum life is from compacted spore bacterium, the long compacted spore of bromegrass, lunata, Chinese sorghum Phoma sp, Chinese sorghum seat branch spore, jowar raw seat branch spore, Chinese sorghum black mole bacterium, head smut of sorghum bacterium (Sphacelothecareiliana), sorghum loose smut bacterium, fine strain of millet hard spore heap ustilago, Chinese sorghum ergot, dry thread Pyrenomycetes, branch top spore is mould, black fruit bacterium (Colletotrichum) (C.sublineolum), Fusarium graminearum, Fusarium oxysporum, etc..

Nucleotide sequence of the present invention can be used to carry out the multiple gene based on nucleic acid amplification and physics drawing practice.Example includes but not limited to the polymorphism analysis (CAPS of allele specific amplification (Kazazian, H.H.jr, 1989, J.Lab.Clin.Med.11:95-96), pcr amplified fragment; Sheffield, V.C., wait people, 1993, Genomics16:325-332), allele-specific connect (Landegren, U., Deng people, 1988, Science241:1077-1080), Nucleotide extension (Sokolov, B.P., 1990, NucleicAcidRes.18:3671), the radiation hybrid mapping (people such as Walter, M.A., 1994, Nat.Genet.7:22-28), fluorescence in situ hybridization (FISH; Svitashev, S.K. and Somers, D.A., 2002, PlantCellTissueOrganCult.69:205-214) and Happy drawing (Dear, P.H. and Cook, P.R., 1989, NucleicAcidRes.17:6795-6807).With regard to these methods, nucleic acid fragment sequence is used for designing and prepares primer pair and be used for amplified reaction or primer extension reaction.The design of this type of primer is well-known to those having ordinary skill in the art.In the method for genetic mapping using PCR-based, the DNA sequence dna difference between the drawing hybrid strain in the region corresponding to nucleotide sequence of the present invention must be identified.But this is generally unnecessary concerning all drawing practices.

Although do not wish the constraint by any theory or theory of operation, those skilled in the art believes that the triterpene level of change has different effects.Can give the resistance of plant to this type of pathogenic agent of at least some in triterpene (as the avenacin) level that usually can suffer to improve in the plant part of fungal pathogen challenge, therefore protective plant also improves its output in fungi stressful environmental.The food deriving from the plant with high triterpene level is considered to have the effect reducing cholesterol, but triterpene level reduces and it is believed that food can be made to have better local flavor.Therefore, the plant in process of growth with AsSCPL1, AsMT1 and/or AsGT2 of change level can contribute to producing the more nutritious and/or better food of local flavor.Therefore, the present invention also comprises the cereal from transgenic plant of the present invention.

Embodiment

The present invention will limit in the following embodiments further, and wherein all numbers and per-cent are by weight and the number of degrees are degree Celsius, unless otherwise indicated.Should be appreciated that, although the embodiments illustrate the preferred embodiments of the invention, only provide in an exemplary manner.According to discussion above and these embodiments, those skilled in the art can determine essential characteristic of the present invention, and without departing from the spirit and scope of the present invention, multiple change and modification can be made to the present invention, be applicable to multiple usage and condition to make it.Therefore, except those herein shown in and describe those except, according to above, various modification of the present invention will be apparent to one skilled in the art.These modification are also intended to belong in the scope of additional claims.

general method

The standard recombinant dna technology used in embodiment and molecule clone technology are known in the art and described by having in such as Publication about Document: (1) Sambrook, J., Fritsch, E.F. and Maniatis, T.MolecularCloning:ALaboratoryManual; ColdSpringHarborLaboratory:ColdSpringHarbor, NY (1989) (Maniatis); (2) T.J.Silhavy, M.L.Bennan and L.W.Enquist, ExperimentswithGeneFusions; ColdSpringHarborLaboratory:ColdSpringHarbor, NY (1984); (3) people such as Ausubel, F.M., CurrentProtocolsinMolecularBiology, publishes (1987) by GreenePublishingAssoc.andWiley-Interscience.

The materials and methods of the maintenance and growth that are applicable to microorganisms cultures is well known in the art.The technology being applicable to embodiment below can at ManualofMethodsforGeneralBacteriology (PhillippGerhardt, R.G.E.Murray, RalphN.Costilow, EugeneW.Nester, WillisA.Wood, NoelR.Krieg and G.BriggsPhillips edits), AmericanSocietyforMicrobiology:Washington, D.C. (1994)); Or ThomasD.BrockinBiotechnology:ATextbookofIndustrialMicrob iology, the 2nd edition, find in the description of SinauerAssociates:Sunderland, MA (1989).For all reagent of the growth of microorganism cells and maintenance, restriction enzyme and material all available from AldrichChemicals (Milwaukee, WI), DIFCOLaboratories (Detroit, MI), GIBCO/BRL (Gaithersburg, or SigmaChemicalCompany (St.Louis MD), MO), unless otherwise indicated.

General molecular cloning has come according to standard method (people such as Sambrook, the same).DNA sequence dna is on ABI automatic sequencer, adopt dye terminator technology (United States Patent (USP) 5,366,860; EP272,007) use the combination of carrier and Insert Fragment Auele Specific Primer to produce.Sequence editor carries out in Sequencher (GeneCodesCorporation, AnnArbor, MI).All sequences covers at least twice in the two directions.Gene order be relatively use DNASTAR software (DNAStar, Inc.) complete.

The implication of abbreviation is as follows: " sec " represents second, " min " expression minute, " h " expression hour, " d " represents sky, " μ L " represents microlitre, and " mL " represents milliliter, and " L " represents liter, " μM " represents micro-molar concentration, " mM " represents millimolar concentration, and " M " represents volumetric molar concentration, and " mmol " represents mmole, " μm ole " represents micromole, " g " expression gram, " μ g " represents microgram, and " ng " represents nanogram, " U " representation unit, " bp " represents base pair and " kB " represents kilobase pair.

embodiment 1

be separated serine carboxypeptidase sample albumen (AsSCPL1), methyltransgerase (AsMT1) and the genome of Transglucosylase (AsGT2) and cDNA fragment

Coding is separated from BAC library by the genomic polynucleotide fragment of the gene that serine carboxypeptidase sample albumen (AsSCPL1), methyltransgerase (AsMT1) affect with Transglucosylase (AsGT2), and this library derives from black oat accession number S75 genomic dna and the cDNA library of following preparation from oat.

BAC library builds from black oat accession number S75 genomic dna ((people such as QiX., 2006, Proc.Natl.Acad.Sci.U.S.A103:18848-18853).DNA probe derives from Sad1 (people such as Osbourn, on March 6th, 2007, US, 7,186,884B2) and Sad2 (people such as Osbourn, US2006-0112448A1), and this probe is for screening complete BAC library.Build the BAC contig of leap one for the biosynthetic gene cluster of avenacin people such as (, 2004, Proc.Natl.Acad.Sci.U.S.A.101:8233-8238) QiX..BAC fingerprint and BAC terminal sequence analysis make us can assemble a contig, and this contig is cloned 462F14,460D15 and 409O10 by BAC and formed.Clone 460D15 comprises Sad1 and Sad2 people such as (, 2006, Proc.Natl.Acad.Sci.U.S.A103:18848-18853) QiX.; Clone 462F14 has the overlapping region of about 70kb and 460D15 in the side near Sad2, but clone 409O10 and clone 460D15 are at the overlapping about 35kb in side near Sad1.Use the end sequence of 409O10 as probe, identify that another BAC clones 341P21.BAC fingerprint and pcr analysis confirm the other end of clone 409O10 and the overlapping about 40kb of clone 341P21.Predict that the length of complete contig is 365kb.

The separation of oat root mRNA and the structure of corresponding cDNA library are described in the people such as HaralampidisK., 2001, Proc.Natl.Acad.Sci.U.S.A.98:13431-13436.In EcoRI/XhoI site, cDNA insertion sequence is cloned in pGADT7 carrier (ClonTechLab, Inc.).To amount to 36,864 clones are stored in 96384 hole microplates.Complete oat root cDNA library lattice is placed on two strainers and is used for hybridization.The insertion sequence DNA cloning 409O10 and 341P21 from BAC is separated by NotI digestion, and used as probe to screen cDNA library.Identify total 60 positive colonies and these insertion sequences of cloning are checked order.Order-checking uses ABI big-Dye ^tMterminatorCycleSequencingReadyReactionKit (AppliedBiosystems) carries out.CDNA sequence is obtained from 52 clones.Except 5 special sequences, remaining 47 cDNA insertion sequences are divided into four genoids.Be further analyzed to identify the longest cDNA clone and the full-length cDNA identifying each class in four classes subsequently to them.

The first kind represents the cDNA of Sad1, therefore confirms that in fact BAC contig does not cross over the gene cluster comprising this gene.

Predict the full-length cDNA encoding serine carboxypeptidase sample albumin A sSCPL1 from Equations of The Second Kind.The nucleotide sequence of this gene is shown in SEQIDNO:1.The aminoacid sequence that the Nucleotide 56 to 1534 of SEQIDNO:1 is derivative is shown in SEQIDNO:2.Nucleotide 1535 to 1537 represents a terminator codon.

3rd class cDNA sequence corresponds to the methyltransgerase of prediction, AsMT1.The nucleotide sequence of this gene is shown in SEQIDNO:3.The aminoacid sequence that the Nucleotide 13 to 1074 of SEQIDNO:3 is derivative is shown in SEQIDNO:4.Nucleotide 1075 to 1077 represents a terminator codon.

4th class cDNA sequence corresponds to the glucosylation enzyme family 1, AsGT2 of a prediction.By obtaining full-length cDNA to the sequential analysis of oat root presumption Transglucosylase EST (refSad1patent).The nucleotide sequence of this gene is shown in SEQIDNO:5.The derived amino acid sequence of the Nucleotide 66 to 1475 of SEQIDNO:5 is shown in SEQIDNO:6.Nucleotide 1458 to 1460 represents a terminator codon.

In order to obtain the genome sequence of said gene, by standard BAC air gun sequencing, 462F14,460D15,409O10 and 341P21 being cloned to the BAC from contig and checking order.Obtain the BAC contig of about 317kb, this contig comprises the gene (beta-amyrin synthase (Sad1), Cytochrome P450 CYP51H10 (Sad2), serine carboxypeptidase sample albumin A sSCPL1, methyltransgerase AsMT1 and Transglucosylase AsGT2) (Fig. 2) of the biosynthetic enzyme of five predictions.Sequence annotation does not disclose more open reading frame widely.Northern blotting and RT-PCR analyze all five genes shown in BAC contig and preferentially express (Fig. 3) in roots.Because avenacin synthesizes at oat root and gathers, and Sad1 and Sad2 is in root-preferred expression, other three Gene A sSCPL1, AsMT1 and AsGT2 also may relate to the biosynthesizing of avenacin.

The comparison of genomic dna sequence and cDNA sequence provides prediction promotor and the genome sequence of three genes of serine carboxypeptidase sample albumen (AsSCPL1), methyltransgerase (AsMT1) and Transglucosylase (AsGT2).The coding genomic polynucleotide fragment of AsSCPL1, AsMT1 and AsGT2 and the 3-kb promoter sequence of prediction are shown in SEQIDNO:7, SEQIDNO:8 and SEQIDNo:9.

embodiment 2

be separated and characterize Sad7 oat mutant

Carry out the seed of mutagenesis diploid oat (black oat) with sodium azide, make the M2 seed germination from single M1 plant and assess root fluorescence to carry out just screening, qualification saponin(e lacks or Sad oat mutant.Candidate's avenacin lacks mutant to be identified based on the root fluorescence reduced, and is confirmed by the methyl alcohol root extract that TLC and HPLC analyzes from M3 seedling of isozygotying.

generate mutant

Substantially according to seed (the accession number S75 of described method sodium azide mutagenesis diploid oat (black oat), from InstituteofGrasslandsandEnvironmentalResearch, Aberystwyth, Wales, UK) (Rines, H.W., 1985, Env.Exp.Bot., 25:7-17).In brief, mutagenesis is carried out as follows.Be immersed in by seed in the Erlenmeyer flask with rubbery stopper sealing in advance, every seed uses 0.5mL water, with 120 circulation per minute vibrations in track platform rocker.Water is poured out after 4 hours in pre-soaking.Be prepared in the 10mM sodium azide solution in 0.1M sodium phosphate, pH3.2, and it is added in seed immediately.As above carrying out vibration after 1 hour, pour out mutagenesis solution and rinse seed 5 to 6 times with water, wherein last three washing times extended to more than 30 minutes.Seed after rinsing is drained moisture, spreads on the paper in stink cupboard dry to it.Make the M2 seed germination from single M1 plant and assess root fluorescence as shown below.

Main avenacin avenacin A-1 comprises N-methyl anthranilic acid, therefore mainly causes the light blue fluorescence (people such as OsbournA.E., 1994, Physiol.Mol.PlantPathol.45:457-467) of oat seedlings root.Avenacin A-1 detects by UV light, and this makes root fluorescence to be used as just screening to identify that saponin(e lacks (Sad) oat mutant.Make the seed germination of single M2 family and assess its root fluorescence.In preliminary screening, represent 1 in screening, after the seedling of 289 M2 families, identify ten and there is the Independent mutants reducing fluorescence, this was reported (1999, Proc.Natl.Acad.Sci.U.S.A.96:12923-1928) by people such as PapadopoulouK..Follow-up screening mutant identifies other 82 independent avenacins carrying out being separated based on the root fluorescence reduced and lacks mutant.

biochemical Characterization

According to the root extract (people such as PapadopoulouK., 1999, Proc.Natl.Acad.Sci.U.S.A.96:12923-1928) of initial ten mutant of described methods analyst.In brief, M3 seed is germinateed 2 days on wet filter paper, gather in the crops the root end 0.5cm part of 20 strain seedling of each strain, and extract in methyl alcohol.Prepare three parts of crude methanol root extracts from M3 seedling and be used for HPLC analysis, on HichromNucleosil5C18 reversed-phase column (4.5 × 250mm), under isocratic condition, in 75% methyl alcohol (flow is 1mL/min), direct analysis 100 μ L aliquots containig, determined wavelength is 225nm.By the standard substance comparison peak region with those concentration known, quantitative four kinds of avenacins.By the extract drying of TLC analysis, be resuspended in 1mL water, and be applied to SepPakC18 reversed-phase column (Waters, Milford, MA), this post is pre-conditioned connects 10mL distilled water for after 10mL methyl alcohol.After by 75% methanol-eluted fractions, by sample drying, be resuspended in 15 μ L100% methyl alcohol, be applied to TLC plate, and use chloroform: methyl alcohol: water (13: 6: 1; V: v: v) be separated.Avenacin A-1 and B-1 and other fluorescent components excite lower video picture at 302nm place UV.Then use aubepine/sulfuric acid/acetic acid (1: 1: 48, V: V: V) to spray TLC plate and at 130 DEG C, toast 5 minutes to detect all four kinds of saponin(es.The root extract deriving from M3 or F3 seedling compares at least 7 kinds of situations, and result is substantially the same.

the genetic analysis of Sad mutant

Hybridization test is carried out between Sad mutant and wild-type A.Whether the saponin(e measuring black oat lacks phenotype due to single sudden change.At least 4 complementation groups of Analysis and Identification to the F2 generation from mutant intermolecular hybrid in initial 10 mutant strains.By these sites called after Sad1 to Sad4 (people such as PapadopoulouK., 1999, Proc.Natl.Acad.Sci.U.S.A.96:12923-1928).Other 4 sites are determined to the further analysis of initial 10 mutant line, by its called after Sad5 to Sad8 (people such as QiX., 2004, Proc.Natl.Acad.Sci.U.S.A.101:8233-8238).

The DNA sequence analysis to 3 new gene comprised in avenacin biosynthesizing (AsSCPL1, AsMT1 and AsGT2) is carried out in Sad5, Sad6, Sad7 and Sad8 mutant.137th amino acid of single core thuja acid change from C to T in AsSCPL1 respectively among mutant #587 (Sad7) and mutant #616 (initial called after Sad5) causes the change from Serine to phenylalanine, the 79th amino acid in #376 (Sad7) causes from proline(Pro) to leucic change (table 1).In AsSCPL1, the new mutant #19.1 of origination point sudden change is also separated by the genomic dna screening other 82 Sad mutant, described mutant is identified by the screening (as mentioned above) of expansion based on the root fluorescence reduced, and uses SurveyorMutationDetectionKit (Transgenomic) to be further analyzed.By pcr amplification target cDNA.Amplified production from two mutant strains is mixed.Heteroduplex formation is carried out and subsequence program detects for mutant according to the specification sheets (TransgenomicCatNo.706025) of manufacturers.Mutant #19.1 comprises a point mutation, predicts that this point mutation causes the 463rd amino acid from Threonine to the change of Isoleucine.Nucleotide change is there is not in AsMT1 and the AsGT2 gene of Sad5, Sad6, Sad7 or Sad8 mutant.The allelomorphism test carried out with mutant #19.1, #616 and #376 shows that all three mutant correspond to a single site, Sad7.These digital proof mutant #616 (it is called Sad5 at first) are corresponding to site Sad7.These results clearly show that Sad7 corresponds to serine carboxypeptidase sample albumin A sSCPL1.AsSCPL1 is required for triterpene skeleton acyl group (be N-methyl anthranilic acid under fluorescence avenacin A-1 and B-1 situation, under non-fluorescence avenacin A-2 and B-2 situation for phenylformic acid) being joined avenacin.

embodiment 3

be separated and characterize Sad9 oat mutant

Initial mutant #616, #825, #376 and #1243 people 1999.PNAS9612923-1928 such as () Papadopoulou does not comprise the change of any Nucleotide in AsMT1 and AsGT2 gene.Sequential analysis is expanded to 82 new Sad mutant, described mutant is separated as described in Example 2.Three mutant M3 strains (#195, #961 and #1310) are accredited as and in AsMT1 gene, there is point mutation (table 2).Any mutant in set does not identify sudden change in AsGT2 gene.DNA sequence analysis confirms a mononucleotide change occurs in the encoding sequence in three mutant (#195, #961 and #1310).Each predicting in the change of these Nucleotide will cause amino acid to change (table 2).

The root of these three mutant lacks the light blue fluorescence relevant to avenacin A-1, excites lower to dark violet fluorescence at UV.A little mutant is called " purple mutant ".Mutant #841 stronger for purple is used and identifies out based on the method (table 2) of TLC from 82 new Sad mutant.Carry out the metabolite analysis of purple mutant as mentioned below.Intercept ten tips of a root (length is 0.5cm) from each strain, and be immersed in 500ul75%MeOH by them, soak time is little between spending the night between one.Be transferred to by supernatant liquor after centrifugation in new pipe, extract is also resuspended in 20ul100%MeOH by drying.Ten ul samples are added on a TLC plate, and described TLC is at ChCl ₃: MeOH:dH ₂development in O (13: 6: 1vol/vol/vol).TLC plate checks under UV excites.The all purple mutant comprising #841 have clearly purple dot on the tlc plate.The sequential analysis of #841 confirms the mononucleotide change occurred in the coding region of this mutant from C to T.Predict that this change causes and become α-amino-isovaleric acid (see table 1) at the L-Ala at amino acid 333 place.

Purple mutant is hybridized each other (#195 × #1310, #961 × #1310, #195 × #961) to test for allelomorphism.By two mutant alleles of sequential analysis in F1 crossbred, confirm heterozygote.All F1 plants remain " purple root " phenotype.These results indicate three purple mutant to be at the mutation allele of homologous genes tic site, are defined as Sad9 now.Gene data, rna expression data (Fig. 3) and metabolite analysis instruction Sad9 corresponds to AsMT1, the methyltransgerase needed in the avenacin biosynthesizing namely in oat.The function of this methyltransgerase may be the o-amino benzoyl acidic group methylated on fluorescence avenacin A-1 and B-1.

embodiment 4

characterize the Transglucosylase in Sad gene cluster

In avenacin gene cluster, 4 in 5 five genes in the BAC contig crossing over Sad gene cluster have shown and have been directly involved in avenacin biosynthetic pathway (people such as Osbourn, on March 6th, 2007, US, 7,186,884B2; The people such as Osbourn, US2006-0112448A1 and embodiment 2 and 3).But even identify multiple mutation alleles of other four genes, the mutant of AsGT2 is not still in the set of 92 minimizing root Fluorescence Mutation of A bodies of our expansion.If AsGT2 is necessary on interpolation one/multiple sugar to avenacin three sugar moieties, the loss function of AsGT2 does not cause the root fluorescence phenotype of minimizing, can not be affected because add fluorophor (N-methyl anthranilic acid).Other possibility has functional superfluous She or AsGT2 sudden change is lethal.Alternatively, AsGT2 can the formation of catalyzing acyl glucose intermediate, and this intermediate is used for the acylations of AsSCPL1 mediation.Therefore the biochemical function of AsGT2 is demonstrated.

In order to test the function of AsGT2, AsGT2cDNA to be cloned in NovagenpET-19b expression vector and as follows at expression in escherichia coli: the Bacillus coli cells comprising described expression construct is inoculated on LB agar, this LB agar supplements 34 μ g/mL paraxin, 50 μ g/mL Gepcillin, 2.5mM trimethyl-glycine and 0.6M sorbyl alcohols.Express cell is 37 degrees Celsius of grow overnight.In order to expressing protein, use the cell on solid medium to be inoculated into ten and there is 50mL supplement in the 250mL flask of the liquid nutrient medium of mentioned component.Culture is at 37 degrees Celsius of shaking culture OD ₆₀₀about 0.6.Then before adding final concentration and being the IPTG inductor of 0.1mM, culture is transferred in 16 degrees Celsius of Shaking Incubatorss and cultivate 30 minutes.Then spend the night at 16 degrees Celsius of shaking culture cultures.

By 4 degrees Celsius, 7, centrifugal 10 minutes of 000 × g results inducing cell.Cell precipitation thing is resuspended in 5 to 10mL to have in the dissolving-binding buffer liquid of Roche without EDTA proteinase inhibitor (every 50mL1 sheet) (300mMNaCl, 50mM phosphorus sodium, 20mM imidazoles, 5% glycerine, pH7.8).Use Frenchpress dissolved cell twice, carry out under cold all the time.By lysate at 4 degrees Celsius, at 10,000g centrifugal 45 minutes.With 0.2 μm of syringe-metre filter supernatant liquor before injection is the FPLC of 0.5mL/ minute for flow velocity.

1mLHiTrap chelating HP post (Amersham) that use is pre-charged with and FPLC system carry out FPLC.Purification column is according to the recommendation 0.1MNiCl of manufacturers ₂be full of.Dissolving-binding buffer liquid pre-equilibration column is used before sample injections.After injection, described post washed 30 minutes to 1 hour with dissolving-binding buffer liquid before the Gradient program with Elution buffer B (300mMNaCl, 50mM sodium phosphate buffer, 700mM imidazoles, 5% glycerine, pH8.0).

table 3: wash-out kind white condition from HiTrap chela meeting HP post

Time (minute)	％B	ML/ minute
			0	8	1.0
20	10	1.0
			40	100	1.0
50	100	1.0

Collect the elute fragment of 1mL on ice, 20mL is for developing result in 10%PAGE system.Concerned fragment merges and dialysed overnight at 4 degrees Celsius, and use 50mM sodium phosphate buffer, pH7.5, has 50% glycerine and 2mMMgCl ₂.Aliquots containig in liquid nitrogen quick freeze and under-80 degrees Celsius store.

The radioassay analytical method of complex receptor substrate is utilized to be used to detect the low-level with pure substrate or concentrated substrate active.This detection analytical method comprises 4mL ¹⁴c-UDP-glucose, 25mL50mM potassium phosphate buffer, pH7.6, the 0.25mL100mM receptor substrate in DMSO, the protein preparation that 5mL dialysed, and 3mL10mMDTT.Reaction, at 28 degrees Celsius, is carried out 2.5 hours under gentle agitation condition, is then added 50mL chloroform: methyl alcohol (2: 1) termination reaction.With chloroform methanol again extracting containing aqueous phase three times, and merge solvent phase.Dry extract be resuspended in 40mL chloroform at 60 c: in methyl alcohol.Also dry laying equal stress on containing aqueous phase is suspended from water.15mL extract is loaded in silica gel tlc plate, with chloroform: methyl alcohol: water (13: 6: 1) carries out chromatography to standard substance.

Tested receptor substrate is beta-amyrin-Arabinoside, phenylformic acid and Whitfield's ointment.Tested saccharide donor is D-Glucose and L-arabinose.

In order to confirm the chemical structure of the product developed by radioassay analytical method, developing a kind of inactive LC-MS and detecting analytical method.This detects analytical method and comprises 10mL20mMUDP-glucose or UDP-pectinose, 60mL50mM potassiumphosphate, pH7.6,2mL1MMgCl ₂, 4mL100mM receptor substrate in DMSO (or 8mL synthesizes substrate, and DMSO only compares thing), 20rnL protein product, 10mL100mMDTT.Reaction cultivates 3.5 hours at 28 degree of gentle agitation.By adding 100% methyl alcohol termination reaction, then at 50 degrees Celsius of Evaporation of methanol from reaction, and use reversed phase chromatography sample (10 μ 1) to analyze by LC/MS/MS, use 100 × 2mm3 μ LunaC18 (2) post (Phenomenex), at 250 μ L.min ^-1, 30 DEG C, with following methyl alcohol+0.1% formic acid, water+0.1% formic acid gradient is run:

table 4: for the methanol gradient of reverse-phase chromatography.

Time (minute)	％MeOH
		0	10
40	95
		41	10
48	10

Checked, with positive ion or negative ion mode in the cycle of operation of separating by UV (214nm, bandwidth 9nm, spectral range 200 to 600nm) and electron spray(ES) MS.Spray chamber's condition is 50 unit sheath gas, without aux/ scanning, and the positive ion spray voltage of 5.2kV, the negative ion mode voltage of 5.0kV, capillary temperature 325 DEG C.The MS of data dependency ²the second scan event carried out with the fragmentation at 35% collision energy place by capturing of the separation width place at 5.0amu.

Result is as shown in table 4.A "+" represents active.

the activity of table 5:AsGT2 para Toluic Acid and salicylate substrate.

Due to chromatography and detect relevant technical problem, the detection analysis of AsGT2 to the activity of beta-amyrin-Arabinoside is indecisive.But phenylformic acid and salicylic result clearly show that AsGT2 has Transglucosylase function to substrate, and described substrate has ring structure, is similar to beta-amyrin.In conjunction with the described fact, be similar to other enzyme in approach, AsGT2 only root express and described gene is arranged in avenacin biological synthesis gene cluster, these results show this genes encoding avenacin synthesis needed for Transglucosylase.

embodiment 5

the recombinant dna construct of AsSCPL1 is expressed in other species

Be hereafter the embodiment of recombinant dna construct, can use them in monocotyledons or dicot plant species, express AsSCPL1, use corn or soybean as embodiment.Use constitutive promoter, those skilled in the art will know, depend on target pathogenic agent or other Consideration, targeted promotor is as the promotor in those embodiments more early described in this article, due to the special whole purposes of described vegetable material, can be identical or even more effective or preferred.Depend on the enzymic activity existed in species and species, can comprise from other gene in biosynthetic pathway to improve expression level.

For comprising the abbreviation of the nucleic acid fragment of different components below using in Examples below:

" RB " and " LB " is corresponding to the right margin of T-DNA and left margin.

" CAMV35SENH " is the promoter region of cauliflower mosaic virus 35 S promoter, and it improves the expression level (people such as BenfeyP.N., 1990, EMBOJ.9:1685-1696) of connected promotor.

" UBIPRO " is the promotor of Maize Ubiquitin gene, carries out description (people such as Christensen, 1992, PlantMol.Biol.18:675-689) to it.

" UBI5 ' UTR " is 5 ' leader region of identical Maize Ubiquitin gene.

" UBIINTRON1 " is the intron of identical ubiquitin gene.Comprise that this intron is verified improves expression level.

" ATTR1 " is at Gateway ^tMthe recombination site (Invitrogen, Carlsbad, California, USA) described in clone's system handbook.

" CCDB " is at Gateway ^tMthe Bacterial Negative selected marker described in clone's system handbook.

" ATTR2 " is at Gateway ^tMthe recombination site described in clone's system handbook.

" PINII " is the Transcription Termination gene from potato proteinase inhibitor II gene.

" CAMV35SPRO " is the promotor of cauliflower mosaic virus 35S gene, and it is constitutive promoter conventional in plant people such as (, 1985, Nature313:810-812) OdellJ.T..

" ADH1INTRON1 " is the intron of corn ADH1 gene.Comprise this intron to have proved to improve expression level (LuehrsenK.R. and WalbotV., 1991, Mol.Gen.Genet.225:81-93).

" BAR " is the antiweed gene being commonly used for corn transformation selected marker.

" SCP1 " is the synthesis constitutive promoter for plant, and it is described in U.S. Patent Publication 6,072, and in 050.

" OMEGA5 ' UTR " is 5 ' leader region of tmv cdna, and its use verified can improve translation skill (people such as Gallie, 1989, MolecularBiologyofRNA, editor Cech (Liss, NewYork), 237-256 page).

" SPC1 " is to provide the coding region of the polypeptide of Spectinomycin resistance, and it allows bacterium to select Svab, Z. and Maliga, P., 1991, Mol.Gen.Genet.228:316-319.

" ColE1ORI " is the functional DNA replication orgin in intestinal bacteria.

the construct of saponin(e biosynthesis gene is expressed in corn

The fragment comprising the open reading frame of AsSCPL1 is obtained respectively from the clone described in embodiment 1.Carry out pcr amplification with the primer causing open reading frame side to be connected to unique restriction sites, described restriction site makes their energy directed clonings to these modified Gateway ^tMin the unique restriction sites of EntryVector (Invitrogen, Carlsbad, California, USA).Described fragment is being connected to Gateway ^tMafter EntryVector, " entry vector " is made up of ATTL1-AsSCPL1-ATTL2, and comprises the kalamycin resistance selected for bacterium.ATTL1 and ATTL2 is at InvitrogenGateway ^tMthe recombination site provided in clone's system (Carlsbad, California, USA).

corn recombinant dna construct 1:E35S-UBI-AsSCPL1-PINII

This builds physical efficiency and is used for single expression AsSCPL1 gene in corn.AsSCPL1 entry vector is used for Gateway ^tMlR and Gateway ^tMreaction (the Komari of modified soil bacillus carrier main chain (modify from pSB1 and obtain), T. people is waited, 1996, PlantJ.10:165-174), this is by adding following component in cos site: RB-CAMV35SENH-UBIPRO-UBI5 ' UTR-UBIINTRON1-ATTR1-CCDB-ATTR2-PINII+CAMV35SENH-CAMV35S PRO-ADH1INTRON1-BAR-PINII-LB-SPC-ColE1ORI.At this Gateway ^tMin reaction, ATTL1 and ATTL2 and ATTR1 and ATTR2 restructuring, being therefore virose by substituting CCDB, CCDB in AsSCPL1 transgenosis to object carrier, and making it possible to as Gateway concerning intestinal bacteria ^tMthe successfully clone of screening described in handbook (Invitrogen, Carlsbad, California, USA).This gained construct comprises a T-DNA, and it will to be transferred in Plant Genome and to comprise RB-CAMV35SENH-UBIPRO-UBI5 ' UTR-UBIINTRON1-ATTB1-AsSCPL1-ATTB2-PINII+CAMV35SENH-CAMV 35SPRO-ADH1INTRON1-BAR-PINII-LB.Region extracellular nucleotides sequence description between RB and LB is in people such as Kormai, T., and opcit., except SPC and ColE1 component.This construct electroporation to be entered in LBA4404 tumefaciens cells wsfe and to be used for transformation experiment as those experiments described in Examples below 8.

the construct of saponin(e biosynthesis gene is expressed in soybean

Obtain AsSCPL1 open reading frame as described above by pcr amplification and be used for corn construct.

soybean recombinant dna construct 1:SCP1-O '-AsSCPL1-PINII

This builds physical efficiency and is used at dicotyledonous middle single expression AsSCPL1 gene.Be connected at the polynucleotide comprising AsSCPL1 open reading frame by and comprise SCP1PRO-ω 5 ' UTR-unique restriction sites, after being same as in the carrier in the AsSCPL1-PINII site of those side joints, linearization plasmid is used for bombardment and from gel, extracts required DNA band.This process also removes the Nucleotide of the amicillin resistance that coding is selected for bacterium.This fragment comprises SCP1PRO-ω 5 ' UTR-AsSCPL1-PINII and for the transformation of soybean as described in Examples below 9.

embodiment 6

the recombinant dna construct of AsMT1 is expressed in other species

Be hereafter the embodiment of recombinant dna construct, can use them in monocotyledons or dicot plant species, express AsMT1, use corn or soybean as embodiment.Use constitutive promoter, those skilled in the art will know, depend on target pathogenic agent or other Consideration, targeted promotor is as the promotor in those embodiments more early described in this article, due to the special whole purposes of described vegetable material, can be identical or even more effective or preferred.Depend on the enzymic activity existed in species and species, can comprise from other gene in biosynthetic pathway to improve expression level.

For comprising the abbreviation of the nucleic acid fragment of different components below using in Examples below:

" RB " and " LB " is corresponding to the right margin of T-DNA and left margin.

" CAMV35SENH " is the promoter region of cauliflower mosaic virus 35 S promoter, and it improves the expression level (BenfeyP.N., waits people, 1990, EMBOJ.9:1685-1696) of connected promotor.

" UBIPRO " is the promotor of Maize Ubiquitin gene, carries out description (people such as Christensen, 1992, PlantMol.Biol.18:675-689) to it.

" UBI5 ' UTR " is 5 ' leader region of identical Maize Ubiquitin gene.

" UBIINTRON1 " is the intron of identical ubiquitin gene.Comprise that this intron is verified improves expression level.

" ATTR1 " is at Gateway ^tMthe recombination site (Invitrogen, Carlsbad, California, USA) described in clone's system handbook.

" CCDB " is at Gateway ^tMthe Bacterial Negative selected marker described in clone's system handbook.

" ATTR2 " is at Gateway ^tMthe recombination site described in clone's system handbook.

" PINII " is the Transcription Termination gene from potato proteinase inhibitor II gene.

" CAMV35SPRO " is the promotor of cauliflower mosaic virus 35S gene, and it is constitutive promoter conventional in plant people such as (, 1985, Nature313:810-812) OdellJ.T..

" ADH1INTRON1 " is the intron of corn ADH1 gene.Comprise this intron to have proved to improve expression level (LuehrsenK.R and WalbotV., 1991, Mol.Gen.Genet.225:81-93).

" BAR " is the antiweed gene being commonly used for corn transformation selected marker.

" SCP1 " is the synthesis constitutive promoter for plant, and it is described in U.S. Patent Publication 6,072, and in 050.

" OMEGA5 ' UTR " is 5 ' leader region of tmv cdna, and its use verified can improve translation skill (people such as Gallie, 1989, MolecularBiologyofRNA, editor Cech (Liss, NewYork), 237-256 page).

" SPC1 " is to provide the coding region of the polypeptide of Spectinomycin resistance, and it allows bacterium to select Svab, Z. and Maliga, P., 1991, Mo1.Gen.Genet.228:316-319.

" ColE1ORI " is the functional DNA replication orgin in intestinal bacteria.

the construct of saponin(e biosynthesis gene is expressed in corn

The fragment comprising the open reading frame of AsMT1 is obtained respectively from the clone described in embodiment 1.Carry out pcr amplification with the primer causing open reading frame side to be connected to unique restriction sites, described restriction site makes their energy directed clonings to these modified Gateway ^tMin the unique restriction sites of EntryVector (Invitrogen, Carlsbad, California, USA).Described fragment is being connected to Gateway ^tMafter EntryVector, " entry vector " is made up of ATTL1-AsMT1-ATTL2, and comprises the kalamycin resistance selected for bacterium.ATTL1 and ATTL2 is at InvitrogenGateway ^tMthe recombination site provided in clone's system (Carlsbad, California, USA).

corn recombinant dna construct 1:E35S-UBI-AsMT1-PINII

This builds physical efficiency and is used for single expression AsMT1 gene in corn.AsMT1 entry vector is used for Gateway ^tMlR and Gateway ^tMreaction (the Komari of modified soil bacillus carrier main chain (modify from pSB1 and obtain), T. people is waited, 1996, PlantJ.10:165-174), this is by adding following component in cos site: RB-CAMV35SENH-UBIPRO-UBI5 ' UTR-UBIINTRON1-ATTR1-CCDB-ATTR2-PINII+CAMV35SENH-CAMV35S PRO-ADH1INTRON1-BAR-PINII-LB-SPC-ColE1ORI.At this Gateway ^tMin reaction, ATTL1 and ATTL2 and ATTR1 and ATTR2 restructuring, being therefore virose by substituting CCDB, CCDB in AsMT1 transgenosis to object carrier, and making it possible to as Gateway concerning intestinal bacteria ^tMthe successfully clone of screening described in handbook (Invitrogen, Carlsbad, California, USA).This gained construct comprises a T-DNA, and it will to be transferred in Plant Genome and to comprise RB-CAMV35SENH-UBIPRO-UBI5 ' UTR-UBIINTRON1-ATTB1-AsMT1-ATTB2-PINII+CAMV35SENH-CAMV35 SPRO-ADH1INTRON1-BAR-PINII-LB.Region extracellular nucleotides sequence description between RB and LB is in people such as Kormai, T., and opcit., except SPC and ColE1 component.This construct electroporation to be entered in LBA4404 tumefaciens cells wsfe and to be used for transformation experiment as those experiments described in Examples below 8.

the construct of saponin(e biosynthesis gene is expressed in soybean

Obtain AsMT1 open reading frame as described above by pcr amplification and be used for corn construct.

soybean recombinant dna construct 1:SCP1-O '-AsMT1-PINII

This builds physical efficiency and is used for single expression AsMT1 gene in dicotyledons.Be connected at the polynucleotide comprising AsMT1 open reading frame by and comprise SCP1PRO-ω 5 ' UTR-unique restriction sites, after being same as in the carrier in the AsMT1-PINII site of those side joints, linearization plasmid is used for bombardment and from gel, extracts required DNA band.This process also removes the Nucleotide of the amicillin resistance that coding is selected for bacterium.This fragment comprises SCP1PRO-ω 5 ' UTR-AsMT1-PINII and for the transformation of soybean as described in Examples below 9.

embodiment 7

the recombinant dna construct of AsGS2 is expressed in other species

Be hereafter the embodiment of recombinant dna construct, can use them in monocotyledons or dicot plant species, express AsGS2, use corn or soybean as embodiment.Use constitutive promoter, those skilled in the art will know, depend on target pathogenic agent or other Consideration, targeted promotor is as the promotor in those embodiments more early described in this article, due to the special whole purposes of described vegetable material, can be identical or even more effective or preferred.Depend on the enzymic activity existed in species and species, can comprise from other gene in biosynthetic pathway to improve expression level.

For comprising the abbreviation of the nucleic acid fragment of different components below using in Examples below:

" RB " and " LB " is corresponding to the right margin of T-DNA and left margin.

" CAMV35SENH " is the promoter region of cauliflower mosaic virus 35 S promoter, and it improves the expression level (BenfeyP.N., waits people, 1990, EMBOJ.9:1685-1696) of connected promotor.

" UBIPRO " is the promotor of Maize Ubiquitin gene, carries out description (people such as Christensen, 1992, PlantMol.Biol.18:675-689) to it.

" UBI5 ' UTR " is 5 ' leader region of identical Maize Ubiquitin gene.

" UBIINTRON1 " is the intron of identical ubiquitin gene.Comprise that this intron is verified improves expression level.

" ATTR1 " is at Gateway ^tMthe recombination site (Invitrogen, Carlsbad, California, USA) described in clone's system handbook.

" CCDB " is at Gateway ^tMthe Bacterial Negative selected marker described in clone's system handbook.

" ATTR2 " is at Gateway ^tMthe recombination site described in clone's system handbook.

" PINII " is the Transcription Termination gene from potato proteinase inhibitor II gene.

" CAMV35SPRO " is the promotor of cauliflower mosaic virus 35S gene, and it is constitutive promoter conventional in plant people such as (, 1985, Nature313:810-812) OdellJ.T..

" ADH1INTRON1 " is the intron of corn ADH1 gene.Comprise this intron to have proved to improve expression level (LuehrsenK.R and WalbotV., 1991, Mol.Gen.Genet.225:81-93).

" BAR " is the antiweed gene being commonly used for corn transformation selected marker.

" SCP1 " is the synthesis constitutive promoter for plant, and it is described in U.S. Patent Publication 6,072, and in 050.

" OMEGA5 ' UTR " is 5 ' leader region of tmv cdna, and its use verified can improve translation skill (people such as Gallie, 1989, MolecularBiologyofRNA, editor Cech (Liss, NewYork), 237-256 page).

" SPC1 " is to provide the coding region of the polypeptide of Spectinomycin resistance, and it allows bacterium to select Svab, Z. and Maliga, P., 1991, Mol.Gen.Genet.228:316-319.

" ColE1ORI " is the functional DNA replication orgin in intestinal bacteria.

the construct of saponin(e biosynthesis gene is expressed in corn

The fragment comprising the open reading frame of AsGS2 is obtained respectively from the clone described in embodiment 1.Carry out pcr amplification with the primer causing open reading frame side to be connected to unique restriction sites, described restriction site makes their energy directed clonings to these modified Gateway ^tMin the unique restriction sites of EntryVector (Invitrogen, Carlsbad, California, USA).Described fragment is being connected to Gateway ^tMafter Entryvector, " entry vector " is made up of ATTL1-AsGS2-ATTL2, and comprises the kalamycin resistance selected for bacterium.ATTL1 and ATTL2 is at InvitrogenGateway ^tMthe recombination site provided in clone's system (Carlsbad, California, USA).

corn recombinant dna construct 1:E35S-UBI-AsGS2-PINII

This builds physical efficiency and is used for single expression AsGS2 gene in corn.AsGS2 entry vector is used for Gateway ^tMlR and Gateway ^tMreaction (the Komari of modified soil bacillus carrier main chain (modify from pSB1 and obtain), T. people is waited, 1996, PlantJ.10:165-174), this is by adding following component in cos site: RB-CAMV35SENH-UBIPRO-UBI5 ' UTR-UBIINTRON1-ATTR1-CCDB-ATTR2-PINII+CAMV35SENH-CAMV35S PRO-ADH1INTRON1-BAR-PINII-LB-SPC-ColE1ORI.At this Gateway ^tMin reaction, ATTL1 and ATTL2 and ATTR1 and ATTR2 restructuring, being therefore virose by substituting CCDB, CCDB in AsGS2 transgenosis to object carrier, and making it possible to as Gateway concerning intestinal bacteria ^tMthe successfully clone of screening described in handbook (Invitrogen, Carlsbad, California, USA).This gained construct comprises a T-DNA, and it will to be transferred in Plant Genome and to comprise RB-CAMV35SENH-UBIPRO-UBI5 ' UTR-UBIINTRON1-ATTB1-AsGS2-ATTB2-PINII+CAMV35SENH-CAMV35 SPRO-ADH1INTRON1-BAR-PINII-LB.Region extracellular nucleotides sequence description between RB and LB is in people such as Kormai, T., and opcit., except SPC and ColE1 component.This construct electroporation to be entered in LBA4404 tumefaciens cells wsfe and to be used for transformation experiment as those experiments described in Examples below 8.

the construct of saponin(e biosynthesis gene is expressed in soybean

Obtain AsGS2 open reading frame as described above by pcr amplification and be used for corn construct.

soybean recombinant dna construct 1:SCP1-O '-AsGS2-PINII

This builds physical efficiency and is used for single expression AsGS2 gene in dicotyledons.Be connected at the polynucleotide comprising AsGS2 open reading frame by and comprise SCP1PRO-ω 5 ' UTR-unique restriction sites, after being same as in the carrier in the AsGS2-PINII site of those side joints, linearization plasmid is used for bombardment and from gel, extracts required DNA band.This process also removes the Nucleotide of the amicillin resistance that coding is selected for bacterium.This fragment comprises SCP1PRO-ω 5 ' UTR-AsGS2-PINII and for the transformation of soybean as described in Examples below 9.

embodiment 8

the conversion of agrobacterium-mediated corn

with the regeneration of transgenic plant

In embodiment 5 to 7, the recombinant dna construct of preparation can be used for the rotaring gene corn plant be prepared as follows.

The method of Zhao (U.S. Patent Publication 5,981,840, and PCT patent disclosure WO98/32326) can be used, with any polynucleotide constructs maize transformation as described in embodiment 5-7.In brief, be separated prematurity plumule and make plumule contact Agrobacterium cell suspension from corn, polynucleotide constructs can be transferred to (step 1: infect step) at least one cell of at least one prematurity plumule by wherein said bacterium.In this step prematurity plumule is immersed in Agrobacterium cell suspension and be used for starting inoculation.Plumule and edaphic bacillus Dual culture for some time (step 2: Dual culture step).After infection step, prematurity plumule is cultivated on solid medium.And then this Dual culture period, optional " dormancy " step is carried out.In this sleep step, plumule is cultivated exists the known microbiotic of at least one to suppress Agrobacterium growth, does not add in the environment of vegetable transformant selective agent simultaneously and carries out (step 3: sleep step).Prematurity plumule is cultivated and there is microbiotic but without on the solid medium of selective agent, for eliminating the dormant stage of edaphic bacillus and infected cell.Next, inoculation plumule is cultivated in the substratum comprising selective agent, and reclaim growth through transformed calli (step 4: select step).Then callus regenerates (step 5: regeneration step) in plant, and the callus grown in Selective agar medium carries out cultivating to regenerate described plant in solid medium.

embodiment 9:

with soybean expression vector soybean transformation somatic embryo culture

and regenerable soybean plant

In embodiment 5 to 7, the recombinant dna construct of preparation can be used for the transgenic soy bean plant be prepared as follows.

culture condition:

Soybean germ generation suspension culture (cv.Jack) is incubated at 35mL liquid SB196 substratum (vide infra), culture condition be 150rpm shaking table cultivate, 26 DEG C and by 16: 8 hours daytime/night photoperiod is with 60-85 μ E/m2/s light intensity cool white light fluorescent lamp.Within every 7 days to two weeks, to be organized in 35mL fresh liquid SB196 by the about 35mg of inoculation and Secondary Culture (preferred subculture intervals for every 7 days) is carried out to culture.

Soybean germ generation suspension culture soybean expression plasmid transforms, all conversions all use DuPontBiolisticPDS1000/HE instrument (helium remodeling), undertaken by Gun Bombardment (people such as Klein, Nature327:70 (1987)) method.

soybean embryo generates the induction of suspension culture:

Soybean culture monthly starts twice, 5 to 7 days, interval between each startup.The beanpod of the band prematurity seed of available soybean plants is gathered after planting in 45 to 55 days.From beanpod, remove seed and be placed in aseptic magenta box.Soybean seed carries out aseptically process in 15 minutes by shake in 5% chlorine bleach liquor and 1 ivory soap (that is, 95mL autoclave distills waterpower 5mL clorox and 1 soap, fully mixes).Seed uses the sterile distilled water rinsing of 21 litre flasks, and the seed those being less than 4mm is placed on single slide.Cut the little end of seed, and cotyledon is extruded seed shell.Cotyledon is transferred in the plate comprising SB1 substratum (every plate 25 to 30 cotyledons).With fiber band by flat board packing and temperature 26 DEG C, cold 16: 8 hours white fluorescent light cycles daytime/night, cultivate eight weeks under intensity of illumination 60 to 80 μ E/m2/s, 4 weeks replaced medium afterwards.On SB1 substratum after inoculation, cut second plumule and put it into SB196 liquid nutrient medium 7 days.

for the preparation of DNA of bombarding:

Complete plasmid or the DNA plasmid fragments comprising concerned gene and selected marker are used to bombardment.The plasmid crossed by gel separating digesting obtains the fragment from soybean expression plasmid, this document describes the structure of described plasmid.In all cases, 100 μ g plasmid DNA are used to 0.5mL certain enzyme mixture hereinafter described.Plasmid is with AscI (100 unit) at NEBuffer4 (20mMTris acetic acid, 10mM magnesium acetate, 50mM potassium acetate, 1mM dithiothreitol (DTT), pH7.9), and 100 μ g/mLBSA, and in 5mM beta-mercaptoethanol, digest 1.5 hours at 37 DEG C.Gained DNA fragmentation is separated by gel electrophoresis (BioWhitakerMolecularApplications) on the SeaPlaqueGTG agarose of 1%, and cuts from sepharose the DNA fragmentation comprising box gene.Use GELase digestive ferment, according to code purify DNA from agarose of manufacturers.

50 μ L aliquots containigs of the sterile distilled water comprising 3mg bronze (3mg gold) are added 30 μ L10ng/ μ LDNA solution (DNA fragmentation of preparation as described herein), 25 μ L5MCaCl ₂, and in 20 μ L0.1M spermidines.Mixture is shaken 3 minutes turbula shaker 3 grades and rotates 10 seconds in desk centrifuge.Remove supernatant liquor, then by 400 μ L100% washing with alcohol and carry out another time simple centrifugal.Remove 400 μ L ethanol, and centrifugation is resuspended in 100% ethanol of 40 μ L.Five μ LDNA suspension are assigned in each floating disc of BiolisticPDS1000/HE instrument dish.Every 5 μ L aliquots containigs are bombarded at every turn, and (such as often coiling) comprises about 0.375mg gold.

tissue preparation and bombarding with DNA:

Seven of about 150 to 200mg day plumule generation in age suspension culture is placed in empty aseptic 60 × 15mm culture dish, and covers this culture dish with plastic wire.Room being emptied to vacuum tightness is 27 to 28 inches of mercury, the tissue bombardment one of each flat board or two, and film rupture pressure is set to 1100PSI.Tissue is placed on distance and retains/stop the about 3.5 inches of places of screen cloth.

transform the selection of embryo:

The plumule (when using acetolactate synthase (ALS) gene as selected marker) using the grand selection of chlorine sulphur transformed.

After bombardment, tissue be placed in brand-new SB196 substratum and cultivate as mentioned above.Latter six to eight days of bombardment, is replaced by SB196 and comprises the grand fresh SB196 substratum of 100ng/mL chlorine sulphur.Selective agar medium upgrades weekly.In four to six weeks after selecting, observe during unconverted downright bad plumule occurs bunch and grow green transforming tissue.Chlorenchyma is separated, is seeded to porous plate, generate suspension culture with the embryo producing conversion that is new, clonal propagation.

the maturation of embryo:

Bunch cultivation four to six week described above in porous plate is there is by from producing the transformed plumule transformed, cultivate at 26 DEG C, in SB196, with the photoperiod of 16: 8 hours and 90 to 120 μ E/m under cold white fluorescence (the cold white EconowattF40/CW/RS/EW of Phillips) and Agro (PhillipsF40Agro) bulb (40 watts) ²the intensity of illumination of s is carried out.After for some time, plumule bunch is moved on in solid agar medium, SB166 and cultivated for one to two week, then 3 to 4 weeks in Secondary Culture to SB103 substratum, obtain ripe plumule.Onboard, in SB103 after maturation, by single plumule from bunch remove, dry and screen desired phenotype.This type of phenotype can be but be not limited to the resistance level at least one fungi of saponin(e level or the change changed.When needs, from some events hereinafter described, obtain plant.

plumule is dry and germinate:

Ripe single plumule dewaters by being placed in empty little culture dish (60 × 15mm) for about four to seven days.Plate fiber band (createing a low humidity chamber) seals.The plumule that will dewater implants SB71-4 substratum, makes its germinating growth under the condition identical with above-mentioned culture condition.From germination substratum, take out the seedling of germination and use water cleaning down, then being implanted in the Redi-Earth in 24 hole pallets, cover with blister pack.Plastic lousing is removed, the plant one week of rehardening after one to two week.If seedling looks hard, then they are transplanted in 10 inches of basins of Redi-Earth, the maximum 3 strain seedling of every basin.After ten to ten six weeks, results mature seed, grinds and for desired phenotype analysis.

culture medium prescription:

sB196-FNLite liquid proliferated culture medium (often liter)

fNLite storing solution

sB1 solid medium (often liter)

The MS salt (Gibco/BRL-Cat.No.11117-066) of 1 packaging

1mLB5 VITAMIN 1000X storing solution

31.5g glucose

2mL2,4-D (20mg/L final concentration)

pH5.7

8gTC agar

sB199 solid medium (often liter)

The MS salt (Gibco/BRL-Cat.No.11117-066) of 1 packaging

1mLB5 VITAMIN 1000X storing solution

30g sucrose

4ml2,4-D (final concentration 40mg/L)

pH7.0

2g solidifying agent

sB166 solid medium (often liter)

The MS salt (Gibco/BRL-Cat.No.11117-066) of 1 packaging

1mLB5 VITAMIN 1000X storing solution

60g maltose

750mgMgCl ₂hexahydrate

5g gac

pH5.7

2g solidifying agent (gelrite)

sB103 solid medium (often liter)

The MS salt (Gibco/BRL-Cat.No.11117-066) of 1 packaging

1mLB5 VITAMIN 1000X storing solution

60g maltose

750mgMgCl2 hexahydrate

pH5.7

2g solidifying agent (gelrite)

sB71-4 solid medium (often liter)

1 bottle of Gamborg ' sB5 salt w/ sucrose (Gibco/BRL-Cat.No.21153-036)

pH5.7

5gTC agar

2,4-D storing solution

PhytotechCat.No.D295 pre-mixing liquor-concentration 1mg/mL

b5 vitamin stock (every 100mL)

Packing is stored in-20 DEG C

10g inositol

100mg nicotinic acid

100mg pyridoxine hydrochloride

1g VitB1

If dissolve rapid not, can by solution heat agitated device in addition low-grade fever.

maturation medium (SHaM) (often liter) is known in the differentiation of SB228-soyabean tissue

Adjustment volume is to 900mL

pH5.8

Autoclaving

Join in cooling substratum (≤30 DEG C):

* glutamine (final concentration 30mM) 4%110mL

* note: after adding glutamine, final volume will become 1010mL.

Because glutamine degradation is quite rapid, it preferably now adds before use substratum.Substratum after glutamine adds 2 weeks expired; Minimum medium not containing glutamine can deposit the longer time.

for FN-liteMacro10X-storing solution #1 (often liter) of SHAM

Add water to final volume

Autoclaving

mSMicro1000X-storing solution #2 (every 1 liter)

Add water to final volume

Autoclaving

feEDTA100X-storing solution #3 (often liter)

Na ₂eDTA* (EDETATE SODIUM) 3.73g

FeSO ₄* 7H ₂o (ferric sulfate heptahydrate) 2.78g

* before adding iron ion, EDTA must dissolve completely.

Add water to final volume

This solution is to photaesthesia.The bottle of splendid attire should with aluminium foil parcel with lucifuge.Autoclaving

ca100X-storing solution #4 (often liter)

CaCl ₂* 2H ₂o (calcium chloride dihydrate) 44g

Add water to final volume

Autoclaving

b5 VITAMIN 1000X-storing solution #5 (often liter)

4% glutamine-storing solution #6 (often liter)

DDI water is heated to 30 DEG C of 900mL

L-glutaminate 40g

Add gradually while stirring and impose low-grade fever.

Be no more than 35 DEG C.

Add water to final volume

Filtration sterilization

Freezen protective *

* note: storing solution freezes 31 DEG C of pyrolysis, and water-bath is to dissolve crystal completely.

the grand storing solution of chlorine sulphur

1mg/mL is in 0.01N ammonium hydroxide

Claims (25)

1. the polynucleotide be separated, described polynucleotide are made up of following sequence:

The nucleotide sequence of (a) coding methyltransferase polypeptides, described polypeptide is made up of the aminoacid sequence of SEQIDNO:4; Or

B nucleotide sequence that () is made up of the total length complementary sequence of (a).

2. the polynucleotide of claim 1, the nucleotide sequence of wherein said coding methyltransgerase is made up of the one in SEQIDNO:3 or 8.

3. comprise the carrier of the polynucleotide of claim 1.

4. recombinant dna construct, described construct comprises the polynucleotide of the first enzyme of the coding triterpene approach of claim 1, and described polynucleotide may be operably coupled to few a kind of regulating and controlling sequence.

5. the recombinant dna construct of claim 4, described construct also comprises at least the second polynucleotide, the polypeptide of at least expression of the second enzyme in described second polynucleotide encoding regulation and control triterpene approach, wherein said the second enzyme is beta-amyrin synthase or CYP51H10.

6., for the method for transformant, described method comprises the recombinant dna construct transformant by claim 4 or claim 5.

7., for the production of the method for transgenic plant, described method comprises recombinant dna construct transformed plant cells by claim 4 or claim 5 and from described transformed Plant cell regeneration transgenic plant.

8. comprise the separation host cell of the recombinant dna construct of claim 4, wherein said host cell is not vegetable cell.

9. the host cell of claim 8, wherein said host cell is selected from yeast cell and bacterial cell.

10. change the method for polypeptide expression level in vegetable cell, described method comprises:

A) with the nucleic acid fragment transformed plant tissue of the separation polynucleotide from claim 1, wherein said polynucleotide can change the expression of natural methyl group transferring enzyme;

B) by described plant tissue regeneration transgenic plant; And

C) assessment is when comparing with the plant of the wild type expression level with corresponding natural methyl group transferring enzyme, and the expression level of the methyltransgerase of described transgenic plant changes,

Wherein said plant is monocotyledons.

11. productions have the method for the plant of resistance at least one fungi, described method comprises:

A. the recombinant dna construct transformed plant cells of the first enzyme of the coding triterpene approach of at least one claim 4 is used;

B. under the condition promoting transgenic plant regeneration, make the transformed plant cell growth from step (a); And

C. the transgenic plant of appraisal procedure (b) are not when with the comparing by the plant that described recombinant dna construct is transformed of same species, improve the resistance of at least one fungi;

Wherein said plant is monocotyledons.

The method of 12. claims 11, wherein said recombinant dna construct also comprises at least the second polynucleotide, the polypeptide of at least expression of the second enzyme in described second polynucleotide encoding regulation and control triterpene approach, wherein said the second enzyme is beta-amyrin synthase or CYP51H10.

The method of 13. claims 11, wherein said recombinant dna construct also comprises at least one polynucleotide, and described polynucleotide encoding is selected from the enzyme of beta-amyrin synthase and CYP51H10.

14. produce the method with the plant of the methyltransgerase of change level, and described method comprises:

A) by the recombinant dna construct transformed plant cells of the first enzyme of the coding triterpene approach of claim 4;

B) under the condition promoting transgenic plant regeneration, make the transformed plant cell growth from step (a); And

C) assessment is when comparing with the amount of the methyltransgerase in not transformed by the described recombinant dna construct plant of same species, and the level of the methyltransgerase of the transgenic plant of step (b) changes;

Wherein said plant is monocotyledons.

The method of 15. claims 14, wherein said recombinant dna construct also comprises at least the second polynucleotide, the polypeptide of at least expression of the second enzyme in described second polynucleotide encoding regulation and control triterpene approach.

The method of 16. claims 14, wherein said recombinant dna construct also comprises at least one polynucleotide, and described polynucleotide encoding is selected from the enzyme of beta-amyrin synthase and CYP51H10.

17. for the production of the method for plant of triterpenoid saponin level with change, and described method comprises:

1. by the recombinant dna construct transformed plant cells of the first enzyme of the coding triterpene approach of at least one claim 4;

2. under the condition promoting transgenic plant regeneration, make the transformed plant cell growth from step (a); And

3. the triterpenoid saponin level of transgenic plant when comparing with the triterpenoid saponin amount in not transformed by the described recombinant dna construct plant of same species of appraisal procedure (b) changes,

Wherein said plant is monocotyledons.

The method of 18. claims 17, wherein said recombinant dna construct also comprises at least the second polynucleotide, the polypeptide of at least expression of the second enzyme in described second polynucleotide encoding regulation and control triterpene approach, wherein said the second enzyme is beta-amyrin synthase or CYP51H10.

The method of 19. claims 17, wherein said recombinant dna construct also comprises at least one polynucleotide, and described polynucleotide encoding is selected from the enzyme of beta-amyrin synthase and CYP51H10.

20. for the production of the method for plant of triterpenoid saponin level with raising, and described method comprises:

A) by the recombinant dna construct transformed plant cells of the first enzyme of the coding triterpene approach of at least one claim 4;

B) under the condition promoting transgenic plant regeneration, make the transformed plant cell growth from step (a); And

C) the triterpenoid saponin level of transgenic plant when comparing with the triterpenoid saponin amount in not transformed by the described recombinant dna construct plant of same species of appraisal procedure (b) improves;

Wherein said plant is monocotyledons.

The method of 21. claims 20, wherein said recombinant dna construct also comprises at least the second polynucleotide, the polypeptide of at least expression of the second enzyme in described second polynucleotide encoding regulation and control triterpene approach, wherein said the second enzyme is beta-amyrin synthase or CYP51H10.

The method of 22. claims 20, wherein said recombinant dna construct also comprises at least one polynucleotide, and described polynucleotide encoding is selected from the enzyme of beta-amyrin synthase and CYP51H10.

23. for the production of the method for plant of triterpenoid saponin level with reduction, and described method comprises:

A) by the recombinant dna construct transformed plant cells of the first enzyme of the coding triterpene approach of at least one claim 4;

B) under the condition promoting transgenic plant regeneration, make the transformed plant cell growth from step (a); And

C) the triterpenoid saponin level of transgenic plant when comparing with the triterpenoid saponin amount in not transformed by the described recombinant dna construct plant of same species of appraisal procedure (b) reduces;

Wherein said plant is monocotyledons.

The method of 24. claims 23, wherein said recombinant dna construct also comprises at least the second polynucleotide, described second polynucleotide encoding regulates and controls the polypeptide of at least the second expression of enzymes in triterpene approach, and wherein said the second enzyme is beta-amyrin synthase or CYP51H10.

The method of 25. claims 23, wherein said recombinant dna construct also comprises at least one polynucleotide, and described polynucleotide encoding is selected from the enzyme of beta-amyrin synthase and CYP51H10.