CN103602691A - Enzymes involved in triterpene synthesis - Google Patents

Abstract

The invention discloses enzymes involved in triterpene synthesis, and particularly relates to isolated polynucleotides. The polynucleotides encode enzymes comprising a carboxypeptidase-like protein, a methyltransferase and a glucosyltransferase. The enzymes are involved in the biosynthesis of beta-amyrin-derived triterpenes in plants and seeds. The invention also relates to construction of recombinant DNA constructs comprising all or a portion of the isolated polynucleotides. The polynucleotides are, in sense or antisense orientation, operably linked to at least one regulatory sequence.

Description

Relate to the synthetic enzyme of triterpene

The application is to be the Chinese patent application 200780053491.4 on June 25th, 2007 dividing an application of " relating to the synthetic enzyme of triterpene " applying date.

Technical field

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

Background technology

Terpenoid is also referred to as isoprenoid, and they have formed maximum natural product family, and such product is existing to be described over 22,000 kinds of individually oriented compounds.Triterpene or terpenoid (hemiterpene, monoterpene, sesquiterpene, diterpene, triterpene, tetraterpene, Polyprenol etc.) play various function in plant, as hormone, Photosynthesis Pigment, electron carrier, polysaccharide fraction conditioning agent and membrane structure component.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, the glycoside that storage form is them, saponin(e (referring to the people such as Price K.R., 1987, CRC Crit.Rev.Food Sci.Nutr.26:27-133).Therefore, for example, the mutant of diploid oat, black oat, it lacks main avenacin, and avenacin A-1 (so-called saponin(e lack or " Sad " mutant) has proved and lacked the disease resistance (people such as Papadopoulou K., 1999, Proc.Natl.Acad.Sci.U.S.A.96:12923-12928).These mutant are easier to be subject to the infringement of many different root fungal infections, comprise gaeumannomyces graminis, and it is normally nonpathogenic concerning oat.Genetic analysis shows that disease susceptibility improves and the reduction of avenacin level has cause-effect relationship.In addition, when inoculation gaeumannomyces graminis, the sad mutant that avenacin level reduces (be about wild-type level 15%) is compared only limited disease symptoms with other mutant that lacks avenacin completely, and this further connects avenacin level and disease resistance.

There are the data of accumulation to show that the saponin(e in diet may be useful (referring to for example Shi, the people such as J.A. (2004) J.Med.Food7:67-78 and Vis, the people such as E.H. (2005) Nutr.Cancer 51:37-44).Same, proved 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 it is slightly damaged that saponin(e only has in the process from soybean to soybean isolate, the saponin(e level in soybean improves and will cause the saponin(e level in isolate to improve (Berhow, the people such as M.A. (2002) Phytochem.Anal.13:343-348; The people (2002) such as Hu J., J.Agric.Food Chem.50:2587-2594).Therefore the saponin(e level improving in soybean will be the effective ways that improve saponin(e amount in human diet.In addition, the raising of saponin(e level can be the compound using in drug development source is provided.

Triterpene and sterol are synthetic 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 ring skeletons, 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).Should between sterol and triterpenoid saponin biosynthetic pathway, form branching-point by the cyclisation event of oxidosqualene cyclase catalysis.Multiple oxidosqualene cyclase is to evolve that relevant (Eur.J.Biochem.256:238-244) and produce multiple three rings, ,He five rings, Fourth Ring structure, this structure can further be modified for Kushiro, the people such as T. (1998).

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

The enzyme of structure comparison (Fig. 1) prediction other class except Cytochrome P450 will be also that beta-amyrin is changed into avenacin A-1 is needed.These enzymes comprise glycosyltransferase (GT), acyltransferase and methyltransgerase (MT).The enzyme that glycosyltransferase belongs to a large enzyme family ，Gai 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 cell processes as Cell wall synthesis and signal transduction (people such as Coutinho PM, 2003, J.Mol.Biol., 328:307-317).In the Qi Shiqige GT of representative leap all zones family, GT family 1 is wherein maximum (Coutinho PM and a Henrissat B, 1999:Carbohydrate active enzymes website http://afmb.cnrs-mrs.fr/CAZY/) family 1 is comprised of the GT of the reversion catalyst mechanism running of shifting via sugar, conventionally act on lower molecular weight acceptor molecule (Vogt T and Jones P, 2000, Trends Plant Sci.5:380-386; Lim E-K and Bowles DJ, 2004, EMBO J23:2915-2922).By with the micromolecular analogy of other glycosylation, the collateralization sugar chain of prediction avenacin A-1 is added in aglycone component synthetic by continuous Jiang Tang unit, it is most probable that to pass through three different glycosyltransferases (GT) active synthetic.Glycosylated first step relates to L-arabinose is added on the C3 hydroxyl of aglycone, transferase mediated by cytosine arabinoside.After this add two D-Glucose molecules, one in the C2 site of pectinose, and another is in C4 site, by (or may two) Transglucosylase mediation (people such as Townsend B, 2006, Phytochemistry Revs.5:109-114).

Acyl group is the common part 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 for example affect other critical process, as ubcellular exchange and sequestering action (taking in or resident mark by acting on vacuole).Had been found that recently the plant acyltransferase that a class is new.These enzyme-serine carboxypeptidase sample acyltransferases-have homology with peptase, but lack peptidase activity, on the contrary can acidylate natural product (Milkowski C & Strack D, (2003), Phytochemistry65:517; People (2005) the Plant Physiology138:1136 such as Fraser CM).Although other plant acyltransferase is generally used coenzyme thioesters as acry radical donor, these SCPL are used 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 contribute to insect-resistant in wild-type tomato (Li AX & Steffens JC, 2000, PNAS97:6902); Arabidopis thaliana enzyme SNGl, it is necessary (people such as Landry LG, 1995, Plant Physiology109:1159 of synthesis of phenyl phenoxy propionic acid mustard seed acyl malate (a kind of UV protective material); The people such as Lehfeldt C, 2000, Plant Cell12:1295); The second Arabidopis thaliana enzyme SNG2, it relates to mustard seed phatidylcholine in synthetic seed people such as (, 2001, Plant J28:83) Shirley AM; With swede type rape acyltransferase BnSCT, the formation of its catalysis mustard seed acid esters, this ester and bitter taste, convergency relevant with seed oil extraction problem (people such as Milkowski C, 2004, Plant J.38:80; The people such as Baumert A, 2005, Phytochemistry66:1334).Although the enzyme and the gene that relate in these are modified still fail to understand its feature, how the natural product of many other important plant origins is prepared by glucose-ester-dependent acyltransferase reaction by biochemical analysis is known.Example is included in antioxidant chlorogenic acid in sweet potato (Ipomoea batatas), the cyanin in wild Radix Dauci Sativae (Daucus carota), the Nutgalls Weibull in Oak Tree (Quercus robur) and at rape (Milkowski C & Strack D (2003) Phytochemistry65:517; People (2005) the Plant Physiology138:1136 such as Fraser CM) the mustard seed acyl in-and the glucosinolate of benzoyl-ester.There are four kinds of avenacins [14] that different structures is relevant.Avenacin A-1 (the main avenacin that is present in oat root) and B-1 carry out acidylate with N-methyl anthranilic acid; and avenacin A-2 and B-2 carry out acidylate (Hostettmann K and Marston A with phenylformic acid; 1995; Saponins; Cambridge University Press; Cambridge, UK).

The O-that SAMe-dependent methyltransgerase relates to many natural plant products methylate (people such as Frick S., 2001, Phytochemistry56:1-4).These enzymes in xylogen precursor and the required compou nd synthesis of other plant defense, play an important role (people such as Gang DR, 2002, Plant Cell14:505-519.

Summary of the invention

The separated polynucleotide of the enzyme relating to during the triterpene that the present invention relates to encode is synthetic.Specifically, the separated polynucleotide of the serine carboxypeptidase sample acyltransferase that the present invention relates to encode new, new methyltransgerase and 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 synthetic enzyme of triterpene in various types of grain will allow their manipulation.The synthetic manipulation of triterpene will cause the change of triterpenoid saponin level or structure.The raising of saponin(e output will cause the raising of Genes For Plant Tolerance insect ability.The food that derives from the plant with high triterpene level is considered to have the effect that reduces cholesterol, yet triterpene level reduces and it is believed that and can make food have better local flavor.Therefore, the transgenic plant with the triterpene level of change can produce resistance to insect, and the food of preparing with the seed that triterpene level or structure change will have nutritive value or the better local flavor of raising.

In another embodiment, the present invention relates to separated polynucleotide, the nucleotide sequence that described polynucleotide comprise encoding serine carboxypeptidase sample isopenicillin-n acyltransferase polypeptides, the aminoacid sequence of described polypeptide, when comparing with SEQ ID NO:2, has at least 95% sequence identity based on Clustal V comparison method; Or the nucleotide sequence of coding Methyl transporters enzyme polypeptide, the aminoacid sequence of described polypeptide, when comparing with SEQ ID NO:4, has at least 95% sequence identity based on Clustal V comparison method; Or the nucleotide sequence of coding Transglucosylase, the aminoacid sequence of described enzyme, when comparing with SEQ ID NO:6, has at least 95% sequence identity based on Clustal V comparison method; Or the nucleotide sequence that comprises (a), (b) or total length complementary sequence (c).

In another embodiment, the present invention relates to comprise the carrier of the separated polynucleotide of the present invention.

In another embodiment, the present invention relates to recombinant DNA construction body, at least a portion of the polynucleotide of the present invention of the first enzyme that this construct comprises coding triterpene approach, described part may be operably coupled to less on a kind of regulating and controlling sequence.

In another embodiment, the present invention relates to recombinant DNA construction body, at least a portion of the polynucleotide of the present invention of the first enzyme that this construct comprises coding triterpene approach, described part may be operably coupled to less on a kind of regulating and controlling sequence, the at least a portion that also comprises at least the second polynucleotide, described the second polynucleotide encoding regulates at least polypeptide of the expression of the second enzyme of triterpene approach.

In another embodiment, the present invention relates to the separated host cell that comprises recombinant DNA construction body 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 the plant transforming, and described plant origin is in the host cell of the conversion of higher plant and seed or cereal, and they derive from this type of plant transforming.These type of transgenic plant comprise the triterpene that those beta-amyrins with level change are derivative or have the plant of modification triterpene.

In another embodiment, the present invention relates to the transgenic plant that comprise recombinant chou of the present invention, wherein regulating sequence is allogeneic promoter, and wherein transgenic plant have the triterpene level of change when the triterpene level with wild-type plant is compared.

The present invention also relates to change the method for expression of polypeptides level in vegetable cell, described method comprises: use the nucleic acid fragment conversion of plant tissue from least a portion separated polynucleotide of the present invention, wherein said polynucleotide can change natural serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase; Described plant tissue is regenerated in transgenic plant; And assess when comparing with the plant with the wild-type expression level of corresponding natural serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase, the expression level of serine carboxypeptidase sample acyltransferase, methyltransgerase or the Transglucosylase of described transgenic plant changes.

The present invention also relates to produce the method at least one fungi to the plant of resistance, described method comprises: by the encode recombinant DNA construction body transformed plant cells of triterpene approach the first enzyme of at least one the present invention; Under the condition that promotes transgenic plant regeneration, make the transformed plant cells growth from step (a); And the transgenic plant of appraisal procedure (b) when with same species be not subject to plant that described recombinant DNA construction body transformed relatively time, the resistance of at least one fungi is improved to situation.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 approach, described enzyme is the oxidosqualene cyclase beta-amyrin synthase (product of Sadl gene; The people such as Haralampidis K., 2001, Proc.Natl.Acad.Sci.U.S.A.98:13431-13436) and/or cytochrome P 450 enzymes CYP51H10 (by Sad2 genes encoding; The people such as Qi X., 2006, Proc.Natl.Acad.Sci.U.S.A.103:18848-18853).

The method that the present invention also relates to produce the plant of serine carboxypeptidase sample acyltransferase, methyltransgerase or the Transglucosylase with change level, described method comprises: by the recombinant DNA construction body transformed plant cells of at least one coding triterpene approach the first enzyme claimed in claim 5; Under the condition that promotes transgenic plant regeneration, make the transformed plant cells growth from step (a); And during the amount comparison of serine carboxypeptidase sample acyltransferase, methyltransgerase or the Transglucosylase of assessment when the plant not transforming with described recombinant DNA construction body with same species in, the expression level change of serine carboxypeptidase sample acyltransferase, methyltransgerase or the Transglucosylase of the transgenic plant of step (b).

The present invention also relates to for the production of the method for plant with the triterpenoid saponin level of change, described method comprises: by the encode recombinant DNA construction body transformed plant cells of triterpene approach the first enzyme of at least one the present invention; Under the condition that promotes transgenic plant regeneration, make the transformed plant cells growth from step (a); And the triterpenoid saponin level of the triterpenoid saponin amount of the transgenic plant of appraisal procedure (b) in the plant not transforming with described recombinant DNA construction body with same species relatively time changes.Recombinant precursor also can comprise at least a portion of at least one the 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 for the production of the method for plant with the triterpenoid saponin level of raising, described method comprises: by the recombinant DNA construction body transformed plant cells of coding triterpene approach the first enzyme of at least one claim 5; Under the condition that promotes transgenic plant regeneration, make the transformed plant cells growth from step (a); And the triterpenoid saponin level of the triterpenoid saponin amount of the transgenic plant of appraisal procedure (b) in the plant not transforming with described recombinant DNA construction body with same species relatively time 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 for the production of the method for plant with the triterpenoid saponin level of reduction, described method comprises: by the encode recombinant DNA construction body transformed plant cells of triterpene approach the first enzyme of at least one the present invention; Under the condition that promotes transgenic plant regeneration, make the transformed plant cells growth from step (a); And the triterpenoid saponin level of the triterpenoid saponin amount of the transgenic plant of appraisal procedure (b) in the plant not transforming with described recombinant DNA construction body with same species relatively time reduces.Recombinant precursor also can comprise at least a portion of at least one the 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 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 the application's a part.

Fig. 1 illustrates the structure of beta-amyrin and avenacin A-1, and explanation a plurality of modifications must occur 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 of the biosynthetic enzyme of five predictions (beta-amyrin synthase (SAD1), Cytochrome P450 CYP51H10 (SAD2), serine carboxypeptidase sample albumin A sSCPL1, methyltransgerase AsMT1, Transglucosylase AsGT2 and GAPDH contrast), and described enzyme is arranged in radical bud, leaf and the flower tissue of oat.

Sequence description summary description accompanying sequence list.In this sequence list, with single letter, represent Nucleotide, with 3 independent letter representation amino acid, as Nucleic Acids Research13:3021-3030 (1985) and Biochemical Journal219 (2): defined in 345-373 (1984) described IUPAC-IUB standards.

SEQ ID NO:1 is that coding is from the cDNA nucleotide sequence of the serine carboxypeptidase sample polypeptide of black oat (AsSCPL1).

SEQ ID NO:2 is the AsSCPL1 aminoacid sequence derived from the genomic fragment showing in the cDNA fragment showing in SEQ ID NO:1 or SEQ ID NO:7.

SEQ ID NO:3 is that coding is from the cDNA nucleotide sequence of the acyl group methyltransgerase of black oat (AsMT1).

SEQ ID NO:4 is the AsMT1 aminoacid sequence derived from the genomic fragment showing in the cDNA fragment showing in SEQ ID NO:3 or SEQ ID NO:8.

SEQ ID NO:5 is that coding is from the cDNA nucleotide sequence of the Transglucosylase of black oat (AsGT2).

SEQ ID NO:6 is the AsGT2 aminoacid sequence derived from the genomic fragment showing in the cDNA fragment showing in SEQ ID NO:5 or SEQ ID NO:9.

SEQ ID NO:7 is the nucleotide sequence of the genomic fragment of coding AsSCPL1 polypeptide.

SEQ ID NO:8 is the nucleotide sequence of the genomic fragment of coding AsMT1.

SEQ ID NO:9 is the nucleotide sequence of the genomic fragment of coding AsGT2.

Embodiment

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

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

Many terms and abbreviation in context disclosed herein, have been used.Provided 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 the formation for the treatment of cell meta-bolites that use or that treat storage of cells, or start another pathways metabolism (thereby being called flow generation step).A lot of these classpaths are all very meticulous, and relate to the product of progressively modifying to make it to form the precise chemical structure structure with expectation to initial substance.

" nucleic acid " used herein means polynucleotide, and comprises strand or double-stranded deoxyribonucleotide or ribonucleotide base polymkeric substance.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 as strand or double-stranded RNA or DNA polymkeric substance, optionally contain nucleotide base synthetic, non-natural or that change.Nucleotide (conventionally existing with their 5 '-monophosphate form) can refer to by following their single-letter title: " 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 " separated " polynucleotide be a kind of by substantially separately or from the biology of natural generation polynucleotide wherein by other polynucleotide of conventional nucleic acid purification method purifying, that is, and other karyomit(e) and extrachromosomal DNA and RNA.The polynucleotide of recombination of polynucleotide and chemosynthesis also contained in this term.Separated polynucleotide of the present invention can comprise all or part of separated polynucleotide, for example, comprise the polynucleotide that are selected from following nucleotide sequence: the total length complementary sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 or this type of nucleotide sequence.

Triterpenoid saponin(e is via Isoprenoid pathway, and by cyclisation 2,3-epoxy squalene forms pentacyclic triterpene based compound, is mainly that volatile oil (beta-amyrin) or dammarane's skeleton are synthetic.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, isomultifiorenol and thalianol." triterpenoid saponin " finger ring triterpene join glycoconjugate, the triterpene except those derive from lanosterol or cycloartenol." steroid saponin(e " refers to derive from lanosterol or cycloartenol glycoside conjugate.Sapogenol is the triterpenoid saponin obtaining via external acid hydrolysis, to their amount that is measured as the triterpenoid saponin existing in the tissue that therefrom extracts saponin(e, provides a relative value.

The level of triterpenoid saponin can cause sapogenol to measure by measurement.The observed value of sapogenol is directly related to the level of triterpenoid saponin.Sapogenol is the triterpenoid saponin obtaining via external acid hydrolysis, to their amount that is measured as the triterpenoid saponin existing in the tissue that therefrom extracts saponin(e, provides a relative value, and this value is directly related to the amount of triterpenoid saponin.

Triterpenoid saponin level can be used technology known in the art to measure.For example, can use HPLC-MS or the HPLC with light scattering detector to measure (referring to for example Rupasinghe, the people such as H.P., (2003), J.Agri.Food Chem.51:5888-5894).Alternatively, can use the HPLC with UV detector to measure (people such as Hubert J, (2005), J.Agric.Food Chem.53:3923-3930).Other method comprises uses GC-FAB.(referring to such as people such as Gee, (1993), J Sci Food Agric.63:201-209).Other method relate to use tlc (TLC) in conjunction with the separated saponin(e of densitometry (referring to for example Oleszek WA. (2002) J.Chromatogr.A967:147-162.; Gurfinkel DM, and Rao AV (2002) J.Agric.Food Chem.50:426-430.

It is also possible using other method to measure triterpenoid saponin.For example, using panimmunity measuring method (for example radioimmunoassay or ELISA) is also suitable (Wang CC, Prasain JK, with Barnes S. (2002) J.Chromatogr.B Analyt.Technol.Biomed.Life Sci.777:3-28, the people such as Ahamed A, (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.For example, " 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 of the polynucleotide that comprise the oxidosqualene cyclase of encoding of the present invention." the triterpenoid saponin level of raising " can be to improve at least 100ppm, 250ppm, and 500ppm, 750ppm, 1000ppm, 1250ppm, 1500ppm, 3000ppm, 6000, or any their integer.

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

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

Term " subfragment with suitable function " and " in function suitable subfragment " are used interchangeably in this article.These terms refer to part or the subsequence of separated nucleic acid fragment, described fragment or the subfragment activated enzyme of whether encoding no matter wherein, and it is all retaining the ability that changes genetic expression or cause certain phenotype.For example, described fragment or subfragment can be used to design mosaic gene, to produce desired phenotype in the plant after conversion.Can be by nucleic acid fragment or subfragment be connected with affiliated promoter sequence with the direction with respect to plant promoter sequence sense or antisense, thereby design the mosaic gene for suppressing, wherein said nucleic acid fragment or the subfragment activated enzyme of no matter whether encoding all can.

One group of amino acid guarding at specific position in the aligned sequences of relevant protein in term " conserved domain " or " motif " fingering.Although the amino acid in other position between homologous protein can change, at the amino acid of specific position high conservative, represent that structure, stability or activity to protein are essential amino acids.Because they can be identified by the high conservative in the aligned sequences of protein homology thing family by them, they can be used as identification marking or " signature " determines whether the protein with new mensuration sequence belongs to the protein families of identifying in the past.

Term " homology ", " homology ", " similar substantially " and " corresponding substantially " are used interchangeably in this article.They refer to that the variation of wherein one or more nucleotide bases can not affect the nucleic acid fragment that the ability of certain phenotype was expressed or produced to nucleic acid fragment mediated gene.These terms also refer to the modification (for example lacking or insert one or more Nucleotide) of nucleic acid fragment of the present invention, with respect to initial not modified nucleic acid fragment, substantially can not change the functional performance of gained nucleic acid fragment.Therefore, just as the skilled artisan will appreciate, these concrete exemplary sequence are not only contained in the present invention.

In addition, technician recognizes, the present invention contain substantially similarly nucleotide sequence also by them at medium stringent condition (as 0.5X SSC, 0.1%SDS, 60 ℃) under, with the ability of exemplified sequence hybridization herein, or the ability of hybridization to any part of nucleotide sequence disclosed herein and hybridization to the sequence suitable with any nucleotide sequence function disclosed herein limits.Can regulate stringency with the similar fragment of screening moderate for example from the homologous sequence of edge biology far away, to the highly similar fragment of screening for example 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 and specific nucleic acid target sequence with than the hybridization of itself and non-target nucleic acid sequence higher can detection level (for example at least 2 times to background) hybridization, and refer to substantially get rid of non-target nucleic acid.The sequence of selective cross has approximately at least 80% sequence identity conventionally each other, or 90% sequence identity, at most and comprise 100% sequence identity (being complete complementary).

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

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

Specificity depends on post-hybridization washing conventionally, and the ionic strength of last washing soln and temperature are key factors.For DNA-DNA crossbred, T _mcan be with the people such as Meinkoth, Anal. Biochem.138:267-284 (1984): T _m=81.5 ℃+16.6 (%GC)-0.61, (log M)+0.41 (%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 that represents with base pair.T _mthe temperature of (under definite ionic strength and pH) complementary target sequence of 50% during with the probe hybridization mating completely.The mispairing of every appearance 1%, T _mreduce approximately 1 ℃; Therefore, adjustable T _mhybridization and/or wash conditions are with the sequence hybridization with having expectation identity.For example, if seek the sequence of have>=90% identity, can be by T _mreduce by 10 ℃.Conventionally, under definite ionic strength and pH, stringent condition is chosen as the melting temperature (Tm) (T than particular sequence and complementary sequence thereof _m) low approximately 5 ℃.Yet utmost point stringent condition can adopt than melting temperature (Tm) (T _m) hybridization and/or washing at the temperature of low 1,2,3 or 4 ℃; Medium stringent condition can adopt than melting temperature (Tm) (T _m) hybridization and/or washing at the temperature of low 6,7,8,9 or 10 ℃; Low stringency condition can adopt than melting temperature (Tm) (T _m) hybridization and/or washing at low 11,12,13,14,15 or 20 ℃ 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 variation of the severity of hybridization and/or washing soln is described in itself.If required mispairing degree causes T _mlower than 45 ℃ (aqueous solution) or 32 ℃ (formamide soln), preferably increase the concentration of SSC to can use higher temperature.Detailed guidance to nucleic acid hybridization can be found in Publication about Document: Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, part i, the 2nd chapter " Overview of principles of hybridization and the strategy of nucleic acid probe assays ", Elsevier, New York (1993); And Current Protocols in Molecular Biology, the 2nd chapter, the people such as Ausubel edit, Greene Publishing and Wiley-Interscience, New York (1995).Hybridization and/or wash conditions can be carried out at least 10,30,60,90,120 or 240 minutes.

" sequence identity " in nucleic acid or peptide sequence context or " identity " refer to that nucleic acid base or the amino-acid residue in two sequences is identical when comparing maximum match in appointment comparison window.

Therefore, " sequence identity per-cent " refers to by the values that relatively sequence of two sections of maximization couplings determines on a comparison window, wherein the part of the polynucleotide in described comparison window or peptide sequence is compared and can be comprised additional or disappearance (being gap) with the reference sequences of (not comprising additional or disappearance), to obtain two sections of maximum match between sequence.The method of calculation of described per-cent are, adding up the quantity in the site that identical nucleic acid base or amino-acid residue occur in two sections of sequences simultaneously counts 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, thereby obtains sequence identity per-cent.The available example of sequence identity per-cent 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.

Sequence contrast and per-cent identity or similarity can be determined with the multiple comparative approach that is designed for detection homologous sequence, these methods include, but is not limited to LASERGENE bioinformation and calculate bag (DNASTAR Inc., Madison, WI) MegAlig ^tMprogram.In the context of present application for patent, should be appreciated that while using sequence analysis software to analyze, except as otherwise noted, otherwise analytical results is by " default value " based on institute's referral procedure.Initial any value or the parameter set loading of software when this " default value " used refers at initializers first.

" Clustal V comparison method " corresponding to be called Clustal V (at Higgins and Sharp, CABIOS, 5:151-153 (1989); And be present in the MegAlign of LASERGENE information biology computation software package (DNASTAR Inc., Madison, WI) Higgins, the people such as D.G. (1992) Comput.Appl.Biosci.8:189-191), ^tMin program.For multiple ratio pair, default value is corresponding to breach point penalty (GAP PENALTY)=10 and notch length point penalty (GAP LENGTH PENALTY)=10.The default parameters that carries out the per-cent identity calculating of comparison in pairs and protein sequence by Clustal method is KTUPLE=1, gap penalty=3, window (WINDOW)=5 and DIAGONALS SAVED=5.And for nucleic acid, these parameters are KTUPLE=2, gap penalty=5, window=4 and DIAGONALS SAVED=4.With after Clustal V program aligned sequences, can be by checking that " sequence distance " table in same program obtains " per-cent identity ".

" BLASTN comparison method ”Shi You NCBI (National Center for Biotechnology Information, NCBI) provide in order to adopt the relatively algorithm of nucleotide sequence of default parameters.

Those skilled in the art is perfectly clear, and the sequence identity of multiple degree can be used for identifying polypeptide from other species, and wherein this class polypeptide has same or analogous function or activity.The available example of identity per-cent includes but not limited to any integer per-cent between 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or 50% to 100%.In fact, any integer amino acid identity 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 separating nucleotide fragment or the complementary sequence of part are also useful.

" codon degeneracy " refers to allow nucleotides sequence to be listed in and do not affect the divergent degree of the genetic code that changes in the situation of aminoacid sequence of coded polypeptide.Therefore, the present invention relates to the nucleic acid fragment of the nucleotide sequence of any aminoacid sequence that comprises encode all or major portion, AsSCPLl, AsMTl and the AsGT2 albumen of described amino acid sequence encode shown in SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.Technician knows " codon bias " that uses the given amino acid whose Nucleotide codon of appointment to show by particular host cell.Therefore,, when synthetic polyribonucleotides is expressed for improving the specific gene of host cell, wish that these polynucleotide of design make its codon usage frequency approach the frequency of the preferred codon of host cell use.

As used herein, " nucleic acid fragment " can be by using the oligonucleotide member of method known to those skilled in the art chemosynthesis to be assembled.These members be connected and anneal to form larger nucleic acid fragment, then its enzyme catalysis can be assembled to build complete required nucleic acid fragment." chemosynthesis " relevant with nucleic acid fragment refers in vitro component Nucleotide be assembled.Can adopt the method for improve setting up to complete the manual chemosynthesis of nucleic acid fragment, or a kind of in the machine that can business obtain by many kinds to complete robotics synthetic.Therefore, the codon bias based on optimization nucleotide sequence with reflection host cell, can customize nucleic acid fragment in order to optimization genetic expression.If codon uses those codons of being partial to host's preference, technician will appreciate that the possibility of successful genetic expression.Determining of preferred codon can be based on to deriving from the detection of the gene (wherein sequence information can obtain) of host cell.

" gene " refers to the nucleic acid fragment that can express specified protein, and it comprises regulating and controlling sequence (5 ' non-coding sequence) before encoding sequence and the regulating and controlling sequence (3 ' non-coding sequence) after encoding sequence." natural gene " refers to the naturally occurring gene with its oneself regulating and controlling sequence." mosaic gene " refers to any gene that is not natural gene, and being included in natural situation is not adjusting sequence and the encoding sequence existing together.Therefore, mosaic gene can comprise regulating and controlling sequence and the encoding sequence that comes from different sources, or comprises regulating and controlling sequence and the encoding sequence that comes from same source but arrange to be different from naturally occurring mode." alien gene " refers to not be present under normal circumstances the gene in host living beings, and it imports host living beings by transgenosis.Alien gene can comprise the natural gene being inserted in non-natural 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 of finding in nucleus, and is included in the organelle DNA of for example, finding in the subcellular component (plastosome, plastid) of cell.

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

" allelotrope " is on karyomit(e), to occupy a kind of in several selective alternative form of the gene of locating point.When on karyomit(e), all allelotrope of given site existence 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 " refer to encode DNA sequence dna of specific amino acid sequence." regulating and controlling sequence " refers to be positioned at upstream (5 ' non-coding sequence), centre or downstream (3 ' non-coding sequence) of encoding sequence, and affects the nucleotide sequence of the transcribing of correlative coding sequence, 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 forms by nearside with compared with far-end upstream element, and the latter often refers to enhanser.Therefore, " enhanser " is the DNA sequence dna that can stimulate promoter activity, and it can be the natural element of promotor, or inserts to strengthen promotor level or tissue-specific allos element.Promotor can wholely come from natural gene, or is comprised of the different elements that comes from different naturally occurring promotors, or even comprises synthetic DNA fragmentation.Those skilled in the art should be appreciated that different promotors can be in different tissues or cell type, or in the different etap, or respond different envrionment conditionss and the expression of guiding gene.It will also be appreciated that the DNA fragmentation of some modification may have identical promoter activity owing in most of the cases can't determining the exact range of regulating and controlling sequence completely.Cause that as a rule the promotor that gene is expressed in most cells type is commonly referred to " constitutive promoter ".Constantly finding can be used for the dissimilar new promotor in vegetable cell at present; In with Publication about Document, can find Multi-instance: Okamuro, J.K.and Goldberg, the Biochemistry ofPlants15:1-82 (1989) that R.B. edits.

" 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 primary transcription process, mRNA stability or the translation efficiency of mRNA.The example (Turner, R. and Foster, G.D., Mol.Biotechnol.3:225-236 (1995)) of translation leader sequence has been described.

" 3 ' non-coding sequence ", " transcription terminator " or " terminator sequence " refers to and is positioned at encoding sequence downstream and comprises that can affect mRNA processes or the polyadenylation recognition sequence of genetic expression and the DNA sequence dna of other sequence encoding conditioning signals.Polyadenylation signal is characterized by conventionally affects the 3 ' end that polyadenylic acid sheet adds mRNA precursor to.Ingelbrecht, the people such as I.L. are exemplified with the purposes Plant Cell1:671-680 (1989) of different 3 ' non-coding sequences.

" rna transcription thing " refers to the produced product of transcribing 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 that while deriving from the RNA sequence of the primary transcript of transcribing post-treatment, it refers to ripe RNA." messenger RNA(mRNA) " or " mRNA " refers to intronless and can be translated into by cell the RNA of protein." cDNA " refers to complementary with mRNA template and uses ThermoScript II from the synthetic DNA of mRNA template.CDNA can be strand, or uses the Klenow fragment of DNA polymerase i to change into two strands.The RNA that " has justice " refer to comprise mRNA and can be in cell or In Vitro Translation become the rna transcription thing of protein.

" sense-rna " refers to all or part of target primary transcript or mRNA complementary, 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, 5 ' non-coding sequence, 3 ' non-coding sequence, intron or encoding sequence are complementary." functional r NA " refers to that but sense-rna, ribozyme rna or other can not translate the RNA to the influential effect of cell processes.Term " complementation " and " reverse complemental " are used interchangeably concerning mRNA transcribes, and in order to define courier's sense-rna.

Term " is operably connected " and refers to the association of the nucleotide sequence 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.For example, when promotor can regulate the expression (that is, this encoding sequence is subject to the control of transcribing of this promotor) of encoding sequence, 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 of the present invention region, no matter directly or indirectly connect, 5 ' end is connected with target mRNA with target mRNA or 3 ' end or be positioned at target mRNA or the first complementary region is 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 in as Publication about Document, have more fully description: Sambrook, J., Fritsch, E.F.and Maniatis, T., Molecular Cloning:A Laboratory Manual; Cold Spring Harbor Laboratory:Cold Spring Harbor, NY (1989).Method for transformation is known by those skilled in the art, and is described in below.

" PCR " or " polymerase chain reaction " is the technology for the synthesis of a large amount of specific DNA fragments, and it comprises the circulation (Perkin Elmer Cetus Instruments, Norwalk, CT) of a series of repetitions.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, then under medium temperature, is extended for two kinds.Above-mentioned three continuous steps of one group are called as one " circulation ".

Term " restructuring " for example refers to the artificial combination of separated sequence fragment originally by chemosynthesis or by handling with genetic engineering technique that separated nucleic acid fragment realizes two.

Term " plasmid ", " carrier " and " box " refer to conventionally to carry the extra-chromosomal element of the gene of the part that does not belong to cell centre metabolism, and are usually the forms of circular double stranded DNA fragment.This class component can be the autonomously replicating sequence, genome integration sequence, phage or the strand that are derived from any source or the nucleotide sequence (linearity or ring-type) of double-stranded DNA or RNA, wherein a plurality of nucleotide sequences have connected or have recombinated and entered in a kind of unique design body, and this unique design body can be introduced the promoter fragment of selected gene product in cell together with corresponding 3 ' end non-translated sequence with DNA sequence dna." conversion box " refers to contain alien gene and except this alien gene, also contains the specific support that is conducive to transform the element that particular host cell transforms." expression cassette " refers to and comprises alien gene, and have the specific support (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 strengthen expression in host.

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

Term used herein " expression " refers to the production of functional end product (for example, mRNA or protein (can be both that precursor can be also mature form)).

Term " importing " refers to be provided nucleic acid (for example expression construct) or albumen to cell.Importing comprises and refers to nucleic acid to be integrated in eucaryon or prokaryotic cell prokaryocyte, can be integrated in the genome of cell, and comprise that finger temporarily offers cell by nucleic acid or albumen in this cell amplifying nucleic acid.Importing comprises and refers to stable or temporary method for transformation and sexual hybridization.Therefore, " importing " that nucleic acid fragment (for example recombinant DNA construction body/expression construct) is inserted in intracellular situation means " transfection " or " conversion " or " transduction ", and comprise and refer to nucleic acid fragment to be integrated in eucaryon or prokaryotic cell prokaryocyte, for example, in this cell amplifying nucleic acid fragment can be integrated into the genome (as karyomit(e), plasmid, plastid or Mitochondrial DNA) of cell, be transformed into autonomous replicon or transient expression (mRNA of transfection).

" maturation " protein refers to the polypeptide (having removed any propetide or the former polypeptide of peptide that are present in initial translation product) through translation post-treatment." precursor " protein refers to the primary translation product of mRNA, and propetide and peptide are former still exists.Propetide and peptide are former can be (but being not limited to) thin inner cellular localization signal.

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

" stable conversion " refers to that the genome that nucleic acid fragment is proceeded to host living beings is to produce stable hereditary property, and this genome comprises nuclear gene group and organelle gene group.On the contrary " instantaneous conversion " refer to nucleic acid fragment to proceed in the core of host living beings or in the organoid that comprises DNA, in the situation that without integrating or causing genetic expression without stable hereditary property.The host living beings that contains transformed nucleic acid fragment is called as " transgenosis " organism.

" transgenosis " refers to that its genome contains plant or the cell of heterologous polynucleotide as used herein.Preferably, heterologous polynucleotide is stably integrated in genome, makes these polynucleotide can be passed to the continuous generation.Heterologous polynucleotide can be individually or is integrated in genome as the part of expression construct.Transgenosis used herein comprises because heterologous nucleic acids existence has changed genotypic any cell, clone, callus, tissue, plant part or plant, comprises and initially carries out the transgenosis of this type of change and those transgenosiss that produce by sexual hybridization or vegetative propagation from initial transgenosis.The genome (chromogene group or karyomit(e) alia gene group) that term " transgenosis " is not contained by conventional plant breeding method or by infecting such as random cross fertilization, non-recombinant virus as used herein, non-recombinant bacteria transforms, the naturally-occurring event non-restructuring swivel base or spontaneous mutation causes changes.

Term " inhibition " refer to when with plant in the detectable enzyme activity level of natural enzyme relatively time, the reduction of detectable enzyme activity level in transgenic plant.In plant, the enzyme activity level of natural enzyme is called " wild-type " activity herein.Term " inhibition " comprises reduction, reduces, lowers, weakens, suppresses, eliminates and stops.This reduction may be due to the level that has reduced natural mRNA and translate into natural enzyme.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 in plant suppresses to complete by several different methods known in the art, comprises following methods." co-suppression " refers to that generation can suppress that identical or fully similar natural gene expresses adopted rna transcription this (United States Patent (USP) 5,231,020).Before this, by being conceived to cross expression with sense orientation, designed the co-suppression construct in plant (referring to people such as Vaucheret with the nucleotide sequence (it causes all RNA with the sequence of crossing expression with homology to reduce) that endogenous mRNA has homology, 1998, Plant J., 16:651-659; And Gura, 2000, Nature404:804-808)." Antisense Suppression " refers to produce the sense-rna transcript that can suppress target protein expression.Can be by plant virus sequence for guiding the inhibition of near-end mRNA encoding sequence (in the open WO98/36083 of disclosed PCT patent on August 20th, 1998).Describe " hair clip " structure and integrated all or part of of mRNA encoding sequence with complementary direction, caused the RNA having expressed to form potential " stem-ring " structure (in the open WO99/53050 of disclosed PCT patent on October 21st, 1999).In this case, the polynucleotide of the genes involved that stem is inserted with sense or antisense direction with respect to promotor by correspondence form, and ring forms by the polynucleotide of some genes involveds, and in construct, these polynucleotide do not have complementary sequence.This has increased co-suppression or reticent frequency in the transgenic plant that obtain.The summary suppressing about hair clip, referring to Wesley, the people such as S.V., 2003, Methods in Molecular Biology, Plant Functional Genomics:Methods and Protocols236:273-286.Wherein stem forms from the Nucleotide for the treatment of suppressor gene by least 30 and encircles by nucleotide sequence forms arbitrarily construct also effectively for suppressing (in disclosed WO99/61632 on December 2nd, 1999).Use the stem in poly--T and poly--A sequence generation stem-ring structure to describe to some extent (in disclosed WO02/00894 on January 3rd, 2002).Yet another kind of modification relates to the formation that promotes the stem in stem-ring structure with synthetic tumor-necrosis factor glycoproteins.The transgenic organism producing with this recombinant dna fragment shows that the level of the polynucleotide of being encoded by the nucleotide fragments that forms loop-stem structure reduces, described in the open WO02/00904 of disclosed PCT patent on January 3rd, 2002.The purposes that produces dsRNA construct has been described.The transcribing of the gene specific sense and antisense RNA that altogether promotor guiding induced gene suppresses in these constructs (referring to for example Shi, the people such as H. (2000) RNA6:1069-1076; Bastin, the people such as P. (2000) J.Cell Sci.113:3321-3328; Giordano, the people such as E. (2002) Genetics160:637-648; LaCount, D.J. and Donelson, J.E. U.S. Patent application 20020182223, is published on December 5th, 2002; Tran, the people such as N. (2003) BMC Biotechnol.3:21; With applicant's U.S. Provisional Application 60/578,404, be filed on June 9th, 2004).

Other method suppressing for enzyme includes but not limited to use and can form catalysis RNA and maybe can have the polynucleotide of ribozyme activity (United States Patent (USP) discloses 4,987,071, be published on January 22nd, 1991), and little RNA (also referred to as miRNA) interferes people (2003) Nature425:257-263 such as () Javier.

For the polynucleotide passage sequence that suppresses and the polynucleotide passage sequence that needs repressed gene to exist, be not 100% identical.For example, use the polynucleotide of the Gene Partial that derives from coding for alpha subunit (United States Patent (USP) discloses 6,362,399) successfully to suppress the subunit that all soybean seed storage protein β-soybean accompanies sphaeroprotein.β-soybean companion sphaeroprotein is a kind of heterogeneous glycoprotein, by three high negative electricity subunits, (is accredited as α, α ' and variation β) forms.The polynucleotide sequence of coding for alpha and α ' subunit is approximately 85% identical each other, yet that the polynucleotide sequence of coding β subunit and α and α ' subunit are respectively is approximately 75% to approximately 80% identical.Therefore the target polynucleotide region, suppressing with need has shown suppressing required target effective at least about 75% identical polynucleotide.Described polynucleotide can be identical at least about 80% with required target sequence, and at least about 90% identical, at least about 95% identical, or approximately 100% is identical.

Natural gene " can suppress express part " refers to part or the subfragment of separated nucleic acid fragment, wherein no matter whether described fragment or subfragment can be translated into activated enzyme, and it is all retaining the ability that changes genetic expression or cause certain phenotype.For example, described fragment or subfragment can be used to design mosaic gene or recombinant dna fragment, to produce desired phenotype in the plant after conversion.Can be by nucleic acid fragment or subfragment be connected with affiliated promoter sequence with the direction with respect to plant promoter sequence sense or antisense, thereby design the mosaic gene for suppressing, no matter whether wherein said nucleic acid fragment or subfragment translate into activated enzyme all can.Recombinant dna fragment can be designed to comprise the nucleic acid fragment that can promote that stem-ring structure forms.In stem-ring structure, ring or stem comprise a part needs repressed gene.Nucleic acid fragment should have at least about 20 ，Gai extensions, extension in abutting connection with Nucleotide identical with the repressed gene of need.Extension in abutting connection with Nucleotide can be any number, from least about 20, or approximately 32, to the size that needs 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 derive from the cell of following material: seed, suspension culture, embryo, mitogenetic region, callus, leaf, root, bud, gametophyte, sporophyte, pollen and sporule.

" filial generation " comprises any follow-up generation of plant.

" plant that at least one fungi is had to resistance " refers to the plant that comprises recombinant DNA construction body of the present invention, when with this plant of fungi infestation, with respect to the plant without recombinant DNA construction body of the present invention, its energy is anti-infective or standing to a greater extent infection, causes infringement minimizing, health degree increase and output lose 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 that does not comprise recombinant DNA construction body of the present invention.The transgenic plant that comprise recombinant DNA construction body of the present invention normally have more the plant of resistance than the same species plant without recombinant DNA construction body of the present invention at least one fungi.

plant expression system, expression cassette and carrier and conversion

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

For driving the source of the promotor that encoding sequence expresses unimportant, as long as it has enough transcriptional activities with by express the interpretable mRNA of desired nucleic acid fragment at reasonable time, in desired host tissue, thereby reaches object of the present invention.(being endogenous) promotor xenogenesis or non-xenogenesis all can be used for implementing the present invention.For example, suitable promotor includes but not limited to: the α of β soybean companion glb promoter causes β subunit, P34/Gly Bd m30K promotor, albumin promoter, Leg A1 promotor and the Leg A2 promotor of subunit, Ku Nizi (Kunitz) trypsin inhibitor 3 promotors, annexin promotor, glycinin Gyl promotor, β soybean companion glb promoter.

Above-mentioned annexin or P34 promotor are described in PCT patent and announce WO04/071178 (being published on August 26th, 2004).The activity level of above-mentioned annexin promotor is suitable with any known strong promoter, and described strong promoter is as (1) CaMV35S promotor (people such as Atanassova, Plant Mol.Biol.37:275-285 (1998); Battraw and Hall, Plant Mol.Biol.15:527-538 (1990); The people such as Holtorf, Plant Mol.Biol.29:637-646 (1995); The people such as Jefferson, EMBO is (1987) J.6:3901-3907; The people such as Wilmink, Plant Mol.Biol.28:949-955 (1995)); (2) Arabidopis thaliana oleosin (oleosin) promotor (people such as Plant, Plant Mol.Biol.25:193-205 (1994); Li, Texas A & M University Ph.D.dissertation, pp.107-128 (1997)); (3) Arabidopis thaliana ubiquitin extended proteins promotor (people such as Callis, J Biol.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 promotor (people such as Schoffl, Mol Gen Genet.217 (2-3): 246-53 (1989)); And (6) corn H3 histone gene promotor (people such as Atanassova, Plant Mol Biol.37 (2): 275-85 (1989)).

Another useful property of above-mentioned annexin promotor is its express spectra in growing seed.The most active in seed in the growth that above-mentioned annexin promotor (is pollinated latter 10 days in) in early days, and substantially keep silent in period subsequently.The express spectra of above-mentioned annexin promotor is all different from many seed specific promoters, and the latter is as conventionally shown the most highly active seed storage protein promotor (people such as Chen, Dev.Genet.10:112-122 (1989) at development later stage; The people such as Ellerstrom, Plant Mol.Biol.32:1019-1027 (1996); The people such as Keddie, Plant Mol.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 the seed specific promoters known from other is different.Therefore, when hope in early days the etap cross when expressing or suppressing a gene, above-mentioned annexin promotor is a very attractive selection.For example, may wish to express gene or a gene that participated in metabolism before seed maturity of a regulation and control early embryonic development.

After identifying the suitable promotor that is applicable to expression encoding sequence, be utilized as the known traditional method of 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 Sambrook, and the people such as J. are at Molecular Cloning:A Laboratory Manual; Second edition; Cold Spring Harbor Laboratory Press:Cold Spring Harbor, New York, 1989 (hereinafter to be referred as " people such as Sambrook; 1989 ") or Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A.and Struhl, K., editor; At Current Protocols in Molecular Biology; John Wiley and Sons:New York, has more detailed description in 1990 (hereinafter to be referred as " people such as Ausubel, 1990 ").

After having built recombinant precursor, can for example,, by the method (transfection, conversion and electroporation) being well known for ordinary skill in the art, be imported vegetable cell.

Also recombinant precursor of the present invention can be imported to 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 can be also 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 (for example 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 plant tissue or the tissue group of the morphology and function different piece that forms plant.Term " genome " refers to: (1) is present in each cell or a complete set of genetic material in virus or organoid (gene and non-coding sequence) of organism; And/or (2) conduct (monoploid) unit is from a complete set of karyomit(e) of parent's heredity.

The method that is used for transforming dicotyledons (mainly utilizing agrobacterium tumefaciens) and obtains transgenic plant has been published in following document: cotton (United States Patent (USP) 5,004,863 together with additive method; United States Patent (USP) 5,159,135); Soybean (United States Patent (USP) 5,569,834; United States Patent (USP) 5,416,011); Those (United States Patent (USP) 5,463,174) for Brassica plants (Brassica); Peanut (people such as Cheng, Plant Cell Rep.15:653-657 (1996); The people Plant Cell Rep. 14:699-703 (1995) such as McKently); Papaya (Ling, the people Bio/technology9:752-758 (1991) such as K.); And pea (the people Plant Cell Rep.15:254-258 (1995) such as Grant).To the summary of other common methods of Plant Transformation referring to Newell, C.A. (Mol.Biotechnol.16:53-65 (2000)).One of these method for transformation have utilized rhizobiaceae (Tepfler, M.and Casse-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 is announced WO92/17598), utilize electroporation (Chowrira, the people such as G.M., Mol.Biotechnol.3:17-23 (1995); Christou, the people such as P., Proc.Natl.Acad.Sci.84:3962-3966 (1987)), utilize microinjection and utilize particle gun bombardment (McCabe, the people such as D.E, Bio/Technology6:923 (1988); The people such as Christou, Plant Physiol.87:671-674 (1988)) carry out.

Exist multiple for the method from plant tissue aftergrowth.The concrete grammar of regeneration will depend on initial plant tissue and concrete plant species to be regenerated.From single plant protoplast transformant or from multiple explant regeneration through transforming, to grow and cultivate plants be (Weissbach and Weissbach (editor) known in the art, be loaded in: Methods for Plant Molecular Biology, (Eds.), Academic:San Diego, CA (1988)).This regeneration and growth method generally include following steps: select the cell transforming and cultivate these individually oriented cells by the common stage of embryonic development and by taking root the plantlet stage.Transgenosis embryo and seed are regenerated in a similar fashion.Subsequently the genetically modified seedling of taking root of gained is planted in the suitable plant growth culture medium such as soil.Preferably, the plant of regeneration is carried out to self-pollination to produce the transgenic plant of isozygotying.Or, the seed bearing plant of product that derives from strain important on the pollen of aftergrowth and agronomy is hybridized.On the contrary, the plant from these important strains is used for pollinating to aftergrowth.Utilize method well-known to those skilled in the art to cultivate the transgenic plant of the present invention that contain required polypeptide.

Except program discussed above, professional is also familiar with having described for the specified conditions of following purpose and the standard source data of program: macromolecular structure, operation and separated (for example DNA molecular, plasmid etc.); The structure of recombinant dna fragment and recombinant expressed body; And clone's screening is with separated.Such as referring to the people such as Sambrook, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor:NY (1989); The people such as Maliga, Methods in Plant Molecular Biology, Cold Spring Harbor:NY (1995); The people such as Birren, Genome Analysis:Detecting Genes, the 1st volume, Cold Spring Harbor:NY (1998); The people such as Birren, Genome Analysis:Analyzing DNA, the 2nd volume, Cold Spring Harbor:NY (1998); Plant Molecular Biology:A Laboratory Manual, editor, Clark, Springer:NY (1997).

The present invention relates to the separated polynucleotide of encoding serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase.As used herein; " polynucleotide " refer to encode polynucleotide of novel serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase, described enzyme relates to the derivative triterpene of beta-amyrin in plant or has the biosynthesizing of the triterpene that changes modification.These separation are called AsSCPLl (serine carboxypeptidase sample acyltransferase), AsMTl (methyltransgerase) and AsGT2 (Transglucosylase) from the enzyme of black oat.

Sad7 mutant can not produce avenacin; can not be accumulated in the triterpene glycoside (people such as Qi X that n-methyl oaminobenzoate (avenacin A-1 and B-1) or benzoate (avenacin A-2 and B-2) acyl group site have hydroxyl; 2004, Proc.Natl.Acad.Sci.U.S.A.101:8233).Therefore there is not triterpene acidylate in them.Four mutant (table 1) with Sad7 phenotype have been identified.Each in them has has infringement to polynucleotide of the present invention, will cause polynucleotide can not express the function mRNA of encode functional protein.These data show that together with biochemical data provided herein the nonmutationed polynucleotide encoding of the present invention is from (ASCPLl) serine carboxypeptidase sample acyltransferase of black oat; this enzyme is responsible for the acidylate of triterpene main chain, and this acylation process does not occur in Sad7 mutant.The cDNA genomic fragment (SEQ ID NO:1 and SEQ ID NO:7) of coding AsSCPLl is disclosed.

table 1: characterize Sad7 mutant.The comparison of the base position changing showing (subscript the 2nd row) based on mutant DNA sequence dna and SEQ ID NO:1.

The methyltransgerase relating in Sad9 gene product and the biosynthesizing of phenylpropionic acid class has homology, be included in and in blade, respond that pathogenic agent attacks and UV stimulates the barley methyltransgerase of the inducing (people such as Christensen A, 1998, Plant Mol.Biol.36:219).Because it is synthetic required that this methyltransgerase is avenacin, its effect may be the anthranilate of avenacin A-1 and the B-1 part (Fig. 1) that methylates.Four mutant (table 2) with Sad9 phenotype have been identified.Each in them has has infringement to polynucleotide of the present invention, will cause polynucleotide can not express the function mRNA of encode functional protein.These data show that together with biochemical data provided herein the nonmutationed polynucleotide encoding of the present invention is from (AsMTl) methyltransgerase of black oat, this enzyme be responsible for methylating anthranilate part of avenacin A-1 and B-1.The cDNA genomic fragment (SEQ ID NO:3 and SEQ ID NO:8) of coding AsMTl is disclosed.

table 2:Sad9 mutant characterizes.The comparison of the base position changing showing (subscript the 2nd row) based on mutant DNA sequence dna and SEQ ID NO:3.

The enzyme of Sad10 Transglucosylase and saccharification Whitfield's ointment and other benzoic acid derivative has homology.CDNA and the genomic fragment (SEQ ID NO:5 and SEQ ID NO:9) of coding from the glucosylation of black oat (AsGT2) disclosed.

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

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

For example; the encode gene of other acyltransferase (those serine carboxypeptidase sample acyltransferases specifically), Transglucosylase or methyltransgerase; can be directly separated with the form of cDNA or genomic dna; the described separated method well known to those skilled in the art of using, by being used all or part of nucleic acid fragment of the present invention to make DNA hybridization probe to screen the library from any required plant.Can design and pass through methods known in the art, based on the synthetic specific oligonucleotide probe of the of the present invention or sequence (people such as Sambrook, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor:NY (1989)).And whole sequence can be directly used in the known method by those of skill in the art, for example random primer DNA marker, nick translation or end-labelling, carry out synthesized dna probe, or synthesize rna probe by obtainable in-vitro transcription system.In addition, can design Auele Specific Primer and use it for and increase partly or the sequence of the present invention of total length.The amplified production of gained can be in amplified reaction process directly mark or after amplified reaction mark, and as cDNA or the genomic fragment of probe separated total length under suitable stringent condition.

In addition, can by two short-movie sections of nucleic acid fragment of the present invention in polymerase chain reaction rules for from DNA or the homogenic longer nucleic acid fragment of RNA amplification coding.Polymerase chain reaction also can carry out with clone's the library of nucleic acid fragment, and the sequence source of one of them primer is from nucleic acid fragment of the present invention, and the sequence of another primer is utilized the existence of polyadenylic acid sheet of 3 ' end of the mRNA precursor of coded plant gene.Alternatively, the sequence that second primer sequence can be based on deriving from cloning vector.For example, technician can be according to RACE rules (people such as Frohman, 1988, Proc.Natl.Acad.Sci.U.S.A., 85:8998-9002), the copy by the region between holding with pcr amplification Single locus and 3 ' or 5 ' in transcript produces cDNA.Primer with 3 ' and 5 ' direction orientation can be used sequences Design of the present invention.3'RACE or 5'RACE system (BRL) that use can business obtains, can 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).Use capable of being combined by 3 ' and the product that generates of 5'RACE program with generate full-length cDNA (Frohman and Martin, 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 that can synthesize aminoacid sequence of the present invention represents part.Can use these peptide immune animals to produce polyclone or monoclonal antibody, described antibody has peptide to comprising described aminoacid sequence or the specificity of albumen.Then can be by these antibody for screening cDNA expression library with the concerned full length cDNA clone of separation (Lerner, 1984, Adv.Immunol.36:1-34; Sambrook).

Can build the plasmid vector that comprises separated polynucleotide of the present invention.The selection of plasmid vector is depended on the method for transformed host cell.Technician knows in order successfully to transform, screen and breed the host cell that comprises mosaic gene, must be present in the genetic elements on plasmid vector.Technician also will recognize, different independent transformation events will cause expression (people such as Jones, 1985, EMBO J., the 4:2411-2418 of different levels and pattern; The people such as De Almeida, 1989, Mol.Gen.Genetics218:78-86), therefore, may with acquisition, show the strain of expectation expression level and pattern by screening multiple events.Such screening can be by the Southern engram analysis of DNA, the Western of the Northern engram analysis of mrna expression, protein expression analyzes or phenotype analytical completes.

Polypeptide of the present invention imported to different cellular compartments with regard to some application or promote that their secretions from cell may be useful.Therefore imagination can further be supplemented recombinant DNA construction body of the present invention, this encoding sequence by target signal in the cell that it is suitable that change is encoded carries out, as encoding transport signals (Keegstra, 1989, Cell56:247-253), there is or do not have the signal sequence (Chrispeels of endoplasmic reticulum retention signal, 1991, Ann.Rev.Plant Phys.Plant Mol.Biol.42:21-53) or removed or do not removed the nuclear localization signal (Raikhel of existing target signal, N., 1992, Plant Phys.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.

For example the expression of chimeric serine carboxypeptidase sample acyltransferase, methyltransgerase or Transglucosylase causes respectively comparing with the level producing in unconverted host cell, and the coded serine carboxypeptidase sample acyltransferase producing in transformed host cell, methyltransgerase or Transglucosylase protein level change.The transgenic plant that comprise polynucleotide of the present invention or plant part also cause plant to have the triterpene level of change under the control of allogeneic promoter, and described polynucleotide are SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 for example.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 for example seed and cereal.Therefore, separated polynucleotide of the present invention can be attached in the recombinant precursor that can import and copy 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 transformed.Also pay close attention to the method for the production of conversion of plant, described method comprises with any polynucleotide transformed plant cells of the present invention and the vegetable cell aftergrowth from transforming.

Can use nucleic acid fragment of the present invention 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 under normal circumstances the cell type of these enzymes or exist in the etap.The effect that this produces triterpene in those cells that change.It is believed that optionally the overexpression of polynucleotide of the present invention of being responsible for the polynucleotide combination of the enzyme of other step in saponin(e biosynthetic pathway with coding improves the resistance at least one fungi.

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

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

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

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

In addition, can build the recombinant precursor that comprises gene of the present invention, wherein except may be operably coupled to the coding region of the promotor that can guide triterpene approach the first expression of enzymes, recombinant precursor also comprises at least one other ，Gai coding region, coding region and may be operably coupled to the promotor that can guide at least one the second expression of enzymes of triterpene approach.The separated gene of the present invention from black oat of expectation and including 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 can expect to reduce or eliminate the expression of serine carboxypeptidase sample acyltransferase of the present invention, methyltransgerase or Transglucosylase in plant applies for some.Suppress polynucleotide of the present invention and can cause plant to produce saponin(e still less, this can improve local flavor then.In order to reach this object; can build the recombinant DNA construction body that is designed for this fermentoid of co-suppression, this structure is by being connected to the polynucleotide of coding serine carboxypeptidase sample acyltransferase of the present invention, methyltransgerase or Transglucosylase in plant promoter sequence.Alternatively, can build the mosaic gene that is designed for the sense-rna of expressing all or part of nucleic acid fragment of the present invention, this structure is by being reversely connected to gene or gene fragment in plant promoter sequence.Can co-suppression or antisense mosaic gene be imported in plant via transforming, wherein reduce or eliminate the expression of corresponding native gene.Also the construct that can prepare the chimeric nucleic acid fragment that causes forming stem-ring structure, wherein polynucleotide of the present invention are partly stem or ring or structure.Using at least a portion preparation of the nucleotide sequence of coding serine carboxypeptidase sample acyltransferase of the present invention, methyltransgerase or Transglucosylase is also possible by the construct that suppresses its expression with RNAi form.Can by any recombinant DNA construction body 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 Haralampidis K., 2001, Proc.Natl.Acad.Sci.U.S.A.98:13431-13436) and/or cytochrome P 450 enzymes CYP51H10 (by Sad2 genes encoding; The people such as Qi X., 2006, Proc.Natl.Acad.Sci.U.S.A.103:18848-18853) with the institute in inhibition approach in steps.The separated gene of the present invention from black oat of expectation and including but not limited to from the example combinations of other gene (can combine to change or reduce the level of triterpenoid saponin) 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;

In certain embodiments, the nucleotide sequence of the present embodiment can be 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.For example, the polynucleotide of described embodiment can carry out stacking with any other polynucleotide or other gene of this embodiment.The combination generating also can comprise any one in a plurality of copies of concerned polynucleotide.The polynucleotide of described embodiment also can occur stacking to produce the plant with multiple desired characteristic with any other gene or the assortment of genes, include but not limited to that the required characteristic of animal-feed is if high oil base for example, because (United States Patent (USP) discloses 6,232,529); Amino acid (for example thionin (United States Patent (USP) discloses 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 the people such as Musumura, (1989), Plant Mol.Biol.12:123)); The digestibility improving (the reserve protein (U.S. Patent Application Serial Number 10/053,410 is filed in November 7 calendar year 2001) of for example modifying; And Trx (U.S. Patent Application Serial Number 10/005,429 is filed in December 3 calendar year 2001)), be above-mentionedly openly incorporated herein by reference.The polynucleotide of described embodiment also can be desired with anti-insect, disease or weedicide characteristic stacking (for example bacillus thuringiensis toxin protein (United States Patent (USP) discloses 5,366,892; 5,747,450; 5,737,514; 5723,756; 5,593,881; The people such as Geiser (1986) Gene48:109); Lectin (people (1994) the Plant Mol.Biol.24:825 such as Van Damme); Fumonisins detoxification (fumonisin detoxification) gene (United States Patent (USP) discloses 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) EMBO J.7 (5): 1241-1248), anti-glutamine synthase inhibitor for example, as glufosinates or basta (bar gene; People (1987) EMBO such as De Block J.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), Pest Management Science61:269-276; The people such as Dufourmantel, (2007) Plant Biotech.J.5:118-133; Also referring to WO1997049816), give the gene (Li and Nicholl, (2005) Pest Managment Science61:277-285) 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 as those be disclosed in the gene in United States Patent (USP) open Shen Qing Publication US2004/0082770, WO02/36782 and WO03/092360)); With process or the desired characteristic of process product for example, as high oil (United States Patent (USP) discloses 6,232,529); Modified oil (for example fatty acid desaturase gene (United States Patent (USP) discloses 5,952,544; WO94/11516)); Treated starch (for example ADPG pyrophosphorylase (AGPase), starch synthase (SS), starch collateralization enzyme (SBE) and starch remove collateralization enzyme (SDBE)); And polymer biological plastics (for example United States Patent (USP) discloses 5.602,321; β-ketothiolase, poly(hydrobutyl ester) synthase and acetoacetyl-CoA reductase enzyme (people (1988) J.Bacteriol.170:5837-5847 such as Schubert), be conducive to express polyhydroxyalkanoatefrom (PHAs)), it is openly incorporated herein by reference.Also can combine the polynucleotide and the polynucleotide that agronomy feature is provided of described embodiment, described agronomy feature as male sterile (for example disclosing 5.583,210 referring to United States Patent (USP)), stalk intensity, flowering time, output improve or transformation technology characteristic for example, as Cycle Regulation or gene target (WO99/61619; WO00/17364; WO99/25821), it is openly incorporated herein by reference.

Can produce by any method these stacking combinations, include but not limited to by any ordinary method or

method or gene transformation cross-breeding plant.If described characteristic is stacking by genetic transformation plant, concerned polynucleotide sequence can combine with any order at any time.For example, can, by the transgenic plant that comprise one or more desired characteristic as target, by follow-up conversion, import more characteristics.Can characteristic and concerned polynucleotide be imported simultaneously by cotransformation rules, described polynucleotide are provided by any combination that transforms box.For example, add two sequences of importing, two sequences can be included in separated conversion box to (trans) or be included in (cis) in same conversion box.Can promote sequence to express by identical promoters or different promoters.In some instances, may expect to import the conversion box of expressing suppressing concerned polynucleotide.This can be used in combination in plant, to generate desired characteristic combination with other any combination that suppresses box or overexpression box.Further recognize and can use locus specificity recombinant chou to tie up to the stacking polynucleotide sequence of required genomic locus.Referring to for example 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.Can use the above-mentioned recombinant precursor that produces the horizontal saponin(e of higher triterpene that causes to produce the plant that resists at least one fungi.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 stem wilt bacterium (Phomopsis sojae), soybean north stem canker, white thin,tough silk bacterium, Cercospora kikuchii, Germ To Soybean Frogeye Leaf Spot, C. dematium (Colletotichum truncatum), the mould fallen leaves of paragutta rod spore germ, Septoria Glycines, phyllosticta bacterium, 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, many spores of clover shell tikka bacterium, clover stemphylium botryosum, common leaf spot of alfalfa bacterium, wheat: wheat bar ustilago, black embryo of wheat germ chain lattice spore, the mould germ of Wheat Black, wheat sickle-like bacteria, fusarium culmorum, wheat loose smut bacterium, wheat shell two spores, 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-based maize ear rot bacterium, dry thread Pyrenomycetes, Rhizoctonia cerealis, gaeumannomyces graminis, Root Rot of Wheat germ, Claviceps purpurea, rye grass Tilletia foetida, 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 stem are put mould 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 (Glomerella graminicola), Diplodia zea bacterium (Diplodia maydis), corn top rot fusarium moniliforme, endogenous colyliform reaping hook is mould, Fusarium graminearum (Gibberella zeae), flavus, southern corn leaf blight, T (southern corn leaf blight), corn circle pinta bacterium I, II & III (Cochliobolus carbonum), Exserohilum turcicum I, II & III, obstruct the shape bacterium that wriggles, 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, the black bud branch of standing grain is mould, lunata, the curved spore such as not, Maize Curvularia, viride, the tough and tensile ustilago of corn, corn dry rot germ, the crazy top sickness bacterium of corn, 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, thick spot shell two spores of Chinese sorghum, 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 the spore bacterium that wriggles, the bromegrass length spore of wriggling, lunata, Chinese sorghum stem point is mould, Chinese sorghum seat branch spore, the raw seat branch of Chinese sorghum spore, Chinese sorghum black mole bacterium, head smut of sorghum bacterium (Sphacelotheca reiliana), sorghum loose smut bacterium, the hard spore heap of fine strain of millet ustilago, Chinese sorghum ergot, dry thread Pyrenomycetes, branch top spore is mould, black fruit bacterium (Colletotrichum) (C.sublineolum), Fusarium graminearum, Fusarium oxysporum, etc..

Can use nucleotide sequence of the present invention to carry out multiple gene and physics drawing practice based on nucleic acid amplification.Example include but not limited to allele specific amplification (Kazazian, H.H.jr, 1989, J.Lab.Clin.Med.11:95-96), the polymorphism analysis (CAPS of pcr amplified fragment; Sheffield, V.C., waits people, 1993, Genomics16:325-332), allele-specific connects (Landegren, U., Deng people, 1988, Science241:1077-1080), Nucleotide extension (Sokolov, B.P., 1990, Nucleic Acid Res.18:3671), the mapping of radiation hybrid (Walter, the people such as M.A., 1994, Nat.Genet.7:22-28), fluorescence in situ hybridization (FISH; Svitashev, S.K. and Somers, D.A., 2002, Plant Cell Tissue Organ Cult.69:205-214) and Happy drawing (Dear, P.H. and Cook, P.R., 1989, Nucleic Acid Res.17:6795-6807).With regard to these methods, nucleic acid fragment sequence is used for designing and preparing primer pair 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 of using PCR-based, must identify the DNA sequence dna difference between the drawing hybrid strain in the region corresponding to nucleotide sequence of the present invention.Yet this is generally unnecessary to all drawing practices.

Although do not wish to be subject to the constraint of any theory or theory of operation, those skilled in the art believes that the triterpene level of change has different effects.Triterpene (as the avenacin) level improving in the plant part that infected by fungal pathogens can be given the resistance of plant at least some these type of pathogenic agent, and protective plant also so improves it and coerces the output in environment fungi.The food that derives from the plant with high triterpene level is considered to have the effect that reduces cholesterol, yet triterpene level reduces and it is believed that and can make food have better local flavor.Therefore, in process of growth, there is AsSCPL1, the AsMT1 of change level and/or the plant of AsGT2 and can contribute to produce 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 be in the following embodiments further limits, wherein all umbers and per-cent be by weight and the number of degrees be degree Celsius, unless otherwise indicated.Should be appreciated that, although these embodiment have illustrated the preferred embodiments of the invention, be only that the mode with illustration provides.According to discussion and these embodiment above, 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, can make multiple change and modification to the present invention, so that it is applicable to multiple usage and condition.Therefore, except those herein shown in and describe those, described in above, various modification of the present invention will be apparent to one skilled in the art.In the scope of claims that these modification are also intended to belong to additional.

general method

The standard recombinant dna technology of using in embodiment and molecule clone technology are known in the art and describe to some extent in as Publication about Document: (1) Sambrook, J., Fritsch, E.F. and Maniatis, T.Molecular Cloning:A Laboratory Manual; Cold Spring Harbor Laboratory:Cold Spring Harbor, NY (1989) is (Maniatis); (2) T.J.Silhavy, M.L.Bennan and L.W.Enquist, Experiments with Gene Fusions; Cold Spring Harbor Laboratory:Cold Spring Harbor, NY (1984); (3) Ausubel, the people such as F.M., Current Protocols in Molecular Biology, publishes (1987) by Greene Publishing Assoc.and Wiley-Interscience.

The materials and methods that maintains and grow that is applicable to microorganisms cultures is well known in the art.Be applicable to that the technology of embodiment can be at Manual of Methods for General Bacteriology (Phillipp Gerhardt below, R.G.E.Murray, Ralph N.Costilow, Eugene W.Nester, Willis A.Wood, Noel R.Krieg and G.Briggs Phillips edit), American Society for Microbiology:Washington, D.C. (1994)); Or Thomas D.Brock in Biotechnology:A Textbook of Industrial Microbiology, the 2nd edition, Sinauer Associates:Sunderland, finds in the description of MA (1989).The all reagent, restriction enzyme and the material that are used for the growth of microorganism cells and maintain are all available from Aldrich Chemicals (Milwaukee, WI), DIFCO Laboratories (Detroit, MI), GIBCO/BRL (Gaithersburg, MD) or Sigma Chemical Company (St.Louis, MO), unless otherwise indicated.

General molecular cloning completes according to standard method (people such as Sambrook, the same).DNA sequence dna is on ABI automatic sequencer, to adopt dyestuff terminator technology (United States Patent (USP) 5,366,860; EP272,007) with the combination of carrier and Insert Fragment Auele Specific Primer, produce.Sequence editor carries out in Sequencher (Gene Codes Corporation, Ann Arbor, MI).All sequences covers at least twice on both direction.Gene order be relatively to use DNASTAR software (DNA Star, Inc.) to 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 to rise, " μ M " represents micro-molar concentration, " mM " represents millimolar concentration, and " M " represents volumetric molar concentration, and " mmol " represents mmole, " μ mole " represents micromole, " g " expression gram, " μ g " represents microgram, " ng " represents nanogram, " U " representation unit, " bp " represents base pair and " kB " expression kilobase pair.

embodiment 1

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

Coding is subject to the separated cDNA library that derives from black oat accession number S75 genomic dna and be prepared as follows from oat from ，Gai library, BAC library of genome polynucleotide passage of serine carboxypeptidase sample albumen (AsSCPL1), methyltransgerase (AsMT1) and the gene of Transglucosylase (AsGT2) impact.

BAC library from black oat accession number S75 genomic dna, build ((people such as Qi X., 2006, Proc.Natl.Acad.Sci.U.S.A103:18848-18853).DNA probe derive from Sad1 (people such as Osbourn, on March 6th, 2007, US, 7,186,884B2) and Sad2 (people such as Osbourn, US2006-0112448A1), this probe is used for screening complete BAC library.Build to cross over one for the biosynthetic gene cluster of avenacin (people such as Qi X., 2004, BAC contig Proc.Natl.Acad.Sci.U.S.A.101:8233-8238).BAC fingerprint and BAC terminal sequence analysis make us can assemble a contig, and this contig clones 462F14,460D15 by BAC and 409O10 forms.Clone 460D15 comprise Sad1 and Sad2 (people such as Qi X., 2006, Proc.Natl.Acad.Sci.U.S.A103:18848-18853); Clone 462F14 has the overlapping region of about 70kb and 460D15 in a side of the most close Sad2, yet clone 409O10 and clone 460D15 stress to fold about 35kb at one of the most close Sad1.Use the end sequence of 409O10 as probe, identify another BAC clone 341P21.BAC fingerprint and pcr analysis are confirmed the other end and the overlapping about 40kb of clone 341P21 of clone 409O10.The length of predicting complete contig is 365kb.

The structure of the separation of oat root mRNA and corresponding cDNA library is described in the people such as Haralampidis K., 2001, Proc.Natl.Acad.Sci.U.S.A.98:13431-13436.In Eco RI/Xho I site, cDNA insertion sequence is cloned into (ClonTech Lab, Inc.) in pGADT7 carrier.To amount to 36,864 clones is stored in 96 384 hole microplates.Complete oat root cDNA library lattice is placed on two strainers for hybridization.By the separated insertion sequence DNA from BAC clone 409O10 and 341P21 of Not I digestion, and used as probe with screening cDNA library.Identified and amounted to 60 positive colonies and these clones' insertion sequence is checked order.ABI is used in order-checking

big-Dye ^tMterminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems) carries out.From 52 clones, obtain cDNA sequence.Except 5 special sequences, remaining 47 cDNA insertion sequences are divided into four genoids.The full-length cDNA that they are further analyzed to identify the longest cDNA clone and identify subsequently each class in four classes.

Therefore the first kind represents the cDNA of Sad1, confirms that in fact BAC contig do not cross over the gene cluster that comprises this gene.

Prediction is from the full-length cDNA encoding serine carboxypeptidase sample albumin A sSCPL1 of Equations of The Second Kind.The nucleotide sequence of this gene is shown in SEQ ID NO:1.Nucleotide 56 to the 1534 derivative aminoacid sequences of SEQ ID NO:1 are shown in SEQ ID NO:2.Nucleotide 1535 to 1537 represents a terminator codon.

The 3rd class cDNA sequence is corresponding to the methyltransgerase of prediction, AsMT1.The nucleotide sequence of this gene is shown in SEQ ID NO:3.Nucleotide 13 to the 1074 derivative aminoacid sequences of SEQ ID NO:3 are shown in SEQ ID NO:4.Nucleotide 1075 to 1077 represents a terminator codon.

The 4th class cDNA sequence is corresponding to the glucosylation enzyme family 1 of a prediction, AsGT2.By oat root being inferred to the sequential analysis of Transglucosylase EST (ref Sad1 patent), obtain full-length cDNA.The nucleotide sequence of this gene is shown in SEQ ID NO:5.The derived amino acid sequence of the Nucleotide 66 to 1475 of SEQ ID NO:5 is shown in SEQ ID NO: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, BAC clone 462F14,460D15,409O10 and 341P21 from contig are checked order.Obtained the BAC contig of about 317kb, the gene of the biosynthetic enzyme that this contig comprises five predictions (beta-amyrin synthase (Sad1), Cytochrome P450 CYP51H10 (Sad2), serine carboxypeptidase sample albumin A sSCPL1, methyltransgerase AsMT1 and Transglucosylase AsGT2) (Fig. 2).Sequence annotation does not disclose more open reading frame widely.Northern blotting and RT-PCR analyze all five genes that show in BAC contig and in root, preferentially express (Fig. 3).Because avenacin synthesizes and gathers at oat root, and Sad1 and Sad2 be in root-preferred expression, and other three Gene A sSCPL1, AsMT1 and AsGT2 also may relate to the biosynthesizing of avenacin.

Prediction promotor and the genome sequence of three genes that serine carboxypeptidase sample albumen (AsSCPL1), methyltransgerase (AsMT1) and Transglucosylase (AsGT2) are relatively provided of genomic dna sequence and cDNA sequence.Coding AsSCPL1, AsMT1 and the genome polynucleotide passage of AsGT2 and the 3-kb promoter sequence of prediction are shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID No:9.

embodiment 2

separation also characterizes Sad7 oat mutant

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

generate mutant

Substantially according to described method, use seed (the accession number S75 of sodium azide mutagenesis diploid oat (black oat), from Institute of Grasslands and Environmental Research, Aberystwyth, Wales, UK) (Rines, H.W., 1985, Env.Exp.Bot., 25:7-17).In brief, carry out as follows mutagenesis.Seed is immersed in the Erlenmeyer flask with rubbery stopper sealing in advance, and every seed is used 0.5mL water, in orbital platform vibrator, with 120 circulation per minutes, vibrates.In pre-soaking, after 4 hours, pour out water.The 10mM sodium azide solution of preparation in 0.1M sodium phosphate, pH3.2, and it is added in seed immediately.After as above vibrating 1 hour, pour out mutagenesis solution water and rinse seed 5 to 6 times, wherein last three washing times extend to more than 30 minutes.Seed after rinsing is drained to moisture, spread on the paper in stink cupboard dry to it.Make also to assess as shown below root fluorescence from the M2 seed germination of single M1 plant.

Therefore main avenacin avenacin A-1 comprises N-methyl anthranilic acid, mainly causes light blue fluorescence people such as (, 1994, Physiol.Mol.Plant Pathol.45:457-467) Osbourn A.E. of oat seedlings root.Avenacin A-1 can detect by UV light, and this makes root fluorescence to be lacked to (Sad) oat mutant as just screening to identify saponin(e.Make the seed germination of single M2 family and assess its root fluorescence.In preliminary screening, in screening, represent after the seedling of 1 ，289Ge M2 family, identified that ten have the independent mutation body that reduces fluorescence, this was reported by people such as Papadopoulou K. (1999, Proc.Natl.Acad.Sci.U.S.A.96:12923-1928).Follow-up screening mutant has identified that other 82 root fluorescence based on reducing carry out separated independent avenacin and lack mutant.

biological chemistry characterizes

According to the root extract of ten initial mutant of described methods analyst (people such as Papadopoulou K., 1999, Proc.Natl.Acad.Sci.U.S.A.96:12923-1928).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.Preparing three parts of crude methanol root extracts from M3 seedling analyzes for HPLC, on Hichrom Nucleosil5C18 reversed-phase column (4.5 * 250mm), under isocratic condition, in 75% methyl alcohol (flow is 1mL/min), direct analysis 100 μ L aliquots containigs, detection wavelength is 225nm.By the standard substance comparison peak region with those concentration known, quantitative four kinds of avenacins.The extract that TLC is analyzed is dried, is resuspended in 1mL water, and is applied to SepPak C18 reversed-phase column (Waters, Milford, MA), and this post is pre-conditioned for connecing 10mL distilled water after 10mL methyl alcohol.With after 75% methanol-eluted fractions, sample is dried, is resuspended in 15 μ L100% methyl alcohol, be applied to TLC plate, and use chloroform: methyl alcohol: water (13: 6: 1; V: v: v) separation.Avenacin A-1 and B-1 and other fluorescent components excite lower video picture at the UV of 302nm place.Then use aubepine/sulfuric acid/acetic acid (1: 1: 48, v: v: v) spray TLC plate and toast 5 minutes to detect all four kinds of saponin(es at 130 ℃.The root extract that derives 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 of measuring black oat lacks phenotype due to single sudden change.To the Analysis and Identification in the F2 generation from mutant intermolecular hybrid at least 4 complementation groups in initial 10 mutant strains.By these sites called after Sad1 to Sad4 (people such as Papadopoulou K., 1999, Proc.Natl.Acad.Sci.U.S.A.96:12923-1928).The further analysis of initial 10 mutant strains has been measured to other 4 sites, by its called after Sad5 to Sad8 (people such as Qi X., 2004, Proc.Natl.Acad.Sci.U.S.A.101:8233-8238).

In Sad5, Sad6, Sad7 and Sad8 mutant, carry out the DNA sequence analysis of 3 new genes to comprising in avenacin biosynthesizing (AsSCPL1, AsMT1 and AsGT2).In AsSCPL1, from the single core thuja acid of C to T, change the 137th amino acid among mutant #587 (Sad7) and mutant #616 (initial called after Sad5) respectively and cause the variation from Serine to phenylalanine, on the 79th amino acid in #376 (Sad7), cause from extremely leucic variation (table 1) of proline(Pro).In AsSCPL1, the new mutant body #19.1 of origination point sudden change also carries out separation by screening the genomic dna of other 82 Sad mutant, the screening (as mentioned above) of described mutant root fluorescence based on reducing by expansion is identified, and is used Surveyor Mutation Detection Kit (Transgenomic) to be further analyzed.By pcr amplification target cDNA.Amplified production from two mutant strains is mixed.According to the specification sheets of manufacturers (Transgenomic Cat No.706025), carry out heteroduplex formation and subsequence program detects for mutant.Mutant #19.1 comprises a point mutation, predicts that this point mutation causes the 463rd variation of amino acid from Threonine to Isoleucine.In the AsMT1 of Sad5, Sad6, Sad7 or Sad8 mutant and AsGT2 gene, do not exist Nucleotide to change.The allelomorphism test of carrying out with mutant #19.1, #616 and #376 shows that all three mutant are corresponding to a single site, Sad7.These digital proof mutant #616 (it is called Sad5 at first) is corresponding to site Sad7.These results clearly show that Sad7 is corresponding to serine carboxypeptidase sample albumin A sSCPL1.AsSCPL1 is essential for the triterpene skeleton that acyl group (be N-methyl anthranilic acid, be phenylformic acid in non-fluorescence avenacin A-2 and B-2 situation) is joined to avenacin in fluorescence avenacin A-1 and B-1 situation.

embodiment 3

separation also characterizes Sad9 oat mutant

Initial mutant #616, #825, #376 and #1243 people 1999.PNAS9612923-1928 such as () Papadopoulou do not comprise any Nucleotide in AsMT1 and AsGT2 gene to be changed.Sequential analysis is expanded to 82 new Sad mutant, and described mutant carries out separation as described in Example 2.Three mutant M3 strains (#195, #961 and #1310) are accredited as and in AsMT1 gene, have point mutation (table 2).Any mutant in set does not identify sudden change in AsGT2 gene.DNA sequence analysis confirms that in the encoding sequence in three mutant (#195, #961 and #1310), a mononucleotide occurring changes.Predict that each in changing of these Nucleotide will cause that an amino acid changes (table 2).

The root of these three mutant lacks the light blue fluorescence relevant to avenacin A-1, is dark violet fluorescence under UV excites.A little mutant are called " purple mutant ".Use the method (table 2) based on TLC to identify out from 82 new Sad mutant a stronger mutant #841 of purple.Carry out as mentioned below the metabolite analysis of purple mutant.Intercepting is from ten tips of a root (length is 0.5cm) of each strain, and they are immersed in 500ul75%MeOH, soak time between one hour between spending the night.After centrifugal, supernatant liquor is transferred in new pipe, is dried and extract is resuspended in 20ul100%MeOH.Ten ul samples are added on a TLC plate, and described TLC is at ChCl ₃: MeOH:dH ₂in 0 (13:6:1vol/vol/v01), develop.TLC plate checks under UV excites.The all purple mutant that comprise #841 have clearly purple dot on TLC plate.The sequential analysis of #841 confirms to occur to change from the mononucleotide of C to T in the coding region of this mutant.Predict that this variation causes that the L-Ala at amino acid 333 places becomes α-amino-isovaleric acid (referring to table 1).

Purple mutant being hybridized each other to (#195 * #1310, #961 * #1310, #195 * #961) tests for allelomorphism.Two mutant alleles by sequential analysis in F1 crossbred, have confirmed heterozygote.All F1 plants have retained " purple root " phenotype.Three purple mutant of these result indications are the mutation alleles in homologous genes tic site, are defined as now Sad9.Gene data, rna expression data (Fig. 3) and metabolite analysis indicate Sad9 corresponding to AsMT1, the methyltransgerase needing in the avenacin biosynthesizing in oat.The function of this methyltransgerase may be the o-amino benzoyl acidic group methylating on fluorescence avenacin A-1 and B-1.

embodiment 4

characterize the Transglucosylase in Sad gene cluster

In avenacin gene cluster, in 5 five genes in the BAC contig of crossing over Sad gene cluster 4 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).Yet, even identified a plurality of mutation alleles of other four genes, the mutant of AsGT2 does not still reduce in the set of root Fluorescence Mutation of A bodies 92 of our expansion.If AsGT2 adds one/a plurality of sugar to necessary on avenacin three sugar moieties, the loss function of AsGT2 does not cause the root fluorescence phenotype of minimizing, because add fluorophor (N-methyl anthranilic acid) not to be affected.Other possibility has functional superfluous She or AsGT2 sudden change is lethal.Alternatively, the formation that AsGT2 can catalyzing acyl glucose intermediate, this intermediate is for the acylations of AsSCPL1 mediation.Therefore proved the biochemical function of AsGT2.

In order to test the function of AsGT2, AsGT2cDNA is cloned in Novagen pET-19b expression vector and as follows at expression in escherichia coli: the Bacillus coli cells that comprises described expression construct is inoculated on LB agar, this LB agar has supplemented 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.For expressing protein, use cell on solid medium to be inoculated into ten and have in the 250mL flask of liquid nutrient medium that 50mL supplemented mentioned component.Culture is at 37 degrees Celsius of shaking culture OD ₆₀₀about 0.6.Then in culture being transferred to 16 degrees Celsius of Shaking Incubatorss before adding final concentration to be the IPTG inductor of 0.1mM, cultivate 30 minutes.Then at 16 degrees Celsius of shaking culture cultures, spend the night.

By 4 degrees Celsius, at centrifugal 10 minutes of 7,000 * g results inducing cell.Cell precipitation thing is resuspended in 5 to 10mL has in dissolving-binding buffer liquid of Roche without EDTA proteinase inhibitor (every 50mL1 sheet) (300mM NaCl, 50mM phosphorus sodium, 20mM imidazoles, 5% glycerine, pH7.8).Use French press dissolved cell twice, under ice-cold condition, carry out all the time.By lysate at 4 degrees Celsius, at 10,000g centrifugal 45 minutes.Before being the FPLC of 0.5mL/ minute in injection for flow velocity by 0.2 μ m syringe-strainer filtering supernatant.

1mL HiTrap chelating HP post (Amersham) and FPLC system that use is pre-charged with are carried out FPLC.Purification column is according to the recommendation 0.1M NiCl of manufacturers ₂be full of.Before sample injection, use dissolving-binding buffer liquid pre-equilibration column.After injection, described post is in use Elution buffer B (300mM NaCl, 50mM sodium phosphate buffer, 700mM imidazoles, 5% glycerine, the use dissolving-binding buffer liquid washing before of gradient program pH8.0) 30 minutes to 1 hour.

table 3: the condition of eluted protein from HiTrap chelating 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 wash-out fragment of 1mL on ice, 20mL is for developing result in 10%PAGE system.Concerned fragment merges and dialysed overnight at 4 degrees Celsius, uses 50mM sodium phosphate buffer, and pH7.5, has 50% glycerine and 2mM MgCl ₂.Aliquots containig is freezing and storage under-80 degrees Celsius rapidly in liquid nitrogen.

Utilize the radioassay analytical method of mixture receptor substrate to be used to detect the low-level activity with pure substrate or concentrated substrate.This detection analytical method comprises 4mL ¹⁴c-UDP-glucose, 25mL50mM potassium phosphate buffer, pH7.6, the 0.25mL100mM receptor substrate in DMSO, the albumen prepared product that 5mL dialysed, and 3mL10mM DTT.Reaction, at 28 degrees Celsius, is carried out 2.5 hours under soft oscillating condition, then adds 50mL chloroform: methyl alcohol (2:1) termination reaction.With chloroform methanol again extracting containing water three times, and merge solvent phase.At 60 degrees Celsius of lower dry extracts and be resuspended in 40mL chloroform: in methyl alcohol.Also be dried containing water and lay equal stress on and be suspended from water.15mL extract is written 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.

For the chemical structure of the product confirming to develop by radioassay analytical method, developed a kind of inactive LC-MS and detected analytical method.This detects analytical method and comprises 10mL20mM UDPG or UDP-pectinose, 60mL50mM potassiumphosphate, pH7.6,2mL1M MgCl ₂, the 4mL100mM receptor substrate in DMSO (or the synthetic substrate of 8mL, DMSO only compares thing), 20mL protein product, 10mL100mM DTT.Reaction was the soft shaking culture of 28 degree 3.5 hours.By adding 100% methyl alcohol termination reaction, then at 50 degrees Celsius, from reaction, evaporate methyl alcohol, and use reversed phase chromatography sample (10 μ 1) to analyze by LC/MS/MS, use 100 * 2mm3 μ Luna C18 (2) post (Phenomenex), at 250 μ Lmin ^-1, 30 ℃, with following methyl alcohol+0.1% formic acid, water+0.1% formic acid gradient is moved:

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

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

By UV (spectral range 200 is to 600nm for 214nm, bandwidth 9nm) and electron spray(ES) MS, check, in the cycle of operation of separating with positive ion or negative ion mode.Spray chamber condition Wei50 unit sheath gas, without aux/ scanning, the positive ion spray voltage of 5.2kV, the negative ion mode voltage of 5.0kV, 325 ℃ of capillary temperatures.The MS of data dependency ²the second scan event by capturing with the fragmentation at 35% collision energy place of the separated width place at 5.0amu, undertaken.

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

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

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

embodiment 5

in other species, express the recombinant DNA construction body of AsSCPL1

Be below the embodiment of recombinant DNA construction body, can use them in monocotyledons or dicotyledons species, to 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 embodiment that more early describe 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 existing in species and species, can comprise from other gene in biosynthetic pathway to improve expression level.

Below using in embodiment below for the abbreviation of the nucleic acid fragment that comprises different components:

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

" CAMV35S ENH " is the promoter region of cauliflower mosaic virus 35 S promoter, and it improves the expression level (people such as Benfey P.N., 1990, EMBO J.9:1685-1696) of connected promotor.

" UBI PRO " is the promotor of corn ubiquitin gene, it carried out to description (people such as Christensen, 1992, Plant Mo1.Bio1.18:675-689).

" UBI5'UTR " is 5 ' leading region of identical corn ubiquitin gene.

" UBI INTRON1 " is the intron of identical ubiquitin gene.Comprise the verified expression level that improves of this intron.

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

" CCDB " is at Gateway ^tMthe negative selectivity mark of bacterium of describing in clone's system handbook is own.

" ATTR2 " is at Gateway ^tMthe recombination site of describing 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 (people such as Odell J.T., 1985, Nature313:810-812) conventional in plant.

" ADH1 INTRON1 " is the intron of corn ADH1 gene.Comprising this intron has proved and can improve expression level (Luehrsen K.R. and Walbot V., 1991, Mo1.Gen.Genet.225:81-93).

" BAR " is commonly used for the antiweed gene that corn transforms selected marker.

" SCP1 " is the synthetic constitutive promoter for plant, and it is described in United States Patent (USP) and discloses 6,072, in 050.

" OMEGA5 ' UTR " is 5 ' leading region of tmv cdna, and verified its use can improve translation skill (people such as Gallie, 1989, Molecular Biology of RNA, editor Cech (Liss, New York), 237-256 page).

" SPCI " 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.

in corn, express the construct of saponin(e biosynthesis gene

From the clone described in embodiment 1, obtain respectively the fragment of the open reading frame that comprises AsSCPL1.With the primer that causes open reading frame side to be connected to unique restriction site, carry out pcr amplification, described restriction site makes them to arrive these modified Gateway by directed cloning ^tMin unique restriction site of Entry Vector (Invitrogen, Carlsbad, California, USA).Described fragment is being connected to Gateway ^tMafter Entry Vector, " entry vector " is comprised of ATTL1-AsSCPL1-ATTL2, and comprises the kalamycin resistance of selecting for bacterium.ATTL1 and ATTL2 are at Invitrogen Gateway ^tMthe recombination site providing in clone's system (Carlsbad, California, USA).

corn recombinant DNA construction body 1:E35S-UBI-AsSCPL1-PINII

This builds physical efficiency and is used at corn single expression AsSCPL1 gene.AsSCPL1 entry vector is used for to Gateway ^tMlR and Gateway ^tMreaction (the Komari of modified soil bacillus carrier main chain (modify and obtain from pSB1), T. wait people, 1996, Plant is J.10:165-174), this is by adding following component in cos site: RB-CAMV35S ENH-UBI PRO-UBI5 ' UTR-UBI INTRON1-ATTR1-CCDB-ATTR2-PINII+CAMV35S ENH-CAMV35S PRO-ADH1 INTRON1-BAR-PINII-LB-SPC-ColE1 ORI.At this Gateway ^tMin reaction, ATTL1 and ATTL2 and ATTR1 and ATTR2 restructuring therefore substitute CCDB by AsSCPL1 transgenosis in object carrier, and CCDB is virose to intestinal bacteria, and makes it possible to as Gateway ^tMscreening described in handbook (Invitrogen, Carlsbad, California, USA) is clone successfully.This gained construct comprises a T-DNA, and it will be transferred in Plant Genome and comprise RB-CAMV35S ENH-UBI PRO-UBI5 ' UTR-UBI INTRON1-ATTB1-AsSCPL1-ATTB2-PINII+CAMV35S ENH-CAMV35S PRO-ADH1INTRON1-BAR-PINII-LB.Outside region between RB and LB, nucleotide sequence is described in Kormai, the people such as T., and op cit, except SPC and ColE1 component.This construct electroporation is entered in LBA4404 Agrobacterium tumefaciens cell and for transformation experiment as those are in the below experiment described in embodiment 8.

in soybean, express the construct of saponin(e biosynthesis gene

By pcr amplification, obtain AsSCPL1 open reading frame for corn construct as mentioned above.

soybean recombinant DNA construction body 1:SCP1-O '-AsSCPL1-PINII

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

embodiment 6

in other species, express the recombinant DNA construction body of AsMT1

Be below the embodiment of recombinant DNA construction body, can use them in monocotyledons or dicotyledons species, to 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 embodiment that more early describe 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 existing in species and species, can comprise from other gene in biosynthetic pathway to improve expression level.

Below using in embodiment below for the abbreviation of the nucleic acid fragment that comprises different components:

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

" CAMV35S ENH " is the promoter region of cauliflower mosaic virus 35 S promoter, and it improves the expression level (Benfey P.N., waits people, and 1990, EMBO J.9:1685-1696) of connected promotor.

" UBI PRO " is the promotor of corn ubiquitin gene, it carried out to description (people such as Christensen, 1992, Plant Mo1.Bio1.18:675-689).

" UBI5'UTR " is 5 ' leading region of identical corn ubiquitin gene.

" UBI INTRON1 " is the intron of identical ubiquitin gene.Comprise the verified expression level that improves of this intron.

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

" CCDB " is at Gateway ^tMthe negative selectivity mark of bacterium of describing in clone's system handbook is own.

" ATTR2 " is at Gateway ^tMthe recombination site of describing 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, it be constitutive promoter conventional in plant (people such as Odell J.T., 1985, Nature313:810-812).

" ADH1INTRON1 " is the intron of corn ADH1 gene.Comprise this intron proved can improve expression level (Luehrsen K.R. and Walbot V., 1991, Mo1.Gen.Genet.225:81-93).

" BAR " is commonly used for the antiweed gene that corn transforms selected marker.

" SCP1 " is the synthetic constitutive promoter for plant, and it is described in United States Patent (USP) and discloses 6,072, in 050.

" OMEGA5 ' UTR " is 5 ' leading region of tmv cdna, and verified its use can improve translation skill (people such as Gallie, 1989, Molecular Biology of RNA, editor Cech (Liss, New York), 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.

" ColEl ORI " is the functional DNA replication orgin in intestinal bacteria.

in corn, express the construct of saponin(e biosynthesis gene

From the clone described in embodiment 1, obtain respectively the fragment of the open reading frame that comprises AsMTl.With the primer that causes open reading frame side to be connected to unique restriction site, carry out pcr amplification, described restriction site makes them to arrive these modified Gateway by directed cloning ^tMin unique restriction site of Entry Vector (Invitrogen, Carlsbad, California, USA).Described fragment is being connected to Gateway ^tMafter Entry Vector, " entry vector " is comprised of ATTLl-AsMTl-ATTL2, and comprises the kalamycin resistance of selecting for bacterium.ATTLl and ATTL2 are at Invitrogen Gateway ^tMthe recombination site providing in clone's system (Carlsbad, California, USA).

corn recombinant DNA construction body 1:E35S-UBI-AsMTl-PINII

This builds physical efficiency and is used at corn single expression AsMTl gene.AsMTl entry vector is used for to Gateway ^tMlR and Gateway ^tMreaction (the Komari of modified soil bacillus carrier main chain (modify and obtain from pSB1), T. wait people, 1996, Plant J.10:165-174), this is by adding following component in cos site: RB-CAMV35S ENH-UBI PRO-UBI5 ' UTR-UBI INTRONl-ATTRl-CCDB-ATTR2-PINII+CAMV35S ENH-CAMV35S PRO-ADHl INTRONl-BAR-PINII-LB-SPC-ColEl ORI.At this Gateway ^tMin reaction, ATTLl and ATTL2 and ATTRl and ATTR2 restructuring therefore substitute CCDB by AsMTl transgenosis in object carrier, and CCDB is virose to intestinal bacteria, and makes it possible to as Gateway ^tMscreening described in handbook (Invitrogen, Carlsbad, California, USA) is clone successfully.This gained construct comprises a T-DNA, and it will be transferred in Plant Genome and comprise RB-CAMV35S ENH-UBI PRO-UBI5 ' UTR-UBI INTRONl-ATTB1-AsMT1-ATTB2-PINII+CAMV35S ENH-CAMV35S PRO-ADH1INTRON1-BAR-PINII-LB.Outside region between RB and LB, nucleotide sequence is described in Kormai, the people such as T., and op cit., except SPC and ColEl component.This construct electroporation is entered in LBA4404 Agrobacterium tumefaciens cell and for transformation experiment as those are in the below experiment described in embodiment 8.

in soybean, express the construct of saponin(e biosynthesis gene

By pcr amplification, obtain AsMTl open reading frame for corn construct as mentioned above.

soybean recombinant DNA construction body 1:SCPl-O '-AsMT1-PINII

This builds physical efficiency and is used at dicotyledons single expression AsMTl gene.At the polynucleotide that comprise AsMTl open reading frame by, be connected to and comprise the unique restriction site of SCPl PRO-ω 5 ' UTR-, after being same as in the carrier in AsMTl-PINII site of those side joints, linearization plasmid is for bombardment and extract required DNA band from gel.This process also removes coding for the Nucleotide of the amicillin resistance of bacterium selection.This fragment comprises SCPl PRO-ω 5'UTR-AsMTl-PINII and for the transformation of soybean as described in embodiment 9 below.

embodiment 7

in other species, express the recombinant DNA construction body of AsGS2

Be below the embodiment of recombinant DNA construction body, can use them in monocotyledons or dicotyledons species, to 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 embodiment that more early describe 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 existing in species and species, can comprise from other gene in biosynthetic pathway to improve expression level.

Below using in embodiment below for the abbreviation of the nucleic acid fragment that comprises different components:

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

" CAMV35S ENH " is the promoter region of cauliflower mosaic virus 35 S promoter, and it improves the expression level (Benfey P.N., waits people, and 1990, EMBO J.9:1685-1696) of connected promotor.

" UBI PRO " is the promotor of corn ubiquitin gene, it carried out to description (people such as Christensen, 1992, Plant Mol.Biol.18:675-689).

" UBI5'UTR " is 5 ' leading region of identical corn ubiquitin gene.

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

" ATTRl " is at Gateway ^tMthe recombination site of describing in clone's system handbook (Invitrogen, Carlsbad, Califomia, USA).

" CCDB " is at Gateway ^tMthe negative selected marker of bacterium of describing in clone's system handbook.

" ATTR2 " is at Gateway ^tMthe recombination site of describing 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 (people such as Odell J.T., 1985, Nature313:810-812) conventional in plant.

" ADHl INTRONl " is the intron of corn ADHl gene.Comprising this intron has proved and can improve expression level (Luehrsen K.R. and Walbot V., 1991, Mo1.Gen.Genet.225:81-93).

" BAR " is commonly used for the antiweed gene that corn transforms selected marker.

" SCPl " is the synthetic constitutive promoter for plant, and it is described in United States Patent (USP) and discloses 6,072, in 050.

" OMEGA5 ' UTR " is 5 ' leading region of tmv cdna, and verified its use can improve translation skill (people such as Gallie, 1989, Molecular Biology of RNA, editor Cech (Liss, New York), the 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.

" ColEl ORI " is the functional DNA replication orgin in intestinal bacteria.

in corn, express the construct of saponin(e biosynthesis gene

From the clone described in embodiment 1, obtain respectively the fragment of the open reading frame that comprises AsGS2.With the primer that causes open reading frame side to be connected to unique restriction site, carry out pcr amplification, described restriction site makes them to arrive these modified Gateway by directed cloning ^tMin unique restriction site of Entry Vector (Invitrogen, Carlsbad, California, USA).Described fragment is being connected to Gateway ^tMafter Entry vector, " entry vector " is comprised of ATTLl-AsGS2-ATTL2, and comprises the kalamycin resistance of selecting for bacterium.ATTLl and ATTL2 are at Invitrogen Gateway ^tMthe recombination site providing in clone's system (Carlsbad, California, USA).

corn recombinant DNA construction body 1:E35S-UBI-AsGS2-PINII

This builds physical efficiency and is used at corn single expression AsGS2 gene.AsGS2 entry vector is used for to Gateway ^tMlR and Gateway ^tMreaction (the Komari of modified soil bacillus carrier main chain (modify and obtain from pSB1), T. wait people, 1996, Plant J.10:165-174), this is by adding following component in cos site: RB-CAMV35S ENH-UBI PRO-UBI5 ' UTR-UBI INTRONl-ATTRl-CCDB-ATTR2-PINII+CAMV35S ENH-CAMV35S PRO-ADHl INTRONl-BAR-PINII-LB-SPC-ColEl ORI.At this Gateway ^tMin reaction, ATTLl and ATTL2 and ATTRl and ATTR2 restructuring therefore substitute CCDB by AsGS2 transgenosis in object carrier, and CCDB is virose to intestinal bacteria, and makes it possible to as Gateway ^tMscreening described in handbook (Invitrogen, Carlsbad, California, USA) is clone successfully.This gained construct comprises a T-DNA, and it will be transferred in Plant Genome and comprise RB-CAMV35S ENH-UBI PRO-UBI5'UTR-UBI INTRONl-ATTB1-AsGS2-ATTB2-PINII+CAMV35S ENH-CAMV35S PRO-ADH1INTRON1-BAR-PINII-LB.Outside region between RB and LB, nucleotide sequence is described in Kormai, the people such as T., and op cit., except SPC and ColEl component.This construct electroporation is entered in LBA4404 Agrobacterium tumefaciens cell and for transformation experiment as those are in the below experiment described in embodiment 8.

in soybean, express the construct of saponin(e biosynthesis gene

By pcr amplification, obtain AsGS2 open reading frame for corn construct as mentioned above.

soybean recombinant DNA construction body 1:SCPl-O '-AsGS2-PINII

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

embodiment 8

the conversion of agrobacterium-mediated corn

regeneration with transgenic plant

The rotaring gene corn plant that the recombinant DNA construction body of preparation can be used for being prepared as follows in embodiment 5 to 7.

Can use the method for Zhao (United States Patent (USP) discloses 5,981,840, and PCT patent is announced WO98/32326), with any polynucleotide constructs maize transformation described in embodiment 5-7.In brief, separated prematurity plumule make plumule contact edaphic bacillus suspension from corn, wherein said bacterium can be transferred to polynucleotide constructs at least one cell of at least one prematurity plumule (step 1: infect step).In this step, prematurity plumule is immersed in edaphic bacillus suspension for starting inoculation.Plumule and edaphic bacillus are cultivated for some time (step 2: be total to culturing step) altogether.After infecting step, prematurity plumule is cultivated on solid medium.And then this common cultivation period, carries out optional " dormancy " step.In this sleep step, plumule is cultivated and to be existed at least one known microbiotic to suppress edaphic bacillus growth, does not add in the environment of vegetable transformant selective agent and carries out (step 3: sleep step) simultaneously.Prematurity plumule is cultivated and to be had microbiotic but on solid medium without selective agent, for eliminating the dormant stage of edaphic bacillus and infected cell.Next, inoculation plumule is cultivated in comprising the substratum of selective agent, and reclaim growth through transformed calli (step 4: select step).Then regeneration in plant of callus (step 5: regeneration step), and the callus of growing in selecting substratum is cultivated the described plant that regenerates in solid medium.

embodiment 9:

with soybean expression vector soybean transformation somatic embryo culture

and the soybean plants of regenerating

The genetically engineered soybean plant that the recombinant DNA construction body of preparation can be used for being prepared as follows in embodiment 5 to 7.

culture condition:

Soybean germ generation suspension culture (cv.Jack) is incubated to 35mL liquid SBl96 substratum (vide infra), culture condition be 150rpm shaking table cultivate, 26 ℃ and by 16:8 hour daytime/noctilucence cycle with 60-85 μ E/m2/s light intensity cool white light fluorescent lamp.Within every 7 days to two weeks, by inoculating about 35mg, be organized in 35mL fresh liquid SB196 the culture cultivation (preferably go down to posterity to cultivate and be spaced apart every 7 days) of going down to posterity.

Soybean germ generation suspension culture transforms with soybean expression plasmid, DuPont Biolistic PDSl000/HE instrument (helium remodeling) is all used in all conversions, by particle gun, bombarding (people such as Klein, Nature327:70 (1987)) method carries out.

soybean embryo generates the induction of suspension culture:

Soybean culture monthly starts twice, and between each startup, interval is 5 to 7 days.After plantation, in 45 to 55 days, gather the beanpod with prematurity seed of available soybean plants.From beanpod, remove seed and be placed in aseptic magenta box.Soybean seed by shaking and within 15 minutes, carry out aseptically process in 5% chlorine bleach liquor and 1 ivory soap (that is, 95mL autoclave distilled water adds 5mL clorox and 1 soap, fully mixes).Seed is used the sterile distilled water rinsing of 21 litre flasks, and the seed that those are less than to 4mm is placed on single slide.Cut the little end of seed, and cotyledon is extruded to seed shell.Cotyledon is transferred in the plate that comprises SB1 substratum (25 to 30 cotyledons of every plate).With fiber band, by flat board packing and 26 ℃ of temperature, at cold daytime/night in 16:8 hour white fluorescent light cycle, under intensity of illumination 60 to 80 μ E/m2/s, cultivate eight weeks, after 4 weeks, change substratum.On SBl substratum, after inoculation, cut second plumule and put it into SB196 liquid nutrient medium 7 days.

preparation for the DNA that bombards:

Complete plasmid or the DNA plasmid fragment that comprises concerned gene and selected marker are used to bombardment.The plasmid of crossing by gel separating digesting obtains the fragment from soybean expression plasmid, this paper describes the structure of described plasmid.In all cases, 100 μ g plasmid DNA are used to 0.5mL certain enzyme mixture hereinafter described.AscI for plasmid (100 unit) NEBuffer4 (20mM Tris acetic acid, 10mM magnesium acetate, 50mM potassium acetate, 1mM dithiothreitol (DTT), pH7.9), 100 μ g/mL BSA, and in 5mM beta-mercaptoethanol, digesting at 37 ℃ 1.5 hours.Gained DNA fragmentation is by gel electrophoresis (BioWhitaker Molecular Applications) separation on 1% SeaPlaque GTG agarose, and cuts from sepharose the DNA fragmentation that comprises box gene.Use GELase digestive ferment, according to the rules of manufacturers purify DNA from agarose.

50 μ L aliquots containigs of the sterile distilled water that comprises 3mg bronze (3mg gold) are added to 30 μ L10ng/ μ L DNA solutions (DNA fragmentation of preparation as described herein), 25 μ L5MCaCl ₂, and in 20 μ L0.1M spermidines.Mixture is shaken 3 minutes and in desk centrifuge, rotated 10 seconds on 3 grades, vortex vibrator.Remove supernatant liquor, then with 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 L DNA suspension are assigned in each floating disc of Biolistic PDS1000/HE instrument dish.Every 5 μ L aliquots containigs are bombarded (for example every dish) at every turn and are comprised about 0.375mg gold.

tissue preparation and bombarding with DNA:

About 150 to 200mg plumule generation in seven day age suspension culture is placed in to empty aseptic 60 * 15mm culture dish, and covers this culture dish with plastic wire.It is 27 to 28 inches of mercury that chamber is emptied to vacuum tightness, and one or two of the tissue bombardment of each flat board, is made as 1100PSI by film rupture pressure.Tissue is placed on to 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) that uses the grand selection of chlorine sulphur to transform.

After bombardment, tissue is placed in to new system SBl96 substratum and cultivates as mentioned above.Latter six to eight days of bombardment, is replaced by SBl96 to comprise the grand fresh SBl96 substratum of 100ng/mL chlorine sulphur.Select substratum to upgrade weekly.The last selecting four to six weeks, observe from unconverted downright bad plumule occurs bunch and grow green transforming tissue.Chlorenchyma is separated, be seeded to porous plate, to produce the embryo of conversion new, clonal propagation, generate suspension culture.

embryo's maturation:

To in porous plate, cultivate as mentioned above four to six weeks from the plumule generation bunch of producing the conversion transforming, cultivation is at 26 ℃, in SB196, under cold white fluorescence (the cold white Econowatt F40/CW/RS/EW of Phillips) and Agro (Phillips F40Agro) bulb (40 watts) with photoperiod and 90 to the 120 μ E/m of 16:8 hour ²the intensity of illumination of s is carried out.After for some time, plumule bunch is moved on in solid nutrient agar, SBl66 and cultivated one to two week, then go down to posterity and cultivate in SB103 substratum 3 to 4 weeks, obtain ripe plumule.Onboard, in SB103 after maturation, by single plumule from bunch remove, be dried and screen desired phenotype.This type of phenotype can be but be not limited to the saponin(e level of change or the resistance level at least one fungi of change.When needs, from some events hereinafter described, obtain plant.

plumule is dried and germinates:

Ripe single plumule dewaters by being placed in empty little culture dish (60 * 15mm) for about four to seven days.Fiber band for plate (createing a low humidity chamber) sealing.The plumule that will dewater is implanted SB71-4 substratum, makes its germinating growth under the condition identical with above-mentioned culture condition.From germination substratum, take out seedling the water cleaning down germinateing, then implanted in the Redi-Earth in 24 hole pallets, with blister pack, cover.After one to two week, remove plastic lousing, the plant one week of rehardening.If seedling looks hard, they are transplanted in 10 inches of basins of Redi-Earth to the maximum 3 strain seedling of every basin.After ten to 16 weeks, results mature seed, grinds and for desired phenotype analysis.

culture medium prescription:

sB196-FN Lite liquid proliferated culture medium (every liter)

fN Lite storing solution

^*first add, stirring and dissolving is in dark bottles

sB1 solid medium (every liter)

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

LmL B5 VITAMIN 1000X storing solution

31.5g glucose

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

pH5.7

8g TC agar

sB199 solid medium (every liter)

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

1mL B5 VITAMIN 1000X storing solution

30g sucrose

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

pH7.0

2g solidifying agent

sB166 solid medium (every liter)

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

1mL B5 VITAMIN 1000X storing solution

60g maltose

750mg MgCl ₂hexahydrate

5g gac

pH5.7

2g solidifying agent (gelrite)

sB103 solid medium (every liter)

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

1mL B5 VITAMIN 1000X storing solution

60g maltose

750mg MgCl2 hexahydrate

pH5.7

2g solidifying agent (gelrite)

sB71-4 solid medium (every liter)

1 bottle of Gamborg ' s B5 salt w/ sucrose (Gibco/BRL-Cat.No.21153-036)

pH5.7

5g TC agar

2,4-D storing solution

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

b5 VITAMIN storing solution (every 100mL)

Packing is stored in-20 ℃

10g inositol

100mg nicotinic acid

100mg pyridoxine hydrochloride

1g VitB1

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

the differentiation of SB228-soyabean tissue and maturation medium (SHaM) (every liter)

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

Because glutamine degraded is quite rapid, it preferably now adds before using substratum.Substratum after glutamine adds 2 weeks expired; Containing the minimum medium of glutamine, can not deposit the longer time.

fN-lite Macro10X-storing solution #1 (every liter) for SHAM

mS Micro1000X-storing solution #2 (every 1 liter)

feEDTA100X-storing solution #3 (every 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 be with aluminium foil parcel with lucifuge.Autoclaving

ca100X-storing solution #4 (every liter)

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

Add water to final volume

Autoclaving

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

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

DDI water is heated to 30 ℃ of 900mL

L-glutaminate 40g

Add gradually while stirring and impose low-grade fever.

Be no more than 35 ℃.

Add water to final volume

Filtration sterilization

Freezing preservation *

* note: storing solution freezes 31 ℃ 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 of separation, described polynucleotide are comprised of following sequence:

(a) nucleotide sequence of coding Methyl transporters enzyme polypeptide, described polypeptide is comprised of the aminoacid sequence of SEQ ID NO:4; Or

(b) nucleotide sequence being formed by the total length complementary sequence of (a).

2. the polynucleotide of claim 1, a kind of form of the nucleotide sequence of wherein said coding methyltransgerase in SEQ ID NO:3 or 8.

3. the carrier that comprises the polynucleotide of claim 1.

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

5. the recombinant DNA construction body of claim 4, described construct also comprises at least the second polynucleotide, the polypeptide of the expression of the second enzyme at least in described the 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 construction body transformant by claim 4 or claim 5.

7. for the production of the method for transgenic plant, described method comprises by the recombinant DNA construction body transformed plant cells of claim 4 or claim 5 and from the vegetable cell regeneration of transgenic plant of described conversion.

8. the separated host cell that comprises the recombinant DNA construction body 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 expression of polypeptides level in vegetable cell, described method comprises:

A) use the nucleic acid fragment conversion of plant tissue from the separated polynucleotide of claim 1, wherein said polynucleotide can change the expression of natural methyltransgerase;

B) by described plant tissue regeneration transgenic plant; And

C) assessment is when comparing with the plant with the wild-type expression level of corresponding natural methyltransgerase, and the expression level of the methyltransgerase of described transgenic plant changes.

11. produce the method at least one fungi to the plant of resistance, and described method comprises:

A. use the recombinant DNA construction body transformed plant cells of the first enzyme of the coding triterpene approach of at least one claim 4;

B. under the condition that promotes transgenic plant regeneration, make the plant cell growth from the conversion of step (a); And

C. the transgenic plant of appraisal procedure (b) when with same species be not subject to plant that described recombinant DNA construction body transformed relatively time, the resistance of at least one fungi is improved;

Wherein said plant is monocotyledons.

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

13. the method for claim 11, wherein said recombinant DNA construction body 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 for the plant of the methyltransgerase with change level, and described method comprises:

A) the recombinant DNA construction body transformed plant cells of the first enzyme of the coding triterpene approach of use claim 4;

B) under the condition that promotes transgenic plant regeneration, make the plant cell growth from the conversion of step (a); And

When c) amount of the methyltransgerase of assessment in the plant not transforming with described recombinant DNA construction body with same species compares, 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 construction body also comprises at least the second polynucleotide, the polypeptide of the expression of the second enzyme at least in described the second polynucleotide encoding regulation and control triterpene approach.

16. the method for claim 14, wherein said recombinant DNA construction body 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 with the triterpenoid saponin level of change, and described method comprises:

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

2. under the condition that promotes transgenic plant regeneration, make the plant cell growth from the conversion of step (a); And

3. the triterpenoid saponin level when triterpenoid saponin amount of the transgenic plant of appraisal procedure (b) in the plant not transforming with described recombinant DNA construction body with same species compares changes,

Wherein said plant is monocotyledons.

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

19. the method for claim 17, wherein said recombinant DNA construction body 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 with the triterpenoid saponin level of raising, and described method comprises:

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

B) under the condition that promotes transgenic plant regeneration, make the plant cell growth from the conversion of step (a); And

Triterpenoid saponin level when c) the triterpenoid saponin amount of the transgenic plant of appraisal procedure (b) in the plant not transforming with described recombinant DNA construction body with same species compares improves;

Wherein said plant is monocotyledons.

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

22. the method for claim 20, wherein said recombinant DNA construction body 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 with the triterpenoid saponin level of reduction, and described method comprises:

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

B) under the condition that promotes transgenic plant regeneration, make the plant cell growth from the conversion of step (a); And

Triterpenoid saponin level when c) the triterpenoid saponin amount of the transgenic plant of appraisal procedure (b) in the plant not transforming with described recombinant DNA construction body with same species compares reduces;

Wherein said plant is monocotyledons.

The method of 24. claims 23, wherein said recombinant DNA construction body also comprises at least the second polynucleotide, the polypeptide of the second expression of enzymes at least in described the second polynucleotide encoding regulation and control triterpene approach, wherein said the second enzyme is beta-amyrin synthase or CYP51H10.

25. the method for claim 23, wherein said recombinant DNA construction body also comprises at least one polynucleotide, and described polynucleotide encoding is selected from the enzyme of beta-amyrin synthase and CYP51H10.

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