CN1293711A - Genes encoding MLO proteins and conferring fungal resistance upon plants - Google Patents

Abstract

This invention describes genes encoding proteins which control resistance of plants to fungal pathogens. The invention also describes transgenic plants resistant to fungal pathogens and methods for making plants resistant to fungal pathogens. The invention further discloses a method to isolate additional genes coding for additional proteins controlling the resistance of plants to fungal pathogens.

Description

Coding MLO protein is also given the gene of the antimycotic resistance of plant

The invention describes the nucleotide sequence of coded protein controlling plant to the resistance of fungal disease.The invention still further relates to the preparation method of the plant of the plant of antimycotic disease and antimycotic disease.

Fungal disease causes about 9,100,000,000 dollars of crop loss every year in the U.S., and they are caused by pathogenic agent widely different on the various biological.Control them with diverse ways traditionally.Now cultivated agricultural and gone up important resistant variety, thereby the multilevel resistance of anti-among a small circle or in a big way pathogen isolation thing or kind is provided.But, this work comprises by genetic cross introduces the long-term arduous process of commercialization strain system with required proterties, and because insect may be evolved into the risk that can overcome the natural phant resistance, needs ongoing effort that new resistance trait is introduced in the commercialization strain.In addition, by applied chemistry mycocide control fungal disease.This method produces effective control action kou usually, but also relevant with issuable resistance pathogenic agent, and may have a negative impact to environment.And, some farm crop, in barley and wheat, be difficulty or unpractical with chemical mycocide control fungal pathogens.Present technology can be better in the interaction of understanding on the molecular level between plant and the pathogenic agent thereof, and part has disclosed resistance mechanism.Model plant Arabidopis thaliana is carried out the major part work of this Molecular Identification, now begun to illustrate the resistance mechanism in the important economic farm crop.

Powdery Mildew is the most of floristic principal disease of influence and has been widely studied.They are characterised in that the white of growing on the plant tissue is to light grey spot or flap, corresponding to mycelium and the cleistothecium of fungi.Powdery Mildew is fungus-caused by several of Erysiphales (Erysiphales).For example, standing grain powdery mildew (Erysiphe graminis) causes the Powdery Mildew of cereal and grass.Though Powdery Mildew is restive in most of farm crop, the barley strain of some anti-most known pathogen isolation bacterium is arranged.Research work shows that unit point is that the mlo site mutation is relevant with resistant phenotype.Mlo resistance mechanism part is illustrated; Be included in the site that contacts of pathogenic agent and form the maxicell wall be called as mastoid process and add body (apposition), wherein mainly comprise callose, but carbohydrate, phenol and protein are also arranged.In the mlo plant, cell walls adds the intrusion that body has stoped pathogenic agent, thereby has produced resistance.

Unfortunately, the strong instrument of this control Powdery Mildew is confined at barley.By fungal disease in the agricultural, especially the problem that causes of Powdery Mildew be it seems, still needs new effective ways controlling these type pathogenic agent in other farm crop, and these methods are attractive and can accept on environment to the farmer economically.

The present invention proposes demand to the novel plant disease control method of passing through gene engineering.More particularly, the present invention relates to control the method for Powdery Mildew, preferably the method for the Powdery Mildew in the important farm crop on the command economy.

The proteinic DNA isolation molecule of the Mlo that the present invention relates to encode, wherein said Mlo protein is given the resistance of plant resistant to fungal pathogens.More particularly, the present invention relates to contain the Mlo protein of the conserved amino acid sequence that inventor of the present invention finds first, and relate to the proteinic DNA isolation molecule of this Mlo of coding.The present invention has also described the carrier that is used for expressing plant dna molecular of the present invention.The invention further relates to the transgenic plant that comprise any dna molecular of the present invention.The present invention has also described the agricultural-food that tool improves the Plant Quarantine characteristic, comprising the transgenic plant of resistant to fungal pathogens by the expression of any dna molecular of the present invention.The present invention also further relates to the method for the plant for preparing antimycotic disease, this method is by changing by corresponding to the expression of gene copy encoded protein matter in source in transgenic plant within any dna molecular of the present invention, or by changing by corresponding to the active of gene copy encoded protein matter in source within any dna molecular of the present invention or stability is carried out.Such transgenic plant as expectedly can be anti-infective the live pathogenic agent of epidermic cell of plant, especially from the fungi (being also known as powdery mildew) of Erysiphales, preferred anti-Erysiphe (Erysiphe) fungi that causes Powdery Mildew, the plant of more preferably anti-standing grain powdery mildew.The present invention has further described and has separated coded protein and have the method for inventing the dna molecular of listed conserved amino acid sequence with same or similar function of dna molecule encode protein of the present invention and code book.

The present invention thereby the new effective ways of controlling fungal disease in the important economic farm crop are provided, thus might reduce the amount of chemicals used that is used for farm crop and reduce the danger that has the pathogenic agent of resistance to occur to control agent.

Thereby the invention provides:

Coding is given the Mlo protein DNA molecule of plant to the resistance of fungal pathogens, wherein said protein comprise with SEQ ID NO:1 or SEQ ID NO:2 at least one identical or similar substantially aminoacid sequence of listed aminoacid sequence, wherein said dna molecular is the cDNA molecule preferably.In preferred embodiments, dna molecular is preferably not from barley and from dicotyledons or following plant: wheat, corn, rice, oat, rye, Chinese sorghum, sugarcane, millet, buy sieve Chinese sorghum (milo) and Palmae.In preferred embodiments, any listed among dna molecular of the present invention and SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7 nucleotide sequence is identical or similar substantially, perhaps identical the or similar substantially Mlo protein of listed Mlo protein among coding and SEQ ID NO:4, SEQID NO:6 or the SEQ ID NO:8.In a more preferred embodiment, comprise the dna molecular of listed nucleotide sequence among SEQ ID NO:3, SEQ ID NO:5 or the SEQ ID NO:7 from wheat.In another embodiment preferred, any listed among dna molecular of the present invention and SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15 or SEQ ID NO:17 nucleotide sequence is identical or similar substantially, perhaps identical the or similar substantially Mlo protein of listed any nucleotide sequence coded Mlo protein among coding and SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16 or the SEQ ID NO:18.In a more preferred embodiment, comprise the dna molecular of listed nucleotide sequence among SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15 or the SEQ ID NO:17 from Arabidopis thaliana.In another preferred embodiment, dna molecular mentioned above is modified the consequently loss of activity of endogenous protein.In particular embodiment of the present invention, said dna modification causes taking place in the aminoacid sequence of respective egg white matter the combination of a kind of, the whole or multiple change in following the change:

-tryptophane (163) is changed into arginine

-proline(Pro) (396) back frameshit

-tryptophane (160) back frameshit

-methionine(Met) (1) is changed into Isoleucine

-glycine (227) is changed into aspartic acid

-methionine(Met) (1) is changed into Xie Ansuan

-arginine (11) is changed into tryptophane

-lose phenylalanine (183), Threonine (184)

-Xie Ansuan (31) is changed into L-glutamic acid

-Serine (32) is changed into phenylalanine

-leucine (271) is changed into Histidine.

In a further preferred embodiment, fungal pathogens preferably infects epidermic cell alive, and more preferably fungal pathogens is from Erysiphales (being also known as powdery mildew), and especially from Erysiphe, most preferably fungal pathogens is the standing grain powdery mildew.

In other embodiments, isolated DNA molecule is the molecule with above-mentioned isolated molecule antisense, as comprising the Mlo protein DNA molecule of at least a aminoacid sequence identical or similar substantially with listed aminoacid sequence among SEQ ID NO:1 or the SEQ ID NO:2 with coding, as the molecule of cDNA molecule antisense, especially with SEQ ID NO:3,5,7,9,11,13, dna molecular shown in 15 or 17 is identical or similar substantially, and coded Mlo protein and SEQ ID NO:4,6,8,10,12,14, the dna molecular of the dna molecular antisense that 16 or 18 listed Mlo protein are identical or similar substantially.

The present invention further provides:

At least comprise a kind of protein of the aminoacid sequence identical or similar substantially with listed aminoacid sequence among SEQ ID NO:1 or the SEQ ID NO:2, wherein said protein is Mlo protein and gives the resistance of plant to fungal pathogens.This protein is preferably from barley, but from dicotyledons or following plant: wheat, corn, rice, oat, rye, Chinese sorghum, sugarcane, millet, buy sieve Chinese sorghum and Palmae.In preferred embodiments, protein of the present invention is by identical or similar substantially with listed any nucleotide sequence among SEQ ID NO:3, SEQ ID NO:5 or the SEQ ID NO:7 nucleotide sequence coded, perhaps identical or similar substantially with listed any Mlo protein among SEQID NO:4, SEQ ID NO:6 or the SEQ ID NO:8.In a more preferred embodiment, said protein is from wheat.In another embodiment preferred, protein of the present invention is by identical or similar substantially with listed any nucleotide sequence among SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15 or the SEQ ID NO:17 nucleotide sequence coded, perhaps identical or similar substantially with listed any Mlo protein among SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 or the SEQ IDNO:18.In a more preferred embodiment, this protein is from Arabidopis thaliana.In another embodiment preferred, preferred fungi pathogenic infection epidermic cell alive, more preferably fungal pathogens is from Erysiphales (being also known as powdery mildew), and especially from Erysiphe, most preferably fungal pathogens is the standing grain powdery mildew.In further preferred embodiment, the present invention also comprises proteinic mutant form or the clipped form by above-mentioned any dna molecule encode.

The present invention further provides:

Contain above-mentioned any dna molecular, as the expression cassette of above-mentioned cDN A, wherein said dna molecular effectively is connected with the termination signal that can express this dna molecular in plant with promotor.In preferred embodiments, said expression cassette is allogenic.In a more preferred embodiment, promotor and termination signal are Eukaryotic.In a more preferred embodiment, promotor and termination signal are allogenic for the coding region.

The present invention also provides:

The carrier that contains above-mentioned any expression cassette.In preferred embodiments, this carrier is used for the conversion of the said expression cassette of plant.In another embodiment preferred, carrier of the present invention is used for the amplification of any above-mentioned dna molecular.

The present invention also provides:

Comprise the expression cassette that contains DNA isolation molecule of the present invention or the cell of its part, this dna molecular in the wherein said expression cassette is effable in said cell.In preferred embodiments, dna molecular is not from barley.In another preferred embodiment, this cell is a vegetable cell.In a more preferred embodiment, this expression cassette stable integration is gone in the genome of this cell, or is included in the autonomously replicationg vector and is retained in the cell as extrachromosomal molecule.

The present invention further provides:

Comprise the expression cassette with DNA isolation molecule of the present invention or the plant of its part.In preferred embodiments, this dna molecular is not from barley.In another preferred embodiment, contained dna molecular is effable in plant in the expression cassette.In another embodiment preferred, described dna molecular stable integration is gone in the Plant Genome, or is included in the self-replacation carrier and is retained in the cell as extrachromosomal molecule.In another embodiment preferred, but the plant resistant to fungal pathogens, the preferred fungal pathogens that infects the epidermic cell of living, more preferably fungal pathogens is from Erysiphales (being also known as powdery mildew), especially from Erysiphe, most preferably fungal pathogens is the standing grain powdery mildew.

The invention still further relates to the seed of said plant, wherein seed randomly processed (as contacted antigenic or coated) and/or packing, as be positioned in the bag and with operation instruction.

The present invention also provides:

The agricultural-food that comprise the plant that contains DNA isolation molecule of the present invention.In preferred embodiments, said agricultural-food are used as feed, food or silage, wherein do not contain the mycotoxins that fungal pathogens produces, as aflatoxin.Therefore, said agricultural-food have the Plant Quarantine characteristic of improvement.

The present invention further provides:

Preparation has the method for the plant of resistance to fungal pathogens, may further comprise the steps:

A) in plant, express rna transcription thing by above-mentioned any dna molecule encode with " justice is arranged " direction; Or

B) in plant, express rna transcription thing by above-mentioned any dna molecule encode with " antisense " direction; Or

C) in plant, express the special cutting of energy by ribozyme corresponding to the messenger RNA(mRNA) transcript of source genes encoding within any above-mentioned dna molecular; Or

D) in plant, express the aptamer be specific to corresponding to the endogenous protein of the coded by said gene of any above-mentioned dna molecular; Or

E) in plant, express the sudden change or the clipped form of above-mentioned any dna molecular, thereby make it can become dominant negative mutant; Or

F) in plant, modify at least one chromosome copies corresponding to the gene of above-mentioned any dna molecular by homologous recombination; Or

G) in plant, modify at least one chromosome copies of regulatory element corresponding to the gene of above-mentioned any dna molecular by homologous recombination.

The present invention further provides:

By the plant that above-mentioned any method obtains, comprise the seed of said plant, wherein seed randomly processed (as contacted antigenic or coated) and/or packing, as be positioned in the bag and with operation instruction.In another embodiment preferred, but the plant resistant to fungal pathogens that obtains is preferably the fungal pathogens that infects the epidermic cell of living, and more preferably fungal pathogens is from Erysiphales (being also known as powdery mildew), especially from Erysiphe, most preferably fungal pathogens is the standing grain powdery mildew.

The present invention also provides:

The agricultural-food of the Plant Quarantine characteristic that the tool that obtains with above-mentioned any method improves.

The present invention further provides:

Separate the method for the dna molecular of coding Mlo protein, may further comprise the steps:

A) will encode at least 6 amino acid whose degenerate oligonucleotides among the SEQ ID NO:1 and be complementary to the degenerate oligonucleotide and the DNA that extracts from plant of the sequence of at least 6 amino acid among the coding SEQ ID NO:2 mix under said degenerate oligonucleotide and the condition that described DNA is hybridized allowing;

B) amplification said DNA of plants dna fragmentation, wherein said dna fragmentation its a left side and right end contain can with said degenerate oligonucleotide annealed nucleotide sequence in the step a); With

C) acquisition contains the full length cDNA clone of the dna fragmentation of step b).

The present invention also provides:

The method of the sudden change copy by " vitro recombination " or " DNA rearrangement " production nucleotide sequence of the present invention.The sudden change copy of nucleotide sequence of the present invention can be used for investing higher fungal pathogens resistance.In preferred embodiments, the sudden change of nucleotide sequence of the present invention copy is used to make plant to resist the more pathogenic agent of wide region.A kind of method like this is described below:

The method of mutagenesis dna molecular of the present invention, wherein said dna molecular have been cut into the double-stranded random fragment of expection size, and this method may further comprise the steps:

A) add one or more strand or double chain oligonucleotide in the double-stranded random fragment group of gained, wherein said oligonucleotide comprises zone identical with the double-stranded template polynucleotide and allogenic zone;

B) the mixture sex change with double-stranded random fragment of gained and oligonucleotide becomes single-chain fragment;

C) causing said single-chain fragment to form under the annealing fragment paired condition in said same area annealing, with gained single-chain fragment group and polysaccharase insulation, wherein said same area is enough to make the member in the pairing to cause duplicating of another member, thereby forms the double-stranded polynucleotide through mutagenesis; And

D) repeat second and at least two circulations of third step again, wherein the mixture that produces of second step in next circulation comprise from last circulation third step through the double-stranded polynucleotide of mutagenesis, and this next circulation has formed the double-stranded polynucleotide of further mutagenesis.

Definition

" DNA isolation molecule " is to be present in outside its natural surroundings so the nucleotide sequence of non-natural product because of manual operation.The separating nucleotide sequence can purified form exists, or is present in the non-natural environment as genetically modified host cell and so on.

" protein " that this paper limits is the whole protein by the corresponding nucleotide sequence encoding, or by the corresponding section encoded protein matter part of nucleotide sequence.

" isolated protein " is the protein by the separating nucleotide sequence encoding, thereby the non-natural product.Isolated protein can exist by purified form, or is present in the non-natural environment, and in genetically modified host cell, wherein said protein is not expressed usually in isogenic non-transgenic host cell or reached with different forms or different scales.

The plant of " resistant to fungal pathogens " does not have by the ability that suppresses or the restriction fungal pathogens is grown on plant or is rarer by this fungus-caused fungi infestation symptom.Fruit-bearing plant grows better, has higher output and produces more seed.

Expression protein relates to the regulation and control of responsible plant to the plant genetic approach of the resistance of fungal pathogens " to give the protein of plant to the resistance of fungal pathogens ".This protein can be positive regulator therein, can strengthen the resistance of plant to fungal pathogens, and perhaps this protein can be down regulator therein, can suppress the resistance of plant to fungal pathogens.Giving plant is Mlo protein to special case of protein of the resistance of fungal pathogens.

" the Mlo protein " of this paper refers in the disease resistance approach basic identity function of tool and protein families (Mlo family) member of some structural homologies is arranged.Structural homology can be, as, this family member has at least one conserved regions.

On the widest meaning, say, term " similar substantially " is when being used herein to nucleotide sequence, the expression with reference to the suitable nucleotide sequence of nucleotide sequence, wherein this suitable sequence encoding and polypeptide with reference to the nucleotide sequence coded essentially identical 26S Proteasome Structure and Function of polypeptide tool, as, at the amino acid place that does not influence the polypeptide function change has taken place only.Preferred similar substantially is nucleotide sequence coded by the nucleotide sequence coded polypeptide of reference.Substantially similar nucleotide sequence and be 80% at least with reference to the homogeny percentage between the nucleotide sequence better is at least 85%, preferably is 90% at least, more preferably is at least 95%, also more preferably is at least 99%.

Term " similar substantially " is when being used herein to protein, expression with reference to the suitable protein of protein, wherein said protein have with reference to the identical substantially 26S Proteasome Structure and Function of protein, as, at the amino acid place that does not influence the polypeptide function change has taken place.When being used for protein or aminoacid sequence, substantially similar protein or aminoacid sequence and be 80% at least with reference to the homogeny percentage between protein or the aminoacid sequence better are at least 85%, preferably are 90% at least, more preferably be at least 95%, also more preferably be at least 99%.

Use the percentage of determining the sequence homogeny based on the computer software of kinetics programmed algorithm.Preferred within the scope of the present invention computer software comprises BLAST (basic local sequence contrast gopher) retrieval software, it be designed to no matter to be checked be that protein or DNA all can retrieve all obtainable sequence libraries.It (is http: ∥ www.ncbi.nlm.nih.gov/BLAST/) at present that BLAST 2.0 versions (incising BLAST) of this gopher have been disclosed on the internet.Wherein use to adopt local sequence contrast but not the correlated gradual algorithm of complete sequence, thereby can survey the relation between the sequence of total separated region only.Specified score value has clear and definite statistics explanation in the BLAST retrieval.Said software is preferably with the optional parameter operation that is set at default value.

Term " gene " refers to encoding sequence and associated adjustment sequence, and wherein encoding sequence is transcribed into RNA, as mRNA, rRNA, tRNA, snRNA, adopted RNA or sense-rna are arranged.The example of regulating sequence is promoter sequence, 5 ' and 3 ' non-translated sequence and terminator sequence.The element that can exist is in addition, for example intron.

" expression " refers in the plant native gene or genetically modifiedly transcribes and/or translate.For example, under the antisense constructs situation, expression can only refer to transcribing of antisense DNA.

The dna sequence dna that " expression cassette " is used for representing instructing the special nucleus nucleotide sequence to express at suitable host cell herein comprises the promotor that effectively is connected with this purpose nucleotide sequence, and this purpose nucleotide sequence also effectively is connected with termination signal.Wherein also comprise the desired sequence of nucleotide sequence correct translation usually.The coding region target protein matter of encoding usually, but also codified has the purpose RNA of function, for example sense-rna or having justice or antisense orientation to suppress the untranslatable rna that special gene is expressed, as sense-rna.The expression cassette that contains the purpose nucleotide sequence can be chimeric, and the meaning is a wherein at least a component component allos another kind of with it.Expression cassette is also naturally occurring, but by the expression cassette that the useful recombinant forms of heterogenous expression is obtained.But, in general, expression cassette and host are allogenic, and promptly the special dna sequence dna in the expression cassette is not natural being present in the host cell, must introduce host cell or host cell precursor with transform mode.The expression of nucleotide sequence can or have only when host cell is exposed to and just cause when some special external stimulates under the inducible promoter control of transcribing at constitutive promoter in the expression cassette.At multicellular organisms, under the situation as plant, promotor can also be that particular tissues or organ or etap are specific.

" allogenic " used herein expression " different natural or synthetic sources " or represent the non-natural state.For example, if use from another organism, the nucleotide sequence transformed host cell of another species especially, then this gene and host cell allos are also with host cell offspring allos with this gene.The nucleic acid that transforms can comprise allogeneic promoter, allogeneic coding sequence or allos terminator sequence.Perhaps, transformed nucleic acid can be allogenic fully, maybe can comprise any possible allos and endogenous nucleic acid combined sequence.Similarly, allos also refers to from same natural initiating cell type and inserts wherein, but the nucleotide sequence that occurs with the non-natural state, as different copy numbers, or under the control of different adjustment element.

Term " promotor " refers to the dna sequence dna of can initial associated dna sequence transcribing.Promoter region also comprises the element that can be used as genetic expression instrumentality such as activating son, enhanser and/or repressor.

" synthetic nucleotide sequence " is used for representing to contain the nucleotide sequence of the non-existent constitutional features of native sequences herein.For example, more be similar to the G+C content of dicotyledons and/or monocotyledons gene and the artificial sequence of normal codon distribution and just be called synthetic.

If the residing position of two sequences makes DNA regulate the expression that sequence influences the dna encoding sequence, promptly claim to regulate dna sequence dna and " effectively connect " or " relevant " with coding RNA or protein DNA sequence.

The sequence that " regulatory element " reference and nucleotide sequence are expressed.Regulatory element contains promotor and the termination signal that effectively is connected with the purpose nucleotide sequence.They also comprise the sequence of nucleotide sequence correct translation needs usually.

" plant " refers to the part of any plant or plant, the spermatophyte of especially any etap.Wherein also comprise cutting, cell or tissue culture and seed.When being used for when of the present invention, term " plant tissue " is including, but not limited to whole strain plant, plant organ, plant seed, protoplastis, callus, cell culture and be organized into structure and/or any vegetable cell group of functional unit.

" vegetable cell " refers to structure and the physiology unit of plant, comprises protoplastis and cell walls.Vegetable cell can be the form of isolating individual cells or artificial culture cell, or as the part as plant tissue or plant organ and so on more senior organization unit.

" conversion " is used for representing nucleic acid is introduced in the cell herein.Especially, the dna molecular stable integration is gone into the genome of purpose organism.

" selective marker " is the gene that is brought the cell selective advantage by the expression in vegetable cell.With the growth phase ratio of non-transformed cell, the selective advantage that has with the selectable marker gene cell transformed may be because the ability that they are grown when existing at negative selective agent (as microbiotic or weedicide).Compare with non-transformed cell, the selective advantage that transformant has also may be because they have compound that utilization adds as the enhanced of nutrition, somatomedin or energy derive or new ability.Selectable marker gene refers to that also the expression in vegetable cell brings the gene or the assortment of genes of cell positive and negative selective advantage.

" selection markers " is not bring the transformant selective advantage by its expression, but the phenotype of transformant and the distinct gene of no transformed cells are given.

Sequence summary in the sequence table

SEQ ID NO:1 conserved amino acid sequence 1

SEQ ID NO:2 conserved amino acid sequence 2

The nucleotide sequence of SEQ ID NO:3 wheat Mlo protein TrMlo1

The protein sequence of SEQ ID NO:4 TrMlo1

The nucleotide sequence of SEQ ID NO:5 wheat Mlo protein TrMlo2

The protein sequence of SEQ ID NO:6 TrMlo2

The nucleotide sequence of SEQ ID NO:7 wheat Mlo protein TrMlo3

The protein sequence of SEQ ID NO:8 TrMlo3

The nucleotide sequence of SEQ ID NO:9 Arabidopsis Mlo protein C IB10259

The protein sequence of SEQ ID NO:10 CIB10259

The nucleotide sequence of SEQ ID NO:11 Arabidopsis Mlo protein C IB10295

The protein sequence of SEQ ID NO:12 CIB10295

The nucleotide sequence of SEQ ID NO:13 Arabidopsis Mlo protein C IB10296

The protein sequence of SEQ ID NO:14 CIB10296

The nucleotide sequence of SEQ ID NO:15 Arabidopsis Mlo protein F19850

The protein sequence of SEQ ID NO:16 F19850

The nucleotide sequence of SEQ ID NO:17 Arabidopsis Mlo protein U95973

The protein sequence of SEQ ID NO:18 U95973

SEQ ID NO:19 oligonucleotide MLO-1

SEQ ID NO:20 oligonucleotide MLO-3

SEQ ID NO:21 oligonucleotide MLO-5

SEQ ID NO:22 oligonucleotide MLO-7

SEQ ID NO:23 oligonucleotide MLO-10

SEQ ID NO:24 oligonucleotide MLO-15

SEQ ID NO:25 oligonucleotide MLO-26

SEQ ID NO:26 oligonucleotide MLO-GSP1

SEQ ID NO:27 oligonucleotide MLO-GSP2

SEQ ID NO:28 oligonucleotide ST27

SEQ ID NO:29 oligonucleotide N37544-1

SEQ ID NO:30 oligonucleotide N37544-2

SEQ ID NO:31 oligonucleotide T22146-1

SEQ ID NO:32 oligonucleotide T22146-2

SEQ ID NO:33 oligonucleotide H76041-1

SEQ ID NO:34 oligonucleotide H76041-2

SEQ ID NO:35 oligonucleotide SAS-1

SEQ ID NO:36 oligonucleotide SAS-2

SEQ ID NO:37 oligonucleotide SAS-3

SEQ ID NO:38 oligonucleotide SAS-4

SEQ ID NO:39 oligonucleotide SAS-5

SEQ ID NO:40 oligonucleotide SAS-6

SEQ ID NO:41 oligonucleotide SAS-7

SEQ ID NO:42 oligonucleotide SAS-8

The preservation bacterial classification

All preservation bacterial classifications all are deposited in the northern area research centre, 1815 NorthernUniversity Street, and Peoria, Illinois 61604, USA.

The Mlo protein DNA molecule that the present invention relates to encode, they give the resistance of plant to fungal pathogens.Inventor of the present invention has identified the conserved amino acid sequence in the Mlo protein first.Conserved amino acid sequence of the present invention is guarded between from three kinds of Mlo protein of wheat and three kinds of Mlo protein from Arabidopis thaliana.These aminoacid sequences are also guarded in the Arabidopsis Mlo of two kinds of suppositions protein.First listed conserved amino acid sequence comprises 13 amino acid among the SEQ ID NO:1.The 4th amino acid among the SEQ ID NO:1 is L, V or I, and five amino acid is V or L, and seven amino acid is F or L.The 13 amino acid among the SEQ ID NO:1 is not I, and is preferably T, S or A.Second listed among SEQID NO:2 conserved amino acid sequence comprises 14 amino acid.First amino acid among the SEQ IDNO:2 is not M and be preferably I, V, S or G.Its 3rd amino acid is F, L or V, and its 6th amino acid is Y or N, and its seven amino acid is A or V, and its 8th amino acid is L or I, and its tenth amino acid is T or S.The present invention includes the separation Mlo protein and the proteinic DNA isolation molecule of this Mlo of coding that contain at least a above-mentioned conserved amino acid sequence.The present invention also comprise contain listed conserved sequence among SEQ ID NO:1 and the SEQ ID NO:2 separate Mlo protein.In preferred embodiments, the proteinic DNA isolation molecule of code book invention Mlo is the cDNA molecule.

In another embodiment, coding contains the Mlo protein DNA molecule of at least a described conserved amino acid sequence not from barley.In another embodiment, said dna molecular from dicotyledons or from wheat, corn, rice, oat, rye, Chinese sorghum, sugarcane, millet, buy sieve Chinese sorghum or Palmae.In preferred embodiments, dna molecular of the present invention and SEQ ID NO:3,5 or 7 or SEQ ID NO:9,11,13,15 or 17 in listed dna molecular identical or similar substantially, or coding and SEQ ID NO:4,6 or 8 or SEQ ID NO:10,12,14,16,18 in the listed identical or similar substantially Mlo protein of any Mlo protein.Dna molecular listed among the SEQ ID NO:3,5 or 7 is from wheat, listed Mlo protein among the SEQ ID NO:4,6 or 8 that encodes respectively.The separation of this dna molecular is further described among the embodiment 1.Dna molecular listed among the SEQ ID NO:9,11,13,15 or 17 is from Arabidopsis, listed Mlo protein among the SEQ IDNO:10,12,14 that encodes respectively, 16 or 18.The separation of this dna molecular is further described among the embodiment 2.

The wheat Mlo protein DNA molecule that the coding of SEQ ID NO:3 is called as TrMlo 1 is preservation with TrMlo 1 and TrMlo 1-5 strain, receives number to be respectively NRRL B-21948 and NRRL B-21949.The wheat Mlo protein DNA molecule that the coding of SEQ ID NO:5 is called as TrMlo 2 is preservation with TrMlo 2 and TrMlo 2-5 strain, receives number to be respectively NRRL B-21950 and NRRL B-21951.The wheat Mlo protein DNA molecule that the coding of SEQ ID NO:7 is called as TrMlo3 is preservation with TrMlo 3 and TrMlo 3-5 strain, receives number to be respectively NRRL B-21952 and NRRL B-21953.TrMlo1 and TrMlo3 comprise the full-length cDNA of corresponding Mlo gene, also comprise the part of corresponding 5 ' and 3 ' non-translational region.TrMlo2 is the longest cDNA clone of the corresponding gene that is repaired.It comprises whole coding region, just lacks first methionine(Met) (initiator codon) of relatively inferring with TrMlo1 and TrMlo3.TrMlo2 also comprises the part of corresponding gene 3 ' non-translational region.

The coding of SEQ ID NO:9 is called as the Arabidopsis Mlo protein DNA molecule of CIB10259 with pCIB 10259 strain preservations, and receiving number is NRRL B-21945.The coding of SEQ IDNO:11 is called as the Arabidopsis Mlo protein DNA molecule of CIB10295 with pCIB 10295 strain preservations, and receiving number is NRRL B-21946.The coding of SEQ ID NO:13 is called as the Arabidopsis Mlo protein DNA molecule of CIB10296 with pCIB10296 strain preservation, and receiving number is NRRL B-21947.CIB10259, CIB10295 and CIB10296 comprise the full-length cDNA of corresponding Mlo gene, also comprise the part of corresponding 5 ' and 3 ' non-translational region.The nucleotide sequence of coding Arabidopsis Mlo protein families member F19850 and U95973 obtains from Gembank.But, all measured the aminoacid sequence of estimating for this two clones and find it and Gembank in the aminoacid sequence estimated be not complementary.The Mlo protein that inventor therefore of the present invention measures is new and non-obvious.Two kinds new estimates that protein all comprise conserved amino acid listed in SEQ ID NO:1 and 2, so they and their the separation cDNA of encoding are included among the present invention.

The Mlo protein of dna molecule encode of the present invention is given the resistance of plant to fungal pathogens, preferably can infect the fungal pathogens of plant epidermis cell alive, more preferably fungal pathogens is from Erysiphales (also being known as powdery mildew) (Agrios G. (1988) plant pathology, the third edition, Academic Press Inc. is especially at the 271st page).Preferably, the Mlo protein of dna molecule encode of the present invention is given plant to the resistance from the pathogenic agent of Erysiphe, and more preferably fungal pathogens is the standing grain powdery mildew.

The present invention also comprises the recombinant vectors that contains any dna molecular of the present invention.In these carriers, said dna molecular preferred package is contained in the expression cassette, and this expression cassette contains the Expression element that is used to express this dna molecular in the host cell that can express said dna molecular.Said regulatory element is promotor and termination signal normally, also preferably includes the element that permission is effectively translated by the protein of dna molecule encode of the present invention.In preferred embodiments, expression cassette is allogenic.The expression cassette that said carrier is used for containing any dna molecular of the present invention is transformed into host cell.In preferred embodiments, the expression cassette stable integration is gone among the DNA of said host cell.In another preferred embodiment, the expression cassette preferred package is contained in the carrier, and this carrier can duplicate in host cell and remain in the host cell as extrachromosomal molecule.In a further preferred embodiment, utilize the said extrachromosomal replication molecule dna molecular of the present invention that in host cell, increases.In preferred embodiments, said host cell is a microorganism, as bacterium, and intestinal bacteria especially.In another preferred embodiment, host cell is an eukaryotic cells, as yeast cell, insect cell or vegetable cell.

In further embodiment, modify dna molecular of the present invention by introducing random mutation with the technology that is known as vitro recombination or DNA rearrangement.This technical description is in the document of incorporating into own forces as a reference: Stemmer etc. herein, in natural 370:389-391 (1994) and the United States Patent (USP) 5605793.With parent nucleotide sequence as herein described serves as that the basis produces millions of nucleotide sequences sudden change copies, and reclaims and have the variant of improved characteristics, as has strengthened and maybe can resist the more pathogenic agent of wide region to the resistance of fungal pathogens.This method comprises from the double-stranded polynucleotide of the double-stranded polynucleotide formation of the template that contains nucleotide sequence of the present invention through mutagenesis, wherein the double-stranded polynucleotide of template have cut into the double-stranded random fragment of expection size, also comprise the steps: to add one or more strand or double chain oligonucleotide in the double-stranded random fragment group of gained, wherein said oligonucleotide comprises zone identical with the double-stranded template polynucleotide and allogenic zone; The mixture sex change of double-stranded random fragment of gained and oligonucleotide is become single-chain fragment; Causing said single-chain fragment to form under the annealing fragment paired condition in said same area annealing, with gained single-chain fragment group and polysaccharase insulation, wherein said same area is enough to make the member in the pairing to cause duplicating of another member, thereby forms the double-stranded polynucleotide through mutagenesis; And repeat second and at least two circulations of third step again, wherein the mixture that produces of second step in next circulation comprise from last circulation third step through the double-stranded polynucleotide of mutagenesis, and this next circulation has formed the double-stranded polynucleotide of further mutagenesis.In preferred embodiments, the single concentration of double-stranded random fragment of planting in the double-stranded random fragment is less than 1% of total DNA weight.In a further preferred embodiment, the double-stranded polynucleotide of template comprise at least about 100 kinds of polynucleotide.In another embodiment, the size of double-stranded random fragment is about 5bp-5kb.In further embodiment, the 4th step of this method comprises repetition second and at least 10 circulations of third step.

The present invention also comprises the cell that contains dna molecular of the present invention, and wherein said dna molecular is not present in its n cell environment.In preferred embodiments, said cell is a vegetable cell.In another preferred embodiment, dna molecular of the present invention can be expressed in said cell, and is contained in and allows it in the expression cassette of this cell inner expression.In preferred embodiments, the expression cassette stable integration is gone among the DNA of this host cell.In another preferred embodiment, expression cassette is included in the carrier, and it can duplicate in cell and remain in the cell as extrachromosomal molecule.

The present invention also comprises the plant that contains above-mentioned vegetable cell.In other embodiments, dna molecular of the present invention can be expressed in the plant, and the resistance of these transgenic plant to fungal pathogens given in any dna molecular of the present invention or the expression of its part in transgenic plant.In preferred embodiments, described fungal pathogens preferably can infect epidermic cell alive, and more preferably fungal pathogens is from Erysiphales (being also known as powdery mildew), and especially from Erysiphe, most preferably fungal pathogens is the standing grain powdery mildew.Therefore the present invention also comprises the transgenic plant that can resist fungal pathogens by the expression of any dna molecular of the present invention or its part.

By plant transformed of the present invention can be monocotyledons or dicotyledons, including, but not limited to corn, wheat, barley, rye, sweet potato, Kidney bean, pea, witloof, lettuce, wild cabbage, Cauliflower, sprouting broccoli, turnip, radish, spinach, asparagus, onion, garlic, pepper, celery, pumpkin, summer squash, hemp, little cucumber, apple, pears, Wen Bai, muskmelon, plum, cherry, peach, nectarine, apricot, strawberry, grape, raspberry, blackberry, blueberry, pineapple, avocado, papaya, mango, banana, soybean, tomato, Chinese sorghum, sugarcane, beet, Sunflower Receptacle, Semen Brassicae campestris (rapeseed), clover, tobacco, Radix Dauci Sativae, cotton, clover, rice, potato, eggplant, cucumber, Arabidopis thaliana and the xylophyta such as softwood tree and deciduous trees, especially corn, wheat or beet.

In case expection Nucleotide has been transformed in the particular variety plant, promptly available traditional breeding technology with its propagation or be transferred in other mutation of same species, especially comprises the commercialization kind in these species.

For they expression in transgenic plant, dna molecular may need to modify and optimize.The codon that all organisms known in the art all have particular preferences to use, the codon in the contained nucleotide sequence of dna molecular of the present invention can be changed when keeping its coded amino acid, to meet the preference of special plant.Have 35%GC content at least, the encoding sequence that preferably surpasses 45%GC content can reach high expression level best in plant.Owing to have ATTTA primitive that can destroy courier's stability and the AATAAA primitive that may cause incorrect polyadenylation, the nucleotide sequence of the low GC content of tool is expressed relatively poor.Although preferred gene order can be expressed in unifacial leaf and the dicotyledonous kind plant fully, because of they preference difference (Murray etc. to codon, nucleic acids research 17:477-498 (1989)), answer special password preference and the GC content preference of modification sequence from satisfying unifacial leaf or dicotyledons.In addition, can screen nucleotide sequence existing with the unreasonable splice site of determining to cause courier's brachymemma.With the method among disclosed patent application EP 0385962, EP 0359472 and the WO 93/07278, cause the institute that requires in the nucleotide sequence as mentioned above to change by well-known site-directed mutagenesis, PCR and synthetic gene constructing technology.

For effective initial translation, may require to modify the sequence contiguous with initial methionine.For example, they can be modified by containing known effective sequence in plant.Joshi has proposed the consensus sequence proper to plant (NAR15:6643-6653 (1987)), and Clontech has proposed further to have translation initiation district (1993/1994 catalogue, the page number 210).These consensus sequences are applicable to nucleotide sequence of the present invention.These sequences are mixed in the structure that contains nucleotide sequence of the present invention, mix degree up to and comprise ATG (simultaneously second amino acid unmodified), or mix degree up to and comprise ATG after GTC (having second amino acid whose possibility of modification transgenosis).

Dna molecular in the transgenic plant is used in the promoters driven that shows function in the plant.The selection of promotor will change with the time and the space requirement of expressing, and also can change according to target species.For protective plant opposing leaf disease substance, preferably in leaf, express; For protective plant opposing grain ear pathogenic agent, preferably in inflorescence, express (as spike, panicle, corn cob etc.); For protective plant opposing root disease substance, preferably in root, express; In order to protect seedling to resist soilborne pathogenic agent, preferably in root and/or seedling, express.But in many cases, the phytopathogen of anti-more than one types of claimed species, thereby be desirably in the multiple tissue and express.Although shown that the promotor of many dicotyledonss can be used for monocotyledons and vice versa, ideal selects the dicotyledons promotor to be used for the expression of dicotyledons, selects Monocotyledon promoter to be used for the expression of monocotyledons.Yet, unrestricted for the source of selected promotor; Expression in the expection cell is just enough as long as they can drive dna molecular.

The preferred promoter of constitutive expression comprises the promotor from Agrobacterium opine synthase gene, as the no promotor, or from the binary promotor (Velten etc. (1984) EMBO J.3:2723-2730) of Agrobacterium Ti-plasmids, or the viral promotors of function arranged in plant, promotor as the gene of CaMV 35S and 19S promotor and coding Actin muscle or ubiquitin.Another preferred promoter is the synthesis type promotor, as Gelvin Super MAS promotor (Ni etc. (1995) plant magazine 7:661-676).Dna molecular of the present invention also can be expressed under the adjusting of the promotor that is subjected to chemical regulation.This is only just synthesizing the protein that causes fungal disease when inducing chemical agent to handle crop plants.The optimization technique that is used for chemical induction genetic expression is specified in disclosed application EP 0332104 and United States Patent (USP) 5614395.The preferred promoter that is used for chemical induction is a tobacco PR-1a promotor.

Preferred promotor kind is that wound is derivable.Many promotors that are expressed in wound site and phytopathogen infection position have been described.Ideally, said promotor should be only have activity infecting the part, position, thus the protein of control fungal disease only accumulate in need synthetic it with in the cell that kills the invasion and attack insect.This type of preferred promoter comprises the promotor described in the following document: Stanford etc., Mol.Gen.Genet.215:200-208 (1989); Xu etc., molecular biology of plants 22:573-588 (1993); Logemann etc., vegetable cell 1:151-158 (1989); Rohrmeier and Lehle, molecular biology of plants 22:783-792 (1993); Firek etc., molecular biology of plants 22:129-142 (1993) and Warner etc., plant magazine 3:191-201 (1993).

Preferred tissue specific expression pattern comprises chlorenchyma specificity, root-specific, stem specificity and flower specific.Be suitable for the promotor that in chlorenchyma expression promoter comprises related gene in many adjusting light compositings, wherein many from monocotyledons and dicotyledons clone.Preferred promotor is the corn PEPC promotor (Hudspeth and Grula, molecular biology of plants 12:579-589 (1989)) from phosphoric acid enol carboxylase gene.The preferred promoter that root-specific is expressed is de Framond (FEBS290:103-106 (1991); EP0452269) described promotor, and further preferred root-specific promoter is the promotor from the T-1 gene that the invention provides.Described in preferred stem specificity promoter such as the United States Patent (USP) 5625136 and drive the promotor of corn trpA genetic expression.

Preferred embodiment of the present invention is the transgenic plant with the different mode expressible dna of Gent molecule.Further preferred embodiment is to induce the transgenic plant of mode expressible dna molecule with wound-induced or pathogenic infection.

Except the selection of suitable promotor, the appropriate transcription terminator of structure needs that is used for the plant marking protein is attached to the heterologous nucleotide sequence downstream.Several said terminators are can obtain in this area and known (as the tml from CaMV, from the E9 of rbcS).The known any obtainable terminator that works in plant can be used in the context of the present invention.

Many other sequences can be mixed in the expression cassette of dna molecular of the present invention.These sequences comprise and show the sequence that can strengthen expression, as intron sequences (as from Adh1 and bronze1) and virus leader sequence (as from TMV, MCMV and AMV).

Preferably with the different cell positions of expression and localization in plant of dna molecular.In some cases, be positioned in the cytosol more satisfactoryly, and in other cases, it is preferred being positioned in some subcellular organelle.Available technology well-known in the art is carried out the Subcellular Localization of the coded enzyme of transgenosis.In general, utilize coding to be blended in the upstream of nucleotide sequence from the DNA of the target peptide of the gene product of known targeted cells device and with it.Many such target sequences are known for chloroplast(id), have shown that they can work in the allos structure.

The carrier that is suitable for Plant Transformation also has description in the other parts of this specification sheets.For agriculture bacillus mediated conversion, binary vector or be suitable with the carrier of a T-DNA edge sequence at least, and for direct transgenosis, any carrier all is suitable, preferably only contains the linear DNA of purpose structure.Under the situation that direct gene shifts, can adopt the conversion or the cotransformation (Schocher etc., biotechnology 4:1093-1096 (1986)) of single DNA kind.For direct transgenosis and agriculture bacillus mediated transfer, usually (but nonessential) and the selective marker that microbiotic (kantlex, Totomycin or methotrexate) or sterilant (glufosinate, glyphosate or proporphyrinogen oxidase inhibitor) resistance can be provided maybe can provide the selective marker (as Phophomannose isomerase gene) of transformant selective advantage to transform together.But, the selection of selective marker is not crucial for the present invention.

In another preferred embodiment, dna molecular of the present invention directly is transformed in the plastom.The plastid transformation technology extensively is described in United States Patent (USP) 5451513,5545817 and 5545818 and institutes of (1994) NAS newspapers 91 such as PCT application WO95/16783 and McBride, among the 7301-7305.The basic fundamental that is used for the chloroplast(id) conversion comprises as the clone's plastid DNA zone that utilizes biolistics or protoplast transformation (as the conversion of calcium chloride or PEG mediation) will be positioned at selective marker and target DNA molecule flank introduces suitable target tissue.The 1-1.5kb flanking sequence that is called as the target practice sequence promoted and plastom between homologous recombination, thereby allow the displacement or the modification in the special district of plastom(e).At first, utilization is given the chloroplast(id) 16S rRNA of anti-spectinomycin of plant and/or streptomycin resistance and the point mutation in the rps12 gene as the selective marker (Svab, Z., Hajdukiewicz, P. and the Maliga that transform, P. institute of (1990) NAS newspaper 87,8526-8530; Staub, J.M. and Maliga, P. (1992) vegetable cell 4,39-45).This causes stable homogeneity transformant, and frequency is about 100 target leaves of every bombardment and produces 1 transformant.The existence of cloning site makes the plastid targeting vector can obtain being used to introduce foreign gene (Staub, J.M. and Maliga, P. (1993) EMBO J.12,601-606 incorporates into own forces as a reference at this) between these marks.Recessive rRNA of bacterium aadA gene substitution or the r-protein antibiotics resistance gene of separating toxenzyme aminoglycoside-3 '-adenylyl transferase with dominant selectable marker-coding spectinomycin can make that transformation efficiency improves (Svab in fact, Z. and Maliga, P. institute of (1993) NAS newspaper 90,913-917).Before this, successfully this mark is used for green alga Reinhard chlamydomonas plastom high frequency transform (Goldschmidt-Clermont, M. (1991) nucleic acids research 19,4083-4089).Other selective markers that can be used for the plastid conversion are well known in the art and are contained in the scope of the invention.In general, need about 15-20 cell division cycle to reach the homoplasmon state after the conversion.By homologous recombination the plastid expression that gene inserts all thousands of copy ring-type plastom in each vegetable cell is compared the advantage with huge copy number with the nuclear expression gene, thereby make expression level can easily surpass 10% of total solubility plant protein.

The present invention also comprises agricultural-food, has the transgenic plant of fungal pathogens resistance or with any method preparation hereinafter described fungal pathogens is had the transgenic plant of resistance comprising the expression by any dna molecular of the present invention.Since these plants are resistant to fungal pathogens, pathogenic agent growth therein is suppressed.Therefore such plant and agricultural-food thereof unlikely comprise the natural generation of many fungal pathogens and may be to the very deleterious mycotoxins of humans and animals.Thereby such agricultural-food have better Plant Quarantine characteristic.In preferred embodiments, such agricultural-food are used as feed, silage or food.

Another object of the present invention provides preparation has the plant of resistance to fungal pathogens method.Give the resistance of plant to fungal pathogens by the Mlo protein of dna molecule encode of the present invention, changing the expression of said protein in its natural host environment is of the present invention one preferred purpose.Another preferred purpose of the present invention is to change the stability of said protein in its natural surroundings or active.The coded protein expression of dna molecular of the present invention, stability or active this change cause plant that the resistance of fungal pathogens is strengthened in the plant.In preferred embodiments, be the negative regulation agent of plant opposing fungal pathogens by the protein of dna molecule encode of the present invention, because it can suppress to be responsible in the plant genetic approach of plant to the resistance of fungal pathogens.Therefore, of the present invention one preferred purpose is the expression of Mlo protein in its natural host environment that reduces by dna molecule encode of the present invention, or reduces the stability of this protein in its natural host environment or active.

" there is justice to suppress "

In preferred embodiments, by " have justice suppress " reduce the coded protein expression of dna molecular of the present invention (referring to as (1996) such as Jorgensen, molecular biology of plants 31,957-973).In this case, all or part of being contained in of dna molecular of the present invention will be introduced in the expression cassette of host cell, the preferred plant cell, and wherein dna molecular is effable.Dna molecular is inserted in the expression cassette with " sense orientation ", be meant that promptly 5 ' the terminal promotor-proximal place of this dna molecular in expression cassette and the coding strand of this dna molecular can be transcribed.In preferred embodiments, dna molecular is interpretable fully, and all contained genetic information can be translated into protein in dna molecular or the part.In another embodiment preferred, dna molecular is that part is interpretable, translates into small peptide.In preferred embodiments, this is to finish by insert at least one precocious terminator codon in dna molecular, causes translation to stop.In another preferred embodiment, dna molecular is transcribed but is not had translation product.This normally by removing the coded proteinic initiator codon of dna molecular, finishes as " ATG ".In a further preferred embodiment, the expression cassette that contains said dna molecular or its part is stabilized and is integrated in the host cell gene group.In another preferred embodiment, the expression cassette that contains said dna molecular or its part is included in the extrachromosomal replication molecule.In containing the transgenic plant of one of above-mentioned expression cassette, the expression of the corresponding gene of contained dna molecular is reduced or eliminates in the expression cassette, causes this protein level reduction or shortage in transgenic plant.But the result is this transgenic plant resistant to fungal pathogens.

" antisense " suppresses

In another preferred embodiment, suppress to reduce the coded protein expression of dna molecular of the present invention by " antisense ".The all or part of of dna molecular of the present invention is contained in the expression cassette, by it dna molecular introduced host cell, the preferred plant cell, and wherein this dna molecular is effable.Dna molecular inserts in the expression cassette with " antisense orientation ", promptly be meant 3 ' terminal promotor-proximal place of this dna molecular in expression cassette, and the noncoding strand of dna molecular can be transcribed.In preferred embodiments, the expression cassette that contains said dna molecular or its part is stabilized and is integrated in the host cell gene group.In another preferred embodiment, the expression cassette that contains said dna molecular or its part is included in the extrachromosomal replication molecule.Some articles of describing this method are incorporated in this so that (Green, P.J. etc., biochemical yearbook 55:569-597 (1986) are described further; Van der Krol, A.R. etc., antisense nucleic acid and protein, pp.125-141 (1991); Abel, P.P. etc., institute of NAS reports 86:6949-6952 (1989); Ecker, J.R. etc., institute of NAS reports 83:5372-5376 (in August, 1986)).

Homologous recombination

In another embodiment preferred, by as be further described in Paszkowski etc., the homologous recombination among the EMBO magazine 7:4021-26 (1988) is at least one is modified corresponding to genome of dna molecular of the present invention copy in the Plant Genome.Said technology utilizes the feature of homologous sequence to discern and use methods known in the art mutually, and reorganization exchanges the nucleotide sequence between them as homology.Take place between the introducing copy nucleotide sequence that homologous recombination can enter cell in the chromosome copies and the conversion of intracellular nucleic nucleotide sequence.Thereby special modification is accurately introduced in the chromosome copies of nucleotide sequence.In one embodiment, the regulatory element of the gene of code book invention protein is modified.Existing regulatory element is by the displacement of different regulatory element, thereby reduced protein expression, perhaps suddenlys change or lacks this controlling element, thereby eliminate protein expression.In another embodiment, come the coding region of modifying protein by deletion entire coded sequence or its part or sudden change.The expression of mutein also can give plant the resistance stronger to fungal pathogens.

In another embodiment preferred, with the end that contains RNA and DNA residue continuous sequence the chimeric oligonucleotide transformant of the duplex conformation of two hair clip caps is arranged, thereby in the dna molecular chromosome copies, introduce sudden change.The supplementary features of oligonucleotide are to exist 2 '-O-to methylate at RNA residue place.Become chromosome copies sequence with dna molecular of the present invention consistent and contain the Nucleotide change of expection the RNA/DNA sequences Design.This technology is further described in the United States Patent (USP) 5501967.

Ribozyme

In another embodiment, invent proteinic RNA with catalytic RNA that is specific to said RNA or ribozyme cutting code book.Ribozyme is expressed in the transgenic plant, cause in the vegetable cell code book to invent proteinic RNA amount and reduce, thereby the protein mass that causes accumulating in the cell reduces and improves the resistance of plant to fungal pathogens.This method is further described in United States Patent (USP) 4987071.

Dominant negative mutation

In another preferred embodiment, the activity of proteins nucleotide sequence coded by the present invention is changed.This is the expression by this proteinic dominant negative mutant in the transgenic plant, causes the endogenous protein loss of activity and finishes.

Aptamer

In another embodiment, but by this proteinic nucleic acid ligands of expression specific combination in transgenic plant, promptly so-called aptamer, thus the coded activity of proteins of dna molecular of the present invention suppressed.Preferably use SELEX (Systematic Evolution of Ligands byExponential Enrichment) method to obtain aptamer.In the SELEX method, the single-chain nucleic acid candidate mixture in tool randomized sequence district is contacted with this protein, those resistatess to the higher nucleic acid of target affinity and candidate's mixture are open.In order to produce the mixture that is rich in part, the nucleic acid that amplification distributes.Repeatedly obtain after repeatedly the nucleic acid of the best affinity of protein tool and be used for the expression of transgenic plant.This method is further described in the United States Patent (USP) 5270163.

The separation method that contains the nucleotide sequence of conserved sequence

The conserved sequence that comprises in the coded Mlo protein of dna molecular of the present invention is used to separate other dna moleculars of this sequence of encoding.In preferred embodiments, produce the mixture of degenerate oligonucleotide, comprising the possible oligonucleotide of sequence described at least a coding SEQ ID NO:1 or the SEQ IDNO:2.Carry out pcr amplification reaction with the oligonucleotide mixture of coding SEQ ID NO:1 described sequence with the sequence complementary oligonucleotide mixture of sequence described in the coding SEQ ID NO:2 with selected template DNA.The mixture of degenerate oligonucleotide is well-known in the art, and the degeneracy degree changes with the need.In preferred embodiments, template DNA is the total DNA sample from plant, and wherein said DNA sample can obtain by method well-known in the art.Separate the amplified fragments that above-mentioned PCR reaction produces with method well-known in the art, and by screening the cDNA library or utilizing the RACE scheme to use it for and separate corresponding full-length cDNA, back two kinds of technology also are that this area is in common knowledge.This method has been represented and has been used to separate the unique and useful strategy of the new gene of giving the resistance of plant to fungal pathogens.

With reference to the embodiment of following detailed description, the present invention will further be described.Provide the purpose of these embodiment just to be used for illustration, but not attempt it is limited to some extent, unless other explanation is arranged.

Embodiment

Embodiment 1: the clone and the order-checking of wheat Mlo gene

Mlo gene with reverse transcription PCR method clone wheat.RNA prepares from the leaf of wheat Cultivar UC703 and utilizes Stratagene RT-PCR test kit that it is carried out reverse transcription.The cDNA that produces is used for the PCR reaction, and primer is as follows:

MLO-26?5’TTC?CAG?CAC?CGG?CAC?AAG?AA?3’(SEQ?ID?No：25)

MLO-10?5’AAG?AAC?TGC?CTG?AAG?AAG?GC?3’(SEQ?ID?No：23)

MLO-7??5’CAG?AAA?CTT?GTC?TCA?TCC?CTG?G?3’(SEQ?ID?No：22)

MLO-5??5’ACA?GAG?ACC?ACC?TCC?TTG?GAA?3’(SEQ?ID?No：21)

MLO-15?5’CAC?CAC?CTT?CAT?GAT?GCT?CA?3’(SEQ?ID?No：24)

Carry out PCR with following listed primer, reaction has produced the amplified fragments of indication size:

MLO-26 and MLO-10 503bp

MLO-26 and MLO-7 1481bp

MLO-5 and MLO-15 650bp

Fragment cloning is gone into pCR2.1 or pCR2.1-TOPO (Invitrogen).Prepare plasmid DNA and carry out dna sequencing from transformant.Order-checking has shown that having three kinds has very high homophylic different cDNA sequences each other.These wheats Mlo gene is called as TrMlo1, TrMlo2 and TrMlo3.

Separate other wheat Mlo clone by the cDNA library that screening is implemented in λ-ZAP II carrier.In order to screen this library, carry out mass excision the library is transformed into cDNA clone group based on Bluescript.These clones are cultivated respectively with the storehouse of 80000 independent clonings and prepare plasmid DNA.In hybrid dna, carry out the PCR reaction with Oligonucleolide primers MLO-5 and MLO-15 (seeing above).Three storehouses have produced the band of 650 base pair expection sizes.By cloning these storehouse fractional separation, all carry out the PCR that plasmid DNA prepares and carry out with primer MLO-5 and MLO-15 each inferior storehouse after per step subsequently with more and more lower density culturing bacterium.After separating, multiple fractionation obtains containing the segmental single clone of insertion of band Mlo sequence.With these clone in two insert sequencing fragment, show that they comprise identical insertion fragment.The 3rd clone's sequence shows wherein inserts fragment except having at 5 ' end 40 additional bases, and two clones' of all the other and other is identical.

The clone who finishes wheat Mlo remainder by the random amplification (RACE) of cDNA end.Carry out the RACE reaction of wheat UC703poly-A+RNA with Marathon cDNA amplification kit (Clontech).Poly-A+RNA is from the total RNA of wheat with oligo-dT cellulose column (Gibco BRL) preparation.The oligonucleotide that is used for the RACE reaction is:

MLO-GSP1??5’TGG?ACC?TCT?TCA?TGT?TCG?ATC?CCA?TCT?G?3’(SEQ?ID?No：26)

MLO-GSP2??5’CCT?GAC?GCT?GTT?CCA?GAA?TGC?GTT?TCA?3’(SEQ?ID?No：27)

5 ' be connected primer and increase and produce the dna fragmentation of about 1300 Nucleotide sizes with what provide in primer MLO-GSP1 and the test kit.Increase with primer MLO-GSP2 and 3 ' linker and to produce the dna fragmentation of about 600 Nucleotide sizes.Fragment cloning is gone into pCR2.1-TOPO, be called TrMlo1-5, TrMlo2-5 and TrMlo3-5, wherein comprise the 5 ' end of wheat Mlo gene TrMlo1, TrMlo2 and TrMlo3 respectively.From said clone, prepare plasmid DNA and plasmid is inserted sequencing fragment.

Embodiment 2: the clone of Arabidopsis Mlo cDNA

Translation product with sequence in software TBLASTN comparison Mlo protein sequence and the database shows that wherein many sequences and Mlo are similar.Wherein, all cloned corresponding to the full-length cDNA of three kinds of Arabidopsis est sequences, accepting number is H76041, N37544 and T22146.For each EST, design oligonucleotides is to be implemented in Arabidopsis cDNA amplified library among the plasmid pFL61 certainly corresponding to sequence (Minet etc. (1992) the Gene Nov16 of said EST; 121 (2): 393-396).Used oligonucleotide is:

N37544-1??5’AAG?ATC?AAG?ATG?AGG?ACG?TGG?AAG?TCG?TGG?3’(SEQ?ID?No：29)

N37544-2??5’AGG?CTG?AAC?CAC?TGG?GGC?GCC?TCT?CAC?CAC?3’(SEQ?ID?No：30)

T22146-1??5’CAA?GTA?TAT?GAT?GCG?CGC?TCT?AGA?GGA?TGA?3’(SEQ?ID?No：31)

T22146-2??5’AGG?TTT?CAC?CAC?TAA?GTC?TCC?TTC?AAT?GGC?3’(SEQ?ID?No：32)

H76041-1??5’GAT?CAT?TCA?AGA?CTT?AGG?CTC?ACT?CAT?GAG?3’(SEQ?ID?No：33)

H76041-2??5’AAC?AGC?AAG?GAA?GAT?TAC?AAA?TGA?TGC?CCA?3’(SEQ?ID?No：34)

The DNA cloning that will prepare from the cDNA library with primer N37544-1 and N37544-2 obtains the fragment of about 500 base pair sizes, and obtains the fragment of about 250 base pair sizes with primer T22146-1 and T22146-2 amplification.Obtain about 350 and two fragments of about 300 base pair sizes with primer H76041-1 and H76041-2 amplification.The fragment of about 300 bases is sizes of estimating by est sequence, and it is used for detecting the existence of cDNA library corresponding to the cDNA of H76041EST subsequently.To be transformed into intestinal bacteria from the DNA in said library, and the clone will be divided into each about 20000 clone's storehouse., subsequently positive storehouse is segmented by the DNA of PCR screening with different primers, so that clone's number is fewer and feweri from each storehouse.By this process is carried through to the end, or in some cases,,, finish the separation of single positive colony by being that probe carries out colony hybridization with the est sequence in case the storehouse size has reached 200 clones or more hour.For EST N37544 and T22146, successfully isolate clone corresponding to EST, will insert sequencing fragment.CDNA sequence corresponding to EST N37544 is named as CIB10295 in plasmid pCIB10295, the plasmid that contains corresponding to the cDNA of EST T22146 (CIB10296) is named as pCIB10296.For these two EST, the form that the Arabidopsis BAC cloned sequence part of GenBank is gone in the preservation recently of corresponding gene group sequence obtains.It is pointed out that by determined these genome sequences of GenBank and estimate that the protein sequence that translates does not conform to the determined sequence of the direct order-checking of cDNA.Therefore, the aminoacid sequence of ESTN37544 and T22146 corresponding gene can not obviously be obtained by the GenBank clauses and subclauses, and can only illustrate by clone and cDNA cloning and sequencing.Found not comprise the gene of H76041, but contained a new Mlo gene family member as inserting fragment with the isolating clone of the primer of H76041 EST.To insert the complete segment order-checking, this Mlo gene family member is named as CIB10259 in plasmid pCIB10259.

Embodiment 3: the structure that is used for the carrier of wheat Mlo genetic expression

Making up two carriers is used for expressing barley Mlo gene at wheat " antisense ".With barley cDNA and primer MLO-5 and MLO-7 ((1) sees above) are carried out PCR, reaction produces the amplified fragments of 1124bp, and it is cloned into pGEM-T (Promega).With Sac II and two kinds of enzymes of Not I this fragment is cut out from pGEM-T.This 1124bp fragment cloning is gone into pBluescript-SK (+).Specifically will insert the pCIB9806 (described in the patent application 08/838219) that fragment cut out and be cloned into BamH I-Sac I-digestion by BamHI and Sac I restriction site, direction of insertion is that the direction of wherein Mlo encoding sequence is opposite with corn ubiquitin promotor.Plasmid is named as pCK01.

" antisense " that be used for the complete Mlo gene of wheat for carrier construction expresses, with primer MLO-1 (5 ' ATG TCG GAC AAA AAA GGG GT3 ' (SEQ ID NO:19)) and MLO-10 ((1) sees above) are carried out PCR, reaction produces the amplified fragments of 635bp, and it is cloned into pCR2.1 (Invitrogen).This fragment is cut out from pCR2.1 as EcoR I fragment, and insert pGEM-9Zf (-) (Promega).To cross over naturally occurring Sac I site among the Mlo to 320 nucleotide fragments of primer sites MLO-10 with Sac I and Bst XI cuts out.With Sac I and Bst XI digestion pCK01, these 320 base fragments are inserted.For the complete structure of Mlo gene in the monocotyledons expression vector, from pGEM-9Zf (-) derivative, cut out the Sac I fragment of 210 Nucleotide.This fragment comprises 5 ' end of Mlo encoding sequence, naturally occurring Sac I site from primer sites MLO-1 to Mlo gene.Insert with Sac I digestion pCK01 derivative and with the fragment of these 210 bases.With primer MLO-1 and MLO-10 by fragment the direction in new carrier construction of pcr analysis clone to determine these 210 bases.The fragment of having only 210 bases wherein produces product corresponding to 530 terminal base pairs of Mlo encoding sequence 5 ' with the clone who inserts with respect to the direction of ubiquitin promotor antisense.The plasmid that produces comprises the complete Mlo encoding sequence that is in " antisense " direction with respect to the ubiquitin promotor, and is named as pCK02.

For the Mlo gene that carrier construction is used for expressing " justice is arranged " direction, use BamH I digested plasmid pCK02 to discharge as inserting segmental Mlo encoding sequence.BamH I fragment is connected back the pCK02 underlying carrier again.Identify that by Sac I digestion institute is reverse bacterium colony with the Mlo encoding sequence with respect to pCK02, generation 1.8kb fragment in such clone by contrast, and obtains 210 base fragments among the clone of pCK02 same structure.Selecting institute is the clone of " justice is arranged " direction with the Mlo encoding sequence with respect to corn ubiquitin promotor, called after pCK03.

Embodiment 4: the structure that is used for expressing at Arabidopis thaliana the carrier of Mlo gene

Mlo among pCIB10259, pCIB10295 and pCIB10296 and the pCK02 (barley Mlo gene) clone is used for the PCR reaction, and the band of generation contains the full-length gene that flank has BamH I restriction site.The primer sequence is as follows: SAS-1:5 ' GGA TTA AGA TCT AAT GGC 3 ' (SEQ ID No:35, be used for pCIB10295) SAS-2:5 ' CAA AGA TCT TCA TTT CTT AAA AG 3 ' (SEQ ID No:36, be used for pCIB10295) SAS-3:5 ' GCG GAT CCA TGT CGG ACA AAA AAG G 3 ' (SEQ ID No:37, be used for barley Mlo) SAS-4:5 ' GCG GAT CCT CAT CCC TGG CTG AAG G 3 ' (SEQ ID No:38, be used for barley Mlo) SAS-5:5 ' GGA TCC ACC ATG GCC ACA AGA TG 3 ' (SEQ ID No:39, be used for pCIB10259) SAS-6:5 ' GGA TCC TTA GTC AAT ATC ATT AGC 3 ' (SEQ ID No:40, be used for pCIB10259) SAS-7:5 ' GCG GAT CCA TGG GTC ACG GAG GAG AAG 3 ' (SEQ ID No:41

Be used for pCIB10269) SAS-8:5 ' GCG GAT CCT CAG TTG TTA TGA TCA GGA 3 ' (SEQ ID No:42,

Be used for pCIB10296)

These bands are cloned into pCR2.1-TOPO, and mensuration produces inserts fragments sequence to confirm that PCR does not introduce sudden change in the plasmid.With BamH I digested plasmid, purifying inserts fragment and is cloned into the pPEH28 of BamH I digestion, a kind of shuttle vectors that contains a copy Arabidopis thaliana ubiquitin gene promoter UBQ3 (Norris etc. (1993) molecular biology of plants 21:895-906) who is located immediately at downstream, BamH I site.Evaluation contains the clone with the Mlo sequence of UBQ3 fusion, carries out restriction enzyme analysis, contains the segmental clone of insertion for " justice is arranged " and " antisense " direction with respect to UBQ3 with discriminating.For each Mlo gene, contain the right way of conduct with the digestion of Xba I and insert segmental clone to inserting segmental clone and containing antisense orientation, purifying inserts fragment and is cloned into the pCIB200 of Xba I digestion.This method is inserted the UBQ3-Mlo genetic fusant between the T-DNA border.

Embodiment 5: the conversion of wheat and the evaluation of expressor

As describing in detail among the patent application WO94/13822, by prematurity embryo's particle bombardment transformed wheat.The plantlet of will in containing the substratum of Basta, the regenerating performing PCR analysis of going forward side by side.For judging the genetically modified existence of Mlo by PCR, the primer is as follows:

MLO-3：????5’ATG?CTA?CCA?CAC?GCA?GAT?CG?3’

ST27：?????5’ACT?TCT?GCA?GGT?CGA?CTC?TA?3’

Primer MLO-3 is corresponding to the genetically modified zone of Mlo, and primer ST27 is positioned at corn ubiquitin promoter sequence.Use Mlo gene and ubiquitin promoter primer to eliminate the false positive of using two Mlo primers to cause in PCR simultaneously, this false positive may be that the Mlo gene chromosome copies from be present in wheat causes.

Affirmation is contained the genetically modified plant of Mlo carry out RNA gel engram analysis, whether contain the chromosome coding wheat Mlo mRNA of change level to determine them.Prepare Poly-A from each transgenic lines ⁺RNA, and trace is to the Hybond-N+ filter membrane.Use corresponding to 530 base fragments of Mlo gene 5 ' end and survey trace.This zone does not exist the pCK01 clone; Therefore, in containing the transgenic lines of pCK01, do not exist and hybridization from the sense-rna of transgene expression.Comprise the pCK02 transgenic lines of this 5 ' terminal fragment for transgenosis wherein, the two bands hybridization of probe and different sizes.Article one, corresponding to the mRNA of about 2.5kb of antisense transgene and 2.0kb mRNA obvious differentiation is arranged from chromosome of wheat coding Mlo gene.The abundance of monitoring 2.0kbmRNA is as the measurement of the gene inhibition efficient that transgenosis reached in each strain system.

Embodiment 6: the disease detection of transgenic wheat strain

Cultivate up to they two weeks genetically modified and unconverted UC703 (contrast) wheat line plant greenhouse big.Plant is transferred in the Percival growth chamber (each circulation is: in the dark 8 hours, in 16 ℃ and the light 16 hours, 20 ℃), and fully inoculates standing grain powdery mildew wheat microspecies by spore.The degree that two week of inoculation back assessment fungal spore forms.Plant is assessed as 1 (almost do not have and do not have the visible sporulation), 2 (some mycelial growths and sporulation are arranged, but plant is shone in comparison few) or 3 (have can compared with the control mycelial growth and sporulation) to basic no mycelia growth.The resistance that the transgenic wheat strain of expression Mlo-construct demonstrates pathogenic agent strengthens.The example as a result that obtains with antisense barley Mlo construct is as hereinafter listed.

The disease resistance of screening transgenic strain R1 and R2 sisters strain

Sisters' strain (T2 seed) of plantation Mlo antisense transformant, inoculation standing grain powdery mildew, and assess disease resistance.

	R1	R2	UC703 (contrast)
The seed of plantation	24	24	24
Germinate	14	24	21
Disease score 1	4	2	0
Disease score 2	0	0	0
Disease score 3	10	22	21

Little percentile R1 and R2 plant show be shown with resistance may be since detected be still with the isolating T2 population of transgenosis.

Embodiment 7: the Arabidopis thaliana strain of expressing the Mlo gene is analyzed

The pCIB200 derivative that will contain the Mlo gene by vacuum infiltration is used for arabidopsis thaliana transformation and belongs to environmental Ws-O (Bechtold, N., Ellis, J. and Pelletier, G. (1993) C.R.Acad.Sci.Paris316，1194-1199)。Select the screening offspring to identify transformant by kantlex.For the Mlo transgenic strain, identify the plant of expressing Mlo by RNA gel engram analysis.For the Mlo gene, analyze the variation of transformant on mRNA accumulation steady state levels by RNA gel engram analysis method.Detection presents target gene the transformant of justice or Antisense Suppression and the change of phytopathogenic fungi two spore powdery mildews and parasitic downy mildew and the pathogenic mutation reaction of bacterial pathogens pseudomonas syringae tomato.Check the leaf of transgenic plant with trypan blue dyeing both macro and micro ground, thereby detect the existence of gangrene.

For the powdery mildew inoculation, spore fully is applied to the Arabidopis thaliana bow structure, plant is remained in the Percival growth chamber at 25 ℃.Inoculate the degree that the assessment fungal spore forms after 10 days.Embodiment 8: utilize similar differentiation between the Mlo sequence from other Mlo gene family member

Expectation is presented at by the contrast of the aminoacid sequence of Mlo genes encoding has the height of many weak points amino acids like the district between all gene products.Design these regional degenerated primers, carry out the PCR reaction with these primers by the recommend method of PCR reagent suppliers.Amplified fragments is as the full-length cDNA or the genomic clone of the new Mlo gene of probe separates.As follows at Mlo protein of the present invention (thick line) and the aminoacid sequence conserved regions that is used between the degenerate oligonucleotide of other Mlo gene isolation:

E???L???M???X1??X2??G???X3???I???S???L???L???L???X4WHEATTrMlo1???GAG?CTC?ATG?CTG?GTG?GGC?TTC?ATCTrMlo2???GAG?CTG?ATG?CTG?GTG?GGG?TTC?ATCTrMlo3???GAG?CTG?ATG?CTG?GTG?GGA?TTC?ATCARABIDOPSISCIB10259?GAG?CTG?ATG?ATT?CTA?GGA?TTC?ATTCIB10295?GAG?CTT?ATG?CTG?TTG?GGA?TTC?ATACIB10296?GAG?CTG?ATG?TTG?TTA?GGG?TTT?ATAF19850???GAG?CTG?ATG?GTT?CTT?GGA?TTC?ATCU95973???GAG?TTG?ATG?TTG?CTG?GGA?CTT?ATA?????5’?GAG?CTB?ATG?MTB?BTR?GGM?TTC?AT?3’????????????X5???T???X6???P???L???X7????X8???X9???V???X10???Q???M???G???SWHEATTrMlo1???????????????????????????GCG??CTC??GTC??ACA??CAG?ATG?GGA?TCATrMlo2???????????????????????????GCG??CTC??GTC??ACA??CAG?ATG?GGA?TCGTrMlo3???????????????????????????GCG??CTA??GTC??ACA??CAG?ATG?GGA?TCAARABIDOPSISCIB10259?????????????????????????GCA??CTA??GTT??ACT??CAG?ATG?GGT?TCACIB10295?????????????????????????GCA??CTT??GTT??ACT??CAG?ATG?GGT?AGTCIB10296?????????????????????????GCC??ATC??GTC??TCA??CAG?ATG?GGA?AGTF19850???????????????????????????GCA??CTC??GTA??ACT??CAG?ATG?GGT?TCTU95973???????????????????????????GTA??ATC??GTT??ACT??CAG?ATG?GGA?TCT???????????????????????????5’?????????WCC??CAT??CTG??AGT??GAC?DAG?BGC?RTA?3’

X1=L, V or I, X2=V or L, X3=F or L, X4=T, S or A.

X5=I, V, S or G, X6=F, L or V, X7=Y or N, X8=A or V, X9=L or I, X10=T or S.

R=A、G，Y=C、T，M=A、C，K=G、T，S=C、G，W=A、T，H=A、C、T，B=C、G、T，V=A、C、G，D=A、G、T，N=A、C、G、T。

Embodiment 9: the modification of encoding sequence and contiguous sequence

In order to express in the transgenic plant host, the dna molecular described in the application can be modified to finish and optimization or their expression of negative regulation.Following problem may run into, and can carry out the modification of these dna moleculars with technology well-known in the art.

(1) codon usage.Preference codon usage and other plant species in some plants are different.Plant evolution generally trends towards having a preference for Nucleotide C and G in the 3rd base position of monocotyledons, and dicotyledons is used Nucleotide A or T usually in this position.Mix for the preferred codon of special objective genetically modified organism by modifying factor, will overcome following many problems about GC/AT content and unreasonable montage.

(2) GC/AT content.The GC content of plant gene surpasses 35% usually.The dna molecular that is rich in A and T Nucleotide can cause many problems in plant.At first, the ATTTA primitive is considered to make courier's instability, has been found in the 3 ' end of many short-lived mRNA.Secondly, the incorrect position existence in the courier is considered to can cause to transcribe in advance end as AATAAA and so on polyadenylation signal.In addition, monocotyledons may be splice site (seeing below) with being rich in the AT recognition sequence.

(3) be close to the sequence of initial methionine.Think that now rrna is attached to courier's 5 ' end and seeks first ATG with initial translation.But, we believe the preference of existence to the contiguous Nucleotide of some ATG, comprise the expression that a new total translation initiation codon can strengthen dna molecular of the present invention at the ATG place.Clontech (1993/1994 catalogue, the 210th page) has proposed a sequence and can be used as total translation initiation district and be used for plant intestinal bacteria uidA expression of gene.In addition, Joshi (NAR 15:6643-6653 (1987)) has compared the plant sequence of many contiguous ATG and disclosed sequence altogether.When the dna molecular expression is met difficulty in the plant, contain one of these sequences at initial ATG place and might promote translation.Under these circumstances, because its second amino acid whose modification, last three Nucleotide of consensus may be not suitable for being contained in by in the modification sequence.The preferred sequence of contiguous initial methionine may be different between the different floristics.It is as follows that the investigation that is arranged in 14 corn genes of GenBank database obtains the result:

The preceding position of initial ATG in 14 corn genes

-10??-9???-8???-7???-6???-5???-4???-3???-2???-1

C????3????8????4????6????2????5????6????0????10???7

T????3????0????3????4????3????2????1????1????1????0

A????2????3????1????4????3????2????3????7????2????3

G????6????3????6????0????6????5????4????6????1????5

Can carry out this to the expection floristics that will insert nucleotide sequence and analyze, and the sequence of contiguous ATG is modified to mix the Nucleotide of preference.

(4) removal of unreasonable splice site.Dna molecular of the present invention also may comprise the primitive that can be identified as 5 ' or 3 ' splice site in the plant, and is cut, thereby produces the courier of brachymemma or disappearance.Can remove these sites with technology well-known in the art.

(5) generation of dominant negative mutant

In addition, dna molecular of the present invention also can comprise to be modified, thus the nucleotide sequence coded reformed molecule of protein active of the present invention.By the expression of protein dominant negative mutant in transgenic plant, cause the loss of activity of endogenous protein, can reach this result.The mutational site that causes this dominant negative mutant to produce in the Mlo nucleotide sequence is listed below.Can introduce the single sudden change or the different combination that suddenlys change that are listed below.

The technology that is used to modify encoding sequence and be close to sequence is well-known in the art.If the initial expression level of dna molecular of the present invention is low and be suitable for changing as mentioned above sequence, then finish the structure of synthetic gene by method well-known in the art.These methods have been described in the patent content of delivering, as EP0385962, EP0359472 and WO93/07278.Under most of situation, preferably use the instantaneous method of inspection (this area is well-known) before gene construct is transferred in the transgenic plant, to detect its expression.

Embodiment 10: the structure of plant conversion carrier

Can obtain many conversion carriers and be used for Plant Transformation, dna molecular of the present invention can be used in combination with any such carrier.The selection of used carrier will be depended on the target species of preferred transformation technology and conversion.For some target species, microbiotic or sterilant selective marker that can be preferably different.The selective marker that routine is used for transforming comprises nptII gene (Vieira and Messing, 1982, the gene 19:259-268 that makes the anti-kantlex of plant, paromycin, Geneticin and associated antibiotic; Bevan etc., 1983, nature 304:184-187), coding aminoglycoside 3 '-adenylyl transferase and give bacterium aadA gene (Goldschmidt-Clermont to the resistance of Streptomycin sulphate or spectinomycin, 1991, nucleic acids research 19:4083-4089), give hph gene (Blochlinger and Diggelmann to the resistance of antibiotic hygromycin, 1984, molecular cytobiology 4:2929-2931) and give dhfr gene (Bourouis and Jarry to the resistance of methotrexate, 1983, EMBO J.2:1099-1104).Other spendable marks comprise phosphinothricin acetyl transferase gene (White etc., 1990, the nucleic acids research 18:1062 that makes the anti-sterilant phosphinothricin of plant; Spencer etc. 1990, theoretical and applied genetics 79:625-631), the sudden change epsp synthase gene (Hinchee etc. of coding glyphosate resistance, 1988, biology/technology 6:915-922), give mutant acetolactate synthetic enzyme (ALS) gene (Lee etc. to the resistance of imidazolone or sulfonylurea, 1988, EMBO is J.7:1241-1248), give the sudden change psbA gene (Smeda etc. that green bristlegrass gone clean resistance, 1993, plant physiology 103:911-917) or as United States Patent (USP) 5767373 described Mutagen protoporphyrinogen oxidase genes.Also can use to produce the positive selective marker of selecting, such as Phophomannose isomerase gene.

Also can finish the evaluation of transformant by the expression of screenable marker gene, such as coding E.C. 2.3.1.28 (CAT), β-glucuronidase (GUS), luciferase and green fluorescent protein (GFP) or any other protein of giving the unique phenotypic character of transformant.

(1) is suitable for the structure of the carrier of Agrobacterium conversion

The many carriers that are used for the agrobacterium tumefaciens conversion are obtainable.These carriers generally carry at least a T-DNA border sequence, and comprise the carrier such as pBIN19 (Bevan, nucleic acids research (1984)) and pXYZ and so on.The structure of two typical carriers has hereinafter been described.

The structure of pCIB200 and pCIB2001

Binary vector pCIB200 and pCIB2001 are used to make up the recombinant vectors that is used for Agrobacterium and make up in the following manner.By digest pTJS75 (Schmidhauser ﹠amp with NarI; Helinski, bacteriology magazine 164:446-455 (1985)) cut out tetracycline resistance gene, insert then and carry NPTII (Vieria; Messing, gene 19:259-268 (1982); Bevan etc., natural 304:184-187 (1983); McBride etc., molecular biology of plants 14:266-276 (1990)) the AccI fragment of pUC4K produces pTJS75kan.With Xho I joint with contain T-DNA left and right sides border sequence, plant nos/npt II can select the EcoRV fragment of the mosaic gene and the pCIB7 of pUC polylinker to be connected (Rothstein etc., gene 53:153-161 (1987)), and Xho I digestion fragment is cloned into the pTJS75kan of Sall digestion, produce pCIB200 (also can be, embodiment 19) referring to EP 0332104.PCIB200 comprises following single polylinker restriction site: EcoR I, Sst I, Kpn I, Bgl II, Xba I and Sal I.PCIB2001 is by inserting the derivative of the pCIB200 that added limitations site polylinker produces.Single restriction site in the pCIB2001 polylinker is EcoR I, Sst I, Kpn I, Bgl II, Xba I, Sal I, Mlu I, Bc II, Avr II, Apa I, Hpa I and Stu I.Except that containing these single restriction sites, pCIB2001 has also that plant and bacterium kantlex are selected gene, are used for the left and right sides T-DNA border sequence of agrobacterium mediation converted, the RK2-that is used for shifting between intestinal bacteria and other hosts derives the trfA function and equally from OriT and the OriV function of RK2.PCIB2001 is applicable to that the clone contains the expression of plants box of self conditioning signal.

PCIB200 and Totomycin thereof are selected the structure of derivative

Binary vector pCIB10 comprises kalamycin resistance encoding gene, the T-DNA left and right sides border sequence that can be used for selecting in the plant, and the sequence of mixing wide spectrum plasmid pRK252, makes its equal reproducible in intestinal bacteria and Agrobacterium.Rothstein etc. (gene 53:153-161 (1987)) have described its structure.Make up a plurality of pCIB10 derivatives, wherein mixed (gene 25:179-188 (1983)) described hygromycin B phosphotransferase genes such as Gritz.These derivatives make it possible to only select transgenic plant cells to Totomycin (pCIB743) or to Totomycin and kantlex the two (pCIB715, pCIB717).

(2) make up the carrier that is suitable for non-Agrobacterium-mediated Transformation

Non-agrobacterium tumefaciens transforms and need not the T-DNA sequence in selected conversion carrier, therefore except that the above-mentioned carrier that contains the T-DNA sequence, also can utilize the carrier that lacks these sequences.The transformation technology that does not rely on Agrobacterium comprises the conversion of being undertaken by particle bombardment, protoplastis absorption (as PEG and electroporation) and microinjection.Selected conversion species are depended in the selection of carrier to a great extent.The structure of some typical carriers has hereinafter been described.

PCIB3064 makes up

PCIB3064 is suitable for selecting to combine the pUC-derivative vector that carries out direct metastatic gene technology with sterilant Basta (or phosphinothricin).Plasmid pCIB246 contains CaMV 35S promoter and the CaMV 35S transcription terminator that merges with intestinal bacteria gus gene operability, and is described in PCT and openly applies among the WO93/07278.The 35S promoter of this carrier contains two ATG sequences at initiation site 5 ' end.With standard round pcr these sites that suddenly change, to remove ATG and to produce restriction site Ssp I and the Pvu II.New restriction site is from single Sal I site 96 and 37bp, from initiation site 101 and 42bp accurately.The pCIB246 derivative that produces is called as pCIB3025.From pCIB3025, downcut gus gene with Sal I and the digestion of Sac I then, mend and flat terminal also the connection again produce plasmid pCIB3060.Plasmid pJIT82 is available from John Innes Centre, and Norwich cuts out to contain from green and produces the 400bp Sma I fragment of bar gene of look Streptomycin sulphate and the Hpa I site (Thompson etc., EMBO J6:2519-2523 (1987)) of inserting pCIB3060).Produced pCIB3064 like this, be used for the polylinker in sterilant selection, ampicillin resistance gene (being used for selecting) and band Sph I, Pst I, Hind III and the single site of BamH I comprising the bar gene under CaMV 35S promoter and terminator control intestinal bacteria.This carrier is suitable for cloning the expression of plants box that contains self conditioning signal.

The structure of pSOG19 and pSOG35

PSOG35 utilizes intestinal bacteria Tetrahydrofolate dehydrogenase (DHFR) as giving the conversion carrier of the selective marker of methotrexate resistance.With pcr amplification 35S promoter (about 800bp), from the intron 6 (about 550bp) of corn Adh1 gene with from the 18bpGUS untranslated leader of pSOG10.Also with the 250bp fragment of pcr amplification coding intestinal bacteria Tetrahydrofolate dehydrogenase type II gene, these two PCR fragments and the SacI-PstI fragment that contains pUC19 carrier framework and nopaline synthase terminator from pBI221 (Clontech) are assembled together.These segmental assemblings produce pSOG19, wherein contain the 35S promoter that merges with intron 6 sequences, GUS leader sequence, DHFR gene and nopaline synthase terminator.Use from corn chlorotic mottle poison (Maize Chlorotic Mottle Virus, the GUS leader sequence generation carrier pSOG35 among leader sequence displacement pSOG19 MCMV).PSOG19 and pSOG35 carry amicillin resistance pUC gene, and have Hind III, Sph I, Pst I and the EcoR I site that can be used for cloning exogenous array.

Embodiment 11: make up the requirement of expression of plants box

At first will be expected at the gene order of expressing in the transgenic plant and be fitted in the expression cassette, be positioned at after the appropriate promotor and the upstream of appropriate transcription terminator.

Promotor is selected

The promotor that is used for expression cassette is selected to determine genetically modified room and time expression pattern in the transgenic plant.Selected promotor will special cells type (as leaf epidermal cell, mesophyll cell, root tegumental cell) or in particular tissues or organ (for example root, leaf or flower) express transgenic, this selection will reflect the biosynthetic desired location of dna molecular of the present invention.Perhaps, selected promotor can drive the expression of gene under photoinduction or the control of other times adjusting promotor.Selection in addition is that selected promotor is by Chemical Regulation.This just might only bring out when needed by chemical inducer processing causing nucleotide sequence expresses.

Transcription terminator

Can obtain multiple transcription terminator and be used for expression cassette.They are responsible for Transcription Termination outside the transgenosis and correct polyadenylation effect thereof.The known suitable transcription terminator that works in plant all can use, and comprises CaMV 35S terminator, tml terminator, nopaline synthase terminator, pea rbcS E9 terminator.They all can use in monocotyledons and dicotyledons.

Be used to strengthen or regulate the sequence of expressing

Found that many sequences can strengthen the genetic expression in the transcriptional units, these sequences can be used in combination with gene of the present invention to improve their expression in transgenic plant.

Shown that multiple intron sequences can strengthen expression, especially in monocot plant cell.For example, found that corn Adh1 gene intron can significantly strengthen the wild type gene expression under its homologous promoter control when introducing maize cell.Find that introne 1 is effective especially, can strengthen expression (Callis etc., gene development 1:1183-1200 (1987)) with the fusion constructs of chloramphenicol acetyl transferasegene.In the identical experiment system, has similar effect (Callis etc. see above) aspect the expression strengthening from the intron of corn bronze1 gene.Intron sequences by in the conventional introduced plant conversion carrier, generally is incorporated in the untranslated leader.

Known many untranslated leaders from virus can strengthen expression, and they are especially effective in the dicotyledons cell.Specifically, shown from tobacco mosaic virus (TMV) (TMV, ' Ω sequence '), the leader sequence of corn chlorotic mottle poison (MCMV) and alfalfa mosaic virus (AMV) strengthen aspect the expression effectively (as Gallie etc., nucleic acids research 15:8693-8711 (1987); Skuzeski etc., molecular biology of plants 15:65-79 (1990)).

The orientation of gene product in the cell

The known number of mechanisms that exists in plant the gene product orientation, the sequence of controlling these machine-processed functions identifies to a certain degree.For example, gene product is oriented to chloroplast(id) by being found in the aminoterminal signal sequence control of multiple proteins, thereby this signal sequence during chloroplast(id) input, cut produce mature protein (as, Comai etc., journal of biological chemistry 263:15104-15109 (1988)).Thereby can merging with heterologous gene products, these signal sequences make allos product input chloroplast(id) (van den Broeck etc., natural 313:358-363 (1985)).Separable 5 ' the end from the following proteins code cDNA of DNA of coding appropriate signals sequence: RUBISCO protein, CAB protein, epsp synthase, GS2 protein and known locations are in many other protein of chloroplast(id).

Some other gene product be positioned in other organoids such as plastosome and peroxysome (as, Unger etc., molecular biology of plants 13:411-418 (1989)).The cDNA that also can handle these products of coding is to be oriented to heterologous gene products in these organoids.Said sequence example is nuclear coding ATPase and mitochondrial specificity aspartic transaminase isoform.The work that is oriented to the cell protein plastid is described by (institute of NAS report 82:6512-6516 (1985)) such as Roger.

In addition, also identified and make gene product be oriented to the sequence of other cellular compartments.The N-terminal sequence is responsible for being oriented to ER, apoplast and aleurone cell's exocytosis (Koehler ﹠amp; Ho, vegetable cell 2:769-783 (1990)).In addition, amino terminal sequence is with the vacuole orientation (Shinshi etc., molecular biology of plants 14:357-368 (1990)) of the responsible gene product of carboxyl terminal sequence.

By above-mentioned suitable target sequence and purpose transgenic sequence are merged, might instruct transgene product to any organoid or cellular compartment.For example, for the chloroplast(id) orientation, the chloroplast(id) signal sequence frame from RUBISCO gene, CAB gene, epsp synthase gene or GS2 gene as one man can be merged with genetically modified N-terminal ATG.Selected signal sequence should comprise known cleavage site, makes up to cut required any amino acid after syzygy should be taken into account cleavage site.In some cases, can reach this requirement by some amino acid that between cleavage site and transgenosis ATG, add in a small amount of amino acid or the displacement transgenic sequence.External translation by the in-vitro transcription construct and use document subsequently (Bartlett etc. are at the method in Edelmann etc. (editor) the chloroplast(id) molecular biology, Elsevier, pp1081-1091 (1982); Wasmann etc., molecule and General Genetics 205:446-453 (1986)) described technology carries out external chloroplast(id) picked-up, and can detect the chloroplast(id) of the syzygy that makes up for the input chloroplast(id) and take in efficient.These constructing technologies are well-known in the art, and can be applied to plastosome and peroxysome equally.May require to select directed for Pesticidal toxins.Normally kytoplasm or chloroplast(id), although also may be mitochondrial or peroxysome in some cases.The expression of nucleotide sequence also may require to be oriented to ER, apoplast or vacuole.

Above-mentioned cell directional mechanism not only can be united use with its homologous promoter, also can unite use with allogeneic promoter, thereby is issued to the purpose of specific cell orientation in the transcriptional regulatory of the promotor of the promotor tool different expression patterns of originating with directional sign.

Embodiment 12: the example that expression cassette makes up

Regardless of the source of promotor how the present invention is included in any plant effable promotor and regulates dna molecular down and express.In addition, the present invention includes the effable promotor of any plant and dna molecular expression any other sequence association use that requires or select.Said sequence includes, but are not limited to, external sequence (as intron [as the Adh introne 1], virus sequence [as TMV-Ω]) that transcription terminator, enhancing are expressed and the sequence that gene product is directed to specific cell device and cellular compartment.

Constitutive expression: CaMV 35S promoter

The structure of plasmid pCGN1761 is described among the disclosed patent application EP 0392225.PCGN1761 comprises ' two ' 35S promoter and tml transcription terminator, and single EcoR I site is arranged between promotor and the terminator, also comprises pUC-type skeleton.Structure has the pCGN1761 derivative of modifying polylinker, also comprises Not I and Xho I site in this polylinker except that having original EcoR I site.This derivative is called as pCGN1761ENX.For gene is expressed under 35S promoter control in transgenic plant, pCGN1761ENX is used in clone's cDNA sequence or gene order (comprising microorganism ORF sequence) in its polylinker.Can cut out the complete 35S promoter-gene order-tml terminator box of this structure by Xba I, BamH I and Bgl I site, with its conversion carrier that is transferred to as described in example 35 above and so on Hind III, Sph I, Sal I and the Xba I site of promotor 5 ' side and terminator 3 ' side.In addition, in order to use other promoter replacement, available Hind III, Sph I, Sal I, Xba I or Pst I are in 5 ' cutting, with any removes two 35S promoter fragments in 3 ' cutting in the polylinker restriction site (EcoR I, Not I or Xho I).

Modify pCGN1761ENX by optimizing translation initiation site

For the described any structure in this part, can around cloning site, modify by introducing the sequence that can strengthen translation.This point is particularly useful in will be from the gene introduced plant expression cassette of microorganism the time, because these genes may not comprise the sequence that is suitable for translation initiation in the plant at contiguous its methionine(Met) place.If the gene from microorganism will be cloned into the expression of plants box at its ATG place, then modifying its insertion site may be more useful to optimize its expression.As the mode example of introducing one of several expression of plants majorizing sequences, referring to the modification (, seeing above) of pCGN1761ENX as Joshi.

But the expression under the control of chemical regulation type promotor

This part has been described with the dual 35S promoter among selected any promoter replacement pCGN1761ENX; An example of describing is the RP-1a promoter replacement with Chemical Regulation.Selected promotor preferably cuts out from its natural origin by restriction enzyme, but or the primer of also available band appropriate end restriction site carry out pcr amplification.If carry out pcr amplification, then promotor should be checked order again after the amplification promotor is cloned into destination carrier has or not the amplification mistake with inspection.From plasmid pCIB1004 (seeing EP 0332104) about the embodiment 21 that makes up but in cut out the tobacco PR-1a promotor of chemical regulation and it be transferred to plasmid pCGN1761ENX.Cut pCIB1004 with NcoI, and mend the linear fragment 3 ' overhang that produces flat by the processing of T4 archaeal dna polymerase.Cut this fragment with the Hind III then, the fragment gel-purified that contains the PR-1a promotor of generation also is cloned into the pCGN1761ENX that dual 35S promoter has excised.This can contain the fragment greatly that contains carrier one terminator that is cloned into the pCIB1004 promoter fragment with cutting of Hind III and separation and finish subsequently by using Xho I cutting and flat with T4 polysaccharase benefit.So just produced the pCGN1761ENX derivative of the insertion polylinker that contains single EcoR I of PR-1a promotor and tml terminator and tool and Not I site.Dna molecular of the present invention can insert in this carrier, and fusion product (being promotor-gene-terminator) can be transferred in the conversion carrier of any selection subsequently, comprises the carrier described in the application.

Constitutive expression: actin promoter

Known several Actin muscle isotypes are expressed in most of cell type, so actin promoter is the good selection of constitutive promoter.Especially, cloned and identified (McElroy etc., vegetable cell 2:163-171 (1990)) from the promotor of rice Act1 gene.Find the 1.3kb fragment of this promotor, comprised all required regulatory elements of expression in the rice protoplastis.In addition, made up many based on Act I promotor, be specifically designed to monocotyledonous expression vector (McElroy etc., molecule and General Genetics 231:150-160 (1991)).Act1-introne 1, Adh1 5 ' flanking sequence and Adh1-introne 1 (from the maize alcohol dehydrogenase gene) have been mixed in them and from the sequence of CaMV 35S promoter.The carrier that shows high expression level is the syzygy of 35S and Act1 intron or the syzygy of Act1 5 ' flanking sequence and Act1 intron.Initial ATG (the GUS reporter gene) optimization of sequence has on every side also strengthened expression.The promoter expression cassettes that McElroy etc. (molecule and General Genetics 231:150-160 (1991)) describe can easily be modified to be used for the expression of dna molecular of the present invention, and it is particularly useful for the monocotyledons host.For example, the fragment that contains promotor can cut out and be used for replacing two 35S promoters of pCGN1761ENX from the McElroy construct, and this carrier can be used for inserting special gene sequence then.The fusion gene of Gou Jianing can be transferred in the suitable conversion carrier subsequently like this.In indivedual reports, found that also rice Act1 promotor and its first intron can instruct the high expression level (Chibbar etc., Plant CellRep.12:506-509 (1993)) in the artificial culture barley cell.

Constitutive expression: ubiquitin promotor

Ubiquitin is another known gene product that accumulates in many cell types, its promotor is cloned in many species, transgenic plant have been used for (as Sunflower Receptacle-Binet etc., plant science 79:87-94 (1991), corn-Christensen etc., molecular biology of plants 12:619-632 (1989)).Corn ubiquitin promotor has been used in the transgenosis monocotyledons system, its sequence and be disclosed among the patent application EP0342926 for monocotyledons transforms constructed carrier.In addition, Taylor etc. (Plant Cell Rep.12:491-495 (1993)) have described the carrier (pAHC25) that contains the corn ubiquitin promotor and first intron, in many monocot plant cell suspended substances high reactivity are arranged when it is introduced by particle bombardment.The ubiquitin promotor is suitable for dna molecular of the present invention expression in transgenic plant, especially monocotyledons fully.Suitable carriers is the pAHC25 derivative modified by introducing suitable ubiquitin promotor and/or intron sequences or any conversion carrier described in the application.

Root-specific is expressed

The preference pattern that nucleotide sequence of the present invention is expressed is that root is expressed.Root is expressed for the soilborne fungal pathogens of control and is particularly useful.Suitable root promotor is the described promotor of de Framond (FEBS290:103-106 (1991)), in disclosed patent application EP0452269 description is arranged also.This promotor is transferred in the suitable carrier such as pCGN1761ENX, so that insert nucleotide sequence and subsequently complete promotor-gene-terminator box is transferred in the purpose conversion carrier.

Wound-induced type promotor

Wound-induced type promotor is particularly useful for the expression of dna molecular of the present invention, because they generally not only bring out the position in wound activity is arranged, and at the phytopathogen infection site activity is arranged also.Many such promotors are described (as Xu etc., molecular biology of plants 22:573-588 (1993), Logemann etc., vegetable cell 1:151-158 (1989), Rohrmeier ﹠amp; Lehle, molecular biology of plants 22:783-792 (1993), Firek etc., molecular biology of plants 22:129-142 (1993), Warner etc., plant magazine 3:191-201 (1993)) and all be applicable to the present invention.Logemann etc. (seeing above) have described 5 ' upstream sequence of dicotyledonous potato wun1 gene.Xu etc. (seeing above) have shown that the wound-induced type promotor from dicotyledonous potato (pin2) is activated in the unifacial leaf rice.In addition, Rohrmeier ﹠amp; Lehle (seeing above) has described and can and can be used to separate the clone of the corn Wip1 cDNA of homologous promoter by standard technique by wound-induced.Similarly, (seeing above) such as Firek etc. (seeing above) and Warner described the wound-induced type gene from monocotyledons Asparagusofficimalis, and it can be expressed in local wound and pathogenic agent is invaded the site.Use clone technology well-known in the art, these promotors can be transferred in the suitable carrier, merge, and be used for expressing these genes in phytopathogen infection position with dna molecular of the present invention.

The marrow preferred expression

Patent application WO93/07278 (Ciba-Geigy) has described the separation of preferred expression in myelocytic corn trpA gene, and has provided and extend to-1726 promoter gene sequence from transcriptional start point.This promotor or its part can be transferred in the carrier such as pCGN1761 with standard molecular biological technique, therein its replaceable 35S promoter and be used to drive the expression of dna molecular of the present invention with the marrow optimal way.In fact, the fragment that contains marrow preferred promoter or its part can be transferred in any carrier and be modified so that use in transgenic plant.

Pollen specific is expressed

Patent application WO93/07278 (Ciba-Geigy) has further described the separation of corn calcium-dependent protein kinase (CDPK) gene that is expressed in the pollen cell.This gene order and promotor reach 1400bp from the transcriptional start point extension.This promotor or its part can be transferred in the carrier such as pCGN1761 with standard molecular biological technique, therein its replaceable 35S promoter and be used to drive the expression of dna molecular of the present invention.In fact, the fragment that contains pollen specific promoter or its part can be transferred in any carrier and be modified so that use in transgenic plant.

Leaf is specific expressed

The corn gene of coding phosphoric acid enol carboxylase (PEPC) is by Hudspeth; Grula (molecular biology of plants 12:579-589 (1989)) describes.The promotor of this gene can be used to drive any expression of gene in leaf specificity mode in transgenic plant by standard molecular biological technique.

The chloroplast(id) orientation expression

Chen ﹠amp; Jagendorf (journal of biological chemistry 268:2363-2367 (1993)) has described successful Application chloroplast transit peptides input heterologous transgene.Used this peptide is from the transit peptides of the rbcS gene of Nicotiana plumbaginifolia (Poulsen etc., molecule and General Genetics 205:193-200 (1986)).With Restriction Enzyme Dra I and Sph I, or Tsp509I and Sph I can cut out the dna sequence dna of this transit peptides of coding and use with arbitrary above-mentioned construct from plasmid prbcS-8B (Poulsen etc. see above).Dra I-Sph I fragment extends to from-58 sites with respect to initial rbcS ATG, and comprise first amino acid (also being a methionine(Met)) that is next to the mature peptide of input after the cleavage site, and TspS09 I-Sph I fragment extends to from-8 sites with respect to initial rbcS ATG, and comprises first amino acid of mature peptide.Like this, these fragments can be inserted the polylinker place of any selected expression cassette rightly, produce with selected promotor (as 35S, PR-1a, Actin muscle, ubiquitin etc.) untranslated leader merges and transcribe syzygy, make the insertion of dna molecular of the present invention can correctly be blended in the downstream of transit peptides simultaneously.This type of is structured in this area is routinely.For example, Dra I end is a flush end, and that 5 ' Tsp509 I site can handle be mended with the T4 polysaccharase is flat, thereby or can be connected the fusion that promotes it and selected promotor with joint or adapter sequence.3 ' Sph I site can keep equally, or can be connected with joint or adapter sequence promoting it to insert in the selected carrier, thereby provides suitable restriction site for inserting dna molecular of the present invention subsequently.The ideal state is first ATG that keeps the ATG of Sph I and comprise dna molecular of the present invention.Chen ﹠amp; Jagendorf (seeing above) provides consensus sequence for the ideal cut of chloroplast(id) input, and methionine(Met) all is preferably placed at first of mature protein in each example.Can there be more change position subsequently, and possible amino acid is not key like this.In any case all available Bartlett etc. (in the method in Edelmann etc. (editor) the chloroplast(id) molecular biology, Elsevier is among the pp1081-1091 (1982)) and the input efficiency of (molecule and General Genetics 205:446-453 (1986)) external assessment fusion constructs of described method such as Wasmann.In general, the best way may be to set up syzygy with the literalness dna molecular of the present invention of N-terminal, and have only when this syzygy can not efficiently be imported chloroplast(id) and just introduce modification, can modify (Chen ﹠amp by the document that provides in this case; Jagendorf sees above; Wasman etc. see above; Ko﹠amp; Ko, journal of biological chemistry 267:13910-13916 (1992)).

For utilizing other GS2 chloroplast transit peptide-coding sequences, can carry out similar operation by other sources (monocotyledons and dicotyledons) or other genes.In addition, can defer to similar flow process to reach the purpose that is oriented to as other subcellular compartments of plastosome and so on.

Q135 ethyl cyclopropyl Q142 ethyl isopropyl Q135 isopropyl phenyl Q142 ethyl ethyl Q142 ethyl cyclopropyl Q136 isopropyl phenyl Q136 ethyl isopropyl Q143 methyl isopropyl Q136 ethyl cyclopropyl Q143 ethyl ethyl Q136 ethyl isopropyl Q143 isopropyl phenyl Q137 methyl isopropyl Q143 ethyl cyclopropyl Q137 isopropyl phenyl Q143 ethyl ethyl Q137 ethyl isopropyl Q144 methyl isopropyl Q137 ethyl cyclopropyl Q144 isopropyl phenyl Q137 ethyl ethyl Q144 ethyl isopropyl Q138 methyl isopropyl Q144 ethyl cyclopropyl Q138 ethyl ethyl Q144 cumene yl ethyl isopropyl Q138 methyl isopropyl Q145 ethyl cyclopropyl Q138 ethyl ethyl Q145 methyl isopropyl Q139 ethyl isopropyl Q145 ethyl ethyl Q145 Q139 Q139 ethyl ethyl cyclopropylmethyl isopropyl Q145 isopropyl phenyl Q139 ethyl cyclopropyl Q146 methyl isopropyl Q139 of ethyl 2 - chloroethyl Q146 ethyl ethyl Q139 ethyl 2; 2,2 - trifluoroethyl Q146 ethyl isopropyl Q139 2 - Chloro of ethyl 2 - chloroethyl Q146 ethyl cyclopropyl Q139 isopropyl phenyl Q146 isopropyl phenyl Q140 methyl isopropyl Q147 methyl isopropyl Q140 ethyl ethyl Q147 ethyl ethyl Q140 ethyl isopropyl Q141 methyl isopropyl Q147 ethyl isopropyl Q147 ethyl cyclopropyl Q141 isopropyl phenyl Q147 ethyl ethyl Q141 ethyl isopropyl Q148 methyl isopropyl yl Q141 ethyl ethyl Q141 ethyl cyclopropyl Q148 isopropyl phenyl Q148 ethyl isopropyl Q142 methyl isopropyl Q148 ethyl ethyl Q148 ethyl cyclopropyl Q142 isopropyl phenyl...

Claims (28)

1. coding Mlo protein DNA molecule, wherein said Mlo protein comprises listed at least a sequence among SEQ ID NO:1 or the SEQ ID NO:2, and said Mlo protein is given the resistance of plant to fungal pathogens.

2. the dna molecular of claim 1, any listed among wherein said dna molecular and SEQ IDNO:3, SEQ ID NO:5 or SEQ ID NO:7 nucleotide sequence is identical or similar substantially, perhaps identical the or similar substantially Mlo protein of listed Mlo protein among coding and SEQ ID NO:4, SEQ ID NO:6 or the SEQ IDNO:8.

3. the dna molecular of claim 1, any listed among wherein said dna molecular and SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or SEQ IDNO:17 nucleotide sequence is identical or similar substantially, perhaps identical with listed Mlo protein among SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 or the SEQ IDNO:18 or the basic similar Mlo protein of coding.

4. each dna molecular among the claim 1-3, wherein said dna molecular is not to derive from barley.

5. each dna molecular among the claim 1-4, wherein said DNA are modified so that the loss of activity of endogenous protein.

6. the dna molecular of claim 5, wherein said dna modification causes that one of following change, whole or various combination are arranged in the respective egg white matter aminoacid sequence:

-tryptophane (163) is changed into arginine

-proline(Pro) (396) back frameshit

-tryptophane (160) back frameshit

-methionine(Met) (1) is changed into Isoleucine

-glycine (227) is changed into aspartic acid

-methionine(Met) (1) is changed into Xie Ansuan

-arginine (11) is changed into tryptophane

-lose phenylalanine (183), Threonine (184)

-Xie Ansuan (31) is changed into L-glutamic acid

-Serine (32) is changed into phenylalanine

-leucine (271) is changed into Histidine.

With claim 1-6 in the dna molecular of each dna molecular antisense.

8. the protein that comprises one of listed sequence among SEQ ID NO:1 or the SEQ ID NO:2 at least, wherein said protein are Mlo protein and give the resistance of plant to fungal pathogens.

9. the protein of claim 8, wherein said protein is by identical or similar substantially with listed any sequence among SEQ ID NO:3, SEQ ID NO:5 or the SEQ ID NO:7 nucleotide sequence coded, and is perhaps identical or similar substantially with listed any protein among SEQ ID NO:4, SEQ ID NO:6 or the SEQ IDNO:8.

10. the protein of claim 8, wherein said protein is by identical or similar substantially with listed any sequence among SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15 or the SEQ ID NO:17 nucleotide sequence coded, and is perhaps identical or similar substantially with listed any protein among SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16 or the SEQ IDNO:18.

11. each protein among the claim 8-10, wherein said protein is not to derive from barley.

12. contain the expression cassette that right requires each dna molecular among the 1-7.

13. contain the carrier of the expression cassette of the dna molecular that comprises claim 12.

14. comprise that containing right requires the expression cassette of each dna molecular among the 1-7 or the cell of its part, wherein the said dna molecular in this expression cassette is effable in said cell.

15. the cell of claim 14, wherein said dna molecular is stabilized in the genome that is integrated into said cell.

16. each cell in claim 14 or 15, wherein said cell is a vegetable cell.

17. comprise that containing right requires the expression cassette of each dna molecular among the 1-7 or the plant of its part, wherein the said dna molecular in this expression cassette is effable in said plant.

18. the plant of claim 17, wherein said dna molecular is stabilized in the genome that is integrated into this plant.

19. comprise containing the agricultural-food that right requires the plant of each DNA isolation molecule among the 1-7, wherein said agricultural-food have the Plant Quarantine characteristic of improvement.

20. the method for the plant of preparation resistant to fungal pathogens may further comprise the steps:

A) in said plant, express among the claim 1-6 each dna molecular with " justice is arranged " direction; Or

B) in said plant, express among the claim 1-6 each dna molecular with " antisense " direction; Or

C) in said plant, express the special cutting of energy by ribozyme corresponding to the coded messenger RNA(mRNA) transcript of the native gene of each dna molecular among the claim 1-6; Or

D) expression is specific to coded protein or its a part of aptamer of each dna molecular among the claim 1-6 in plant; Or

E) in plant, express the sudden change or the clipped form of each dna molecular among the claim 1-6; Or

F) in plant, modify at least one chromosome copies corresponding to the gene of each dna molecular among the claim 1-6 by homologous recombination.

21. plant with the resistant to fungal pathogens of the method for claim 20 preparation.

22. the plant of claim 21, wherein said fungal pathogens can infect epidermic cell alive.

23. the plant of claim 21, wherein said fungal pathogens is from Erysiphales.

24. the plant of claim 21, wherein said fungal pathogens is from Erysiphe.

25. the plant of claim 21, wherein said fungal pathogens is the standing grain powdery mildew.

26. the tool that obtains with the method for claim 20 improves the agricultural-food of Plant Quarantine characteristic.

27. a method of separating the dna molecular of coding Mlo protein may further comprise the steps:

A) will encode at least 6 amino acid whose degenerate oligonucleotides among the SEQ ID NO:1 and be complementary to the degenerate oligonucleotide and the DNA that extracts from plant of at least 6 amino acid whose sequences among the coding SEQ ID NO:2 mix under said degenerate oligonucleotide and the condition that described DNA is hybridized allowing;

B) amplification said DNA of plants dna fragmentation, wherein said dna fragmentation its a left side and right end contain can with said degenerate oligonucleotide annealed nucleotide sequence in the step a); With

C) acquisition contains the full length cDNA clone of the dna fragmentation of step b).

28. the method for the dna molecular of mutagenesis claim 1, wherein said dna molecular have been cut into the double-stranded random fragment of expection size, this method may further comprise the steps:

A) add one or more strand or double chain oligonucleotide in the double-stranded random fragment group of gained, wherein said oligonucleotide comprises zone identical with the double-stranded template polynucleotide and allogenic zone;

B) the mixture sex change with double-stranded random fragment of gained and oligonucleotide becomes single-chain fragment;

C) causing said single-chain fragment to form under the annealing fragment paired condition in said same area annealing, with gained single-chain fragment group and polysaccharase insulation, wherein said same area is enough to make the member in the pairing to cause duplicating of another member, thereby forms the double-stranded polynucleotide through mutagenesis; With

D) repeat second and at least two circulations of third step again, wherein the mixture that produces of second step in next circulation comprise from last circulation third step through the double-stranded polynucleotide of mutagenesis, and this next circulation has formed the double-stranded polynucleotide of further mutagenesis.