CN1114694C - Procedures and materials for conferring disease resistance in plants - Google Patents

Abstract

Immunostimulating 7,8-disubstituted guanine derivatives that also contain a morpholinyl group as a position-9 substituent are disclosed whose structures are represented by formula I I wherein X is O or S; R1 is a hydrocarbyl or substituted hydrocarbyl group having a length of about one to about seven carbon atoms; R2 is a hydrogen or C1-C8 acyl; R3 is hydrogen, a C1-C5 aliphatic hydrocarbyl, aralkyl or substituted aralkyl group; and the pharmaceutically acceptable base addition salts thereof. Also disclosed are compositions containing an immunostimulating substituted guanine derivative and processes for using the same.

Description

The method of conferring disease resistance in plants and material

This is the U.S. Patent application No.08/567 of the December 4 nineteen ninety-five application of not authorizing together, 375 renewal application, and No.08/567, the 375th, U.S. Provisional Application No.60/004,645 renewal application.The U.S. Patent application No.08/475 that it is not still applied for together the June 7 nineteen ninety-five of authorization, 891 renewal application, and No.08/475, the 891st, the U.S. Patent application No.08/373 of the application on January 17 nineteen ninety-five of not authorizing together, 374 renewal application.These applications are all quoted as a reference at this.

Invention field

The present invention relates generally to molecular biology of plants.Particularly, it relates to the nucleic acid and the method for conferring disease resistance in plants.

Statement to the invention right of under the research and development of government-funded, making

The present invention is under the lot number GM47907 of NIH and USDA lot number No.9300834, finishes under the subsidy of government.There is certain right in government to this invention.

Background of invention

The locus of giving disease resistance is identified in many plant speciess.Interactional genetic analysis between many plant-pathogenic agent is shown plant contains the locus of giving at the concrete kind of pathogenic agent (containing the complementary nontoxic gene) resistance.The qualitative means of giving various different crops disease resistances that provide to the molecular level of these genes.

Those molecular level qualitatively resistant gene in plant be divided into 4 classes.A kind of gene, the Hml in the corn, a kind of reductase enzyme of encoding, it to fungal pathogens Cochliobolus Carbonum effectively (people such as Johal, Science, 258:985-987 (1992)).In tomato, the Pto gene is given the resistance to the pseudomonas syringae (Pseudomonas syringae) of expressing the avrPto nontoxic gene, (people such as Martin, Science, 262:1432 (1993)).The Pto genes encoding serine threonine protein kinase of estimating.Tomato Cf-9 gene give to the resistance of the dark yellow branch of the fungi of carrying nontoxic gene Avr9 spore (Cladosporium fulvum) kind (people such as Jones, Science, 266:789-793 (1994)).The outer LRR albumen of the born of the same parents that tomato Cf-9 genes encoding is inferred.At last, the RPS2 gene of Arabidopis thaliana (Arabidopsis thaliana) give to the resistance of the pseudomonas syringae of expressing the avrRpt2 nontoxic gene (people such as Bent, Science, 265:1856-1860 (1994)).The protein that the RPs2 coding has LRR motif and P-cyclic group preface.

The bacterium blight that is caused by xanthomonas (Xanthomonas spp.) influences nearly all farm crop, and causes crop loss widely in the world wide.Rice (Oryza sativa) the bacterium blight that is caused by rice Xanthomonas campestris (Xanthomonas oryzae pv.oryzae (Xoo)) is the principal disease of this crop.Identified the Xoo kind of in having distinguished resistance (Xa) trans-genetic hybrid rice Cultivar induction of resistance or susceptibility reaction.A kind of resistance source (Xa21) in rice (Oryza longistaminata) wild species, identify (people such as Khush, Proceedings of the International Workshop on Bacterial Blight of RicePeople such as (International Rice Research Institue, 1989) and Ikeda, Jpn J.Breed40 (Suppl.1): 280-281 (1990)).Xa21 is a dominance resistant gene seat, and it gives the resistance to all known Xoo strain isolateds, and unique be the Xa gene that carries Xoo 6 resistances qualitatively.To the heredity of Xa21 locus and physical analysis identified in a large number closely linked mark on No. 11 karyomit(e) (people such as Ronald, Mol.Gen. Genet.236:113-120 (1992)).Yet Xa21 gives the molecular mechanism of this pathogen resistance not clear.

In order to clone the plant gene of giving various different bacterium, fungi and virus disease resistance, carried out suitable effort.Only there is a kind of pest resistance genes to be cloned in the monocotyledons.Because monocot crops is supported most in the world humans and animals, so the disease resistence gene that identifies in these plants is a particularly important.The present invention is directed to these and other demand.

Summary of the invention

The invention provides isolating nucleic acid construct thing, it contains RRK polymerized nucleoside acid sequence, and this sequence is hybridized with SEQ.ID.No.1 or SEQ.ID.No.3 under stringent condition.Representational RRK polymerized nucleoside acid sequence is the Xa21 sequence of the Xa21 polypeptide of coding as shown in SEQ.ID.No.4.RRK polynucleotide encoded protein matter is rich in leucic repetition motif and/or cytoplasm protein kinase domain.Nucleic acid construct thing of the present invention also contains the promotor that operationally is connected in RRK polymerized nucleoside acid sequence.Promotor can be organizing specific type promotor or constitutive promoter.

The present invention also provides a kind of nucleic acid construct thing, it contain be connected in allos polymerized nucleoside acid sequence, from the promoter sequence of RRK gene.Representational allos polymerized nucleoside acid sequence comprises gives the structure gene of plant to pathogen resistance.

The present invention also provides a kind of transgenic plant that contain the expression cassette of reorganization, this expression cassette contain operationally be connected in the polymerized nucleoside acid sequence, from the promotor of RRK gene; And a kind of transgenic plant that contain recombinant expression cassettes, this expression cassette contains the promotor of the plant that operationally is connected in RRK polymerized nucleoside acid sequence.Although any plant all can be used for the present invention, what conveniently use is paddy rice and tomato.

The present invention also provides and has strengthened the method for plant to the Xanthomonas campestris resistance.This method comprises: with the expression cassette introduced plant of reorganization, this expression cassette contains the plant promoter that operationally is connected in RRK polymerized nucleoside acid sequence.This method can be advantageously used in paddy rice and tomato plants.

Definition

Term " plant " comprises whole plant, plant organ (as leaf, stem, root etc.), seed and vegetable cell and their filial generation.The kind that can be used for the plant of the inventive method generally can be wide to the higher plant type that can carry out transformation technology, comprises unifacial leaf and dicotyledons.

" heterologous sequence " is from sequence not of the same race, if perhaps from a kind of then be to its original form sequence through fully modifying.For example, the promotor that operationally is connected in allos structure gene can be the kind that obtains this structure gene from being different from, and perhaps, if from a kind of, then one of them or both have carried out sufficient modification to their original form.

" RRK gene " be in the new disease resistence gene of a class of the coding RRK polypeptide that contains the outer LRR structural domain of born of the same parents, membrane spaning domain and cytoplasmic protein kinase domain the member (as for example as shown in RLK5, Pto and the Fen (people such as Martin, Vegetable cell6:1543-1552 (1994)).As used herein, the LRR structural domain is as shown in Figure 1 and the zone of 24 the residue repeating units of having an appointment found in Cf-9 and RLK5.)。With the nucleic acid hybridization and/or the amplification technique of sequence disclosed herein and standard, the technician can identify the member of this genoid.For example, can detect in 58 paddy rice recombinant inbred strains and the isolating polymorphism of blast (Pyricularia oryzae) resistant gene (Pi7) from the nucleic acid probe of Xa21 gene.Identical probe also can detect polymorphism in the near homogenic system of carrying xa5 and Xa10 resistant gene.

In some preferred example, the member of this class disease resistence gene can by they can by degeneracy, amplify corresponding to the PCR primer of LRR and kinase domain and to identify.For example, used primer to separate homologous gene in the tomato.The representational primer that is used for this purpose is tcaag caaca atttg tcagg nca a/gat a/c/t cc (for LRR structural domain sequence GQIP) and taaca gcaca ttgct tgatt tnan g/a tcncg g/atg (kinase domain sequence HCDIK).

" Xa21 polymerized nucleoside acid sequence " is the subsequence or the total length polymerized nucleoside acid sequence of Xa21 gene such as paddy rice Xa21 gene, can will give plant to the resistance of xanthomonas (as X.oryzae) when it is present in the transgenic plant.Representational polynucleotide of the present invention comprises the coding region of SEQ.ID.No.3.The Xa21 polynucleotide is typically long at least about 3100-6500 Nucleotide, is generally about 4000-4500 Nucleotide.

" Xa21 polypeptide " is the gene product of Xa21 polymerized nucleoside acid sequence, and it has the activity of Xa21, promptly can give the resistance to xanthomonas.The same with other RRK polypeptide, the feature of Xa21 polypeptide is to exist to contain to be rich in leucic repeated fragment (leucine rich repeats, ectodomain LRR) and/or cytoplasmic protein kinase domain.Representational Xa21 polypeptide of the present invention is included among the SEQ.ID.No.4.

In genetically modified expression, the technician will appreciate that the polymerized nucleoside acid sequence of insertion needn't be identical, and can be and the gene order " basic identical " that originates from.As described below, these varients are specifically contained by this term.

Thereby transcribed and translate under the situation that produces functional r RK polypeptide at the polymerized nucleoside acid sequence that inserts, the technician will appreciate that, because the degeneracy of codon, a large amount of polymerized nucleoside acid sequences identical polypeptide of can encoding is arranged.These varients are all specifically contained by term " RRK polymerized nucleoside acid sequence ".In addition, term specifically comprised total length, with the sequence of RRK gene order basic identical (method of determining is as described below), and the proteinic sequence of the reservation RRK protein function of encoding out.Therefore, under the situation of disclosed herein paddy rice RRK gene, above-mentioned term comprises such variation polymerized nucleoside acid sequence, and basic identical and encoded protein mass-energy enough makes the transgenic plant that contain this sequence that the resistance of anti-Xanthomonas campestris or other plant disease or insect is arranged to these sequences with disclosed sequence herein.

When arranging by following maximum correspondence, if Nucleotide or amino acid residue sequence in two sequences are respectively identical, these two polynucleotides or polypeptide just be known as " identical " so.Term " is complementary to " and is used to herein refer to that complementary sequence is identical with all or part of of polymerized nucleoside acid sequence of reference.

Sequence between two (or a plurality of) polynucleotides or polypeptide more generally is performed such, and promptly compares the order of two sequences in a fragment or " comparison window ", to differentiate or to compare the sequence similarity of regional area.The fragment that is used for the comparison purpose is at least about 20 continuous positions, be generally about 50-200, more commonly be about 100-150, wherein after two sequences press the best mode arrangement, with this sequence and only have the canonical sequence of the continuous position of similar number to compare.

For comparing, the carrying out of the optimal arrangement of sequence can be used Smith and Waterman Senior applied mathematics (Adv.Appl.Math.)Local homology's algorithm of 2:482 (1981), Needleman and Wunsch Molecule Biology magazine (J.Mol.Biol.)The homology algorithm of 48:443 (1970), Person and Lipman U.S. section Institute of institute newspaper( Proc.Natl.Acad.Sci.)(U.S.A.) the similarity searching method of 85:2444 (1988), or finish these algorithms (GAP with computer, BESTFIT, FASTA, and TFASTA, in WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI), perhaps by range estimation.

" sequence same percentage " is definite by the sequence that compares two optimal arrangement in comparison window, wherein, the polymerized nucleoside acid sequence in comparison window part can comprise for two sequences of optimal arrangement and interpolation or the disappearance (as breach) of control sequence (it does not contain insert or disappearance) when comparing.Per-cent is to calculate like this: determine the nucleic acid base identical in two sequences or the position number of amino-acid residue, with the sum of the position number that mates divided by position in the comparison window, then the result be multiply by 100, obtain the sequence same percentage.

" basic identical " of term polynucleotide refers to canonical parameter and above-mentioned program (preferred BESTFIT) with canonical sequence relatively the time, the sequence that the polymerized nucleoside acid sequence contains has at least 60% sequence identical, preferably at least 80% sequence is identical, more preferably at least 90% sequence is identical, and at least 95% sequence is identical best.It will be understood by those skilled in the art that factors such as degeneracy by considering codon, amino acid similarity, reading frame position, can carry out suitable adjustment to these numerical value, to determine by two proteinic homogenies that nucleotide sequence is coded.For these purposes, the basic identical of aminoacid sequence refers generally at least 40%, and preferably at least 60%, more preferably at least 90%, at least 95% sequence is identical best.The polypeptide of " similar substantially " has aforesaid sequence, except replacing with the conservative amino acid change residue position inequality.Conservative amino acid is replaced the phase trans-substitution of the residue refer to have similar side chain.For example, one group of amino acid with aliphatic lateral chain is glycine, L-Ala, Xie Ansuan, leucine, Isoleucine; One group of amino acid with aliphatic hydroxide radical side chain is Serine, Threonine; One group of amino acid with amide side chains is l-asparagine and glutamine; One group of amino acid with aromatic side chains is phenylalanine, tyrosine, tryptophane; One group of amino acid with basic side chain is Methionin, arginine and Histidine; And one group of amino acid with sulfur-containing side chain is halfcystine and methionine(Met).Preferred conservative amino acid set of permutations is: Val-Leu-Isoleucine, phenylalanine-tyrosine, Methionin-arginine, L-Ala-Xie Ansuan and l-asparagine-glutamine.

The essentially identical sign of another nucleotide sequence is that two molecules can be in phase mutual cross under the appropriate condition.Appropriate condition can be high tight or low stringent condition, and is different under different situations.Generally, the optional selection of bit sequencing of stringent condition be listed in low about 5-20 ℃ of fixed ionic strength and the pyrolysis chain temperature (Tm) under the pH.Temperature when Tm is about 50% target sequence with the probe hybridization of coupling fully (under the fixed ionic strength and pH).Generally, tight wash conditions is at pH7, and salt concn is that about 0.02 mole and temperature are at least about 60 ℃.Yet the nucleic acid of phase mutual cross still may not be essentially identical under stringent condition, if their encoded polypeptides are essentially identical.This can take place when for example producing the nucleic acid copy with the maximum codon degeneracy that genetic code allowed.For Southern hybridization, high tight wash conditions can comprise at least washs once in 0.1 * SSC at 65 ℃.

The available standards program also is used as probe with nucleic acid disclosed herein (as SEQ.ID.No.1 or 3), identifies nucleic acid of the present invention from cDNA or genomic library.Low tight hybridization conditions typically is included in 65 ℃ with 2 * SSC washing at least once.Preferably wash with 1 * SSC after the washing at 65 ℃.

As used herein, the homologue of specific RRK gene (paddy rice Xa21 gene as disclosed herein) is second kind of encoding amino acid sequence has at least 25% identical or proteinic gene (in same species or different plant species) of 45% similar (as above determining) with the peptide sequence of first kind of gene product.Believe that generally homologue has common evolution origin.

The accompanying drawing summary

Fig. 1 is the proteinic comparison of being rich in the leucine repeated fragment.

Fig. 2 A-F shown contain can with the BAC in the zone of Xa21-specific probe hybridization and clay clone's part restriction map.

Fig. 3 has shown the restriction map of pB822 (most active copy).

Fig. 4 has shown in the transgenic plant that contain the Xa21 gene of cloning from pB822, the test result of measurement Xanthomonas campestris resistance.

Fig. 5 has shown the site plan of TRK1.

Fig. 6 has shown the site plan of TRL1.

The description of preferred embodiment

The present invention relates to plant RRK gene, as the Xa21 gene of paddy rice.From the RRK gene especially nucleotide sequence of Xa21 gene, can be used in plant, give resistance to Xanthomonas campestris or other pathogenic agent.The present invention is used in all higher plants that can be subjected to pathogenic infection and gives resistance.Therefore, the present invention can be used for the vegetation type of a large amount of different range, comprise kind: white walnut (Juglans) from following dependent of dead military hero, Fragaria (Fragaria), Lotus (Lotus), Medicago (Medicago), donkey Macroptilium (Onobrychis), Clover (Trigonella), Semen Trigonellae belongs to (Trigonella), Vigna (Vigna), both citrus (Citrus), linum (Linum), Geranium (Geranium), cassava (Manihot), Daucus (Daucus), Arabidopsis (Arabidopsis), Btassica (Brassica), Rhaphanus (Raphanus), the Europe sinapsis alba belongs to (Sinapis), Atropa (Atropa), Capsicum (Capsicum), Datura (Datrua), poison tobacco (Hyoscyamus), tomato belongs to (Lycopersicon), Nicotiana (Nicotiana), Solanum (Solanum), green winter Solanum (Petunia), Digitalis (Digitalis), Majorana, Cichorium (Ciahorium), Helianthus (Helianthus), Lactuca (Lactuca), Brome (Bromus), Asparagus (Asparagus), antirrhinum (Antirrhinum), Heterocallis, Nemesis, Pelargonium (Pelargonium), Panicum (Panieum), Pennisetum (Pennisetum), Ranunculus (Ranunculus), Senecio (Senecio), (Salpiglossis), Cucumis (Cucumis), Browaalia, Glycine (Glycine), Pisum (Pisum), Phaseolus (Phaseolus), lolium (Lolium), Zea (Zea), Avena (Avena), Hordeum (Hordeum), Secale (Secale), Trisetum (Triticum) and sorghum (Sorghum).

Following description is the universal method of representational separation Xa21 gene and other RRK genes to the separation of Xa21 gene in the paddy rice and the embodiment chapters and sections of qualitative process.Isolating gene can be used for making up recombinant vectors then, so that give transgenic plant with RRK genetic expression.

Generally, term in following recombinant DNA technology and experimental arrangement be know in this area with normally used.Use standard technique for clone, DNA isolation and RNA, amplification and purifying.Generally, the enzyme reaction that relates to dna ligase, archaeal dna polymerase, restriction enzyme etc. is carried out according to the explanation of manufacturers.These technology and various other technologies are generally according to people's such as Sambrook " molecular cloning-laboratory manual ", ColdSpring Harbor Laboratory, and Cold Spring Harbor, New York, carry out (1989).

Xa21 and separating of relevant RRK gene can finish with multiple technologies.For example, can use based on the oligonucleotide probe of sequence disclosed herein and differentiate gene required in cDNA or the genome dna library.In order to make up genomic library, can produce the big fragment of genomic dna by random fracture (as passing through restriction enzyme), form the concatermer that can be wrapped in the suitable carrier thereby be connected with carrier DNA then.In order to prepare the cDNA library, from required organ such as leaf, isolate mRNA, contain the CDNA library of RRK genetic transcription thing then with the mRNA preparation.Perhaps, can be from its hetero-organization of expressing RRK gene or homologue extracting and mRNA prepare cDNA.

Then, use the RRK gene probe of paddy rice Xa21 gene as disclosed herein, screen cDNA or genomic library based on the clone.Available probe and genomic dna or cDNA sequence hybridization are to isolate the homologous gene in the identical or different plant species.

Perhaps, available amplification technique amplifies interested nucleic acid from nucleic acid samples.For example, from genomic dna, cDNA, genomic library or cDNA library, directly amplify the sequence of RRK and related gene with polymerase chain reaction (PCR) technology.PCR and other amplification in vitro method also can be used for, and for example, clones coding is treated the nucleotide sequence of expressed protein, and preparation is used as nucleic acid, nucleic acid sequencing or other purposes of the probe that whether the required mRNA of detection exists in sample.

Be used for identifying the suitable primer and the probe of RRK sequence, can draw by sequence more provided herein from tissue.For the overall review of PCR, can referring to PCR Protocols:A Guide to Methods and Applications.(Innis, M, Gelfand, D., Sninsky, J. and White, T. edits), AcademicPress, San Diego (1990), the document is incorporated herein by reference.

The also available technology of knowing is as the technology described in scientific and technical literature, synthetic polynucleotide.Referring to people such as for example Carruthers, Cold Spring Harbor Symp.Quant.Biol.People such as 47:411-418 (1982) and Adams, J.Am.Chem.Soc.105:661 (1983).Then or by synthetic complementary strand, under appropriate condition, with chain annealing together, perhaps add complementary strand by archaeal dna polymerase with suitable primer sequence, can obtain double chain DNA fragment.

Then, the separation sequence for preparing as describe herein is used in and provides RRK genetic expression in the required plant, thereby the Xanthomonas campestris resistance is provided.The technician will appreciate that, coding has the proteic nucleic acid of RRK (as SEQ.ID.No.2 and 4) of function needn't have and the identical sequence of disclosed representative gene herein.In addition, the same with other protein, have the different structure territory of carrying out difference in functionality by the polypeptide of RRK genes encoding.Therefore, the RRK gene order needs not to be total length, as long as required protein function structural domain is expressed.As described below protein of the present invention contains the leucic repeated fragment structural domain that is rich in outside the born of the same parents like that, and the interior kinase domain of born of the same parents.Available various recombinant DNA technologies well known to those skilled in the art are designed the protein chain of modification easily.For example, can make chain be different from naturally occurring sequence in the primary structure level by amino-acid substitution, insertion, disappearance etc.Modify and also can comprise, will exchange from proteinic structural domain of the present invention and dependency structure territory from other pest resistance genes.For example, the proteinic ectodomain of the present invention (comprising the zone of being rich in the leucine repeated fragment) can be replaced by the ectodomain of tomato Cf-9 gene, thereby the resistance to the rice fungus diseases substance is provided.These modified forms array configuration in a large number use, thereby produce the protein chain of final modification.

In order to use isolating RRK sequence in above-mentioned technology, preparation is fit to the recombinant DNA carrier of transformed plant cells.The technology that is used to transform various higher plant kind is well-known, and is described in technology and the scientific literature.For example, referring to people such as Weising, Ann.Rev.Genet.22:421-477 (1988).

The encode dna sequence dna of required RRK polypeptide, the cDNA or the genome sequence of the full length protein of for example encoding can be used for making up the recombinant expression cassettes that can introduce required plant.An expression cassette typically comprises the RRK polynucleotide, and this polynucleotide operationally is connected in transcribing of instructing that the RRK gene order transcribes in transforming the required tissue of plant and translation initiation is regulated sequence.

For example, can adopt can regeneration plant in a organized way middle finger lead the plant promoter fragment that RRK expresses.This promotor is called as " composing type " promotor herein, and it enlivens under the stage in most envrionment conditionss and growth or cytodifferentiation.The example of constitutive promoter comprises cauliflower mosaic virus (CaMV) 35S transcription initiation zone, from 1 of the T-DNA of Agrobacterium tumefaciems (Agrobacterium tumafaciens) ' or 2 ' promotor and other transcription initiation zones from various different plant genes known to the skilled.

Perhaps, plant promoter can instruct the expression of RRK gene in particular organization, perhaps is in more accurate environment or grows control down.This promotor is called as " induction type " promotor herein.Can influence the envrionment conditions that inducible promoter transcribes comprises pathogenic agent invasion and attack, anaerobic condition or has light.

Be in the example of growing control promotor down only comprise some tissue as leaf, root, really, seed or spend in the initiation promotor of transcribing.The operation of promotor also can be different, and this depends on its position in genome.Therefore, inducible promoter is all or part of composing type in some position.

Endogenesis promoter from RRK gene of the present invention can be used for instructing expression of gene.These promotors also can be used for the expression of directing heterologous structure gene.Therefore, promotor can be used in the recombinant expression cassettes driving expression of gene, and this gene can be given the resistance that any pathogenic agent is comprised fungi, bacterium etc.

Whether in order to differentiate promotor, analyzing clone's described herein 5 ' part has the sequence signature of promoter sequence.For example, the promoter sequence element comprises TATA box consensus sequence (TATAAT), and it is usually located at 20-30 base pair place, transcription initiation site upstream.In plant, in the more upstream of TATA box, typically there is a promoter element at-80 to-100 places in the position, and it has a series of VITAMIN B4 round three polynucleotide G (or T) NG.People such as J.Messing, Genetically engineered plant (Genetic Engineering in Plants),Pp.221-227 (Kosage, Meredith and Hollaender edit, and 1983).

Suitable if desired expression of polypeptides should be introduced the polyadenylation zone at 3 of RRK coding region ' end so.The polyadenylation zone can be from natural gene, or from various other plant gene, or from T-DNA.

The carrier that contains from the RRK gene order typically contains marker gene, and this marker gene makes vegetable cell that selectable phenotype be arranged.For example, the marker gene biocide resistance of can encoding, especially to microbiotic as resistance to kantlex, G418, bleomycin, Totomycin, to the resistance of weedicide as resistance to chlorosluforon or Basta.

These DNA constructions can be introduced in the genome of required plant host with various routine techniquess.For example, plant protoplast or embryo callus are used such as technology such as electroporation, PEG perforation, partickle bombardment and microinjections, the DNA construction directly can be imported the genomic dna of vegetable cell, perhaps use impact method such as DNA partickle bombardment, can be with the direct introduced plant tissue of DNA construction.Perhaps, the DNA construction can merge with suitable T-DNA flank region, imports in the conventional Agrobacterium tumefaciems host carrier then.When cell during by infectation of bacteria, Agrobacterium tumefaciems host's virulence function can instruct construction and adjacent mark to be inserted in the plant cell dna.

Transformation technology is to know in this area, and sufficient description is arranged in science and patent documentation.The method of introducing the DNA construction with polyoxyethylene glycol is described in people such as Paszkowski, Europe molecular biology association magazine (Embo J.)2717-2722 (1984).Electroporation technology is described in people such as Fromm, Institute of American Academy of Sciences newspaper, 82:5824 (1985).The bombardment transformation technology is described in people such as Klein, Nature, 327:70-73 (1987).Use big metering method, can transform cereal kind such as rye (people such as de la Pena, Nature, 325:274-276 (1987)), corn (people such as Rhodes, Science, 240:204-207 (1988)) and paddy rice (people such as Shimamoto, Nature, 338:274-276 (1989) uses electroporation; People such as Li, Plant Cell Rep.12:250-255 (1993) uses the bombardment technology).

The transformation technology of Agrobacterium tumefaciems mediation is fully described in scientific literature.Referring to people such as for example Horsch, Science, people such as 233:496-498 (1984) and Fraley, Institute of American Academy of Sciences newspaper, 80:4803 (1983).Although edaphic bacillus is mainly used in dicotyledonous, the also available edaphic bacillus of some monocotyledons transforms.People such as Hiei for example, Plant J.Described among the 6:271-282 (1994) and used the edaphic bacillus rice transformation.

With above-mentioned any transformation technology derive and the plant transformed cell, can be cultured to the complete plant of regeneration, this plant has the genotype of conversion, thereby has the phenotype of required RRK control.This regeneration techniques depends on certain plants functions of hormones in the tissue culture growth substratum, typically depends on RRK nucleotide sequence involved biocide and/or weedicide mark.Be described in people such as Evan from the protoplast regeneration plant of cultivating, Protoplasts Isolation and Culture, Handbook of Plant Cell Culture, pp.124-176, MacMillilan Publishing Company, New York, 1983; And Binding, Regeneration of Plants, Plant Protoplasts,Pp.21-73, CRC Press, Boca Raton, 1985.Also can regenerate from plant callus, explant, organ or its part.This regeneration techniques has comprehensive description people such as Klee among the Ann.Rev.Of Plant Phys.38:467-486 (1987).

The inventive method is specially adapted to the RRK polynucleotide to introduce in the plant transformed under the mode that do not have at nature and the situation.Particularly, the RRK polynucleotide can be different from the feature of natural phant and express often or in large quantities.

The technician recognizes that also after expression cassette stably was introduced into transgenic plant and confirms to operate, it can be introduced into other plant by sexual hybridization.Can use any technology in a large amount of standard hybridization techniques, this depends on kind to be hybridized.

The modification effect of RRK genetic expression can increase or reduce and determine by detecting in the mRNA level, for example by the Northern blotting.In addition, the phenotype effect of genetic expression can detect by the lesion length of measuring in the plant.The analytical procedure of suitable definite resistance is described below.

Provide the following example in the elaboration mode, they do not provide constraints.

Embodiment 1

Also can use and come the separating plant gene based on the cloning of collection of illustrative plates.This method comprises identifying with the closely linked dna marker of target gene.The requirement that big chromosomal region is carried out succeeing based on the clone and the physical analysis of collection of illustrative plates is to obtain containing the segmental gene library of the big insertion of genomic dna.Recently, Shizuya.H. etc. have described a kind of bacterial artificial chromosome (BAC) system at newspaper (Proc.Natl.Acad.Sci.) 89 of institute of American Academy of Sciences among the 8794-8797 (1992), and the big fragment of genomic dna is used to clone people.This system uses the carrier based on F-factor, can comprise the human genome DNA greater than 300kb.DNA can high-efficient cloning in intestinal bacteria (E.coli.), easy and simple to handle, preserve stable.Hereinafter be to utilize of the explanation of this technical point from gene of the present invention.

Carry the BAC of Xa21 correlated series and separating of clay clone

The preparation of BAC clone A. materials and methods paddy rice high-molecular-weight DNA

With the paddy rice system of international paddy rice research association (IRRI), the IR-BB21 that carries Xa-21 is as vegetable material.Plant grew in greenhouse for 3 to 5 weeks.Collect leaf texture, use distilled water wash, pulverize then.According to Hatano, S. wait at (plant science magazine) (Plant Scince), 83,55-64 (1992) and Zhang, people such as H.B. are in plant (Plant), 7, described in the 175-184 (1994), carry out following modification, from rice tissue, extract high-molecular-weight DNA: in liquid nitrogen, with cold mortar and pestle will be about 20g leaf texture grinding powder.The powder stirring suspension is extracted (NE) damping fluid (1MM spermidine, 1mM spermine, 10mM Na at the cold nuclear of 200ml ₂EDTA, 10mm Trizma alkali, 80mM KCl, 0.15%Triton-X100 and 0.4M sucrose, pH9.4) in.This mixture is filled in the GSA bottle by two-layer cheesecloth, 4 ℃ with 1200g centrifugal 20 minutes.Abandoning supernatant is resuspended in nucleus precipitation (light green) in the cold NE damping fluid of 50ml.Precipitation after resuspended is filled in the 50ml test tube to remove green fragment of tissue through 80 microns sieve, with 1000g centrifugal 10 minutes then.To precipitate resuspendedly and centrifugal as mentioned above once more, but filter without 80 microns sieve.Nucleus precipitation (about 5 * 10 ⁸Individual/ml) be resuspended in 2.5ml SCE damping fluid (the 1M sorbyl alcohol, the 0.1M Trisodium Citrate, 60mM EDTA, pH7.0) in, imbed in 2.5ml 1% low melting point (LMP) agarose (Ultrapure).The piece of 80 μ l was cultivated 2 days in 50 ℃ at the ESP of 25ml solution (0.5M EDTA, pH9.3,1% sarcosyl, 5mg/ml Proteinase K, Boehringer Mannheim), during change a damping fluid.Contain 5 μ g DNA in each piece.The part of high-molecular-weight DNA digests and utilizes the size separation of PFGE

The agar sugar is at 50 ℃ of TE (10mm Tris-HCl and 1mM EDTA to being added with 1mm PMSF (phenyl methyl fluorosulfonyl); PH8.0) carry out twice dialysis of 1 hour; at room temperature use HindIII damping fluid (50mM NaCl, 10mM Tris-HCl, 10mM MgCl then ₂With the 1mM dithioerythritol, pH7.9) carry out 1 hour balance twice.Piece is melted with 15 minutes at 65 ℃, 37 ℃ of insulations 5 minutes, part digestion then.(NEB USA), was hatched 30 minutes at 37 ℃ to add 5 HindIII to 7 units/piece in dna solution.The 0.5%EDTA termination reaction that adds 1/10 volume pH8.0.With the sharp mouth transfer pipet that cuts into the 2mm internal diameter the postdigestive DNA of part is added in the 0.8%LMP agarose at once, utilize PFGE (CHEF DR II system, BioRad USA) to separate.Used two kinds of different PFGE methods to make up the library.First kind, gel is at the 150V electrophoresis, and the initial sum of using 8 seconds stops switching time, carries out 16 hours at 14 ℃.Unsegregated DNA (〉=200kb) be concentrated into a fillet band.Second kind, gel is at the 150V electrophoresis, and increased to 90 second by 60 seconds switching time, carried out 16 hours at 14 ℃.The gel that contains the DNA of part digestion in two kinds of methods is downcut, be immersed among the TE, use the marker swimming lane of ethidium bromide staining gel simultaneously.From gel, cut and contain greater than 200kb fragment (first kind of PFGE method) or contain the agarose bar of 250-350kb fragment (second method).The agarose bar is put into the test tube of 1.5ml in 4 ℃ of balances 2 hours in TE, and 60 ℃ of fusings 10 minutes, (Epicenter, USA) (every 100g agarose adds 1 unit enzyme) digestion was hatched 1 hour at 45 ℃ with gel enzyme (Gelase).This dna solution is directly used in ligation.The preparation of carrier with separate and ligation

Carrier pBeloBAC II, (California Institute ofTechnology USA) provides to win soil by H.Shizuya and M.Simon.This carrier has inserted the lacz gene in pBAC108L.Shizuya etc. (1992).A single colony inoculation is contained to 5ml in the LB substratum of 12.5 μ g/ml paraxin,, add 6 milliliters of LB substratum then 37 ℃ of growths 4 to 5 hours.Inoculum is about 16 hours of 37 ℃ of growths, until OD _600nmReach 1.3-1.5.Plasmid maximum separation test kit (Qiagen, USA) separation quality grain with Qiagen.Carrier DNA is by 45, and the cesium chloride of 000RPM/ethidium bromide equilibrium centrifugation carried out purifying in 60 hours.(Beckman USA), is decelerated to 35 with rotor, and centrifugal 1 hour of 000RPM makes gradient loose to use fixed angle rotor 70.1.With HindIII fully, analyze with gel electrophoresis plasmid digestion.Carrier is terminal, and (Epicenter, USA) 30 ℃ of dephosphorylations 1 hour, per 1 μ g carrier DNA used the enzyme of 1 unit with the HK Phosphoric acid esterase.Made HK Phosphoric acid esterase inactivation in 30 minutes 65 ℃ of heating.Carry out ligation in 100 μ l, (NEB, the paddy DNA of USA) will about 40ng selecting through size (about 85 μ l) are connected (mol ratio about 10 to 1, carrier is excessive) with the carrier (1 μ l) that 10ng HindIII digests wherein to use the T4 dna ligase of 400 units.Before the conversion, ULTRAFREE-MC filter test tube (Millipore, USA) in, ligation liquid with TE in 4 ℃ of dialysis all night.BAC transforms

According to following setting, utilize Cell-Porator (GIBCO-BRL, USA) with the intestinal bacteria DH10B cell of electroporation transformed competence colibacillus (GIBCOBRL, USA): voltage: 400; Electric charge speed: fast; Add compressive resistance: 4,000; Electric capacity: 300 μ; Resistance: low.Electroporation mixes 13 μ l competent cells with 0.5-1.0 μ l ligation liquid each time.Behind the electroporation, cell transfer is arrived 1ml SOC solution (2% bacteriotrypsin peptolysis, 0.5% bacterium yeast extract, 10MM NaCl, 2.5mM KCl, 10mM MgCL ₂, 10mM MgSO ₄, 20mM glucose, pH7.0) in, cultivated 45 minutes in 37 ℃ of slight vibrations (90-95RPM).Cell is dispersed in the LB culture plate that contains paraxin (12.5 μ g/ml), X-gal (40 μ g/ml) and IPTG (sec.-propyl sulphur-β-D-galactoside) (0.072 μ g/ml).Plate was cultivated 24 hours at 37 ℃.To contain paddy DNA and insert segmental white colony and choose on the fresh LB culture plate, carry out the dithering second time.With BAC clone transfer to 384 lattice droplet plates (Genetix, UK) on, contain the freezing damping fluid of 60 μ l LB (36mM K in the lattice ₂HPO ₄, 13.2mM KH ₂PO ₄, 1.7mM citric acid, 0.4MM MgSO ₄, 6.SmM (NH ₄) ₂SO ₄, 4.4%v/v glycerine, 12.5 μ g/ml paraxin LB), were cultivated 24 hours at 37 ℃.Because the bacterium colony more than 95% still is white when programmed screening, only once screens in following experiment, directly white colony is chosen on the 384 lattice droplet plates.The library is duplicated twice, be kept in two different-80 ℃ of refrigerators.The preparation of filter membrane

BAC bacterium colony in each lattice on the 384 lattice droplet plates is all copied to Hybond N ⁺Filter membrane (Amersham, USA) on.Filter membrane is placed in the plastics casing that contains LB/ agarose and 12.5 μ g/ml paraxin, and box reaches about 2 to 3mm 37 ℃ of incubated overnight until colony diameter.As Nizetic, people such as D are at nucleic acids research (Nucl.Acids Res.) 19,182 (1990); Hoheisel, people such as J.D. are at cell (Cell) 73, and 109-120 handles filter membrane described in (1993).Hybridization and wash conditions are as described in the people such as Hoheisel (1993).React label probe with random primer extension.Feinberg, A.P. and Vogelstein, B., biochemical analysis (Anal.Biochem.) 132,6-13 (1983); Appendix 137,266-267 (1984).B. result

Above-mentioned BAC library comprises 11,000 clones.Make up this library with two kinds of methods.Preceding half library with 7269 clones is to use Ramsay, M and Wicking, C., at people's molecular genetics method (Protocols inHuman Molecular Genetics), the compression zone band method described in the 197-221 (1991) select to make up through size once and forms.Back half library with 3731 clones is to utilize DNA to part digestion to carry out twice size to select to make up and form.But big or small twice selection can not improve DNA and insert segmental mean size.Obviously, in the dna solution of selecting through size (only isolating 250 to 350kb DNA), still there is small molecule DNA to exist.Below experiment showed, be used for connecting 350 to 500kb between twice of DNA size selected to have improved BAC clone's the big average fragment of inserting.Selecting 54 at random among the BAC clone from the library, wherein 50 contain paddy DNA (93.0%).Part clone does not contain insertion fragment (7%).DNA inserts segmental size between 30 to 250kb, and mean value is 125kb.

Be used to make up the rice cell nuclear of the high-molecular-weight DNA separation in BAC library from purifying.Low-speed centrifugal (＜1000g) removed most chloroplast(id) and plastosome.The low probability (＜0.3%) of finding chloroplast(id) or plastosome clone in the new BAC library has reduced the possibility that organelle DNA/nucleus DNA connects.

The BAC library be used to make up one group cross over the Xa21 locus adjoin clone (adjoin (sequence) crowd).With two dna markers that Xa21 is chain, RG103 (1kb, referring to people such as Ronald, molecular genetics (Mol.Gen.Genet.) 236:113-120 (1992)) and pTA818 (1.2kb is equivalent to people's such as Ronald RAPD818) screening BAC library.Find that in containing the strain of Xa21 RG103 has 8 copies, and with this strain in 8 genome HindIII dna fragmentations hybridization.These fragments are all in heredity and physically chain with Xa21 disease resistence gene seat.PTA818 and 2 dna fragmentation hybridization, a wherein minimum fragment and Xa21 locus are chain.People such as Ronald (1992).

With pTA818 (2 copies) and RG103 (8 copies) probe and 7296 BAC clone hybridizations.Identifying 7 BAC clone and 5 BAC with pTA818 hybridization with RG103 hybridization respectively clones.From these clones, separate BAC DNA, and digest with HindIII.With PFGE DNA isolation fragment.Southern analyzes demonstration, and 7 BAC with RG103 hybridization clone and carry the segmental 4 kinds of different copies of RG103 genome HindIII.Probe and one section 4.3kb and the hybridization of 9.5kb fragment are with one section 9.6kb and the hybridization of 6.2kb fragment.The size of dna fragmentation is to calculate from the λ DNA with HindIII digestion.

Isolate 4 and carry the segmental BAC clone of a kind of pTA818 HindIII, and identify a BAC clone who carries another kind of copy.Contain the pTA818 of BAC and mark PTA24g (RAPD248 that is equivalent to Ronald etc. (1992)) hybridization, this confirms, the RAPD marker gene that these two quilts are cloned and comprises all in 60kb mutually.People such as Ronald (1992).

Identifying 12 BAC with 2 kinds of cloned dna sequences (corresponding to 10 dna fragmentations in the rice genome) hybridization clones, this is a little less than screening (7296 bacterium colonies according to the 2x genome equivalent, 450, the 000kb genome on average inserts big or small 125kb) 20 bacterium colonies estimating.Specifically, this part in the library, excessively performance of pTA818 sequence and 4 RG103 hybridization sequences (in 8 4).On the contrary, other 4 RG103 sequences performance is not enough.These clones' DNA inserts clip size between 40 to 140kb.

The preparation of high molecular (HMW) DNA in the clay clone A. materials and methods paddy rice leaf

The paddy rice of carrying the Xa21 locus is 1188 vegetable materials as separation HMW DNA.Collect the leaf texture in 4 to 6 ages in week of 120g, in liquid nitrogen, grind to form fine powder with cold mortar and pestle.Then with the powder stirring suspension at cold H damping fluid [the 4mM spermidine of 800ml; the 1mM spermine; 10mM EDTA, 10mMTris-HCl, 80mM KCl; 0.5M sucrose; 1mM PMSF (phenyl methyl alkylsulfonyl chlorine adds before using), 0.5% (v/v) Triton-X100; 1/1000 (v/v) β mercaptoethanol (adding before using), pH9.5] in.Mixture is filled in the GSA bottle through 80 microns sieve, is resuspended in precipitation in the 400ml H damping fluid and filtration once more.Merge twice filtrate, 4 ℃ with 3500rpm centrifugal 10 minutes.Precipitation is resuspended in the 300ml lavation buffer solution (identical with the H damping fluid, but do not contain PMSF and β mercaptoethanol), 4 ℃ with 3500rpm centrifugal 10 minutes.Precipitating washed twice again, is light green until sedimentary color.Precipitation is resuspended in the 40ml lavation buffer solution, adds isopyknic lysis buffer (2% sarcosyl, 100mM Tris-HCl, 0.5M EDTA, pH9.5) lysing cell nuclear that contains 2mg/ml Proteinase K (Boehringer Mannhein).50 ℃ of reactions 5 hours, at room temperature use isopyknic phenol-chloroform-primary isoamyl alcohol (24: 24: 1) extraction 30 minutes (gentle reversing) then, remove protein thus.Gentle 3M sodium acetate (pH5.5) and one deck 2 volume of ethanol that add one deck 1/10 volume, and gentle reversing is for several times, precipitates HMW DNA thus.At last, draw DNA with heavy caliber point mouth transfer pipet from ethanol, use 70% washing with alcohol, drying is dissolved in 1mlTE (10mM Tris-HCl, 1mM EDTA, pH8.0) middle standing over night.Usually, can from the 120g leaf, separate and obtain 250 μ g HMW DNA.Insert the part digestion of preparation (A) the HMW DNA of DNA

Trial test.30 μ g (70 μ l) HMW DNA is mixed with 10 μ l10 * Sau3AI damping fluid (NEB), 37 ℃ of preheatings 5 minutes.In dna solution, add 20 μ l (2 unit) Sau3AI then, slightly mix with heavy caliber point mouth suction pipe, 37 ℃ of cultivations.0,5, take out 15 μ l of equivalent in the time of 10,20,30 and 70 minutes, mixing with termination reaction with 0.5M EDTA 5 μ l (pH8.0) on ice immediately.Then in freezer compartment, in 0.3% agarose/TBE gel, with 2V/cm gel length electrophoretic analysis sample.

Repeat trial test, the best when using 37 ℃ is cultivated 20 minutes intervals, can obtain large-scale DNA and partly digest.(B) size is selected

In the SW27 rotor, at 20 ℃, carry out 26,5 to 40% sucrose gradient centrifugations of 000rpm 13 hours, the DNA of separate part digestion.Kapillary is stretched into the centrifuge tube bottom,, carefully collect each part solution (20ml altogether) of 0.8ml thus with the gradient solution of speed absorption very slowly.Every duplicate samples get 20 μ l on 0.3% sepharose with 2V/cm gel length electrophoresis 36 hours.35 to 50kb DNA component is pooled together.With isopyknic water dilution sucrose solution, use 2 times of volume of ethanol deposit D NA then.Use standard method to carry out incomplete reaction.Connection, packing and transfection

Cosmid vector pHC80 is provided by Scot doctor Hulbert.Carrier is connected with 2 to 1 molar ratios with insertion DNA, and ultimate density is 0.8 μ g/ μ l.600 T4 of unit dna ligases are used in ligation, and (NEB USA) spends the night at 16 ℃.(Stratagene USA) according to the explanation of Stratagene, packs DNA after the connection, then transfection competent cell intestinal bacteria NM554 at external use GigapackII packaging extract.Library screening

With 61440 the clay bacterium colonies (greater than 5 genome equivalents) on 160 384 lattice droplet plates transfer to Hybond N+ filter membrane (Amersham, USA) on, form two kinds of density.In first method, (Genetix, U.K.) low density (1536 bacterium colonies/11.5 * 15cm filter membrane) is duplicated clay clone, grow overnight on the LB/ agar that contains 10 μ g/ml penbritins with manual copying instrument.Cover whole cosmid library with 40 filter membranes.In the second approach, use Beckman Biomek ^TMMobile operation instrument in a row high-density duplicates clay, uses method same as described above to grow.Use 3 * 3 rows, on the filter membrane with 3456 colony lift to one 8.5 * 12cm.In order on negative background, accurately to locate positive bacterium colony, cultivate one with reference to clay bacterium colony (comprising the RG103 marker gene) in first position of each 3 * 3 grid.Thereafter the bacterium colony from 8 droplet plates of cosmid library is cultivated in 8 positions.At this moment, the filter membrane of 20 8.5 * 12cm can cover whole library.For with a special probe hybridization, RG103 and this special probe were with 1: 4 mixed, formation is with reference to pattern.

Bacterium and the immobilization of utilization on the water vapour bath method cracking filter membrane of following modification: bacterium colony is being dipped in cracked solution (0.5M NaOH towards last, 1.5M on two 3MM Whatman NaCl), placed 4 minutes under the room temperature, the plastics casing that filter membrane is housed was hatched 6 minutes in 85 ℃ water vapour is bathed, then filter membrane is transferred to and be immersed in neutralization solution (1M Tris-HCl (pH7.4), 1.5M on the 3MM Whatman Nacl), placed 4 minutes.Be immersed in the Proteinase K solution (50mM Tris-HCl (pH8.0), 50mM EDTA (pH8.0), 100MmNaCl, 1% (v/v) sarcosyl, 250 μ g/ μ l Proteinase Ks) and hatched removal protein and cell debris 20 minutes in 37 ℃.Filter membrane is at room temperature used the gentle washing of 2 * SSC solution 5 minutes, and handled 2.5 minutes with UV apart from 10cm dry back.

Hybridize according to following standard method: filter membrane prehybridization solution (7%SDS, 0.5MNa ₂PO ₄(pH7.2), 1mM EDTA, 100 μ g/ml ssDNA) handle 2 hours to all night at 65 ℃.With random primer extension method label probe, 65 ℃ of vibration hybridization all night.Wash (40mMNa under the room temperature slightly ₂PO ₄(pH7.2), 1%SDS) filter membrane is hatched filter membrane 20 minutes in 65 ℃ of gentle vibrations in identical solution then.B. result

With three Xa21 linked markers (RG103, RAPD248 and RAPD818) screening cosmid library.Genome Southern analyzes demonstration, and three copy numbers that are marked in the resistant strain are respectively 8,1 and 2 (unexposed results).Identify 6 positive clays and clone, and confirm through further Southern analysis with the hybridization of RG103 mark.But, do not identify the positive colony that contains RAPD248 and RAPD818.

Embodiment 2

The evaluation of Xa21 gene

Isolating 5 clays clone and 1 BAC clone further identify with the Restriction Enzyme collection of illustrative plates in embodiment 1.Fig. 2 A to 2E is clay clone's a part restriction map.Fig. 2 F is BAC clone's a part restriction map.

Identify an opening code-reading frame (SEQ.ID.No.1) among a clone pB806 therein.It comprises promoter region, the intron of inferring and a part 3 ' sequence.SEQ.ID.No.2 shows the aminoacid sequence of inferring.The intron of inferring excises.

The aminoacid sequence of inferring has disclosed proteinic two characteristics, these two characteristics show that it is by a collection of novel type disease resistance of plant gene, claims the RRK genes encoding herein.The first, comprise about 23 series connection by its extracellular domains of protein of these genes encodings and be rich in leucine tumor-necrosis factor glycoproteins (LRR), mean length is 24 amino acid.The LRR motif be considered in multiple proteins with protein-proteins react and part in conjunction with relevant.Extracellular domain also comprises the zone between LRR and signal peptide, wherein comprises the SWNTS motif, and this motif is guarded in numerous protein, comprising Df-9, PGIP and RLK5.In addition, this albumen also comprises a zone, with PLPK5 and TMK1 and so on receptor-like protein kinase (PLPK) (people such as Walker, plant magazine (Plant J.) 3:451 (1993); People's vegetable cells such as Chang (Plant Cell) 4:1263 (1992); People such as Valon, molecular biology of plants Plant Molec.Biol.) 23:415 (1993)) and tomato resistant gene product P to people such as (, science (Scince) 262:1432 (1993)) Martin have the sequence identity of height.Signal domain, extracellular domain (comprising the LRR district), membrane-spanning domain and kytoplasm kinases territory have been determined among the SEQ.ID.No.2.

Fig. 3 is another clone, the Restriction Enzyme figure of pB822, and this clone is used for the hereinafter used plasmid of the transformation experiment described in the embodiment 3 of basis.Xa21 gene among this clone is also by order-checking (SEQ.ID.No.3).Putative amino acid sequence (SEQ.ID.No.4) shows the motif that identifies among the identical SEQ.ID.No.2.

The protein kinase territory carries 11 subdomains, comprises 15 conservative protein kinase characteristic residues in the subdomain, and both sides are respectively also film territory (aa 677-707) and C-terminal territories of a 33aa.Between the residue P of the intron of inferring two high conservatives in inferring catalytic domain and the E (aa892 and aa893).Consensus sequence shows that obviously Xa21 has serine/threonine kinase (opposite with tyrosine) activity among subdomain VI (DIKSSN) and the VIII (GTGYAAPE).

Previous studies have shown that, people such as (, science (Scince), 266:793-795 (1994)) Stone reacts in the kinase reaction territory (KID) of the RLK5 albumen of phosphorylation and 2C type serine-threonine protein Phosphoric acid esterase.KID and phosphorylation contain RLK5 and the proteinic LRR of TMK1 combines, but can not combine with S associated receptor kinases ZmpK1 and RLK4.This result shows that Arabidopis thaliana KID is similar with the SH2 structural domain of animal proteinum on function.The sequence of Arabidopis thaliana receptor-like kinase enzyme RLK5, TMK1 and Xa21 arranged show round a serine residue one group of conservative amino acid (N/Q) X (L/V) S (G/S) is arranged (L/A) (F/V) (P/E), this serine residue is the carboxyl terminal of last residue (arginine), in all proteins kinases camber conservative (Xa21 gene product 999).The Tyrosine O-phosphate carboxyl terminal is similar among carboxyl terminal position that has in these protein and the sarcoma viral oncoprotein pp60 of the Rous c-Src, and the latter is necessary with the protein bound that contains the SH2 structural domain.Not with KID bonded S associated receptor kinases ZmpK1, lack these conservative amino acid in RLK4 and SRK6 and the iuntercellular kinases.So this zone plays a part and KID albumen high affinity and specific binding site.So can utilize the aminoacid sequence that changes the Xa21 zone to change the proteinic affinity to KID, the part of regulating the LRR structural domain thus is in conjunction with the intracellular signal transmission that causes.

Embodiment 3

Use the Xa21 gene-transformed plant

Available above-mentioned Xa21 gene transformation rice plant proves that this gene can make the susceptible plant have the Xanthomonas campestris resistance.To people such as Li, the method correct among vegetable cell report (Plant Cell Rep.) 12:250-255 (1993) imports gene the rice plant of susceptible with this.In brief, carry out cotransformation with Totomycin construction pMON410 (Monsanto provides) and the Bluscreipt carrier that contains target sequence.In addition, in the pTA818 carrier, this carrier is from Invitrogen carrier pcr1000 and contain the fragment RAPD818 (people such as Ronald, the same) of 1kb with the Kpn fragment cloning of pB822.The plasmid that forms claims pC822.Select plant with Totomycin (30mg/L), screen resistance then Xoo kind 6.

Utilize standard method to test the Xanthomonas campestris resistance of transformant.According to people such as Kaufma, the method among plant disease report (Plant Disease Rep.) 57:537-541 (1973) experimentizes.In brief, Xoo kind 6 was grown 3 days on the PSA plate.Scrape and get bacterium, be suspended in the water, regulating the OD value is 10 ⁹Colony-forming unit/ml.Scissors is soaked in suspension, shear the leaf of transformed plant (bombarding back 4 months) at distance blade tip 5cm place.Just inoculating the appearance of back 11 days lesions evaluates plant.

Fig. 4 shows the lesion length data, contains the experiment that the expression vector of pC822 cloned genes carries out from use.The individuality 106 ,-9 ,-22,11 ,-17 ,-1 ,-12 ,-4,16 and 29 that is developed into by different transformants carries the pC822 construction, and, to compare with the rice plant that transforms with carrier (1-15) with the unconverted contrast (IR24) of susceptible, resistance improves.

Embodiment 4

From tomato, separate the RRK gene

As mentioned above, the Xa21 sequence can be used for utilizing degenerated primer or low stringency hybridizing method to separate the RRK gene from other plant species.Present embodiment has illustrated and has utilized the degenerated primer method to separate from tomato and identify two RRK genes.

The preparation degenerated primer makes sequence between PCR (polymerase chain reaction) product amplification LRR and kinases territory, crosses over membrane-spanning domain thus.Forward primer is from the conservative property motif in the LRR district of Xa21 and several other vegetable-protein (for example, cf-9, RLK5 and PGIP).Reverse primer is from the conservative property motif of Xa21 kinases territory and other plant serine-threonine kinase territory (for example, RLK5, Pto and Fen) people such as (, vegetable cell (PlantCell) 6:1543-1551 (1994)) Martin.

The degenerated primer that is used for the amplification of PCR product is as follows: the 1.LRR district

TCA AGC AAC AAT TTG TCA GGN CA (A/G) AT (A/C/T) CC (SEQ.ID.No.5) is the kinases district 2.

TAA?CAG?CAC?ATT?GCT?TGA?TTT?NAN(G/A)TC?NCG(G/A)TG(SEQ.ID.No.6)

TAA?CAG?CAC?ATT?GCT?TGA?TTT?NAN(G/A)TC(G/A)CA(G/A)TG(SEQ.ID.No.7)

TAA?CAG?CAC?ATT?GCT?TGA?TTT?NAN(G/A)TC(T/C)CT(G/A)TG(SEQ.ID.No.8)

PCR

PCR condition following (reactions of 20 microlitres):

First circulation:

94 ℃, 30 seconds (sex change)

55 ℃, 30 seconds (annealing)

72 ℃, 1 minute (prolongation)

19 taking turns in the circulation thereafter, every round-robin annealing temperature of taking turns descends 1 ℃.The 20th take turns loop ends after, reaction solution was hatched 10 minutes at 72 ℃.

State in the use after the first amplification of primer, use following Auele Specific Primer to carry out the amplification second time:

TAAGCAACAATTTG(SEQ.ID.No.9)

TAACAGCACATTGCTTGA(SEQ.ID.No.10)

This time the condition of amplification is as follows:

94 ℃, 15 seconds

55 ℃, 15 seconds

72 ℃, 15 seconds

35 take turns circulation after, reaction solution was hatched 10 minutes at 72 ℃.

Clone PCR products is as the probe in screening tomato cDNA library.Tomato cDNA and oligomerization dT primer hybridization are connected with the EcoRI adapter, are cloned in the λ GT11 carrier, make up the library thus.

Above primer is used to separate two kinds of tomato PCR products and the cDNA that belongs to disease resistence gene RRK family.First kind of clone TRK1 (tomato receptor kinase 1) is the PCR product of a 250bp, is used to separate part cDNA.Dna sequence dna is shown in SEQ.ID.No.11.The putative amino acid sequence of TRK1 is shown in SEQ.ID.No.12.

This is cloned in one or two copy in the tomato dna group, one of them is positioned at chromosomal galianconism No. 1 on cleavage map, near Xanthomonas campestris campestris pv.vesicatoria resistant gene (Rxl) (people such as Zu, heredity (Genetics) 141:675-682 (1995)) (referring to Fig. 5).

Second clone TRL1 (tomato acceptor sample 1) is the PCR product of one section 496bp.Dna sequence dna is shown in SEQ.ID.No.13.The aminoacid sequence of inferring is SEQ.ID.No.14.TRL1 is in several cM of the sudden change mcn on No. 3 karyomit(e) on the cleavage map, and sudden change mcn causes the spotty necrosis of plant, and this is a kind of typical defensive phenotype.

Above result shows that the Xa21 gene can be used for separating the RRK gene from other plant species.For example, separating the TRK1 and the TRL1 gene that obtain is the important composition that causes the interior signal pipeline of plant of defense response.These genes are used in tomato and other plant species and produce disease resistance with gene engineering research.

Above embodiment is used to illustrate the present invention rather than to the restriction of its scope.Concerning persons skilled in the art, it is conspicuous that of the present invention other changes form, and these are all within appended claim scope.Whole open source literatures, plasmid and the patent disclosure that this paper the quotes reference that all is cited.

SEQ.ID.No.1

RRK-FaagctttctaaattatttaactctaagtctgttattatccccaagtacatcatcatcatacataatatttcatattcacgacatccttaagctagatgcttttggccattctcttatctttttaaagaaattctctcccaattaagatgagagtgtcttctagcaatttgccagtttttacaatgtctttgagtcctcacacattttcatgatgttaccaataaattacggacgccgtgtttagttctaaagtttttcttcaaacttacaacttttcaatcgcatcaaaactttctcctacacacacaaactttcaacttttccatcacatcgttccaatttcaaccaaacttccaattttggtatgaactaaacacagccgaaaacaaaatctgtgtgttatggccctgtttagattctaacttttccattacatcaaactttcctacatacacgaactttcaacttttccgtcacatcgtttcaattttttaaaacttccatttttaacgtggaactaaacacaacctatataacggaatttgtcaaaaactcaatggtgaaagtcacacctcacaggaagggcgcgctctagtcaagacatcattaaacaggtacacaggttgtactagcttgtcatgtttatcttgcgtctgcgagacgtaaatccatgccaaacaaaagtgcttctatagagatatcataaggatatggtttggggccatatccaactgctcaggagagatctcgttcggaggtgaggttagatgttcacctctccacacataacgaaggcgatcttcttcgcatatgattaggcattagataaaataaccttaaaaaataaatcaatatgatttttttagaaaaaaattatatacactaagtataagcattgtcaaggaggaagaaacacacactcccatatagagagatagaaacatagctataggtagtgtcactgagtattttccatcacgcatatccatataaaattagggggtgttacatccataggtgtaaagttttggcatgttatatcgagtattacgtagaatgccgtattaggtgtccgggcactaataaaaaaataattacagaatccgttagtaaaccgcgagataaatttattaagcctaattaatcccatcattaacaaatgtttaccgtagcaccacattgtcaaatcatggagcaattaggtttaaaagattcgtctcgcaaattagtcataatctgtgcaattagttatttttagactatatttaagacttcgtacaggtgttcaaacgttcgatgtgacatggtgcaaaattttagggtgtcatctagacactcccttaattagaaagttaggaagaggcggtaaagaacgcagcatgactgaaactttgaaaatttgataaggtacaccaactggagtatcttttattttcattgaagactttgaccagaagagcttgacccgtttttcttggagtagccagtaatgtttcattcttttccttttgctgggacttctttttattttttttgacaggagccatttgttgggacttgggatccctttactgttataggaccagtgcttgaatccaaacactgcattgatcagctcagctcattgtagcgcactcctccgcatgcATGGCGAGATCACCAACGTCGGTCATGATCTCTTCTTTGCTGCTGCTGCTGTTGATCGGCCCAGCGAGCAGTGACGATGATGCTGCTGCTGCTGCTGCTCGTACCAGTACAGGCGGCGTCGCGGCGACGAACTCGCGCTGCTCTCTTTCAAGTCATCCCTGCTACACCAGGGGGGCTTGTACGCTGGCATCTTGGAACACGTCCGGCCACGGCCAGCACTGCACATGGGTGGGTGTTGTGTGCGGCCGCGCGCGCCGGCACCCACACAGGGTGGTGAAGCTGCTGCTGCGCTCGTCCAACCTGTCCGGGATCATCTCGCCGTCGCTGGGCAACCTGTCCTTCCTCAGGGAGCTGGACCTCAGCGACAACTACCTCTCCGGCGAGATACCACCGGAGCTCAGCCGTCTCAGCAGGCTTCAGCTGCTGGAGCTGAGCGGTAACTCCATCCAAGGGAGCATCCACGCGGCCATTGGAGCATGCACCAAGTTGACATCGCTAGACCTCAGCCACAACCAACTGAGATTGGTGCCAGCTGAAACATCTCTCGAATTTGTACCTTCACACCAATGGTTATGTCAGGAGAGATTCCATCTGATTTTGGGCAATCTCACTACGCCTTCAGTATTTGATTTGACCTGCAACAGATTATCACGGAGCTATACCTTCATCGCTAGGGCAGCTCAGCAGCAGTCTATTGACTATGAATTTTGTGCTACGAACAATCTAACTGGCATGATCCCCAATTCTATCTGGAACCTTTCGTCTCTAGCAGCGTTTAGCTGTCAAGCGAAAAACAAGCTAGGTGGTATGATCCCTACAAATGCATTCAAAACCCTTCACCTCCTCGAGGTGGTAGATATGGGCACTAACCGATTCCATGGCAAAATCCCTGCCTCAGTTGCTAATGCTTCTCATCTGACACGGCTTCAGATTGATGGCAACTTGTTCAGTGGAATTATCACCTCGGGGTTTGGAAGGTTAAGAAATCTCACAACACTGTATCTCTGGAGAAATTTGTTTCAAACTAGAGAACAAGAAGATTGGGGGTTCATTTCTGACCTAACAAATTGCTCCAAATTACAAACATTGGACTTGGGAGAAAATAACCTGGGGGGAGTTCTTCCTAATTCGTTTTCCAATCTTTCCACTTCGCTTAGTTTTCTTGCACTTGATTTGAATAAGATCACAGGAAGCATTCCAAAGGATATTGGCAATCTTATTGGCTTACAACATCTCTATCTCTGCAACAACAATTTCAGAGGGTCACTTCCATCATCGTTGGGCAGGCTTAGAAACTTAGGCATTCTAGTCGCCTACGAAAACAACTTGAGCGGTTCGATCCCATTGGCCATAGGAAATCTTACTGAACTTAATATCTTACTGCTCGGCACCAACAAATTCAGTGGTTGGATACCATACACACTCTCAAACCTCACAAACTTGTTGTCATTAGGCCTCTCGCACCTCGCACCACAATCAGGGTTGGATACCTACACATCTCAACCTCACAACTGTGTCATAGCCTTCACTATACCTAGTGGGTCCCAAATACCCCAGGTGAAATTAATTCAAATAGTCCAAACACCTATCAAAAAGATGATCAATGTATCAAAAAATACACTTGGAGGGATCAGATACCCACAAGAAATAGGGCATCTCAAAAATCTAGTAGAATTCATGCAGAATCGAATAGATATCAGTAAAATCCCTAACACGCTTGGTGATTGCCAGCTCTTACGGTATCTTTATCTGCAAAATAATTTGTTATCTGGTAGCATCCCATCAGCCTTGGGTCAGCTGAAAGGTCTCGAAACTCTTGATCTCTCAAGCAACAATTTGTCAGGCCAGATACCCACATCCCTTAGCAGATATTACTATGCTTCATTCCTTGAACCTTTCTTTCAACAGCTTTGTGGGGGAAGTGCCAACCATTGCGTGCTTTCGCAGATGCATCCGGGATCTCAATCCAAGGCAATGCCAAACTCTGTGGTGGAATACCTGATCTACATCTGCCTCGATGTTGTCCCATTACTAGAGAACAGAAAGCATTTTCCAGCTCTACCTATTTCTGTTTCTCTGGTCGCAGCACTGGCCATCCTCTCATCACTCTACTTGCTTATAACCTGGAACAAGAGAACTAAAAAGGGAGCCCCTTCAAGAACTTCCATGAAAGGCCACCCATTGGTCTCTTATCCGCAGTTGGTAAAAGCAACAGATGGTTTCGCGCCGACCAATTTGTTGGGTTCTGGATCATTTGCCTCAGTATACAAACGAAAGCTTGAAAATCCTAAGGCACTCAAGAGTTTCACTGCCGAATGTGAAGCACTACGAAATATGCGACATCGAAATCTTGTCAAGATAGTTACAATTTGCTCGAGCATTGATAACAGAGGGAACGATTTCAAAGCAATTGTGTATGACTTCATGCCCAACGGCAGTCTGGAAGATTGGATACACCCTGAAACAAATGATCAAGCAGACCAGAGGCACTTGAATCTGCATCGAAGAGTGACCATACTACTTGATGTTGCCTGTGCATTGGACTATCTTCACCGCCATGGCCCTGAACCTGTTGTACACTGTGATGTTAAATCAAGCAATGTGCTGTTAGATTCTGATATGGTAGCGCATGTTGGAGATTCTGGGCTTGCAAGAATACTTGTTGATGGGACCTCATTGATACAACAGTCAACAAGCTCGATGGGATTTAGAGGGACAATTGGCTATGCAGCACCAggtcagcaagtccttccagtattttgcattttctgatctctagtgctatatgaaatagtttttacctctagtgaaactgatggagaatataagtaattaattgaactaattaaattgcacaaaaataagattatttgccatatctattcagatgctaaatatagctagttcatagaggtacatattttttttatataggaatctagagctactacacactcaaatcaaattatgggtgttttctgctctacactgcaatatgaaatgattatcagaaggatcaaatttgagtaaatttgtcaattctacatttaagaaacacttttttttgtatgtactagttattacaattttttatttcaagaacttgcattgaccatgaaaagtacttggtactacttctaattcccacatggaggtggtgaaaataatatagatacaaaaacgaagtatcatatgttgtgtgatatactataatcacaatgaacacaaacaggattcgtacaaaagtaattggccatcatagcaactgattgcttggggtaactgtatagcacaatcataccaaatttctttagatatgtatttgtaaattagattcttaaagttaaatatgaaatttcattggtatttatgtttctttatataataaaaattaatccaacctttacatctaccatttgtccagccatccttgttatttgtgatatttaacacgtaattttacataattatacatccaagttctttttatttaacactggaaatttgaaatcgtatttcctactcaaacaGAGTATGGCGTCGGGCACATTGCATCAACACATGGAGATATTTACAGCTATGGAATTCTAGTGCTGGAAATAGTAACCGGGAAGCGGCCAACTGACAGTACATTCAGACCCGATTTGGGCCTCCGTCAGTACGTTGAACTGGGCCTACATGGCAGAGTGACGGATGTTGTTGACACGAAGCTCATTTTGGATTCTGAGAACTGGCTGAACAGTACAAATAATTCTCCATGTAGAAGAATCACTGAATGCATTGTTTCGCTGCTTAGACTTGGGTTGTCTTGCTCTCAGGATTTNCCATTGAGTAGACGCCACCCGGAGATATCACCGACGAACTGAatgccatcaaacagaatctctccggagttgtttccagtgtgtgaaggtgcgagcctcgaattctgatgttatgtcttgtaatgttttattgccactagtcttcagattggaatgctcttccgatcagacttcttcagtggtatctaccacacgatcactaaagtcatcgtggctatttcctgatccagcatatctgatcatgcatgttctgtgttttatacctgtattttactctgaattgccacacctcaaccctgcctctgtttgtttggcatacaaaagatagtgatgagtatattgtttcaggggcttcctagttggcgtgtgtgcttaccggcacgcacgcagcccgagggtgggtttctttttttttccattgttattccgttgcttttttccaccacggtagattttttttttctggatttccattttttccgttgtttttctctatcgcttatgctggcggatttttttccgtggtttttttttcaagacgagtatatctaatgtaactaacatgttacttttagataacgatggttattaagataagatttttttctggaagatttttgtaagtaaatggtaaaaaatatggaaatggaaacggaaatagttttgctgttataccgatcgtttccatatttaccgtattcttatagaaattaccgtntcttataatatggtaattaccgtatttctaaatatgttgatatcgattttgctatatatttgtcgacSEQ.ID.No.2

RRK-FMARSPTSVMISSLLLLLLIGPASSDDDAAAAAARTSTGGVAATNSRCSLSSHPCYTRGACTLASWNTSGHGQHCTWVGVVCGRARRHPHRVVK

LLLRSS N LSGIISPSLGNLSFLRELDLSD NYLSGEIPPELSRLSRLQLLELSG NSIQGSIHAAIGACTKLTSLDLSH NQLRLVPAETSLEFVPSHQWLCQERFHLILGNLTTPSVFDLTC NRLSRSYTFIARAAQQQSIDYEFCAT NNLTGMIPNSIWNLSSLAAFSCQAKNKLGGMIPTNAFKTLHLLEVVDMGT NRFHGKIPASVANASHLTRLQIDG NLFSGIITSGFGRLRNLTTLYLWR NLFQTREQEDWGFISDLTNCSKLQTLDLGE NNLGGVLPNSFSNLSTSLSFLALDL NKITGSIPKDIGNLIGLQHLYLCN NNFRGSLPSSLGRLRNLGILVAYE NNLSGSIPLAIGNLTELNILLLGT NKFSGWIPYTLSNLTNLLSLGLSHLAPQSGLDTYTSQPHNCVIAFTIPS GSQIPQVKLIQIVQTPIKKMINVSK NTLGG IRYPQEIGHLKNLVEFMQNRID ISK IPNTLGDCQLLRYLYLQN NLLSGSIPSALGQLKGLETLDLSS NNLSGQIPTSLSRYYYASFLEPFFQQLCGGSANHCVLSQMHPGSQSKAMPNSVVEYLIYICLDVVPLLENRKHFPALPISVSLVAALAILSSLYLLITWNKRTKKGAPSRTSMKGHPLVSYPQLVKATDGFAPTNLLGSGSFASVYKRKLENPKALKSFTAECEALRNMRHRNLVKIVTICSSIDNRGNDFKAIVYDFMPNGSLEDWIHPETNDQADQRHLNLHRRVTILLDVACALDYLHRHGPEPVVHCDVKSSNVLLDSDMVAHVGDSGLARILVDGTSLIQQSTSSMGFRGTIGYAAPEYGVGHIASTHGDIYSYGILVLEIVTGKRPTDSTFRPDLGLRQYVELGLHGRVTDVVDTKLILDSENWLNSTNNSPCRRITECIVSLLRLGLSCSQDL ^*(F) PLSRRHPEISPTNZ ^*L：“N”＝“G”F：“N”＝“C”SEQ.ID.No.3：ORF 3918 ( 3075bp ( 843bp ) ) ATGATATCACTCCCATTATTGCTCTTCGTCCTGTTGTTCTCTGCGCTGCTGCTCTGCCCTTCAAGCAGTGACGACGATGGTGATGCTGCCGGCGACGAACTCGCGCTGCTCTCTTTCAAGTCATCCCTGCTATACCAGGGGGGCCAGTCGCTGGCATCTTGGAACACGTCCGGCCACGGCCAGCACTGCACATGGGTGGGTGTTGTGTGCGGCCGCCGCCGCCGCCGGCACCCACACAGGGTGGTGAAGCTGCTGCTGCGCTCCTCCAACCTGTCCGGGATCATCTCGCCGTCGCTCGGCAACCTGTCCTTCCTCAGGGAGCTGGACCTCGGCGACAACTACCTCTCCGGCGAGATACCACCGGAGCTCAGCCGTCTCAGCAGGCTTCAGCTGCTGGAGCTGAGCGATAACTCCATCCAAGGGAGCATCCCCGCGGCCATTGGAGCATGCACCAAGTTGACATCGCTAGACCTCAGCCACAACCAACTGCGAGGTATGATCCCACGTGAGATTGGTGCCAGCTTGAAACATCTCTCGAATTTGTACCTTTACAAAAATGGTTTGTCAGGAGAGATTCCATCCGCTTTGGGCAATCTCACTAGCCTCCAGGAGTTTGATTTGAGCTTCAACAGATTATCAGGAGCTATACCTTCATCACTGGGGCAGCTCAGCAGTCTATTGACTATGAATTTGGGACAGAACAATCTAAGTGGGATGATCCCCAATTCTATCTGGAACCTTTCGTCTCTAAGAGCGTTTAGTGTCAGAGAAAACAAGCTAGGTGGTATGATCCCTACAAATGCATTCAAAACCCTTCACCTCCTCGAGGTGATAGATATGGGCACTAACCGTTTCCATGGCAAAATCCCTGCCTCAGTTGCTAATGCTTCTCATTTGACAGTGATTCAGATTTATGGCAACTTGTTCAGTGGAATTATCACCTCGGGGTTTGGAAGGTTAAGAAATCTCACAGAACTGTATCTCTGGAGAAATTTGTTTCAAACTAGAGAACAAGATGATTGGGGGTTCATTTCTGACCTAACAAATTGCTCCAAATTACAAACATTGAACTTGGGAGAAAATAACCTGGGGGGAGTTCTTCCTAATTCGTTTTCCAATCTTTCCACTTCGCTTAGTTTTCTTGCACTTGAATTGAATAAGATCACAGGAAGCATTCCGAAGGATATTGGCAATCTTATTGGCTTACAACATCTCTATCTCTGCAACAACAATTTCAGAGGGTCTCTTCCATCATCGTTGGGCAGGCTTAAAAACTTAGGCATTCTACTCGCCTACGAAAACAACTTGAGCGGTTCGATCCCGTTGGCCATAGGAAATCTTACTGAACTTAATATCTTACTGCTCGGCACCAACAAATTCAGTGGTTGGATACCATACACACTCTCAAACCTCACAAACTTGTTGTCATTAGGCCTTTCAACTAATAACCTTAGTGGTCCAATACCCAGTGAATTATTCAATATTCAAACACTATCAATAATGATCAATGTATCAAAAAATAACTTGGAGGGATCAATACCACAAGAAATAGGGCATCTCAAAAATCTAGTAGAATTTCATGCAGAATCGAATAGATTATCAGGTAAAATCCCTAACACGCTTGGTGATTGCCAGCTCTTACGGTATCTTTATCTGCAAAATAATTTGTTATCTGGTAGCATCCCATCAGCCTTGGGTCAGCTGAAAGGTCTCGAAACTCTTGATCTCTCAAGCAACAATTTGTCAGGCCAGATACCCACATCCTTAGCAGATATTACTATGCTTCATTCCTTGAACCTTTCTTTCAACAGCTTTGTGGGGGAAGTGCCAACCATTGGTGCTTTCGCAGCTGCATCCGGGATCTCAATCCAAGGCAATGCCAAACTCTGTGGTGGAATACCTGATCTACATCTGCCTCGATGTTGTCCATTACTAGAGAACAGAAAACATTTCCCAGTTCTACCTATTTCTGTTTCTCTGGCCGCAGCACTGGCCATCCTCTCATCACTCTACTTGCTTATAACCTGGCACAAGAGAACTAAAAAGGGAGCCCCTTCAAGAACTTCCATGAAAGGCCACCCATTGGTCTCTTATTCGCAGTTGGTAAAAGCAACAGATGGTTTCGCGCCGACCAATTTGTTGGGTTCTGGATCATTTGGCTCAGTATACAAAGGAAAGCTTAATATCCAAGATCATGTTGCAGTGAAGGTACTAAAGCTTGAAAATCCTAAGGCGCTCAAGAGTTTCACTGCCGAATGTGAAGCACTACGAAATATGCGACATCGAAATCTTGTCAAGATAGTTACAATTTGCTCGAGCATTGATAACAGAGGGAACGATTTCAAAGCAATTGTGTATGACTTCATGCCCAACGGCAGTCTGGAAGATTGGATACACCCTGAAACAAATGATCAAGCAGACCAGAGGCACTTGAATCTGCATCGAAGAGTGACCATACTACTTGATGTTGCCTGCGCACTGGACTATCTTCACCGCCATGGCCCTGAACCTGTTGTACACTGTGATATTAAATCAAGCAATGTGCTGTTAGATTCTGATATGGTAGCCCATGTTGGAGATTTTGGGCTTGCAAGAATACTTGTTGATGGGACCTCATTGATACAACAGTCAACAAGCTCGATGGGATTTATAGGGACAATTGGCTATGCAGCACCAGGTCAGCAAGTCCTTCCAGTATTTTGCATTTTCTGATCTCTAGTGCTATATGAAATAGTTTTTACCTCTAGTGAAACTGATGGAGAATATAAGTAATTAATTGAACTAATTAAATTGCACAAAAATAAGATTATTTGCCATATCTATTCAGATGCTAAATATAGCTAGTTCATAGAGGTACAGATTTTTTTATATAGGACTCTAGAGCTACCACACACTCAAATCAAATTATGGGTGTTTTCTGCTCTACACTGCAATATGAAATGATTATTACTTCTACATGAACTGATGGAGGAGTTTCAGAAGGATCAAATTTGAGTAAATTTTTTCAATTCTACATTTAAGAAACACTTTTTTTTCATATGCTAGTTACATTTTTTTATTTCACGAGCTTACATTGACCATGAAAAATACTTGGCACTACTTACTAATTCCCACATGGAGGTAGTGAAAATAATATAGATACAAAAACGAAATATCCTATGTTGTGTGATATACTATAATCACAATGAACACAAACAGGATTCGTACAAAAGTAATTAGCCATCATAGCAACTGATTGCTTGGGGTAACTGTATAGCACAATCATACCAAATTTCTTTAGATATGTATCTGTAAATTAGATTCTTAAAGTTAAATATGAAATTTCATTGGTATTTATGTTTCTTTATATAATAAAAATTAATCCAGCCTTTGCATCTATCATTTGTCCAGACATCCTTGTTATTTGTGATATTTAACACGTAAATTTACATAATTATACATCCAAGTTCTTTTTATTTAACACTGTAAATTTCAAATCGTACATGTTATAAAGAATGTACTATATTTCCTGCTCAAACAGAGTATGGCGTTGGGCTCATTGCATCAACGCATGGAGATATTTACAGCTATGGAATTCTAGTGCTGGAAATAGTAACCGGGAAGCGGCCAACTGACAGTACATTCAGACCCGATTTGGGCCTCCGTCAGTACGTTGAACTGGGCCTACATGGCAGAGTGACGGATGTTGTTGACACGAAGCTCATTTTGGATTCTGAGAACTGGCTGAACAGTACAAATAATTCTCCATGTAGAAGAATCACTGAATGCATTGTTTGGCTGCTTAGACTTGGGTTGTCTTGCTCTCAGGAATTGCCATCGAGTAGAACGCCAACCGGAGATATCATCGACGAACTGAATGCCATCAAACAGAATCTCTCCGGATTGTTTCCAGTGTGTGAAGGTGGGAGCCTTGAATTCTGASEQ.ID.No.41025A MISLPLLLFVLLFSALLLCPSSS 23B DDDGDAAGDELALLSFKSSLLYQGGQSLASWN 55

TSGHGQHCTWVGVVCGRRRRRHPHR 80C VVK?LLLRSSN?LSGIISPS 98

LGNLSFLRE?LDLGDNY?LSGEIPPE 122

LSRLSRLQL?LELSDNS?IQGSIPAA 146

IGACTKLTS?LDLSHNQ?LRGMIPREI 171

GASLKHLSN?LYLYKNG?LSGEIPSA 195

LGNLTSLQE?FDLSFNR?LSGAIPSS 219

LGQLSSLLT?MNLGQNN?LSGMIPNS 243

IWNLSSLRA?FSVRENK?LGGMIPTNA 268

FKTLHLLEV?IDMGTNR?FHGKIPAS 292

VANASHLTV?IQIYGNL?FSGIITSG 316

FGRLRNLTE?LYLWRNL?FQTREQDDWGFISD 346

LTNCSKLQT?LNLGENN?LGGVLPNSF 371

SNLSTSLSF?LALELNK?ITGSIPKD 395

IGNLIGLQH?LYLCNNN?FRGSLPSS 419

LGRLKNLGI?LLAYENN?LSGSIPLA 443

IGNLTELNI?LLLGTNK?FSGWIPYT 467

LSNLTNLLS?LGLSTNN?LSGPIPSE 491

LFNIQTLSIMINVSKNN?LEGSIPQE 516

IGHLKNLVE?FHAESNR?LSGKIPNT 540

LGDCQLLRY?LYLQNNL?LSGSIPSA 564

LGQLKGLET?LDLSSNN?LSGQIPTS 588

LADITMLHS?LNLSFNS?FVGEVPT 611

IGAFAAASG?ISIQGNAKLCGGIP 634D DLHLPRCCPLLENRKH 650E FPVLPISVSLAAALAILSSLYLLITW 676F HKRTKK 682G GAPSRTSMKGHPLVSYSQLVKATDG 707H FAPTNLLGSGSFGSVYKGKLNIQDHVAVKVLKLENPKALKSFTA 751

ECEALRNMRHRNLVKIVTICSSIDNRGNDFKAIVYDFMPNGSLE 795

DWIHPETNDQADQRHLNLHRRVTILLDVACALDYLHRHGPEPVV 839

HCDIKSSNVLLDSDMVAHVGDFGLARILVDGTSLIQQSTS 879

SMGFIGTIGYAAPEYGVGLIASTHGDIYSYGILVLEI 916

VTGKRPTDSTFRPDLGLRQYVELGLHGRVTDVVDTKLILDSENW 960

LNSTNNSPCRRITECIVWLLRLGLSCSQELPSSRTPTGDIIDEL 1004I NAIKQNLSGLFPVCEGGSLEF 1025SEQ.ID.No.5TCAAGCAACAATTTGTCAGGNCA A/G AT A/C/T CCSEQ.ID.No.6TAACAGCACATTGCTTGATTTNAN G/A TCNCG G/A TGSEQ.ID.No.7TAA CAG CAC ATT GCT TGA TTT NAN ( G/A ) TC ( G/A ) CA ( G/A ) TGSEQ.ID.No.8TAA CAG CAC ATT GCT TGA TTT NAN ( G/A ) TC ( T/C ) CT ( G/A ) TGSEQ.ID.No.9TAAGCAACAATTTGSEQ.ID.No.10TAACAGCACATTGCTTGASEQ.ID.No.11TRK1 ( DNA ) TCGACGTCGAACAATCGCTTGTCTGGTGCACTTCCTAGTGCTATTGGAAACTATTCAGGGCTGAAGAATCTTGTGTTAACTGGAAATGGTTTCTCAGGTGATATCCCTTCTGATATTGGCAGACTAAAGAGCATCTTAAAGCTGGACCTGAGTAGAAACAACTTCTCTGGCACAATCCCTCCTCAGATTGGTAACTGTCTTTCCTTAACTTACTTGGATTTGAGCCAAAATCAACTTTCTGGTCCTATCCCAGTTCAAATTGCTCAAATTCACATCTTAAATTACATCAATATTTCCTGGAATCACTTCAACGAGAGCCTTCCCGCGGAGATTGGCTTGATGAAGAGTTTAACTTCAGCAGATTTTTCCCACAATAACTTATCTGGATCAATACCTGAAACAGGCCAATATTTATATTTCAACTCAACTTCCTTCACCGGCAACCCTTATCTCTCTGGATCCGACTCGACTCCTAGCAACATTACATCCAACTCACCGTCAGAACTTGGAGACGGAAGTGACAGCAGAACTAAGGTTCCTACAATATACAAGTTCATATTTGCATTTGGGCTCTTATTCTGCTCCCTCATTTTCGTTGTCTTAGCAATAATCAAGACAAGAAAGGGGAGTAAGAATTCAAATTTGTGGAAGCTGACAGCATTTCAGAAGCTTGAGTTCGGAAGTGAAGACGTCTTGCAGTGCTTGAAAGACAACAACGTCATAGGGAGAGGTGGAGCAGGGATAGTGTATAAGGGAACTATGCCAAATGGTGATCATGTCGCGGTGAAGAAATTGGGAATAAGCAAAGGCTCACATGATAACGGCCTATCTGCTGAACTTAACACATTAGGGAAGATCAGGCATAGGTACATTGTGAGACTGCTCGCGTTTTGTTCAAACAAGGAAGTCAACTTGCTAGTTTATGAGTACATGCTAAATGGAAGCTTAGGTGAAGTGCTTCATGGGAAGAACGGCGGGCAACTCCAATGGGAAACTAGGCTAAAAATAGCCATAGAAGCTGCCAAGGGCCTTTCTTATTTGCACCACGATTGCTCCCCTATGATAATCCACCGCGATGTCAAGTCCAACAATATATTGTTGAACTCTGAACTTGAAGCTCATGTTGCAGATTTTGGATTAGCCAAGTACTTTCGTAACAATGGTACCTCTGAGTGCATGTCTGCAATTGCAGGATCTTATGGCTACATTGCTCCAGAATATGCATACACGCTGAAAATTGATGAGAAAAGCGATGTGTATAGCTTTGGAGTGGTGTTGTTGGAGCTTATAACAGGACGAAGGCCAGTAGGAAATTTTGGAGAAGAAGGAATGGACATTGTACAATGGGCGAAAACGGAGACAAAATGGAGCAAAGAAGGGGTGGTGAAAATCTTGGATGAGAGGCTAAAAAATGTTGCAATTGTTGAAGCTATGCAAGTATTTTTTGTAGCAATGCTTTGTGTTGAAGAGTACAGCATTGAGAGGCCTACAATGAGGGAAGTAGTCCAAATGCTTTCTCAAGCTAAACAACCAAATACTTTCCAAATCCAATAASEQ.ID.No.12STSNNRLSGALPSAIGNYSGLKNLVLTGNGFSGDIPSDIGRLKSILKLDLSRNNFSGTIPPQIGNCLSLTYLDLSQNQLSGPIPVQIAQIHILNYINISWNHFNESLPAEIGLMKSLTSADFSHNNLSGSIPETGQYLYFNSTSFTGNPYLSGSDSTPSNITSNSPSELGDGSDSRTKVPTIYKFIFAFGLLFCSLIFVVLAIIKTRKGSKNSNLWKLTAFQKLEFGSEDVLQCLKDNNVGRGGAGIVYKGTMPNGDHVARSAGFAAASSRGGIVYKGTMPNGDHVAVKKLGISKGSHDNGLSAELNTLGKIRHRYIVRLLAFCSNKEVNLLVYEYMLNGSLGEVLHGKNGGQLQWETRLKIAIEAAKGLSYLHHDCSPMIIHRDVKSNNILLNSELEAHVADFGLAKYFRNNGTSECMSAIAGSYGYIAPEYAYTLKIDEKSDVYSFGVVLLELITGRRPVGNFGEEGMDIVQWAKTETKWSKEGVVKILDERLKNVAIVEAMQVFFVAMLCVEEYSIERPTMREVVQMLSQAKQPNTFQIQ SEQ.ID.No.13TRL1 DNATCAAGCAACAATTTRTCAGGACAAATACCTTCAGGCTTGGCCAATGTGACCACACTGGCAGCATTTAACGTTTCTTTCAATAATCTGTCTGGGCCACTGCCTCTTAACAAAGATTTGATGAAGTGTAATAGTGTTCAGGGAAACCCCTTTCTGCAATCGTGCCATGTATTTTCTCTATCAACACCTTCTACAGATCAGCAGGGAAGAATAGGGGACTCACAAGATTCTGCTGCGTCTCCTTCAGGTTCAACCCAGAAAGGAGGGTGCAGCGGTTTCAACTCCATAGAGATTGCATCCATAACATCTGCGGCAGCTATTGTGTCAGTTCTTCTTGCTCTGATAGTCCTGTTCTTTTACACCAGAAAATGGAATCCAAGATCTAGAGTTGCTGGATCTACCAGGAAAGAAGTCACAGTGTTTACAGAAGTTCCGGTTCCTTTAACATTTGAAAATGTAGTGCGGGCCACAGAGATCTCAAATCAAGCAATGTGCTGTT

Claims (13)

1. an isolating nucleic acid construct thing is characterized in that, it contains the polymerized nucleoside acid sequence of disease resistence gene, and this polynucleotide contains the Xa21 gene, the Xa21 polypeptide shown in described Xa21 genes encoding SEQ.ID.No.2 or the SEQ.ID.No.4.

2. nucleic acid construct thing as claimed in claim 1 is characterized in that, the polymerized nucleoside acid sequence is the gene of total length.

3. nucleic acid construct thing as claimed in claim 1 is characterized in that described polymerized nucleoside acid sequence comprises the opening code-reading frame of SEQ.ID.No.1 or SEQ.ID.No.3.

4. nucleic acid construct thing as claimed in claim 1 is characterized in that, also contains the promotor of the polymerized nucleoside acid sequence that operationally is connected in disease resistence gene.

5. nucleic acid construct thing as claimed in claim 4 is characterized in that, promotor is an organizing specific type promotor.

6. nucleic acid construct thing as claimed in claim 4 is characterized in that promotor is a constitutive promoter.

7. a genetically modified vegetable cell is characterized in that it contains recombinant expression cassettes, and this expression cassette contains the plant promoter that operationally is connected in the described Xa21 polymerized nucleoside of claim 1 acid sequence.

8. vegetable cell as claimed in claim 7 is characterized in that plant promoter is an allogeneic promoter.

9. vegetable cell as claimed in claim 7 is characterized in that, this vegetable cell is the rice plants cell.

10. a method that increases plant to the Xanthomonas campestris resistance is characterized in that, this method comprises that with the recombinant expression cassettes introduced plant, this expression cassette contains the plant promoter that operationally is connected in the described disease resistence gene polymerized nucleoside of claim 1 acid sequence.

11. method as claimed in claim 10 is characterized in that, this plant tissue is from paddy rice.

13. method as claimed in claim 10 is characterized in that, this promotor is an organizing specific type promotor.

14. method as claimed in claim 10 is characterized in that, this promotor is a constitutive promoter.

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