CN101050232A - Pi15 resistance gene of rice blast, and application - Google Patents

Abstract

This invention discloses rice Magnaporthe grisea resistance gene Pi15 nucleotide sequence; amino acid polypeptide sequence coded by rice Magnaporthe grisea resistance gene Pi15, and its application. Magnaporthe grisea resistance gene Pi15 is a member of resistance gene family nonTIR-NBS-LRR, and is a constitutive expression gene. Magnaporthe grisea resistance gene Pi15 can be used to breed disease-resistant rice or plants through genetic transformation, or used as a molecular marker in plant breeding.

Description

Pi 15 resistance gene of rice blast and application thereof

Technical field

The present invention relates to the genetically engineered field, be specifically related to a kind of clone and application thereof of pi 15 resistance gene of rice blast.

Background technology

Plant usually is subjected to the infringement of multiple pathogen in the process of growth, plant then takes multiple defence policies to protect self, avoids being subjected to it to attack.A most important defense mechanism is exactly the defence answering system that can discern the existence of obligate pathogenic microorganism and start self in the plant.Plant is mediated by disease-resistant gene the identification of pathogenic bacteria.Therefore, the analysis of disease-resistant gene product structure and research are the bases of understanding plant disease-resistant mechanism, also have important directive significance for the prevention and the control of Plant diseases.

So far, from 3 kinds of monocotyledonss and 5 kinds of dicotyledonss, separated more than 40 disease-resistant gene.To the structure of these disease-resistant genes and discovering of product, though host plant difference, the pathogen of institute's antagonism also has fungi, bacterium, differences such as virus and nematode, but the structure of disease-resistant gene and product have many common constitutional featuress, there is rich leucine tumor-necrosis factor glycoproteins (leucine-rich repeat as the C-end, LRR), there is nucleotide binding site (nucleotidebinding site in the N-end, NBS), and leucine zipper (leucine zipper, LZ), coiled coil structural domain (coiled-coil, CC), and membrane spaning domain (transmembrane domain, TM), protein kinase (protein kinase, PK), and fruit bat Toll albumen and Mammals interleukin-1 receptor (Toll and interleukin-1 receptor, TIR) etc.According to they coded proteic constitutional featuress, disease-resistant gene can be divided into 7 class (Hammond-Kosack﹠amp; Jones, 1997; Dangl﹠amp; Jones 2001; Iyer﹠amp; McCouch 2004).

The first kind, toxin reduction enzyme disease-resistant gene.As corn disease-resistant gene Hm1, it is the 1st plant disease resistance genes that quilt is cloned, and it is responsible for the resistance to fungi Cochliobolus carbonum microspecies 1.Hm1 coding separate the HC toxin that toxenzyme can the passivation pathogenic fungi be produced, and the HC toxin is the virulence factor that fungi C.carbonum microspecies 1 produce, it determines this germ can only infect some genotype of corn (Johal etc., 1992).Second class, NBS-LRR class disease-resistant gene.The nearly N end of their encoded protein is NBS, and nearly C end then is made up of LRR.As RPS2 (Bent etc., 1994), RPM1 (Grant etc., 1995), I2 (Simon etc., 1998); RPP5 (Parker etc., 1997), N (Dodd etc., 2001), L6 (Lawrence etc., 1995), Mla1 (Zhou etc., 2001), Mla6 (Halterman etc., 2001); Disease-resistant gene of paddy rice such as Xa1 (Yoshimura etc., 1998), Pib (Wang etc., 1999), Pita (Bryan etc., 2000) etc.The 3rd class, PK class disease-resistant gene.As tomato Pto gene, its product is one and is positioned at intracellular serine-threonine protein kinase enzyme, do not have LRR structural domain (Martin etc., 1993).The 4th class, LRR-TM class disease-resistant gene.Gene C f-2 (Dixon etc., 1996), Cf-4 (Thomas etc., 1997), Cf-5 (Dixon etc., 1998), the Cf-9 (Jones etc., 1994) of the different physiological strains of the anti-leaf mold of tomato, and the gene Hs1 of the anti-Cyst nematode of beet ^Pro-1(Cai etc., 1997) etc.The 5th class, LRR-TM-PK class disease-resistant gene is representative with rice bacterial blight resistance gene Xa21 (Song etc., 1995).The 6th class is representative with the RPW8 of Arabidopis thaliana, and its encoded protein only contains complete CC and NBS structural domain (Xiao etc., 2001).The 7th class is representative with the xa5 gene of paddy rice, and its encoded protein is a transcription factor (TFIIA γ) (Iyer﹠amp; McCouch, 2004).

The disease-resistant proteic disease-resistant gene of coding NBS-LRR class is a class disease-resistant gene maximum in the plant disease resistance genes, constructional feature according to the disease-resistant protein N terminal of NBS-LRR class, this genoid can be divided into two big class TIR-NBS-LRR (TNL) and CC-NBS-LRR (CNL) (Meyers etc. again, Pan etc., 2000; Cannon etc., 2002; Richly etc., 2002).TNL class disease-resistant gene is mainly found in dicotyledons, does not also find (Bai etc., 2002 so far in the monocotyledons genome; Meyers etc., 2002).The disease-resistant gene that in monocotyledons, identifies the at present disease-resistant albumen of CNL class of mainly encoding, also there is a large amount of CNL class disease-resistant genes in the dicotyledons, comparatively speaking, the CNL class disease-resistant gene in the monocotyledons is than more rich and varied (Cannon etc., 2002) in the dicotyledons.

Studies show that NBS, CC, TIR structural domain may participate in signal conduction (Hammond-Kosack ﹠amp; Jones 1997), although this class R albumen does not have the intrinsic kinase activity, NBS can activate kinases or G albumen, NBS has in conjunction with ATP or GTP and hydrolytic enzyme activities (Traut, 1994).The signal conduction (Jones, 1994) in the disease-resistant defense response of TIR structure possibility involved in plant downstream.Nearest evidence shows that the L family diversity of flax selects to also occur in the TIR zone, and this zone forms specificity (Luck etc., 2000) with corresponding LRR zone coevolution.The function of LRR structural domain be mainly concerned with protein-protein and with interaction (the Jones ﹠amp of part; Jones, 1996; Kajava, 1998), be product or the indirect interactional position of product (Bent, 1996 that the disease-resistant gene product is direct and the pathogenic bacteria nontoxic gene is encoded by inference; Baker etc., 1997).Jia etc. (2000) are processed as one 176 amino acid whose activated protein AVR-Pita by yeast two-hybrid proof AVR-Pita encoded protein ₁₇₆The specific exciton of microspecies is delivered to plant cytoplasm and combines with the LRD zone of Pita acceptor specifically, thus the defense response of Pita mediation in the activating cells.Av-rPita after the Ala among the Pita becomes Ser ₁₇₆Can not combine with LRD, thereby show susceptible.This result has proved that directly the LRR structural domain may be exactly the zone of pathogenic bacteria identification; Mutual " gene pairs gene " relation of making to have verified from molecular level for the first time paddy rice and Pyricularia oryzae of Pita and AvrPita.

Paddy rice is one of most important food crop in the world, and it is staple food with rice that population over half is arranged approximately.(no condition: Pyriculariagrisea Sacc.) rice blast that causes is that Rice Production is endangered one of severe diseases, annually all causes serious grain loss by pathogenic fungi Magnapothe grisea Barr..From the viewpoint of Sustainable development of environment protection with agricultural, breed and the utilization of disease-resistant variety is the safe and effective procedure of control rice blast.But, because the diversity and the volatility of rice blast fungus population, people lack effective utilization of antagonism gene in addition, and antagonism mechanism lacks understanding fully, so that the susceptibleization problem of disease-resistant variety not only is not resolved, and becomes thremmatology man stubborn problem the most because of the shortage of effective anti-source gene and short-livedization of disease-resistant variety on the contrary.Therefore, excavate, identify and clone disease-resistant gene and reasonably be applied to the breeding for disease resistance plan and become the preferential major issue that solves in the agri-scientific research.

Along with molecular biological fast development, so far, have at least more than 80 rice blast master to imitate resistant gene and be in the news, wherein located for 60 by molecule.At present, also not identified the rice blast master on the 3rd karyomit(e) of paddy rice imitates the resistant gene, all identified the main resistant gene site of imitating on remaining 11 karyomit(e), and contain a plurality of rice blast resistances site on the karyomit(e) that has, and the resistant gene of some gene locus is that cluster exists.It is worthy of note in the localized resistant gene of numerous quilt successes, to have only two resistant genes of resistant gene Pib and Pita successfully to be cloned, they lay respectively on the 2nd karyomit(e) and the 12nd karyomit(e) of paddy rice.Before proposing, the present patent application also on paddy rice the 1st karyomit(e), do not clone the report of rice blast resistance gene.

Clone's disease-resistant gene is the prerequisite to the research of paddy rice resistance mechanism, discloses the molecule mechanism of rice anti-rice blast and can control and reduce the harm of Pyricularia oryzae to paddy rice better.Simultaneously,, can control and increase the disease resistance of plant artificially, widen the anti-spectrum of plant clone's the modification and the transformation of disease-resistant gene.These aspects are that employing conventional plant breeding and improving technology institute are inaccessiable.

Summary of the invention

A rice blast resistance gene that the objective of the invention is to carry among the separating clone rice varieties GA25 and the dna fragmentation that comprises the promotor of regulating and control this gene.

Another object of the present invention provides the coded protein of above-mentioned rice blast resistance gene.

Another object of the present invention provides the above-mentioned carrier that contains above-mentioned resistant gene.

Another object of the present invention provides above-mentioned carrier transgenic plant transformed.

Another object of the present invention provides the application of above-mentioned protein in preparation resisting rice blast bacteria medicine.

Further purpose of the present invention provides molecule marker that above-mentioned resistant gene produces and rice blast is had application in the paddy rice of disease resistance in seed selection.

The present invention relates to separate and use a kind of dna fragmentation of the Pi15 of comprising gene, this fragment is given plant the caused disease of Pyricularia oryzae (Magnaporthe grisea) is produced specific disease resistance response.This invention is applicable to all plants to this pathogenic bacteria sensitivity.These plants comprise monocotyledons and dicotyledons.Wherein, described fragment perhaps is equivalent to the dna sequence dna shown in the SEQ ID NO:1 basically shown in sequence table SEQ IDNO:1, and perhaps its function is equivalent to the subfragment of sequence shown in the SEQ ID NO:1.A kind of NBS-LRR proteinoid of this dna sequence encoding, its aminoacid sequence such as SEQ ID NO:2 are listed, and structure is as shown in Figure 6.Dna fragmentation shown in the present is constitutive expression at the leaf tissue of paddy rice.

The Pi15 resistant gene coding NBS-LRR albumen that separates, clones.This albumen comprises two main structural domain: NBS and LRR zone, and wherein the NBS structural domain contains conservative kinase 1a:IVGPVGFGKT, is positioned at 198-207 amino-acid residue of this polypeptide; Kinase 2a:LIIIDS is positioned at 275-280 amino-acid residue of this polypeptide; Kinase3a:SKIIVTTH is positioned at 303-310 amino-acid residue of this polypeptide; GLPL::GDPLFR is positioned at 347-352 amino-acid residue of this polypeptide; MHD:MHN is positioned at this polymorphic 503-505 amino-acid residue; And the 600-858 of this a proteic C-end amino-acid residue is 11 LRR repetitions, and its leucine content is 17.0%, is one section sequence that is rich in Ser immediately following the LRR multiple, and length is 168 amino-acid residues.

The nucleotide fragments of coding NBS or LRR may have independently function in the Pi15 gene.Fragment and the reorganization of other nucleic acid fragments with coding different structure territory in the Pi15 gene can constitute mosaic gene or protein, make it to have new function.The Pi15 gene is modified or transformed, can change or increase certain function of gene.For example, the LRR zone of this gene is replaced with the structural domain of other resistant genes, maybe the NBS structural domain with this gene carries out rite-directed mutagenesis, may cause the change of the anti-spectrum of the forfeiture of gene resistance or gene.

The present invention comprises that equally the primary structure part with the Pi15 resistant gene effectively connects the upward formed mosaic gene of proper regulation sequence, and comprises the plant of this gene and the seed of this kind of plant in genome.As this gene can be natural or chimeric.For example, will comprise the fragment of this gene and the promotor of a constitutive expression and be connected, this promotor can and be expressed under any condition cytocerastic any period.The promotor of this constitutive expression comprises the promotor of cauliflower mosaic virus 35S etc.On the other hand, also promotor that can the promotor of this gene and a tissue specific expression or developmental stage is specific expressed or accurately the promotor of environmental induction be connected, these promotors are referred to as inducible promoter.Like this, the change of environment, the difference of developmental stage can change this expression of gene, and is same, also this expression of gene can be limited in some tissues, makes by this gene induced disease resistance response and obtains artificial control.Wherein envrionment conditions comprises attack, anaerobic condition and the light etc. of pathogenic bacteria, and tissue and developmental stage comprise leaf, fruit, seed and flower etc.

According to Pi15 gene order information provided by the invention (SEQ ID NO:1), those skilled in the art can easily obtain the gene that is equal to Pi15 by the following method: (1) obtains by database retrieval; (2) with the Pi15 gene fragment be genomic library or the acquisition of cDNA library of probe Screening of Rice or other plant; (3) according to Pi15 gene order information design oligonucleotides primer, from genome, mRNA and the cDNA of paddy rice or other plant, obtain with the method for pcr amplification; (4) on the basis of Pi15 gene order, obtain with the gene engineering method transformation; (5) method with chemosynthesis obtains this gene.

Rice blast resistance gene P15 provided by the invention has important use and is worth.One of application is that described Pi15 gene order is connected to any plant conversion carrier, with any method for transformation the Pi15 disease-resistant gene is imported paddy rice or other plant cell, can obtain the transgenosis disease-resistant variety of expressing said gene, thereby be applied to produce.Of the present invention gene constructed in plant conversion carrier, can suitably modify described gene or its regulating and controlling sequence, also can before its transcription initiation codon, replace the original promotor of described gene, thereby widen and strengthen the resistance of plant pathogenic bacteria with other promotor.

The Another application of resistant gene provided by the invention is to produce specific molecule marker according to described gene order information, includes but not limited to SNP (mononucleotide polymorphic), SSR (simple sequence repeats polymorphic), RFLP (restriction enzyme length is polymorphic), CAP (the cutting amplified fragments is polymorphic).Can identify the resistant gene type of paddy rice or other plant with this mark, be used for the molecular marker assisted selection breeding, thereby improve the efficiency of selection of breeding.

The present invention has following beneficial effect: change clone's disease-resistant gene over to susceptible plant, help to produce new disease-resistant plants.Particularly can be with transformation technology a plurality of disease-resistant genes that in plant, add up, and can not produce the chain problem of bad gene in the genome of following appearance in the traditional breeding technology, and can shorten breeding time.The clone of disease-resistant gene can overcome the problem that can not shift disease-resistant gene in the traditional breeding method between plant species.

In addition, disease-resistant transfer-gen plant that the present invention can further provide or the above-mentioned dna fragmentation of applications exploiting obtains and corresponding seed, and with gene of the present invention or based on the recombinant chou plants transformed of this gene or the seed that obtains by this class plant.Can gene of the present invention be changed over to other plant with the mode of sexual hybridization.

Description of drawings

Fig. 1 is the map based cloning synoptic diagram of pi 15 resistance gene of rice blast;

Fig. 2 is the high-res heredity and the electronics physical map of pi 15 resistance gene of rice blast;

Fig. 3 is the predictive genes figure of Pi15 candidate region;

Fig. 4 be pi 15 resistance gene of rice blast the RNAi transformant based on the segmental PCR detected result of hygromycin gene figure;

Fig. 5 a is pi 15 resistance gene of rice blast RNAi T ₀The resistance of transformed plant is identified figure;

Fig. 5 b is pi 15 resistance gene of rice blast RNAi T ₁The expression analysis figure of transformed plant;

Fig. 6 is the allelic gene structure figure of the anti-sense of pi 15 resistance gene of rice blast;

Fig. 7 is a pi 15 resistance gene of rice blast amino acids coding peptide sequence structure iron;

Fig. 8 is that the RT-PCR of pi 15 resistance gene of rice blast and allelotrope pi15 expression characterization thereof detects figure;

The Pi15 loci gene type that Fig. 9 identifies parent and disease plant for molecule marker CRG4 is figure as a result;

Wherein, a represents the physical map of pi 15 resistance gene gene among Fig. 2, the recombination event that takes place between two marks of the numeral between mark; B represents to make up the contig of Pi15 gene physical map, and short sea line is represented the fine BAC/PAC clone of japonica rice variety Japan that IRGSP announces, vertical line is represented mark place clone's relative position.C represents the physical map of resistant gene Pi5 and Pi3 gene, the recombination event that takes place between two marks of the numeral between mark;

The arrow of band oblique line is represented the candidate gene predicted among Fig. 3;

Swimming lane 1 is molecular weight marker DL2000 among Fig. 4; The RNAi that swimming lane 2-8 is respectively pi 15 resistance gene transforms seedling S1, S2, S4, S5, S6, S7, the pcr amplification product of the hygromycin gene of R1; Swimming lane 9 is the non-transformant of acceptor Q3 (GA25); Swimming lane 10 is the clear water contrast.

GA25 is the donor kind of pi 15 resistance gene among Fig. 5 a, shows as disease-resistant; Remaining is the RNAi conversion seedling of pi 15 resistance gene.GA25-S1 wherein, GA25-S2, GA25-S3 and GA25-S4 show as susceptible; It is disease-resistant that GA25-S5 shows as moderate.

The 1st swimming lane is the DL2000 standard molecular weight among Fig. 5 b; The 2nd swimming lane is the Actin gene amplification product that comes genomic dna; The 3-9 swimming lane is the Actin gene amplification product from cDNA; The 10-16 swimming lane is the Pi15 gene amplification product from cDNA.

Green square box is represented the exon of pi 15 resistance gene among Fig. 6, and the square box of band oblique line is respectively 5 ' and 3 ' non-translational region, and fine rule is represented intron.Fig. 6 a is the gene structure figure of pi 15 resistance gene; Fig. 6 b is the gene structure figure from the susceptible allelotrope pi15 of kind LTH; Fig. 6 c is the gene structure figure from the susceptible allelotrope pi15 of kind Q61.

3 zones that the letter of band underscore is inferred NBS for composition among Fig. 7, and GLPL zone and MHD zone; The amino-acid residue that independently list the lower section is the LRR zone and is rich in the Ser sequence.

Fig. 8 the 1st swimming lane is the DL2000 standard molecular weight; The 2nd, 6,10 swimming lanes are the RT-PCR amplified production of pi 15 resistance gene; The 2nd, 7,11 swimming lanes are the RT-PCR product from the susceptible allelotrope pi15 of kind LTH; The 4th, 8,12 swimming lanes are the RT-PCR product from the susceptible allelotrope pi15 of kind Q61; The 5th, 8,13 swimming lanes are the RT-PCR product from the susceptible allelotrope pi15 of Q1063; The 14th swimming lane is the contrast from genomic dna.

Q13 is susceptible parent among Fig. 9; GA25 is disease-resistant parent; R1-R7 is disease-resistant individuality; S1-S15 is disease-resistant individuality; The arrow indication is mark CRG4.

Embodiment

Embodiment 1: the genetic analysis of pi 15 resistance gene of rice blast and Primary Location

The present invention is to 3 F by the cross combination origin of japonica rice disease-resistant variety GA25 and 3 long-grained nonglutinous rice susceptible variety AS20-1, IR36 and Q61 ₂Colony inoculates bacterial strain CHL0416 and CHL0670 to the clearly demarcated non-affinity/compatible host response of parents' kind performance respectively, and the result shows this 3 F ₂Disease-resistant plant was all controlled by a pair of dominant gene with separating than all meeting 3R:1S, infer GA25 thus the resistance to above-mentioned 3 inoculating strains that is showed of disease plant in the colony.To these 3 F ₂The resistant gene of colony's correspondence carries out linkage analysis, and the result shows that they are controlled by same dominant gene, has therefore made up from these 3 F ₂Colony's origin, totally 76 disease-resistant individualities and 504 big mapping populations that susceptible individual is formed.

Utilize RAPD (random amplified polymorpgic DNA, RAPD) and STS (sequence tagged site) molecular marking technique, in conjunction with based on segregating population analytical method (bulked-sgregant analysis, BSA) recessive population analysis method (recessive-classanalysis, RCA), this goal gene has been carried out linkage analysis.The result shows, goal gene Pi15 is positioned the interval of about 0.7cM on the 9th karyomit(e), wherein RAPD mark BAPi15 ₇₈₂And BAPi15 ₄₈₆With the Pi15 close linkage, genetic distance is respectively 0.35cM and 1.1cM.In order to determine the chromosomal region of Pi15 gene on the reference kind is Japanese fine apace, we are to RAPD mark BAPi15 ₇₈₂And BAPi15 ₄₈₆Clone and check order, and utilize bioinformatic analysis tools BLAST N to carry out homology analysis and find RAPD mark BAPi15 ₇₈₂Be positioned on the 9th karyomit(e) RGP pac clone AP005811, its physical location is 52485-53000, RAPD mark BAPi15 ₄₈₆Land on the 9th chromosomal BAC clone AP005879, its physical location is 79624-79106.

Embodiment 2: the structure that covers the electronics contig in Pi15 site

Because RAPD mark BAPi15 ₇₈₂And BAPi15 ₄₈₆With Pi15 site close linkage, therefore, after the RAPD mark has been realized chromosome landing, with the fine genome sequence of reference kind Japan as a reference, made up the contig that covers the Pi15 site, it is by 4 (Fig. 2) that the clone is constituted such as BAC clone AP005879, pac clone AP005593, AP005811 and AP005700.

Embodiment 3: the Fine Mapping of pi 15 resistance gene of rice blast

For further Fine Mapping Pi15 site, we are according to the contig that has made up, in the goal gene regionl development mark of PCR-based technology.Utilize online Software tool ORF Finder and BLASTP that this regional sequence is searched for, obtained 9 ORF with LRR or NBS structure.Then, these 9 ORF as CRG, have been designed 9 pairs of Auele Specific Primers at its flanking sequence separately and carried out pcr amplification respectively.Original adoption the rTaq archaeal dna polymerase all can't amplify band.Process is groped, and we adopt cocktail, and Ex Taq and Probest Taq archaeal dna polymerase are combined use, solve unmatched problem between primer and the template DNA preferably, make each CRG all obtain amplification.The result shows: CRG1, and CRG2, CRG3, CRG4,6 CRG marks such as CRG5 and CRG6 show specificity (target stripe only occurring) preferably, and 3 other CRG marks are then abandoned because of its specificity is bad.In the good CRG mark of 6 specificitys, mark CRG1 shows polymorphism between anti-sense parent; And mark CRG2, CRG3, CRG4, CRG5 and CRG6 then also show good polymorphism (table 1) after enzyme is cut.

On the other hand, at BAPi15 ₄₈₆And between the CRG6, existing a SSR mark RM7364, it is positioned on the RGP pac clone AP005593.Therefore, SSR mark RM7364, together with above 6 polymorphism mark CRG1, CRG2, CRG3, CRG4, CRG5 and CRG6 together, (Fig. 2 is a) to be used for next step linkage analysis.

At first, we use SSR mark RM7364 and CRG mark CRG1 to 504 extremely susceptible F ₂Individuality has carried out linkage analysis.The result shows, has occurred 6 recombinant chous between Pi15 site and the mark RM7364, and genetic distance is 0.6cM; And 7 reorganization have taken place between Pi15 and the mark CRG1, genetic distance is 0.7cM.Because at the detected recombinant chou in mark RM7364 site and at the detected recombinant chou in mark CRG1 site is diverse, can judge in view of the above, mark RM7364 and CRG1 lay respectively at the both sides in Pi15 site, wherein RM7364 is positioned at the Pi15 site near a centric side, and CRG1 then is positioned at the side of Pi15 site near telomere.So 13 recombinant chous between RM7364 and CRG1 are used to next step linkage analysis (table 2).The result shows, 5 reorganization have taken place between CRG6 and the Pi15 site one side in the kinetochore, and genetic distance is 0.5cM (table 2); 4 reorganization have taken place between CRG5 and the Pi15 site, and genetic distance is 0.4cM (table 2).And in telomere one side, the recombinant chou in CRG2 and Pi15 site is reduced to 1, and genetic distance is 0.1cM (table 2).Utilize CRG3 and CRG4 to proceed chromosome walking, find they and Pi15 site be divided into fully from, that is (table 2) takes place in reorganization.Therefore explanation, chromosome walking has ideally arrived terminal point (Fig. 2).This just means that also the Pi15 site is defined between CRG5 and the CRG2, and genetic distance therebetween is 0.5cM.According to the result of chromosome walking, can infer that in 6 analyzed CRG, having only CRG3 and CRG4 may be the candidate gene of Pi15.

According to the genetic distance between Pi15 and Pi5 and the grappling mark G103, Pan etc. (2003) have integrated and have contained Pi15, Pii, the genetic map of the resistant gene of Pi5 and Pi3 bunch.Because the P.C.Ronald study group of Univ California-Davis USA has also made up the physical map (Jeon, 2003) of Pi5/Pi3.Therefore, be necessary it is incorporated into the electronics physical map in the Pi15 site of this research and establishment.Analyze by Paiwise BLAST, will with chain 5 marks of Pi5 land on contig (Fig. 2 c).This figure shows, although Pi15 and Pi5/Pi3 are inconsistent with respect to the genetic distance between the G103, and, they geographically but are that partly overlapping (Fig. 2 is a).

Embodiment 4: the predictive genes in goal gene zone

Do not slip through the net in order to guarantee candidate's goal gene to greatest extent, the zone of being defined with the nearest flank mark of Pi15 gene locus (producing the mark of minimum recombinant chou) is as goal gene zone (Fig. 3), utilize the fine sequence of order-checking kind Japan as the reference sequence, carried out the forecast analysis of candidate's goal gene by 3 kinds of gene annotation softwares.

According to the note of Gramene Software tool, 6 genes are contained in the goal gene zone, for the convenience of narrating, with they difference called after R15-a, R15-b, R15-c, R15-d, 6 genes such as R15-L1 and R15-L2.Wherein, R15-L1 (being positioned at the 50-56kb place) for having the resistant gene of inferring of NBS-LRR structure, is labeled the grappling of CRG5 institute; R15-L2 (being positioned at the 57-66kb place) for have LRR and protein kinase (protein Kinase, PK) infer resistant gene, be labeled the grappling of CRG4 institute.Since CRG3 and CRG4 and Pi15 site be divided into fully from, and have the conserved structure of resistant gene, so R15-L1 and R15-L2 are confirmed as the candidate gene (Fig. 3) of Pi15.

On the other hand, according to the prediction of RiceGAAS and Softberry Software tool, there are 6 genes in the goal gene zone, and wherein, R15-L3 (being positioned at the 50-64kb place) has the resistance protein gene in NBS-LRR zone for coding.Because R15-L3 has comprised the R15-L1 of Gramene institute note and the zone at R15-L2 place, and by the common grappling of CRG3 and CRG4 institute.Therefore, R15-L3 also is confirmed as the candidate gene (Fig. 3) of Pi15.

In sum, be reference with the fine sequence of Japan, there are 3 genes to be confirmed as the candidate gene of Pi15 in the candidate region, they are R15-L1, R15-L2 and R15-L3.Wherein, candidate gene R15-L3 has comprised 2 candidate gene R15-L1 and R15-2.

Embodiment 5: the affirmation of candidate's goal gene

In order to confirm the result of predictive genes, designed Auele Specific Primer at the exon region of candidate gene and carried out the RT-PCR amplification and analyze.Particularly, with genomic dna in contrast, still be the PCR product of cDNA to distinguish from genomic dna; Select the positive internal reference of actin, to detect the quality that the RNA reverse transcription becomes cDNA.Because candidate gene R15-L3 region has comprised R15-L1 and two genes of R15-L2, so we have designed a pair of primer, forward primer is positioned on the exon of R15-L1, reverse primer is positioned on the exon of R15-L2, candidate gene R15-L3 has been carried out RT-PCR, and amplified production cloned and checked order.The result shows that this amplified production is the sequence of goal gene for the big or small 1100bp of expection, illustrates that predicting the outcome of RiceGAAS and Softberry Software tool is correct, and R15-L3 is the candidate gene of pi 15 resistance gene.

The acquisition of embodiment 6:Pi15 full length gene cDNA and the derivation of gene structure

Employing is moved one's steps, and method is anti-to Pi15, the allelic dna sequence dna of sense is measured.Utilize 5 ' and 3 ' RACE reaction, obtained that Pi15 is anti-, the allelic full-length cDNA of sense and it is checked order.Sequence alignment is the result show, the pi 15 resistance gene full-length cDNA is 3505bp, and the coding region is 3078bp, contain 5 exons, size is respectively 314bp, 1151bp, 1317bp, 160bp and 137bp, and contain 4 introns, size is respectively 976bp, 198bp, 454bp and 862bp, 5 ' and 3 ' non-translational region are respectively 144bp and 296bp (Fig. 6).

Susceptible allelotrope pi15 full-length cDNA from LTH is 3655bp, contain 4 exons, size is respectively 314bp, 2662bp, 160bp and 137bp, and contain 3 introns, size is respectively 971bp, 442bp and 862bp (Fig. 6), wherein, " TAA " that exist on the 2nd exon causes translating premature termination.

Susceptible allelotrope pi15 full-length cDNA from Q61 is 2511bp, contains 5 exons, and size is respectively 314bp, 1147bp, 510bp, 243bp, 320bp and 137bp, and contain 5 introns, size is respectively 971bp, 198bp, 563bp, 443bp and 862bp (Fig. 6), wherein, " TGA " that exist on the 3rd exon causes translating premature termination.

Explanation thus, the different cut modes of mRNA have caused the premature termination of protein translation in the susceptible variety, thereby have caused the difference of anti-sense phenotype.

The expression analysis excessively of embodiment 7:Pi15 gene

Full length cDNA sequence design 1 pair of primer (comprise respectively and contain Kpn I and Sal I restriction enzyme site) according to the Pi15 gene, cDNA to GA25 increases, obtain the complete coding region of Pi15, and be cloned among the binary vector pCAMBIA1300S, then, imported agrobacterium strains EHA105.Will height susceptible variety Q1063 mature seed evoked callus on inducing culture.The EHA105 that contains the goal gene conversion carrier was cultivated 1 day for 28 ℃ at the YM nutrient agar, be collected in the MB liquid nutrient medium of the Syringylethanone that contains 100 μ mol/L.The rice callus tissue was immersed bacterium liquid 20 minutes, blot and transfer to the MB nutrient agar behind the bacterium liquid and cultivate.Shift callus to the culture medium culturing that contains the 50mg/L Totomycin, subculture was 1 time in per 14 days, subculture 2 times.Behind the resistance screening, change regeneration culture medium over to and differentiate the conversion seedling, obtained 140 strains of conversion seedling respectively.Resistance qualification result show candidate gene R15-L3 has showed the identical resistance with resistant gene donor GA25 under susceptible variety Q1063 genetic background, show that pi 15 resistance gene has successfully been cloned.

The RNAi of embodiment 8:Pi15 gene analyzes

The RT-PCR product cloning of above-mentioned R15-L3 to binary vector pCAMBIA1300RS, has been imported agrobacterium strains EHA105.With disease-resistant variety GA25 mature seed evoked callus on inducing culture.The EHA105 that contains the goal gene conversion carrier was cultivated 1 day for 28 ℃ at the YM nutrient agar, be collected in the MB liquid nutrient medium of the Syringylethanone that contains 100 μ mol/L.The rice callus tissue was immersed bacterium liquid 20 minutes, blot and transfer to the MB nutrient agar behind the bacterium liquid and cultivate.Shift callus to the culture medium culturing that contains the 50mg/L Totomycin, subculture was 1 time in per 14 days, subculture 2 times.Behind the resistance screening, change regeneration culture medium over to and differentiate the conversion seedling, obtained 60 strains of conversion seedling respectively.The resistance qualification result shows have been lost the resistance of resistant gene donor GA25 by transformed plant (Fig. 5 a) has shown that also pi 15 resistance gene has successfully been cloned.

The PCR that transformed plant has been carried out hygromycin gene identifies (Fig. 4).Detected result proof target gene fragment has imported these transformant.To these T ₁The expression analysis of transformed plant finds that Pi15 expression of gene amount is lower than disease-resistant plant (Fig. 5 b) in the disease plant.

The structure of embodiment 9:Pi37 resistance protein

The protein sequence of Pi15 genes encoding is shown in SEQ ID NO:2 in the sequence table.The Pi15 resistant gene coding NBS-LRR albumen that separates, clones.This albumen comprises two main structural domain: NBS and LRR zone, and wherein the NBS structural domain contains conservative kinase 1a:IVGPVGFGKT, is positioned at 198-207 amino-acid residue of this polypeptide; Kinase 2a:LIIIDS is positioned at 303-310 amino-acid residue GLPL:GDPLFR that 275-280 amino-acid residue kinase 3a:SKIIVTTH of this polypeptide be positioned at this polypeptide and is positioned at 347-352 amino-acid residue MHD:MHN of this polypeptide and is positioned at this polymorphic 503-505 amino-acid residue; And the 600-858 of this a proteic C-end amino-acid residue is 11 LRR repetitions, and its leucine content is 17.0%, is one section sequence that is rich in Ser immediately following the LRR multiple, and length is 168 amino-acid residues (Fig. 7).

Embodiment 10:Pi15 expression of gene specificity analysis

With RT-PCR Pi15 expression of gene pattern is analyzed.From disease-resistant variety GA25 and susceptible variety LHT, extract total RNA in the blade of Q61 and Q1063, utilize reverse transcription test kit SuperScript ^TMReverse Transcriptase II carries out the synthetic of reverse transcription cDNA article one chain.The RT-PCR primer is

L2RTF:5 '-CCAGCAGTTTAAGATCAG-3 ' and

L2RTR：5’-GGTGAAGCTGTGCTGCAATTTC-3’。The PCR reaction is: 94 ℃ of pre-sex change 4min; Next be 35 circulations, cycling program is as follows: 94 ℃ of sex change 30sec, and 56 ℃ of annealing 30sec, 72 ℃ are extended 1min; Last 72 ℃ are extended 7min, and temperature drops to 4 ℃ and promptly finishes amplification.Experimental result shows, the RNA reverse transcription template of the leaf tissue before and after the inoculation of resistant variety and susceptible variety all can amplify special fragment, illustrate that Pi15 and allelotrope pi15 thereof all can express in leaf tissue, promptly belong to the gene (Fig. 8) of constitutive expression.

The application of embodiment 11:Pi15 gene order in the molecular marker assisted selection breeding

Utilize Pi15 gene order information provided by the invention can produce molecule marker, be used to identify that the range gene type that has on the Pi15 site is the plant of Pi15Pi15, Pi15pi15 and pi15pi15, (table 1 can be applied in the molecular marker assisted selection breeding process; Fig. 9).

Embodiment 12: the resistance feature of pi 15 resistance gene

Pi 15 resistance gene is to separating the response type from Thailand, Japan and Chinese rice blast fungi isolates, significantly with Piz, and Piz ^t, Piz ⁵And Pik ^mDifferent etc. main disease-resistant gene, therefore, Pi15 can be used as replenishing of these high resistance ospc genes, is widely used in breeding for disease resistance (table 3) in the works.

Table 1 is at Pi15 candidate resistance gene (candidate resistatance gene, the CRG) mark of target region exploitation

The mark title	Primer sequence (5 '-3 ') a	Annealing temperature (℃)	Enzyme	Testing conditions b
					CRG1 CRG2 CRG3 CRG4 CRG5 CRG6 CRG7 CRG8 CRG9	F：ctctagcagacatgggaccttac R：gcccattcaactgagattgtccc F：cacaggacactcaacccttcac R：tgtatcagcacttcagcaggc F：cggacctgtcattgcctttg R：accttccttggctccttgatgc F：ggcgatgagagctggtttcctg R：cctccgcagaatgatgcagaaag F：gaggcagagcaggcaatggcaac R：ctatcgcgtggtcagcaagaaggc F：agcgcgtcgtccagatcgtagc R：gagatacgcttggatgtggtgg F：ccaccgggcagccagatgatc R：cgccaggtcgagactttgagc F：cgcctccgtacgcctctctat R：catccccgcgtgcaataaagta F：cgccgactaatcgaacgaagact R：ccatccacatccacatccgtatc	60 56 58 60 64 62 60 66 50	-- Hinf I Hinf I Dra I Alu I HaeIII/HhaI -- -- --	A B B B B A - - -

^aF: forward primer; R: reverse primer; ^bThe A=2% sepharose; The B=6% polyacrylamide gel.

The recombinant chou of 13 goal gene flanks of table 2 is in the gene type assay of 7 marker sites of target region

Recombinant type b	Mark a
								RM7364	CRG6	CRG5	CRG4	CRG3	CRG2	CRG1
	A B C D E	H H H S S	H H S S S	H S S S S	S S S S S	S S S S S	S S S S H								S S S H H

^aS: genotype is that susceptible parent is homozygous; H: genotype is parents' a heterozygous;

^bA: recombinant chou I275, I285, I356 and I379; B: recombinant chou I55; C: recombinant chou I93; D: recombinant chou I182, I230, I260, I261, I410 and I528; E: recombinant chou I393.

Table 3 pi 15 resistance gene is to the anti-spectrum analysis of 10 Pyricularia oryzae bacterial strains from Japan, Thailand and China

Cultivar	Gene	Japanese isolates a			Thai isolates			Chinese isolates
												Hoku
		1	Ai75-61	Ai74-134	THL63	THL156	THL170	CHL0416	CHL0540	CHL0638	CHL0668
GA25 Fujisaka
	5 C104PKT Fukunishiki Toride 1 C101A51 PiNo.4 Tsuyuake	Pi15 Pii Pi3 Piz Piz t Piz 5 Pita 2 Pik m	R b S NT c R R NT R R	R R NT R S NT R S	R S NT S S NT R S	S S S R R R R S	S S S R R R R R	R S S R R R R S	R R S S S R S S	R R S R R S R R	R R S R S R R R	R R S R S R S R

a，Isolates selected from Japan，Thailand，and China were tested in respectivecountry in 1996，1998，and 2001.

b，R，resistant，S，susceptible.

c，NT，not tested.

Pi 15 resistance gene of rice blast and application sequence table thereof

SEQUENCE LISTING

＜110〉Agricultural University Of South China

＜120〉pi 15 resistance gene of rice blast and application thereof

<130>

<160>2

<170>PatentIn version 3.2

<210>1

<211>3505

<212>Full length cDNA

＜213〉Oryza paddy rice (Orysa sativa L.)

<220>

<221>5’UTR

<222>(1)..(143)

<220>

<221>exon

<222>(144)..(3221)

<220>

<221>3’UTR

<222>(3222)..(3505)

<400>1

TCGACGATTC GACTGGAGCA CGGGACACTG ACATGGACTG AAGGAGTAGA AAACCTTCTC 60

TCTTCTCGTT CTCCTATTTC ACAAATCACA ACCGGATTGC TTTCTTCCTT CCTCCGGTCT 120

GGTCCCCAAC CTCCGCGGCA GCC ATG GTT GGC GCC GAG ATG CTT GTG GCC GCG 173

Met Val Gly Ala Glu Met Leu Val Ala Ala

5 10

GCG GTG AGC CAG GTC GCC CGG AAG ATC MAC GAC ATC GTG GGG GTC GCG 221

Ala Val Ser Gln Val Ala Arg Lys Ile Asn Asp Ile Val Gly Vsl Ala

15 20 25

CAG GGC GAG GTG AAG CTG TGC TGC AAT TTC AGC GAC GAT TTG GAG GGC 269

Gln Gly Glu Val Lys Leu Cys Cys Asn Phe Ser Asp Asp Leu Glu Gly

30 35 40

ATC AAG GAT ACC CTT GTG TAC CTG GAA ACC TTG CTG AAA AAT GCG GAG 317

Ile Lys Asp Thr Leu Val Tyr Leu Glu Thr Leu Leu Lys Asn Ala Glu

45 50 55

AAT AAC TCC TTC GGA AGC GAC AGG GCC AAC CTG CGC CAC TGG CTT GGC 365

Asn Asn Ser Phe Gly Ser Asp Arg Ala Asn Leu Arg His Trp Leu Gly

60 65 70

CAG ATC AAG TCC CTG GCT TAC GAT ATC GAA GAT ATC GTT GAT GGG TAC 413

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

75 80 85 90

TAC TCT TCC AAG GAG CAG TTC GAT GGG GGC AGC TAT GCA CAG AAG GGG 461

Tyr Ser Ser Lys Glu Gln Phe Asp Gly Gly Ser Tyr Ala Gln Lys Gly

95 100 105

TCA TTA TTC TGC TCG CTA TCC AAT CCA ATG CTT CTG AAA GGT AGC ATG 509

Ser Leu Phe Cys Ser Leu Ser Asn Pro Met Leu Leu Lys Gly Ser Met

110 115 120

GTT TAT AAG ATG AAA TCC AAG AGA GAG ATG CTA CAG CAA AGC CAA CAG 557

Val Tyr Lys Met Lys Ser Lys Arg Glu Met Leu Gln Gln Ser Gln Gln

125 130 135

TTG CCC AAT CAG TAT CAT TTC CTT TCA TAT ATC AAT TCA GCT GTG CAT 605

Leu Pro Asn Gln Tyr His Phe Leu Ser Tyr Ile Asn Ser Ala Val His

140 145 150

TAT TTT GAG GAG AAG CAA ACA ACA TCA TAC AGA AAT ACT GAC ATT GCA 653

Pi 15 resistance gene of rice blast and application sequence table thereof

Tyr Phe Glu Glu Lys Gln Thr Thr Ser Tyr Arg Asn Thr Asp Ile Ala

155 160 165 170

ATT GTC GGG AGG GAT GCT GAT TTG GAT CAT CTC ATG GAT CTT TTA ATG 701

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

175 180 185

CAA AAC AGC GCT GAA GAG CTT TGT ATT ATA CCC ATA GTT GGG CCT GTA 749

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

190 195 200

GGT TTT GGA AAG ACA AGC CTT GCA CAG TTA GTT TTC AAT GAT ACA AGA 797

Gly Phe Gly Lys Thr Ser Leu Ala Gln Leu Val Phe Asn Asp Thr Arg

205 210 215

ACA GAG GTA TTC AGC TTT AGG ATA TGG GTT CAT GTT TCC ATG GGT AAT 845

Thr Glu Val Phe Ser Phe Arg Ile Trp Val His Val Ser Met Gly Asn

220 225 230

ATC AAC CTT GAA AAA ATT GGG AGA GAT ATA GTT TCA CAA ACT ACA GAA 893

Ile Asn Leu Glu Lys Ile Gly Arg Asp Ile Val Ser Gln Thr Thr Glu

235 240 245 250

AAA ATT GAG GGA AAT ATG CAG CTG CAG TCA ATC AAG AAT GCT GTT CAG 941

Lys Ile Glu Gly Asn Met Gln Leu Gln Ser Ile Lys Asn Ala Val Gln

255 260 265

CGT GTG CTA AAT AAA TAT AGT TGC TTG ATC ATA ATA GAC AGC CTT TGG 989

Arg Val Leu Asn Lys Tyr Ser Cys Leu Ile Ile Ile Asp Ser Leu Trp

270 275 280

GGA AAG GAT GAA GAA GTG AAT GAA TTG AAG CAG ATG TTG CTT ACA GGT 1037

Gly Lys Asp Glu Glu Val Asn Glu Leu Lys Gln Met Leu Leu Thr Gly

285 290 295

AGA CAC ACA GAA AGC AAG ATC ATA GTG ACC ACT CAT AGC AAT AAA GTA 1085

Arg His Thr Glu Ser Lys Ile Ile Val Thr Thr His Ser Asn Lys Val

300 305 310

GCT AAG CTG ATT TCC ACC GTT CCA CTG TAC AAG TTG GCA GCT TTA TCT 1133

Ala Lys Leu Ile Ser Thr Val Pro Leu Tyr Lys Leu Ala Ala Leu Ser

315 320 325 330

GAG GAT GAT TGT TTA AAA ATA TTC TCT CAA AGG GCA ATG ACA GGT CCG 1181

Glu Asp Asp Cys Leu Lys Ile Phe Ser Gln Arg Ala Met Thr Gly Pro

335 340 345

GGT GAC CCG TTG TTC AGG GAA TAT GGA GAA GAA ATC GTT AGA AGG TGT 1229

Gly Asp Pro Leu Phe Arg Glu Tyr Gly Glu Glu Ile Val Arg Arg Cys

350 355 360

GAA GGC ACA CCC TTG GTA GCC AAT TTT CTC GGT TCT GTG GTG AAT GCT 1277

Glu Gly Thr Pro Leu Val Ala Asn Phe Leu Gly Ser Val Val Asn Ala

365 370 375

CAA CGA CAA AGG CGT GAG ATT TGG CAA GCT GCA AAG GAT AAA GAA ATG 1325

Gln Arg Gln Arg Arg Glu Ile Trp Gln Ala Ala Lys Asp Lys Glu Met

380 385 390

TGG AAG ATA GAG GAA GAT TAT CCC CAA GAC AAA ACT TCA CCA CTA TTT 1373

Trp Lys Ile Glu Glu Asp Tyr Pro Gln Asp Lys Thr Ser Pro Leu Phe

395 400 405 410

CCA TCA TTC AAG ATA ATA TAT TAT AAT ATG CCC CAT GAG CTA AGA TTA 1421

Pro Ser Phe Lys Ile Ile Tyr Tyr Asn Met Pro His Glu Leu Arg Leu

415 420 425

TGC TTT GTA TAT TGT TCA ATC TTC CCT AAA GGA ACT GTT ATA GAA AAG 1469

Cys Phe Val Tyr Cys Ser Ile Phe Pro Lys Gly Thr Val Ile Glu Lys

430 435 440

AAG AAA CTT ATT CAG CAA TGG ATT GCA CTT GAC ATG ATT GAG TCC AAA 1517

Leu Ile Gln Gln Trp Ile Lys Lys Ala Leu Asp Met Ile Glu Ser Lys

445 450 455

Pi 15 resistance gene of rice blast and application sequence table thereof

CAT GGA ACC TTG CCA CTT GAT GTA ACT GCG GAG AAA TAT ATT GAT GAA 1565

His Gly Thr Leu Pro Leu Asp Val Thr Ala Glu Lys Tyr Ile Asp Glu

460 465 470

CTT AAA GCA ATC TAT TTC CTT CAA GTT TTA GAG CGG TCT CAG AAT GAT 1613

Leu Lys Ala Ile Tyr Phe Leu Gln Val Leu Glu Arg Ser Gln Asn Asp

475 480 485 490

GCA GAA AGA TCC AGT GCT TCT GAG GAA ATG CTT CGC ATG CAT AAC TTG 1661

Ala Glu Arg Ser Ser Ala Ser Glu Glu Met Leu Arg Met His Asn Leu

495 500 505

GCT CAT GAT CTT GCT AGA TCG GTT GCT GGT GAA GAT ATC CTT GTT ATT 1709

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

510 515 520

TTA GAT GCC GAG AAC GAG CGC AAC GCC AGA TAT TGC GAT TAC CGT TAT 1757

Leu Asp Ala Glu Asn Glu Arg Asn Ala Arg Tyr Cys Asp Tyr Arg Tyr

525 530 535

GCA CAG GTG TCT GCT TCT AGT TTA GAG CCA ATC GAT CGC AAG GCA TGG 1805

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

540 545 550

CCT TCC AAG GCA AGG TCA CTA ATT TTC AAG AAT AGT GGT GCG GAC TTT 1853

Pro Ser Lys Ala Arg Ser Leu Ile Phe Lys Asn Ser Gly Ala Asp Phe

555 560 565 570

GAG CGT GTC AGT GAA GTT CTT TCA GTG AAC AAA TAC CTG CGT GTT TTG 1901

Glu Arg Val Ser Glu Val Leu Ser Val Asn Lys Tyr Leu Arg Val Leu

575 580 585

GAT CTC AGT GGA TGT TGT GTT CAA GAT ATT CCA TCT CCT ATC TTT CAG 1949

Asp Leu Ser Gly Cys Cys Val Gln Asp Ile Pro Ser Pro Ile Phe Gln

590 595 600

CTG AAA CAA TTG AGA TAC CTC GAC GTT TCA TCT TTA TCT ATT ACA GCA 1997

Leu Lys Gln Leu Arg Tyr Leu Asp Val Ser Ser Leu Ser Ile Thr Ala

605 610 615

CTC CCT CTG CAA ATT AGT AGC TTT CAT AAG TTA CAA ATG TTG GAT CTT 2045

Leu Pro Leu Gln Ile Ser Ser Phe His Lys Leu Gln Met Leu Asp Leu

620 625 630

TCA GAA ACT GAA CTA ACA GAG TTG CCA CCC TTT ATA AGC AAC TTA AAA 2093

Ser Glu Thr Glu Leu Thr Glu Leu Pro Pro Phe Ile Ser Asn Leu Lys

635 640 645 650

GGA CTG AAT TAT TTG AAT CTC CAA GGT TGC CAG AAA CTT CAA CGA TTG 2141

Gly Leu Asn Tyr Leu Asn Leu Gln Gly Cys Gln Lys Leu Gln Arg Leu

655 660 665

AAT AGC CTT CAT TTG TTG CAT GAT CTA CAT TAC CTA AAC TTG TCA TGC 2189

Asn Ser Leu His Leu Leu His Asp Leu His Tyr Leu Asn Leu Ser Cys

670 675 680

TGC CCT GAA GTT ACT AGT TTT CCT GAA TCT ATT GAA AAT CTG ACC AAA 2237

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

685 690 695

CTC CGT TTC TTG AAT CTT TCT GGA TGC TCT AAG CTT TCA ACA TTA CCT 2285

Leu Arg Phe Leu Asn Leu Ser Gly Cys Ser Lys Leu Ser Thr Leu Pro

700 705 710

ATC AGA TTT TTG GAA TCA TTT GCT AGC CTC TGT TCT TTG GTA GAT CTT 2333

Ile Arg Phe Leu Glu Ser Phe Ala Ser Leu Cys Ser Leu Val Asp Leu

715 720 725 730

AAC TTA AGT GGC TTT GAA TTC CAA ATG TTG CCC GAC TTT TTT GGC AAC 2381

Asn Leu Ser Gly Phe Glu Phe Gln Met Leu Pro Asp Phe Phe Gly Asn

735 740 745

ATA TAT TCA CTT CAG TAT TTA AAT CTG TCA AAA TGT TTG AAA CTT GAG 2429

Ile Tyr Ser Leu Gln Tyr Leu Asn Leu Ser Lys Cys Leu Lys Leu Glu

Pi 15 resistance gene of rice blast and application sequence table thereof

750 755 760

GTA TTA CCA CAA TCT TTT GGC CAA CTT GCA TAT CTG AAA AGC CTA AAT 2477

Val Leu Pro Gln Ser Phe Gly Gln Leu Ala Tyr Leu Lys Ser Leu Asn

765 770 775

CCT TCA TAT TGT TCT GAT CTT AAA CTG CTG GAA TCC TTT GAA TGC CTT 2525

Pro Ser Tyr Cys Ser Asp Leu Lys Leu Leu Glu Ser Phe Glu Cys Leu

780 785 790

ACC TCT CTT CGG TTT TTG AAT CTC TCG AAC TGC TCT AGG CTT GAA TAT 2573

Thr Ser Leu Arg Phe Leu Asn Leu Ser Asn Cys Ser Arg Leu Glu Tyr

795 800 805 810

TTG CCG TCG TGC TTT GAC AAG CTT AAT AAT TTA GAG TCT CTG AAT TTA 2621

Leu Pro Ser Cys Phe Asp Lys Leu Asn Asn Leu Glu Ser Leu Asn Leu

815 820 825

TCA CAA TGT CTT GGA CTT AAA GCA CTA CCT GAA TCA CTT CAA AAC CTT 2669

Ser Gln Cys Leu Gly Leu Lys Ala Leu Pro Glu Ser Leu Gln Asn Leu

830 835 840

AAA AAT CTT CAG CTT GAT GTT TCT GGG TGT CAG GAT TGT ATA GTA CAA 2717

Lys Asn Leu Gln Leu Asp Val Ser Gly Cys Gln Asp Cys Ile Val Gln

845 850 855

TCC TTT TCT CTA AGT ACC AGA AGT TCC CAG TCC TGC CAA CGG TCG GAG 2765

Ser Phe Ser Leu Ser Thr Arg Ser Ser Gln Ser Cys Gln Arg Ser Glu

860 865 870

AAA GCT GAG CAG GTC AGA TCA AGA AAC AGT GAA ATT TCA GAG ATC ACT 2813

Lys Ala Glu Gln Val Arg Ser Arg Asn Ser Glu Ile Ser Glu Ile Thr

875 880 885 890

TAT GAG GAA CCT GCT GAG ATT GAA CTT TTA AAG AAT AAC CCA AGT AAA 2861

Tyr Glu Glu Pro Ala Glu Ile Glu Leu Leu Lys Asn Asn Pro Ser Lys

895 900 905

GAT TTG GCC TCC ATC TCA CAC CTA AAT GAG GAT AGA ATT GAG GAG CCT 2909

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

910 915 920

GAA GTT GTC ACT GAG CCA AGT GCA ACT AGA GGT ATG GTA CAA CAG ATT 2957

Gln Ile Pro Gly Asn Gln Leu Ser Ser Pro Ser Ser His Leu Ser Ser

925 930 935

CCA GGA AAC CAG CTC TCA TCG CCT TCA TCT CAT CTT TCT TCC TTT GCA 3005

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

940 945 950

TCA AGC TCA GCG CCA TTT GCA TCC TCC TCT TCG GAC ACC TCA ACA AGT 3053

Ser Ser Ser Ala Pro Phe Ala Ser Ser Ser Ser Asp Thr Ser Thr Ser

955 960 965 970

GAG CAT CCA GTG CCT AAT GAA GAG GCG GCA GCT TTG ACA GTT CCT CGG 3101

Glu His Pro Val Pro Asn Glu Glu Ala Ala Ala Leu Thr Val Pro Arg

975 980 985

TCC AAC GAG AAA TGC GAC AAC ACT CCC ATG CCG GTA AAA GAT GGC CTG 3149

Ser Asn Glu Lys Cys Asp Asn Thr Pro Met Pro Val Lys Asp Gly Leu

990 995 1000

ATA TCT GAA GAT GAT GCA CCG GTA CAT CTG CAT CAG AAG CCC CTG CAG 3197

Ile Ser Glu Asp Asp Ala Pro Val His Leu His Gln Lys Pro Leu Gln

1005 1010 1015

GCG ACA GCC ATG GCA GCC ATA TGA CTGACCTGT AATCCTACAA 3240

Ala Thr Ala Met Ala Ala Ile ***

1020 1026

GAAGCCAACT GAAGATTCAT ATGTGGACTG AGTGAAATTA TGAAAGTTAT TGGAATAAAT 3300

TGTTGCTCTG TATGTGAGAG CAACCTTCAG TCCGTTAGCC TGGTTCCTTT TAGTAGTGTT 3360

CTACTATTGG GAGATCTTCA TCAACATTTT ACATGAAACG TAATGTAATG AACCTCTGTT 3420

Pi 15 resistance gene of rice blast and application sequence table thereof

AATTGTTAAT TGTCAGAGCT CTAGTTTTTT GTGGTATAAA AAAAAAAAAA AAAAAAAAAA 3480

AACACTGTCA TGCCGTTACG TAGCG 3505

<210>2

<211>1026

<212>NBS-LRR

＜213〉Oryza paddy rice (Orysa sativa L.)

<400>2

Met Val Gly Ala Glu Met Leu Val Ala Ala Ala Val Ser Gln Val Ala Arg Lys

6 12 18

Ile Asn Asp Ile Val Gly Val Ala Gln Gly Glu Val Lys Leu Cys Cys Asn Phe

24 30 36

Ser Asp Asp Leu Glu Gly Ile Lys Asp Thr Leu Val Tyr Leu Glu Thr Leu Leu

42 48 54

Lys Asn Ala Glu Asn Asn Ser Phe Gly Ser Asp Arg Ala Asn Leu Arg His Trp

60 66 72

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

78 84 90

Tyr Ser Ser Lys Glu Gln Phe Asp Gly Gly Ser Tyr Ala Gln Lys Gly Ser Leu

96 102 108

Phe Cys Ser Leu Ser Asn Pro Met Leu Leu Lys Gly Ser Met Val Tyr Lys Met

114 120 126

Lys Ser Lys Arg Glu Met Leu Gln Gln Ser Gln Gln Leu Pro Asn Gln Tyr His

132 138 144

Phe Leu Ser Tyr Ile Asn Ser Ala Val His Tyr Phe Glu Glu Lys Gln Thr Thr

150 156 162

Ser Tyr Arg Asn Thr Asp Ile Ala Ile Val Gly Arg Asp Ala Asp Leu Asp His

168 174 180

Leu Met Asp Leu Leu Met Gln Asn Ser Ala Glu Glu Leu Cys Ile Ile Pro Ile

186 192 198

Val Gly Pro Val Gly Phe Gly Lys Thr Ser Leu Ala Gln Leu Val Phe Asn Asp

204 210 216

Thr Arg Thr Glu Val Phe Ser Phe Arg Ile Trp Val His Val Ser Met Gly Asn

222 228 234

Ile Asn Leu Glu Lys Ile Gly Arg Asp Ile Val Ser Gln Thr Thr Glu Lys Ile

240 246 252

Glu Gly Asn Met Gln Leu Gln Ser Ile Lys Asn Ala Val Gln Arg Val Leu Asn

258 264 270

Lys Tyr Ser Cys Leu Ile Ile Ile Asp Ser Leu Trp Gly Lys Asp Glu Glu Val

276 282 288

Asn Glu Leu Lys Gln Met Leu Leu Thr Gly Arg His Thr Glu Ser Lys Ile Ile

294 300 306

Val Thr Thr His Ser Asn Lys Val Ala Lys Leu Ile Ser Thr Val Pro Leu Tyr

312 318 324

Lys Leu Ala Ala Leu Ser Glu Asp Asp Cys Leu Lys Ile Phe Ser Gln Arg Ala

330 336 342

Met Thr Gly Pro Gly Asp Pro Leu Phe Arg Glu Tyr Gly Glu Glu Ile Val Arg

348 354 360

Arg Cys Glu Gly Thr Pro Leu Val Ala Asn Phe Leu Gly Ser Val Val Asn Ala

366 372 378

Gln Arg Gln Arg Arg Glu Ile Trp Gln Ala Ala Lys Asp Lys Glu Met Trp Lys

384 390 396

Ile Glu Glu Asp Tyr Pro Gln Asp Lys Thr Ser Pro Leu Phe Pro Ser Phe Lys

402 408 414

Ile Ile Tyr Tyr Asn Met Pro His Glu Leu Arg Leu Cys Phe Val Tyr Cys Ser

420 426 432

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

438 444 450

Ala Leu Asp Met Ile Glu Ser Lys His Gly Thr Leu Pro Leu Asp Val Thr Ala

456 462 468

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

474 480 486

Ser Gln Asn Asp Ala Glu Arg Ser Ser Ala Ser Glu Glu Met Leu Arg Met His

492 498 504

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

510 516 522

Leu Asp Ala Glu Asn Glu Arg Asn Ala Arg Tyr Cys Asp Tyr Arg Tyr Ala Gln

528 534 540

Resistance gene of rice blast Pil5 and application sequence table thereof

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

546 552 558

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

564 570 576

Leu Ser Val Asn Lys Tyr Leu Arg Val Leu Asp Leu Ser Gly Cys Cys Val Gln

582 588 594

Asp Ile Pro Ser Pro Ile Phe Gln Leu Lys Gln Leu Arg Tyr Leu Asp Val Ser

600 606 612

Ser Leu Ser Ile Thr Ala Leu Pro Leu Gln Ile Ser Ser Phe His Lys Leu Gln

618 624 630

Met Leu Asp Leu Ser Glu Thr Glu Leu Thr Glu Leu Pro Pro Phe Ile Ser Asn

636 642 648

Leu Lys Gly Leu Asn Tyr Leu Asn Leu Gln Gly Cys Gln Lys Leu Gln Arg Leu

654 660 666

Asn Ser Leu His Leu Leu His Asp Leu His Tyr Leu Asn Leu Ser Cys Cys Pro

672 678 684

Glu Val Thr Ser Phe Pro Glu Ser Ile Glu Asn Leu Thr Lys Leu Arg Phe Leu

690 696 702

Asn Leu Ser Gly Cys Ser Lys Leu Ser Thr Leu Pro Ile Arg Phe Leu Glu Ser

708 714 720

Phe Ala Ser Leu Cys Ser Leu Val Asp Leu Asn Leu Ser Gly Phe Glu Phe Gln

726 732 738

Met Leu Pro Asp Phe Phe Gly Asn Ile Tyr Ser Leu Gln Tyr Leu Asn Leu Ser

744 750 756

Lys Cys Leu Lys Leu Glu Val Leu Pro Gln Ser Phe Gly Gln Leu Ala Tyr Leu

762 768 774

Lys Ser Leu Asn Pro Ser Tyr Cys Ser Asp Leu Lys Leu Leu Glu Ser Phe Glu

780 786 792

Cys Leu Thr Ser Leu Arg Phe Leu Asn Leu Ser Asn Cys Ser Arg Leu Glu Tyr

798 804 810

Leu Pro Ser Cys Phe Asp Lys Leu Asn Asn Leu Glu Ser Leu Asn Leu Ser Gln

816 822 828

Cys Leu Gly Leu Lys Ala Leu Pro Glu Ser Leu Gln Asn Leu Lys Asn Leu Gln

834 840 846

Leu Asp Val Ser Gly Cys Gln Asp Cys Ile Val Gln Ser Phe Ser Leu Ser Thr

852 858 864

Arg Ser Ser Gln Ser Cys Gln Arg Ser Glu Lys Ala Glu Gln Val Arg Ser Arg

870 876 882

Asn Ser Glu Ile Ser Glu Ile Thr Tyr Glu Glu Pro Ala Glu Ile Glu Leu Leu

888 894 900

Lys Asn Asn Pro Ser Lys Asp Leu Ala Ser Ile Ser His Leu Asn Glu Asp Arg

906 912 918

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

924 930 936

Gln Ile Pro Gly Asn Gln Leu Ser Ser Pro Ser Ser His Leu Ser Ser Phe Ala

942 948 954

Ser Ser Ser Ala Pro Phe Ala Ser Ser Ser Ser Asp Thr Ser Thr Ser Glu His

960 966 972

Pro Val Pro Asn Glu Glu Ala Ala Ala Leu Thr Val Pro Arg Ser Asn Glu Lys

978 984 990

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

996 1002 1008

Pro Val His Leu His Gln Lys Pro Leu Gln Ala Thr Ala Met Ala Ala Ile ***

1014 1020 1026

Claims (9)

1, a kind of pi 15 resistance gene of rice blast encoded protein matter, it is characterized in that its aminoacid sequence shown in SEQ ID NO:2, or this sequence is replaced, disappearance or add one or several amino-acid residue and the amino acid polypeptide with identical function that forms.

2, protein according to claim 1 is characterized in that aminoacid sequence is shown in SEQ IDNO:2.

3, the nucleotide sequence of the described proteinic pi 15 resistance gene of rice blast of coding claim 1.

4, pi 15 resistance gene of rice blast according to claim 3, its nucleotide sequence is shown in SEQ IDNO:1.

5, a kind of carrier that contains claim 3 or 4 described genes.

6, the described carrier transgenic plant transformed of claim 5.

7, the molecule marker of claim 3 or 4 described genes generations.

8, claim 1 or the 2 described protein application in preparation resisting rice blast bacteria medicine.

9, the described molecule marker of claim 7 has application in the paddy rice of disease resistance in seed selection to rice blast.

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