CN1831008A - Rice blast fungus non-toxin gene Avr-pii and its application - Google Patents

Abstract

The invention discloses the nontoxic gene Avr-Pii of the nice blast of the paddy and its appliance. The invention provides the nucleotide sequence and the coded amino acid polypeptide sequence of the magnaporthe grisea nontoxic gene Avr-Pii; the gene is a gene expressed by the forming mould. The invention also deals with the design of the molecular target point according to the frame and the function of the gene; the gene and the corresponding gene against disease are inducted into the host planet such as the paddy covalently to cultivate the diseaseresistant race. The invention deals with the appliance of the molecule sign produced from the gene sequence to monitoring the rice blast colony in the field and the appliance to instruct the reasonable layout of the diseaseresistant race according to the monitoring result.

Description

Rice blast fungus non-toxin gene Avr-pii and application thereof

Technical field

The present invention relates to gene engineering technology field, be specifically related to a kind of separating clone and application of rice blast fungus non-toxin gene Avr-pii.

Background technology

By the microbial rice blast of rice blast is one of destructive disease of tool on world's Rice Production, and this bacterium can also be infected more than 50 kind of grasses such as wheat class, cereal.Can the Pyricularia oryzae bacterial strain be by making decision mutually between the product of the nontoxic gene of this pathogenic bacteria bacterial strain and the host's disease-resistant gene product in growth on the certain species.Therefore, the analysis of nontoxic gene product structure and research are to understand the biochemical basis that pathogenic bacteria microspecies and host's kind specialization are done mutually, also have important directive significance for the prevention and the control of Plant diseases.

The mutual relationship of Pyricularia oryzae and paddy rice is (Flor, 1947 that meet " gene pairs gene " hypothesis; Silueet al., 2000; Bryan et al., 2000; Orbach et al.2000), that is to say, susceptibleization of disease-resistant variety be the nontoxic gene that disease-resistant gene is corresponding, Pyricularia oryzae had held owing to original and disease-resistant variety to pathogenic direction taken place to suddenly change cause (Kiyosawa 1966; Joosten et al., 1994; Zeigler et al., 1994; Bryan et al., 2000; Orbach et al.2000).Therefore, want fundamentally to solve the susceptibleization problem of disease-resistant variety, just must concentrate on focus on disease-resistant gene and the nontoxic gene, based on molecular biology method, further investigate their mechanism of action separately in conjunction with plant genetics and breeding, plant pathology and microbiological basic theory and technology, and interaction between the two.Need not query, these researchs are in case the essence of the gene pairs gene relationship that the acquisition breakthrough will make people profoundly understand to be existed between plant and the pathogenic bacteria provides brand-new theory and approach (de Wit, 1992 for its control then; Bryan et al., 2000; Orbachet al.2000).

5 avirulence gene of rice blast of having cloned so far, can be divided into two classes according to its function.

Specific exciton between first kind nontoxic gene coding is planted.This class has comprised non-specific PWL (the pathogenicity on weeping lovegrass) gene family that causes a disease of weeping love grass performance.In this gene family, PWL2 at first is cloned into by the method for chromosome walking, and this gene plays a part to stop rice blast fungi isolates to infect weeping love grass (Eragrostis curvula) (Sweigard et al., 1995).Transforming with PWL2 has pathogenic wild type strain to weeping love grass, and transformant loses pathogenic to weeping love grass, has but kept pathogenic to other host fully.Although this explanation PWL2 is the gene of a decision host range, its function is the same with the nontoxic gene of classical definition.The PWL2 coding is rich in the hydrophilic protein of glycine, includes a signal peptide.Research finds that also PWL2 allelotrope is unstable in heredity; generation has pathogenic spontaneous mutant to weeping love grass through regular meeting; and there is the polymorphism of height in the PWL2 site in the paddy rice bacterial strain of different geographic origin, and this also understands the variability of rice blast fungus nontoxic gene from gene level Shanghai Stock Exchange.Kang etc. (1995) have further studied the 26S Proteasome Structure and Function of PWL gene family PWL1, PWL3 and PWL4.They and PWL2 have 75%, 51% and 57% homology respectively on amino acid levels, but not on same site.PWL1 is isolating from the ragimillet bacterial strain, with PWL2 function homology, stops germ pathogenic to weeping love grass; PWL3 and PWL4 are non-functional under natural situation, but PWL4 are placed after the promotor of PWL1 or PWL2, and just becoming has function, show that PWL is the gene family of a tachytely.

The second class nontoxic gene is the nontoxic gene on the ordinary meaning in the rice blast fungus, the exciton of varietY specificity (specialization) of promptly encoding.This genoid comprises AVR-Pita (claiming AVR2-YAMO in the past), ACE1 and AVR1-CO39.AVR-Pita is the nontoxic gene with the special mutual work of paddy disease-resistant gene Pita.The AVR-Pita metalloprotease of encoding although still there is not direct biochemical foundation, changes an amino acid in this metalloprotease structural domain, and it just can not do (Jia et al., 2000) mutually with disease-resistant gene Pita.Functional analysis shows, AVR-Pita ₁₇₆Directly with rice cell in (defense response that excites Pita to regulate and control causes the disease resistance response (Jia et al., 2000) of paddy rice for Leucine-rich-domain, LRD) combination in the proteic rich leucine of Pita zone.This is the directly mutual example of doing of product of second nontoxic gene and disease-resistant gene coding, also is the hypothesis that has confirmed to meet between avirulence gene of rice blast and the paddy disease-resistant gene gene pairs gene for the first time from molecular level.Be different from AVR-Pita, polyketide synthase (Polyketide synthase corresponding to a macromolecule of the nontoxic gene ACE1 of disease-resistant gene Pi33 coding, PKS) and non-ribosomal polypeptide synthetic enzyme (non-ribosomalpeptide synthase, NRPS) complex body (Berruyer et al., 2003; Bohnert at al., 2004).Utilize the Ace1-GFP fusion rotein to discover, Ace1 is positioned in the tenuigenin of appressorium, shows that Ace1 discerns by secreting outside the born of the same parents and by vegetable cell.No longer show nontoxicly by the point mutation in the B-polyketide synthases zone of Ace1 being discovered point mutation cause the protein product loss of activity of Ace1p, the existence that shows ACE1 is the prerequisite of paddy disease-resistant kind identification fungi signal.Different with the fungi nontoxic gene of having reported, the not direct and paddy rice effect of ACE1 encoded protein enzyme, but the meta-bolites of Ace1 is by paddy rice identification (Fudal et al., 2002).Though another nontoxic gene AVR1-CO39 is cloned, but its function is not determined (Farman and Leong, 1998) at present as yet, and is not superfluous at this.

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: Pyricularia grisea Sacc.) rice blast that causes is one of destructive disease of tool on world's Rice Production by pathogenic fungi Magnapothe grisea Barr., the annual rice yield loss (Baker et al., 1997) that all causes 10-30%.From the viewpoint of environment protection with the Sustainable development of agricultural, using disease-resistant variety is the most economical effective and environmentally safe measure of control rice blast.But, because the diversity and the volatility of rice blast fungus population, make the resistance instability of disease-resistant variety.So that the susceptibleization problem of disease-resistant variety never is resolved.Set about from the pathogenic bacteria nontoxic gene, be expected to explain the interaction of Pyricularia oryzae and paddy rice, and the pathogenesis of pathogenic bacteria, thereby, for the rice blast breeding for disease resistance is laid a solid foundation.

Along with molecular biological fast development, at present, have at least more than 40 avirulence gene of rice blast to be located by molecule.Wherein, PWL1, PWL2, Avr1-CO39, Avr-Pita and ACE1 are by clone (B hnert et al.2004; Farman and Leong 1998; Kang et al.1995; Orbach et al.2000; Sweigard et al.1995).Before proposing, the present patent application also do not have the report that avirulence gene of rice blast is cloned on Pyricularia oryzae the 6th karyomit(e).

Summary of the invention

A blast fungus non-toxin gene Avr-pii that the objective of the invention is to carry among the separating clone Pyricularia oryzae bacterial strain CHL346 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 avirulence gene of rice blast.

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

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

Another object of the present invention provides the application of said gene in design pesticide molecule target spot.

Another object of the present invention provides the method for utilizing said gene to cultivate disease-resistant plants.

Further purpose of the present invention provides said gene and is setting up the Molecular Detection system, the application in the monitoring plant rice blast incidence.

The present invention relates to separate and use a kind of dna fragmentation that comprises gene A vr-Pii, this fragment is given Pyricularia oryzae (Magnaporthe grisea) produces specificity (specialization) to paddy rice non-virulent reaction.Wherein, described fragment perhaps is equivalent to the dna sequence dna shown in the SEQ ID NO:1 basically shown in sequence table SEQ ID NO:1, and perhaps its function is equivalent to the subfragment of sequence shown in the SEQ ID NO:1, and structure as shown in Figure 4.The aminoacid sequence of this dna sequence encoding is 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.Dna fragmentation shown in the present is constitutive expression in the Pyricularia oryzae mycelium.The partial sequence homology of Avr-Pii nontoxic gene encoded protein of separating, cloning and Pyricularia oryzae telomere unwindase gene.The Avr-Pii gene is modified or transformed, can change or increase certain function of gene.Rite-directed mutagenesis is carried out in coding region to this gene, may cause avirulent forfeiture of gene or change.

The present invention comprises equally and being connected comprising the fragment of this gene and the promotor of a constitutive expression that this promotor can be expressed with the different times of invading plant under any condition.The promotor of this constitutive expression comprises the promotor of cauliflower mosaic virus 35S etc.On the other hand, also the promotor of this gene and a tissue specific expression or the promotor of accurate environmental induction can be connected, these promotors are referred to as inducible promoter.Like this, the change of environment, the different times of invading plant can change this expression of gene.Wherein envrionment conditions comprises the upgrowth situation of plant, temperature, and humidity etc., the different times of invading plant comprises that spore germination, appressorium form, infect the nail differentiation and infect mycelia expansion etc.

According to Avr-Pii 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 Avr-Pii by the following method: (1) obtains by database retrieval; (2) with the Avr-Pii gene fragment be genomic library or the acquisition of cDNA library of probe screening Pyricularia oryzae or other pathogenic bacteria; (3) according to Avr-Pii gene order information design oligonucleotides primer, from genome, mRNA and the cDNA of Pyricularia oryzae or other pathogenic bacteria, obtain with the method for pcr amplification; (4) on the basis of Avr-Pii gene order, obtain with the gene engineering method transformation; (5) method with chemosynthesis obtains this gene.

Blast fungus non-toxin gene Avr-pii provided by the invention has important use and is worth.One of using is the molecular target that structure and function thereof according to this gene design novel agrochemical.

Two of application is that described Avr-Pii gene order is connected to any conversion carrier, with any method for transformation Avr-Pii nontoxic gene and corresponding disease-resistant gene Pii covalency is imported paddy rice or other plant cell.That is to say, place pathogenic bacteria to invade the promotor of abduction delivering nontoxic gene, and corresponding disease-resistant gene is placed under the promotor of constitutive expression, import in the host plant together, when the host is subjected to pathogen infection, nontoxic gene is just by the abduction delivering nontoxic protein, nontoxic protein is as exciton then, excite its special disease-resistant gene to express, produce corresponding receptor protein, identification mutually causes the host to produce anaphylaxis between them, with stop the invasion pathogenic bacteria decide grow and expand.Because this engineered plant is based on the comprehensive expression and the effect of various defense responses behind host's disease resistance response, therefore, this novel resistance is stable and persistent.

The Another application of nontoxic 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 monitor the physiological strain of field rice blast fungus population and the dynamic change of genetic construction thereof with these marks, and the distribution situation of this nontoxic gene in the natural population of field; Help the disease resistance evaluation of rice varieties and the evaluation of Pyricularia oryzae microspecies; Also help the rational deployment of disease-resistant variety and by turns, more effectively to control the generation of rice blast.

The avirulent transgenosis bacterial strain that the present invention can further provide or the above-mentioned dna fragmentation of applications exploiting obtains, and with the bacterial strain of gene transformation of the present invention.Also can gene of the present invention be changed over to other bacterial strain with the mode of sexual hybridization.

Beneficial effect of the present invention: the present invention helps to disclose the molecule mechanism that specificity is done and evolved mutually between Pyricularia oryzae microspecies and rice varieties by clone and functional analysis thereof to avirulence gene of rice blast.Can be in practice according to the structure of this gene and the molecular target of functional design novel agrochemical thereof; This gene and corresponding disease-resistant gene covalency can be imported host plant cultivation permanent disease-resistant kinds such as paddy rice; Help to set up the Molecular Detection system of the pathogenic variation of Pyricularia oryzae natural population, the distribution situation of research avirulence gene of rice blast in the natural population of field, the composition and the variation characteristics of microspecies in the announcement rice blast fungus population; Also help the disease resistance evaluation of rice varieties and rational deployment thereof and by turns, so that more effectively control the generation of rice blast.

Description of drawings

Fig. 1 is the map based cloning synoptic diagram of rice blast fungus non-toxin gene Avr-pii;

Fig. 2 is the high-res genetic map and the electronics physical map of rice blast fungus non-toxin gene Avr-pii;

Fig. 3 is the PCR (3a) and Southern (3b) the detected result figure of selective marker hygromycin gene of the partial resistance transformant of rice blast fungus non-toxin gene Avr-pii;

Fig. 4 is the gene structure figure of rice blast fungus non-toxin gene Avr-pii;

Fig. 5 is that the RT-PCR of rice blast nontoxic gene Avr-Pii expression characterization detects figure;

The Avr-Pii loci gene type that Fig. 6 identifies parent and offspring's individuality thereof for molecule marker MS6-1 is figure as a result; Wherein, A is the high-res genetic map in Avr-Pii site among Fig. 2.Sea line is represented chromosomal region, and the top is the SSR mark, and bracket inner digital is the recombinant chou number of each marker site, and the below numeral is the genetic distance (cM) between mark; B is the electronics physical map of Avr-Pii site areas.Long sea line is represented chromosomal region, and short sea line is represented the BAC clone, and vertical line is represented the landing position of linked marker; C is the electronics physical map in telomere zone, Avr-Pii site; Sea line is represented genome area, and the arrow of black is depicted as 2 candidate's nontoxic genes.

Swimming lane 1-5 is the pcr amplification product of hygromycin gene of the conversion bacterial strain of nontoxic gene Avr-Pii among Fig. 3 a; Swimming lane 6 is the non-transformant of F-strain CHL42; Swimming lane 7 is the PCR product of the hygromycin gene of carrier pBHt1 plasmid; M is molecular weight marker DL2000.Swimming lane 1-4 is the Southern hybrid belt of hygromycin gene of the conversion bacterial strain of nontoxic gene Avr-Pii among Fig. 3 b; Swimming lane 5 is the non-transformant of F-strain CHL42; Swimming lane 6 is the Southern hybrid belt of the hygromycin gene of carrier pBHt1 plasmid; M is molecular weight marker DL2000.

Green square box is represented the exon of nontoxic gene AvrPii among Fig. 4, and the square box of band oblique line is respectively 5 ' and 3 ' non-translational region, and straight line is then represented intron.

Fig. 5 is avirulent strains CHL346 and healthy paddy rice, the nontoxic gene expression of inoculation back 24h, 128h; Tubullin is an internal reference.

P1 is nontoxic parent CHL346 among Fig. 6; P2 is poisonous parent CHL42, and A is nontoxic thecaspore offspring individuality, and V is deleterious thecaspore offspring individuality; M: molecular weight marker DL2000.

Embodiment

Embodiment 1: the genetic analysis of rice blast fungus non-toxin gene Avr-pii and Primary Location

The present invention utilizes the map based cloning method to clone nontoxic gene Avr-Pii.At first, in order to excavate and identify the new nontoxic gene of Pyricularia oryzae, 242 offspring's thecaspore inoculation rice varieties Fujisaka 5 (No. 5, rattan slopes that utilization is obtained by CHL346 (MAT1-1) and CHL42 (MAT1-2) hybridization, contain disease-resistant gene Pii), the result shows, non-pathogenic bacteria strain and pathogenic strains separates than meeting 1: 1 in this mapping population.Infer that thus the non-virulent to rice varieties Fujisaka 5 that CHL346 showed is controlled by a pair of dominant gene.

In order to determine the chromosome position of nontoxic gene apace, utilize the reference sequences (reference sequence) of reference strain 75-10, develop and made up the genetic map of forming by 121 microsatellite markers (SSR, simple sequencerepeat).Then, (bulked-segregantanalysis BSA), has screened whole 121 SSR marks by population mixture compartment analysis method, obtained 7 with nontoxic gene chain be positioned at SSR mark on the 6th karyomit(e), further carried out linkage analysis with 242 offspring's bacterial strains.The result shows that this nontoxic gene site is positioned in the 6th chromosomal telomere 11.For this nontoxic gene of Fine Mapping, utilize the 6th chromosomal end sequence and special tumor-necrosis factor glycoproteins (TTAGGG) the n design primer of telomere of reference strain 70-15, obtained telomeric sequence by LR-PCR (long-range PCR) technology.

Embodiment 2: the Fine Mapping of blast fungus non-toxin gene Avr-pii and electronics physical mapping

In order to determine the position in Avr-Pii site subtly, we utilize the telomeric sequence of acquisition to develop 2 candidate's nontoxic gene mark (candidate avirulence gene, CAG), the result show one of them CAG mark CAG6-1 and goal gene be divided into fully from, therefore, the Avr-Pii site by Fine Mapping at telomere 11.In order to make up the physical map in this site, we utilize bacterial artificial chromosome (the bacterial artificial chromosome of reference strain 70-15, BAC) and the telomeric sequence that obtains of this research, by bioinformatic analysis (bioinformatics analysis, BIA), made up the electronics physical map in this site.The result shows, the Avr-Pii site by physical positioning in the zone of about 8.0kb (Fig. 2).

Embodiment 3: the prediction note and the sequential analysis of blast fungus non-toxin gene Avr-pii candidate gene

In order to determine the candidate gene of Avr-Pii, we utilize the reference sequences of bacterial strain 70-15, FGENESH (http://www.softberry.com) by predictive genes software Softberry has carried out predictive genes and note analysis to the goal gene zone, candidate's nontoxic gene of tentatively having determined Avr-Pii is Avr-Pii-L1 and Avr-Pii-L2, therefore, by following its function of functional complementation experimental verification.

Embodiment 4: the conversion of blast fungus non-toxin gene Avr-pii is identified with the nontoxicity that transforms bacterial strain

Utilize the LR-PCR technology increased respectively the 1.9kb of 2 candidate genes and 2.0kb fragment and be cloned into binary vector transformation system pBHt1, imported agrobacterium strains AGL1 then.The AGL1 that contains the goal gene conversion carrier minimum medium (minimal medium, MM) in 28 ℃ cultivated 2 days; Also (induction medium IM) washes twice, and these thalline suspend again with the inducing culture (IM+AS) that contains 200 μ M Syringylethanones (AS) then, cultivates 6h down for 28 ℃ with inducing culture to collect bacterial cell; Conidial suspension (1 * 10 with above-mentioned suspension and isopyknic Pyricularia oryzae ⁶Individual spore/ml) mix.The mixed solution of getting 200 μ l is coated in the common substratum that adds AS (200 μ M), and (co-cultivationmedium on nitrocellulose filter CM), cultivates 48h under 25 ℃ altogether; Collect fungi and bacterial cell on the nitrocellulose filter with the MM liquid nutrient medium, getting common cultivation bacterium liquid that 200 μ l collect then coats and contains hygromycin B (300 μ g/ml), screening culture medium (the screening medium of cefotaxime sodium (200 μ g/ml) and Pyocianil (250 μ g/ml), SM) on, cultivate 7-10d down for 25 ℃; Change single bacterium colony over to screening culture medium once more and carry out postsearch screening; 3-4 grow down after week, the bacterium colony of surviving is changed in the PDA test tube slant substratum for 25 ℃.Treat that mycelia covers with the inclined-plane, the rice straw of sterilization is positioned on the cap; 2 weeks back taking-up rice straw is wiped the mycelia on the rice straw; Behind the 36h, the single conidium on the picking rice bar is the transformant bacterial strain.

100 Avr-Pii-L1 conversion bacterial strains and 150 Avr-Pii-L2 conversion bacterial strains are being carried out pathogenic evaluation.The result shows to have only 30 Avr-Pii-L2 to transform bacterial strain under the genetic background of toxic strain CHL42, has showed the identical nontoxicity with nontoxic gene F+strain CHL346.Explanation thus, candidate gene Avr-Pii-L2 is exactly nontoxic gene Avr-Pii.

The PCR and the Southern blot that the conversion bacterial strain of having realized having complementary functions have been carried out hygromycin gene identify that the result shows that target gene fragment has imported these transformant (the part individuality is seen Fig. 3).The nontoxicity of explanation conversion bacterial strain is produced by the expression of nontoxic gene Avr-Pii thus.Above presentation of results, nontoxic gene Avr-Pii has successfully been cloned.

The structure of embodiment 5:Avr-Pii gene

The employing method of moving one's steps is measured the dna sequence dna of Avr-Pii.Utilize 5 ' and 3 ' RACE reaction, obtained the full-length cDNA of Avr-Pii and it is checked order.SEQ ID NO:1 in the sequence table is the dna sequence dna of Avr-Pii.Avr-Pii gene DNA length is 1938bp, and its full-length cDNA is 750bp, contains the opening code-reading frame of a 174bp, and 5 ' and 3 ' non-translational region is respectively 136bp and 441bp.By icp gene group DNA and cDNA, find that the opening code-reading frame of this gene has only 1 exon, and 3 ' non-translational region contains 2 introns (Fig. 4).

The structure of embodiment 6:Avr-Pii nontoxic protein

The protein sequence of Avr-Pii genes encoding is shown in SEQ ID NO:2 in the sequence table.Nontoxic gene Avr-Pii 1 protein polypeptide of forming by 57 amino-acid residues of encoding.Database is relatively found the partial sequence homology of this nontoxic gene and telomere unwindase gene.

Embodiment 7: rite-directed mutagenesis

The sequence of Avr-Pii gene is shown in SEQ ID NO:1, through sequential analysis relatively, the 251st base finding the dna sequence dna of avirulent strains CHL346 is T, and virulent strain CHL42 then is C, finally causes this site amino acid to become tryptophane (Ser) by leucine (Leu).Therefore, according to a pair of complementary primer of the sequences Design in this site of avirulent strains, the mutational site design on primer, by the amplification of overlapping extension twice PCR, is utilized the base mispairing of pcr amplification, the C of toxic strain is sported the T of avirulent strains, the fragment cloning that contains the mutational site that will obtain at last and carries out genetic transformation and the checking that has complementary functions to the pBHt1 carrier, experimental result shows, toxic strain is after carrying out rite-directed mutagenesis, and phenotype reverts to nontoxicity.

Embodiment 8:Avr-Pii expression of gene specificity analysis

Utilize the RT-PCR technology that Avr-Pii expression of gene pattern is analyzed.From avirulent strains CHL346 with extracting total RNA the rice leaf of avirulent strains CHL346 inoculation, utilize reverse transcription test kit SuperScript ^TMReverse TranscriptaseIII carries out the synthetic of reverse transcription cDNA article one chain.The RT-PCR primer is: For:5 ' atcgaagttggtccagggc3 '; Rev:5 ' ttatacgggttccgggg 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 45sec, 72 ℃ are extended 1min; Last 72 ℃ are extended 7min, and temperature drops to 4 ℃ and promptly finishes amplification.Experimental result shows, avirulent strains CHL346 and all can amplify special fragment with the RNA reverse transcription template of its postvaccinal leaf tissue illustrates that Avr-Pii is the gene (Fig. 5) of constitutive expression.

The application of embodiment 9:Avr-Pii gene

Utilize the sequence information of Avr-Pii gene provided by the invention, according to the structure of this gene and the molecular target of functional design novel agrochemical thereof; This gene and corresponding disease-resistant gene covalency are imported host plant cultivation disease-resistant varieties such as paddy rice; The molecule marker that produces according to this gene order is in field rice blast fungus population Application in Monitoring (Fig. 6); And instruct application in the disease-resistant variety rational deployment according to the result of monitoring.

Rice blast fungus non-toxin gene Avr-pii and application sequence table thereof

SEQUENCE?LISTING

＜110〉Agricultural University Of South China

＜120〉rice blast fungus nontoxic gene AvrPii and application thereof

<130>

<160>2

<170>PatentIn?version?3.2

<210>1

<211>1938

<212>DNA

＜213〉Magnaporthe grisea belongs to Pyricularia oryzae (Magnaporthe grisea Barr. (no condition: Pyricularia grisea Sacc.))

<220>

<221>5’UTR

<222>(1)..(135)

<220>

<221>CDS

<222>(136)..(306)

<220>

<221>exon

<222>(136)..(309)

<220>

<221>gene

<222>(136)..(309)

<220>

<221>3’UTR

<222>(310)..(873)

<400>1

ggacggtggg?tagaggtggc?aggggacgag?ggacagttcc?ggttgcgaac?gggggcggat 60

tgcgacaaaa?aaagggtgtt?gtttgaggtg?gttgcaggat?ttcaaaacca?ggcgccggag 120

ttgttccggg?aggaa?atg?aaa?acg?agg?atg?ggg?gat?agg?cat?tgg?gat?gga 171

Met?Lys?Thr?Arg?Met?Gly?Asp?Arg?His?Trp?Asp?Gly

1 5 10

aaa?ttc?gaa?ggg?agg?gag?gcg?tgg?aaa?cgg?atg?gga?aag?cgg?gtt?aaa 219

Lys?Phe?Glu?Gly?Arg?Glu?Ala?Trp?Lys?Arg?Met?Gly?Lys?Arg?Val?Lys

15 20 25

tgg?gga?agg?atg?gaa?acg?aac?gag?ttg?tgt?ata?tta?ttt?ttt?cga?tta 267

Trp?Gly?Arg?Met?Glu?Thr?Asn?Glu?Leu?Cys?Ile?Leu?Phe?Phe?Arg?Leu

30 35 40

gtt?gaa?att?tgg?agc?agg?gag?atg?gag?ggc?ggt?ggg?att?taa 309

Val?Glu?Ile?Trp?Ser?Arg?Glu?Met?Glu?Gly?Gly?Gly?Ile

45 50 55

atgcgtaagt?attaacaatt?tgtaaatgga?tatatataaa?taaaataaac?gcagataaaa 369

taaaagtaat?tagcaaaaaa?ggcaaataaa?attaaaatcg?aagttggtcc?agggccgtga 429

atataaaatt?agcaaacacg?aatattaaaa?tgataaatat?gttttaaaag?cgcagaaaga 489

cgaaaaatag?tgattataag?ggtaaagggc?cgggaaggtt?agggttagaa?gtggttgtgg 549

ttaaagaaat?gcaaagcgtc?cccgtaaccc?gtttaatggc?gcggagcgcc?catgtccggt 609

ggcgtggagg?ccggaaggtt?agggttaaaa?ataattatgg?tttaaaaaag?cgcggagcgc 669

ccccggaacc?cgtataatgg?cggcggagcg?cccatgtccg?gcggcgtgga?ggccggaagg 729

ttagggttaa?aagtgattgt?ggtttaaaaa?aacacggagc?gtcccatgtt?cggcggcgtt 789

Rice blast fungus non-toxin gene Avr-pii and application sequence table thereof

gaggccggaa?ggttagggtt?atatggaggc?gggagggtga?gggttatata?agatgcgttg 849

ggattaaaat?agtggaagta?taacgaaaat?agtgcagaaa?agttgaaatt?gaaataaagg 909

ggaaaatata?ataaatcaaa?taatagaaaa?aagttgaggg?gattaaaaaa?acatatggaa 969

aaataaattt?gaagttgtta?taaattatga?ttgggcgaaa?tgaaatgcgg?tcaaattggg 1029

aaagcaaaag?gagttaaaat?ataaaagcaa?attacaaaca?agtattatac?cagtaaggga 1089

aaatgttgaa?atatgacaaa?ataaaagaga?aatataaaaa?caatcgaaaa?agaatatttt 1149

ttaatatgtg?gtgtgaaaag?ataataaaac?ataaaatagg?tataaaataa?tatttaataa 1209

tacgtatata?aaataaatgt?tatatggatg?gaatataaac?caaatataac?caaaagaaaa 1269

gaaaatataa?aatagcgcaa?ttacggtaat?ataaaccgtt?taatggttat?atataaaaac 1329

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ataaaatggt?aagaagtatt?gaaagttgca?ataagtccgg?gttatatata?acgcaaaaaa 1449

ataaacgaaa?aaaataaaaa?aggaaagggt?atgaattatt?ttgtaaaaaa?gtaaaaatat 1509

aaaaaaacgt?taaaaataaa?attgtaaaaa?atggcgttat?aaggcccggg?cgcgaagcgc 1569

gtaaccaatc?gagcagggcg?gcaaaggtaa?aaagagagtt?aaaataatcc?gtgggcgcga 1629

agcgcggcca?attatattcc?aaaaagccag?ccgcaaatgg?cacgcgtttt?atatatttga 1689

tttaatggtt?aaaaaaagtg?gaaggaagga?aatagcatgg?ttaaaaaccc?gatacggttt 1749

acggatttga?cgaaataaag?cacggaaaaa?gaaaaaagac?gttggcggat?aaaacggcgt 1809

attggtttaa?aattgcaatt?aaagcaaata?ttggacgaaa?aaacggagtt?ttgcggagtg 1869

taaaacccgg?cgttataaat?aattatacag?agaaccagcc?cggtaataat?ggtagtaggt 1929

acgagtggc 1938

<210>2

<211>57

<212>PRT

＜213〉Magnaporthe grisea belongs to Pyricularia oryzae (Magnaporthe grisea Barr. (no condition: Pyricularia grisea Sacc.))

<400>2

Met?Lys?Thr?Arg?Met?Gly?Asp?Arg?His?Trp?Asp?Gly?Lys?Phe?Glu?Gly

1 5 10 15

Arg?Glu?Ala?Trp?Lys?Arg?Met?Gly?Lys?Arg?Val?Lys?Trp?Gly?Arg?Met

20 25 30

Glu?Thr?Asn?Glu?Leu?Cys?Ile?Leu?Phe?Phe?Arg?Leu?Val?Glu?Ile?Trp

35 40 45

Ser?Arg?Glu?Met?Glu?Gly?Gly?Gly?Ile

50 55

Claims (9)

1, rice blast fungus non-toxin gene Avr-pii encoded protein matter, its aminoacid sequence are shown in SEQ IDNO: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, its aminoacid sequence is shown in SEQ ID NO:2.

3, the nucleotide sequence of the described proteinic rice blast fungus non-toxin gene Avr-pii of coding claim 1.

4, rice blast fungus non-toxin gene Avr-pii according to claim 3, its nucleotide sequence is shown in SEQ ID NO:1.

5, the carrier that contains claim 3 or 4 described genes.

6, claim 3 or the 4 described genes application in design pesticide molecule target spot.

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

8, the method for utilizing claim 3 or 4 described genes to cultivate disease-resistant plants.

9, claim 3 or 4 described genes are being set up the Molecular Detection system, the application in the monitoring plant rice blast incidence.

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