CN102517297A - Rice rhizoctonia solani effector gene AG1IA06910 and application thereof - Google Patents

Abstract

The invention provides a rice rhizoctonia solani effector gene AG1IA06910 and application thereof. The rice rhizoctonia solani effector gene AG1IA06910 has a nucleotide sequence shown in the formula of SEQ ID NO.1. Results of the research on the rice rhizoctonia solani effector gene AG1IA06910 show that in practice, a molecular target of a novel pesticide can be designed according to a structure and functions of the rice rhizoctonia solani effector gene AG1IA06910; a receptor protein gene of an effector protein of host cell such as a rice cell and the like can be knocked out or be subjected to mutation so that a breed with lasting disease resistance is obtained; the rice rhizoctonia solani effector gene AG1IA06910 is conducive to establishing of a molecular detection system for detecting pathogenic variation of a natural rhizoctonia solani colony, research of the distribution of the rice rhizoctonia solani effector gene AG1IA06910 in a natural colony of a field, and revealing of composition and variation of microspecies of a rhizoctonia solani colony, is also conducive to disease resistance identification, reasonable distribution and rotation of rice, and is convenient for effective control of banded sclerotial blight.

Description

Paddy rice dry thread Pyrenomycetes effector Gene A 1IA06910 and application thereof

Technical field

The present invention relates to gene engineering technology field, specifically, relate to a kind of paddy rice dry thread Pyrenomycetes effector Gene A 1IA06910 and application thereof.

Background technology

By the microbial rice sheath blight disease of miliary damping-off is a kind of fungal disease and rice blast, bacterial leaf-blight paddy rice three big diseases arranged side by side.It is wide that dry thread Pyrenomycetes is infected scope, comprises various crop such as paddy rice, corn, wheat, potato, herbage, soybean.Dry thread Pyrenomycetes can cause that various crop generation disease like this is closely-related with its secretion effector (effector) molecule.This molecule can be regulated host's congenital immunity and strengthen and parasitize.Now generally believed that these effector are pathogenic determinative (Kamoun, 2007 that strengthen the key that parasitizes; Hogenhout et al., 2009).

Dry thread Pyrenomycetes is got in touch the also accurate vegetable cell of controlling with its host plant closely through secretion effector molecule.Effector targeted plants molecule changes the plant process.In phytopathogen interacted, effector was main pathogenic determinative, and congenital immunity and reinforcement that they reconcile plant parasitize.Now, in international conference, genome tissue, evolution, exchange and the functional study of thread pathogenic bacteria effector become very active research discussion field.

Thread pathogenic bacteria secretory protein is not novel especially with the host's that upsets their and invade notion; But the research of this respect is accelerated; Because it makes a whole set of secretory protein catalogue that generates in certain specific pathogen fungus kind become possible (Dean et al., 2005; Kamper et al., 2006; Haas et al., 2009).This research is on the one hand promoted by the cloud computing prediction of the arrival in gene order-checking epoch and the secretion signal followed and other effector sequence motifs diagnostor.One of characteristic of some pathogenic fungi effector families is to contain linear motif or contain the short sequence pattern relevant with specific function, such as inserting in the host cell or target host cell nuclear.

RXLR is the most outstanding example in these effector molecules, and it has defined host's traffic structure territory in the oomycetes RXLR family.The RXLR of prediction also is used in the high-throughout screening, to confirm their new functionally active as early as possible and effectively.ATR1 and ATR13 be the model plant Arabidopis thaliana oomycetes pathogenic bacteria Hyaloperonospora arabidopsidis have active two effector of nontoxic gene.ATR1 and ATR13 are that the RXLR effector length of secretor type is respectively 310 and 150 amino acid, and bring out resistance (Allen et al., 2004 of RPP1-Nd/WsB and PP13-Nd mediation respectively; Rehmany et al., 2005).This interactional conciliation has altogether formed allele (Allen et al., 2008 of nontoxic gene different, tachytely and resistant gene; Hall et al., 2009).ATR13 also comprises 7 conservative leucines/Isoleucine Tumor-necrosis factor glycoproteins except the characteristic with signal peptide and RXLR motif, this Tumor-necrosis factor glycoproteins is by RPP13 identification necessary (Allen et al., 2008).In fact the sudden change of this Tumor-necrosis factor glycoproteins is not evolved and is meaned that it is a performance toxic action necessary (Allen et al., 2008).In addition, no cytotoxic activity also depends on the variable amino acid (Allen et al., 2008) of ATR13C-end.It should be noted that in RPP13 family, to have at least a gene to discern the allelotrope of ATR13 with all ignorant mode up to now, this has stressed nontoxic gene and resistant gene complex interactions network (Hall et al., 2009).ATR1 and ATR13 highly variable and their the homogenic variety in Arabidopis thaliana in H.arabidopsidis means that these effector possibly extremely help the flexibility of pathogenic bacteria.ATR1 and the ATR13 effect in the resistance path that suppresses the basis is through carrying out heterogenous expression and be transported in the host cell to set up (Sohn et al., 2007) in plant pathogenic bacteria Pseudomonas syringae DC3000.When being transported in the Arabidopis thaliana by P.syringae DC3000, ATR1 and ATR13 have increased toxicity, and have reduced the accumulation of callose in the Arabidopis thaliana (Sohn et al., 2007).

AVR3a is the tenuigenin RXLR effector albumen of Phytophthora infestans.The interesting characteristic of AVR3a is its double activity in mechanism of causing a disease.AVR3a brings out anaphylaxis (HR) (Armstrong et al., 2005) in the plant of expressing the R3a gene.But in the plant that lacks the R3a gene, AVR3a suppresses P.infestans INF1 latent ground albumen inductive necrocytosis (Bos et al., 2006).These different activity can be separated on structure level.The AVR3a that deletion or sudden change have taken place at C-terminal amino acid residue tyrosine 147 places has kept the anaphylaxis of R3a mediation, but can not suppress the necrocytosis of INF1 inductive (Bos et al., 2006; 2009).In addition, two main allelotrope encoded protein matter of AVR3a have only two amino acid whose difference at effector structural domain place, but active difference is clearly but arranged.Be AVR3a ^K80/I103Rather than AVR3a ^E80/M103Activation R3a and be more intense inhibition ((Bos et al., 2006) of INF1 inductive necrocytosis.

Except RXLR effector, another tenuigenin effector family, i.e. Crinkler have been confirmed in the research of the secretory protein of P.infestans again.Crinkler is big, the diversified effector of the another one of oomycetes a family.High-throughout functional screening has disclosed two necrocytosis inducible protein CRN1 and CRN2 (crinkling and necrosis), when they can cause the shrinkage phenotype (Torto et al., 2003) of blade during systematic express in plant.Utilizability along with the P.infestans genome sequence; Research to the rare zone of gene has disclosed except RXLR effector gene; The CRN gene family is positioned the repetition rich region altogether; And the Phytophthora gene family with other is compared the theatrical expansion of CRN gene family (genes of 193 predictions) (Haas et al., 2009) in P.infestans.The expansion of this gene family is through the inner recombination event generation of poly-gene duplication and gene, and this incident has finally caused big, a diversified chimeric effector system (Haas et al., 2009).CRN effector is to be the modular protein of characteristic with the N-of prediction end secreting signal peptide; The back is that a quilt is guarded but is not the structural domain (Haas et al., 2009) that constant LXLFLAK motif (the proteic main defined feature of CRN) defines.Most of CRN carry a DWL structural domain (Haas et al., 2009) with the ending of HVLVXXP motif behind the LXLFLAK structural domain.Haas et al. (2009) proposes the reorganization behind reorganization, especially the HVLVXXP motif between the CRN, has produced extremely diversified C-end structure territory (being to be determined by 36 different amino acid in P.infestans).Be similar to several RXLR albumen, the induced expression in vegetable cell of number of C RN C-end (the N-end transfer organization territory that therefore lacks prediction) necrocytosis (Haas et al., 2009).Yet the dependency of CRN inductive necrocytosis remains unclear in disease.

Yoshida et al. (2009) has found three candidate effector gene Pex22 from the peculiar sequence of Pyricularia oryzae lna168, excavating; Pex31 and Pex33; With three nontoxic phenotype Avr-Pia, Avr-Pik/km/kp and Avr-Pii have perfect contact respectively.Genetic transformation experiment confirm subsequently be effector Pex22, Pex31 and Pex33 have given the nontoxicity that the paddy rice of expressing Pia Pik/km/kp and Pii resistant gene is respectively cultivated system.Learn that through positional cloning being accredited as is that the Pex22 of Avr-Pia is independently (Miki et al., 2009).Pex22, Pex31 and Pex33 are very little, on length, are respectively 85,70 and 113 amino acid, comprise secretion signal, and are identified in the tenuigenin in rice cell, this means that they are transported in the vegetable cell in the process that infects.What is interesting is that Pex22 forms a little family that comprises four homologues; This little family is with the characteristics that appear as of two sequence motifs: motif 1 is [LI] xAR [SE] [DSE]; Be similar to the LxAR motif of Avr-Piz-t, can suppress the necrocytosis (Li et al., 2009) of BAX mediation; Motif 2 is [RK] CxxCx12H, shows the similarity (Yoshida et al., 2009) of the C2H2 zinc-finger motif relevant with protein-protein interaction.

From the transfer effectorAvrL567 family of flax rust bacterium Melampsora lini be through with the resistance protein L5 of flax, (Dodds et al., 2006) that the interaction that L6 and L7 are directly connect is realized.This system is special, because the crystalline structure of AvrL567-A and AvrL567-D is illustrated (Wang et al., 2007).This just allows under the protein structure background, and the effect with the relevant polymorphum residue of molecular recognition event on the gene pairs gene basis detects (Wang et al., 2007).Two kinds of albumen all do not have near known structure homologue.Four important height polymorphum residues are in 50,56,90 and 96 positions of AvrL567, and they are crucial for activating R albumen, and are positioned at the proteic surface of these effector.The distribution of four residues that what is interesting is AvrL567 shows the interaction between the proteic rich leucine repeating structure of AvrL567 and the R territory across whole protein surface, and requiring to interact for integral body has the multiple tie point of storage effect.This keeps in coevolution or develops toxicity function (is favourable for pathogenic agent) but the detection of evading the host simultaneously possibly be important.Proteic structure has also disclosed two positive charge surface points that combine DNA, and experiment in vitro subsequently shows that these protein binding are to nucleic acid.This shows in the time from initial sequence, almost can not obtaining useful clue, and structure biology is the functional study that how to promote effector.

Use map based cloning strategy clone from Leptosphaeria maculans, to obtain AvrLm1, three nontoxic genes of AvrLm6 and AvrLm4-7 (Gout et al., 2006; Fudal et al., 2007; Parlange et al., 2009).From 122 to 205 little amino acid whose secretory proteins of their coded prediction all do not have similarity with any albumen in the public database.AvrLm6 and AvrLm4-7 comprise 6 and 8 cysteine residues respectively.These halfcystines possibly stablized AvrLm6 and AvrLm4-7 albumen in the plastid external enwergy.Yet AvrLm1 has only a cysteine residues, considers that the outer effector of all plastids that are described is rich cysteine protein so far, so AvrLm1 is transported in the host cell (Kamoun, 2007) probably.Although we know that very they have avirulent activity, the mechanism of AvrLm identification, corresponding Rlm resistant gene and their action site remain unclear.Effector fungi with other fungi of great majority of being cloned into is at present compared, and these three L.maculans genes have lower GC content.They all are single copy genes, are positioned at the 60kb (AvrLm4-7) of rich AT and rich transposon, 133kb (AvrLm6), the class heterochromatin zone of 269kb (AvrLm1).These effector are positioned in unique genome environment; Be fungi karyomit(e) (the van der Does and Rep that is independent of with good conditionsi; 2007), the inferior telomere zone (Pain et al., 2008) of Plasmodium; Perhaps traces of the rare zone of gene (Haas et al., 2009) in P.infestans.This is believed to accelerate gene evolution and quickens flexibility (van der Does and Rep, 2007 of pathogenic agent to the host; Pain et al., 2008; Haas et al., 2009).

The nematophyte pathogenic bacteria has been evolved out and has had the effector albumen (Kamoun, 2006) of arrestin enzymic activity to protect several kinds of host's hydrolytic enzyme activities.Recently Misas-Villamil and van der Hoorn have done these effector and their host's target and have estimated (2008).

Host's specialization toxin (HST) is the effector molecule with Chemical Diversity, it be by phytopathogenic fungi produce as virulence factor performance function.These pathogenic factor of determinations only just have activity (Wolpert et al., 2002) in to the responsive host plant of the pathogenic bacteria that produces this toxin.What is interesting is; Quite a few kinds of protein HST; PtrToxA such as detritus nutrition fungal species Pyrenophora tritici-repentis and Stagonospora nodorum; SnTox1, SnTox2 and SnTox4 show susceptibility (Liu et al., 2004 that bright dipping relies on the necrosis induction of pattern and promoted host's wheat plant of toxin susceptibility; Friesen et al., 2007; Abeysekara et al., 2009; Manning et al., 2009).These effector performance functions are necessary, the host gene Tsn1 of corresponding dominance, Snn1; Snn2; Snn4 is plotted on the responsive host's of toxin the map, and the product of these genes is considered to the direct or indirect interaction receptor of HST (Liu et al., 2004; Friesen et al., 2007; Abeysekara et al., 2009; Manning et al., 2009).It should be noted that in HST, to show the interaction different that acceptor molecule is a resistance (Wolpert et al., 2002) necessary rather than in the nontoxic-resistant gene of classics interacts in HST with the gene pairs gene.PtrToxA is in the HST of P.triticirepentis and S.nodorum, study the most thorough.It has a modular structure that contains N-end secretion signal, and secretion signal is excised to form maturation protein, is that the host transports necessary RGD structural domain and C-end effector structural domain (Sarma et al., 2005 behind the secretion signal; Manning et al., 2007).It is reported that PtrToxA is positioned at chloroplast(id) and follows chloroplast protein ToxABP1 interaction (Manning et al., 2007).In case it is transported in the chloroplast(id), the mode that PtrToxA just relies on light promotes virulence and final induced activity oxygen species accumulation (Manning et al., 2009) through I of stray light system and photosystem II.

Class Nep1 albumen (NLP) is bacterium, fungi and oomycetes toxin (Pemberton and Salmond, 2004 of inducing dicotyledons downright bad; Ottmann et al., 2009).Although their distributions in different taxonomical groups are varied; NLP has a common fold characteristics; Promptly; One seven peptide (GHRHDWE) motif and two conservative halfcystines are arranged, show and the sea anemone cytolysin of sea life generation and structural similarity (Gijzen and Nurnberger, 2006 of synocytotoxin; Ottmann et al., 2009).The detritus vegetative phase in later stage that is expressed in host infection of NLP gene (NPP1 and PiNPP1.1) is (Qutob et al., 2002 of raising in the oomycetes pathogenic agent P.sojae of half biotroph and P.infestans; Kanneganti et al., 2006).These NLP have the death that helps host tissue under their dissolved cell activity effect, and therefore in detritus trophopathy substance process of growth, promote its intrusion (Qutob et al., 2002; Kanneganti et al., 2006).Although known that now some NLP promote virulence as toxin, and all members that may not be certain this family have the activity of synocytotoxin in their host.

The CBAC of our still unclear a lot of effector and they are the successful reproductions that how to strengthen pathogenic agent at present.We also know the target of thread pathogenic agent effector hardly, particularly transfer to the inner effector of host cell.

Summary of the invention

The objective of the invention is effector gene that carries among the separating clone dry thread Pyrenomycetes strains A G1IA and the dna fragmentation that comprises the promotor of regulating and control this gene.

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

Further purpose of the present invention provides this gene and is setting up the Molecular Detection system, detects the application in the plant hypochnus incidence.

Further purpose of the present invention provides the application of this gene in improving the paddy disease-resistant breeding.

Effector Gene A 1IA06910 among the dry thread Pyrenomycetes strains A G1IA of separating clone of the present invention, its nucleotide sequence is shown in SEQ ID NO.1.This nucleotides sequence is listed in the dry thread Pyrenomycetes mycelia and is constitutive expression.

The Argine Monohydrochloride sequence of said gene is shown in SEQ ID NO.2.Be to be understood that; Under the prerequisite that does not influence protein-active (promptly not in proteic active site), those skilled in the art carry out various replacements, interpolation to the aminoacid sequence shown in the SEQ ID NO.2 and/or lack the aminoacid sequence that one or several amino acid obtains to have same function.

In addition, consider the degeneracy of codon, for example can be in its coding region, under the condition that does not change aminoacid sequence, or at its non-coding region under the condition that does not influence protein expression, above-mentioned proteic gene order is modified to encoding.Therefore, the present invention also comprises replacement, the interpolation that the above-mentioned proteic gene order of encoding is carried out and/or lacks one or more amino-acid residues and the aminoacid sequence with identical function that forms.The present invention also comprises just sequence or the antisense sequences based on said gene, comprises the host cell that contains said nucleotide sequence or its segmental cloning vector or expression vector, contain said carrier, contains said nucleotide sequence or its segmental plant transformed nucleus transgene carrier.

The present invention comprises that equally the coding region with this gene is connected with an inducible promoter, and this promotor can be induced down at IPTG, in Bacillus coli cells, expresses this gene.Inducible promoter comprises the promotor of tissue specific expression and the promotor of accurate environmental induction.The present invention comprises that also the promotor with the coding region of this gene and a constitutive expression is connected, and this promotor can be expressed under any condition and at the different times of invading tissue.The promotor of this composing type comprises the promotor of cauliflower mosaic virus 35S etc., and the nucleotide sequence of this promotor is shown in SEQ ID NO.3.

AG1IA06910 expresses be connected back adding inductor with inducible promoter after, does not add inductor and does not express; With need not add the just sustainable expression of any inductor after constitutive promoter is connected.

Express.Wherein envrionment conditions comprises the upgrowth situation, temperature, humidity of plant etc., and the different times of invading plant comprises that spore germination, appressorium form, invade the nail differentiation and infect mycelia expansion etc.

Dry thread Pyrenomycetes effector gene 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 said gene order is connected to any conversion carrier that contains fluorescence protein gene, imports paddy rice or other vegetable cell with any method for transformation effector gene and fluorescence protein gene covalency.Utilize the fluorescence co-focusing transmission electron microscope can observe in paddy rice or other vegetable cell migration and the location of effector albumen in vegetable cell with the GFP amalgamation and expression.Utilize this effector to angle out in paddy rice or other plant and the protein bound receptor protein of this effector for bait protein.

Three of application is to utilize one or more bases that gene engineering method knocks out this receptor gene in paddy rice or other vegetable cell or deletion, interpolation, sudden change this receptor gene with disappearance or change the function of acceptor gene, obtains the resistant plant of anti-a certain effector.

The Another application of effector gene provided by the invention is according to the specific molecule marker of said gene order information generating, includes but not limited to SNP (SNP), SSR (simple sequence repetition polymorphum), RFLP (restriction enzyme length polymorphism), CAP (cutting amplified fragments polypeptide).Can detect the physiological strain of field dry thread Pyrenomycetes colony and the dynamic change of genetic construction thereof with these marks, and the distribution situation of this effector gene in the natural population of field; Help the disease resistance evaluation of rice varieties and the evaluation of dry thread Pyrenomycetes microspecies; The rational deployment that also helps disease-resistant variety with by turns so that control the generation of hypochnus effectively.

The present invention can further provide or the transgenic bacterial strain of the no corresponding effector gene function that 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 dry thread Pyrenomycetes microspecies and rice varieties through clone and functional analysis thereof to dry thread Pyrenomycetes effector gene.Can be in practice according to the structure of this gene and the molecular target of functional design novel agrochemical thereof; Can the proteic receptor protein gene of this effector in the host cells such as paddy rice be knocked out or suddenlys change, to obtain the permanent disease-resistant kind; Help to set up the Molecular Detection system of the pathogenic variation of dry thread Pyrenomycetes natural population, the distribution situation of research dry thread Pyrenomycetes effector gene in the natural population of field, the composition and the variation characteristics of microspecies in the announcement dry thread Pyrenomycetes colony; Also help disease resistance evaluation and the rational deployment thereof of rice varieties with by turns so that control the generation of hypochnus effectively.

Description of drawings

Fig. 1 is the map based cloning synoptic diagram of dry thread Pyrenomycetes effector Gene A 1IA06910;

Fig. 2 is the PCR detected result figure of dry thread Pyrenomycetes effector Gene A 1IA06910;

Fig. 3 is the SDS-PAGE detected result figure of dry thread Pyrenomycetes effector Gene A 1IA06910 expressing protein;

Wherein, M is a molecular weight marker among Fig. 2, is followed successively by 5000bp, 3000bp, 2000bp, 1000bp, 750bp (adding bright band), 500bp, 250bp, 100bp; Swimming lane 1,3,4 is the PCR product of AG1IA06910; Swimming lane 2 negative contrast (ddH ₂O ₂); Swimming lane 5-8 is the PCR product of the transformant bacterial strain of AG1IA06910 gene, and wherein 5,6 is false positive transformant bacterial strain.

Swimming lane M is a molecular weight marker among Fig. 3, is followed successively by 80kD, 60kD, 40kD (adding bright band), 30kD, 20kD, 12kD; Swimming lane 1,2 the purpose band occurs for the protein band after inducing at the 30kD place; Swimming lane 3 is inductive bacterium liquid eggs informal voucher band not.

Embodiment

Below in conjunction with embodiment, specific embodiments of the invention describes in further detail.Following examples are used to explain the present invention, but are not used for limiting scope of the present invention.These embodiment unless otherwise indicated, all scientific and technical terminologies among the application all have and the identical implication of one skilled in the art's common sense of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually adopt for example people such as Sambrook of normal condition; Molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the method for advising according to manufacturer.

Embodiment 1: the clone of the effector gene of prediction

The AG1IA bacterial strain be by the Sichuan Agricultural University paddy rice separation and purification from the field water sheath and culm blight of rice disease plant voluntarily, be ubiquitous fungal bacterial strain on the rice plant, extensively be distributed in the area of rice cropping.(the AG1IA bacterial strain has been published in Sichuan Agricultural University, Master's thesis, the analysis of dry thread Pyrenomycetes AG1IA inducing maize differential protein, author Li Yun)

Under Bechtop, with the mycelia of a small amount of AG1IA of tweezers picking, insert in the 50ml PDA substratum, smash mycelia to pieces, 28 ℃, 200r/m cultivates two days later, collects mycelia with four layers of filtered through gauze, extracts total RNA with liquid nitrogen grinding.Do primer with oligodT, reverse transcription obtains cDNA.Sequences Design primer according to prediction is following:

5’ATGTGCAGCGCACTTGTGTCCAGACCATGTGATA?AGATCTGG3’

5 ' TCACGTGGCCGTCGAGTCGGCAGGTTTCGGAGGTGCATGCGGAAT3 ' is that the template clone obtains goal gene, the gene of being cloned into such as Fig. 2 with cDNA.

Structure, conversion and the expression of embodiment 2:effector gene prokaryotic carrier

The not assorted band of the goal gene electrophoresis detection that PCR obtains; Can be according to TransGen Biotech Peasy-E1kit explanation; Be connected to goal gene on the expression vector pEASY-E1, identical through sequenced genes AG1IA06910 and forecasting sequence, base sequence is shown in SEQ ID NO.1.Conversion condition is identical with the normal intestinal bacteria conversion condition, the picking positive transformant, 37 ℃ be cultured to the OD value and be 0.6 after, change 28 ℃ over to, IPTG1mM induced 7 to 11 hours, expressed effector albumen.The SDS-PAGE of expressing protein detects figure like Fig. 3.

Embodiment 3: the pathogenic detection of expressing protein

The thalline ultrasonic disruption of collecting after AG1IA06910 expresses obtains crude protein, notes not making protein denaturation (broken time 5S is advisable, and power is no more than 200W, remains sample in the shattering process under condition of ice bath) in the shattering process.Choose the rice leaf in the tri-leaf period of the consistent hot-house culture of growth, be placed on and keep the moistening so that blade of filter paper to preserve moisture in the sterilization petridish of built-in two layers of filter paper.Toothpick with sterilization is made a call to an aperture in rice leaf central authorities, covers on the aperture with the filter paper of the 0.5cm * 0.5cm of three layers of sterilization, and the amount of inoculating crude protein is 50 microlitres.Blade after infecting is placed on 28 degrees centigrade of illumination boxs, illumination 12h, dark 12h, RH (disease index) 80%.A couple of days observes the blade incidence and takes pictures continuously, the record course of disease.Infect the blade that back 6 days reference proteins infect and do not have macroscopic variation, jaundice and Kong Bianda around the blade aperture that the effector protein product infects; Infecting blade that back 7 days effector protein products infect, obviously to compare the blade that infects according to albumen yellow, and black appears in regional area; Infect the blade that back 8 days reference proteins infect and still keep green, then big area jaundice of the blade that the effector protein product infects, downright bad (PCD).

Embodiment 4:effector expression of gene specificity analysis

Utilize the RT-PCR technology that AG1IA06910 expression of gene pattern is analyzed.Total RNA from the inoculation rice leaf and the mycelia of inoculation rice leaf are extracted after reverse transcription obtains cDNA, utilizes forecasting sequence design primer (primer sequence is with the primer sequence among the embodiment 1), all can be cloned into goal gene, like Fig. 2.The PCR program is: 94 ℃ of preparatory sex change 4min; 94 ℃ of sex change 50sec, 55 ℃ of annealing 50sec, 72 ℃ are extended 1min, 35 circulations; Extend 10min after 72 ℃.Explain that this gene is the gene of constitutive expression.

The application of embodiment 5:effector Gene A 1IA06910 gene

Utilize the sequence information of AG1IA06910 gene provided by the invention, according to the structure of this gene and the molecular target of functional design novel agrochemical; The staple of this novel agrochemical can be the analog of AG1IA010188 gene proteins; Can be attached to the target site in the paddy rice with AG1IA010188 gene proteins competitiveness; But can not make the further downstream delivery of signal path of rice pathogenesis; Thereby make the AG1IA010188 gene proteins not can be incorporated into the target site in the paddy rice, make the blocking-up of morbidity signal path at the receptor protein position.Can also with this gene proteins in paddy rice receptor protein gene or other gene silencing in the signal path or knock out; Cultivate the new rice variety of anti-hypochnus; Make sheath blight fungus form appressorium or to infect pad, but can not further invade plant tissue inside on the plant surface; Even it is inner perhaps to invade plant tissue; Because other gene in receptor protein gene or the signal path by reticent or knock out and make morbidity signal path blocking-up such as anaphylaxis, alleviates illness and can only be confined to certain sub-fraction zone in rather than develop into and put in order the strain plant.

Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.

Claims (10)

1. paddy rice dry thread Pyrenomycetes effector Gene A 1IA06910, its nucleotide sequence is shown in SEQ ID NO.1.

2. the albumen of the said genes encoding of claim 1.

3. albumen as claimed in claim 2, its aminoacid sequence is:

1) aminoacid sequence shown in the SEQ ID No.2; Or

2) aminoacid sequence shown in the SEQ ID No.2 is through replacing, lack and/or increasing one or more amino acid and have equal active aminoacid sequence.

4. the carrier and the host cell that contain the said gene of claim 1.

5. gene as claimed in claim 1 is characterized in that its function combines to regulate and control with transcription factor through promotor.

6. gene as claimed in claim 5 is characterized in that, said promotor is inducible promoter or constitutive promoter.

7. the application of the said gene of claim 1 in design pesticide molecule target spot.

8. the said gene of claim 1 is being set up the Molecular Detection system, detects the application in the plant hypochnus incidence.

9. the application of the said gene of claim 1 in the paddy disease-resistant breeding.

10. application as claimed in claim 9 is characterized in that, said breeding for disease resistance be protein product with the said gene of claim 1 in paddy rice receptor protein gene or other gene silencing in the signal path or knock out.

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