CN102021186A - Magnaporthe grisea MoPPF3 gene and function and application of coding protein of magnaporthe grisea MoPPF3 gene - Google Patents

Abstract

The invention discloses a magnaporthe grisea MoPPF3 gene and function and application of coding protein of the magnaporthe grisea MoPPF3 gene. The gene can control formation and pathogenicity of a magnaporthe grisea infection nail, the gene and cDNA (complementary deoxyribonucleic acid) thereof and protein coded by the gene respectively have the nucleotide or amino acid sequence shown as SEQ ID No. 1, No. 2 and No. 3 in a sequence list. The gene can be expressed in conidium, appressorium and infected hypha of magnaporthe grisea and is necessary for fatty acid metabolism. The MoPPF3 gene is knocked out, so that formation rate of magnaporthe grisea is reduced to be 1/3 that of wild type, osmotic pressure endurance capacity is obviously reduced, and capability to infect rice leaves is completely lost. Expression or modification of the protein coded by the MoPPF3 gene or/and homogenous protein thereof in other pathogenic fungi can be taken as important candidate targets and can be used for designing and screening antifungal novel medicament. The invention discloses a magnaporthe grisea gene and function and application of the coding protein of the magnaporthe grisea MoPPF3 gene, wherein the gene can control formation and pathogenicity of the magnaporthe grisea infection nail.

Description

The function of rice blast fungus MoPPF3 gene and proteins encoded thereof and purposes

Technical field

The present invention relates in microbiological genetic engineering and the plant protection field formation and the pathogenic gene and the application of proteins encoded thereof of control fungal infection nail.

Background technology

Rice blast fungus (Magnaporthe oryzae) belongs to the Ascomycotina fungi, can infect multiple grasses such as paddy rice, wheat, barley, causes seasonal febrile diseases.Especially this bacterium is infected the rice blast that paddy rice causes all there is generation every year in each cultivated rice district in the world, and harm is extensively serious.Generally speaking, the harm of rice blast can make paddy rice underproduction 5-10%, and the grave illness field can cause the paddy rice total crop failure.Rice blast was once repeatedly popular in China, also was one of main disease of China paddy rice.

Rice blast fungus originates in conidium to infecting of plant.Conidia germination attached to plant leaf forms germ tube, the top of germ tube is differentiated to form appressorium successively and infects nail, infecting nail relies on the huge turgescence of ripe appressorium accumulation to sting the plant epidermis cell, in vegetable cell, form and infect mycelia, infecting mycelia expands and grows surely with iuntercellular in vegetable cell, finally forming diameter on blade is the grey or the beige scab of 2-3 millimeter, infectivity mycelia in the scab penetrates plant tissue and is differentiated to form conidiophore in the air, further forms conidium.Conidium descends to discharge and adheres in the effect of wind, rain, causes infecting again plant.Rice blast fungus is attached to the cycle that conidium produces again from conidium and was generally 3-5 days, at vegetative season, if condition is suitable, can repeatedly infect, and plant is caused serious harm.It is essential to infect in the infection processs that is formed on rice blast fungus of nail, and can the decision rice blast fungus that has or not that infect nail invaded plants interior tissue cell, and then finishes and infect circulation and work the mischief; And infect the height of following closely rate of formation, determine the hazard rating of rice blast.If can block the formation of infecting nail, just can control the generation of rice blast or reduce the hazard rating of rice blast.Therefore, the molecule mechanism that the research rice blast fungus infects nail formation not only helps to disclose rice blast fungus and the relevant pathogenic molecular mechanism of filamentous fungus, and comprises that for research and development control the medicament of the fungal diseases of plants of rice blast has significant application value.

Is the process of a meticulous adjusting from formation, the maturation of appressorium to the formation of infecting nail, and this process is regulated and control by cell cycle, and is associated with conidial autophagy death, needs not oligogenic participation.But it is at present fewer about the report of in this respect gene clone and molecule mechanism.Identify these genes, resolve their molecular function and the influence pathogenic thereof, might therefrom find can be used as the albumen of sterilant target, for the efficient medicament of exploitation control rice blast and other fungal diseases of plants is established the theory and technology basis rice blast fungus.

Summary of the invention

Purpose of the present invention aims to provide fungal infection nail and forms and a pathogenic essential new gene and encoded protein matter thereof.

Fungal infection nail provided by the present invention formation and pathogenic essential gene source are in rice blast fungus, and name is called MoPPF3, can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna; SEQ ID № in the sequence table: 1 by 3118 based compositions, comprise the promoter region and the coding region of gene.There are 2 exons the MoPPF3 coding region, lays respectively at SEQID №: 15 ' end the 1501st between 1867 bit bases and the 1958th between 2628 bit bases; 5 ' end the 1st to 1500 bit bases be promoter sequence.

2) SEQ ID № in the sequence table: the polynucleotide encoding sequence of 3 protein amino acid sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits;

4) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 80% above homology, and the identical function protein DNA sequence of encoding.

The eDNA of MoPPF3 gene also belongs to protection scope of the present invention, can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna, by 1038 based compositions,

2) SEQ ID № in the code sequence tabulation: the polynucleotide sequence of 3 protein amino acid sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the polynucleotide sequence of the 2 dna sequence dnas hybridization that limit;

4) with sequence table in SEQ ID №: 2 dna sequence dnas that limit have 80% above homology, and the identical function protein DNA sequence of encoding.

The rigorous condition of described height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

Utilize the promotor of MoPPF3 gene, the expression vector that coding region sequence makes up and clone and the host bacterium that obtains by this carrier conversion all to belong to protection scope of the present invention.

With the nucleotide sequence in arbitrary zone in MoPPF3 gene design primer, and be used for detecting that the MoPPF3 expression of gene also belongs within protection scope of the present invention under the compound treatment situation by pcr amplification.

The protein MoPpf3 of MoPPF3 genes encoding also belongs to the protection domain of this invention, is the protein with one of following aminoacid sequence:

1) the SEQ ID № in the sequence table: 3; Its sequence is made up of 345 amino acid.

2) with SEQ ID № in the sequence table: 3 aminoacid sequence through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with hypha,hyphae growth or generation of conidium and pathogenic relevant protein.The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.

Have with MoPpf3 or with it 40% or above conforming homologous protein in the aminoacid sequence design polypeptide in arbitrary zone, and preparation antibody is used for detecting, and the proteic expression of MoPpf3 also belongs within protection scope of the present invention under the compound treatment situation.

The present invention has proved that the sudden change of MoPPF3 gene or disappearance cause rice blast fungus to infect nail rate of formation and pathogenic remarkable reduction.Illustrate that the MoPPF3 gene is that rice blast fungus causes the necessary gene of rice blast.Therefore, screening can stop this genetic expression and its protein expression, modification and localized compound, can effectively control the generation of conidial generation of rice blast fungus and rice blast, and the candidate's novel drugs that can be used as new type bactericide is directly utilized.That is to say that the important use of MoPPF3 provided by the present invention is that this expression of gene and its protein expression, modification and location can be used as screening and the design that important candidate's target site is used for antifungal medicine (particularly anti-rice blast fungus medicament).Further resolve the signal transduction path that infects nail formation that this gene participates in, can find also that therefrom candidate's target site is used for the screening and the design of antifungal medicine (particularly anti-rice blast fungus medicament).In addition, also can separate this sequence again as probe or as the basis of PCR design of primers in rice blast fungus with a certain section of this gene nucleotide series, what also can be used to screen, separate other fungi has the sequence of certain sequence homology with this gene.

Description of drawings

Accompanying drawing 1. wild type strain P131 and mutant MS5087 infect the comparison of nail formation situation.The conidium of wild type strain P131 and mutant MS5087 inoculates onion epidermis cell respectively, and dark is preserved moisture and cultivated 24 hours, and the formation situation of nail is observed and relatively infected to microscopically.The white arrow indication is for infecting nail among the figure, and scale is 25 microns.

The foreign aid inserts mark hygromycin gene on position synoptic diagram in the MoPPF3 gene among the accompanying drawing 2. mutant MS5087.The inverted triangle indication inserts the insertion site of mark hygromycin gene for the foreign aid.

Accompanying drawing 3. gene M oPPF3 knock out.A) knock out tactful synoptic diagram.The forward and reverse primer of left arm be respectively upf (5 '-CACTAGTCGTCATCGTATCGAC-3 ', 5 ' end contains the SpeI restriction enzyme site) and upr (5 '-CGAATTCAATGGGTATTTGATC-3 ', 5 ' end contains the EcoRI restriction enzyme site), the forward and reverse primer of right arm be respectively downf (5 '-ACTCGAGCATTCATCAGTCCAC-3 ', 5 ' end contains the XhoI restriction enzyme site) and downr (5 '-AGGTACCGGACAGTTCAAGGTC-3 ', 5 ' end contains the KpnI restriction enzyme site); The pcr amplified fragment of left and right two arms is respectively with Spe I+EcoR I and Xho I+Kpn I digestion, is connected respectively to the both sides of hygromycin phosphotransferase gene in the pKOV21 carrier (hph) according to the upstream and downstream order, constitutes the gene substitution knockout carrier.Primer out and hphup are used for PCR and are just screening and knock out body, and primer inf and inr are used for negative screening and knock out body.S represents SacI.B) MoPPF3 knocks out the checking of body.Left side figure is the Southern hybridization checking that MoPPF3 knocks out body.Wild type strain P131 digests through SacI respectively with the genomic dna that knocks out body CD5, transfer on the nylon membrane, with A) shown in probe1 be that hybridization probe (being the right arm of knockout carrier) is hybridized, the hybrid belt of wild-type P131 is 2745bp, the hybrid belt that knocks out body CD5 is 6534bp.Right figure is the RT-PCR checking that MoPPF3 knocks out body.Wherein P131 is a wild-type, and CD5 is the body that knocks out of MoPPF3, and CX6 is a complement.ACTIN is as the internal reference thing.

The body that knocks out of accompanying drawing 4. wild type strains and MoPPF3 gene infects the comparison of following closely rate of formation.The conidium that knocks out body CD5 of wild type strain P131 and MoPPF3 inoculates onion epidermis cell respectively, and dark is preserved moisture and cultivated 24 hours, and nail formation situation is observed and add up, relatively infected to microscopically.Left side figure is for infecting situation relatively, and right figure is for infecting the nail rate of formation relatively.The experiment triplicate; Scale is 25 microns.

Accompanying drawing 5. wild type strains and MoPPF3 gene knock out body to paddy rice, the pathogenic comparison of barley.Left figure and middle figure are respectively the conidial suspension of rice blast fungus, and (concentration is 5 * 10 ⁴Individual/milliliter) spray inoculation paddy rice and the typical photo of barley leaves after 5 days; The typical photo of rice leaf after 5 days that the inoculated by hypha block that right figure is a rice blast fungus scratches.P131 and CD5 represent wild-type respectively and knock out body.

The appressorium that knocks out body of accompanying drawing 6. wild type strains and MoPPF3 gene is compared the induction of osmotic pressure.The reagent that forms osmotic pressure is 30%PEG-8000.The experiment triplicate; P131 and CD5 represent wild-type respectively and knock out body.

The comparison that knocks out body colonial morphology under the Different Nutrition condition of accompanying drawing 7. wild type strains and MoPPF3 gene.The body CD5 that knocks out of wild type strain P131 and MoPPF3 is containing 50mM glucose (Glucose), or 50mM sodium-acetate (Sodium acetate), or the minimum medium (MM) of 50mM sweet oil (Olive oil) is gone up in the colonial morphology of 25 ℃ of illumination cultivation after 7 days relatively.

The cluster analysis of accompanying drawing 8.MoPpf3 and its homologous protein.Illustrate: FG09633, NCU07263, AnAcuH, ScCrc1, UM05869, DmColt, HsCact1 and AtBou are for deriving from Fusarium graminearum (Fusariumgraminearum) respectively, coarse arteries and veins born of the same parents enzyme (Neurospora crassa), Aspergillus nidulans (Aspergillusnidulans), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), smut of maize bacterium (Ustilagomaydis), fruit bat (Drosophila melanogas ter), the MoPpf3 homologous protein in human (Homo sapiens) and the Arabidopis thaliana (Arabidopsis thalanian).The albumen cluster analysis is to use NJplot and MEGA software to finish.

Dynamic and the Subcellular Localization of the expression of accompanying drawing 9.MoPpf3 in rice blast fungus.(concentration is 2 * 10 will to contain the conidial suspension of the transformant CG11 of MoPPF3-GFP fusion vector ⁵Individual/milliliter) front that point is received fresh onion entocuticle, inoculate back 0 hour, 12 hours, examined under a microscope conidium, appressorium respectively in 24 hours and infected the expression of MoPPF3-GFP in the mycelia.Illustrate: the left column photo is to take under the bright field, and right row photo is to take under the dark field.Last row is a conidium; The appressorium that middle row conidium inoculation onion epidermis formed after 12 hours; Following row inoculates the mycelia of infecting that onion epidermis forms after 24 hours for conidium.Scale is 20 microns.

Embodiment

For a better understanding of the present invention, illustrate further by the following examples, but be not limitation of the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.

Embodiment 1, gene M oPPF3 separation and evaluation

1) infects the nail rate of formation and reduce mutant choice.Conidium (2 * 10 with each transformant in the REMI transformant storehouse of wild-type rice blast fungus bacterial strain P131 ⁵) be inoculated into onion epidermis cell respectively, after 25 ℃ of dark were preserved moisture 24 hours, be 10 * 40 microscopically observation and add up the nail that infects that forms in magnification, screen one and infect the mutant MS5087 that the nail rate of formation reduces (being about 27%).This mutant and wild-type P131 form the diversity ratio that infects nail and see Fig. 1 on onion epidermis.

2) genetic analysis of mutant MS5087.With mutant MS5087 with do not have hygromycin resistance, the wild type strain S1528 sexual hybridization that mating type is opposite, picking filial generation thecaspore carries out hygromycin resistance and infects the analysis of nail rate of formation, discovery is in 35 thecaspores being measured, the thecaspore of 18 tidal mycins all shows as wildtype phenotype, the thecaspore of 17 moisture resistance mycins all shows as mutant phenotype (infecting the nail rate of formation reduces), show the mutant phenotype of mutant MS5087 and foreign aid insert the mark hygromycin gene be divided into from, and hygromycin gene unit point in mutant gene group DNA inserts.

3) evaluation of the influenced gene of function among the mutant MS5087.Choose and insert that pUC18 does not partly have the restriction enzyme HindIII of restriction enzyme site to clear up the genome of mutant fully among the plasmid pUCATPH, carry out from connection, the competent cell of transformed into escherichia coli JM109 with the T4DNA ligase enzyme behind the ethanol sedimentation.Extract the plasmid of transformant, and carry out enzyme and cut evaluation.Enzyme is cut in the correct plasmid of evaluation and the sequence of pUC18 part, also contain the genomic sequence that part is inserted the site side in being contained pUCATPH.With itself and Pyricularia oryzae genome ( Http:// www.broadinstitute.org/annotation/genome/magnaporthe_gri sea/) compare, predict, find that the insertion site of exogenous plasmid among the mutant MS5087 is arranged in first exon of MGG_10492.5, is positioned at ATG downstream 116bp (Fig. 2), called after MoPPF3.This position of inserting the site is corresponding to SEQ ID №: 1 the 1616th.Extract the predicted amino acid sequence of MoPPF3 genes encoding and carry out the BLASTP comparison on NCBI, the albumen of finding this genes encoding may be one and be positioned the mitochondrial fatty acyl carnitine carrier of fungi.

4) clone of gene M oPPF3 cDNA.Extract total RNA in the wild-type Pyricularia oryzae P131 mycelia with the Trizol method, and clear up wherein DNA with DNasI.With Oligo (dT) is primer, carries out reverse transcription, obtains article one chain of MoPPF3cDNA.According to the cDNA sequences Design RT-PCR primer rtf (5 '-ATGTCAGCATCTTCGCAAC-3 ') of supposition MoPPF3 in the international Pyricularia oryzae database and rtr (5 '-TCAGTCAAACACCTTGTTC-3 ').Article one chain with cDNA is a template, the cDNA of amplification MoPPF3 gene.Gained PCR product is connected to the T-carrier, after sequence verification is correct, has obtained the cDNA sequence of gene M oPPF3, and its nucleotide sequence comprises SEQ ID №: the base of the 1st to the 1038th shown in 2.

Embodiment 2, gene M oPPF3 infect the effect of following closely in the forming process at Pyricularia oryzae

1) MoPPF3 gene complementation mutant MS5087

A. the structure of complementary carrier

From plasmid pSM331, downcut neomycin phosphotransferase gene with XbaI, it is connected with the pBlueScriptKS (+) of XbaI enzyme cutting, obtain plasmid pKN.According to the nucleotide sequence of gene M oPPF3, designed respectively forward primer hbup (5 '-AGGTACCTGACAGCAGTCCGTG-3 ', KpnI) and reverse primer hbdown (5 '-ACTCGAGAGTTTGTGGCGTCTG-3 ', XhoI).Genomic dna with wild type strain P131 is a template, go out the dna fragmentation of a 3118bp with fidelity LA-Taq enzymatic amplification, this fragment comprises the promoter sequence of MoPPF3 gene and upstream 1.5kb thereof, and its sequence is corresponding to SEQ ID № in the sequence table: the 1-3118bp in 1.This fragment is connected on the carrier pKN that cuts with same enzyme after cutting with KpnI and XhoI enzyme.Enzyme is cut evaluation, and after sequence verification is correct, obtains including the MoPPF3 gene of prediction and the complementary plasmid vector pCPPF3 of selective marker neomycin phosphotransferase gene.

B. the conversion of rice blast fungus

Adopt CaCl ₂The method of the conversion fungi protoplastis of/PEG mediation, with complementary carrier pCPPF3 random integration in the genome of mutant MS5087.Concrete operations following (being aseptic technique):

With the mycelia of mutant MS5087,150 milliliters of liquid CM substratum (yeast extracts 0.1% in 500 milliliters of triangular flasks of spore mixture inoculation, enzymic hydrolysis casein food grade 0.05%, acid hydrolysis casein food grade 0.05%,, glucose 1%, nitrocalcite 0.1%, potassium primary phosphate 0.02%, sal epsom 0.025%, sodium-chlor 0.015%), under 26 ℃, 100 rev/mins conditions, shake training 36 hours.Three layers of sterilization lens wiping paper filter and collect mycelium, mycelium is transferred in 50 milliliters of centrifuge tubes after washing with the 0.7M sodium chloride solution, the enzyme penetrating fluid that per 1 gram mycelia adding is 1 milliliter (contains 20 mg/ml driselases, with the preparation of 0.7M sodium-chlor), 26-28 ℃, under 100 rev/mins of conditions during enzymolysis 3-4 after, with 0.7M sodium-chlor washing mycelium, filter through three layers of sterilization lens wiping paper, collect protoplastis, 4,000 rev/min centrifugal 15 minutes, earlier with 25 milliliters of STC (1.2M sorbyl alcohol, 10mM Tris-Cl, pH 7.5,50mM calcium chloride) the washing protoplastis is once washed 2 times with 10 milliliters of STC respectively then, with STC protoplastis concentration is transferred to 1 * 10 after the centrifugation ⁸Individual/milliliter.

The protoplastis of mutant MS5087 is sub-packed in 50 milliliters of centrifuge tubes, every pipe 150 microlitres, add isopyknic linearizing complementary carrier pCPPF3 (about 2 micrograms) and STC mixed solution, placed on ice 20 minutes, dropwise slowly add 2 milliliters/pipe PTC solution (60% poly-hexylene glycol 3350 then, 10mM Tris-pH 7.5,50mM calcium chloride), static 20 minutes, add the ice-cold STC of 25 milliliters/pipe on ice, slow mixing, 4,000 rev/mins, 4 ℃ centrifugal 15 minutes, remove supernatant, every then pipe adds 3 milliliters LR substratum (0.1% yeast extract, 0.1% enzymic hydrolysis casein food grade, 1M sucrose), room temperature leaves standstill cultivated after 12 hours, change culture dish over to, add about 12 liters of SR (LR+1.6% agar) that are cooled to about 50 ℃, mixing, treat its solidify dry up after, spread 0.7% agar (be cooled to about 50 ℃, contain the Xin Meisu of 400 mcg/ml) of 12 milliliters of one decks above.Cultivated 4～6 days for 26 ℃, the transformant that occurs is gone on the CM solid medium that contains 400 mcg/ml Xin Meisus, after the postsearch screening single bacterium colony changed on the rolled oats tomato substratum and cultivate, and carry out monospore and separate.

C. complement and wild-type P131 infect the comparison of nail rate of formation

Conidium (2 * 10 with complementary transformant ⁵) be inoculated into onion epidermis cell, after 25 ℃ of dark were preserved moisture 24 hours, be 10 * 40 microscopically observation and add up the nail that infects that forms in magnification.Found that the nail rate of formation that infects of complementary transformant is about 84%, has reached wild type strain and has infected the level of following closely rate of formation (being about 86%).Mutant phenotype among this explanation mutant MS5087 is that the normal function owing to gene M oPPF3 is affected and causes, and gene M oPPF3ATG upstream 1.5kb fragment has comprised functional promotor.SEQ ID № in the functional promoter sequence of MoPPF3 gene such as the sequence table wherein: the 1-1500bp in 1.

2) the MoPPF3 gene knocks out analysis

A. the structure of knockout carrier

Gene knockout adopts the method for homologous recombination, and with the coding region that hygromycin phosphotransferase gene is replaced MoPPF3 gene among the wild-type P131, specific strategy is seen accompanying drawing 3.To from vector plasmid pCB1004, downcut hygromycin phosphotransferase gene and be connected to pBlueScriptKS (+) connection of cutting, obtain new plasmid vector pKNH with the same restrictions restriction endonuclease.Genomic dna with wild-type P131 is a template, with primer upf (5 '-CACTAGTCGTCATCGTATCGAC-3 ', 5 ' end contains the SpeI restriction enzyme site) and upr (5 '-CGAATTCAATGGGTATTTGATC-3 ', 5 ' end contains the EcoRI restriction enzyme site) fragment that amplifies is as left arm, with primer downf (5 '-ACTCGAGCATTCATCAGTCCAC-3 ' 5 ' end contain XhoI restriction enzyme site) and downr (5 '-AGGTACCGGACAGTTCAAGGTC-3 ', 5 ' hold contain the KpnI restriction enzyme site) fragment that amplifies is as right arm.Left arm is with SpeI and FcoRI double digestion, and right arm is with XhoI and KpnI double digestion, is connected respectively to the both sides of hygromycin phosphotransferase gene among the carrier pKNH according to the upstream and downstream order, obtains the knockout carrier pKOC5 of gene M oPPF3.

B. the conversion of rice blast fungus and the acquisition that knocks out body

Adopt above-mentioned CaCl ₂The method for transformation of/PEG mediation is transformed into knockout carrier pKOC5 among the wild type strain P131, and what finally obtain gene M oPPF3 knocks out body CD5, and this knocks out body and has carried out verifying (Fig. 3) with Southern hybridizing method and RT-PCR method respectively.The Southern results of hybridization shows, is that hybridization probe (being the right arm of knockout carrier) is hybridized with the probe1 shown in Fig. 3 A, and wild-type P131 shows the hybrid belt of 2.7kb, knocks out the hybrid belt that body CD5 shows 6.5kb.RT-PCR result shows, can amplify a large amount of transcripts of MoPPF3 gene among the wild-type P131, and in knocking out body CD5 without any amplified band.So CD5 is the body that knocks out of MoPPF3 gene.

C. knock out the comparison that body and wild-type P131 infect the nail rate of formation

With conidial suspension (2 * 10 ⁵) the some front of receiving fresh onion entocuticle, observe and Taking Pictures recording infects the forming process of nail and infects the rate of formation of nail in microscopically after 24 hours.Found that, MoPPF3 knock out body CD5 infect the nail rate of formation be 26%, and wild-type P131 infect the nail rate of formation 86% (Fig. 4).The mutant MS5087 that knocks out among body CD5 and the present invention of MoPPF3 has similar defective infecting on the nail rate of formation, and all the nail rate of formation that infects than wild-type P131 significantly reduces.This explanation MoPPF3 gene is essential by the formation that rice blast fungus infects nail.

Embodiment 3, the effect of MoPPF3 gene in rice blast fungus is pathogenic

(concentration is 5 * 10 with the conidial suspension that knocks out body CD5 of wild-type P131 and MoPPF3 ⁴Individual/milliliter) spray inoculation is in four leaves, one heart stage rice leaf front equably, an or leaf one heart stage barley leaves front, or mycelia piece point received four leaves, one heart stage rice leaf front, lucifuge was cultivated 36 hours under 26 ℃ and 100% relative humidity, see the wet cultivation of the follow-up continuation of insurance of light 3 days, observe incidence.Found that, the knocking out body CD5 and on host plant paddy rice and barley, all can not form scab of MoPPF3 gene, on the rice leaf that scratches, can not form scab, and under the same terms, wild-type P131 all can form typical rice blast scab (Fig. 5) on paddy rice and barley leaves.This explanation, with respect to wild-type P131, the virulence that knocks out body CD5 of MoPPF3 significantly reduces, and MoPPF3 is the pathogenic necessary gene of rice blast fungus.

Embodiment 4, the effect of MoPPF3 gene in anti-osmotic pressure ability of rice blast fungus and fatty acid metabolism

1) wild-type and the comparison that knocks out the anti-osmotic pressure ability of body

(concentration is 2 * 10 with the conidial suspension that knocks out body CD5 of wild-type P131 and MoPPF3 gene ⁵Individual/milliliter) to put and receive on the cover glass, dark is preserved moisture and was cultivated 24 hours.Then cover glass being immersed in 30% PEG-8000 (w/v) solution 15 minutes, is that 10 * 40 microscopically is observed and the appressorium number of statistics distortion in magnification.Found that the ratio that knocks out body CD5 distortion appressorium of MoPPF3 is about 52%, and the ratio of wild bacterium P131 distortion appressorium is about 27% (Fig. 8).Knocking out of this explanation MoPPF3 significantly descends the ability of Pyricularia oryzae tolerance osmotic pressure.

2) wild-type and the comparison that knocks out body colonial morphology under the Different Nutrition condition

The body CD5 25 ℃ of illumination cultivation on the minimum medium that contains 50mM glucose, 50mM sodium-acetate and 50mM sweet oil respectively that knock out of wild-type P131 and MoPPF3 gene are compared colonial morphology after 7 days.Found that MoPPF3 knocks out body CD5 in that to contain on the minimum medium of sodium-acetate growth extremely slow, containing on the minimum medium of sweet oil, can not grow, all there were significant differences (Fig. 7) with wild bacterium P131 for this.This explanation gene M oPPF3 is that fatty acid metabolism is necessary.

The phylogenetic analysis of embodiment 5, MoPpf3

With the aminoacid sequence of rice blast fungus MoPpf3 NCBI ( Http:// www.ncbi.nlm.nih.gov/) database carry out blastp retrieval, obtain the homologous protein F609633 of MoPpf3, NCU07263, AnAcuH, ScCrc1, UM05869, DmColt, HsCact1 and AtBou, derive from Fusarium graminearum (Fusarium graminearum) respectively, coarse arteries and veins born of the same parents enzyme (Neurospora crassa), Aspergillus nidulans (Aspergillus nidulans), yeast saccharomyces cerevisiae (Saccharomyces cerevisiae), smut of maize bacterium (Ustilago maydis), fruit bat (Drosophilamelanogaster), human (Homo sapiens) and Arabidopis thaliana (Arabidopsis thalanian).The AtBou in Arabidopis thaliana, the consistence of the aminoacid sequence of other homologous protein and the aminoacid sequence of MoPpf3 is all more than 40%.Wherein, the aminoacid sequence of NCU07263, FG09633 and UM05869 is respectively shown in SEQ IDNo:4,5 and 6 in the sequence table.With NJplot above-mentioned proteinic aminoacid sequence being compared, with MEGA4 constructing system evolutionary tree (Fig. 8), found that the MoPpf3 proteinoid is prevalent in fungi, the plant and animal, is a conservative albumen.By comparison albumen MoPpf3 and analogue thereof; also find carnitine-fatty acyl carnitine translocator of MoPPF3 predictive genes coding; it contains the mitochondrial transport superfamily protein structural domain of three supposition, and this structural domain may participate in the transfer of mitochondrial inner membrane energy or the integration of other eukaryotic cell device (for example peroxysome) film.

Expression performance analysis and the Subcellular Localization of embodiment 6, MoPpf3

1) structure of MoPPF3-GFP fusion vector and conversion:

From plasmid vector pCam35s-GFP, downcut the GFP gene with NcoI, it is connected into the pKN that cuts with the NcoI enzyme, obtain plasmid pKNTG.With primer crclgfPf (5 '-ATTAGGTACCTGCATATCCGATGTAGCTTG-3 ') and crc1gfpr (5 '-ATATCTCGAGGTCAAACACCTTGTTCATG-3 '), genomic dna with wild-type P131 is a template, amplification obtains comprising the dna fragmentation ((genome sequence that contains gene M oPPF3 promoter region 1292bp and coding region) of 2417bp, after cutting with KpnI and XhoI enzyme, be connected on the carrier pKNTG that cuts with same enzyme, obtain containing the plasmid vector pKGPPF3 of fusion gene MoPPF3-GFP and selective marker neomycin phosphotransferase gene.Use above-mentioned CaCl ₂The method for transformation of/PEG mediation is transformed into knocking out among the body CD5 of gene M oPPF3 with pKGPPF3, obtain the transformant CG11 of MoPPF3-GFP fusion vector, the nail rate of formation (84.5 ± 2.4%) that infects of this transformant does not have significant difference with wild type strain P131, illustrates that MoPPF3-GFP can exercise the function of gene M oPPF3.

2) the expression performance analysis of gene M oPPF3-GFP:

(concentration is 2 * 10 will to contain the conidial suspension of the transformant CG11 of MoPPF3-GFP fusion vector ⁵Individual/milliliter) point receive fresh onion entocuticle the front.In inoculation back 0 hour respectively, 12 hours, 24 hours microscopicallies were observed and Taking Pictures recording conidium, appressorium and infect the expression of MoPPF3-GFP in the mycelia.Found that, conidium, the appressorium of CG11 with infect the green fluorescence that all can observe GFP in the mycelia, illustrate that gene M oPPF3-GFP all has expression (Fig. 9) in the whole infection processs of rice blast fungus.

3) Subcellular Localization of MoPpf3

Fluorescent microscope is observed the distribution of MoPPF3-GFP in conidium among the transformant CG11 down, and the result shows that this fusion rotein may be positioned at plastosome (Fig. 9).

Claims (9)

1. control is pathogenic in the rice blast fungus follows closely the indispensable gene MoPPF3 that forms with infecting, and it is characterized in that having SEQ ID №: 1 dna sequence dna, perhaps SEQ ID № in the code sequence tabulation: the polynucleotide sequence of 3 protein sequence.

2. rice blast fungus according to claim 1 is pathogenic and infect indispensable gene MoPPF3 encoded protein matter MoPpf3 that nail forms and have SEQ ID № in the sequence table: the amino acid residue sequence shown in 3.

3. rice blast fungus according to claim 1 is pathogenic and infect the cDNA that follows closely the indispensable gene MoPPF3 that forms, and it is characterized in that having SEQ ID № in the sequence table: the dna sequence dna shown in 2.

4. utilize the pathogenic expression vector that makes up with the indispensable gene MoPPF3 that infects nail formation of the described rice blast fungus of claim 1.

5. utilize right 4 described expression vectors to transform the clone and the host bacterium of gained.

6. according to claim 2, by rice blast fungus is pathogenic and infect indispensable protein MgPpf3 that nail forms and lack or suddenly change or modifies and it is infected follow closely rate of formation or/and the application in the virulence generation defective.

7. according to claim 6, pathogenic and infect indispensable protein MgPpf3 that nail forms with rice blast fungus as the utilization of target in design and screening antifungal drug.

8. the indispensable protein MoPpf3 that nail forms is infected in control in the described rice blast fungus of claim 2, homologous protein in other plant pathogenic fungi Fusarium graminearum (Fusarium graminearum) and Ustilago maydis (D C.) Corola. (Ustilago maydis) is respectively FG09633 and UM05869, its feature has SEQ ID № in the sequence table respectively: the aminoacid sequence shown in 5,6.

9. described according to Claim 8, in the rice blast fungus control control pathogenic and infect indispensable protein MoPpf3 that nail forms in the other plant pathogenic fungi homologous protein FG09633 and UM05869 as the utilization of target in design and screening antifungal drug.

Images