CN1699411A - Fungus conidium shape protein associated with pathogenicity and genes encoding same and use thereof - Google Patents

Abstract

The invention discloses a fungus conidium shape protein associated with pathogenicity and genes encoding same and use, wherein the protein has one of the following amino acid sequences, (1) SEQ ID No.1 in the sequence table, 3, (2) protein related to fungus conidiospore shape and pathogenicity obtained through subjecting SEQ ID No.3 amino acid sequences in the sequence table to one or several amino acid sequences substitution and/or deletion and/or addition. The amino acid sequence of the fungus conidiospore shape and pathogenicity related protein and the amino acid sequence of the encoding gene can be used as target position spot for the screening and design of antifungal medicaments, a certain segment of the nucleotide sequence can also be used as probes for separating the sequence again from Magnaporthe grisea, it can also be used for screening, separating the sequences of other fungus with a finite sequence consanguinity with the gene.

Description

Fungus conidium shape and pathogen-relatedprotein and encoding gene thereof and application

Technical field

The present invention relates to fungus conidium shape and pathogen-relatedprotein and encoding gene and application in microbiological genetic engineering and the plant protection field.

Background technology

(Magnaporthe grisea) is the fungi of Ascomycotina to Magnaporthe grisea, can infect paddy rice, wheat, barley, grain and other multiple grass, 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, harm 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.1993, rice blast was very popular in China, still underproduction over ten billion jin under the situation of control comprehensively.Magnaporthe grisea also can infect the turfgrass that Herba Stenotaphri helferi, Bermuda grass, eremochloa ophiuroides etc. belong to, and causes big area withered, is the common disease of turfgrass.

The conidium of Magnaporthe grisea resembles a pear in shape, and is made up of three cells.General 3 to 5 conidiums produce with the mode of the sympodium top from a conidiophore.After conidium discharges, on conidiophore, stay the scar of the shape of going down on one's knees.Magnaporthe grisea originates in conidial formation to infecting of plant, after its process comprises that conidium is attached on the plant leaf, through sprouting, appressorium forms with ripe, the generation and the accumulation of turgor pressure in the appressorium, infect nail and produce and directly the penetrating of plant epidermis, formation, the branch of infectivity mycelia in the vegetable cell, the infectivity mycelia is between vegetable cell and several steps such as expansion between tissue and field planting.When Magnaporthe grisea infects susceptible host, grey or the beige scab of infectivity mycelia more than the formation of the expansion between plant tissue diameter 2-3 millimeter, the mycelia of infecting in these scabs penetrates plant tissue and is differentiated to form conidiophore in the air, further forms conidium.Conidium descends to discharge, adheres to washing away of wind, rain, causes infecting again of plant.It is 3-5 days that Magnaporthe grisea is attached to the general required time of cycle that conidium produces again from conidium; At vegetative season, if condition is suitable, can repeatedly infect, work the mischief.Therefore, the conidium of Magnaporthe grisea is main primary source of infection and source of infection again, infects in the circulation indispensable in disease.The severity of rice blast and Magnaporthe grisea form that conidial what are directly related, so, the situation that people often measure the prediction disease to take place by conidium in the monitoring air.Research Magnaporthe grisea conidium forms and the molecular genetic of morphogenesis not only has important significance for theories for disclosing the pathogenic molecular mechanism of Magnaporthe grisea, and has significant application value for the control of instructing rice blast.

Report aspect formation of Magnaporthe grisea conidium and variation is fewer at present, even the general content that does not also all have about gene clone and molecule mechanism of report is arranged.

Hamer in 1989 etc. have reported Smo-mutant (Hamer, J.E., Valent, B., and Chumley, F.G., Mutations at the SMO genetic locus affect the shape of diversecell types in the rice blast fungus, Genetics, 1989 (122): 351-361.) be relevant Magnaporthe grisea conidium morphologic variation aspect report early.To be them at utilization Teflon film screen and obtain this mutant obtains in the process of defective Magnaporthe grisea mutant on appressorium forms, the mutant of this type can not form the conidium and the ascus of normal morphology, can form the thecaspore (can not sprout although the thecaspore of some Smo-is arranged) of normal morphology.The Smo-sudden change does not influence bacterial strain and nourishes and grows, forms conidium, reduction division and infecting host plant normally.

Shi in 1993 and Leung report that they handle the 70-15 bacterial strain with electroporation, from the bacterium that can survive, be separated to Con1-mutant (Shi, Z.X., and Leung, H.Genetic analysis and rapidmapping of a sporulation mutation in Magnaporthe grisea, MPMI, Vol.7, No.1,1993, pp.113-120.), it is different that this mutant and wild-type form conidial form, on each conidiophore, only produce a conidium of holding three cells of the elongation of giving birth to.Growth and the conidial formation of Con1-mutant on substratum has only 73% and 2.3% of wild-type respectively, can not form normal appressorium on slide glass or onion epidermis, and does not have pathogenic on original susceptible rice varieties.After 2 years, Shi and leung report that they use REMI to obtain a series ofly to form relevant mutant Con2--Con7-(Shi with conidium, Z.X., and Leung, H.Genetic analysis of sporulationin Magnaporthe grisea by chemical and insertional mutagenesis, MPMI, Vol.8, No.6,1995, pp.949-959.).CON1-7 (CON1 that comprises 1993 reports) is controlling a series of processes such as generation aerial hyphae, conidiophore, formation conidium, the normal conidium of formation form respectively.

Mutant con5-and con6-has completely lost conidium and has formed ability, and con1-, con2-, con4-and con7-act on the downstream influences of con5-and con6-and produce spore ability and conidial growth, and con4-and con7-sporulation quantity reduce about 35%.Con1-and con7-can not form appressorium, thereby have lost pathogenic to paddy rice, and con2-and con4-appressorium rate of formation obviously reduce, thereby pathogenicly obviously weaken.By the genetic affinity analysis revealed between them, Con1 and Con2 are chain, and Con5 and Con6 are chain, and have: the superordination of Con5＞Con6＞Con7 and Con2＞Con1.

1998, they have obtained a kind of Magnaporthe grisea mutant that forms conidial deviant Forms by REMI reports such as Lau, they call ACR1-(Lau to it, G.W., and Hamer, J.E.Acropetal:Agenetic locus required for conidiophore architecture and pathogenicity inthe rice blast fungus, Fungal Genetics and Biology, 1998 (24): 228-239.).Magnaporthe grisea normally produces three to five conidiums from the top of a conidiophore, sympodium mode difference therewith, the conidium of ACR1-in the mode of bunchiness and is born on the conidiophore, these conidial apical cells can not produce top spore mucus (STM), therefore can not adhere to hydrophobic surface.The ability of the generation of conidium appressorium of ACR1-also reduces greatly, can sprout in the conidium that produces germ tube and have only 5% can form appressorium, so virulence also weakens greatly.Lau etc. have also cloned corresponding gene, and its open reading frame comprises 633 amino acid whose zones of transcribing of coding, and 3 ' end comprises the non-coding region of long about 1.3kb.Discover that this comprises 633 about 2kb of amino acid open reading frame, comprise the ORFs of a plurality of weak points, all lack homology with known protein in the present database.

More than the research report shows that it is a very complicated incident that conidial formation of Magnaporthe grisea and form thereof make up, and comprises many relevant with it gene.Clone these genes, separate bright their molecular function and the effect in conidial formation of Magnaporthe grisea and morphogenesis, thereby study their influences pathogenic to Magnaporthe grisea, to help to make clear the pathogenic molecular mechanism of Magnaporthe grisea, and might therefrom find can be used as albumen and the gene and the promotor of sterilant target, for the effective chemical control approach of exploitation rice blast and other plant seasonal febrile diseases is established the theory and technology basis.

Summary of the invention

The purpose of this invention is to provide fungus conidium shape and pathogen-relatedprotein and encoding gene thereof.

Fungus conidium shape provided by the present invention and pathogen-relatedprotein, name is called MgCOM1, derives from Magnaporthe grisea, is the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 3;

2) with SEQ ID № in the sequence table: 3 amino acid residue sequence through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with fungus conidium shape and pathogenic relevant protein.

Sequence 3 in the sequence table is made up of 774 amino-acid residues.

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.

The encoding gene of MgCOM1 (MgCOM1) also belongs to protection scope of the present invention.

The genomic gene of MgCOM1 can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein 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 90% above homology, and the identical function protein DNA sequence of encoding.

Sequence 1 in the sequence table is by 4276 based compositions, comprise three exons and two introns, from the 786th of 5 of sequence 1 ' end to 956 bit bases is first exon, from the 957th of 5 of sequence 1 ' end to 1222 bit bases is first intron, from the 1223rd of 5 of sequence 1 ' end to 1485 bit bases is second exon, from the 1486th of 5 of sequence 1 ' end to 1556 bit bases is second intron, is the 3rd exon from the 1557th of 5 of sequence 1 ' end to 3447 bit bases; Its coding region the 786th of 5 of sequence 1 ' end between 3447 bit bases.From the 1st of 5 of sequence 1 ' end to 785 bit bases is promoter sequence.

The cDNA gene of MgCOM1 can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

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

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

4) with sequence table in SEQ ID №: 2 dna sequence dnas that limit have 90% above homology, and the identical function protein DNA sequence of encoding.

Sequence 2 in the sequence table is by 2591 based compositions, and its encoding sequence is to 2425 bit bases from the 101st of 5 of sequence 1 ' end.

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.

Contain the MgCOM1 expression carrier, clone and host bacterium all belong to protection scope of the present invention.

Arbitrary segmental primer is also within protection scope of the present invention in the amplification MgCOM1 gene.

The sudden change that experimental results show that the MgCOM1 gene causes Magnaporthe grisea to produce elongated conidium, form the ability that infects nail and infectivity mycelia and weaken, and the virulence on the affinity rice varieties weakens.Screening can destroy expression, modification and the localized medicine of this genetic expression, shearing and product thereof, is an important use of the present invention.The nucleotide sequence of MgCOM1 and the aminoacid sequence of MgCOM1 can be used as target site and are applied in the screening and design of antifungal medicine (particularly anti-Magnaporthe grisea medicament), also can separate this sequence again as probe in Magnaporthe grisea with a certain section of this nucleotide sequence, what also can be used to screen, separate other fungi has the sequence of certain sequence homology with this gene.

The present invention proves that the variation of Magnaporthe grisea MgCOM1 can make conidium become slender type by the pears type, and causes virulence significantly to weaken.Therefore, can utilize the dependency screening sterilant of this conidial morphologic variation and pathogenic variation.

Description of drawings

Fig. 1 is that the conidium form of the insertion mutant of wild strain 70-15 and three MgCom1 compares

Fig. 2 A is that insertion mutant X54 conidial germination rate on onion epidermis of wild strain 70-15 and MgCom1 compares

Fig. 2 B is that the rate of formation of insertion mutant X54 appressorium on onion epidermis of wild strain 70-15 and MgCom1 compares

Fig. 2 C is the insertion mutant X54 of wild strain 70-15 and MgCom1 infects nail on onion epidermis rate of formation comparison

Fig. 2 D is that the rate of formation of insertion mutant X54 infectivity mycelia on onion epidermis of wild strain 70-15 and MgCom1 compares

Fig. 3 is that insertion mutant X54, H3035 and the H3587 of wild strain 70-15 and three MgCom1 forms scab relatively on affinity paddy rice CO39 blade

Fig. 4 A is the synoptic diagram of complementary carrier pKNS

Fig. 4 B is the synoptic diagram of gene replacement vector pPSH

Fig. 5 is that the conidium form of complementary transformant CX54 and wild strain 70-15 compares

Fig. 6 A is that insertion mutant X54 conidial germination rate on onion epidermis of complementary transformant CX54 and wild strain 70-15 and MgCom1 compares

Fig. 6 B is that the rate of formation of insertion mutant X54 appressorium on onion epidermis of complementary transformant CX54 and wild strain 70-15 and MgCom1 compares

Fig. 6 C is the insertion mutant X54 of complementary transformant CX54 and wild strain 70-15 and MgCom1 infects nail on onion epidermis rate of formation comparison

Fig. 6 D is that the rate of formation of insertion mutant X54 infectivity mycelia on onion epidermis of complementary transformant CX54 and wild strain 70-15 and MgCom1 compares

Fig. 7 is insertion mutant X54, H3035, H3587 and corresponding complementary transformant CX54, CH3035, the CH3587 of wild strain 70-15 and three MgCom1 pathogenic comparison to the rice varieties CO39 of affinity

Fig. 8 compares for the conidium form of gene substitution transformant K204 and wild strain 70-15

Fig. 9 A is wild strain 70-15 and gene substitution transformant K204 infect the nail rate of formation on onion epidermis comparison

Fig. 9 B is the comparison of wild strain 70-15 and gene substitution transformant K204 infectivity mycelia rate of formation on onion epidermis

Formation was infected and is followed closely and the difference of infectivity mycelia when Figure 10 inoculate back 24 hours for wild strain 70-15 and gene substitution transformant K204 on onion epidermis

Figure 11 is wild strain 70-15 and mutant X54, H3035, and H3587 and gene substitution transformant K204 are to the pathogenic comparison of the rice varieties CO39 of affinity

Figure 12 Aa is the structural representation of complementary carrier pKNSP1.8

Figure 12 Ab is the conidium form with the transformant that obtains behind the pKNSP1.8 transforming gene displacement transformant K204

Figure 12 Ba is the structural representation of complementary carrier pKNSP1.0

Figure 12 Bb is the conidium form with the transformant that obtains behind the pKNSP1.0 transforming gene displacement transformant K204

Figure 12 Ca is the structural representation of complementary carrier pKNSP0.8

Figure 12 Cb is the conidium form with the transformant that obtains behind the pKNSP0.8 transforming gene displacement transformant K204

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.

Percentage composition among the following embodiment if no special instructions, is the quality percentage composition.

The acquisition of embodiment 1, fungus conidium shape and pathogen-relatedprotein MgCOM1 and encoding gene thereof

One, mutant choice and evaluation

1, the structure in the REMI transformant storehouse of Magnaporthe grisea bacterial strain 70-15

Extract pUCATPH (Lu in a large number by alkaline lysis, S., Lyngholm, L., Yang, G., Bronson, C., Yoder, O.C.1994.Tagged mutant s at the Toxl locus of Cochliobolusheterostrophus by restriction enzyme-mediated integration.Proc.Natl.Acad.Sci.USA91:1264-12653) plasmid DNA, cut its linearizing with restriction enzyme HindIII, KpnI, SacI and SmaI enzyme respectively, press method (Shi Z., Christian D. ， ﹠amp that REMI transforms; LeungH., Enhanced transformation in Magnaporthe grisea by restriction enzymemediated integration of plasmid DNA.Phytopathology, 1995,85:329-333.) the Magnaporthe grisea bacterial strain 70-15 of conversion wild-type.Add linearizing plasmid of 2 micrograms and corresponding restriction enzyme in each transformation system, wherein KpnI, HindIII, SacI and SmaI adopt 30U, 45U, 30U and 15U respectively.Concrete step is seen the conversion of the second section Magnaporthe grisea of embodiment two.

2, conidial preparation

The mycelia of each REMI transformant of Magnaporthe grisea bacterial strain 70-15 is fully interrupted, be uniformly applied on Tomato juice's medium oatmeal (every liter contain 150ml Tomato juice, 30-50 gram rolled oats boiled crossed leaching filtrate, 20 gram agar in 30 minutes) flat board, 26 ℃ of-28 ℃ of cultivations, when the visible newborn mycelia of naked eyes grows media surface, gently mycelia is washed with cotton swab, and water is rinsed well, cover the individual layer gauze, in 26 ℃ of-28 ℃ of illumination cultivation 48 hours, at the promptly visible a large amount of Magnaporthe grisea spore of media surface.

3, conidium morphologic variation mutant choice

In magnification is that 10 * 40 microscopically is observed the ripe conidial form of Magnaporthe grisea bacterial strain, and each bacterial strain is measured 50 conidial length of maturation and wide respectively.The result obtains the mutant X54 of three analogous shape variations, H3035, H3587 altogether.The conidium of these three mutant is growing and narrow (Fig. 1, A are 70-15, and B is X54, and C is H3035, and D is H3587) in shape than the conidium of the Magnaporthe grisea 70-15 of wild-type.Three mutant and the diversity ratio of wild-type bacteria on conidial length and width are as table 1.

The conidial length and the width of three mutant of table 1. and wild-type bacteria

	Long (unit: micron)		Wide (unit: micron)
					On average	The significance of difference	On average	The significance of difference
	??70-15 ??X54 ??H3035 ??H3587	??23.41 ??29.55 ??29.03 ??29.49	??A ??B ??B ??B	??9.74 ??8.24 ??8.14 ??7.95					??A ??B ??B ??B

Annotate: the significance of difference is between two groups of B does not have difference in 5% remarkable scope, the significance of difference be between A and B two groups in 5% remarkable scope significant difference.

4, pathogenic mensuration

Adopt the described method of step 2 to cultivate the conidium of Magnaporthe grisea, spore washes the back and filters in 50 milliliters centrifuge tube with double-deck lens wiping paper, and 4000 rev/mins of room temperatures were collected spore in centrifugal 5 minutes, were suspended in then in the polysorbas20 of 0.25 ‰ (volume ratio).Adjust conidium concentration to every milliliter 5 * 10 ⁴Individual spore, spray inoculation is in the face of blade of the affinity rice varieties CO39 seedling of one heart stage of five leaves equably.Using the same method prepares conidial suspension, adjusts conidium concentration to every milliliter 5 * 10 ⁵Individual spore, spray inoculation is in the front of fresh onion entocuticle equably.Back 2 hours respectively in inoculation, 6 hours, 12 hours, 24 hours, 36 hours, conidial sprouting is observed in microscopically in 48 hours and 72 hours, appressorium, infects the processes such as formation of nail and infectivity mycelia, and calculates conidial germination rate and appressorium, infects the rate of formation of nail and infectivity mycelia.

It is lower than the wild-type that the result shows that on onion epidermis three mutant form the ratio that infects nail and infectivity mycelia, wherein wild-type 70-15 and mutant X54 on onion epidermis, form infect follow closely and the ratio of infectivity mycelia shown in Fig. 2 A-Fig. 2 D and table 2.

The ratio of nail and infectivity mycelia is infected in table 2.70-15 and X54 formation in 24,36 and 48 hours behind the inoculation onion epidermis

The inoculation back time (hour)	Infect nail rate of formation (/ 100 appressorium)		Infectivity mycelia rate of formation (/ 100 appressorium)
					??70-15	??X54	??70-15	??X54
	??24 ??36 ??48	??30.0 ??86.3 ??89.9	??24.9 ??61.5 ??72.1	??0.0 ??59.4 ??72.5					??0.0 ??10.2 ??21.3

On affinity rice varieties CO39 blade, the virulence of three mutant is also lower than the wild-type, and wherein, three mutant and wild type strain form the comparative result of number of extendability scab and foxiness on the CO39 blade as shown in table 3; Wild strain 70-15 and three mutant X54, H3035 and H3587 form scab as shown in Figure 3 on affinity paddy rice CO39 blade, A is 70-15 among the figure, and B is X54, and C is H3035, and D is H3587.

Three mutant of table 3. and wild type strain form the comparison of the number of extendability scab and foxiness on the CO39 blade

	Expansion scab number (individual/10 centimetre blade)	Brown some scab number (individual/10 centimetre blade)	Total scab number (individual/10 centimetre blade)
				??70-15 ??X54 ??H3035 ??H3587	??44.4 ??18.3 ??0.7 ??1.5	??159.4 ??101.8 ??54.9 ??72.1	??203.8 ??120.1 ??55.6 ??73.6

Two, mutation type surface changes and inserts the mark coseparation analysis

Method being divided into genetic cross to insertion mark hygromycin gene and mutant phenotype in the mutant from analyzing, these three mutant do not had mutant phenotype and hygromycin resistance with one respectively, and the Magnaporthe grisea bacterial strain GUY11 that mating type is opposite is hybridized, analyze its thecaspore offspring's conidium shape and hygromycin resistance situation, concrete grammar is as follows:

With said mutation body X54, H3035 and H3587 respectively with do not have hygromycin resistance, the normal Magnaporthe grisea bacterial strain of the conidium form Guy11 cultivation that on the tomato medium oatmeal, stands facing each other.At first be cultured to colony edge and be about to receive together, then it is moved to 20 ℃ of illumination cultivation, about about 20 days perithecium in the intersection formation black projection of bacterium colony in 25 ℃.The sophisticated perithecium of picking squeezes gently in sterilized water and breaks, and discharges the ascus in the shell, and ascus suspension is coated on the water agar plate, and the single mycelia of picking ascus endoascus spore germination is cultivated on the tomato medium oatmeal after 24 hours.Add up these thecaspores offspring's hygromycin resistance and conidium form.The result is as shown in table 4, shows in the thecaspore offspring who measures, and the offspring of conidium shape sudden change has hygromycin resistance, illustrates that the phenotype of the conidium shape sudden change of these three mutant is isolating altogether with inserting the mark hygromycin gene.In the filial generation of mutant X54 and Guy11, the offspring of moisture resistance mycin is mutant phenotype entirely, and in the filial generation of mutant H3035 and H3587 and Guy11, the offspring of moisture resistance mycin is mutant phenotype a bit, some then is the wild-type phenotype, explanation is inserted the mark hygromycin gene and only is inserted in a genomic site in mutant X54, may be inserted in genomic two or more different sites and insert the mark hygromycin gene in mutant H3035 and H3587.

Hygromycin resistance and the conidium form of table 4.Guy11 and three mutant filial generations

	General Logistics Department's algebraically	The moisture resistance mycin			Moisture resistance mycin not
								Wild-type	Mutant	Add up to	Wild-type	Mutant	Add up to
		??X54×Guy11 ??H3035×Guy11 ??H3587×Guy11	??52 ??31 ??20	??0 ??7 ??3	??30 ??21 ??11	??30 ??28 ??14	??22 ??3 ??6							??0 ??0 ??0	??22 ??3 ??6

Three, the clone of the MgCOM1 gene relevant with the Magnaporthe grisea conidium shape

1. plasmid rescue and genome TAC library screening

By to inserting the rescue of plasmid in three mutant, obtain the genome sequence of corresponding insertion site side respectively, and determined the on position of plasmid in three mutant.Concrete grammar is as follows: choose and insert plasmid pUCATPH (Lu, S., Lyngholm, L., Yang, G., Bronson, C., Yoder, O.C.1994.Tagged mutants at the Toxl locus of Cochliobolus heterostrophusby restriction enzyme-mediated integration.Proc.Natl.Acad.Sci.USA91:1264-12653) pUC18 does not partly have the restriction enzyme of restriction enzyme site to clear up the genome of mutant fully in, and mutant X54 and H3587 select for use restriction enzyme BamHI to clear up, and mutant H3035 selects for use restriction enzyme EcoRV to clear up.Carry out from connecting the competent cell JM109 of transformed into escherichia coli with the T4DNA ligase enzyme the refining back of 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.In database, retrieve then identifying that correct plasmid checks order, analyze near the scope of the gene in insertion site and possible exon.The result shows that in mutant X54 plasmid is inserted in the restriction enzyme site place of a restricted type restriction endonuclease SmaI, corresponding to the 174th among the SEQ ID NO.1; In mutant H3035 and H3587, plasmid is inserted in the restriction enzyme site place of a restricted type restriction endonuclease HindIII, corresponding to the 1098th among the 2120th of SEQ ID NO.1 and the SEQ ID NO.2.With the official website of the side sequence that obtains and international rice blast fungus genome association ( Http:// www.riceblast.org/) Magnaporthe grisea announced genome database relatively, these two are inserted sites and all are positioned at chromosomal contig2.217 I number.Dna segment (sequence 4) with one section 1392bp between these two sites is a probe, (its average cloned sequence size is 50kb in the genome TAC library of screening Magnaporthe grisea 70-15, cover complete genomic 16-18 altogether doubly, this genome TAC library is according to document scholar's Wei equality (Construction and Evaluation of a TAC Library of Magnaporthe grisea, Plant Pathology, 2003,33:57-62) method of describing makes up), obtain four positive colonies, they all comprise the genomic gene (nucleotide sequence of the 1st to the 4276th shown in the SEQ ID NO.1) of MgCOM1.

Restriction enzyme that relates in the plasmid rescue process and T4DNA ligase enzyme are produced by precious biotechnology (Dalian) company limited, consult and use specification sheets and use.

2, RT-PCR and 5 ' RACE obtain the cDNA gene of MgCOM1

Collecting the product spore time according to the conidial method of 2 preparations of being mentioned in the step 1 is the conidium of 24 hours Magnaporthe grisea bacterial strain 70-15, guanidinium isothiocyanate (GT) method extracts total RNA, clearing up DNA wherein, is primer SuperscriptII with poly (T) then ^TMThermoScript II carry out reverse transcription, obtain article one chain of the total cDNA of conidium.Utilize Britain Exeter university foundation plant pathogenic fungi est database ( Http:// cogeme.ex.ac.uk/), following two couples of RT-PCR primer: 609 ': the 5 '-CTTTTGCCATAACTCTGAGC-3 ' of design and down6428:5 '-GGTGCCCTGACAGTAGTTTCC-3 ' from 3 ' non-translational region and two different exons; 2069:5 '-ACACCACTTGCTAAGAAGTTC-3 ' and prepoly3:5 '-CACCTGGTATAGGAGATCCG-3 ', 3 ' end cDNA sequence of amplification gene.The cDNA fragment that amplification is obtained is connected to back order-checking on the T-carrier, 5 ' sequences Design 5 ' the RACE primer of holding that utilizes order-checking to obtain: comprise gene specific reverse transcription primer RT primer:5 '-CTCATCTTCGTC-3 ' and following two couples of PCR primer: A1:5 '-TTCGGATCGGTAACGTAGAGG-3 ' and S1:5 '-CAACCCGACGATGCCAATA-3 '; A2:5 '-CTGTGTTGCCGCCGTAGAGGA-3 ' and S2:5 '-CAAGAAGCCCAAGTACATGCC-3 ', the cDNA sequence of amplification gene 5 ' end.The experimental technique of 5 ' RACE is with reference to 5 ' Full RACE Core Set of precious biotechnology (Dalian) company limited.Equally the cDNA fragment that is obtained is connected to back order-checking on the T-carrier, and splices the 3 ' end of the gene that records respectively and the cDNA sequence of 5 ' end.The result shows that the cDNA gene of MgCOM1 has the base pair of the 1st to the 2591st shown in the SEQ ID NO.2.Its encoding sequence be from SEQ ID NO.2 the 101st to the 2425th bit base, coding has the protein MgCOM1 of the amino acid residue sequence of sequence 3 in the sequence table.The Protein Data Bank of MgCOM1 and NCBI is compared, this protein belongs to a kind of new protein, the similarity of it and all known protein matter is all very low, only the putative protein (accession number is EAA67862.1) with gibberella has 36% consistence, with the predicted protein (accession number is EAA35776.1) of Neurosporaintemedia 33% consistence is arranged.

The nucleotide sequence of the genomic gene of sequence 1 and the cDNA sequence of sequence 2 are compared, the result shows in the genomic gene of MgCOM1 and comprises two introns, the shearing of these two introns all meets " GT-AG " principle, from the 786th of 5 of sequence 1 ' end to 956 bit bases is first exon, from the 957th of 5 of sequence 1 ' end to 1222 bit bases is first intron, from the 1223rd of 5 of sequence 1 ' end to 1485 bit bases is second exon, from the 1486th of 5 of sequence 1 ' end to 1556 bit bases is second intron, is the 3rd exon from the 1557th of 5 of sequence 1 ' end to 3447 bit bases; Its coding region the 786th of 5 of sequence 1 ' end between 3447 bit bases.

The affirmation of embodiment 2, MgCOM1 and encoding gene function thereof

Comprise complementation test and gene substitution experiment.Comprise the structure of complementary carrier and gene replacement vector in this section in the content and, obtain the recombinant conversion body of Magnaporthe grisea in the above-mentioned two class carriers importing Magnaporthe grisea.The structure of complementary carrier is meant that the dna segment of genomic gene (Nucleotide of the 4370th of contig2.217 the 14885 in Nucleotide to the in the Magnaporthe grisea genome database) of total length that will comprise the MgCOM1 of prediction links to each other with a carrier that has neomycin resistance gene, here neomycin resistance gene is not a limitation of the present invention, other can cause the gene of antibiotics resistance, and promptly the gene that causes fungus resistant outside the hygromycin gene can reach same effect.The Magnaporthe grisea F-strain that complementary carrier is imported is that three mutant are Magnaporthe grisea X54, H3035 and H3587.The structure of gene replacement vector is meant that the genomic gene (Nucleotide of the 4370th of contig2.217 the 14885 in Nucleotide to the in the Magnaporthe grisea genome database) of total length that will comprise the MgCOM1 of prediction is connected in the carrier, replaces the major part of the genomic gene of MgCOM1 then with hygromycin gene.The Magnaporthe grisea bacterial strain that gene replacement vector imported is wild-type Magnaporthe grisea 70-15, gene replacement vector passes through the flanking sequence and the genomic corresponding sequence generation of the wild type strain homologous recombination of gene both sides, thereby genomic gene partial sequence and the hygromycin gene of corresponding site MgCOM1 in the genome are replaced.

One, the structure of complementary carrier and knockout carrier

1. the prediction of gene

At first plasmid among three mutant X54, H3035 and the H3587 is inserted the place, site contig2.217 sequence with software GENESCAN ( Http:// genes.mit.edu/GENSCAN.html) predict, in conjunction with the official website of international rice blast fungus genome association ( Http:// www.riceblast.org/) in to the prediction of gene in the Magnaporthe grisea genome, the insertion site of plasmid all is included in the genomic gene (Nucleotide of the 4370th of contig2.217 the 14885 in Nucleotide to the in the Magnaporthe grisea genome database) of the MgCOM1 of same prediction on the minus strand among three mutant X54, H3035 and the H3587.Although it is inconsistent in the official website of three kinds of predictive modes (Arabidopsis, Maize and Vertebrate) and international rice blast fungus genome association among the GENESCAN to the location and the exon-intron structure of this gene, the sequence between two restriction enzyme SphI sites among the contig2.217 but (from the Nucleotide of 14885 in the 4370th the Nucleotide to the of contig2.217) can both comprise the scope that this several modes is predicted this gene, and no longer comprises other gene except this gene.

2. the structure of complementary carrier

At first the primer of the restricted property of anamorphic zone restriction endonuclease XbaI recognition sequence amplifies the restriction enzyme that neomycin phosphotransferase gene is connected into pBlueScriptKS (+) and cuts between the XbaI of site the plasmid vector called after KN that obtains from cloned plasmids carrier pS65T-C1 (gi:1019893). then from embodiment 1, downcut the dna segment of the genomic gene (nucleotides of the 4370th of contig2.217 the 14885 in nucleotides to the in the Magnaporthe grisea genome database) of the MgCOM1 that includes prediction in 1 of step 3 in the resulting positive colony with restriction enzyme SphI; The KN that cuts with restriction enzyme EcoRV enzyme behind the end-filling is connected, and obtains including the genomic gene (nucleotides of the 4370th of contig2.217 the 14885 in nucleotides to the in the Magnaporthe grisea genome database) of the MgCOM1 of prediction and the complementary carrier pKNS (Fig. 4 A) of selected marker neomycin phosphotransferase gene. Restriction enzyme that relates in the building process and ligase enzyme, the end-filling enzyme is produced by precious biotechnology (Dalian) company limited, consults and uses specification sheets and uses.

3. the structure of knockout carrier (gene replacement vector).

With reference to three kinds of predictive mode (Arabidopsis among the GENESCAN, Maize and Vertebrate) and the official website of international rice blast fungus genome association in the structure of exon-intron of genomic gene of the MgCOM1 that predicted, sequence between two restriction enzyme XhoI and the ApaI site (from the Nucleotide of 13228 in the 6332nd the Nucleotide to the of contig2.217) can comprise the major part (sequence that comprises about 4049bp of this section sequence upstream in the 1-2847 position of sequence 1 and the genome) of the genomic gene of MgCOM1 and not comprise any other gene.The dna segment that from embodiment 1, includes on the one hand the genomic gene (Nucleotide of the 4370th of contig2.217 the 14885 in Nucleotide to the in the Magnaporthe grisea genome database) of the MgCOM1 of prediction in 1 of step 3 in the resulting positive colony with restriction enzyme SphI cutting-out, the pUC18 that cuts with same restriction enzyme SphI enzyme is connected called after PS2; From plasmid vector pUCATPH, downcut hygromycin phosphotransferase gene with restriction enzyme XbaI on the other hand and be connected the plasmid vector called after p7h6 that obtains with the pGEM7zf (+) that same restriction enzyme is cut; Downcutting hygromycin phosphotransferase gene with restriction enzyme XhoI and ApaI from plasmid p7h6 then is connected with the PS2 that same enzyme is cut, obtain gene replacement vector pPSH (Fig. 4 B), the XhoI of the genomic gene of the MgCOM1 of the prediction among the plasmid vector pKNS (Nucleotide of the 4370th of contig2.217 the 14885 in Nucleotide to the in the Magnaporthe grisea genome database) and the sequence between the ApaI site are just replaced by hygromycin phosphotransferase gene like this, stay the side sequence of genomic gene of the MgCOM1 of 1958bp and 1657bp respectively in the both sides of hygromycin phosphotransferase gene.

Two, the conversion of Magnaporthe grisea.

The method of the first-selected REMI of the conversion of Magnaporthe grisea.The original meaning of REMI is the integration of restriction enzyme mediation, adopt and do not destroy the intragentic restriction enzyme of carrier the carrier linearizing, under the mediation of restriction enzyme, transform the protoplastis of Magnaporthe grisea, the plasmid that the is transformed place, point of contact that is incorporated into corresponding restriction enzyme in the genome at random.In the present invention, omitted the application of restriction enzyme in the conversion of Magnaporthe grisea, transformation efficiency can be lower than the method for adding restriction enzyme, do not transformed plasmid integration in genome but do not influence.The preparation and the method for transformation of protoplastis are as follows:

1. the preparation of protoplastis

500 milliliters of triangular flasks, the 150 milliliters of liquid CM substratum (yeast extracts 0.1% of packing into, enzymic hydrolysis casein food grade 0.05%, glucose 1%, nitrocalcite 0.1%, potassium primary phosphate 0.02%, sal epsom 0.025%, sodium-chlor 0.015%), insert Magnaporthe grisea 70-15 respectively, X54,1 * 106 conidium of H3035 and H3587, at 26-28 ℃, shake training 30-32 hour under 100 rev/mins of conditions, three layers of sterilization lens wiping paper filter collects mycelium, mycelium is transferred in 50 milliliters of centrifuge tubes of sterilization 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 after enzymolysis 3-4 hour, with 0.7M sodium-chlor washing mycelium, filter through three layers of sterilization lens wiping paper, collect protoplastis, 4,000 rev/mins centrifugal 15 minutes, earlier with 25 milliliters of STC (1.2M sorbyl alcohols, 10mM Tris-Cl, pH 7.5,50mM calcium chloride) the washing protoplastis is once, wash 2 times with 10 milliliters of STC respectively then, with STC protoplastis concentration is transferred to 0.5-1 * 10 after the centrifugation ⁸Individual/milliliter.

2. Magnaporthe grisea transforms

Respectively with Magnaporthe grisea 70-15, X54, H3035 and H3587 protoplastis are sub-packed in 50 milliliters of centrifuge tubes of sterilization, every pipe 150 microlitres, add isopyknic linearizing carrier (about 2 micrograms respectively, add in the centrifuge tube of Magnaporthe grisea 70-15 with the linearizing gene replacement vector pPSH of Restriction enzyme Sma I, Magnaporthe grisea X54, add linearizing complementary carrier pKNS in the centrifuge tube of H3035 and H3587 respectively with restriction enzyme NotI) and the 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 on ice 20 minutes, add the ice-cold STC of 25 milliliters/pipe, slow mixing, 4,000 rev/min, 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 12-13 hour, change culture dish over to, add 12 milliliters of SR (LR+1.6% agar) that are cooled to 50 ℃, mixing, treat its solidify dry up after, 0.7% top-layer agar of spreading 12 milliliters of one decks above (is cooled to 50 ℃, contains the G418 (complementation test) of 400 mcg/ml or the Totomycin of 300 mcg/ml (U.S. Roche company produces, the gene substitution test).Cultivated 4-6 days for 28 ℃, (the complementary transformant of Magnaporthe grisea three mutant X54, H3035 and H3587 is respectively CX54, CH3035 and CH3587 with the transformant that occurs; The gene substitution transformant K204 of Magnaporthe grisea 70-15) goes to solid CM substratum (0.6% yeast extract, 0.3% enzymic hydrolysis casein food grade, 0.3% acid hydrolysis casein food grade, 1% sucrose, 1.6% agar) (contain on the G418 (complementation test) of 400 mcg/ml or the Totomycin of 250 mcg/ml (gene substitution test), after the postsearch screening single bacterium colony changed on the rolled oats tomato substratum and cultivate, and carry out monospore and separate.

The result of complementation test shows that the ectopic integration of complementary carrier in Magnaporthe grisea three mutant X54, H3035 and H3587 genome makes conidial shape of these three mutant recover wild form.Complementary transformant CX54, the CH3035 of three mutant and conidial length of CH3587 and width and wild-type bacteria 70-15's is more as shown in table 5.The complementary transformant CX54 of mutant X54 and the conidium form of wild-type bacteria 70-15 as shown in Figure 5, scale is 5 microns among the figure.

Conidial length and the width of complementary transformant of table 5. and wild-type bacteria 70-15

	Long (unit: micron)		Wide (unit: micron)
					On average	The significance of difference	On average	The significance of difference
	??70-15 ??CX54 ??CH3035 ??CH3587	??23.41 ??23.83 ??24.19 ??23.68	??A ??A ??A ??A	??9.74 ??9.45 ??9.44 ??9.41					??A ??A ??A ??A

Annotate: the significance of difference is between two groups of A does not have difference in 5% remarkable scope.

The ectopic integration of complementary carrier in three mutant gene groups makes the virulence of these three mutant also return to the level of wild-type, be included in the rate of formation that infects nail and infectivity mycelia on the onion epidermis, and the number that on affinity rice varieties CO39 blade, forms scab.Wherein, mutant X54 and corresponding complementary transformant CX54 inoculation back 24 hours, 36 hours and on onion epidermis, formed in 48 hours the ratio that infects nail and infectivity mycelia and with the comparison of wild-type bacteria shown in table 6 and Fig. 6 A-Fig. 6 D; Wild strain 70-15 and three MgCom1 insert mutant X54, H3035, H3587 and corresponding complementary transformant CX54, CH3035, CH3587 form scab on affinity rice varieties CO39 blade number as shown in Figure 7.Among Fig. 7, A is 70-15, and B is X54, and C is H3035, and D is H3587, and E is CX54, and F is CH3035, and G is CH3587.

Table 6.70-15, X54 and CX54 form the ratio that infects nail and infectivity mycelia on onion epidermis

The inoculation back time (hour)	Infect nail rate of formation (/ 100 appressoriums)			Infectivity mycelia rate of formation (/ 100 appressoriums)
							??70-15	??X54	??CX54	??70-15	??X54	??CX54
	??24	??30.0	??24.9	??44.0	??0.0	??0.0							??0.0

??36 ??48

??86.3 ??89.9

??61.5 ??72.1

??85.6 ??86.7

??59.4 ??72.5

??10.2 ??21.3

??59.8 ??71.6

The portion gene group gene order that the result of gene substitution test shows MgCOM1 and conidial shape that the hygromycin gene PERMUTATION OF SEQUENCES causes transformant K204 to be produced have with the present invention in three mutant X54, the H3035 variation the same with H3587.The conidial length of gene substitution transformant K204 and width and as shown in table 7 with the comparative result of wild-type bacteria 70-15, mutant X54, H3035, H3587.Wherein, the conidium form of gene substitution transformant K204 and wild strain 70-15 more as shown in Figure 8, among the figure, A is 70-15, B is K204, scale is 25 microns.The portion gene group gene order of MgCOM1 and hygromycin gene PERMUTATION OF SEQUENCES also cause the virulence of transformant to weaken, be included in the rate of formation that infects nail and infectivity mycelia on the onion epidermis, and the number that on affinity rice varieties CO39 blade, forms scab.Wherein, gene substitution transformant K204 and wild-type bacteria 70-15 inoculate back 24 hours, 36 hours on onion epidermis and formation in 48 hours is infected the ratio of nail and infectivity mycelia shown in table 8 and Fig. 9 A and Fig. 9 B.Form when wild strain 70-15 and gene substitution transformant K204 inoculate back 24 hours on onion epidermis infect nail and infectivity mycelia difference as shown in figure 10, A is 70-15 among the figure, B is K204, scale is 30 microns.Gene substitution transformant K204 and wild-type bacteria 70-15 form the quantity of extendability scab and foxiness on the CO39 blade as shown in table 9.Wild strain 70-15 and mutant X54, H3035, H3587 and gene substitution transformant K204 are as shown in figure 11 pathogenic to the rice varieties CO39's of affinity, and A is that 70-15, B are that X54, C are that H3035, D are that H3587, E are K204 among the figure.

The mitogenetic spore of table 7. transformant K204 and wild-type bacteria 70-15, mutant X54, H3035 and H3587

The length of son and width are relatively

	Long (unit: micron)		Wide (unit: micron)
					On average	The significance of difference	On average	The significance of difference
	??70-15 ??X54 ??H3035 ??H3587 ??K204	??23.41 ??29.55 ??29.03 ??29.49 ??29.05	??A ??B ??B ??B ??B	??9.74 ??8.24 ??8.14 ??7.95 ??8.81					??A ??B ??B ??B ??B

Annotate: the significance of difference is between two groups of B does not have difference in 5% remarkable scope, the significance of difference be between A and B two groups in 5% remarkable scope significant difference.

Table 8. gene substitution transformant K204 and wild-type bacteria 70-15 are in inoculation back 24 hours, 36 hours and 48 little

The time form the ratio infect nail and infectivity mycelia

The inoculation back time (hour)	Infect nail rate of formation (/ 100 appressorium)		Infectivity mycelia rate of formation (/ 100 appressorium)
					??70-15	??K204	??70-15	??K204
	??24 ??36 ??48	??30.0 ??86.3 ??89.9	??0.8 ??3.4 ??33.7	??0.0 ??59.4 ??72.5					??0.0 ??0.3 ??16.1

Table 9. gene substitution transformant K204 and wild-type bacteria 70-15 form extendability scab and brown on the CO39 blade

The quantity of spot

	Expansion scab number (individual/10 centimetre blade)	Total scab number (individual/10 centimetre blade)
			??70-15 ??K204	??44.4 ??1.1	??203.8 ??45.0

Determining of embodiment 3, promoter region

One, promoter region determines

By the total length of the MgCOM1 genomic gene of the cDNA gene order of the MgCOM1 of sequence 2 and prediction (Nucleotide of the 4370th of contig2.217 the 14885 in Nucleotide to the in the Magnaporthe grisea genome database) is compared, the latter comprises the sequence of gene start codon precontract 6.4kb, and do not predict in the sequence of gene start codon precontract 2kb in this section sequence conservative promoter sequence and cis-acting elements ( Http:// bimas.dcrt.nih.gov/molbio/proscan/).Therefore by seek can complementary gene displacement transformant the shortest sequence determine promoter sequence.In this embodiment, made up altogether and comprised 1814bp before the gene start codon, the complementary carrier that the sequence of three kinds of different lengthss such as 983bp and 785bp links to each other with the MgCOM1 gene as promoter sequence.

At first made up complementary carrier pKNSP1.8 (Figure 12 Aa that the segment (from the Nucleotide of 10211 in the 4912nd the Nucleotide to the of contig2.217) of 5299bp between two restriction enzyme PstI sites links to each other with the neomycin phosphotransferase mark in the present embodiment, the segment of from complementary carrier pKNS, downcutting 5299bp with PstI be connected with the KN that same enzyme is cut obtain), this carrier comprises the terminator sequence of 829bp after the promoter sequence of the preceding 1814bp of genomic gene initiator codon of MgCOM1 and the terminator codon.With pKNSP1.8 transforming gene displacement transformant K204, the result shows the conidium form (Figure 12 Ab) of the transformant of gained and the level that virulence can return to wild-type.

Present embodiment is on the basis of complementary carrier pKNSP1.8, select for use restriction enzyme cut progressively disappearance fall 831bp (HindIII and EcoRI enzyme cut that pKNSP1.8 reclaims 919bp comprise the promotor part, cut the MgCOM1 Gene Partial that pKNSP1.8 reclaims 3553bp with EcoRI and PstI enzyme, being connected into HindIII jointly is connected with the KN that the PstI enzyme is cut) and 1029bp (the KpnI enzyme is cut pKNSP1.8, between PstI that will lack and KpnI the part of 1029bp, the part that reclaims other connects), obtain comprising the complementary carrier pKNSP1.0 (Figure 12 Ba) and the pKNSP0.8 (Figure 12 Ca) of the promoter sequence of 983bp and 785bp before the genomic gene initiator codon of MgCOM1 respectively.With these two carriers transforming genes displacement transformant K204 respectively, the result shows that these two carriers transform the level that the conidium form of resulting transformant (Figure 12 Bb and Figure 12 Cb) and virulence can both return to wild-type.The promotor that proof can normally start the genomic gene of MgCOM1 should comprise at least from the sequence of the 785bp of translation initiation codon upstream, i.e. the 1st to the 785th nucleotide sequence shown in SEQ ID NO.1.

Two, the gene that started of two kinds of different lengths promoter sequences is invaded the expression of different developmental phases in the right process at Magnaporthe grisea

The at first synthetic primer that has restriction enzyme EcoRI recognition sequence respectively amplifies the restriction enzyme that green fluorescence protein gene (GFP) is connected into pBlueScriptKS (+) and cuts between the EcoRI of site the plasmid vector called after KSG that obtains from cloned plasmids carrier phGFP-S65T (gi:1289374).Increase from the pKNSP1.8 promotor of 983bp of the synthetic primer that has restriction enzyme HindIII and EcoRV restriction enzyme site respectively partly is connected between the HindIII and EcoRV restriction enzyme site of KSG, obtains including the carrier pNpSal of 983bp promotor and GFP gene; The 785bp promoter sequence that the synthetic primer that has restriction enzyme KpnI and EcoRV restriction enzyme site respectively increases from pKNSP1.8 partly is connected between the KpnI and EcoRV restriction enzyme site of KSG, obtains including the carrier pKPG of 785bp promotor and GFP gene; PNpSal and pKPG are transformed wild type strain 70-15 respectively, and the result shows that pNpSal and pKPG transform transformant that wild type strain 70-15 obtains respectively in conidium, and germ tube all has the expression of varying strength GFP in appressorium and the infectivity mycelia.

Sequence table

<160>1

<210>1

<211>4276

<212>DNA

＜213〉Magnaporthe grisea (Magnaporthe grisea)

<400>1

ggtacccggc?cggaacgcct?ggcccctgca?actcaccaac?tccgccctcc?actgacgttc????60

ccactccatt?tctcgtgcct?ctttcgttgc?tcgctccacc?cacccatcca?ttctttgcct????120

gcgtgcgtgt?cggggcgatc?ccaggtgcat?cttccgtacg?acctctttcg?ccccgggttt????180

gctttgtact?gcctttgctc?tgcttgctca?tcactgcttg?cgagtgagtg?tggtgtctgt????240

acgccctctt?tggaaacaac?tgttggatca?cttcttgctc?cgtccttctt?ttttcccggc????300

cattgttctt?tggatctgcc?aatttacttg?ttacctcatc?actgaactgg?caaccccctg????360

ttcctctcgt?ttctttgctt?tactctaccc?gctctgcatc?ctgctcccag?ttattcggtt????420

ttctttggac?cgacattaac?actttccctt?tcgatatcga?ccgcctcctt?agctcgccag????480

gaacctccag?aagatctatc?ggcatacaat?gaggtgaaga?gtgctgcacc?acggaaggat????540

actggcagca?ttgaaagcct?gaagaacgtc?tgtcggcttc?ggccgcccgc?caccctcggc????600

tggtctgaca?actgacgtcc?tcttaccccc?tttactctct?tgacgaaacc?aacaacaacc????660

cattcttttt?cgactaatgc?caatcgacct?gaccacgacc?gaccacgata?acgagccagc????720

ggaattctcg?ttgtgaattg?aactcggacg?aaaagcaaga?cggccgagcc?gagttgccca????780

tcaagatgtc?tctcgtcatt?ccggcagggg?gtctggagct?ggagacgtcg?gtcgacaaga????840

tgaccagtct?cccgctccag?gcttttgcca?taaccctgag?cgatgacatg?cttgaggacc????900

tgatagacag?cttccaaaat?ggccaggaga?tcgagttgtc?cctggggcag?tcaccggtag????960

gtttattttg?gatcgaaccc?caatagtgat?gcctctttgg?aggtaccttg?ctgtttgcct????1020

ttgcccatca?attgcctgga?tacaatcaga?ataattcttg?tgattgtgaa?caatagcttt????1080

agaagaaaag?ctggtctctg?tgcgctcccg?caacctgctt?ggcgaagata?gcatttagcc????1140

ctaaatctcg?accatgcatg?aaccataact?cacctatccg?tcaacttacg?atccactgac????1200

actattctgt?tcgtatctca?aggctttcct?ctacggcggc?aacacagcac?ccatcactcg????1260

aatccccgag?aatttctcct?acgacctcta?cgttaccgat?ccgaactcgc?ccgaaacggc????1320

tagccttgca?cccaacccga?cgatgccaat?attcaagaaa?caacacctca?acttggtcaa????1380

gaagcccaag?tacatgccaa?agggcttcat?tgacgaagat?gaggccccgg?agctgggagc????1440

tttagagatc?atcgagaacc?cggaaaagtc?tcagacgtcc?tcacagtatg?cattctgtgt????1500

ccaactgacc?tttttaccca?gtggtcaggt?aactaacggt?agagctcact?ttaaaggtcc????1560

aagagctctt?cacttcaacc?tcagtctgcg?ctgaacaaga?agccgaagtc?tgcgacggcc????1620

gggaagaaga?cggcggctag?taatgcgata?accatgtcca?cacaggctcg?ctcgcgtccg????1680

acaagcccgg?caatcagcgc?cgttggctct?cctcttcccg?agtcgtcgat?cgaatcttcg????1740

caccagcaaa?tcgtgaagga?ggctaaagaa?ctacgcgcac?cgctcatcca?cgccctcgct????1800

gttcgggaga?tgacatatga?cgaactctgg?gagaagtggg?gcaagggtga?tgatgacgag????1860

tcgaggcggg?agttccgtaa?tattctgagc?aaggtcgccg?agcaggtcaa?gaactctaac????1920

aagtacatga?tgaagaagaa?ccactggaag?gagttggatg?tctggaatca?caattacgac????1980

tcggattcgg?accgccaaac?cgccatcgac?aatgctgtgc?ggcactttga?caagatgcga????2040

attggtgcct?cggagccaga?gtggcagaag?ctgttgccgt?ttgacgaccg?tggaaagggc????2100

aagtgtctga?gcaagcttca?agcttcgtta?gccaggggcc?cgaaaggact?cactgttcac????2160

gttcagaatg?cagacgatac?gagcggagct?ggttcgccag?acacggacaa?ccgctctatc????2220

agcgggtcgc?agcccatgtc?caggtcgagc?tcacaaaaca?agccaaagaa?ggcagtcgaa????2280

aagaaaccag?ctgtttcagc?accgaaaaag?ccgaccgctc?ccaaggtttc?tccctccaag????2340

cccgccgcca?agccggcagc?caagacagga?gccagaggtc?cgctatcaaa?agaaatcatc????2400

accgattctg?acgaatccag?tgacgagatc?ccgctttcgc?agacaaagaa?cgtggtcaaa????2460

aaacaagcgg?cacctgcggt?gagggctccc?aagcctccaa?tatctgcacc?agtttcgggt????2520

ccttcgtccc?ttcccaagaa?gcccccgcca?cctgcggcac?gcgagccagc?caagccgcag????2580

atcacagcaa?agccgccggt?gaagagacca?cgcgaggagg?aggacagcag?ttccagctcg????2640

ggcacaccac?ttgctaagaa?gttcaaggtc?aaggagccag?tgagggcacc?aaaagagccc????2700

atcagggcac?cgaaagaacc?tatcagggcg?ccaaaggaaa?ctactgtcag?ggcacccaag????2760

gaggtcatcc?gtgcgccacg?agagacgaag?ccgaccaagg?aacccaaggc?tgcgcctttg????2820

cctgtcagca?agccccgtcc?cgcagactcg?agtcagagca?cgtcgaggac?cggaagctcg????2880

aatatatcgt?tcaaccggtc?gaagaacaca?tcgcctgcca?agtcttcgcc?tcttgcatca????2940

tcgcccccaa?ccaatgcttc?cgatatcgat?ccggctgagg?aggccatgat?tgctaatgcc????3000

aatcgcaaac?gcaaggctga?cgcatactac?aacgactcat?cgtcgaccac?tagcagcagc????3060

agcagcaacg?tacaaacttc?gggcaagaac?tcgattaaaa?agcgcaccca?cgacgatgac????3120

gtgagcgtga?gcagaagagg?cgctggtagc?aagcttcctc?cggatgttgt?agccaaagca????3180

cgccgcttca?aagaggcgta?ctcggattac?gagcgactgc?actatgagct?ttcgggaatg????3240

aacaaccccg?aggagagcaa?gctcaatgag?ctcatgaaca?tgcaccgcag?gttggaaaag????3300

atgaagaagg?agatatactc?gaccacaggt?gctacttaca?atggggaccg?tgaccgtgct????3360

cacaagtctg?gagagcagaa?gcggagtagc?gccgtggcca?gcagccgccg?cgagcgcgac????3420

gagtacaacg?actatggccg?gcattgagat?ttgaggaggc?gatcaaagtt?gagcgatact????3480

gcgccgataa?tattattctt?ggcagcgatt?cgctccttcg?catctcgaac?ctgttgctga????3540

aacaagtcga?ttgactcggt?gtgcttctgc?tcccaagact?cggatcgcaa?aggcggatct????3600

cctataccag?gtggccgatg?tgtcttgatt?atttcccgag?aagtgttctt?tttttaacac????3660

aactttgttt?tgcttttcaa?tgatcgttat?actgcatatt?tctcaagttt?taccccggag????3720

tgctccacag?aagcaactct?tgcacatctc?attccatcat?tttatggcgc?accttggttc????3780

tggagttcat?gggcggcata?taacaaatga?cagcatcttt?tgtcttgatg?gaaaatggtc????3840

ttttggatta?actactcccc?ttgtcctctc?tttcctttgc?attaatcata?tacaaacacc????3900

tggggagcat?atcatgatcc?tttatcgttt?actcttcggg?acaggcaggg?gcatttgaaa????3960

ttaagcgata?ccacaccagg?atatacttgg?tatttcagtc?ccactaggag?aactgaatgg????4020

cggagggggt?cgaaaacagg?actggaaaga?ttgtgaggga?tgacgatagg?ggtttcaaag????4080

gttgctacta?tgcttaacta?gttgacagtt?tgaatcagct?acctgacgtt?ttggttcctt????4140

tgcttgcatt?tgactcaagg?actcttgttg?gttttcatgt?agtctttttt?tcaacgtctc????4200

tagcaggttt?agagcaaggg?ttcatttagc?taccgaattg?aacacagaaa?gttaagctca????4260

cgaccgcgaa?ctgcag????????????????????????????????????????????????????4276

<210>2

<211>2591

<212>DNA

＜213〉Magnaporthe grisea (Magnaporthe grisea)

<220>

<221>CDS

<222>(101)..(2425)

<400>2

gacctgacca?cgaccgacca?cgataacgag?ccagcggaat?tctcgttgtg?aattgaactc????60

ggacgaaaag?caagacggcc?gagccgagtt?gcccatcaag?atgtctctcg?tcattccggc????120

agggggtctg?gagctggaga?cgtcggtcga?caagatgacc?agtctcccgc?tccaggcttt????180

tgccataacc?ctgagcgatg?acatgcttga?ggacctgata?gacagcttcc?aaaatggcca????240

ggagatcgag?ttgtccctgg?ggcagtcacc?ggctttcctc?tacggcggca?acacagcacc????300

catcactcga?atccccgaga?atttctccta?cgacctctac?gttaccgatc?cgaactcgcc????360

cgaaacggct?agccttgcac?ccaacccgac?gatgccaata?ttcaagaaac?aacacctcaa????420

cttggtcaag?aagcccaagt?acatgccaaa?gggcttcatt?gacgaagatg?aggccccgga????480

gctgggagct?ttagagatca?tcgagaaccc?ggaaaagtct?cagacgtcct?cacagtccaa????540

gagctcttca?cttcaacctc?agtctgcgct?gaacaagaag?ccgaagtctg?cgacggccgg????600

gaagaagacg?gcggctagta?atgcgataac?catgtccaca?caggctcgct?cgcgtccgac????660

aagcccggca?atcagcgccg?ttggctctcc?tcttcccgag?tcgtcgatcg?aatcttcgca????720

ccagcaaatc?gtgaaggagg?ctaaagaact?acgcgcaccg?ctcatccacg?ccctcgctgt????780

tcgggagatg?acatatgacg?aactctggga?gaagtggggc?aagggtgatg?atgacgagtc????840

gaggcgggag?ttccgtaata?ttctgagcaa?ggtcgccgag?caggtcaaga?actctaacaa????900

gtacatgatg?aagaagaacc?actggaagga?gttggatgtc?tggaatcaca?attacgactc????960

ggattcggac?cgccaaaccg?ccatcgacaa?tgctgtgcgg?cactttgaca?agatgcgaat????1020

tggtgcctcg?gagccagagt?ggcagaagct?gttgccgttt?gacgaccgtg?gaaagggcaa????1080

gtgtctgagc?aagcttcaag?cttcgttagc?caggggcccg?aaaggactca?ctgttcacgt????1140

tcagaatgca?gacgatacga?gcggagctgg?ttcgccagac?acggacaacc?gctctatcag????1200

cgggtcgcag?cccatgtcca?ggtcgagctc?acaaaacaag?ccaaagaagg?cagtcgaaaa????1260

gaaaccagct?gtttcagcac?cgaaaaagcc?gaccgctccc?aaggtttctc?cctccaagcc????1320

cgccgccaag?ccggcagcca?agacaggagc?cagaggtccg?ctatcaaaag?aaatcatcac????1380

cgattctgac?gaatccagtg?acgagatccc?gctttcgcag?acaaagaacg?tggtcaaaaa????1440

acaagcggca?cctgcggtga?gggctcccaa?gcctccaata?tctgcaccag?tttcgggtcc????1500

ttcgtccctt?cccaagaagc?ccccgccacc?tgcggcacgc?gagccagcca?agccgcagat????1560

cacagcaaag?ccgccggtga?agagaccacg?cgaggaggag?gacagcagtt?ccagctcggg????1620

cacaccactt?gctaagaagt?tcaaggtcaa?ggagccagtg?agggcaccaa?aagagcccat????1680

cagggcaccg?aaagaaccta?tcagggcgcc?aaaggaaact?actgtcaggg?cacccaagga????1740

ggtcatccgt?gcgccacgag?agacgaagcc?gaccaaggaa?cccaaggctg?cgcctttgcc????1800

tgtcagcaag?ccccgtcccg?cagactcgag?tcagagcacg?tcgaggaccg?gaagctcgaa????1860

tatatcgttc?aaccggtcga?agaacacatc?gcctgccaag?tcttcgcctc?ttgcatcatc????1920

gcccccaacc?aatgcttccg?atatcgatcc?ggctgaggag?gccatgattg?ctaatgccaa????1980

tcgcaaacgc?aaggctgacg?catactacaa?cgactcatcg?tcgaccacta?gcagcagcag????2040

cagcaacgta?caaacttcgg?gcaagaactc?gattaaaaag?cgcacccacg?acgatgacgt????2100

gagcgtgagc?agaagaggcg?ctggtagcaa?gcttcctccg?gatgttgtag?ccaaagcacg????2160

ccgcttcaaa?gaggcgtact?cggattacga?gcgactgcac?tatgagcttt?cgggaatgaa????2220

caaccccgag?gagagcaagc?tcaatgagct?catgaacatg?caccgcaggt?tggaaaagat????2280

gaagaaggag?atatactcga?ccacaggtgc?tacttacaat?ggggaccgtg?accgtgctca????2340

caagtctgga?gagcagaagc?ggagtagcgc?cgtggccagc?agccgccgcg?agcgcgacga????2400

gtacaacgac?tatggccggc?attgagattt?gaggaggcga?tcaaagttga?gcgatactgc????2460

gccgataata?ttattcttgg?cagcgattcg?ctccttcgca?tctcgaacct?gttgctgaaa????2520

caagtcgatt?gactcggtgt?gcttctgctc?ccaagactcg?gatcgcaaag?gcggatctcc????2580

tataccaggt?g?????????????????????????????????????????????????????????2591

<210>3

<211>774

<212>PRT

＜213〉Magnaporthe grisea (Magnaporthe grisea)

<400>3

Met?Ser?Leu?Val?Ile?Pro?Ala?Gly?Gly?Leu?Glu?Leu?Glu?Thr?Ser?Val

1???????????????5???????????????????10??????????????????15

Asp?Lys?Met?Thr?Ser?Leu?Pro?Leu?Gln?Ala?Phe?Ala?Ile?Thr?Leu?Ser

20??????????????????25??????????????????30

Asp?Asp?Met?Leu?Glu?Asp?Leu?Ile?Asp?Ser?Phe?Gln?Asn?Gly?Gln?Glu

35??????????????????40??????????????????45

Ile?Glu?Leu?Ser?Leu?Gly?Gln?Ser?Pro?Ala?Phe?Leu?Tyr?Gly?Gly?Asn

50??????????????????55??????????????????60

Thr?Ala?Pro?Ile?Thr?Arg?Ile?Pro?Glu?Asn?Phe?Ser?Tyr?Asp?Leu?Tyr

65??????????????????70??????????????????75??????????????????80

Val?Thr?Asp?Pro?Asn?Ser?Pro?Glu?Thr?Ala?Ser?Leu?Ala?Pro?Asn?Pro

85??????????????????90??????????????????95

Thr?Met?Pro?Ile?Phe?Lys?Lys?Gln?His?Leu?Asn?Leu?Val?Lys?Lys?Pro

100?????????????????105?????????????????110

Lys?Tyr?Met?Pro?Lys?Gly?Phe?Ile?Asp?Glu?Asp?Glu?Ala?Pro?Glu?Leu

115?????????????????120?????????????????125

Gly?Ala?Leu?Glu?Ile?Ile?Glu?Asn?Pro?Glu?Lys?Ser?Gln?Thr?Ser?Ser

130?????????????????135?????????????????140

Gln?Ser?Lys?Ser?Ser?Ser?Leu?Gln?Pro?Gln?Ser?Ala?Leu?Asn?Lys?Lys

145?????????????????150?????????????????155?????????????????160

Pro?Lys?Ser?Ala?Thr?Ala?Gly?Lys?Lys?Thr?Ala?Ala?Ser?Asn?Ala?Ile

165?????????????????170?????????????????175

Thr?Met?Ser?Thr?Gln?Ala?Arg?Ser?Arg?Pro?Thr?Ser?Pro?Ala?Ile?Ser

180?????????????????185?????????????????190

Ala?Val?Gly?Ser?Pro?Leu?Pro?Glu?Ser?Ser?Ile?Glu?Ser?Ser?His?Gln

195?????????????????200?????????????????205

Gln?Ile?Val?Lys?Glu?Ala?Lys?Glu?Leu?Arg?Ala?Pro?Leu?Ile?His?Ala

210?????????????????215?????????????????220

Leu?Ala?Val?Arg?Glu?Met?Thr?Tyr?Asp?Glu?Leu?Trp?Glu?Lys?Trp?Gly

225?????????????????230?????????????????235?????????????????240

Lys?Gly?Asp?Asp?Asp?Glu?Ser?Arg?Arg?Glu?Phe?Arg?Asn?Ile?Leu?Ser

245?????????????????250?????????????????255

Lys?Val?Ala?Glu?Gln?Val?Lys?Asn?Ser?Asn?Lys?Tyr?Met?Met?Lys?Lys

260?????????????????265?????????????????270

Asn?His?Trp?Lys?Glu?Leu?Asp?Val?Trp?Asn?His?Asn?Tyr?Asp?Ser?Asp

275?????????????????280?????????????????285

Ser?Asp?Arg?Gln?Thr?Ala?Ile?Asp?Asn?Ala?Val?Arg?His?Phe?Asp?Lys

290?????????????????295?????????????????300

Met?Arg?Ile?Gly?Ala?Ser?Glu?Pro?Glu?Trp?Gln?Lys?Leu?Leu?Pro?Phe

305?????????????????310?????????????????315?????????????????320

Asp?Asp?Arg?Gly?Lys?Gly?Lys?Cys?Leu?Ser?Lys?Leu?Gln?Ala?Ser?Leu

325?????????????????330?????????????????335

Ala?Arg?Gly?Pro?Lys?Gly?Leu?Thr?Val?His?Val?Gln?Asn?Ala?Asp?Asp

340?????????????????345?????????????????350

Thr?Ser?Gly?Ala?Gly?Ser?Pro?Asp?Thr?Asp?Asn?Arg?Ser?Ile?Ser?Gly

355?????????????????360?????????????????365

Ser?Gln?Pro?Met?Ser?Arg?Ser?Ser?Ser?Gln?Asn?Lys?Pro?Lys?Lys?Ala

370?????????????????375?????????????????380

Val?Glu?Lys?Lys?Pro?Ala?Val?Ser?Ala?Pro?Lys?Lys?Pro?Thr?Ala?Pro

385?????????????????390?????????????????395?????????????????400

Lys?Val?Ser?Pro?Ser?Lys?Pro?Ala?Ala?Lys?Pro?Ala?Ala?Lys?Thr?Gly

405?????????????????410?????????????????415

Ala?Arg?Gly?Pro?Leu?Ser?Lys?Glu?Ile?Ile?Thr?Asp?Ser?Asp?Glu?Ser

420?????????????????425?????????????????430

Ser?Asp?Glu?Ile?Pro?Leu?Ser?Gln?Thr?Lys?Asn?Val?Val?Lys?Lys?Gln

435?????????????????440?????????????????445

Ala?Ala?Pro?Ala?Val?Arg?Ala?Pro?Lys?Pro?Pro?Ile?Ser?Ala?Pro?Val

450?????????????????455?????????????????460

Ser?Gly?Pro?Ser?Ser?Leu?Pro?Lys?Lys?Pro?Pro?Pro?Pro?Ala?Ala?Arg

465?????????????????470?????????????????475?????????????????480

Glu?Pro?Ala?Lys?Pro?Gln?Ile?Thr?Ala?Lys?Pro?Pro?Val?Lys?Arg?Pro

485?????????????????490?????????????????495

Arg?Glu?Glu?Glu?Asp?Ser?Ser?Ser?Ser?Ser?Gly?Thr?Pro?Leu?Ala?Lys

500?????????????????505?????????????????510

Lys?Phe?Lys?Val?Lys?Glu?Pro?Val?Arg?Ala?Pro?Lys?Glu?Pro?Ile?Arg

515?????????????????520?????????????????525

Ala?Pro?Lys?Glu?Pro?Ile?Arg?Ala?Pro?Lys?Glu?Thr?Thr?Val?Arg?Ala

530?????????????????535?????????????????540

Pro?Lys?Glu?Val?Ile?Arg?Ala?Pro?Arg?Glu?Thr?Lys?Pro?Thr?Lys?Glu

545?????????????????550?????????????????555?????????????????560

Pro?Lys?Ala?Ala?Pro?Leu?Pro?Val?Ser?Lys?Pro?Arg?Pro?Ala?Asp?Ser

565?????????????????570?????????????????575

Ser?Gln?Ser?Thr?Ser?Arg?Thr?Gly?Ser?Ser?Asn?Ile?Ser?Phe?Asn?Arg

580?????????????????585?????????????????590

Ser?Lys?Asn?Thr?Ser?Pro?Ala?Lys?Ser?Ser?Pro?Leu?Ala?Ser?Ser?Pro

595?????????????????600?????????????????605

Pro?Thr?Asn?Ala?Ser?Asp?Ile?Asp?Pro?Ala?Glu?Glu?Ala?Met?Ile?Ala

610?????????????????615?????????????????620

Asn?Ala?Asn?Arg?Lys?Arg?Lys?Ala?Asp?Ala?Tyr?Tyr?Asn?Asp?Ser?Ser

625?????????????????630?????????????????635?????????????????640

Ser?Thr?Thr?Ser?Ser?Ser?Ser?Ser?Asn?Val?Gln?Thr?Ser?Gly?Lys?Asn

645?????????????????650?????????????????655

Ser?Ile?Lys?Lys?Arg?Thr?His?Asp?Asp?Asp?Val?Ser?Val?Ser?Arg?Arg

660?????????????????665?????????????????670

Gly?Ala?Gly?Ser?Lys?Leu?Pro?Pro?Asp?Val?Val?Ala?Lys?Ala?Arg?Arg

675?????????????????680?????????????????685

Phe?Lys?Glu?Ala?Tyr?Ser?Asp?Tyr?Glu?Arg?Leu?His?Tyr?Glu?Leu?Ser

690?????????????????695?????????????????700

Gly?Met?Asn?Asn?Pro?Glu?Glu?Ser?Lys?Leu?Asn?Glu?Leu?Met?Asn?Met

705?????????????????710?????????????????715?????????????????720

His?Arg?Arg?Leu?Glu?Lys?Met?Lys?Lys?Glu?Ile?Tyr?Ser?Thr?Thr?Gly

725?????????????????730?????????????????735

Ala?Thr?Tyr?Asn?Gly?Asp?Arg?Asp?Arg?Ala?His?Lys?Ser?Gly?Glu?Gln

740?????????????????745?????????????????750

Lys?Arg?Ser?Ser?Ala?Val?Ala?Ser?Ser?Arg?Arg?Glu?Arg?Asp?Glu?Tyr

755?????????????????760?????????????????765

Asn?Asp?Tyr?Gly?Arg?His

770

<210>4

<211>1392

<212>DNA

＜213〉Magnaporthe grisea (Magnaporthe grisea)

<400>4

gaattctcgt?tgtgaattga?actcggacga?aaagcaagac?ggccgagccg?agttgcccat????60

caagatgtct?ctcgtcattc?cggcaggggg?tctggagctg?gagacgtcgg?tcgacaagat????120

gaccagtctc?ccgctccagg?cttttgccat?aaccctgagc?gatgacatgc?ttgaggacct????180

gatagacagc?ttccaaaatg?gccaggagat?cgagttgtcc?ctggggcagt?caccggtagg????240

tttattttgg?atcgaacccc?aatagtgatg?cctctttgga?ggtaccttgc?tgtttgcctt????300

tgcccatcaa?ttgcctggat?acaatcagaa?taattcttgt?gattgtgaac?aatagcttta????360

gaagaaaagc?tggtctctgt?gcgctcccgc?aacctgcttg?gcgaagatag?catttagccc????420

taaatctcga?ccatgcatga?accataactc?acctatccgt?caacttacga?tccactgaca????480

ctattctgtt?cgtatctcaa?ggctttcctc?tacggcggca?acacagcacc?catcactcga????540

atccccgaga?atttctccta?cgacctctac?gttaccgatc?cgaactcgcc?cgaaacggct????600

agccttgcac?ccaacccgac?gatgccaata?ttcaagaaac?aacacctcaa?cttggtcaag????660

aagcccaagt?acatgccaaa?gggcttcatt?gacgaagatg?aggccccgga?gctgggagct????720

ttagagatca?tcgagaaccc?ggaaaagtct?cagacgtcct?cacagtatgc?attctgtgtc????780

caactgacct?ttttacccag?tggtcaggta?actaacggta?gagctcactt?taaaggtcca????840

agagctcttc?acttcaacct?cagtctgcgc?tgaacaagaa?gccgaagtct?gcgacggccg????900

ggaagaagac?ggcggctagt?aatgcgataa?ccatgtccac?acaggctcgc?tcgcgtccga????960

caagcccggc?aatcagcgcc?gttggctctc?ctcttcccga?gtcgtcgatc?gaatcttcgc????1020

accagcaaat?cgtgaaggag?gctaaagaac?tacgcgcacc?gctcatccac?gccctcgctg????1080

ttcgggagat?gacatatgac?gaactctggg?agaagtgggg?caagggtgat?gatgacgagt????1140

cgaggcggga?gttccgtaat?attctgagca?aggtcgccga?gcaggtcaag?aactctaaca????1200

agtacatgat?gaagaagaac?cactggaagg?agttggatgt?ctggaatcac?aattacgact????1260

cggattcgga?ccgccaaacc?gccatcgaca?atgctgtgcg?gcactttgac?aagatgcgaa????1320

ttggtgcctc?ggagccagag?tggcagaagc?tgttgccgtt?tgacgaccgt?ggaaagggca????1380

agtgtctgag?ca????????????????????????????????????????????????????????1392

Claims (10)

1, fungus conidium shape and pathogen-relatedprotein are the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 3;

2) with SEQ ID № in the sequence table: 3 amino acid residue sequence through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and with fungus conidium shape and pathogenic relevant protein.

2, the encoding gene of described fungus conidium shape of claim 1 and pathogen-relatedprotein.

3, gene according to claim 2 is characterized in that: the genomic gene of described fungus conidium shape and pathogen-relatedprotein has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 3 protein 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 90% above homology, and the identical function protein DNA sequence of encoding.

4, gene according to claim 3 is characterized in that: the coding region of the genomic gene of described fungus conidium shape and pathogen-relatedprotein from the 786th of 5 of sequence 1 ' end between 3447 bit bases.

5, gene according to claim 2 is characterized in that: the cDNA gene of described fungus conidium shape and pathogen-relatedprotein can have one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna;

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

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

4) with sequence table in SEQ ID №: 2 dna sequence dnas that limit have 90% above homology, and the identical function protein DNA sequence of encoding.

6, gene according to claim 5 is characterized in that: the encoding sequence of the cDNA gene of described fungus conidium shape and pathogen-relatedprotein for from the 101st of 5 of sequence 2 ' end to 2425 bit bases.

7, the expression vector that contains the encoding gene of described fungus conidium shape of claim 1 and pathogen-relatedprotein, clone and host bacterium.

8, the promotor of the encoding gene of fungus conidium shape and pathogen-relatedprotein has from 5 of sequence 1 ' the 1st nucleotide sequence to 785 bit bases of end.

9, described fungus conidium shape of claim 1 and pathogen-relatedprotein and encoding gene thereof the application in the screening antifungal medicine.

10, application according to claim 9 is characterized in that: described antifungal medicine is anti-Magnaporthe grisea medicament.

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