CN1763086A - ANK protein for controlling fungus colony growth and pathogenicity and its coding gene and utilization - Google Patents

Abstract

The present invention provides one new kind of gene controlling fungus growth and virulence and its utilization, and its coded protein has ankyrin repeat module. The gene in pyricular fungus is named as MgMBP1, and its whole length, cDNA, coded protein and promoter have the nucleotide sequences and amino acid sequences as shown. The gene is expressed in different hypha and infection stages, and its elimination results in obviously reduced pyricular fungus growth, infection default and virulence. It is also found that protein with consistency over 40 % to MgMBP1 exists also in grey botrytis, corn stem rot fungus, wheat bakanae fungus, etc. and may have the same function. The expression and modification of MgMBP1 and its homogenous protein, the cutting of the gene product and the nucleotide sequence of the promoter may be used as target sites in designing and screening new antifungal preparations.

Description

ANK albumen and the encoding gene and the utilization of new control fungal colony growth and virulence

Technical field

The present invention relates to control in the fields such as Pesticide Science, plant pathology and mycology the function and the application of fungal colony growth and virulence albumen and encoding gene and promotor.

Background technology

Ankyrin repeat (ANK, ankyrin repeat) is a kind of block that extensively utilizes in the organism, refers to 33 conservative relatively amino acid whose repeating.This module is at first found (Breeden and Nasmyth in two yeast cell cycle modulin Swi6 and Cdc10,1987), subsequently, in cytoskeleton ankyrin (ankyrins), also found to contain the structure of 24 this tumor-necrosis factor glycoproteinss, therefore this structure is called ankyrin repeat die body (ANK repeat motif) (Lux et al, 1990).Crystalline structure shows, this die body is folded into β 2 α 2 secondary structures, spatially then form L type structure, beta hairpin (galianconism of " L ") extends in outside antiparallel two α spirals (" L's " is long-armed), almost vertical with the plane that the α spiral constitutes, form a successive antiparallel β lamella by beta hairpin between the ANK die body that faces mutually each other, the ANK that number does not wait is together in series, rely on hydrogen bond and hydrophobic interaction, form tight stable structure territory, form numerous but the ANK protein molecule that function is different of kind.This protein is widely distributed, to human body, to extracellular medium report is arranged all from nucleus, tenuigenin from virus, prokaryotic organism, eukaryote.In the SMART database, contain 19,276 ANK in fixed 3608 protein and repeat (NRDB; Schultz et al, 1998; Letunic et al.2002).ANK structural domain mediating protein and protein interaction, can with multiple part combination, exercise the biological function of numerous and complicated, comprise that signal conduction, cytoskeleton form, transcribe and cell cycle regulating, (Steven G such as inflammation generation and development, membranin and toxin, 1999, Leila K.Mosavi, 2004).

Do mutually aspect the research in plant and microorganism, Delaney etc. screen the mutant nim1 of a no induction of immunity reaction on Arabidopis thaliana, salicylic accumulation after being induced by pathogen, this mutant is arranged, but but can not induce the SAR expression of gene, show that this sudden change blocked the downstream of SA (Delaney et al, 1995), this NIM1 albumen suppresses sub-I κ B with the Mammals transcription factor that contains ANK and has similarity (Ryals et al, 1997).People such as Cao have screened one to inducing the unresponsive mutant npr1 of SAR (Cao et al.1994) on Arabidopis thaliana, after find NPR1 gene regulating SAR again, and the new ANK multiple albumen (Cao et al.1997) that contains of encoding.Discover that further NPR1 is the key gene of the disease-resistant signal transduction path of Arabidopis thaliana downstream control SA accumulation, it also is the center part of SA system's resistance of bringing out, by the regulating effect of the special signal control NPR1 of cause of disease in multipath, synergy activates a series of disease-resistant related gene generation system's resistance (Dempsey etc., 1999) thereby NPR1 and other protein take place optionally.In the SA approach, the NPR1 gene is induced the proteic generation of PR by WRKY70, thereby starts SAR (Clarte et al.1998,2000 of plant; Li et al.2004).In addition, research is also found: the coded product of this gene can mediate protein-protein interaction (Thomma etc., 1999 with transcription factor TGA family member; Zhou etc., 1999), and this mutual work molecular basis of NPR1 effect just.Therefore, infer that NPR1 albumen reacts by being used for SA activated plant defense with other albumen.

In the yeast cell, containing ANK multiple YAR1 gene is the indispensable gene (Deborah etal.1996) of cell proliferation.Eukaryotic growth course is divided G1, S, G2, M four periods, and the transition of G1-S phase is the center regulation and control stage, and in many cells, mitotic division signal and cell size are responsive unusually in this stage.In the fission yeast, the promoter region of many genes in this stage is connecting some cis-regulating element, as SCB (Swi4-Swi6 cell cycle box, CACGAAA) and MCB (MluI cell cycle box, ACGCGTNA).Comprise Swi4 albumen and Swi6 albumen with SCB bonded transcription factor SBF (SCB-binding factor), activate G1 cell cycle gene, cell walls biosynthesis gene and HO gene transcription; And comprising Swi6 albumen and Mbp1 albumen with MCB bonded transcription factor MBF (MCB-binding factor), some must gene transcription (Ho et al, 1999 to activate the DNA synthetic; Koch et al, 1993).Among the Swi6/Cdc10 family protein member, Swi6, Swi4, Mbp1, Res1/Sct1, Res2/Pct1, Cdc10 etc. have the core texture territory that the ANK sequence is formed, and (Xu et al.1996 plays an important role in the signal conduction that participates in the whole cell cycle; Lan etal.1997; Steven et al.1998).MBP1 and SWI4 have similarity on sequence, though it lacks separately and yeast is caused death cause the unconventionality expression of DNA synthetic gene, but the bacterial strain that lacks MBP1 and SWI4 gene simultaneously then can not be survived.And the regulation and control of Swi4-Mbp1 transcription factor have high conservative in ascomycetous yeast, even may be whole Eukaryotic universals (Koch et al, 1993).

The rice blast that is caused by Magnaporthe grisea (Magnaporthe grisea) is global rice disease, also is one of main disease of China paddy rice, and the grave illness field can cause the paddy rice total crop failure.This disease almost all has big generation every year in China part provinces and cities, also once is very popular for several times in the whole country.Recent national big area is popular to be 1993.Under the situation of control comprehensively, rice blast still causes paddy underproduction over ten billion jin then.2004, provinces and cities such as Chongqing and Sichuan broke out large-area rice blast, and only the Hechuan city in Chongqing just has 20,000 mu of paddy rice that rice blast takes place, wherein nearly 5000 mu of paddy rice total crop failure, and the important agricultural problem of Ceng Zuowei is in the news.Magnaporthe grisea is a kind of thread ascomycetes fungi, except that paddy rice, can also infect more than the 50 kind of grass that comprises wheat, barley and some turfgrasss, causes seasonal febrile diseases.Simultaneously, this bacterium has the common infection processs of pathogenic fungi of many important crops, comprises that generation of conidium and sprouting, appressorium form, invade, infect the expansion of mycelia.In addition, Magnaporthe grisea can carry out genetic cross and conversion.For this reason, the investigator of many developed countries with Magnaporthe grisea as the pattern pathogenic fungi, comprehensively, in depth carrying out the molecular mechanism research of plant pathogenic bacterium pathogenicity and pathotype variation, expectation is studied by this type of and is sought the fungi specific target and be marked with the design novel agrochemical.Especially recently the Magnaporthe grisea whole genome sequence of finishing is measured, and has established solid foundation for carrying out the pathogenic functional genome research of pathogenic fungi.

Information biology studies show that: contain nearly 51 of the proteic gene of ANK motif in Magnaporthe grisea (Magnaporthe grisea) genome, the albumen that much contains the ANK motif is also arranged in other plant pathogenic fungi, but the functional study of relevant ANK protein gene in filamentous fungus yet there are no report.The present invention finds by bioinformatic analysis, a proteic gene of ANK is arranged Magnaporthe grisea, gibberella saubinetii (Gibberella zeae), corn stalk rot disease bacterium (beading gibberella, Gibberella moniliformis), more conservative between Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Botrytis cinerea bacterium (Botrytis cinerea), wheat glume blight bacterium fungies such as (Phaeosphaeria nodorum), its protein reaches more than 40% in the consistence on the amino acid levels, the temporary in the present invention called after MBP1 of this albumen.For biological function and the molecular mechanism of effect, the especially effect in growth, growth and the pathogenic course of pathogenic fungi of understanding this ANK gene and proteins encoded thereof, the present invention is an example with MgMBP1, has carried out gene knockout and functional study.Found that: the gene knockout of MgMBP1 causes number of drawbacks such as Magnaporthe grisea poor growth, and virulence significantly weakens.Therefore, this ANK motif albumen and expression thereof can be used as target, are used for the design and the screening of novel agrochemical, in the control of fungal diseases of plants significant application value are arranged.

Summary of the invention

It is research mode that the present invention is intended to the Magnaporthe grisea, provide and come from Magnaporthe grisea (Magnaporthe grisea), gibberella saubinetii (Gibberella zeae), corn stalk rot disease bacterium (beading gibberella, Gibberella moniliformis), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Botrytis cinerea bacterium (Botrytis cinerea), the sequence of a conservative relatively ANK gene and proteins encoded thereof in the wheat glume blight bacterium plant pathogenic fungis such as (Phaeosphaeria nodorum), the promoter sequence of this gene and this gene effect and the expression characteristic in control colony growth and virulence.

ANK albumen provided by the present invention is MgMBP1 in Magnaporthe grisea, for having the protein of one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 3; Its sequence is made up of 796 amino-acid residues.

2) with SEQ ID № in the sequence table: 3 amino acid residue sequence is through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and to fungal colony growth and the influential protein of virulence.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 MgMBP1 (MgMBP1) also belongs to protection scope of the present invention.MgMBP1 is meant the dna sequence dna with one of following nucleotide sequence:

1) as SEQ ID № in the sequence table: the genomic dna sequence shown in 1; Its sequence signature is: by 5690 based compositions, comprise four exons and three introns, from 5 ' the 1498th at end to 1553 bit bases are first exon, from 5 ' the 1554th at end to 1932 bit bases are first intron, from 5 ' the 1933rd at end to 2191 bit bases are second exon, from 5 ' the 2192nd at end to 2495 bit bases are second intron, from 5 ' the 2496th at end to 2659 bit bases are the 3rd exon, from 5 ' the 2660th at end to 2794 bit bases are the 3rd intron, are the 4th exon from the 2795th at 5 ' end to 4674 bit bases; Its coding region is positioned at SEQ ID №: 15 ' end the 1498th between 4674 bit bases.From SEQ ID №: the 1st at 5 ' end of 1 to 1497 bit bases are promoter sequence.

2) SEQ ID № in the code sequence tabulation: the polynucleotide sequence of 3 protein sequences, or it is through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and to the corresponding polynucleotide sequence of fungal colony growth and the influential protein sequence of virulence.

3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 80% above homology, and encode same molecular and biological function protein DNA sequence.

The cDNA of MgMBP1 involved in the present invention is meant to have one of following characteristics nucleotide sequence:

1) SEQ ID № in the sequence table: 2 dna sequence dna; This sequence is by 2391 based compositions.

2) SEQ ID № in the code sequence tabulation: 3 protein or its are through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and to fungal colony growth and the influential proteinic polynucleotide sequence of virulence.

3) with sequence table in SEQ ID №: 2 dna sequence dnas that limit have 80% above homology, and encode same molecular or biological function protein DNA sequence.

The promoter sequence of MgMBP1 involved in the present invention is characterized in that having one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 4 dna sequence dna; This sequence is characterized in that having the ability of driving purposes genetic expression by 1318 based compositions.

2) SEQ ID № in sequence table: on 4 the basis through the replacement of one or more bases and or disappearance and/or interpolation and deutero-DNA variant sequence is characterized in that having the ability of driving purposes genetic expression.

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

The expression vector, clone and the host bacterium that utilize MgMBP1 gene, cDNA and promotor to make up all belong to protection scope of the present invention.

Protection scope of the present invention also comprises: come from Botrytis cinerea bacterium (Botrytis cinerea), corn stalk rot disease bacterium (beading gibberella; Gibberella moniliformis), in the gibberella saubinetii (Gibberella zeae), wheat glume blight bacterium (Phaeosphaeria nodorum), Sclerotinia sclerotiorum plant pathogenic fungis such as (Sclerotinia sclerotiorum) with MgMBP1 homologous albumen, name is respectively BcMBP1, GmMBP1, GzMBP1, PnMBP1 and SsMBP1.It is characterized in that: itself and MgMBP1 have 40% or above consistence respectively on aminoacid sequence; Its sequence is respectively SEQ ID №: 5, SEQ ID №: 6, SEQ ID №: 7, SEQ ID №: 8 and SEQ ID №: the aminoacid sequence shown in 9.

With the nucleotide sequence in arbitrary zone in MgMBP1 gene design primer, and be used for detecting that the MgMBP1 expression of gene also belongs within protection scope of the present invention under the compound treatment situation by pcr amplification.

Have with MgMBP1 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 MgMBP1 also belongs within protection scope of the present invention under the compound treatment situation.

Knocking out of MgMBP1 gene involved in the present invention causes the Magnaporthe grisea colony growthing slow, and conidium germinates and appressorium forms slowly, form the ability that infects nail and infectivity mycelia weakens, and the virulence on susceptible rice varieties weakens.The most important purposes of the present invention is: use above-mentioned achievement, design and screening can destroy the compound of the expression of this genetic expression, shearing and proteins encoded thereof; Or the localized compound of nuclear of MgMBP1 can be modified or block to design and screening to the aminoacid sequence of MgMBP1; Or design and screening can be blocked and the conjugated protein bonded compound of nucleotide sequence of MgMBP1 promotor; Thereby the antifungal medicament of development of new.In addition, purposes of the present invention also comprises: as probe separated DNA sequence again in Magnaporthe grisea, perhaps isolating in other fungi, have the sequence of certain sequence homology with this gene with a certain section of the DNA of the nucleotide sequence of MgMBP1 promotor or MgMBP1 gene or cDNA.

The invention still further relates to and come from gibberella saubinetii (Gibberella zeae), corn stalk rot disease bacterium (beading gibberella, Gibberella moniliformis), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Botrytis cinerea bacterium (Botrytis cinerea), wheat glume blight bacterium plant pathogenic fungis such as (Phaeosphaeria nodorum), have on aminoacid sequence with Magnaporthe grisea MgMBP1 and to be higher than 40% conforming homologous protein, they may have identical biological function with Magnaporthe grisea MgMBP1.Therefore, the promotor of these homologous proteins and encoding gene thereof also can be used as target site, is used to design and screen the medicine of anti-these fungies.

Being expressed as target, carrying out purposes such as the screening of antifungal medicine and design and also belong within protection scope of the present invention of above-mentioned MgMBP1 of utilization and homologous protein thereof.

Description of drawings

Accompanying drawing 1. comes from Magnaporthe grisea (Magnaporthe grisea, Mg), gibberella saubinetii (Gibberella zeae, Gz), corn stalk rot disease bacterium (Gibberella moniliformis, Gm), Sclerotinia sclerotiorum (Sclerotiniasclerotiorum, Ss), Botrytis cinerea bacterium (Botrytis cinerea, Bc), wheat glume blight bacterium (Phaeosphaerianodorum, the sibship of MBP1 proteinoid Pn).

The structure and the proof of accompanying drawing 2. Magnaporthe grisea paddy rice wild type strain P131MgMBP1 gene knockout bodies.Illustrate: last figure is the synoptic diagram that knockout carrier makes up; Figure below is the Southern hybridization affirmation figure that knocks out body, and left side figure is to be probe with MgMBP1, and right side figure is to be probe with the metathetical hygromycin gene.

The colonial morphology that knocks out body Q877 of accompanying drawing 3. Magnaporthe grisea paddy rice wild type strain P131 and MgMBP1 gene, colony growth velocity ratio are.Illustrate: upward be 3-7 days colony diameter, middle colony growth speed is the in kind photo of cultivation after 7 days down.The used substratum of colony growth is an oat tomato substratum, 25 ℃ of illumination cultivation.Initial inoculum size is identical.

Accompanying drawing 4. Magnaporthe grisea paddy rice wild type strain P131 with and the comparison of MgMBP1 gene knockout transformant Q877 conidial germination rate different time sections on onion epidermis.Illustrate: upward photo is the photo in kind behind the inoculation 4hr, and a left side is a wild-type, and is right for knocking out body; Down for inoculating the percentage of back 4-36hr spore germination.

Accompanying drawing 5. Magnaporthe grisea paddy rice wild type strain P131 with and the comparison of MgMBP1 gene knockout transformant Q877 appressorium rate of formation different time sections on onion epidermis.Illustrate: upward be the photo in kind behind the inoculation 10hr, a left side is a wild-type, and is right for knocking out body; Down for inoculating the percentage that back 4-36hr appressorium forms.

Accompanying drawing 6. Magnaporthe grisea paddy rice wild type strain P131 with and the MgMBP1 gene knockout transformant Q877 comparison of on onion epidermis, infecting the success ratio different time sections.Illustrate: upward be the photo in kind behind the inoculation 16hr, a left side is a wild-type, and is right for knocking out body; Infect the percentage that nail forms for inoculation back 8-36hr down.

Accompanying drawing 7. Magnaporthe grisea paddy rice wild type strain P131 with and the comparison of MgMBP1 gene knockout transformant Q877 infectivity mycelial growth situation on onion epidermis.Picture is the photo in kind behind the inoculation 36hr, and a left side is a wild-type, and is right for knocking out body; Figure below is to have the percentage that branch infects the appressorium of mycelia.

Accompanying drawing 8. Magnaporthe grisea paddy rice wild type strain P131 and and MgMBP1 gene knockout transformant Q877 virulence comparison.Illustrate: last figure is inoculation paddy rice and the in kind photo of barley after 96 hours, and a left side is the inoculation paddy rice, and the right side is the inoculation barley; Figure below is the mean length and the width comparison of the susceptible rice varieties Lijiang xintuanheigu of inoculation total scab and extended pattern scab quantity and scab after 96 hours.The live body spray inoculation is taked in inoculation; Inoculum density is 1.5 * 10 ⁴Individual spore/milliliter; The 4th the leaf phase Lijiang xintuanheigu seedling; Inoculate the cultivation of preserving moisture of back 36 hours dark, then see light, 25 ℃.

The comparison of five complementary transformant C804, the C805 of accompanying drawing 9. Magnaporthe grisea paddy rice wild type strain P131 and MgMBP1 gene knockout transformant Q877, C811, C812, C817 colony growth diameter.Illustrate: go up colony diameter on oat tomato substratum, cultivating the 3rd day to the 7th day; Be the 5th day photo in kind of cultivation down.

The conidia germination rate of five complementary transformant C804, the C805 of accompanying drawing 10. Magnaporthe grisea paddy rice wild type strain P131 and MgMBP1 gene knockout transformant Q877, C811, C812, C817 relatively.Illustrate: the conidia germination percentage of inoculation onion epidermis 4-36hr.

The comparison of accompanying drawing 11. wild type strain P131 and five complementary transformant C804, C805, C811, C812, C817 appressorium rate of formation.Illustrate: the percentage of inoculation onion epidermis 4-36hr appressorium rate of formation.

Accompanying drawing 12. wild type strain P131 and five comparisons that complementary transformant C804, C805, C811, C812, C817 infect the nail rate of formation.Illustrate: inoculation onion epidermis 8-36hr infects the percentage of nail rate of formation.

Accompanying drawing 13.MgMBP1 mycelia, spore, appressorium, infect the nail and the infectivity mycelia in expression figure.Illustrate: left column is that the bright field is captured, and the centre is classified as under the dark field blue light captured, and the captured photo stacking diagram in light and shade visual field is classified on the right side as.

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.

Embodiment 1: the bioinformatic analysis of ANK albumen MBP1 in the plant pathogenic fungi genome.

At first, the est database of the plant pathogenic fungi that Britain Exeter university is set up ( Http:// cogeme.ex.ac.uk/) retrieve, analyze, found that 3 codings contain the EST of ANK domain protein (sequence number is respectively MagUK14180864, MagUK30406229 and MagUKCon[8983]).The result shows that ANK albumen is the class expressing protein in the pathogenic fungi.Thereby, respectively with the whole genome sequence of these 3 EST and Magnaporthe grisea bacterial strain 70-15 ( Http:// www.riceblast.org./) compare analysis, find to contain 51 ANK albumen that infer, that function is different in the Magnaporthe grisea.Further, utilize gibberella saubinetii (Gibberella zeae), corn stalk rot disease bacterium (beading gibberella, Gibberella moniliformis), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), Botrytis cinerea bacterium (Botrytis cinerea), the wheat glume blight bacterium important existing genomic datas of pathogenic fungi such as (Phaeosphaeria nodorum), the ANK albumen that wherein may exist is analyzed, is predicted.Found that: in the Magnaporthe grisea a kind of ANK albumen in these several pathogenic bacterias, all have a correspondence, consensus amino acid sequence is higher than 40% homologous protein.The albumen of this genes encoding is made up of 796 amino acid in the Magnaporthe grisea, has 27% consistence with zymic MBP1 albumen at aminoacid sequence, and the consistence that wherein combines the territory with the DNA of MBP1 reaches 56%, so with its called after MgMBP1.The homologous protein of MgMBP1 in above-mentioned other fungal pathogen then distinguished called after GzMBP1, GmMBP1, SsMBP1, BcMBP1 and PnMBP1.The proteic sibship of MBP1 as shown in Figure 1 in the several plant pathogenic fungi.

Embodiment 2: the knocking out of gene

1. the structure of gene substitution transformant.The structure of gene replacement vector is meant that two flank sequence orientations with this gene of prediction are connected in the carrier, and this carrier has been connected into a neomycin resistance gene in advance, is connected into hygromycin gene (seeing the synoptic diagram in the accompanying drawing 2) between two flank sequences.

2. gene replacement vector transforms Magnaporthe grisea.Adopt and do not destroy the intragentic restriction enzyme of carrier, transform the protoplastis of Magnaporthe grisea the carrier linearizing.The preparation and the method for transformation of protoplastis are as follows:

(1), conidial preparation.The Magnaporthe grisea mycelia of fully interrupting is uniformly applied on the medium oatmeal 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, the individual layer gauze covered in 26 ℃ of-28 ℃ of illumination cultivation about 48 hours, at the promptly visible a large amount of Magnaporthe grisea spore of media surface.

(2), the preparation of protoplastis.500 milliliters of triangular flasks pack into 150 milliliters of CM substratum (yeast extract 0.1%, 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%), insert about 10 ⁶Individual conidium, 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 ℃, enzymolysis is after 3 ~ 4 hours under 100 rev/mins of conditions, 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, 10mMTris-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 0.5 ~ 1 * 10 after the centrifugation ⁸Individual/milliliter.

(3), Magnaporthe grisea transforms: protoplastis is sub-packed in 50 milliliters of centrifuge tubes of sterilization, every pipe 150 microlitres, add isopyknic linearizing carrier (about 2 micrograms) and STC mixed solution, placed on ice 20 minutes, dropwise slowly add 2 milliliters/pipe PTC solution (60% poly-hexylene glycol 3350,10mM Tris-pH 7.5,50mM calcium chloride) then, 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 hours, changed culture dish over to, added about 12 milliliters of SR (LR+1.6% agar) that are cooled to about 50 ℃, mixing, treat its solidify dry up after, 0.7% top-layer agar of 12 milliliters of the one decks in shop (is cooled to about 50 ℃ the Totomycin of 300 mcg/ml above.Cultivated 4 ~ 6 days for 28 ℃, the transformant that occurs is gone on the CM substratum (containing the Xin Meisu of 350 mcg/ml and the Totomycin of 250 mcg/ml) of two kinds of resistances simultaneously, will be containing on the Xin Meisu substratum long change over to and cultivate on the rolled oats tomato substratum, collect mycelia and extract DNA and carry out pcr amplification and further determine the gene substitution transformant at single bacterium colony long on the hygromycin resistance substratum.By this screening, obtained 7 altogether and knocked out body.

Embodiment 3: gene substitution transformant phenotype is observed

1. gene substitution transformant monospore is separated, lay several bacterium cakes with the 5mm punch tool and be inoculated on the tomato oat medium flat board and cultivate in 25-26 ℃ of illumination box knocking out the monospore bacterium colony of one of body Q877 and wild-type bacterium colony, measured colony diameter in the 3rd day to the 7th day, and deducted the former bacterium cake of 5mm diameter and be true expansion diameter.What second day colony diameter deducted the day before yesterday is the bacterium colony spreading rate.Table 1 be Q877 and the 3rd day to the 7th day diversity ratio of P131 colony diameter, table 2 be the bacterium colony spreading rate relatively.

Table 1, knock out body Q877 and wild type strain P131 bacterium colony expansion diameter difference

	Bacterium colony expansion diameter average (mm)
			P131	Q877
	The 3rd day	14.5625			2.875
The 4th day			22.8125	6.40625
	The 5th day	31.375			11.9375
The 6th day			39.75	16.75
	The 7th day	45.1875			20.875
The significance of difference			A	B

Annotate: the significance of difference be between A and B two groups in 5% remarkable scope significant difference.

Table 2, knock out body Q877 and wild type strain P131 bacterium colony spreading rate difference

	The average spreading rate of bacterium colony average (mm)	The significance of difference
			P131	7.65625	A
Q877	4.5	B

Annotate: the significance of difference be between A and B two groups in 5% remarkable scope significant difference.

2. the observation that gene substitution transformant infects on the onion entocuticle.Embodiment 1 described conidium is washed the back filter in 50 milliliters centrifuge tube with double-deck lens wiping paper, 4000 rev/mins of room temperatures collection in centrifugal 5 minutes spore is suspended in 0.25 ‰ the polysorbas20 then.Adjust conidium concentration to every milliliter of 1.5-2 * 10 ⁴Spore, spray inoculation is on the onion entocuticle that is placed on the 1.2% solid water agar equably, and the 25-26 ℃ of dark culturing 36 hours of preserving moisture sees that then light cultivates.Time segment investigation counting.Table 3 is the difference that knocks out body Q877 and each time period spore germination rate of wild type strain P131 spore inoculating onion entocuticle, appressorium rate of formation and infect the nail rate of formation.

Annotate: the significance of difference be between A and B two groups in 5% remarkable scope significant difference.The significance of difference analysis of the data the when significance of difference refers to 36h in this table.

3. pathogenic mensuration.Adopt embodiment 1 described method 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 collection in centrifugal 5 minutes spore is suspended in 0.25 ‰ the polysorbas20 then.Adjust conidium concentration to every milliliter of 1.5-2 * 10 ⁴Spore, spray inoculation is in the face of blade of four leaves, one heart stage rice seedling equably.The 25-26 ℃ of dark culturing 36 hours of preserving moisture sees that then light cultivates.96 hours " Invest, Then Investigate " scabs.Table 4 is for knocking out the difference of body Q877 and wild type strain P131 inoculation rice leaf scab number, and table 5 is for knocking out the difference of body Q877 and wild type strain P131 inoculation rice leaf scab size.

Table 3. knocks out body Q877 and each time period spore germination rate of wild type strain P131 spore inoculating onion entocuticle, appressorium rate of formation and infects the difference of nail rate of formation

	Spore germination rate average (%)		Appressorium rate of formation average (%)		Infect nail rate of formation average (%)
							P131	Q877	P131	Q877	P131	Q877
	4h	91.883	66.483	74.067	13.117	0								0
8h							94.717	73.550	92.150	50.530	5.4	3.567
	12h	95.917	70.967	94.500	64.850	29.033							9.533
24h							97.583	78.383	95.083	72.750	91.967	28.067
	36h	98.400	79.033	93.400	74.667	94.217							41.25
The 36h significance of difference							A	B	A	B	A	B

Table 4. knocks out the difference of body Q877 and wild type strain P131 inoculation rice leaf scab number

	Scab sum average (individual)	Expansion spot number average (individual)	The significance of difference
				P131	47.5	25.5	A
Q877	6.8	2.4	B

Annotate: the significance of difference be between A and B two groups in 5% remarkable scope significant difference.

Table 5. knocks out the difference of body Q877 and wild type strain P131 inoculation rice leaf scab size

	Scab length (mm)		Scab width (mm)
					Mean value	The significance of difference	Mean value	The significance of difference
	P131	2.642857	A	1.761905					A
Q877					1.083333	B	0.833333	B

Annotate: the significance of difference be between A and B two groups in 5% remarkable scope significant difference.

Embodiment 4: the clone of Magnaporthe grisea control colony growth and virulence gene M gMBP1.

1.PCR amplification.According to sequence in the database and software analysis, draw this gene Position Approximate and possibility exon, the design primer carries out pcr amplification, and amplified production is connected on the T carrier.

The comparison of table 6. wild type strain P131 and five complementary transformant c804, c805, c811, c812, c817 bacterium colony expansion diameter on the tomato oat medium

	Bacterium colony expansion diameter average (mm)
							P131	C804	C805	C811	C812	C817
	The 3rd day	16.50	14.00	16.00	14.50	14.00							15.25
The 4th day							24.75	23.25	25.00	24.75	24.50	25.00
	The 5th day	32.50	31.75	32.50	30.75	30.25							32.75
The 6th day							42.25	39.75	40.75	40.00	41.75	41.75
	The 7th day	47.00	45.00	46.00	44.00	46.25							47.25
The significance of difference							A	A	A	A	A	A

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

2. choosing suitable enzyme cuts above-mentioned fragment and is connected on the complementary carrier, this complementation carrier is connected with neomycin resistance gene, this carrier linearizing changed over to knock out in the body, method for transformation is as described in the above-mentioned embodiment 1, the picking transformant is carried out colony growth and pathogenic experiment, all reply wild-type, prove that this amplified fragments has comprised this complete gene.

Table 6 is the comparison of wild type strain P131 and five complementary transformant c804, c805, c811, c812, c817 bacterium colony expansion diameter on the tomato oat medium.Table 7 is wild type strain P131 and five complementary transformant c804, c805, c811, c812, spore germination rate, the appressorium rate of formation of c817 spore inoculating onion entocuticle and the comparisons of infecting the nail rate of formation.

3.RT-PCR。The collection product spore time is 24 hours conidium, and guanidinium isothiocyanate (GT) method extracts total RNA, carries out reverse transcription with the ThermoScript II of SuperscriptII TM, obtains article one chain of the total cDNA of conidium.Design the cDNA total length of primer amplification gene according to the possible exon of software analysis.

Embodiment 5: the expression of gene period analysis

1, the structure of carrier.The promotor of gene linked to each other with the GFP gene be connected in the carrier that has neomycin phosphotransferase gene.

2, expression vector transforms Magnaporthe grisea.Press method for transformation described in the embodiment 1, adopt and do not destroy the intragentic restriction enzyme of carrier, transform the protoplastis of Magnaporthe grisea the carrier linearizing.Picking has the transformant of neomycin resistance to be observed under fluorescent microscope, find this gene mycelia, spore, appressorium, infect the nail and the infectivity mycelia stage expression is all arranged.

Table 7 wild type strain P131 and five complementary transformant c804, c805, c811, c812, spore germination rate, the appressorium rate of formation of c817 spore inoculating onion entocuticle and the comparisons of infecting the nail rate of formation.

		P131	C804	C805	C811	C812	C817
								Spore germination rate %	4h	93.15	93.28	92.10	91.80	91.79	92.30
8h	93.91	95.68	95.58	95.58	96.49	93.00
							12h		97.74	98.15	98.45	97.95	98.13	97.40
24h	99.13	97.97	98.01	98.09	98.24	98.91
							36h		98.22	98.22	98.66	97.51	98.20	98.33
The 36h significance of difference	A	A	A	A	A	A
							Appressorium rate of formation %		4h	69.22	62.61	70.48	65.65	60.51	62.18
8h	91.77	92.37	90.88	93.34	92.23	93.49
								12h	95.84	96.62	95.25	95.57	95.58	93.82
24h	96.74	94.40	95.17	93.84	95.10	94.64
								36h	92.15	94.89	96.06	95.28	95.27	95.58
The 36h significance of difference	A	A	A	A	A	A
								Infect nail rate of formation %	8h	6.68	7.09	7.28	8.37	8.16	6.55
12h	39.88	34.99	32.91	31.33	33.31	39.93
							24h		92.83	93.59	93.29	94.23	93.86	92.07
36h	94.76	94.25	93.67	93.99	94.64	95.31
							The 36h significance of difference		A	A	A	A	A	A

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

Sequence table

＜110〉China Agricultural University

＜120〉ANK albumen and the encoding gene and the utilization of new control fungal colony growth and virulence

<160>9

<210>1

<211>5690

<212>DNA

＜213〉Magnaporthe grisea (Magnaporthe grisea)

<400>1

gatatcttag taatcctctc gagttaaggt aggtatacta atcggtagtt ataacttctt 60

ccatccattt caatcctgtt ctcaggatga ggatggctga aacgaggata aacatcccaa 120

gcaagttacc aacgggtagc tggccgtata cattctttga tttgtttggc tgcaaccttg 180

catccatcgt aaccattttg gagggatatt ttgcttggcc ctcctgactg acttgcgagt 240

tcgcctggta ccaaggccca aagccgcagg atgatcggat tactaccaaa ttcaatccac 300

cagatcgatc gcattgtcat caagttgtct gagccgctga ctacattctt ggggcacggt 360

caagagaggc atggcatgca tgccacaact aattccatgt ctgccactcc cgagtctcgc 420

actgcagcca aatacgggag aactcggact ggacttttgt atcacacggg cggaaagtaa 480

acaagactgc gagtggttgc aaaagctaga gcatgggatc tggttggtag aaaagcgtac 540

cgtggttcgc tgaaggtatt tatactatac ttctacaaag tgaactagcg acttgttgtc 600

ccctgaaatc ggccacctga acgcctcatg acacccgtgt agaatcgtat taagtagcag 660

gaaagggaga aaaaaccaca aatgaaagga aataagtcat cgtacagtac aagaccgaac 720

catgactgca aatagtgagg attctagtcc cgaggcaaaa aacccagcta tcaaggtcat 780

ggggcgcaac tatacatgcc atatttgtct acatgctgcg acggtggtga agttatgggc 840

gacccccgag aggacactgg cgagaatcct ggtgacagat cgacccaggc aggaggagac 900

gatgacctcc aaagcggtga accaaaagac gcatggtcaa cacgggttgg gcagtcgccg 960

gcatgaggca attgtagcgg aggcgtgcgg cacaaagcta atgttccggc cgtgctctac1020

agagccgatg ttggtggcgc aaggtccatc gtctcgccac ccaccaggcc gtttgttact1080

gatgatgggc cgtcggcggt actatttacc atgccatccg acacctcttc ttccatgata1140

gcaacaagct cctccaggtt gtcgtcgagg ttctccgcca aggggcctaa acagttccta1200

aggagtcgcc ggtattggtc gatcttctcg cccgtgccga ccatgctgag tgcctcaatg1260

tactccgtct ccgccatttg ctgctcgcct aaaagatagc gaagctcgtt ggccaaagcg1320

agcctctcct cgatgctgtt ggtgttcccg ttggaatcac cgttcatgct tgcgtccagc1380

tcccgctcaa ctaggccttc gacatgaaga cggttcagtg tgcaaacaac tgccttgacc1440

tttccggtgg ctgtctcgag ttcaccgtca ataccaaaag cttgctctgg cggcaacagc1500

tgcgacccca gctccgcaag cttgtttgtt aaagaggtgt gctcagcttg tgcgttcgac1560

aatattcgtg tcgcctcgcg ctcggcttcg tctttgatcg tgaactcgtc ttcgtatgac1620

ctggcaagat cgtggaactt ctcaaagacc aaaggtgtaa ttcggttctg cacggtgagg1680

gcggcctctg acttgaggct ggctgtcatt ttgttgctgt ggctgttggc cgcgccaatg1740

gtcatcgcgc cgacatcacc agccaacgca tcgcgaaagg aaacctgtct tctagccgat1800

tcaggtgcaa atggagagga tgatctttgt ggcacagagc gcagcttctt gctggcattg1860

agctccttga tcaaatctgc agcaatcatg ccctcgttgt ttgtaatgtc agtgctggca1920

ccacggccca ggagggcacg aatgcatttg ctcgaaccgc gctgcgccgc aagatgaaca1980

gctgtgttgc catcattgtc ctgggcgtcg aggagctgct gaacaaagtt ggggtcgtgt2040

tgggtctcct gcaacctatt gaggatgttg tccagataat accgtgaaca cgttggactg2100

tggacgcgac cgcccttgag aatggccgca tgatgaatgg cggtacaacc tgacaggtcg2160

cggatgttga ttgtgtggaa gagctccttg agcacgacgg ggaaggtttg cttatcgtag2220

cagttggtga acgtgaccgc cctcatgagc ggcgtctcac ccctcacgtt cctcgaatct2280

ggctgagcat tgaactgaaa aagctgtttg ataatgtcga catcgcccat cgaggcggcc2340

caatggaggg ctgtatgctg gtccgcgtct atggggaagt ttggcctgaa gttgacggga2400

ggctcaggac gcatggctgg cgtcggctgg ttcctagaca gcaggaaata gtctaataac2460

tcgtcaccgt agattctatg ctgctgctcg atcatactct cggtgggctc ttcgcgcttg2520

cgtttcctgt gtccggttga gaagtgcgac aggtccgggc ggtcgtcttc agccatgtaa2580

gaagctgaag caacagtcag gttatctggt gtgtcgtcct cgtcaaccaa catctctccg2640

ttctcgtatg catcttgagg gtgtagagag ggctggtagt gcagtggtgg tggtggtgcc2700

tgcttggcta ctgcgcgggc tttgttgttg aatctaggca cggctggctt ctttggttgc2760

ttgggtttgc tcgtgtgtcg tggagccggg ggcgggctat caggccccgg agaaaactcg2820

aagatgggcc gtaacctgtc gaagatgttg tttcggtggg ccaaagcctc acctgcttcc2880

agaggaatcc atgtgcctga gagggtttca aattatgagg gtcagtaatc cggtttgtga2940

gacgatcctc aggcggcagt cggtagccga gtcgccggcc gcagatgaaa gtacacacct3000

tggtactttc catatccgcc ctgaaccttc tcatgctgat ccttttgtac ctcgcgttcc3060

aaaatgcgag ttcttgcggg tttgtcaaag ccggcggcct tgaggatatg tgttgcgttg3120

atccagtcgt cgacacgtcg gcgcattaca tgctctttga gatctactcc gaactggtac3180

tcatagacag gtatctattg gattgtgtga ctgttagaca aaggttttct cacaaccttg3240

gagggtatgt ctcttgcagg aattcgggaa tatgtagaaa gaagcagatg cgagacccag3300

atcacaagcc gacaagggag cagcaaggga aaagaggtga gcgtattgcg ccatggcggg3360

acgagtgata gatgggattg acggcgcaaa ggcatcgcaa tcatgcaggt agggaagaaa3420

gcggcggccc acatgataga ccagaaagcc tcccgtgtgc cagcgtgcgc aaatacaaat3480

agggaagatg gtaacgcacc ccgctgtagg tcgcactgta gatgccaggc ccagtcgggg3540

cactggctgc tgctgctgct gccttgacca tggtctggcc tggcggtggc cggagccgga3600

aaggatcggg gtagccggcg ggagacacga tgatgtctgg tttcgcacga ggatgacgct3660

cggggggcaa tggaggtcgc cagtgatggt gatgatcgaa cgactggggg aaagcaatgc3720

aagtaatctg ggtttgctgc tttggtggaa tagggtcgct gtatacagtt catgcactag3780

ggtactcgta aaaggtattt gatggtttaa tggaaggcga tgaacagctt aattggcaaa3840

atgggactaa agaagggatc aaagctccag ctccaggttt agcaaggctg ttggtggtgg3900

tcgcaccgga ttgcgacgag gcgaccgatc ggtcgatagc aaccgagtca atttttggat3960

tttcttcaat tgctgggccg acggcgctct tgtcgattgc gcgcggtctg gtctggtctg4020

gtggagcgag tgcaagggtc aaattgggaa atgggggaag aaagagaggt ttgacgcgaa4080

ggaagcgtgt ctgacgcgat ggagaaggtg tagaccgcgt ttccaattta cctttaccgg4140

attttcaagc tcgtgcggtc gatcgcctga ggcacgcggg atttccccac catgccgtgc4200

gccaacccag agtgataagc aagctaaggc accgcatgga aatccatgat cgcgcgaaat4260

tgagacgtat tttcacgtga ctgtacgtag gtacctacaa ggagggacct gacccatcgc4320

atcccaaacg ccaaccccgc aaaagacccg acgccggtcg tcttctccca cccgttgact4380

tgtgagatgt ttctgaatac cttggtaggt attcaagccg caattggctc aatcaattgg4440

ctgtggactc ggtcaccatc gggcatctac ccgagatttt tttgtttcgt cggccccaac4500

gccgtcctca caaaatgacg tctacgaaca cgagcctaag tcgagtacct taaaaggtaa4560

ccccgcgtcc gagccctttt tgactttcgg ggaccttcaa ccccacagtg acgccggctg4620

cttaggagat agatatttgg aattgtaagt gtcacaagtc ctgtatattt atcccatgga4680

ctcaccgaga taaacggcgc gaatctgagt ctgacctttt gtcacaacgg cgaacagcta4740

gctaccgtac ctacctaaac aataacatcg tagcctcttg cgaacacctc gaccgacacg4800

gttggccacc atgcctctca tcgcacagaa ccccaccaac agggtcatcc ttggcctgat4860

gacctttggc ccggacgagg ctacaggtgc ccgcattaca tccgtggacg agttcggcaa4920

ggtcctcgac atcctccaga agcgcggata caacgaggtg gacactgccc gcatgtacat4980

tggtggcaag caggaggcat tcactcgtga ggttggctgg aagcaacgcg gccttacttt5040

ggcgaccaag gttcaatacc cgtcagaata tggcatgaat gctcctgata aagtcaagga5100

gtcggtggac ctcagtttga aagagctggg cactgactgc gtcgatgtaa gctttatctg5160

ccttttgttc cttatacact ccagttccat actatgatgc aacaaaacag tcatgtatgc5220

tatgatctta gtggctgaca gaacttacat cttccagctc ctctatcttc atgccgccga5280

ccgaggtaca ccctttgccg agacactccg cgccatcaac gacctacaca aggccggcaa5340

attcgtcaac tttggcattt ccaactttgc tgcatacgag gttgccgaga tcgtcatgac5400

atgcgtgcaa aacaactggg tgcgaccaac tgtttaccag gccatgtaca atgtcatcac5460

acggtctatc gaggcagaac tgattcctgc atgtcgccga tacggacttg acctggtggt5520

gtacaatccc attgccggcg gtcttttcag cggcaagatc aagacccagg atatggtacc5580

tgctgagggt cgattcagcg actcgactac cagtatgggt aagatgtacc gcaacaggta5640

cttcaaggag accacattca aagccctcca aacgatagag gctgcggttg5690

<210>2

<211>2391

<212>DNA

＜213〉Magnaporthe grisea (Magnaporthe grisea)

<400>2

atggtgggga aatcccgcgt gcctcaggcg atcgaccgca cgagcttgaa aatccgcgac 60

cctattccac caaagcagca aacccagatt acttgcattg ctttccccca gtcgttcgat120

catcaccatc actggcgacc tccattgccc cccgagcgtc atcctcgtgc gaaaccagac180

atcatcgtgt ctcccgccgg ctaccccgat cctttccggc tccggccacc gccaggccag240

accatggtca aggcagcagc agcagcagcc agtgccccga ctgggcctgg catctacagt300

gcgacctaca gcgggatacc tgtctatgag taccagttcg gagtagatct caaagagcat360

gtaatgcgcc gacgtgtcga cgactggatc aacgcaacac atatcctcaa ggccgccggc420

tttgacaaac ccgcaagaac tcgcattttg gaacgcgagg tacaaaagga tcagcatgag480

aaggttcagg gcggatatgg aaagtaccaa ggcacatgga ttcctctgga agcaggtgag540

gctttggccc accgaaacaa catcttcgac aggttacggc ccatcttcga gttttctccg600

gggcctgata gcccgccccc ggctccacga cacacgagca aacccaagca accaaagaag660

ccagccgtgc ctagattcaa caacaaagcc cgcgcagtag ccaagcaggc accaccacca720

ccactgcact accagccctc tctacaccct caagatgcat acgagaacgg agagatgttg780

gttgacgagg acgacacacc agataacctg actgttgctt cagcttctta catggctgaa840

gacgaccgcc cggacctgtc gcacttctca accggacaca ggaaacgcaa gcgcgaagag900

cccaccgaga gtatgatcga gcagcagcat agaatctacg gtgacgagtt attagactat960

ttcctgctgt ctaggaacca gccgacgcca gccatgcgtc ctgagcctcc cgtcaacttc1020

aggccaaact tccccataga cgcggaccag catacagccc tccattgggc cgcctcgatg1080

ggcgatgtcg acattatcaa acagcttttt cagttcaatg ctcagccaga ttcgaggaac1140

gtgaggggtg agacgccgct catgagggcg gtcacgttca ccaactgcta cgataagcaa1200

accttccccg tcgtgctcaa ggagctcttc cacacaatca acatccgcga cctgtcaggt1260

tgtaccgcca ttcatcatgc ggccattctc aagggcggtc gcgtccacag tccaacgtgt1320

tcacggtatt atctggacaa catcctcaat aggttgcagg agacccaaca cgaccccaac1380

tttgttcagc agctcctcga cgcccaggac aatgatggca acacagctgt tcatcttgcg1440

gcgcagcgcg gttcgagcaa atgcattcgt gccctcctgg gccgtggtgc cagcactgac1500

attacaaaca acgagggcat gattgctgca gatttgatca aggagctcaa tgccagcaag1560

aagctgcgct ctgtgccaca aagatcatcc tctccatttg cacctgaatc ggctagaaga1620

caggtttcct ttcgcgatgc gttggctggt gatgtcggcg cgatgaccat tggcgcggcc1680

aacagccaca gcaacaaaat gacagccagc ctcaagtcag aggccgccct caccgtgcag1740

aaccgaatta cacctttggt ctttgagaag ttccacgatc ttgccaggtc atacgaagac1800

gagttcacga tcaaagacga agccgagcgc gaggcgacac gaatattgtc gaacgcacaa1860

gctgagcaca cctctttaac aaacaagctt gcggagctgg ggtcgcagct gttgccgcca1920

gagcaagctt ttggtattga cggtgaactc gagacagcca ccggaaaggt caaggcagtt1980

gtttgcacac tgaaccgtct tcatgtcgaa ggcctagttg agcgggagct ggacgcaagc2040

atgaacggtg attccaacgg gaacaccaac agcatcgagg agaggctcgc tttggccaac2100

gagcttcgct atcttttagg cgagcagcaa atggcggaga cggagtacat tgaggcactc2160

agcatggtcg gcacgggcga gaagatcgac caataccggc gactccttag gaactgttta2220

ggccccttgg cggagaacct cgacgacaac ctggaggagc ttgttgctat catggaagaa2280

gaggtgtcgg atggcatggt aaatagtacc gccgacggcc catcatcagt aacaaacggc2340

ctggtgggtg gcgagacgat ggaccttgcg ccaccaacat cggctctgta g2391

<210>3

<211>796

<212>PRT

＜213〉Magnaporthe grisea (Magnaporthe grisea)

<400>3

Met Val Gly Lys Ser Arg Val Pro Gln Ala Ile Asp Arg Thr Ser Leu

5 10 15

Lys Ile Arg Asp Pro Ile Pro Pro Lye Gln Gln Thr Gln Ile Thr Cys

20 25 30

Ile Ala Phe Pro Gln Ser Phe Asp His His His His Trp Arg Pro Pro

35 40 45

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

50 55 60

Pro Ala Gly Tyr Pro Asp Pro Phe Arg Leu Arg Pro Pro Pro Gly Gln

65 70 75 80

Thr Met Val Lys Ala Ala Ala Ala Ala Ala Ser Ala Pro Thr Gly Pro

85 90 95

Gly Ile Tyr Ser Ala Thr Tyr Ser Gly Ile Pro Val Tyr Glu Tyr Gln

100 105 110

Phe Gly Val Asp Leu Lys Glu His Val Met Arg Arg Arg Val Asp Asp

115 120 125

Trp Ile Asn Ala Thr His Ile Leu Lys Ala Ala Gly Phe Asp Lys Pro

130 135 140

Ala Arg Thr Arg Ile Leu Glu Arg Glu Val Gln Lys Asp Gln His Glu

145 150 155 160

Lys Val Gln Gly Gly Tyr Gly Lys Tyr Gln Gly Thr Trp Ile Pro Leu

165 170 175

Glu Ala Gly Glu Ala Leu Ala His Arg Asn Asn Ile Phe Asp Arg Leu

180 185 190

Arg Pro Ile Phe Glu Phe Ser Pro Gly Pro Asp Ser Pro Pro Pro Ala

195 200 205

Pro Arg His Thr Ser Lys Pro Lys Gln Pro Lys Lys Pro Ala Val Pro

210 215 220

Arg Phe Asn Asn Lys Ala Arg Ala Val Ala Lys Gln Ala Pro Pro Pro

225 230 235 240

Pro Leu His Tyr Gln Pro Ser Leu His Pro Gln Asp Ala Tyr Glu Asn

245 250 255

Gly Glu Met Leu Val Asp Glu Asp Asp Thr Pro Asp Asn Leu Thr Val

260 265 270

Ala Ser Ala Ser Tyr Met Ala Glu Asp Asp Arg Pro Asp Leu Ser His

275 280 285

Phe Ser Thr Gly His Arg Lys Arg Lys Arg Glu Glu Pro Thr Glu Ser

290 295 300

Met Ile Glu Gln Gln His Arg lle Tyr Gly Asp Glu Leu Leu Asp Tyr

305 310 315 320

Phe Leu Leu Ser Arg Asn Gln Pro Thr Pro Ala Met Arg Pro Glu Pro

325 330 335

Pro Val Asn Phe Arg Pro Asn Phe Pro Ile Asp Ala Asp Gln His Thr

340 345 350

Ala Leu His Trp Ala Ala Ser Met Gly Asp Val Asp Ile Ile Lys Gln

355 360 365

Leu Phe Gln Phe Asn Ala Gln Pro Asp Ser Arg Asn Val Arg Gly Glu

370 375 380

Thr Pro Leu Met Arg Ala Val Thr Phe Thr Asn Cys Tyr Asp Lys Gln

385 390 395 400

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

405 410 415

Asp Leu Ser Gly Cys Thr Ala Ile His His Ala Ala Ile Leu Lys Gly

420 425 430

Gly Arg Val His Ser Pro Thr Cys Ser Arg Tyr Tyr Leu Asp Asn Ile

435 440 445

Leu Asn Arg Leu Gln Glu Thr Gln His Asp Pro Asn Phe Val Gln Gln

450 455 460

Leu Leu Asp Ala Gln Asp Asn Asp Gly Asn Thr Ala Val His Leu Ala

465 470 475 480

Ala Gln Arg Gly Ser Ser Lys Cys Ile Arg Ala Leu Leu Gly Arg Gly

485 490 495

Ala Ser Thr Asp Ile Thr Asn Asn Glu Gly Met Ile Ala Ala Asp Leu

500 505 510

Ile Lys Glu Leu Asn Ala Ser Lys Lys Leu Arg Ser Val Pro Gln Arg

515 520 525

Ser Ser Ser Pro Phe Ala Pro Glu Ser Ala Arg Arg Gln Val Ser Phe

530 535 540

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

545 550 555 560

Asn Ser His Ser Asn Lys Met Thr Ala Ser Leu Lys Ser Glu Ala Ala

565 470 475

Leu Thr Val Gln Asn Arg Ile Thr Pro Leu Val Phe Glu Lys Phe His

580 585 590

Asp Leu Ala Arg Ser Tyr Glu Asp Glu Phe Thr Ile Lys Asp Glu Ala

595 600 605

Glu Arg Glu Ala Thr Arg Ile Leu Ser Asn Ala Glu Ala Glu His Thr

610 615 620

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

625 630 635 640

Glu Gln Ala Phe Gly Ile Asp Gly Glu Leu Glu Thr Ala Thr Gly Lys

645 650 655

Val Lys Ala Val Val Cys Thr Leu Asn Arg Leu His Val Glu Gly Leu

660 665 670

Val Glu Arg Glu Leu Asp Ala Ser Met Asn Gly Asp Ser Asn Gly Asn

675 680 685

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

690 695 700

Leu Leu Gly Glu Gln Gln Met Ala Glu Thr Glu Tyr Ile Glu Ala Leu

705 710 715 720

Ser Met Val Gly Thr Gly Glu Lys Ile Asp Gln Tyr Arg Arg Leu Leu

725 730 735

Arg Asn Cys Leu Gly Pro Leu Ala Glu Asn Leu Asp Asp Asn Leu Glu

740 745 750

Glu Leu Val Ala Ile Met Glu Glu Glu Val Ser Asp Gly Met Val Asn

755 760 765

Ser Thr Ala Asp Gly Pro Ser Ser Val Thr Asn Gly Leu Val Gly Gly

770 775 780

Glu Thr Met Asp Leu Ala Pro Pro Thr Ser Ala Leu

785 790 795

<210>4

<211>1318

<212>DNA

＜213〉Magnaporthe grisea (Magnaporthe grisea)

<400>4

GCGCCAACCC AGAGTGATAA GCAAGCTAAG GCACCGCATG GAAATCCATG ATCGCGCGAA 60

ATTGAGACGT ATTTTCACGT GACTGTACGT AGGTACCTAC AAGGAGGGAC CTGACCCATC120

GCATCCCAAA CGCCAACCCC GCAAAAGACC CGACGCCGGT CGTCTTCTCC CACCCGTTGA180

CTTGTGAGAT GTTTCTGAAT ACCTTGGTAG GTATTCAAGC CGCAATTGGC TCAATCAATT240

GGCTGTGGAC TCGGTCACCA TCGGGCATCT ACCCGAGATT TTTTTGTTTC GTCGGCCCCA300

ACGCCGTCCT CACAAAATGA CGTCTACGAA CACGAGCCTA AGTCGAGTAC CTTAAAAGGT360

AACCCCGCGT CCGAGCCCTT TTTGACTTTC GGGGACCTTC AACCCCACAG TGACGCCGGC420

TGCTTAGGAG ATAGATATTT GGAATTGTAA GTGTCACAAG TCCTGTATAT TTATCCCATG480

GACTCACCGA GATAAACGGC GCGAATCTGA GTCTGACCTT TTGTCACAAC GGCGAACAGC540

TAGCTACCGT ACCTACCTAA ACAATAACAT CGTAGCCTCT TGCGAACACC TCGACCGACA600

CGGTTGGCCA CCATGCCTCT CATCGCACAG AACCCCACCA ACAGGGTCAT CCTTGGCCTG660

ATGACCTTTG GCCCGGACGA GGCTACAGGT GCCCGCATTA CATCCGTGGA CGAGTTCGGC720

AAGGTCCTCG ACATCCTCCA GAAGCGCGGA TACAACGAGG TGGACACTGC CCGCATGTAC780

ATTGGTGGCA AGCAGGAGGC ATTCACTCGT GAGGTTGGCT GGAAGCAACG CGGCCTTACT840

TTGGCGACCA AGGTTCAATA CCCGTCAGAA TATGGCATGA ATGCTCCTGA TAAAGTCAAG900

GAGTCGGTGG ACCTCAGTTT GAAAGAGCTG GGCACTGACT GCGTCGATGT AAGCTTTATC960

TGCCTTTTGT TCCTTATACA CTCCAGTTCC ATACTATGAT GCAACAAAAC AGTCATGTAT1020

GCTATGATCT TAGTGGCTGA CAGAACTTAC ATCTTCCAGC TCCTCTATCT TCATGCCGCC1080

GACCGAGGTA CACCCTTTGC CGAGACACTC CGCGCCATCA ACGACCTACA CAAGGCCGGC1140

AAATTCGTCA ACTTTGGCAT TTCCAACTTT GCTGCATACG AGGTTGCCGA GATCGTCATG1200

ACATGCGTGC AAAACAACTG GGTGCGACCA ACTGTTTACC AGGCCATGTA CAATGTCATC1260

ACACGGTCTA TCGAGGCAGA ACTGATTCCT GCATGTCGCC GATACGGACT TGACCTGG 1318

<210>5

<211>684

<212>PRT

＜213〉Botrytis cinerea bacterium (Botrytis cinerea)

<400>5

Met Val Asn Thr Gly Ala Lys Ser Gly Pro Gly Ile Tyr Ser Ala Thr

1 5 10 15

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

20 25 30

Glu His Val Met Arg Arg Arg Gln Asp Asp Trp Ile Asn Ala Thr His

35 40 45

Ile Leu Lys Ala Ala Gly Phe Asp Lys Pro Ala Arg Thr Arg Ile Leu

50 55 60

Glu Arg Glu Val Gln Lys Glu Glu His Glu Lys Ile Gln Gly Gly Tyr

65 70 75 80

Gly Lys Tyr Gln Gly Thr Trp Val Pro Leu Asp Lys Gly Gln Ser Leu

85 90 95

Ala Gln Arg Asn Asn Ile Tyr Glu Lys Leu Arg Ala Ile Phe Glu Tyr

100 105 110

Thr Pro Gly Glu Phe Ser Pro Pro Pro Ala Pro Lys His Ala Thr Asn

115 120 125

Lys Pro Lys Val Lys Lys Pro Ala Val Pro Lys Trp Gly Ala Lys Pro

130 135 140

Val Pro Ile Gln Gln Ala Ala Val Thr Arg Gln Val Glu Glu Asn Tyr

145 150 155 160

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

165 170 175

Val Thr Val Ala Ser Ala Ser Tyr Met Ala Glu Asp Asp Arg Phe Asp

180 185 190

Ile Ser Gln Pro Pro Thr Gly His Arg Lys Arg Lys Arg Gly Glu Arg

195 200 205

Asp Asp Phe His Glu Asp Asp Pro Val Asp Val Arg Ala Gln Ala His

210 215 220

Leu Met Trp Ala Asp Glu Leu Leu Asp Tyr Phe Met Ala Pro Pro Thr

225 230 235 240

Asp Ala Asn Ser Ile Asp Arg Arg Pro Glu Pro Pro Ile Asn Phe Glu

245 250 255

Pro Asn Phe Ile Ile Asp Thr Asp Gly His Thr Gly Leu His Trp Ala

260 265 270

Ala Ser Ile Gly Asp Val Asp Ile Leu Lys Gln Leu Lys Arg Phe Gly

275 280 285

Ala Asn Thr Thr Cys Arg Asn Ser Arg Gln Glu Thr Pro Leu Ile Arg

290 295 300

Ser Val Leu Phe Thr Asn Ser Tyr Asp Lys Gln Asn Phe Pro Leu Ile

305 310 315 320

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

325 330 335

Thr Val Leu His His Ala Ala Ala Thr Thr Asn Met Lys Gln Lys Phe

340 345 350

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

355 360 365

Ile Tyr His Pro Asp Glu Val Gln Arg Leu Leu Asp Ala Arg Asp Ile

370 375 380

Asn Gly Asn Thr Ala Ile His Ile Ala Ala Lys Asn Lys Ala Arg Lys

385 390 395 400

Cys Val Arg Ala Leu Met Gly Arg Gly Ala Ser Thr Asp Ile Leu Asn

405 410 415

Asp Met Gly Glu Thr Ala Glu Glu Ile Ile Gln Asp Leu Asn Ala Ser

420 425 430

Arg Arg Ser Glu Arg His Gln Gln Ala Ala Ser Ser Ser Pro Phe Ala

435 440 445

Pro Asp Ser Ala Arg His Ile Ser His Tyr Asp Asp Leu Glu Gln Glu

450 455 460

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

465 470 475 480

Asn Asn Ala Glu Pro Ile Leu Leu Glu Arg Leu His Ala Leu Ala Cys

485 490 495

Ser Phe Asp Glu Glu Leu Ala Glu Lys Glu Asn Ala Glu Ala Asp Ser

500 505 510

Lys Arg Ile Leu Glu Gly Val Lys Met Glu Leu Ala Gly Val Arg Glu

515 520 525

Gln Ala Arg Gln Leu Ala Glu Glu Glu Glu Gly Glu Glu Asn Ser Ile

530 535 540

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

545 550 555 560

Val Glu Arg Gln Gln Gln Leu Leu Leu Leu Ser Arg Thr Gln His Glu

565 570 575

Gly Ser Lys Ile Asn Gly His Gly Gly Gly Gln Val Asn Gly Ser Ala

580 585 590

Asp Leu Glu Glu Ser Glu Asn Glu Lys Ile Met Val Ala Gln His Leu

595 600 605

Gln Glu Glu Ile Glu Arg Arg Lys Ala Leu Val Gly Glu Tyr Ile Gly

610 615 620

Ala Leu Gly Val Ala Gly Gly Gly Asp Glu Gly Glu Leu Tyr Lys Arg

625 630 635 640

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

645 650 655

Asp Glu Gly Leu Asp Ala Leu Ile Ala Asn Leu Glu Glu Glu Arg Gly

660 665 670

Gly Val Gly Leu Glu Arg Glu Met Gly Asp Glu Leu

675 680

<210>6

<211>676

<212>PRT

＜213〉corn stalk rot disease bacterium (beading gibberella, Gibberella moniliformis)

<400>6

Met Val Lys Ala Asn Ala Asn Ala Ala Ala Ala Ala Ala Ala Ser Gly

1 5 10 15

Pro Gly Val Tyr Ser Ala Val Tyr Ser Gly Ile Pro Val Tyr Glu Phe

20 25 30

Gln Phe Gly Gly Asp Leu Lys Glu His Val Met Arg Arg Arg His Asp

35 40 45

Asn Trp Ile Asn Ala Thr His Ile Leu Lys Ala Ala Gly Phe Asp Lys

50 55 60

Pro Ala Arg Thr Arg Ile Leu Glu Arg Asp Val Gln Lys Asp Val His

65 70 75 80

Glu Lys Ile Gln Gly Gly Tyr Gly Lys Tyr Gln Gly Thr Trp Ile Pro

85 90 95

Leu Glu Ser Gly Gln Ala Leu Ala Glu Arg His Ser Val Phe Asp Arg

100 105 110

Leu Arg Pro Ile Phe Glu Tyr Val Ala Gly Ala Glu Ser Pro Pro Pro

115 120 125

Ala Pro Arg His Ala Ser Lys Pro Lys Gly Pro Lys Ser Lys Pro Pro

130 135 140

Leu Pro Lys Trp Asn Asn Pro Pro Pro Ala Pro Ala Pro Ala Pro Val

145 150 155 160

Ile His Asp Asp Pro Asp Thr Val Met Gly Asp Glu Asp Thr Pro Asp

165 170 175

Asn Leu Thr Val Ala Ser Ala Ser Tyr Met Ala Glu Asp Glu Arg Phe

180 185 190

Glu Met Pro Gln Val Pro Gly Thr Gly Arg Lys Arg Arg Arg Asp Asp

195 200 205

Ser Asn Leu Gln Asp Leu Thr Glu Gln Gln His Ala Leu Tyr Gly Asp

210 215 220

Glu Leu Leu Asp Tyr Phe Leu Leu Ser Lys Thr Asp Gln Pro Ala Val

225 230 235 240

Lys Pro Asp Pro Pro Ala Asn Phe Gln Pro Asn Trp Pro Ile Asp Ala

245 250 255

Glu Asp His Thr Ala Leu His Trp Ala Ser Ala Met Gly Asp Val Asp

260 265 270

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

275 280 285

Ile Arg Gly Glu Thr Pro Phe Met His Ser Val Asn Phe Thr Asn Cys

290 295 300

Tyr Glu Lys Gln Ser Phe Pro Val Val Met Lys Glu Leu Phe Glu Thr

305 310 315 320

Phe Asp Ala Arg Asp Asn Met Gly Cys Thr Val Ile His His Ala Ala

325 330 335

Val Met Lys Asn Gly Arg Val Phe Asn Ser Ser Cys Ser Arg Tyr Tyr

340 345 350

Leu Asp Asn Ile Leu Asn Lys Leu Gln Glu Gly Leu Glu Pro Ser Ala

355 360 365

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

370 375 380

His Leu Ala Ala Gln Arg Asn Ala Arg Lys Cys Ile Arg Ala Leu Leu

385 390 395 400

Gly Arg Asn Ala Ser Ser Asp Leu Thr Asn Asn Asp Gly Ile Arg Ala

405 410 415

Glu Asp Leu Ile Met Asp Leu Asn Ala Thr Lys Lys Glu Arg Gly Pro

420 425 430

Gln Arg Ser Ser Ser Pro Phe Ala Pro Glu Ser Gln Arg His Ala Ser

435 440 445

Phe Lys Asp Ala Leu Leu Glu Lys Ala Asn Arg Gln Pro Pro Val Ala

450 455 460

Phe Gln Ser Ala Ala Ala Asn Thr Val Gln Ser Arg Ile Thr Pro Leu

465 470 475 480

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

485 490 495

Arg Glu Lys Asp Val Ala Glu Ala Glu Ala Lys Arg Ile Leu Ala Asn

500 505 510

Thr Gln Ala Asp Leu Asn Val Leu Arg Gln Asn Ile Thr Asp Ala Glu

515 520 525

Gly Gln Leu Glu Ser Glu Glu Ala Ala Ser Lys Ash Asn Gln Glu Ala

530 535 540

Asn Leu Ala Lys His Gln Val Leu Ser Leu Ile Thr His Gln Asn Arg

545 550 555 560

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

565 570 575

Asp Gly Gly Val Gln Glu Glu Ser Tyr Glu Glu Arg Leu Ser Leu Ala

580 585 590

Arg Gln Leu Ser Thr Leu Leu Ala Glu Gln Arg Thr Ala Glu Thr Asp

595 600 605

Tyr Val Glu Ala Leu Ser Met Val Gly Thr Gly Glu Lys Ile Glu Lys

610 615 620

Tyr Arg Lys Leu Leu Lys Arg Cys Leu Asp Pro Lys Glu Gly Asp Ser

625 630 635 640

Leu Asp Thr Asn Leu Asp Ser Leu Ile Glu Met Met Glu Glu Glu Arg

645 650 655

Asp Glu Asp Gly Met Val Gly Val Met Glu Pro Gln Pro Met Glu Leu

660 665 670

Ser Gly Gly Ile

675

<210>7

<211>675

<212>PRT

＜213〉gibberella saubinetii (Gibberella zeae)

<400>7

Met Val Lys Ala Asn Ala Asn Ala Ala Pro Pro Ala Thr Ala Pro Gly

1 5 10 15

Val Tyr Ser Ala Val Tyr Ser Gly Ile Pro Val Tyr Glu Phe Gln Phe

20 25 30

Gly Ala Glu Leu Lys Glu His Val Met Arg Arg Arg Ser Asp Asp Trp

35 40 45

Ile Asn Ala Thr His Ile Leu Lys Ala Ala Gly Phe Asp Lys Pro Ala

50 55 60

Arg Thr Arg Ile Leu Glu Arg Asp Val Gln Lys Asp Val His Glu Lys

65 70 75 80

Ile Gln Gly Gly Tyr Gly Lys Tyr Gln Gly Thr Trp Ile Pro Leu Glu

85 90 95

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

100 105 110

Pro Ile Phe Glu Tyr Val Gln Gly Thr Glu Thr Pro Pro Pro Ala Pro

115 120 125

Lys His Ala Ser Lys Pro Lys Gly Pro Lys Ser Arg Pro Pro Leu Pro

130 135 140

Lys Trp Asn Asn Pro Pro Pro Pro Pro Pro Ala Pro Ala Pro Val Ile

145 150 155 160

Gln Asp Asp Gly Asp Thr Leu Met Gly Asp Glu Asp Thr Pro Asp Asn

165 170 175

Leu Thr Val Ala Ser Ala Ser Tyr Met Ala Glu Asp Glu Arg Phe Glu

180 185 190

Met Pro Gln Ala Pro Gly Thr Gly Arg Lys Arg Arg Arg Asp Asp Asn

195 200 205

Asn Leu Gln Asp Leu Thr Glu Gln Gln His Ala Leu Tyr Gly Asp Glu

210 215 220

Leu Leu Asp Tyr Phe Leu Leu Ser Lys Thr Asp Gln Pro Ala Val Lys

225 230 235 240

Pro Asp Pro Pro Ala Asn Phe Gln Pro Asn Trp Pro Ile Asp Ala Glu

245 250 255

Asp His Thr Ala Leu His Trp Ala Ser Ala Met Gly Asp Leu Asp Val

260 265 270

Val Lys Gln Leu Lys Arg Phe Asn Ala Ser Ser Thr Val Lys Asn Ile

275 280 285

Arg Gly Glu Thr Pro Phe Met His Ser Val Asn Phe Thr Asn Cys Tyr

290 295 300

Glu Lys Gln Ser Phe Pro Met Val Met Lys Glu Leu Phe Glu Thr Phe

305 310 315 320

Asp Ala Arg Asp Asn Met Gly Cys Thr Val Ile His His Ala Ala Val

325 330 335

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

340 345 350

Asp Asn Ile Leu Asn Lys Leu Gln Glu Thr Leu Glu Pro Ser Ala Phe

355 360 365

Gln Gln Leu Leu Asp Ile Gln Asp Asn Glu Gly Asn Thr Ala Leu His

370 375 380

Leu Ala Ala Gln Arg Asn Ala Arg Lys Cys Ile Arg Ala Leu Leu Gly

385 390 395 400

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

405 410 415

Asp Leu Ile Met Asp Leu Asn Ala Thr Lys Lys Asp Arg Gly Pro Gln

420 425 430

Arg Ser Ser Ser Pro Phe Ala Pro Glu Ser Gln Arg His Ala Ser Phe

435 440 445

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

450 455 460

Gln Ser Ala Ala Ala Asn Thr Val Gln Ser Arg Ile Ser Pro Leu Ile

465 470 475 480

Met Glu Lys Phe Gln Asp Leu Ala Lys Ser Tyr Glu Asp Glu Phe Arg

485 490 495

Glu Lys Asp Ile Ala Glu Ser Glu Ala Lys Arg Leu Leu Ser Asn Thr

500 505 510

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

515 520 525

Gln Leu Glu Pro Glu Glu Ala Ala Ser Lys Gln Ser Thr Glu Ala Asn

530 535 540

Leu Ala Lys His Gln Val Leu Ser Leu Ile Thr His Gln Ser Arg Leu

545 550 555 560

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

565 570 575

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

580 585 590

Glu Leu Ser Ser Leu Leu Ala Glu Gln Arg Lys Ala Glu Ala Glu Tyr

595 600 605

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

610 615 620

Lys Lys Leu Leu Asn Arg Cys Leu Asp Thr Lys Glu Ala Glu Ser Leu

625 630 635 640

Asp Thr Asn Leu Asp Ser Leu Ile Glu Met Met Glu Glu Glu Arg Asp

645 650 655

Glu Asn Gly Met Val Gly Ala Met Asp Pro Glu Pro Met Glu Leu Ser

660 665 670

Val Gly Ile

675

<210>8

<211>693

<212>PRT

＜213〉wheat glume blight bacterium (Phaeosphaeria nodorum)

<400>8

Met Pro Pro Ala Pro Asp Gly Lys Ile Tyr Ser Ala Thr Tyr Ser Asn

1 5 10 15

Val Pro Val Tyr Glu Cys Asn Val Asn Gly Asn His Val Met Arg Arg

20 25 30

Arg Ala Asp Asp Trp Ile Asn Ala Thr His Ile Leu Lys Val Ala Asp

35 40 45

Tyr Asp Lys Pro Ala Arg Thr Arg Ile Leu Glu Arg Glu Val Gln Lys

50 55 60

Gly Val His Glu Lys Val Gln Gly Gly Tyr Gly Lys Tyr Gln Gly Thr

65 70 75 80

Trp Ile Pro Leu Glu Glu Gly Arg His Leu Ala Glu Arg Asn Gly Val

85 90 95

Leu His Lys Met Arg Pro Ile Phe Asp Tyr Val Pro Gly Asp Arg Ser

100 105 110

Pro Pro Pro Ala Pro Lys His Ala Thr Ala Ala Ser Asn Arg Met Lys

115 120 125

Pro Pro Arg Gln Ser Ala Ala Ala Ala Ala Ala Ala Arg Asn Ser Lys

130 135 140

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

145 150 155 160

Ser Gln Gln Ser Gln Val Ser Glu Asp Pro Tyr Glu Ala Ser Gln Thr

165 170 175

Arg Ser Gln Ile Phe Arg Glu Glu Thr Pro Asp Asn Glu Thr Val Val

180 185 190

Ser Glu Ser Met Leu Gly Asp His Asp Met Leu Asp Ala Ser Gln Tyr

195 200 205

Ser Thr Gly Ser Arg Lys Arg Lys Arg Gly Val Asp Gln Met Ser Met

210 215 220

Leu Asp Gln Gln His Gln Ile Trp Ala Asp Ala Leu Leu Asp Tyr Phe

225 230 235 240

Met Leu Leu Asp His Glu Ala Ala Cys Ala Trp Pro Glu Pro Pro Pro

245 250 255

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

260 265 270

His Trp Ala Ala Ala Met Gly Asp Val Gly Val Val Lys Glu Leu Ile

275 280 285

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

290 295 300

Leu Met Arg Ala Val Met Phe Thr Asn Asn Phe Asp Lys Glu Thr Met

305 310 315 320

Pro Ser Met Val Lys Ile Phe Gln Gln Thr Val His Arg Thr Asp Trp

325 330 335

Phe Gly Ser Thr Val Phe His His Ile Ala Ala Thr Thr Ser Ser Ser

340 345 350

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

355 360 365

Leu Ser Glu Thr Trp Ile Pro Glu Glu Val Thr Arg Leu Leu Asn Ala

370 375 380

Gln Asp Gln Asn Gly Asp Thr Ala Ile Met Ile Ala Ala Arg Asn Gly

385 390 395 400

Ala Arg Lys Cys Val Arg Ser Leu Leu Gly Arg Ser Val Ser Val Asp

405 410 415

Ile Pro Asn Lys Lys Gly Glu Thr Ala Asp Asp Leu Ile Arg Glu Leu

420 425 430

Asn Gln Arg Arg Arg Met His Gly Arg Thr Arg Gln Ala Ser Ser Ser

435 440 445

Pro Phe Ala Pro Pro Leu Glu His Arg Leu Asn Gly His Ile Ala Ala

450 455 460

Leu Asp Gly Gly Pro Leu Leu Pro Val Pro Phe Pro Ser Met Val Ala

465 470 475 480

Arg Glu Pro Val Gln Tyr Arg Ser Gln Thr Ala Ser His Leu Met Thr

485 490 495

Lys Val Ala Pro Thr Leu Leu Glu Lys Cys Glu Glu Leu Ala Ala Ala

500 505 510

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

515 520 525

Arg Val Val Lys Arg Arg Gln Ala Glu Leu Glu Ala Val Arg Lys Gln

530 535 540

Val Ala Glu Leu Gln Asn Met Gly Asn Gly Leu His Ile Asp Leu Asn

545 550 555 560

Asp Asp Glu Ala Asp Gly Gln Gln Glu Glu Glu Leu Arg Met Leu Val

565 570 575

Glu Glu Ala Glu Ser Leu Leu Glu Ile Glu Gln Lys Ala Glu Leu Arg

580 585 590

Arg Leu Cys His Asn Thr Pro Gln Pro Asn Lys Thr Asn Ser Pro Val

595 600 605

Asp Val Ser Glu Lys Leu Arg Leu Ala Leu Leu Leu His Arg Ala Gln

610 615 620

Leu Glu Arg Arg Glu Leu Val Arg Glu Val Val Gly Asn Leu Ser Val

625 630 635 640

Ala Gly Met Ser Glu Lys Gln Gly Thr Tyr Lys Lys Leu Ile Ala Lys

645 650 655

Ala Leu Gly Glu Arg Glu Glu Asp Val Glu Leu Met Leu Pro Glu Ile

660 665 670

Leu Gln Glu Leu Glu Glu Ala Glu Thr Gln Glu Arg Ala Glu Gly Leu

675 680 685

Asp Gly Ser Pro Leu

690

<210>9

<211>610

<212>PRT

＜213〉Sclerotinia sclerotiorum (Sclerotinia sclerotiorum)

<400>9

Met Arg Arg Arg His Asp Asp Trp Ile Asn Ala Thr His Ile Leu Lys

1 5 10 15

Ala Ala Gly Phe Asp Lys Pro Ala Arg Thr Arg Ile Leu Glu Arg Glu

20 25 30

Val Gln Lys Glu Glu His Glu Lys Ile Gln Gly Gly Tyr Gly Lys Tyr

35 40 45

Gln Gly Thr Trp Val Pro Leu Glu Lys Gly Gln Ala Leu Ala Gln Arg

50 55 60

Asn Asn Ile Tyr Glu Lys Leu Arg Ala Ile Phe Glu Tyr Thr Pro Gly

65 70 75 80

Glu Leu Ser Pro Pro Pro Ala Pro Lys His Ala Thr Asn Lys Pro Lys

85 90 95

Val Asp Ser Thr Asn Arg Ile Glu Pro Val Pro Ile Gln Gln Ala Val

100 105 110

Ile Ala Arg Gln Val Glu Glu Asn Tyr Asp Asn Ile Ser Thr Gln Leu

115 120 125

Asn Asp Asp Glu Ser Val Ala Asp Asp Val Thr Val Ala Ser Ala Ser

130 135 140

Tyr Met Ala Glu Asp Asp Arg Phe Asp Met Pro Gln Pro Pro Thr Gly

145 150 155 160

His Arg Lys Arg Lys Arg Asp Arg Glu Arg Asp Glu Phe Gln Gln Asp

165 170 175

Asp Pro Val Asp Leu Arg Ala Gln Ala His Leu Met Trp Ala Asp Glu

180 185 190

Leu Leu Asp Tyr Phe Met Ala Pro Pro Thr Asp Ala Asn Leu Met Asp

195 200 205

Arg Arg Pro Glu Pro Pro Ile Asn Phe Glu Pro Asp Phe Ile Ile Asp

210 215 220

Thr Asp Gly His Thr Gly Leu His Trp Ala Ala Ser Ile Gly Asp Leu

225 230 235 240

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

245 250 255

Asn Asn Arg Gln Glu Thr Pro Leu Met Arg Ser Val Leu Phe Thr Asn

260 265 270

Ser Tyr Asp Lys Gln Asn Phe Pro Leu Ile Val Lys Glu Leu Ile Asn

275 280 285

Thr Ala His Glu Ile Asp Ser Cys Gly Ala Thr Val Leu His His Ala

290 295 300

Ala Ala Thr Thr Asn Met Lys Gln Lys Phe Arg Cys Ala Gln Tyr Tyr

305 310 315 320

Leu Asp Val Leu Leu Glu Lys Leu Ile Glu Ile Phe His Pro Asp Glu

325 330 335

Val Gln Arg Leu Leu Asp Ala Arg Asp Ile Asn Gly Asn Thr Ala Ile

340 345 350

His Ile Ala Ala Lys Asn Lys Ala Arg Lys Cys Val Arg Ala Leu Met

355 360 365

Gly Arg Gly Ala Ser Thr Asp Ile Leu Asn Asp Met Gly Glu Thr Ala

370 375 380

Glu Glu Ile Ile Gln Asp Leu Asn Ala Ser Arg Arg Ser Glu Arg Tyr

385 390 395 400

Gln Gln Ala Ala Ser Ser Ser Pro Phe Ala Pro Asp Ser Ala Arg His

405 410 415

Ile His Ala Leu Ala Cys Ser Phe Asp Glu Glu Leu Ala Glu Lys Glu

420 425 430

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

435 440 445

Leu Ala Gly Val Arg Glu Gln Ala Arg His Leu Ala Gly Glu Glu Glu

450 455 460

Gly Glu Glu Asn Ala Ile Ala Ala Lys Ala His Leu Val Gly Leu Gln

465 470 475 480

Glu Arg Val Gln Ser Leu Val Glu Arg Gln Gln Gln Leu Leu Leu Leu

485 490 495

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

500 505 510

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

515 520 525

Leu Ala Ala Gln Gln Leu His Glu Glu Ile Gln Arg Arg Lys Ala Leu

530 535 540

Val Gly Glu Tyr Ile Gly Ala Leu Gly Val Ala Gly Gly Gly Asp Glu

545 550 555 560

Gly Glu Leu Tyr Lys Arg Val Ile Val Arg Asp Leu Gly Val Asp Glu

565 570 575

Gly Glu Gly Gly Leu Ser Asp Glu Gly Leu Asp Ala Leu Ile Ala Asn

580 585 590

Leu Glu Glu Glu Arg Gly Gly Val Gly Leu Glu Arg Glu Val Gly Asp

595 600 605

Glu Leu

610

Claims (12)

1, a kind of mycoprotein of controlling fungal colony growth and virulence contains ankyrin duplicate domain (ANK duplicate domain), and being up to 26% known functional protein with its consistence on aminoacid sequence is zymic MBP1.In yeast, MBP1 controls the cell cycle, is different from proteic function of the present invention.In Magnaporthe grisea, the albumen called after MgMBP1 that this is new.MgMBP1 and homologous protein thereof are the protein with one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: sequence shown in 3.

2) with SEQ ID № in the sequence table: the replacement of one or several amino-acid residue of amino acid residue sequence process of 3 and/or disappearance and/or interpolation and the protein that causes fungal colony poor growth, virulence to weaken.

3) with sequence table in SEQ ID №: 3 have the protein that consistence is higher than 40% pathogenic fungi Botrytis cinerea bacterium (Botrytiscinerea), corn stalk rot disease bacterium (Gibberella moniliformis), gibberella saubinetii (Gibberella zeae), wheat glume blight bacterium (Phaeosphaeria nodorum), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), SEQ ID № in its aminoacid sequence such as the sequence table: 5,6,7,8,9.

2, the proteic encoding gene of described control fungal colony growth of claim 1 and virulence.

3, gene according to claim 2 is characterized in that: described control fungal colony growth and the proteic genomic gene of virulence 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: 3 protein sequences or be higher than 40% proteinic polynucleotide with its consistence.

4, gene according to claim 2 is characterized in that: the growth of described control fungal colony is positioned at the № from sequence SEQ ID with the coding region of the proteic genomic gene of virulence: 15 ' hold the 1017th between 4193 bit bases.

5, gene according to claim 2 is characterized in that: described control fungal colony growth and the proteic cDNA of virulence can have one of following nucleotide sequence:

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

2) 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.

6, gene according to claim 5 is characterized in that: the encoding sequence of the growth of described control fungal colony and the proteic cDNA of virulence is for from SEQ ID №: the 1st at 5 ' end of 2 is to 2391 bit bases.

7, expression vector, clone and the host bacterium that utilizes described control fungal colony growth of claim 1 and the proteic encoding gene of virulence to make up.

8, the promotor of the MgMBP1 gene of control Magnaporthe grisea colony growth and virulence has the № from SEQ ID: the 1st nucleotide sequence to 1318 bit bases of 5 ' end of 4.

9, the proteic expression of MBP1 of described control fungal colony growth of claim 1 and virulence and modification are as the application of target in design and screening antifungal medicine.

10, the shearing of the described gene transcript of claim 2 is as the application of target in design and screening antifungal medicine

11, the promotor of the described MgMBP1 gene of claim 8 and conjugated protein as the application of target in design and screening antifungal medicine.

12, claim 9,10,11 described antifungal medicines are the medicament of anti-Magnaporthe grisea (Magnaporthe grisea), gibberella saubinetii (Gibberella zeae), corn stalk rot disease bacterium (Gibberella moniliformis), Sclerotinia sclerotiorum (Sclerotinia sclerotirum), Botrytis cinerea bacterium (Botrytis cinerea), wheat glume blight bacterium (Phaeosphaena nodorum).

Images