CN101050463A - Pathogenicity gene mgATG5 of fungus from rice blast germ, and application - Google Patents
Pathogenicity gene mgATG5 of fungus from rice blast germ, and application Download PDFInfo
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Abstract
本发明公开了一种源于稻瘟病菌的真菌致病性基因mgATG5，其核苷酸序列或其互补链的核苷酸序列为SEQ ID NO：1。基因mgATG5编码的cDNA序列具有SEQ ID NO：2所示的核苷酸序列。基因mgATG5编码的蛋白质具有SEQ ID NO：3所示的氨基酸序列。基因mgATG5的启动子具有SEQ ID NO：4所示的核苷酸序列。分别利用基因mgATG5的表达作为靶标、利用上述蛋白质的表达和修饰作为靶标、结合利用上述蛋白质以及启动子作为靶标，在设计和筛选抗真菌药物中的应用。
The invention discloses a fungal pathogenic gene mgATG5 derived from Magnaporthe grisea, its nucleotide sequence or the nucleotide sequence of its complementary chain is SEQ ID NO:1. The cDNA sequence encoded by the gene mgATG5 has the nucleotide sequence shown in SEQ ID NO:2. The protein encoded by the gene mgATG5 has the amino acid sequence shown in SEQ ID NO:3. The promoter of the gene mgATG5 has the nucleotide sequence shown in SEQ ID NO:4. Using the expression of the gene mgATG5 as the target, the expression and modification of the above protein as the target, and the combination of the above protein and the promoter as the target, the application in the design and screening of antifungal drugs.
Description
Technical field
The invention belongs to plant pathology, Pesticide Science and microbiological genetic engineering field, provide one derive from Pyricularia oryzae, nourish and grow, spore produces, appressorium forms with pathogenic in promotor and the nucleotide sequence of coding region and the aminoacid sequence of coded protein thereof of the new gene mgATG5 that plays an important role.Nucleotide sequence provided by the invention and aminoacid sequence can be used as in screening that target is applied to antifungal medicine and the design, and a certain section that also can utilize this nucleotide sequence is as probe or having among the clone of gene of certain homology with this sequence of utilizing that the antibody of this protein Preparation is applied to Pyricularia oryzae and other fungies.
Background technology
The rice blast that is caused by Pyricularia oryzae (Magnaporthe grisea) is the destructive disease on global a kind of paddy rice.The annual in the world rice yield that is caused by rice blast is lost between 10～30%.The almost annual popular outburst that Pyricularia oryzae is all arranged of China.Nearest 2005, Sichuan Province and Chongqing City were subjected to the most serious rice blast over 10 years, and rice blast takes place in 2,300,000 mu of rice fields that Sichuan Province has 20 cities, 127 counties, and the Chongqing City has more than 100 ten thousand mu of rice fields Pyricularia oryzae takes place; Current rice blast is popular rapidly, hazard rating is heavy, the morbidity rice varieties is many, it is serious that rice yield is influenced.Except paddy rice, Pyricularia oryzae also can infect other more than 50 kinds of grasses, also millet (Eleusine coracana), barley (Hordeum vulgare), wheat important farm crop such as (Triticum aestivum) is caused serious harm.Pyricularia oryzae also is the interactional model animals of a kind of research plant pathogenic fungi-host plant, have the total plant of many pathogenic fungies and cause a disease and infect circulation, comprise that spore generation, sprouting, appressorium form, infect bolt and form, invade pathogenic courses such as mycelial growth; Its pathogenic molecular mechanism is being studied by many developed countries, and expectation goes out the medicine target of novel pesticide screening test by this type of research and design, thereby develops, designs the novel anti fungi-medicine.
Include sexual stage and imperfect stage the life history of Pyricularia oryzae.Perfect stage produces ascus and thecaspore after by the strain mating of 2 different mating types.The mating type of Pyricularia oryzae is by mating gene M AT1-1 and MAT1-2 control.Imperfect stage is that the vegetative hyphae differentiation produces conidiophore and pyriform conidium.At nature, Pyricularia oryzae is mainly finished the life history by the imperfect stage, and conidium is the main intrusion source of plant.
After the Pyricularia oryzae asexual spore was fallen the rice leaf surface, infection processs began.After spore and water chance, the viscose glue in separate on the spore top discharges, and makes spore tightly stick to the rice leaf surface.The rice leaf surface has one deck wax cuticle, and this film has very big hydrophobicity, and viscose glue makes spore stick to this hydrophobic repellency surface.Adhere in back 2 hours, spore germination also produces germ tube.Germ tube stretches out and grows from an apical cell of spore usually.A kind of stickies is also secreted on the germ tube top, makes germ tube tightly stick to the plant leaf surface, prevents to be washed away by water droplet.In 4 hours, the germ tube growth stops, and the top crozier forms and expands and forms appressorium.The hardness of matrix is an important factor of germ tube differentiation, only forms appressorium at hard solid surface as magnaporthe grisea spore, and can not form appressorium at fluid surface or soft stromal surface.And the matrix hydrophobic surface also the effect of the wax by being similar to the rice leaf surface excite the formation of appressorium.Soon, the cell that produces germ tube carries out a mitotic division after spore germination.A nucleus is stayed in the spore, and another nucleus moves by germ tube, transfers to appressorium in the formation with the tenuigenin content of spore and germ tube.These tenuigenin contents comprise that the fat that is stored in the big vacuole of appressorium central authorities of growing drips.Then, between appressorium and germ tube, form barrier film.Along with the appressorium maturation, melanin deposition appears in the appressorium cell walls, finally forms the melanochrome layer.The melanochrome layer allows water molecules freely to pass through, but the material that is dissolved in water can not freely pass through, thereby allows appressorium set up and keep turgescence in the very big cell.After the melanochrome process of appressorium was finished, appressorium produced a narrow and small mycelia--infect bolt.Infecting bolt is accomplished penetrating owing to appressorium produces huge turgescence of rice leaf.Turgescence will stop appressorium to penetrate at artificial surface or rice leaf surface as long as reduce a little.Measuring result shows that the appressorium turgescence reaches 8.0Mpa, is equivalent to the pressure of 40 times of doughnuts.The effect of appressorium melanochrome layer has been to limit the perviousness of cell walls, helps the accumulation of permeate substance in the cell and the generation of turgescence.Glycerine is a kind of soluble substance, and the osmotic potential of generation is enough to make appressorium to produce huge turgescence.In the turgescence production process, glycerine surpasses 3M in cell inner accumulation concentration.
After the intrusion, infect bolt and be divided into and infect mycelia, growth and infect other cell in rice leaf rapidly.Invade after 72 hours, the pathogenic bacteria biomass has reached and has infected 10% of blade.After 5～7 days, differentiate a large amount of new conidiums on the conidiophore, and discharge from scab.The sporocyst malaria of these new formation takes contiguous plant to and begins new infection processs.In winter, Pyricularia oryzae is survived the winter on straw and paddy with mycelia and conidial form, finishes and infects circulation.
The pathogenic course of Pyricularia oryzae is the molecular process of a complexity.Cloned at present many genes relevant with rice blast bacterium pathogenicity, as: MPG1, CPKA, PMK1, MAGB, PLS1, SMO1, PDE1, MPS1, PTH11, CBP1, ICL1, BUF1, ALB1, ACR1, RSY1, HEX1.It is relevant that wherein most and appressorium form, as PMK1, MAGB, MAC1 etc.; The melanic formation of subparticipation and accumulation (ALB1, RSY1, BUF1 etc.) and glycerine synthetic relevant (ICL1 etc.); Minority relevant with penetrating of Pyricularia oryzae (MPS1, PLS1, PDE1 etc.).A kind of hydrophobin of Pyricularia oryzae germ tube excretory by the MPG1 genes encoding, participates in the mutual work of mycelia and paddy rice leaf table, for forming normal appressorium institute necessary (Talbot, 1999).The cAMP cyclase of the proteic alpha subunit of allos trimerization G of MAGB genes encoding and MAC1 coding also is to form appressorium necessary (Liu and Dean, 1997; Choiand Dean, 1997; Kulkarni and Dean, 2004).The PKA-c of catalytic subunit of the cAMP deopendent protein kinase A of CPKA genes encoding is that appressorium penetrates necessary (Mitchell ﹠amp; Hamer, 1995; Xu et al, 1997), and the PKA activity in the germ tube that is breaking up is for forming also very crucial (the Adachi ﹠amp of normal appressorium; Hamer1998).Equally, mitogen activated protein kinase (Mitogen-activated protein kinase, MAPK) gene PMK1 required (the Xu ﹠amp that is that appressorium forms; Hamer 1996).Melanochrome synthesizing series gene also proves appressorium and penetrates indispensable (Chumley and Valent, 1990; Motoyama etc., 1998).Found also that in various sudden change word banks some muton disappearances are pathogenic, as: a kind of proteic ABC1 gene (Urban etc. relevant with ATP carrier (ATP-bindingcassette transporters) encode, 1999), the PDE1 gene (Balhadere etc. of coding P-type ATP enzyme, 1999,2001) and the coding the proteic PLS1 gene of tetraspannin sample (Clergeot etc., 2001).However, the molecule mechanism of causing a disease of Pyricularia oryzae still is unclear.
The pathogenic gene of evaluation, clone plant pathogenic fungi, especially relevant with invasion procedure gene can be the target site that design, screening antifungal drug provide usefulness.Having proved at present the target spot of some medicines in comprising some fungies of Pyricularia oryzae, is a kind of very important Pyricularia oryzae sterilant as tricyclazole, and its effect is to suppress melanic synthetic, and target spot is three hydroxyl naphthalene reductase enzymes of Pyricularia oryzae; The action target spot of polypeptide sterilant sorphen A be mould demethylase (CYP51).Therefore, utilize the pathogenic gene that in the pathogenic course of Pyricularia oryzae, has critical function that Protocols in Molecular Biology is identified, the clone is new, can provide the drug target of usefulness for design, the new antifungal drug of screening.
Summary of the invention
The technical problem to be solved in the present invention provides a kind of new, mycelial growth to fungies such as Pyricularia oryzaes, generation of conidium, conidium and germinates, adheres to spore and form and infect that bolt forms and pathogenic gene mgATG5 that material impact is arranged and uses thereof.
In order to solve the problems of the technologies described above, the invention provides a kind of epiphyte pathogenic gene mgATG5 that comes from Pyricularia oryzae, the nucleotide sequence of this gene or its complementary nucleotide sequence are SEQ ID NO:1.
The present invention provides the cDNA sequence of said gene mgATG5 coding simultaneously, and this cDNA sequence has the nucleotide sequence shown in the SEQ IDNO:2.
The present invention provides said gene mgATG5 encoded protein matter simultaneously, and this protein has the aminoacid sequence shown in the SEQ ID NO:3.
The present invention provides the promotor of said gene mgATG5 simultaneously, and this promotor has the nucleotide sequence shown in the SEQ ID NO:4.
The present invention also provides the expression that utilizes said gene mgATG5 as target, the application in design and screening antifungal drug.
The present invention also provides and has utilized above-mentioned protein expression and modify as target, the application in design and screening antifungal drug.
The present invention also provides in conjunction with utilizing above-mentioned protein and promotor as target, in the middle application of design and screening antifungal drug.
The protein sequence of gene mgATG5 of the present invention and this genes encoding is a probe with its full length nucleotide, separates the gene with identical function that comes from other pathogenic fungi by hybridization; Or, separate the gene that comes from other pathogenic fungi by PCR with identical function with the sequences Design primer; Or with its encoded protein matter, hybridization separates the albumen with identical function that comes from other pathogenic fungi.Above-mentioned fungi comprises: Fusarium graminearum (Fusarium graminearum), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), wheat glume blight bacterium (Stagonospora nodorum).
The alleged antifungal drug of the present invention comprises the medicine of resisting rice blast bacteria (Magnaporthe grisea), Fusarium graminearum (Fusarium graminearum), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), wheat glume blight bacterium (Stagonospora nodorum).
The present invention utilizes gene knockout and gene complementation to prove gene mgATG5 that the process that penetrates and the virulence of Pyricularia oryzae appressorium are had key effect in the Pyricularia oryzae.The promotor of this gene, the expression of proteins encoded and modification can be used as the drug target of novel pesticide design and screening.
Gene mgATG5 of the present invention clones from Pyricularia oryzae by the cDNA subtracted library.Detailed process comprises: the SSH subtracted library by screening Pyricularia oryzae cDNA obtains mycelia or adheres to the est sequence of differential expression in the spore.By RT-PCR checking goal gene at Pyricularia oryzae mycelia, spore, adhere to the situation that exists in the spore.From the Pyricularia oryzae genomic dna, obtain goal gene by the high-fidelity long range PCR.By PCR or with the est sequence of this gene is that probe is from checking of Pyricularia oryzae cDNA library and the complete cDNA that separates goal gene.The functional verification of goal gene and note are finished by knocking out with complementary recovery of goal gene.The action site of goal gene in Pyricularia oryzae determined in the position of cell by the fusion rotein of goal gene and reporter gene GFP.The mensuration of gene promoter is that promotor is connected with reporter gene GFP, and GFP carries out in the expression of each etap of Pyricularia oryzae under promotor instructs by observing.
Pyricularia oryzae SSH subtracted library involved in the present invention is meant and utilizes SSH technology (Suppression subtractivehybridization, SSH) Pyricularia oryzae cDNA library that make up, that contain some independent clonings.
RT-PCR involved in the present invention analyzes and is meant according to cDNA sequences Design primer, the cDNA that genetically deficient muton and the complementary mRNA reverse transcription that recovers muton are formed carries out pcr amplification, verify respectively genetically deficient muton and gene complementation recover genetically deficient in the muton and recover after situations such as transcript.
Goal gene clone involved in the present invention is meant the est sequence that obtains according in the SSH library, screens the cDNA clone with this designing probe from the cDNA library, and the cDNA clone who obtains is checked order; And utilize dna sequence dna in the cDNA sequence alignment genome database, and design the dna sequence dna of primer with this from Pyricularia oryzae genomic dna amplification acquisition gene, be cloned in the T carrier, check order; Utilize its transcription sequence of programanalysis according to the dna sequence dna that obtains, and design from Pyricularia oryzae cDNA library, the increase cDNA sequence of acquisition gene of primer, be connected in the T carrier, carry out sequencing analysis with this.
Shouthern hybridization analysis involved in the present invention is meant that the dna sequence dna that utilizes the gene flank is as probe, gene knockout transformant and the gene recovery transformant that is obtained carried out Southern hybridization respectively, knock out the genetically deficient situation of transformant and the insertion copy number of knockout carrier with analyzing gene, and the complementary transformant gene that recovers of analyzing gene inserts situations such as copy number.
The function of goal gene involved in the present invention is determined knocking out and recover to carry out by mgATG5.The process of knocking out of concrete mgATG5 gene comprises the structure of knockout carrier, changes knockout carrier over to Pyricularia oryzae, obtains the gene knockout muton; The complementary recovery process of the gene knockout muton of mgATG5 comprises the structure of complementary carrier, and complementary carrier quiding gene knocks out the muton bacterial strain, obtains the transformant that gene recovers.The structure of gene knockout carrier is meant that the section of DNA fragment that will wait to knock out two flanks of gene is connected to the both sides of the hygromycin gene that contains the hygromycin gene carrier separately.The structure of gene complementation carrier be meant with contain target gene total length dna fragmentation with have a carrier that is different from the selection markers (being the Glufosinate ammonium resistant gene among the present invention) of hygromycin gene and be connected.The preferred method that in the present invention foreign vector is imported Pyricularia oryzae is a protoplast transformation, and the experimenter also can select other method such as ATMA method, particle bombardment etc. for use according to the situation of oneself.Among the present invention, the Pyricularia oryzae bacterial strain that gene knockout carrier imported is wild type strain Guy11, the experimenter also can select other Pyricularia oryzae wild type strain for use, and genetically deficient makes the phenotype of muton be different from the variation of original wild-type, to the pathogenic forfeiture of plants such as paddy rice.Among the present invention, the bacterial strain that the gene complementation carrier is imported is the resulting genetically deficient muton of the present invention, and the ectopic integration of complementary carrier in this genetically deficient muton genome makes the phenotype of this muton recover normal.
The cDNA clone process of mgATG5 provided by the present invention comprises: screening contains the cDNA clone of target gene mgATG5 from the SSH subtracted library, obtain its est sequence, utilize its est sequence from the Pyricularia oryzae genome database, to obtain corresponding section of DNA sequence, dna fragmentation at the target gene that from the Pyricularia oryzae genomic dna, increases according to its sequences Design primer, order-checking obtains its dna sequence dna, utilizes GenScan (http://genes.mit.edu/genscan.html) to carry out the prediction of promotor, coding region and tailing point.According to the transcript sequences Design primer of prediction, and by the PCR acquisition of increasing from Pyricularia oryzae cDNA library, its sequence is shown in SEQ IDNO:2.
The present invention also provides mgATG5 encoded protein matter, and it has shown in SEQ ID NO:3 aminoacid sequence or has aminoacid sequence sequence identity and that have identity function more than 42% with the sequence shown in the SEQ IDNO:2.This albumen can come from Pyricularia oryzae, also can come from other pathogenic fungi.The invention provides Fusarium graminearum (Fusariumgraminearum), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), wheat glume blight bacterium (Stagonosporanodorum) in have the aminoacid sequence that has the homologous protein of 42% above consensus amino acid sequence with mgATG5 albumen.
Gene involved in the present invention is because the genetically deficient sudden change that homologous recombination causes causes the decline of Pyricularia oryzae sporulation quantity, spore germination and appressorium to form delay, the reduction of appressorium turgescence and invades the bolt rate of formation and reduce, and the disappearance of the virulence on susceptible rice varieties.Therefore, 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 expression of gene, shearing and proteins encoded thereof, or the compound that can modify this proteic aminoacid sequence of design and screening, thereby the antifungal drug that exploitation makes new advances.In addition, purposes of the present invention comprises that also the DNA that utilizes this gene or cDNA sequence separate with this gene as probe and have the sequence of certain sequence homology in other fungi.
With the expression of above-mentioned institute clone gene proteins encoded, be modified to target; design, screening novel anti fungi-medicine; perhaps to have shown in SEQ ID No:3 sequence or to have the aminoacid sequence design polypeptide in arbitrary zone in the albumen 42% above sequence identity and that have identity function with SEQ ID No:3; and preparation antibody is used for detecting proteic expression under the compound treatment situation, all belongs within protection scope of the present invention.
Promotor with above-mentioned institute clone gene is a target, and design and screening novel anti fungi-medicine also belong within protection scope of the present invention.
Therefore, the present invention has the following advantages:
1, appressorium is the important structure that Pyricularia oryzae is invaded host plant, the appressorium turgescence is the main mechanical forces that Pyricularia oryzae penetrates the host plant horny layer of epidermis, the mgATG5 gene participates in the generation of Pyricularia oryzae appressorium turgescence, mgATG5 gene and Pyricularia oryzae pathogenic closely related, mgATG5 inactivation or disappearance, cause Pyricularia oryzae appressorium turgescence to descend pathogenic forfeiture.Therefore the mgATG5 gene acts on great in the rice blast pathogenic process.
2, Pyricularia oryzae is propagated between plant leafs such as paddy rice by conidium, and conidium is a link in the rice blast pathogenic circulation, is to prevent effective measure of germ popular thereby eliminate the pathogenic circulation of conidium cut-out.MgATG5 participates in the forming process of rice blast pathogen conidiospore, and mgATG5 inactivation or disappearance cause the rice blast pathogen conidiospore rate of formation significantly to descend (be about wild-type 4.5%), can effectively suppress the popular of rice blast and outburst.Therefore the mgATG5 gene acts on great in the rice blast pathogenic process.
3, mgATG5 provides special drug effect target for developing novel antifungal medicines, at the target medicine target of mgATG5 as drug development, by a large amount of compounds are screened, can find the material that can work with the medicine target, these materials can be used as the prevention fungal infection.Because the specificity of medicine target, the antifungal drug that screens is generally less for the toxicity of animal and plant.
Description of drawings
Fig. 1 is wild type strain Guy11 and mgATG5 genetically deficient muton Δ ATG5 and the pathogenic comparison diagram of the complementary transformant ATG5-HB1 of mgATG5 on paddy rice susceptible variety CO39.Guy11 is a wild type strain, and Δ ATG5 is a mgATG5 genetically deficient muton, and ATG5-HB1 recovers transformant for the mgATG5 gene complementation, and gelatin is the gelatin contrast solution of 0.2% (w/v).The spore concentration of spray inoculation is 1 * 10
5/ ml inoculates back 7 days and takes pictures.
Fig. 2 is that the complementary transformant HB24 of wild type strain Guy11 and mgATG5 genetically deficient muton Δ ATG5 and mgATG5 invades bolt formation comparison diagram on the barley epidermis.Guy11 is a wild type strain, and Δ ATG5 is a mgATG5 genetically deficient muton; 24h, 48h, 72h, 96h are inoculation back photo opporunity.The spot-inoculated spore concentration that exsomatizes is 1 * 10
5/ ml.
Fig. 3 is that gene knockout carrier makes up synoptic diagram.
Fig. 4 is a gene complementation vector construction synoptic diagram.
Fig. 5 is gene knockout position and process synoptic diagram.
Fig. 6 is the big or small comparison diagram of the appressorium turgescence that forms of the complementary muton ATG5-HB1 of wild type strain Guy11 and mgATG5 genetically deficient muton Δ ATG5 and mgATG5 (appressorium subside rate), is the cell of spore inoculating 24h postadhesion born of the same parents in different glycerol concentration solution (1M, 2M, 3M and the 4M) rate of subsiding.
Fig. 7 is the conidium rate of formation comparison diagram of wild type strain Guy11 and mgATG5 genetically deficient muton Δ ATG5 and the complementary muton ATG5-HB1 of mgATG5.The Pyricularia oryzae bacterial strain is that the punch tool of 0.7CM is beaten and got the mycelia sheet with diameter after cultivating 11 days on the CM solid medium, adds up the quantity of spore; Unit is * 10
4Individual spore/sheet.
Fig. 8 is spore germination rate (%) comparison diagram of wild type strain Guy11 and mgATG5 genetically deficient muton Δ ATG5 and the complementary transformant ATG5-HB1 of mgATG5.
Fig. 9 be wild type strain Guy11 and mgATG5 genetically deficient muton Δ ATG5 and the complementary transformant ATG5-HB1 of mgATG5 adhere to spore rate of formation (%) comparison diagram.
Figure 10 is wild type strain Guy11 and mgATG5 genetically deficient muton Δ ATG5 and the speed of growth comparison diagram of the complementary transformant ATG5-HB1 of mgATG5 on MM substratum, nitrogen stress substratum, scarce charcoal substratum.MM is the MM substratum, and MM-N is the nitrogen stress substratum, and MM-C is for lacking the charcoal substratum; The colony diameter of measuring is the colony diameter of growth in the time of 12 days.
Figure 11 is the tenuigenin location map of mgATG5 gene coded protein.The mgATG5-GFP fusion rotein is distributed in the tenuigenin, is spot distribution in some position, and the left side is to take under the bright field, and the right side is to take under the dark field.Upper left (A) is the spore photo under the common light microscopic, upper right (B) is the spore photo under the fluorescent microscope, (C) is the mycelia photo under the common light microscopic in the left side, (D) is the mycelia photo under the fluorescent microscope in the right side, lower-left (E) is the appressorium photo under the common light microscopic, and bottom right (F) is the appressorium photo under the fluorescent microscope.
Figure 12 is the glycogenosome comparison diagram (electromicroscopic photograph) in the spore that forms of the complementary transformant ATG5-HB1 of wild type strain Guy11 and mgATG5 genetically deficient muton Δ ATG5 and mgATG5.Last figure (A) is wild type strain Guy11, and middle figure (B) is mgATG5 genetically deficient muton Δ ATG5, and figure below (C) is the complementary transformant ATG5-HB1 of mgATG5.
Figure 13 is wild type strain Guy11 and the mgATG5 genetically deficient muton Δ ATG5 comparison diagram that autophagic vacuole forms in the nitrogen stress substratum.Last figure is the photo that wild type strain Guy11 bacterial strain autophagic vacuole forms, and middle figure is the photo that mgATG5 genetically deficient muton bacterial strain autophagic vacuole forms, and figure below is the photo that the complementary transformant ATG5-HB1 of mgATG5 autophagic vacuole forms.
Figure 14 be the proteic sequence comparison diagram of ATG5 in several pathogenic fungies (Phylip tree, NJ).Illustrate: mgATG5, fgATG5, ssATG5, snATG5 are respectively the ATG5 albumen from Pyricularia oryzae (Magnaporthe grisea), Fusarium graminearum (Fusarium graminearum), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), wheat glume blight bacterium (Stagonospora nodorum).
Embodiment
Below in conjunction with accompanying drawing the specific embodiment of the present invention is described in further detail.
Embodiment 1: the clone of the gene relevant with rice blast bacterium pathogenicity:
1, SSH library screening: the difference that Pyricularia oryzae appressorium cDNA and sporogenous hyphae cDNA difference subtract the back acquisition subtracts cDNA, be cloned into the T carrier, order-checking obtains the cDNA sequence, the sequence and the Pyricularia oryzae database that obtain are compared, determine the difference expression gene of acquisition, and verify the differential expression specificity with RT-PCR.
2, the clone of mgATG5 cDNA: the appressorium cDNA library of the Pyricularia oryzae Guy11 bacterial strain of existence-20 ℃ of going bail for, gene cDNA sequence design synthetic pcr primer thing according to prediction, amplification comprises whole gene coding region at interior gene cDNA fragment, be cloned into the T carrier, order-checking obtains the sequence of mgATG5 cDNA, and its sequence is shown in SEQ ID:2.
3, the clone of mgATG5 genomic dna: the genomic dna of the Pyricularia oryzae Guy11 bacterial strain of existence-20 ℃ of going bail for, gene DNA sequence design synthetic pcr primer thing according to prediction, amplification comprises the dna fragmentation of whole full-length gene, be cloned into the T carrier, order-checking obtains the sequence of mgATG5 genomic dna, and its sequence is shown in SEQ ID:1.
The acquisition and the evaluation of embodiment 2:mgATG5 genetically deficient muton
1, the structure of gene knockout carrier: the dna fragmentation that is about 500～2000bp in mgATG5 upstream region of gene and downstream is inserted into the both sides of hygromycin gene in the carrier that contains hygromycin gene respectively, as Fig. 3 and shown in Figure 5.Specific as follows: the method for employing high-fidelity PCR is the dna fragmentation that is about 500～2000bp in amplification mgATG5 upstream region of gene and downstream from Pyricularia oryzae DNA genome respectively, is connected to earlier on the T carrier; The upstream dna fragmentation downcuts from the T carrier with restriction enzyme XhoI and SalI then, is inserted on the same restriction enzyme site of the pBShph1 carrier that contains hygromycin gene; Then the downstream DNA fragment is also downcut from the T carrier with restriction enzyme SpeI and XbaI from the T carrier, is inserted on the SpeI and XbaI site of the above-mentioned pBShph1 carrier that has inserted the upstream dna fragmentation, promptly obtains the mgATG5 gene knockout carrier.The building process that contains the pBShph1 carrier of hygromycin gene: adopt earlier the method for high-fidelity PCR from the pCB1003 plasmid, increase hygromycin gene (HPH or HygB
r), be connected on the T carrier; Downcut with restriction enzyme HindIII and SalI then, and be inserted in the same restriction enzyme site of pBS plasmid, promptly obtain the pBShph1 carrier.
2, the preparation of Pyricularia oryzae protoplasma carrier and DNA transform: the Pyricularia oryzae bacterial strain is earlier growth on the CM flat board about 6 days.Cut the bacterium colony of two about 3cm of diameter, place 150mL CM liquid nutrient medium, it is smashed to pieces with triturator; Evenly divide then to install in two bottles of 200mL CM liquid nutrient mediums, 28 ℃, 125rpm cultivated 48 hours.Four layers of filtered through gauze are collected mycelia, with aseptic water washing twice, moisture are pressed dry.Mycelia in enzymolysis solution (Glucanex, enzyme liquid concentration 7.5mg/mL, 0.7M NaCl solution) enzymolysis 2-3hr 30 ℃, 80rpm.Take out enzymolysis solution, three metafiltration paper filter, and residue is removed in 0.7M NaCL washing, and filtrate collection is in the centrifuge tube of 50mL.4 ℃, 3000rpm, centrifugal 10min.With 10-20mL STC (1.2M Sorbitol, 10mM Tris-HCL PH7.5,20mM CaCL
2) precipitation suspends.4 ℃, 3000rpm, centrifugal 10min; Repeat twice.Get an amount of STC suspension precipitation, making final concentration is 0.5～1.0 * 10
8Individual/mL.Each 50ml centrifuge tube packing 150 μ l protoplastis adds 2 μ g linearizing DNA, places 25min under the room temperature.Dropwise add 1mL PTC (60%PEG4000,10mM Tris-HCL PH7.5,20mMCaCL
2), mixing, room temperature is placed 25min.Slowly add 5mL OCM liquid nutrient medium (adding 1.2M Sorbitol/1M Sucrose in the CM liquid nutrient medium), shake up gently, place 28 ℃, 100rpm shakes and spends the night.Add the solid OCM Top substratum that contains 200 μ g/mL Totomycin in the protoplastis of every pipe incubated overnight and (add 20%Sucrose in the CM liquid nutrient medium, agar powder 1g), mixing, pour into and be covered with OCM Bottom substratum and (add 20%Sucrose in the CM liquid nutrient medium, agar powder 1.5g) flat board, three flat boards in every pipe shop.Dark, 28 ℃, to cultivate about an about week, the picking transformant is inoculated on the solid CM that contains 200 μ g/mL Totomycin.
3, the evaluation of mgATG5 genetically deficient muton: extract transformant DNA, with method validation transformants such as PCR, Southern hybridization with hygromycin resistance.
The complementation of embodiment 3, mgATG5 genetically deficient muton recovers:
The full length DNA sequence of mgATG5 gene is connected in the carrier that has the Glufosinate ammonium resistant gene, as shown in Figure 4.Be specially: adopt high-fidelity long segment PCR method (LD PCR) from the Pyricularia oryzae genomic dna, to increase and obtain containing the dna fragmentation in mgATG5 full length gene coding region, promoter region and zone, terminator, be inserted into earlier in the pCR-XL-TOPO carrier, and then from the TOPO carrier, downcut this dna fragmentation with restriction enzyme NotI and MluI, be inserted into the NotI and the MluI site of the pBARKS1 carrier that contains the Glufosinate ammonium resistant gene, the complementation that promptly is built into the mgATG5 gene recovers carrier---pBAR-ATG5 carrier.Before the protoplasma that carries out DNA transformed, the pBARATG5 carrier was being used restriction enzyme NotI linearization for enzyme restriction.
Then according to the method for embodiment 2, the complementary carrier of linearizing this mgATG5 gene is transformed mgATG5 genetically deficient bacterial strain protoplastis.The transformant that picking has the Glufosinate ammonium resistance is inoculated on the solid CM that contains Glufosinate ammonium.Extraction has the transformant DNA and the RNA of Glufosinate ammonium resistance, with method validation transformants such as RT-PCR, Southern hybridization.
The changing condition that has compared phenotype after gene recovers by the following method: on perfect medium, compared mgATG5 transgenation and the complementary sporulation quantity variation (see figure 7) of recovering transformant (being complementary muton or complementary transformant); MgATG5 transgenation and the complementary sprouting variation (see figure 8) of recovering the transformant spore have been compared; MgATG5 transgenation and the complementary variation (see figure 9) of adhering to the spore rate of formation of recovering transformant have been compared; The turgescence of adhering to spore that has compared mgATG5 transgenation and the formation of complementary recovery transformant changes (see figure 6); The mycelia autophagic vacuole that has compared mgATG5 transgenation and the formation of complementary recovery transformant forms situation (seeing Figure 13); The glycogen content that has compared the spore of mgATG5 transgenation and the formation of complementary recovery transformant changes (seeing Figure 12); On MM substratum, nitrogen stress substratum, scarce carbon substratum, compare mgATG5 transgenation and the complementary growth change (see figure 10) of recovering transformant; Fig. 1 is seen in the pathogenic recovery of complementary transformant.
The pathogenic mensuration of embodiment 4:mgATG5 genetically deficient muton and complementary muton:
1, spray inoculation: barley or paddy rice CO39 seed kind are cultivated 8 days (barley) or 14 days (the paddy rice CO39 plant of 3～4 leaf phases) in flowerpot earth.Wash the spore of getting Pyricularia oryzae wild type strain, muton and complementary transformation from the CM flat board of cultivating 12 days, spore concentration is diluted to 1 * 10 with the gelatin of 0.2% (wt/v)
5/ ml.With miniature airbrush with the spore suspension spray inoculation to the plant leaf surface.Simultaneously, with 0.2% gelatin as the negative control spray inoculation.The inoculation plant is placed in the moist chest, preserves moisture in 25 ℃ of dark surrounds and cultivates 24 hours (barley) or 48 hours (paddy rice).Inoculate plant then and put into and continue to be cultured to the 4th day (barley) or the 7th day, the 14th day (paddy rice) in the growth cabinet (25 ℃, 80% above humidity, 12 hours periodicity of illuminations), the plant illness is fully shown.The rice blast symptom of Taking Pictures recording plant leaf, and according to (1986) reported method such as Bonman records with estimate the disease serious degree of plant.Simultaneously, tried to select the plant a slice the most serious infected blade, the Pyricularia oryzae scab on the record blade in one section zone of 5cm length from every strain.The results are shown in Figure 1.
2, in vitro inoculation: the barley seed kind was cultivated 7～10 days in flowerpot earth.Wash the spore of getting Pyricularia oryzae wild type strain, muton and complementary transformation from the CM flat board of cultivating 12 days, being diluted to spore concentration is 5 * 10
4/ ml or 1 * 10
5/ ml.Spore liquid 20 μ l one dropping point is inoculated in the barley leaves upper surface that exsomatizes.In growth cabinet, preserve moisture illumination cultivation (illumination/dark, 12h: 12h), 25 ℃.Cultivating 24h respectively, 48h behind 72h and the 96h, takes out partial blade and observes.Observe scab earlier and form situation, and Taking Pictures recording.Clip part scab is put into methanol solution and is fixed and slough chlorophyll then.The scab blade is fixed 24 hours (room temperature) in methanol solution after, (lactophenol oil: 95% ethanol=1: 2) room temperature is fixed 1 hour to move into the fixing transparent liquid of lactophenol oil.The lactophenol oil formula is: lactic acid 20ml, phenol 20ml, 20% glycerine 40ml, distilled water 20ml.The scab blade is used tongue phenol indigo plant/aniline blue (0.01% tongue phenol indigo plant/0.06% aniline blue, lactophenol oil) dyeing 5～10 minutes again.The dyeing blade decolours in lactophenol oil.Examine under a microscope the scab development, the appressorium penetration coefficient of statistics 24h and 48h and the growth of intrusion mycelia and the production of secondary spore, and Taking Pictures recording.The results are shown in Figure 2.
The Subcellular Localization of embodiment 5:mgATG5:
The complete coding region sequence of GFP gene is connected to the downstream of the mgATG5 gene coding region of not being with terminator codon, is built into fusion gene and is connected in the carrier that has hygromycin gene.According to the method for embodiment 2, linearizing this carrier is transformed wild-type Guy11 bacterial strain protoplastis.Picking has the transformant of hygromycin resistance and observe the distribution of mgATG5-GFP fusion rotein in the Pyricularia oryzae cell under fluorescent microscope.The result shows that mgATG5 is positioned in the tenuigenin, and quantity is bigger in some organoid.The results are shown in Figure 11.
Defining and expression analysis of embodiment 6:mgATG5 promotor:
After the complete coding region sequence of GFP gene is connected to the mgATG5 gene coding region upstream section of DNA sequence of (not containing coding region sequence), is built into fusion gene and is connected in the carrier that has hygromycin gene.According to the method for embodiment 2, linearizing this carrier is transformed wild-type Guy11 bacterial strain protoplastis.GFP albumen under the mgATG5 promotor of the transformant that picking has a hygromycin resistance observing under fluorescent microscope instructs is at the expression of Pyricularia oryzae different developmental phases.The mgATG5 promoter sequence is seen shown in the SEQ ID NO:4.
Embodiment 7: the proteic bioinformatic analysis of fungi ATG5:
For whether clear and definite ATG5 albumen is guarded in other pathogenic fungies, at following pathogenic fungi: Fusarium graminearum (Fusarium graminearum), Sclerotinia sclerotiorum (Sclerotinia sclerotiorum), wheat glume blight bacterium (Stagonospora nodorum), the proteic aminoacid sequence of mgATG5 is carried out the Blastp retrieval to the protein sequences of these fungies, obtain the aminoacid sequence of the homologous protein of above-mentioned pathogenic fungi, its sequence is respectively as SEQ ID 5, shown in 6 and 7, and with these albumen difference called afters fgATG5, ssATG5 and snATG5.Sequential analysis shows that the ATG5 albumen of above-mentioned fungi reaches 59%, 54% and 42% respectively with the proteic consistence of mgATG5 on aminoacid sequence.Utilize the proteic homologous relationship figure of ATG5 (Phylip tree) of the above-mentioned fungi of clustalw program (http://www.ebi.ac.uk/clustalw/) drafting to see Figure 14.
Embodiment 8: utilize the proteic expression of mgATG5 or modify as target the screening antifungal medicament.
Pyricularia oryzae is cultivated in the liquid perfect medium in a large number, be divided into some aliquots after collecting mycelia, adding compound to be screened or drug candidate in each part continues to cultivate a few hours at perfect medium, in the nitrogen stress substratum, (also add compound or drug candidate to be screened simultaneously) then and continue inducing culture about 2 hours, get mycelia is observed mycelia under phase microscope autophagic vacuole formation situation (Figure 13), perhaps mycelia utilizes conventional electron microscope slice production method to make the formation situation of observing autophagic vacuole behind the ultrathin section(ing) under electron microscope.If mgATG5 albumen takes place to be modified incident by drug candidate and lose activity, then mycelia does not form autophagic vacuole.The compound that obtains utilizes the method for embodiment 4 again, measures the wild-type Pyricularia oryzae and under the condition that this compound exists whether the pathogenic of paddy rice is weakened, and further determines the anti-mycotic efficiency of compound.
Embodiment 9: the expression that utilizes mgATG5 is screened antifungal medicament as target.
Pyricularia oryzae is cultivated in the liquid perfect medium in a large number, be divided into some aliquots after collecting mycelia, adding compound to be screened or drug candidate in each part continues to cultivate a few hours at perfect medium, in the nitrogen stress substratum, (also add compound or drug candidate to be screened simultaneously) then and continue inducing culture about 2 hours, extract the RNA of mycelia, adopt the expression situation of the method detection mgATG5 of real-time quantitative PCR.If the expression of mgATG5 is suppressed by drug candidate, then there are not the transcript of mgATG5 or the transcript of mgATG5 significantly to reduce in the hyphal cell.The compound that obtains utilizes the method for embodiment 4 again, measures the wild-type Pyricularia oryzae and under the condition that this compound exists whether the pathogenic of paddy rice is weakened, and further determines the anti-mycotic efficiency of compound.
Embodiment 10: the promotor of utilizing mgATG5 is screened antifungal medicament as target.
The promotor of mgATG5 and fluorescin GFP are built into the fusion rotein carrier, or the promotor of mgATG5 is built into the fusion rotein carrier with fluorescin GFP and mgATG5 albumen, method by embodiment 2 is transferred in the Pyricularia oryzae, then the Pyricularia oryzae transformant bacterial strain of expressing at the GFP under the promoter regulation of mgATG5 is cultivated in the liquid perfect medium in a large number, be divided into some aliquots after collecting mycelia, add compound to be screened or drug candidate in each part and continue to cultivate a few hours to a couple of days, get mycelia is observed the GFP of mycelia under fluorescent microscope green fluorescence expression at perfect medium.If the combined thing of the promotor of mgATG5 suppresses and loses activity, thereby then GFP albumen is not expressed mycelia and is lost green fluorescence.The compound that obtains utilizes the method for embodiment 4 again, measures the wild-type Pyricularia oryzae and under the condition that this compound exists whether the pathogenic of paddy rice is weakened, and further determines the anti-mycotic efficiency of compound.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Sequence table
SEQ?ID?NO：1
CTCTAGGGGG?TTGCAGTGCG?TTCGTCACGC?GTAAGGCTTC?CTCGGCCAGT?AAGATGGTGT 60
GCTGGGGGGA?CTTTTCTTTC?TGACCTGGAA?TCACTCACTG?CTGGTTGCTT?GCCAAGCTTG 120
CTTGCTCGGA?AAGGGGTTAA?TACAAAACCA?GCTAGAAAAA?AAAAAAGATC?TACAAATGGT 180
GCATGTCTCA?TCGCGGGAAC?GTGAATGGTT?CGTTTTGCTG?GCTGTAGTCT?GTACCTCTAA 240
TTTCTTTTTT?TTTCTTTTTT?CCCCGGTGGC?TTTTTGGATT?CCCTCACTGC?AAAAAAGAGA 300
TGGAAGCGAG?TGCAACCTGA?TAGTGGCTAC?ATGCCAACCT?AGCTAGCTAT?ACATCCTTTT 360
ACAGCCTGGG?CCATTTGAAT?TCGGCGGGCA?AGCGGGGTTA?ATGGTACTCT?CTCTGGTTTT 420
TGGTAGTGGT?TCCCTGGTTT?GCGGAGGTGA?AATGGCAGCT?GTCATCCCAA?ACATACGGAG 480
TAAACTGATC?ACAGCAAAGT?GAGAAGGGGG?GATGTGATCC?GGATCACGGC?AATGAGCTAG 540
TCCCTACCTA?CAGGCAGTGC?CTGCCTACCA?ATAGCTCGTA?CAGAAAATCC?CTGCCCCTCT 600
CTCTGTTGTG?TCTCCGAGTT?GCTTTTCTCG?TCCTCTCTCC?AAACCCCGCA?TCGAAAAAGT 660
AAAGCCCGAT?GCTCACCCAC?CCCTGTCTGC?AGGTTCCCCA?TCGCCAAGTT?ATCTTGTGTC 720
TCTTGAGCTG?CAAAAGCAAC?CCATTGAAGC?AACCATCAAC?AGCGCCGATT?AAATTTGATA 780
TCGACAAGGT?AAAAAAGGAT?AAGAACAACA?AGAAGAAAAA?GAGATCAGAA?AAAAAAGGAA 840
ATCGCCCAAA?GTTTTACGGT?TCCCTCAGCT?TTCAAGACGG?TCGCGCCATT?GTGAAAACGG 900
TGGCACGGCT?TCACGACTTA?ACGGCTTGAA?CGGGTAACCG?CACCCAGGAG?GGTCGGCTGG 960
ATAATTGATT?CTCGGCCGGT?CCAGAACTAA?AAAAAAAAAA?AAAAAAAAAA?GGCTGCCTAG 1020
AGTAACATTT?GAACTACCTA?CGAATAATCG?GCGGGGTACT?GCATAGGTAG?TACAATAGTC 1080
GGGAGATATT?TAGTCTTTTT?GGGTTGGTTT?CATTTTCGTT?ACCTTTTTTT?TTACACCCCA 1140
CGTGCAGCTC?GTCGTTTTCG?TCCCCTAAAA?AAAACGCGAC?CCGGTTCTTG?GGCCGTTTCT 1200
TCGAGACCAG?GAAAAAGAGA?GACAAGGGTG?TGTGGGGGGA?AAACCACAAC?CGACGAGAAG 1260
GCATTCGGCA?GATCGCTATC?TCCCTACGGA?TAATCCAAAT?AAGAGAAATC?GTGATCGGGG 1320
AACCCGGATA?TTTCAAACTC?ACCGCTTCTG?ACTGAACACA?CCCTACTGTA?TCTTGTTGTT 1380
GCCGTCAGAA?TTACCGTTTA?CGTTTGCAGA?CAACCGTCAC?TGCTACCACC?CGACAGGGGG 1440
TCGCATGGGT?GTTTAAATGG?CTTCGCCGCG?CCGATCAGAA?GCCGACCTCG?CCAGCCCGGC 1500
ACGCGGGCCG?GGTTCGGGCC?CGCCATCCCC?ACCTCATCCC?GGTCCCGACT?CGGCAAGGCC 1560
ACTATCAAGA?ATCACGTCGG?CGGCAGCGTC?ACCACCACCA?CCGGGACAAC?GAACCGCGGA 1620
ACCACCCACA?ACCATCTCTA?TCCCCCGCAC?CCTCTGGCGC?CTCAGCATCC?CTTTGTACAT 1680
CACTCACCCC?TCCCTCCCCA?ACACACCCTT?CATAACCTCC?CTCCCGCGCG?TATCCTACCT 1740
GTCTCTTCTT?CTCCCCCGGA?TCCGCGCCTT?CCTGCCCCCA?TCCATCCCAG?CCCCGACCAG 1800
CTTCCACCAC?GAAGGGATCG?CGCTGCGCGC?CCTGCCGCTC?GGCCTGCTCG?TCGACCTGTA 1860
CCAGCCCACG?CTGCCGTGGC?GCCTGACGGT?CGACCAAGGG?GACGACTGGC?ACGTGGGCGA 1920
CACCTTCCTC?AACGGCGTCA?AGGAGGCCGA?CTTTGTGCGC?AACGGACACG?CCAAGCAGAT 1980
TATGGGCATG?AGCAAGGCCG?ACACCACGGC?GCTGTGGAAC?GCCGTCCGCG?ACGCAGACTA 2040
CCCGGCTTGG?GCCCGCATCA?ACGCCCGTCT?GCTCAACCCT?AGCACCCCGA?TCAAGCACGT 2100
GCCCCTGCGC?ATCTACATCC?CCAGCAGCGG?TGGTGGTGGC?GCCGGGGGAG?CAACGCCCGC 2160
CGGGTCTTTT?AGGGTCATGC?AGACCCTCGT?TCCACCCCGG?ACGGCAAACC?GTGAGTAAAC 2220
CATCAGCCTC?GACTTGCTTT?TTGCCGCCAT?TCAACTCTTT?AAAGTTTTTT?TTTTTTTTTT 2280
TTTTTTTTTG?AGAGCACGGA?ACCGAGCCTG?ACGTGTCGCT?GACTTGGTTT?GCTTCCACAG 2340
GTATTCCGCA?GACTCTCGGC?CCAACACTAC?GTGACTTGCT?CCCTGTCTTG?TTCCCGAGCA 2400
GCAGAGATCC?CGTACTGGCC?AACGTGGTGC?TTCACGGCGC?TCCCGTGCCA?TTCTCGGCCC 2460
CGCTAGAGGG?CTTGATGCGC?GAGGCCGCCT?ATCCAGACGG?CTGGCTTTGT?TTAACCGTCG 2520
TGCCATTATG?ATTTTTTGAC?AGGGGCAAAA?ACAGTAGTCT?TGAGCTCAAG?GGCTAAAAAC 2580
TTGACCGTTG?TTTCCCTCCA?ACTAGAACTA?GATGGTGATT?TGAGCGACGT?TCGATTTCAC 2640
GTGCATTGAA?TTTGGTTTCA?CAATTCTTGG?AGCTGAAAAG?ATGCAAGGAT?GGCCTTTGAT 2700
GTTTATTGCC?TCACAAAGAG?ATTCAAGAGA?CCAAAAAAGA?ACAACATGTC?TTATCCAACA 2760
AAGTACTTCA?AGATCAAATA?TGATCCACCC?TCAACCAGCC?ATCTATATAT?GCAACATGTA 2820
CTTGCAACGG?ACCTGTCCGC?CGCGCAGCTA?TCACTTCGTA?CCACCTCGCA?ATAAAAAAAG 2880
AAAAAGGAAT?ATACCATCAT?TAGAGTATGC?GTGTATAACA?TAACTCAGAA?AGTGAAAAGA 2940
AAATCAAAGA?AAAAGCCGAC?CTAGTAAATG?GGTATGTATG?CAGGTACGCT?TCATCCAAGA 3000
AGATAAACAA?AGAATACAAA?ACGTCGCTGA?GCATGATACT?TGTTGCCTTT?GCCATAAAAC 3060
AGCTTCATCC?ACATGAGGGG?CCGTCATTAT?CTTGTCAAGT?CGGAAAGGAA?AAGAACATGA 3120
AACAAGTATG?ATCTTAAATG?CAAGCAAGAT?GCCAGCCAGA?AAAAAAGGGA?GAAGCTGATC 3180
CTAATCCAGA?AGAAAATACG?AGGGTGTTTA?AATGGCAAAA?TATTAGCGAG?ATAATTATGT 3240
GGTAGAGCTC?AACTGCAATC?CTTTGCAGCG?CGAGCTTTCC?CAAGTAATTA?AATGCAGCGA 3300
GCCCTCTGTC?GCCACGGCAA?ATCAGGCCAT?GTCGGGTCCG?GTCTTGAACA?TTCGCCAAAC 3360
TTGCTTTTCG?GGCGAGCCCT?GTCAACATGA?CACAGCAGGT?AGAATCTGAA?GGGAAGCGAG 3420
GCTTAGCAGG?AGCGTACTGC?AACAGCCTGT?TTCTTGCTAG?CAGAGAACCT?TGATGGTGTG 3480
GTGAATGCGA?GTTCCGCTTT?CCTGGGAGCA?CTTTCATCGA?GGTACCTGAA?CATCCACTGC 3540
TCCGTTGGGT?GTTGGATGGG?GCGGCTGATG?AGGATGAGGA?GAGTAAGAGA?ACGGCGGT 3598
SEQ?ID?NO：2
AGGGGGTCGC?ATGGGTGTTT?AAATGGCTTC?GCCGCGCCGA?TCAGAAGCCG?ACCTCGCCAG 60
CCCGGCACGC?GGGCCGGGTT?CGGGCCCGCC?ATCCCCACCT?CATCCCGGTC?CCGACTCGGC 120
AAGGCCACTA?TCAAGAATCA?CGTCGGCGGC?AGCGTCACCA?CCACCACCGG?GACAACGAAC 180
CGCGGAACCA?CCCACAACCA?TCTCTATCCC?CCGCACCCTC?TGGCGCCTCA?GCATCCCTTT 240
GTACATCACT?CACCCCTCCC?TCCCCAACAC?ACCCTTCATA?ACCTCCCTCC?CGCGCGTATC 300
CTACCTGTCT?CTTCTTCTCC?CCCGGATCCG?CGCCTTCCTG?CCCCCATCCA?TCCCAGCCCC 360
GACCAGCTTC?CACCACGAAG?GGATCGCGCT?GCGCGCCCTG?CCGCTCGGCC?TGCTCGTCGA 420
CCTGTACCAG?CCCACGCTGC?CGTGGCGCCT?GACGGTCGAC?CAAGGGGACG?ACTGGCGCGT 480
GGGCGACACC?TTCCTCAACG?GCGTCAAGGA?GGCCGACTTT?GTGCGCAACG?GACACGCCAA 540
GCAGATTATG?GGCATGAGCA?AGGCCGACAC?CACGGCGCTG?TGAAACGCCG?TCCGCGACGC 600
AGACTACCCG?GCTTGGGCCC?GCATCAACGC?CCGTCTGCTC?AACCCTAGCA?CCCCGATCAA 660
GCACGTGCCC?CTGCGCATCT?ACATCCCCAG?CAGCGGTGGT?GGTGGCGCCG?GGGGAGCAAC 720
GCCCGCCGGG?TCTTTTAGGG?TCATGCAGAC?CCTCGTTCCA?CCCCGGACGG?CAAACCGTAT 780
TCCGCAGACT?CTCGGCCCAA?CACTACGTGG?CTTGCTCCCT?GTCTTGTTCC?CGAGCAGCAG 840
AGATCCCGTA?CTGGCCAACG?TGGTGCTTCA?CGGCGCTCCC?GTGCCATTCT?CGGCCCCGCT 900
AGAGGGCTTG?ATGCGCGAGG?CCGCCTATCC?AGACGGCTGG?CTTTGTTTAA?CCGTCGTGCC 960
ATTATGATTT?TTTGACAGGG?GCAAAAACAG?TAGTCTTGAG?CTCAAGGGCT?AAAAACTTGA 1020
CCGTTGTTTC?CCTCCAACTA?GAACTAGATG?GTGATTTGAG?CGACGTTCGA?TTTCACGTGC 1080
ATTGAATCTG?GTTTCACAAT?TCTTGGAGCT?GAAAAGATGC?AAGGATGGCC?TTTGATGTTT 1140
ATTGCCTCAC?AAAGAGATTC?AAGAGACCA 1169
SEQ?ID?NO：3
Met?Ala?Ser?Pro?Arg?Arg?Ser?Glu?Ala?Asp?Leu?Ala?Ser?Pro?Ala
1 5 10 15
Arg?Gly?Pro?Gly?Ser?Gly?Pro?Pro?Ser?Pro?Pro?His?Pro?Gly?Pro
20 25 30
Asp?Ser?Ala?Arg?Pro?Leu?Ser?Arg?Ile?Thr?Ser?Ala?Ala?Ala?Ser
35 40 45
Pro?Pro?Pro?Pro?Gly?Gln?Arg?Thr?Ala?Glu?Pro?Pro?Thr?Thr?Ile
50 55 60
Ser?Ile?Pro?Arg?Thr?Leu?Trp?Arg?Leu?Ser?Ile?Pro?Leu?Tyr?Ile
65 70 75
Thr?His?Pro?Ser?Leu?Pro?Asn?Thr?Pro?Phe?Ile?Thr?Ser?Leu?Pro
80 85 90
Arg?Val?Ser?Tyr?Leu?Ser?Leu?Leu?Leu?Pro?Arg?Ile?Arg?Ala?Phe
95 100 105
Leu?Pro?Pro?Ser?Ile?Pro?Ala?Pro?Thr?Ser?Phe?His?His?Glu?Gly
110 115 120
Ile?Ala?Leu?Arg?Ala?Leu?Pro?Leu?Gly?Leu?Leu?Val?Asp?Leu?Tyr
125 130 135
Gln?Pro?Thr?Leu?Pro?Trp?Arg?Leu?Thr?Val?Asp?Gln?Gly?Asp?Asp
140 145 150
Trp?His?Val?Gly?Asp?Thr?Phe?Leu?Asn?Gly?Val?Lys?Glu?Ala?Asp
155 160 165
Phe?Val?Arg?Asn?Gly?His?Ala?Lys?Gln?Ile?Met?Gly?Met?Ser?Lys
170 175 180
Ala?Asp?Thr?Thr?Ala?Leu?Trp?Asn?Ala?Val?Arg?Asp?Ala?Asp?Tyr
185 190 195
Pro?Ala?Trp?Ala?Arg?Ile?Asn?Ala?Arg?Leu?Leu?Asn?Pro?Ser?Thr
200 205 210
Pro?Ile?Lys?His?Val?Pro?Leu?Arg?Ile?Tyr?Ile?Pro?Ser?Ser?Gly
215 220 225
Gly?Gly?Gly?Ala?Gly?Gly?Ala?Thr?Pro?Ala?Gly?Ser?Phe?Arg?Val
230 235 240
Met?Gln?Thr?Leu?Val?Pro?Pro?Arg?Thr?Ala?Asn?Arg?Ile?Pro?Gln
245 250 255
Thr?Leu?Gly?Pro?Thr?Leu?Arg?Asp?Leu?Leu?Pro?Val?Leu?Phe?Pro
260 265 270
Ser?Ser?Arg?Asp?Pro?Val?Leu?Ala?Asn?Val?Val?Leu?His?Gly?Ala
275 280 285
Pro?Val?Pro?Phe?Ser?Ala?Pro?Leu?Glu?Gly?Leu?Met?Arg?Glu?Ala
290 295 300
Ala?Tyr?Pro?Asp?Gly?Trp?Leu?Cys?Leu?Thr?Val?Val?Pro?Leu
305 310
SEQ?ID?NO：4
CTCTAGGGGG?TTGCAGTGCG?TTCGTCACGC?GTAAGGCTTC?CTCGGCCAGT?AAGATGGTGT 60
GCTGGGGGGA?CTTTTCTTTC?TGACCTGGAA?TCACTCACTG?CTGGTTGCTT?GCCAAGCTTG 120
CTTGCTCGGA?AAGGGGTTAA?TACAAAACCA?GCTAGAAAAA?AAAAAAGATC?TACAAATGGT 180
GCATGTCTCA?TCGCGGGAAC?GTGAATGGTT?CGTTTTGCTG?GCTGTAGTCT?GTACCTCTAA 240
TTTCTTTTTT?TTTCTTTTTT?CCCCGGTGGC?TTTTTGGATT?CCCTCACTGC?AAAAAAGAGA 300
TGGAAGCGAG?TGCAACCTGA?TAGTGGCTAC?ATGCCAACCT?AGCTAGCTAT?ACATCCTTTT 360
ACAGCCTGGG?CCATTTGAAT?TCGGCGGGCA?AGCGGGGTTA?ATGGTACTCT?CTCTGGTTTT 420
TGGTAGTGGT?TCCCTGGTTT?GCGGAGGTGA?AATGGCAGCT?GTCATCCCAA?ACATACGGAG 480
TAAACTGATC?ACAGCAAAGT?GAGAAGGGGG?GATGTGATCC?GGATCACGGC?AATGAGCTAG 540
TCCCTACCTA?CAGGCAGTGC?CTGCCTACCA?ATAGCTCGTA?CAGAAAATCC?CTGCCCCTCT 600
CTCTGTTGTG?TCTCCGAGTT?GCTTTTCTCG?TCCTCTCTCC?AAACCCCGCA?TCGAAAAAGT 660
AAAGCCCGAT?GCTCACCCAC?CCCTGTCTGC?AGGTTCCCCA?TCGCCAAGTT?ATCTTGTGTC 720
TCTTGAGCTG?CAAAAGCAAC?CCATTGAAGC?AACCATCAAC?AGCGCCGATT?AAATTTGATA 780
TCGACAAGGT?AAAAAAGGAT?AAGAACAACA?AGAAGAAAAA?GAGATCAGAA?AAAAAAGGAA 840
ATCGCCCAAA?GTTTTACGGT?TCCCTCAGCT?TTCAAGACGG?TCGCGCCATT?GTGAAAACGG 900
TGGCACGGCT?TCACGACTTA?ACGGCTTGAA?CGGGTAACCG?CACCCAGGAG?GGTCGGCTGG 960
ATAATTGATT?CTCGGCCGGT?CCAGAACTAA?AAAAAAAAAA?AAAAAAAAAA?GGCTGCCTAG 1020
AGTAACATTT?GAACTACCTA?CGAATAATCG?GCGGGGTACT?GCATAGGTAG?TACAATAGTC 1080
GGGAGATATT?TAGTCTTTTT?GGGTTGGTTT?CATTTTCGTT?ACCTTTTTTT?TTACACCCCA 1140
CGTGCAGCTC?GTCGTTTTCG?TCCCCTAAAA?AAAACGCGAC?CCGGTTCTTG?GGCCGTTTCT 1200
TCGAGACCAG?GAAAAAGAGA?GACAAGGGTG?TGTGGGGGGA?AAACCACAAC?CGACGAGAAG 1260
GCATTCGGCA?GATCGCTATC?TCCCTACGGA?TAATCCAAAT?AAGAGAAATC?GTGATCGGGG 4320
AACCCGGATA?TTTCAAACTC?ACCGCTTCTG?ACTGAACACA?CCCTACTGTA?TCTTGTTGTT 1380
GCCGTCAGAA?TTACCGTTTA?CGTTTGCAGA?CAACCGTCAC?TGCTACCACC?CGACAGGGGG 1440
TCGCATGGGT?GTTTAA 1456
SEQ?ID?NO：5
＞fgATG5 Fusarium graminearum Fusarium graminearum
Met?Ser?Ser?Pro?Ile?Pro?Gln?Ala?Leu?Trp?Ser?Ala?Gln?Ile?Pro
1 5 10 15
Leu?His?Ile?Thr?His?Pro?Ala?Ser?Pro?Thr?Thr?Pro?Phe?Ile?Thr
20 25 30
Ser?Ile?Pro?Arg?Phe?Ser?Tyr?Leu?Ala?Leu?Leu?Ile?Pro?Arg?Leu
35 40 45
Ser?Thr?Phe?Phe?Asn?Ser?Pro?Cys?Ser?Ser?Phe?His?Phe?Glu?Asp
50 55 60
Val?Gln?Leu?Arg?Asn?Leu?Ala?Val?Gly?Leu?Leu?Val?Asp?Leu?Tyr
65 70 75
Gln?Pro?Ala?Leu?Pro?Trp?Lys?Leu?Thr?Val?Asn?Asp?Gly?Val?Gly
80 85 90
Trp?Asp?Ile?Ala?Asp?Thr?Phe?Leu?Asn?Cys?Val?Lys?Glu?Ala?Asp
95 100 105
Phe?Val?Arg?Asn?Gly?Asn?Ala?Asn?Gln?Ile?Met?Lys?Met?Ser?Lys
110 115 120
Glu?Asn?Thr?Thr?Gln?Leu?Trp?Asn?Ala?Val?Ile?Asp?Asn?Asp?His
125 130 135
Pro?Ser?Phe?Asn?Arg?Ile?Asn?Ser?His?Leu?Leu?Asn?Ala?Pro?Thr
140 145 150
Ala?Leu?Lys?His?Val?Pro?Ile?Arg?Ile?Tyr?Val?Pro?Thr?Ser?Gly
155 160 165
Pro?Asp?Ser?Ser?Ala?Thr?His?Pro?Glu?His?Ala?Thr?Phe?Lys?Val
170 175 180
Ile?Gln?Ser?Leu?Met?Ala?Ala?Thr?Ser?Ser?Asp?Arg?Arg?Pro?Lys
185 190 195
Leu?Leu?Gly?Gln?Ala?Leu?Lys?Glu?Val?Leu?Pro?Gly?Leu?Phe?Pro
200 205 210
Ser?Ser?Arg?Asp?Pro?Ile?Leu?Ala?Lys?Val?Val?Met?His?Gly?Ala
215 220 225
Gly?Val?Pro?Phe?Asp?Ala?Pro?Leu?Glu?Asp?Leu?Met?Arg?Glu?Ala
230 235 240
Ala?Tyr?Pro?Asp?Gly?Trp?Leu?Cys?Leu?Val?Val?Ile?Val?Leu
245 250
SEQ?ID?NO：6
＞ssATG5 Sclerotinia sclerotiorum Sclerotinia sclerotiorum
Met?Gln?Ser?Leu?Ile?Trp?Ala?Ser?Ala?Ile?Pro?Leu?Tyr?Ile?Thr
1 5 10 15
His?Ser?Ser?Ser?Thr?Ile?Pro?Tyr?Leu?Ile?Asn?Val?Pro?Arg?Val
20 25 30
Ser?Tyr?Leu?Ala?Leu?Leu?Phe?Pro?Arg?Leu?Thr?Ser?Phe?Phe?Gly
35 40 45
Glu?Asn?Val?Ser?Ser?Phe?Ser?Tyr?Glu?Gly?Ile?Leu?Leu?Lys?Asn
50 55 60
Leu?Pro?Val?Gly?Leu?Leu?Cys?Asp?Leu?Tyr?Gln?Pro?Glu?Leu?Pro
65 70 75
Trp?Arg?Ile?Glu?Leu?Gly?Asp?Gly?Pro?Leu?Phe?Asp?Ile?His?Asp
80 85 90
Thr?Phe?Ile?Asn?Ser?Val?Lys?Glu?Ala?Asp?Phe?Met?Arg?Asn?Gly
95 100 105
Asn?Ala?Lys?Gly?Ile?Met?Ser?Met?Ser?Lys?Glu?His?Ser?Thr?Gln
110 115 120
Leu?Trp?Asn?Ser?Val?Gln?Asp?Asn?Asp?Phe?Ser?Thr?Tyr?His?Lys
125 130 135
Ile?Ser?Thr?Ile?Leu?Leu?Asn?Pro?Ala?Thr?Ala?Leu?Lys?His?Ile
140 145 150
Pro?Leu?Arg?Ile?Tyr?Leu?Pro?Ser?Ser?Ser?Thr?Pro?Ser?Ser?Thr
155 160 165
Pro?His?Pro?Gly?Ser?Ser?Gly?Ser?Ser?Lys?Ala?Pro?Ser?Thr?Ala
170 175 180
Ser?Pro?Pro?Ser?Pro?Leu?Phe?Thr?Phe?Lys?Thr?Ile?Gln?Thr?Leu
185 190 195
Ile?Gln?Pro?Gln?Thr?Thr?Ser?Arg?Glu?Pro?Gln?Thr?Leu?Gly?Gly
200 205 210
Ala?Leu?Asn?Ser?Val?Leu?Pro?Thr?Leu?Phe?Pro?Ser?Lys?Arg?Asp
215 220 225
Ala?Ile?Leu?Ala?Glu?Val?Ile?Leu?His?Gly?Ala?Thr?Val?Pro?Phe
230 235 240
Lys?Ala?Val?Leu?Glu?Asp?Leu?Met?Arg?Glu?Ala?Ser?Tyr?Ala?Asp
245 250 255
Gly?Trp?Leu?Asn?Val?Cys?Val?Val?Met?Leu?Asn
260 265
SEQ?ID?NO：7
＞snATG5 Stagonospora nodorum wheat glume blight bacterium
Met?Ser?Ser?Arg?Glu?Val?Thr?Ser?Arg?Leu?Arg?Glu?Lys?Val?Trp
1 5 10 15
Asn?Gly?Ser?Val?Pro?Leu?Glu?Ile?Arg?Leu?His?Lys?Gly?Asp?Cys
20 25 30
Arg?Thr?Tyr?Asp?Asp?Ser?Asp?Ala?Tyr?Leu?Ile?Gln?Phe?Pro?Arg
35 40 45
Leu?Ser?Tyr?Leu?Ala?Leu?Leu?Ile?His?Lys?Leu?His?Ala?Phe?Phe
50 55 60
Ala?Pro?Ser?Leu?Ile?Tyr?Pro?Asp?Ile?His?Pro?Ser?Asp?Leu?Trp
65 70 75
Phe?Ser?Tyr?Glu?Gly?Val?Pro?Leu?Lys?Trp?His?Tyr?Pro?Leu?Gly
80 85 90
Leu?Leu?Tyr?Asp?Leu?Tyr?Ser?Gly?Ala?Glu?Pro?Tyr?His?Pro?Ser
95 100 105
Asp?Ser?Pro?Pro?Pro?Ser?Pro?Thr?Thr?Pro?Ser?Lys?Gln?Asp?Ser
110 115 120
Lys?Gln?Pro?Leu?Pro?Trp?Arg?Leu?Thr?Leu?His?Thr?Ser?Ala?Tyr
125 130 135
Pro?Thr?Thr?Gln?Leu?Ile?Pro?Leu?Asp?Asn?Asn?Asn?Leu?Gln?Ile
140 145 150
His?Asp?Leu?Phe?Ile?His?Ser?Val?Lys?Glu?Ala?Asp?Tyr?Leu?Arg
155 160 165
Thr?Gly?Thr?Gly?Lys?Thr?Val?Met?Phe?Leu?Ser?Gln?Ala?Asp?Ser
170 175 180
Thr?Gln?Leu?Trp?Asp?Ala?Val?Val?Lys?His?Asp?Phe?Ala?Leu?Phe
185 190 195
Asn?Pro?Ile?Asn?Gln?Lys?Leu?Leu?Asn?Pro?Gln?Gly?Val?Asn?Leu
200 205 210
Arg?His?Leu?Pro?Val?Arg?Leu?Tyr?Leu?Pro?His?Ala?Gly?Val?Asp
215 220 225
Glu?Glu?Asp?Arg?Gly?Met?Gly?Ser?Val?Arg?Val?Val?Gln?Ser?Leu
230 235 240
Val?Lys?Val?Glu?Val?Gly?Ser?Arg?Gln?Pro?Gln?Thr?Ile?Gly?Thr
245 250 255
Ala?Leu?Asn?Gln?Ile?Leu?Pro?Thr?Leu?Phe?Pro?Ser?Arg?Arg?Ser
260 265 270
Ala?Leu?Leu?Ala?Gln?Ala?Val?Leu?His?Gly?Ala?Val?Val?Pro?Leu
275 280 285
Gly?Ala?Ser?Val?Glu?Glu?Leu?Ile?Arg?Ser?Val?Ala?Tyr?Leu?Asp
290 295 300
Gly?Trp?Leu?His?Ile?Ala?Ile?Val?Met?Met?Gly
305 310
Claims (7)
1, a kind of epiphyte pathogenic gene mgATG5 that comes from Pyricularia oryzae is characterized in that: the nucleotide sequence of this gene or its complementary nucleotide sequence are SEQ ID NO:1.
2, the cDNA sequence of gene mgATG5 coding as claimed in claim 1, it is characterized in that: described cDNA sequence has the nucleotide sequence shown in the SEQID NO:2.
3, gene mgATG5 encoded protein matter as claimed in claim 1, it is characterized in that: described protein has the aminoacid sequence shown in the SEQ IDNO:3.
4, the promotor of gene mgATG5 as claimed in claim 1 is characterized in that: described promotor has the nucleotide sequence shown in the SEQ ID NO:4.
5, the expression that utilizes gene mgATG5 as claimed in claim 1 is as target, in the application that designs and screen in the antifungal drug.
6, utilize protein expression as claimed in claim 3 and modification as target, the application in design and screening antifungal drug.
7, in conjunction with utilizing protein as claimed in claim 3 and promotor as claimed in claim 4, in the application that designs and screen in the antifungal drug as target.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710067584 CN101050463A (en) | 2007-03-16 | 2007-03-16 | Pathogenicity gene mgATG5 of fungus from rice blast germ, and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200710067584 CN101050463A (en) | 2007-03-16 | 2007-03-16 | Pathogenicity gene mgATG5 of fungus from rice blast germ, and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101050463A true CN101050463A (en) | 2007-10-10 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200710067584 Pending CN101050463A (en) | 2007-03-16 | 2007-03-16 | Pathogenicity gene mgATG5 of fungus from rice blast germ, and application |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935660A (en) * | 2010-09-02 | 2011-01-05 | 北京大学 | Promoter induced by Magnaporthe oryzae and its application |
| CN104964865A (en) * | 2015-01-16 | 2015-10-07 | 湖南省茶叶研究所（湖南省茶叶检测中心） | Cotton blue dyeing method for section of diseased leaf |
| CN106047901A (en) * | 2016-06-08 | 2016-10-26 | 浙江省农业科学院 | Fungus pathogenic gene Moarrdc1 derived from Magnaporthe Oryzae and application |
| CN108157385A (en) * | 2017-12-01 | 2018-06-15 | 昆明理工大学 | A kind of purposes of aspartic endopeptidase gene |
| CN108546708A (en) * | 2018-04-19 | 2018-09-18 | 贵州大学 | A kind of screening technique of filamentous fungi knock out mutants body |
-
2007
- 2007-03-16 CN CN 200710067584 patent/CN101050463A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101935660A (en) * | 2010-09-02 | 2011-01-05 | 北京大学 | Promoter induced by Magnaporthe oryzae and its application |
| CN104964865A (en) * | 2015-01-16 | 2015-10-07 | 湖南省茶叶研究所（湖南省茶叶检测中心） | Cotton blue dyeing method for section of diseased leaf |
| CN106047901A (en) * | 2016-06-08 | 2016-10-26 | 浙江省农业科学院 | Fungus pathogenic gene Moarrdc1 derived from Magnaporthe Oryzae and application |
| CN108157385A (en) * | 2017-12-01 | 2018-06-15 | 昆明理工大学 | A kind of purposes of aspartic endopeptidase gene |
| CN108157385B (en) * | 2017-12-01 | 2020-04-10 | 昆明理工大学 | Application of aspartic acid endopeptidase gene |
| CN108546708A (en) * | 2018-04-19 | 2018-09-18 | 贵州大学 | A kind of screening technique of filamentous fungi knock out mutants body |
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