CN107200773A - Come from pathogenic gene MoSNT2 of Pyricularia oryzae and application thereof - Google Patents

Abstract

The invention discloses a pathogenic protein MoSNT2 derived from Magnaporthe grisea, the protein is composed of the amino acid sequence shown in SEQ ID No: 1; the invention also discloses the gene MoSNT2 encoding the pathogenic protein, the gene It consists of the nucleotide sequence shown in SEQ ID No:2. The invention also discloses the use of the above-mentioned gene or protein as a target for designing or screening antifungal drugs. The invention provides an important target gene or target protein for the control of rice blast. The drug developed by using the gene or protein as a target can destroy the gene expression or protein function of the rice blast fungus, and can effectively control the pathogenicity and prevalence of the rice blast fungus.

Description

Pathogenic gene MoSNT2 derived from rice blast fungus and its use

technical field

The invention belongs to the field of microbial genetic engineering, and specifically relates to a pathogenic gene MoSNT2 derived from rice blast fungus, and also relates to the use of the gene.

Background technique

Rice blast (having another name called rice fever) is one of the important diseases of rice, which can cause a substantial reduction in production, and when serious, reduce production by 40% to 50%, or even fail to harvest. Rice blast occurs in all rice regions in the world, and most of them occur on leaves and nodes, especially the early and severe occurrence of panicle blast or node blast, which can cause white ears and even extinction. The pathogen that causes rice blast is Magnaporthe oryzae, a semi-living parasitic fungus (Magnaporthe oryzae). It has the characteristics of many physiological races, rapid reproduction and variation, and strong prevalence, which poses a huge threat to rice production. In order to ensure the safety of rice production, it is urgent to find effective ways to control rice blast.

The infection process of rice blast fungus to host rice is mainly divided into the following stages: conidia adhere to the host, spores germinate germ tubes and differentiate to produce appresses that infect organs, appresses produce infection nails and penetrate plant epidermis, invade Infected hyphae proliferate in plant tissues and secrete effector proteins to inhibit host immunity, mycelia kill plant tissues to form necrotic spots, form conidiophores and release conidia (Wilson et al., Nature Reviews Microbiology, 2009, 7(3) :185-195). Many genes play an important role in the process of rice blast infecting the host rice, regulating the development of infected organs and the proliferation of infection hyphae, thus determining the pathogenicity and infectivity of rice blast. Elucidating the molecular functions of these pathogenicity-related genes will help to develop and utilize new targets for rice blast control.

Dean et al. (Dean et al., 2005, 434(7036): 980-986) completed the genome sequence determination of Magnaporthe grisea strain 70-15. Seoul National University in South Korea constructed a mutant library of Magnaporthe grisea and reported 202 pathogenic genes (Jeon et al., Nature Genetics, 2007(39): 561-565). Chinese researchers have sequenced and interpreted the genome of the epidemic strains of Magnaporthe grisea, and identified many genes related to the pathogenicity of Magnaporthe grisea. The transcription factors, secreted effector proteins, and secretory regulatory factors encoded by these genes , plays a crucial role in the process of blast fungus infection of host rice (Dong et al., 2015, PLoS Pathogens 11(4):e1004801). Scholars at home and abroad have identified many pathogenicity-related genes (Yan et al., Current Opinion in Microbiology, 2016, 34:147–153). The metabolic and signaling pathways involved in these pathogenic genes mainly include: melanin synthesis, fatty acid β-oxidation, trehalose metabolism, glyoxylate cycle, cell autophagy, cell secretion, cAMP/PKA, G protein, MAPK signaling pathway, etc. . However, for these metabolic and signaling pathways, currently available fungal-specific control targets are still very limited.

At present, the method of controlling plant pathogenic fungi is mainly chemical control. Identifying and analyzing the pathogenicity genes of plant pathogenic fungi, especially the functional genes related to the host invasion process, can provide important target sites for the design and screening of antifungal drugs. The targets of traditional antifungal drugs mainly include important enzymes related to cell wall synthesis, synthetases of key components such as sterols and sphingomyelin in cell membranes, and synthesis machinery of fungal proteins and nucleic acids, etc. (Yu Xinxu, 2013, Master of Fujian Agriculture and Forestry University Dissertation; Liu Xiaohuan, Journal of Pharmaceutical Analysis, 2015, 35(2): 193-202). Tricyclazole is widely used in the control of rice blast fungus. It can destroy the penetrating function of appressoria in infected organs, and its target is trihydroxynaphthalene reductase related to cell wall melanin synthesis. Rice blast fungus is prone to mutation, rapid reproduction and strong adaptability. Long-term use of a single antifungal drug will easily cause the pathogen to accumulate drug resistance, resulting in a decline in control efficacy. Therefore, the use of molecular biology techniques to clone and identify new disease-causing genes that play an important role in the infection process of the rice blast fungus can provide important drug targets for the design and screening of new antifungal agents. Comprehensive prevention and control has very important theoretical significance and application value.

Contents of the invention

Aiming at the current problem that rice blast is seriously harmful and lacks effective control methods, the purpose of the present invention is to provide a pathogenic protein derived from rice blast fungus.

Another object of the present invention is to provide a gene encoding the above-mentioned pathogenic protein. The gene plays an important role in the mycelium vegetative growth, conidia production and germination, appressorium formation and pathogenicity of Magnaporthe grisea.

The third object of the present invention is to provide an expression vector containing the above gene.

The fourth object of the present invention is to provide the use of the above-mentioned pathogenic protein.

The fifth object of the present invention is to provide the use of the above-mentioned genes.

The sixth object of the present invention is to provide the use of the above expression vector.

To achieve the above object, the technical scheme of the present invention is as follows:

The present invention provides a pathogenic protein derived from Magnaporthe grisea, named as MoSnt2, the amino acid sequence of the protein is as follows:

(1), having the amino acid sequence shown in SEQ ID No: 1;

(2) An amino acid sequence formed by adding, replacing or deleting the amino acid sequence shown in SEQ ID No: 1 by one or several amino acid residues, and having the pathogenicity function of rice blast.

The present invention also provides a gene encoding the above-mentioned pathogenic protein, named as MoSNT2, the gene has the following nucleotide sequence (a), (b) or (c):

(a) have the nucleotide sequence shown in SEQ ID No: 2;

(b) a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID No:2 under stringent conditions;

(c) The nucleotide sequence shown in SEQ ID No: 2 is formed by adding, deleting or replacing one or several bases, and encodes a nucleotide sequence having the function of rice blast pathogenic protein.

The present invention also provides expression vectors containing the above genes.

The present invention also provides the application of the above-mentioned pathogenic protein as a target for designing or/and screening antifungal drugs.

The present invention also provides the application of the above gene as a target for designing or/and screening antifungal drugs.

The fungus mentioned in the above application refers to Magnaporthe oryzae and the like.

The present invention also provides the application of the above expression vector in screening antifungal drugs.

The fungus mentioned in the above application refers to Magnaporthe oryzae and the like.

The present invention has the advantages and beneficial effects: the present invention provides an important target gene or target protein for preventing and controlling the rice blast fungus. The rice blast pathogenic protein of the present invention has important effects on various growth and development stages such as mycelia vegetative growth, conidia production and germination, appressorium formation, and pathogenicity of the rice blast fungus. The gene or protein is used as a target to develop The drug can destroy the gene expression or protein function of Femurora oryzae, and can effectively control the pathogenicity and prevalence of Magnaporthe grisea.

Description of drawings

Figure 1. Histograms of MoSNT2 gene expression levels at different developmental stages; 1 is liquid vegetative hyphae; 2 is conidiophores; 3 is conidia; 4 is epispores developed for 4 hours; 5 is developed for 8 hours Epispora; 6: Episporium developed for 12 hours; 7: Episporium developed for 16 hours; 8: Episporium developed for 20 hours; 9: Episporium developed for 24 hours; 10 was infection hyphae growing on rice leaf sheaths; Infectious hyphae that stain rice leaves.

Figure 2. Schematic diagram of the construction process of MoSNT2 gene knockout mutants.

Figure 3. The PCR detection electrophoresis pattern of the MoSNT2 knockout mutant using P1 and P2 as primers; 1 is the DNA molecular weight standard; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is △Mosnt2.3; 5 △Mosnt2.4; 6 is △Mosnt2.5; 7 is wild-type Guy11.

Figure 4. The PCR detection electrophoresis pattern of the MoSNT2 knockout mutant with P3 and P4 as primers; 1 is the DNA molecular weight standard; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is △Mosnt2.3; 5 △Mosnt2.4; 6 is △Mosnt2.5; 7 is wild-type Guy11.

Figure 5. The southern hybridization pattern of the MoSNT2 knockout mutant; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is △Mosnt2.3; 5 is △Mosnt2.4; 6 is △ Mosnt2.5.

Figure 6. Photographs of colonies and aerial hyphae of MoSNT2 knockout mutants on CM plate medium; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is Compl.

Fig. 7. Micrographs of aerial hyphae growth and sporulation of MoSNT2 knockout mutants; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is Compl.

Fig. 8. Histogram of equal sporulation of MoSNT2 knockout mutants; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is Compl.

Figure 9. Micrographs of isospore morphology of MoSNT2 knockout mutants; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is Compl.

Figure 10. Photographs of the colony morphology of MoSNT2 knockout mutants and others under the stress of the cell wall inhibitor CFW; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is Compl.

Figure 11. Photographs of colony morphology of MoSNT2 knockout mutants and others under H ₂ O ₂ oxidative stress; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is Compl.

Figure 12. Microphotographs of the development of epispora infecting organs such as MoSNT2 knockout mutants; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is Compl.

Figure 13. Photos of the pathogenicity of MoSNT2 knockout mutants to host rice leaves; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is Compl.; blank control.

Figure 14. Photos of the pathogenicity of MoSNT2 knockout mutants to host rice roots; 1 is Guy11; 2 is △Mosnt2.1; 3 is △Mosnt2.2; 4 is Compl.; blank control.

detailed description

The present invention will be further explained and illustrated by the following examples, but the protection scope of the present invention is not construed as any limitation. The methods used in the following examples are conventional methods unless otherwise specified.

Example 1: Isolation and cloning of the MoSNT2 gene

(1) The SNT2 protein plays an important role in the oxidative stress response of yeast (Baker et al., Molecular and Cellular Biology, 2013, (33) 19:3735–3748), but the function of this protein in Magnaporthe grisea is unknown. There is a certain similarity between the protein sequences of yeast and Magnaporthe oryzae, so first search the yeast SNT2 protein sequence from the yeast genome database (http://www.yeastgenome.org/), and then use blastP to search the genome of Magnaporthe oryzae Database ( http://www.broadinstitute.org/annotation/genome/magnaporthe_grisea/MultiHome.html ) was searched for homologous sequences, and a predicted protein MGG_04421 of Magnaporthe grisea was obtained, named MoSnt2. SEQ ID NO: 1.

(2) Cloning of the MoSNT2 gene: for cloning the cDNA sequence of MoSNT2, first use the RNeasy Plant MiniKit kit (Qiagen Company) to extract total RNA from the wild-type strain Guy11 of Magnaporthe grisea, and then use the SuperScript III Reverse Transcriptase kit (Invitrogen Company), the total RNA was reverse-transcribed to generate the first-strand cDNA, and then SNT2-cDNA-For and SNT2-cDNA-Rev were used as primers, and the MoSNT2 gene coding sequence was cloned from the first-strand cDNA using Phusion high-fidelity enzyme (ThermoFisher Company) , a total of 5382bp, and finally connected to the cloning vector pEASY-Blunt (Quanshijin Company) for sequencing analysis to obtain the clone of the MoSNT2 gene; see SEQ ID NO: 2 for the sequence of the MoSNT2 gene.

The PCR reaction system (50 μl): Guy11 strain cDNA 0.5 μl (50ng), Phusion DNA polymerase 0.5 μl (2U/μl), 5×Phusion HF buffer 10 μl, dNTP (25mmol/L, each) 1 μl, upstream primer (50 μmol/L) 0.5 μl, downstream primer (50 μmol/L) 0.5 μl, ddH ₂ O 37 μl. The PCR reaction program was: 98°C for 30s; 35 cycles of 98°C for 10s, 60°C for 30s, 72°C for 3.5min; 72°C for 10min, 4°C for heat preservation. Described primer SNT2-cDNA-For (upstream primer) and SNT2-cDNA-Rev (downstream primer) are:

SNT2-cDNA-For: 5'-ATGGCTCAAAAGGGTTCAGGTG-3' (SEQ ID NO: 3);

SNT2-cDNA-Rev: 5'-AGACAGTAGATTTCGCAACGAAG-3' (SEQ ID NO: 4).

Example 2: Quantitative RT-PCR detection of MoSNT2 gene expression levels at different developmental stages

(1) Preparation of media and solutions:

(1) CM liquid medium: its composition and proportion are: D-Glucose 10g, Peptone 2g, YeastExtract 1g, Casamino Acids 1g, 20×Nitrate Salts ^* 50ml, Vitamin Solution ^# 1ml, TraceElements ^& 1ml, add ddH ₂ O to 1000ml, and adjust the pH value to 6.5 with 1mol/L NaOH solution.

(2) CM agar plate: its composition and proportion are: D-Glucose 10g, Peptone 2g, YeastExtract 1g, Casamino Acids 1g, 20×Nitrate Salts ^* 50ml, Vitamin Solution ^# 1ml, TraceElements ^& 1ml, agar powder 15g, Add ddH ₂ O to 1000ml, and adjust the pH value to 6.5 with 1mol/L NaOH solution; sterilize with damp heat at 121°C for 20min.

(3) ^* 20×Nitrate Salts (1000ml) composition and proportion are: NaNO ₃ 120g, KCl 10.4g, MgSO ₄ 7H ₂ O 10.4g, KH ₂ PO ₄ 30.4g, add ddH ₂ O to 1000ml, 121 ℃ damp heat sterilization for 20min.

(4) ^# Vitamin Solution (1000ml) composition and proportion are: Biotin 0.1g, Pyridoxin 0.1g, Thiamine 0.1g, Riboflavin 0.1g, PABA(p-aminobenzoic acid) 0.1g, NicotinicAcid 0.1g, add ddH ₂ O To 1000ml, sterilized by filtration, and stored in the dark at 4°C.

(5) ^& Trace Elements (100ml) composition and proportion are: ZnSO ₄ 7H ₂ O 2.2g, H ₃ BO ₃ 1.1g, MnCl ₂ 4H ₂ O 0.5g, FeSO ₄ 7H ₂ O 0.5g, CoCl ₂ ·6H ₂ O 0.17g, CuSO ₄ ·5H ₂ O 0.16g, Na ₂ MoO ₄ ·2H ₂ O 0.15g, add ddH ₂ O to 100ml, sterilize by filtration, store in dark at 4°C.

(2) Test method:

The wild-type strain Guy11 of Magnaporthe grisea was used as material to collect vegetative mycelia in CM liquid medium, conidiophores and conidia in the sporulation stage on the CM agar plate, bacteria at different time points in the appressorium development stage, and Colonize rice leaf sheaths or leaf infection hyphae; use RNeasy Plant Mini Kit (Qiagen) to extract total RNA from the collected cells, and then use PrimeScript RT Reagent Kit with gDNA Eraser (TAKARA) to reverse transcribe Generate cDNA, then use RP1 and RP2 as primers, use SYBR Premix Ex Taq (TAKARA company) to carry out quantitative RT-PCR, and use the expression level of rice blast fungus gene β-tublin for normalization correction. The primers RP1 and RP2 are:

RP1: 5'-CAGGATTATGGAGTTCTTG-3' (SEQ ID NO: 5);

RP2: 5'-CTAGTATCGTTCACCTTAC-3' (SEQ ID NO: 6).

Results (see Figure 1) The expression level of MoSNT2 gene was the highest in conidiophores and conidia at the sporulation stage; during appressorium development, the expression level of MoSNT2 gene reached the highest value 12 hours after spore inoculation, infecting hyphae It is close to the expression level in vegetative hyphae. The above results indicated that the MoSNT2 gene was expressed in all stages of growth and development of Magnaporthe grisea.

Example 3: Construction of Magnaporthe grisea MoSNT2 gene knockout mutant

Proceed as follows:

A knockout mutant of the gene MoSNT2 was constructed using the Split-Marker gene knockout method (reference: Kershaw et al., P Natl Acad Sci USA, 2009, 106(37):15967-15972) (see FIG. 2 ). The coding sequence of about 2.5kbp of the gene MoSNT2 was selected as the targeting site. During the gene knockout process, the homologous recombination event replaced the targeting site sequence with the hygromycin selection marker gene HYG, thereby forming a gene knockout mutant. The specific construction steps are as follows:

(1) Using the genomic DNA of the blast fungus wild-type Guy11 as a template, using SNT2-LF-For and SNT2-LF-Rev, or SNT2-RF-For/SNT2-RF-Rev as primers, DNA polymerase KOD- Plus (Kangwei Century Biotechnology Co., Ltd.) performed PCR amplification to obtain the left-wing LF (SEQ ID NO: 7) or right-wing RF fragment (SEQ ID NO: 8) of the MoSNT2 gene. The PCR reaction system (50μl) is: Guy11 genomic DNA 0.5μl (100ng), Taq polymerase 0.5μl (5U/μl), 10×buffer (+MgCl ₂ , 25mmol/L) 5μl, dNTP (25mmol/L, each ) 0.5 μl, upstream primer (50 μmol/L) 0.5 μl, downstream primer (50 μmol/L) 0.5 μl, ddH ₂ O 42.5 μl. The PCR reaction program was as follows: 94°C for 5 min; 35 cycles of 94°C for 30s, 58°C for 30s, 72°C for 1 min; 72°C for 10 min, 4°C for heat preservation. The primers SNT2-LF-For and SNT2-LF-Rev are:

SNT2-LF-For: 5'-AATCCCTGCGGGCTACTT-3' (SEQ ID NO: 9);

SNT2-LF-Rev: 5'-CCTTCAATATCATTCTTCTGTCGACCTCACAAAGGCACCGCTG-3 (SEQ ID NO: 10).

The primers SNT2-RF-For and SNT2-RF-Rev are:

SNT2-RF-For: 5'-CCCAGCACTCGTCCGAGGGCAAAGGAATAGGCATCTCCAAGTTCGGCTCA-3' (SEQ ID NO: 11);

SNT2-RF-Rev: 5'-ACAACCGCAACCCTGGGA-3' (SEQ ID NO: 12).

(2) Using HYG-For and HY-split, or YG-split and HYG-Rev as primers to amplify the fragment HY of the hygromycin selection marker gene HYG (see SEQ ID NO: 17) or respectively from the plasmid template pCB1004 Fragment YG (see SEQ ID NO: 18). Described primer HYG-For and HY-split are:

HYG-For: 5'-GTCGACAGAAGATGAT-3' (SEQ ID NO: 13);

HY-split: 5'-TACTTCGAGCGGAGGCATCC-3' (SEQ ID NO: 14).

Described primer YG-split and HYG-Rev are:

YG-split: 5'-CGTTGCAAGACCTGCCTGAA-3' (SEQ ID NO: 15);

HYG-Rev: 5'-CTATTCCTTTGCCCTCGGACG-3' (SEQ ID NO: 16).

The above PCR reaction system (50 μl) is: pCB1004 plasmid template 0.5 μl (10ng), Taq polymerase 0.5 μl (5U/μl), 10×buffer (+MgCl ₂ , 25mmol/L) 5μl, dNTP (25mmol/L, each) 0.5 μl, upstream primer (50 μmol/L) 0.5 μl, downstream primer (50 μmol/L) 0.5 μl, ddH ₂ O 42.5 μl. The reaction program of PCR is: 94°C for 5min; 35 cycles of 94°C for 30s, 58°C for 30s, 72°C for 1min and 30s; 72°C for 10min, 4°C for heat preservation.

(3) Use agarose gel electrophoresis to recover the four fragments LF, RF, HY and YG amplified above; through fusion PCR, use the primer pair SNT2-LF-For and HY-split to connect the fragments LF and HY to form a fusion Fragment LF-HY (see SEQ ID NO: 19); simultaneously use primer pair YG-split and SNT2-RF-Rev to ligate fragments YG and RF to form fusion fragment YG-RF (see SEQ ID NO: 20).

The above PCR reaction system (50 μl) for obtaining the fusion fragment LF-HY or YG-RF is: 1 μl (about 50 ng) of the upstream fragment, 1 μl (about 50 ng) of the downstream fragment, 0.5 μl (5 U/μl) of Taq polymerase, 10× 5 μl of buffer (+MgCl ₂ , 25 mmol/L), 0.5 μl of dNTP (25 mmol/L, each), 0.5 μl of upstream primer (50 μmol/L), 0.5 μl of downstream primer (50 μmol/L), and 41 μl of ddH ₂ O. The PCR reaction program was: 94°C for 5min; 35 cycles of 94°C for 30s, 58°C for 30s, 72°C for 1min and 30s; 72°C for 10min, 4°C for heat preservation.

(4) Obtaining of MoSNT2 knockout mutants: DNA fragments LF-HY and YG-RF were recovered by agarose gel electrophoresis, while referring to the method described by Tablot et al. (Tablot et al., The Plant Cell, 1993, 5: 1575-1590) Protoplasts of the wild-type strain Guy11 of Magnaporthe grisea were prepared, LF-HY and YG-RF fragments were co-transformed into the protoplasts, and fungal transformants were screened on CM agar plates containing hygromycin. As a result, MoSNT2 gene knockout transformants (or called MoSNT2 knockout mutants) were obtained. In the gene replacement process (as shown in Figure 2), most of the exogenous DNA is inserted into the genome of Magnaporthe grisea, because the LF and RF sequences of the fusion fragment are homologous to the MoSNT2 site sequence on the genome, so there will be Homologous substitutions occurred to obtain MoSNT2 knockout mutants.

Example 4 Identification of Magnaporthe grisea MoSNT2 Gene Knockout Mutant

Proceed as follows:

(1) Extract the genomic DNA of the transformant obtained in Example 3, and use P1 and P2, or P3 and P4 as primers to carry out PCR amplification respectively, wherein the randomly inserted fragments cannot produce amplified bands, only at the MoSNT2 gene locus Transformants with homologous substitutions can produce amplified bands. The PCR reaction system (25 μl) for the detection of transformants is: 0.5 μl (50 ng) of genomic DNA of transformants, 0.3 μl (5 U/μl) of Taq polymerase, 2.5 μl of 10×buffer (+MgCl ₂ , 25 mmol/L), dNTP (25 mmol/L, each) 0.2 μl, upstream primer 0.2 μl, downstream primer 0.2 μl, ddH ₂ O 21.1 μl. The PCR reaction program was: 94°C for 5 min; 35 cycles of 94°C for 30 s, 58°C for 30 s, 72°C for 2.5 min; 72°C for 10 min, 4°C for heat preservation. Wherein said primers P1 and P2 are:

P1: 5'-GGTAGGAGAAAGGCTGATGG-3' (SEQ ID NO: 21);

P2: 5'-GCTTCTGCGGGCGATTTGTGTA-3' (SEQ ID NO: 22).

Described primers P3 and P4 are:

P3: 5'-CAGCGAGAGCCTGACCTATTGC-3' (SEQ ID NO: 23);

P4: 5'-CTTCCTGACGCTTCCGATAA-3' (SEQ ID NO: 24).

Results The size of the amplification product obtained by using P1 and P2 as primers was 2.8 kbp (see Figure 3), and the size of the amplification product obtained by using P3 and P4 as primers was 2.2 kbp (see Figure 4). The five transformants were knockout mutants produced by homologous substitution, named △Mosnt2.1, △Mosnt2.2, △Mosnt2.3, △Mosnt2.4 and △Mosnt2.5 respectively.

(2) Southern hybridization identification: Genomic DNA of the wild-type strain Guy11 and 5 knockout mutants were extracted, and then digested with the restriction endonuclease SphI, and the digested products were subjected to agarose gel electrophoresis and then transferred to a membrane. Digoxigenin DNA labeling and detection kit (Roche Company) was used in Southern hybridization test. The hybridization probe is an LF fragment, and the PCR reaction system for synthesizing the probe is the same as the aforementioned operation method.

Results (see Figure 5) wild-type Guy11 strain has hybridization signals at 1.2kbp and 4.1kbp, and the knockout mutant ΔMosnt2 makes a SphI restriction site in the coding frame disappear due to homologous substitution (as shown in Figure 2 ), resulting in hybridization signals at 4.1kbp and 5.5kbp, the southern identification results confirmed that △Mosnt2.1, △Mosnt2.2, △Mosnt2.3, △Mosnt2.4 and △Mosnt2.5 are all correct MoSNT2 knockout Remove mutants.

Example 5 Construction of MoSNT2 Gene Complementary Vector and Reverted Bacterial Strain

In order to verify that the phenotype of the mutant is caused by the deletion of the gene MoSNT2, the complementary vector pNEB was constructed according to the yeast cell recombination method described by Oldenburg et al. (Oldenburg et al., 1997, Nucleic Acids Research, 25:451-452) -MoSNT2-GFP-BAR, the C-terminus of the MoSnt2 protein in the carrier is labeled with green fluorescent protein GFP to facilitate the analysis of the protein subcellular localization of MoSnt2. The method for constructing the carrier is as follows:

(1) Use the genomic DNA of Guy11 as a template, and use SNT2-For1 and SNT2-Rev1, SNT2-For2 and SNT2-Rev2, SNT2-For3 and SNT2-Rev3, or SNT2-For4 and SNT2-Rev4 as primers for PCR amplification, respectively. The amplified product contains four overlapping fragments of the promoter and coding frame of the full-length MoSNT2 gene; at the same time, GFP-SNT2-For and GFP-SNT2-Rev are used as primers to amplify the coding frame and terminator of green fluorescent protein; Use BAR-For and BAR-Rev as primers to amplify the resistance selection marker gene BAR of glufosinate-ammonium; the six DNA fragments obtained above were combined with the vector pNEB-Nat linearized by SacI and HindIII double enzyme digestion (He et al., Plos One, 2012, 7(3):e33270) together, co-transform yeast competent cells, under the action of the recombination repair system in yeast cells, DNA fragments and vectors can be connected to form recombinant vectors, named pNEB-MoSNT2-GFP-BAR. The primers SNT2-For1 and SNT2-Rev1 described therein are:

SNT2-For1:

5'-AACTGTTGGGAAGGGCGATCGGTGCGGGCCACTAGTTCATTGTTACCGAAAGAAGCC-3' (SEQ ID NO: 25);

SNT2-Rev1: 5'-AACCCTTTTGAGCCATGAATTCGACGACCAGGCAGGCAAATC-3' (SEQ ID NO: 26).

The primers SNT2-For2 and SNT2-Rev2 are:

SNT2-For2: 5'-CCTGCCTGGTCGTCGAATTCATGGCTCAAAAGGGTTCAGGTG-3' (SEQ ID NO: 27);

SNT2-Rev2: 5'-AGTGGATTCCCAGGTAGCG-3' (SEQ ID NO: 28).

The primers SNT2-For3 and SNT2-Rev3 are:

SNT2-For3: 5'-TGTACCATGCCAGCCTCTG-3' (SEQ ID NO: 29);

SNT2-Rev3: 5'-TGACGCTTCCGATAATGTTCT-3' (SEQ ID NO: 30).

The primers SNT2-For4/SNT2-Rev4 are:

SNT2-For4: 5'-ACCCAACCAGCATTCTTCCC-3' (SEQ ID NO: 31);

SNT2-Rev4: 5'-AGACAGTAGATTTCGCAACG-3' (SEQ ID NO: 32).

The primers GFP-SNT2-For and GFP-SNT2-Rev are:

GFP-SNT2-For: 5'-GCCAGCCCTTCGTTGCGAAATCTACTGTCTcccgggATGGTGAGCAAGGGCGAGGA-3' (SEQ ID NO: 33);

GFP-SNT2-Rev:

5'-AGTGCTCTTCAATATCATTCTTCTGTCGACGGCCGGCCGTGGAGATGTGGAGTGGGC-3' (SEQ ID NO: 34).

Described primer BAR-For and BAR-Rev are:

BAR-For: 5'-GTCGACAGAAGATGATATTG-3' (SEQ ID NO: 35);

BAR-Rev:

5'-TTCACACAGGAAACAGCTATGACCATGATTATTTAAATACGCGTGGCCGGCCGTCGACCTAAATCTCGGTGACGG-3' (SEQ ID NO: 36).

(2) Transform the recombinant vector pNEB-MoSNT2-GFP-BAR obtained in step (1) into the mutant △Mosnt2.1 protoplast to obtain a revertant, and name the revertant: Compl. (Compl. is English The abbreviated form of the word complement means supplement and restoration).

Example 6: Comparative Analysis of MoSNT2 Knockout Mutant △Mosnt2 and Wild Type and Reverted Strains

(1) Colony growth test of knockout mutant △Mosnt2

The wild-type Guy11, the knockout mutants △Mosnt2.1, △Mosnt2.2 and the revertant strain Compl. were inoculated on CM agar medium, cultured at 25°C for 10 days, and then the colonies were observed.

The results (see Figure 6) the bacterial colonies of the knockout mutants △Mosnt2.1 and △Mosnt2.2 were significantly smaller than those of the wild-type strain Guy11 and the reverting strain Compl.; in addition, the knockout mutants △Mosnt2.1 and △Mosnt2.2 The aerial hyphae of the colony were few, but the aerial hyphae of the wild-type Guy11 and the revertant strain Compl. were dense. It shows that the deletion of MoSNT2 gene affects the colony morphology and growth rate of Magnaporthe grisea.

(2) Detection of sporulation of knockout mutant △Mosnt2

(a) The wild-type strain Guy11, the knockout mutants △Mosnt2.1 and △Mosnt2.2, and the revertant strain Compl. were inoculated on CM agar medium, 16 hours of light/8 hours of darkness, and grown at 25°C After 10 days, the production of conidia was observed under a microscope.

Results (see Figure 7) A large number of conidia were formed on the aerial hyphae of the wild-type Guy11 and the revertant strain Compl., but very few conidia were formed on the aerial hyphae of the knockout mutants △Mosnt2.1 and △Mosnt2.2 Conidia were formed; it indicated that the deletion of MoSNT2 gene greatly reduced the conidia production.

(b) Scrape the conidia on the surface of the colony with 5 ml of sterile water, filter the spore suspension with a lens tissue, and then count the number of spores under a microscope.

Results (see Figure 8) The number of spores produced by the knockout mutants △Mosnt2.1 and △Mosnt2.2 was significantly less than that of the wild-type strain Guy11 and the revertant strain Compl. It shows that MoSNT2 plays a key role in the sporulation process.

(c) The spore morphology of the knockout mutant △Mosnt2 was observed under a microscope.

Results (see Figure 9) the spores of the wild-type Guy11 and the revertant strain Compl. presented a normal pear shape, while the conidia of the knockout mutants △Mosnt2.1 and △Mosnt2.2 were abnormal in shape, elongated and round shape or stick. It shows that MoSNT2 plays an important role in the process of spore morphogenesis.

The above results indicated that the MoSNT2 gene played an important role in the sporulation and spore morphogenesis of Magnaporthe grisea.

(3) Sensitivity test of knockout mutant △Mosnt2 to cell wall stress

Fluorescent whitening agent (Calcofluor white, CFW) can bind fungal cell wall component chitin, so it will affect the assembly and structure of fungal cell wall, causing cell wall stress to fungi. The wild-type Guy11, the knockout mutants △Mosnt2.1, △Mosnt2.2 strains and the reverting strain Compl. were inoculated on CM agar plates containing 200 μg/ml fluorescent whitening agent and grown for 5 days, and then the colony morphology was observed.

Results (see Figure 10): Compared with the wild-type Guy11 and the revertant strain Compl., the knockout mutants △Mosnt2.1 and △Mosnt2.2 significantly weakened the colony growth on the CFW stress plate and were more sensitive to CFW. This suggests that MoSNT2 affects the cell wall integrity of M. oryzae.

(4) Sensitivity test of knockout mutant △Mosnt2 to oxidative stress

Add hydrogen peroxide (H ₂ O ₂ ) to the CM agar medium to make the final concentration 2mM, 3mM or 5mM respectively, so as to form an oxidative stress growth environment. Wild-type Guy11, knockout mutants ΔMosnt2.1, Δ The Mosnt2.2 strain and the reverting strain Compl. were inoculated on CM agar medium supplemented with hydrogen peroxide (H ₂ O ₂ ), grown at 25° C. for 10 days, and then the colony morphology was observed.

The results (see Figure 11) hydrogen peroxide can significantly inhibit the colony growth of each bacterial strain, but its inhibitory degree to the knockout mutants △Mosnt2.1 and △Mosnt2.2 is significantly higher than that of the wild-type Guy11 and the reversion strain Compl.; When the hydrogen peroxide concentration was 5mM, both Guy11 and the revertant strain Compl. could grow, but the knockout mutants △Mosnt2.1 and △Mosnt2.2 could not grow at all. It shows that the knockout mutants are highly sensitive to oxidative stress, so MoSNT2 is an important factor regulating the oxidative stress response of M. oryzae.

(5) Observation test of epispores in organs infected by knockout mutant △Mosnt2

Conidia were collected from colonies grown on CM agar medium, prepared as a spore suspension, and then the spore suspension was inoculated onto a hydrophobic glass slide (product of Fisher Scientific), left at 25°C for 0, 10, and 24 hours, and then placed in Observe under a microscope.

The results (see Figure 12) both the wild-type Guy11 and the revertant strain Compl. could germinate and differentiate to form melanized appressoria on hydrophobic slides 10 hours after inoculation, but the knockout mutants △Mosnt2.1 and △Mosnt2. 2 Although they could germinate, they could not differentiate into epispores; 24 hours after inoculation, the epispores of wild-type Guy11 and the revertant strain Compl. matured, and the conidia collapsed due to autophagy, but the knockout mutant △Mosnt2.1 And △Mosnt2.2 still can't form epispora, and the conidia keep intact without collapsing. The above results suggest that MoSNT2 plays a key role in the formation of appressocytes.

Embodiment 7: Comparison test of rice blast pathogenicity of MoSNT2 gene knockout mutant

Proceed as follows:

(1) The wild-type strain Guy11 of Magnaporthe grisea, the knockout mutants △Mosnt2.1, △Mosnt2.2 and the reverting strain Compl. were inoculated on CM agar medium respectively, and grown at 25°C for 10 days, and then the growth was positive The agar block of the mycelia was punched. The rice variety CO39 (the rice material is preserved in the Rice Research Institute of Sichuan Agricultural University) is used as the host material.

(2) Bacteria blocks were inoculated on the leaves of CO39 grown for 21 days, and the leaves were inoculated with uninoculated agar blocks as a control. The incidence of rice leaves was observed 5 days after inoculation.

Results (see Figure 13) typical rice blast lesions were formed on rice leaves inoculated with wild-type Guy11 and the revertant strain Compl., but not on rice leaves inoculated with knockout mutants △Mosnt2.1 and △Mosnt2.2 Typical rice blast lesions, only form acute brown spots caused by hypersensitivity reactions.

(3) The bacteria blocks were inoculated on the roots of CO39 grown for 21 days, and the roots inoculated with uninoculated agar blocks were used as a control, and the disease was observed 7 days after the inoculation.

The results (see Figure 14) wild-type Guy11 and the revertant strain Compl. can make the roots necrotic and turn black, but the knockout mutants △Mosnt2.1 and △Mosnt2.2 cannot make the rice roots produce black necrotic spots, thus losing the ability to control Pathogenicity in rice roots.

The above results indicated that the MoSNT2 gene is the key gene of the pathogenicity of Magnaporthe grisea.

What have been listed above are only some specific embodiments of the present invention. But the present invention is not limited by the above embodiments, those skilled in the art can directly derive or associate many deformations from the content disclosed in the present invention, and any other changes, modifications, Substitution, combination, and simplification should all be regarded as equivalent replacement methods, and should all be included within the protection scope of the present invention.

SEQUENCE LISTING

<110> Sichuan Agricultural University

<120> Pathogenic gene MoSNT2 derived from Magnaporthe grisea and its use

<160> 36

<170> PatentIn version 3.5

<210> 1

<211> 1794

<212> PRT

<213> Magnaporthe oryzae

<400> 1

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

1 5 10 15

Thr Thr Thr Ser Ala His Lys Gly Thr Ala Glu Ser Lys Gln Ser Ser

20 25 30

Asn Ala Pro Ser Ser Gly Pro Ser Thr Thr Ser Ser Ser Ser Ala Ser

35 40 45

Val Leu Pro Lys Met Ser Asp Pro Gly Pro Pro Gln Ala Leu Gly Ser

50 55 60

Pro Ala Ser Glu Ser Thr Thr Lys Ser Ser Ser Thr Ala Lys Asp Ala

65 70 75 80

Thr Ala Gly Thr Thr Ala Ser Pro Tyr Gly Thr Arg Ser Arg Asn Arg

85 90 95

Gly Gly Asn Ser Arg Pro Asn Tyr Ala Glu Asp Lys Asp Ile Asp Met

100 105 110

Asp Ile Phe Glu Gln Leu His Pro Gln Lys Arg Asp Asp Asp Ser Lys

115 120 125

Lys Thr Ser Arg Gln Asn Ala Ser Ser Ser Ala Thr Asn Thr Gly Asp

130 135 140

Thr His Asn Thr Pro Pro Pro Pro Pro Arg Thr Thr Asn Gly Leu Ser

145 150 155 160

Ser Arg Lys Pro Leu Pro Met Asp Asn Lys Gln Ser Gln Ala Ala Ala

165 170 175

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

180 185 190

Thr Gly Ser Thr Gln Thr Thr Ser Lys Lys Arg Lys Ala Ala Ser Gln

195 200 205

Thr Ser Ser Asn Gln Pro Pro Ala Thr Glu Ser Thr His Val Pro Ala

210 215 220

Asn Ala Lys Lys Pro Ser Asn Asn Asn His Asn Ser Asn Ala Ala Ser

225 230 235 240

Ser His Gly Ala Asp Lys Gly Tyr Ala Ala Thr Asn Leu Leu Thr Phe

245 250 255

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

260 265 270

Gly Thr Tyr Leu Glu Val Asn Asp His Val Tyr Leu Val Cys Glu Pro

275 280 285

Pro Gly Glu Pro Tyr Tyr Leu Ala Arg Ile Met Glu Phe Leu His Ala

290 295 300

Lys Asn Asp Pro Ser Gln Pro Val Asp Ala Leu Arg Val Asn Trp Tyr

305 310 315 320

Tyr Arg Pro Lys Asp Ile Ala Arg Lys Val Asn Asp Thr Arg Ala Val

325 330 335

Phe Ala Thr Met His Ser Asp Ile Ser Pro Leu Thr Ser Leu Arg Gly

340 345 350

Lys Cys Thr Ile Lys His Lys Ala Glu Ile Lys Gly Lys Leu Glu Glu

355 360 365

Tyr Arg Lys Asn Pro Asp Cys Phe Trp Phe Glu Lys Leu Tyr Asp Arg

370 375 380

Tyr Ile Gln Lys Asn Tyr Glu Val Ile Pro Thr Phe Gln Ile Ile Asn

385 390 395 400

Val Pro Glu Lys Val Lys Lys Val Leu Asp Glu Arg Trp Lys Tyr Ile

405 410 415

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

420 425 430

Cys Arg Arg Cys Ser Gly Tyr Cys Ala Ser Asn Asp Ser Val Asp Cys

435 440 445

Ala Val Cys Glu His Thr Tyr His Met Asn Cys Val Arg Pro Pro Leu

450 455 460

Leu Lys Lys Pro Ser Arg Gly Phe Ala Trp Ser Cys Ala Ala Cys Ser

465 470 475 480

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

485 490 495

Leu Asp Pro Asn Ala Glu Ala Glu Glu Glu Glu Phe Phe Asp Glu Glu

500 505 510

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

515 520 525

Ala Asn Asp Met His Ile Pro Ala Thr Glu Glu Gln Met Tyr His Ala

530 535 540

Ser Leu Trp Pro Tyr Arg Tyr Leu Gly Ile His Cys Lys Val Glu Asp

545 550 555 560

Ala Leu Asp Tyr Asp Asp Arg Ile Tyr Pro Arg Ala Ser Thr Arg Val

565 570 575

Gly Pro Arg His Gln Ala Thr Val Leu Asp Trp Pro Gly Arg Pro Val

580 585 590

Gln Tyr Val Lys Ala Pro Glu Ile Glu Ile Lys Lys Thr Gly Arg Lys

595 600 605

Asp Gly Lys Leu Asn Lys Glu Ala Gln Ala Ala Leu Glu Ala Glu Lys

610 615 620

Val Ala Lys Ala Lys Arg Pro Lys Trp Ile Gln Asp Glu Pro Pro Gly

625 630 635 640

Tyr Val Pro Arg Gly Glu Asp Tyr Pro Asn Asp Asp Pro Arg Asn Thr

645 650 655

Ala Gln Leu His Trp Arg Pro Pro Glu Leu Asp Leu Pro Glu Glu Ser

660 665 670

Gly Pro Glu Glu Ala His Ile Ser Glu Ser Glu Ile Asp Lys Tyr Met

675 680 685

Glu Gln Ala Lys Gly Met Ala Leu Asp Leu Asp Leu Pro Glu His Ser

690 695 700

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

705 710 715 720

Asp Ala Glu Arg Ala Leu Glu Glu Leu Pro Lys Leu Ser Lys Glu Ala

725 730 735

Phe Asp Glu Pro Gln Leu Thr Ala Ala Glu Leu Lys Lys Phe Glu Glu

740 745 750

Gly Ile Ser Lys Phe Gly Ser Glu Leu Tyr Ser Val Lys Lys His Ile

755 760 765

Lys Thr Val Lys Pro Gly Thr Leu Val Arg Phe Tyr Tyr Thr Trp Lys

770 775 780

Lys Thr Glu Arg Gly Lys Gln Val Trp Gly Asn Tyr Ser Gly Arg Lys

785 790 795 800

Ser Lys Lys Glu Ala Lys Glu Ala Lys Lys Ala Glu Thr Ala Ser Gln

805 810 815

Asn Lys Met Gln Asp Asp Val Ala Asp Asp His Asp Asp Ser Ala Phe

820 825 830

Asp Ala Ala Lys Ala Ala Glu Lys Lys Arg Ser Phe Ile Cys Lys Phe

835 840 845

Cys Asn Thr Lys Ser Ser Arg Gln Trp Arg Arg Ala Pro Asn Ala Ser

850 855 860

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

865 870 875 880

Gly Val Gln Tyr Val Val Ala Leu Cys Arg Arg Cys Ala Glu Leu Trp

885 890 895

Arg Arg Tyr Ala Ile Gln Trp Glu Asp Val Asp Gln Leu Tyr Ser Lys

900 905 910

Val Ala Gln Ala Gly Gly Arg Ala Trp Lys Lys Lys Ile Asp Glu Glu

915 920 925

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

930 935 940

Ser Ser Ser Gly Ala Ala Thr Pro Pro Ser Asn Thr Thr Pro Ala Pro

945 950 955 960

Ala Ser Thr Gln Pro Ala Ala Ser Gly Gln Glu Pro Ala Arg Lys Lys

965 970 975

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

980 985 990

Thr Glu Pro Val Gly Thr Thr Thr Thr Thr Ala Pro Ala Ser Lys Lys Lys

995 1000 1005

Glu Lys Ala Ser Leu Glu Lys Glu Lys Glu Lys Glu Lys Glu Lys

1010 1015 1020

Glu Lys Glu Lys Glu Lys Glu Lys Glu Pro Val Lys Glu Lys Lys

1025 1030 1035

Glu Ala Pro Ala Pro Pro Pro Val Pro Glu Ile Pro Lys Pro Arg

1040 1045 1050

Thr Met Pro Cys Asp Ile Cys Arg Gln Leu Glu Pro Leu Gly Asp

1055 1060 1065

Gln His Ile Thr Cys Lys Glu Cys Arg Met Thr Val His Arg Asn

1070 1075 1080

Cys Tyr Gly Val Val Asp Asn Arg Asn Pro Gly Lys Trp Val Cys

1085 1090 1095

Asp Met Cys Ile Asn Asp Arg Ser Pro His Val Ser Ile His Tyr

1100 1105 1110

Lys Cys Val Leu Cys Pro Val Glu Tyr Thr Glu His Asp Phe Val

1115 1120 1125

Glu Pro Pro Lys Val Ser His Lys Lys Lys Thr Glu Lys Asp Arg

1130 1135 1140

Glu Arg Glu Arg Gln Glu Arg Glu Ala Ala Val Asn Ala Ala Glu

1145 1150 1155

His Tyr Arg Lys Arg Gln Glu Glu Leu Asn Arg Pro Val Asn Pro

1160 1165 1170

Arg Glu Pro Leu Lys Arg Thr Ala Asp Asn Asn Trp Val His Val

1175 1180 1185

Thr Cys Ser Val Trp Thr Pro Glu Val Lys Phe Gly Asn Ala Lys

1190 1195 1200

Ala Leu Glu Pro Ser Glu Gly Ile Pro Ser Ile Pro Arg Ser Arg

1205 1210 1215

Tyr Ser Glu Val Cys Glu Val Cys Lys Ser Thr Gly Gly Ala Cys

1220 1225 1230

Thr Asn Cys Pro Gln Cys Lys Ala Ser Val His Val Glu Cys Ala

1235 1240 1245

His Gln Ser Asp Asp Tyr Val Leu Gly Phe Glu Ile Thr Pro Val

1250 1255 1260

Lys Gly Ser Arg Arg Asp Gln His Asn Ile Val Thr Ile Gly Gly

1265 1270 1275

Glu Ser Gly Ser Met Ser Ala Ser Val Trp Cys Lys Leu His Ala

1280 1285 1290

Pro Lys Lys Thr Val Val His Gln Met Tyr Asp Val Val Asp Glu

1295 1300 1305

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

1310 1315 1320

Ala Asp Leu Thr Leu Thr Gly Cys Ala Arg Lys Ala Asn Leu Ile

1325 1330 1335

Ser Thr Ala Ala Arg Met Ser Asn Pro Ile Val Thr Thr Thr Thr Thr

1340 1345 1350

Ala Val Asn Arg Arg Ala Ser Thr Thr Thr Thr Val Ser Thr Thr Thr Thr

1355 1360 1365

Pro Ser Ala Met His Ile His Ser Leu Leu Asn Gly Asp Ser Pro

1370 1375 1380

Gly Asp Pro His Asp Leu Ala Val Pro Gly Gly Lys Ile Cys Ile

1385 1390 1395

Thr Cys Gly Val Asp Val Ser Pro Arg Trp Tyr Pro Ile Ser Asp

1400 1405 1410

Ser His Glu Arg Glu Leu Ala Asn Gly His Tyr Gly Ala Leu Gly

1415 1420 1425

Thr Glu Ala Gln Lys Phe Ala Glu Gln Arg His Phe Gln Cys His

1430 1435 1440

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

1445 1450 1455

Pro Pro Pro Pro Gln Asp Pro Pro Pro Val Ala Ala Asn Thr Ile

1460 1465 1470

Asn Pro Ser Gly Pro Glu Ala Thr Ala His Ser Ala Ala Val Met

1475 1480 1485

Thr Asn Gly Ile Asp His Gly Pro Asn Gly Val Asp Ala Ala Val

1490 1495 1500

Arg Arg Gly Thr Pro Pro Leu Thr Ser Pro Arg Gln Pro Glu His

1505 1510 1515

Asp His Ile Pro Gly Arg Pro Ser Pro Tyr Leu Trp Gln Ser Gly

1520 1525 1530

Leu Gln Ala Pro His Pro Thr Gly Leu Val His Pro Thr Val Pro

1535 1540 1545

Ile Gln Gly Pro Pro Pro Pro Met Gln Ala Pro Pro Leu Gln Pro

1550 1555 1560

Pro Pro Ile Ala Pro Pro Pro Met Ala Arg Ser Met Ser Gly Arg

1565 1570 1575

Gly Gln Thr Gly Val Gln Pro Pro Gly Pro Val Pro Pro Ser Gln

1580 1585 1590

Ala Tyr Gln Pro Leu Pro Pro Pro Pro Pro Pro Thr His Ser Ala Pro

1595 1600 1605

Ser Gly Pro Tyr Gly Asp Trp His Arg Thr Thr His His Gly Pro

1610 1615 1620

Pro Met Asn Gly Arg Pro Pro Ser Arg Ala Ser Arg Ile Ser Pro

1625 1630 1635

Ile Ile Pro Pro Leu Ala Pro Pro Ala Leu Arg Pro Pro Ser Leu

1640 1645 1650

His His Ser Pro His Ala Pro His Ala His Leu Thr Asn Gly His

1655 1660 1665

Met Val Asn Gly Ala Gly Ala Pro Gly Arg Arg Leu Ser Gly Pro

1670 1675 1680

Pro Pro Pro Pro Ser Arg Asp Gly Gln Gly Pro Tyr Met Gly Ser

1685 1690 1695

Tyr His Ser Pro Ala Pro Tyr His Ser Pro Ala Pro His Gln Ser

1700 1705 1710

Asn Gly Thr Met Val Pro Pro Arg Ile Asp His Ala Phe Ala Ser

1715 1720 1725

Val Leu Asn Pro Pro Arg Ala Tyr Gly Asn Ser Gly Ser Val Gln

1730 1735 1740

Pro Pro Ala His Met Ser Pro Ala Val Ala Arg Asp Ala Pro Ile

1745 1750 1755

Ser Arg Asp Gly Pro Leu Leu Ser Gln Pro Pro Pro Pro Ala Arg

1760 1765 1770

Ala Pro Glu Ser Arg Pro Ala Thr Gly Ala Ser Ala Ser Pro Ser

1775 1780 1785

Leu Arg Asn Leu Leu Ser

1790

<210> 2

<211> 5382

<212>DNA

<213> Magnaporthe oryzae

<400> 2

atggctcaaa agggttcagg tggctcagat gccgcaacac ctaataccac gacgacgtcg 60

gctcacaaag gcaccgctga gagcaagcaa tcttccaatg cgccctcttc tgggccatcg 120

acgacatcgt cgtcatctgc atcggttctg cccaaaatgt cagatcccgg tccaccacag 180

gccctaggaa gtccagcgtc agaatcgaca accaagtcga gttcgactgc caaggacgcg 240

acggccggca ccacggcatc tccttatggc acgcgatctc gcaacagggg cggcaactct 300

cgtcccaatt atgcagagga caaggacatt gatatggaca tttttgagca gttgcatcct 360

cagaagcgag atgacgacag caagaagacg tcgcgccaaa acgcatcgtc gtcagccacc 420

aacactggcg atacacacaa cacacctccg ccaccacctc gcaccactaa cgggttgtcg 480

tccagaaagc cgctgcccat ggataacaag cagtcccagg ctgcagccgc caaagagtca 540

tcctcaaatg ctaaccaggc aaccggcgct ggctcaactg gatccacaca gaccacatcg 600

aaaaagcgaa aagctgcatc acaaactagc agtaatcagc caccagcgac ggaatcgacg 660

cacgtgccag ccaacgccaa gaagcccagt aataataatc acaacagcaa cgctgcatcg 720

agccacggcg ccgacaaggg atatgctgct actaatttgc tcactttcga aaactgtggc 780

gcgcggccca aagatggcaa attgattgct gacgatggca catatcttga agtcaatgac 840

catgtctacc tggtttgcga gcccccaggc gaaccctatt atctcgccag gattatggag 900

ttcttgcatg caaagaacga cccttcgcag ccggtcgatg ctttgcgtgt gaattggtac 960

taccgcccaa aagatattgc ccgtaaggtg aacgatacta gggcggtttt tgcaaccatg 1020

cactcggaca ttagcccttt gacatcgctc cgcggcaaat gcacgatcaa gcacaaggca 1080

gagatcaagg gcaagctcga agagtaccgc aaaaatcccg attgtttctg gttcgagaaa 1140

ctctacgaca ggtacatcca gaaaaattac gaggtgatcc cgacgtttca gatcatcaat 1200

gtacccgaga aggtcaaaaa ggtccttgac gagaggtgga aatacatcct ggtcgagcag 1260

ggccgcggca aggagctcac cagcgccgtc aagacgtgca ggaggtgctc tggctattgc 1320

gcaagtaacg attctgtcga ctgcgctgtg tgtgagcaca cgtaccacat gaactgtgtg 1380

cgaccacccc tgctcaagaa gccatcacgt ggctttgcct ggtcctgtgc cgcctgcagt 1440

agagcccagg agcgcaagct ggaggctcgc aacacgccaa atgtcagcct ggatccgaat 1500

gcagaggccg aggagggagga gtttttcgat gaggaggagg aggacgctgg tctcgacacc 1560

ggccgcacca gtcctgccga tggcgccaac gatatgcaca tccctgcaac cgaggagcag 1620

atgtaccatg ccagcctctg gccatatcgc tacctgggaa tccactgcaa ggtggaggac 1680

gccctcgatt acgacgaccg aatatatccc cgtgcatcga ctcgtgttgg cccaagacac 1740

caggcgacgg tgcttgactg gcctggtcga ccggtccaat acgtcaaggc tcccgaaatt 1800

gaaataaaaa agacgggtcg taaagacggg aagctgaaca aggaagcgca agctgccctc 1860

gaggccgaaa aggtcgccaa ggccaaacgt cccaagtgga tccaggatga accccctggc 1920

tatgtgcccc gtggtgagga ctaccccaat gacgatcccc ggaataccgc ccagcttcac 1980

tggagacctc ctgagcttga cctcccggag gaatcgggtc cggaggaggc gcatatttcc 2040

gaatcggaga tcgacaagta catggagcaa gcgaaaggaa tggcgctaga cctcgacctc 2100

ccggagcact ctacgaatct gctggaccag gcgctccgac ttctctacga acacggttac 2160

gacgcggagc gtgcactgga agagctcccc aagctgagta aggaagcttt tgacgaacca 2220

cagctcacag ctgccgagct caaaaagttc gaggaaggca tctccaagtt cggctcagag 2280

ctttacagcg tgaagaaaca tatcaagacc gtcaaaccag ggacgcttgt tcgtttctac 2340

tacacatgga agaagacaga gcgcggaaag caggtgtggg gcaactactc tggccgcaag 2400

agcaagaagg aggccaagga agccaagaaa gcggagacag cctcacagaa taagatgcag 2460

gacgacgtcg cagacgacca cgatgactcg gctttcgacg cagctaaagc ggcggaaaag 2520

aaacggtctt tcatttgcaa gttttgcaac accaagagct cgcgtcagtg gagacgcgcg 2580

ccaaatgcat caggcgctct ggtcacggag agcgggggca agggtgccaa caaggacaag 2640

ggcgttcagt atgtcgtggc attgtgtcga agatgtgctg agctctggcg ccgctatgcc 2700

attcagtggg aggatgtcga ccagctctac agcaaggtgg cacaggccgg tggccgtgcg 2760

tggaagaagaaga agatcgacga ggagctgctc aaggagatcg ttgctgctga gcaaagaagc 2820

aagaacacgc caagcagtag cggtgcggct actccgccat cgaacaccac gcctgcgccc 2880

gcttccactc aaccagcagc ttcgggtcaa gaaccggcgc ggaagaaaca gaagacaacg 2940

cagccgcctc aagacaagga tgtggaaatg accggcacgg agcctgttgg cacgacaaca 3000

acggcgcccg catccaagaa aaaggagaag gccagcttgg aaaaggagaa agagaaggaa 3060

aaggagaaag agaaggaaaa ggagaaggag aaagaaccag tcaaagaaaa gaaggaagcg 3120

ccggctcctc cccctgttcc ggagataccc aagccgcgaa caatgccttg cgatatctgt 3180

cggcagctgg aacctctggg cgaccagcat atcacgtgca aagaatgtcg catgacagtg 3240

catcggaact gctatggcgt cgtcgacaac cgcaaccctg ggaagtgggt gtgtgacatg 3300

tgcatcaacg acagaagtcc gcatgtctcc attcattaca aatgtgtctt gtgtcctgtt 3360

gaatacaccg agcacgattt tgtggaaccg ccaaaggtat ctcataaaaa gaagacagaa 3420

aaggatcgcg agcgcgagcg ccaagaacgt gaggctgcgg tcaacgcagc agaacattat 3480

cggaagcgtc aggaagagct gaaccgcccg gtcaatcctc gtgagcctct caaacgaaca 3540

gccgacaaca attgggtaca tgtcacttgc tcggtgtgga ctcctgaggt caagtttggc 3600

aatgccaaag ctctcgagcc cagcgaaggg atcccttcta tcccgagatc gaggtactcg 3660

gaggtttgcg aggtctgcaa atctacgggc ggtgcctgca caaactgccc tcagtgcaag 3720

gcatcagtgc atgtcgagtg tgcccaccag tcagacgact acgtcctag ttttgagatc 3780

actcccgtca agggatcacg ccgcgatcag cacaacatcg taactattgg tggtgagagc 3840

ggctccatga gcgcgtctgt ctggtgcaag cttcatgcac cgaagaagac tgtcgtgcat 3900

caaatgtacg atgttgtcga cgaagcggga acgaacgctt tacaactata cgtgcagaac 3960

ttcaagaagg cagatcttac tctcactggc tgcgcacgaa aggccaactt gataagcact 4020

gctgcgcgca tgtccaaccc gatagtgacg acgactacgg cggtgaatcg cagagcatcg 4080

accacgacgg tttcaacaac caccccttcg gcaatgcata ttcacagtct cctcaacggg 4140

gacagccctg gagaccctca cgacctagca gtcccaggtg gcaagatatg tataacatgc 4200

ggagtcgacg tcagccctcg atggtatccc atcagcgaca gtcacgagcg ggagcttgca 4260

aacggtcact atggtgctct cggcactgag gcgcagaagt ttgctgagca gcgacatttc 4320

cagtgtcaca aatgcaagaa gctcaacaag caacccaagt ctcacgtgcc accgccgccg 4380

ccgccgcaag atcctccgcc ggttgctgca aatacgataa atccctccgg gcctgaagca 4440

acagcgcatt cagcagctgt aatgacgaac ggcatagatc acggtcccaa tggcgtcgat 4500

gcagcggttc gtcgaggcac accacctcta acaagccctc gccaaccaga gcacgatcat 4560

ataccgggta ggcctagccc gtacctatgg caatccggac ttcaggctcc acatccgacc 4620

ggcttggtgc atcctacggt ccctatacag gggccgccac ccccgatgca ggcaccgcct 4680

ctccaaccac cgccgatagc tccgccgccg atggcacgta gtatgtcagg ccgcggtcaa 4740

accggtgtac agcctccggg gccagtaccg ccctcccaag catatcaacc cttgccgccg 4800

cctcctccca ctcattcggc accatctggc ccctacggcg attggcatcg gacgactcat 4860

cacggacccc caatgaatgg tcgtcctcct tcacgcgcat cacggatttc cccgatcata 4920

ccaccctctgg caccaccagc acttcgtcca ccgtctctcc accactctcc tcatgctcct 4980

cacgcgcatc tgaccaacgg ccacatggtc aacggtgcgg gtgcaccagg aagacgatta 5040

agcgggccgc caccgccacc ttcgcgagat ggtcagggcc catacatggg aagctatcat 5100

tctcctgcac cataccatag cccagcgccg caccaaagca acgggacaat ggtgccacct 5160

cgcatagatc atgcttttgc gtcagtcctc aaccctccga gagcatatgg aaacagcggg 5220

agcgtgcagc cgcctgcgca catgagccca gctgtggcga gggacgctcc tatttctagg 5280

gatggaccac ttctttccca gccaccgcca ccggccaggg cacctgagtc gagacctgca 5340

acagggtgcca gcgccagccc ttcgttgcga aatctactgt ct 5382

<210> 3

<211> 22

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-cDNA-For

<400> 3

atggctcaaa agggttcagg tg 22

<210> 4

<211> 23

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-cDNA-Rev

<400> 4

agacagtaga tttcgcaacg aag 23

<210> 5

<211> 19

<212>DNA

<213> Artificial Sequence

<220>

<223> RP1

<400> 5

caggattatg gagttcttg 19

<210> 6

<211> 19

<212>DNA

<213> Artificial Sequence

<220>

<223>RP2

<400> 6

ctagtatcgt tcacccttac 19

<210> 7

<211> 1167

<212>DNA

<213> Artificial Sequence

<220>

<223> LF

<400> 7

aatccctgcg ggctactttg acgatactac gcaccgcaga gcctgcaaaa agctgtagct 60

ggcggtccca tacaccggac ccatatattc gaagctagct cactaaatcg attggacaag 120

ggtgtggggt cccacccagg gactgatgta ggaagtgcaa aaaaatggac gagccttgcg 180

ggcgccgcca gcccttgttg ctttctgcgt gccgggcaag ccagccagcc agtacgggac 240

gtccccagtt gggtttggtc ttccatgtct ctttcttgtc ttgggggcgt gtggtccgtc 300

catcatatcc gcctttctgg gacatgcatc cattgaccat ctttctctaa atcctttctt 360

cgcggcactc catctctttc ctcactttcc acttttccgc cttccaagag gtcagtcgtt 420

gtgcactccc acgcgccccc gacgaaggac atttttgctt ccctctcggc accaggcaag 480

caagccagcc agtccctctc tacatgatcg gattaccagc ccggtttggg ttcccagcgc 540

cacaacgctt gtaactcgcg cgaagggctg ctggccttca aactcgattc ctcataacat 600

ttgctcgtgc agatctaact caccgggcat ctcgggggac acgaacaccc gcccacttcc 660

ttaacttctt tgtcaatcgc ccatatatct ctccttttcc tattccattc ccccccacac 720

aattcaaacc ttgcaccttt ctcacccatt tcgacataaa ctgggtccga ctttctttcc 780

tttcctcctt gtcacccaac cttgggtttc gaaaccttcg atttccatct gtaccccatca 840

atctcttctg gcatgcatta tccgccacac gctcacctga atctgggagt ctcatcactg 900

tttacgtcgc gcattgcggc ctgcccagag cagttgtatt ctgcttcttg cggctgcgcc 960

gcctaccttt ccttcccata gttgtttctt tgcagcggaa ggcgaaattt gatcgacccg 1020

gcctctgggt ctcacttggg accttataaa cagcgcccac gtccagcaga tttgcctgcc 1080

tggtcgtcat ggctcaaaag ggttcaggtg gctcagatgc cgcaacacct aataccacga 1140

cgacgtcggc tcacaaaggc accgctg 1167

<210> 8

<211> 1026

<212>DNA

<213> Artificial Sequence

<220>

<223> RF

<400> 8

gcatctccaa gttcggctca gagctttaca gcgtgaagaa acatatcaag accgtcaaac 60

cagggacgct tgttcgtttc tactacacat ggaagaagac agagcgcgga aagcaggtgt 120

ggggcaacta ctctggccgc aagagcaaga aggaggccaa ggaagccaag aaagcggaga 180

cagcctcaca gaataagatg caggacgacg tcgcagacga ccacgatgac tcggctttcg 240

acgcagctaa agcggcggaa aagaaacggt ctttcatttg caagttttgc aacaccaaga 300

gctcgcgtca gtggagacgc gcgccaaatg catcaggcgc tctggtcacg gagagcgggg 360

gcaagggtgc caacaaggac aagggcgttc agtatgtcgt ggcattgtgt cgaagatgtg 420

ctgagctctg gcgccgctat gccattcagt gggaggatgt cgaccagctc tacagcaagg 480

tggcacaggc cggtggccgt gcgtggaaga agaagatcga cgaggagctg ctcaaggaga 540

tcgttgctgc tgagcaaaga agcaagaaca cgccaagcag tagcggtgcg gctactccgc 600

catcgaacac cacgcctgcg cccgcttcca ctcaaccagc agcttcgggt caagaaccgg 660

cgcggaagaa acagaagaca acgcagccgc ctcaagacaa ggatgtggaa atgaccggca 720

cggagcctgt tggcacgaca acaacggcgc ccgcatccaa gaaaaaggag aaggccagct 780

tggaaaagga gaaagagaag gaaaaggaga aagagaagga aaaggagaag gagaaagaac 840

cagtcaaaga aaagaaggaa gcgccggctc ctccccctgt tccggagata cccaagccgc 900

gaacaatgcc ttgcgatatc tgtcggcagc tggaacctct gggcgaccag catatcacgt 960

gcaaagaatg tcgcatgaca gtgcatcgga actgctatgg cgtcgtcgac aaccgcaacc 1020

ctggga 1026

<210> 9

<211> 18

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-LF-For

<400> 9

aatccctgcg ggctactt 18

<210> 10

<211> 42

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-LF-Rev

<400> 10

ccttcaatat catcttctgt cgacctcaca aaggcaccgc tg 42

<210> 11

<211> 50

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-RF-For

<400> 11

cccagcactc gtccgagggc aaaggaatag gcatctccaa gttcggctca 50

<210> 12

<211> 18

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-RF-Rev

<400> 12

acaaccgcaa ccctggga 18

<210> 13

<211> 16

<212>DNA

<213> Artificial Sequence

<220>

<223> HYG-For

<400> 13

gtcgacagaa gatgat 16

<210> 14

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> HY-split

<400> 14

tacttcgagc ggaggcatcc 20

<210> 15

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> YG-split

<400> 15

cgttgcaaga cctgcctgaa 20

<210> 16

<211> 21

<212>DNA

<213> Artificial Sequence

<220>

<223> HYG-Rev

<400> 16

ctattccttt gccctcggac g 21

<210> 17

<211> 1148

<212>DNA

<213> Artificial Sequence

<220>

<223> HY

<400> 17

gtcgacagaa gatgatattg aaggagcact ttttgggctt ggctggagct agtggaggtc 60

aacaatgaat gcctattttg gtttagtcgt ccaggcggtg agcacaaaat ttgtgtcgtt 120

tgacaagatg gttcatttag gcaactggtc agatcagccc cacttgtagc agtagcggcg 180

gcgctcgaag tgtgactctt attagcagac aggaacgagg aattattat catctgctgc 240

ttggtgcacg ataacttggt gcgtttgtca agcaaggtaa gtgaacgacc cggtcatacc 300

ttcttaagtt cgcccttcct ccctttattt cagattcaat ctgacttacc tattctaccc 360

aagcaacgct tcgattagga agtaaccatg aaaaagcctg aactcaccgc gacgtctgtc 420

gagaagtttc tgatcgaaaa gttcgacagc gtctccgacc tgatgcagct ctcggagggc 480

gaagaatctc gtgctttcag cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat 540

agctgcgccg atggtttcta caaagatcgt tatgtttatc ggcactttgc atcggccgcg 600

ctcccgattc cggaagtgct tgacattggg gaattcagcg agagcctgac ctattgcatc 660

tcccgccgtg cacagggtgt cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt 720

ctgcagccgg tcgcggaggc catggatgcg atcgctgcgg ccgatcttag ccagacgagc 780

gggttcggcc cattcggacc gcaaggaatc ggtcaataca ctacatggcg tgatttcata 840

tgcgcgattg ctgatcccca tgtgtatcac tggcaaactg tgatggacga caccgtcagt 900

gcgtccgtcg cgcaggctct cgatgagctg atgctttggg ccgaggactg ccccgaagtc 960

cggcacctcg tgcacgcgga tttcggctcc aacaatgtcc tgacggacaa tggccgcata 1020

acagcggtca ttgactggag cgaggcgatg ttcggggatt cccaatacga ggtcgccaac 1080

atcttcttct ggaggccgtg gttggcttgt atggagcagc agacgcgcta cttcgagcgg 1140

aggcatcc 1148

<210> 18

<211> 731

<212>DNA

<213> Artificial Sequence

<220>

<223> YG

<400> 18

cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca 60

tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc 120

aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg 180

tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg 240

atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt 300

tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg 360

aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt 420

tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat 480

cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg 540

ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat 600

ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg 660

atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg 720

caaaggaata g 731

<210> 19

<211> 2315

<212>DNA

<213> Artificial Sequence

<220>

<223> LF-HY

<400> 19

aatccctgcg ggctactttg acgatactac gcaccgcaga gcctgcaaaa agctgtagct 60

ggcggtccca tacaccggac ccatatattc gaagctagct cactaaatcg attggacaag 120

ggtgtggggt cccacccagg gactgatgta ggaagtgcaa aaaaatggac gagccttgcg 180

ggcgccgcca gcccttgttg ctttctgcgt gccgggcaag ccagccagcc agtacgggac 240

gtccccagtt gggtttggtc ttccatgtct ctttcttgtc ttgggggcgt gtggtccgtc 300

catcatatcc gcctttctgg gacatgcatc cattgaccat ctttctctaa atcctttctt 360

cgcggcactc catctctttc ctcactttcc acttttccgc cttccaagag gtcagtcgtt 420

gtgcactccc acgcgccccc gacgaaggac atttttgctt ccctctcggc accaggcaag 480

caagccagcc agtccctctc tacatgatcg gattaccagc ccggtttggg ttcccagcgc 540

cacaacgctt gtaactcgcg cgaagggctg ctggccttca aactcgattc ctcataacat 600

ttgctcgtgc agatctaact caccgggcat ctcgggggac acgaacaccc gcccacttcc 660

ttaacttctt tgtcaatcgc ccatatatct ctccttttcc tattccattc ccccccacac 720

aattcaaacc ttgcaccttt ctcacccatt tcgacataaa ctgggtccga ctttctttcc 780

tttcctcctt gtcacccaac cttgggtttc gaaaccttcg atttccatct gtaccccatca 840

atctcttctg gcatgcatta tccgccacac gctcacctga atctgggagt ctcatcactg 900

tttacgtcgc gcattgcggc ctgcccagag cagttgtatt ctgcttcttg cggctgcgcc 960

gcctaccttt ccttcccata gttgtttctt tgcagcggaa ggcgaaattt gatcgacccg 1020

gcctctgggt ctcacttggg accttataaa cagcgcccac gtccagcaga tttgcctgcc 1080

tggtcgtcat ggctcaaaag ggttcaggtg gctcagatgc cgcaacacct aataccacga 1140

cgacgtcggc tcacaaaggc accgctggtc gacagaagat gatattgaag gagcactttt 1200

tgggcttggc tggagctagt ggaggtcaac aatgaatgcc tattttggtt tagtcgtcca 1260

ggcggtgagc acaaaatttg tgtcgtttga caagatggtt catttaggca actggtcaga 1320

tcagccccac ttgtagcagt agcggcggcg ctcgaagtgt gactcttatt agcagacagg 1380

aacgaggaca ttattatcat ctgctgcttg gtgcacgata acttggtgcg tttgtcaagc 1440

aaggtaagtg aacgacccgg tcataccttc ttaagttcgc ccttcctccc tttatttcag 1500

attcaatctg acttacctat tctacccaag caacgcttcg attaggaagt aaccatgaaa 1560

aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc 1620

tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga 1680

gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat 1740

gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa 1800

ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac 1860

ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc 1920

gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt 1980

caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg 2040

caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg 2100

ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac 2160

aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc 2220

ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg 2280

gagcagcaga cgcgctactt cgagcggagg catcc 2315

<210> 20

<211> 1757

<212>DNA

<213> Artificial Sequence

<220>

<223> YG-RF

<400> 20

cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca 60

tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc 120

aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg 180

tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg 240

atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt 300

tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg 360

aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt 420

tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat 480

cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg 540

ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat 600

ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg 660

atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg 720

caaaggaata ggcatctcca agttcggctc agagctttac agcgtgaaga aacatatcaa 780

gaccgtcaaa ccagggacgc ttgttcgttt ctactacaca tggaagaaga cagagcgcgg 840

aaagcaggtg tggggcaact actctggccg caagagcaag aaggaggcca aggaagccaa 900

gaaagcggag acagcctcac agaataagat gcaggacgac gtcgcagacg accacgatga 960

ctcggctttc gacgcagcta aagcggcgga aaagaaacgg tctttcattt gcaagttttg 1020

caacaccaag agctcgcgtc agtggagacg cgcgccaaat gcatcaggcg ctctggtcac 1080

ggagagcggg ggcaagggtg ccaacaagga caagggcgtt cagtatgtcg tggcattgtg 1140

tcgaagatgt gctgagctct ggcgccgcta tgccattcag tgggaggatg tcgaccagct 1200

ctacagcaag gtggcacagg ccggtggccg tgcgtggaag aagaagatcg acgaggagct 1260

gctcaaggag atcgttgctg ctgagcaaag aagcaagaac acgccaagca gtagcggtgc 1320

ggctactccg ccatcgaaca ccacgcctgc gcccgcttcc actcaaccag cagcttcggg 1380

tcaagaaccg gcgcggaaga aacagaagac aacgcagccg cctcaagaca aggatgtgga 1440

aatgaccggc acggagcctg ttggcacgac aacaacggcg cccgcatcca agaaaaagga 1500

gaaggccagc ttggaaaagg agaaagagaa ggaaaaggag aaagagaagg aaaaggagaa 1560

ggagaaagaa ccagtcaaag aaaagaagga agcgccggct cctccccctg ttccggagat 1620

acccaagccg cgaacaatgc cttgcgatat ctgtcggcag ctggaacctc tgggcgacca 1680

gcatatcacg tgcaaagaat gtcgcatgac agtgcatcgg aactgctatg gcgtcgtcga 1740

caaccgcaac cctggga 1757

<210> 21

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> P1

<400> 21

ggtaggagaa aggctgatgg 20

<210> 22

<211> 22

<212>DNA

<213> Artificial Sequence

<220>

<223> P2

<400> 22

gcttctgcgg gcgatttgtg ta 22

<210> 23

<211> 22

<212>DNA

<213> Artificial Sequence

<220>

<223> P3

<400> 23

cagcgagagc ctgacctatt gc 22

<210> 24

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> P4

<400> 24

cttcctgacg cttccgataa 20

<210> 25

<211> 57

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-For1

<400> 25

aactgttggg aagggcgatc ggtgcgggcc actagttcat tgttaccgaa agaagcc 57

<210> 26

<211> 42

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-Rev1

<400> 26

aacccttttg agccatgaat tcgacgacca ggcaggcaaa tc 42

<210> 27

<211> 42

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-For2

<400> 27

cctgcctggt cgtcgaattc atggctcaaa agggttcagg tg 42

<210> 28

<211> 19

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-Rev2

<400> 28

agtggattcc caggtagcg 19

<210> 29

<211> 19

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-For3

<400> 29

tgtaccatgc cagcctctg 19

<210> 30

<211> 21

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-Rev3

<400> 30

tgacgcttcc gataatgttc t 21

<210> 31

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-For4

<400> 31

acccaaccag cattcttccc 20

<210> 32

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> SNT2-Rev4

<400> 32

agacagtaga tttcgcaacg 20

<210> 33

<211> 56

<212>DNA

<213> Artificial Sequence

<220>

<223> GFP-SNT2-For

<400> 33

gccagccctt cgttgcgaaa tctactgtct cccgggatgg tgagcaaggg cgagga 56

<210> 34

<211> 57

<212>DNA

<213> Artificial Sequence

<220>

<223>GFP-SNT2-Rev

<400> 34

agtgctcctt caatatcatc ttctgtcgac ggccggccgt ggagatgtgg agtgggc 57

<210> 35

<211> 20

<212>DNA

<213> Artificial Sequence

<220>

<223> BAR-For

<400> 35

gtcgacagaa gatgatattg 20

<210> 36

<211> 75

<212>DNA

<213> Artificial Sequence

<220>

<223> BAR-Rev

<400> 36

ttcacacagg aaacagctat gaccatgatt atttaaatac gcgtggccgg ccgtcgacct 60

aaatctcggt gacgg 75

Claims (8)

1. a kind of pathogenicity proteins for coming from Pyricularia oryzae, it is characterised in that the amino acid sequence of the albumen is as follows：

(1), with SEQ ID No:Amino acid sequence shown in 1；

(2), by SEQ ID No:Amino acid sequence shown in 1 is by the addition of one or several amino acid residues, replacement or scarce Become homeless it is being formed and with rice blast cause a disease sexual function amino acid sequence.

2. encode the gene of the pathogenicity proteins described in claim 1, it is characterised in that the gene have following (a), (b) or (c) nucleotide sequence：

(a) there is SEQ ID No:Nucleotide sequence shown in 2；

(b) under high stringency conditions with SEQ ID No:The nucleotide sequence of nucleotide sequence complementation shown in 2；

(c) by SEQ ID No:Addition, missing or replacement of the nucleotide sequence Jing Guo one or several bases shown in 2 is formed And coding with rice blast pathogenicity proteins function nucleotide sequence.

3. the expression vector containing gene described in claim 2.

4. the pathogenicity proteins described in claim 1 are used as the application for designing or/and screening antifungal drug target.

5. the gene described in claim 2 is used as the application for designing or/and screening antifungal drug target.

6. the application according to claim 4 or 5, it is characterised in that described fungi refers to Pyricularia oryzae (Magnaporthe oryzae)。

7. application of the expression vector on screening antifungal drug described in claim 3.

8. application according to claim 7, it is characterised in that described fungi refers to Pyricularia oryzae (Magnaporthe oryzae)。