| Magnaporthe graea(Hebeet) Barr Comb. Nov(anamorph.P>r/'cM/ana oryzae Cavara) is a haploid heterothallic ascomycete. The fungus cause rice blast, one of the most damaging worldwide diseases of rice. Among the system of rice blast disease, the interaction between host and pathogen copes with gene-for-gene relationship. Clone of avirulence gene and other pathogenesis-related genes contribute to understanding the pathogenic specificity and infectious mechanism.Restriction enzyme-mediated integration (REMI) was used to construct the biggest inserted mutant library of Magnaporthe grisea up to now, which contained 27063 transformants. The aim fungus, M.grisea Y34 strain, was used to infect 19 differentiated cultivars, the result showed that there were 13 avirulence genes in the fungus. pV2, a binary vector, which contains the hygromycin B phosphotransferase gene linked to Aspergillus nidulans regulatory sequences as selective marker, was constructed. 8 restriction enzymes, Hindlll, Sail, SacI, Smal, EcoRV,ApaI,XhoI,KpnI, can be used for transforming M.grisea with the vector of pV2. Mycelial pellets, collected after shaking conididium in liquid completive medium 40 hours, was hydrolyzed with Drislease for about 3 hours, The best protoplasts were acquired. To acquire the excellent transformation efficiency, the proper time of bathing on ice before and after dripping PTC are 20 and 28 minutes respectively, the appropriate concentration of the 8 restriction enzyme above is 30,20,20,20,20,20,30,20 units per dish respectively. Results showed that different enzymes could produce different transformation efficiency, and the efficiency with blunt-end enzymes was less than that with cohesive-end enzymes. The best transformation efficiency of the 8 enzymes above was 48, 48, 35, 17, 14, 39, 30, 27 transformants per dish respectively. 3256, 2956 ,2720, 1443, 2038 ,2996.2689,2728 transformants of each of the 8 enzyme above were acquired.Southern analysis was performed to evaluate the inserted mutant library. 13 transformants, chosen random from the transformants mediated with each of the 8 enzyme above, were digested with Bglll and the restriction enzyme used for transformation. The results showed that plasmids inserted exist with 4 types, that is, multisites with multicopies, singlesite with multicopies, singlesite with single copy and plasmid deletion insertion. Among the transformants from the 8 enzymes above, the percentage of single site insertion is 84%. 83%, 61%, 92%, 77%, 77%, 61%, 92% respectively. The rate of single copy insertion is 69%, 83%, 54%, 23%, 15%, 46%, 92%. There were no relativity between the transformation efficiency and the type of insertion. Single copy and REMI insertion among the transformants from the cohesive end enzymes are higher than that from the blunt end enzymes. Total 77% single site insertion indicate that the inserted mutant library are suit to isolate genes.In addition, plasmids pSacI-1 and pCB1004 were used for transforming the Magnaporthe gresea 131 and Y34 strains, 3395 and 2012 transformants were acquired respectively.Molecular genetics analysis was carried out with the mutant H6 which was from the Magnaporhte grisea 131 REMI transformants.The phenotype of sporulation deficiency and slow growth was confirmed after comparing its phenotype with the wild type phenotype of 131 strain. Southern analysis showed that the mutation was the result of a single copy plasmid insertion. Cross H6 with opposite mate strain 1528, 21 ascospores were isolated. The results of identifying the rate between the mutation phenotype and the wild type phenotype among the 21 ascospores and assaying the co-separation with the HyB resistance showed that the mutation was controlled by a single gene and the gene was disrupted by inserted plasmid. The frank fragment of the fungal genomic DNA was separated by plasmid rescue. Sequenced part of the fragment and homologous comparison showed that there was no homology with the genes in the Genebank, which implicated that the gene was a new one. Genomic library of th...
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