Construction Of Restriction Enzyme-mediated Integration Mutant Library Of Magnaporthe Grisea And Mutant Screening

Rice blast disease caused by Magnaporthe grisea is one of the most important diseases of rice. Rice blast fungus has become a model organism to investigate the interactions between plants and plant-pathogens fungi. Construction of insertion mutant library and screening mutants redound to studying functional genome and the mechanism of pathogenicity of M. grisea.In this study, an insertion mutant library of M.grisea P131 and Y34 which contained 10334 transformants was generated using the approach of Restriction Enzyme Mediated Integration (REMI). In the process of library construction, PV2, a transformation vecter was linearized with XhoI and used to transform a blast fungus race Y34, The transformation generated 2200 transformants. PV2 was linearized with XhoI, ApaI , EcoRV respectively and used to transform P131, the transformations generated 2659, 2517 and 228 transformants respectively by the three individual enzyme-mediated insertion. The plasmid pCB1004 was linearized with EcoR I and used to transform P131. The transformation generated 2750 transformants. Results showed that the transformation efficiency among the cohesive-end enzymes XhoI, Apal and EcoRI has no significant difference,they can generate 35-50 transformants per culture dish. But the blunt-end enzyme EcoRV generated 7 transformants per culture dish, the transformation efficiency was very low.The method of inoculating onion epidermis and rice leaves was used to screen mutants from the insertion mutant library. The author obtained 18 mutants from about 7000 transformants. As compared with the wild type P131, the sporulation deficiency mutant Mo938 showed 5% in the total amount of spores on plate, the conidium distortion mutant Mol745 showed that conidia were variform and produced less penetration pegs on onion epidermis, the mutants Mo2424, EI2445 decreased in penetration pegs formation on onion epidermis, the rates of penetration pegs formation were less than 20% and 1%, respectively. Mol745, EI2445 did not form lesion on rice leaves and completely lost their pathogenicity, the mutant Mo2424 formed few lesion on rice leaves, and the amount reduced 86.9% than the amount of P131 formed lesion.Co-segregation analysis revealed that Mol745, Mo2424, EI2445 were insertional mutants and Mo938 was not, its mutant phenotype was not due to the plasmid insertion. Furthermore, the genomic Southern blot analysis was used to verify the sites of plasmids insertion in co-segregated mutants genome. The results showed that the plasmids inserted in Mol745 was singlesite with multicopies, and two sites in Mo2424, these results were accordant with co-segregation analysis. But Southern blot revealed that plasmids inserted in EI2445 were multisites and different from co-segregation analysis. These studies were the base of plasmid rescue and gene cloning.