Mutant Library Construction By Using Of Promoter Trapping In Magnaporthe Grisea

Magnaporthe grisea is a causal agent of rice blast and diseases of many other different grasses. Understanding of the molecular basis of this disease is not only benificial for rice blast control, but also can serve as a model for revealing of other fungal pathogen-plant interactions. Obviously, identification of genes required for pathogenicity is a key step toward achieving this goal. The parastic interaction between the pathogenic fungus and its plant host is a complex biological process. Mutation analysis of pathogen is a powerful strategy in study of this process. To elucidate the mechanisms of the pathogenesis, we clone genes by using of insertional mutagenesis. As we know, strategies, such as REMI, ATMT and trasnposon tagging have cloned many interesting genes in many plant pathogenic fungi so far. However, those methods will not permit isolation of genes without clear mutant phenotypes during development either because of gene redundancy or due to the gene product not being essential for development under the conditions in the laboratory. One way to gain access to such genes is to use a method called promoter trapping, in which expression of a reporter gene can be initiated only from an endogenous promoter because the reporter gene lacks its own promoter. In this method, it is not necessary to obtain the obvious phenotype mutants. Here we discribe this promoter trapping method for M. grisea that depends upon restriction enzyme-mediated insertion of a vector carrying a promoterless eGFP (green fluorescent protein) gene. New promoter trapping vectors pEGFP-HPH and pCB 1003-EGFP have been developed to trap genes in M. grisea. The redults are shown as follows:1. New promoter trapping vectors pEGFP-HPH and pCB1003-EGFP have been constructed which possess the marker hygromycin gene and a promoterless eGFP gene;2. Transformation system is stabilized. The frequency of the transformation is 40-60mutants/ug DNA;3. We constructed a library in Magnaporthe grisea which consisted of 1077mutants. eGFP expression was detected by fluoroscopy. eGFP expression in 489 mutants. The frequency of the eGFP expression is 45.4%. By analysing of the 489 mutants, 3 mutants grow slowly, 9 mutants'clony are white, the spore production of 35 mutants are decreased and 18 mutants deduced in pathogenecity. We use Southern blot analysis to check the library and get the different signals. These data indicated that the promoter trapping technique has a good future. Byanalyzing the mutants, we can get the useful mutants which have defects inpathogenicity, metabolism, and alterations in morphology.