Cloning And Functional Analysis Of CAMK, And CHK1 Gene In Magnaporthe Oryzae

Magnaporthe oryzae is a well-known ascomycetous fungus that causes rice blast. Understanding of the molecular basis of this disease is not only beneficial for rice blast control, but also can serve as a model for revealing other fungal pathogen-plant interactions. Identification of pathogenicity-relative genes is a key step towards this goal. Mutation analysis of pathogen is a powerful strategy in study of the parasitic interaction between the pathogenic fungus and its plant host, which might make the identification of pathogenicity-relative genes possible. In this study, CAMK and CHK1 gene from M. oryzae were cloned. The results are showed as follows:1. CAMK was found from a library of mature appressorium cDNA and its cDNA clones were obtained by PCR strategy, while the sequence homologues were found by using the yeast amino acid sequence of CAMK and CHK1 to search from M. oryzae genome databases, assigned as CAMK and CHK1 The genomic DNA clones covered the full length of ORF and cDNA clones were obtained by PCR strategy and the nucleotide sequences of the genomic DNA and cDNA were analyzed.2. Hygromycin-resistance transformants were obtained by CAMK and CHK1gene replacement vector construction and fungal transformation. Knockout mutants were identified by PCR and Southern blot. Single copy was detected by genomic Southern blot in the genome of Guy11 for each gene.3. The colony modality ofΔCAMK was different from Guy11. Compared to Guy11, the growth rate of the mutants on CM, CM-C, CM-N and CM with 1M NaCl had no obvious change. Conidia germination and appressoria formation of mutantΔCAMK was different from that of wild type.4. Several eGFP fusion expressing vectors, including PHNSCHK1E, PHNECHK1 and PHCHK1PE were constructed.5 The colony modality ofΔCAMK was different from Guy11. Compared to Guy11, the growth rate of the mutants on CM, CM-C, CM-N and CM with 1M NaCl had no obvious change. Conidia germination and appressoria formation of mutantΔCHK1 was suppressed,compared to wild type. The pathogenicity ofΔCHK1 mutant wasn't reduced on barley. Guy11, the mutant was UV irradiation, the growth rate is different.