The Analysis Of P-ATPase Gene Family And The Research Of MoCTAl、MoCTA3in The Rice Blast Fungus Magnaporthe Oryzae

Magnaporthe oryzae is one of the most severe diseases of cultivated ricethroughout the world. And it is also the principal model organism for research oninteraction between fungal pathogen and host plants. Research for the fungalpathogenicity can facilitate the disease integrated management and the rice breedingfor durable resistance. There are many kinds of P-ATPases in the plasma membrane,which participat in cation trans-membrane transport, cell signal transduction, andstability of the cell membrane. Existing research indicate that P-ATPase genes arenecessary for appressorium morphogenesis and pathogenicity in the Rice BlastFungus Magnaporthe oryzae. In this research, we identified twenty-three P-ATPasegenes in Magnaporthe oryzae. To analyze the role of P-ATPase in pathogenesis ofMagnaporthe oryzae, A P-ATPase gene named MoCTA3was cloned fromMagnaporthe oryzae, and Targeted gene replacement of MoCTA1was gained. Theresults are showed as following:1. Twenty-three P-ATPase genes were identified in Magnaporthe grisea, the largestnumber yet identified in fungus kingdom, based on the genome-wide CodingSequence（CDS）blast of Magnaporthe grisea using P-ATPases amino sequencefrom protein database. Phylogenetic analysis distinguished4distinct families,and7subfamilies. Six motifs were found in amino sequences of more than90%genes, analyzed by MEME software. GC fraction analysis revealed that theaverage GC content of P-ATPase gene was0.519-0.628, some higher than0.516,average GC content of Magnaporthe grisea genome. It was also found there wasno evident GC gradient change for these P-ATPase gene.2. We cloned a P-ATPase gene named MoCTA3from Magnaporthe oryzae.Sequence analysis and amino acid sequence alignment were done to analyze thegene. Simultaneously quantitative RT-PCR was performed at0h,48h,72h,96hpost-inoculation and the hyphal phase to examine the MoCTA3expression. Theresults showed that full length of the MoCTA3was3747bp, which contained6extrons,5introns and encoded1096amino acids. The gene contained7trans-membrane domains, including the conservative calcium ion transportingdomain at the site of24-98. The similarity of the MoCTA3amino acids sequencebetween Magnaporthe oryzae, and the Nectria haematococca, Fusariumoxysporum, Metarhizium anisopliae, Glomerella graminicola were77%,78%,83%and79%, respectively. Gene expression analysis suggested that the geneexpression level of hyphal phase was significantly higher than that of the infection stage. The gene expression increased gradually with time during theinfection phase, and the highest appeared in72hpi, then decreased. Theseindicated that MoCTA3was involved in Magnaporthe oryzae’s plant infectionand played an important role in the pathogenicity of the rice blast fungus.3. We gained a mutant of gene MoCTA1, using the technology of targeted genereplacement. Phenotype analysis results show that the aerial hypha of ΔMoCTA1was prosperity in CM and PDA mediums, but rarefaction in RDC medium,compared with Guy11, the wild strain of the ΔMoCTA1. The growth rate ofΔMoCTA1was decreased distinctly in CM and PDA mediums, but no significantdeviation in RDC medium. Expression of MoCTA1-GFP combined proteinresults indicated that the encoding product of MoCTA1was concentrated onbarrier diaphragm in sporus. Pathogenicity assay discovered that ΔMoCTA1cannot form the normal affection, and the pathogenicity was decreased.