Bioloical Function Analysis Of The TRNA-splicing Endonuclease SEN54Gene (MoTSEN54) In Magnaporthe Oryzage

Rice is one of the most important crops worldwide, and rice production directlyaffects global social and economic development. Rice blast, caused by the rice blastfungus,Magnaporthe oryzae, leads to serious damage to rice production. Because ofcomplexity physiological races, enhanced resistance and environmental problems,chemicals and disease-resistant varieties can not meet the prevention needs.Pathogenicity study of M.oryzae on the molecular level can help us to understand thedeeper interaction between rice and M.oryzae, can help us to cultivate more excellentrice varieties, and can help us to develop more effective new environment-friendly andlow toxicity fungicides. So research the function of pathogenic gene on M.oryzae isimminent.Firstly, we built a T-DNA random insertion mutants library of rice blast fungus byATMT technology. Then we screened an T-DNA insertion mutant which was extremesensitive to hydrogen peroxide and decreased virulence by analyzing the degree ofvirulence and hydrogen peroxide stress. Through Tail-PCR technology we amplified theflanking sequence. Blasting the sequence, we found that the T-DNA fragment wasinserted into a hypothetical protein which matched to MGG₁2745of M.oryzae. Thisgene is located on four chromosome in M.oryzae genome, the total length is2247bp,encoding506amino acid，and no introns. The protein contains a conserved domain,named the tRNA splicing endonuclease SEN54. The subunit is one component of tRNAsplicing enzyme,and deletion of this gene can lead to abnormal embryonic developmentin animals such as zebrafish. So SEN54gene plays a important role on embryonicdevelopment in animals, but the function in rice blast fungus is not yet known.In order to explore function of MoTSEN54gene, we knockouted the target gene inthe wild-type strain Y34by ATMT technology, and obtained the target gene disruption transformants successfully. We used the knockout strains as complementary strain andacquired complementary transformants of MoTSEN54gene using ATMT method.Comparing Y34strain、knockout strain and complementary strain, we found that theyhave many significant differences in melanin formation, spores, appressoria formationand the presence of ROS stress biology, etc. Compared with Y34strain, we found thatthe melanin of knockout strain was disappeared, the number of spores was decreased,and knockout strain basically does not form appressoria. Knockout strain was sensitiveto hydrogen peroxide and congo red, and it could not infect barley. Although thecomplementary strain could not fully recover the defects, the phenotypes can consistentwith the Y34strain.These results suggest that MoTSEN54gene is associated with pathogenicity, andplays an important role in the regulation of the appressoria formation and pathogenicityin M.oryzae.