Screening And Verification Of The Target Sites Of Transcription Factor StMR1 Regulating Melanin Synthesis In Setosphaeria Turcica

Melanin is a key pathogenic factor of Setosphaeria turcica to invade the host,and its synthesis pathway is regulated by the transcription factor StMRl.StMRl protein has unique Cys2His2 and Zn(Ⅱ)2Cys6 double zinc finger domains.So far,a total of six melanin synthesis regulatory genes of plant pathogenic fungi have been successfully cloned,all of which encode the same type of transcription factors.Transcription factors can specifically bind to DNA to perform transcription function.The specific characterization of transcription factor DNA binding is the key to analyze the regulatory function and evolution of transcription factors.However,it is not clear that how these transcription factors regulate DHN melanin synthesis and target sites.In this study,we created complementation mutants of StMR1 gene based on the knockout mutants of StMR1 gene,further proved the relationship between StMR1 and DHN melanin synthesis.The yeast one-hybrid technique was used to verify the direct regulatory relationship between StMR1 and key genes in the melanin synthesis pathway.MEME was used to predict the specific recognize motifs of StMR1.The above results could lay the foundation for the study of the molecular mechanism of StMR1 regulating the melanin synthesis in S.turcica.The main research results were as follows:1.The complementation vector of StMR1 gene was builded using the pBS and 3FLAG+6HA-pUC57 vectors,which integrated with the glufosinate gene(Bar).The complementation strains C.△Stmr1-1 and C.△Stmr1-2 were created based on the knockout mutant △Stmr1 by using PEG-mediated transformation of protoplasm.2.Analysis of the complementation strains C.△Stmr1-1 and C.△Stmr1-2 showed that the melanin synthesis ability in the complementation strains was 9-10 fold higher than that in △Stmr1,and the infection efficiency of the complementation strains were increased distinctly.3.Real-time quantitative PCR technology was used to analyze the expression of key genes(St3HNR,St4HNR,StPKS.StSCD,StLAC1,and StLAC2)in the melanin synthesis pathway.Compared with the knockout mutant △Stmr1,the expressions levels of synthetase gene StPKS,reductase genes St3HNR and St4HNR,dehydratase gene StSCD and laccase genes StLAC1 and StLAC2 in the complementary strains were significantly increased.The gene expression levels of St3HNR and St4HNR in the complementation strains was increased by 4-6 times compared with △Stm1;the gene expression level of StSCD was increased 2-3 fold;StPKS was increased 0.8-1 fold;both StLAC1 and StLAC2 were increased by a fold of 1-2.4.The yeast one-hybrid technique was used to study the interaction between the transcription factor StMRl and the key genes of the melanin synthesis pathway.The results showed that StMR1 interacts with the promoters of the target genes St3HNR,St4HNR,StPKS and StLAC2,but not with StSCD and StLAC1,indicating that StMR1 directly regulates the expression of the melanin synthase genes StPKS,St4HNR,St3HNR and StLAC2 and thus affects melanin synthesis in S.turcica.5.The sequence characteristics of the promoter regions of StPKS,St4HNR,St3HNR and StLAC2 genes were analyzed by MEME online analysis.It was predicted that the core sequence recognized by StMR1 may be CAT(G/T),and yeast one-hybrid test were used to corfirm the sequence binds to StMR1.6.Taking target gene StPKS as an example,the role of Cys2His2 zinc finger motif and Zn(Ⅱ)2Cys6 zinc cluster motif in StMR1 gene function was verified by yeast one-hybrid.The results showed that StPKS-pAbAi only interacted with AD-StMR1,which has double zinc finger domain,and did not interact with AD-StMR1△C2H2,AD-StMR1△C6 and ADStMR1△C2H2/C6.These results indicated that the double zinc finger domain is necessary for StMR1 gene function.