The Regulation Of StHog1 To Transcription Factor StMsn2 And The Screening And Verification Of Target Genes Of StMsn2 In Setosphaeria Turcica

In our previous study,the ? St HOG1 strain was obtained by employing Agrobacterium-mediated transformation technology.Through the analysis of?St HOG1 strain,it was found that St HOG1 gene was involved in the regulation of hyperosmotic stress response of Setosphaeria turcica.We speculated that St Msn2 is a key transcription factor downstream of St Hog1,and its activity is regulated by phosphorylation modification,but its specific molecular mechanism is still unclear.Based on the previous studies,the transcription of St MSN2 and the expression level of its coding protein were analyzed by RT-q PCR and Western blot.The protein dephosphorylation test showed that the transcriptional activity of St Msn2 was regulated by phosphorylation modification under 0.4 M Na Cl hypertonic condition.The target genes regulated downstream of St Msn2 transcription factor were screened and validated by chromosome immunoprecipitation(Ch IP),electrophoretic mobility shift analysis(EMSA)and double fluorescence reporting system.Therefore,it is important to clarify the regulatory relationship between transcription factor St Msn2 and St Hog1 and the target genes regulated downstream of St Msn2 for improving the HOG-MAPK signaling pathway and elucidating the molecular mechanism of its pathway regulating hypertonic stress response of Setosphaeria turcica.The main results were as follows:1.St MSN2 contains an open reading frame(ORF)with a length of 1617 bp.Conservative domain analysis shows that St Msn2 protein has two conserved zinc finger domains(Zn F-C2H2),located at 418~441 and 447~469 sites of N-terminal amino acids,respectively.Phylogenetic analysis shows that St Msn2 is closely related to Msn2 in Aspergillus parasiticus.2.Three polyclonal antibodies(Msn2-1,Msn2-2 and Msn2-3)against St Msn 2 were prepared by using antigen epitopes.The specificity of the antibodies was detected by Western blot.The results showed that only a clear hybridization band appeared,which was about 58.8 k Da.It was indicated that the specificity of Msn2-2 antibody was strong.3.The ?St HOG1 strain was analyzed by fluorescence quantitative RT-q PCR and Western blot.It was found that the transcription level of St MSN2 gene in the mutants was very low and the protein encoded by St MSN2 did not express.This indicated that St Hog1 was located in the upstream of St Msn2 and regulated its expression.4.Western blot technique was used to detect the expression level of St Msn2 under hypertonic condition.The results showed that the expression level of St Msn2 was significantly increased and two distinct bands appeared,with a mass fraction difference of 10 k Da.Further treatment of protein with dephosphorase showed that the bands with larger mass fraction narrowed and smaller bands widened,which proved that St Msn2 protein.Phosphorylation occurred under hyperosmotic stress and participated in the hyperosmotic stress response of the pathogen.5.According to the results of Ch IP-Seq data analysis,nine target candidate genes regulated by St Msn2 were screened out.Bioinformatics analysis showed that there were Motif sequences binding to St Msn2 in the promoter regions of the nine genes.Furthermore,Ch IP-q PCR technique was used to verify that St Msn2 transcription factor can bind to promoter regions of genes St ATA1 and St ODA1,indicating that St ATA1 and St ODA1 are downstream target genes regulated by St Msn2.6.Expression and purification of St Msn2-His recombinant protein in prokaryotic expression system,and EMSA technique has proved that St Msn2 can directly bind to STRE elements in promoter regions of genes St ATA1 and St ODA1.Further transcriptional activation experiments in the tobacco system showed that St Msn2 transcription factor could bind to STRE elements in the promoter regions of genes St ATA1 and St ODA1 and activate the expression of reporter genes.