Molecular Mechanism Of TasMYB27 Transcription Factor Regulating Trichoderma Against Alternaria Alternata

Trichoderma is a biocontrol fungi,its antagonistic properties are based on the activation of multiple mechanisms.In his study,TasMYB27 gene were overexpressed in Trichoderma asperellum by transgenosis method and they sre examined in terms of both morphology and physiology.Secondly,TasMYB27 gene was prokaryotic expressed protein and purified successfully.Thirdly,the downstream genes of TasMYB27 were analyzed by Yeast single hybridization.To explore the related functions of this gene and lay a foundation for exploring the detoxification regulatory network of T.asperellum by the above methods.The results are as follows.Fifteen MYB transcription factors are obtained from T.asperellum genome and varieties of response elements are found in promoter region by bioinformatics analysis.In addition,TasMYB27 and its 18 homologous sequences all contain the Myb_DNA-Bind₆ conserved domain.The replacement of the first leucine with phenylalanine may help to identify the new target gene.What’s more,the prediction results of protein secondary structure shows that TasMYB27 protein has the most random coil.The tertiary structure is modeled by 1h88.1.C and the core structure of TasMYB27 is β-folded surrounded by three α-helices.The full length sequence of TasMYB27 gene is obtained by cloning with specific primers.TasMYB27 is a hydrophilic protein with 717 bp CDS length,encoding 238 aa and 27.39 kDa molecular weight.Then,TasMYB27 contains multiple reactive elements across motif and promoter regions analysis,qRT-PCR results shows that TasMYB27 is significantly expressed at 24 h under the toxin stress of Alternaria alternata.Therefore,TasMYB27 gene may be involved in the regulation of T.asperellum resistance to toxin stress.Two TasMYB27 overexpression lines are obtained by protoplast transformation by PEG-CaCl2.The concentric rings of Tas-OE are more obvious than Tas-WT,but there is no difference in the morphology of the phialide and conidia.The inhibition ability and toxin tolerance of Tas-OE are enhanced,and the activities of POD and CAT enzymes are also higher than Tas-WT.Finally,The prokaryotic expression system of TasMYB27 is established,and the optimal expression conditions are 0.1 mmol/L,37℃,3 h,and 69.5 kDa soluble TasMYB27 protein is successfully purified.Then,TasMYB27 transcription factor is self-activated by Yeast two-hybrid assay.In addition,Six DNA sequences sre screened from yeast random DNA library,and the core base sequence of one of the unknown new elements "TCGGGATGTA" is determined as "GATG".From what has been discussed above,a comprehensive bioinformatics analysis is carried out on the MYB transcription factor family of T.asperellum,and the temporal response pattern analysis,gene function study and cis-acting elements identified by TasMYB27 transcription factor under toxin stress are conducted.We expect that all the results and conclusions can provide meaningful clues for signal transduction networks in T.asperellum response to toxin stress.