Molecular Mechansum Of Resistance Regulate Of Trichoderma MYB36 Transcription Factors To Alternaria Alternata Toxin

Trichoderma as an Bio-control fungi has been widely used in the biological control of forest diseases.In order to better play its biocontrol role,it is particularly important to understand the intrinsic mechanism of Trichoderma in resisting the stress of pathogenic fungi.In this study,we investigated the molecular mechanism of MYB36 transcription factor of Trichoderma asperellum CGMCC11653 strain in regulating the toxin stress of Alternaria alternata,this study carried out a series of molecular biology experiments,such as transcriptome sequencing,establishment of genetic transformation system,yeast single hybridization,etc.Firstly,the transcriptome of Trichoderma under the stress of A.alternata toxin was established.After analyzing the transcriptome data.it was found that the genes and biological processes regulated by Trichoderma were very different at different time points.The expression of various disease-resistant genes was not changed significantly at 0-12 h,but after 48 h of toxin stress,Trichoderma regulated the most response of disease-resistant genes,such as:ABC transporter ABC1(3.44 times),Chitinase gene Chitinase5(1.87 times),Cytochrome P450 gene Cyp4(4.38 times),heat stress protein gene Hsp1(2.41 times).Glucan gene Glucan16(2.91 times).Efflux protein gene EP2(3.90 times)and hydrolytic enzyme gene Hydrolase79(5.94 times).When T.asperellum were stressed by toxin,the expression level of the MYB family transcription factors of T.asperellum was significantly changed,indicating that MYB transcription factors played an important role in response to pathogen toxin stress.The expression level of transcription factor MYB118 reached the highest at 48 h under toxin stress.which was 1.64 times than that of the control group.The 42 MYB transcription factors homologous to TasMYB36 in 5 Trichoderma genomes were analyzed.The phylogenetic tree showed that these MYB transcription factors were distributed in 11 branches.The 42 MYB transcription factors contained 62 DNA-binding regions,and their amino acid sequences were conserved and different from those found in plants.The semi-quantitative results showed that the expression of the five MYB transcription factors(MYB25,MYB27,MYB36,MY38 and MYB58)was significantly different,and the five MYB transcription factors were further verified by qRT-PCR.The results showed that the ex pression of MYB transcription factor was consistent with the transcriptome data.In the fluorescence quantitative data,the expression level of transcription factor MYB36 was always down-regulated before 12 h of toxin stress,and reached the lowest level at 12 h with transcription peak of 2.25 times.It was then up-regulated.reaching highest level at 48 h,with transcription maximum of 2.39 times.The optimal protoplasm preparation system was optimized:the 24-36h fresh mycelium was selected.After cleaning with sterilizing water,the mycelium should be cleaned again with NaCl solution.The concentration of the enzyme was 10 mg/mL,and 4 h was the best time to prepare protoplasts by dissolving mycelium.At the same time,the genetic transformation system of Trichoderma CGMCC11653 was established by protoplast precipitation method:the optimal concentration of protoplast transformation was 107 protoplast/L and the combination mode of 100 ?L protoplast+30 ?L plasmid had the highest conversion efficiency.After transformation,the optimal culture time for protoplast regeneration was 12 h.Using the improved preparation method and transformation system,4 positive inverters(T1,T2,T3,T4)were obtained.The DNA sequences that specifically bound to transcription factors were screened by yeast one-hybrid.6 DNA sequences were identified by TasMYB36 transcription factor,including an unknown new element "TCCCATCGAT".In addition,we also found that TasMYB36 transcription factor binding element "MRS1" is very different from the elements that can bind in plants,"MRS1" element and MYB36 protein binding core base is "ACAT".At the same time,we calculated the disease-resistant gene promoters of MRS1 elements in the genome of Trichoderma.These MRS1 elements were widely distributed in the promoter regions of relevant disease-resistant genes.such as ABC transporters and a large number of hydrolytic enzymes.In summary,the conclusions and results obtained in this study lay a foundation for further understanding of the signaling network of MYB transcription factor in the regulation of toxin stress.