Transcriptome Analysis Of Trichoderma Harzianum E15 And Analysis Of Key Gene Family Of Bursaphelenchus Xylophilus

Trichoderma spp.Fungi are a type of soil habitual bacteria that widely exist in humid habitats such as forests,grasslands,and farmland.Trichoderma harzianum is one of the earliest species used in biological control in the genus Trichoderma spp.Pine wilt disease is a devastating disease caused by Bursaphelenchus xylophilus that causes the pine plants to die quickly.The disease has caused significant losses to China's forestry.T.harzianum has an inhibitory effect on soil nematodes,but the virulence factors of T.harzianum to B.xylophilus have been rarely studied.In this study,T.harzianum E15 was subjected to biologically repeated nematode stress treatment,and stress-treated hypha samples were sampled and transcriptome sequenced in a sterile environment.Through analysis and mining of transcriptome data,differentially expressed genes were found.And further screened out the subtilase protease family(peptidase S8)gene and the alcohol dehydrogenase gene that may be related to the response of T.harzianum to B.xylophilus.The bioinformatics method was used to analyze the genes of the two families.The experiment mainly draws the following conclusions:1.A total of 502953500 clean reads were detected in the six sample groups of the transcriptome sequencing,and a total of 75.45G sequencing data was obtained.The lowest unique map rate of the transcriptome sequencing data and the T.harzianum reference genome was higher than 90%,indicating that the E15 strain was closely related to the reference genome and the accuracy of gene annotation was higher.A total of 5645 differentially expressed genes were screened for differential expression analysis of genes,of which 2857 genes were up-regulated and 2788 genes were down-regulated.The top 10 pathways in the KEGG metabolic pathway enrichment analysis are the ribosome pathway(tre03010),the eukaryotic ribosome biogenic pathway(tre03008),the aminoacyl-tRNA biosynthesis pathway(tre00970),and the RNA polymerase pathway.(Tre03020),spliceosome pathway(tre03040),RNA transport pathway(tre03013),phenylalanine,tyrosine and tryptophan biosynthetic pathway(tre00400),porphyrin and chlorophyll metabolism pathway(tre00860),?-Alanine metabolism pathway(tre00410),glycolysis/gluconeogenesis pathway(tre00010).According to the GO annotation enrichment analysis,the peptidase S8 gene was significantly enriched in the protein metabolic process(GO:0019538).Based on the results of gene function annotation and gene differential expression analysis results,12 genes from the subtilisin family and the alcohol dehydrogenase family were screened for significantly up-regulated expression.These genes are most likely related to the anti-nematode response of T.harzianum,and subsequent bioinformatics analysis of these genes was performed.2.The 12 genes selected from the subtilisin family were named ThSPl-ThSP12.Phylogenetic trees of eight species of Trichoderma subtilisin genes were constructed.The analysis results show that ThSP1 belongs to the Peptidase S8 family subfamily 7 subfamily(cd07491);ThSP2,ThSP3-ThSP7 belong to the S8 and S53 family,but ThSP2 and ThSP3-ThSP7 belong to different subfamilies(c110459,cd00306);ThSP8 to ThSP12 belong to Peptidase S8 family subfamily 5 subfamily(cd07489).Amino acid sequence alignment and protein motif analysis helped us determine the location of the subtilisin conserved domain and find the location of the active site.The cis-acting elements of the subtilisin family were predicted and analyzed,and cis-acting elements that might be related to jasmonic acid,salicylic acid,low temperature response and drought induction were found.3.The selected gene of the alcohol dehydrogenase family was named ADH1-ADH12.Phylogenetic trees of three Trichoderma alcohol dehydrogenase genes were constructed,and the results showed that the seven genes ADH1,ADH2,ADH4,ADH6,ADH8,ADH9,and ADH10 belong to the same subfamily,while ADH3,ADH5,ADH7,and ADH12 are more common.Close kinship.The results of amino acid multiple sequence alignment and protein motif analysis showed that each alcohol dehydrogenase monomer has two main domains:a catalytic domain and a coenzyme binding domain.By aligning,we located the two domains and found the most conserved motifs in their sequences.The homology modeling method was used to predict the tertiary structure of T.harzianum dehydrogenase,and the sequence identity was 68.19%.A visual analysis of the predicted results was performed by analyzing the structure of soft nails.