Screening Of Spore-producing Mutants Of Trichoderma And Analysis Of Differentially Expressed Genes

Trichoderma harzianum is an important biocontrol fungi.The study of the key functional genes of Trichoderma harzianum can improve the understanding of the growth,sporulation and biocontrol mechanism of Trichoderma harzianum,and provide theoretical basis for the study of high quality biocontrol agents.At present,there are many commercial Trichoderma preparations,mainly conidia preparations,but the production of Trichoderma conidia is greatly affected by the environment.How to obtain a large number of spores stably is still one of the limiting factors in the industrial production.Therefore,it is of great significance to excavate the key genes of Trichoderma conidia to increase the conidia yield and improve the application effect of biocontrol.In this study,we select Trichoderma harzianum T2-16 as the research object,establish the EMS mutagenesis technology system of Trichoderma and construct its mutant library,screen the mutants with different spore production by phenotypic determination of conidia and growth potential,and further carry out transcriptome analysis of sporulation differentially expressed genes.The purpose of this study is to provide theoretical support for excavating the key causes of conidia production of Trichoderma and enriching and perfecting the mechanism of conidia production.The main results of the experiment are as follows:1.Mutagenic receptors is the spore concentration of 10⁶cfu·m L^-1Trichoderma harzianum T2-16,treating with 0.4 mol·L^-1EMS for 4 hours,an efficient and stable mutagenesis system of Trichoderma harzianum T2-16was established and the mutant library was constructed.Through analysis of growth potential,sporulation ability and antagonistic ability,it was found that some mutants had obvious changes in colony color,colony expansion speed,sporulation ability and antagonistic ability.Among the 418 mutant strains with stable inheritance,the positive mutation rate of growth potential was 46.20%,and the canonical mutant strain with relative inhibition rate of more than 30%accounted for 11.96%.2.It was found that the mutants T91,T120,T215,T240 and T383 had obvious changes in colony color,conidia number and conidiophores by screening sporulation mutants in PD medium and PDA medium.After systematic observation,Trichoderma harzianum T2-16 and mutant T215were used for transcriptome sequencing.3.Based on the comparative analysis of transcriptome sequencing of Trichoderma harzianum T2-16 and mutant t215,the results showed that the pathways related to conidia production of Trichoderma harzianum T2-16 may include amino acid biosynthesis,glycometabolism of amino sugar and nucleotide,secondary metabolite biosynthesis,tyrosine metabolism,etc.Among them,the cysteine synthase gene（GME2374＿g）and the cysteine dioxygenase gene（GME7986＿g）are mainly enriched in the biosynthesis of amino acids,are mainly enriched in the sugar metabolism of amino sugars and the endochitinase gene（GME8986＿g）and the chitinase gene（GME10321＿g）,mainly enriched in secondary metabolite biosynthesis the threonine aldolase gene（GME286＿g）and the glycerol-3-phosphate dehydrogenase gene（GME3499＿g）and the aldehyde dehydrogenase gene（GME426＿g）and the fumaroylacetoacetase gene（GME3224＿g）,which are mainly enriched in tyrosine metabolism,may be genes regulating conidia production.