The Function Of G?? Gene Thga3 In Trichoderma Harzianum

The research on the mechanism of signal transduction and regulation in Trichoderma spp.is important for exploring the biocontrol mechanisms and further improving the biocontrol effection.G protein is a GTP binding protein-mediated cell transmembrane signaling system which plays an important key role in regulating the growth,sporulation,metabolism and pathogenicity of fungi.The typical G proteins are formed by G?,G?,and G? subunits,among which G? subunit includes G??,G??,G??.Generally,it is believed that the function of G?III subunit is mainly to affect the enzymes activities involving the cell wall structureion,cell wall structure,pathogenicity of strains and so on,and maybe negatively regulate the sporulation of fungi.In this study,though the methods and techniques of gene knockout,reverse mutation technique,and transcriptome sequencing analysis,the function of G?III subunit were explored for T.harzianum.thga3 gene was obtained from Th-33 genome sequence.Sequence number in Th-33 genome is Tha₀8474.thga3 gene was amplified by PCR and analyzed.The open reading frame of the complete thga3 was 1068 bp,encoding 355 amino acids.GenBank accession number is KY937956.The identity of thga3 and G?? subunit of T.virens?XP₀13955540.1?was 99%,while the identity between thga3 and TGA3 of T.atroviride?XP₀13940666.1L?was 97%.The knockout mutants and reverse mutants of Th-33 were obtained.Firstly,knockout vector pkh-ko?thga3 was constructed and transformed into Th-33 protoplasts by PEG-mediated protoplast transformation,then ?thga3-17 named ?thga3 and ?thga3-19,two thga3 knockout mutants were screened.Then,reverse mutant vector pkh-ko-G418-thga3 was constructed and successfully transformed into the ?thga3 protoplasts,the reverse mutant Rthga3 was get.The biological characteristic of wild-type Th-33,mutant ?thga3,and Rthga3 were analyzed.Compared to wild-typeTh-33,the sporulation of ?thga3 were decreased by 94%,the growth rate became slowlier with15%,the inhibition rate to Rhizoctonia solani was reduced by 26%,the chitinase activity was decreased by 23%,at the same time,the mycoparasitic ability and hydrophobicity were all lost completely.qRT-PCR showed that the expression of Tha₀9745 connecting with hydrophobicity function was significantly reduced 80% compared with the wide type,which was similar for Rthga3.These phenomenons reflected thga3 could not only regulate the growth,sporulation,antagonism,mycoparasitism,hydrophobic and chitinase activity,but also regulate the expression of the hydrophobin gene Tha₀9745.Under the same condition of PDA medium and being cultured for 7 days with three biological replicates,transcriptome sequencing of T.harzianum Th-33 and ?thga3 were done by Illumina Hiseq2500.Through the sequencing,different expression of G protein signal system was checked,which were involving with the cell wall degradation enzymes and sporulation related gene.Compared to the wild-type Th-33,three G protein receptor proteins Tha₀2907,Tha₀3034 and Tha₀6700 were down-regulated,one G protein signal transduction protein RGS1 Tha₀7915 was up-regulated,three chitinase genes Tha₀0180,Tha₀2554and Tha₀9915 were down-regulated;Sporulation related gene Tha₀9974 brlA was down-regulated,which hypothesized that thga3 may regulate sporulation by regulating the expression of brlA.What's more,thga3 may regulate the chitinase activity by regulating the expression of chitinase genes Tha₀0180,Tha₀2554 and Tha₀9915,it was speculated that thga3 may affect the antagonism and mycoparasitism.Moreover,thga3 also could regulate the mycelial hydrophobicity by regulating the expression of Tha₀9745.Though thga3 could affect the expressions of multiple G protein receptor proteins and regulatory proteins in the G protein signaling network,and further regulate a series of biological characteristics in Trichoderma,many works need to be further explored.