Cloning Of Trichodermin Biosynthesis Gene Cluster(tri)and Functional Characterization Of Tri3?tri11?tri4 Genes

Trichodermin,one of trichothecenes compounds,produced mainly by Trichoderma brevicompactum,it has strongly antifungal activity on many kinds of plant pathogenic fungi.To preliminary clarify the function of tri cluster on trichodermin biosynthesis by T.brevicompactum,we did a study in three aspects,the main results and conclusions are listed as follows:The results revealed that T.brevicompactum has a 24793 bp cluster that includes orthologs of tri6,tri4,tri3,tri10,tri11,tri12,tri14 genes in the Fusarium TRI cluster,but that the TRI5 orthologue is not within this core cluster.High orthologues of tri cluster have been identified between 0248 and the two reported strains T.brevicompactum IBT 40841 and T.arundinaceum IBT 40837.The deduced amino acid sequences of tri genes orthologues exhibited 90% identity.The most notable difference between the three strains' tri clusters is the size of the tri11-tri12 intergenic region.The influence of three carbon sources on the trichodermin production as well as the expression levels of tri genes in T.brevicompactum were investigated.The result showed that the expression levels of tri genes under high trichodermin-producing condition were much higher than that of trichodermin-nonproducing or low trichodermin-producing condition after 40 h incubation.The expression level of tri genes was positively correlated with the yield of trichodermin.By using homologous recombination technique,we constructed tri3?tri11 ? tri4 gene knockout vectors,and name them pKT3,pKT11,pKT4,respectively.By using Agrobacterium tumefaciens-mediated transformation,the three genes were deleted,select gene knockout transformants and name them ? tri3,? tri11,? tri4.The influence of genes deletion on the trichodermin production as well as the expression levels of tri genes in T.brevicompactum were investigated.Targeted ? tri3 knockout mutants exhibited a sharp decline in production of trichodermin,according to the chemical structure of trichodermin as well as the homologies analysis of tri3 gene.Our results demonstrated that the Tbtri3 gene responsible for the biosynthesis of trichodermin,the Tbtri3 gene encoded enzyme catalyzes acetylation reactions of the hydroxy at C-4 of the trichodermol skeleton.q RT-PCR results suggested that twice feedback regulation may have occurred after Tbtri3 gene was deleted.It's worth nothing that in ?tri3 mutants,small amount of trichodermin still be detected,one hypothesis for the reduced level of trichodermin is the presence of acetylation isozymes.There was no trichodermin was detected in the Tbtri11 gene knockout mutants,sequence analysis revealed that Tbtri11 gene encoded cytochrome P450 monooxygenases,and contained all the conserved structural features described for this family of proteins.Our results demonstrated that the Tbtri11 gene encoded enzyme catalyzes C-4 hydroxylation and generate trichodermol.Also there was no trichodermin was detected in the Tbtri4 gene knockout mutants,this means that Tbtri4 gene directly responsible for the biosynthesis of trichodermin,the Tbtri4 gene encoded cytochrome P450 monooxygenase catalyzes oxygenation of trichodiene at three carbons,C-2,C-11,and C-13,and generated isotrichodol.