Cloning And Function Analysis Of Tril4from Trichoderma Brevicompactum0248

Trichoderma brevicompactum, widely existed in soil and plant, is abiocontrol fungus for plant pathogenic fungi. Trichodermin, one of trichothecenes,is the important active compound in the metabolites of T. brevicompactum. It is ofgreat significance to research trichodermin biosynthesis pathway for the industrialproduction and agricultural application of trichodermin. The genes that involve inthe biosynthesis of trichothecenes are located in the tri cluster and thefunctions of them have been clarified. While the function of tri14gene, alsolocated in the tri cluster, remains unknown. The function of tri14gene intrichodermin biosynthesis pathway need further study.The function of tri14gene in T. brevicompactum0248was studied in thispaper. According to the transcriptome data, the fragment of tri14gene wasamplified and confirmed. By RACE cDNA amplification kit, the full cDNAsequence of tri14was obtained, which contains1331bp. There was a59bpintron in the DNA sequence of tri14. It was speculated that tri14mightencodes a Ca2+dependent dolioform protein which contains364AA.pMD18-T, pSilent-1and pCAMBIA0380were used as original plasmidsto construct pCAMBIA0380-H-Si vector which was used for silencing theexpression of tri14. First, an interference fragment expression unit wasconstructed into pSilent-1which was named as pSilent-Si. Secondly, the hphgene cassette and the interference fragment expression unit from pSilent-Siwere inserted into pCAMBIA0380respectively. The final plasmid wastransformed into strain0248by Agrobacterium tumefaciens AGL-1. A totalof67transformants were isolated from the hygromycin B plate.GC was used for measuring the yield of trichodermin and quantitativereal-time PCR was used for detecting the genes expression levels. Threetransformants (ZH31, ZH52and ZH58) which show lower expression level of tri14were selected from67transformants for next study. Both strain0248and the three transformants were cultured in BD medium. The yields oftrichodermin and the expression levels of tri14, tri4, tri5, tri11in thesestrains showed several results as below. One, the expression levels of tri14intransformants were lower than that in strain0248means that the expressionof tri14has been silenced successfully in transformants. Two, comparingwith strain0248, there were similar expression levels of tri4, tri5and similaryields of trichodermin, but higher expression levels of tri11, in thetransformants. It was speculated that tri14may participates the pathway oftrichodermin indirectly and may regulate the expression of other genes whichwere involved in the trichodermin biosynthesis, such as tri11. Three, base onthe other reports, it was considered that the transcription of tri5, not tri4ortri11, was probably a limiting step of trichodermin biosynthesis.