Cloning And Expression Of Gene Chit37 And Gene Thi4 From Trichoderma Harzianum

Trichoderma harzianum is a filamentous soil fungus known as an effective biocontrol agent for a wide range of economically important airborne and soilborne plant pathogens. It plays a key role in biocontrol and environmental protection. To discover biocontrol mechanism in the level of genome and explore genes associated with biocontrol, an induced cDNA library of T. harzianum T88 mycelium was constructed by using colloidal chitin as sole carbon source and 1,386 ESTs were obtained (accession no. DY761370~DY762921). A novel endochitinase gene Chit37 and a novel thiazole biosynthetic enzyme gene Thi4 were obtained successfully by this means. The sequences of two genes were published on the GenBank with the accession numbers of DQ647957 and DQ647956.The animo acids sequence of gene Chit37 shared higher sequence identities with chitinase 18 family and contained a highly conserved motif (FDGIDIDIE) which corresponded to a catalytic domain. This family hydrolyze chitin so that it palys a very important role in agricultural biocontrol, ocean wastes degradation and resources recycling. The exploitation of novel chitinase gene Chit37 will supply the valuable materials for construction of efficient engineering strains. The gene Chit37 was expressed in E. coli BL21 (DE3) and the optimal induced conditions was 0.1mmol/L IPTG, 8h. This result will be significant to the preceeding research and large scale fermentation. Heterologous expression of the gene in S. cerevisiae H158 was carried out. Northern blotting analysis demonstrated that the gene was expressed at the transcription level. Considering the possible effects of untranslated regions (UTRs), two kinds of fragments of Chit37 were used in this study, the full-length gene (Chit37-I) and the coding region of the gene (Chit37-II). Semi-quantitative RT-PCR indicated that Chit37-I exhibited high expression level on 96h and 108h after galactose induction, while Chit37-II showed high mRNA transcription level on 84h and 96h. Both of the expressed products secreted to the medium and were measured in active forms. The optimal reaction temperature, pH and substrate concentration of CHIT37-I and CHIT37-II were 40℃, 5.0 and 5 %. However, the optimal culture time was 108h and 96h respectively. The CHIT37-II showed higher activity than CHIT37-I throughout and indicated the advantages in large scale fermentation. Discussions on the action of UTRs will benefit the reasonable utilization in the future.Expression vector pPKTCP was constructed and was transformed into T. harzianum mediated by A. tumefaciens. Molecular analysis showed that the T-DNA was integrated into the genome of T. harzianum with a single insert of T-DNA. The transformants were stable through mitotic and meiotic cell divisions. There was significant difference in antifungal activities between original strain of T. harzianum and its transformants. The conidial germination and mycelial growth of S. sclerotiorum, R. solani, F. oxysporum, S. rolfsii, F. moniliforme and B. cinerea were significantly inhibited in varying degrees. This result indicated that CHIT37 had antifungal activity and played a key role in biocontrol of T.harzianum. Besides, transformants could be used as inocula against plant diseases. This work has important theoretical significance and practical application value and will promote the industrialization process of biocontrol. Gene Thi4 was found sharing higher sequence identities with Thi4 family. Its function associated with abiotic stress and involved in organismic metabolism as the key enzyme of vitamin B1 biosynthesis. In addition, it was widely used in agricultural production as the framework of many pesticide and herbicide thiazole. Therefore, its molecular cloing and analysis have important significant for mass production of vitamin B1, super-effective pesticides by biological method and environmental sustainable development. The gene Thi4 was expressed in E. coli BL21 (DE3) and the optimal induced conditions was 0.1mmol/L IPTG, 4h. Heterologous expression of the gene in S. cerevisiae H158 was carried out. Northern blotting analysis demonstrated that the gene was expressed at the transcription level. The construction of efficient engineering strain supplied materials and basis for the industrial production of enzyme preparation.