Heterologous Expression Of Protease Gene SS42from Trichoderma Atroviride And Characterization Of Recombinant Protein In Biocontrol Progress

The plant disease is a great influence on agriculture and forestry production. Fungal disease is accounting for80%of plant disease. Chemical pesticides play an important role in the prevention and treatment in traditional which is a huge damage to the environmental and human beings health. Biological control of plant fungal pathogen is much safer so that it is the prime candidate in the search for reducing dependency on chemical pesticides. Several Trichoderma strains have been reported to be effective in controlling plant diseases, and the action of hydro lytic enzymes is considered as the main mechanism involved in the antagonistic process. The fungus Trichoderma atroviride is a well known biocontrol agent. It produces subtilisin-like serine protease （SS）, which is important in the development of biological control.In this study, a subtilisin-like serine protease gene SS42has been cloned from biocontrol T.atroviride ACCC30153. The SS42gene contains an open reading frame （ORF）,1328bp in length, interrupted by three short introns, and encodes a protein of434amino acids with a molecular weight of42.55kDa and a pI of5.53. BlastP search indicated that SS42protease is a close homologue of Peptidases_S8_S53superfamily, and sharing the highest similarity of98%with T. reesei （XP₀06966748）. Furthermore, signalP prediction showed that the SS42amino acid sequence was cleaved by signal peptidase between positions21and22（AAA|||MP）. We also predicted a oval three-dimensional structure of the proteins with Swissmodel.The expression of SS42gene was analyzed by RT-qPCR after T. atroviride ACCC30153cultured in nine different medias:MM, C starvation, N starvation,1%stem powder,1%leaf powder.1%Cytospora chrysosperma or1%Alternaria alternata cell wall,5%C. chrysosperma or5%A. alternata fermentation liquid culture condition. The results indicated that the expression of the SS42gene was all up-regulated regulated by treatments of different culture condition, and showed the highest expression at4h,177.29times higher than pretreatment with1%A. alternata cell wall. The results of RT-qPCR indicated that both plants and phytopathogen could interact with Trichoderma ACCC30153, and SS42gene highly expressed in the process.The cDNA of SS42was inserted into the pGEX-4T-2vector and transformed into Escherichia coli BL21for heterologous expression induced by IPTG. A clearly visible band with molecular mass about68kDa in the SDS-PAGE gel indicated that the transformant harboring the gene SS42had been successfully translated in E.coli and produced a recombinant protein. The bacterial was cultured under different concentrations of IPTG, different temperature, different OD and so on, of which the product was also inclusion body protein. Finally, the protein was purified by power showing a single clear, high concentration gel on SDS-PAGE.We carried out a study of enzyme activity on serine proteinase rSS42by Folin method. Recombinant serine proteinase remains relatively stable enzyme activity at30-60℃and pH4-8.5. When transformant BL21-SS42was induced with1mM IPTG for4h, the serine protease exhibited its maximal activity of almost20U/mL at40℃and pH9.0.Dual culture with Trichoderma atroviride and five plant pathogens （Fusarium, Sclerotinia, blight fungus, Cytospora, Rhizoctonia solani）,the research showing that Trichoderma always occupy an absolute advantage of living space and nutrition resulting to inhibit the growth of pathogens. It was carried out of fungi-repressing test with recombinant serine proteinase rSS42, the result found that rSS42inhibited the growth of five plant pathogens,which indicated the recombinant serine proteinase had the capability of biocontrol.In conclusion, the cloning of Trichoderma atroviride ACCC30153SS42gene had laid the foundation for the research on function and protein property. The proteinase was secreted into the culture medium by Escherichia coli BL21in a functionally activity form, and the expression serine proteinase could inhibit the growth of some fungal pathogen.It provided a material basis for developing a biological pesticide of recombinant protein SS42.