Functional Characterization Of A Glucanase Gene From Trichoderma Asperellum

Trichoderma is a class of important fungi that control plant diseases.Trichoderma can produce a lot of antimicrobial substances,Among them,some studies have shown that glucanase can significantly inhibit the growth of plant disease pathogens.In this study,a glucanase gene cDNA has been cloned from biocontrol Trichoderma asperellum ACCC30536 strain.The full length of the cDNA was 984 bp.The cDNA encodes a protein of 327 amino acids with a molecular weight of 35.44 kDa and a theoretical pI of 4.76,and the protein is a hydrophilic transmembrane protein.BlastP search indicated that glucanase is a close homologue of Glyco-hydro-12 superfamily.The expression of glucanase gene was analyzed by RT-qPCR after T.asperellum ACCC30536 cultured in eleven different medias:MM,C starvation,N starvation,1%CMC powder,1%root powder,1%stem powder,1%leaf powder,1%Septoria tritici or 1%Alternaria alternata cell wall,1%S.tritici or 1%A.alternata fermentation liquid culture condition.The results indicated that the glucanase gene is differentially regulated by treatments of different culture condition.The expression of the glucanase gene was down-regulated during the C starvation culture and the 1%CMC powder culture,and showed lowest expression at24,12h.2^-2.8,2^-4.4 times lower than pretreatment.Glucanase gene is mainly up-regulated under N starvation,MM,1%root powder,1%stem powder,1%leaf powder,1%Alternaria alternata cell wall,1%A.alternata fermentation liquid culture condition,1%Cytospora chrysosperma cell wall,1%C.chrysosperma fermentation liquid culture condition,and showed peak expression level at 72,12,24,12,12,24,48,24,48h respectively.The peak expression was higher 2^3.8,2^3.6,2^5.5,2^3.4,2^3.1,2^2.9,2^3.3,2^2.6,2^3.1 times than before treatment,respectively.The results of RT-qPCR indicated that the glucanase gene is differentially regulated by plants and phytopathogen,and glucanase gene highly expressed in the process.To further study the function of T.asperellum glucanase gene,the prokaryotic expression vector pGEX-4T-2-Glu of T.asperellum glucanase gene was successfully constructed and transferred into Escherichia coli BL21 to obtain prokaryotic recombinant strain.The gene was expressed in E.coli BL21 by induction culture with final concentration IPTG of 1.0 mmol/L.A clearly visible band with molecular mass about 61.44 kDa in the SDS-PAGE gel indicated that the transformant harboring the glucanase gene had been successfully translated in E.coli and produced a recombinant protein.The longer the induction time of IPTG,the more expression of recombinant protein.We used the DNS method to determine the enzymatic activity of recombinant glucanase.The results showed that the enzyme activity of recombinant glucanase was very much affected by pH and the optimum pH was 4.5.The activity of recombinant dextranase was less affected by temperature,and the activity of glucanase was relatively high at 40-50oC,and the optimum temperature was 45oC.In conclusion,a glucanase gene cDNA has been cloned from biocontrol T.asperellum ACCC30536 strain.Bioinformatics analysis of glucanase The differences in the expression of glucanase during the interaction between T.asperellum ACCC30536 and pathogens or plants were explored.The prokaryotic expression vector pGEX-4T-2-Glu of T.asperellum glucanase gene was successfully constructed and transferred into E.coli BL21 to obtain prokaryotic recombinant strain.It provided the theoretical foundation and technical support for the preparation of a large amount of glucanase and developping the glucanase biocontrol value.