Expreesion Of Recombinant Chi46 In Pichia Pastoris And Preliminary Study On Its Anti-Pathogenic Activity

Chaetomium globosum is an important plant biocontrol fungus with wide application. C. globosum possesses many important enzymes genes that have tremendous value in biocontrol, one of which is a 46kD endochitinase gene (chi46). Chitinase (Ec.3.2.14), a group of enzymes capable of degrading pathogenic fungi chitin directly to chitin oligosaccharides or N-acetylglucosamine, are produced by a wide variety of organisms including plants, microorganism and animals, and contribute to many physiological reaction. Chitinase have received increased attention because of their potential application in biocontrol of plant-pathogenic fungi and pests, as well as in bioconversion of shellfish chitin wastes.In order to use the biocontrol potential of chi46 to produce broad-spectrum resistant Biopesticide, this study used Pichia pastoris as expression system,which was easy to culture and expressed high-level recombinant proteins. After amplifying the chitinase gene from pGEX-chi with designed primers, The chi46 gene and expression vector pPIC9 were digested with EcoR I and Not I,and ligation was carried out in vitro. Detected by PCR, double enzyme digestion and sequence, the recombinant expression plasmid with correct reading frame pPIC9-chi6 was constructed.The constructed plasmid pPIC9-chi6 was linearized with a restriction enzyme Stu I and transformed into Pichia GS115 and KM71 competent cell by electransformation method. Detected by Methanol-utilizing phenotype screening and PCR analysis, two GS115 (Mut+) transformants and three KM71 (Muts) transformants was got. The parent vector was linearized with the same restriction enzyme and transformed to GS115 and KM71 as control. Induced by methanol, these integrants were analyzed expression levels every 24 hours of interest protein by SDS-PAGE and the engineering strains with high chitinase expression level, such as G6-5 and K6-9 was determined.We carried out some study on bioactivities of chitinase including enzyme activity, some enzyme characteristics and fungi-repressing test. Research found that the optimum fermentation days for GS115 transformant of G6-5 was 3 days later than that for KM71transformant of K6-9, but enzyme activity of G6-5 was 8.24% higher than K6-9. Recombinant CHI46 remains relatively stable enzyme activity at 35℃-65℃and pH3-10, which indicates its good prospect of application in biocontrol.The best combination of enzyme system was gotten, that was Cu2+ 5mmol/L, 45℃, pH 5.0, and in the condition of which the recombinant CHI46 activity with N,O-carboxymethyl chitosan as the substrate was 30.19U Measureing enzyme cativity with different substrates, we found that recombinant CHI46 activities with fungi cell wall as the substrate were much higher than those with compounds.Recombinant CHI46 inhibited the growth of Rhizoctonia solani, Alternaria alternata and Cytospora chrysosperma, that indicatd the recombinant CHI46 had the capability of degrading cell wall of fungal pathogen.The research of CHI46 has great value in economy and theory. The Chaetomium globosum chitinase was secreted into the culture medium by the yeast Pichia pastoris in a functionally activity form, and the expression chitinase can inhibit the growth of some fungi. It implied the expression chitinase could be applied widely for the chitin industry and biological fields. Therefore, we hope to reconstruct the chi46 gene, exploration of appropriate methord of fermentation and enzyme activity stabilization to obtain the yeast engineering strains suitable for industrial application and produce bio-pesticide for biological control of plant diseases.