Prokaryotic Expression Of Chitinase Genes Chi31 And Chi32 From Limonium Bicolor And Recombinase Activity Analysis

Chitinase (Ec.3.2.14) is a kind of glycosidase catalyzing the degredation of chitin, an important constituent of fungal cell wall. Chitinase have roles in suppressing the growth of fungi and enhancing the resistant ability to fungal pathogens in plants. 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.Two chitinase genes chi31 and chi32 have been isolated from Limonium bicolor and deposited in GenBank with the accession number DQ431248 and DQ431249,respectively. In this study, the transcript patterns of two chitinase genes to response fungal phytopathogen Rhizoctonia solani kuhn were researched by using real-time RT-PCR. These two chitinase genes respose synergistically in different tissues and periods of infection, each plays indispensible roles in the pathogen resistant system, constitute an integrated defence system to outer stimuli.The cDNA conding region of chitinase genes chi31 and chi32 were inseted into the expression vector pET-52b(+), respectively; and then, the prokayotic intracellular expression vector pET-chi31in and pET-chi32in were transformed into E. coli BL21;the last, transformant BL21-chi31in and BL21-chi32in were obtained. The recombinant chitinases were identified by SDS-PAGE examination. The CHI31in and CHI32in enzymatic activities were determined by the modified Schales method.In the prokayotic intracellular expression, the optimal reaction condition for the activity of CHI31in is 5mmol/L of Mn2+,40℃, and the pH of 5.0. Under this condition, the CHI31in activity was 0.772U using the Septoria tritici cell wall as substrate. Correspondingly, the optimal condition of the recombinant CHI32in is 5mmol/L of Ba2+,45℃, and the pH of 5.0. The CHI32in activity is 0.792U with the substrate of Cytospora chrysosperma cell wall and under this condition.Simultaneously, prokayotic extracellular expression vectors pET-chi31ex and pET-chi32ex were constructed. The optimal reaction condition for the activity of prokayotic extracellular CHI31ex is 5mmol/L of Zn2+, the temperature of 40℃, and the pH of 5.0. Under this condition, the CHI31ex activity in the crude fermentation broth was 0.921U using the Septoria tritici cell wall as substrate. Correspondingly, the optimal condition of the recombinant CHI32ex is 5mmol/L of K+, the temperature of 45℃, and the pH of 5.0. And with the substrate of Cytospora chrysosperma cell wall, the activity of CHI32ex is 0.897U in the crude fermentation media under this condition. The activity of extracellular recombinant enzymes to fungal cell wall and compounds are generally higher than that of the intracellular recombinant enzymes in the crude fermentation broth. The extracellular CHI31ex activity is 0.921U, but the intracellular CHI31in activity of is 0.499U, using substrate of Cytospora chrysosperma cell wall. With the substrate of C. chrysosperma cell wall, the CHI32ex activity is 0.897U but the activity of intracellular CHI32in is 0.792U. However, the recombinant CHI31in and CHI32in of the extracellular prokaryotic expression demonstrated better hydrolytic ability to cell walls of different fungal than synthetic chitins, suggesting a high potentiality utilization of the extracellular prokaryotic expression on biocontrol. The recombinant CHI31in andCHI32in showed a high capacity of degrading the cell wall of Phytophthora sojae.The prokayotic intracellular and extracellular recombinant chitinases have much value in controlling plant pathogen in agriculture and forestry. Production of recombinat enzyme can decrease the cost of production and the expense on control plant pathogen. This study also provides solid foundation for the further application of chitinase genes in pathogen-resistant plant molecular breeding.