Cloning And Expression Of Rice Chitinase Genes

Genome of rice were extracted from young leaves and used as temple. Two chitinase genes chAB and ch24 were amplified by PCR with primer A1/A2 and B1/B2 separately. The two genes were then cloned into pUC18-T vector and sequenced. The chitinase gene chAB is 861bp in size and is the member of the family classⅢ, while ch24 is 972bp in size and belong to the chitinase family classⅠ.The two genes chAB and ch24 were cloned into pET-32a vector separately to constuct prokaryotic expression vector pAB-32a and p24-32a. The conditions for protein expression were tested by orthogonal test. The optimum protocol for expression of pAB-32a involves: 0.6mM IPTG induction, the pH of the medium is 7.0, oscillate fermenting 5h with 200rpm under 30℃. The optimum protocol for expression of pAB-32a involves: 0.6mM IPTG induction, the pH of the medium is 6.0, oscillate fermenting 6h with 200rpm under 35℃. The recombinant protein pAB-32a and p24-32a were mainly existed in inclusion body. The inclusion bodies were refolded by urea gradient dialysis refolding method and then purified with Nickel column. The enzymatic activity of the recombinant proteins pAB-32a and p24-32a were 0.17U/mg and 0.12U/mg separately after renaturation.In order to realize the soluble and secretive expression of the recombinant chitinase, the two genes chAB and ch24 were cloned into the vector pMBP-P separately to construct expression vector pAB-MBP(P) and p24-MBP(P). The results showed that the optimal inducible concentration of IPTG was 0.6mM for the expression of the two recombinant proteins. The results of antifungal test showed that the fungistasis effect of pAB-MBP(P) and p24-MBP(P) to R. solani sclerotia are better than pAB-32a and p24-32a.