为深入研究大肠杆菌谷胱甘肽转运系统的蛋白质结构和功能,对该系统中的gsiB基因进行了克隆和表达条件的优化。根据大肠杆菌谷胱甘肽转运系统中底物结合蛋白gsiB基因序列,利用PCR方法扩增到该基因的编码区序列,利用SLIC(Sequence and ligation–independent cloning)方法直接将其插入pWaldo-GFPe中,成功构建了重组表达质粒pWaldo-GFP-GsiB。将重组质粒转化不同的大肠杆菌表达菌株进行诱导表达,通过改变培养温度和IPTG浓度等条件,得到了能够大量表达目标蛋白的重组子。结果表明:大肠杆菌BL21(DE3)是gsiB基因表达的最佳宿主菌;18℃低温诱导培养有利于gsiB基因的大量表达;0.1mmol/LIPTG足够诱导gsiB基因表达,增加IPTG浓度(0.1mmol/L～1.0mmol/L)并不能明显地促进gsiB基因的表达。Western blotting结果显示目标蛋白质有表达,其分子量大小与预期相符。
Sequence analysis of the chitinase gene cht1 of Bacillus circulans showed that it contained 2151 nucleotides,which codes the precursor of chitinase CHT1 with 717 amino acid residues.The nucleotide and deduced amino acid sequences of cht1 showed 81% and 95% homology with those of ChiA of B.circulans WL-12,respectively.The cht1 gene was cloned into the Escherichia coli-Bacillus subtilis shuttle vector pSUGV4 and two recombinant plasmids, named pUSCH1 and pUSCH2 which contained 2.9kb and 4.0 kb insert respectively,were obtained.The recombinant plasmids were transformed into B.subtilis DB104 and WB600.Chitinase activity was detected both in transformed E.coli and B.subtilis.The DB104/pUSCH1 strain was found to be effective in the bio-controlling the infection of Magnaporthe grisea under greenhouse condition,which showed 71.67% decrease in rice disease incidence.
