苏云金芽孢杆菌沉默基因cry2Ab3在大肠杆菌中表达及杀虫活性研究

Expression and Insecticidal Activities Analysis of Silent Gene cry2Ab3 form Bacillus thuringiensis C002 Strain

对苏云金芽孢杆菌C0 0 2菌株cry2Ab基因阳性克隆pHT3 1 5 2Ab进行亚克隆和序列测定 ,在CenBank注册后经国际Bt杀虫蛋白基因委员会正式命名为cry2Ab3。序列分析表明该基因含有芽孢杆菌特异的RBS序列 ,但没有功能性启动子 ,为沉默基因。根据大肠杆菌T7表达载体pET 2 1b克隆位点和cry2Ab3开放阅读框架 (ORF)两端序列 ,设计合成一对特异引物L2ab5和L2ab3 ,高保真PCR扩增获得cry2Ab3完整ORF ,经酶切、连接构建了重组表达质粒pET 2Ab3。表达质粒导入大肠杆菌BL2 1 (DE3 ) ,IPTG诱导后 ,SDS PAGE电泳证实了cry2Ab3的表达。生物测定显示诱导培养物对棉铃虫初孵幼虫和小菜蛾二龄幼虫具有杀虫活性 ,能明显抑制二化螟二龄幼虫生长 ,但对甜菜夜蛾和玉米螟没有明显活性。进一步提取Cry2Ab3蛋白 ,生测结果表明其对棉铃虫LC50 为 3 2 5 5 μg g。

A cry2Ab gene carried on the pHT315-2Ab from C002 strain, Chinese native isolate of Bt was subcloned, sequenced and registered in GenBank, then designated as cry2Ab3 by the Bt cry gene Nomenclature Committee. Sequence analysis revealed that the cry2Ab3 was a silent gene with the RBS sequence but lack of functional promoter. One pair of PCR primer L2ab5/L2ab3 was designed according to the sequence of E.coli T7 phage expression vector pET21b MCS and the cry2Ab3 ORF. The full ORF sequence was amplified with Taq plus DNA polymerase and inserted into the BamHⅠ/EcoRⅠ site of pET21b, and the recombinant expression plasmid was designated as pET-2Ab3. The result of SDS-PAGE analysis proved that Cry2Ab3 was expressed in E.coli BL21(DE3) induced by IPTG. Bioassay results showed that E.coli BL21(DE3) harbored pET-2Ab3 had insecticidal activities against H.armigera 1st larve and P.xylostella 2nd instar larvae, the corrected mortality (CM%) was 68.3 (7d) and 34.9(4d) respectively; and strong inhibition to the growth of C.suppresalis 2nd star larvae, while no actvity against S.exitera and O.furnacalis. Furthermore, Cry2Ab3 protein was extracted and the LC 50 against H.armigear was determined as 32.55μg/g。