Bt杀虫蛋白基因cry8Ea2的克隆、表达和活性

Cloning,Expression and Insecticidal Activity of cry8Ea2 Gene from Bacillus thuringiensis Strain B-DLL

根据已知Btcry8类基因的全长序列设计一对特异性引物JJX5和JJX3,以Bt菌株B-DLL的质粒DNA为模板扩增出3.5 kb大小的片段;将该片段插入大肠杆菌表达载体pET-21b中,并完成了该片段的全序列测定。该基因编码区为3 495 bp,编码的蛋白质由1 164个氨基酸残基组成,相对分子质量为131.8 kDa,等电点为pH 4.71,为弱酸性蛋白。该基因(GenBank:EU047597)编码的氨基酸序列与Cry8Ea1的氨基酸序列同源性高达99.31%,被国际Bt基因命名委员会正式命名为cry8Ea2。经IPTG诱导后该基因在大肠杆菌BL21(DE3)中能够正常表达130 kDa的蛋白,表达产物对暗黑鳃金龟、琉璃弧丽金龟和柳蓝叶甲具有活性,在浓度为8.98μg/g时对暗黑鳃金龟、琉璃弧丽金龟一龄幼虫14 d的校正死亡率分别为46.67%和55.56%;在浓度为89.8μg/mL时对柳蓝叶甲三龄幼虫96 h的校正死亡率为33.33%。

The full length cry8Ea gene was amplified by PCR using a pair of special primers, JJX5 and JJX3, designed according to cry8-type genes sequences,and inserted into E. coli expression vector pET-21b to obtain the recombinant plasmid. Nucieic acid sequence analysis showed that this gene was 3 495 base pairs encoding 1 164 amino acids, which were homolog of 99.31% compared with Cry8Eal, the molecular weight of the protein was 131.8 kDa with isoelectric point pH4.71 .This gene sequence had been registered in GenBank（accession number was EU047597）,and named cry8Ea2 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The result of SDS-PAGE indicated that cry8Ea2 gene could be expressed as a 130 kDa protein in E. coli BL21 （DE3） strain induced by IPTG. Bioassay of the expression product from the crySEa2 gene showed evident toxic to the larvae of Popillia flavosellata, Holotrichia paraUela and Plagiodera versicolora, with the corrected mortality of 46.67 % , 55.56 % and 33.33 % , respectively.