摘要： 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%。

关键词: Bt, cry8Ea2基因, 基因克隆, 蛋白表达, 杀虫活性

Abstract: The full length cry8Ea gene was amplified by PCR using a pair of special primers,JJX5 and JJX3,de-signed according to cry8-type genes sequences,and inserted into E. coli expression vector pET-21b to obtain the recom 2 binant plasmid.Nucleic acid sequence analysis showed that this gene was 3 495 base pairs encoding 1 164 amino acids, which were homologof 99.31 % compared with Cry8Ea1,the molecular weight of the protein was 131.8 kDa with isoelec-tric point pH4.71. This gene sequence had been registered in GenBank(accession number was EU047597),and named cry8Ea-as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The result of SDS-PAGE in- dicated that cry8Ea-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 cry8Ea-gene showed evident toxic to the larvae of Popillia flavosella-ta, Holotrichia parallela and Plagiodera versicolora,with the corrected mortality of 46.67 %,55 1 56 % and 33.33 %,re-spectively.

Key words: Bacillus thuringiensis, cry8Ea2 gene, Cloning, Protein expression, Insecticidal activity

中图分类号: 

• Q785