苏云金芽孢杆菌B-Pr-88菌株中cry2Ab4基因的表达和杀虫活性研究

Expression and Insecticidal Activity of a Novel Gene cry2Ab4 from Bacillus thuringiensis Strain B-Pr-88

摘要:

以我室自行分离的对鳞翅目夜蛾科害虫具有高毒力的Bt菌株B-Pr-88为材料，用PCR-RFLP方法从其质粒DNA文库中筛选到含cry2Ab基因的一个阳性克隆pZF858，序列测定发现，该片段含有cry2Ab全长基因，开放读码框为1902bps，编码由633个氨基酸组成的70.7kD蛋白，氨基酸同源性与已公布的cry2Ab基因同源性均为99.8％，经Bt基因国际命名委员会正式命名为cry2Ab。根据cry2Ab基因开放阅读框架(ORF)两端序列，设计合成一对特异引物L2ab5和L2ab3，PCR扩增获得cry2Ab 完整ORF，与大肠杆菌表达载体pET-21b连接，构建了重组表达质粒pET-2Ab4，质粒导入大肠杆菌BL21（DE3），IPTG诱导后，SDS-PAGE电泳证实该基因表达了60kD的蛋白，生物测定表明，Cry2Ab4对棉铃虫和大豆食心虫具有高毒力，同时对小菜蛾和二化螟有一定的杀虫活性，而对亚洲玉米螟和甜菜夜蛾没有杀虫活性。

Abstract:

The full length cry2Abgene was cloned by PCR-RFLP method from Bt strain B-Pr-88，which was isolated in China with high toxicity to the Lepidopteran insect pests. Nucleic acid sequence analysis showed that this gene was 1902 base pairs encoding 633 amino acids. This cry gene was named cry2Ab4 as a novel gene by Bacillus thuringiensis Delta Endotoxin Nomenclature Committee. The full open reading frame sequence of the cry2Ab4 gene was amplified with a pair of PCR primers L2ab5/L2ab3 designed according to its DNA sequence，and inserted into the BamHⅠ/EcoRⅠ sites of E. coli expression vector pET21b to obtain the recombinant plasmid pET-2Ab4. The result of SDS-PAGE proved that Cry2Ab4 could be expressed as a 60kD protein in E. coli BL21（DE3）strain induced by IPTG. Bioassay of the expressed product of the cry2Ab4 gene showed that Cry2Ab4 was highly toxic to the larvae of Helicoverpa armigera and Leguminivora glycinivorella，moderately active to the larvae of Plutella xylostella and Chilo suppressalis，but not insecticidal to the larvae of Spodotera exigua and Ostrinia furnacalis. Our result indicated that cry2Ab4 gene could be used as a novel gene for generation of transgenic plants and engineered microorganism.