对粉纹夜蛾高毒力cry9Ea基因的克隆及表达

Cloning and characterization of a novel gene cry9Ea7 encoding crystal protein with high toxicity against Trichoplusia ni

摘要:

摘要：【目的】从本实验室分离的Bt4菌株中克隆cry9Ea基因，并研究其表达和杀虫活性。【方法】以PCR-RFLP方法鉴定Bt4菌株含有cry9基因，然后以菌株Bt4的质粒为模板，利用全长引物F9EA /R9EA 进行PCR扩增全长基因。【结果】将目的片段插入到表达载体pET21b，得到大肠杆菌重组表达质粒pETcry9Ea。转化E. coli BL21(DE3)，诱导后表达130 kDa的蛋白，再将cry9Ea7基因连接到穿梭载体pSXY422b，电激转化HD73-(cry-)，得到工程菌BioHD9Ea7，提取Cry9Ea7晶体蛋白，并进行生物活性测定。生物活性测定结果显示Cry9Ea7蛋白对粉纹夜蛾（Trichoplusia ni）初孵幼虫具有高毒力，LC50为0.044 μg/ml，而对甜菜夜蛾（Spodoptera exigua）和棉铃虫（Helicoverpa armigera）初孵幼虫未显示活性。【结论】克隆和表达了一个对粉纹夜蛾高毒力的基因cry9Ea7，并成功构建了工程菌BioHD9Ea7。

Abstract:

Abstract: [Objective] We cloned a novel cry9Ea gene encoding a Cry9Ea protein with a high insecticidal activity against Trichoplusia ni. [Methods] We identified a cry9-type gene from Bt strain (Bt4) by PCR-RFLP. The full length cry9Ea gene was amplified by PCR with a pair of primers F9EA/R9EA. [Results] We cloned the complete cry9Ea7 gene into pET21b. The SDS-PAGE results showed that the 130 kDa Cry9Ea7 protein was expressed in E. coli BL21(DE3). We also constructed an engineering strain BioHD9Ea7 by transforming a shuttle vector pSXY422b containing the cry9Ea7 gene into Bt acrystalliferous mutant HD73-(cry-). The bioassay results indicated that the Cry9Ea7 protein was highly toxic against Trichoplusia ni neonates, and the LC50 value was 0.044 μg/mL. However, the Cry9Ea7 protein showed no activity against Spodoptera exigua and Helicoverpa armigera neonates. [Conclusion] The novel cry9Ea7 gene encoding Cry9Ea7 is highly toxic against Trichoplusia ni neonates.