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【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130kDa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0.044μg/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。
【Objective】 The cry9Ea gene was cloned from Bt4 strain isolated in this laboratory and its expression and insecticidal activity were studied. 【Method】 The Bt4 strain was identified as containing cry9 gene by PCR-RFLP. Then the full-length gene was amplified by PCR using the full-length primer F9EA / R9EA using the plasmid of strain Bt4 as a template. 【Result】 The target fragment was inserted into the expression vector pET21b to obtain the recombinant plasmid pETcry9Ea of E. coli. The recombinant protein was transformed into E.coli BL21 (DE3), and the protein was expressed at 130kDa after induction. The cry9Ea7 gene was ligated into the shuttle vector pSXY422b and electroporated HD73- (cry-) to obtain the engineered bacteria BioHD9Ea7. The protein of Cry9Ea7 was extracted and bioactive Determination. Bioassay results showed that the Cry9Ea7 protein was highly virulent to newly hatched larvae of Trichoplusia ni with an LC50 of 0.044 μg / mL, whereas the newly hatched larvae of Spodoptera exigua and Helicoverpa armigera No activity shown. 【Conclusion】 A cry9Ea7 gene with high virulence to Trichoplusia ni was cloned and expressed, and BioHD9Ea7 was successfully constructed.