Prokaryotic expression and bioactivity analysis of the fusion insecticidal protein of Bacillus thuringiensis Cry1A.301-Vip3A
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摘要: 【目的】 对苏云金芽孢杆菌（Bacillus thuringiensis，Bt）的杀虫蛋白基因（Cry1A.301和Vip3A）进行原核融合表达及生物活性分析，为玉米抗虫基因资源发掘和品种创制提供科学参考。【方法】 利用连接肽构建不同杀虫机理的Cry1A.301和Vip3A融合基因，构建其原核表达载体，在大肠杆菌中进行诱导表达，对融合蛋白进行理化性质和结构域预测分析及定量检测，并采用人工饲料混合饲喂法测定融合蛋白对亚洲玉米螟（Ostrinia furnacalis）、棉铃虫（Helicoverpa armigera）和草地贪夜蛾（Spodoptera frugiperda）的杀虫活性。【结果】 构建的融合基因基本骨架为5'-Cry1A.301-Vip3A-3'，中间由编码连接肽（8个氨基酸）的核苷酸序列（TCCACCTGCTCCACCTGCTCCACC）组装而成，经诱导表达形成融合蛋白Cry1A.301-Vip3A。该融合蛋白含有1409个氨基酸，分子量约为157 kD，为稳定的酸性蛋白，包含Cry1A.301和Vip3A蛋白家族的4个特征结构域。Cry1A.301-Vip3A融合蛋白在大肠杆菌BL21（DE3）细胞中成功表达，且Cry1A.301和Vip3A蛋白的表达量无显著差异（P>0.05，下同）。饲喂Cry1A.301-Vip3A融合蛋白7 d后，亚洲玉米螟、草地贪夜蛾和棉铃虫幼虫校正死亡率达100.00%。Cry1A.301-Vip3A融合蛋白与Cry1A.301蛋白对亚洲玉米螟的杀虫活性无显著差异，均显著高于Vip3A蛋白（P<0.05，下同）。Cry1A.301-Vip3A融合蛋白、Vip3A蛋白和Cry1A.301蛋白对棉铃虫的杀虫活性无显著差异。Cry1A.301-Vip3A融合蛋白和Vip3A蛋白对草地贪夜蛾的杀虫活性无显著差异，均显著高于Cry1A.301蛋白。【结论】 原核表达的Cry1A.301-Vip3A融合蛋白结构稳定，对亚洲玉米螟、棉铃虫和草地贪夜蛾均具有较好的杀虫活性。
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关键词:
- 苏云金芽孢杆菌（Bt） /
- 融合蛋白 /
- 原核表达 /
- 生物活性
Abstract: 【Objective】 To conduct prokaryotic fusion expression and bioactivity analysis on the insecticidal protein genes（Cry1A.301 and Vip3A）of Bacillus thuringiensis（Bt），so as to provide scientific reference for the exploration of insect-resistant gene resources and the creation of maize varieties. 【Method】 Utilized the connecting peptides to construct the fusion genes of Cry1A.301 and Vip3A with different insecticidal mechanisms. Then，constructed their prokaryotic expression vectors and induced expression in Escherichia coli. Prediction analysis on the physicochemical properties and domains of the fusion proteins as well as quantitative detection was conducted. Moreover，the insecticidal activities of the fusion proteins against Ostrinia furnacalis，Helicoverpa armigera and Spodoptera frugiperda were detected by the artificial diet mixed feeding method. 【Result】 The basic framework of the constructed fusion gene was 5'-Cry1A. 301-Vip3A-3'，with the nucleotide sequence（TCCACCTGCTCCACCTGCTCCACC）encoding the connecting peptide（8 amino acids） assembled in the middle. Through induced expression，the fusion protein Cry1A.301-Vip3A was formed. This fusion protein contained 1409 amino acids，with a molecular weight of approximately 157 kD. It was a stable acidic protein and encompassed 4 characteristic domains of the Cry1A.301 and Vip3A protein families. The fusion protein Cry1A.301-Vip3A could be successfully expressed in E. coli strain BL21（DE3），and there was no significant difference in the expression levels of Cry1A.301 and Vip3A proteins（P>0.05，the same below）. After feeding the fusion protein Cry1A.301-Vip3A for 7 d，the corrected mortality rates of O. furnacalis，H. armigera and S. frugiperda reached 100.00%. The insecticidal effect of the fusion protein Cry1A.301-Vip3A on O. furnacalis was not significantly different from that of the Cry1A.301 protein，but both were significantly higher than that of the Vip3A protein（P<0.05，the same below）. The insecticidal activity of the fusion protein Cry1A.301-Vip3A on H. armigera was not significantly different from those of Vip3A protein and Cry1A.301 protein. The insecticidal activity of the fusion protein Cry1A.301-Vip3A on S. frugiperda was not significantly different from that of Vip3A protein，but was significantly higher than that of Cry1A.301 protein. 【Conclusion】 The Cry1A.301-Vip3A fusion protein expressed in prokaryotes has a stable structure and exhibits good insecticidal activities against O. furnacalis，H. armigera and S. frugiperda. -
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