粘虫颗粒体病毒增效蛋白在苏云金芽胞杆菌中的表达及增效活性

Expression and synergistic activity of enhancin from Pseudaletia unipuncta granulovirus-Ps in Bacillus thuringiensis

摘要:

【目的】在苏云金芽胞杆菌(Bacillus thuringiensis, Bt)中表达截短后的转宿主粘虫颗粒体病毒(Pseudaletia unipuncta granulovirus-Ps, PuGV-Ps)增效蛋白，为构建增效Bt工程菌提供理论基础。【方法】通过对截短后增效蛋白的密码子进行优化，构建增效蛋白及其融合蛋白表达载体，分析不同启动子指导下增效蛋白表达量的变化，明确增效蛋白对Bt的增效活性。【结果】本研究构建了表达载体pHTP_cry1AcCoEn81、pHTRHCoEn81和pHTNCCoEn81，SDS-PAGE结果显示pHTP_cry1AcCoEn81和pHTNCCoEn81分别可以产生81 kDa和134 kDa的重组蛋白。启动子P_cry1Ac和P_cry8E指导下的增效蛋白表达量和重组增效蛋白产量均无显著性差异。生物测定结果表明，重组增效蛋白可以显著增加Bt对小菜蛾的杀虫活性。【结论】研究结果表明，密码子优化的PuGV-Ps增效蛋白可以在Bt中表达并具有显著增效活性，为高效苏云金芽胞杆菌工程菌的构建及应用提供理论指导。

Abstract:

[Objective] To express the truncated fragments of enhancin gene from Pseudaletia unipuncta granulovirus-Ps (PuGV-Ps) in Bacillus thuringiensis (Bt) and provide a theoretical basis for the construction of Bt engineering bacteria. [Methods] The codon of the truncated fragments of enhancin gene was optimized for the construction of the expression vectors of enhancin and the expression of fusion proteins. Then, the expression levels of enhancin under the guidance of two promoters were analyzed, and the synergistic activity of enhancin on Bt was determined. [Results] The expression vectors pHTP_cry1AcCoEn81, pHTRHCoEn81, and pHTNCCoEn81 were constructed in the study. SDS-PAGE showed that pHTP_cry1AcCoEn81 and pHTNCCoEn81 produced recombinant proteins of 81 kDa and 134 kDa, respectively. There was no significant difference in the expression level of enhancin or the yield of recombinant protein under the guidance of promoters P_cry1Ac and P_cry8E. Furthermore, the recombinant enhancin significantly increased the insecticidal activity of Bt against Plutella xylostella. [Conclusion] The codon-optimized enhancin of PuGV-Ps can be expressed in Bt and has significant synergistic activity, which provides theoretical guidance for the construction and application of Bt engineering bacteria with high efficiency.