苏云金芽胞杆菌LM1212菌株cry基因启动子的活性分析

Activity of cry gene promoter of Bacillus thuringiensis LM1212

摘要:

【背景】前期发现一株具有独特分化表型的苏云金芽胞杆菌（Bacillus thuringiensis，Bt） LM1212，该菌株共有14种杀虫基因，组成了10个转录单元。利用源自菌株LM1212的晶体产生细胞调控因子（crystal producing cell regulator，CpcR）以及其可以激活的cry35-like基因启动子，已在典型Bt菌株HD73中成功构建了非芽胞杀虫蛋白表达体系。【目的】比较菌株LM1212的不同杀虫基因启动子的转录活性，明确可被转录因子CpcR激活的且转录活性较高的启动子，以此为基础优化非芽胞杀虫蛋白表达体系。【方法】将10个启动子区域分别与lacZ报告基因融合构建在pHT304-18Z载体上，得到了10个重组质粒；然后将cpcR基因及其启动子（P_cpcR-cpcR）分别反向构建在选取的启动子区域与lacZ报告基因的上游，得到可以表达CpcR且与上述构建相对应的10个重组质粒，将这些重组质粒分别转入不含CpcR的菌株HD73，即获得20个可用于测定β-半乳糖苷酶活性的重组菌株。通过光学显微镜观察和SDS-PAGE检测明确杀虫蛋白表达的情况。【结果】在菌株HD73中，β-半乳糖苷酶活性检测结果显示，启动子P₁、P₃、P₄、P₅、P₆、P₇和P₈转录均可被转录因子CpcR激活，启动子P₁₀的转录受到转录因子CpcR抑制。在CpcR存在时，P₇、P₈启动子的转录活性相对较高。利用转录因子CpcR和P₇、P₈启动子成功表达了对草地贪夜蛾有较高杀虫活性的Vip3Aa11蛋白。【结论】筛选到的高转录活性的启动子可用于优化非芽胞杀虫蛋白表达体系，从而构建新型工程菌，为草地贪夜蛾的生物防治提供新思路。

Abstract:

[Background] Bacillus thuringiensis (Bt) LM1212 with a unique differentiation phenotype has been identified. It carries 14 insecticidal genes, which comprise 10 transcription units. With the crystal producing cell regulator (CpcR) derived from the LM1212 strain and the cry35-like gene promoter activated by CpcR, the non-sporulating expression system of insecticidal protein has been successfully established in the typical Bt strain HD73. [Objective] To compare the transcriptional activities of different insecticidal gene promoters of strain LM1212, identify the promoters with high transcriptional activity and activated by the transcription factor CpcR, and optimize the non-sporulating expression system. [Methods] Ten promoter regions were respectively fused with lacZ reporter gene on the pHT304-18Z vector, and 10 recombinant plasmids were obtained. The cpcR gene and its promoter (P_cpcR-cpcR) were reversely integrated in the upstream region of each selected promoter region and the lacZ reporter gene, and 10 recombinant plasmids capable of expressing CpcR and corresponding to the above construction were obtained. Subsequently, these recombinant plasmids were separately transferred into the HD73 strain without CpcR, and thus 20 recombinant strains were obtained for the determination of β-galactosidase activity. The expression of insecticidal protein was determined by microscope observation and SDS-PAGE. [Results] In HD73 strain, the transcription of the promoters P₁, P₃, P₄, P₅, P₆, P₇, and P₈ could be activated by CpcR, while that of the promoter P₁₀ was inhibited by CpcR. In the presence of CpcR, P₇ and P₈ showed high transcriptional activities. Vip3Aa11 protein with strong activity against Spodoptera frugiperda was successfully expressed by CpcR combined with P₇ and P₈. [Conclusion] The screened promoter with high transcriptional activity can be used to optimize the non-sporulating expression system of insecticidal protein, so as to construct novel bacteria for the biocontrol of S. frugiperda.