College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, 163319, China.
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, 100193, China.
Appl Microbiol Biotechnol. 2018 Apr;102(8):3687-3699. doi: 10.1007/s00253-018-8836-5. Epub 2018 Mar 8.
Bacillus thuringiensis bacteria show insecticidal activities that rely upon the production of insecticidal crystal proteins, which are encoded by cry or cyt genes and can target a variety of insect pests. It has been shown that cry1Ac is the only cry gene in B. thuringiensis subsp. kurstaki HD73 (B. thuringiensis HD73) and its expression is controlled by both σ and σ. Here, we report a novel σ-dependent strong promoter of a non-cry gene (HD73_5014), which can direct strong cry1Ac gene expression in B. thuringiensis HD73. We constructed an E. coli-B. thuringiensis shuttle vector (pHT315-P -1Ac) for cry1Ac gene expression, using the HD73_5014 gene promoter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis showed that expression of the cry1Ac gene directed by the HD73_5014 gene promoter was at the same level as that directed by the previously known strongest cry promoter, P . However, this strain did not form typical bipyramidal crystals in mother cells, as observed by transmission electron microscopy and atomic force microscope. The strain with Cry1Ac protein expression under the control of the HD73_5014 gene promoter (P -cry1Ac) showed insecticidal activity against Plutella xylostella similar to that under the control of the orf1cry8E gene promoter (P -cry1Ac). Collectively, these results suggest that the HD73_5014 gene promoter, as a non-cry gene promoter, would be an efficient transcriptional element for cry gene expression. These data also show the possibility for improving Cry production by searching for transcriptional elements in not only cry genes, but also non-cry genes.
苏云金芽孢杆菌细菌表现出杀虫活性，这依赖于杀虫晶体蛋白的产生，这些蛋白由 cry 或 cyt 基因编码，可以针对多种害虫。已经表明，cry1Ac 是苏云金芽孢杆菌亚种 kurstaki HD73（苏云金芽孢杆菌 HD73）中唯一的 cry 基因，其表达受 σ 和 σ 控制。在这里，我们报告了一个非 cry 基因（HD73_5014）的新型 σ 依赖性强启动子，该启动子可以在苏云金芽孢杆菌 HD73 中指导强 cry1Ac 基因表达。我们构建了一个用于 cry1Ac 基因表达的大肠杆菌-苏云金芽孢杆菌穿梭载体（pHT315-P -1Ac），使用了 HD73_5014 基因启动子。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和 Western blot 分析表明，由 HD73_5014 基因启动子指导的 cry1Ac 基因表达水平与先前已知的最强 cry 启动子 P 相同。然而，通过透射电子显微镜和原子力显微镜观察，该菌株在母细胞中没有形成典型的双金字塔形晶体。在 HD73_5014 基因启动子控制下表达 Cry1Ac 蛋白的菌株（P -cry1Ac）对小菜蛾的杀虫活性与在 orf1cry8E 基因启动子控制下（P -cry1Ac）相似。总的来说，这些结果表明，HD73_5014 基因启动子作为一个非 cry 基因启动子，将是 cry 基因表达的有效转录元件。这些数据还表明，通过不仅在 cry 基因中，而且在非 cry 基因中搜索转录元件，有可能提高 Cry 的产量。
