Zhou Changmei, Zheng Qingyun, Peng Qi, Du Lixin, Shu Changlong, Zhang Jie, Song Fuping
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. 2014 Sep;98(18):7901-9. doi: 10.1007/s00253-014-5874-5. Epub 2014 Jun 14.
To optimize the expression of cry genes in a Bacillus thuringiensis sigK mutant failing in crystal releasing, the transcriptional activity of the cry promoters cry1A, cry3A, cry4A, and cry8E was compared using lacZ gene fusions. A beta-galactosidase assay indicated that the cry8E promoter showed the highest transcriptional activity. A novel Escherichia coli-B. thuringiensis shuttle vector pHT315-8E21b was constructed for cry gene expression using the cry8E promoter and the multiple cloning sites from vector pET21b, based on vector pHT315. SDS-PAGE analysis showed that the expression of the cry1Ac gene directed by the cry8E promoter was increased by approximately 2.4-fold over the expression directed by the cry3A promoter. The cry1Ba gene was expressed in the sigK mutant with the constructed vector pHT315-8E21b. Normal bipyramidal crystals encapsulated in mother cell were observed by transmission electron microscopy (TEM). The encapsulated Cry1Ba protein expressed in the sigK mutant showed activity against Ostrinia furnacalis and Plutella xylostella similar to that of the released Cry1Ba protein expressed in the acrystalliferous strain HD73 and can be protected from inactivation by UV light. All these results suggest that the cry8E promoter can be an efficient transcriptional element for cry gene expression in sigK mutants and can be utilized for the construction of a genetically engineered strain.
为了优化在晶体释放方面存在缺陷的苏云金芽孢杆菌sigK突变体中cry基因的表达，利用lacZ基因融合比较了cry1A、cry3A、cry4A和cry8E启动子的转录活性。β-半乳糖苷酶测定表明，cry8E启动子显示出最高的转录活性。基于载体pHT315，利用cry8E启动子和来自载体pET21b的多克隆位点，构建了一种用于cry基因表达的新型大肠杆菌-苏云金芽孢杆菌穿梭载体pHT315-8E21b。SDS-PAGE分析表明，cry8E启动子指导的cry1Ac基因表达比cry3A启动子指导的表达增加了约2.4倍。cry1Ba基因在构建的载体pHT315-8E21b的sigK突变体中表达。通过透射电子显微镜（TEM）观察到母细胞中包裹着正常的双金字塔形晶体。在sigK突变体中表达的包裹型Cry1Ba蛋白对亚洲玉米螟和小菜蛾显示出与无晶体菌株HD73中表达的释放型Cry1Ba蛋白相似的活性，并且可以免受紫外线灭活。所有这些结果表明，cry8E启动子可以作为sigK突变体中cry基因表达的有效转录元件，并可用于构建基因工程菌株。
