苏云金芽胞杆菌sigK 基因插入失活突变体的特点及其对cry3A基因启动子活性的影响

Characterization of Bacillus thuringiensis sigK disruption mutant and its influence on activation of cry3A promoter

摘要:

摘要: 【目的】构建苏云金芽胞杆菌(Bacillus thuringiensis，简称Bt) sigK 基因插入失活突变体，分析突变体特点并明确其对cry3A 基因启动子的影响。【方法】采用同源重组技术在苏云金芽胞杆菌HD-73 菌株sigK 基因中插入卡那霉素抗性基因，构建了sigK 基因插入失活突变体。通过生长曲线测定、扫描电子显微镜观察晶体、芽胞形成情况和芽胞计数及SDS-PAGE 等方法分析了突变体的特点; 构建了遗传恢复菌株对上述性状进行了功能验证; 利用启动子融合lacZ 技术检测了cry3A 基因启动子的转录活性。【结果】获得了苏云金芽胞杆菌HD-73 菌株sigK 基因突变体，生长曲线测定表明，突变体较出发菌株在稳定期后期生长较慢; 扫描电子显微镜观察和芽胞计数分析显示，突变体丧失了形成芽胞和晶体的能力; SDS-PAGE 分析表明突变体中伴胞晶体蛋白的表达量明显低于出发菌株和恢复菌株。利用载体pHT315 携带sigK 基因及其启动子在突变株中表达，所获得的遗传恢复菌株恢复了突变株产生芽胞和晶体的能力; sigK 基因的突变可以提高cry3A 基因启动子在产胞后期的转录活性，对cry3A 启动子指导的Cry 蛋白表达量没有显著影响。【结论】本研究证明sigK 基因为苏云金芽胞杆菌芽胞形成所必需，并影响伴胞晶体蛋白的产量; sigK 基因功能的丧失有利于cry3A 基因启动子在产胞后期的转录。

Abstract:

Abstract: ［Objective］ To construct and characterize a sigK gene disruption mutant of Bacillus thuringiensis and to study influence of sigK gene disruption on the activation of cry3A gene promoter.[Methods] We constructed the sigK gene disruption mutant HD△sigK by inserting kanamycin resistance gene via homologous recombination． Scanning electron microscopy and spore formation analysis were used to detect the abilities of sporulation and crystal protein formation of boththe mutant and the wild-type strain． SDS-PAGE analysis was used to detect the expression of crystal protein． Betagalactosidase assay of cry3A'-lacZ gene fusion was performed to analyze the influence of sigK gene disruption on the activation of cry3A promoter． ［Results］ The growth curve showed that mutant grew slowly in late stationary phase compared to the wild-type strain． Scanning electron microscopy and spore formation analysis indicated that no spore was produced in sigK disruption mutant． SDS-PAGE results exhibited that the expression of cry gene was significantly decreased in the mutant． Beta-galactosidase assay showed that the activation of cry3A promoter was stronger in the mutant than that in HD-73 during late stationary phase，but the disruption of sigK gene had no significant influence on the production of Cry1Ac which was initiated by cry3A gene promoter． ［Conclusion］ These results indicated that sigK gene was one of the essential genes during the sporulation of Bacillus thuringiensis，and influenced the expression of crystal protein． The expression of crystal protein which was initiated by cry3A gene promoter in sigK disruption mutant could be used to develop high-efficiency and safe biological pesticides.