苏云金芽胞杆菌spoIIID基因缺失突变株的特点

Characterization of Bacillus thuringiensis spoIIID gene Mutantation

摘要:

摘要：【目的】构建苏云金芽胞杆菌spoIIID基因缺失突变株，并研究其与出发菌株的表型及性质差异。【方法】采用基因同源重组技术敲除了苏云金芽胞杆菌HD-73菌株中的spoIIID基因，构建了spoIIID缺失突变株，测定生长曲线，并通过扫描电子显微镜观察，芽胞计数分析及SDS-PAGE 蛋白电泳比较突变株与出发菌株的差异。构建遗传互补菌株，观察菌株性状的回复情况。【结果】通过温敏载体同源重组敲除技术获得了苏云金芽胞杆菌HD-73菌株spoIIID基因缺失突变株，生长曲线测定表明，突变株较出发菌株在平稳期后期生长较缓和；扫描电子显微镜观察和芽胞计数分析显示，突变株基本丧失了形成芽胞的能力，但依然形成晶体。SDS-PAGE结果显示，在 SSM培养基中，突变株对伴胞晶体蛋白的形成量影响并不显著；在营养较富集的Luria-Bertani培养基中，突变株中伴胞晶体蛋白的形成量较野生型和互补株明显降低。利用载体pHT315携带spoIIID操纵子互补突变株，互补株恢复了产生晶体和芽胞的能力。【结论】本研究证明spoIIID基因是苏云金芽胞杆菌芽胞形成所必需，同时与晶体蛋白的表达相关。

关键词:关键词：苏云金芽胞杆菌;芽胞;杀虫晶体蛋白;spoIIID基因;突变

Abstract:

Abstract: [Objective] Construction and characterization of a spoIIID gene deletion mutant of Bacillus thuringiensis. [Methods] Scanning electron microscopy and spore formation analysis were used to detect the ability of sporulation and formation of crystal protein in both the mutant and the wild strain. SDS-PAGE analysis was used to detect the expression of crystal protein. [Results] Scanning electron microscopy and spore formation analysis showed that spores were hardly produced and the crystal existed in the spoIIID deletion strain. SDS-PAGE results showed that the expression of cry gene in the mutant was decreased in Luria-Bertani medium, but not affected obviously in Schaeffer’s sporulation medium (SSM). [Conclusion]This indicated that the spoIIID gene was one of the essential genes for the sporulation of Bacillus thuringiensis, and influenced the expression of crystal protein.

Key words:Key words: Bacillus thuringiensis; spoIIID; spore; crystal protein