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

【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。

[ Objectivel 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. [Resultsl 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.