论文部分内容阅读
【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。
【Objective】 The purpose of this study was to construct the mutant strain of spo Ⅲ D gene of Bacillus thuringiensis and to study its phenotype and the difference of its nature with the original strain. 【Method】 The spoⅢD gene was deleted from the Bacillus thuringiensis HD-73 strain by homologous recombination technique. The sp Ⅲ D deletion mutant was constructed and the growth curve was determined. The growth curve was analyzed by scanning electron microscopy, sporocytosis analysis and SDS-PAGE protein electrophoresis Compare the differences between the mutant and the starting strain. Construction of genetic complementary strains to observe the recovery of strains traits. 【Result】 The deletion mutant of spo Ⅲ D gene of Bacillus thuringiensis strain HD-73 was obtained by homologous recombination knockdown technique. The growth curve of the mutant strain showed that the mutant strains grew more slowly than the starting strains at the late stationary phase. The results of scanning electron microscopy And spore count analysis showed that the mutants basically lost the ability to form spores, but still form crystals. The result of SDS-PAGE showed that in the SSM medium, the effect of the mutants on the formation of the congealed crystal protein was not significant. In the Luria-Bertani medium, Compared with wild-type and complementary strains significantly reduced. Using the vector pHT315 to carry the spo Ⅲ D operon complementary mutants, the complementation restored the ability to produce crystals and sprouts. 【Conclusion】 This study demonstrated that spo Ⅲ D gene is necessary for the formation of Bacillus thuringiensis spores and is related to the expression of crystal proteins.