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【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。
【Objective】 The purpose of this study was to construct the in-inserted mutant of sigK gene of Bacillus thuringiensis (Bt), analyze the characteristics of the mutant and clarify its effect on the promoter of cry3A gene. 【Method】 The kanamycin resistance gene was inserted into the sigK gene of Bacillus thuringiensis strain HD-73 by homologous recombination, and the sigK gene insertional inactivation mutant was constructed. The characteristics of the mutant were analyzed by growth curve, scanning electron microscopy (SEM), sporulation and sporocyst count, SDS-PAGE and other methods. The genetic recovery strains were constructed to validate the above traits. The promoter lacZ fusion protein The cry3A gene promoter transcriptional activity. 【Result】 The sigK gene of Bacillus thuringiensis strain HD-73 was obtained. The growth curve of the mutant showed that the mutant grew slower than the original strain at the late stationary phase. The results of scanning electron microscopy and spore counting showed that the mutant lost its formation Spore and crystal. The SDS-PAGE analysis showed that the expression of crystal protein of the companion cells in the mutant was significantly lower than that of the original strain and the recovered strain. The sigk gene and its promoter were expressed in the mutant strain by using pHT315 vector. The obtained genetic restorer strain restored the ability of mutant strains to produce spores and crystals. The mutation of sigK gene could increase the transcriptional activity of cry3A gene promoter , No significant effect on cry3A promoter-directed Cry protein expression. 【Conclusion】 The present study demonstrated that sigK gene is necessary for the formation of Bacillus thuringiensis spores and affects the production of crystal protein. The loss of function of sigK gene is beneficial to the transcription of cry3A gene promoter during the late-culture stage.