炭疽芽孢杆菌sigF缺失株的构建及其对芽孢形成的影响

Construction of sigF deletion mutant of Bacillus anthracis and its effect on formation of spores

目的构建炭疽芽孢杆菌sigF基因缺失株,以及缺失株的sigF回复株,分析缺失株、回复株表型特点并明确sigF基因对芽孢形成的影响,为后续研究提供基础。方法采用同源重组技术在炭疽菌A16D2(pXO1+pXO2^-)菌株sigF基因处插入大观霉素抗性基因,取代sigF基因,构建了sigF基因缺失突变株。通过构建回复株质粒并电转到sigF基因缺失株中,构建了sigF基因的回复株。通过生长曲线测定、糖代谢能力比较、显微镜观察芽孢形成及微流控实验实时观察细胞生长等方法分析突变株和回复株的特点。结果获得炭疽菌A16D2菌株sigF基因缺失突变株和相应的回复株。生长曲线测定表明,突变株较出发菌株在繁殖体阶段生长状态无明显差异;糖代谢实验表明,突变株与出发菌株在对碳水化合物的利用上差异不显著;显微镜观察显示,突变株丧失了形成芽孢的能力、回复株恢复了部分形成芽孢的能力;微流控实验观察显示,缺失株细胞保持在不对称分裂的状态,回复株能形成芽孢。结论成功获得了炭疽菌A16D2sigF基因缺失突变株。该研究证明sigF基因为炭疽菌形成芽孢所必需,但不是细胞生长所必需的因子。

Objective To construct sigF deletion mutant of Bacillus anthracis and the complementary strain of sigF deletion mutant in order to analyze the effect of losing sigF on formation of spores. Methods The spectinomycinadenyltransferase gene（ spc） was inserted to replace sigF of B. anthracis by homologous recombination. A plasmid which contained sigF and sigF promotor was constructed and then transferred to the mutant to get a complementary strain of sigF deletion mutant. The characters of the mutant were analyzed by measuring growth curves,the ability of carbohydrate metabolism was compared,and spore formation was observed under a microscope. Results The sigF deletion strain A16D2 △sigF was constructed from A16D2,which had a similar growth rate to the wild type A16D2 in logarithmic phase,but was not significantly different from the initial strain in the ability to use carbohydrates,although unable to form spores. The strain was found to maintain the state of asymmetric division by microfluidics experiment. Conclusion It is showed by this study that sigF is the essential gene of B. anthracis for spore formation,but not essential for vegetative growth.