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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 gene-deficient strain of Bacillus anthracis as well as the sigF-repletion strain of the deleted strain, and to analyze the phenotypic characteristics of the deleted and recovered strains and to determine the effect of the sigF gene on the formation of spores, so as to provide the basis for subsequent research. Methods The spectinomycin resistance gene was inserted into the sigF gene of the anthrax A16D2 (pXO1 + pXO2 ~ -) strain by homologous recombination, and the sigF gene was substituted for the sigF gene to construct the sigF gene deletion mutant. The constructed sigF gene was constructed by constructing the plasmids of revertants and electroporation into sigF gene-deleted strains. The characteristics of mutant and recovery strains were analyzed by growth curve determination, glucose metabolism ability, microscopic observation of sporulation and microfluidic experiments in real-time observation of cell growth. Results The sigF gene deletion mutant and the corresponding recovery strain of the anthrax A16D2 strain were obtained. The results of growth curve showed that there was no significant difference in the growth status between the mutant strain and the original strain in the vegetative stage. The glucose metabolism test showed that there was no significant difference in carbohydrate utilization between the mutant strain and the original strain. Microscopic observation showed that the mutant strain lost its formation Bacillus spore ability, the recovery of strains restored part of the ability to form spores; Microfluidic experimental observations show that the deletion of the cells remain in the asymmetric division of the state, the recovery strain can form spores. Conclusion The mutant of A16D2sigF gene was successfully obtained. This study demonstrated that the sigF gene is necessary for anthrax to form spores, but is not an essential factor for cell growth.