炭疽芽孢杆菌sigF缺失株的构建及其对芽孢形成的影响

发布时间：2018-05-15 05:11

  本文选题：芽孢杆菌 + 炭疽　； 参考：《军事医学》2017年03期

【摘要】：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。
[Abstract]:Objective to construct the sigF gene deletion strain of Bacillus anthracis and the sigF recovery strain of the deletion strain, to analyze the phenotypic characteristics of the deleted strain and to clarify the effect of sigF gene on the spores formation, and to provide a basis for further study. Methods the gene of spectinomycin resistance was inserted into the sigF gene of anthrax strain A16D2(pXO1 pXO _ 2 by homologous recombination technique, replacing the sigF gene, and the mutant of sigF gene deletion was constructed. By constructing the plasmid of the sigF gene and transferring it to the sigF gene deletion strain, the sigF gene recovery strain was constructed. The characteristics of mutants and recovers were analyzed by means of growth curve determination, comparison of glucose metabolism ability, observation of spores formation by microscope and real-time observation of cell growth by microfluidic experiments. Results the sigF gene deletion mutants and corresponding recovers of anthrax A16D2 strain were obtained. The growth curve test showed that there was no significant difference in the growth status of the mutant strain compared with the original strain in the reproductive phase, the glucose metabolism test showed that there was no significant difference in the utilization of carbohydrate between the mutant strain and the original strain, and the microscopic observation showed that there was no significant difference between the two strains in the utilization of carbohydrates. The mutant lost the ability to form spores, and the recovery strain recovered part of the ability to form spores, and the microfluidic experiments showed that the cells of the missing strain remained in the state of asymmetric division, and the recovered strain could form spores. Conclusion the mutant of A16D2sigF gene deletion of anthrax was successfully obtained. This study demonstrated that the sigF group is necessary for the formation of spores by anthrax, but is not an essential factor for cell growth.
【作者单位】： 上海海洋大学食品学院;军事医学科学院生物工程研究所病原微生物生物安全国家重点实验室;
【基金】：国家自然科学基金资助项目(81271785)
【分类号】：R378.72

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