鳗弧菌rpoS功能的研究

IOCAS-IR > 海洋环流与波动重点实验室

	鳗弧菌rpoS功能的研究
其他题名	The study of rpoS in Vibrio anguillarum
	郭东升
学位类型	博士
	2009-05-31
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	鳗弧菌 Rpos Rpos突变株毒力抗逆性
摘要	rpoS 基因编码RpoS因子（RNA聚合酶的σ亚基），能增强细菌细胞对外界不良环境的抗逆性和适应能力。我们从鳗弧菌（Vibrio anguillarum）M3 Fosmid 文库中得到rpoS 基因序列，全长999bp，同源分析发现与大肠杆菌（Escherichia coli）有71%的相似性，与霍乱弧菌（Vibrio cholerae ）有78%的相似性。为了研究rpoS在鳗弧菌中的作用，我们利用in-frame deletion技术构建了rpoS 基因的非极性缺失突变株，同时利用细菌的接合转移，以低拷贝克隆质粒pSUP202为载体，构建了突变株的互补株。在TSB培养基中，rpoS 基因的缺失减缓了鳗弧菌对数期的生长，但是对稳定期的生长没有影响。对数期鳗弧菌在1M蔗糖（渗透压胁迫）和5%（v/v）乙醇中的生长减缓，而在18%(v/v)乙醇中的生长及42℃热击时的存活率相对于野生型没有变化。不同生长时期的鳗弧菌对15mM H2O2的反应有所不同，rpoS 的缺失使对数期的鳗弧菌对15mM H2O2的反应更加敏感。在rpoS基因互补株中，上述表型几乎恢复到野生型水平。我们通过感染实验发现，rpoS 基因的缺失使鳗弧菌的LD50提高了20倍。 RpoS的突变对鳗弧菌的泳动和胞外酶的产生也有影响。突变株泳动圈直径是野生型的73.8%，在明胶平板和酪蛋白平板上的透明圈直径分别为野生型的61%和69%。通过azocaseion检测胞外产物ECP酶活发现，突变株的酶活是野生型的35.5%。以上数据表明了RpoS在鳗弧菌对数期应对外界不良环境时起到了重要作用，同时参与了鳗弧菌的致病过程。
其他摘要	The sigma factor RpoS has been described as a general stress response regulator that controls the expression of genes which confer increased resistance to various stresses in some gram-negative bacteria. In this study ,we cloned the whole rpoS gene and the flanking rigions from the Fosmid library of Vibrio anguillarum strain M3. Sequence analysis revealed a 999-bp open reading frame which is predicted to have 71% identity to Escherichia coli and 78% identity to Vibrio cholerae. To elucidate the role of RpoS in physiology and pathogenesis of V.anguillarum,we constructed a non-polar rpoS mutant by in-frame deletion technology .A complemented srtain was also constructed by using low copy plasmid pSUP202 into which the rpoS gene of M3 was introduced. The rpoS mutant strain was subjected to various environmental stresses, and the resistance phenotypes of the this mutant strain was investigated. When cultured in TSB, the rpoS mutant strain grew slower in the phase of logarithm than the wild type . In addition, the rpoS mutant in log phase was more sensitive to1M sucrose(osmotic stress), 5%（v/v）ethanol , 42℃ heat stress and 15mM H2O2 compared with the wild type strain.When the rpoS mutant strain was complemented with pSUP202 carried the rpoS gene,the mutant restored the phenotypes almost similar to those of the wild type strain.Interestingly, the rpoS mutant strain in stationary phase had no difference with the wild type when exposed to 15mM H2O2. The role of rpoS was also assessed in the motility and the activities of several extracellular proteases. The motility of the rpoS mutant strain on the soft agar was severely diminished.In addition, the mutant strain exhibited a diminished protease activities in extracellular products (ECP). The gelatinase and casinase activies of ECPs of the rpoS mutant strain decrease by 73.8% and 69%, respectively, when compared with to those of the wild type strain. The activity of ECP of the mutant against azocasin was only 35.5% of that of the wild type. The complemented strain restored the proteases activities almost to the wild-type. At the time,infection experiments showed that the rpoS mutant increased at least twenty times in LD50 compared to the wild-type in turbot fish. Overall, these studies suggested that RpoS in Vibrio anguillarum is important for adaptation to environmental changes and maybe have a role in virulence.
页数	57
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/239
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	郭东升. 鳗弧菌rpoS功能的研究[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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