【作者】 魏鉴腾；

【导师】 陈吉祥；

【作者基本信息】 中国海洋大学 ， 遗传学， 2008， 硕士

【摘要】 溶藻胶弧菌（Vibrio alginolyticus）是一种条件致病菌,它能感染人类和海洋动物。自然界中的细菌能够适应各种各样的不利环境,并且能在一定条件下进入活的非可培养（VBNC）状态。rpoS基因编码RpoS因子,是细菌稳定期一个调控因子,当细菌进入稳定期时,RpoS因子能够增强细胞对外界不良环境的抗逆性和适应能力。本论文目的是研究RpoS因子在溶藻胶弧菌处于恶劣环境中或诱导其进入VBNC状态过程中的作用。首先克隆了溶藻胶弧菌VIB283的rpoS基因,利用同源重组构建了该菌rpoS基因的突变菌株。通过对突变菌株和野生菌株的比较发现,在正常条件下培养,野生株和突变株都能很好的在2216E液体培养基中生长,但突变株的最终细菌浓度是野生株浓度的1/2。突变菌株对过氧化氢的应答敏感,两菌的存活率差异显著,而在高渗透压条件下,两株菌的存活率没有明显差异。将细菌寡营养条件下培养12天后,野生菌株的活菌数为原来的53.50%,突变株为原来的1.96%,两菌差异显著。在4℃寡营养条件下培养野生菌株在69天进入VBNC状态,而突变株则在37天即可进入。将野生菌株和突变菌株注射斑马鱼,检测其致病性的变化,注射的斑马鱼都有不同程度的死亡,野生菌株和突变菌株半数致死量(LD₅₀)相似,分别是5.78×10⁷ CFU/mL和2.05×10⁷ CFU/mL,rpoS基因突变对溶藻胶弧菌致病性没有明显影响。更多还原

【Abstract】 Vibrio alginolyticus was an opportunistic pathogen of human being and marine animals. The bacterium was able to adapt to a variety of environmental stresses and enter into the viable but nonculturable (VBNC) state. The alternate sigma factor RpoS, which was a regulator during the stationary phase, played an important role in surviving under these stressful situations in many bacteria. The aim of this study was to evaluate the roles of RpoS of V. alginolyticus in adapting to environmental stresses and entering into the VBNC state under starvation stresses. The rpoS gene of V. alginolyticus VIB283 was cloned and an rpoS mutant was constructed by homologous recombination. The mutant strain was more sensitive to environmental stresses such as high osmolarity and long-term starvation. Both wild-type and the mutant strains of V. alginolyticus grew well in 2216E broth at 28℃, with a little decrease of the viable plate count in the rpoS mutant (2.5×10~9 CFU/mL ) compared with the wild-type strain (5×10~9 CFU/mL) during the stationary phase. The mutant strain was more sensitive to oxidative stress than the wild-type counterparts. When the wild type and the mutant cells were exposed to hyperosmolarity (2.4 M NaCI), there were no difference between the mutant and the wild type. The viable counts of the wild-type strain maintained to 53.50%, whereas the viable counts of the mutant strain reduced to 1.96% after 12 days of carbon starvation. The mutant V. alginolyticus entered into the VBNC state within 37 days at 4℃, whereas the wild-type strain entered the VBNC state within 69 days under the same conditions. Resuscitation of the rpoS mutant in VBNC state could also be obtained. Disruption of rpoS in V. alginolyticus VIB283 did not obviously affect its virulence to zebra fish. These results suggested that rpoS of V. alginolyticus is important for adaptation to environmental stresses.更多还原