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[摘要]
研究乙醇对副溶血性弧菌(Vibrio parahaemolyticus)直接耐热溶血素(thermostable direct hemolysin,TDH)产生和它的编码基因tdh表达的影响。以0.5%、1.0%、2.0%、4.0%、8.0%乙醇处理2株带有tdh副溶血性弧菌ATCC33847和SZ32,研究对副溶血性弧菌有氧或者无氧生长的影响。选取1.0%乙醇处理来研究TDH产量和tdh表达变化。使用TDH抗血清试剂盒测定副溶血性弧菌培养液上清中TDH水平;使用荧光定量PCR方法分析tdh基因表达状况。低浓度(0.5%、1.0%、2.0%)乙醇存在时,副溶血弧菌菌株的生长未受到显著影响,乙醇浓度4.0%时副溶血性弧菌生长受到明显抑制,8%时未见有细菌生长。1.0%乙醇处理副溶血性弧菌培养液上清中TDH水平较未处理显著上升。胞内tdh表达水平升高,ATCC33847在有氧和无氧时分别升高到6.6倍和5.7倍,SZ32在有氧和无氧时分别升高到5.9倍和8.6倍。乙醇能够促进tdh基因表达从而使得TDH蛋白产量升高。本研究还比较了甲醇、乙醇、正丙醇对tdh表达的影响,发现它们对tdh表达均具有促进作用但相互之间没有明显差异。
[Key word]
[Abstract]
The effect of ethanol on thermostable direct hemolysin (TDH) production and corresponding tdh gene expression by Vibrio parahaemolyticus was studied. Two tdh-positive V. parahaemolyticus strains, ATCC33847 and SZ32 were separately treated with 0.5%, 1.0%, 2.0%, 4.0%, and 8.0% ethanol solutions. The effects on aerobic or anaerobic growth of V. parahaemolyticus were monitored, while changes in TDH production and tdh gene expression were studied with 1.0% ethanol treatment. TDH concentration in V. parahaemolyticus culture supernatant was quantitatively analyzed using a TDH detection kit (KAP-RPLA, Denka Seiken Co., Japan), while tdh gene expression was analyzed by real-time fluorescence-based quantitative polymerase chain reaction. The results showed that V. parahaemolyticus growth was not influenced by the presence of ethanol at low concentrations (0.5%, 1.0%, and 2.0%). On the other hand, 4.0% and 8.0% ethanol caused significant and total inhibition of bacterial growth, respectively. With ethanol treatment, TDH titers of culture supernatants showed a significant increase under aerobic as well as anaerobic conditions and intracellular tdh expression was also elevated. Moreover, ATCC33847 strain showed an increase in tdh expression by 6.6- and 5.7-fold, while SZ32 strain showed an increase by 5.9- and 8.6-fold under aerobic and anaerobic conditions, respectively. Thus, ethanol treatment enhanced tdh gene expression, which may have resulted in an increase in TDH production. The effects of methanol, ethanol, and n-propanol on tdh gene expression were also compared and the results showed increased tdh gene expression by all three alcohols, however, the effects were not significantly different.
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[基金项目]
广东省自然科学基金研究团队项目(S2012030006235);广州市科技计划项目(201300000074)