食蚊鱼P-糖蛋白基因克隆及三氯生对其mRNA表达的影响
Cloning of P-Glycoprotein Gene and Effects of Triclosan on Its mRNA Expression in Mosquitofish (Gambusia affinis)
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摘要: 三氯生（TCS）是一种广谱高效抗菌剂，在水环境和生物体内均不同程度检出，对水生生物具有潜在风险。P-糖蛋白（P-gp）是生物体多型异源物质抗性防御系统中重要的"膜解毒蛋白"，对水生生物体内的有毒物质和代谢产物具有重要的外排和转运作用。为探究P-gp在鱼类免疫中的作用，克隆了食蚊鱼（Gambusia affinis）P-gp基因的cDNA，检测了不同浓度（50、100和150 □g·L-1） TCS暴露12 h、1 d、3 d、5 d、7 d和9 d后，P-gpmRNA相对表达量的变化。实验获得的食蚊鱼P-gpcDNA共5 452 bp，编码1 294个氨基酸，具有ABC转运蛋白家族典型的跨膜结构、功能区域和作用位点，与其他鳉形目的鱼类P-gp氨基酸序列同源性较高。TCS对P-gpmRNA表达的影响呈现倒"U"型的时间-剂量效应，50 □g·L-1和100 □g·L-1 TCS胁迫后P-gp表达量先升高后下降，100 □g·L-1暴露组表达高峰在暴露1 d时，50 □g·L-1 TCS暴露组表达高峰延迟至3 d，而150 □g·L-1暴露组P-gp表达无显著变化。结果表明，P-gp基因参与了TCS胁迫的解毒过程，有助于食蚊鱼抵抗外源污染物的毒性作用。Abstract: Triclosan (TCS) is a broad-spectrum antibacterial agent that has been detected in different environmental media and organisms at concentrations ranging from ng·L-1 to □g·L-1, which indicates the high ecological risk to aquatic organisms. P-glycoprotein (P-gp) is an important "membrane detoxification protein" in multixenobiotic resistance system, and plays an important role on the efflux and transport of toxic substances and metabolites in aquatic organisms. To investigate how P-gp functions in fish immunization, the cDNA of P-gp in Gambusia affinis was cloned, and the relative mRNA expression of P-gp was also tested after TCS exposure at different concentrations (50, 100 and 150 □g·L-1) for 12 h, 1 d, 3 d, 5 d, 7 d and 9 d. The newly identified P-gp gene of G. affinis was 5 452 bp in length with an open reading frame (ORF) of 3 885 bp encoding a polypeptide of 1 294 amino acids, it contained the typical transmembrane domains, conserved motifs and functional sites of the ABC transporter family and showed high identities with the P-gp amino acid sequences of other fishes in Cyprinodontiformes. P-gp mRNA expression increased firstly and then decreased, showing a time-dose effect pattern with inverted "U"-type changes in the 50 □g·L-1 and 100 □g·L-1 TCS group. The highest expression of P-gp in 100 □g·L-1 TCS group occured after 1 d exposure, while the expression peak in the 50 □g·L-1 group was delayed to 3 d, and the P-gp expression in the 150 □g·L-1 group had no significant change. Taken together, the P-gp was involved in the detoxification process of TCS exposure and contributed to the resistance of G. affinis against pollutants.
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Key words:
- triclosan /
- Gambusia affinis /
- P-glycoprotein gene /
- cDNA cloning /
- mRNA expression
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