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)

作　　者：宋晓红[1,2,3] 陆爱金 杜士林 陈双凤 陈旺光 梁延鹏 黄亮亮[1,2,3] 曾鸿鹄 Song Xiaohong;Lu Aijin;Du Shilin;Chen Shuangfeng;Chen Wangguang;Liang Yanpeng;Huang Liangliang;Zeng Honghu(College of Environmental Science and Engineering,Guilin University of Technology,Guilin 541004,China;Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology,Guilin 541004,China;Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area,Guilin 541004,China)

机构地区：[1]桂林理工大学环境科学与工程学院,桂林541004 [2]广西环境污染控制理论与技术重点实验室,桂林541004 [3]岩溶地区水污染控制与用水安全保障协同创新中心,桂林541004

出　　处：《生态毒理学报》2020年第4期167-176,共10页Asian Journal of Ecotoxicology

基　　金：国家自然科学基金资助项目(51578171);广西科技计划项目(桂科AD18126018);广西自然科学基金资助项目(2018GXNSFAA281022)。

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(Pgp)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c DNA,检测了不同浓度(50、100和150μg·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 452bp,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,50μg·L-1和100μg·L-1TCS胁迫后P-gp表达量先升高后下降,100μg·L-1暴露组表达高峰在暴露1 d时,50μg·L-1TCS暴露组表达高峰延迟至3 d,而150μg·L-1暴露组Pgp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。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 μg·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-gpin Gambusia affiniswas cloned,and the relative mRNA expression of P-gpwas also tested after TCS exposure at different concentrations(50,100 and 150 μg·L-1) for 12 h,1 d,3 d,5 d,7 d and9 d.The newly identified P-gpgene of G.affiniswas 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 Pgp amino acid sequences of other fishes in Cyprinodontiformes.P-gpmRNA expression increased firstly and then decreased,showing a time-dose effect pattern with inverted "U"-type changes in the 50 μg·L-1 and100 μg·L-1 TCS group.The highest expression of P-gpin 100 μg·L-1 TCS group occured after 1 d exposure,while the expression peak in the 50 μg·L-1 group was delayed to 3 d,and the P-gpexpression in the 150 μg·L-1 group had no significant change.Taken together,the P-gpwas involved in the detoxification process of TCS exposure and contributed to the resistance of G.affinisagainst pollutants.

关 键 词：三氯生 食蚊鱼 P-糖蛋白基因 CDNA克隆 MRNA表达 

分 类 号：X171.5[环境科学与工程—环境科学]