拟穴青蟹Na^+/H^+-exchanger基因克隆及其在盐度胁迫下的表达分析  

Cloning and expression analysis of Na^+/H^+-exchanger in Scylla paramamosain

作　　者：刁乐 张凤英[1] 宋炜[1] 马春艳[1] 王伟[1] 谌微[1] 马凌波[1] 赵明[1] 乔振国[1] 蒋科技[1] DIAO Le;ZHANG Feng-ying;SONG Wei;MA Chun-yan;WANG Wei;CHEN Wei;MA Ling-bo;ZHAO Ming;QIAO Zhenguo;JIANG Ke-ji(Key Laboratory of Oceanic and Polar Fisheries, Ministry of Agriculture and Rural Affairs, East China Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Shanghai 200090, China;College of Fishery and Life Science, Shanghai Ocean University, Shanghai 201306, China)

机构地区：[1]中国水产科学研究院东海水产研究所,农业农村部远洋与极地渔业创新重点实验室,上海200090 [2]上海海洋大学水产与生命学院,上海201306

出　　处：《海洋渔业》2020年第2期214-224,共11页Marine Fisheries

基　　金：中国水产科学研究院东海水产研究所基本科研业务费项目(2018M08,2014Z01);宁波市科学技术局农业重大科技专项(2016C11003);农委“科技兴农”项目(2018-02-08-00-07-F01550);现代农业产业技术体系专项资金(CARS-48)。

摘　　要：克隆了拟穴青蟹(Scylla paramamosain)Na^+/H^+-exchanger基因,其cDNA序列全长3624 bp,开放阅读框(ORF)长2886 bp,可编码961个氨基酸,预测蛋白质分子量和等电点分别为110.8 kDa和7.42,具有信号肽和典型的Na^+/H^+-exchanger蛋白结构域,含12个跨膜α螺旋。拟穴青蟹Na^+/H^+-exchanger的氨基酸序列与三疣梭子蟹(Portunus trituberculatus)的一致度最高,达到84.9%;该基因在卵巢中表达量最高,其次是精巢、鳃等;该基因在受精卵时期表达水平最高,大眼幼体时期次之。拟穴青蟹大眼幼体在低盐(盐度7和17)胁迫过程中,Na^+/H^+-exchanger基因在20 min表达水平最高,之后基本恢复到正常表达水平;在高盐(盐度37)胁迫(20 min^2 h)期间,该基因表达水平先下降再逐渐上升。Na^+/H^+-exchanger基因在拟穴青蟹大眼幼体阶段盐度适应中发挥着重要作用,且低盐显著诱导Na^+/H^+-exchanger基因的高表达,推测拟穴青蟹Na^+/H^+-exchanger基因在低盐环境下发挥重要的渗透调节功能。Na^+/H^+-exchanger is a membrane-associated enzyme responsible for the active transport of Na^+and H^+ions across cell membranes and generating chemical and electrical gradients.It plays an important role in salinity adaptation process of aquatic crustacean.Scylla paramamosain is a mud crab commonly consumed in the Southeast Asia,whose growth,development and immunity are significantly affected by salinity variations.In order to investigate the function of Na^+/H^+-exchanger in S.paramamosain under salinity stress,a full-length of Na^+/H^+-exchanger cDNA sequence from S.paramamosain was obtained.The full length of Na^+/H^+-exchanger cDNA was 3624 bp,with a predicted 2886 bp open reading frame(ORF)encoding 961 amino acids with a predicted molecular weight of 107.1 kDa.Comparison with homologous proteins showed that the deduced Na^+/H^+-exchanger sequence had the highest sequence identity to Portunus trituberculatus(84.9%),and the two sequences were clustered into one group by phylogenetic analysis.Typical domains including one signal peptide,one Na^+/H^+-exchanger domain and twelve transmembrane alpha helixes were found in amino acid sequence of Na^+/H^+-exchanger.Furthermore,the RT-qPCR revealed that Na^+/H^+-exchanger mRNA was ubiquitously expressed in all of the detected tissues,with the highest expression in the ovary,followed by gill,testes and gill.During the larval development,the expression of Na^+/H^+-exchanger was the highest in fertilized eggs,followed by megalopa stage.During salinity stress,the expression of Na^+/H^+-exchanger in low salinity groups(7 and 17)increased significantly during 20 min.Subsequently,the expression was down-regulated during 1 h,then gradually rose and returned to normal during 2-6 h.The expression of Na^+/H^+-exchanger in high salinity group(37)declined first and then rose gradually during 20 min-2 h,and the expression level significantly declined during 6 h.Taken all those things together,it is hypothesized that Na^+/H^+-exchanger of S.paramamosain plays a vital role mainly in

关 键 词：拟穴青蟹 Na^+/H^+-exchanger 盐度胁迫 表达分析 

分 类 号：S917[农业科学—水产科学]