中国明对虾NHE3基因克隆及其在pH胁迫下的表达  

Molecular cloning and sequence analysis of Na^+/H^+-exchanger isoform 3 in Fenneropenaeus chinensis and its expression in response to pH stress

作　　者：李政道 李健[1,3] 葛倩倩 王佳佳[1] 何玉英[1] 王培春[4] LI Zhengdao;LI Jian;GE Qianqian;WANG Jiajia;HE Yuying;WANG Peichun(Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences;Key Laboratory of Sustainable Development of Marine Fisheries,Ministry of Agriculture and Rural Affairs,Qingdao 266071,China;National Demonstration Center for Experimental Fisheries Science Education(Shanghai Ocean University),Shanghai 201306,China;Functional Laboratory of Marine Fisheries Science and Food Production Processes,Qingdao National Laboratory for Marine Science and Technology,Qingdao 266071,China;Haichen Aquatic Products Co.Ltd.,Rizhao 276805,China)

机构地区：[1]中国水产科学研究院黄海水产研究所,农业农村部海洋渔业可持续发展重点实验室,山东青岛266071 [2]水产科学国家级实验教学示范中心(上海海洋大学),上海201306 [3]青岛海洋科学与技术国家实验室,海洋渔业科学与食物产出过程功能实验室,山东青岛266071 [4]日照海辰水产有限公司,山东日照276805

出　　处：《中国水产科学》2018年第5期958-966,共9页Journal of Fishery Sciences of China

基　　金：国家虾产业技术体系项目(CARS-48);2015年山东省泰山领军人才工程高效生态农业创新类计划项目(LJNY2015002);中国水产科学研究院黄海水产研究所基本科研业务费项目(20603022016009);青岛海洋科学与技术国家实验室鳌山科技创新计划项目(2015ASKJ02);国家自然科学基金青年科学基金项目(31702319).

摘　　要：为研究钠/氢交换体(Na+/H+-exchanger,NHE)在中国明对虾(Fenneropenaeus chinensis)响应pH胁迫过程中发挥的作用,首先采用静水毒性实验方法确定了中国明对虾酸碱半致死pH,然后利用RACE技术克隆了中国明对虾Na+/H+-exchanger isoform 3(命名为FcNHE3)基因,并通过荧光定量PCR及RNA干扰技术分析了其在pH胁迫下的表达特征及功能。结果显示,72 h酸性半致死pH和碱性半致死pH分别为5.2和9.1。克隆获得FcNHE3基因(Gen Bank:MF373587)cDNA序列全长3508 bp,开放阅读框2805 bp,编码934个氨基酸,具有信号肽和12个跨膜结构域;蛋白同源分析发现,FcNHE3与青蟹(Carcinus maenas)同源性最高,达到74%;系统进化分析显示,FcNHE3与三疣梭子蟹(Portunus trituberculatus)和青蟹亲缘关系最近。荧光定量PCR分析表明,FcNHE3基因在鳃组织中表达量显著高于其他组织(P<0.05);酸性半致死pH(pH 5.2)胁迫下,FcNHE3基因在整个胁迫过程中显著上调表达(P<0.05);碱性半致死pH(pH 9.1)胁迫下,FcNHE3基因在前48 h显著下调表达(P<0.05),12 h表达量最低,仅在72 h出现上调表达。RNA干扰后,FcNHE3基因表达受到抑制,pH 5.2胁迫下对虾存活率相比对照组显著下降。研究表明相较于高pH胁迫,FcNHE3基因在中国明对虾响应低pH胁迫过程中可能发挥更重要的调节作用。Na^+/H^+-exchanger is a membrane-associated enzyme responsible for the active transport of Na^+and H+ions across cell membranes,and generates chemical and electrical gradients.It plays an important role in the aquatic adaptation of aquatic crustaceans.To investigate the function of the Na^+/H^+-exchanger in Fenneropenaeus chinensis under pH stress,Na^+/H^+-exchanger 3 cDNA of Fenneropenaeus chinensis,named FcNHE3(Gen-Bank:MF373587),was cloned from the gill tissues of the animal by Reverse Transcription-Polymerase Chain Reaction(RACE).The full-length of FcNHE3 cDNA was 3508 bp(base pairs),including a 2805 bp open reading frame(ORF)encoding a 934-amino acid peptide with one signal peptide and 12 transmembrane domains.Comparison with homologous proteins showed that the deduced FcNHE3 amino acid sequence has the highest sequence identity with Carcinus maenas(74%),and along with Portunus trituberculatus,were clustered into one group by phylogenetic analysis.Results of RT-qPCR showed that FcNHE3 expression level in the gills was significantly higher than in other tissues(P<0.05).The expression level of FcNHE3 in the gills was up-regulated under low pH stress(pH 5.2)during the entire duration of exposure.The expression level of FcNHE3 in the gills was down-regulated during the first 48 h and up-regulated at 72 h under high pH stress(pH 9.1).After the knockdown of FcNHE3 expression by RNA interference(RNAi),shrimp mortality was found to be significantly higher under low pH stress when compared with the control group.The results suggest that FcNHE3 may play a more important role in regulating acid-base balance under low pH stress than under high pH stress.

关 键 词：中国明对虾 钠/氢交换体(NHE) 基因克隆 PH胁迫 RNA干扰 

分 类 号：S963[农业科学—水产养殖]