中华绒螯蟹水通道蛋白1基因的克隆、表达及RNA干扰研究  

Study on Cloning,Expression and RNA Interference of Aquaporin 1 Gene of Eriocheir sinensis

作　　者：杨志刚[1,2] 张龙[1,2] 陈春宇 成永旭[1,2] 夏冬梅 YANG Zhigang;ZHANG Long;CHEN Chunyu;CHENG Yongxu;XIA Dongmei(Key Laboratory of Freshwater and Aquatic Germplasm Resources, Ministry of Agriculture, Shanghai Ocean University, Shanghai 201306, China;Research Center for Fish Nutrition and Environmental Ecology, Ministry of Agriculture,Shanghai Ocean University, Shanghai 201306, China;Fujian Key Laboratory of Functional Aquafeed and Culture Environment Control, Zhangzhou, Fujian 363000, China)

机构地区：[1]上海海洋大学农业部淡水水产种质资源重点实验室,上海201306 [2]上海海洋大学农业部鱼类营养与环境生态研究中心,上海201306 [3]福建省水产功能性饲料与养殖环境调控重点实验室,福建漳州363000

出　　处：《复旦学报（自然科学版）》2021年第1期93-101,共9页Journal of Fudan University：Natural Science

基　　金：国家重点研发计划(2018YFD0900603);现代农业产业技术体系专项(CARS-48);上海市科委工程技术中心能力提升项目(19DZ2284300);黄河三角洲人才工程(DYRC20190210)。

摘　　要：采用cDNA末端快速扩增(Rapid-Amplification of cDNA Ends,RACE)技术从中华绒螯蟹的后鳃中克隆获得水通道蛋白1(AQP1)的全长序列,利用实时荧光定量(qRT-PCR)构建中华绒螯蟹AQP1基因组织表达谱,并进行生物信息学分析.EsAQP1的cDNA全长序列为1646bp,开放阅读框(ORF)为1119bp,编码373个氨基酸.理化分析预测其分子量为39.367kDa,等电点为5.1.序列比对结果显示EsAQP1的氨基酸序列分别与三疣梭子蟹(Portunus trituberculatus)、可口美青蟹(Callinectes sapidus)、罗氏沼虾(Macrobrachium rosenbergii)和斑节对虾(Penaeus monodon)具有74%、71%、64%和63%的同源性.不同组织荧光定量结果显示,EsAQP1基因在中华绒螯蟹肠和鳃组织中表达丰度较高,在肝胰腺和血细胞中表达丰度较低.分析发现盐度胁迫会显著影响中华绒螯蟹肠道和鳃组织中EsAQP1基因的表达,进一步通过RNA干扰EsAQP1基因表达之后,对干扰后Na^(+)-K^(+)-Cl^(-)协同转运蛋白(NKCC)基因NKCC和Na^(+)/K^(+)-ATPase基因Na^(+)/K^(+)-ATP的表达进行分析,发现NKCC和Na+/K+-ATP表达显著上调.以上研究结果表明,EsAQP1基因可能在中华绒螯蟹的渗透压调节系统中发挥重要作用.The full-length sequence of aquaporin 1 was cloned from the gill tissues of Eriocheir sinensis.The complete cDNA sequence of AQP1 was 1646bp in length,and the open reading frame(ORF)was 1119bp,corresponding to 373 amino acids.The molecular mass of the predicted protein is 39.367kDa with an estimated PI of 5.1.Sequence alignment showed that the amino acid sequence of EsAQP1 shares 74%,71%,64%and 63%identity with that of the Portunus trituberculatus,the Callinectes sapidus,the Macrobrachium rosenbergii and the Penaeus monodon,respectively.Quantitative real-time PCR analysis revealed that EsAQP1 were detected in a wide range of tissues,with highest expression in intestine and gill,and weakest expression in hepatopancreas and blood cells.By analyzing the expression of EsAQP1 gene under salinity stress,it was found that salinity stress can significantly change the expression pattern of EsAQP1 gene in the intestine and gills of E.sinensis.Through RNA interference of the expression of AQP1,the expressions of Na^(+)-K^(+)-Cl^(-)cotransporter(NKCC)gene NKCC and Na^(+)/K^(+)/ATPase gene Na^(+)/K^(+)-ATP were investigated after interference.It was found that NKCC and Na^(+)/K^(+)-ATP were significantly up-regulated.These results indicate that AQP1 may play an important role in the osmotic pressure regulation system of E.sinensis.

关 键 词：中华绒螯蟹 水通道蛋白 基因克隆 RNA干扰 盐度胁迫 

分 类 号：R392[医药卫生—免疫学]