三疣梭子蟹F型ATP酶β亚基(F-ATPaseβ)基因的克隆、组织表达及在家系近交中的变化  

cDNA Cloning and Expression Analysis of F-ATPaseβ Subunit Gene in Portunus trituberculatus and Its Variation in Family Inbreeding

作　　者：王竹青 任宪云[2] 高保全[2] 刘萍[2] 张小辉[1,2] 张杰 于旋[2] WANG Zhuqing;REN Xianyun;GAO Baoquan;LIU Ping;ZHANG Xiaohui;ZHANG Jie;YU Xuan(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306;Key Laboratory of Sustainable Development of Marine Fisheries,Ministry of Agriculture,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]农业部海洋渔业可持续发展重点实验室,中国水产科学研究院黄海水产研究所,青岛266071

出　　处：《渔业科学进展》2018年第1期97-106,共10页Progress in Fishery Sciences

基　　金：国家自然科学基金面上项目(41576147)、泰山领军人才工程高效生态农业创新类计划项目(LJNY2015002)和中央级公益性科研院所基本科研业务费专项资金资助项目(20603022015016)共同资助

摘　　要：采用RACE技术(c DNA末端快速扩增技术)克隆获得三疣梭子蟹(Portunus trituberculatus)F型ATP酶b亚基(F-ATPaseβ)基因,命名为pt F-ATPaseβ。该基因c DNA全长为1965 bp,5￠和3￠非编码区分别为571 bp和341 bp,开放阅读框为1053 bp,推测编码350个氨基酸,预测分子量为37.9 k Da,理论等电点为4.86。pt F-ATPaseβ氨基酸序列含有F1-ATPaseβ标志性蛋白结构域、AAA结构域和ATP-synt-ab-C结构域。同源性及系统分析显示,pt F-ATPaseβ氨基酸序列与斑节对虾(Penaeus monodon)、凡纳滨对虾(Litopenaeus vannamei)同源性高达89%。实时荧光定量PCR显示,pt F-ATPaseβ基因在肝胰腺、肌肉、心脏、鳃、胃、肠、精巢和卵巢中均有表达,其中,在肝胰腺和心脏中表达量最高,在肠中最少。随着近交系数的增加,各代pt F-ATPaseβ基因的表达量在肝胰腺和心脏中均下降且显著低于F_0代(P<0.05)。酶活检测结果显示,心脏中的ATP合酶活性从F6代开始出现下降且显著低于F_0代(P<0.05),但肝胰腺中ATP合酶活性无显著变化。本研究结果表明,近交造成了三疣梭子蟹pt F-ATPaseβ基因表达及ATP合酶活力的衰退。A full-length cDNA sequence of F-ATPaseβsubunit gene from Portunus trituberculatus(ptF-ATPaseβ)was cloned by rapid amplification of cDNA ends(RACE).The sequence of ptF-ATPaseβwas 1965 bp,containing a 571 bp 5'UTR,341 bp 3'UTR,and a 1053 bp open reading frame(ORF)that encodes 350 amino acids polypeptides.The isoelectric point(pI)was 4.86 and the molecular mass was 37.9 kDa.The amino acid sequence analysis demonstrated that ptF-ATPaseβhas an F1-ATPaseβdomain,an AAA domain,and an ATP-synt-ab-C domain.Homology and phylogenetic analysis revealed that the amino acid sequence of ptF-ATPaseβshared a high similarity(89%)with Penaeus monodon and Litopenaeus vannamei.ptF-ATPaseβmRNA level was detected in all tested tissues including the hepatopancreas,muscle,heart,gill,stomach,intestine,testis,and ovary.The ptF-ATPaseβhad the highest level in the hepatopancreas and heart,and the lowest expression in the intestine.With the increase of inbreeding coefficient,ptF-ATPaseβexpression decreased significantly in the hepatopancreas and heart(P<0.05).The ATP synthase activity in the heart began to fall from F6 generation and was significantly lower than F0 generation(P<0.05),but there was no significant change in the hepatopancreas.The results illustrate that inbreeding gradually reduces the expression of ptF-ATPaseβand the ATP synthase activity in P.trituberculatus.

关 键 词：三疣梭子蟹 F-ATPaseβ 基因克隆 表达分析 近交 

分 类 号：S917.4[农业科学—水产科学]