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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
王竹青1,2, 任宪云2, 高保全2, 刘 萍2, 张小辉1,2, 张 杰1,2, 于 旋2
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
摘要:
采用RACE技术(cDNA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ptF-ATPaseβ。该基因cDNA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 kDa,理论等电点为4.86。ptF-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,ptF-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,ptF-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ptF-ATPaseβ基因的表达量在肝胰腺和心脏中均下降且显著低于F0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F0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ptF-ATPaseβ基因表达及ATP合酶活力的衰退。
关键词:  三疣梭子蟹  F-ATPaseβ  基因克隆  表达分析  近交
DOI:
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基金项目:
cDNA Cloning and Expression Analysis of F-ATPaseβ Subunit Gene in Portunus trituberculatus and Its Variation in Family Inbreeding
WANG Zhuqing1,2, REN Xianyun2, GAO Baoquan2, LIU Ping2, ZHANG Xiaohui1,2, ZHANG Jie1,2, YU Xuan2
1.College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306;2.Key Laboratory of Sustainable Development of Marine Fisheries, Ministry of Agriculture, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071
Abstract:
A full-length cDNA sequence of F-ATPase β subunit gene from Portunus trituberculatus (ptF-ATPaseβ) was cloned by rapid amplification of cDNA ends (RACE). The sequence of ptF-ATPaseβ was 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-ATPaseβ has an F1-ATPaseβ domain, an AAA domain, and an ATP-synt-ab-C domain. Homology and phylogenetic analysis revealed that the amino acid sequence of ptF-ATPaseβ shared a high similarity (89%) with Penaeus monodon and Litopenaeus vannamei. ptF-ATPaseβ mRNA level was detected in all tested tissues including the hepatopancreas, muscle, heart, gill, stomach, intestine, testis, and ovary. The ptF-ATPaseβ had the highest level in the hepatopancreas and heart, and the lowest expression in the intestine. With the increase of inbreeding coefficient, ptF-ATPaseβ expression 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-ATPaseβ and the ATP synthase activity in P. trituberculatus.
Key words:  Portunus trituberculatus  ptF-ATPaseβ  Gene cloning  Expression analysis  Inbreeding