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Na+-K+-ATPase α1基因全长cDNA的克隆及序列分析和表达 [中文引用][英文引用]

Full length cDNA cloning and tissue expression of the Na+-K+-ATPase α1 subunit from Cyprinus carpio var. jian

关键词(英文):Cyprinus carpio var. jian  Na+-K+-ATPase α1  clone  sequence analysis  tissue expression 
分类号:Q959
出版年·卷·期(页码):2012·19·第4期(594-603)
DOI: 10.3724/SP.J.1118.2012.00594
-----摘要:-------------------------------------------------------------------------------------------

为了解Na+-K+-ATPase α1基因的分子结构及其在建鲤盐度调控过程中起到的作用, 采用同源克隆和末端快速扩增(RACE)的方法分离克隆了建鲤(Cyprinus carpio var. jian)Na+-K+-ATPase α1基因全长cDNA, 得到3 397 bp的全长cDNA, 包括3 081 bp的开放阅读框(ORF), 232 bp 的5¢末端非编码区(UTR)以及219 bp的3¢末端非编码区(UTR)。对推测的该基因氨基酸序列进行同源性比对和系统分析显示: 建鲤与其他鱼类该基因的氨基酸序列相似度为90.22%~95.52%, 与斑马鱼(Danio rerio)相似度最高(95.52%), 与虱目鱼(Chanos chanos)相似度偏低, 其次是平鲷(Rhabdosargus sarba)、萨罗罗非鱼(Sarotherodon melanotheron)、底鳉(Fundulus heteroclitus)、黑鲷(Acanthopagrus schlegelii)、马苏大马哈鱼(Oncorhynchus masou)和虹鳟(Oncorhynchus mykiss), 与大西洋鲑(Salmo salar)的相似度最低(90.22%), 与非洲爪蟾(Xenopus laevis)和人(Homo sapiens)的相似度分别为89.62%和89.51%。以上结果表明, 不同物种间的Na+-K+-ATPase α1基因的氨基酸序列极为保守。用实时定量PCR (RT-PCR)检测该基因在建鲤鳃、肾、肠、肝、脑和脾的相对表达量, 其中脑最高, 其次是鳃、脾、肾、肠, 肝最低, 这表明脑、鳃和脾是建鲤Na+-K+-ATPase α1基因主要的表达器官。本研究旨为进一步探索建鲤的盐度调控机制奠定分子基础。

-----英文摘要:---------------------------------------------------------------------------------------

Na+-K+-ATPase is a ubiquitous plasma membrane enzyme that actively transports Na+ out of and K+ into animal cells. Its function is coupled to the hydrolysis of ATP and Na+-K+-ATPase plays a central role in the regulation of membrane potential, cell ion content, and the excitability and contractility of cells. Structurally, the Na-K-ATPase consists of two major polypeptides, a large catalytic α-subunit and a smaller glycosylated β-subunit. The α-subunit contains an intracellular ATP binding site, a phosphorylation site, and an extracellular binding site for cardiac glycosides, whereas the glycosylated β-subunit is thought to be a necessary component for maintaining stability and correct membrane orientation of the sodium pump. Four isoforms of the α-subunit and three isoforms of the β-subunit have been identified in mammals. The α1-subunit is expressed ubiquitously among tissues and is thought to play a ‘‘housekeeping’’ role in maintaining Na+ and K+ gradients and cell viability. We investigated the molecular mechanisms regulating Na+-K+-ATPase gene expression and osmoregulation in Cyprinus carpio var. jian. The full-length cDNA encoding Cyprinus carpio var. jian Na+-K+-ATPase α1 was cloned from Cyprinus carpio var. jian using homology cloning and RACE PCR. The Na+-K+-ATPase α1 was 3 397 bp in length, including a 232 bp 5’terminal UTR, a 3 081 bp encoding region, and a 219 bp 3’terminal UTR. There are eight transmembrane-spanning regions in the α1 subunit. Alignment of the deduced amino acid sequences of the Na+-K+-ATPase α1 and those of other vertebrates demonstrated that they shared many protein features that are common among fish and mammals. Phylogenetic analysis using MEGA 4 software revealed that the putative Na+-K+-ATPase α1 amino acid and that of Danio rerio had high similarity (95.52%). Similarly, the similarity was 92.65%, 91.97%, 91.38%, 90.52%, 90.42%, 90.23%, 90.22%, 89.62%, and 89.51% with Chanos chanos, Rhabdosargus sarba, Sarotherodon melanotheron, Fundulus heteroclitus, Acanthopagrus schlegelii, Oncorhynchus masou, Oncorhynchus mykiss, Salmo salar, Xenopus laevis, and Homo sapiens, respectively. Real-time quantitative PCR revealed that the Na+-K+-ATPase α1he Na+-K+-ATPase α1 transcript was detected at a high level in the brain, at moderate levels in the gills, spleen, kidney, and intestines, and at low levels in the liver. gene was expressed in all 6 tissues: brain, liver, kidney, intestines, spleen, and gills. T

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中文著录格式: 刘珊1,2,,李冰2,,张成锋2,,魏可鹏1,2.Na+-K+-ATPase α1基因全长cDNA的克隆及序列分析和表达.中国水产科学.2012;19(4):594-603.
英文著录格式: LIU,Shan1,,2,,LI,Bing2,,ZHANG,Chengfeng2,,WEI,Kepeng1,2,,ZHU,Jian1,,2.Full length cDNA cloning and tissue expression of the Na+-K+-ATPase α1 subunit from Cyprinus carpio var. jian.No Title Settings.2012;19(4):594-603.

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