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]

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。

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