Cloning, Sequence Analysis Of The Na~+-k~+-ATPase α1and β1Gene In The Cyprinus Carpio VAR. Jian And Effects Of Salinities On The Transcriptional Expression

Na+-K+-ATPase is a ubiquitous plasma membrane enzyme that actively transports Na+out of and K+into animal cells. By its continuous function coupled to the hydrolysis of ATP, Na+-K+-ATPase plays a central role in the regulation of membrane potential, cell ion content as well as excitability and contractility of the cells. Structurally, the Na+-K+-ATPase consists of two major polypeptides, a large catalytic α-subunit and a smaller glycosylated β-subunit. In fish species, the cDNAs of Na+-K+-ATPase α1and β1subunit have been cloned from a variety of sources including the white sucker Catostomus commersoni, European eel Anguilla Anguilla, zebrafish Danio rerio, tilapia Oreochromis mossambicus, Atlantic Salmon Salmo Salar and other teleosts.To reveal the molecular mechanisms of the osomregulation in Cyprinus carpio var.jian and its regulation of Na+-K+-ATPase gene expression, the full-length cDNA encoding Cyprinus carpio var.jian Na+-K+-ATPase α1, α1.3and β1subunit were cloned from Cyprinus carpio var.jian using homology cloning and RACE PCR approach. The Na+-K+-ATPase α1was3397bp in length, including232bp5’terminal UTR,3081bp encoding region and219bp3’terminal UTR and there are eight transmembrane-spanning regions in the α1subunit; the Na+-K+-ATPase α1.3was3265bp in length, including77bp5’terminal UTR,3075bp encoding region and113bp3’terminal UTR and there are eight transmembrane-spanning regions in the α1.3subunit, too; the Na+-K+-ATPase β1was2198bp in length, including108bp5’terminal UTR,906bp encoding region and1184bp3’terminal UTR and there are only one transmembrane-spanning regions in the β1subunit, at the same time, one secretion signal peptide was detected in this subunit.Alignment of deduced amino acid sequences of the Na+-K+-ATPase α1and those of other vertebrates demonstrated that they shared many protein features common in fish and mammalians. Phylogenetic analysis using MEGA4software showed that the putative Na+-K+-ATPase α1amino acid and that of Danio rerio were close to each other in similarity of95.52%. For other animals, the similarity was92.65%、91.97%、91.38%、 90.52%、90.42%.90.23%、90.22%、89.62%、89.51%with Chanos chanos, Rhabdosargus sarba, Sarotherodon melanotheron, Fundulus heteroclitus, Acanthopagrus schlegelii, Oncorhynchus masou, Oncorhynchus mykiss, Salmo salar, Xenopus laevis, Homo sapiens, respectively. For Na+-K+-ATPase α1.3, the similarity was89.06%、87.29%、86.02%、85.34%、85.14%、84.26%、84.12%、83.92%、83.87%、83.79%、83.79%、83.58%、83.48%、83.46%、82.39%、82.23%、81.51%and81.45%from the other animals.The similarity between Na+-K+-ATPase β-subunit of Cyprinus carpio var.jian and other animals was76.08%、66.67%、64%、63.74%、61.2%、59.2%with Danio rerio, Rhabdosargus sarba, Oncorhynchus masou, Oreochromis mossambicus, Homo sapiens, Xenopus laevis, respectively.Real-time quantitative PCR results indicated that the Na+-K+-ATPase al, Na+-K+-ATPase α1.3and Na+-K+-ATPase β1genes were expressed in all the7tissues-brain, liver, kidney, intestines, spleen, heart and gills. The Na+-K+-ATPase α1transcript was detected at a high level in intestines, gills and brain, moderate in liver, heart, and kidney, and low in spleen. The Na+-K+-ATPase a1.3was highly expressed in gill and heart, the expression in kidney, intestines and brain were marginally lower, intermediate in liver and hardly expressed in spleen. The levels of β1mRNA are highest in the spleen, with intermediate levels in the liver and intestines, and low levels in the gill, heart, kidney and brain.The levels of Na+-K+-ATPase α1and Na+-K+-ATPase β1mRNA in Cyprinus carpio var.jian gill during adaptation to different Na+environments were detected at different times. The levels of Na+-K+-ATPase β1-subunit mRNA were increased in all the experimental groups while the Na+-K+-ATPase α1was decreased. In addition to this, a certain variation trend of Na+-K+-ATPase α1was observed during acclimation to various environments which composed of3stages, that is, stress response period, actively regulation period and adapting period. The Na+-K+-ATPase β1has a different way of change due to different functions of the two subunits.