低盐度可诱导鲈鱼胞浆型PEPCK基因表达

磷酸烯醇式丙酮酸羧激酶(PEPCK)催化草酰乙酸生成磷酸烯醇式丙酮酸,是糖异生途径的第1个限速酶.本研究用SMARTRACE技术从鲈鱼肝脏中分离克隆了PEPCK基因的全长cDNA序列.该基因全长2215bp,包含1个123bp的5′非翻译区和217bp的3′非翻译区,开放阅读框为1875bp,编码1个由624个氨基酸组成的蛋白质,该蛋白理论分子量为69.1kD,等电点为5.87.氨基酸序列分析表明,与其它动物的胞浆型PEPCK相似性很高,与黑鲷为94.2%,与大西洋鲑为86.4%,与人为75.9%,而与该鱼线粒体型PEPCK氨基酸同源性只有70.6%.系统发育分析显示,该蛋白首先与其它动物的cPEPCK聚成一支,然后再与鱼类的mPEPCK成簇,认为该PEPCK属于胞浆型.同时用RT-PCR分析了PEPCK基因在10个组织中的表达,结果表明只有在肝脏、消化道和肾脏有较高的表达.将鲈鱼从盐度为25的海水转入盐度为12的海水48h后,肝脏和肾脏的PEPCK基因表达有增加.实验结果表明,本实验克隆的为鲈鱼胞浆型PEPCK,低盐度可诱导其表达.

Low Salinity Induces Cytosolic PEPCK Expression in Sea Perch (Lateolabrax japonicus)

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate into phosphoenolpyruvate, the first rate-limiting step in gluconeogenesis. The full-length 2 215 bp PEPCK cDNA amplified from the liver of sea perch (Lateolabrax japonicus) by SMART RACE consists a 123 bp 5′ UTR, a 217 bp 3′ UTR and an open reading frame (ORF) of 1 875 bp encoding a polypeptide of 624 amino acids with a predicted molecular weight of 69.1 kD and electric point of 5.87. The sea perch PEPCK has high amino acid homology with the cytosolic PEPCK from other species, 94.2% with Acanthopagrus schlegelii, 86.4% with Salmon salar, and 75.9% with Homo sapiens, as shown by BLAST analyses. It shares a 70.6% identity with the mPEPCK. Semi-quantitative RT-PCR was used to determine the expression of PEPCK in muscle, heart, eye, brain, gill, liver, intestine, kidney, fat and spleen. The results showed that PEPCK was expressed in liver, intestine and kidney. The PEPCK mRNA in the liver and kidney could be induced after the fish was transferred to low salinity for 48 hours.These results indicated that our obtained PEPCK was cytosolic and was inducible by low salinity.