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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.
Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes oxaloacetate to generate phosphoenolpyruvate, which is the first rate-limiting enzyme in gluconeogenesis.In this study, the PEPCK gene was isolated and isolated from sea bass by SMARTRACE The full-length cDNA was 2215bp in length and contained a 123bp 5 ’untranslated region and a 217bp 3’ untranslated region. The open reading frame was 1875bp and encoded a protein consisting of 624 amino acids. The theoretical molecular weight was 69.1 kD and the isoelectric point was 5.87. Amino acid sequence analysis revealed a high similarity to the cytosolic PEPCK of other animals, 94.2% with black sea bream, 86.4% with Atlantic salmon and 75.9% with human, whereas And only 70.6% homology with the mitochondrial PEPCK amino acid.Phylogenetic analysis showed that the protein first clustered with cPEPCK of other animals and then clustered with the mPEPCK of fish, which was considered to be cytoplasmic. The expression of PEPCK gene in 10 tissues was analyzed by RT-PCR and the results showed that the expression of PEPCK gene was only found in liver, digestive tract and kidney.Sea perch was transferred from seawater with salinity 25 to seawater with salinity 12 for 48h After the liver and kidney PEPCK gene expression increased The experimental results show that the experimental cloning of perch cytoplasm PEPCK, low salinity can induce its expression.