乌克兰鳞鲤丙酮酸激酶基因全长cDNA克隆及组织表达分析

Molecular Cloning and Tissue Distribution of Pyruvate kinase(PK) Full-length cDNA from Ukraine Scale Carp(Cyprinus carpio)

采用RT-PCR和RACE方法克隆乌克兰鳞鲤丙铜酸激酶基因全长cDNA序列,试验结果表明,乌克兰鳞鲤丙酮酸羧激酶基因cDNA全长1913bp,其中开放阅读框1617bp,编码538个氨基酸,预测蛋白分子质量为58.72ku,等电点为6.57,3′非翻译区长154bp及多聚腺苷酸尾巴,5′非翻译区长143bp。具有活性位点和结构域边界。乌克兰鳞鲤丙酮酸羧激酶基因保守性较好,与已知的草鱼、斑马鱼和绿河鲀的相似性为84%~92%,与哺乳类(人类丙酮酸羧激酶)的相似度为68%。以β-actin基因为内标,采用半定量RT-PCR方法对丙酮酸羧激酶基因在肝胰脏、脑、肾脏、心脏、肌肉、肠道等组织的表达进行研究,结果表明,低糖组丙酮酸羧激酶基因在各组织中均有表达,在肝胰脏、脑和心脏中表达较丰富;高糖组丙酮酸羧激酶基因在肝胰脏、脑、肾脏和肠道中有明显表达,其中在肝胰脏和脑中的表达量较丰富,在心脏和肌肉中的表达很微弱。

A full-length cDNA encoding pyruvate kinase（PK）was cloned from liver cells of Ukraine scale carp by RT-PCR and rapid amplification of cDNA ends（RACE）methods.The analysis on PK gene cDNA sequence（1913 bp）indicated that this gene contained a 1617 bp open reading frame（ORF）and encoded a 538-AA polypeptide.The PK protein had a calculated molecular weight of 58.72 ku and a PI of 6.57,with active site and domain boundaries.The PK of Ukraine scales carp had high conservatism,with identity of as high as 84%~92% with other fishes,and 68% between Ukraine scales carp and mammals.The tissue distribution of PK mRNA in liver,brain,kidney,heart,muscle and intestine of Ukraine scale carp was analyzed by semi-quantitative RT-PCR method usingβ-actin gene as the internal control.The results showed that in the fish fed low-carbohydrate diet group,PK were expressed in each tissue,highly expressed in liver,brain and heart;the fish fed high-carbohydrate diet,PK were highly expressed in liver and brain,and weakly expressed in heart and muscle.