虹鳟脑型肌酸激酶基因cDNA全长的克隆与序列分析

The cloning and analysis of CKB full length cDNA derived from Oncorhynchus mykiss

肌酸激酶(Creatine kinase,CK)能够催化磷酸基团在二磷酸腺苷(ADP)和磷酸肌酸间的可逆性转移,在细胞能量代谢过程中发挥重要作用。以虹鳟(Oncorhynchus mykiss)为研究材料,使用RT-PCR和RACE法分离和克隆了虹鳟脑型肌酸激酶(CKB)基因cDNA的全长序列(GenBank登录号:FJ548753)。序列全长1493bp,其中5′端非翻译区81bp,3′端非翻译区266bp,开放性阅读框1146bp,编码381个氨基酸。虹鳟鱼CKB蛋白存在两个重要功能结构域,分别为EF-hand结构域和ATP:guanido磷酸转移酶结构域。构建的系统进化树证实所克隆的肌酸激酶CK基因属于脑型肌酸激酶基因CKB。虹鳟鱼CKB蛋白序列与大西洋鲑(Salmo salar)的CKB在进化树上最先聚为一支,这与两者同属鲑科鱼类这一事实是一致的。虹鳟鱼CKB蛋白序列与大西洋鲑的CKB蛋白同源性高达99%,与已报道的哺乳动物的CKB蛋白同源性均在80%以上相符,表明CKB基因在进化过程中是高度保守的,在细胞能量发生与转移过程中发挥着重要作用。

ATP levels in vertebrate cells are largely regulated by creatine kinase （ EC 2.7.3.2 ） that reversibly catalyze the transfer of phosphate between ATP and various phosphogens. In this paper, RT-PCR and RACE （ Rapid Amplification of eDNA Ends） methods were used for the isolation of the full length cDNA of CKB gene （ GenBank accession number ： FJ548753 ） from brain of Oncorhynchus mykiss. Sequence analysis revealed a 1 493 bp eDNA containing the 81 bp 5＇-untranslated region, 266 bp 3＇-untranslated region and 1 146 bp open reading frame encoding 381 amino acids. O. mykiss CKB has two important protein function domains, namely, EF-hand domain and ATP： guanido phosphotransferase domain. Evolutionary tree of all types of creatine kinases was then constructed, and it was determined that this cDNA of O. mykiss creatine kinase from brain belonged to brain type CK. CKB protein of O. mykiss and CKB protein of S. salar first clustered into a branch, because they all belong to the same salmon. Sequence alignment between O. mykiss and S. salar exhibited 99% identity rate of amino acid, and the more than 80% identity rate of amino acid with mammal. The result indicated the CKB gene is highly conservative in the progress of evolution and plays an important role in cell energy generation and transport process.