虹鳟Ndufb2基因全长cDNA序列的克隆与分析

Cloning and sequence analysis of Ndufb2 full-length cDNA derived from Oncorhynchus mykiss

从虹鳟鱼(Oncorhynchus mykiss)的脑组织中提取RNA,经逆转录-聚合酶链反应及cDNA末端快速扩增技术克隆出Ndufb2基因全长cDNA序列(GenBank登录号:FJ534641),并对其序列进行分析.扩增结果表明:Ndufb2基因的cDNA序列全长899bp,其中5′端非翻译区长152bp,3′端非翻译区长441bp,开放阅读框长306bp,编码101个氨基酸;其N端前50个氨基酸为线粒体靶序列.将翻译的虹鳟Ndufb2蛋白序列与大西洋鲑(Salmo salar)的Ndufb2蛋白序列比对发现,其同源性高达98%,且虹鳟Ndufb2蛋白序列与已报道的哺乳动物的Ndufb2蛋白序列同源性均在55%以上.表明Ndufb2基因在进化过程中是高度保守的,推测其在线粒体呼吸链组成中发挥着重要作用.

Rainbow trout belongs to Salmonidae aerobic fish, and it is necessary for high dissolved oxygen content of living water environment. If the dissolved oxygen content of living water is less than 5 mg/L, it will cause the increase of respiratory rate, which is the so called ＂aquaculture floating head＂ phenomenon. Because the fish lives in hypoxia environment and the 90o/oo oxygen consumption is in the mitochondria, the transmission mechanism in composition and electronic respiratory chain may be different from the terrestrial animal. At the mitochondrial inner membrane, electrons from NADH and succinate pass through the electron transport chain to oxygen, which is reduced to water. Complex I is one of the main sites at which premature electron leakage to oxygen occurs, thus being one of the main sites of production of harmful superoxide. The first isolation of mitochondrial complex I since 1961, its composition and structure have had a primary understanding, but the specific mechanism of its participation in respiration, especially the function of each subunit is not clear. The protein encoded by Ndufb2 gene is a subunit of the multisubunit NADH ： ubiquinone oxidoreductase （complex I）. Mammalian complex I is composed of 45 different subunits. This protein has NADH dehydrogenase activity and oxidoreductase activity. It plays an important role in transferring electrons from NADH to the respiratory chain. Reverse transcription PCR （RT-PCR） and rapid amplification of eDNA ends （RACE） methods were used for the isolation of the whole eDNA of Ndufb2 gene from brain of Oncorhynchus mykiss. The assembly taskes of 3r and 5^-RACE sequence were completed by DNAman program. A pair of gene specific primers were designed to amplify the full-length eDNA sequence. ClustalX 1.81 and MEGA 3.0 software were used to calculate the amino acid sequence differences, and then the phylogenetic relationships of rainbow trout Ndufb2 gene sequence with other species were analyzed. Protein phosphorylation sites and protein kinase phosphorylation sites were predicted using online software NetPhosK 1 and NetPhos 2. Sequence analysis results showed that the rainbow trout Ndufb2 eDNA was 899 bp in length, 5~ untranslated region （5t UTR） 152 bp, 3r untranslated region （3r UTR） 441 bp, open reading frame （ORF） 306 bp, encoding 101 amino acids. The protein molecular mass was 11.4 ku and isoelectric point was 5.31. This gene sequence had been submitted to the GenBank database （accession number： FJ534641）. Mitochondrial target sequence of 50 amino acid motif appeared from the 1st to 50th amino acid site. Two antigenic sites （65-ILWHCWHDPD-74 and 23-QKIVIRK-29） of the Ndufb2 protein were found, and one serine phosphorylation site （aa49）, two threonine phosphorylation sites （aal5, aa47） and seven kinase phosphorylation sites were also found. Sequence alignment exhibited 98 % identity of amino acids between O. mykiss and Salmo salar, and more than 55 ~ identity of amino acids between O. rnykiss and mammal. Phylogenetic tree showed that the Ndufb2 of rainbow trout had the closest relationship with those of Atlantic salmon and zehrafish, then the amphibians, birds, marsupials and mammals. The phylogenetic analysis showed that the constructed Ndufb2 protein phylogenetic tree was consistent with the traditional species classification tree. The above results indicate that the Ndufb2 gene is relatively conservative in the progress of evolution and play an important role in electron transport process of mitochondrial respiratory chain. The cloned full-length cDNA sequence of mitochondrial respiratory chain gene Ndufb2 of rainbow trout lays a theoretical foundation for the structure and function of clarifying fish mitochondrial complex I.