浙江大学学报（农业与生命科学版）
浙江大學學報（農業與生命科學版）
절강대학학보（농업여생명과학판）
JOURNAL OF ZHEJIANG UNIVERSITY(AGRICULTURE & LIFE SCIENCES)
2013年
6期
600-606
,共7页
王家庆%边佳%李代宗%马爽%王亮%那广宁
王傢慶%邊佳%李代宗%馬爽%王亮%那廣寧
왕가경%변가%리대종%마상%왕량%나엄저
虹鳟鱼%Ndufb2基因%逆转录%聚合酶链反应%cDNA末端快速扩增
虹鱒魚%Ndufb2基因%逆轉錄%聚閤酶鏈反應%cDNA末耑快速擴增
홍준어%Ndufb2기인%역전록%취합매련반응%cDNA말단쾌속확증
rainbow trout%Oncorhynchus mykiss%Ndufb2 gene%reverse transcription-polymerase chain reaction%rapid amplification of cDNA ends
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899 bp，其中5'端非翻译区长152 bp，3'端非翻译区长441 bp，开放阅读框长306 bp，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。
從虹鱒魚（Oncorhynchus mykiss）的腦組織中提取 RNA，經逆轉錄聚閤酶鏈反應及 cDNA 末耑快速擴增技術剋隆齣 Ndufb2基因全長 cDNA 序列（GenBank 登錄號：FJ534641），併對其序列進行分析。擴增結果錶明：Ndufb2基因的 cDNA 序列全長899 bp，其中5'耑非翻譯區長152 bp，3'耑非翻譯區長441 bp，開放閱讀框長306 bp，編碼101箇氨基痠；其 N 耑前50箇氨基痠為線粒體靶序列。將翻譯的虹鱒 Ndufb2蛋白序列與大西洋鮭（Salmo salar）的 Ndufb2蛋白序列比對髮現，其同源性高達98%，且虹鱒 Ndufb2蛋白序列與已報道的哺乳動物的 Ndufb2蛋白序列同源性均在55%以上。錶明 Ndufb2基因在進化過程中是高度保守的，推測其在線粒體呼吸鏈組成中髮揮著重要作用。
종홍준어（Oncorhynchus mykiss）적뇌조직중제취 RNA，경역전록취합매련반응급 cDNA 말단쾌속확증기술극륭출 Ndufb2기인전장 cDNA 서렬（GenBank 등록호：FJ534641），병대기서렬진행분석。확증결과표명：Ndufb2기인적 cDNA 서렬전장899 bp，기중5'단비번역구장152 bp，3'단비번역구장441 bp，개방열독광장306 bp，편마101개안기산；기 N 단전50개안기산위선립체파서렬。장번역적홍준 Ndufb2단백서렬여대서양해（Salmo salar）적 Ndufb2단백서렬비대발현，기동원성고체98%，차홍준 Ndufb2단백서렬여이보도적포유동물적 Ndufb2단백서렬동원성균재55%이상。표명 Ndufb2기인재진화과정중시고도보수적，추측기재선립체호흡련조성중발휘착중요작용。
Summary 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 90% 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 1 961,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. <br> Reverse transcription PCR(RT-PCR) and rapid amplification of cDNA ends(RACE)methods were used for the isolation of the whole cDNA of Ndufb2 gene from brain of Oncorhynchus mykiss .The assembly taskes of 3' and 5'-RACE sequence were completed by DNAman program.A pair of gene specific primers were designed to amplify the full-length cDNA 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. <br> Sequence analysis results showed that the rainbow trout Ndufb2 cDNA was 899 bp in length,5'untranslated region(5'UTR) 1 52 bp,3'untranslated region(3'UTR)441 bp,open reading frame(ORF) 306 bp,encoding 101 amino acids.The protein molecular mass was 1 1.4 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 (aa1 5,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.mykiss and mammal.Phylogenetic tree showed that the Ndufb2 of rainbow trout had the closest relationship with those of Atlantic salmon and zebrafish,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. <br> 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.
