草鱼过氧化氢酶全长cDNA的克隆、序列同源分析与组织表达  

Full-length cDNA Cloning,Sequence Homology Analysis and Tissue Expression of a Catalase Gene from Grass Carp(Ctenopharyngodon idellus)

作　　者：郑清梅[1] 韩春艳[1] 温茹淑[1] 钟艳梅[1] 姚琼凤[1] 侯雨文[1] 

机构地区：[1]嘉应学院生命科学学院,梅州514015

出　　处：《基因组学与应用生物学》2011年第5期529-538,共10页Genomics and Applied Biology

基　　金：广东高校优秀青年创新人才培育项目(LYM08093);广东省自然科学基金项目(S2011010003451)共同资助

摘　　要：过氧化氢酶(catalase,CAT)是生物体内抗氧化防御系统的关键酶之一,在清除过氧化氢而避免机体产生氧化应激的过程中起重要作用。本研究从草鱼(Ctenopharyngodon idellus)肝胰脏中克隆了CAT完整编码序列(complete coding sequence,CDS)。该CAT序列(GenBank登陆号:FJ560431)全长2263bp,包括完全开放阅读框(ORF)1575bp、5'非编码区(UTR)118bp和3'UTR570bp。其ORF编码525个氨基酸残基,理论分子量为59.59kD,等电点为7.02。在草鱼CAT cDNA的终止密码子附近,其3'UTR具有长且完整的AC重复序列,与斑马鱼、鲢鱼及啮齿类动物CAT的3'UTR AC重复序列相似。序列比较表明,草鱼CAT的核苷酸及推测氨基酸序列与其它多种物种的一致性均较高,其一致性分别为93.4%～43.0%和98.1%～63.3%。同时,草鱼CAT cDNA的推测氨基酸序列具有与其它动物高度保守的特征性基序,包括亚铁血红素结合信号序列"RLFSYPDTH"、酶活性中心序列"FDRERIPERVVHAKGA"及3个催化位点残基His74、Asn147和Tyr357。此外,草鱼CAT还具有保守的亚铁血红素结合口袋与NADPH结合位点。根据草鱼CAT基因的上述特征,推测其属于CAT基因家族中的单功能或典型CAT基因亚群。采用实时荧光定量PCR(Q-PCR)检测草鱼CAT的组织表达特征。结果显示,草鱼CATmRNA在所检测的11种组织器官中均有表达,其中在肝中表达水平量较高,在红肌、白肌和脂肪中表达量较低。本研究结果将有助于进一步探讨鱼类CAT基因的结构与功能,并为研究其抗氧化分子机理奠定基础。Catalase is a key enzyme in the antioxidant systems of living organisms that plays an important role in the against oxidative stress by eliminating hydrogen peroxide.The full-length catalase cDNA was cloned from hepatopancreas of grass carp（Ctenopharyngodon idellus）.The gene CAT（GenBank Accession No.FJ560431） was 2 263 base-pairs（bp）,including a complete protein coding region（ORF） of 1 575 bp,a 5＇ untranslated region（UTR） of 118 bp and 3＇ UTR of 570 bp.The ORF encoded 525 amino acid（aa） residues and the molecular mass of the predicted protein was 59.59 kD with an estimated pI of 7.02.The 3＇ UTR of the cDNA contained a dinucleotide repeat near the termination codon consisting of a near perfect and long CA array,which had the same characteristic of catalase from zebrafish,silver carp and rodents.Sequence comparison showed that the nucleotide sequence and the deduced amino acid sequence of grass carp CAT shared 93.4%~43.0% and 98.1%~63.3%,respectively,identity with that of other selected species.Several highly conserved motifs including the proximal heme-ligand signature sequence ＂RLFSYPDTH＂,the proximal active site signature ＂FDRERIPERVVHAKGA＂,and the three catalytic amino acid residues of His74,Asn147 and Tyr357 were identified in the deduced amino acid sequence of CAT from grass carp.Besides the CAT also had highly conserved heme binding pocket and NADPH binding site.Accordingly above-mentioned sequence signature,the grass carp CAT was presumed to be monofunctional CAT or typical CAT in the CAT family.CAT transcripts were detected in the tissue of grass carp.And variance mRNA abundance was detected in different tissue.Real-time quantitative PCR（Q-PCR） method was used to analyze the CAT mRNA expression characterization in tissues of grass carp.The results showed expression of CAT mRNA was found in all detected 11 species of tissues and which was high in the tissues of liver and low in the tissues of red muscle,white muscle and fat.Therefore,this study would be contributed

关 键 词：草鱼 过氧化氢酶 克隆 序列分析 组织表达 

分 类 号：S917.4[农业科学—水产科学] Q78[生物学—分子生物学]