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当前位置: 首页 > 文章 > 鲮β-肌动蛋白基因的分子克隆及其作为分子内标的可靠性分析 中国水产科学 2006,13 (5) 708-713
Position: Home > Articles > Molecular cloning and characterization of the β-actin gene of mud carp and its reliability as endogenous control Journal of Fishery Sciences of China 2006,13 (5) 708-713

鲮β-肌动蛋白基因的分子克隆及其作为分子内标的可靠性分析

作  者：
张殿昌;江世贵;邵艳卿;黄燕琴
单  位：
中国水产科学研究院南海水产研究所水产养殖与生物技术研究室
关键词：
β-actin;鲮;系统发生;分子内标
摘  要：
-βactin是actin家族的一员,在维持细胞结构、细胞内运动和细胞分裂等细胞生理活动方面发挥着重要的作用。克隆的鲮(Cirrhinus molitorella)-βactin基因的cDNA全长1 804 bp,开放阅读框长1 128 bp,编码375个氨基酸,5’和3’末端非翻译区域(UTR)分别为94 bp和582 bp。RT-PCR结果表明,-βactin在未经LPS刺激的和经LPS刺激的鲮心脏、肾脏、肌肉、肝脏、脾脏、肠、脑和皮肤等8种组织中广谱表达,表达水平没有明显差异,可以作为基因表达调控研究的分子内标。鲮与其他鱼类-βactin基因编码区核苷酸序列同源性在85.9%~99.6%。分子系统进化树分析表明,可将鱼类分成鲤形目,鲈形目,鲑形目和合鳃目4大支,与传统分类一致,-βactin基因核苷酸序列是研究分子进化的一个很好的分子标记。
译  名：
Molecular cloning and characterization of the β-actin gene of mud carp and its reliability as endogenous control
作  者：
ZHANG Dian-chang~(1),JIANG Shi-gui~(1),SHAO Yan-qing~(1,2),HUANG Yan-qin~(1,2)(1.South China Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Guangzhou 510300,China;2.Shanghai Fisheries University,Shanghai 200090,China)
关键词：
β-actin;Cirrhinus molitorella;phylogeny;endogenous control
摘  要：
Attempts to develop quantitative assays of mRNA samples have led to the common use of housekeeping gene expression to normalize the data.Assays based on Northern blots and semiquantitative reverse transcription polymerase chain reaction(RT-PCR) techniques are characterized by multiple steps,each introducing variation from sample to sample.In order to relate the results to each other and to the samples from which they originated,molecular biology assays need endogenous control,which are similarly influenced by the efficiency of each step.β-actin is a member of the actin family of genes,which play important roles in maintaining cytoskeletal structure,cell motility,cell division,intracellular movements and contractile processes,its expression is constitutively and ubiquitously high in all tissues,and therefore β-actin is the most widely used housekeeping gene.However,recent researches demonstrated that expressions of β-actin maybe were significantly different in the different environmental or physiological states,so the housekeeping gene need to be carefully validated before its use in such quantitative mRNA assays.In the present study,in order to quantitatively assay expression patterns of immuno-related genes of mud carp(Cirrhinus molitorella),we need to investigate a reliable molecular endogenous control to normalize mRNA levels.So a full-length cDNA of mud carp β-actin gene was cloned using reverse transcription polymerase chain reaction(RT-PCR) and RACE(rapid amplification of cDNA ends) polymerase chain reaction(PCR)(GenBank accession NO.DQ007446).The cDNA sequence of this gene is 1?804?bp in length and contains an open reading frame(ORF) of 1?128 nt encoding a 375 amino acid protein.The 5' and 3' untranslated regions of the messenger are 94 and 582 nucleotides long,respectively.RT-PCR showed β-actin gene of mud carp in heart,kidney,muscle,liver,spleen,intestine,brain and skin was expressed similarly in healthy and LPS-stimulated mud carp,suggesting an ubiquitous expression pattern and suitable for endogenous control to normalize mRNA levels.Nucleotide identities of the β-actin between mud carp and other fish species are 85.9%-99.6%.Phylogenetic tree showed all the fishes compared were divided into four branches,which was similar to the traditional taxa.The β-actin may be a good phylogenetic marker in fish taxonomy.

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