缢蛏β-ACTIN-1基因的分子特性及其表达分析

Molecular characteristics and expression analysis of β-ACTIN 1 gene from Sinonovacula constricta

摘要:

为研究缢蛏功能基因的表达调控,利用SMART cDNA文库构建试剂盒成功构建了缢蛏肝脏组织的标准化cDNA文库。对随机选取的5 679个克隆进行随机测序,比对、筛选出2条β-actin同源序列,对其中一条EST序列两端进行扩增、测序,然后进行5′RACE(rapid amplification of cDNA ends,RACE)扩增、测序,拼接得到全长cDNA序列,命名为β-ACTIN 1。该序列全长为1 552 bp,包括73 bp的5′非翻译区和348 bp的3′非翻译区,以及1 131 bp的开放阅读框。阅读框共编码376个氨基酸,推算分子量约为41.95 ku,理论等电点为5.23。与其他7种软体动物的氨基酸序列进行比对发现,β-ACTIN 1基因的氨基酸序列中Ile179、Glu229、Ser232、Pro236、Ile248、Asn272、Cys273、Val283、Ser320、Ser325、Val330、Pro339等12个氨基酸残基具有特异性;同时发现缢蛏β-ACTIN 1氨基酸序列与其他软体动物的相似性高达97%以上。系统进化分析显示,缢蛏首先与软体动物聚在一起,然后与节肢动物聚在一起,再依次与鱼类、两栖类、哺乳类聚在一起。荧光定量PCR检测结果显示,β-ACTIN 1基因在缢蛏各组织中的表达及鳗弧菌诱导后的表达均不稳定,不适合作为内参基因。

关键词:缢蛏; cDNA文库; β-ACTIN 1基因; 序列分析; 基因表达

Abstract:

The normalized cDNA library from liver of Sinonovacula constricta was constructed using the SMART cDNA construction kit and large numbers of colonies were randomly picked and sequenced.Two EST sequences with high homology with β-actin gene of other species were found and then the complete express sequence of one from S.constricta was obtained by PCR and 5′RACE, named β-ACTIN 1.The cDNA of this gene was 1 552 bp,which consists of a 73 bp 5′ untranslated region(UTR),a 1 131 bp open reading frame(ORF)and a 348 bp 3′ UTR.The translated protein is composed of 376 amino acids,with 41.95 ku molecular weight,and its calculated isoelectric point was 5.23.Compared with the other 7 molluscs of amino acid sequences,the amino acid sequence of β-ACTIN 1 inS.constricta has twelve specific amino acid residues:Ile179,Glu229,Ser232,Pro236,Ile248,Asn272,Cys273,Val283,Ser320,Ser325,Val330,Pro339,respectively.Meanwhile,the amino acids sequence of β-ACTIN 1 in S.constricta shared the high similarity with the other 7 molluscs(more than 97%).Phylogenetic analysis suggested that S.constricta clustered with mollusca firstly,and then clustered with arthropoda,finally clustered with fish,amphibians,mammals.The quantitative reverse transcriptase(qRT-PCR)analyses showed that the expression level of β-ACTIN 1 gene was not stable in different tissues and after the Vibrio anguillarum induced in S.constricta. So this β-actin gene was not suitable as an internal control.

Key words:Sinonovacula constricta; cDNA library; β-ACTIN 1 gene; sequence analysis; gene expression