粘虫β-actin基因cDNA的克隆、序列分析及表达量检测

Molecular cloning,sequence analysis and expression detection of β-actin gene in the oriental armyworm,Mythimna separata

β-actin基因作为actin家族的一员,在基因定量实验中常用作内参基因。本实验运用RT-PCR和RACE技术,以粘虫Mythimna separata(Walker)cDNA为模板,对β-actin基因进行克隆获得全长cDNA序列,并利用生物信息学方法,对β-actin基因全长cDNA序列及推测得到的β-actin蛋白序列进行分析。结果表明,获得的粘虫核β-actin基因cDNA序列长度为1 472 bp,其中包括68 bp的5′非编码区、273 bp的3′非编码区和1 131 bp的开放阅读框,编码一个376个氨基酸蛋白,具有actin蛋白家族典型特征。推测得到的粘虫β-actin蛋白理论分子量为41.7497 ku,等电点为5.29,富含6种类型的特定功能位点。该蛋白序列与其他动物β-actin蛋白序列具有97.9%～99.7%高度同源性。β-actin表达量检测结果显示β-actin在6种不同组织间表达无显著差异(P>0.05),表明β-actin可作为研究粘虫不同基因表达水平高低的可靠内参基因。该基因的cDNA序列已经递交GenBank并获得登录号为GQ856238。

β-actin,a member of the actin family,is often used as an internal standard in quantitative gene analysis.In this study,a full-length cDNA of the β-actin gene from Mythimna separata（Walker） was cloned using RT-PCR and the RACE technique.The results show that this cDNA is 1 472 base pairs（bp） long and contains a 5＇-untranslated region（5＇-UTR） of 68 bp and a 3＇-untranslated region（3＇-UTR） of 273 bp.The open reading frame（ORF） of 1 131 bp encodes a 376 amino acid protein with a predicted molecular weight of 41.7497 ku and PI value of 5.29.This predicted protein contains 6 functional sites,shares the typical actin family signature and 97.9 %～99.7% identity with actin from other animals.Quantitative assays indicate that there was no significant difference（P0.05） between 6 different tissues,suggesting that β-act in is a reliable internal control in M.separata.The cloned cDNA has been submitted to GenBank（Accession number：GQ856238）.