粘虫甘油醛-3-磷酸脱氢酶基因cDNA的克隆、序列分析及表达量检测

Molecular Cloning,Sequence and Expression Detection Analysis of Glyceraldehyde-3-phosphate dehydrogenase Gene in Mythimna separata(Walker)

运用RT-PCR和RACE技术,以粘虫(Mythimna separata(Walker))cDNA为模板,对甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因进行克隆获得全长cDNA序列,并利用生物信息学方法,对GAPDH全长cDNA序列及推测得到的GAPDH蛋白序列进行分析.结果表明,获得的粘虫GAPDH基因cDNA序列长度为1 317 bp,其中包括80 bp的5′非编码区、238 bp的3′非编码区和999 bp的开放阅读框(Open Reading Frame,ORF),编码一个332个氨基酸蛋白,具有GAPDH蛋白家族的两个功能结构域.该GAPDH蛋白理论相对分子质量为35.498 6 kDa,等电点为7.63,富含6种类型的特定功能位点.该蛋白序列与其他动物GAPDH蛋白序列具有77.4%～92.9%高度同源性.GAPDH基因表达量检测结果显示GAPDH在粘虫6种不同组织间表达量无显著差异(P>0.05),表明GAPDH可作为研究粘虫功能基因表达量分析的可靠内参基因.该基因的cDNA序列已经递交GenBank并获得登录号为HM055756.

A full-length cDNA of glyceraldehyde-3-phosphate dehydrogenase（GAPDH） gene from Mythimna separata（Walker） was cloned using RT-PCR and RACE technique.The GAPDH cDNA is composed of 1 317 base pairs（bp）,including a 5′-untranslated region（5′-UTR） of 80 bp,a 3′-untranslated region（3′-UTR） of 238 bp and an open reading frame（ORF） of 999 bp.A 332 amino acid protein with a predicted molecular weight of 35.498 6 kDa and PI value of 7.63 is encoded by the ORF.The predicted protein are composed of 6 function sites and two typical GAPDH family functional Domains,sharing 77.4%～92.9% identity with those of other animals.The quantitative assays indicated that there is no significantly difference（P 〉0.05） among 6 different tissues,suggesting GAPDH is a reliable internal control in Mythimna separata（Walker）.This cDNA has been submitted to GenBank（Accession number： HM055756）.