米蛾β-actin基因cDNA片段的克隆及表达量检测  

Molecular Cloning and Expression Levels of β-actin Gene in Rice Moth [Corcyra cephalonica(Stainton)]

作　　者：丛斌[1] 张明珠[1] 胡志凤[1] 段立佳 王晓静[1] 张统书[1] 董辉[1] 

机构地区：[1]沈阳农业大学植物保护学院,沈阳110161

出　　处：《沈阳农业大学学报》2016年第1期24-28,共5页Journal of Shenyang Agricultural University

基　　金：国家自然科学基金项目(30971962);教育部博士点基金项目(20112103110008);辽宁省教育厅项目(L2013267)

摘　　要：为克隆米蛾[Corcyra cephalonica(Stainton)]β-actin基因cDNA片段,建立米蛾β-actin基因实时荧光定量PCR(qRT-PCR)方法,并评估其在实时荧光定量研究中作为内参基因的可靠性,利用反转录PCR(RT-PCR)技术克隆获得米蛾β-actin基因cDNA片段,采用SYBR GreenⅠ染料法建立qRT-PCR方法,检测在米蛾卵、幼虫、蛹和成虫4个虫态中β-actin基因的表达量。获得了长为822bp的米蛾β-actin基因片段(Gen Bank accession:KJ599569),编码273个氨基酸残基;荧光定量标准曲线的Ct值检测范围为13~28,扩增效率为90.1%,相关系数R^2=0.992,溶解曲线分析结果显示产物为特异的单峰,其Tm值为(84±0.5)℃,符合定量要求;实时荧光定量结果表明,β-actin基因在米蛾不同发育历期中的表达水平无显著性差异(p>0.05)。成功建立了米蛾β-actin基因qRT-PCR方法 ,β-actin基因可以作为米蛾基因表达定量研究中的可靠内参基因。研究结果为β-actin作内参基因进行米蛾功能基因表达差异研究奠定了分子基础。We cloned the cDNA of β-actin gene fragment from Corcyra cephalonica(Stainton) and evaluated the reliability of this gene as a reference gene in the Real-time fluorescent quantitative PCR(qRT-PCR) researchs. β-actin gene fragment was cloned by using reverse transcription PCR(RT-PCR) technique, qRT-PCR was performed using SYBR Green I to detect the expression of β-actin in four different stages of C. cephalonica. The results showed that the β-actin cDNA gene fragment had 822 base pairs in length(Gen Bank accession: KJ599569) and encoded 273 amino acid residues. The Ct value range of fluorescence quantitative standard was about 13-28, the amplification efficiency was 90.8%, R^2=0.993, Tm value was(84±0.5)℃. These numbers conform to the requirements of the quantitative. The qRT-PCR results showed that expression level was close in different stages of C.cephalonica. The method of qRT-PCR of β-actin from C. cephalonica was established successfully, β-actin gene could be used as a reliable reference gene in the quantitative analysis of gene expression in C. cephalonica. The research of β-actin laid the molecular foundation for the further research on the different expression of function genes in C. cephalonica.

关 键 词：米蛾 Β-ACTIN基因 反转录PCR 实时荧光定量PCR 

分 类 号：S433.4[农业科学—农业昆虫与害虫防治]