印度谷螟实时荧光定量PCR内参基因的选择  

Selection of reference genes in quantitative real-time PCR of Plodia interpunctella(Lepidoptera: Pyralidae)

作　　者：唐培安[1] 张棋麟[2] 薛昊[1] 袁明龙[3] 

机构地区：[1]南京财经大学食品科学与工程学院,江苏省现代粮食流通与安全协同创新中心,南京210023 [2]南京大学生命科学学院,演化发育生物学研究所,南京210023 [3]兰州大学草地农业科技学院,草地农业生态系统国家重点实验室,兰州730020

出　　处：《中国科学：生命科学》2016年第10期1201-1209,共9页Scientia Sinica(Vitae)

基　　金：粮食公益性行业科研专项(批准号:201413007-2);国家科技支撑计划(批准号:2013BAD17B01);国家自然科学基金(批准号:31000828);江苏省高校优势学科建设工程资助

摘　　要：实时荧光定量PCR(qRT-PCR)是研究基因表达的重要手段之一,而选择合适的内参基因是获得可靠研究结果的基础.本研究以世界性分布的重要仓储害虫印度谷螟(Plodia interpunctella)为研究对象,旨在筛选出该虫不同发育阶段及不同品系qRT-PCR的最适内参基因,并利用两个独立软件(ge Norm和Norm Finder)对8个备选内参基因的表达稳定性进行了评价.结果表明,不同发育阶段最稳定表达的内参基因为Ef1a,而不同品系最稳定的是b-Actin和Ef1a.利用筛选到的Ef1a,b-Actin及两者组合对目标基因(过氧化物酶POD)进行标准化,印度谷螟不同品系间的POD基因表达量存在显著差异,而以稳定性最差的SD作为内参时,不同品系间POD的相对表达量并无显著差异.这表明,在开展qRT-PCR研究时评价内参基因的稳定性非常必要,且基于筛选到的最佳基因作为内参时,才能准确获得目标基因的相对表达水平.这些结果将有助于更好地研究印度谷螟发育及品系特异相关基因的功能,并为筛选印度谷螟在其他条件下的最适内参基因提供参考.Quantitative real-time PCR（qRT-PCR） is an important tool for studying gene expression, whereas selection of reliable reference genes（RGs） is the basis for obtaining accurate qRT-PCR results. Plodia interpunctella is an important stored-product pest with worldwide distribution. In this study, we screened out reliable RGs of P. interpunctella for different developmental stages and strains, and evaluated expression stability of eight candidate RGs by using two independent softwares（ge Norm and Norm Finder）. The results indicated that the most stable expression RG was Ef1a under different developmental stages, whereas b-Actin and Ef1a showed the most stable expression for different strains. Expression pattern of a target gene（peroxidase, POD） exhibited significant discrepancies between different strains based on normalization of Ef1a, b-Actin, as well as the combination of these two genes, but no significant difference was found with SD that was the least stability RG. Therefore, it is an essential step for evaluating stability of candidate RGs before qRT-PCR analyses for target genes, and accurate expression levels of target genes could be acquired based upon normalization of the most reliable RGs selected by experiments. These results will be helpful for better studying function of development-related and strain-specific genes in P. interpunctella, and provide valuable information for further selecting optional RGs in qRT-PCR analyses of this pest under other conditions.

关 键 词：印度谷螟 仓储害虫 实时定量PCR 基因表达 内参基因 稳定性评价 

分 类 号：S379.5[农业科学—农产品加工]