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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.
Real-time quantitative PCR (qRT-PCR) is one of the important means to study gene expression, and to select the appropriate internal reference gene is the basis for obtaining reliable research results.In this study, Plodia interpunctella, an important storage pest distributed worldwide, The objective of this study was to screen out the most suitable internal control genes for qRT-PCR in different developmental stages and different strains of the pest and to use two independent softwares (ge Norm and Norm Finder) for the stability of expression of eight candidate internal reference genes The results showed that Ef1a was the most stable internal control gene in different developmental stages, and b-Actin and Ef1a were the most stable in different strains.The Ef1a, b-Actin, POD), there was a significant difference in POD gene expression between different strains of Indian rice stem borer, but the relative expression of POD in different strains did not show significant difference when SD was the worst stability, It is necessary to evaluate the stability of the reference gene during the qRT-PCR study, and the relative expression level of the target gene can be accurately obtained based on the selected best gene as an internal reference. Help to better study the Indian meal moth development and strain-specific genes function, and provide a reference for the screening of Indian meal moth optimum reference gene under other conditions.