Journal of Stored Products Research ( IF 2.7 ) Pub Date : 2021-08-20 , DOI: 10.1016/j.jspr.2021.101877 Yue Zhang 1 , Jia-peng Yang 1 , Ren-huai Dai 1 , Yi Yan 1, 2 , Wen-jia Yang 2 , Da-ming Hu 3
Lasioderma serricorne (Fabricius) is an important insect pest of stored products worldwide. The real-time quantitative polymerase chain reaction (RT-qPCR) is a reliable technique commonly used to analyze gene expression across various biological processes. For accurate gene expression analyses using RT-qPCR, selection of stable reference genes to normalize RT-qPCR data is a prerequisite. However, the lack of studies on validation of reference genes in L. serricorne limits application of RT-qPCR in L. serricorne. In this study, the expression stability of eleven candidate reference genes (Actin, ARF1, β-Tubulin, EF1α, SYN6, TBP1, RPL3, RPL12, RPL13a, RPL32, 18S rRNA using five algorithms (geNorm, NormFinder, BestKeeper, ΔCt and RefFinder) in L. serricorne under different experimental and biotic conditions was assessed. The results showed that the best combinations of reference genes that could be used as internal controls in L. serricorne were 18S rRNA and RPL13a for different development stages; 18S rRNA and RPL3 for different larval tissues; RPL3 and RPL13a for different temperatures; RPL32, RPL12 and Actin for starvation. These results provided for the first time a comprehensive list of stable reference genes for the normalization of RT-qPCR analyses in L. serricorne and will benefit the in-depth molecular biology research in the future.
中文翻译:
丝角激光 (F.) 中用于 RT-qPCR 归一化的候选参考基因的稳定性评估
Lasioderma serricorne (Fabricius) 是世界范围内储藏产品的重要害虫。实时定量聚合酶链反应 (RT-qPCR) 是一种可靠的技术,常用于分析各种生物过程中的基因表达。为了使用 RT-qPCR 进行准确的基因表达分析,选择稳定的参考基因以标准化 RT-qPCR 数据是先决条件。然而,缺乏对L. serricorne中参考基因验证的研究限制了 RT-qPCR 在L. serricorne 中的应用。本研究中,11个候选参考基因(肌动蛋白、ARF1、β-微管蛋白、EF1α、SYN6、TBP1)的表达稳定性,RPL3,RPL12,RPL13A,RPL32,18S rRNA的使用五个算法(geNorm,NormFinder,BestKeeper,ΔCT和RefFinder)在L.烟草甲不同的实验和生物的条件下进行了评估。结果表明,可以在被用来作为内部对照的参照基因的最佳组合L.烟草甲分别为18S rRNA基因和RPL13A用于不同发育阶段; 不同幼虫组织的18S rRNA和RPL3;不同温度的RPL3和RPL13a;RPL32 ,RPL12和肌动蛋白用于饥饿。这些结果首次为L. serricorne RT-qPCR分析的标准化提供了完整的稳定参考基因列表,将有利于未来分子生物学的深入研究。