Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae).
Evaluation of Reference Genes for Reverse Transcription Quantitative PCR Studies of Physiological Responses in the Ghost Moth, Thitarodes armoricanus (Lepidoptera, Hepialidae).
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DOI：
10.1371/journal.pone.0159060
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Han R
中科院分区：
文献类型：
--
作者：
Liu G;Qiu X;Cao L;Zhang Y;Zhan Z;Han R
逆转录实时定量聚合酶链反应（RT-qPCR）是在内源性调控的基础上定量检测靶基因表达水平的灵敏方法。一个合适的标准化参考基因集对于可靠的结果是必不可少的。鬼蛾Thitarodes armoricanus是鳞翅目昆虫中重要的经济成员，是药用真菌Ophiocorphins sinensis的寄主。近年来的研究主要集中在该物种对高原环境的适应机制和宿主对O.在这些研究中，通常使用中华绒螯蟹感染和RT-qPCR来破译生理功能的遗传基础。然而，缺乏对Thitarodes属中候选参考基因的全面评估。在此，对八个候选参考基因（ACT、EF、EIF 4A、GAPDH、G6 PDH、RPL 13 A、TUB和18 S）在T.分析了七龄幼虫不同发育阶段和不同身体部位的armoricanus，以及沿着低温饲养的幼虫、暴露于两种真菌感染的幼虫和饲喂不同饲料的幼虫。三个已建立的软件程序Bestkeeper，geNorm和NormFinder的，用于计算处理之间的变化。结果表明，不同处理的参比基因存在差异，其中EF、EIF 4A和GAPDH最适合不同发育阶段和幼虫身体部位; EF、EIF 4A和RPL 13 A最适合低温攻毒; EF、EIF 4A和TUB最适合真菌感染和饲料处理。因此，本研究进一步有助于建立一种准确的方法，用于标准化T的RT-qPCR结果。armoricanus，并作为相关昆虫物种的基因表达研究的参考。
Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the sensitive method to quantify the expression levels of target genes on the basis of endogenous control. An appropriate reference gene set for normalization is essential for reliable results. The ghost moth, Thitarodes armoricanus, a host species of a medicinal fungus, Ophiocordyceps sinensis, is an economically important member of the Lepidoptera. Recent studies have focused on the mechanism of adaptation of this species to its high-altitude environment and host immune response to O. sinensis infection and RT-qPCR is commonly used in these studies to decipher the genetic basis of physiological functions. However, a thorough assessment of candidate reference genes in the genus Thitarodes is lacking. Here, the expression levels of eight candidate reference genes (ACT, EF, EIF4A, GAPDH, G6PDH, RPL13A, TUB and 18S) in T. armoricanus at different developmental stages and in different body parts of the seventh instar larvae were analyzed, along with larvae kept under low temperatures, larvae exposed to two fungal infections and larvae fed different diets. Three established software programs–Bestkeeper, geNorm and NormFinder–were employed to calculate variation among the treatments. The results revealed that the best-suited reference genes differed across the treatments, with EF, EIF4A and GAPDH found to be the best suited for the different developmental stages and larvae body parts; EF, EIF4A and RPL13A found to be the best suited for low-temperature challenge; and EF, EIF4A and TUB found to be the best suited for the fungal infections and dietary treatments. This study thus further contributes to the establishment of an accurate method for normalizing RT-qPCR results for T. armoricanus and serves as a reference for gene expression studies of related insect species.