Reference gene selection for gene expression studies using RT-qPCR in virus-infected planthoppers.
Reference gene selection for gene expression studies using RT-qPCR in virus-infected planthoppers.
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DOI：
10.1186/1743-422x-8-308
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发表时间：
2011-06-16
期刊：
影响因子：
4.8
通讯作者：
del Vas M
中科院分区：
文献类型：
--
作者：
Maroniche GA;Sagadín M;Mongelli VC;Truol GA;del Vas M
飞虱不仅在取食时对作物造成严重的机械破坏，而且还是几种植物病毒的传播媒介。分析持续感染的飞虱的基因表达可能揭示病毒传播的分子基础。实时定量RT-PCR （Quantitative real-time RT-PCR, RT-qPCR）是目前用于基因表达定量分析的最准确、最灵敏的方法。为了使结果的定量数据归一化，需要在实验过程中表达恒定的内参基因。首次从能持续传播植物固原病毒Río Cuarto病毒（MRCV）的飞虱中分离到常用内参基因actin （ACT）、α1-微管蛋白（TUB）、甘油醛3-磷酸脱氢酶（GAPDH）、延伸因子1 α (EF1A)、核糖体蛋白S18 （RPS18）和多泛素C （UBI）的部分序列。设计特异性RT-qPCR引物，利用geNorm、Normfinder和BestKeeper工具检测这些基因在mrcv感染和naïve飞虱中的表达稳定性。综合分析表明，UBI基因最适合作为mrcv感染飞虱定量基因表达的内对照，其次是18S和ACT基因，而TUB和EF1A基因变化最大。此外，MRCV感染可上调EF1A。建立了mrcv感染稻飞虱载体的RT-qPCR基因表达分析平台。我们的工作是首次报道病毒感染昆虫的内参基因选择，并可能为未来MRCV和其他病毒-飞虱病理系统的基因表达研究提供先例。
Planthoppers not only severely affect crops by causing mechanical damage when feeding but are also vectors of several plant virus species. The analysis of gene expression in persistently infected planthoppers might unveil the molecular basis of viral transmission. Quantitative real-time RT-PCR (RT-qPCR) is currently the most accurate and sensitive method used for quantitative gene expression analysis. In order to normalize the resulting quantitative data, reference genes with constant expression during the experimental procedures are needed. Partial sequences of the commonly used reference genes actin (ACT), α1-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1A), ribosomal protein S18 (RPS18) and polyubiquitin C (UBI) from Delphacodes kuscheli, a planthopper capable of persistently transmitting the plant fijivirus Mal de Río Cuarto virus (MRCV), were isolated for the first time. Specific RT-qPCR primers were designed and the expression stability of these genes was assayed in MRCV-infective and naïve planthoppers using geNorm, Normfinder and BestKeeper tools. The overall analysis showed that UBI, followed by 18S and ACT, are the most suitable genes as internal controls for quantitative gene expression studies in MRCV-infective planthoppers, while TUB and EF1A are the most variable ones. Moreover, EF1A was upregulated by MRCV infection. A RT-qPCR platform for gene expression analysis in the MRCV-infected planthopper vector Delphacodes kuscheli was developed. Our work is the first report on reference gene selection in virus-infected insects, and might serve as a precedent for future gene expression studies on MRCV and other virus-planthopper pathosystems.