Reference Gene Selection and Evaluation for Gene Expression Studies Using qRT-PCR in the White-Backed Planthopper, Sogatella furcifera (Hemiptera: Delphacidae)
Reference Gene Selection and Evaluation for Gene Expression Studies Using qRT-PCR in the White-Backed Planthopper, Sogatella furcifera (Hemiptera: Delphacidae)
复制标题
使用 qRT-PCR 在白背飞虱、白背飞虱（半翅目：飞虱科）中进行基因表达研究的参考基因选择和评估
DOI：
10.1093/jee/tov333
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发表时间：
2016-04-01
影响因子：
2.2
通讯作者：
Liu, Yu-di
中科院分区：
文献类型：
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作者：
An, Xing-kui;Hou, Mao-lin;Liu, Yu-di
白背飞虱（半翅目，飞虱科）是对水稻危害最大的害虫之一。为了更好的控制策略，人们利用反转录定量实时聚合酶链反应（qRT-PCR）进行了各种遗传学研究。适当应用qRT-PCR需要可靠的内源对照；然而，关于白背飞虱这方面的研究还很缺乏。本研究采用qRT-PCR方法，对延长因子1- α (ef1 - α)、多泛素（UB）、核糖体蛋白S18 （RPS18）、肌动蛋白1 （ACT）、α -1微管蛋白（TUB）、甘油醛-3-磷酸（GAPDH）、核糖体蛋白L9 （RPL9）、核糖体蛋白L10 （RPL10）、18S核糖体RNA （18S）等9个常用内参基因在不同发育阶段、南方水稻黑条矮缩病毒（SRBSDV）在不同组织和不同温度胁迫下的获取。使用四个软件程序（geNorm、NormFinder、BestKeeper和delta Ct法）和基于web的综合工具RefFinder对候选内参基因进行比较和排序。综合上述4个软件程序的RefFinder分析结果显示，在不同发育阶段和不同温度胁迫下，TUB被评为最合适的内参基因，GAPDH和RPL9分别在SRBSDV和不同组织中表现出最高的获取稳定性。这些结果将为今后白背飞虱基因表达的研究提供坚实的基础，也将为其他相关昆虫建立标准化的qRT-PCR程序提供帮助。
The white-backed planthopper, Sogatella furcifera (Hemiptera, Delphacidae), is one of the most devastating rice pests. For a better control strategy, various genetic studies have been conducted using reverse-transcription quantitative real-time polymerase chain reaction (qRT-PCR). The appropriate application of qRT-PCR requires reliable endogenous controls; however, studies on this aspect of the white-backed planthopper are lacking. In the present study, nine commonly used reference genes, elongation factor 1-alpha (EF1-alpha), polyubiquitin (UB), ribosomal protein S18 (RPS18), actin 1 (ACT), alpha-1 tubulin (TUB), glyceraldehyde-3-phosphate (GAPDH), ribosomal protein L9 (RPL9), ribosomal protein L10 (RPL10), and 18S ribosomal RNA (18S), were evaluated by qRT-PCR for their expression stability under four different experimental conditions (different developmental stages, acquisition of Southern rice black-streaked dwarf virus (SRBSDV), different tissues, and different temperature stress). These results were analyzed using four software programs (geNorm, NormFinder, BestKeeper, and the delta Ct method) and a Web-based comprehensive tool RefFinder to compare and rank candidate reference genes. According to the results of RefFinder analysis, which integrates the abovementioned four software programs, TUB was ranked as the most suitable reference gene at different developmental stages and under different temperature stress, and GAPDH and RPL9 showed the highest stability for acquisition of SRBSDV and different tissues, respectively. These results will provide a solid foundation for future gene expression study on the white-backed planthopper, and also will give aids in establishing a standardized qRT-PCR procedure for other related insects.