Yuan Miao, Lu Yanhui, Zhu Xun, Wan Hu, Shakeel Muhammad, Zhan Sha, Jin Byung-Rae, Li Jianhong
Laboratory of Pesticide, College of Plant Science & Technology, Huazhong Agricultural University, Wuhan, China.
Laboratory of Insect Molecular Biology and Biotechnology, Department of Applied Biology, College of Natural Resources and Life Science, Dong-A University, Busan, Korea.
PLoS One. 2014 Jan 23;9(1):e86503. doi: 10.1371/journal.pone.0086503. eCollection 2014.
The brown planthopper (BPH), Nilaparvata lugens (Hemiptera, Delphacidae), is one of the most important rice pests. Abundant genetic studies on BPH have been conducted using reverse-transcription quantitative real-time PCR (qRT-PCR). Using qRT-PCR, the expression levels of target genes are calculated on the basis of endogenous controls. These genes need to be appropriately selected by experimentally assessing whether they are stably expressed under different conditions. However, such studies on potential reference genes in N. lugens are lacking. In this paper, we presented a systematic exploration of eight candidate reference genes in N. lugens, namely, actin 1 (ACT), muscle actin (MACT), ribosomal protein S11 (RPS11), ribosomal protein S15e (RPS15), alpha 2-tubulin (TUB), elongation factor 1 delta (EF), 18S ribosomal RNA (18S), and arginine kinase (AK) and used four alternative methods (BestKeeper, geNorm, NormFinder, and the delta Ct method) to evaluate the suitability of these genes as endogenous controls. We examined their expression levels among different experimental factors (developmental stage, body part, geographic population, temperature variation, pesticide exposure, diet change, and starvation) following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guidelines. Based on the results of RefFinder, which integrates four currently available major software programs to compare and rank the tested candidate reference genes, RPS15, RPS11, and TUB were found to be the most suitable reference genes in different developmental stages, body parts, and geographic populations, respectively. RPS15 was the most suitable gene under different temperature and diet conditions, while RPS11 was the most suitable gene under different pesticide exposure and starvation conditions. This work sheds light on establishing a standardized qRT-PCR procedure in N. lugens, and serves as a starting point for screening for reference genes for expression studies of related insects.
褐飞虱（Nilaparvata lugens，半翅目，飞虱科）是最重要的水稻害虫之一。利用逆转录定量实时PCR（qRT-PCR）对褐飞虱进行了大量的遗传学研究。使用qRT-PCR，根据内参基因来计算靶基因的表达水平。这些基因需要通过实验评估它们在不同条件下是否稳定表达来进行适当选择。然而，目前缺乏对褐飞虱潜在内参基因的此类研究。在本文中，我们对褐飞虱的八个候选内参基因进行了系统探索，即肌动蛋白1（ACT）、肌肉肌动蛋白（MACT）、核糖体蛋白S11（RPS11）、核糖体蛋白S15e（RPS15）、α2-微管蛋白（TUB）、延伸因子1δ（EF）、18S核糖体RNA（18S）和精氨酸激酶（AK），并使用四种替代方法（BestKeeper、geNorm、NormFinder和ΔCt法）来评估这些基因作为内参基因的适用性。我们按照MIQE（定量实时PCR实验发表的最低信息）指南，检测了它们在不同实验因素（发育阶段、身体部位、地理种群、温度变化、农药暴露、饮食变化和饥饿）中的表达水平。基于RefFinder的结果，该软件整合了四个当前可用的主要软件程序来比较和排名测试的候选内参基因，发现RPS15、RPS11和TUB分别是不同发育阶段、身体部位和地理种群中最合适的内参基因。RPS15是不同温度和饮食条件下最合适的基因，而RPS11是不同农药暴露和饥饿条件下最合适的基因。这项工作为在褐飞虱中建立标准化的qRT-PCR程序提供了思路，并作为筛选相关昆虫表达研究内参基因的起点。
