Dai Tian-Mei, Lü Zhi-Chuang, Liu Wan-Xue, Wan Fang-Hao
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, PR China.
Agricultural Genome Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, PR China.
PLoS One. 2017 Mar 21;12(3):e0173821. doi: 10.1371/journal.pone.0173821. eCollection 2017.
The Bemisia tabaci Mediterranean (MED) cryptic species has been rapidly invading to most parts of the world owing to its strong ecological adaptability, which is considered as a model insect for stress tolerance studies under rapidly changing environments. Selection of a suitable reference gene for quantitative stress-responsive gene expression analysis based on qRT-PCR is critical for elaborating the molecular mechanisms of thermotolerance. To obtain accurate and reliable normalization data in MED, eight candidate reference genes (β-act, GAPDH, β-tub, EF1-α, GST, 18S, RPL13A and α-tub) were examined under various thermal stresses for varied time periods by using geNorm, NormFinder and BestKeeper algorithms, respectively. Our results revealed that β-tub and EF1-α were the best reference genes across all sample sets. On the other hand, 18S and GADPH showed the least stability for all the samples studied. β-act was proved to be highly stable only in case of short-term thermal stresses. To our knowledge this was the first comprehensive report on validation of reference genes under varying temperature stresses in MED. The study could expedite particular discovery of thermotolerance genes in MED. Further, the present results can form the basis of further research on suitable reference genes in this invasive insect and will facilitate transcript profiling in other invasive insects.
烟粉虱地中海隐种（MED）由于其强大的生态适应性，已迅速入侵到世界大部分地区，它被认为是研究快速变化环境下胁迫耐受性的模式昆虫。选择合适的内参基因用于基于qRT-PCR的胁迫响应基因表达定量分析，对于阐明耐热性的分子机制至关重要。为了在MED中获得准确可靠的标准化数据，分别使用geNorm、NormFinder和BestKeeper算法，在不同热胁迫条件下对八个候选内参基因（β-肌动蛋白、甘油醛-3-磷酸脱氢酶、β-微管蛋白、延伸因子1-α、谷胱甘肽S-转移酶、18S核糖体RNA、核糖体蛋白L13A和α-微管蛋白）进行了不同时间段的检测。我们的结果表明，β-微管蛋白和延伸因子1-α是所有样本组中最佳的内参基因。另一方面，18S核糖体RNA和甘油醛-3-磷酸脱氢酶在所研究的所有样本中稳定性最差。β-肌动蛋白仅在短期热胁迫情况下被证明具有高度稳定性。据我们所知，这是关于MED在不同温度胁迫下内参基因验证的第一份全面报告。该研究可加快MED耐热性基因的特定发现。此外，目前的结果可为进一步研究这种入侵昆虫中合适的内参基因奠定基础，并将有助于其他入侵昆虫的转录谱分析。
