Selection and validation of reference genes for qRT-PCR analysis during biological invasions: The thermal adaptability of Bemisia tabaci MED.
Selection and validation of reference genes for qRT-PCR analysis during biological invasions: The thermal adaptability of Bemisia tabaci MED.
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生物入侵期间 qRT-PCR 分析参考基因的选择和验证：烟粉虱 MED 的热适应性
DOI：
10.1371/journal.pone.0173821
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Wan FH
中科院分区：
文献类型：
--
作者：
Dai TM;Lü ZC;Liu WX;Wan FH
地中海烟粉虱（MED）隐种因其强大的生态适应性而迅速入侵世界大部分地区，被认为是快速变化环境下抗逆性研究的模式昆虫。选择合适的内参基因进行基于 qRT-PCR 的定量应激反应基因表达分析对于阐明耐热性的分子机制至关重要。为了获得 MED 中准确可靠的标准化数据，分别使用 geNorm、NormFinder 和 BestKeeper 算法在不同时间段的不同热应力下检查八个候选参考基因（β-act、GAPDH、β-tub、EF1-α、GST、18S、RPL13A 和 α-tub）。我们的结果表明，β-tub 和 EF1-α 是所有样本集中的最佳参考基因。另一方面，18S 和 GADPH 在所有研究的样品中表现出最低的稳定性。 β-act 被证明仅在短期热应力的情况下高度稳定。据我们所知，这是 MED 中第一份关于在不同温度胁迫下验证参考基因的综合报告。该研究可以加速 MED 中耐热基因的特定发现。此外，目前的结果可以为进一步研究这种入侵昆虫中合适的参考基因奠定基础，并将有助于其他入侵昆虫的转录谱分析。
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.