Nucleus of Applied Biology, Embrapa Maize and Sorghum, Sete Lagoas, MG, 35701-970, Brazil.
Laboratory of Plant-Pest Molecular Interaction, Embrapa Genetic Resources and Biotechnology, Brasília, DF, 70770-917, Brazil.
Mol Biol Rep. 2020 Jul;47(7):4989-5000. doi: 10.1007/s11033-020-05550-z. Epub 2020 Jun 27.
The relative quantification of gene expression is mainly realized through reverse transcription-quantitative PCR (RT-qPCR). However, the accuracy of this technique is deeply influenced by the expression stability of the reference genes used for data normalization. Therefore, the selection of suitable reference genes for a given experimental condition is a prerequisite in gene expression studies. Dichelops melacanthus (Hemiptera: Pentatomidae) is an important phloem sap-sucking insect pest of soybean, wheat, and maize in Brazil. Most of the genetic and molecular biology studies require gene expression analysis. Nevertheless, there are no reports about reference genes for RT-qPCR data normalization in D. melacanthus. In this study, we evaluated the expression stability of nine candidate reference genes (nadh, sdhb, gapdh, fau, ef1a, rpl9, ube4a, gus and rps23) in different developmental stages, body parts, sex, starvation-induced stress and dsRNA exposure by RefFinder software that integrates the statistical algorithms geNorm, NormFinder, BestKeeper, and ΔCt method. Our results showed that ef1a and nadh are the most stable reference genes for developmental stages, fau and rps23 for sex, ube4a and rps23 for body parts, rpl9 and fau for starvation stress, and nadh and sdhb for dsRNA exposure treatment. The reference genes selected in this work will be useful for further RT-qPCR analyses on D. melacanthus, facilitating future gene expression studies that can provide a better understanding of the developmental, physiological, and molecular processes of this important insect pest. Moreover, the knowledge gained from these studies can be helpful to design effective and sustainable pest management strategies.
基因表达的相对定量主要通过逆转录定量 PCR（RT-qPCR）来实现。然而，该技术的准确性受到用于数据归一化的参考基因表达稳定性的深刻影响。因此，为特定实验条件选择合适的参考基因是进行基因表达研究的前提。巴西的豆野螟（半翅目：蝽科）是大豆、小麦和玉米重要的韧皮部汁液取食性害虫。大多数遗传和分子生物学研究都需要进行基因表达分析。然而，在豆野螟中，还没有关于 RT-qPCR 数据归一化的参考基因的报道。在这项研究中，我们通过 RefFinder 软件评估了 9 个候选参考基因（nadh、sdhb、gapdh、fau、ef1a、rpl9、ube4a、gus 和 rps23）在不同发育阶段、身体部位、性别、饥饿诱导应激和 dsRNA 暴露下的表达稳定性，该软件整合了 geNorm、NormFinder、BestKeeper 和 ΔCt 方法等统计算法。我们的结果表明，ef1a 和 nadh 是发育阶段最稳定的参考基因，fau 和 rps23 是性别最稳定的参考基因，ube4a 和 rps23 是身体部位最稳定的参考基因，rpl9 和 fau 是饥饿应激最稳定的参考基因，nadh 和 sdhb 是 dsRNA 暴露处理最稳定的参考基因。本研究中选择的参考基因将有助于对豆野螟进行进一步的 RT-qPCR 分析，为研究该重要害虫的发育、生理和分子过程提供便利。此外，从这些研究中获得的知识有助于设计有效和可持续的害虫管理策略。
