Laboratory of Molecular Analysis (LAM), Life Sciences Department, Faculty of Medicine, Federal University of Tocantins, Palmas, TO, Brazil.
Postgraduate Program in Biodiversity and Biotechnology, Rede Bionorte, Federal University of Tocantins, Palmas, TO, Brazil.
Sci Rep. 2024 Jan 31;14(1):2556. doi: 10.1038/s41598-024-52948-x.
Relative gene expression analysis through RT-qPCR is an important molecular technique that helps understanding different molecular mechanisms, such as the plant defense response to insect pests. However, the use of RT-qPCR for gene expression analysis can be affected by factors that directly affect the reliability of the results. Among these factors, the appropriate choice of reference genes is crucial and can strongly impact RT-qPCR relative gene expression analyses, highlighting the importance in correctly choosing the most suitable genes for the success of the analysis. Thus, this study aimed to select and validate reference genes for relative gene expression studies through RT-qPCR in hybrids of Eucalyptus tereticornis × Eucalyptus camaldulensis (drought tolerant and susceptible to Leptocybe invasa) under conditions of inoculation by the Beauveria bassiana fungus and subsequent infestation by L. invasa. The expression level and stability of eleven candidate genes were evaluated. Stability was analyzed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper, and Delta-Ct algorithms. The selected reference genes were validated through the expression analysis of the transcriptional factor EcDREB2 (dehydration-responsive element-binding protein 2). For all treatments evaluated, EcPTB, EcPP2A-1, and EcEUC12 were the best reference genes. The triplets EcPTB/EcEUC12/EcUBP6, EcPP2A-1/EcEUC12/EcPTB, EcIDH/EcSAND/Ecα-TUB, EcPP2A-1/Ecα-TUB/EcPTB, and EcPP2A-1/EcUPL7/EcSAND were the best reference genes for the control plants, mother plants, plants inoculated with B. bassiana, plants infested with L. invasa, and plants inoculated with B. bassiana and subsequently infested with L. invasa, respectively. The best determined reference genes were used to normalize the RT-qPCR expression data for each experimental condition evaluated. The results emphasize the importance of this type of study to ensure the reliability of relative gene expression analyses. Furthermore, the findings of this study can be used as a basis for future research, comprising gene expression analysis of different eucalyptus metabolic pathways.
通过 RT-qPCR 进行相对基因表达分析是一种重要的分子技术，有助于理解不同的分子机制，如植物对昆虫害虫的防御反应。然而，RT-qPCR 用于基因表达分析的效果可能会受到直接影响结果可靠性的因素的影响。在这些因素中，适当选择参考基因至关重要，并且会强烈影响 RT-qPCR 相对基因表达分析，突出了正确选择最适合分析成功的基因的重要性。因此，本研究旨在通过 RT-qPCR 从相对基因表达研究中选择和验证参考基因，研究对象是在被球孢白僵菌接种和随后被舞毒蛾侵害的情况下，杂交桉树（耐旱且易感舞毒蛾）的杂种。评估了 11 个候选基因的表达水平和稳定性。使用 RefFinder 工具分析稳定性，该工具整合了 geNorm、NormFinder、BestKeeper 和 Delta-Ct 算法。通过对转录因子 EcDREB2（脱水响应元件结合蛋白 2）的表达分析验证了所选的参考基因。对于所有评估的处理，EcPTB、EcPP2A-1 和 EcEUC12 是最佳的参考基因。对于对照植物、母本植物、接种球孢白僵菌的植物、感染舞毒蛾的植物以及接种球孢白僵菌后感染舞毒蛾的植物，EcPTB/EcEUC12/EcUBP6、EcPP2A-1/EcEUC12/EcPTB、EcIDH/EcSAND/Ecα-TUB、EcPP2A-1/Ecα-TUB/EcPTB 和 EcPP2A-1/EcUPL7/EcSAND 是最佳的参考基因组合。在评估的每种实验条件下，最佳确定的参考基因用于对 RT-qPCR 表达数据进行归一化。研究结果强调了进行此类研究以确保相对基因表达分析的可靠性的重要性。此外，本研究的结果可作为未来研究的基础，包括对不同桉树代谢途径的基因表达分析。
