Bharati Rohit, Sen Madhab Kumar, Kumar Ram, Gupta Aayushi, Sur Vishma Pratap, Melnikovová Ingrid, Fernández-Cusimamani Eloy
Department of Crop Sciences and Agroforestry, The Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Kamýcká 129, 165 00 Prague 6, Czech Republic.
Department of Agroecology and Crop Production, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, 165 00 Prague 6, Czech Republic.
Plants (Basel). 2022 Oct 27;11(21):2878. doi: 10.3390/plants11212878.
L. (rosemary) is known to have a wide range of pharmacological effects including antidiabetic, anticarcinogenic, and antitumorigenic properties owing to its secondary metabolites. Studies aiming to elevate these metabolites have utilized various elicitors and stresses under in vitro conditions, although underlying molecular mechanisms remain unexplored. Gene expression studies using RT-qPCR might provide valuable information regarding how plant and plant cells interact and perceive various treatments and elicitors. However, despite being able to calculate accurate fold changes, the accuracy of the RT-qPCR data highly depends on the expression of reference genes. To the best of our knowledge, there is no information available on the stable reference genes in rosemary under in vitro conditions. Thus, in this paper, we assessed the stability of seven commonly used reference genes under different elicitor and stress conditions using RT-qPCR. Thereafter, the five most commonly used software and algorithms (comparative ΔCt, BestKeeper, NormFinder, geNorm, and RefFinder) were used to rank the candidates based on their expression stabilities. In conclusion, we recommend using a combination of , and to normalize the gene expression experiments in rosemary under in vitro conditions. The selected reference genes were verified using , a pharmacologically important gene, whose expression might alter under nanoparticle treatment. Additionally, reference genes for several plant tissues, elicitors, and stresses are also proposed. The conclusions obtained from this current study will accelerate the future molecular work in and other related species.
迷迭香已知具有广泛的药理作用，包括由于其次生代谢产物而具有的抗糖尿病、抗癌和抗肿瘤特性。旨在提高这些代谢产物的研究在体外条件下利用了各种诱导子和胁迫，尽管潜在的分子机制仍未被探索。使用RT-qPCR进行的基因表达研究可能会提供有关植物和植物细胞如何相互作用以及感知各种处理和诱导子的有价值信息。然而，尽管能够计算准确的倍数变化，但RT-qPCR数据的准确性高度依赖于参考基因的表达。据我们所知，在体外条件下，关于迷迭香中稳定参考基因的信息尚无可用。因此，在本文中，我们使用RT-qPCR评估了七个常用参考基因在不同诱导子和胁迫条件下的稳定性。此后，使用五个最常用的软件和算法（比较ΔCt、BestKeeper、NormFinder、geNorm和RefFinder）根据候选基因的表达稳定性对其进行排名。总之，我们建议使用 、 和 的组合来标准化体外条件下迷迭香的基因表达实验。使用 （一个药理学上重要的基因）对所选参考基因进行了验证，其表达在纳米颗粒处理下可能会改变。此外，还提出了几种植物组织、诱导子和胁迫的参考基因。从本研究中获得的结论将加速未来在 及其他相关物种中的分子研究工作。
