School of Pharmacy, Jiangsu Health Vocational College, Nanjing, Jiangsu, China.
College of Horticulture, Hunan Agricultural University, Changsha, Hunan, China.
Sci Rep. 2021 Sep 23;11(1):18993. doi: 10.1038/s41598-021-98434-6.
Angelica decursiva is one of the lending traditional Chinese medicinal plants producing coumarins. Notably, several studies have focused on the biosynthesis and not the RT-qPCR (quantitative real-time reverse transcription polymerase chain reaction) study of coumarins. This RT-qPCR technique has been extensively used to investigate gene expression levels in plants and the selection of reference genes which plays a crucial role in standardizing the data form the RT-qPCR analysis. In our study, 11 candidate reference genes were selected from the existing transcriptome data of Angelica decursiva. Here, four different types of statistical algorithms (geNorm, NormFinder, BestKeeper, and Delta Ct) were used to calculate and evaluate the stability of gene expression under different external treatments. Subsequently, RefFinder analysis was used to determine the geometric average of each candidate gene ranking, and to perform comprehensive index ranking. The obtained results showed that among all the 11 candidate reference genes, SAND family protein (SAND), protein phosphatase 2A gene (PP2A), and polypyrimidine tract-binding protein (PTBP) were the most stable reference genes, where Nuclear cap binding protein 2 (NCBP2), TIP41-like protein (TIP41), and Beta-6-tubulin (TUBA) were the least stable genes. To the best of our knowledge, this work is the first to evaluate the stability of reference genes in the Angelica decursiva which has provided an important foundation on the use of RT-qPCR for an accurate and far-reaching gene expression analysis in this medicinal plant.
当归属植物是产生香豆素的传统中药之一。值得注意的是，已有多项研究集中于香豆素的生物合成，而非实时荧光定量聚合酶链反应（qRT-PCR）研究。该 qRT-PCR 技术已广泛用于研究植物中的基因表达水平，以及选择在标准化 qRT-PCR 分析数据中起着关键作用的参考基因。在我们的研究中，从当归属的现有转录组数据中选择了 11 个候选参考基因。这里，使用了四种不同的统计算法（geNorm、NormFinder、BestKeeper 和 Delta Ct）来计算和评估不同外部处理下基因表达的稳定性。随后，使用 RefFinder 分析来确定每个候选基因排名的几何平均值，并进行综合指数排名。获得的结果表明，在所有 11 个候选参考基因中，SAND 家族蛋白（SAND）、蛋白磷酸酶 2A 基因（PP2A）和多嘧啶 tract 结合蛋白（PTBP）是最稳定的参考基因，而核帽结合蛋白 2（NCBP2）、TIP41 样蛋白（TIP41）和 Beta-6-微管蛋白（TUBA）是最不稳定的基因。据我们所知，这项工作首次评估了当归属中参考基因的稳定性，为该药用植物中准确而深远的基因表达分析提供了重要基础。
