Isatis indigotica, a traditional Chinese medicine, produces a variety of active ingredients. However, little is known about the key genes and corresponding expression profiling involved in the biosynthesis pathways of these ingredients. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful, commonly-used method for gene expression analysis, but the accuracy of the quantitative data produced depends on the appropriate selection of reference genes. In this study, the systematic analysis of the reference genes was performed for quantitative real-Time PCR normalization in I. indigotica. We selected nine candidate reference genes, including six traditional housekeeping genes (ACT, α-TUB, β-TUB, UBC, CYP, and EF1-α), and three newly stable internal control genes (MUB, TIP41, and RPL) from a transcriptome dataset of I. indigotica, and evaluated their expression stabilities in different tissues (root, stem, leaf, and petiole) and leaves exposed to three abiotic treatments (low-nitrogen, ABA, and MeJA) using geNorm, NormFinder, BestKeeper, and comprehensive RefFind algorithms. The results demonstrated that MUB and EF1-α were the two most stable reference genes for all samples. TIP41 as the optimal reference gene for low-nitrogen stress and MeJA treatment, while ACT had the highest ranking for ABA treatment and CYP was the most suitable for different tissues. The results revealed that the selection and validation of appropriate reference genes for normalizing data is mandatory to acquire accurate quantification results. The necessity of specific internal control for specific conditions was also emphasized. Furthermore, this work will provide valuable information to enhance further research in gene function and molecular biology on I. indigotica and other related species.

中文翻译：

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板蓝根财富中基因表达的定量实时PCR标准化参考基因的选择。

板蓝根（Itistis indigotica）是一种传统中药，可产生多种有效成分。然而，对于这些成分的生物合成途径中涉及的关键基因和相应的表达谱了解甚少。定量实时聚合酶链式反应（qRT-PCR）是用于基因表达分析的强大，常用的方法，但所产生的量化数据的准确性取决于参考基因的适当的选择。在这项研究中，对参考基因进行了系统分析，以进行靛蓝假单胞菌定量实时PCR标准化。我们选择了9个候选参考基因，包括6个传统管家基因（ACT，α-TUB，β-TUB，UBC，CYP和EF1-α）和3个新近稳定的内部控制基因（MUB，TIP41和RPL）。靛蓝虫的转录组数据集 并使用geNorm，NormFinder，BestKeeper和全面的RefFind算法评估了它们在不同组织（根，茎，叶和叶柄）和暴露于三种非生物处理（低氮，ABA和MeJA）中的叶子的表达稳定性。结果表明，MUB和EF1-α是所有样品中两个最稳定的参考基因。TIP41是低氮胁迫和MeJA处理的最佳参考基因，而ACT在ABA处理中排名最高，而CYP最适合不同组织。结果表明，选择和验证合适的参考基因以标准化数据对于获得准确的定量结果是必不可少的。还强调了针对特定条件进行特定内部控制的必要性。此外，