Yi Xiao-Zhe, Wu Lan, Xiang Li, Wang Meng-Yue, Chen Shi-Lin, Shi Yu-Hua, Liu Xia
School of Chemistry, Chemical Engineering and Life Sciences, Wuhan University of Technology Wuhan 430070, China.
Key Laboratory of Beijing for Identification and Safety Evaluation of Chinese Medicine, Institute of Chinese Meteria Medica, China Academy of Chinese Medical Sciences Beijing 100700, China Artemisinin Research Center, Institute of Chinese Meteria Medica, China Academy of Chinese Medical Sciences Beijing 100700, China.
Zhongguo Zhong Yao Za Zhi. 2022 Feb;47(3):659-667. doi: 10.19540/j.cnki.cjcmm.20210919.101.
Artemisia Argyi Folium, a traditional Chinese medicine of important medicinal and economic value, sees increasing demand in medicinal and moxibustion product market. Screening stable and reliable reference genes for quantitative real-time PCR(qRT-PCR) is a prerequisite for the analysis of gene expression in Artemisia argyi. In this study, eight commonly used reference genes, Actin, 18s, EF-1α, GAPDH, SAND, PAL, TUA, and TUB, from the transcriptome of A. argyi, were selected as candidate genes. The expression of each gene in different tissues(roots, stems, and leaves) of A. argyi and in leaves of A. argyi after treatment with methyl jasmonate(MeJA) for different time(0, 4, 8, 12 h) was detected by qRT-PCR. Then, geNorm, NormFinder, BestKeeper, ΔCT, and RefFinder were employed to evaluate their expression stability. The results demonstrated that Actin was the most stable reference gene in different tissues and in leaves treated with MeJA, and coming in the second was SAND. Furthermore, the expression of DXS and MCT which are involved in terpenoid backbone biosynthesis was detected in different tissues and after MeJA treatment. The results showed that the expression patterns of DXS and MCT in different tissues and under MeJA treatment calculated with Actin and SAND as internal reference genes were consistent, which validated the screening results. In conclusion, Actin is the most suitable reference gene for the analysis of gene expression in different tissues of A. argyi and after MeJA treatment. This study provides valuable information for gene expression analysis in A. argyi and lays a foundation for further research on molecular mechanism of quality formation of Artemisia Argyi Folium.
艾叶是一种具有重要药用和经济价值的传统中药，在药用和艾灸产品市场上的需求日益增加。筛选用于定量实时PCR（qRT-PCR）的稳定可靠的内参基因是分析艾叶基因表达的前提条件。本研究从艾叶转录组中选取了8个常用的内参基因Actin、18s、EF-1α、GAPDH、SAND、PAL、TUA和TUB作为候选基因。通过qRT-PCR检测了每个基因在艾叶不同组织（根、茎和叶）以及茉莉酸甲酯（MeJA）处理不同时间（0、4、8、12小时）后的艾叶叶片中的表达情况。然后，使用geNorm、NormFinder、BestKeeper、ΔCT和RefFinder评估它们的表达稳定性。结果表明，Actin是不同组织和经MeJA处理的叶片中最稳定的内参基因，其次是SAND。此外，检测了参与萜类骨架生物合成的DXS和MCT在不同组织以及MeJA处理后的表达情况。结果显示，以Actin和SAND作为内参基因计算得出的DXS和MCT在不同组织以及MeJA处理下的表达模式一致，验证了筛选结果。综上所述，Actin是分析艾叶不同组织以及MeJA处理后基因表达最适合的内参基因。本研究为艾叶基因表达分析提供了有价值的信息，为进一步研究艾叶品质形成的分子机制奠定了基础。
