艾Artemisia argyi实时荧光定量PCR内参基因筛选  

Screening of reference genes for quantitative real-time PCR in Artemisia argyi

作　　者：易小哲 邬兰[2,3] 向丽[2,3] 王梦月 陈士林 师玉华[2,3] 刘霞[1] 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)

机构地区：[1]武汉理工大学化学化工与生命科学学院,湖北武汉430070 [2]中国中医科学院中药研究所中药鉴定与安全性评估北京市重点实验室,北京100700 [3]中国中医科学院中药研究所青蒿素研究中心,北京100700

出　　处：《中国中药杂志》2022年第3期659-667,共9页China Journal of Chinese Materia Medica

基　　金：中国中医科学院优秀青年科技人才(创新类)培养专项(ZZ13-YQ-101,ZZ13-YQ-106-C1);武汉理工大学专业学位硕士研究生团队指导项目(201701);国家自然科学基金青年基金项目(81903758)。

摘　　要：艾叶是中国传统中药,在药用和灸用产品的市场需求日益增加,具有重要的药用价值和经济价值。筛选稳定可靠的qRT-PCR内参基因是开展艾的基因表达差异分析研究的必要前提。该研究从艾的转录组数据中选取Actin、18s、EF-1α、GAPDH、SAND、PAL、TUA、TUB 8个常用的内参基因作为候选基因,利用实时荧光定量技术(qRT-PCR)检测各个候选基因在艾的不同组织(根、茎、叶)中和茉莉酸甲酯(MeJA)处理后不同时间点(0、4、8、12 h)的表达水平,然后应用geNorm、NormFinder、BestKeeper、ΔCT和RefFinder综合分析候选内参基因表达的稳定性,结果表明在艾的不同组织中和MeJA处理后表达最稳定的内参基因均是Actin,SAND次之。进一步检测萜类合成酶途径DXS和MCT基因在艾的不同组织中和MeJA处理后的表达水平,结果表明以Actin和SAND作为内参基因计算出的DXS、MCT的表达趋势一致,验证了筛选结果的可靠性。综上,该研究发现Actin是最适合艾的不同组织和MeJA处理后基因表达研究的内参基因,将为艾叶的基因表达分析提供科学参考,同时为艾叶品质形成的分子机制研究奠定基础。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.

关 键 词：艾 艾叶 实时荧光定量PCR 内参基因 

分 类 号：R284.1[医药卫生—中药学]