毛叶芋兰不同组织qRT-PCR内参基因选择与验证  

Selection and Validation of qRT-PCR Reference Genes in Different Tissues of Nervilia plicata

作　　者：池珈仪 刘舒璇 向思诗 詹若挺[1] 何瑞[1] CHI Jiayi;LIU Shuxuan;XIANG Sishi;ZHAN Ruoting;HE Rui(Research Center of Chinese Herbal Resource Science and Engineering,Guangzhou University of Chinese Medicine/Key Laboratory of Chinese Medicinal Resource from Lingnan,Ministry of Education,Guangzhou University of Chinese Medicine/School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine,Guangzhou,Guangdong 510006,China)

机构地区：[1]广州中医药大学中药资源科学与工程研究中心/广州中医药大学岭南中药资源教育部重点实验室/广州中医药大学中药学院,广东广州510006

出　　处：《热带作物学报》2025年第1期35-43,共9页Chinese Journal of Tropical Crops

基　　金：2022年省级乡村振兴战略专项资金种业振兴项目“广东省南药种业创新园项目”(No.2022-NJS-00-002,粤财农[2022]184号)。

摘　　要：毛叶芋兰(Nervilia plicata)是一种珍稀南药,目前对其有效成分及其分子调控机理鲜有报道。实时荧光定量PCR(real-time quantitative PCR,qRT-PCR)在植物基因表达分析中得到了广泛应用,但在毛叶芋兰中缺少研究,限制了相关工作的开展。在前期工作基础上,本研究从毛叶芋兰转录组数据库中筛选出Actin、GAPDH、TUA、UBC、UBQ、EF-1α、EF-1β、CYP、RPL共9个常见管家基因,以毛叶芋兰叶片、叶柄和球茎3个组织部位为材料,开展qRT-PCR内参基因的筛选工作。经过9个基因在不同组织的表达水平、稳定性和综合分析,筛选出最适合的内参基因,并选择毛叶芋兰花青素合成途径关键基因NpDFR、Np3GT进行验证。结果发现:EF-1α和CYP的表达稳定性相近,整体稳定性最好。2个内参基因单独或共同使用时,NpDFR、Np3GT在不同组织的表达情况与转录组测序结果基本一致,表明其均可用作qRT-PCR的内参基因。本研究结果为进一步开展毛叶芋兰药效相关基因的分子作用机理研究提供依据。Nervilia plicata is a rare southern Chinese herb.The study on the plant at molecular level is hardly seen.Real-time quantitative PCR(qRT-PCR)has been widely used in plant gene expression analysis,but there is a lack of research on it in N.plicata,which limits the progress of related work.Based on the previous researches,nine candidate reference genes,including Actin,GAPDH,TUA,UBC,UBQ,EF-1α,EF-1β,CYP and RPL,were screened out from the transcriptomic sequencing data.In order to select suitable reference gene in N.plicata,qRT-PCR technique was em-ployed to detect the expression levels of the candidate reference genes in leaf,petiole and corm tissues of the plant.Af-ter analyzing the expression levels,the stability and comprehensive analysis of nine genes in different tissues,the most suitable reference gene was obtained.Finally,the most suitable reference gene was validated using NpDFR,Np3GT that related to anthocyanin synthesis.Results showed that the expression stability of EF-1αand CYP was similar,with the best overall stability.When using either EF-1αor CYP,or the combination of them,as reference,the expression patterns of NpDFR and Np3GT in different tissues were consistent with the transcriptome sequencing results.This indicates that both EF-1αand CYP can be used as reference genes in N.plicata for qRT-PCR.This study would provide a basis for fur-ther research on the molecular mechanism of genes related to the pharmacological effects of N.plicata.

关 键 词：毛叶芋兰 内参基因 实时荧光定量PCR 表达稳定性 

分 类 号：S567.2[农业科学—中草药栽培]