何首乌实时荧光定量PCR内参基因筛选  

Reference gene screening for Real-time quantitative PCR in Polygonum multiflorum

作　　者：王浩 蔡启忠 刘露 杨全 周良云 WANG Hao;CAI Qi-zhong;LIU Lu;YANG Quan;ZHOU Liang-yun(Guangdong Engineering Research Center of Good Agricultural Practice,Comprehensive Development for Cantonese Medicinal Materials,Guangzhou Comprehensive Experimental Station of National Industrial Technology System for Chinese Materia Medica,Key Laboratory of State Administration of Traditional Chinese Medicine for Production,Development of Cantonese Medicinal Materials,School of Traditional Chinese Medicine,Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]广东药科大学中药学院国家中医药管理局岭南药材生产与开发重点研究室国家中药材产业技术体系广州综合试验站广东省南药规范化种植与综合开发工程技术研究中心,广东广州510006

出　　处：《中国中药杂志》2021年第1期80-85,共6页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2017YFC1700704);2017年广东省岭南中药材保护资金专项(粤财社[2017]60号);广东省普通高校青年创新人才类项目(2018KQNCX133);广东省科技项目(2017A020213023)。

摘　　要：为筛选适宜何首乌Polygonum multiflorum不同组织基因表达分析的内参基因,从何首乌转录组数据中选取Actin,GAPDH,SAND,PP2A,TIP41常用的5个候选内参基因,设计特异性引物。通过实时荧光定量PCR,利用geNorm,NormFinder,Best-Keeper,Delta CT以及RefFinder等方法对5个候选内参基因在何首乌不同组织中的表达稳定性进行分析,为何首乌基因差异表达研究提供可靠的内参基因,以确保分析结果的可靠性。结果表明,各候选内参基因在何首乌不同组织中表达水平和稳定性有较大差异,其中对各候选内参基因的表达水平(Ct)的分布分析显示,以Actin和GAPDH基因在各组织中表达水平相对较高,而SAND,PP2A,TIP41表达水平较低;通过不同方法对各候选内参基因稳定性进行分析,geNorm分析结果以PP2A,GAPDH表达最为稳定,SAND表达稳定性最差,NormFinder和Delta CT法均以PP2A稳定性最高,SAND稳定性最低,而BestKeeper分析以Actin最稳定。通过RefFinder对候选基因稳定性进行综合评价分析得出以PP2A基因稳定性最高,其后依次为GAPDH,Actin,TIP41,SAND,以SAND基因稳定性最差。因此,PP2A基因为何首乌不同组织基因表达分析比较理想的内参基因。To select suitable references gene of Polygonum multiflorum for gene expression analysis in different tissues,five candidate reference genes like Actin,GAPDH,SAND,PP2A,TIP41 were selected from the transcriptome data of P.multiflorum,then the specific primers were designed.The expression stability of the five reference genes in different tissues of P.multiflorum was analyzed by Real-time quantitative PCR through avilable analysis methods such as geNorm,NormFinder,BestKeeper,Delta CT and RefFinder,to ensure the reliability of the analysis results.The results showed that there were significant differences in the expression levels and stability of candidate genes in different tissues of P.multiflorum.Ct distribution analysis of the expression levels of candidate genes showed that the expression levels of Actin and GAPDH genes were relatively high in different tissues,while the expression levels of SAND,PP2A and TIP41 were lower.The stability of each candidate gene was analyzed by different methods.The results of geNorm analysis showed that the expression of PP2A and GAPDH was the most stable,the expression stability of SAND was the worst,the stability of PP2A was the highest in both NormFinder and Delta CT,the stability of SAND was the lowest,and the stability of Actin was the most stable in BestKeeper analysis.Through the comprehensive evaluation and analysis of the stability of candidate genes by RefFinder,it is con-cluded that the stability of PP2A gene is the highest,followed by GAPDH,Actin,TIP41,SAND,and SAND gene is the worst.Therefore,the PP2A gene is an ideal reference gene for the analysis of gene expression in different tissues of P.multiflorum.

关 键 词：何首乌 内参基因 实时荧光定量PCR 

分 类 号：S567.239[农业科学—中草药栽培]