摘要
筛选合适的内参基因是应用实时荧光定量PCR(qRT-PCR)技术分析基因表达的前提。为黄草乌Aconitum vilmori-nianum中基因表达分析研究提供可靠的内参基因,基于黄草乌转录组数据,筛选出19个候选内参基因(ACT1、ACT2、ACT3、aTUB1、aTUB2、bTUB、18S rRNA、UBQ、eIF2、eIF3、eIF4、eIF5、CYP、GAPDH1、GAPDH2、PP2A1、PP2A2、ACP、EF1α),用qRT-PCR技术检测各候选内参基因在黄草乌根、茎、叶和花4个器官中的表达情况,Ct值分析结果表明,18S rRNA在各器官中表达丰度过高,GAPDH2的波动范围较大,二者均不宜作为黄草乌的内参基因。对其他候选基因的表达稳定性进行分析,geNorm和NormFinder分析结果基本一致,稳定性排名前三的内参基因均为PP2A1、EF1α和CYP,BestKeeper分析结果表明,稳定性排名前三的内参基因为EF1α、ACT3和PP2A1。不同软件分析得出的候选内参基因表达稳定性排名存在差异,因此,采用几何平均值法综合分析黄草乌各候选内参基因的表达稳定性,结果表明,PP2A1、EF1α和ACT3的稳定性最佳。基于geNorm、NormFin-der、BestKeeper和几何平均值法综合分析结果,PP2A1和EF1α在黄草乌不同器官中的表达最稳定,是黄草乌不同器官基因表达分析的最佳内参基因。
Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis.To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum,this study selected 19 candidate re-ference genes(ACT1,ACT2,ACT3,aTUB1,aTUB2,bTUB,18S rRNA,UBQ,eIF2,eIF3,eIF4,eIF5,CYP,GAPDH1,GAPDH2,PP2A1,PP2A2,ACP,and EF1α)based on the transcriptome data of A.vilmorinianum.qRT-PCR was conducted to profile the expression of these genes in the root,stem,leaf,and flower of A.vilmorinianum.The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes.NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1,EF1α,and CYP as the highly stable ones.However,BestKeeper suggested EF1α,ACT3,and PP2A1 as the top stable genes.In view of the different results from different softwares,the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes,the results of which indicated that PP2A1,EF1α,and ACT3 were the most stable.Based on the comprehensive analysis results of geNorm,NormFinder,BestKeeper,and geometric mean method,PP2A1 and EF1αpresented the most stable expression in different organs of A.vilmorinianum.PP2A1 and EF1αwere the superior reference genes for gene expression profiling in different organs of A.vilmorinianum.
作者
曾礼芳
李国栋
王宝婕
王一博
程景平
曹小青
管丽娜
朱灵英
钱子刚
马晓惠
ZENG Li-fang;LI Guo-dong;WANG Bao-jie;WANG Yi-bo;CHENG Jing-ping;CAO Xiao-qing;GUAN Li-na;ZHU Ling-ying;QIAN Zi-gang;MA Xiao-hui(Yunnan Key Laboratory of Southern Medicinal Resource,School of Chinese Materia Medica,Yunnan University of Chinese Medicine,Kunming 650500,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2021年第12期3116-3122,共7页
China Journal of Chinese Materia Medica
基金
中央本级重大增减支项目(2060302)。
关键词
黄草乌
内参基因
实时荧光定量PCR
Aconitum vilmorinianum
reference gene
quantitative Real-time PCR
