镉胁迫下草果实时荧光定量PCR内参基因筛选

Selection of Reference Gene for Quantitative Real-Time PCR in Amomum tsaoko under Cadmium Stress

为筛选镉胁迫下草果不同组织中稳定表达的内参基因,采用实时荧光定量PCR法,以50μmol/L镉溶液培养的草果幼苗为试验材料,对GAPDH、tubulin、60S、actin、EIF、EIF2α、UBC、UBCE2、UBQ共9个看家基因在草果根、茎、叶中的表达情况进行检测,并采用geNorm、NormFinder和BestKeeper软件及在线工具RefFinder,对9个候选内参基因的表达稳定性进行分析。结果表明,各候选内参基因在草果根、茎、叶中的表达丰度存在较大差异,Ct值为15~28,其中UBQ的Ct值最小,其基因表达丰度较高。geNorm、NormFinder和BestKeeper 3个软件的分析结果略有不同,RefFinder综合分析结果显示EIF是最稳定表达的内参基因。本研究确定镉胁迫下草果RT-qPCR分析的最适内参基因为EIF,对今后镉胁迫下草果相关基因的表达分析奠定基础。

In order to screen the stable internal reference genes of Amomum tsaoko under cadmium stress,9 housekeeping genes(GAPDH,tubulin,60S,actin,EIF,EIF2α,UBC,UBCE2 and UBQ)were screened and the expression of them in roots,stems and leaves of seedlings of A.tsaoko(treated with 50μmol/L CdCl2)was assessed by the method of real-time quantitative PCR(RT-qPCR).Three softwares(geNorm,NormFinder and BestKeeper)and the RefFinder online tool were used to analyze the expression stability of 9 candidate genes.The results showed that the expression abundance of the candidate internal reference genes is quite different.The Ct value was between 15~28,and the Ct value of UBQ was the smallest,the expression abundance of it was higher.The analysis results of geNorm,NormFinder and BestKeeper were slightly different.EIF was considered as the most stably expressed internal reference gene by the comprehensive analysis of RefFinder.In this study,we determined that EIF was the most suitable internal reference gene for RT-qPCR analysis of A.tsaoko under cadmium stress.Our study provided a theoretical foundation for the expression analysis of related genes in A.tsaoko under cadmium stress.