实时荧光定量PCR分析蒙古韭低温诱导叶片中内参基因的选择

实时荧光定量PCR (real-time fluorescence quantitative PCR,qRT-PCR)是广泛应用于基因表达分析的实验技术。在基因表达分析过程中,选择稳定表达的内参基因对实验结果的准确性非常重要。以低温诱导24 h和72 h蒙古韭(Allium mongolicum)的叶片为材料,无处理0 h叶片为对照,使用荧光定量PCR法分析了Am5S-rRNA、AmActin、AmGAPDH和AmEF1-α4个看家基因的表达情况。通过ge Norm和NormFinder程序分析,发现AmGAPDH稳定性最好,Am5S-r RNA和AmActin次之,AmEF1-α稳定性最差,因此选择AmGAPDH作为蒙古韭基因表达分析的内参基因。本研究通过qRT-PCR方法分析稳定表达的内参基因,对后续蒙古韭低温诱导基因表达分析有重要的意义。

Selection of Reference Genes Using Real-time Quantitative PCR in Low-temperature Induced Leaves of Allium mongolicum

Real-time quantitative PCR is a commonly used technology to analyze the gene expression profile.It is important to select an appropriate reference gene for accuracy of experimental data in gene expression analysis.We used geNorm and NormFinder program to evaluate the gene expression stabilities of four reference genes(Am5 S-rRNA,AmActin,AmGAPDH and AmEF1-α) using low temperature induced leaves at 24 h and 72 h as materials,and 0 hour as control.The results showed that the best reference gene was AmGAPDH,and Am5 S-rRNA and AmActin were second,and the worst stability of AmEF1-α.So AmGAPDH would be used as reference gene in gene expression analysis,and this result has important significance for the subsequent analysis of low-temperature induced gene expression in Allium mogolicum.