毛竹qRT-PCR分析中内参基因的选择

摘要

实时荧光定量PCR(quantitative Real-time PCR,qRT-PCR)是目前研究基因表达的常用方法,而在特定的实验材料及条件下选择合适的内参基因是准确分析目标基因相对表达量的首要条件.本研究以毛竹(Phyllostachys edulis)的根、茎、叶和笋等器官和组织为实验材料,通过qRT-PCR技术对5种常用内参基因:甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)、肌动蛋白(actin,ACT)、热休克蛋白(heat shock protein,HSP)、18S核糖体RNA(18S ribosomal RNA,18S rRNA)和真核起始因子4α(eukaryotic initiation factor 40,eIF-4α)在毛竹不同器官和组织中的表达情况进行了分析.经GeNorm、NormFinder和BestKeeper 3个软件对比分析发现,对5种常用内参基因而言,在利用qRT-PCR分析比较毛竹不同叶片之间的基因表达差异时,可选取eIF-4α作为校正内参基因;比较笋不同部位之间的基因表达差异时,可选取18S rRNA或ACT作为校正内参基因;而比较毛竹根、茎、叶不同器官组织分化中的基因表达差异时,可选取18S rRNA作为校正内参基因.而上述发现与多数文献中把ACT作为毛竹唯一内参基因的研究不一致.本研究为在毛竹qRT-PCR分析中选择合适的内参基因提供了理论依据.%Quantitative Real-time PCR (qRT-PCR) is a commonly-used method for studying gene expression currently,in which choosing appropriate reference genes for specific materials or particular conditions is a prerequisite for accurately analyzing relative expressions of target genes.This study took the roots,stems,leaves and bamboo shoots of Phyllostachys edulis as experimental materials and analyzed the expressions of 5 kinds of widely used reference genes-glyceraldehyde-3-phosphate dehydrogenase (GAPDH),actin (ACT),heat shock protein (HSP),18S ribosomal RNA (18S rRNA) and eukaryotic initiation factor 4α (eIF-4α) in different tissues or organs by the aid of the qRT-PCR.Three programs including GeNorm,NormFinder and BestKeeper were used to determine the expression stability of these reference genes.The results showed that the most stable reference genes of P.edulis had many differences in the various experimental conditions.After the comparative analysis using GeNorm,NormFinder and BestKeeper,eIF-4α had the highest expression stability and was viewed as the corrective reference gene when the genes expression in different leaves of P.edulis were analyzed and compared by qRT-PCR.When the differences of genes expression were compared in different parts of bamboo shoots,ACT or 18S rRNA could be chosen as the corrective reference gene in different parts of bamboo shoots.In order to compare the differences of gene expression among the roots,stems and leaves of P.edulis,the expression stability of 18S rRNA was the best on GeNorm and BestKeeper and was second on NormFinder.Therefore,18S rRNA could be chosen as corrective reference gene in different tissues of P.edulis.These findings were inconsistent with those of previous studies,which ACT was used as the only one reference gene.This study suggested that suitable reference genes should be selected on the basis of specific requirements,experiment conditions,and the characteristics of experimental material in practical applications.The results of this study would provide a theoretical basis for selecting the appropriate reference gene of P.edulis in the analysis of qRT-PCR.