青钱柳实时荧光定量PCR内参基因的筛选

Selection of Reference Genes for Quantitative Real-Time PCR in Cyclocarya paliurus

为筛选适用于青钱柳(Cyclocarya paliurus(Batal.)Iljinskaja)不同组织器官的稳定内参基因,本研究利用青钱柳花芽的转录组测序数据,选取Actin、CYP、EIF、GAPDH、TUB、TEF和18S r RNA共7个看家基因作为候选内参基因;以根、茎、叶、雌花和雄花为材料,通过实时荧光定量PCR(RT-qPCR)技术对各候选内参基因进行扩增。结合Norm Finder、ge Norm和BestKeeper等软件分析各候选基因的表达稳定性,用RefFinder综合分析获得适宜的内参基因。结果表明,不同组织中各内参基因表达稳定性具有一定差别,稳定性综合排序从高到低为EIF>18S r RNA>TEF>GAPDH>CYP>Actin>TUB。但由于EIF较18S r RNA的CT值差异较大,即18S r RNA在各组织中的表达量更高,因此本研究确定18S r RNA为青钱柳荧光定量分析的最适内参基因,为后续相关基因功能性研究奠定理论基础。

In order to select the stable reference genes suitable for various tissues of Cyclocarya paliurus(Batal.)Iljinskaja,7 housekeeping genes,i.e.Actin,CYP,EIF,GAPDH,TUB,TEF and 18 S rRNA were screened as candidate reference genes for Quantitative real-time PCR(RT-qPCR),based on transcriptome data of floral buds in C.paliurus.Total RNA extracted from roots,stems,leaves,female flowers,and male flowers of C.paliurus were amplified by real-time quantitative RT-qPCR.Then,the expression stability of each reference gene was analyzed by NormFinder,ge Norm and Bestkeeper,and the suitable reference gene(s)were selected comprehensively based on RefFinder.The results showed that the expression stability of each candidate reference gene varied depending on various tissues of C.paliurus,and the comprehensive ranking of expression stability was EIF>18 S rRNA>TEF>GAPDH>CYP>Actin>TUB.Nevertheless,the expression level of 18 S rRNA was higher than EIF in each tissue,so the 18 S r RNA was identified as the preferred reference gene for RT-qPCR analysis in C.paliurus,that could provide the theory basis for subsequent studies on related genes expression.