桂花组织基因表达中荧光定量PCR内参基因的筛选

Reference gene selection for quantitative real-time polymerase chain reaction(qRT-PCR) normalization in the gene expression of sweet osmanthus tissues

为了筛选桂花Osmanthus fragrans不同组织基因表达分析中适合的内参基因,以桂花‘堰虹桂’O.fragrans‘YanhongGui’花蕾、盛开花序、嫩叶、成熟叶和1年生茎等5个不同组织为材料,利用实时荧光定量聚合酶链式反应(qRT-PCR)检测7个候选内参基因的表达水平,并利用geNorm,NormFinder和BestKeeper软件对各候选内参基因的表达稳定性进行评价,最后利用桂花不同组织中OfCRTISO1基因相对表达水平验证筛选的内参基因的可靠性。结果表明:综合3个软件的评价排序,确定不同组织中最佳内参基因为OfRAN1和OfUBC2,而Of18S是最差内参基因。OfCRTISO1基因相对表达水平分析证实,不同组织中qRT-PCR分析使用OfRAN1和OfUBC2等2个表达最稳定的基因组合,即可获得更为精确的基因表达结果。旨在为桂花组织间重要基因的定量表达分析提供科学依据。

To select the suitable reference gene(s) for gene expression analysis in different tissues of Osmanthus fragrans, five tissues: buds, fully-opened inflorescences, young leaves, adult leaves, and one-year-old stems,from O. fragrans ‘Yanhong Gui' were used as materials to detect the expression levels of seven candidate reference genes by quantitative real-time polymerase chain reaction(q RT-PCR). Then, an aggregated analysis of gene expression stability for these candidates was conducted using three software programs: ge Norm,Norm Finder, and Best Keeper. Finally, the relative expression level of OfCRTISO1 in different tissues was analyzed to verify the reference genes selected in this study. Results of the aggregated analysis showed that OfRAN1 and OfUBC2 were the best reference genes from the different tissues, and Of18 S was the worst reference gene. In addition, analysis of the relative expression level for OfCRTISO1 confirmed that OfRAN1 and OfUBC2 were the two most stable reference genes in the q RT-PCR analysis of the tissues, and the application of OfRAN1 and OfUBC2 was enough to achieve more accurate and reliable results in these tissues. Thus, the present study has provided an important reference for analyzing the expression of key genes in different tissues of O. fragrans.