Reference Gene Selection for Real-time Quantitative PCR Normalization in Tree Peony (Paeonia suffruticosa Andr.) | 牡丹实时定量PCR分析中内参基因的选择

Chinese. 实时荧光定量PCR(qPCR)是目前研究基因定量表达的重要方法，根据特定实验材料及条件选择qPCR分析中合适的内参基因对于准确校正目的基因的表达至关重要。本研究以牡丹 (Paeonia suffruticosa Andr.) 不同组织(根、茎、叶和花瓣)、切花开放不同时期及不同处理(乙烯或葡萄糖处理)的花瓣为材料，利用qPCR技术探讨了5种常用看家基因：β-微管蛋白基因(β-tubulin)、泛素基因(ubiquitin)、甘油醛-3-磷酸-脱氢酶基因(GAPDH)、肌动蛋白基因(actin)及18S核糖体RNA(18S rRNA)的表达情况，各看家基因均能特异扩增并显示较高的扩增效率。经geNorm和NormFinder程序统计学计算处理，综合分析结果显示，在牡丹不同组织或切花开放不同时期花瓣中，ubiquitin表达最为稳定；在不同处理的切花花瓣中，GAPDH表达最为稳定，二者分别适宜作为相应条件下的内参基因。本研究认为，各实验条件下使用ubiquitin和GAPDH两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。本研究结果对牡丹中关键基因的定量表达分析具有重要的实用价值。

English. Real-time quantitative PCR(qPCR) has been widely used in gene expression analysis to date, and selection of suitable reference genes for qPCR according to specific experimental materials or conditions is very important for accurate normalization of target gene expression. However, only a few studies on reference genes have been done in plants. The present research here was conducted to identify appropriate reference gene(s) for normalization in future expression studies on tree peony (Paeonia suffruticosa Andr.). In this study, expression of five commonly used housekeeping genes such as β-tubulin, ubiquitin, glyceraldehyde- 3-phosphate dehydrogenase(GAPDH), Actin and 18S ribsomal RNA(18S rRNA) were assessed by qPCR in various tissues(roots, stems, leaves and petals) of tree peony and the petals of this cut flower during different opening phases or under different treatments(with ethylene or glucose). All the candidate reference genes could amplify specifically with high efficiency in qPCR reaction. To determine the expression stability of these genes, qPCR data were determined by two commonly used applets(geNorm and NormFinder), which produced highly comparable results depending on the experimental parameters. According to the analysis of geNorm program, the widely used reference genes ubiquitin and GAPDH were top ranked under all above conditions, which demonstrated they were both in the highest stability, whereas others including β-tubulin, actin and 18S rRNA were poorly ranked. Similarly, the results from NormFinder showed that ubiquitin was the most stable gene in the first two experimental conditions, while GAPDH was the most stable one in the petals of cut flower. As a result, for various tissues, as well as petals at different opening stages of tree peony cut flower, ubiquitin could be chosen as the proper reference gene, while compared to differences of gene expression in petals of this cut flower among diverse treatments, GAPDH was considered as the most appropriate reference gene. Besides, given using a normalization factor that was the combination of multiple housekeeping genes, the analysis recommended two most suitable reference genes (ubiquitin and GAPDH) were enough to achieve more accurate and reliable results among all investigated experimental conditions. The present study has provided an important reference for analysis the expression of critical genes in tree peony.