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选择合适的内参基因是保证实时荧光定量PCR分析(quantitative Real-time PCR,q RT-PCR)准确性的前提条件。本研究选取了‘凤丹’(Paeonia ostii‘Feng Dan’)不同颜色变异个体的花瓣、不同组织(茎、叶片、花萼、花瓣)、不同发育时期的种子及‘凤丹’、西施’(Paeonia ostii‘Xi Shi’)、‘雀好’(Paeonia ostii‘Que Hao’)3个品种花发育不同时期的花瓣共33份材料,利用q RT-PCR检测了来自于牡丹(Paeonia suffruticosa)转录组数据库的16个候选内参基因AMPDS、ARFA1C、ERVTP、FCF2PRP、GATA24、GRF9、MBF1A、MHCRP、PAD1FP、PIN1AT、PKSP、PP2A、PP2CFP、PTRP、PUF1639、RPS9以及6个看家基因ARP2、IWS1、GAPDH、TBCC、TUA5和UBC28在四组实验条件下的表达水平,并利用ge Norm和Norm Finder程序对各基因进行稳定性评价。综合分析结果表明,在不同组的实验条件下最适合作为牡丹基因表达的内参基因各不相同。在以凤丹、西施和雀好3个品种花发育不同阶段花瓣为材料时,最稳定的内参基因为ERVTP、PP2CFP;在以不同花色梯度凤丹花瓣为材料时,最稳定的内参基因为RPS9和ARFA1C;在以不同组织(茎、叶、花萼和花瓣)为材料时,AMPDS和PUF1639表达变化最为稳定;对于不用发育阶段的种子而言,RPS9和PUF1639为最适合的内参基因。另外,PUF1639、MBF1A、PP2CFP和RPS9四个新筛选的内参基因较所选传统看家基因表现更优。研究结果表明,通过牡丹转录组数据来筛选稳定表达的内参基因是可靠且高效的。本研究为牡丹基因表达分析提供了稳定可靠的内参基因。
Choosing the appropriate internal reference gene is a prerequisite for ensuring the accuracy of quantitative RT-PCR (q RT-PCR). In this study, we selected the petals, tissues (stems, leaves, calyx, petals), seeds at different developmental stages and ’Paeonia’ (Paeonia ’Feng Dan’ A total of 33 petals of three varieties of flower, “ostii’Xi Shi” and “Paeonia ostii’Que Hao”, were developed. Paeonia suffruticosa transcriptome database was detected by q RT-PCR 16 candidate internal reference genes AMPDS, ARFA1C, ERVTP, FCF2PRP, GATA24, GRF9, MBF1A, MHCRP, PAD1FP, PIN1AT, PKSP, PP2A, PP2CFP, PTRP, PUF1639, RPS9 and six housekeeping genes ARP2, IWS1, GAPDH, , TUA5 and UBC28 under the four experimental conditions, and the stability of each gene was evaluated by ge Norm and Norm Finder programs. The results of comprehensive analysis showed that the most suitable internal control genes for peony gene expression under different experimental conditions were different. The most stable internal reference gene was petunia, and the most stable internal reference gene was ERVTP and PP2CFP. The most stable internal reference gene was RPS9 And ARFA1C. The most stable changes of AMPDS and PUF1639 expression were observed in different tissues (stem, leaf, calyx and petals). For seedless stage, RPS9 and PUF1639 were the most suitable internal control genes. In addition, the four newly screened internal control genes of PUF1639, MBF1A, PP2CFP and RPS9 performed better than the selected traditional housekeeping genes. The results show that it is reliable and efficient to screen the stable expression of the reference genes through the peony transcriptome data. This study provided a stable and reliable reference gene for peony expression analysis.