牡丹不同发育阶段种子和花瓣组织实时荧光定量PCR中内参基因的挖掘与筛选

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摘要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, qRT-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qRT-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geNorm和NormFinder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。

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	刘洪峰
	高乐旋
	胡永红

关键词 ：牡丹, 实时荧光定量PCR, 内参基因

Abstract：The selection of a suitable reference gene is an important prerequisite for successful gene expression analysis by quantitative Real-time PCR (qRT-PCR). In this study, expression stability of 22 endogenous candidate genes including 16 new candidate reference genes form transcriptome data of tree peony, including AMPDS, ARFA1C, ERVTP, FCF2PRP, GATA24, GRF9, MBF1A, MHCRP, PAD1FP, PIN1AT, PKSP, PP2A, PP2CFP, PTRP, PUF1639, RPS9 and 6 traditional housekeeping genes, including ARP2, IWS1, GAPDH, TBCC, TUA5 and UBC28 were assessed by qRT-PCR in a diverse set of 33 tissue samples representing different floral development stages of ‘Feng Dan’(Paeonia ostii ‘Feng Dan’), ‘Xi Shi’(Paeonia ostii ‘Xi Shi’) and ‘Que Hao’(Paeonia ostii ‘Que Hao’), different color of ‘Feng Dan’, different samples (stems, leaves, sepals and flowers), different development stages of seeds. The results showed that the most suitable candidate reference genes of tree peony had many differences in the various experimental condition groups. Though there were some minor differences, the results of two programs were similar in the same experimental condition group. ERVTP, PP2CFP, FCF2PRP and UBC28 had the highest expression stability in flowers of different floral development stages in Feng Dan, Xi Shi and Que Hao. RPS9 and ARFA1C were the best genes in flowers of different color of Feng Dan. AMPDS, PUF1639 and MBF1A were the most stable reference genes which were suitable for different samples (stems, leaves, sepals, petals). In seeds of different development stages, RPS9 and PUF1639 had the stable expression. Further, four newly selected reference genes (PUF1639, MBF1A, PP2CFP and RPS9) of tree peony were superior to traditional ones in terms of their expression stability. Identifying stably expressed genes for candidate reference genes from transcriptome database was found to be reliable and efficient. The reference genes selected in this study will be helpful to improve the quality of gene expression studies in tree peony.

Key words： Paeonia ostii Quantitative Real-time PCR Reference genes

收稿日期: 2015-03-24 出版日期: 2015-10-11

基金资助:江南牡丹花色素苷形成的分子机理及新花色品种培育;初探O-甲基转移酶（ OMT）家族基因

通讯作者: 胡永红 E-mail: huyonghong@csnbgsh.cn

引用本文:

刘洪峰高乐旋胡永红. 牡丹不同发育阶段种子和花瓣组织实时荧光定量PCR中内参基因的挖掘与筛选[J]. , 2015, 23(12): 1639-1648.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/ 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2015/V23/I12/1639

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