荷花花瓣着色过程实时荧光定量PCR内参基因的筛选及验证. (Chinese)

[Objectives] Selection of reliable reference genes is the primary step for accurate results of quantitative real-time PCR (RT-qPCR) analysis. The present study here was conducted to identify appropriate reference genes for normalization in future expression studies on lotus (Nelumbo nucifera). [Methods] Expression of 8 commonly used housekeeping genes (ACT, EF1α, GAPDH, FPGS, TUA, LEU, CUL and TRY) was detected by RT-qPCR in petals from lotus cultivars with diverse flower colors and at different developmental stages in this study. Expression stabilities of these housekeeping genes were evaluated by geNorm, NormFinder and Bestkeeper, respectively. Furthermore, relative expression levels of chalcone synthase (CHS) were normalized to the 8 candidate reference genes, which would be used to confirm their stability. [Results] geNorm analysis indicated that ACT and EF1α were both the most stable genes in diverse flower color cultivars and during different developmental stages. According to the analysis of NormFinder and BestKeeper, EF1α and ACT were in the high stability in diverse flower color cultivars. During different flower developmental stages, ACT was the most stable gene and EF1α also top ranked by NormFinder, while EF1α was the most stable gene but ACT was poorly ranked by BestKeeper. Meanwhile, GAPDH and TRY were not suitable for reference. Although different algorithms of three softwares might lead to the inconsistent results, EF1α and ACT were considered to be the most stable genes, which was confirmed by the expression analysis of CHS. [Conclusions] For lotus cultivars with diverse flower colors and at different developmental stages, two most suitable reference genes (EF1α and ACT) are enough to achieve more accurate and reliable results. The present study has provided an important reference for analysis the expression of critical genes in lotus during petal coloration. [ABSTRACT FROM AUTHOR]

[目的]选择稳定的内参基因是准确分析实时荧光定量PCR（RT-qPCR）结果的重要前提，本文旨在筛选荷花花瓣着色过程中稳定的内参基因，使目标基因的定量更加准确。[方法]以荷花4种不同花色（红、黄、粉、白）品种、不同花发育期（蕾期、初花期、盛花期、末花期）的花瓣为试材，利用RT-qPCR技术检测8个常用看家基因（ACT、EF1α、GAPDH、FPGS、TUA、LEU、CUL和TRY）的表达水平，并结合geNorm、NormFinder和BestKeeper软件对其表达稳定性进行评价。为了进一步验证8个候选基因的稳定性，分别以它们作为内参基因，检测目的基因查耳酮合成酶基因（CHS）的表达。[结果]geNorm软件分析表明，不同荷花花色品种及不同花发育期间，EF1α和ACT表达稳定性最好。NormFinder和BestKeeper软件显示在不同花色品种中，EF1a和ACT的表达均较稳定；在不同花发育期间，NormFinder软件分析显示ACT表达最稳定，EF1α稳定性也相对较高，而BestKeeper软件分析显示EF1α表达最稳定，但ACT稳定性较差。同时研究发现，GAPDH和TRY在所有样品中稳定性都较差。不同软件之间结果的差异可能是由算法不同造成。拟定EF1α和ACT为最适的内参基因，进一步利用CHS的表达分析验证了EF1α和ACT的稳定性。[结论]在荷花不同花色品种及不同花发育期间，使用EF1α和ACT 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。本研究结果对荷花花瓣着色过程中关键基因表达的RT-qPCR分析具有重要的实用价值。 [ABSTRACT FROM AUTHOR]

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