[1]王彦杰,陈叶清,薛泽云,等.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[J].南京农业大学学报,2017,40(3):408-415.[doi:10.7685/jnau.201609033]
 WANG Yanjie,CHEN Yeqing,XUE Zeyun,et al.Selection and validation of reference genes for RT-qPCR normalization in lotus(Nelumbo nucifera)during petal coloration[J].Journal of Nanjing Agricultural University,2017,40(3):408-415.[doi:10.7685/jnau.201609033]
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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()
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《南京农业大学学报》[ISSN:1000-2030/CN:32-1148/S]

卷:
40卷
期数:
2017年3期
页码:
408-415
栏目:
OA栏目
出版日期:
2017-05-01

文章信息/Info

Title:
Selection and validation of reference genes for RT-qPCR normalization in lotus(Nelumbo nucifera)during petal coloration
作者:
王彦杰1 陈叶清1 薛泽云1 周华2 金奇江1 徐迎春1
1. 南京农业大学园艺学院, 江苏 南京 210095;
2. 江西省观赏植物遗传改良重点实验室/江西省科学院生物资源研究所, 江西 南昌 330096
Author(s):
WANG Yanjie1 CHEN Yeqing1 XUE Zeyun1 ZHOU Hua2 JIN Qijiang1 XU Yingchun1
1. College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China;
2. Key Laboratory of Horticultural Plant Genetic and Improvement of Jiangxi/Institute of Biology and Resources, Jiangxi Academy of Sciences, Nanchang 330096, China
关键词:
花色荷花实时荧光定量PCR内参基因
Keywords:
flower colorNelumbo nuciferaquantitative real-time PCRreference gene
分类号:
S682.2
DOI:
10.7685/jnau.201609033
摘要:
[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、CULTRY)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,EF1αACT表达稳定性最好。NormFinder和BestKeeper软件显示在不同花色品种中,EF1aACT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,GAPDHTRY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EF1αACT为最适的内参基因,进一步利用CHS的表达分析验证了EF1αACT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EF1αACT 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:
[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.

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备注/Memo

备注/Memo:
收稿日期:2016-09-24。
基金项目:国家自然科学基金项目(31400600);江苏省自然科学基金项目(BK20140695);江西省观赏植物遗传改良重点实验室开放基金(2013-KLB-03)
作者简介:王彦杰,讲师。
通信作者:徐迎春,副教授,主要从事荷花逆境生理研究,E-mail:xyc@njau.edu.cn。
更新日期/Last Update: 2017-05-16