Journal of Nuclear Agricultural Sciences ›› 2019, Vol. 33 ›› Issue (9): 1707-1716.DOI: 10.11869/j.issn.100-8551.2019.09.1707

• Induced Mutations for Plant Breeding·Agricultural Biotechnology • Previous Articles     Next Articles

Selection and Validation of Reference Genes for Quantitative Real-Time PCR Analysis in Iris bulleyana During Flower Color Variation

MA Lulin1,2, CUI Guangfen1, WANG Xiangning1, JIA Wenjie1, DUAN Qing1, DU Wenwen1, WANG Jihua1,*, CHEN Fadi2,*   

  1. 1 Flower Research Institute of Yunnan Academy of Agricultural Sciences/Yunnan Flower Breeding Key Lab/National Engineering Research Center For Ornamental Horticulture, Kunming, Yunnan 650205;
    2 College of Horticulture of Nanjing Agricultural University, Nanjing, Jiangsu 210095
  • Received:2018-05-21 Revised:2018-08-10 Online:2019-09-09 Published:2019-07-23

西南鸢尾花色变异实时定量PCR内参基因的筛选与验证

马璐琳1,2, 崔光芬1, 王祥宁1, 贾文杰1, 段青1, 杜文文1, 王继华1,*, 陈发棣2,*   

  1. 1 云南省农业科学院花卉研究所/云南省花卉育种重点实验室/国家观赏园艺工程技术研究中心,云南 昆明 650205;
    2 南京农业大学园艺学院,江苏 南京 210095
  • 通讯作者: *王继华,男,研究员,主要从事观赏植物病理学研究。E-mail: wangjh0505@sohu.com;陈发棣,男,教授,主要从事花卉遗传育种研究。E-mail: chenfd@njau.edu.cn。同为通讯作者。
  • 作者简介:马璐琳,男,副研究员,主要从事观赏植物分子生物学研究。E-mail: malulin@sohu.com
  • 基金资助:
    云南省农业科学院应用基础研究重点项目(YJZ201701),云南省科技领军人才计划(2016HA005),云南省现代农业花卉苗木产业技术体系建设(2017KJTX0010)

Abstract: In order to screen the appropriate reference genes for real-time quantitative polymerase chain reaction (RT-qPCR) analysis of the main pigments synthesis related genes in the blue/white flower buds of Iris bulleyana, in this study, 6 traditional reference genes (ɑ-TUBβ-TUBAQPACTGAPDHUBQ) were selected based on RNA-seq data of I. bulleyana Dykes and its white form-I. bulleyana Dykes f. alba Y. T. Zhao. lily 18S was used as the control reference gene. The expression of all 7 candidate reference genes were examined by reverse transcriptional PCR (RT-PCR) and RT-qPCR, and the stability were analyzed by geNorm, NormFinder and BestKeeper programs. The RT-PCR results showed that the primers of the 7 candidate reference genes were all specifically amplified, and no significant difference was detected on the gene expression level among the 6 different samples. The RT-qPCR analysis showed a significant difference among the expression level of 7 reference genes, which displayed the highest level for 18S and the lowest level for UBQ. The analyses of geNorm, NormFinder and BestKeeper showed that Actin was the most stable reference gene and β-TUB displayed the lowest stability, indicating Actin was suitable for reference gene. The RNA-seq data was exactly consistent with the RT-qPCR analyses of the partial relative genes in the two biosynthetic pathways of flavonoid/anthocyanin and carotenoid which using Actin as reference gene. This research gives an example of the reference gene screen for the relative genes expression analysis in the flavonoid/anthocyanin and carotenoid biosynthetic pathway of Iris, and provides a theoretical basis for plant resource utilization and flower color breeding.

Key words: Iris bulleyana, flower color variation, real-time quantitative PCR, reference gene

摘要: 为筛选适用于不同花色西南鸢尾花色合成途径相关基因表达分析的内参基因,本研究根据西南鸢尾及其白花变型白花西南鸢尾花蕾组织的转录组测序数据,筛选了6个常用内参基因(ɑ-TUBβ-TUBAQPACTGAPDHUBQ),同时以百合18S做为对照内参基因,在西南鸢尾及其白花变型白花西南鸢尾的花蕾组织中,分别通过反转录PCR(RT-PCR)初筛和RT-qPCR检测表达量,并利用geNorm、NormFinder和BestKeeper软件对7个候选内参基因的稳定性进行评价。RT-PCR初筛结果显示,7个候选内参基因引物的特异性均较好,在6个样品间没有明显差异;RT-qPCR分析表明,7个候选内参基因的表达量存在一定差异,其中18S表达量最高,UBQ最低;geNorm、NormFinder和BestKeeper分析结果表明,ACT表现最稳定,最适合做为内参基因,β-TUB相对稳定性最低;以Actin为内参对西南鸢尾类黄酮/花青素和类胡萝卜素2类色素合成途径中部分相关基因的RT-qPCR分析结果与转录组测序结果相一致。本研究为鸢尾属植物花色素合成相关基因表达分析内参基因的筛选以及鸢尾属植物资源利用和花色育种研究提供了理论参考。

关键词: 西南鸢尾, 花色变异, 实时定量PCR, 内参基因