Journal of Nuclear Agricultural Sciences ›› 2021, Vol. 35 ›› Issue (1): 60-71.DOI: 10.11869/j.issn.100-8551.2021.01.0060

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

Cloning and Selection Evaluation of Reference Gene for Quantitative Real-Time PCR in Hibiscus esculentus L.

LI Yongping, YE Xinru, WANG Bin, CHEN Mindong, LIU Jiangting, ZHU Haisheng*, WEN Qingfang*   

  1. Fujian Key Laboratory of Vegetable Genetics and Breeding/Fujian Engineering Research Center for Vegetables/Crops Research Institute, Fujian Academy of Agricultural Sciences/Vegetable Research Center, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350013
  • Received:2019-06-04 Online:2021-01-10 Published:2020-11-16
  • Supported by:
    福建省属公益类科研院所基本科研专项(2019R1031-4),中央引导地方科技发展专项(2018L3005),福建省农业科学院科技创新团队建设项目(STIT2017-1-2),国家大宗蔬菜产业技术体系福州综合试验站(CARS-23-G-53)

黄秋葵实时荧光定量PCR内参基因的克隆与筛选评价

李永平, 叶新如, 王彬, 陈敏氡, 刘建汀, 朱海生*, 温庆放*   

  1. 福建省蔬菜遗传育种重点实验室/福建省农业科学院作物研究所/福建省农业科学院蔬菜研究中心/福建省蔬菜工程技术研究中心,福建 福州 350013
  • 通讯作者: *朱海生,男,研究员,主要从事蔬菜生物技术与育种研究。E-mail:zhso246@163.com;温庆放,男,研究员,主要从事蔬菜育种与栽培研究。E-mail:fjvrc@163.com。同为通讯作者。
  • 作者简介:李永平,女,副研究员,主要从事蔬菜生物技术与育种研究。E-mail:248937256@qq.com

Abstract: The study was aimed at selecting the stable reference genes to ensure the reliability and accuracy in gene expression analysis of Hibiscus esculentus L.. The ORF sequences of 18SrRNA, ACT, EF-1a, TUA, TUB and GAPDH were screened and verified(obtained) from ‘Green White No. 1’ according to the RNA-seq database of Hibiscus esculentus L.. By quartitative real-time technology, combined with GeNorm, NormFinder and BestKeeper software analysis, we evaluated the expression stability of six reference genes in different tissue, different development stages of fruit and leaf and different stress treatments (low temperature, high temperature, drought). The results showed that six genes can express in different tissues, development stages and stresses, but the expression stabilities were not the same. Among them,EF-1a was the most stable under pod development and high temperature stress. 18SrRNA was the most stable under various tissue, leaf development and drought stress. ACT was the most stable under low temperature stress. In addition, with all 29 treatments, the expression of 18SrRNA, EF-1a and ACT was relatively stable and can be used for fluorescence quantitative expression analysis. This study provides a basis for the study of gene function and regulation mechanism in Hibiscus esculentus L.

Key words: Hibiscus esculentus L, reference gene, stress, different tissues

摘要: 为筛选黄秋葵实时荧光定量PCR的稳定内参基因,本研究以绿白1号为试验材料,根据黄秋葵RNA-seq数据库,筛选并验证获得18SrRNAACTEF-1αTUATUBGAPDH等6个内参基因ORF序列;以黄秋葵不同组织、不同发育时期果实、不同发育时期叶片和低温、高温、干旱胁迫处理的叶片为材料,利用实时荧光定量PCR技术分析测定基因表达量,并结合GeNorm、NormFinder和BestKeeper软件评价6个内参基因的稳定性。结果表明,6个基因在不同组织、各发育阶段及不同胁迫下均有表达,但表达稳定性不尽相同,其中,EF-1α在黄秋葵果荚发育、高温胁迫下表达稳定性最好;18SrRNA在黄秋葵各组织、叶发育和干旱胁迫下表达稳定性最优;ACT在低温胁迫下表现最稳定。此外,在29个处理中,18SrRNAEF-1αACT表达均较稳定,可以用于荧光定量表达分析。本研究结果为黄秋葵基因功能分析和调控机理研究提供了基础。

关键词: 黄秋葵, 内参基因, 胁迫, 不同组织