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https://www.ahs.ac.cn/images/0513-353X/images/top-banner12.jpg|#|菊花

园艺学报 ›› 2022, Vol. 49 ›› Issue (11): 2489-2501.doi: 10.16420/j.issn.0513-353x.2021-0763

• 新技术新方法 • 上一篇    下一篇

秋石斛RT-qPCR内参基因的筛选与验证

侯天泽1, 易双双2,3, 张志群2,3, 王健1,*(), 李崇晖2,3,*   

  1. 1海南大学园艺学院,海口 570208
    2中国热带农业科学院热带作物品种资源研究所,农业农村部华南作物基因资源与种质创制重点实验室,海口 571101
    3海南省热带观赏植物种质创新利用工程技术研究中心,海南儋州 571737
  • 收稿日期:2021-10-15 修回日期:2022-04-30 出版日期:2022-11-25 发布日期:2022-11-25
  • 通讯作者: 王健,李崇晖 E-mail:blchh@sina.com
  • 基金资助:
    国家重点研发计划项目(2018YFD1000405);海南省基础与应用基础研究计划(自然科学领域)高层次人才项目(2019RC308);中央级公益性科研院所基本科研业务费专项资金项目(1630032020035);中央级公益性科研院所基本科研业务费专项资金项目(1630032017024)

Selection and Validation of Reference Genes for RT-qPCR in Phalaenopsis- type Dendrobium Hybrid

HOU Tianze1, YI Shuangshuang2,3, ZHANG Zhiqun2,3, WANG Jian1,*(), LI Chonghui2,3,*   

  1. 1College of Horticulture,Hainan University,Haikou 570208,China
    2Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China,Ministry of Agriculture and Rural affairs,Tropical Crops Genetic Resources Institute,Chinese Academy of Tropical Agricultural Sciences,Haikou 571101,China
    3The Engineering Technology Research Center of Tropical Ornamental Plant Germplasm Innovation and Utilization,Hainan Province,Danzhou,Hainan 571737,China
  • Received:2021-10-15 Revised:2022-04-30 Online:2022-11-25 Published:2022-11-25
  • Contact: WANG Jian,LI Chonghui E-mail:blchh@sina.com

摘要:

为了筛选出秋石斛中稳定表达的内参基因用于实时荧光定量PCR(RT-qPCR)分析,以不同品种和不同发育阶段花芽(包括花瓣、唇瓣和萼片)为材料,使用geNorm、NormFinder 和BestKeeper软件计算了常用的7个候选内参基因表达的稳定性,使用RefFinder对其稳定性进行综合评价,并对筛选出的内参基因进行了验证。结果表明,各内参基因在秋石斛花芽中的表达稳定性差异较大,在不同品种同一发育阶段的花芽中β-actinTUAGADPH表达最稳定;在花芽发育过程中β-actinTUACYP表达最稳定;而对于花器官不同组织,β-actinTUAGADPH在萼片中,β-actinCYPPGK在花瓣中,CYPTUAPGK在唇瓣中表达最稳定。

关键词: 秋石斛, 花芽, 发育阶段, 组织, 实时荧光定量PCR, 内参基因, 基因表达

Abstract:

In order to screen reference genes stably expressing in Phalaenopsis-type Dendrobium hybrid for analysis of real-time fluorescent quantitative PCR,the whole floral buds,including the sepals,petals and labellums,from various cultivars and the floral bud at different development stages of dendrobium were used as materials. The expression stability of seven candidate gene was calculated by three commonly used software(geNorm,NormFinder and BestKeeper),comprehensive stability rankings were merged by RefFinder and the selected reference genes were verified. The results suggested that the expression stability of each reference gene in dendrobium differed greatly. In floral buds of different cultivars at the same developmental stage,β-actinTUA,and GADPH were the most stable reference genes. The expression of β-actinTUA,and CYP were the most stable during the development of the floral buds. For different tissues of floral organ,β-actinTUA,and GADPH were the most stable reference genes in the sepals,β-actinCYP,and PGK were in the petals and CYPTUA,and PGK were in the labellum.

Key words: Phalaenopsis-type Dendrobium hybrid, floral bud, developmental stage, tissues, RT-qPCR, reference gene, gene expression

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