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园艺学报 ›› 2015, Vol. 42 ›› Issue (10): 1983-1992.doi: 10.16420/j.issn.0513-353x.2015-0180;

• 观赏植物 • 上一篇    下一篇

牡丹泛素延伸蛋白基因ubiquitin克隆及其作为内参基因的研究

刘传娇1,2,王顺利2,*,薛璟祺2,朱富勇2,任秀霞2,李名扬1,**,张秀新2,**   

  1. 1西南大学园艺园林学院,重庆市花卉工程技术研究中心,重庆 400715;2中国农业科学院蔬菜花卉研究所,农业部园艺作物生物学与种质创制重点实验室,北京 100081
  • 出版日期:2015-10-25 发布日期:2015-10-25
  • 基金资助:

    国家自然科学基金项目(31501800,31572156);国家公益性行业(农业)科研专项(201203071);国家‘863’计划项目
    (2011AA10020703);中国农业科学院科技创新工程项目(CAAS-ASTIP-IVFCAAS);中国农业科学院院所基金项目(1610092015002-01)

Molecular Cloning of Ubiquitin Protein Gene and Study on This Gene as Reference Gene in Tree Peony

LIU Chuan-jiao1,2,WANG Shun-li2,*,XUE Jing-qi2,ZHU Fu-yong2,REN Xiu-xia2,LI Ming-yang1,**,and ZHANG Xiu-xin2,**   

  1. 1College of Horticulture and Landscape Architecture,Southwest University,Chongqing Engineering Research Center for Floriculture,Chongqing 400715,China;2Key Laboratory of Biology and Genetic Improvement of Horticultural Crops,Ministry of Agriculture,P. R. China,Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China
  • Online:2015-10-25 Published:2015-10-25

摘要:

以牡丹品种‘洛阳红’(Paeonia suffruticosa‘Luoyanghong’)为试验材料,采用RT-PCR方法获得一个牡丹泛素延伸蛋白基因Psubiquitin(PsUBI),其cDNA开放阅读框(ORF)长度为447 bp,编码148个氨基酸,GenBank登录号为KP742952。序列比对发现,该基因与其他23种植物泛素延伸蛋白核苷酸序列的相似性均在81%以上,氨基酸序列的相似性达96%。进化分析表明,牡丹泛素延伸蛋白与棉花泛素延伸蛋白的亲缘关系最近。实时荧光定量PCR结果显示,在牡丹不同组织器官中,相对于其他5个常用看家基因(GAPDH、GAPDH1、tubulin、tubulin2、18S rRNA),ubiquitin的PCR扩增曲线的CT值最为恒定,尤其是在不同花器官中的CT值完全一致。进一步分析发现该基因在牡丹的根、茎、叶片、花、雄蕊和心皮组织中均恒定表达,特别是花器官中的表达量几乎完全一致。以ubiquitin作为内参基因探讨控制花器官发育的基因PsAG的表达情况,结果显示PsAG的表达模式与其作用位点相吻合,ubiquitin更适宜作为牡丹花器官研究的内参基因。

关键词: 牡丹, ubiquitin, 基因克隆, 表达分析, 内参基因

Abstract:

One ubiquitin protein gene was obtained from tree peony(Paeonia suffruticosa‘Luoyanghong’)by RT-PCR. It was designated as PsUBI and the GenBank accession number was KP742952. The open reading frame(ORF)of PsUBI was 447 bp,and could encode 148 amino acids.Homologous alignment shows that it shared over 81% nucleotide sequence similarity and over 96% amino acid sequence similarity with ubiquitins in other plants. The phylogenetic tree analysis suggested that the relationship of ubiquitin between P. suffruticosa and Gossypium hirsutum was close,based on the amino acid sequences. Real time quantitative PCR analysis revealed that compared to other five commonly used housekeeping genes(GAPDH,GAPDH1,tubulin,tubulin2,18S rRNA),ubiquitin has the most constant CT values of PCR amplification curve in different tissues of tree peony,especially in floral organs. Further analysis of the results showed that PsUBI was constantly expressed in various organs of tree peony such as roots,stems,leaves,petals,stamens and carpels,especially in floral organs. We used ubiquitin as the reference gene,and found that the expression pattern of PsAG in floral organs of tree peony was consistent with its action sites. Thus,PsUBI can be more appropriate for floral organ study of tree peony.

Key words: tree peony, ubiquitin, gene clone, expression analysis, reference gene

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