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园艺学报 ›› 2019, Vol. 46 ›› Issue (2): 365-374.doi: 10.16420/j.issn.0513-353x.2018-0207

• 研究报告 • 上一篇    下一篇

牡丹休眠相关基因PsGRASs筛选、克隆和表达特性分析

吴 红,刘春英,付祥梅,盖树鹏,张玉喜*   

  1. 青岛农业大学生命科学学院,山东省高校植物生物技术重点实验室,山东青岛 266109
  • 出版日期:2019-02-25 发布日期:2019-02-25
  • 基金资助:
    国家自然科学基金面上项目(31471908,31692194)

Screening,Cloning and Expression Patterns Analysis of PsGRASs Associated with Dormancy Release in Tree Peony(Paeonia suffruticosa)

WU Hong,LIU Chunying,FU Xiangmei,GAI Shupeng,and ZHANG Yuxi*   

  1. Qingdao Agricultural University,College of Life Sciences,Key Lab of Plant Biotechnology in Universities of Shandong Province,Qingdao,Shandong 266109,China
  • Online:2019-02-25 Published:2019-02-25

摘要: 通过筛选、克隆牡丹PsGRASs基因并进行表达模式分析,初步解析其是否参与牡丹休眠解除,以期为牡丹反季节催花提供候选基因。利用HMM(Hidden Markov Model)模型,基于GRAS基因家族的种子(Pfam号为PF03514)序列,利用BioEdit软件对牡丹花芽休眠的454测序转录组数据进行本地检索,筛选到2个具有GRAS结构域的序列。通过RACE扩增得到了其全长cDNA,其中PsGRAS1序列为2 308 bp,开放阅读框(open reading frame,ORF)为1 848 bp,编码615个氨基酸;PsGRAS2为2 034 bp,ORF为1 560 bp,编码518个氨基酸,两个编码PsGRASs 蛋白的同源性为19.87%。进化树结果表明,PsGRAS1蛋白与拟南芥GRAS家族中的DELLA亚家族的GAI和RGLs聚到一个大的分支,PsGRAS2蛋白与拟南芥HAMs(AtSCL6/15/22/26/27)并为一支,说明PsGRAS1和PsGRAS2来自GRAS家族的两个不同分支。组织表达结果显示PsGRAS1和PsGRAS2在牡丹初花期不同组织中表达水平不同,PsGRAS1在叶中表达水平最高,在雄蕊中最低;而PsGRAS2在苞片中表达水平最高,在花瓣中最低。在牡丹花芽休眠解除过程中,PsGRAS1呈下调趋势,低温28 d表达水平最低,而PsGRAS2呈先上调后下调趋势,低温14 d表达水平最高。在外源GA3处理7 d的牡丹花芽中,PsGRAS1的表达显著下降,而PsGRAS2显著提高。这表明PsGRAS1和PsGRAS2都参与了休眠过程,但功能不同。

关键词: 牡丹, GRAS, 休眠, 赤霉素, 表达模式

Abstract: By screening and cloning the PsGRASs gene of peony and analyzing its expression pattern,the author preliminarily analyzed whether it participated in the dormancy release of peony,in order to provide candidate genes for anti-seasonal flower urge of peony. Based on the seed sequence of the GRAS gene family(Pfam No. PF03514),local blast was run to search GRAS gene family members in 454 sequencing transcriptomic database using the Hidden Markov Model(HMM)model and BioEdit software.As a result,two ESTs with GRAS domain were found. The full-length cDNAs were obtained by RACE. The full cDNA sequence of PsGRAS1 was 2 308 bp,the open reading frame(ORF)was 1 848 bp,encoding 615 amino acids;the PsGRAS2 was 2 034 bp,and the ORF was 1 560 bp,encoding 518 amino acids. The homology of the two PsGRAS proteins was 19.87%. The results of phylogenetic tree showed that PsGRAS1 protein and GA1,RGLs of DELLA subfamily in Arabidopsis were clustered into a large branch,PsGRAS2 protein is in a branch with Arabidopsis HAMs(AtSCL6/15/22/26/27),indicating that two GRAS members are from two different branches of the GRAS family. The tissue expression results showed that PsGRAS1 and PsGRAS2 were expressed in different tissues at the early stage of peony flowering. The expression level of PsGRAS1 was the highest in leaves and the lowest in stamens,but that of PsGRAS2 in bracts was the highest,and the lowest in the petals. PsGRAS1 genes showed a down-regulation pattern during the dormancy release,and had the lowest expression level after 28 d chilling treatment,while PsGRAS2 showed an up-regulation trend with the highest transcript level after 14 d chilling treatment and followed by a downward trend. After being treated with exogenous GA3 for 7 days,the PsGRAS1 gene showed a dramatically decreasing trend and the PsGRAS2 gene was significantly increased. These results indicated that both PsGRAS1 and PsGRAS2 were involved in the dormancy process with different functions.

Key words: tree peony, GRAS, dormancy, gibberellin, expression pattern

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