《植物生理学报》 2017, 53(4): 677-686

芍药PlGA20ox基因的克隆及其在芽内休眠解除进程中的表达分析

韩璐璐^*, 李俊杰^*, 马燕, 国静, 郭先锋^**

山东农业大学林学院, 山东泰安271018

通信作者：韩璐璐；E-mail: guoxf@sdau.edu.cn

摘　要：

以芍药‘大富贵’芽为试材, 采用RT-PCR结合cDNA末端快速扩增(RACE)技术, 克隆得到GA20ox基因的cDNA全长, 命名为PlGA20ox (GenBank登录号为KU886552)。该基因全长1 351 bp, 开放阅读框1 146 bp, 共编码381个氨基酸, 含有高度保守的20G-Fe(II)-Oxy蛋白结构域、Fe²⁺结合位点(His-247、Asp-249和His-303)和2-酮戊二酸结合位点(Arg-313和Ser-315)。PlGA20ox蛋白分子量为43 209.1 Da, 为稳定蛋白, 无跨膜结构域, 无信号肽, 属于C19-GAoxs。氨基酸序列同源性及系统进化树分析表明: PlGA20ox与牡丹PsGA20ox同源性高达96%, 亲缘关系最近。采用农杆菌介导法将PlGA20ox基因于本生烟叶片中瞬时表达, 观察显示: PlGA20ox蛋白定位于细胞质中。该基因在芍药各器官中均有表达, 其中, 在芽中表达最高, 其次花瓣, 在根、萼片、叶片和茎中表达微弱。实时荧光定量PCR和高效液相色谱(HPLC)结果显示: 在低温解除芍药芽内休眠进程中, PlGA20ox表达水平呈先上升后总体下降趋势, 且与内源GA₃含量呈显著正相关(r=0.901*); 外施赤霉素会明显增加内源GA₃含量, 但同时抑制PlGA20ox的表达, 说明PlGA20ox调控GA₃的合成, 同时受植物体内活性GA₃含量的负反馈调节。

关键词：芍药; PlGA20ox; 克隆; 亚细胞定位; 表达; 芽

收稿：2017-02-10   修定：2017-03-31

资助：山东省自然科学基金(ZR2014CM028)。

Cloning and expression analysis of PlGA20ox gene in peony (Paeonia lactiflora) buds during endodormancy release

HAN Lu-Lu^*, LI Jun-Jie^*, MA Yan, GUO Jing, GUO Xian-Feng^**

Colloge of Forestry, Shandong Agricultural University, Taian, Shandong 271018, China

Corresponding author: HAN Lu-Lu; E-mail: guoxf@sdau.edu.cn

Abstract:

The full-length cDNA of PlGA20ox gene (GenBank accession number: KU886552) was cloned from Paeonia lactiflora ‘Da fugui’ buds by RT-PCR and rapid-amplification of cDNA ends (RACE) methods. Cloned PlGA20ox gene is 1 351 bp in length, containing 1 146 bp open reading frame (ORF) encoding 381 amino acids. Amino acid sequence analysis indicated that PlGA20ox possesses conserved 20G-Fe(II)-Oxy protein domain, highly conserved Fe²⁺ and 2-oxoglutaric acid binding sites. PlGA20ox is a kind of stable protein with a predicted molecular weight of 43 209.1 Da and belongs to C19-GAoxs without transmembrane domain or signal peptide. The homology between PlGA20ox and GA20ox from P. suffruticosa is as high as 96%, indicating the closest genetic relationship. Subcellular localization showed that the PlGA20ox protein was localized in the cytoplasm. Semi-quantitative PCR showed that PlGA20ox was expressed in each organ of P. lactiflora but differentially. It was extremely high in buds, followed by petals, and weakly present in the leaves, stems, sepals and roots. The expression level of PlGA20ox showed an overall downward trend with an initial rise, which is positively correlated to endogenous GA₃ content during bud endodormancy release induced by chilling. The exogenous GA₃ could increase the endogenous GA₃ content and inhibit the expression of PlGA20ox significantly, indicating that PlGA20ox can regulate the synthesis of GA₃ meanwhile receive negative feedback regulation from active GA₃ in plants.

Key words: Paeonia lactiflora; PlGA20ox; cloning; subcellular location; expression; buds