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

发布时间：2018-05-20 14:13

本文选题：芍药 + PlGAox　；参考：《植物生理学报》2017年04期

【摘要】：以芍药‘大富贵’芽为试材,采用RT-PCR结合c DNA末端快速扩增(RACE)技术,克隆得到GA20ox基因的c DNA全长,命名为Pl GA20ox(Gen Bank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(I I)-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)。Pl GA20ox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:Pl GA20ox与牡丹Ps GA20ox同源性高达96%,亲缘关系最近。采用农杆菌介导法将Pl GA20ox基因于本生烟叶片中瞬时表达,观察显示:Pl GA20ox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,Pl GA20ox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Pl GA20ox的表达,说明Pl GA20ox调控GA3的合成,同时受植物体内活性GA3含量的负反馈调节。
[Abstract]:Using Paeonia lactiflora 'Dafugui' bud as test material, the full length of c DNA of GA20ox gene was cloned by RT-PCR and rapid amplification of c DNA terminal. The full length of c DNA was named Pl GA20ox(Gen Bank accession number KU886552. The total length of the gene is 1 351 BP, open reading frame 1 146 BP, encoding 381 amino acids, containing highly conserved 20G-Fe(I I)-Oxy domain His-247 Asp-249 and His-303), 2-ketoglutaric acid binding site Arg-313 and Ser-315).Pl GA20ox protein with molecular weight of 43 209.1 Daa, which is a stable protein. No transmembrane domain, no signal peptide, belonging to C19-GAoxs. The analysis of amino acid sequence homology and phylogenetic tree showed that the homology of the amino acid sequence and the peony Ps GA20ox was 96%, and the phylogenetic relationship was the most recent. Using Agrobacterium tumefaciens mediated expression of Pl GA20ox gene in tobacco leaves, it was observed that the protein of Pl GA20ox was located in the cytoplasm. The gene was expressed in all organs of Paeonia lactiflora, the highest in bud, the second in petals, and weak in roots, sepals, leaves and stems. The results of real-time fluorescence quantitative PCR and high performance liquid chromatography (HPLC) showed that the expression of Pl GA20ox increased first and then decreased during the process of releasing dormancy of Paeonia lactiflora at low temperature. Gibberellin significantly increased the content of endogenous GA3, but inhibited the expression of Pl GA20ox at the same time, indicating that Pl GA20ox regulated the synthesis of GA3 and was negatively regulated by the content of active GA3 in plant.
【作者单位】：山东农业大学林学院;
【基金】：山东省自然科学基金(ZR2014CM028)~~
【分类号】：Q943.2;S682.12

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