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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。
The full-length c cDNA of GA20ox gene was cloned by RT-PCR combined with rapid amplification of c DNA ends (RACE), and named as Pl GA20ox (Gen Bank Accession No. KU886552). The gene was 1 351 bp in length and 1 146 bp in open reading frame, encoding a total of 381 amino acids. The gene contained a highly conserved 20G-Fe (II) -Oxy protein domain. The Fe2 + binding site (His-247, Asp-249 and His-303) and 2-oxoglutarate binding sites (Arg-313 and Ser-315). Pl GA20ox has a molecular weight of 43 209.1 Da, a stable protein, no transmembrane domain and no signal peptide, belonging to C19-GAoxs. Amino acid sequence homology and phylogenetic tree analysis indicated that the homology of Pl GA20ox and Peony Ps GA20ox was as high as 96%, and the genetic relationship was the closest. The Agrobacterium-mediated method was used to transiently express Pl GA20ox gene in Nicotiana benthamiana leaves. The results showed that Pl GA20ox protein localized in the cytoplasm. The gene was expressed in various organs of Paeonia lactiflora. Among them, it was the highest expression in buds, followed by the petals, which were weakly expressed in roots, sepals, leaves and stems. Real-time PCR and high performance liquid chromatography (HPLC) showed that the expression level of Pl GA20ox firstly decreased and then decreased, and positively correlated with the content of endogenous GA3 (r = 0.901 *). The application of gibberellin could significantly increase the content of endogenous GA3, but at the same time inhibited the expression of Pl GA20ox, indicating that GA3 was regulated by Pl GA20ox and negatively regulated by GA3 content in plants.