目的 克隆珍稀濒危药用植物铁皮石斛Dendrobium officinale 赤霉素3-氧化酶(gibberellin 3-oxidases,GA3ox)基因(DoGA3ox),并进行生物信息学和表达分析。方法 采用RACE 和RT-PCR 技术获得DoGA3ox 基因cDNA 全长;利用生物信息学软件预测其编码蛋白的理化性质、结构域及亚细胞定位等分子特性;用DNASTAR 7.0 和MEGA 6.0 软件分别对其进行氨基酸多序列比对和进化关系分析;借助实时荧光定量PCR(qRT-PCR)检测基因表达模式。结果 克隆到DoGA3ox基因(GenBank 注册号KT597694),cDNA 全长1 318 bp,编码一条由353 个氨基酸组成的多肽,相对分子质量为39 052.5,等电点6.21;DoGA3ox 蛋白不含跨膜域和信号肽,具有GA3ox 的保守结构域DIOX_N(40~130)和2OG-FeII_Oxy(197~299);DoGA3ox 与大葱、油棕、海枣、野茶树及蓝猪耳GA3ox 蛋白一致性分别为55%、56%、54%、51%、50%,与单子叶植物大葱、油棕和海枣的亲缘关系较近。qRT-PCR 实验结果显示DoGA3ox 基因在铁皮石斛种子共生(非共生)萌发1~3级时,其相对表达量呈现出先升高后降低再升高的趋势,分别为未萌发种子的13.44(5.21)、7.28(2.32)和9.40(6.21)倍,由此可知,该基因在共生萌发种子中的表达量高于非共生萌发种子。结论 首次克隆得到1 个DoGA3ox 基因的全长cDNA,其转录本在共生和非共生不同萌发级别种子中的表达特性说明该基因在铁皮石斛种子萌发中起着重要调控作用。

Objective To clone the gibberellin 3-oxidase gene DoGA3ox from an important and endangered medicinal plant Dendrobium officinale (Orchidaceae), followed by bioinformatic and expression analysis. Methods RACE and RT-PCR were used to isolate the full-length gene. The physicochemical properties, conserved domains, and subcellular localization of DoGA3ox protein were determined using a series of bioinformatic tools. The phylogenetic analyses were performed using DNASTAR 7.0 and MEGA 6.0 software. Real time quantitative PCR was employed for gene expression analysis. Results The full-length cDNA of DoGA3ox (GenBank registration number KT597694) was 1 318 bp in size, and encoded a 353-amino acid peptide chain with a molecular weight of 39 052.5 and an isoelectric point (pI) of 6.21; The deduced DoGA3ox protein without transmembrane or signal peptide residues, contained the gibberellin 3-oxidases conserved domains that DIOX_N (40-130) and 2OG-FeII_Oxy (197-299). DoGA3ox had 51%-56% similarity with GA3ox proteins from various plants, and was closely related to the monocot Allium fistulosum, Elaeis guineensis, and Phoenix dactylifera. The relative transcript levels of DoGA3ox were increased at stage 1, then decreased (stage 2), and increased again (stage 3) during D. officinale symbiotic/asymbiotic seed germination, the fold change to ungerminated seeds was 13.44 (symbiotic)/5.21 (asymbiotic), 7.28/2.32 and 9.40/6.21, respectively. Moreover, its transcript level was higher in symbiotic germination than that in the asymbiotic status. Conclusion The full-length DoGA3ox gene is cloned for the first time, and its expression in different stages during seed germination indicates that DoGA3ox gene plays a crucial role in the regulation of seed germination in D. officinale.

教育部博士点基金优先发展领域(20131106130002);药植所创新团队发展计划(PIRTI-IT1302);中央公益性科研院所基本科研业务费专项(YZ-12-14);中医药行业科研专项(201407005)