目的 克隆珍稀濒危药用植物铁皮石斛转录因子基因DoWRKY3，并进行生物信息学和表达模式分析。方法 采用RT-PCR和RACE技术获取基因全长；利用生物信息学软件预测蛋白的理化性质、结构域和亚细胞定位等分子特性；用软件DNASTAR 6.0和MEGA 6.0分别进行氨基酸多序列比对和进化关系分析；借助定量PCR检测基因表达模式。结果 分离到DoWRKY3基因（GenBank注册号KT957549），cDNA全长2 065 bp，编码1条由509个氨基酸组成的多肽，相对分子质量55 580，等电点6.58；推定的DoWRKY3氨基酸序列具有植物WRKY蛋白家族保守的2个WRKY结构域（217～279、381～449）、2个WRKYGQK位点和2个C₂H₂型锌指结构元件（C-X₄-C-X_22-23-H-X₁-H）；该蛋白预测无信号肽或跨膜域，定位在细胞核；DoWRKY3蛋白与多种植物WRKY蛋白一致性较高（46.3%～57.4%），与拟南芥AtWRKY3、AtWRKY4和丹参SmWRKY54蛋白等亲缘关系近，聚在WRKY分子进化树的Group 1分支；DoWRKY3基因具有组织表达特异性，其转录本在石斛根和叶中表达量较高，分别为茎中的2.32倍和1.69倍。结论 DoWRKY3基因全长的分子克隆与特征分析，为深入研究该基因在铁皮石斛生长发育、逆境生理以及次级代谢调控中的生物学功能奠定基础。

Objective To isolate and characterize a WRKY transcription factor encoding gene DoWRKY3 in a rare endangered medicinal orchid species Dendrobium officinale, followed by bioinformatics analysis and expression pattern detection. Methods RT-PCR and RACE technologies were used to isolate the full length cDNA of DoWRKY3. Characteristics of physiochemical properties, conserved domains, and subcellular localization of the deduced DoWRKY3 protein were determined by a series of bioinformatics tools. The analyses of multiple alignment and phylogenetic tree were performed using DNASTAR 6.0 and MEGA 6.0 softwares, respectively. Quantitative PCR was used for gene expression analysis. Results The full length cDNA of DoWRKY3 (GenBank accession KT957549) was 2 065 bp in length, and encoded a 509-aa protein with a molecular weight of 55 580 and an isoelectric point of 6.58; The deduced DoWRKY3 protein sequence had two WRKYGQK motifs, two WRKY domains (217—279, 381—449), and two C₂H₂-type zinc-finger signatures (C-X₄-C-X_22-23-H-X₁-H), which are all conserved among the WRKY proteins. DoWRKY3 protein did not contain a signal peptide or a transmembrane region, and was predicted to locate in nucleus; DoWRKY3 had high identities (46.3%—57.4%) with various WRKY proteins from several plants; DoWRKY3 was closely related to Arabidopsis AtWRKY3, AtWRKY4, and Salvia miltiorrhiza SmWRKY54 proteins, and belonged to the Group 1 of the WRKY evolutionary tree; DoWRKY3 gene was differentially expressed in the three included organs. The transcripts were more abundant in the roots and leaves, with 2.32 and 1.69 fold, respectively, over that in the stems. Conclusion Molecular cloning and characterization of the full length DoWRKY3 gene will be useful for further functional determination of the gene involving in the growth and development, physiological stress adaptations, and secondary metabolic regulations of D. officinale.

国家自然科学基金资助项目（31101608）；陕西省自然科学基金项目（2017JM8030）；陕西省青年科技新星项目（2012KJXX-44）