Molecular cloning and characterization of a transcription factor gene dowrky3 in dendrobium officinale / 中草药

ABSTRACT

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-X22-23-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.