Molecular Cloning and Expression Analysis of the Transcription Factor AsWRKY25 in Aquilaria sinensis (Lour.) Gilg / 中国药学杂志

ABSTRACT

OBJECTIVE: To clone AsWRKY25, a transcription factor gene of Aquilaria sinensis, for bioinformatic analysis and tissue expression analysis, and express its protein in prokaryotic cells, thus to lay a foundation for further study of the biological function of AsWRKY25. METHODS: With the cDNA isolated from A. sinensis callus as template, the full-length coding sequence (CDS) of AsWRKY25 was amplified using PCR method. The recombinant vector pET-28a-AsWRKY25 was transformed into Escherichia coli BL21 (DE3) for prokaryotic expression. The physiochemical properties and bioinformatic characters of AsWRKY25 were calculated by a series of bioinformatics tools and softwares. The expression patterns in different tissues were detected by RT-PCR. RESULTS: The full-length coding sequence of AsWRKY25 transcription factor was cloned from the callus of A. sinensis. The CDS of AsWRKY25 was 1 728 bp in length and contained a 1 728 bp open reading frame (ORF) encoding 575 amino acids. The CDS sequence was codon-optimized and then ligated into the pET-28a expression vector. The optimal induction condition of recombinant pET-28a-AsWRKY25 was 1 mmol•L-1 IPTG at 37℃ for 6 h in E. coli BL21 (DE3). The result of tissue expression analysis showed that AsWRKY25 had the highest expression level in the agarwood layer and the lowest level in roots, stems and branches. CONCLUSION: The WRKY transcription factor AsWRKY25 is successfully cloned from A. sinensis and expressed in E. coil. It is speculated that AsWRKY25 may be involved in the wound-induced agarwood-formation process in A. sinensis.