Overexpression of transcriptional factor ORCA3 increases the accumulation of catharanthine and vindoline in Catharanthus roseus hairy roots

Abstract

The AP2/ERF-domain transcription factor ORCA3 of Catharanthus roseus has been recognized earlier as a regulator of the expression of some important genes involved in the terpenoid indole alkaloid (TIA) biosynthetic pathway in C. roseus cells. Therefore, this factor was postulated to play an important role for the biosynthesis of secondary metabolites in plants. To investigate the effect of ORCA3 overexpression on the TIA biosynthesis in C. roseus hairy roots, biotransformation of Orca3 gene was conducted with the disarmed Agrobacterium rhizogenes C58C1 harboring pCAMBIA1304+-Orca3, a plasmid that contains an Orca3 gene, a Gus gene, and a hygromycin-resistance (hyg) gene, all driven separately by the cauliflower mosaic virus 35S promoter (35SCaMV). Putative transgenic hairy root lines were induced and subcultured. The T-DNA fragment of pCAMBIA1304+-Orca3 was proved to be integrated into C. roseus hairy root genome by genomic-PCR analysis, real-time quantitative PCR analysis, and GUS staining assay. Metabolite analysis using HPLC established that the average content of catharanthine and vindoline in the transgenic hairy root extracts was increased 2.49-fold and 4.2-fold in comparison to the control hairy root lines, respectively. However, vinblastine content could not been detected by HPLC method in transgenic and control hairy root cultures.