Abstract

The AP2/ERF-domain transcription factor ORCA2 from Catharanthus roseus was demonstrated earlier to regulate the expressions of Str gene, an important gene involved in the terpenoid indole alkaloids biosynthetic pathway in C. roseus cells. Therefore, the factor was postulated to play an important role in the production of secondary metabolites in plants. To investigate the effect of over expression of ORCA2 on the TIAs biosynthesis in C. roseus hair roots, transformation of ORCA2 gene was conducted with the disarmed Agrobacterium rhizogenes C58C1 harboring pCAMBIA1304⁺, a plasmid that contains the Orca2 gene, a Gus gene and an Hpt gene all under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Transgenic hairy root cultures expressing Orca2 gene were obtained and demonstrated by genomic- polymerase chain reaction (PCR) analysis for the integration of the Orca2 gene in the C. roseus genome, by real-time quantitative PCR (RT-QPCR) and β-glucuronidase (GUS) staining for the expression of the foreign genes. Metabolite analysis using high performance liquid chromatography(HPLC) analysis established that the average content of catharanthine and vindoline in the transgenic hairy root extracts was increased up to 2.03 and 3.67-fold in comparison to the control lines, respectively. However, vinblastine could not been detected in the transgenic and control hairy root cultures by HPLC.

Key words: Catharanthus roseus, ORCA2, hairy root, overexpression, terpenoid indole alkaloids (TIAs), AP2/ERF-domain transcription factor.

Abbreviation

TIAs, Terpenoid indole alkaloids; PCR, polymerase chain reaction;MS, Murashige and Skoog; ORCA, octadecanoid-responsive CatharanthusAP2/ERF-domain protein; CTAB, cetyltrimethyl ammonium bromide; HPLC, high performance liquid chromatography; RT-QPCR, real-time quantitative PCR; GUS,β-glucuronidase; STR, strictosidine synthase; TDC, tryptophan decarboxylase.