Preliminary characterization of Brugmansia candida h6h gene

MARCONI, PATRICIA; STUMPO, RITA; CARDILLO, ALEJANDRA BEATRIZ; GIULIETTI, ANA MARIA

Basel

Congreso; 11th European Congress on Biotechnology; 2003

Objetives: Brugmansia candida is a native South-American plant which produces tropane alkaloids mainly scopolamine. This alkaloid is synthesized from hyoscyamine. Hyoscyamine 6b-hydroxilase (H6H) catalyzes hydroxylation of hyoscyamine leading to scopolamine formation. The aim of this work is to evaluate the presence of h6h gene in this regional plant specie for further cloning purposes. Different organs (tips of roots, stem, leaves and mother cells of microespors) of mature plants that were harvested at the immature flowering stage in the Jardin Botanico of Buenos Aires (Argentina). Hairy roots culture were obtained from seedlings transformed with Agrobacterium rhizogenes LBA 9402 according to Giulietti et al. (1993).    Total RNA was isolated from organs and HR mentioned above with Trizol-Reagent as previously described (Stumpo et al. 1999) and compared to the extraction with RNeasy Plant Kit (Qiagen). After cDNA synthesis using Superscript II reverse transcriptase (Life Technologies), PCR reaction was done on bases of Hyoscyamus niger h6h gene specific primer design due to high homology founded for the transcript among Solanaceae family. Results: The results indicate the existence of h6h gene in native B. candida plants. PCR reaction showed a specific band of 1.1 Kb in the case of root tips of mature plant, and the same but stronger specific signal from mother cells of microespors. However, no signal was detected in the case of HR cultures material. Previously data obtain in our lab showed evidences in the kinetics different patterns of production of hyoscyamine and scopolamine alkaloids in HR cultures elicited with fungal extracts (Pitta et al., 2003). These results suggest an underlined gene regulatory mechanism and strength the current PCR results. Further experimental work is being performed in order to develop H6H cDNA cloning and expression strategies for biotransformation purposes.