Study of expression of H6H gene in Brugmansia candida hairy root cultures

MARCONI, PATRICIA; CARDILLO, ALEJANDRA BEATRIZ; ALVAREZ, ALEJANDRA; GIULIETTI, ANA MARÍA

Bariloche

Congreso; SAIB 2003; 2003

The tropane alkaloids hyoscyamine and scopolamine are valuable drugs employed as antispasmodics and in the treatment of motion sickness. Their production by biotechnology processes is under study in Brugmansia candida hairy roots (HR) cultures. Elicitation is a strategy used to increase secondary metabolites including alkaloids. In previous work, we reported that acetate buffer is able to increase hyoscyamine (700%) and scopolamine (200%) in these HR cultures. Hyoscyamine-6-b-hydroxylase (H6H) is the enzyme responsible for converts hyoscyamine into scopolamine. In order to understand the difference levels of alkaloids reached after elicitation in this work, we study the expression of H6h mRNA in HR cultures elicited with acetate buffer. B. candida hairy root cultures were obtained from seedlings transformed with Agrobacterium rhizogenes LBA9402 according to Giulietti et al. (1993). In elicitation experiments 0, 1.0 and 2.0 mM of acetate buffer (Ac-) was added to the culture medium. The samples were collected at 0, 6, 12 and 24 h after subculture. Total RNA was obtained with RNeasy Plant Kit (Qiagen). cDNA synthesis was done 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. The results obtained shown the presence of H6hmRNA after 24 h of elicited treatment. Strong expression of h6hgene was detected with 2.0 mM of Ac- buffer while a weak expression was detected at 1.0 mM of Ac- buffer.