Enhanced Secretory Activity of Atropa belladonna Hairy Root Culture Over-expressing ADP-Ribosylation Factor Gene(Pharmacognosy)

Agrobacterium-mediated transformation of leaf tissues of Atropa belladonna with an adenosine 5'-diphosphate (ADP)-ribosylation factor gene of carrot, arf-001, was performed employing pBCR82 as an expression vector. This vector co-expresses rot gene cluster together with arf-001, and thus, the transformed host cells were obtained as hairy roots. Two cell lines of the transformed belladonna were established as the liquid cultures of hairy root tissues, and expression of arf-001 and accumulation of its product in the cells were confirmed by RT-PCR and Western blot analyses, respectively. A marked increase in extracellular protein concentrations was observed in the transformed belladonna root cultures as compared with the controls transformed with an empty vector. However, the secretion of the proteins of the transformants was markedly reduced in the presence of a physiological concentration of monensin. These results suggest that over-expression of arf-001 in belladonna results in the enhancement of secretory activity in the transformed cells.