Metabolic research for development of opium poppy producing high amount of morphine

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To develop opium poppy producing high amount of morphine, I attempted to investigate the metabolic mechanism of morphine in opium poppy and clone genes encoding morphine-metabolizing enzymes. Consequently, I demonstrated that in response to wounding, morphine is metabolized into bismorphine by cell wall peroxidases. After purification of these enzymes using various chromatography, the purified enzymes were analyzed by the amino-add sequencer, but I could obtain no information on the primary structures of the enzymes. The cloning of morphine-metabolizing enzymes was achieved by PCR reaction. As a first step, degenerate PCR was camed out using primers designed from the conserved regions in the plant peroxidases. Finally, three genes encoding full-length regions were isolated by 5'-RACE and 3'-RACE. Analysis of their amino add sequences demonstrated that these genes encode peroxidases. Although heterogeneous expression of them using E. coil was attempted, I could not express these enzymes in active forms. Now I try to express these genes using insect cells.