RESUMO
We report on the silencing of codeinone reductase (COR) in the opium poppy, Papaver somniferum, using a chimeric hairpin RNA construct designed to silence all members of the multigene COR family through RNA interference (RNAi). After gene silencing, the precursor alkaloid (S)-reticuline-seven enzymatic steps upstream of codeinone-accumulated in transgenic plants at the expense of morphine, codeine, oripavine and thebaine. Methylated derivatives of reticuline also accumulated. Analysis verified loss of Cor gene transcript, appearance of 22-mer degradation products and reduction of enzyme activity. The surprising accumulation of (S)-reticuline suggests a feedback mechanism preventing intermediates from general benzylisoquinoline synthesis entering the morphine-specific branch. However transcript levels for seven other enzymes in the pathway, both before and after (S)-reticuline, were unaffected. This is the first report of gene silencing in transgenic opium poppy and of metabolic engineering to cause the high-yield accumulation of the nonnarcotic alkaloid reticuline.
Assuntos
Oxirredutases do Álcool/deficiência , Alcaloides/metabolismo , Benzilisoquinolinas/metabolismo , Engenharia Genética/métodos , Morfina/metabolismo , Papaver/genética , Papaver/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , Oxirredutases do Álcool/genética , Alcaloides/genética , Células Cultivadas , Álcool Oxidorredutases Dependentes de NAD(+) e NADP(+) , Plantas Geneticamente Modificadas/genéticaRESUMO
The opium poppy is a source of the pharmaceuticals codeine, morphine and their derived analgesics. Here we describe the initial characterization of the poppy mutant known as top1 (for 'thebaine oripavine poppy 1'), which accumulates the morphine and codeine precursors thebaine and oripavine and does not complete their biosynthesis into morphine and codeine. The original discovery of top1 stimulated a re-engineering of the opioid industry in the island state of Tasmania, which grows over 40% of the world's licit opiates, in order to produce thebaine and oripavine efficiently from morphine-free poppy crops to provide precursors for highly effective analgesics and for treatment of opioid addiction.