Isolation and characterization of latex-specific promoters from Papaver somniferum L.
Raymond, Michelle Jean
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The pharmacologically important alkaloids morphine and codeine are found in latex of opium poppy (Papaver somniferum). Latex is harbored in laticifers, a specialized vascular cell-type. Isolation and characterization of latex-specific genes may provide a useful tool to metabolically engineer increased alkaloid production. Previous research in the Nessler laboratory identified genes that exhibit latex-specific gene expression. Latex-specific genes were an 2-oxoglutarate-dioxygense (DIOX), involved in hydroxylation, desaturation and epoxidation reactions, and two of the major latex proteins, MLP146 and MLP149. MLP-like proteins function in fruit ripening in various species that do not have the laticifer cell type. The latex-specific promoters (LSPs) for the three genes were sequenced. The 2.5 kb DIOX promoter was fused to the reporter gene Β-glucuronidase (GUS) to characterize its expression pattern. To assess the functional sites within the DIOX promoter, deletions were made 1.5 kb and 0.14 kb upstream of the ATG start codon, fused to GUS, and transformed into opium poppy, Arabidopsis thaliana, and tobacco (Nicotiana tabacum). The 2.5 kb DIOX:GUS and 1.5 kb EcoRIDIOX:GUS reporter gene constructs showed vascular specific expression in opium poppy, Arabidopsis, and tobacco. The 0.14 kb SpeIDIOX promoter deletion construct showed no activity in opium poppy, and limited expression in the shoot apical meristem and root hypocotyl axis in Arabidopsis. These results indicate that the minimum active DIOX promoter is greater than 0.14 kb. Over 1 kb of the LSPs were sequenced and analyzed for regulatory elements using the Plant cis-acting regulatory DNA elements database, PLACE (http://www.dna.affrc.go.jp/PLACE). Knowledge of the cis-elements and regulatory regions of LSPs would serve as a tool for metabolic engineering of poppy alkaloids. Sixty-five elements were conserved among 2 of the 3 LSPs. Among the cis-elements identified, some are associated with basic functions such as: light regulation, carbon metabolism and plant defense. Other elements include: WRKY elements that are binding sites of transcription factors known for signaling plant defense genes, a vascular cis-element, and a fruit specific element. The presence of plant defense and vascular cis-elements in the LSPs, correlate with the concept that latex is a protective defense mechanism found in the vascular system. The latex-specific promoters isolated and cis-elements identified in this research are potential tools for driving increased alkaloid production in opium poppy.
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