RESUMEN
A cDNA clone, designated SdGGPPS2, was isolated from young seedlings of Scoparia dulcis. The putative amino acid sequence of the translate of the gene showed high homology with geranylgeranyl diphosphate synthase (GGPPS) from various plant sources, and the N-terminal residues exhibited the characteristics of chloroplast targeting sequence. An appreciable increase in the transcriptional level of SdGGPPS2 was observed by exposure of the leaf tissues of S. dulcis to methyl jasmonate, yeast extract or Ca(2+) ionophore A23187. In contrast, SdGGPPS1, a homologous GGPPS gene of the plant, showed no or only negligible change in the expression level upon treatment with these stimuli. The truncated protein heterologously expressed in Escherichia coli in which the putative targeting domain was deleted catalyzed the condensation of farnesyl diphosphate and isopentenyl diphosphate to liberate geranylgeranyl diphosphate. These results suggested that SdGGPPS2 plays physiological roles in methyl jasmonate and yeast extract-induced metabolism in the chloroplast of S. dulcis cells.
Asunto(s)
Acetatos/farmacología , Ciclopentanos/farmacología , Farnesiltransferasa/genética , Oxilipinas/farmacología , Scoparia/genética , Activación Transcripcional , Secuencia de Aminoácidos , Farnesiltransferasa/química , Farnesiltransferasa/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Fosfatos de Poliisoprenilo/metabolismo , Scoparia/efectos de los fármacos , Scoparia/enzimología , Alineación de Secuencia , Sesquiterpenos/metabolismo , LevadurasRESUMEN
We constructed a recombinant antibody fragment--single chain fragment-variable (scFv) antibody--derived from hybridoma cell lines to control the concentration of solasodine glycosides in hairy root cultures of Solanum khasianum transformed by the anti-solamargine (As)-scFv gene. The properties of the As-scFv protein expressed in Escherichia coli were almost identical to those of the parent monoclonal antibody (MAb). Up to 220 ng recombinant As-scFv was expressed per milligram of soluble protein in transgenic hairy root cultures of S. khasianum. The concentration of solasodine glycosides was 2.3-fold higher in the transgenic than in the wild-type hairy root, as reflected by the soluble As-scFv level and antigen binding activities. These results suggested that the scFv antibody expressed in transgenic hairy roots controlled the antigen level, thus representing a novel plant breeding methodology that can produce secondary metabolites.
Asunto(s)
Glicósidos/biosíntesis , Raíces de Plantas/genética , Alcaloides Solanáceos/biosíntesis , Alcaloides Solanáceos/genética , Solanum/metabolismo , Secuencia de Aminoácidos , Anticuerpos/genética , Anticuerpos/metabolismo , Secuencia de Bases , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación de la Expresión Génica de las Plantas , Glicósidos/química , Glicósidos/inmunología , Hibridomas , Fragmentos de Inmunoglobulinas/inmunología , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alcaloides Solanáceos/química , Alcaloides Solanáceos/inmunología , Alcaloides Solanáceos/metabolismo , Solanum/genéticaRESUMEN
We identified a novel metabolic system of morphine in the opium poppy (Papaver somniferum L.). In response to stress, morphine is quickly metabolized to bismorphine consisting of two morphine units, followed by accumulation in the cell wall. This bismorphine binds predominantly to pectins, which possess high galacturonic acid residue contents, through ionical bonds. Our newly developed method using artificial polysaccharides demonstrated that bismorphine bridges are formed between the two amino groups of bismorphine and the carboxyl groups of galacturonic acid residues, resulting in cross-linking of galacturonic acid-containing polysaccharides to each other. The ability of bismorphine to cross-link pectins is much higher than that of Ca2+, which also acts as a cross-linker of these polysaccharides. Furthermore, we confirmed that cross-linking of pectins through bismorphine bridges leads to resistance against hydrolysis by pectinases. These results indicated that production of bismorphine is a defense response of the opium poppy. Bismorphine formation is catalyzed by anionic peroxidase that pre-exists in the capsules and leaves of opium poppies. The constitutive presence of morphine, together with bismorphine-forming peroxidase, enables the opium poppy to rapidly induce the defense system.