RESUMO
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.
Assuntos
Glicosídeos/biossíntese , Raízes de Plantas/genética , Alcaloides de Solanáceas/biossíntese , Alcaloides de Solanáceas/genética , Solanum/metabolismo , Sequência de Aminoácidos , Anticorpos/genética , Anticorpos/metabolismo , Sequência de Bases , Linhagem Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Glicosídeos/química , Glicosídeos/imunologia , Hibridomas , Fragmentos de Imunoglobulinas/imunologia , Dados de Sequência Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alcaloides de Solanáceas/química , Alcaloides de Solanáceas/imunologia , Alcaloides de Solanáceas/metabolismo , Solanum/genéticaRESUMO
Solasodine glycosides were analyzed for the selection of higher-producing natural resources in the fruits of Solanum spp. by western blotting and ELISA using anti-solamargine monoclonal antibody (MAb). Western blotting of Solanum khasianum showed a correlation with the analysis by ELISA. The results indicated that the fruits of S. khasianum contained the highest level of solasodine glycosides, compared to other species having 53.43 +/- 3.90 microg/mg dry wt. The high level of solasodine glycosides in S. khasianum suggests that it may be suitable for commercial production.
Assuntos
Anticorpos Monoclonais , Fitosteróis/imunologia , Solanaceae/química , Alcaloides de Solanáceas/análise , Alcaloides de Solanáceas/imunologia , Western Blotting , Calibragem , Sequência de Carboidratos , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Frutas/química , Glicosídeos/análise , Humanos , Dados de Sequência MolecularRESUMO
Solakhasoside (1), a novel steroidal saponin, was isolated from the fruit of Solanum khasianum. Its structure was determined as (23S, 25S)-spirot-5-en-3beta,17alpha, 23-triol-3-O-[alpha-L-rhamnopyranosyl-(1-->2)[beta-D-xylopyranosyl-(1 -->3)]-beta-D-galactopyranoside] (1) by spectroscopic analysis.
Assuntos
Oligossacarídeos/isolamento & purificação , Solanaceae/química , Configuração de Carboidratos , Sequência de Carboidratos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Oligossacarídeos/química , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
Immunoaffinity column using anti-solamargine monoclonal antibody for separation of solasodine glycosides was established. This method was specific for solasodine glycosides which was detected by thin layer chromatography and the western blotting. Total solasodine glycosides have been separated directly from the crude extract of Solanum khasianum fruit by the newly established immunoaffinity column.
RESUMO
A method of determination for solasodine glycosides by using thin-layer chromatography (TLC) immunostaining was investigated. Solasodine glycosides separated by silica gel TLC were transferred to a polyvinylidene difluoride membrane. The membrane was treated with sodium periodate solution followed by bovine serum albumin (BSA), resulting in a solasodine glycoside-BSA conjugate. Conjugation was confirmed by matrix-assisted laser desorption/ionization mass spectrometry. Individual spots were stained by monoclonal antibody against solamargine. Immunostaining of solasodine glycosides was more sensitive compared to other stainings. The newly established immunostaining method can be extended to analysis of the distribution of solasodine glycoside in the plant body.
