RESUMO
The conditions of high efficient chicory transformation with Mycobacterium tuberculosis antigene ESAT6 have been determined. Transformation frequency was up to 86% when the cotyledons were cultivated within 3 days without cefotaxime and then 1 day without kanamycine. DNA PCR-analysis has shown the presence both of selective nptII and target esxA genes in all analysed plants. At the same time RT-PCR has shown the presence of nptII transcripts for eight analysed lines and esxA transcripts for only five analysed lines.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Cichorium intybus/genética , Mycobacterium tuberculosis/imunologia , Plantas Geneticamente Modificadas/genética , Transformação Genética , Antígenos de Bactérias/biossíntese , Proteínas de Bactérias/biossíntese , Cichorium intybus/crescimento & desenvolvimento , DNA de Plantas/genética , Engenharia Genética , Vetores Genéticos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Reação em Cadeia da Polimerase , Vacinas contra a Tuberculose/genéticaRESUMO
Green fluorescent protein (GFP) is commonly used as a reporter protein in a wide range of biological experiments. The efficient protocol of Agrobacterium-mediated transient expression in Nicotiana excelsior was applied for quick preparative production of recombinant GFP. The protein purification scheme has been developed and included ammonium sulfate precipitation and Q-sepharose anion-exchange chromatography. It results in obtaining of a fraction with about 85% GFP homogeneity and the protein yield of about 75%.
Assuntos
Proteínas de Fluorescência Verde , Nicotiana/genética , Proteínas Recombinantes , Rhizobium/genética , Eletroforese em Gel de Poliacrilamida , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/isolamento & purificação , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Agrobacterium-mediated transient expression may be regarded as a promising method for inexpensive large-scale production of recombinant proteins. We optimized the protocol of transient expression in Nicotiana benthamiana and compared six Australian species of Nicotiana as hosts for transient expression. The transient expression of GFP under 35S CaMV promoter was observed in all species tested, although the GFP content in leaves of N. benthamiana, N. exigua, and N. excelsior was significantly higher (3.8, 3.7, and 2.0% TSP, respectively). Usage of viral-based expression system resulted in considerable increase of GFP accumulation in N. excelsior and N. benthamiana (63.5 and 16.2% TSP, respectively). We displayed that N. excelsior has the best characteristics in regard to biomass yield as well as GFP accumulation level for both types of the expression cassettes tested.
Assuntos
Genes Reporter , Vetores Genéticos , Nicotiana/genética , Folhas de Planta/genética , Rhizobium , Transgenes , Proteínas do Capsídeo/genética , Regulação da Expressão Gênica de Plantas/genética , Vírus de Plantas/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas/genética , Rhizobium/genéticaRESUMO
Hairy root cultures of Nicotiana benthamiana have been obtained by co-cultivation of leaf explants with Agrobacterium rhizogenes strain A4 harboring a binary vector plasmid, and transgenic nature of the obtained cultures was confirmed by PCR analysis. Transgenic plants were regenerated from hairy roots. The biomass yield of transgenic plants grown in vitro was almost two-fold higher than those of wild-type N. benthamiana plants. They differed from untransformed plants by short internodes, reinforced stem, thick and wrinkled leaves and more developed root system. The level of Agrobacterium-mediated transient expression of green fluorescent protein (GFP) in the regenerated plants was similar to that of untransformed plants.