RESUMEN
The antifungal activity of hevein-like proteins has been associated with their chitin-binding activities. Pn-AMP1 and Pn-AMP2, two hevein homologues from Pharbitis nil, show in vitro antifungal activities against both chitin and non-chitin containing fungi. Purified Pn-AMPs retained antifungal activities only under non-reducing conditions. When Pn-AMP2 cDNA was constitutively expressed in tomato (Lycopersicon esculentum) plants under the control of CaMV35S promoter, the transgenic plants showed enhanced resistance against both the non-chitinous fungus Phytophthora capsici, and the chitin-containing fungus Fusarium oxysporum. Thus, the chitin component in the fungal cell wall is not an absolute requirement for Pn-AMP's antifungal activities. These results when considered together suggest that Pn-AMPs have the potential for developing transgenic plants resistant to a wide range of phytopathogenic fungi.
Asunto(s)
Antifúngicos/química , Antifúngicos/farmacología , Convolvulaceae/química , Fusarium/efectos de los fármacos , Phytophthora/efectos de los fármacos , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Solanum lycopersicum/microbiología , Secuencia de Aminoácidos , Antifúngicos/aislamiento & purificación , Antifúngicos/metabolismo , Northern Blotting , Quitina/metabolismo , Electroforesis en Gel de Poliacrilamida , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Pruebas de Sensibilidad Microbiana , Peso Molecular , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/aislamiento & purificación , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
In order to produce a human lactoferrin (hLf) protein in cultured plant cells, we developed Korean ginseng (Panax ginseng) cell line using an oxidative stress-inducible peroxidase (SWPA2) promoter and characterized the production of human lactoferrin in cultured cells. A construct containing a targeting signal peptide from tobacco endoplasmic reticulum fused to human lactoferrin cDNA under the control of SWPA2 promoter was engineered. Transgenic Korean ginseng cell lines that produced a recombinant hLf protein were successfully generated and confirmed by PCR and Southern blot analyses. Western blot and ELISA analyses showed that hLf protein was synthesized in the transgenic cells. The production of hLf showed a maximal level (up to 3.0% of total soluble protein) in the stationary phase of callus cultures. These results suggest that the transgenic cell lines in this study will be biotechnologically useful for the commercial production of hLf protein in cell cultures, with no need for purification.
Asunto(s)
Lactoferrina/biosíntesis , Lactoferrina/genética , Nicotiana/genética , Nicotiana/metabolismo , Panax , Fitoterapia , Células Cultivadas , Humanos , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
Transgenic Nicotiana tabacum cell lines were developed expressing the human lactoferrin gene driven by the oxidative stress-inducible peroxidase (SWPA2) promoter. Western blot analysis showed the accumulation of both the full-length human lactoferrin protein as well as a immuno-reactive truncated fragment. Accumulation of human lactoferrin as monitored by ELISA increased proportionally to cell growth and reached a maximal (up to 4.3% of total soluble proteins) at the stationary phase of growth. Protein extracts from transgenic tobacco cells exhibited antibacterial activity.