RESUMO
A synthetic DNA construct containing cholera toxin B subunit, genetically fused to the surface glycoprotein of rabies virus was expressed in tobacco plants from a seed specific (legumin) promoter. Seed specific expression was monitored by real-time PCR, GM1-ELISA and Western blot analyses. The fusion protein accumulated in tobacco seeds at up to 1.22% of the total seed protein. It was functionally active in binding to the GM1-ganglioside receptors, suggesting its assembly into pentamers in seeds of the transgenic plants. Immunoblot analysis confirmed that the approximately 80.6 kDa monomeric fusion polypeptide was expressed in tobacco seeds and accumulated as an approximately 403 kDa pentamer. Evaluation of its immunoprotective ability against rabies and cholera is to be examined.
Assuntos
Toxina da Cólera/metabolismo , Glicoproteínas/metabolismo , Nicotiana/genética , Vírus da Raiva/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sementes/genética , Proteínas Virais/metabolismo , Western Blotting , Clonagem Molecular , DNA de Plantas/genética , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica de Plantas , Genoma de Planta/genética , Immunoblotting , Plantas Geneticamente Modificadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transgenes/genéticaRESUMO
BACKGROUND: Spodoptera litura (F.) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). Constitutive expression of δ-endotoxin Cry1EC gives protection against S. litura, as reported earlier. In this study, insect bites and salicylic acid induced high-level expression of Cry1EC was achieved in peanut. In order to achieve this, the expression of pathogenesis responsive promoter PR-1a was enhanced by placing it downstream of the CaMV35S promoter in the pCAMBIA 1300 backbone. The resultant promoter CaMV35S(r)PR-1a expressed a high level of insecticidal δ-endotoxin Cry1EC. The Gus expression under the control of CaMV35S(r)PR-1a served as a convenient marker for evaluation of promoter response to different treatments. RESULTS: Transgenic events that showed a very low level of uninduced expression and no expression in seeds were selected. The Cry1EC expression in leaves increased nearly eightfold in the selected event, following induction by salicylic acid. Both the salicylic-acid-treated and the S. litura-bitten leaves showed the highest expression after 2 days. Leaves from salicylic-acid-induced transgenic plants caused 100% mortality of S. litura at all stages of larval development. CONCLUSION: The results suggest that high expression of inducible promoters provides a good strategy for the development of safer transgenic food and feed crops.
Assuntos
Arachis/imunologia , Proteínas de Bactérias/imunologia , Endotoxinas/imunologia , Expressão Gênica , Proteínas Hemolisinas/imunologia , Controle de Insetos/métodos , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas/imunologia , Regiões Promotoras Genéticas , Spodoptera/efeitos dos fármacos , Animais , Arachis/genética , Arachis/parasitologia , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/genética , Proteínas de Bactérias/farmacologia , Endotoxinas/genética , Endotoxinas/farmacologia , Regulação da Expressão Gênica de Plantas , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/farmacologia , Doenças das Plantas/parasitologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/parasitologia , Spodoptera/fisiologiaRESUMO
The tobacco cutworm (Spodoptera litura) is a polyphagous foliage insect and a major pest on peanut (Arachis hypogaea L.). S. litura is susceptible to the chimeric delta-endotoxin Cry1EC reported earlier. De-embryonated cotyledon explants of peanut were transformed using Agrobacterium tumefaciens strain EHA101 harboring a synthetic cry1EC gene driven by the CaMV 35S promoter. Transgenic plants of peanut with a single copy insertion of cry1EC were selected in the T(0) generation by Southern blot hybridization. Real-time PCR, Western blot and ELISA analysis indicated that expression of the cry1EC gene was higher in single copy T(1) plants. Immunoassay showed expression of Cry1EC up to 0.13% of total soluble protein in T(1) plants. Leaf feeding bioassay on highly expressing transgenic lines showed 100% killing of larvae at the 2(nd) instar stage of S. litura. This is the first report of transgenic peanut plants with resistance to S. litura.