RESUMEN
Rice stripe virus (RSV) causes dramatic losses in rice production worldwide. In this study, two monoclonal antibodies (MAbs) 16E6 and 11C1 against RSV and a colloidal gold-based immunochromatographic strip were developed for specific, sensitive, and rapid detection of RSV in rice plant and planthopper samples. The MAb 16E6 was conjugated with colloidal gold and the MAb 11C1 was coated on the test line of the nitrocellulose membrane of the test strip. The specificity of the test strip was confirmed by a positive reaction to RSV-infected rice plants and small brown planthopper (SBPH), and negative reactions to five other rice viruses, healthy rice plants, four other vectors of five rice viruses, and non-viruliferous SBPH. Sensitivity analyses showed that the test strip could detect the virus in RSV-infected rice plant tissue crude extracts diluted to 1:20 480 (w/v, g/mL), and in individual viruliferous SBPH homogenate diluted to 1:2560 (individual SPBH/μL). The validity of the developed strip was further confirmed by tests using field-collected rice and SBPH samples. This newly developed test strip is a low-cost, fast, and easy-to-use tool for on-site detection of RSV infection during field epidemiological studies and paddy field surveys, and thus can benefit decision-making for RSV management in the field.
Asunto(s)
Anticuerpos Monoclonales/química , China , Cromatografía de Afinidad/métodos , Colodión/química , Coloides/química , Oro Coloide/química , Ensayo de Materiales , Membranas Artificiales , Oryza/virología , Enfermedades de las Plantas/virología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Especificidad de la Especie , Tenuivirus/aislamiento & purificaciónRESUMEN
The non-structural NS3 protein gene from the rice hoja blanca virus (RHBV) was fused to the glutathione-S-transferase carboxilic end and expressed in Escherichia coli strain JM83. Large quantities of fusion protein were produced in insoluble form. The fusion protein was fractionated in SDS-PAGE and purified by electroelution, polyclonal antibodies were raised in rabbit and the antiserum was absorbed with bacterial crude extract. A band of similar size as that of NS3 protein was observed in Western blots using extracts from RHBV-infected rice plants. Immunoelectron microscopy with colloidal gold-labeled antibodies against NS3 protein and the viral nucleocapsid protein revealed in situ accumulation of NS3 protein in the cytoplasm but not in the viral inclusion bodies, vacuoles or chloroplasts of RHBV-infected plants, following the same pattern of distribution as the RHBV nucleocapsid protein.
Asunto(s)
Animales , Conejos , Expresión Génica , Oryza/virología , Proteínas no Estructurales Virales/genética , Tenuivirus/química , Virus de Plantas/genética , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Microscopía Inmunoelectrónica , Proteínas no Estructurales Virales/metabolismo , Virus de Plantas/metabolismo , Western BlottingRESUMEN
Plant regeneration from seven-week-old callus cultures derived from mature embryos of several indica rice cultivars was achieved with frequencies of morphogenic calli from 10 to 47 percent. Three media were tested both for callogenesis and plant regeneration. For 3 of the 7 genotypes examined, the best combination of media for plant regeneration was Murashige & Skoog basal medium: MSC (callogenesis) and MSR (regeneration). The rates of callogenesis were not related to the capacity for plant regeneration. Two genotypes CR-1113 and CR-5272 produced the highest number of regenerated green plants. The results of this study suggest that genetic differences could be directly linked to the ability to regenerate in these plant cultivars