Caracterização molecular parcial e localização subcelular de um isolado brasileiro do vírus do mosaico do feijoeiro do sul dos E.U.A
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2001-02-22
Autores
Moreira, Andréia Estela [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O presente trabalho descreve a caracterização molecular parcial e a localização subcelular de um isolado brasileiro do vírus do mosaico do feijoeiro do sul dos E.U.A. (southern bean mosaic virus, SBMV, gênero Sobemovirus) encontrado no Estado de São Paulo e aqui intitulado SBMV-BSP. O SBMV-BSP infectou sistemicamente plantas de Phaseolus vulgaris L. 'Rosinha induzindo mosaico, distorção foliar e redução do tamanho da planta. O SBMV-BSP foi purificado à partir de folhas de feijoeiros, cv. Rosinha, infectados produzindo preparações de bom grau de pureza e concentração viral satisfatória (50-70 mg/Kg de folhas infectadas). Por eletroforese em gel desnaturante SDS-PAGE, foi determinada a massa molecular da proteína capsidial com valor estimado em 30 kDa. Um anti-soro para a proteína capsidial foi obtido de galinhas imunizadas com preparações purificadas do vírus (anti-SBMV), sendo este específico e de alto título (1:128.000). Em SDS-PAGE, uma análise comparativa de extratos de planta sadia e infectada evidenciou a presença de uma banda a mais neste último que foi identificada por Western blot utilizando o anti-SBMV como sendo correspondente à proteína capsidial do SBMV-BSP. RNAs de vários tamanhos (4,2 Kb; 3,1 Kb; 2,65 Kb; 2,15 Kb; 1,64 Kb; 1,36 Kb; e 1,0 Kb) foram obtidos do SBMV-BSP purificado, confirmando a heterogeneidade dos RNAs presentes nos virions. Uma sonda molecular não radioativa que reconhece especificamente os RNAs do SBMV-BSP e não reconhece RNAs de planta sadia foi produzida. Através de Northern blot e hibridização com essa sonda todos os fragmentos de RNAs foram detectados também em extratos de plantas infectadas pelo vírus, comprovando pela primeira vez a presença dos RNAs heterogêneos na planta hospedeira. Duas espécies de RNAs de fita dupla foram extraídas a partir de extratos de plantas de feijoeiro infectadas pelo SBMV-BSP,...
The present work describes the partial molecular characterization and the subcellular localization of a Brazilian isolated of Southern bean mosaic virus (SBMV, genus Sobemovirus) found in the São Paulo State and designated SBMV-BSP. The SBMV-BSP infected systemically plants of Phaseolus vulgaris L Rosinha inducing mosaic, leaf distortions and reduction of the size of the plant. The SBMV-BSP was purified from infected bean leaves yielding preparations with a good degree of purity and satisfactory virus concentration (50-70 mg/kh of infected leaves). By denaturing gel electrophoresis (SDS-PAGE), the molecular mass of the coat protein was determined to have an estimated value of 30 kDa. An antiserum for the coat protein was obtained from hens immunized with purified viral preparation (anti-SBMV), having high specificity and title (1:120,000). In SDS-PAGE a comparative analysis of infected and healthy plants showed the presence of an extra band in extracts of infected plants identified by Western blot, using anti-SBMV, as the coat protein of the SBMV-BSP. RNAs of many sizes (4.2 Kb; 3.1 Kb; 2.65 Kb; 2.15 Kb; 1.64 Kb; 1.36 Kb; e 1.0 Kb) were detected in SBMV-BSP purified preparations, confirming the heterogeneity of the RNAs found in virions. A non-radioactive molecular probe was produced which recognizes specifically viral RNAs but does not recognize RNAs from healthy plants. Using Northern blot and hybridization with the specific probe it was shown that the heterogenous RNA fragments were also found in extracts of infected plants providing, for the first time, evidence of their presence in the host plant. Two species of double stranded RNAs (dsRNA) were obtained from extracts of SBMV-BSP infected plants, with sizes of 4.2 Kbp and 1.0 Kbp. In bean leaves, SBMV-BSP particles were seen dispersed in the cytoplasm and sometimes arranged in almost crystalline patterns...(Complete abastract click electronic access below)
The present work describes the partial molecular characterization and the subcellular localization of a Brazilian isolated of Southern bean mosaic virus (SBMV, genus Sobemovirus) found in the São Paulo State and designated SBMV-BSP. The SBMV-BSP infected systemically plants of Phaseolus vulgaris L Rosinha inducing mosaic, leaf distortions and reduction of the size of the plant. The SBMV-BSP was purified from infected bean leaves yielding preparations with a good degree of purity and satisfactory virus concentration (50-70 mg/kh of infected leaves). By denaturing gel electrophoresis (SDS-PAGE), the molecular mass of the coat protein was determined to have an estimated value of 30 kDa. An antiserum for the coat protein was obtained from hens immunized with purified viral preparation (anti-SBMV), having high specificity and title (1:120,000). In SDS-PAGE a comparative analysis of infected and healthy plants showed the presence of an extra band in extracts of infected plants identified by Western blot, using anti-SBMV, as the coat protein of the SBMV-BSP. RNAs of many sizes (4.2 Kb; 3.1 Kb; 2.65 Kb; 2.15 Kb; 1.64 Kb; 1.36 Kb; e 1.0 Kb) were detected in SBMV-BSP purified preparations, confirming the heterogeneity of the RNAs found in virions. A non-radioactive molecular probe was produced which recognizes specifically viral RNAs but does not recognize RNAs from healthy plants. Using Northern blot and hybridization with the specific probe it was shown that the heterogenous RNA fragments were also found in extracts of infected plants providing, for the first time, evidence of their presence in the host plant. Two species of double stranded RNAs (dsRNA) were obtained from extracts of SBMV-BSP infected plants, with sizes of 4.2 Kbp and 1.0 Kbp. In bean leaves, SBMV-BSP particles were seen dispersed in the cytoplasm and sometimes arranged in almost crystalline patterns...(Complete abastract click electronic access below)
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Palavras-chave
Virus do mosaico do feijão, Fitopatologia
Como citar
MOREIRA, Andréia Estela. Caracterização molecular parcial e localização subcelular de um isolado brasileiro do vírus do mosaico do feijoeiro do sul dos E.U.A. 2001. 105 f. Dissertação (mestrado) - Universidade Estadual Paulista, Instituto de Biociências, Letras e Ciências Exatas, 2001.