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1.
Biosci. j. (Online) ; 31(4): 1133-1142, july/aug. 2015.
Artigo em Inglês | LILACS | ID: biblio-964569

RESUMO

In this work, a virus isolate collected from pumpkin plants (Cucurbita pepo L.), showing severe symptoms of mosaic and leaf deformation, grown in Cuba, was analyzed using indicator plants, electron microscopy, and phylogenetic analysis. Plants of pumpkin, cv. Caserta, inoculated with this virus isolate showed mosaic, leaf distortion and blistering symptoms, whereas papaya plants were immune and did not show any symptoms. A transmission electron microscopic examination of leaf dip preparations made from infected pumpkin leaves revealed the presence of elongated and flexuous particles, approximately 780-800 x 12 nm in size. Genomic fragments containing the coat protein (CP) and HC-Pro genes, amplified by specific primers for Papaya ringspot virus, W strain (PRSV-W), showed amino acid identities of both genes higher than 94% when compared to other PRSV-W isolates from America. In the phylogenetic tree, this virus isolate has grouped with other virus isolates from America, Australia, and India and was more distant from the Asian isolates. Taken together, the analyses allow the conclusion that this virus isolate is a W strain of PRSV, detected for the first time in Cuba.


Neste trabalho um isolado viral coletado em planta de abóbora (Cucurbita pepo L), apresentando sintomas severos de mosaico e deformação foliar, proveniente de uma lavoura localizada em Cuba, foi analisado utilizando plantas indicadoras, microscopia eletrônica de transmissão e análise filogenética. Plantas de abóbora cv. Caserta, inoculadas com este isolado do vírus mostrou mosaico, distorção foliar e bolhas, enquanto que as plantas de mamão foram imunes e não apresentaram sintoma. Exame ao microscópio eletrônico de tranmissão de telas preparadas com a técnica leaf dip, empregando o extrato de folhas de abóbora infectadas revelou a presença de partículas alongadas e flexuosas, medindo cerca de 780-800 x 12 nm. A análise de fragmentos genômicos contendo os genes da proteína capsidial (CP) e HC-Pro, amplificados por primers específicos para Papaya ringspot virus, estirpe W (PRSV -W), mostrou identidades de aminoácidos superiores a 94 % quando ambos os genes foram comparados a outros isolados americanos de PRSV -W. Na árvore filogenética, este isolado estudado se agrupou com os isolados de PRSV-W da América, Austrália e Índia, ficando mais distante dos isolados asiáticos. Tomadas em conjunto, as análises permitem concluir que este isolado viral pertence à estirpe W do PRSV, detectada pela primeira vez em Cuba.


Assuntos
Filogenia , Cucurbita pepo , Cuba , Cucurbita , Proteínas do Capsídeo , Biologia Molecular
2.
Electron. j. biotechnol ; 10(2): 178-190, Apr. 15, 2007. ilus, graf
Artigo em Inglês | LILACS | ID: lil-499183

RESUMO

Gene silencing, also called RNA interference (RNAi) is a specific mechanism of RNA degradation involved in gene regulation, development and defense in eukaryotic organisms. It became an important subject in the teaching programs of molecular biology, genetics and biotechnology courses in the last years. The aim of this work is to provide simple and inexpensive assays to understand and teach gene silencing using plants as model systems. The use of transient and permanent transgenic plants for expressing reporter genes, like those derived from jellyfish green fluorescent protein (gfp) encoding gene, provides a nice, colorful and conclusive image of gene silencing. Three experimental approaches to evidence RNA silencing are depicted. In the first approach gene silencing is demonstrated after transient expression of reporter genes in non-transgenic plants. In the second, silencing is triggered against a reporter gene stably integrated into a transgenic plant. The third approach involves the triggering of RNA silencing against endogenous genes using viral vectors. In addition we illustrate systemic gene silencing showing how the silencing signal is spread over a plant and finally it is also demonstrated the suppression of gene silencing. The first group of experiments is recommended to be tough on undergraduate courses, the following two sections are recommended for graduate courses. Hopefully, it will help students to understand this important phenomenon and to unravel the importance of gene silencing as a key gene regulation mechanism and as a molecular and biotechnological tool.


Assuntos
RNA de Plantas/genética , Inativação Gênica , Interferência de RNA , Ensino , Biotecnologia/educação , Proteínas de Fluorescência Verde , Modelos Genéticos , Plantas Geneticamente Modificadas/genética , Interferência Viral
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