Caracterização molecular e biológica de begomovírus de soja (Glycine max) e leiteiro (Euphorbia heterophylla) e resistência a vírus mediada por RNA interferente em plantas transgênicas de soja

• Autor(a) principal: Fernandes, Fernanda Rausch
• Data de Publicação: 2009
• Tipo de documento: Tese
• Idioma: por
• Título da fonte: LOCUS Repositório Institucional da UFV
• Texto Completo: http://locus.ufv.br/handle/123456789/1023
• topic: Soja; Resistência a doenças; Begomovirus; Glycine max; Euphorbia heterophylla; Soybean; Resistance to diseases; Begomovirus; Glycine max; Euphorbia heterophylla; CNPQ::CIENCIAS AGRARIAS::AGRONOMIA::FITOSSANIDADE::FITOPATOLOGIA

Molecular characterization of begomoviruses is of fundamental importance to identify the mechanisms of genetic variability of these important plant pathogens. Genome analysis allows for an understanding of mutation and recombination mechanisms which lead to evolution of new viral species. In Brazil, only a few reports of begomovirus infection of soybean have been made to date. Foliar samples of soybean plants with typical symptoms of begomovirus infection were collected in a commercial field at Santo Antônio de Goiás (Goiás state) and analyzed for the presence of a begomovirus. Following confirmation of viral infection by PCR, the entire viral genome was amplified from infected samples by rolling circle amplification, cloned and sequenced. Sequence analysis indicated infection by three distinct begomoviruses: Bean golden mosaic virus (BGMV), Sida micrantha mosaic virus (SiMMV) and Okra mottle virus (OMoV), the latter having been recently described in okra plants in Brazil, and now for the first time in soybean. A begomovirus isolate detected in Euphorbia heterophylla, a common weed in soybean fields, was also cloned and shown to comprise an isolate of the recently described novel species, Euphorbia mosaic Peru virus (EuMPV). The carlavirus Cowpea mild mottle virus (CpMMV) is a plant pathogen of relatively recent occurrence in soybean, representing a potential threat to the crop under Brazilian conditions. RNA silencing is a natural plant defense against viruses which has been widely employed to generate virus- resistant transgenic plants. In this work, RNA silencing was targeted to a sequence of the CpMMV capsid protein (CP) gene in an attempt to generate resistant soybean plants. In another construct, fragments of the SiMMV Rep gene and of the CP gene of the potyvirus Soybean mosaic virus (SMV) were also used in an attempt to generate transgenic soybean plants displaying resistance to both viruses. The pCARLA vector contains a 493 bp fragment of the CpMMV CP gene, and was used to transform soybean embryos by biolistics. The pGEMPOTY vector contains a 303 bp fragment of the SMV CP gene and a 404 bp fragmento of the SiMMV Rep gene. Plants generated from the bombardment with the pGEMPOTY vector were regenerated in selective medium and are currently being tested for the presence of the transgenes. Five transformants were obtained with the pCARLA vector, and were challenged with CpMMV. The transgenic plants did not show any visible symptoms of virus infection. The progeny of the transgenic plants will be challenged with CpMMV to further confirm these results and to verify stable inheritance and expression of the transgene.