Data/hora: 31/05/2024 08:05:45

Biblioteca(s): | Embrapa Hortaliças.
Data corrente: | 05/12/2007
Data da última atualização: | 24/01/2024
Autoria: | FERREIRA, P. de T de O.; LEMOS, T. O.; NAGATA, T.; INOUE-NAGATA, A. K.
Afiliação: | PAULO DE TARSO DE OLIVEIRA FERREIRA; THAÍS OLIVEIRA LEMOS, UNIVERSIDADE CATÓLICA DE BRASÍLIA; TATSUYA NAGATA, UNIVERSIDADE CATÓLICA DE BRASÍLIA; ALICE KAZUKO INOUE NAGATA, CNPH.
Título: | One-step cloning approach for construction of agroinfectious begomovirus clones.
Ano de publicação: | 2008
Fonte/Imprenta: | Journal of Virological Methods, Amsterdam, v. 147, n. 2, p. 351-354, Feb. 2008.
ISSN: | 1879-0984
DOI: | 10.1016/j.jviromet.2007.10.001
Idioma: | Inglês
Conteúdo: | Most begomoviruses have a bipartite genome containing two circular ssDNA segments (DNA-A and DNA-B). Routine infectious clone construction relies upon cloning of the whole genome, which is then subcloned as a tandem one-and-half or two genome- (containing two replication origins) cassette into a vector prior to agro-inoculation. The construction of cassettes containing two replication origins is, however, a time-consuming process. Here an improved method for rapid construction of agroinfectious begomovirus clones is described. Total DNA was extracted from a tomato plant infected with Tomato golden vein virus and viral ssDNA molecules were amplified using phi-29 bacteriophage polymerase. High molecular weight DNA was partially digested with a single cutting restriction endonuclease (BamHI) and DNA fragments containing genome dimers were ligated into pCAMBIA0380, and used to transform Escherichia coli cells. This transformation yielded clones containing either DNA-A or DNA-B dimers. One clone each was used to transform Agrobacterium tumefaciens cells. DNA-A and DNA-B transformants were mixed and inoculated into test plants. All inoculated plants (tomato and Nicotiana benthamiana) became infected, confirming the infectivity of the clones. This approach was proven to be extremely fast and useful for the production of infectious clones.
Palavras-Chave: | Cultivar Santa Clara.
Thesagro: | Agrobacterium Tumefaciens; Clonagem; Tomate.
Thesaurus Nal: | Begomovirus; Cloning (plants); Solanum lycopersicum.
Categoria do assunto: | --
Registro original: | Embrapa Hortaliças (CNPH)
Exemplares: | CNPH - (33031-1) - UPC - AP - DD - 017135 -  - 017135 - DIS