Caracterização Molecular Do Plasmídeo pLK39 Extraído De Uma Bactéria Endofítica Isolada De Solanum Lycocarpum

• Ano de defesa: 2002
• Autor(a) principal: OLIVEIRA, Vera Lúcia Cardoso de
• Orientador(a): BATAUS, Luiz Artur Mendes
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Goiás
• Programa de Pós-Graduação: Mestrado em Biologia
• Departamento: Ciências Biolóicas
• País: BR
• Palavras-chave em Português: Bactéria; Endofítico; DNA; Plasmídeo; 1.Microorganismos Endofíticos 2.Plasmídeos 3.Plasmídeo pLK39
• Palavras-chave em Inglês: Bactéria; Endophytic; DNA; Plasmid
• Área do conhecimento CNPq: CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA
• Link de acesso: http://repositorio.bc.ufg.br/tede/handle/tde/1287

The characterization of the plasmid pLK39.was the aim of work.pLK39 was isolated from a endophytic Gram-negative, bactéria isolated from leaves form Solanum lycocarpum (Lobeira). In order to obtain a selection marker which would characterization in Escherichia coli the kanamycin, resistence gene from pUC4K. was subcloned into the PstIsite of pLK39. The characterization of PLK39 was basedon studies of stability, incompatibility, determination of the copy number and through DNA sequencing. The stability was carried out in Escherichia coli XL10 cells. Cells transformed with pLK39 were inoculated in Lúria broth containing Kanamycin (50μg/ml) during24 hours. After 24 hours of incubation one sample of the culture was in medim with and without selective pressure, and inoculated in Lúria broth without pressure médium, for 240 generations. After 240 generations 81,5% of cells maintained the plasmid, showing that pLK39is highly stable in Escherichia coli to make the incompatibility test, cells of Escherichia coli XL10 were co-transformed with pLK39/pUC18; pBR322 and pLK39/pACYC184. After 240 generations, the plasmid pLK39 iof detected in all systems used, showing the compatibility of pLK39 with the plasmids tested. The copý number pLK39 was estimated by the intensity of plasmidial bands in agarose gel, electrophoresis using a photodocumentation and analysis system (KODAK EDAS). The copý number of pLK39 was estimated as 25 copies per cell. PLK39 was digested with Sau3AI restriction and subcloned into the BamHI site of plasmid pUC18. The recombinant clones were sequenced and 1637 pb reading fragment was obtained. This sequence showed high homology with pSW200, pSW100, pEC3, pUCD5000 and pBERT, which were isolated from Gram-negative bacteria. This sequence possesses two possible genes. The ORF I showed high homology with mobB gene from plasmid pSW200 (96%), pSW100 (96%), pEC3 (94%), pUCD5000 (94%) and pBERT (87%). The ORF II showed homology with mobD gene from plasmids pSW200 (92%), pSW100 (90%), pEC3 (90%) and PUCD5000 (89%).