Expressão e análise de proteínas recombinantes de Xanthomonas axonopodis pv. citri em E. coli, visando análise estrutural por cristalografia
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Data
2007-01-30
Autores
Bortolossi, Julio Cesar [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O cancro cítrico doença causada pela bactéria gram negativa Xanthomonas axonopodis pv. citri (Xac), representa muitos prejuízos para citricultura brasileira e mundial. Em 2002, a Xac teve todo seu genoma seqüenciado. Uma biblioteca de mütantes foi constituída e os mutantes foram analisados por inoculação in planta, deste modo tres mutantes foram selecionados por apresentarem diferenças nos sintomas, quando comparados com a estirpe selvagem Xac. Os genes nocauteados nos mutantes codificam três proteínas diferentes: DGTP pirofosfato tiamina sintase, envolvida na biossíntese de vitamina B 1, a subunidade E da enzima PHA sintase, que atua na síntese de polihidroxialcanoatos. que acumulados na forma de grânulos intracelulares constituem substâncias de reserva da célula, e podem formar biopolímeros e, ainda o terceiro gene nocauteado, que codifica uma proteína hipotética. As curvas de crescimento revelaram que o mutante da DGTP pirofosfohidrolase tiamina sintase não se desenvolve na planta hospedeira. O mutante da PHA sintase cresce menos que a Xac selvagem nos dois primeiros dias de avaliação, já o mutante da proteína hipotética sofre uma fase de declínio a partir do oitavo dia de avaliação experimental. A subunidade E da PHA sintase da Xac, gene codificador desta proteína foi donado em vetor de expressão e a proteína recombinante foi produzida em E.coli na forma solúvel.
The citric canker disease caused for the bacterium gram negative Xantlzomonas axonopodis pv. citri (Xac), presents many damages for Brazilian and world-wide citriculture. In 2002, aiming to know the mechanisms of attack of the disease, the Xac had alI its sequenced genome. A library of mutants was constituted and the mutants had been analyzed by inoculation in plant, three mutants had been isolated for not to show differences in the symptoms when compared with the wild lineage Xac. The mutants codify three different proteins: DGTP pyrophosphate tiamine synthase involved in biosynthesis B 1 vitamin; enzyme PHA synthase subunit E that is involved in the synthesis of polyhydroxyalkanoate, in the intracellular granule f01111, they constitute substances of reserve of the cell an can still to form biopolymer; and hypothetical proteins. The profile of growth of the wild Xac and the mutated Xacs in the three cases of the mutants had been evaluated in order to compare the profile of growth of the bacterial wild and mutant during the infection in citric host. The growth curves showed that the mutant of the DGTP pyrophosphate tiamine synthase does not develop the disease in the host plant. The PHA synthase grows less than the wild Xac in the first days of evaluaction, however the mutant of the hypotetical, shown a decline period for eighth day of the experimental evaluaction. The subunit E the PHA synthase of the Xac, the code gene of this protein was cloned in expression vector and the recombinant protein was produced in E.coli in the soluble form.
The citric canker disease caused for the bacterium gram negative Xantlzomonas axonopodis pv. citri (Xac), presents many damages for Brazilian and world-wide citriculture. In 2002, aiming to know the mechanisms of attack of the disease, the Xac had alI its sequenced genome. A library of mutants was constituted and the mutants had been analyzed by inoculation in plant, three mutants had been isolated for not to show differences in the symptoms when compared with the wild lineage Xac. The mutants codify three different proteins: DGTP pyrophosphate tiamine synthase involved in biosynthesis B 1 vitamin; enzyme PHA synthase subunit E that is involved in the synthesis of polyhydroxyalkanoate, in the intracellular granule f01111, they constitute substances of reserve of the cell an can still to form biopolymer; and hypothetical proteins. The profile of growth of the wild Xac and the mutated Xacs in the three cases of the mutants had been evaluated in order to compare the profile of growth of the bacterial wild and mutant during the infection in citric host. The growth curves showed that the mutant of the DGTP pyrophosphate tiamine synthase does not develop the disease in the host plant. The PHA synthase grows less than the wild Xac in the first days of evaluaction, however the mutant of the hypotetical, shown a decline period for eighth day of the experimental evaluaction. The subunit E the PHA synthase of the Xac, the code gene of this protein was cloned in expression vector and the recombinant protein was produced in E.coli in the soluble form.
Descrição
Palavras-chave
Citric canker, Mutant, Polyhydroxyalkanoate, Tiamine synthase, Hypotethical, Cancro cítrico, Tiamina, Xanthomonas, Mutantes, Proteínas recombinantes
Como citar
BORTOLOSSI, Julio Cesar. Expressão e análise de proteínas recombinantes de Xanthomonas axonopodis pv. citri em E. coli, visando análise estrutural por cristalografia. 2007. x, 46 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Ciências Agrárias e Veterinárias, 2007.