Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2007. / Submitted by Aline Jacob (alinesjacob@hotmail.com) on 2010-01-06T22:52:25Z
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Previous issue date: 2007-02-23 / Os genes cry1Ca, cry2Ab e cry10Aa de diferentes estirpes brasileiras de Bacillus thuringiensis foram amplificados por PCR, clonados em um vetor de clonagem e seqüenciados. As análises das seqüências mostraram alta identidade com outros genes cry já descritos. Os genes foram removidos dos vetores de clonagem e introduzidos em um plasmídeo vetor de transferência (pSynXIVVI+X3) para construção de baculovírus recombinantes por recombinação homóloga. Os vírus recombinantes foram purificados por diluição seriada em placa de 96 poços e usados para infectar células de Trichoplusia ni (BTI-Tn5B1-4) e larvas de Spodoptera frugiperda. A análise transcricional dos genes cry2Ab e cry10Aa foi realizada por RT-PCR, a partir de mRNA extraído de células de inseto infectadas (72 h p.i.), para confirmação da presença de um transcrito específico para os genes. Extratos de larvas infectadas (96 h p.i.) com os vírus vSyncry1Ca, vSyncry2Ab e vSyncry10Aa foram usados para purificação dos cristais das proteínas recombinantes por ultracentrifugação. Em SDS-PAGE, os extratos apresentaram polipeptídeos de aproximadamente 65, 65 e 74 kDa, correspondentes, respectivamente, ao tamanho das proteínas Cry1Ca, Cry2Ab e Cry10Aa. Estes mesmos cristais foram analisados por microscopia de luz e eletrônica e mostraram-se na forma de grandes cristais cubóides. A toxicidade dos cristais das proteínas recombinantes foram verificadas para larvas de segundo instar de S. frugiperda e Anticarsia gemmatalis (Cry1Ca, sendo a CL de 114,44 e 19,49 ng/mL, respectivamente), e a proteína 50 Cry2Ab recombinate foi tóxica para larvas de S. Frugiperda (CL de 3,40 g/mL). No 50 entanto, a proteína Cry10Aa recombinante foi altamente tóxica para larvas neonatas de de 7,12 g /mL. Neste trabalho, foi demonstrado que proteínas A. grandis com CL 50 Cry recombinants são similares às proteínas naturais, possuindo alta toxicidade para diferentes insetos-praga, o que confirma a utilidade do sistema de expressão baseado em baculovírus e células de inseto para o estudo de proteínas Cry. _______________________________________________________________________________ ABSTRACT / The cry1Ca, cry2Ab and cry10Aa genes from different Brazilian strains of Bacillus thuringiensis were amplified by PCR, cloned into a plasmid cloning vector and sequenced. Sequence analysis showed high identity to previous known cry genes. The genes were removed from the cloning vector and introduced into a transfer vector (pSynXIVVI+X3) for the construction of recombinant baculoviruses by homologous recombination. The recombinant viruses were purified by serial dilution in 96 well plates and used to infect Trichoplusia ni (BTI-Tn5B1-4) insect cells and Spodoptera frugiperda larvae. Transcritptional analysis of the cry2Ab and cry10Aa genes was carried out by RT-PCR, using mRNA extracted from infected insect cells (72 h p.i.), in order to confirm the presence of the gene specific transcript. Recombinant virus (vSyncry1Ca, vSyncry2Ab and vSyncry10Aa) infected insect extracts (96 h p.i.) were used for the purificaton of crystals, made of recombinant proteins, by ultracentrifugation. In SDS-PAGE, the insect extracts showed polypeptides of approximately 65, 65 and 74 kDa, corresponding, respectively to the sizes of the proteins Cry1Ca Cry2Ab e Cry10Aa. These same crystals were analysed by light and electron microscopy and showed the shape of big cuboidal crystals. Furthermore, the crystals preparations were toxic to second instar S. frugiperda and Anticarsia gemmatalis larvae, with a CL of 114,44 and 19,49 ng/mL, respectively (from 50 vSynCry1Ca-infected insect extracts), to second instar S. frugiperda, with a CL de 50 3,40 g/mL (from vSynCry2Ab-infected insect extracts) and neonate A. grandis larvae, of 7,12 g /mL (from vSynCry10Aa-infected insect extracts). This work with a CL 50 showed that recombinant Cry proteíns are similar to their natural couterpart, showing the high toxicity to different insect pests, which demonstrate the utility of the baculovirus expression system for the study of Cry proteins.
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.unb.br:10482/2994 |
| Date | 23 February 2007 |
| Creators | Aguiar, Raimundo Wagner de Souza |
| Contributors | Ribeiro, Bergmann Morais, Monnerat, Rose Gomes |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Source | reponame:Repositório Institucional da UnB, instname:Universidade de Brasília, instacron:UNB |
| Rights | info:eu-repo/semantics/openAccess |
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