06251nam a2200145 a 450000100080000000500110000800800410001910000190006024501150007926001100019450000930030452056660039765000270606365000150609016298562015-11-17       2008    bl uuuu  m   00u1 u    #d1 aGALVÃO, P. G.  aAjuste metodológico para sequenciamento de plasmídeos de Bacillus thuringiensis subsp. kurstaki estirpe S76.  a2008. 136 p. Dissertação (Mestrado) - Universidade Federal do Rio de Janeiro, Rio de Janeiro, RJ.c2008  aParceria: UFRJ. Orientador: José Ivo Baldani; Co-orientador: Jean Luiz Simões Araújo.  a0 Bacillus thuringiensis deposita, no citoplasma da celula em esporula~ao, inclus5es cristalinas de natureza proteica com atividade para larvas de insetos de diversas ordens, nemat6ides, acaros e protozoarios. Os genes cry, que codificam as delta-endotoxinas contidas nesses cristais, estao localizados em plasmideos conjugativos de alto peso molecular, que se replicam com baixo numero de c6pias e possuem notavel estabilidade segregacional. Diversos aspectos do complexo perfil de elementos extracromossomais de B. thuringiensis, que pode representar ate 20% do genoma da bacteria, tern sido estudados desde a decada de 1970. Entretanto, 0 seqiienciamento se restringe a regi5es contendo os genes de delta-endotoxinas e ffagmentos adjacentes. Estudos recentes utilizando a estirpe brasileira 876 de B. thuringiensis kurstaki, indicaram uma alta atividade entomopatogenica contra 0 genero Diatraea. 8abe-se que nas estirpes da subespecie kurstaki os genes responsaveis pel a produ~ao das delta-endotoxinas com atividade para Lepidoptera estao localizados em plasmideos de aproximadamente 44 e 110 MDa. Entretanto, pouco e conhecido a respeito dos outros plasmideos de Btk 876. 0 seqiienciamento de plasmideos desta estirpe podera fomecer uma serie de novas informa~5es a respeito do DNA extracromossomal desta bacteria, explicando, inclusive, a alta mortalidade causada por esta estirpe. Ate 0 momento, existem poucos estudos caracterizando os plasmideos de B. thuringiensis, principalmente os maiores. Por isso, a otimiza~ao da metodologia utilizada para extra~ao e purifica~ao dessas moIeculas tomou-se extremamente necessaria. Os plasmideos de BtK 876 foram extraidos e purificados atraves de ultracentrifuga~ao em gradiente de CsCI e com 0 kit comercial QIAGE~, sendo que ambas as tecnicas apresentaram urn perfil plasmidial semelhante. Foi selecionado 0 plasmideo denominado pBt20 de BtK 876, que foi extraido do gel e posteriormente eletroeluido e amplificado com 0 kit comercial GenomiPhiTM DNA Amplification, visando aumentar a mBSSB de DNA plasmidial para as futuras etapas do trabalho. 0 DNA plasmidial do pBt20 foi ffagmentado atraves de nebuliza~ao e os ftagmentos obtidos (100 a 2000 pb) geraram uma bibiioteca com aproximadamente 6.600 clones. Foram seqiienciados 1.440 clones, que possibilitaram a montagem de 56 contigs, totalizando 33,7 kb. A compara~ao desses contigs com 0 banco de dados revelou 41% com valores de e-value nao-significativos, 7% que codificam para proteinas que nao apresentaram similaridade com 0 banco de dados, 18% que codificam para proteinas hipoteticas conservadas e 34% que possuem fun~5es putativas. Dentre as proteinas similares ao banco de dados destacam-se HxlR, Doc, Mob, Rep, alem das enzimas pertencentes as familias beta-Iactamases, exoribonuclease e fosfotransferases. A ocorrencia de redundancia observada na biblioteca, e consequentemente, na montagem das sequencias, nao permitiu 0 fechamento do sequenciamento do pBt20. Bacillus thuringiensis deposits in the sporulation cell cytoplasm, crystalline protein inclusions with activity for larvae of various insect orders, nematodes, mites and protozoa. The cry genes, which encode the delta-endotoxins are located in high molecular weight plasmids that replicate with low copy number and show remarkable segregational stability. Different aspects of the B. thuringiensis plasmid complex profile, which can represent up to 20% of the bacteria genome, have been studied since the 1970 decade. However, the sequencing is restricted to the regions containing the delta-endotoxins genes and adjacent fTagments. Recent studies using the Brazilian 576 strain of B. thuriflgieflsis kurstaki, showed high activity against the entomopatogenic genera Diatraea. It is known that in kurstaki strains the genes responsible for the production of delta-endotoxins with activity for Lepidoptera are located in plasmids of approximately 44 and 110 MDa. However, little is known about the other plasmids of Btk S76. The sequencing of these plasmids can provide a range of new information about the extracromossomal DNA of this bacterium, even explaining the high mortality caused by this strain. So far, there are few studies characterizing the plasmids of B. thuringiens/s, especially the larger ones. Therefore, the optimization of the extraction and purification methodology for these molecules has become extremely necessary. The plasmids were extracted fTom BtK S76 and purified through ultracentrifugation in CsCI gradient and the commercial kit QIAGEN@, and both techniques showed a similar plasmid profile. It was selected the plasmid called pBt20 of BtK S76, which was extracted fTom the gel and then electroeluted and amplified through the commercial GenomiPhiTM DNA Amplification kit, to increase the plasmid DNA mass for next steps of this work. The plasmid DNA of pBt20 was fragmented through nebulization and a library of approximately 6.600 clones was generated with fragments between 100 to 2000 bp. It was sequenced 1.440 clones, which allowed to assembly 56 contigs, totaling 33.7 Kb. The comparison of these contigs with the database revealed 7% that showed no similarity with the database, 18% that encode for conserved hypothetical proteins and 34% that show putative functions, 41 % with non-significant e-value. Among the proteins similar to the data base there are HxIR, Doc, Mob, Rep, in addition to the enzymes belonging to the beta-Iactamase, exoribonuclease and fosfotransferases families. The occurrence of redundancy found in the library and consequently the assembling of the sequences did not allow to close the sequencing of the pBt20 plasmid.  aBacillus Thuringiensis  aPlasmídeo