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Previous issue date: 2013-04-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The screening for genes in metagenomic libraries from soil creates opportunities to explore the enormous genetic and metabolic diversity of microorganisms. Rivers are ecosystems with high biological diversity, but few were examined using the metagenomic approach. With this objective, a metagenomic library was constructed from DNA soil samples collected at three different points along the Jundia?-river (Rio Grande do Norte-Brazil). The points sampled are from open area, rough terrain and with the direct incidence of sunlight. This library was analyzed functionally and based in sequence. For functional analysis Luria-Bertani solid medium (LB) with NaCl concentration varied from 0.17M to 0.85M was used for functional analysis. Positives clones resistant to hypersaline medium were obtained. The recombinant DNAs were extracted and transformed into Escherichia coli strain DH10B and survival curves were obtained for quantification of abiotic stress resistance. The sequences of clones were obtained and submitted to the BLASTX tool. Some clones were found to hypothetical proteins of microorganisms from both Archaea and Bacteria division. One of the clones showed a complete ORF with high similarity to glucose-6-phosphate isomerase which participates in the synthesis of glycerol pathway and serves as a compatible solute to balance the osmotic pressure inside and outside of cells. Subsequently, in order to identify genes encoding osmolytes or enzymes related halotolerance, environmental DNA samples from the river soil, from the water column of the estuary and ocean were collected and pyrosequenced. Sequences of osmolytes and enzymes of different microorganisms were obtained from the UniProt and used as RefSeqs for homology identification (TBLASTN) in metagenomic databases. The sequences were submitted to HMMER for the functional domains identification. Some enzymes were identified: alpha-trehalose-phosphate synthase, L-ectoina synthase (EctC), transaminase L-2 ,4-diaminobutyric acid (EctB), L-2 ,4-diaminobutyric acetyltransferase (EctA), L-threonine 3 dehydrogenase (sorbitol pathway), glycerol-3-phosphate dehydrogenase, inositol 3-phosphate dehydrogenase, chaperones, L-proline, glycine betaine binding ABC transporter, myo-inositol-1-phosphate synthase protein of proline simportadora / PutP sodium-and trehalose-6-phosphate phosphatase These proteins are commonly related to saline environments, however the identification of them in river environment is justified by the high salt concentration in the soil during prolonged dry seasons this river. Regarding the richness of the microbiota the river substrate has an abundance of halobacteria similar to the sea and more than the estuary. These data confirm the existence of a specialized response against salt stress by microorganisms in the environment of the Jundia? river / A busca por genes baseada na constru??o e an?lise de bibliotecas metagen?micas a partir de solo gera oportunidades para explorar uma enorme diversidade gen?tica e metab?lica de microrganismos. Os rios s?o ecossistemas com alta diversidade biol?gica, mas ainda pouco explorados por meio de metagen?mica. Com o objetivo de explorar a diversidade microbiana, uma biblioteca metagen?mica foi constru?da a partir de DNA extra?do de substrato de rio em tr?s pontos ao longo do rio Jundia? (Rio Grande do Norte-Brasil). Os pontos de amostragem s?o derivados de ?rea aberta, terreno acidentado e com a incid?ncia direta da luz solar. Esta biblioteca foi analisada funcionalmente e tamb?m com base em sequ?ncias. Para a an?lise funcional foi utilizado meio de cultura s?lido LB com concentra??o de NaCl variando de 0,17M a 0,85M. Foram obtidos 15 clones positivos com caracter?sticas halotolerantes. Os DNAs recombinantes foram extra?dos e retransformados em cepa de Escherichia coli DH10B e curvas de sobreviv?ncia foram obtidas para confirma??o e quantifica??o da resist?ncia ao estresse abi?tico. As sequ?ncias dos clones foram obtidas e submetidas a ferramenta BLASTX e assim foi comprovado que alguns clones codificavam prote?nas hipot?ticas. Um dos clones apresentou uma ORF completa com elevada similaridade de glucose-6-fosfato-isomerase que participa na s?ntese do precursor de glicerol, sendo um soluto compat?vel para equilibrar a press?o osm?tica no interior e no exterior das c?lulas. Posteriormente, para identifica??o de genes que codificam osm?litos relacionados com halotoler?ncia e identifica??o da diversidade microbiol?gica, amostras de DNA ambiental do substrato do rio e da coluna d??gua do estu?rio e oceano foram coletadas e pirosequenciadas. As sequ?ncias de osm?litos de diferentes microrganismos foram obtidas a partir do UniProt e utilizadas como RefSeqs para a identifica??o por homologia (TBLASTN) nos bancos de dados metagen?micos. As sequ?ncias identificadas nos bancos de dados ambientais foram submetidas ao programa HMMER com o fim de identificar dom?nios funcionais. Foram identificadas as enzimas: alfa-trealose-fosfato sintase, L-ectoina sintase (ectC), transaminase do ?cido L-2,4-diaminobut?rico (EctB), ?cido L-2 ,4-diaminobut?rico acetiltransferase (EctA), L-treonina 3-desidrogenase (via de s?ntese do sorbitol), Glicerol-3-fosfato desidrogenase, inositol-3-fosfato desidrogenase, chaperonas, L-prolina glicina beta?na liga??o transportador ABC, mio-inositol-1-fosfato sintase, a prote?na simportadora de prolina/s?dio -PutP e trealose-6-fosfato fosfatase. Estas s?o enzimas que participam da s?ntese de osm?litos comumente relacionados a ambientes salinos, no entanto a identifica??o desses solutos em ambiente de rio ? justificada pela elevada concentra??o salina no solo durante prolongadas esta??es de seca neste rio. Quanto ? riqueza da microbiota foi identificado que o substrato do rio possui uma abund?ncia de halobact?rias semelhante a do mar e superior a do estu?rio. Esses dados confirmam a exist?ncia de uma resposta especializada contra o estresse salino por microrganismos no ambiente do rio Jundia?
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/12654 |
| Date | 16 April 2013 |
| Creators | Silva, Uaska Bezerra e |
| Contributors | CPF:00297997750, http://lattes.cnpq.br/1083882171718362, Quirino, Betania Ferraz, CPF:58483543168, http://lattes.cnpq.br/3916535995785654, Uchoa, Adriana Ferreira, CPF:58024867320, http://lattes.cnpq.br/6644671747055211, Theodoro, Raquel Cordeiro, CPF:22405839830, http://lattes.cnpq.br/0977453259767928, Thompson, Claudia Elizabeth, CPF:97930563049, http://lattes.cnpq.br/8413464882207319, Lima, Lucymara Fassarela Agnez |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Biotecnologia, UFRN, BR, Biotecnologia Industrial; Biotecnologia em Agropecu?ria; Biotecnologia em Recursos Naturais; Biotecn |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | Portuguese |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/doctoralThesis |
| Format | application/pdf |
| Source | reponame:Repositório Institucional da UFRN, instname:Universidade Federal do Rio Grande do Norte, instacron:UFRN |
| Rights | info:eu-repo/semantics/openAccess |
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