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Previous issue date: 2009-02-27 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The total number of prokaryotic cells on Earth has been estimated at 4 to 6x1030 and only about 1% of microorganisms present in the environment can be cultivated by standard techniques of cultivation and plating. Therefore, it is a huge biological and genetic pool that can be exploited, for the identification and characterization of genes with biotechnological potential. Within this perspective, the metagenomics approach was applied in this work. Functional screening methods were performed aiming to identify new genes related to DNA repair and / or oxidative stress resistance, hydrocarbon degradation and hydrolytic activities (lipase, amylase and protease). Metagenomic libraries were built utilizing DNA extracted from soil samples collected in Jo?o C?mara RN. The libraries were analyzed functionally using specific substrate containing solid medium (hydrolytic activity), supplemented with H2O2 (DNA repair and / or resistance to oxidative stress) and liquid medium supplemented with light Arabian oil (activity, degradation of hydrocarbons). After confirmation of activity and exclusion of false-positive results, 49 clones were obtained, being 2 positive for amylase activity, 22 resistant to oxidative stress generated by H2O2 and 25 clones active for hydrocarbons degradation. Analysis of the sequences showed hypothetical proteins, dienelactona hydrolase, DNA polymerase, acetyltransferase, phosphotransferase, methyltransferase, endonucleases, among other proteins. The sequence data obtained matched with the functions tested, highlighting the success of metagenomics approaches combined with functional screening methods, leading to very promising results / O n?mero total de c?lulas procari?ticas na Terra tem sido estimado em 4 a 6x1030 sendo que apenas cerca de 1% dos microrganismos presentes no meio ambiente pode ser cultivado, atrav?s de t?cnicas padr?o de cultivo e plaqueamento, se apresentando, portanto, como um enorme pool biol?gico e gen?tico que pode ser explorado, visando a identifica??o e a caracteriza??o de genes com potencial biotecnol?gico. Dentro desta perspectiva, a abordagem metagen?mica foi aplicada neste trabalho a partir de metodologias de sele??o funcional visando ? identifica??o de novos genes relacionados ao reparo de DNA e/ou resist?ncia a estresse oxidativo, genes relacionados com atividade de degrada??o de hidrocarbonetos, e atividade hidrol?tica (lipase, amilase e protease). Com esse objetivo, uma biblioteca metagen?mica, constru?da a partir de amostras de solo coletadas no munic?pio de Jo?o C?mara RN, foi analisada funcionalmente utilizando meios s?lidos contendo substratos espec?ficos (atividade hidrol?tica), suplementados com H2O2 (reparo de DNA e/ou resist?ncia a estresse oxidativo) e meio liquido suplementado com ?leo ?rabe leve (atividade de degrada??o de hidrocarbonetos). Ap?s confirma??o da atividade e exclus?o de falsos-positivos foram obtidos 49 clones, sendo 2 positivos para atividade amilase, 22 resistentes ao estresse oxidativo gerado por H2O2 e 25 com atividade de degrada??o de hidrocarbonetos, cuja an?lise das seq??ncias revelou,al?m de prote?nas hipot?ticas, dienolactona hidrolase, DNA polimerases, acetiltransferase, fosfotransferase, metiltransferase, endonucleases entre outras, cuja coer?ncia com as fun??es ensaiadas, ressalta o sucesso da abordagem metagen?mica aliada a metodologias funcionais e, os resultados obtidos bastante promissores.
PALAVRAS CHAVE: Metagenoma, Biotecnologia
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.ufrn.br:123456789/16763 |
| Date | 27 February 2009 |
| Creators | Silva, Rita de C?ssia Barreto da |
| Contributors | CPF:00297997750, http://lattes.cnpq.br/1083882171718362, Menck, Carlos Frederico Martins, CPF:00639606806, http://lattes.cnpq.br/8043136069525312, Scortecci, K?tia Castanho, CPF:13451112825, http://lattes.cnpq.br/4808910380593455, Lima, Lucymara Fassarela Agnez |
| Publisher | Universidade Federal do Rio Grande do Norte, Programa de P?s-Gradua??o em Gen?tica e Biologia Molecular, UFRN, BR, Gen?tica e Biologia Molecular |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| 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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