Clonagem e caracterização do gene da enzima Δ6-dessaturase de ácido graxo e análise de parte do genoma funcional de Thalassiosira fluviatilis

AUTOR(ES)
DATA DE PUBLICAÇÃO

2007

RESUMO

Omega 3 polyunsaturated fatty acids are molecules found in oils that are of fundamental importance in the maintenance of human health, that need to be consumed by the humans. Special attention is given to the omega 3 long chain polyunsaturated fatty acids (LCPUFAs), like eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) due to their influence in the prevention and modulation of certain pathological conditions like obesity and cardiovascular diseases and by their essentiality to brain and retina development. The main sources of LCPUFAs are fishes, specially those of deep and cold marine waters. Several studies have demonstrated that these oils found in the fishes come from their zooplancton ingestion of organisms, which contain microalgae that produce large amounts of LCPUFAs. Among these microalgae are those of the diatomacea family, and due to these are good candidates for the study of the omega 3 biosynthetic pathway and corresponding genes. Here we report a partial functional genome analysis of the diatomacea Thalassiosira fluviatilis, with the objective of contributing to the further understanding of the biosynthetic pathway of LCPUFAs. To accomplish that, two cDNA libraries of two omega 3 diatomacea producers were constructed, Thalassiosira fluviatilis e Chaetoceros muelleri. It is our goal in the future to do a comparative functional genome analysis of the two microalgae with others reported in the literature. With the objective of generating molecular tools for molecular engineering of LCPUFAs in plants, we report the cloning and characterization of the gene encoding ?6- desaturase, the first enzyme envolved in the synthesis of EPA and DHA, responsible for the production of the intermediate stearidonic acid from a- linolenic acid. T. fluviatilis cDNA, originally used to make the cDNA library, was used as a template to isolate and clone the gene encoding ?6-desaturase using PCR and RACE approaches. The gene cloned has an ORF of 1455 nucleotides, encoding a putative protein of 484 aminoacid residues. The total sequence includes a 3UTR of 120 nucleotides. The predicted protein has a cytochrome b5 domain fused at the N-terminal and three histidine-box conserved motifs as described in other desaturases; the predicted protein has also high identity with the ?6-desaturase from Thalassiosira pseudonana. Regarding the analysis of the cDNA libray of T. fluviatilis, 1920 ESTs were sequenced starting from the 5 end and the sequences validated and analyzed using the Program Sistema Genoma (www.genoma.embrapa.br). Those, 1090 sequences were validated generating 775 clusters, comprehending 628 singletons and 147 contigs. Within the clusters, 165 presented identity with proteins deposited in the GenBank, 109 presented identity with nucleotide sequences of T. pseudonana and 36 clusters had identity with EST sequences from GenBank. A totality of 579 clusters did not have identity with any sequence within the bank and algorithms analyzed, being exclusives to T. fluviatilis. In the analysis were identified 07 genes envolved in the fatty acid metabolism, 04 of them been comprehending genes encoding enzymes envolved in the biosynthesis of omega 3 polyunsaturated fatty acids, indicating that it massive sequencing of EST is a good approach to cloning genes envolved in the omega 3 pathway in microalgae.

ASSUNTO(S)

genoma funcional biologia molecular clonagem molecular Ácidos graxos polinsaturados omega-3

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