Análise do transcritoma de etiquetas de sequências expressas da hipófise e parte do cérebro do tambaqui (Colossoma macropomum) e expressão do cDNA do hormônio de crescimento em Pichia pastoris

Abstract

The tambaqui, Colossoma macropomum, is a fish of great commercial value to the Amazon region and represents an emblematic species taking into account the studies carried out on its biology, physiology and genetics. Due to its importance, the aim of this study was to construct and characterize a cDNA library of mixed parts of the hypophysis and the brain in order to identify transcripts by genome annotation using bioinformatics tools for categorization of the transcripts. Genes of biotechnological interest, followed by the expression and secretion of one of them in recombinant form were also identified. A total of 1728 clones were sequenced that resulted in 1060 ESTs grouped into 184 contig s and 12 singlets. Most of the known genes found in this analysis is related to the ribosomal genes, followed by mitochondrial genes. In addition, other important transcripts were identified and the novelty index was 43%., it was possible to identify and isolate growth hormone gene (GH) and then start the study of expression and secretion of recombinant growth hormone in tambaqui (tGH) in the Pichia pastoris. The structural gene region of growth hormone was constructed by chemical synthesis and inserted into pPIC9. The recombinant plasmid named tGH was introduced into cells of P. pastoris by electroporation, recombinant clones were selected by auxotrophic marker gene His4. The highest expression of tGH by P. pastoris GS115 was reached after 72 h of culture. The molecular mass estimated by SDS-PAGE 15% revealed that the size of tGH was 22 kDa, similar to the size of the growth hormone of other species of vertebrates described in the literature. Thus, the tGH of this important species for aquaculture can then be applied in experimental tests in the near future as a factor iforaccelerating the fish growth. These results open perspectives for a broader study, both in the genome and in the application of recombinant piscine growth hormone in fish farming.