RESUMO
Ferritin is a major intracellular iron storage protein in higher vertebrates and plays an important role in iron metabolism. In this study, ferritin H subunit was cloned from the larvae of yellow snapper, Lutjanus argentiventris, by rapid amplification of cDNA ends (RACE) following in silico transcriptome analysis. The full-length cDNAs of the LaFeH was 1231 bp in length encoding 177 amino acids with a predicted molecular mass (MW) about 20.82 kDa and theoretical isoelectric point (pI) of 5.79. Amino acid alignment revealed that LaFeH shared high similarity with other known ferritins. It shared high degree identity to the ferritin H subunits of Lates calcarifer (99%), Takifugu rubripes (97%) and Dicentrarchus labrax (97%), and low identity to that of human (82%) and mouse (84%). By real-time PCR assays, the mRNA transcripts of LaFeH was found to be higher expressed in head-kidney, eye, heart and brain. Moreover, mRNA expression levels of LaFeH was measured by real-time PCR in larvae exposed with graded levels of iron (6.8 µg/ml and 13.6 µg/ml (Fe2x and Fe4x, respectively) and an iron chelation assay. Results showed that the expression of the LaFeH mRNA increased gradually with Fe2x in water. The LaFeH gene expression declined with increasing iron exposure levels at Fe4x. Finally, we can observe a high expression of LaFeH gene in larvae exposed to iron chelation therapy at 2 h; however this increase was gradually decreasing over time. In summary, the LaFeH gene expression for larvae yellow snapper showed a dose-depend increase following the iron treatment. These data indicated that iron bioavailability regulates LaFeH at transcriptional level in larvae yellow snapper. Further studies are necessary to ascertain their role in the immune response in teleost fish.
Assuntos
Apoferritinas/genética , Proteínas de Peixes/genética , Regulação da Expressão Gênica , Ferro/metabolismo , Perciformes/genética , Sequência de Aminoácidos , Animais , Apoferritinas/química , Apoferritinas/metabolismo , Sequência de Bases , DNA Complementar/genética , DNA Complementar/metabolismo , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Estrutura Molecular , Especificidade de Órgãos , Perciformes/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Alinhamento de Sequência/veterináriaRESUMO
The aim of this study was to evaluate the single or combined effects of Lactobacillus sakei with inulin suitable for immunological in vivo studies in farmed fish. By in vitro assays, L. sakei strain 5-4 showed antibacterial activities against all assayed fish pathogens (except the Vibrio harveyi strain CAIM-1793). L. sakei was able to survive at high fish bile concentrations. Fermentation of the agave inulin resulted in a large increase in number of lactobacilli. For the in vivo study, fish were fed for 8 weeks four practical diets: control diet (control), L. sakei 5-4 (10(7) CFU/g), inulin (1% or 10 g/kg) and L. sakei + inulin (10(7) CFU/g + 10 g/kg). The weight gain showed clearly the synergistic effect of L. sakei 5-4 and inulin at 6 and 8 weeks of treatments. Leopard grouper fed with L. sakei alone or combined with inulin have significantly increased the assayed physiological and humoral immune parameters. By real-time PCR assays, the mRNA transcripts of immunoglobulin M (IgM) were found to be higher expressed in intestine, head kidney, mucus, gill, spleen and skin. Moreover, mRNA expression levels of IgM in head kidney and anterior intestine were measured by real-time PCR. L. sakei 5-4 and L. sakei + inulin supplemented diet up-regulated the expression of IgM at week 4 and 8 in intestine and head kidney, respectively. These results support the idea that the L. sakei 5-4 alone or combined with agave inulin improved growth performance and stimulates the immune system of leopard grouper.