RESUMEN
Interferon regulatory factors (IRFs) play a key role in mediating the host response against pathogen infection and other important biological processes. This is the first report of an IRF family member in blunt snout bream Megalobrama amblycephala. The complete cDNA of M. amblycephala (Ma) IRF1 gene has 1422 nucleotides (nt.), with an open reading frame of 858 nt, encoding a polypeptide of 285 amino acids. The putative MaIRF1 polypeptide shared significant structural homology with known IRF1 homologs: a conserved IRF domain was found at the N-terminal and an IRF association domain 2 at the C-terminal. Phylogenetic analysis showed that MaIRF1 amino acid sequence clustered with other teleost IRF1s, with a grass carp ortholog exhibiting the highest similarity. MaIRF1 mRNA expression patterns were studied using quantitative real-time PCR in healthy fish tissues and after a challenge with Aeromonas hydrophila bacterium. It was constitutively expressed in all examined tissues: the highest in blood, the lowest in muscle. The expression after A. hydrophila challenge was up-regulated in liver, spleen and kidney, but down-regulated in intestine and gills. At the protein level, similar expression patterns were observed in liver and gills. Patterns differed in intestine (up-regulation), spleen (down-regulation) and kidney (expression mostly unchanged). This study indicates that MaIRF1 gene plays an important role in the blunt snout bream immune system, hence providing an important base for further studies.
Asunto(s)
Cyprinidae/genética , Cyprinidae/inmunología , Enfermedades de los Peces/inmunología , Factor 1 Regulador del Interferón/genética , Factor 1 Regulador del Interferón/metabolismo , Aeromonas hydrophila/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cyprinidae/clasificación , ADN Complementario/genética , ADN Complementario/metabolismo , Regulación hacia Abajo , Enfermedades de los Peces/microbiología , Proteínas de Peces/química , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Infecciones por Bacterias Gramnegativas/inmunología , Infecciones por Bacterias Gramnegativas/microbiología , Factor 1 Regulador del Interferón/química , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Regulación hacia ArribaRESUMEN
Interferon regulatory factors (irfs) are a family of genes that encode transcription factors with important roles in regulating the expression of Type I interferons (IFNs) and other genes associated with related pathways. irfs have multitudinous functions in growth, development and regulation of oncogenesis. In this study, three irf family members (irf2, irf7, irf9) were identified and characterized in Megalobrama amblycephala at the mRNA and amino acid levels. M. amblycephala irfs share a high sequence homology with other vertebrate irfs. Constitutive expression levels of the three genes were detected (using qPCR) in all studied tissues: low to medium in kidney, gills, heart and muscle, and high in liver, spleen, intestine and blood. qPCR was also used to analyze the dynamic expression patterns of irfs in different embryonic development stages: irf2 is not activated during the embryonic development, whereas irf9 appears to play important roles around hatching and during the larval development. Transcripts of all three studied irfs were upregulated after stimulation by Aeromonas hydrophila bacterium in liver, spleen, head kidney and trunk kidney, whereas downregulation was observed in intestine and gills. The results show that these three irfs are likely to be important factors in the blunt snout bream immune system. They also provide a foundation for studying the origin and evolution of the innate immune system in the blunt snout bream.
Asunto(s)
Aeromonas hydrophila/inmunología , Cyprinidae/inmunología , Enfermedades de los Peces/inmunología , Proteínas de Peces/metabolismo , Infecciones por Bacterias Gramnegativas/inmunología , Factores Reguladores del Interferón/metabolismo , Animales , Evolución Biológica , Clonación Molecular , Proteínas de Peces/genética , Regulación del Desarrollo de la Expresión Génica , Inmunidad Innata , Factor 2 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/genética , Factores Reguladores del Interferón/genética , Subunidad gamma del Factor 3 de Genes Estimulados por el Interferón/genética , Filogenia , ARN Mensajero/genética , Alineación de SecuenciaRESUMEN
The innate immunity serves as the primary line of defense against pathogen invasion. Toll-like receptors (TLRs) play a pivotal role in the innate immunity by sensing specific pathogen-associated molecular patterns and activating immune responses. In this study, 14 TLRs (TLR1, 2, 3, 4, 5, 7, 8a, 8b, 9, 18, 19, 20, 21 and 22) were identified and characterized in a cyprinid fish Megalobrama amblycephala. A majority of MaTLRs possessed the typical structural features of the TLR protein family: LRR domain, TM region and TIR domain. Phylogenetic analyses confirmed the existence of six TLR families and revealed close relationships with other cyprinid orthologs. All TLRs were constitutively expressed in all eight examined tissues. After Aeromonas hydrophila challenge, the expression profiles of MaTLR mRNAs were analyzed in liver, spleen and head kidney: MaTLR1, 2, 5, 9, 21 and 22 were up-regulated, MaTLR3, 7, 8a, 8b and 19 were down-regulated, and MaTLR4, 18 and 20 expression patterns varied among tissues. These results indicate that most of MaTLRs are likely to be involved in the immune responses against bacterial infection.