RESUMO
The fibroblast growth factor receptor (FGFR) belongs to the tyrosine kinase family consisting of four members (FGFR1-4). This study involved identification and characterization of FGFR1 and FGFR3 from mud crab Scylla paramamosain for the first time. The obtained cDNAs of SpFGFR1 and SpFGFR3 were 2,380 bp and 2,982 bp in length with a 1,503 bp and 2,310 bp open reading frame, respectively. The predicted SpFGFR1 protein included three immunoglobulin domains and a transmembrane region, while SpFGFR3 protein possessed a typical TyrKc (Tyrosine kinase, catalytic) domain. Real-time PCR analysis showed that SpFGFR1 and SpFGFR3 were highly expressed in the hepatopancreas. Furthermore, the expression levels of SpFGFR1 and SpFGFR3 in the hepatopancreas were enhanced following challenges with Vibro alginolyticus, Staphylococcus aureus, Poly (I:C) and White spot syndrome virus, which shows the involvement of SpFGFR1 and SpFGFR3 in innate immune response to infections from bacteria and virus. There was significant suppression of six antimicrobial peptide genes (SpALF1-5 and SpCrustin) and three NF-κB members (SpDorsal, SpIKK and SpRelish) when SpFGFR1 and SpFGFR3 was interfered in vivo. Also, treatment of the hemocytes with specific inhibitor of SpFGFR for 24 h consistently down-regulated SpDorsal, SpRelish and AMPs. These results suggested that SpFGFR1 and SpFGFR3 played important roles in regulating the Toll signaling pathway and immune deficiency (IMD) pathway through NF-κB signaling pathway. These findings may provide new insights into the role of FGFRs in the innate immune function of crustaceans.
Assuntos
Braquiúros , Animais , NF-kappa B/metabolismo , Proteínas de Artrópodes , Receptores de Fatores de Crescimento de Fibroblastos/genética , Filogenia , Imunidade Inata/genética , Transdução de Sinais , Poli I-C/farmacologia , Proteínas Tirosina Quinases/genéticaRESUMO
Transforming growth factor-ß type III receptor (TßR3), as a co-receptor of TGF-ß superfamily, plays critical roles in development and growth as well as some disease pathogeneses by presenting ligands to other receptors in vertebrates. However, the identification and functional characterization of TßR3 had not been reported yet in invertebrates. In the present study, TßR3 was first identified and characterized in mud crab Scylla paramamosain. The obtained cDNA length of SpTßR3 was 2, 424 bp with a 1, 854 bp open reading frame, which encoded a putative peptide of 617 amino acids containing a typical transmembrane region and a Zona pellucida (ZP) domain. Real-time PCR results showed that SpTßR3 was predominantly expressed at early embryonic development stage and early postmolt stage, suggesting its participation in development and growth. We report, for the first time in invertebrates, the challenge of both Vibro alginolyticus and Poly (I:C) could alter the expression patterns of SpTßR3. Notably, the expression levels of SpIKK, two NF-κB members (SpRelish and SpDorsal), and five antimicrobial peptide genes (SpCrustin and SpALF1-4) were significantly suppressed when SpTßR3 was interfered in vivo. Secondly, the overexpression of SpTßR3 in vitro could activate NF-κB signaling through the dual-luciferase reporter assays. Furthermore, the bacterial clearance assay after SpTßR3 was silenced in vivo highlighted the potential of SpTßR3 in activating the innate immune responses. These results implied the involvement of SpTßR3 in the innate immune responses by regulating the NF-κB pathway. This study first indicated that TßR3 was present in invertebrate, and it participated in not only the development and growth but also the innate immunity of S. paramamosain. It also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.
Assuntos
Braquiúros/genética , Braquiúros/imunologia , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Transdução de Sinais/genética , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Poli I-C/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/química , Alinhamento de Sequência , Vibrio alginolyticus/fisiologiaRESUMO
Interleukin-1 receptor associated kinases (IRAK) is the most important downstream kinases of TLRs/IL-1R signaling pathway for signal transduction and activation of inflammatory response against pathogen infections. However, the molecular identification and function characterization of IRAK2 homologs in lower vertebrate remains obscure. In this study, three IRAK2 genes (AdIRAK2a, AdIRAKb and AdIRAK2c) and their respective transcripts were identified from the Chinese giant salamander Andrias davidianus. This is the first evidence that three different IRAK2 genes exist in an ancient amphibian species, which has never been reported in other vertebrates. The complete open reading frames (ORFs) of AdIRAK2a, AdIRAK2b and AdIRAK2c were 2112 bp, 1917 bp and 816 bp encoding deduced proteins of 703 amino acids (aa), 628 aa and 271 aa, respectively. All three AdIRAK2 proteins contained two predicted conserved functional domains, including a death domain (DD) and a serine/threonine protein kinases domain (KD). Phylogenetic analysis showed that the three AdIRAK2s clustered together with other known IRAK2 of vertebrates. The three AdIRAK2s were ubiquitously expressed in all tested tissues with a similar tissues distribution pattern. After challenge of Aeromonas hydrophila (A. hydrophila), Staphylococcus aureus (S.aureus), giant salamander iridovirus (GSIV, belonging to the genus Ranavirus in the family Iridoviridae) and polyinosinic:polycytidylic acid (poly(I:C)), the expression levels of all AdIRAK2s in blood were significantly altered, however, they exhibited distinct response patterns. Moreover, the results of over-expression and RNAi of AdIRAK2s implied the involvement of AdIRAK2s in triggering NF-κB-mediated signaling pathways and inflammatory responses. This study might provide a better understanding of the presence and immune regulation function of IRAK2 in amphibians and even in vertebrates.
Assuntos
NF-kappa B , Transdução de Sinais , Animais , NF-kappa B/genética , FilogeniaRESUMO
This study aimed to investigate whether different salinity stresses (0, 2, 4, 6, or 8 ppt NaCl) affect the histoarchitecture, serum biochemical indices, antioxidant responses, and transcriptome analysis of the red swamp crayfish Procambarus clarkii. Transmission electron microscopy results showed that the degree of damage to the nuclei and mitochondria in the hepatopancreas increased with increasing salinity. Scanning electron microscopy results showed that the degree of gill wrinkles was enhanced under salinity stress. Serum biochemical indices demonstrated that the cholesterol significantly decreased while the triglyceride, aspartate transaminase, and alanine transaminase contents significantly increased with increasing salinity. The antioxidant parameters, including catalase, total antioxidant capacity, and glutathione peroxidase, significantly decreased, while the malondialdehyde content significantly increased under salinity stress. Transcriptome analysis revealed that the expression pattern of immune-related genes showed a downward trend. These findings enrich our knowledge about the salinity stress response of farmed organisms and provide a theoretical base for salinity domestication and saline soil cultivation of P. clarkii, which might contribute to income improvement, employment generation, food security, and environmental safety.
Assuntos
Antioxidantes , Astacoidea , Animais , Antioxidantes/metabolismo , Astacoidea/fisiologia , Perfilação da Expressão Gênica , Hepatopâncreas , Estresse Salino , TranscriptomaRESUMO
The epidermal growth factor receptor (EGFR) is a pleiotropic glycoprotein which plays a role in regulating cell proliferation, migration and differentiation. However, the genetic diversity of EGFR in crustaceans as well as its function, such as whether it is involved in immune regulation, remains obscure. In this study, two EGFR genes, including EGFR1 and EGFR2, and three transcripts were identified and characterized in Scylla Paramamosain for the first time. To our knowledge, this is the first time that more than one EGFR gene was identified in a single species. The complete open reading frames (ORFs) of SpEGFR1, SpEGFR2a and SpEGFR2b were 4377 bp, 4404 bp and 4341 bp encoding deduced proteins of 1458 amino acids (aa), 1467 aa and 1446 aa, respectively. All EGFR had a signal peptide region and two Recep_L_domain region, followed by a transmembrane region and a conserved tyrosine kinase domain (TyrKc), and phylogenetic analysis demonstrated three SpEGFRs clustered together with invertebrate EGFR branch. Tissue specific expression analysis depicted that all SpEGFRs presented similar transcription patterns. The expression levels of SpEGFR1 and SpEGFR2s in hepatopancreas and gills were significantly altered after the stimulation of bacterial and viral pathogens including Staphylococcus aureus, Vibrio alginolyticus, White spot syndromre virus and Polycytidylinic acid. The in vivo RNA interference assays demonstrated that expression levels of SpIKK, two members of NF-κB (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF1-5) were significantly reduced when SpEGFR1 or SpEGFR2 was silenced, respectively. The transcription patterns of SpIKK, SpRelish, SpDorsal and AMPs exhibited similar down- or up-regulation trend when the primary cultured hemocytes were treated with EGFR antagonist or agonist for 24 h. These results suggested that SpEGFR might play an important role in innate immune responses to bacterial and viral infections by regulating the NF-κB pathway. It also provided a better understanding of the origin or evolution of EGFR in crustaceans and even invertebrates.
Assuntos
Braquiúros , Genes erbB-1 , Animais , Aminoácidos/genética , Proteínas de Artrópodes/metabolismo , Receptores ErbB/genética , Regulação da Expressão Gênica , Imunidade Inata/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Filogenia , Sinais Direcionadores de Proteínas/genéticaRESUMO
Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-ß promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-ß promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-ß promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.