Isolation and expression of grass carp toll-like receptor 5a (CiTLR5a) and 5b (CiTLR5b) gene involved in the response to flagellin stimulation and grass carp reovirus infection

Toll-like receptor 5 (TLR5), a member of Toll-like receptors (TLRs) family and is responsible for the bacterial flagellin recognition in vertebrates, play an important role in innate immunity. In the study, two TLR5 genes of grass carp (Ctenopharyngodon idellus), named CiTLR5a and CiTLR5b, were cloned and analyzed. Both CiTLR5a and CiTLR5b are typical TLR proteins, including LRR motif, transmembrane region and TIR domain. The full-length cDNA of CiTLR5a is 3054 bp long, with a 2646 bp open reading frame (ORF), 78 bp 5' untranslated regions (UTR), and 330 bp 3' UTR. The full-length cDNA of CiTLR5b is 3326 bp, with a 2627 bp ORF, 95 bp 5' UTR, and 594 bp 3' UTR. Phylogenetic analysis showed that CiTLR5a and CiTLR5b were closed to the TLR5 of cirrhinus mrigala, cyprinus_carpio, and danio redo. Subcellular localization indicated that CiTLR5a and CiTLR5b shared similar localization pattern and may locate in the plasma membrane of transfected cells. Real-time quantitative PCR revealed CiTLR5a and CiTLR5b were constitutively expressed in all examined tissues, whereas the highest expressed tissue differed. Following exposure to flagellin and GCRV, CiTLR5a and CiTLR5b were up-regulated significantly. Moreover, the downstream genes of TLR5 signal pathway such as MyD88, NF-kappa B, IRF7, IL-1 beta, and TNF-alpha also up-regulated significantly, whereas the I kappa B gene was down-regulated, suggesting that CiTLR5a and CiTLR5b involved in response to flagellin stimulation and GCRV infection. The results obtained in the study would provide a new insight for further understand the function of TLR5 in teleost fish. (C) 2015 Elsevier Ltd. All rights reserved

Global gene expression patterns of grass carp following compensatory growth

Background: Compensatory growth is accelerated compared with normal growth and occurs when growth-limiting conditions are overcome. Most animals, especially fish, are capable of compensatory growth, but the mechanisms remain unclear. Further investigation of the mechanism of compensatory growth in fish is needed to improve feeding efficiency, reduce cost, and explore growth-related genes

Cloning and preliminary functional studies of the JAM-A gene in grass carp (Ctenopharyngodon idellus)

Grass carp (Ctenopharyngodon idellus) is a very important aquaculture species in China and other South-East Asian countries; however, disease outbreaks in this species are frequent, resulting in huge economic losses. Grass carp hemorrhage caused by grass carp reovirus (GCRV) is one of the most serious diseases. Junction adhesion molecule A (JAM-A) is the mammalian receptor for reovirus, and has been well studied. However, the JAM-A gene in grass carp has not been studied so far. In this study, we cloned and elucidated the structure of the JAM-A gene in grass carp (GcJAM-A) and then studied its functions during grass carp hemorrhage. GcJAM-A is composed of 10 exons and 9 introns, and its full-length cDNA is 1833 bp long, with an 888 bp open reading frame (ORE) that encodes a 295 amino acid protein. The GcJAM-A protein is predicted to contain a typical transmembrane domain. Maternal expression pattern of GcJAM-A is observed during early embryogenesis, while zygote expression occurs at 8 h after hatching. GcJAM-A is expressed strongly in the gill, liver, intestine and kidney, while it is expressed poorly in the blood, brain, spleen and head kidney. Moreover, lower expression is observed in the gill, liver, intestine, brain, spleen and kidney of 30-month-old individuals, compared with 6-month-old. In a GcJAM-A-knockdown cell line (CIK) infected with GCRV, the expression of genes involved in the interferon and apoptosis pathways was significantly inhibited. These results suggest that GcJAM-A could be a receptor for GCRV. We have therefore managed to characterize the GcJAM-A gene and provide evidence for its role as a receptor for GCRV. Crown Copyright (C) 2013 Published by Elsevier Ltd. All rights reserved.Grass carp (Ctenopharyngodon idellus) is a very important aquaculture species in China and other South-East Asian countries; however, disease outbreaks in this species are frequent, resulting in huge economic losses. Grass carp hemorrhage caused by grass carp reovirus (GCRV) is one of the most serious diseases. Junction adhesion molecule A (JAM-A) is the mammalian receptor for reovirus, and has been well studied. However, the JAM-A gene in grass carp has not been studied so far. In this study, we cloned and elucidated the structure of the JAM-A gene in grass carp (GcJAM-A) and then studied its functions during grass carp hemorrhage. GcJAM-A is composed of 10 exons and 9 introns, and its full-length cDNA is 1833 bp long, with an 888 bp open reading frame (ORE) that encodes a 295 amino acid protein. The GcJAM-A protein is predicted to contain a typical transmembrane domain. Maternal expression pattern of GcJAM-A is observed during early embryogenesis, while zygote expression occurs at 8 h after hatching. GcJAM-A is expressed strongly in the gill, liver, intestine and kidney, while it is expressed poorly in the blood, brain, spleen and head kidney. Moreover, lower expression is observed in the gill, liver, intestine, brain, spleen and kidney of 30-month-old individuals, compared with 6-month-old. In a GcJAM-A-knockdown cell line (CIK) infected with GCRV, the expression of genes involved in the interferon and apoptosis pathways was significantly inhibited. These results suggest that GcJAM-A could be a receptor for GCRV. We have therefore managed to characterize the GcJAM-A gene and provide evidence for its role as a receptor for GCRV. Crown Copyright (C) 2013 Published by Elsevier Ltd. All rights reserved

Identification, characterization and the interaction of Tollip and IRAK-1 in grass carp (Ctenopharyngodon idellus)

Tollip and IRAK-1 are key components of the TLR/IL-1R signaling pathway in mammals, which play crucial roles as mediators of the TLR/IL-1R signal transduction pathways. Although several TLRs have been found in fish, molecular associations, protein protein interactions or the role of the TLR signaling pathway in infection-induced immunity in fish has received little attention. In this study. Tollip and IRAK-1 sequences of grass carp were isolated from a head kidney cDNA library. Full length transcripts and sequences of promoter regions were obtained by 3' and 5' RACE and genome walking, respectively. Reporter gene-promoter constructs and real-time RT-PCR analysis was used to determine grass carp Tollip and IRAK-1 transcription pattern in tissues. Recombinant proteins were used for antibodies production. Phylogenetically, the grass carp loci clustered with previously reported Tollip and IRAK-1genes, respectively, and their sequences shared the highest identity with the genes of zebrafish (Danio rerio). The promoter region of grass carp Tollip and IRAK-1 proved to be active. After viral infection transcript levels of both loci were upregulated in most immune-related tissues in a time-dependent manner. Using antibodies produced in this study, immunofluorescence analysis indicated that Tollip and IRAK-1 were uniformly distributed and co-localized in the cytoplasm of CIK cells. After viral infection, however, Tollip and IRAK-1 both trended toward the cell membrane. Our results demonstrate the existence of Tollip and IRAK-1 proteins in teleost species, and suggest that Tollip-IRAK-1 complexes are being recruited to receptor complexes after stimulation with virus. These results provide novel insights into the role of the TLR signaling pathway in teleosts, especially the action of teleost Tollip and IRAK-1 and the interaction of these molecules as part of this pathway. (C) 2012 Elsevier Ltd. All rights reserved.Tollip and IRAK-1 are key components of the TLR/IL-1R signaling pathway in mammals, which play crucial roles as mediators of the TLR/IL-1R signal transduction pathways. Although several TLRs have been found in fish, molecular associations, protein protein interactions or the role of the TLR signaling pathway in infection-induced immunity in fish has received little attention. In this study. Tollip and IRAK-1 sequences of grass carp were isolated from a head kidney cDNA library. Full length transcripts and sequences of promoter regions were obtained by 3' and 5' RACE and genome walking, respectively. Reporter gene-promoter constructs and real-time RT-PCR analysis was used to determine grass carp Tollip and IRAK-1 transcription pattern in tissues. Recombinant proteins were used for antibodies production. Phylogenetically, the grass carp loci clustered with previously reported Tollip and IRAK-1genes, respectively, and their sequences shared the highest identity with the genes of zebrafish (Danio rerio). The promoter region of grass carp Tollip and IRAK-1 proved to be active. After viral infection transcript levels of both loci were upregulated in most immune-related tissues in a time-dependent manner. Using antibodies produced in this study, immunofluorescence analysis indicated that Tollip and IRAK-1 were uniformly distributed and co-localized in the cytoplasm of CIK cells. After viral infection, however, Tollip and IRAK-1 both trended toward the cell membrane. Our results demonstrate the existence of Tollip and IRAK-1 proteins in teleost species, and suggest that Tollip-IRAK-1 complexes are being recruited to receptor complexes after stimulation with virus. These results provide novel insights into the role of the TLR signaling pathway in teleosts, especially the action of teleost Tollip and IRAK-1 and the interaction of these molecules as part of this pathway. (C) 2012 Elsevier Ltd. All rights reserved

Characterizations of four toll-like receptor 4s in grass carp Ctenopharyngodon idellus and their response to grass carp reovirus infection and lipopolysaccharide stimulation

In this study, the subcellular localization, tissue distribution and response to grass carp reovirus (GCRV) infection and lipopolysaccharide (LPS) stimulation of four grass carp Ctenopharyngodon idellus toll-like receptor 4 (tlr4) genes were investigated. All four genes were constitutively expressed in all tissues studied, but the subcellular localization and tissue exhibiting the highest expression differed for each protein. Following GCRV infection, all the four tlr4s were upregulated in all tissues examined, and stimulation of C. idellus kidney (CIK) cells with LPS resulted in downregulation of all four tlr4s. These results provide a foundation for further investigation of tlr4 genes in bony fishes. (C) 2015 The Fisheries Society of the British Isle

Isolation and analysis of a novel grass carp toll-like receptor 4 (tlr4) gene cluster involved in the response to grass carp reovirus

The mammalian response to lipopolysaccharide (LPS) is mainly mediated by Toll-Like Receptor 4 (TLR4). Fish and mammalian TLR4 vary; fish TLR4 ligands are unknown. Isolation of fish tlr4 genes is difficult due to their complex genomic structure. Three bacterial artificial chromosome (BAC) clones containing grass carp tlr4 were obtained. Four tlr4 genes, with a varied genomic structure and different protein domains were subsequently isolated by constructing a subcloned library and rapid amplification of cDNA ends (RACE). The four tlr4 genes were expressed during development from 12 h post-fertilization, in all healthy adult fish tissues tested, and significantly increased in grass carp reovirus (GCRV)-infected liver and muscle, suggesting the tlr4 genes play a role in GCRV infection. This study effectively separated each gene in the tlr4 gene cluster, implies that grass carp TLR4 proteins have different ligand recognition specificities to mammalian TLRs, and provides information on the functional evolution of TLRs. (C) 2012 Elsevier Ltd. All rights reserved.The mammalian response to lipopolysaccharide (LPS) is mainly mediated by Toll-Like Receptor 4 (TLR4). Fish and mammalian TLR4 vary; fish TLR4 ligands are unknown. Isolation of fish tlr4 genes is difficult due to their complex genomic structure. Three bacterial artificial chromosome (BAC) clones containing grass carp tlr4 were obtained. Four tlr4 genes, with a varied genomic structure and different protein domains were subsequently isolated by constructing a subcloned library and rapid amplification of cDNA ends (RACE). The four tlr4 genes were expressed during development from 12 h post-fertilization, in all healthy adult fish tissues tested, and significantly increased in grass carp reovirus (GCRV)-infected liver and muscle, suggesting the tlr4 genes play a role in GCRV infection. This study effectively separated each gene in the tlr4 gene cluster, implies that grass carp TLR4 proteins have different ligand recognition specificities to mammalian TLRs, and provides information on the functional evolution of TLRs. (C) 2012 Elsevier Ltd. All rights reserved

Cloning and characterization of the grass carp (Ctenopharyngodon idella) Toll-like receptor 22 gene, a fish-specific gene

Toll-like receptor 22 (TLR22) is a fish-specific TLR which recognizes double-strand (ds) RNA and participates in the innate immune response through the Toll-IL-1R homology domain-containing adaptor protein 1 (TICAM-1). To further investigate how the innate immune system of teleosts responds to viral infections, we cloned the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) TLR22 (CiTLR22). The complete cDNA sequence of CiTLR22 was 3647 bp and encodes a polypeptide of 954 amino acids. Analysis of the deduced amino acid sequence indicated that CiTLR22 has typical structural features of proteins belonging to the TLR family. These included 17 LRR domains (residues 88-634) and one C-terminal LRR domain (LRR-CT, residues 694-745) in the extracellular region, and a TIR domain (residues 801-944) in the cytoplasmic region. Comparison with homologous proteins showed that the deduced CiTLR22 has the highest sequence identity to common carp TLR22 (82.9%). Genomic DNA of CiTLR22 was obtained by long-distance (Ld) PCR and structure analysis revealed that the CiTLR22 gene is encoded by uninterrupted exons. Reverse transcriptase-PCR (RT-PCR) revealed that CiTLR22 is a non-maternal gene. It is prominently expressed in immune relevant tissues such as spleen and head kidney. Quantitative RT-PCR analysis showed that CiTLR22 transcripts were upregulated significantly in immune relevant tissues and blood following grass carp reovirus (GCRV) infection. In the whole genomic sequence, nine single nucleotide polymorphisms (SNPs) were detected. Seven of them were sited in the coding region, and the other two located in the 5' and 3' untranslated region (UTR) respectively. None of the SNPs was associated with the resistance of grass carp to GCRV. These results suggested a role for CiTLR22 in mediating immune protection against viral infection in grass carp. (C) 2012 Elsevier Ltd. All rights reserved.Toll-like receptor 22 (TLR22) is a fish-specific TLR which recognizes double-strand (ds) RNA and participates in the innate immune response through the Toll-IL-1R homology domain-containing adaptor protein 1 (TICAM-1). To further investigate how the innate immune system of teleosts responds to viral infections, we cloned the full-length cDNA sequence of grass carp (Ctenopharyngodon idella) TLR22 (CiTLR22). The complete cDNA sequence of CiTLR22 was 3647 bp and encodes a polypeptide of 954 amino acids. Analysis of the deduced amino acid sequence indicated that CiTLR22 has typical structural features of proteins belonging to the TLR family. These included 17 LRR domains (residues 88-634) and one C-terminal LRR domain (LRR-CT, residues 694-745) in the extracellular region, and a TIR domain (residues 801-944) in the cytoplasmic region. Comparison with homologous proteins showed that the deduced CiTLR22 has the highest sequence identity to common carp TLR22 (82.9%). Genomic DNA of CiTLR22 was obtained by long-distance (Ld) PCR and structure analysis revealed that the CiTLR22 gene is encoded by uninterrupted exons. Reverse transcriptase-PCR (RT-PCR) revealed that CiTLR22 is a non-maternal gene. It is prominently expressed in immune relevant tissues such as spleen and head kidney. Quantitative RT-PCR analysis showed that CiTLR22 transcripts were upregulated significantly in immune relevant tissues and blood following grass carp reovirus (GCRV) infection. In the whole genomic sequence, nine single nucleotide polymorphisms (SNPs) were detected. Seven of them were sited in the coding region, and the other two located in the 5' and 3' untranslated region (UTR) respectively. None of the SNPs was associated with the resistance of grass carp to GCRV. These results suggested a role for CiTLR22 in mediating immune protection against viral infection in grass carp. (C) 2012 Elsevier Ltd. All rights reserved