Transcriptome analysis of head kidney in grass carp and discovery of immune-related genes.

BMC Veterinary Research (Impact Factor: 1.86). 07/2012; 8(1):108. DOI: 10.1186/1746-6148-8-108
Source: PubMed

ABSTRACT BACKGROUND: Grass carp (Ctenopharyngodon idella) is one of the most economically important freshwater fish, but its production is often affected by diseases that cause serious economic losses. To date, no good breeding varieties have been obtained using the oriented cultivation technique. The ability to identify disease resistance genes in grass carp is important to cultivate disease-resistant varieties of grass carp. RESULTS: In this study, we constructed a non-normalized cDNA library of head kidney in grass carp, and, after clustering and assembly, we obtained 3,027 high-quality unigenes. Solexa sequencing was used to generate sequence tags from the transcriptomes of the head kidney in grass carp before and after grass carp reovirus (GCRV) infection. After processing, we obtained 22,144 tags that were differentially expressed by more than 2-fold between the uninfected and infected groups. 679 of the differentially expressed tags (3.1%) mapped to 483 of the unigenes (16.0%). The up-regulated and down-regulated unigenes were annotated using gene ontology terms; 16 were annotated as immune-related and 42 were of unknown function having no matches to any of the sequences in the databases that were used in the similarity searches. Semi-quantitative RT-PCR revealed four unknown unigenes that showed significant responses to the viral infection. Based on domain structure predictions, one of these sequences was found to encode a protein that contained two transmembrane domains and, therefore, may be a transmembrane protein. Here, we proposed that this novel unigene may encode a virus receptor or a protein that mediates the immune signalling pathway at the cell surface. CONCLUSION: This study enriches the molecular basis data of grass carp and further confirms that, based on fish tissue-specific EST databases, transcriptome analysis is an effective route to discover novel functional genes.

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    ABSTRACT: Trunk kidney is a vital organ for excretion in teleosts. There have been sporadic reports of processing pathogens for the immune function in trunk kidney. However, molecular processes of pathogen recognition receptors (PRRs) responding to virus and viral/bacterial pathogen-associated molecular patterns (PAMPs) are poorly elucidated in trunk kidney. In the present study, we investigated transcriptional profiles of twelve representative immune-related genes (TLRs (TLR3, TLR7 and TLR22); RLRs (RIG-I, MDA5 and LGP2); NLRs (NOD1 and NOD2); adaptor molecules (MyD88 and IPS-1); effector molecule type I interferon (IFN-I) and immunoglobulin M (IgM)) in trunk kidney tissue of grass carp (Ctenopharyngodon idella) (designated as Ci) injection of grass carp reovirus (GCRV) utilizing quantitative real-time RT-PCR (qRT-PCR). Furthermore, mRNA expression patterns of these genes (IgM excepted) were examined post GCRV infection and polyinosine-polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS) or peptidoglycan (PGN) stimulation in primary trunk kidney cells of grass carp. The relative values of CiTLR3, CiTLR22 and CiMyD88 were increased post GCRV challenge and viral/bacterial PAMPs stimulation. The mRNA transcriptions of CiTLR7 were obviously activated with GCRV challenge. Remarkably, the mRNA expressions of CiRIG-I, CiMDA5, CiLGP2 and CiIPS-1 were largely up-regulated with GCRV challenge and viral/bacterial PAMPs stimulation. Interestingly, the expression tendencies of CiNOD1 and CiNOD2 were differential not only in GCRV challenge and poly(I:C) stimulation, but also in LPS and PGN stimulation. It was demonstrated that CiIFN-I induced powerful anti-viral and anti-bacterial effects in trunk kidney. In addition, the expression of CiIgM was induced at 72 h post GCRV injection in vivo. Collectively, these results suggest that trunk kidney of grass carp serves as an important immune organ, and plays crucial roles in triggering anti-viral and anti-bacterial immune responses both in vivo and in vitro.
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    ABSTRACT: Interleukin-8 (IL-8) is a CXC chemokine that plays key regulatory roles in the immune and inflammatory responses implicated in many human diseases. In this study, we identified and characterized an IL-8 homologue from the grass carp, Ctenopharyngodon idellus. A sequence alignment of the full-length cDNA and genomic DNA showed that the exon/intron organization of grass carp IL-8 (gcIL-8) is identical to those of other known CXC chemokine genes. A multiple alignment analysis showed that gcIL-8 is an ELR(-)CXC chemokine, and its deduced amino acid sequence shares 81% and 36% identity with common carp IL-8s L1 (GenBank ID: ABE47600) and L2 (GenBank ID: AB470924), respectively, suggesting that it belongs to the lineage 1 group of fish IL-8 proteins. On a phylogenetic tree, gcIL-8 clustered with other teleost IL-8 proteins to form a fish-specific clade, clearly distinct from those of bird, mammal, and amphibian proteins. Real-time quantitative PCR analysis indicated that gcIL-8 is differentially expressed in various tissues under normal conditions and that the expression of gcIL-8 mRNA in immune-related tissues is clearly upregulated by Aeromonas hydrophila infection. To explore the biological effects of gcIL-8, we produced a recombinant protein, rgcIL-8, in a prokaryotic expression system. Purified rgcIL-8 was confirmed to be chemoattractive for head kidney neutrophils and mononuclear leukocytes in vitro. Our histopathological study also revealed that rgcIL-8 exerts proinflammatory effects by inducing neutrophil infiltration and erythrocyte extravasation. Overall, these results suggest that IL-8 is crucially involved in the inflammatory responses of fish.
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