Dataset Information

Characterization and expression analysis of common carp Cyprinus carpio TLR5M.

ABSTRACT: TLR5 is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the TLR5M gene of common carp using the rapid amplification of cDNA ends (RACE) method. The TLR5M cDNA was 3182?bp in length and contained a 2658-bp open reading frame, which encoded a protein of 885 amino acids (aa). The entire coding region of the TLR5M gene was successfully amplified from genomic DNA and contained a single exon. The aa sequence of carp TLR5M showed the highest similarity (84.46%) to Cirrhinus mrigala. Tissue-specific expression analysis of the TLR5M gene by quantitative real-time polymerase chain reaction revealed its broad distribution in various organs and tissues; however, the highest level of TLR5M expression was noted in the liver. TLR5M gene expression was examined after flagellin stimulation and showed highly significant (p<0.01) induction in the spleen, heart, liver and kidney. The induction of TLR5M was analyzed in various organs infected with Aeromonas hydrophila. TLR5M gene expression in the kidney and spleen was significantly (p<0.01) increased. Concurrently, modulation of TLR5M gene expression and the induction of IFN-?, IL-1?, IL-10 and TNF-?4 were analyzed in peripheral blood leucocytes after lipopolysaccharide, concanavalin A, and flagellin stimulation. In the treated group, significant induction of these genes was noted, although the intensity varied between the tissues. These findings may indicate a crucial role for TLR5M in the innate immunity of common carp in response to pathogenic invasion.

SUBMITTER: Duan D

PROVIDER: S-EPMC3785217 | biostudies-literature | 2013 Oct

REPOSITORIES: biostudies-literature

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Characterization and expression analysis of common carp Cyprinus carpio TLR5M.

Duan Duo D Sun Zhen Z Jia Shengmei S Chen Yilong Y Feng Xiangru X Lu Qiang Q

DNA and cell biology 20130809 10

TLR5 is responsible for the recognition of bacterial flagellin in vertebrates. In this study, we cloned the TLR5M gene of common carp using the rapid amplification of cDNA ends (RACE) method. The TLR5M cDNA was 3182 bp in length and contained a 2658-bp open reading frame, which encoded a protein of 885 amino acids (aa). The entire coding region of the TLR5M gene was successfully amplified from genomic DNA and contained a single exon. The aa sequence of carp TLR5M showed the highest similarity (8 ...[more]

PMID: 23930591

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Project description:BackgroundAquaporins (Aqps) are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. Among vertebrate species, Aqps are highly conserved in both gene structure and amino acid sequence. These proteins are vital for maintaining water homeostasis in living organisms, especially for aquatic animals such as teleost fish. Studies on teleost Aqps are mainly limited to several model species with diploid genomes. Common carp, which has a tetraploidized genome, is one of the most common aquaculture species being adapted to a wide range of aquatic environments. The complete common carp genome has recently been released, providing us the possibility for gene evolution of aqp gene family after whole genome duplication.ResultsIn this study, we identified a total of 37 aqp genes from common carp genome. Phylogenetic analysis revealed that most of aqps are highly conserved. Comparative analysis was performed across five typical vertebrate genomes. We found that almost all of the aqp genes in common carp were duplicated in the evolution of the gene family. We postulated that the expansion of the aqp gene family in common carp was the result of an additional whole genome duplication event and that the aqp gene family in other teleosts has been lost in their evolution history with the reason that the functions of genes are redundant and conservation. Expression patterns were assessed in various tissues, including brain, heart, spleen, liver, intestine, gill, muscle, and skin, which demonstrated the comprehensive expression profiles of aqp genes in the tetraploidized genome. Significant gene expression divergences have been observed, revealing substantial expression divergences or functional divergences in those duplicated aqp genes post the latest WGD event.ConclusionsTo some extent, the gene families are also considered as a unique source for evolutionary studies. Moreover, the whole set of common carp aqp gene family provides an essential genomic resource for future biochemical, toxicological, physiological, and evolutionary studies in common carp.

Project description:BackgroundImmunoglobulins (Igs) distributed among systemic immune tissues and mucosal immune tissues play important roles in protecting teleosts from infections in the pathogen-rich aquatic environment. Teleost IgZ/IgT subclasses with different tissue expression patterns may have different immune functions.ResultsIn the present study, a novel secreted IgZ heavy chain gene was cloned and characterized in common carp (Cyprinus carpio). This gene exhibited a different tissue-specific expression profile than the reported genes IgZ1 and IgZ2. The obtained IgZ-like subclass gene designated CcIgZ3, had a complete open reading frame contained 1650 bp encoding a protein of 549 amino acid residues. Phylogenetic analysis revealed that CcIgZ3 was grouped with carp IgZ2 and was in the same branch as IgZ/IgT genes of other teleosts. Basal expression detection of the immunoglobulin heavy chain (IgH) in healthy adult common carp showed that CcIgZ3 transcripts were widely expressed in systemic immune tissues and mucosal-associated lymphoid tissues. CcIgZ3 was expressed at the highest levels in the head kidneys, gills, and gonads, followed by the spleen, hindgut, oral epithelium, liver, brain, muscle, foregut, and blood; it was expressed at a very low level in the skin. The transcript expression of CcIgZ3 in leukocytes isolated from peripheral blood cells was significantly higher than that in leukocytes isolated from the spleen. Different groups of common carp were infected with Aeromonas hydrophila via intraperitoneal injection or immersion. RT-qPCR analysis demonstrated that significant differences in CcIgZ3 mRNA levels existed between the immersion and injection groups in all the examined tissues, including the head kidney, spleen, liver, and hindgut; in particular, the CcIgZ3 mRNA level in the hindgut was higher in the immersion group than in the injection group. The different routes of A. hydrophila exposure in common carp had milder effects on the IgM response than on the CcIgZ3 response. Further study of the relative expression of the IgH gene during the development of common carp showed that the tissue-specific expression profile of CcIgZ3 was very different from those of other genes. RT-qPCR analysis demonstrated that the CcIgZ3 mRNA level increased gradually in common carp during the early larval development stage from 1 day post fertilization (dpf) to 31 dpf with a dynamic tendency similar to those of IgZ1 and IgZ2, and IgM was the dominant Ig with obviously elevated abundance. Analyses of the tissue-specific expression of IgHs in common carp at 65 dpf showed that CcIgZ3 was expressed at mucosal sites, including both the hindgut and gill; in contrast, IgZ1 was preferentially expressed in the hindgut, and IgZ2 was preferentially expressed in the gill. In addition to RT-qPCR analysis, in situ hybridization was performed to detect CcIgZ3-expressing cells and IgM-expressing cells. The results showed that CcIgZ3 and IgM transcripts were detectable in the spleens, gills, and hindguts of common carp at 65 dpf.ConclusionsThese results reveal that CcIgZ3 gene transcripts are expressed in common carp during developmental stage not only in systemic tissues but also in mucosal tissues. CcIgZ3 expression can be induced in immune tissues by A. hydrophila challenge via immersion and intraperitoneal injection with significantly different expression profiles, which indicates that CcIgZ3 is involved in the antimicrobial immune response and might play an important role in gut mucosal immunity.

Dataset Information

Characterization and expression analysis of common carp Cyprinus carpio TLR5M.

Publications

Characterization and expression analysis of common carp Cyprinus carpio TLR5M.

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