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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:Omega-3 long chain polyunsaturated fatty acids (n-3 LC-PUFA), particularly docosahexaenoic acids (22:6n-3, DHA), have positive effects on multiple biologic and pathologic processes. Fish are the major dietary source of n-3 LC-PUFA for humans. Growing evidence supports acyl-coenzyme A (acyl-CoA) synthetase 6 (acsl6) being involved in cellular DHA uptake and lipogenesis in mammals, while its molecular function and regulatory mechanism remain unknown in fish. The present study focused on investigating the molecular characterization and transcription regulation of the acsl6 gene in the freshwater teleost common carp (Cyprinus carpio). First, the full length of acsl6 cDNA contained a coding region of 2148 bp for 715 amino acids, which possessed all characteristic features of the acyl-CoA synthetase (ACSL) family. Its mRNA expression was the highest in the brain, followed by in the heart, liver, kidney, muscle, and eyes, but little expression was detected in the ovary and gills. Additionally, a candidate acsl6 promoter region of 2058 bp was cloned, and the sequence from -758 bp to -198 bp was determined as core a promoter by equal progressive deletion and electrophoretic mobility shift assay. The binding sites for important transcription factors (TFs), including stimulatory protein 1 (SP1), CCAAT enhancer-binding protein (C/EBP?), sterol-regulatory element binding protein 1c (SREBP1c), peroxisome proliferator activated receptor ? (PPAR?), and PPAR? were identified in the core promoter by site-directed mutation and functional assays. Furthermore, the intraperitoneal injection of PPAR? agonists (balaglitazone) increased the expression of acsl6 mRNA, coupling with an increased proportion of DHA in the muscle, while opposite results were obtained in the injection of the SREBP1c antagonist (betulin). However, the expression of acsl6 and DHA content in muscle were largely unchanged by PPAR? agonist (fenofibrate) treatment. These results indicated that acsl6 may play an important role for the muscular DHA uptake and deposition in common carp, and PPAR? and SREBP-1c are the potential TFs involved in the transcriptional regulation of acsl6 gene. To our knowledge, this is the first report of the characterization of acsl6 gene and its promoter in teleosts.

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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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