Molecular cloning, characterization and expression analysis of TLR9, MyD88 and TRAF6 genes in common carp (Cyprinus carpio)

Pawapol Kongchum, Eric M. Hallerman, Gideon Hulata, Lior David, Yniv Palti*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

95 Scopus citations

Abstract

Induction of innate immune pathways is critical for early host defense, but there is limited understanding of how teleost fishes recognize pathogen molecules and activate these pathways. In mammals, cells of the innate immune system detect pathogenic molecular structures using pattern recognition receptors (PRRs). TLR9 functions as a PRR that recognizes CpG motifs in bacterial and viral DNA and requires adaptor molecules MyD88 and TRAF6 for signal transduction. Here we report full-length cDNA isolation, structural characterization and tissue mRNA expression analysis of the common carp (cc) TLR9, MyD88 and TRAF6 gene orthologs. The ccTLR9 open-reading frame (ORF) is predicted to encode a 1064-amino acid (aa) protein. We found that MyD88 and TRAF6 genes are duplicated in common carp. This is the first report of TRAF6 duplication in a vertebrate genome and stronger evidence in support of MyD88 duplication is provided. The ccMyD88a and b ORFs are predicted to encode 288-aa and 284-aa peptides, respectively. They share 91% aa sequence identity between paralogs. The ccTRAF6a and b ORFs are both predicted to encode 543-aa peptides sharing 95% aa sequence identity between paralogs. The ccTLR9 gene is contained in a single large exon. The ccMyD88a and ccMyD88b coding sequences span five exons. The TRAF6b gene spans six exons. PCR amplification to obtain the entire coding sequence of ccTRAF6a gene was not successful. The 2104-bp fragment amplified covers the 3' end of the gene and it contains a partial sequence of one exon and three complete exons. The predicated protein domains of the ccTLR9, ccMyD88 and ccTRAF6 are conserved and resemble orthologs from other vertebrates. Real-time quantitative PCR assays of the ccTLR9, MyD88a and b, and TRAF6a and b gene transcripts in healthy common carp indicated that mRNA expression varied between tissues. Differential expression of duplicate copies were found for ccMyD88 and ccTRAF6 in white and red muscle tissues, suggesting that paralogs may have evolved and attained a new function. The genomic information we describe in this paper provides evidence of sequence and structural conservation of immune response genes in common carp.

Original languageAmerican English
Pages (from-to)361-371
Number of pages11
JournalFish and Shellfish Immunology
Volume30
Issue number1
DOIs
StatePublished - Jan 2011

Bibliographical note

Funding Information:
This study was supported by a grant from the US–Israel Binational Agricultural Research and Development Fund (Project No. IS-3997-07). P.K. was also supported by a Royal Thai Government doctoral scholarship . E.H. is supported by Virginia Agricultural Experimental Station through the USDA-NIFA Hatch Program. We thank Kristy Shewbridge, Roseanna Long, Brian Smith, Meghan Manor and Dr. Beth Cleveland for their technical assistance. We are very grateful to Naparat Prapaiwong and Mark Peterman at the E. W. Shell Fisheries Center, Auburn University, AL, USA for their help with the experimental fish. We also thank Dr. Gregory Wiens for reviewing this manuscript. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Keywords

  • Gene duplication
  • Innate immunity
  • MyD88
  • TLR9
  • TRAF6

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