Identification, mRNA expression and genomic structure of TLR22 and its association with GCRV susceptibility/resistance in grass carp ( Ctenopharyngodon idella)

Abstract

Toll-like receptor 22 ( TLR22) plays a crucial role in response to virus infection by recognizing double stranded RNA (dsRNA) in aquatic animals. In the present study, a TLR22 homologue gene was identified and characterized from grass carp ( Ctenopharyngodon idella) ( CiTLR22). CiTLR22 genomic sequence comprises 4754 base pairs (bp), containing one intron. The cDNA sequence consists of 3831 bp, encoding a protein of 954 amino acid residues. CiTLR22 was constitutively expressed in all 15 investigated tissues, highly in gill and lowly in liver and spleen. The expression profile of CiTLR22 in spleen was rapidly and significantly up-regulated at 6 h (456.13-fold, P < 0.05), then rapidly recovered to normal level at 12 h ( P > 0.05) post-injection of grass carp reovirus (GCRV). The expression levels of CiTLR22 were rapidly elevated post-poly(I:C) stimulation in dose- and time-dependent manners in CIK ( C. idella kidney) cell line. After GCRV infection, CiTLR22 transcripts were inhibited at the early stage, then were up-regulated and reached a peak at 24 h post-infection, latterly down-regulated in CIK cell culture. In the whole genomic sequence, six single nucleotide polymorphisms (SNPs) were detected. Five of them were sited in the coding region and all synonymous, and another located in the 5′ untranslated region (UTR). The following SNP analysis revealed that 2406 C/T was just a mutation. Only 417 G/T was significantly associated with the resistance of grass carp to GCRV both in genotype ( P = 0.013) and allele ( P = 0.015). −8 A/T and 2574 C/T, 863 C/T and 1923 G/T, 863 C/T and 2574 C/T were pairwise linkage disequilibrium. None of the haplotype was associated with the resistance of grass carp to GCRV. The results indicate that CiTLR22 plays an important role in the responses to dsRNA and GCRV, and is partially inhibited by GCRV in vitro. The potential molecular marker lays foundation for the selective breeding of the GCRV-resistant grass carp.