C. Idella Kidney Research Articles

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

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.

Myeloid differentiation factor 88 gene is involved in antiviral immunity in grass carp Ctenopharyngodon idella

A full-length MyD88 cDNA (CiMyD88) was cloned and characterized from grass carp Ctenopharyngodon idella. CiMyD88 was found to be broadly expressed and was up-regulated by grass carp reovirus (GCRV) and CiMyD88 transcripts in vitro were rapidly elevated in C. idella kidney (CIK) cells after challenge with poly(I:C). These results suggest that CiMyD88 may be involved in the antiviral immune defence in C. idella.

Evaluation of internal control genes for qRT-PCR normalization in tissues and cell culture for antiviral studies of grass carp ( Ctenopharyngodon idella)

Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) has become one of the most commonly used techniques for RNA expression. To obtain more reliable results with biological significance, it requires data normalization using an appropriate internal control gene. Here, we cloned partial sequence of elongation factor 1α ( EF1α) gene from grass carp ( Ctenopharyngodon idella). The stabilities of four commonly used internal control genes encoding 18S rRNA, β-actin, EF1α, and glyceraldehyde-3-phosphate dehydrogenase ( GAPDH) were integratedly assessed using the geNorm, NormFinder and BestKeeper programs. Integrative analyses of qRT-PCR data indicated that the stability ranking of the examined genes was 18S rRNA > EF1α > GAPDH > β-actin in gill, head kidney, heart, intestine, liver, muscle, skin, spleen, and trunk kidney tissues in untreated grass carp. When the same calculations were done in spleen tissue at different time points post grass carp reovirus (GCRV) infection, the gene ranking was 18S rRNA > β-actin > GAPDH > EF1α. The rank ordering of expression stability was EF1α > β-actin > 18S rRNA > GAPDH in C. idella kidney ( CIK) cell culture stimulated by poly(I:C). The recommended ranking was EF1α > GAPDH > β-actin > 18S rRNA in CIK cells infected by GCRV. The results indicated that 18S rRNA was the best invariant internal control gene in individual level in grass carp, EF1α was the most suitable in CIK cell culture stimulated by poly(I:C) or infected by GCRV. As an assay, EF1α was employed to examine the changes of Toll-like receptor 3 ( TLR3) and melanoma differentiation associated gene 5 ( MDA5) after virus infection in CIK cells. These data laid the foundation for more precise results in qRT-PCR studies of gene expression in grass carp.