| In recent years, great economic losses were caused by aquatic epidemic in worldwide. As lower vertebrates, fish rely mainly on innate immunity against pathogens infection for the defective adaptive immune system. Grass carp (Ctenopharyngodon idella) is considered one of the economically important aquaculture species in China with the largest production among the"Great Four Cultured Fish". Despite its delicate taste, fast growth and low production cost, the toughest challenge is that grass carp is susceptible to a wide variety of diseases, and the annual loss reaches billion yuan. Grass carp hemorrhage was caused by Grass Carp Reovirus (GCRV) (a dsRNA virus), which was one of the most serious epidemics in grass carp. Up to now, no effective method was appearent for the prevention and treatment of this disease. And the available vaccines were less effective for fish's defective adaptive immunity. Thus, identifying critical molecular and polymorphism sites which were associated with resiatance to GCRV in the innate immunity networks will shed new light on the disease control in aquaculture.TLR (Toll-like receptors) NLR (Nod-like receptors) and RLR (RIG-I-like receptors) signaling pathways were the remarkable examples of homologs executing antiviral roles which were revolutionarily conserved in metazoans. The TLR pathway is considered to be one of the most crucial antiviral mechanisms in multicellular animals. At least 13 members have been found in mammal and more members have been found in aquatic animals. Different members identify various pathogens associated molecular patterns (PAMP) and eventually produce a series of reponse to disease. Therefore clarify the role of TLR family member in the antiviral response and found the association with the disease resistance is of great value.In the present study, the primers were designed according to the gDNA sequence of CiTLR3 we got previously. The single nucleotide polymorphism sites were got through the sequencing and multiple sequence alignment. The resistant and susceptible dividuals were classfied in the infection experiment and all of them genotyped with PCR-RFLP. The result was analysed withχ2 test. For the CiTLR22 study, degenerate primers were designed according to multiple sequence alignement to get core amplicons, and then RACE (Rapid amplification of cDNA end), semi-quantitative RT-PCR (sqRT-PCR), qRT-PCR were processed for cloning and expression analysis. Results were as follows:(1) Polymorphisms in CiTLR3 and their association with the susceptibility/resistance to grass carp reovirusWe sequenced the whole sequence including coding sequence and non-coding sequence with twelve pairs of primers, and twelve polymorphic loci (-764 G/T, -613 A/C, -543 A/G, -488 G/T, -304 A/G, -106 G/T, -11 A/G, 1811 A/G, 3917 G/T, 4116 G/T, 4683 G/T and 4731 C/T) and an ins-del (1 --/AT) mutation were found. Seven sites located in the 5'UTR and the first intron. Only three of them located in the coding sequence and all of them were synonymous mutation, and the other three located in the 3'UTR. The statistical result indicated that only -764 G/T was significantly associated with the resistance of grass carp to GCRV both in genotype (P=0.040) and allele (P=0.025). In the confirmation experiment the mortality in the -764 GG genotype individuals was significantly lower than GT genotype and TT genotype. Linkage disequilibrium analysis revealed -543 A/G, -488 G/T, 4116 G/T and 4731 C/T were linkage disequilibrium, and haplotype analysis revealed that haplotype GTTT frequency in susceptible group was significantly higher than that in the resistant group. All the results indicated that haplotype GTTT and genotype -764 TT and -764 GT individuals were susceptible to GCRV while -764 GG was resistant, which could be the optional markers for selective breeding for the GCRV-resistant grass carp in future.(2) The association between CiTLR22 and the resistance to grass carp reovirusToll-like receptor 22 (TLR22) is one of the special members of TLR family in teleost, which recognizes double strand RNA (dsRNA) as PAMP in the innate immunity and induce IFN. In the present study, we cloned grass carp (Ctenopharyngodon idella) Toll-like receptor 22 (CiTLR22) gene by homology cloning and rapid amplification of cDNA ends polymerase chain reaction (RACE-PCR). CiTLR22 was composed of 3831 base pairs and translated into a protein of 954 amino acid residues. The predicted protein was composed of a signal peptide (1-30aa) 17 Leucine-rich repeats (LRR) motifs a transmembrane (TM) domain and a Toll-interleukin 1-resistance (TIR) domain. Analysis of the homology between CiTLR22 and other known TLR22 has revealed significant similarities to common carp goldfish and zebrafish. The tissue-specific expression pattern detection indicated that CiTLR22 was constitutively expressed in all tested tissues. The highest level was detected in gill and the lowest was in brain liver and spleen. The expression profile of CiTLR22 both in spleen and liver was up-regulated after infection of grass carp reovirus (GCRV). Similarly, the expression level was regulated in treatment of GCRV in the CIK cell. The expression level was obviously modulated in treatments of poly I:C at the density of 5μg/ml, 10μg/ml, 25μg/ml in the CIK cell. While the CiTLR22 expression level on the treatment of complexed poly I:C at 5μg/ml was lower than other treatments at the same time point. 6 single nucleartide polymorphism loci (-8 A/T, 417 G/T, 863 C/T, 1923 G/T, 2406 C/T,2574 C/T) were detected in CiTLR22 gene. 2406 C/T was just a mutation in the following PCR-RFLP analysis. The single nucleotide polymorphism loci located in the coding sequence were synonymous mutation. The statistical result in the PCR-RFLP indicated that 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). Linkage disequilibrium analysis revealed -8 A/T and 2574 C/T, 863 C/T and 1923 G/T, 863 C/T and 2574 C/T were linkage disequilibrium, while no significant association was found between the haplotype and the resistance to GCRV.All the results indicated that CiTLR22 was induced in the antivirus response. The single nucleotide polymorphism -764 G/T the haplotype GTTT among -543 A/G, -488 G/T, 4116 G/T and 4731 C/T in CiTLR3 and the polymorphism 417 G/T in CiTLR22 were associated with the susceptility/resistance of grass carp to GCRV. The present study promisingly supplied the optional markers for selective breeding for the GCRV-resistant grass carp in future. |
