Function Analysis Of Single Nucleotide Polymorphisms(Snps)in Porcine Tlr3Gene

Toll-like receptor3(TLR3), a member of TLRs family, can specifically recognize double stranded RNA (dsRNA) that is the intermediate product during multiplication of virus. Unpon binding to dsRNA, TLR3can initiate a complex signal cascade to modulate downstream cytokine, including type I interferon, interleukin6(IL-6), IL-8, production through the activation of interferon regulatory factor3(IRF3) and nuclear factor kappa B (NF-κB). Studies on human and mouse have shown that TLR3is an important candidate gene for disease resistance, and polymorphisms in it have been associated with susceptibility/resistance to numerous diseases.This study was designed to analyze the functional significance of three nonsynonymous SNPs, C.800C>T (p. T266M), c.933C>G (p.I1311M), and c.2129C>G (p.T710S), which were selected based on our previous research, in porcine TLR3gene in transiently transfected PK-15cells by using site-directed mutagenesis, eukaryotic expression, dual-luciferase reporter system, and western blot methods. The main results are as follows:1. The complete coding sequence of porcine TLR3gene which is2718bp in length was obtained by RT-PCR method. The predicted polypeptide is composed of905amino acids. Compared with porcine TLR3mRNA deposited in GenBank (No, DQ266435.1), there are3synonymous SNPs, C.36C>T, c.2643T>C and c.2649A>G, in cloned sequence;2. Site-directed mutation was introduced into porcine TLR3gene cloned using PCR-based method and TLR3variants with point mutation at positions800(C>T),933(C>G) and2129(C>G) in CDS were obtained. The recombinant pcDNA3.1+plasmids carrying TLR3gene (wild or mutant type), pcDNA3.1-TLR3W, pcDNA3.1-C800T-TLR3V pcDNA3.1-C933G-TLR3V and pcDNA3.1-C2129G-TLR3V, were constructed successfully;3. Compared with wild tylpe TLR3gene, SNP c.933A>G significantly decreased the ability of receptor to induce luciferase activity (p<0.01), which indicating that the mutation has an influence on ligand recognition and signal transduction;4. SNP c.933A>G has no effect on the translation of TLR3, which was revealed by western-blot analysis.