Molecular Cloning,Sequence Analysis And Expression Quantity Of Toll-like Receptor 3,5 And 7 Genes In Wild Duck

Toll-like receptors(TLRs)are an important family of pattern recognition receptors(PRRs),which are distributed in almost all multicellular organisms.When the organism encounters pathogen invasion,TLRs can quickly recognize pathogen-associated molecular patterns(PAMPs),activate signaling pathways,and induce the expression of proinflammatory factors and interferons.It can activate natural immune response and mediate specific immune response to resist pathogens.At present,at least 10 species of TLRs have been identified,and TLR3/5/7 is homologous to mammals and has similar functions.TLR3 and TLR7 are distributed in cells and play an important role in the process of antiviral immune response.TLR3 recognizes dsRNA and poly(I:C),while TLR7 recognizes ssRNA and quinoline imidazole derivatives.TLR5 distributes on cell surface,mainly recognizes flagellin of bacteria,and plays an important role in bacterial infection.However,the current research on TLRs is mainly focused on poultry,while few studies on wild birds have been reported.Wild duck,as a repository of many important avian viruses,has strong tolerance to many pathogens,and TLRs is closely related to its natural immune mechanism against virus.Therefore,it is important to study its complex antiviral mechanism for the prevention and control of some avian diseases.In this study,specific primers were designed according to the conserved sequence of avian TLR3/5/7 gene.The ORF full-length genes of wild duck(mallard)TLR3/5/7 were cloned for the first time.They are 2688 bp,encoding 895 amino acids;2580 bp,encoding 859 amino acids;and 3144 bp,encoding 1047 amino acids.The prediction of protein domains of SMART program showed that TLR3/5/7 had typical TLRs family structure characteristics,at the same time,the structure of LRRs in the extracellular domain of each gene was significantly different.DNA Star program was used to analyze gene sequence and amino acid homology.The results showed that TLR3/5/7 gene had similar homology with birds,fish and mammals,from high to low,birds,mammals and fish,respectively.It was found by using MrBayes v.3.2 program that TLR3/5/7 gene has similar evolutionary relationships among birds,mammals and fish,and the evolutionary relationship among the same species is highly conservative.In addition,the fluorescence quantitative PCR detection methods for wild duck TLR3,TLR5 and TLR7 were established for the first time.The expression of TLR3/5/7 in tissues and viscera of different wild ducks was quantitatively analyzed by fluorescence quantitative PCR.The results showed that the expression level of wild duck species was higher than that of domestic duck,and the gene expression level in spleen was generally higher.The expression of TLR5 and TLR7 in the same and different tissues of different wild ducks was analyzed by one-way ANOVA.The difference of TLR5 and TLR7 expression was more significant than that of TLR3 in different wild duck species and domestic ducks.In this study,the full-length genes of Toll-like receptors 3,5 and 7 were successfully cloned and preliminarily analyzed by bioinformatics.An efficient and specifie fluorescenee quantitative PCR method for detection of wild duck TLRs was established.The significant relationship between the expression of Toll-like receptor subfamilies of wild ducks was revealed.These enrich our understanding of Toll-like receptors in wild ducks and provide theoretical support for further exploring the complex antiviral mechanism of wild ducks.