Molecular Characteristic,Tissue Distribution And Signaling Pathway Of TLR5(Toll-like Receptor 5)of Duck(Cairna Moschata)

Toll-like receptors(TLRs)are a class of typical pattern recognition receptors(PRRs)that play a key role in host defense against invading pathogens.They can trigger innate immune responses by specifically recognizing conservative pathogen associated molecular patterns(PAMPs)found in pathogenic microorganisms.TLR5,a key member of the TLR family,initiate innate immunity to bacterial infection by recognizing and binding to bacterial flagellin,and further transmitting the signal of antigen into the cells via certain pathways.Although TLR5 of mammalian animals,such as human and mouse,were well studied,little is known about structure and functions of the duck TLR5.Herein,the present study conducted following researches:(1)By using molecular methods,the complete CDS of Cairna moschata TLR5 was cloned and sequenced.The molecular characterizations of the gene and encoded product were analyzed by bioinformatics methods.(2)Expression of the TLR5 in different tissue and organs were quantitatively evaluated.(3)In vivo expression of TLR5 and involved cytokines were determined after infection of ducking by flagellin-producing bacteria.Subsequently,in vivo expression of TLR5,important signal molecules and cytokines were also studied after stimulation of PBMC by flagellin.The main research methods and results of this study are presented as follows.1.Molecular cloning,characterization of Cairna moschata Toll-like receptor 5To amplify complete CDS of Cairna moschata TLR5 gene,3 sets of primers were designed in the conserved regions by comparison of TLR5 genes from different species,including mouse,chicken and goose.PCR products obtained from 3 primer pairs were separately cloned into T vector and sequenced.After assemble of the PCR products,the complete CDS of the TLR5 was successfully obtained.Bioinformatics techniques were used to analyze molecular characterizations of the TLR5 gene and encoded product.The results demonstrated that the CDS is 2580bp in length and encodes a protein with 859 amino acid.The putative amino acid sequence of Cairna moschata TLR5 consisted of a transmembrane domain,11 leucine-rich repeat(LRR)domains,a leucine-rich repeat C-terminal(LRR-CT)domain.The amino acid sequence of Cairna moschata TLR5 shared highest identity with Anas platyrhynchos(99.7%),97.7%with Tadorna tadorna,93.9%with goose.It suggested that TLR5 is highly conserved among the species.2.Development of a real-time PCR for duck TLR5 and quantitative anaysis of duck TLR5 expression in tissuesBased on the Cairna moschata TLR5 gene sequence obtained,a specific primer was designed and a SYBR Green I real-time RT-PCR for quantitative analysis of duck TLR5 mRNA expression was developed.Furthermore,the expression of different tissues TLR5 mRNA of Cairna moschata was detected by this method.The results showed that the developed real-time PCR have good sensitivity with the lowest detection detection limit of 6 copies/?l and the linear range was 6×103?6×108copies/?l(R2=0.998).Repeatability test results showed that the inter-assay variations were between 1.02%to 1.77%,intra-assay variations were between 0.15%to 2.0%.The TLR5 mRNA was expressed in a broad range of tissues and the strongest signals was obtained with spleen,liver,lung,and bursa.3.Selection of stable reference genes for quantitative analysis of mRNA expression by real-time PCRTo select stable reference genes for normalization of data from real-time PCR studies involved in afterward,in this study,the real-time PCRs targeting six reference genes(ACTB?GAPDH?18S rRNA?ALB?HSP90?RPL4)were developed.The expression levels of these genes in 13 kinds of tissues of E.coli-infected ducks were determined at Oh?12 h?24 h and 36h post-infection.The GeNorm software was used to evaluate the stability of the reference genes and determine the reference numbers necessary for accurate normalization of the data.The results showed that there were differences in the stability of internal reference genes in different tissues and organs after infection with E.coli.For kidney,the combination of three internal reference genes RPL4?18S rRNA and HSP90 are necessary to normalize the PCR data;while for spleen,lung,duodenum,jejunum,ileum,cecum,rectum and bursa of Fabricius,two reference genes should be used,that is,HSP90 and RPL4,18S rRNA and RPL4,RPL4 and 18S rRNA,RPL4 and 18S rRNA,RPL4 and 18S rRNA,RPL4 and 18SrRNA,RPL4 and 18S rRNA,RPL4 and HSP90,respectively.In the heart,liver,pancreas and brain,one reference gene is enough for PCR data normalization.They are HSP90,RPL4,RPL4 and 18S rRNA.The selected reference genes will be used in subsequent studies to ensure the reliability of the real-time PCR data.4.Response of TLR5 and inflammatory molecules against E.coli infection in ducksTo investigate the role of the TLR5 in the process of inflammatory against flagellin-producing bacteria,10-day-old healthy ducklings were orally infected by duck-derived E.coli O46 isolate SYW004,and the expression of TLR5,TNF-a,IL-1?and other inflammatory cytokines was measured using real-time RT-PCR at different time points post infection.Histopathological analysis was also performed.The results showed that the expression of TLR5 mRNA increased over time in most organs including the spleen,bursa of Fabricius,duodenum and cecum.The expression of TNF-a and IL-1? mRNA was also upregulated to varying degrees at corresponding time points.The expression of IL-6 mRNA was upregulated at 24 h after infection.Pathological analysis showed that varying degrees of hyperemia,hemorrhage and inflammatory cell infiltration were noted in all the tissues.Inflammation appeared the earliest and most severe in the spleen,bursa of Fabricius,duodenum and cecum.The results showed that the spleen,bursa of Fabricius,duodenum and cecum may be involved in TLR5-mediated inflammatory immune response against E.coli.5.TLR5-induced signaling mechanism of inflammatory response against bacterial infection in ducksPeripheral blood mononuclear cells(PBMCs)were isolated and prepared and stimulated with commercial flagellin.The dynamic expression of TLR5 and related signaling molecules was determined using real-time PCR.The results showed that the expression of TLR5,MyD88,NF-?b,TNF-a and IL-1? was upregulated at 4h after flagellin stimulation,peaked at 8h,and decreased thereafter.This suggests that TLR5,after recognizing and binding to flagellin,induces innate immunity to bacteria via MyD88-dependent pathway.In this study,the full-length CDS of Cairna moschata TLR5 was obtained for the first time,and its molecular characteristics were analyzed comprehensively.Quantitative RT-PCR was used to quantitatively analyze TLR5 expression in different tissues.The study proved,for the first time,that the expression of TLR5 is related to the degree of inflammatory response.TLR5 binds to flagellin and mediates the inflammatory response of bacterial infection by MyD88-dependent pathway.In conclusion,the present study explored molecular characterizations and functions of the duck TLR5,and has important significance to finally clarify the mechanism of innate immunity against bacterial infection induced by TLR5.