Genes Expression Analysis Of Toll-like Receptor Signaling Pathway In Northeast Chinese Lamprey(Lethenteron Morii)

Northeast Chinese lamprey(Lethenteron morii)is an ancient jawless animal that evolved between invertebrates and vertebrates.Research on its immune system has theoretically significant implications for all organism.Toll-like receptors are most of the non-specific pattern-recognition receptors(PRRs)that recognize pathogens and activate the innate immunity.In this study,the full-length cDNA of TLR7 a,TLR7b,TLR14 d and TLR2 d were cloned,and tissue distribution in juvenile and adult and the gene expression in the tissues(supraneural body,gill,intestine,kidney)of adult challenged by Pseudomonas aeruginosa were studied by real-time fluorescence quantitative PCR(qPCR).Eukaryotic expression vector were constructed and detect the localization of TLR in HEK293 T cells.In order to further explore the function of the TLR gene and its signal transduction process,the adaptor protein(MyD88 and TRAF6)in the TLR signal pathway and the downstream transcription factor NF-кB family members and their promoters were studied.The results as following: 1.The cloning and bioinformatics analysis of the TLR gene in Northeast Chinese lampreyThe cDNA sequences of the TLR7 a,TLR7b,TLR14 d and TLR2 d were obtained by cloning technology.The open reading frame(ORF)of the four genes are 3252 bp,3117 bp,2241 bp,2439 bp;encoding1083,1038,746,812 amino acids,respectively.TLR7 a,TLR7b and TLR2 d have LRR domain,transmembrane region,and TIR domain,but TLR14 d lacked of LRR domain.Phylogenetic tree analysis showed that TLR7 a and TLR7 b were clustered with TLR7 in mammals and fish,respectively.TLR14 d and TLR2 d were clustered with TLR2 subfamily in mammals,birds,amphibians and teleosts,and TLR14 d had the closest phylogenetic relationship with TLR14 in Japanese Pufferfish(Takifugu rubripes).2.Expression analysis of TLR gene in Northeast Chinese lampreyThe results of qPCR showed that the TLR gene was expressed in the tissues detected in juvenile and adult.The four TLR genes had the highest expression levels in the juvenile heart;TLR7b and TLR14 d had the highest expression levels in the adult brain.In addition,these four TLR genes had higher expression levels in immune-related tissues such as supraneural body,liver,and skin.The expression of TLR in adult challenged by P.aeruginosa was analyzed with qPCR.The result showed that the TLR genes were induced by bacteria,but expression pattern of different tissues at different time were different.The expression of TLR7 a was significantly up-regulated in supraneural body,gill,intestine and kidney(P<0.05);the expression of TLR7 b was significantly up-regulated in supraneural body and kidney(P<0.05);the expression of TLR14 d was significantly up-regulated in supraneural body,gill and kidney(P<0.05);the expression of TLR2 d was significantly up-regulated in supraneural body,gill and kidney(P<0.05),but it was significantly lower in the intestine(P<0.05).3.The subcellular localization of TLR gene in Northeast Chinese lampreyThe pCMV-TLR recombinant plasmid was transfected into HEK293 T cells to observe the location of TLR.The results showed that TLR14 d was localized in the nucleus,cytoplasm and multiple organelles;TLR2d was localized in the cytoplasm and multiple organelles.4.The cloning and bioinformatics analysis of MyD88 and TRAF6 genes in Northeast Chinese lampreyThe cDNA sequences of MyD88 and TRAF6 genes were obtained by cloning.The ORF of MyD88 is 852 bp,encoding 283 amino acids,its sequence contains one death domain and one TIR domain.The ORF of TRAF6 is 1785 bp and encoded 594 amino acids,its sequence contains one RING domain,two zinc fingers,one coiled-coil region and one MATH domain.The phylogenetic tree showed that MyD88 and TRAF6 were clustered with MyD88 and TRAF6 in mammals,birds,amphibians and teleosts,respectively.5.Expression of MyD88 and TRAF6 genes in Northeast Chinese lampreyThe results of qPCR showed that MyD88 and TRAF6 were constitutively expressed in all tissues of juvenile and adult.Both the two genes had higher level expression in skin and gill in the juvenile.In the adult,MyD88 in kidney and muscle as well as TRAF6 in the kidney and gill had higher level expression.qPCR showed that the expression of MyD88 was significantly up-regulated in the gill,kidney and supraneural body after 12 h,24 h,and 48 h of adult challenged by P.aeruginosa(P<0.05).Similarly,the expression of TRAF6 was significantly up-regulated in supraneural body after 12 h and in gill,kidney and intestine after 24 h(P<0.05).6.The cloning and bioinformatics analysis of NF-кB family genes in Northeast Chinese lampreyThe cDNA sequences of four Rel genes were obtained with cloning technology.The ORF of Rel-a,Rel-b,Rel-c and Rel-d are 1011,999,900 and 999 bp,encoding 336,332,299 and 332 amino acids,respectively.Each Rel gene contains one RHD domain and one IPT domain.The phylogenetic tree showed that the four Rel genes were clustered with Rel in mammals and teleosts.7.Expression of the orthologous genes of NF-кB family in Northeast Chinese lampreyThe results of qPCR showed that in the juvenile,the expression of Rel-a and Rel-b had higher level in gill,and Rel-c and Rel-d had higher level in skin.In the adult,the expression of Rel-a and Rel-c had higher level in muscle and heart,Rel-b and Rel-d had higher level in gill.8.Promoter activity of NF-кB family in Northeast Chinese lampreyThe DNA sequences of the four Rel promoters were obtained with cloning techniques,the results showed that the promoter had important transcription factor binding sites(TFBS),such as Oct-1,GATA-1,AP-1,NF-1,SP-1.All the promoters except Rel-c had the TATA Box and CAAT Box that were closely related to transcription.The results of the dual luciferase reporter gene showed that the Rel-b promoter had the highest activity,which was 8.77 folds than the control group.9.Effect of TLR gene in Northeast Chinese lamprey after overexpression on the activity of Rel-b PromoterThe results of the dual luciferase reporter gene showed that TLR14 d and TLR2 d inhibited the activity of Rel-b promoter.However,TLR14 d and TLR2 d co-transfected with the adaptor molecule Myd88 could enhance the activity of Rel-b promoter significantly.In this study,the full-length cDNA of four TLR genes(TLR7a,TLR7 b,TLR14d and TLR2d)were cloned for the first time.The expressions of the four TLR genes of Northeast Chinese lamprey in different tissues at different growth stages as well as chanllenged by bacteria were analyzed.The location of TLR14 d and TLR2 d in HEK293 T cells were determined.In order to further study the function of TLR genes,the adaptor protein(MyD88 and TRAF6)in TLR signal pathway,downstream transcription factor NF-кB family members and their promoters were also identified.The activity of Rel-b promoter in overexpressing TLR gene was detected.It is expected to provide a theoretical basis for the study of TLR and its function in signal transduction.