Cloning And Function Of TLR5 And Some Genes In NF-?B Family In Large Yellow Croaker

Based on the previously investigation of the two types of TLR5(LcTLR5M,LcTLR5S)of large yellow croaker,Larimichthys crocea,in our lab,we cloned the promotors of TLR5,and analyzed the structures and transcriptional binding sites of them.Furthermore,their transcriptional binding sites were verified by fragment deletion and site-directed mutagenesis.The subcellular localization plasmids of LcTLR5 M and LcTLR5 S were constructed and their expression profiles were studied.We also constructed the over-expression plasmids of LcTLR5 M and LcTLR5 S and detected their over-expression effects on the activity of p65 promoter by co-transfection with NF-?B-p65 subunit promoter,respectively.The full-length cDNA sequence of the RelB gene of the NF-?B family of large yellow croaker(LcRelB)was obtained,and its molecular structure and tissue expression profile of LcRel B were analyzed,as well as its spatiotemporal expression in Larimichthys crocea kidney(LCK)cell line was detected after the pathogen-related molecular pattern(PAMP)stimulation.We analyzed the subcellular localization of LcRelB.In addition,the effects of overexpression of LcRel B on some cytokines(e.g.TNF-?)and the regulation mechanism with I?B-? were analyzed,respectively.The promoter sequence of p65 gene was amplified and its activity was detected.And we studied the effect of p65 over-expression on the promoter activity of the downstream cytokines(IL-1?,TNF-? and IFN-I)and the regulatory mechanism of I?B-? for p65.The results are as follows:(1)The promoter of Lc TLR5 M was from-1829 bp to +189bp,including CEBP,Oct-1,GATA-1,AP1,and any other conserverative immune-related transcriptional binding sites,but lacking CpG island.A series fragment deletion analysis indicated that the core promoter region-417 to +189 might contain cis-elements.While the negative regulatory elements may be from-799 to-417.The LcTLR5 S promoter region was from-1932 bp to +106bp,the transcriptional binding sites including CEBP,Sp1,Oct-1,AP1,NF-?B were predicted in the promotor but no CpG island was predicted.The synergistic effect of promoter activity may be from-808 to +106.Oct-1 and NF-?B binding sites have positive effects on LcTLR5 S promoter activity.The activity of LcTLR5 S promoter could be activated by over-expression of NF-?B-p65.Subcellular localization analysis showed that LcTLR5 M was mainly located in cytomembrane while LcTLR5 S was mianly located in cytoplasm.Over-expression of LcTLR5 M did not show significantly affect on the activity of NF-?B-p65 promoter,while over-expression of LcTLR5 S could up-regulated NF-?B-p65 promoter activity significantly(p<0.05).(2)The full-length of RelB gene of large yellow croaker(LcRelB)was 1946 bp,containing 1788 bp open reading frame(ORF)which encoding 595 amino acids,including RHD-DNA-bind domain and IPT domain.The theoretical molecular weight of LcRelB amino acids was 66.5 KDa,with isoeletric point of 5.6.The phylogenetic analysis showed that the LcRelB was closely related to RelB from Takifugu rubripes.LcRelB transcripts were widely distributed in most determined tissues,with the highest expression in the blood,followed by spleen and head kidney,but the lowest expression in the skin.The expression of LcRelB in the LCK cell line was significantly up-regulated after stimulation with LPS and poly I:C but down-regulated after PGN challenge(p<0.05).The LcRelB protein was located in the nucleus.Over-expression of LcRelB significantly down-regulated the activity of IL-1? promoter,while the promoter activity of TNF-? and IFN-I were significantly up-regulated(p<0.05).And when the NF-?B family inhibitor Lc I?B-? and LcRelB co-transfected,the TNF-? promoter activity can be significantly inhibited.(3)p65 promoter is located between-1695 bp to +111bp,containing a(GT)n repeat region,a CpG island and some transcriptional binding sites,including USF,AP1,SRF,IRF-1,NF-?B.Our findings showed that the activity of Lcp65 promoter could be induced by the some PAMPs stimulation(LPS,PGN,Flagellin),among which,the induction of Flagellin was the most significant(p<0.01).Subcellular localization indicated that Lcp65 protein was mainly located in the nucleus.Over-expression of Lcp65 could significantly increase the promoter activity of IL-1?,TNF-? and IFN-I,and the activity of TNF-? was the strongest(p<0.05).And when the NF-?B family inhibitor Lc I?B-? and Lcp65 co-transfected,the TNF-? promoter activity can be significantly inhibited.