| The first line defense ofcell innate immunity is receptors protein which can recognize conservative constituents of pathogenic bacteria.Transcriptome data in grass carp spleen injected with Aeromonas hydrophila revealed that there were six Toll-like receptors(TLRs)genes differentially expressed.The expression of these differentially expressed TLR genes insusceptible groupswas higher than that in resistance groups.Tissue exprssion profileshad revealed that TLR21 gene was highly expressed in skin,spleen and intestine.The expression of TLR18 gene was higher in spleen,gill and heart,and TLR22 gene expressionwas higher in spleen and head kidney.Those results suggested that TLRs genes were involved in immnune reponse in grass carp.In this paper,TLRs genes family members wereidentified,and comprehensive genes expression profiles of them had been examined based ongenome-wide level.Functional analysis of TLR18,TLR20 and TLR21 genes had been studied.Results were as follows:A complete set of 20 TLR genes were identified from the grass carp genome by comprehensive data mining of existing genomic resources,and they were classified into six subfamilies.Extensive comparative genomic analyses suggested that most TLR genes were conserved among vertebrates,while some were lineage-specific.Expression profiles of the TLR genes were determined in healthy grass carp tissues and individuals after A.hydrophila infection.The TLR genes were,overall,expressed at relatively low levels at the entire cellular transcriptome level,as the total RPKM value of the 20 TLR genes was approximately 144.73.Nine TLR genes were differentially expressed after bacterial infections in SG/RG grass carp.After A.hydrophila infection,six TLR genes were upregulated,and two genes were differentially expressed at all time-points.The extent of induction or suppression for all differentially expressed genes was 2.0-12.9 folds increased or decreased after the bacterial infection.Among these significantly upregulated genes,TLR20.2 was most significantly upregulated by approximately 12.9 times at 48 h post-challenge.Genome-wide identification and comprehensive gene expression profiling of TLR gene family members in grass carp would provide opportunities for functional analysis,to unravel their exact roles in host pathogen interactions.In vitro,after stimulation of C.idella kidney(CIK)cells with purified flagellin from S.typhimurium,lipopolysaccharide(LPS)and polyinosinic-polycytidylic acid(poly(I:C)),it was found that gctlr18 expression showed a tendency to rise firstly and then decrease,and reached the peak at 24h(9.78 fold),48h(8.87 fold)and 12h(8.92 fold),respectively.Overexpression of gctlr18 gene upregulated mRNA expression of il-8,inf-1 and tnf-?,the expressions of which increased by 1.2 folds,10.15 foldsand 3.61 folds,respectively,except foril-8.Meanwhile,it activated the NF-?B fluorescence reporter system,and defenses the bacterial invasion.The number of bacterial invasion of gctlr18-overexpressed cell group was 83.4% less than that of the control group.Moreover,gctlr18 might recognize flagellin,LPS and poly(I: C)pathogenic components,and gctlr18 overexpression could induce inflammatory factor secretion,which might be induced by NF-?B signaling pathway to remove exogenous pathogens.The TLR20 cDNA sequence was classified as gctlr20.2 according to the grass carp genome data.gctlr20.2 gene was includedin the differentially expressed genes in spleen transcriptome infected with A.hydrophila.In vitro stimulation experiments showed that flagellin,LPS and poly(I: C)could stimulate the expression of gctlr20.2.The expressions of gctlr20.2 reached 3.35 and 2.8 times at 24 h after stimulation of flagellin and poly(I: C)groups respectively,and whose expression reached highest expression of the 2.07 timesat 6h for LPS stimuluswhen.Overexpression and interference of gctlr20.2 gene had a regulational effect on downstream immune-related genes.gctlr20.2 overexpression induced increase of il1?,il8 and tnf-? transcription,il1? up to 1.46 times,il8 up to 1.45 times,tnf-? up to 1.91 times.SiRNA-tlr20.2 inhibited the efficiency of gctlr20.2 mRNA by 65.7% and down-regulated the transposition of ifn and il8 genes by 0.77 and 0.14 times,respectively.Thus,gctlr20.2 gene may also recognize flagellin,LPS and poly(I: C),and whose overexpression activates the NF-?B signaling pathway to cause inflammatory responses.Down-regulation of gctlr20.2 gene could reduce the secretion of inflammatory factors,and finally helped the body to keep an immune balance.In vitro,CIK cells stimulated with flagellin and LPS,it was found that gctlr21 expression was regulated,while overexpression and interference of this gene had a regulational effect on the downstream immune-related genes.In LPS infection,the gctlr21 expression rose to 2.45 times to reach the peak at 8.63 times,then slowly decreased from 6h to 48 h.In FLA-ST infection,the gctlr21 transcription level rose to 2.7 times at 6h,reached peak value 5.61 times at 24 h,and then back to 2.22 times at 48 h.Gctlr21 overexpression induced a significant up-regulation of il1? and ifn expression at 2.12 and 1.66 folds,respectively,and which activated the NF-?B fluorescence reporter system.The inhibitory effect of Sitlr21-2 on gctlr21 expression was 0.48 times,and the expression of il1?,il8 and tnf-? were significantly inhibited.Screening of target-regulated microRNAs revealed that Ci-miR-3,let-7i and Ci-miR-14 inhibited the transcription of gctlr21 gene expression.Agomir of microRNAs also could regulate immune-related genes.Thus,gctlr21 might also be involved in flagellin and LPS recognition processes,suggesting that gctlr21 overexpression could activate NF-?B regulation of inflammatory factor transcription.The effect of RNA interference was similar with that of Ci-miR-3,let-7i and Ci-miR-14,and it was speculated that gctlr21 could down-regulate the inflammatory reaction to avoid excessive injury. |
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