草鱼TLR基因鉴定及三个TLR基因免疫功能初步研究
发布时间：2018-09-12 15:12
【摘要】：细胞先天性免疫的第一道防线是那些能识别病源菌保守成分的受体。研究发现草鱼感染嗜水气单胞菌引发细菌性败血症。在感染转录组数据中发现TLR受体(Toll-like receptor)基因是差异表达基因,这说明TLR基因家族参与机体抵御外源性致病菌的入侵。另外组织表达谱结果显示,大部分TLR基因在免疫组织中高表达,比如皮肤、脾脏、肝和鳃等。从转录组水平和组织表达模式都说明TLR参与与机体免疫反应,因此对TLR基因全面、系统化的研究尤为重要。本文基于草鱼基因组、嗜水气单胞菌感染脾脏转录组、以及嗜水气单胞菌抗病组和易感组转录组数据深度剖析TLR基因具体信息,对已完成TLR18,TLR20和TLR21cDNA克隆的基础上进行功能性探究。主要结果如下:草鱼基因组有20种TLR基因,分属于6个亚家族,基因组中尚未发现TLR14,TLR16,TLR19和TLR26基因,却发现草鱼特有的TLR27基因。系统发育和共线性分析显示TLR11亚家族是最大的分支,TLR11家族基因共线性分析结果和洞穴鱼、斑马鱼、河豚和罗非鱼相似。TLR基因组序列分析,大部分TLR基因没有内含子,属于一个亚分支上的TLR基因一般有相似的内含子/外显子结构和数目。正常组织表达谱表明TLR5b,TLR5a,TLR1和TLR25表达量相对比较高,但总体TLR基因RPKM比较低,约为144.73。草鱼嗜水气单胞菌易感和抗病转录组中有9个TLR基因差异表达;脾脏转录组中6个TLR基因上调,上调或抑制的程度为2到12.9倍。其中TLR20.2上调最为显著,在感染48h时,该基因上调了12.9倍。体外gctlr18诱导表达,利用鞭毛蛋白,LPS和poly(I:C)分别刺激CIK细胞系,发现gctlr18的表达量均呈现先上升后下降的趋势,分别在24h,48h和12h表达量到达峰值,诱导倍数分别是9.78倍,8.87倍和8.92倍。过表达gctlr18基因对il-8,inf-1和tnf-α有调控作用,分别上升1.2倍,10.15倍和3.61倍,其中对il-8没有显著诱导性。过表达gctlr18能激活NF-κB荧光报告系统,以及抵御细菌入侵的作用,细菌侵染数比对照组少83.4%。因此,gctlr18可能识别鞭毛蛋白,LPS和poly(I:C)致病成分,gctlr18过表达能诱导炎症因子分泌,可能是通过NF-κB信号通路引发炎症以清除外源性病原菌。将TLR20 cDNA序列根据草鱼基因组数据库比对得到该基因属于gctlr20.2,感染嗜水气单胞菌草鱼脾脏转录组中属于差异表达基因。体外刺激实验表明,鞭毛蛋白,LPS和poly(I:C)能刺激诱导gctlr20.2的表达,鞭毛蛋白和poly(I:C)在24h达到峰值3.35倍和2.8倍,LPS刺激后6h时表达量最高2.07倍。过表达和干扰该基因对下游免疫相关基因有调控作用,gctlr20.2过表达诱导il1β,il8和tnf-α转录增加,il1β上调1.46倍,il8上调1.45倍,tnf-α上调1.91倍。siRNA-tlr20.2对gctlr20.2mRNA有65.7%的抑制效率,并且下调ifn和il8基因的转录,分别为0.77倍和0.14倍。因此,gctlr20.2也能识别鞭毛蛋白,LPS和poly(I:C)致病成分,gctlr20.2过表达激活NF-κB信号通路引发炎症反应。下调gctlr20.2能够减少炎症因子分泌,有助于机体恢复免疫平衡。鞭毛蛋白和LPS体外刺激gctlr21表达,同时过表达和干扰该基因对下游免疫相关基因有调控作用。LPS感染6h时,表达量上升到2.45倍,到24h到达峰值8.63倍,随后缓慢下降。FLA-ST感染6h时,转录水平上升至2.7倍,24h时出现峰值5.61倍,48h后下降到2.22倍。Gctlr21过表达诱导il1β和ifn表达显著性上调,分别为2.12倍和1.66倍,并且激活NF-κB荧光报告系统。Sitlr21-2对gctlr21表达抑制效果为0.48倍,并显著性抑制下游免疫因子il1β,il8和tnf-α的表达,对ifn抑制效果不明显。筛选靶调控microRNA,发现Ci-miR-3,let-7i和Ci-miR-14对gctlr21基因转录有抑制作用。过表达microRNA对免疫相关基因也有调控作用。因此,gctlr21基因可能也参与鞭毛蛋白和LPS的识别过程,推测gctlr21过表达能激活NF-κB调控炎症因子的转录。RNA干扰的效果与Ci-mi R-3,let-7i和Ci-miR-14也可以靶调控gctlr21的表达效果类似,推测gctlr21转录下调能减弱炎症反应避免机体过度损伤。
[Abstract]:The first line of defense against cellular innate immunity is those receptors that recognize the conserved components of pathogenic bacteria. Studies have found that grass carp infected with Aeromonas hydrophila cause bacterial sepsis. In addition, the results of tissue expression profiles showed that most TLR genes were highly expressed in immune tissues, such as skin, spleen, liver and gill. The transcriptome level and tissue expression patterns showed that TLR was involved in the immune response of the organism. Therefore, it is particularly important to study the TLR gene comprehensively and systematically. The transcriptome data of Aeromonas spp. infected spleen, Aeromonas hydrophila resistant and susceptible groups were analyzed in depth to explore the function of TLR gene based on the cloning of TLR18, TLR2 0 and TLR21. TLR14, TLR16, TLR19, and TLR26 genes were found to be unique to grass carp. Phylogenetic and collinear analyses showed that the TLR11 subfamily was the largest branch. The TLR11 family was similar to cave fish, zebrafish, puffer fish and tilapia in gene analysis. Normal tissue expression profiles showed relatively high levels of TLR5b, TLR5a, TLR1 and TLR25, but the overall TLR gene RPKM was relatively low, about 144.73. Nine TLR genes were differentially expressed in the susceptible and resistant transcripts of Aeromonas hydrophila. TLR genes were up-regulated and up-regulated by 2 to 12.9 times. TLR20.2 was up-regulated most significantly, and up-regulated by 12.9 times at 48h after infection. The expression of gctlr18 was induced by flagellin, LPS and poly (I:C) in vitro. The expression of gctlr18 increased first and then decreased, respectively, at 24h, 48h and 1h after infection. Overexpression of gctlr18 gene could regulate il-8, inf-1 and TNF-a by 1.2 times, 10.15 times and 3.61 times, respectively. overexpression of gctlr18 could activate the NF-kappa B fluorescence reporting system and resist bacterial invasion. Therefore, gctlr18 may recognize flagellin, LPS and poly (I:C) pathogenic components. Overexpression of gctlr18 can induce the secretion of inflammatory factors, possibly through the NF-kappa B signaling pathway to cause inflammation to eliminate exogenous pathogens. Flagellin, LPS and poly (I: C) stimulated the expression of gctlr20.2 in vitro. Flagellin and poly (I: C) peaked at 3.35 and 2.8 times at 24 h, and the highest expression was 2.07 times at 6 h after LPS stimulation. Overexpression and interference with the gene were immune to the downstream. Overexpression of gctlr20.2 induced an increase in the transcription of IL-1 beta, IL-8 and tnf-a, up-regulation of IL-1 beta by 1.46 times, up-regulation of IL-8 by 1.45 times and up-regulation of TNF-a by 1.91 times. siRNA-tlr20.2 inhibited the expression of gctlr20.2 mRNA by 65.7%, and down-regulated the transcription of IFN and IL-8 genes by 0.77 and 0.14 times, respectively. LPS and poly(I:C) pathogenic components, gctlr20.2 overexpression activates NF-kappa B signaling pathway to trigger inflammation. Downregulation of gctlr20.2 can reduce the secretion of inflammatory factors and help to restore immune balance. Flagellin and LPS stimulate the expression of gctlr21 in vitro, and over-expression and interference of the gene may regulate the downstream immune-related genes in LPS infection. At 6 h, the expression increased to 2.45 times, reached a peak value of 8.63 times at 24 h, and then decreased slowly. At 6 h, the transcriptional level of FLA-ST increased to 2.7 times, reached a peak value of 5.61 times at 24 h, and decreased to 2.22 times at 48 h. Overexpression of Gctlr21 induced significant up-regulation of IL-1 beta and IFN expression, 2.12 times and 1.66 times respectively, and activated the NF-kappa B fluorescence reporting system. 2 inhibited the expression of gctlr21 by 0.48 times, and significantly inhibited the expression of downstream immune factors IL-1 beta, IL-8 and tnf-a, but did not inhibit the expression of ifn. The Target-regulated microRNA screening showed that Ci-Mi-3, let-7i and CI-Mi-14 inhibited the transcription of gctlr21 gene. Overexpression of microRNA also regulated the expression of immune-related genes. Genes may also be involved in the recognition of flagellin and LPS, suggesting that the overexpression of gctlr21 can activate NF-kappa B to regulate the transcription of inflammatory factors. RNA interference is similar to that of CI-mi R-3, let-7i and CI-microRNA-14 in targeting the expression of gctlr21, suggesting that the down-regulation of gctlr21 transcription can attenuate inflammation and avoid excessive injury.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943
本文编号：2239421
[Abstract]:The first line of defense against cellular innate immunity is those receptors that recognize the conserved components of pathogenic bacteria. Studies have found that grass carp infected with Aeromonas hydrophila cause bacterial sepsis. In addition, the results of tissue expression profiles showed that most TLR genes were highly expressed in immune tissues, such as skin, spleen, liver and gill. The transcriptome level and tissue expression patterns showed that TLR was involved in the immune response of the organism. Therefore, it is particularly important to study the TLR gene comprehensively and systematically. The transcriptome data of Aeromonas spp. infected spleen, Aeromonas hydrophila resistant and susceptible groups were analyzed in depth to explore the function of TLR gene based on the cloning of TLR18, TLR2 0 and TLR21. TLR14, TLR16, TLR19, and TLR26 genes were found to be unique to grass carp. Phylogenetic and collinear analyses showed that the TLR11 subfamily was the largest branch. The TLR11 family was similar to cave fish, zebrafish, puffer fish and tilapia in gene analysis. Normal tissue expression profiles showed relatively high levels of TLR5b, TLR5a, TLR1 and TLR25, but the overall TLR gene RPKM was relatively low, about 144.73. Nine TLR genes were differentially expressed in the susceptible and resistant transcripts of Aeromonas hydrophila. TLR genes were up-regulated and up-regulated by 2 to 12.9 times. TLR20.2 was up-regulated most significantly, and up-regulated by 12.9 times at 48h after infection. The expression of gctlr18 was induced by flagellin, LPS and poly (I:C) in vitro. The expression of gctlr18 increased first and then decreased, respectively, at 24h, 48h and 1h after infection. Overexpression of gctlr18 gene could regulate il-8, inf-1 and TNF-a by 1.2 times, 10.15 times and 3.61 times, respectively. overexpression of gctlr18 could activate the NF-kappa B fluorescence reporting system and resist bacterial invasion. Therefore, gctlr18 may recognize flagellin, LPS and poly (I:C) pathogenic components. Overexpression of gctlr18 can induce the secretion of inflammatory factors, possibly through the NF-kappa B signaling pathway to cause inflammation to eliminate exogenous pathogens. Flagellin, LPS and poly (I: C) stimulated the expression of gctlr20.2 in vitro. Flagellin and poly (I: C) peaked at 3.35 and 2.8 times at 24 h, and the highest expression was 2.07 times at 6 h after LPS stimulation. Overexpression and interference with the gene were immune to the downstream. Overexpression of gctlr20.2 induced an increase in the transcription of IL-1 beta, IL-8 and tnf-a, up-regulation of IL-1 beta by 1.46 times, up-regulation of IL-8 by 1.45 times and up-regulation of TNF-a by 1.91 times. siRNA-tlr20.2 inhibited the expression of gctlr20.2 mRNA by 65.7%, and down-regulated the transcription of IFN and IL-8 genes by 0.77 and 0.14 times, respectively. LPS and poly(I:C) pathogenic components, gctlr20.2 overexpression activates NF-kappa B signaling pathway to trigger inflammation. Downregulation of gctlr20.2 can reduce the secretion of inflammatory factors and help to restore immune balance. Flagellin and LPS stimulate the expression of gctlr21 in vitro, and over-expression and interference of the gene may regulate the downstream immune-related genes in LPS infection. At 6 h, the expression increased to 2.45 times, reached a peak value of 8.63 times at 24 h, and then decreased slowly. At 6 h, the transcriptional level of FLA-ST increased to 2.7 times, reached a peak value of 5.61 times at 24 h, and decreased to 2.22 times at 48 h. Overexpression of Gctlr21 induced significant up-regulation of IL-1 beta and IFN expression, 2.12 times and 1.66 times respectively, and activated the NF-kappa B fluorescence reporting system. 2 inhibited the expression of gctlr21 by 0.48 times, and significantly inhibited the expression of downstream immune factors IL-1 beta, IL-8 and tnf-a, but did not inhibit the expression of ifn. The Target-regulated microRNA screening showed that Ci-Mi-3, let-7i and CI-Mi-14 inhibited the transcription of gctlr21 gene. Overexpression of microRNA also regulated the expression of immune-related genes. Genes may also be involved in the recognition of flagellin and LPS, suggesting that the overexpression of gctlr21 can activate NF-kappa B to regulate the transcription of inflammatory factors. RNA interference is similar to that of CI-mi R-3, let-7i and CI-microRNA-14 in targeting the expression of gctlr21, suggesting that the down-regulation of gctlr21 transcription can attenuate inflammation and avoid excessive injury.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943
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