赤眼鳟与草鱼TLR19结构及功能差异初探
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S 917.4

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国家自然科学基金面上项目(31972787);国家自然基金青年项目(31802288)


Preliminary analysis of the structural and functional differences between Squaliobarbus curriculus and Ctenopharyngodon idella TLR19s
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National Natural Science Foundation of China (General Program, Grant No. 31972787);Science Fund for Creative Research Groups of the National Natural Science Foundation of China (Grant No. 31802288)

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    摘要:

    为探索赤眼鳟TLR19(ScTLR19)与已报道草鱼TLR19(CiTLR19)的结构及功能差异,实验克隆了ScTLR19基因全长并分析其结构特点,利用实时荧光定量PCR(qPCR)分析其组织表达及草鱼呼肠孤病毒(GCRV)感染后的表达特征,同时采取过表达实验揭示其对TLR19信号通路下游功能基因的激活特性。结果显示,ScTLR19基因编码957个氨基酸,与CiTLR19氨基酸序列具有高度相似性(94.04%);但ScTLR19胞外区由9个亮氨酸重复序列(LRR)组成,比CiTLR19在654~677氨基酸位点之间明显多1个LRR,且在三级结构上相应多2个α-螺旋和6个β-折叠。ScTLR19广泛表达于赤眼鳟各组织,GCRV攻毒后,其表达水平在免疫组织肾脏中显著上调。赤眼鳟鳍条细胞系过表达ScTLR19并接受GCRV攻毒后,干扰素调节因子IRF7在攻毒期间的表达水平与对照组无显著差异,而干扰素调节因子IRF3在攻毒12和24 h的表达水平显著上调,且衔接蛋白MyD88和TRIF的表达水平均显著上调。研究表明,与CiTLR19相比,ScTLR19同样只对IRF3而不对IRF7进行调控,但不同于CiTLR19单一调控TRIF的模式,其同时对MyD88和TRIF二者进行调控,可能介导更多样的抗病毒免疫反应。

    Abstract:

    In order to explore the structural and functional differences between a TLR19 homolog (designated as ScTLR19) from S. curriculus and the reported TLR19 (designated as CiTLR19) from C. idellus, we cloned the full-length cDNA sequence of ScTLR19 gene and analyzed its sequence and structural characteristics, investigated its expression patterns of various tissues and the responses to grass carp reovirus (GCRV) infection with real-time fluorescent quantitative PCR (qPCR) technique, and revealed its distinct regulation on the downstream functional genes of TLR19 signaling pathway by overexpression experiments in this study. The results showed that the ScTLR19 gene encoded 957 amino acids, whose sequence shared high similarity (94.04%) with that of CiTLR19. The extracellular region of ScTLR19 protein consisted of nine leucine-rich repeats (LRRs), and harbored one more LRR than that for CiTLR19 at the amino acid sites of 654~677. The ScTLR19 also owned two more α-helixes and six more β-folds than that of CiTLR19 for the three dimensional structure. The transcriptional expressions of ScTLR19 were widely detected in various tissues of S. curriculus, and significantly up-regulated in kidney after GCRV infection. When the S. curriculus fin cells overexpressed ScTLR19 and were then challenged with GCRV, the transcriptional expressions of interferon regulatory factor 7 (IRF7) did not significantly change (P>0.05), while the transcriptional expressions of interferon regulatory factor 3 (IRF3) and both MyD88 and TRIF that were reported as two important adaptor proteins in TLR signal pathways, were significantly up-regulated at 12 h and 24 h post GCRV infection, compared to the control group (P<0.05). According to the previous study on CiTLR19, ScTLR19, just like CiTLR19, only regulated IRF3, but not IRF7 signal pathway during GCRV infection, but unlike CiTLR19 only regulating TRIF signal pathway, ScTLR19 could regulate both MyD88 and TRIF signal pathways, which might enable ScTLR19 with more antiviral immune functions to enhance the resistance against GCRV for S. curriculus. These results shed light on the differences in the structure and function between two TLR19 genes from S. curriculus and C. idellus.

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袁文玲,吕钊,刘益,刘小燕,肖调义.赤眼鳟与草鱼TLR19结构及功能差异初探[J].水产学报,2020,44(12):1948~1959

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  • 收稿日期:2020-01-14
  • 最后修改日期:2020-05-04
  • 录用日期:2020-05-07
  • 在线发布日期: 2020-12-11
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