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引用本文:孙娇,聂力,苗亮,陈炯.香鱼(Plecoglossus altivelis)脾脏酪氨酸激酶SYK的鉴定及其介导的MAPK信号通路研究.海洋与湖沼,2020,51(5):1182-1193.
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香鱼(Plecoglossus altivelis)脾脏酪氨酸激酶SYK的鉴定及其介导的MAPK信号通路研究
孙娇1, 聂力1, 苗亮1, 陈炯1,2
1.宁波大学海洋学院 农产品质量安全危害因子与风险防控国家重点实验室 宁波 315211;2.宁波大学海洋学院 应用海洋生物技术教育部重点实验室 宁波 315832
摘要:
脾脏酪氨酸激酶(SYK)是一种非受体型酪氨酸激酶(non-receptor tyrosine kinase,NRTK)。在哺乳动物中,SYK是重要的细胞信号通路接头分子,其在免疫细胞活化,胞外刺激的信号转导和病原体识别等多种生命过程中扮演重要角色,然而在鱼类中却鲜有报道。本研究以香鱼(Plecoglossus altivelis)为研究对象,探讨了其SYK(PaSYK)在应对病原微生物感染及在活化免疫信号通路中的作用。我们克隆获得了香鱼SYK的cDNA序列,并利用多重序列比对、构建进化树等生物信息学方法分析PaSYK的进化地位;采用实时荧光定量PCR(RT-qPCR)方法分析PaSYK在健康组织及鳗弧菌(Vibrio anguillarum)感染后的表达变化;亚细胞定位检测PaSYK在HEK293T细胞的分布情况;在香鱼头肾单核/巨噬细胞(Monocyte/macrophage,MO/Mφ)中过表达PaSYK,研究其对炎症细胞因子表达的调控作用并在HEK293T细胞及香鱼头肾MO/Mφ中研究PaSYK对MAPK信号通路的活化作用。多重序列比对结果显示,SYK的蛋白质序列和结构域在进化过程中是高度保守的。系统进化树结果表明,PaSYK与河鳟的亲缘关系密切。RT-qPCR结果表明,SYK基因mRNA在检测的所有组织中均有表达,鳗弧菌感染后免疫相关组织中PaSYK表达量均上调。此外,PaSYK能显著诱导香鱼头肾MO/Mφ中促炎因子TNF-α和IL-1β的表达,但对抑炎因子TGF-β和IL-10的表达起到略微的抑制作用。Western blot结果证明PaSYK可以激活MAPK信号通路。综上所述,SYK分子在进化上高度保守,香鱼SYK与其哺乳动物中的同源物具有保守的功能,均能活化MAPK信号通路并在炎症反应中发挥作用。
关键词:  脾脏酪氨酸激酶(SYK)  香鱼  鳗弧菌感染  炎症细胞因子  MAPK信号通路
DOI:10.11693/hyhz20191200249
分类号:Q78
基金项目:国家自然科学基金项目,31702374号,31972821号,31772876号;浙江省自然科学基金项目,LZ18C190001号,LQ17C190001号;宁波市科技创新团队项目,2015C110018号
MOLECULAR CHARACTERIZATION OF A SYK HOMOLOGUE FROM AYU, PLECOGLOSSUS ALTIVELIS: INSIGHTS INTO ITS CONSERVED ROLE IN MAPK SIGNALING ACTIVATION
SUN Jiao1, NIE Li1, MIAO Liang1, CHEN Jiong1,2
1.State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Ningbo University, Ningbo 315211, China;2.Key Laboratory of Applied Marine Biotechnology of Ministry of Education, Ningbo University, Ningbo 315832, China
Abstract:
Spleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase (NRTK). In mammals, SYK is an important adaptor that plays an essential role in the activation of immune cells, signal transduction of extracellular stimuli, and pathogen recognition etc. However, study regarding the function of this molecule in fish is limited. Therefore, the role of ayu Plecoglossus altivelis SYK (PaSYK) in pathogen infection and the activation of immune signaling pathways was studied. The cDNA sequence of PaSYK was retrieved; Multiple sequence alignment and phylogenetic tree analysis were conducted to analyze the evolutionary status of PaSYK. The expressions of SYK in healthy and Vibrio anguillarum infected tissues of ayu were revealed in real-time fluorescence quantitative PCR (RT-qPCR). Subcellular localization was conducted in HEK293T cells to detect the cellular distribution of PaSYK. We overexpressed PaSYK in HEK293T cells and ayu head kidney monocyte/macrophage (MO/Mφ), and its regulatory role on cytokine expression and the activation of MAPK signaling pathway were deciphered. Multiple sequence alignment indicated that the protein sequences and functional domains of SYK were highly conserved during evolution. PaSYK was closely related to its homologue of river trout (Salmo trutta) as indicated in the phylogenetic tree. RT-qPCR results showed that the mRNA of SYK gene was expressed in all tissues examined, and the expression of PaSYK was up-regulated in all tissues detected after V. anguillarum infection. Furthermore, PaSYK significantly induced the expression of the pro-inflammatory factors TNF-α and IL-1β, and slightly inhibited the expression of anti-inflammatory factors TGF-β and IL-10. Western blot results demonstrated that, like its homologues in mammals, PaSYK could activate the MAPK signaling pathway. Therefore, SYK is highly conserved during the evolution. PaSYK and its mammalian homologues have conserved functions, and both could activate MAPK signaling pathway and play a role in inflammatory response.
Key words:  spleen tyrosine kinase (SYK)  ayu Plecoglossus altivelis  Vibrio anguillarum infection  cytokines  MAPK signaling pathway
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