红鳍东方鲀感染哈氏弧菌前后组织中socs3基因的表达差异分析
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摘要
为研究红鳍东方鲀Takifugur ubripes免疫系统相关细胞因子信号传导抑制因子-3(Suppressor of cytokine signaling,SOCS3)的表达情况,采用相对实时荧光定量PCR方法比较了体质量为(290.34±5.13)g的健康红鳍东方鲀脑、脾脏、肠、鳃、肌肉和肝脏6个不同组织中socs3基因表达量,以及感染哈氏弧菌Vibrio harveyi后肝脏、脾脏和肠组织socs3基因表达量的变化。结果表明:socs3基因在健康红鳍东方鲀肝脏组织、肌肉组织中均有显著表达(P<0.05),在鳃、脑、脾脏、肠组织中表达不显著(P>0.05);感染哈氏弧菌的红鳍东方鲀,在感染后的24、48 h时,肝脏组织socs3的表达量显著高于0 h(P<0.05),分别约为初始时的10倍和8倍,其他组织变化不明显(P>0.05);利用NCBI的ORF Finder程序鉴定所得红鳍东方鲀SOCS3(NP_001072096.1)序列的潜在开放阅读框(ORF),通过模块化结构研究工具来鉴定保守结构域和信号肽,表明socs3的cDNA序列全长1533 bp,其中5′端非编码区为215 bp,开放阅读框(ORF)长度为606 bp,3′端非编码区为713 bp,共编码201个氨基酸;利用MEGA 6.0软件进行氨基酸序列比对并构建系统发育树,结果显示,硬骨鱼类单独聚为一支,与哺乳动物、两栖动物及鸟类分开,硬骨鱼类中的SOC3b单独聚为一支,红鳍东方鲀与其同科的黑青斑河豚Tetraodon nigroviridis的SOC3聚在一起,并与三刺鱼Gasterosteus aculeatus属于同一分支。本研究结果可为红鳍东方鲀免疫应答的研究提供数据支持。
The expression of suppressor of cytokine signaling 3(socs3) gene in six tissues was comparatively investigated in healthy redfin puffer Takifugu rubripes and in liver of the redfin puffer challenged with Vibrio harveyi using real-time quantitative fluorescence PCR in order to probe for immune function of redfin puffer. The gene socs3 was shown to be significantly expressed in liver and muscle of healthy redfin puffer(P<0.05), without significant expression in gill, brain, spleen and intestine(P>0.05). There was significantly higher expression level of socs3 in liver of the infected redfin puffer for 24 h and 48 h than for 0 h(P<0.05), as much about ten times as the initial concentration in 24 h and as much eight times as the initial concentration in 48 h, without significant differences in other tissues(P>0.05). The NCBI's ORF Finder procedure revealed the potential Open Reading Frame(ORF) of SOCS3 sequence(NP_001072096), and conserved domain and signal peptide were identified by modular structural research tools. MEGA 6.0 revealed that the total length of sequence of socs3 cDNA was 1533 bp, SOCS3 protein including the 5′ end non-coding region length of 215 bp, the length ORF of 606 bp, the 3′ end non-coding region length of of 713 bp and coding 201 amino acids. The phylogenetic analysis of SOCS3 indicated that the teleost fishes were grouped into one, in the same branch as Tetradon nigrovividis, and Gasterosteus aculeatus. separated from mammals, amphibians and birds. The findings indicate that the immune expression and cytokine of gene socs3 play an important role in immune regulation process in redfin puffer, promoting understanding of immune function in redfin puffer.
The expression of suppressor of cytokine signaling 3(socs3) gene in six tissues was comparatively investigated in healthy redfin puffer Takifugu rubripes and in liver of the redfin puffer challenged with Vibrio harveyi using real-time quantitative fluorescence PCR in order to probe for immune function of redfin puffer. The gene socs3 was shown to be significantly expressed in liver and muscle of healthy redfin puffer(P<0.05), without significant expression in gill, brain, spleen and intestine(P>0.05). There was significantly higher expression level of socs3 in liver of the infected redfin puffer for 24 h and 48 h than for 0 h(P<0.05), as much about ten times as the initial concentration in 24 h and as much eight times as the initial concentration in 48 h, without significant differences in other tissues(P>0.05). The NCBI's ORF Finder procedure revealed the potential Open Reading Frame(ORF) of SOCS3 sequence(NP_001072096), and conserved domain and signal peptide were identified by modular structural research tools. MEGA 6.0 revealed that the total length of sequence of socs3 cDNA was 1533 bp, SOCS3 protein including the 5′ end non-coding region length of 215 bp, the length ORF of 606 bp, the 3′ end non-coding region length of of 713 bp and coding 201 amino acids. The phylogenetic analysis of SOCS3 indicated that the teleost fishes were grouped into one, in the same branch as Tetradon nigrovividis, and Gasterosteus aculeatus. separated from mammals, amphibians and birds. The findings indicate that the immune expression and cytokine of gene socs3 play an important role in immune regulation process in redfin puffer, promoting understanding of immune function in redfin puffer.
引文
[1] 杜佳垠.红鳍东方鲀健康养殖之四健康养殖科学治病[J].齐鲁渔业,2004(9):54-56.
[2] 孙鲲.哈氏弧菌种特异性分子标记的发现及其在哈氏弧菌快速诊断和免疫防治中的应用[D].青岛:中国科学院研究生院(海洋研究所), 2009.
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[4] 王保坤,余俊红,李筠,等.花鲈弧菌病病原菌(哈维氏弧菌)的分离与鉴定[J].中国水产科学,2002,9(1):52-55.
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[7] 马爱军,李伟业,王新安,等.红鳍东方鲀养殖技术研究现状及展望[J].海洋科学,2014,38(2):116-121.
[8] 胡梦红.抗生素在水产养殖中的应用、存在的问题及对策[J].水产科技情报,2006,33(5):217-221.
[9] Croker B A,Kiu H,Nicholson S E.SOCS regulation of the JAK/STAT signalling pathway[J].Seminars in Cell & Developmental Biology,2008,19(4):414-422.
[10] Larsen L,R?pke C.Suppressors of cytokine signalling:SOCS[J].APMIS:Acta Pathologica,Microbiologica,Et Immunologica Scandinavica,2010,110(12):833-844.
[11] Jin H J,Shao J Z,Xiang L X,et al.Global identification and comparative analysis of SOCS genes in fish:insights into the molecular evolution of SOCS family[J].Molecular Immunology,2008,45(5):1258-1268.
[12] Banks A S,Li J Z,Mckeag L,et al.Deletion of SOCS7 leads to enhanced insulin action and enlarged islets of Langerhans[J].Journal of Clinical Investigation,2005,115(9):2462-2471.
[13] 冷俊楠,赵素梅,高士争.Leptin介导的SOCS3信号通路研究进展[J].云南农业大学学报,2011,26(3):405-411.
[14] Lejeune D,Demoulin J B,Renauld J C.Interleukin 9 induces expression of three cytokine signal inhibitors:cytokine-inducible SH2-containing protein,suppressor of cytokine signalling(SOCS)-2 and SOCS-3,but only SOCS-3 overexpression suppresses interleukin 9 signalling[J].Biochemical Journal,2001,353(1):109-116.
[15] 刘春来,刘红雨,陈军.SOCS家族分子在肿瘤发生发展中的作用[J].中国肺癌杂志,2016,19(9):620-625.
[16] 李景,肖光雄.三阴性乳腺癌中SOCS3及IL-6的表达[J].现代肿瘤医学,2016,24(6):917-920.
[17] Jin H J,Xiang L X,Shao J Z.Identification and characterization of suppressor of cytokine signaling 1 (SOCS-1) homologues in teleost fish[J].Immunogenetics,2007,59(8):673-686.
[18] Jager L D,Dabelic R,Waiboci L W,et al.The kinase inhibitory region of SOCS-1 is sufficient to inhibit T-helper 17 and other immune functions in experimental allergic encephalomyelitis[J].Journal of Neuroimmunology,2011,232(1-2):108-118.
[19] Boyle K,Zhang J G,Nicholson S,et al.Deletion of the SOCS box of suppressor of cytokine signaling 3 (SOCS3) in embryonic stem cells reveals SOCS box-dependent regulation of JAK but not STAT phosphorylation[J].Cellular Signalling,2009,21(3):394-404.
[20] Walker D G,Whetzel A M,Lue L F.Expression of suppressor of cytokine signaling genes in human elderly and Alzheimer’s disease brains and human microglia[J].Neuroscience,2015,302:121-137.
[21] Naka T,Narazaki M,Hirata M,et al.Structure and function of a new STAT-induced STAT inhibitor[J].Nature,1997,387(6636):924-929.
[22] Liang J,Wang D M,Renaud G,et al.The stat3/socs3a pathway is a key regulator of hair cell regeneration in zebrafish stat3/socs3a pathway:regulator of hair cell regeneration[J].Journal of Neuroscience,2012,32(31):10662-10673.
[23] Studzinski A L,Almeida D V,Lanes C F C,et al.SOCS1 and SOCS3 are the main negative modulators of the somatotrophic axis in liver of homozygous GH-transgenic zebrafish (Danio rerio)[J].General and Comparative Endocrinology,2009,161(1):67-72.
[24] Elsaeidi F,Bemben M A,Zhao X F,et al.Jak/stat signaling stimulates zebrafish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq[J].Journal of Neuroscience,2014,34(7):2632-2644.
[25] Zhang M,Xiao Z Z,Sun L.Suppressor of cytokine signaling 3 inhibits head kidney macrophage activation and cytokine expression in Scophthalmus maximus[J].Developmental & Comparative Immunology,2011,35(2):174-181.
[26] Babon J J,Nicola N A.The biology and mechanism of action of suppressor of cytokine signaling 3[J].Growth Factors,2012,30(4):207-219.
[27] Wang T H,Secombes C J.Rainbow trout suppressor of cytokine signalling (SOCS)-1,2 and 3:molecular identification,expression and modulation.[J].Molecular Immunology,2008,45(5):1449-1457.
[28] Meng J,Yao X G,Zhou L,et al.GW25-e0422 relationship between polymorphism of SOCS-3 and dyslipidemia in China Xinjiang Uygur[J].Journal of the American College of Cardiology,2014,64(16):C28.
[29] 郝连旭.半滑舌鳎CC型趋化因子和SOCS的系统发育和表达模式分析[D].青岛:中国科学院研究生院(海洋研究所),2016.
[30] 肖占刚.鲤免疫相关基因SOCS-3、Prothymosin α和Thymosin β-like的分子克隆及相关特性研究[D].北京:中国科学院大学,2010.
[31] 朱建国,江福能,毕学成,等.细胞因子通路抑制因子3在前列腺癌中的表达及其意义[J].中华实验外科杂志,2012,29(6):1047-1049.
[32] Ru P,Steele R,Hsueh E C,et al.Anti-miR-203 upregulates SOCS3 expression in breast cancer cells and enhances cisplatin chemosensitivity[J].Genes & Cancer,2011,2(7):720-727.
[2] 孙鲲.哈氏弧菌种特异性分子标记的发现及其在哈氏弧菌快速诊断和免疫防治中的应用[D].青岛:中国科学院研究生院(海洋研究所), 2009.
[3] Austin B,Zhang X H.Vibrio harveyi:a significant pathogen of marine vertebrates and invertebrates[J].Letters in Applied Microbiology,2010,43(2):119-124.
[4] 王保坤,余俊红,李筠,等.花鲈弧菌病病原菌(哈维氏弧菌)的分离与鉴定[J].中国水产科学,2002,9(1):52-55.
[5] Jayasree L,Janakiram P,Madhavi R.Characterization of Vibrio spp. associated with diseased shrimp from culture ponds of Andhra Pradesh (India)[J].Journal of the World Aquaculture Society,2006,37(4):523-532.
[6] 邓欢,陈俅,刘权恕,等.养殖海湾扇贝弧菌病的研究[J].应用与环境生物学报,2003,9(5):517-521.
[7] 马爱军,李伟业,王新安,等.红鳍东方鲀养殖技术研究现状及展望[J].海洋科学,2014,38(2):116-121.
[8] 胡梦红.抗生素在水产养殖中的应用、存在的问题及对策[J].水产科技情报,2006,33(5):217-221.
[9] Croker B A,Kiu H,Nicholson S E.SOCS regulation of the JAK/STAT signalling pathway[J].Seminars in Cell & Developmental Biology,2008,19(4):414-422.
[10] Larsen L,R?pke C.Suppressors of cytokine signalling:SOCS[J].APMIS:Acta Pathologica,Microbiologica,Et Immunologica Scandinavica,2010,110(12):833-844.
[11] Jin H J,Shao J Z,Xiang L X,et al.Global identification and comparative analysis of SOCS genes in fish:insights into the molecular evolution of SOCS family[J].Molecular Immunology,2008,45(5):1258-1268.
[12] Banks A S,Li J Z,Mckeag L,et al.Deletion of SOCS7 leads to enhanced insulin action and enlarged islets of Langerhans[J].Journal of Clinical Investigation,2005,115(9):2462-2471.
[13] 冷俊楠,赵素梅,高士争.Leptin介导的SOCS3信号通路研究进展[J].云南农业大学学报,2011,26(3):405-411.
[14] Lejeune D,Demoulin J B,Renauld J C.Interleukin 9 induces expression of three cytokine signal inhibitors:cytokine-inducible SH2-containing protein,suppressor of cytokine signalling(SOCS)-2 and SOCS-3,but only SOCS-3 overexpression suppresses interleukin 9 signalling[J].Biochemical Journal,2001,353(1):109-116.
[15] 刘春来,刘红雨,陈军.SOCS家族分子在肿瘤发生发展中的作用[J].中国肺癌杂志,2016,19(9):620-625.
[16] 李景,肖光雄.三阴性乳腺癌中SOCS3及IL-6的表达[J].现代肿瘤医学,2016,24(6):917-920.
[17] Jin H J,Xiang L X,Shao J Z.Identification and characterization of suppressor of cytokine signaling 1 (SOCS-1) homologues in teleost fish[J].Immunogenetics,2007,59(8):673-686.
[18] Jager L D,Dabelic R,Waiboci L W,et al.The kinase inhibitory region of SOCS-1 is sufficient to inhibit T-helper 17 and other immune functions in experimental allergic encephalomyelitis[J].Journal of Neuroimmunology,2011,232(1-2):108-118.
[19] Boyle K,Zhang J G,Nicholson S,et al.Deletion of the SOCS box of suppressor of cytokine signaling 3 (SOCS3) in embryonic stem cells reveals SOCS box-dependent regulation of JAK but not STAT phosphorylation[J].Cellular Signalling,2009,21(3):394-404.
[20] Walker D G,Whetzel A M,Lue L F.Expression of suppressor of cytokine signaling genes in human elderly and Alzheimer’s disease brains and human microglia[J].Neuroscience,2015,302:121-137.
[21] Naka T,Narazaki M,Hirata M,et al.Structure and function of a new STAT-induced STAT inhibitor[J].Nature,1997,387(6636):924-929.
[22] Liang J,Wang D M,Renaud G,et al.The stat3/socs3a pathway is a key regulator of hair cell regeneration in zebrafish stat3/socs3a pathway:regulator of hair cell regeneration[J].Journal of Neuroscience,2012,32(31):10662-10673.
[23] Studzinski A L,Almeida D V,Lanes C F C,et al.SOCS1 and SOCS3 are the main negative modulators of the somatotrophic axis in liver of homozygous GH-transgenic zebrafish (Danio rerio)[J].General and Comparative Endocrinology,2009,161(1):67-72.
[24] Elsaeidi F,Bemben M A,Zhao X F,et al.Jak/stat signaling stimulates zebrafish optic nerve regeneration and overcomes the inhibitory actions of Socs3 and Sfpq[J].Journal of Neuroscience,2014,34(7):2632-2644.
[25] Zhang M,Xiao Z Z,Sun L.Suppressor of cytokine signaling 3 inhibits head kidney macrophage activation and cytokine expression in Scophthalmus maximus[J].Developmental & Comparative Immunology,2011,35(2):174-181.
[26] Babon J J,Nicola N A.The biology and mechanism of action of suppressor of cytokine signaling 3[J].Growth Factors,2012,30(4):207-219.
[27] Wang T H,Secombes C J.Rainbow trout suppressor of cytokine signalling (SOCS)-1,2 and 3:molecular identification,expression and modulation.[J].Molecular Immunology,2008,45(5):1449-1457.
[28] Meng J,Yao X G,Zhou L,et al.GW25-e0422 relationship between polymorphism of SOCS-3 and dyslipidemia in China Xinjiang Uygur[J].Journal of the American College of Cardiology,2014,64(16):C28.
[29] 郝连旭.半滑舌鳎CC型趋化因子和SOCS的系统发育和表达模式分析[D].青岛:中国科学院研究生院(海洋研究所),2016.
[30] 肖占刚.鲤免疫相关基因SOCS-3、Prothymosin α和Thymosin β-like的分子克隆及相关特性研究[D].北京:中国科学院大学,2010.
[31] 朱建国,江福能,毕学成,等.细胞因子通路抑制因子3在前列腺癌中的表达及其意义[J].中华实验外科杂志,2012,29(6):1047-1049.
[32] Ru P,Steele R,Hsueh E C,et al.Anti-miR-203 upregulates SOCS3 expression in breast cancer cells and enhances cisplatin chemosensitivity[J].Genes & Cancer,2011,2(7):720-727.