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Volume 43 Issue 11
Oct.  2019
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Molecular evolution analysis of interleukin-8 in great blue-spotted mudskipper (Boleophthalmus pectinirostris) and the effect of different pathogen stimulus on its expression

  • Corresponding author: Ge SHI, sg2610105@126.com
  • Received Date: 2018-11-22
    Accepted Date: 2019-03-17
  • Great blue-spotted mudskippers (Boleophthalmus pectinirostris) have lived in mudflats along the coasts of Japan and South China for a long time. Compared with the fish living in water, B. pectinirostris, as an amphibious fish, is more vulnerable to terrestrial and aquatic pathogenic factors. Interleukin-8 is a CXC chemokine that plays an important role in regulating the inflammatory response. In this study, we screened the interleukin-8 gene from skin transcriptome of B. pectinirostris, then cloned and verified its coding region. The results indicated that the open reading frame(ORF) of B. pectinirostris IL-8 gene was 306 bp, coding 101 amino acids, including a typical signal peptide of 18 amino acids, and a SCY domain of 62 amino acids, which also possessed the four conserved cysteine residues, namely Cys-30, Cys-32, Cys-57 and Cys-73. Like most fish, B. pectinirostris interleukin-8 amino acid sequence lacked the ELR motif which was the receptor binding site and it was replaced by Asn-Ser-His (NSH) in great blue-spotted mudskipper. In all known fish amino acid sequences, rules of this motif were as follows: the first amino acid was never the amino acid with positive charge, the second amino acid was generally those amino acids with no charge, and most of third amino acid were amino acids with positive charge. Phylogenetic analysis showed that purification selection affected the diversity of this motif in fish. Tissue expression analysis by RT-qPCR indicated that interleukin-8 gene was widely expressed in healthy tissues of B. pectinirostris and it had the highest expression in the gill and brain. Bacteria and poly (I∶C) injection experiments showed that expression of IL-8 in liver, spleen and brain tissues was up-regulated after the infection, indicating that IL-8 plays an important role in the inflammatory response and immune response of liver, spleen and brain tissues. This study provided important reference for the research of immune genes of B. pectinirostris, thus having more comprehensive understanding of the evolution process and function of fish immune genes.
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Molecular evolution analysis of interleukin-8 in great blue-spotted mudskipper (Boleophthalmus pectinirostris) and the effect of different pathogen stimulus on its expression

    Corresponding author: Ge SHI, sg2610105@126.com
  • 1. College of Marine Science, Zhejiang Ocean University, Zhoushan 316000, China
  • 2. School of Marine Sciences, Ningbo University, Ningbo 315000, China

Abstract: Great blue-spotted mudskippers (Boleophthalmus pectinirostris) have lived in mudflats along the coasts of Japan and South China for a long time. Compared with the fish living in water, B. pectinirostris, as an amphibious fish, is more vulnerable to terrestrial and aquatic pathogenic factors. Interleukin-8 is a CXC chemokine that plays an important role in regulating the inflammatory response. In this study, we screened the interleukin-8 gene from skin transcriptome of B. pectinirostris, then cloned and verified its coding region. The results indicated that the open reading frame(ORF) of B. pectinirostris IL-8 gene was 306 bp, coding 101 amino acids, including a typical signal peptide of 18 amino acids, and a SCY domain of 62 amino acids, which also possessed the four conserved cysteine residues, namely Cys-30, Cys-32, Cys-57 and Cys-73. Like most fish, B. pectinirostris interleukin-8 amino acid sequence lacked the ELR motif which was the receptor binding site and it was replaced by Asn-Ser-His (NSH) in great blue-spotted mudskipper. In all known fish amino acid sequences, rules of this motif were as follows: the first amino acid was never the amino acid with positive charge, the second amino acid was generally those amino acids with no charge, and most of third amino acid were amino acids with positive charge. Phylogenetic analysis showed that purification selection affected the diversity of this motif in fish. Tissue expression analysis by RT-qPCR indicated that interleukin-8 gene was widely expressed in healthy tissues of B. pectinirostris and it had the highest expression in the gill and brain. Bacteria and poly (I∶C) injection experiments showed that expression of IL-8 in liver, spleen and brain tissues was up-regulated after the infection, indicating that IL-8 plays an important role in the inflammatory response and immune response of liver, spleen and brain tissues. This study provided important reference for the research of immune genes of B. pectinirostris, thus having more comprehensive understanding of the evolution process and function of fish immune genes.

  • IL-8是CXC趋化因子家族的典型成员之一,又被称为CXCL8,其可在内源性和外源性的刺激下,由多种类型细胞(包括巨噬细胞、中性粒细胞、血管内皮细胞、上皮细胞、成纤维细胞、T淋巴细胞等)产生[1-2],通过刺激中性粒细胞释放溶菌酶并发生形态变化,同时触发呼吸爆发[3],因此在急性炎症反应特别是细菌感染中发挥着重要作用[4]。此外,IL-8还具有多种作用,如增强人巨细胞病毒在纤维母细胞中的复制,通过促进血管生成,在中性粒细胞氧化破裂和促进伤口愈合中发挥关键作用[5]。最新的研究发现,大黄鱼(Larimichthys crocea)在被副溶血性弧菌(Vibrio parahemolyticus)感染后,脾脏、头肾和肝脏中IL-8的表达升高,同时,将IL-8基因导入HEK293T细胞中并进行细胞内信号检测,确定IL-8介导的ERK磷酸化信号活化,表明IL-8在介导细胞内ERK1/2磷酸化信号通路方面发挥着重要作用[6]。1986年Kownazki等[7]发现了一种由单核细胞产生的中性粒细胞趋化因子,并于1988年的一次国际大会上将其正式命名为白细胞介素-8,随着白细胞介素-8研究的不断深入,许多鱼类IL-8基因被克隆和分析,包括牙鲆(Paralichthys olivaceous)[8]、虹鳟(Oncorhynchus mykiss)[9]、带状角鲨(Triakis scyllia)[10]、黑线银鲛(Chimaera phantasma)[11]、鲤(Cyprinus carpio)[12]、黑线鳕(Melanogrammus aeglefinus)[13]、草鱼(Ctenopharyngodon idella)[14]、大黄鱼[15]、香鱼(Plecoglossus altivelis)[16]和鳜(Siniperca chuasti)[17]等。由于至今尚未在无脊椎动物中发现趋化因子或受体,因此一般认为包括IL-8在内的趋化因子是从脊椎动物开始出现的[1]。研究表明IL-8的结构保守,所有IL-8的共同特征是包含至少2个半胱氨酸残基构成的二硫键,具有相似的三维结构,包括至少3个β折叠和1个α螺旋[18]。哺乳动物IL-8是一种ELR+蛋白[19],与哺乳动物不同的是,除鳕形目(Gadiformes)外的大多数鱼类都缺乏ELR基序[13],该基序被其他氨基酸替代,比如DLR、LLR、EMH、EQH和SLH等,目前,对鱼类IL-8基因分子进化分析的研究较少,造成这种差异的原因还需要进一步研究。近年来,随着各种鱼类全基因组和转录组信息的不断增加,IL-8同源基因在鱼类中的鉴定越来越多,在系统发育上,硬骨鱼的IL-8趋化因子可分为CXCL8_L1、CXCL8_L2和CXCL8_L3 3组[20],Zhou等[21]最近的研究克隆了大黄鱼CXCL_L3基因,证实了大黄鱼CXCL_L3能增强头肾和脾脏巨噬细胞的呼吸爆发活性,并在炎症反应中发挥作用。

    大弹涂鱼(Boleophthalmus pectinirostris)属虾虎鱼科(Gobiidae),大弹涂鱼属(Boleophthalmus),栖息在中国南方和日本沿海潮间带的泥滩中,作为一种高营养价值的美食而被食用。同时,其生存的复杂环境也使其具有较高的研究价值。poly(I∶C)是一种免疫刺激剂,经常被用来模拟病毒感染[22]。本研究旨在克隆并鉴定大弹涂鱼IL-8编码区基因,并进行分子进化分析和同源基因比对,通过荧光定量PCR分析其mRNA在健康组织和受感染的肝、脾组织中的表达情况,为后续大弹涂鱼免疫基因的研究奠定理论基础。

    • 本研究中的所有健康大弹涂鱼购自浙江宁波山下村,自购买日起暂养于300 L充气水箱中,海水盐度为30,禁食,每日早晚换水。鳗弧菌(V. anguillarum)菌种购自中国工业微生物菌种保藏管理中心(CICC),poly(I∶C)购自上海源叶生物科技有限公司。RNA提取试剂盒购自TaKaRa(上海)公司,cDNA反转录试剂盒及荧光定量(q-PCR)试剂盒购自全式金(北京)试剂公司。PCR扩增仪购自Eppendorf(德国)公司,高通量实时荧光定量PCR仪LC 480购自罗氏仪器公司(瑞士)。

    • 在暂养的大弹涂鱼中挑选大小均一的360尾鱼,平均体质量(20±1) g,随机分为对照组、鳗弧菌注射组和poly(I∶C)注射组,每组120尾,分在3个养殖箱中。大弹涂鱼被MS-222麻醉后,对照组腹腔注射200 μL生理盐水,鳗弧菌注射组腹腔注射200 μL浓度为1×107 CFU/mL的鳗弧菌液(用生理盐水稀释),poly(I∶C)注射组腹腔注射200 μL浓度为0.15 μg/μL的poly(I∶C)稀释液(用生理盐水溶解)。实验过程中禁食,保持水温为23~26 ℃,全程充氧。注射后分别在0、6、12、24、36、48和72 h取肝脏、脾脏和脑组织,液氮速冻后放入−80 ℃超低温冰箱中保存备用。在暂养箱中选取健康大弹涂鱼,取肝脏、脾脏、肾脏、肠、脑、鳃、皮肤和肌肉组织,液氮速冻后放入−80 °C超低温冰箱中保存。以上组织样本每个时间点每组取3条。从上述冷冻组织中提取大弹涂鱼RNA,并反转录成cDNA,放入−20 ℃冷冻保存。

    • 大弹涂鱼IL-8基因编码区全长筛选自大弹涂鱼皮肤组织转录组。用大弹涂鱼肝脏cDNA对其序列进行PCR克隆验证,所用引物序列见表1。PCR条件包括95 °C 初始变性5 min,95 °C 30 s、59 °C 45 s、72 °C 1 min 20 s,35个循环,最后72 °C延伸15 min。扩增产物通过琼脂糖凝胶电泳进行验证,选择单一明亮条带的样品送测,测序公司为上海生工生物工程股份有限公司。测序结果通过blastn在线程序(http://www.ncbi.nlm.nih.gov/BLAST/)和MEGA 5[23]软件进行比对。

      名称
      primer
      引物序列(5′-3′)
      sequence (5′-3′)
      IL-8 F1 TCCTCGCTAATGGCATGTCTCC
      IL-8 R1 ACCCAGGATGACTGTGGATTCA
      IL-8 QF2 CTTGGGACCCTCTTTGTCCTC
      IL-8 QR2 TGGCACCAGTTTGATGCTCC
      β-actin QF1 AGGGTGTGATGGTCGGTATGGG
      β-actin QR1 CGCAGCTCGTTGTAGAAGGTGT

      Table 1.  Primers used in this study

    • 本研究所有其他鱼类相关基因序列均从美国国家生物技术信息中心(https://www.ncbi.nlm.nih.gov/nucleotide/)检索获取(表2表3)。序列相似性和一致性通过MatGAT 2.01程序进行分析[24],使用DNAMAN 6.0软件[25]和蛋白质预测程序(https://www.predictprotein.org)进行蛋白质预测,IL-8蛋白质结构域特征和基序通过SMART(http://smart.embl-heidelberg.de/)[26]和Motifscan(https://myhits.isb-sib.ch/cgi-bin/motif_scan/)[27]程序预测。使用Mega 5[23]和GeneDoc[28]对大弹涂鱼和其他物种的IL-8基因进行了多重序列比对。蛋白质同源结构建模由SWISS-MODEL程序(https://swissmodel.expasy.org/)[29]生成。使用Mega 5[23]建立大弹涂鱼IL-8基因和其他物种CXCL8同源基因的系统发生树。

      基因名
      gene name
      拉丁学名
      Latin name
      中文物种名
      species
      NCBI登录号
      NCBI accession no.
      IL-8 Anoplopoma fimbria 裸盖鱼 BT082568.1
      IL-8 Notothenia coriiceps 革首南极鱼 XM_010770775.1
      IL-8 Oplegnathus fasciatus 条石鲷 KC522965.1
      IL-8 L. crocea 大黄鱼 KP202400.1
      IL-8 Pundamilia nyererei 奈里朴丽鱼 XM_005751068.1
      IL-8 B. pectinirostris 大弹涂鱼 MG490978
      CXCL8_L3 Oryzias latipes 青鳉 XM_004079853.3
      CXCL8_L3 Cynoglossus semilaevis 半滑舌鳎 XM_008323613.1
      CXCL8_L3 Osmerus mordax 美洲胡瓜鱼 BT075428.1
      CXCL8_L3 Astyanax mexicanus 墨西哥丽脂鲤 XM_007238388.2
      CXCL8_L1 O. kisutch 银大麻哈鱼 XM_020475138.1
      CXCL8_L1 L. crocea 大黄鱼 KP202400.1
      CXCL8_L1 C. carpio AJ421443.1
      CXCL8_L2 C. carpio AB470924.1

      Table 2.  Homology analysis of genes used in phylogenetic tree

      拉丁学名
      Latin name
      中文物种名
      species
      NCBI登录号
      NCBI accession no.
      B. pectinirostris 大弹涂鱼 MG490978
      Salmo salar 大西洋鲑 NM001140710.2
      O. mykiss 虹鳟 AY160981.1
      C. idella 草鱼 JN255694.1
      Gadus morhua 大西洋鳕 AY614593.1
      Latris lineata 条纹婢䱵 FJ532283.1
      M. aeglefinus 黑线鳕 AJ566335.2
      S. chuatsi JX157147.1
      Takifugu rubripes 红鳍东方鲀 AB125645.1
      Thunnus maccoyii 蓝鳍金枪鱼 JX157147.1
      Scophthalmus maximus 大菱鲆 HQ872499.1
      Trachinotus ovatus 卵形鲳鲹 KT922006.1
      Rachycentron canadum 军曹鱼 KY312038.1
      Acanthopagrus schlegelii 黑鲷 DQ000611.1
      Pagrus major 真鲷 HM453864.1
      Sparus aurata 大西洋鲷 JX976619.1
      Amphiprion ocellaris 眼斑双锯鱼 XM023287849.1
      Neolamprologus brichardi 布氏新亮丽鲷 XM006805505.1
      Maylandia zebra 斑马拟丽鱼 XM004570701.2
      P. olivaceus 牙鲆 AF216646.1
      Chiloscyllium griseum 灰斑竹鲨 HG964665.1
      Triakis scyllium 皱唇鲨 AB063299.1
      Gekko japonicus 多疣壁虎 XM015416527.1
      Anas platyrhynchos 绿头鸭 AB236334.1
      Mustela putorius furo 雪貂 KJ831208.1
      Oryctolagus cuniculus 穴兔 KT216053.1
      Macaca nemestrina 豚尾猴 U19851.1
      Homo sapiens BC013615.1

      Table 3.  Taxonomy of IL-8 sequences used in this study

    • 系统发生树由MrBayes 3.1程序构建[30],使用文献中作者推荐的参数最多的通用模型GTR+I+G。为保证系统发生树的准确性,程序运行1 000 000+代,直到相对标准差(RSD)显著小于0.01,然后在采样1 000棵进化树之后,丢弃老化样本的前四分之一部分。重建的系统发生树用位点模型(site-model)探究IL-8基因的分子进化模式,通过似然比检验,评估IL-8基因密码子是否存在正选择。但正选择不一定对所有的位点起作用,所以需要使用分枝—位点模型(branch-site model)来分析特定的当前或祖先谱系的正向选择。

    • 大弹涂鱼IL-8基因在肝和脾组织中的表达情况通过荧光定量PCR进行检测。荧光定量PCR引物IL-8 QF和IL-8 QR(表1)被用于扩增一段长度为132 bp的片段,引物β-actin QF和β-actin QR用于扩增大弹涂鱼内参基因β-actin的一个片段作为参照。实验在20 μL体系中进行,包括10 μL的2×TransStart® Tip Green qPCR SuperMix预混液,0.4 μL的cDNA模板,0.4 μL的上下游引物和8.8 μL的双蒸水(ddH2O)。qPCR程序设定:95 °C 5 min,95 °C 20 s、60 °C 25 s、72 °C 25 s,共40个循环。所有样品均进行重复扩增,扩增产物的解离曲线分析在每个循环结束时进行,确定只有一个特殊产物被扩增和检测,并自动设定基线。IL-8基因相对表达量的计算使用2−ΔΔCt 方法[31]

    2.   结果
    • 大弹涂鱼IL-8基因编码区全长306 bp(图1),编码了1条由101个氨基酸组成的多肽链(图2),其中前18个氨基酸残基为信号肽序列(MKLCVAVMLGTLFVLANGM),分泌之后在图1所示的蛋氨酸和丝氨酸之间位置切割,形成成熟肽。IL-8基因编码的肽链结构域SCY由62个氨基酸组成(图2)。

      Figure 1.  Nucleotide and deduced amino acid sequence of B. pectinirostris IL-8 cDNA

      Figure 2.  Multiple sequence alignments of B. pectinirostris IL-8 with other known IL-8 amino acids sequences

      多氨基酸序列比对结果显示,有4个保守的半胱氨酸分别位于第30、32、57和73位氨基酸残基处,其中第30位和第57位半胱氨酸,第32位和第73位半胱氨酸分别以同样的方式组成了二硫键(图2)。在SCY结构域中发现了CXC基序(Cys-30、Arg-31和Cys-32,即CRC),大弹涂鱼IL-8多肽的分子量为11.126 ku,等电点为9.33。三级结构的预测如图3所示,大弹涂鱼的IL-8多肽与人、斑马拟丽鱼和眼斑双锯鱼IL-8多肽的三级结构非常相似,并且在C-末端都有一个α-螺旋结构。

      Figure 3.  Predicted tertiary structures of B. pectinirostris (a), H. sapiens (b), M. zebra (c) and A. ocellaris (d)

    • 在已知的IL-8序列中,大弹涂鱼IL-8氨基酸序列与斑马拟丽鱼氨基酸序列的一致性(71.6%)和相似性(88.2%)高于其他鱼类,与两栖类、鸟类和哺乳类的一致性(29%~33.7%)和相似性(55.3%~60%)较低(表4)。

      物种
      species
      大弹涂鱼
      B. pectinirostris
      斑马拟丽鱼
      M. zebra
      大西洋鲑
      S. salar
      大西洋鳕
      G. morhua
      卵形鲳鲹
      T. ovatus
      大西洋鲷
      S. aurata
      绿头鸭
      A. platyrhynchos
      雪貂
      M. putorius furo
      穴兔
      O. cuniculus

      H. sapiens
      大弹涂鱼
      B. pectinirostris
      71.6 29.2 30.3 31.8 31.4 33.7 30.5 29.0 30.2
      斑马拟丽鱼
      M. zebra
      88.2 31.8 31.8 36.1 32.1 30.5 31.1 26.4 25.9
      大西洋鲑
      S. salar
      57.4 57.8 57.4 59.6 51.5 35.2 32.4 31.4 36.0
      大西洋鳕
      G. morhua
      53.5 52.9 79.2 66.3 60.4 35.2 31.4 32.4 33.0
      卵形鲳鲹
      T. ovatus
      60.4 56.9 77.8 86.1 74.7 42.5 34.0 34.0 37.6
      大西洋鲷
      S. aurata
      56.4 51.0 73.2 77.2 84.8 38.7 34.3 34.0 32.7
      绿头鸭
      A. platyrhynchos
      55.3 54.4 66.0 63.1 62.1 61.2 48.1 48.1 51.9
      雪貂
      M. putorius furo
      60.4 52.0 55.4 54.5 57.4 54.5 66.0 80.2 73.3
      穴兔
      O. cuniculus
      57.4 54.9 57.4 55.4 57.4 58.4 66.0 92.1 79.2
      人 H. sapiens 57.4 57.8 60.6 54.5 56.6 58.6 67.0 87.1 84.2
      *.数值以百分比表示
      *. values are given as percentages

      Table 4.  Similarities (bottom-left) and identities (top-right) of IL-8 amino acid sequences between         B. pectinirostris and other species

      大弹涂鱼IL-8基因与奈里朴丽鱼、大黄鱼、条石鲷、革首南极鱼和裸盖鱼的IL-8基因聚为一支,随后该分支与半滑舌鳎、青鳉、美洲胡瓜鱼和墨西哥丽脂鲤的CXCL8_L3基因聚为一个大支。银大麻哈鱼、鲤和大黄鱼的CXCL8_L1基因聚为另一支,鲤的CXCL8_L2基因独立为一支(图4)。

      Figure 4.  Phylogenetic tree of fish IL-8 homologous genes

    • 本研究构建了以2种软骨鱼为外群的系统发育树(图5),并以此来分析IL-8基因的进化关系。通过系统发育树发现,大弹涂鱼和眼斑双锯鱼首先聚在一起,然后与布氏新亮丽鲷和斑马拟丽鱼聚为一支。在另一支中,大西洋鲑和虹鳟首先聚在一起,然后与草鱼聚为一支。随后,2种鳕形目鱼,黑线鳕和大西洋鳕单独聚为一支。最后,红鳍东方鲀与其他鲈形目形成一支。

      Figure 5.  Phylogenetic tree of fish IL-8 sequence

      通过分子进化分析,探索硬骨鱼IL-8基因受到的选择压力(表5)。位点模型(site-model)表明鱼类IL-8基因处于纯化选择,因为M1a-M2a和M7-M8模型均未检测到任何正选择位点。采用分支—位点模型检测大弹涂鱼,仍然没有发现正选择位点。

      模型   
      model   
      参数
      NP
      自由参数
      free parameters
      模型比较
      model comparison
      −lnL
      −lnL
      LRT检验值
      2Δ(lnL)
      P
      P-value
      正选择位点
      (BEB)
      位点模型
      site model
      M0 42 ω=0.228 M3 vs. M0 2 929.884 91.19 0.0
      none
      M3 46 p0=0.223, p1=0.586, p2=0.190 ω0=0.021, ω1=0.215, ω2=0.661 2 884.288
      M1a 43 p0=0.813, p1=0.187 ω0=0.176, ω1=1.000 M1a vs. M2a 2 903.547 0.0 1.0 未通过
      not allowed
      M2a 45 p0=0.813, p1=0.095, p2=0.092 ω0=0.176, ω1=1.000, ω2=1.000 2 903.547
      M7 43 p=0.872, q=2.512 M7 vs. M8 2 885.112 0.0 1.0 未通过
      not allowed
      M8 45 p0=0. 998, p=0.875, p1=0.002 q=2.532, ω= 1.000 2 885.112
      分支—位点模型
      branch-site model
      1. null-B. pectinirostris 44 1 vs. 2 2 903.548 0.0 1.0 未通过
      not allowed
      2. B. pectinirostris 45 2 903.548

      Table 5.  Results of site model and branch-site models on IL-8 genes

    • 采用实时荧光定量PCR分析健康的大弹涂鱼肝脏、脾脏、肾脏、皮肤、肠、肌肉、鳃和脑组织中IL-8基因表达的情况,结果显示该基因在所有组织中广泛表达,其中在脑组织中表达量最高,其次是脾脏、鳃和肝脏,在肌肉、肾脏、肠中表达量较低,在皮肤中表达量最低(图6)。

      Figure 6.  Temporal expression profiles of B. pectinirostris IL-8 expression in different healthy tissues

      在大弹涂鱼肝脏组织中,生理盐水注射组的IL-8基因的表达一直呈平稳状态,无显著性差异。在鳗弧菌注射组,IL-8基因在肝中的表达量显著上调,并在感染后12 h达到选取时间点的最高水平,为对照组的30倍,随后呈现下降的趋势,在36~72 h呈平稳的状态;在poly(I∶C)注射组,IL-8基因在肝中的表达量同样显著上调,并在36 h达到选取时间点最高水平,数值约为对照组的45倍,之后逐渐下降(图7-a)。

      Figure 7.  Temporal expression profiles of B. pectinirostris IL-8 expression injected with V. anguillarum or poly (I∶C) in liver (a), spleen (b) and brain (c)

      在大弹涂鱼脾脏组织中,生理盐水注射组的IL-8基因的表达同样一直呈平稳状态,无显著性差异。但在鳗弧菌注射组,在0~36 h内,IL-8的表达量较为平稳,为对照组的4倍左右,随后在48 h上升,最后在72 h达到选取时间点的最高水平,为对照组的12倍;在poly(I∶C)注射组,IL-8的表达量显著上升,在12 h达到选取时间点的顶点,为对照组的14倍,随后在24 h后下降,并维持在平稳的状态(图7-b)。

      在大弹涂鱼脑组织中,生理盐水组注射后IL-8基因表达呈平稳状态,鳗弧菌注射组,0~12 h内表达平稳,在24~48 h内处于上升状态,于48 h达到所有测定时间点的最高水平,约为对照组的20倍,随后下降;在poly(I∶C)注射组,0~48 h内,IL-8基因的表达呈上升状态,于48 h达到所有测定时间点的最高水平,约为对照组的5倍,随后下降(图7-c)。

    3.   讨论
    • IL-8是趋化因子家族中的一种低分子质量细胞因子,在炎症反应中起着相当重要的作用。本研究从大弹涂鱼皮肤组织转录组中获得大弹涂鱼IL-8基因的cDNA序列,并进行序列验证。该基因ORF区域全长为306 bp,预测编码101个氨基酸。有4个保守的半胱氨酸残基,分别位于第30、32、57和73位氨基酸残基处,前2个半胱氨酸残基和1个精氨酸形成了一个CXC基序,在其他趋化因子的研究中同样也发现了类似的基序[16]。这4个半胱氨酸形成了2对二硫键,对于许多趋化因子的三级结构和功能来说至关重要[32],比如CXCL13[33]

      同草鱼和大西洋鲑等鱼类一样,大弹涂鱼IL-8氨基酸序列缺少谷氨酸—亮氨酸—精氨酸(ELR)基序,该基序被认为是引导中性粒细胞的重要基序[34]。在已研究的鱼类IL-8序列中,该关键基序通常被LLR[15]、EMH[17]、EQH[35]、SLH[8]和DPR[12]等其他基序所取代。与草鱼和大西洋鲑等鱼类不同的是,大弹涂鱼该基序被3个不同的氨基酸残基完全取代,分别为天冬酰胺、丝氨酸和组氨酸,并不含有谷氨酸、亮氨酸或精氨酸,这可能表明了大弹涂鱼IL-8基因在整个进化系统中处于一个特殊的位置。ELR基序通常在哺乳动物、鸟类和爬行动物中被发现,而在硬骨鱼中,该基序只在黑线鳕和大西洋鳕中被发现,这2种鱼均属于鳕鱼科[13,36],因此需要进一步研究该基序在鳕鱼科与其他鱼类中的功能。在通常的研究中,缺乏ELR基序的CXC趋化因子被认为能够吸引淋巴细胞和单核白细胞,然而在一些其他的研究中显示,鱼类含有的DLR等其他基序的CXC趋化因子即使缺乏ELR基序也能吸引中性粒细胞[9,37],这显示了鱼类CXC趋化因子的特异性。并且,早期学者的研究发现,当哺乳动物的ELR基序转变为DLR时,它们仍然能够吸引中性粒细胞[38]。后期更深入的研究还发现,当ELR突变为DLR时,其蛋白生物活性降低了100倍。这些研究表明,虽然DLR基序具有一定的功能性,但其能力明显弱于ELR基序。根据另一篇研究报道,当大菱鲆IL-8的EMH基序被置换为3个丙氨酸(Ala)时,IL-8蛋白趋化能力显著降低,相比之下,当EMH基序突变为ELR时,突变的IL-8蛋白趋化活性没有改变[39],由此推测该基序可能与靶细胞受体的相互作用有关。从氨基酸种类角度分析,这三联基序规律如下:第一个氨基酸为极性氨基酸、非极性氨基酸或带负电荷的酸性氨基酸,第二个氨基酸为非极性氨基酸或不带电荷的极性氨基酸,第三个氨基酸为带正电荷的精氨酸或组氨酸,目前还没有发现赖氨酸。

      鱼类IL-8同源基因系统发生树表明,本实验中大弹涂鱼IL-8基因与大黄鱼、条石鲷和革首南极鱼等IL-8基因同源性较高,与大黄鱼、青鳉、半滑舌鳎和鲤等鱼类CXCL8_L3的同源性较低,且在进化过程中鱼类的IL-8基因出现了分化,形成了CXCL8_L1、CXCL8_L2和CXCL8_L3这3个同源基因亚群。在本实验室现有的转录组数据中尚未发现大弹涂鱼IL-8的同源基因及多拷贝,因此,大弹涂鱼IL-8是否存在同源基因及多拷贝现象还需进一步研究。

      通过本实验构建的系统发生树可以看出,大弹涂鱼与眼斑双锯鱼亲缘关系较近,与鳕形目,鲤形目(Cypriniformes)亲缘关系较远,虽然大弹涂鱼属于虾虎鱼科,眼斑双锯鱼属于雀鲷科(Pomacentridae),但是在进化过程中,这2种鱼类的祖先首先分化出来,形成了各自不同的分支。多序列比对和序列相似性分析表明,大弹涂鱼IL-8与斑马拟丽鱼的序列相似性最高,为88.2%,与其他鱼类序列相似性较低(50%~60%),这与系统进化树的结果相符。而且,大弹涂鱼IL-8氨基酸序列与非鱼类的序列相似性也为50%~60%,这说明大弹涂鱼在进化过程中可能处于一个特殊的位置。无论是位点还是分支—位点模型均未检测到任何正选择位点,说明硬骨鱼类IL-8基因处于强烈的纯化选择下,在进化过程中高度保守,不同硬骨鱼类的IL-8结构、功能和作用机理均未发生较大突变,由此可知,在大弹涂鱼乃至硬骨鱼类中,该基因可能扮演了不可替代的地位。

      其他鱼类IL-8的研究结果显示,IL-8在鱼类各组织中的特异表达水平并不一致。本实验中实时荧光定量PCR结果表明,大弹涂鱼IL-8基因广泛表达于各健康组织中,在脑中的表达量最高,其次是脾脏和鳃,皮肤中的表达量最低,肝脏中的表达量不高,这与鳙(Aristichthys nobilis)IL-8的组织谱相似[40]。在未受刺激的大弹涂鱼脾脏和鳃中,免疫基因半乳糖凝集素-8的表达量高于其他组织,肝脏组织中的表达量最低[41]。对大黄鱼的研究表明,IL-8在大黄鱼未受感染的肝脏组织中表达量最高,在脑、鳃和脾脏中表达量最低[6,15],在半滑舌鳎中也出现了类似的表达结果[42]。牙鲆IL-8在健康脾脏和头肾中高表达[43]。IL-8由一系列细胞产生,如单核细胞和巨噬细胞,以及内皮细胞和成纤维细胞[2],造成不同鱼类IL-8组织表达差异的原因可能是由于在进化过程中,不同鱼类的这些器官中的一部分免疫细胞类型发生了改变。但在已有的鱼类研究中未发现健康脑组织中表达量最高的现象,分析原因可能和鳙鳃组织中表达量最高的原因相同[40],大弹涂鱼脑组织需要更多的IL-8来促进中性粒细胞向炎症部位迁移以适应复杂的滩涂环境。

      实时荧光定量PCR检测了感染后大弹涂鱼IL-8基因在肝脏、脾脏和脑组织中的表达情况,结果显示,2种致病原刺激后,IL-8基因在肝脏和脾脏中表达量上调,这与黑线鳕、大西洋鳕和虹鳟等肝脏和脾脏中的免疫应答结果一致[13,36,44],免疫细胞的补充对于促进不同器官对病原体的免疫过程十分重要[45],因此,在肝脏、脾脏中,IL-8在细菌和病毒入侵时刺激诱导免疫细胞迁移或激活免疫应答的过程中发挥着重要作用。肝脏组织中IL-8在鳗弧菌刺激后的12 h表达水平最高,随后逐渐恢复,这与大黄鱼受副溶血性弧菌感染后的研究结果相似[6],而脾脏中72 h才表现为所有时间点的最高表达水平,这与大黄鱼被副溶血性弧菌感染后,脾脏组织中6 h时出现最高表达水平的结果不一致,造成该结果的原因可能是这2种鱼类脾脏中免疫细胞的组成类型不同,大弹涂鱼脾脏中IL-8基因应答较慢,可能存在着免疫细胞迁移的现象。poly(I∶C)注射后,肝组织36 h IL-8基因表现为所有时间点的最高表达水平,随后逐渐恢复,而大黄鱼在poly(I∶C)刺激后,肝脏中IL-8基因表达量呈现下降的趋势[15],黑线鳕在poly(I∶C)刺激后的24 h内,肝脏中IL-8的表达量呈现上升的趋势[36],这可能说明不同鱼类肝脏对poly(I∶C)刺激后的响应方式不同,大黄鱼肝脏受到poly(I∶C)刺激后的短暂时间内,IL-8基因在免疫应答中受到了抑制,而大弹涂鱼和黑线鳕受刺激后,其IL-8基因通过吸引中性粒细胞等免疫细胞,在肝脏中发挥着重要作用。对于脾脏而言,poly(I∶C)注射后12 h时,IL-8基因表达水平最高,说明大弹涂鱼在poly(I∶C)感染后,脾脏的免疫应答早于肝脏,脾脏是重要的淋巴和造血器官,说明在此阶段,淋巴和造血器官在抗病毒感染的反应中发挥着重要作用。

      白细胞介素-8除了具有趋化中性粒细胞的作用外,还被认为是中枢神经系统髓鞘形成的积极调节因子[46],星形胶质细胞和小胶质细胞在受刺激后可以产生IL-8[47]。鳗弧菌注射后,脑中IL-8基因的表达量先处于平稳状态,后于48 h升高至对照组的20倍;poly(I∶C)注射后,先缓慢上升,并于48 h达到对照组的5倍,表明IL-8基因在脑组织的炎性反应中也起到了重要作用。在以往的研究中,重度颅脑损伤患者脑髓液中IL-8显著升高[48],因此推测在本实验中大弹涂鱼可能由于细菌刺激出现了一定程度的脑损伤,且IL-8的表达不仅来自脑组织中的巨噬细胞等免疫细胞,还可能来自胶质细胞。

      综上所述,本实验首次分析了大弹涂鱼IL-8的cDNA的序列特征,构建了系统发生树,进行了分子进化分析,通过荧光定量技术分析了IL-8基因在不同组织中的表达和在不同病原体刺激下在肝脏、脾脏和脑组织中的表达,表明IL-8基因在大弹涂鱼肝脏、脾脏和脑组织的炎性反应和免疫应答中发挥着重要作用,为进一步了解大弹涂鱼和其他虾虎鱼类的免疫基因提供理论参考。

      感谢刘桓君、杨立国和周建波等同学在实验、本文构思和撰写过程中给予的帮助。

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