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花鲈b2m基因cDNA的克隆及表达分析

葛婉仪 雷丽娜 蒋昕彧 李霞 孙兆盛 王伟 高谦

葛婉仪, 雷丽娜, 蒋昕彧, 李霞, 孙兆盛, 王伟, 高谦. 花鲈b2m基因cDNA的克隆及表达分析[J]. 南方水产科学. doi: 10.12131/20200031
引用本文: 葛婉仪, 雷丽娜, 蒋昕彧, 李霞, 孙兆盛, 王伟, 高谦. 花鲈b2m基因cDNA的克隆及表达分析[J]. 南方水产科学. doi: 10.12131/20200031
Wanyi GE, Lina LEI, Xinyu JIANG, Xia LI, Zhaosheng SUN, Wei WANG, Qian GAO. cDNA cloning and expression pattern analysis of b2m from spotted sea bass (Lateolabrax maculatus)[J]. South China Fisheries Science. doi: 10.12131/20200031
Citation: Wanyi GE, Lina LEI, Xinyu JIANG, Xia LI, Zhaosheng SUN, Wei WANG, Qian GAO. cDNA cloning and expression pattern analysis of b2m from spotted sea bass (Lateolabrax maculatus)[J]. South China Fisheries Science. doi: 10.12131/20200031

花鲈b2m基因cDNA的克隆及表达分析

doi: 10.12131/20200031
基金项目: 国家重点研发计划“蓝色粮仓科技创新”重点专项 (2019YFD0900604)
详细信息
    作者简介:

    葛婉仪 (1999—),女,本科生,研究方向为鱼类免疫学与病害防控。E-mail:1103617638@qq.com

    通讯作者:

    高 谦 (1968—),男,博士,研究员,从事鱼类免疫学与病害防控研究。E-mail: qgao@shou.edu.cn

  • 中图分类号: S 917.4

cDNA cloning and expression pattern analysis of b2m from spotted sea bass (Lateolabrax maculatus)

  • 摘要: 为探究花鲈 (Lateolabrax maculatus) b2m (Beta-2 microglobulin) 基因对细菌和病毒感染的响应,该研究通过RACE-PCR扩增获得了花鲈b2m基因,该基因cDNA全长为1 383 bp,开放阅读框 (Open reading frame, ORF) 为357 bp,编码118个氨基酸。所推测的编码蛋白与已报道的鱼类B2m的氨基酸序列相似度为44.8%~63.2%。基因共线性分析显示,b2m基因座位置在鱼类基因组中高度保守。利用实时荧光定量PCR检测花鲈b2m基因在各组织器官中的分布,结果表明花鲈b2m在所检测组织器官中均有表达,脾脏中表达量最高,鳃、头肾和脑中次之。通过腹腔注射脂多糖 (Lipopolysaccharide, LPS)、聚肌苷-脱氧胞苷酸 (Poly I:C) 和迟缓爱德华氏菌 (Edwardsiella tarda, Edw) 研究花鲈感染后b2m的表达变化,结果显示3种刺激物均可诱导花鲈b2m在鳃、肠、头肾、脾脏组织中表达上调,推测b2m基因参与了花鲈抗细菌、抗病毒感染的免疫应答过程。
  • 图  1  花鲈b2m的核酸和氨基酸序列

    起始密码子 (ATG) 和终止密码子 (TAA) 用阴影覆盖;预测的信号肽用黑色单下划线标示;预测的Igc1结构域用红色双下划线标示;红色框标记为酪氨酸磷酸化位点;黑色框标记为苏氨酸磷酸化位点

    Figure  1.  Nucleotide and deduced amino acid sequence from b2m gene of L. maculatus

    Translation start (ATG) and stop (TAA) codens are shaded. The predicted signal peptide is marked with a single black underline. The predicted Igc1 domain is marked with a red double underscore. The red box is the tyrosine phosphorylation site. The black box is the threonine phosphorylation site.

    图  2  花鲈B2m与其他物种B2m的氨基酸序列比对

    双向箭头标注为信号肽;红色三角形区间标注为免疫球蛋白样结构域Igc1;*表示高度保守的半胱氨酸位点;黑色表示此位点氨基酸完全一致;深灰色和浅灰色表示此位点氨基酸相似度由75%到50%

    Figure  2.  Multiple alignment of B2m amino acid sequences from L. maculatus and other species

    Two-way arrows are labeled as signal peptides; the red triangle region is marked as Igc1; *. A highly conserved cysteine site; Black. The identical amino acids; dark gray, light gray. The similarities change form 75% to 50%.

    图  3  依B2m氨基酸序列构建的系统进化树

    系统发育进化树由MEGA X软件以最大似然法构建

    Figure  3.  Phylogenetic tree of B2m from various species

    Phylogenetic trees are constructed with the maximum likelihood method by MEGA X software.

    图  4  花鲈B2m二级和三级结构预测

    A. 花鲈B2m二级结构,粉色:α螺旋, 黄色:β折叠,灰色:无规则卷曲结构;B. 花鲈B2m三级结构卡通模型;C. 花鲈B2m三级结构空间立体球模型

    Figure  4.  Prediction of secondary structure and tertiary structure of L. maculatus B2m

    A. Secondary structure of B2m from L. maculatus; pink. α-helix; yellow. β-sheet; gray. Coils; B. Cartoon model of tertiary structure B2m from L. maculatus; C. Spacefill model of tertiary structure of B2m from L. maculatus

    图  5  花鲈和齿舌鲈的b2m基因结构比较

    Figure  5.  Comparison of b2m gene structure between L. maculatus and D. labrax

    图  6  智人、珍珠鸟、红原鸡、抹香鲸、斑马鱼、半滑舌鳎和花鲈中b2m基因的共线性分析图

    Figure  6.  Synteny diagram of b2m gene loci in H. sapiens, T. guttata, G. gallus, P. catodon, D. rerio, C. semilaevis and L. maculatus

    图  7  花鲈b2m的组织表达模式

    Figure  7.  Expression level of L. maculatus b2m mRNA in different tissues

    图  8  腹腔注射LPS、Poly (I:C)、Edw对花鲈b2m表达调控的影响

    通过比较处理组 [腹腔注射LPS、Poly (I:C)、Edw] 和对照组的b2m基因表达水平,计算基因表达的倍数变化;数据以“平均值±标准差” (n=3) 表示;*. 显著性相关 (P<0.05);**. 极显著性相关 (P<0.01)

    Figure  8.  Effect of intraperitoneal injection with LPS, Poly (I:C) and Edw on regulation of b2m expression in L.maculatus

    Fold change of gene expression was calculated by comparing the level of LPS, Poly (I:C) and Edw groups with that of control group. The data are presented as "$\overline X \pm {\rm{SD}} $" (n=3); *. Significant at 0.05 level (P<0.05); **. Very significant at 0.01 level (P<0.01)

    表  1  实验中用到的引物

    Table  1.   Primers used in this experiment

    引物名称
    Primer name
    引物序列 (5'–3')
    Primer sequence
    用途
    Purpose
    b2m-F CATGAAGATGTTTGTCTGCGCA 中间片段克隆
    b2m-R GAGAACTGGCACTACCACCT 中间片段克隆
    b2m-5'R1 AGTGTTCGACAAGCCCAACAC 5'RACE-PCR
    b2m-5'R2 TCTGCTCTGCCTCCTGGCGCTTT 5'RACE-PCR
    b2m-3'F1 AGGAGGACTGGCACTACCACCT 3'RACE-PCR
    b2m-3'F2 ATGGAGAGTTAATACCTGAAGCC 3'RACE-PCR
    b2m-CF AGAGCGACTGAAGGTGAAG 序列验证
    b2m-CR GGTGTACATTTGGAGATAAATGG 序列验证
    b2m-qF TCGACAAGCCCAACACCT qPCR
    b2m-qR CAAAGGAGACGACTACGCCT qPCR
    EF1α-qF ATCTCTGGATGGCACG GAGA qPCR
    EF1α-qR CAGTGTGGTTCCGCTA GCAT qPCR
    下载: 导出CSV

    表  2  用于序列比对和系统进化树构建所用物种的信息

    Table  2.   Information of species used for sequence alignment and construction of phylogenetic tree

    物种
    Species
    氨基酸数量
    Number of amino acids
    相似度
    Similarity/%
    登录号
    Accession No.
    C. carpio 116 47.20 Q03422.1
    犀角金线鲃 S. rhinocerous 116 48.00 XP_016430127.1
    齐口裂腹鱼 S. prenanti 116 49.60 QEE82339.1
    斑马鱼 D. rerio 116 45.60 Q04475.1
    斑点叉尾鮰 I. punctatus 116 50.40 O42197.1
    青鳉 O. latipes 116 44.80 Q90ZJ6
    M. miiuy 116 48.00 ADV36787.1
    白斑狗鱼 E. lucius 116 44.80 ACO14540.1
    大西洋鲑 S. salar 116 50.40 AAG17537.1
    虹鳟 O. mykiss 116 52.00 AAB04663.1
    玻璃梭鲈 S. vitreus 116 49.60 AAW65850.1
    欧洲齿舌鲈 D. labrax 116 48.80 CBJ56267.1
    半滑舌鳎 C. semilaevis 118 48.00 ACR38919.1
    大黄鱼 L. crocea 118 63.20 XP_010753212.1
    智人 H. sapiens 119 38.40 NP _004039.1
    草原田鼠 M. ochrogaster 119 36.80 XP_005364420.1
    下载: 导出CSV
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  • 收稿日期:  2020-02-22
  • 修回日期:  2020-05-29
  • 网络出版日期:  2020-11-19

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