松江鲈（Trachidermus fasciatus）髓样分化因子MyD88基因克隆与组织表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2015.02.020

摘要/Abstract

摘要： 髓样分化因子（MyD88）是TOLL样受体介导的信号通路中的一个关键接头分子，通过激活核转录因子（nuclear factor-kappaB，NF-κB）而参与机体的先天免疫。克隆了松江鲈的MyD88基因（命名为TfMyD88），并对该基因进行了生物信息学和表达模式分析。结果显示，TfMyD88 cDNA序列全长1 555 bp，5' UTR长89 bp，3' UTR长599 bp；开放阅读框长度为867 bp，编码288个氨基酸。SMART软件预测TfMyD88分子的N端为一个保守的死亡结构域（death domain，DD），C端存在典型的TIR（Toll/interleukin-1 receptor）结构域。TfMyD88与其它脊椎动物MyD88的氨基酸相似性达57.58%-82.64%，系统进化树分析表明TfMyD88与同属鲈形总目的花鲈和鳜鱼聚在一起，所有鱼类MyD88聚为一支。Real-time PCR检测显示TfMyD88广泛表达于松江鲈各组织，但在鳃中的相对表达量最高；其次为脾脏和皮肤。LPS（lipopolysaccharide）刺激后，TfMyD88在松江鲈的血液、肝脏、皮肤、脾脏均出现明显上调。刺激2 h后，在血液TfMyD88表达量升高了近60倍，在皮肤中的表达量也升高了27倍。上述结果表明TfMyD88可能参与松江鲈先天免疫。

关键词: 松江鲈, MyD88, 基因克隆, 先天免疫, LPS

Abstract: Myeloid differentiation factor 88（TfMyD88） is a key adaptor protein in the Toll-madiated signaling passway. In this study, we cloned the full-length cDNA from the roughskin soulpin, Trachidermus fasciatus. The full-length of MyD88 was 1 555 bp, which contained an open reading frame（ORF） of 867 bp encoding a polypeptide of 288 amino acids. The deduced amino acid sequence has a conserved death domain（DD） at the N-terminal and a typical Toll/interleukin-1 receptor（TIR） domain at the C-terminal. The mRNA expression patterns of TfMyD88 in healthy and LPS challenged roughskin soulpin were detected using quantitative Real-time PCR. MyD88 was expressed broadly in the blood, heart, liver, gill, intestine, skin, musle, kidney, spleen, brain, with the highest expression in the gill. The expression of TfMyD88 post challenge with LPS（lipopolysaccharide） was detected in blood, liver, gill, skin and spleen. The up-regulation of the expression levels of TfMyD88 in all the detccted tissues after chanllaged by LPS suggests that MyD88 plays an important role in roughskin soulpin defenses against bacterial infection.

Key words: Trachidermus fasciatus, MyD88, gene clone, innate immunity, LPS

毕彩红, 张秋霞, 于珊珊, 陈学昭, 刘春影, 祝茜. 松江鲈（Trachidermus fasciatus）髓样分化因子MyD88基因克隆与组织表达分析[J]. 生物技术通报, 2015, 31(2): 135-142.

Bi Caihong, Zhang Qiuxia, Yu Shanshan, Chen Xuezhao, Liu Chunying, Zhu Qian. Molecular Cloning and Expression Analysis of MyD88 in Roughskin Soulpin，Trachidermus fasciatus[J]. Biotechnology Bulletin, 2015, 31(2): 135-142.

参考文献

[1] Kiyoshi T, Shizuo A. TLR signaling pathways[J]. Seminars in Immunology, 2004, 16（1）：3-9.
[2] Ruslan M, Paula PH, Elizabeth K, et al. MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways[J]. Molecular Cell, 1998, 2（2）：253-258.
[3] Adachi O, Kawai T, Takeda K, et al. Targeted disruption of the My-D88 gene results in loss of IL-1-and IL-18-mediated function[J]. Immunity, 1998, 9（1）：143-150.
[4] Kimberly B, Fabio M, Christoph E, et al. MyD88, an adapter protein involved in Interleukin-1 signaling[J]. The Journal of Biological Chemistry, 1998, 273（20）：12203-12209.
[5] Kawai T, Akira S. Signaling to NF-kappaB by Toll-like receptors [J]. Trends in Molecular Medicine, 2007, 13（11）：460-469.
[6]Bowie AG, Haga IR. The role of Toll-like receptors in the host response to viruses[J]. Molecular Immunol, 2005, 42（8）：859-867.
[7]Relling N, Ehlers S, Holscher C. MyDths and un-TOLLed truths：Sensor, instructive and effector immunity to tuberculosis[J]. Immunology Letters, 2008, 116（1）：15-23.
[8]李强, 李学伟, 朱砺, 等. 猪MyD88基因的克隆及组织表达谱分析[J]. 畜牧兽医学报, 2009, 40（10）：1429-1434.
[9]满初日嘎, 谢少林, 杜丽, 等. 海南坡鹿MYD88cDNA的克隆及其生物信息学分析[J].吉林农业大学学报, 2011, 33（3）：319-323.
[10]郭智莉, 周作勇, 聂奎. 鸭髓样分化因子MyD88部分cDNA克隆及组织表达图谱分析[J]. 中国兽医杂志, 2012, 48（4）：33-36.
[11]朱炳林, 李俊, 方维焕, 等. 中华鳖MyD88部分序列克隆及其在组织中的表达差异分析[J]. 水产学报, 2010, 34（7）：1018-1024.
[12]Jault C, Pichon L, Chluba J. Toll- like receptor gene family and TIR-domain adapters in Danio rerio[J]. Molecular Immunology, 2004, 40：759-771.
[13]Purcell MK, Smith KD, Hood L, et al. Conservation of toll-like receptor signaling pathwaysin teleost fish[J]. Comparative Biochemistry and Physiology Part D：Genomics and Proteomics, 2006, 1（1）：77-88.
[14]Yao CL, Kong P, Wang ZY, et al. Molecular cloning and expression of MyD88 in large croaker Pseudosciaena crocea[J]. Fish & Shellfish Immunol, 2009, 26：249-255.
[15]沙珍霞, 王娜, 王启龙, 等. 半滑舌鳎髓样分化因子的克隆和表达分析[J]. 中国水产科学, 2010, 17（4）：659-670.
[16]Ilson W, Youngdeuk L, Hyowon K, et al.Characterization and exp-ression analysis of the myeloid differentiation factor 88（MyD88） in rock bream Oplegnathus fasciatus[J]. Molecular Biology Reports, 2011, 38：3911-3920.
[17]韦友传, 高谦, 昌明先, 等. 斜带石斑鱼MyD88基因的克隆与表达[J]. 基因组学与应用生物学, 2011, 30（3）：288-295.
[18]Takano T, Kondo H, Hirono I. Identification and characterization of a myeloid differentiation factor 88（MyD88） cDNA and gene in Japanese flounder, Paralichthys olivaceus[J]. Developmental and Comparative Immunology, 2006, 30（9）：807-816.
[19]Liu YY, Yu SS, Chai YM, et al. Lipopolysaccharide-induced gene expression of interleukin-1 receptor-associated kinase 4 and interleukin-1b in roughskin sculpin（Trachidermus fasciatus）[J]. Fish & Shellfish Immunology, 2012, 33：690-698.
[20] Yu SS, Yang H, Chai YM, et al. Molecular cloning and characte-rization of a C-type lectin in roughskin sculpin（Trachidermus fasciatus）[J]. Fish & Shellfish Immunol, 2013, 34：582-592.
[21]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCt method[J]. Methods, 2001, 25（4）：402-408.
[22]Hidenori O, Hideh ito T, Zenichiro K, et al. Structural basis for the multiple interactions of the MyD88 TIR domain in TLR4 signaling[J]. PNAS, 2009, 106：10260-10265.
[23]Sun D, Ding A. MyD88-mediated stabilization of interferon-γ-induced cytokine and chemokine mRNA[J].Nature Immunology, 2006, 7（4）：375-381.
[24]Tomokazu T, Hidehiro K. Identification and characterization of a myeloid differentiation factor 88（MyD88） cDNA and gene in Japanese flounder, Paralichthys olivaceus[J]. Developmental and Comparative Immunology, 2006, 30：807-816.
[25] Yan Y, Cui HC, Wei JG, et al. Functional genomic studies on an immune-and antiviral-related gene Of Myd88 in orange-spotted Grouper, Epinephelus coioides[J]. Chinese Science Bulletin, 2012, 57（25）：3277-3287.
[26] Tang D, Gao YH, Wang RX, et al. Characterization, genomic organization, and expression profiles of MyD88, a key adaptor molecule in the TLR signaling pathways in miiuy croaker（Miichthys miiuy）[J]. Fish Physiology and Biochemistry, 2012, 38（6）：1667-1677.