猪Toll样受体4基因SNPs功能分析

doi:10.3724/SP.J.1005.2012.01050

遗传 ›› 2012, Vol. 34 ›› Issue (8): 1050-1056.doi: 10.3724/SP.J.1005.2012.01050

猪Toll样受体4基因SNPs功能分析

杨秀芹¹, 陈月婵¹, 汪亮², 李海涛¹, 刘娣², 关庆芝¹, 付博²

1. 东北农业大学动物科技学院, 哈尔滨 150030 2. 黑龙江省农业科学院, 哈尔滨 150086

收稿日期:2012-01-03 修回日期:2012-03-15 出版日期:2012-08-20 发布日期:2012-08-25
通讯作者: 杨秀芹 E-mail:xiuqin163@163.com
基金资助:
黑龙江省教育厅科学技术研究项目(编号：11551033)资助

Functional analysis of SNPs in porcine TLR4 gene

YANG Xiu-Qin¹, CHEN Yue-Chan¹, WANG Li-ang², LI Hai-Tao¹, LIU Di², GUAN Qing-Zhi¹, FU Bo²

1. College of Animal Science & Technology, Northeast Agricultural University, Harbin 150030, China 2. Heilongjiang Academy of Agricultural Science, Harbin 150086, China

Received:2012-01-03 Revised:2012-03-15 Online:2012-08-20 Published:2012-08-25

摘要/Abstract

摘要： Toll样受体4(Toll-like receptor 4, TLR4)在机体的免疫反应中发挥重要作用, 该基因突变会影响受体的信号转导能力和机体的疾病抗性/易感性。文章在前期工作的基础上, 进一步分析c.611 T>A (p.Leu204His)、c.1027 C>A (p.Gln343Lys) 和c.1605 G>T (p.Leu535Phe) 3个错义突变对猪TLR4功能的影响。利用RT-PCR方法克隆猪TLR4基因全长编码区并引入定点突变; 利用真核表达、双荧光素酶报告系统和Western blotting方法在瞬时转染的PK-15细胞内研究3个单核苷酸多态(Single nucleotide polymorphisms, SNPs)对猪TLR4配体识别和信号转导能力的影响; 同时, 利用创造酶切位点PCR-RFLP方法分析对TLR4活性有显著影响的点突变在民猪、大白、长白和中国东北野猪4个群体中的分布。结果, 成功获得了民猪TLR4基因的全长编码区和3个单碱基变异体, 构建了不同等位基因的真核表达载体, 在PK-15细胞内确定了c.1605 G>T变异导致TLR4向下游传递信号的能力显著降低(P<0.01), 该SNP只存在于民猪和野猪中且频率较高。猪TLR4基因c.1605 G>T变异影响Toll样受体的信号传递, 可能和机体的疾病抗性/易感性有关。

关键词: 猪, TLR4, SNP, 功能分析

Abstract: Toll-like receptor 4 (TLR4) plays an important role in immune response and the polymorphism in it might affect protein signaling and host resistance/susceptibility to disease. This study was designed to characterize the functional relevance of 3 nonsynonymous single nucleotide polymorphisms (SNPs), c.611 T>A (p.Leu204His), c.1027 C>A (p.Gln343Lys), and c.1605 G>T (p.Leu535Phe), which were selected based on our previous studies. RT-PCR method was used to clone the complete coding sequence of porcine TLR4 gene and the PCR-based method was used to introduce the point mutation. The effects of 3 SNPs on the ligand recognition and signaling of porcine TLR4 were investigated in transiently transfected PK-15 cells using dual-luciferase reporter system and Western blotting method. At the same time, the distribution of c.1605 G>T among pig populations composed of Min pig, Yorkshire, Landrace, and Wild boar from north-eastern China was studied by created restriction site PCR-RFLP method. The complete coding sequence of TLR4 gene in Min pig and 3 variants with single point mutations were obtained. Eukaryotic expression vectors containing differ-ent alleles of porcine TLR4 were constructed. SNP c.1605 G>T significantly decreased the TLR4 signaling (P<0.01) and the polymorphism only existed in Min pig and Wild boar from northeastern China with high frequencies. SNP c.1605 G>T in porcine TLR4 might affect the receptor function and host resistance/susceptibility to diseases.

Key words: porcine, TLR4, SNP, functional analysis

杨秀芹，陈月婵，汪亮，李海涛，刘娣，关庆芝，付博. 猪Toll样受体4基因SNPs功能分析[J]. 遗传, 2012, 34(8): 1050-1056.

YANG Xiu-Qin, CHEN Yue-Chan, WANG Li-ang, LI Hai-Tao, LIU Di, GUAN Qing-Zhi, FU Bo. Functional analysis of SNPs in porcine TLR4 gene[J]. HEREDITAS, 2012, 34(8): 1050-1056.

参考文献

[1] Chang ZL. Important aspects of Toll-like receptors, ligands and their signaling pathways. Inflamm Res, 2010, 59(10): 791-808.
[2] Takeuchi O, Akira S. Pattern recognition receptors and inflammation. Cell, 2010, 140(6): 805-820.
[3] Uenishi H, Shinkai H. Porcine Toll-like receptors: The front line of pathogen monitoring and possible implications for disease resistance. Dev Comp Immunol, 2009, 33(3): 353-361.
[4] Chow JC, Young DW, Golenbock DT, Christ WJ, Gusovsky F. Toll-like receptor-4 mediates lipopolysaccharide-induced signal transduction. J Biol Chem, 1999, 274(16): 10689-10692.
[5] Lien E, Means TK, Heine H, Yoshimura A, Kusumoto S, Fukase K, Fenton MJ, Oikawa M, Qureshi N, Monks B, Finberg RW, Ingalls RR, Golenbock DT. Toll-like receptor 4 imparts ligand-specific recognition of bacterial lipopolysaccharide. J Clin Invest, 2000, 105(4): 497-504.
[6] Takeuchi O, Hoshino K, Kawai T, Sanjo H, Takada H, Ogawa T, Takeda K, Akira S. Differential roles of TLR2 and TLR4 in recognition of gram-negative and gram-positive bacterial cell wall components. Immunity, 1999, 11(4): 443-451.
[7] Rallabhandi P, Bell J, Boukhvalova MS, Medvedev A, Lorenz E, Arditi M, Hemming VG, Blanco JC, Segal DM, Vogel SN. Analysis of TLR4 variants: new insights into TLR4/MD-2/CD14 stoichiometry, structure, and signaling. J Immunol, 2006, 177(1): 322-332.
[8] Latha M, Vaidya S, Movva S, Chava S, Govindan S, Govatati S, Banoori M, Hasan Q, Kodati VL. Molecular pathogenesis of endometriosis; Toll-like receptor-4 A896G (D299G) polymorphism: a novel explanation. Genet Test Mol Biomarkers, 2011, 15(3): 181-184.
[9] Verma A, Prasad KN, Gupta RK, Singh AK, Nyati KK, Rizwan A, Pandey CM, Paliwal VK. Toll-like receptor 4 polymorphism and its association with symptomatic neurocysticercosis. J Infect Dis, 2010, 202(8): 1219-1225.
[10] Buraczynska M, Baranowicz-Gaszczyk I, Tarach J, Ksiazek A. Toll-like receptor 4 gene polymorphism and early onset of diabetic retinopathy in patients with type 2 diabetes. Hum Immunol, 2009, 70(2): 121-124.
[11] Kathrani A, House A, Catchpole B, Murphy A, German A, Werling D, Allenspach K. Polymorphisms in the TLR4 and TLR5 gene are significantly associated with inflammatory bowel disease in German shepherd dogs. PLoS One, 2010, 5(12): e15740.
[12] Kataria RS, Tait RG Jr, Kumar D, Ortega MA, Rodiguez J, Reecy JM. Association of toll-like receptor four single nucleotide polymorphisms with incidence of infectious bovine keratoconjunctivitis (IBK) in cattle. Immunogenetics, 2011, 63(2): 115-119.
[13] Zhang GW, Wang HZ, Chen SY, Li ZC, Zhang WX, Lai SJ. A reduced incidence of digestive disorders in rabbits is associated with allelic diversity at the TLR4 locus. Vet Immunol Immunopathol, 2011, 144(3-4): 482-486.
[14] Leveque G, Forgetta V, Morroll S, Smith AL, Bumstead N, Barrow P, Loredo-Osti JC, Morgan K, Malo D. Allelic variation in TLR4 is linked to susceptibility to Salmonella enterica serovar Typhimurium infection in chickens. Infect Immun, 2003, 71(3): 1116-1124.
[15] Ye X, Avendano S, Dekkers JC, Lamont SJ. Association of twelve immune-related genes with performance of three broiler lines in two different hygiene environments. Poult Sci, 2006, 85(9): 1555-1569.
[16] Shinkai H, Tanaka M, Morozumi T, Eguchi-Ogawa T, Okumura N, Muneta Y, Awata T, Uenishi H. Biased distribution of single nucleotide polymorphisms (SNPs) in porcine Toll-like receptor 1 (TLR1), TLR2, TLR4, TLR5, and TLR6 genes. Immunogenetics, 2006, 58(4): 324-330.
[17] Palermo S, Capra E, Torremorell M, Dolzan M, Davoli R, Haley CS, Giuffra E. Toll-like receptor 4 genetic diversity among pig populations. Anim Genet, 2009, 40(3): 289-299.
[18] 潘章源, 叶兰, 朱璟, 杜子栋, 黄小国, 朱国强, 包文斌, 吴圣龙. 猪TLR4基因外显子1新等位基因的分离及遗传变异分析. 遗传, 2011, 33(2): 163-167.
[19] 翟春媛, 李海涛, 杨秀芹, 李金玲, 刘娣. 猪TLR4基因的变异位点分析. 中国畜牧杂志, 2011, 47(3): 18-21.
[20] 杨秀芹, 翟春媛, 李海涛, 刘娣. 猪TLR4基因可变剪接体的鉴定. 畜牧兽医学报, 2011, 42(8): 1051-1056.
[21] 赵春江, 李宁, 邓学梅. 应用创造酶切位点法检测单碱基突变. 遗传, 2003, 25(3): 327-329.
[22] Medzhitov R, Preston-Hurlburt P, Janeway CA Jr. A human homologou of the Drosophila Toll protein signals activation of adaptive immunity. Nature, 1997, 388(6640): 394-397.
[23] Miyake K. Roles for accessory molecules in microbial recognition by Toll-like receptors. J Endotoxin Res, 2006, 12(4): 195-204.
[24] Sun J, Duffy KE, Ranjith-Kumar CT, Xiong J, Lamb RJ, Santos J, Masarapu H, Cunningham M, Holzenburg A, Sarisky RT, Mbow ML, Kao C. Structural and functional analyses of the human Toll-like receptor 3. Role of glycosylation. J Biol Chem, 2006, 281(16): 11144-11151.
[25] Kim HM, Park BS, Kim JI, Kim SE, Lee J, Oh SC, Enkhbayar P, Matsushima N, Lee H, Yoo OJ, Lee JO. Crystal structure of the TLR4-MD-2 complex with bound endotoxin antagonist Eritoran. Cell, 2007, 130(5): 906-917.
[26] Park BS, Song DH, Kim HM, Choi BS, Lee H, Lee JO. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature, 2009, 458(7242): 1191- 1195.
[27] Kimura M. The neutral theory of molecular evolution and the world view of the neutralists. Genome, 1989, 31(1): 24-31.

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猪Toll样受体4基因SNPs功能分析

Functional analysis of SNPs in porcine TLR4 gene

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