中国荷斯坦牛TLR_1基因SNPs快速筛查及蛋白功能预测
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摘要
为探究中国荷斯坦牛Toll样受体1(TLR_1)基因与中国荷斯坦牛乳房炎抗性的关联性,以中国荷斯坦牛为试验对象,构建DNA混合池,采用PCR扩增和直接测序法对中国荷斯坦牛TLR_1基因进行单核苷酸多态性(SNP)检测,从而筛选出对中国荷斯坦牛免疫方面有显著影响的SNPs位点,同时应用在线软件对中国荷斯坦牛TLR_1基因编码的蛋白质进行功能预测。结果表明,中国荷斯坦牛TLR_1基因编码727个氨基酸,组成了一种不稳定的水溶性蛋白,蛋白质二、三级结构均以α-螺旋为主;PCR扩增后,在扩增片段处共发现8个SNPs位点,分别为A~(61)T-TLR_1、C~(632)A-TLR_1、C~(1408)T-TLR_1、C~(1451)T-TLR_1、A~(1461)G-TLR_1、A~(1475)C-TLR_1、G~(1550)A-TLR_1、G~(1596)A-TLR_1,其中A~(61)T-TLR_1、C~(632)A-TLR_1、C~(1408)T-TLR_1、A~(1461)G-TLR_1、G~(1596)A-TLR_1属错义突变,分别由原来的赖氨酸(Lys)、苯丙氨酸(Phe)、丙氨酸(Ala)、异亮氨酸(Ile)、缬氨酸(Val)突变为甲硫氨酸(Met)、亮氨酸(Leu)、缬氨酸(Val)、缬氨酸(Val)、异亮氨酸(Ile),SNPs位点的等位基因频率在突变前后均存在差异。DNA池结合直接测序技术检测到TLR_1基因8个SNPs位点,可作为中国荷斯坦牛乳房炎的遗传标记进行深入研究;蛋白质功能预测结果表明,有多种与免疫相关的因子几率较高。本研究结果为中国荷斯坦牛在分子领域的抗病育种提供了理论依据。
In order to investigate the association between Toll like receptor 1(TLR_1) gene and mastitis resistance in Chinese Holstein cattle, a DNA pooling was constructed in Chinese Holstein cattle. The single nucleotide polymorphism(SNP) of TLR_1 gene of Chinese Holstein cattle was detected by PCR amplification and direct sequencing, and the SNPs loci which had a significant effect on the immunity of Chinese Holstein cattle were screened. At the same time, the online software was used to predict the function of the protein encoded by the TLR_1 gene of Chinese Holstein cattle. The results showed that Chinese Holstein TLR_(1 )gene encodes 727 amino acids and constitutes an unstable water-soluble protein. After PCR amplification, a total of 8 SNPs loci were found in the amplified fragments, namely A~(61)T-TLR_1, C~(632)A-TLR_1,C~(1408)T-TLR_1, C~(1451)T-TLR_1, A~(1461)G-TLR_1, A~(1475)C-TLR_1, G~(1550)A-TLR_1 and G~(1596)A-TLR_1. Among them, C~(632)A-TLR_1, C~(1451)T-TLR_1, A~(1461)G-TLR_1, A~(1475)C-TLR_1 and G~(1596)A-TLR_1 resulted in missense mutation. The allele frequencies of SNPs loci with five missense mutations were mutated from the original lysine(Lys), phenylalanine(Phe), alanine(Ala), isoleucine(Ile), andvaline(Val) to methionine(Met), leucine(Leu), valine(Val), valine(Val) and isoleucine(Ile), and the allele frequencies of the five missense mutation SNPs were slightly different before and after the mutation, and the rest are significantly different. The DNA pooling combined with direct sequencing technology to detect 8 SNPs of TLR_1 gene, which can be used as a genetic marker for Chinese Holstein cow mastitis. The results of protein function prediction show that there are many factors associated with immune related factors. The results of this study provide a theoretical basis for the resistance breeding of Chinese Holstein cattle in molecular field.
In order to investigate the association between Toll like receptor 1(TLR_1) gene and mastitis resistance in Chinese Holstein cattle, a DNA pooling was constructed in Chinese Holstein cattle. The single nucleotide polymorphism(SNP) of TLR_1 gene of Chinese Holstein cattle was detected by PCR amplification and direct sequencing, and the SNPs loci which had a significant effect on the immunity of Chinese Holstein cattle were screened. At the same time, the online software was used to predict the function of the protein encoded by the TLR_1 gene of Chinese Holstein cattle. The results showed that Chinese Holstein TLR_(1 )gene encodes 727 amino acids and constitutes an unstable water-soluble protein. After PCR amplification, a total of 8 SNPs loci were found in the amplified fragments, namely A~(61)T-TLR_1, C~(632)A-TLR_1,C~(1408)T-TLR_1, C~(1451)T-TLR_1, A~(1461)G-TLR_1, A~(1475)C-TLR_1, G~(1550)A-TLR_1 and G~(1596)A-TLR_1. Among them, C~(632)A-TLR_1, C~(1451)T-TLR_1, A~(1461)G-TLR_1, A~(1475)C-TLR_1 and G~(1596)A-TLR_1 resulted in missense mutation. The allele frequencies of SNPs loci with five missense mutations were mutated from the original lysine(Lys), phenylalanine(Phe), alanine(Ala), isoleucine(Ile), andvaline(Val) to methionine(Met), leucine(Leu), valine(Val), valine(Val) and isoleucine(Ile), and the allele frequencies of the five missense mutation SNPs were slightly different before and after the mutation, and the rest are significantly different. The DNA pooling combined with direct sequencing technology to detect 8 SNPs of TLR_1 gene, which can be used as a genetic marker for Chinese Holstein cow mastitis. The results of protein function prediction show that there are many factors associated with immune related factors. The results of this study provide a theoretical basis for the resistance breeding of Chinese Holstein cattle in molecular field.
引文
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[11] 赵杰,游新勇,徐贞贞,陈爱亮,赵燕,何雯菁,杨曙明.SNP检测方法在动物研究中的应用[J].农业工程学报,2018,34(4):299-305
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[13] 李树珍,万慧荣,杨光.DNA 池结合 DHPLC 和直接测序技术在江豚SNPs检测中的应用[J].兽类学报,2009,29(2):185-190
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[17] Vahana B M,Raj G D,Pawer R M C.Expression profile of toll like receptors in a range of water buffalo tissues (Bubalus bubalis)[J].Vet Immunopatho,2008,126(1):149-155
[18] Wang J,Zhang Z,Liu J.Structural characterization and evolutionary analysis of fish-specific TLR27[J].Fish & Shellfish Immunology,2015,45(2):940-945
[19] Yang C A,Scheibenbogen C,Bauer S L.Afrequent Toll-like receptor 1 gene polymorphism affects NK and T cell IFN-Υ production and is associated with Helicobacter pylori-induced gastric disease [J].Helicobacter,2013,18(1):13-21
[20] Hawn T R,Misch E A,Dunstan S J.The polymorphism P315L of human toll-like receptor 1 impairs innate immune sensing of microbial cell wall components[J].Journal of Immunology,2007,178(10):6387-6394
[21] Omueti K O,Mazur D J,Thompson K S.A common human TLR1 polymorphism regulates the innate immune response to lipopeptides[J].European Journal Immunology,2007,37(8):2280-2289
[22] Schumann R R,Tapping R I.Genomic variants of TLR1ittakes (TLR-) two to tango[J].European Journal Immunology,2007,37 (8):2059-2062
[23] Moisan J,Camateros P,Thuraisingam T.TLR7 lig-and prevents allergen-induced airway hyperresponsiveness and eosinophilia in allergic asthma by a MYD88-dependent and MK2 -independent pathway [J].Am J Physiol Lung Cell Mol Physiol,2006,290 (5):L987-L995
[24] Higgs R,Cormican P,Cahalane S,Allan B,Lliyd A T,Meade K,James T,Lynn D J,Babiuk L A,Farrelly C O.Induction of a novel chicken toll-like receptor following salmonella enterica serovar typhinurium infection[J].Infection and Immunity,2006,74(3):1692-1698
[25] 储明星,李春苗,石万海,安永福,陈宏权,狄冉,方丽.荷斯坦母牛TLR6基因多态性及其与体细胞评分关系的研究[J].畜牧兽医学报,2009,40(11):1621-1629
[26] 周秀敏,李强子,毕英杰,任卫合,张丽.荷斯坦牛TLR6基因多态性分析[J].中国草食动物科学,2017,37(1):1-3
[27] 农伟伦,毕英杰,李宏旭,张丽.甘肃黑猪H-FABP基因克隆、SNPs筛查及生物信息学分析[J].浙江农业学报,2018,30(3):378-385
[28] 贾浩,张小白,宋晓峰.人类胞内蛋白半衰期与其亚细胞定位的相关性研究[J].计算机与应用化学,2011,28(4):411-414
[29] Arnold J N,Wormald M R,Sim R B.The impact of glycosylation on the biological function and structure of human immunoglobulins [J].Annual Review Immunology,2007,25(2):21-50
[30] Calarese D A,Scanlan C N,Zwick M B L.Antibody domain exchange is an immunological solution to carbohydrate cluster recognition[J].Science,2003,300(5628):2065-2071
[2] 王梦琦,倪炜,张慧敏,杨章平,王西朴,蒋彦森,毛永江.中国荷斯坦牛TLR1基因启动子区SNP多态与乳房炎抗性和泌乳性状的关联分析[J].农业生物技术学报,2017,25(3):397-404
[3] Russell C D,Widdison S,Leigh J A.Identification of single nucleotide polymorphisms in the bovine Toll- like receptor 1 gene and association with health traits in cattle[J].Veterinary Research,2012,43:17-28
[4] 李春苗,石万海,储明星,安永福,陈宏权,狄冉,方丽.荷斯坦母牛TLR1基因多态性及其与体细胞评分关系[J].中国农业科学,2009,42(6):2118-2125
[5] 杨永江,吴恩芸,任稳稳,马博妍,李宏旭,高成宏,刘丽霞,张丽.中国荷斯坦牛TLR2基因SNPs快速筛查及等位基因频率估算[J].中国奶牛,2018,5(20):20-23
[6] 许家磊,王宇,后猛,李强.SNP检测方法的研究进展[J].分子植物育种,2015,13(2):475-482
[7] 姚红伟,张立冬,孙金阳,刘霄霞.DNA分子标记技术概述[J].河北渔业,2010,199(7):42-46
[8] 李娜,焦浈,秦广雍.DNA分子标记技术及其在小麦育种及遗传研究中的应用[J].核农学报,2005,19(4):322-326
[9] Aitken N,Smith S C,Morin P.Single nucleotide polymorphism (SNP) discovery in mammals:A targeted-gene approach[J].Molecular Ecology,2004,13(6):1423-1431
[10] 张玉,韩明星,曹明月,赵亚运,李娟,薛良义.大黄鱼FST基因SNP筛选及与生长性状相关性分析[J].核农学报,2018,32(5):883-891
[11] 赵杰,游新勇,徐贞贞,陈爱亮,赵燕,何雯菁,杨曙明.SNP检测方法在动物研究中的应用[J].农业工程学报,2018,34(4):299-305
[12] 初芹,李东,侯诗宇,石万海,刘林,王雅春.基于DNA池测序法筛选奶牛高信息量SNP标记的可行性[J].遗传,2014,36(7):691-696
[13] 李树珍,万慧荣,杨光.DNA 池结合 DHPLC 和直接测序技术在江豚SNPs检测中的应用[J].兽类学报,2009,29(2):185-190
[14] 李强子,朱国强,刘吴鑫,刘丽霞,张丽.荷斯坦牛TLR6基因CDS区的生物信息学分析[J].江苏农业学报,2016,32(3):608-614
[15] 周蕾,顾建新.N-糖基化位点鉴定方法和非经典N-糖基化序列[J].生命科学,2011,23(6):605-611
[16] Litynska A,Przybylo M,Pochec E.Comparison of the lectin-binding pattern in different human melanoma cell lines[J].Melanoma Rsearch,2001,11(3):205-212
[17] Vahana B M,Raj G D,Pawer R M C.Expression profile of toll like receptors in a range of water buffalo tissues (Bubalus bubalis)[J].Vet Immunopatho,2008,126(1):149-155
[18] Wang J,Zhang Z,Liu J.Structural characterization and evolutionary analysis of fish-specific TLR27[J].Fish & Shellfish Immunology,2015,45(2):940-945
[19] Yang C A,Scheibenbogen C,Bauer S L.Afrequent Toll-like receptor 1 gene polymorphism affects NK and T cell IFN-Υ production and is associated with Helicobacter pylori-induced gastric disease [J].Helicobacter,2013,18(1):13-21
[20] Hawn T R,Misch E A,Dunstan S J.The polymorphism P315L of human toll-like receptor 1 impairs innate immune sensing of microbial cell wall components[J].Journal of Immunology,2007,178(10):6387-6394
[21] Omueti K O,Mazur D J,Thompson K S.A common human TLR1 polymorphism regulates the innate immune response to lipopeptides[J].European Journal Immunology,2007,37(8):2280-2289
[22] Schumann R R,Tapping R I.Genomic variants of TLR1ittakes (TLR-) two to tango[J].European Journal Immunology,2007,37 (8):2059-2062
[23] Moisan J,Camateros P,Thuraisingam T.TLR7 lig-and prevents allergen-induced airway hyperresponsiveness and eosinophilia in allergic asthma by a MYD88-dependent and MK2 -independent pathway [J].Am J Physiol Lung Cell Mol Physiol,2006,290 (5):L987-L995
[24] Higgs R,Cormican P,Cahalane S,Allan B,Lliyd A T,Meade K,James T,Lynn D J,Babiuk L A,Farrelly C O.Induction of a novel chicken toll-like receptor following salmonella enterica serovar typhinurium infection[J].Infection and Immunity,2006,74(3):1692-1698
[25] 储明星,李春苗,石万海,安永福,陈宏权,狄冉,方丽.荷斯坦母牛TLR6基因多态性及其与体细胞评分关系的研究[J].畜牧兽医学报,2009,40(11):1621-1629
[26] 周秀敏,李强子,毕英杰,任卫合,张丽.荷斯坦牛TLR6基因多态性分析[J].中国草食动物科学,2017,37(1):1-3
[27] 农伟伦,毕英杰,李宏旭,张丽.甘肃黑猪H-FABP基因克隆、SNPs筛查及生物信息学分析[J].浙江农业学报,2018,30(3):378-385
[28] 贾浩,张小白,宋晓峰.人类胞内蛋白半衰期与其亚细胞定位的相关性研究[J].计算机与应用化学,2011,28(4):411-414
[29] Arnold J N,Wormald M R,Sim R B.The impact of glycosylation on the biological function and structure of human immunoglobulins [J].Annual Review Immunology,2007,25(2):21-50
[30] Calarese D A,Scanlan C N,Zwick M B L.Antibody domain exchange is an immunological solution to carbohydrate cluster recognition[J].Science,2003,300(5628):2065-2071
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