乌珠穆沁绵羊RIPK2基因多态性与生长性状的关联

doi:10.3864/j.issn.0578-1752.2016.07.015

摘要/Abstract

摘要： 【目的】基于前期绵羊肉用性状GWAS研究结果，旨在探讨RIPK2基因对乌珠穆沁绵羊生长性状的影响,找到该基因中与绵羊生长性状相关的分子标记，同时对GWAS结果进行验证。【方法】在343只乌珠穆沁绵羊试验群体中，选取其中30个个体的血液DNA样本，以相同浓度组成池DNA，设计引物对RIPK2基因外显子及其上下游1 000bp的调控区进行PCR扩增，PCR产物检测为目的条带后测序。使用DNAMAN和Chromas2软件对测序峰图进行分析，采用飞行质谱方法对检测到的SNP位点及前期GWAS得到的SNP位点进行基因型分型。使用Haploview软件对多态位点构建单倍型及连锁不平衡分析。使用SPSS （22.0）软件进行RIPK2基因SNP位点与生长性状间的关联分析。【结果】共发现了4个多态位点，A5536G的同义突变rs01位于第四外显子内，在该位点检测到三种基因型，AA基因型频率为0.42，AG基因型频率为0.45，GG基因型频率为0.13,优势等位基因为A，其频率为0.65；在第七外显子上检测到T8952C错义突变rs02，在该突变位点检测到3种基因型，其中TT基因型频率为0.82，TC基因型频率为0.16，CC基因型频率为0.02，T为优势等位基因，基因频率为0.9；在第十外显子检测到 T2836C的突变rs05，在该位点检测到两种基因型TT和CC，没有检测到杂合子基因型，TT基因型频率为0.25，CC基因型频率为0.75；上游调控区发现一个G5181C的突变rs30，有3种基因型，其中GC基因型频率为0.50，CC基因型频率为0.18，GG基因型频率为0.32，G为优势等位基因，其频率为0.58; GWAS得到的SNP位点是T4456C，在本研究中被命名为rs34，在该位点有3种基因型，其中TT基因型频率为0.49，TC基因型频率为0.37，CC基因型频率为0.14，优势等位基因T的频率为0.68。χ²适合性检验发现，除rs34位点外，其余位点均处于Hardy-Weinberg平衡状态(P＞0.05)。rs02位点处于低度多态(PIC＜0.25)，其余位点处于中度多态(0.25＜PIC＜0.5)。连锁不平衡分析发现，rs01和rs02位点强连锁，在群体中共构建了3种单倍型，AT 为优势单倍型，其频率为0.645，这两个位点共构建了6 种基因型组合，其中AATT为优势基因型组合，频率为0.425。单标记关联分析表明：rs01位点的AA、AG基因型的个体和GG基因型个体在4月龄体重、胸围上差异显著（P＜0.05），在6月龄体高、胸围上差异显著(P＜0.05)。rs02位点CC基因型个体在4月龄体重、体高、胸围和6月龄胸宽上显著优于TT、TC基因型个体（P＜0.05),在4月龄体斜长、6月龄体高和体斜长上极显著优于TT、TC基因型的个体（P＜0.01）。rs30位点上，GG基因型的个体在4月龄体重、胸围与其他两种基因型个差异显著(P＜0.05)，3个基因型的个体在6月龄胸围上均有显著差异（P＜0.05）。rs34位点的3个基因型的个体仅在4月龄体重上差异显著(P＜0.05)。rs01-rs02组合基因型关联分析发现：GGCC基因型组合的个体在4月龄体重、体高、体斜长、胸围上与其他基因型之间差异显著（P＜0.05）,GGCC基因型组合的个体在6月龄体高、体斜长、胸宽上与其他基因型组合的个体差异显著（P＜0.05）。【结论】RIPK2基因对绵羊生长性状具有较显著的影响，其SNPs作为分子标记对乌珠穆沁绵羊生长性状的选育具有一定的指导意义。

关键词: RIPK2基因；生长性状, 多态性；关联分析；乌珠穆沁绵羊

Abstract: 【Objective】Based on the results of the previous genome-wide association on the sheep meat traits, we studied the possibility of the RIPK2 gene to be a novel candidate gene for the sheep growth traits, in order to confirm the GWAS results, as well as to find new molecular markers related to the sheep growth traits.【Method】In the 343 Ujumqin sheep experiment group, 30 DNA samples with a fixed concentration were selected to construct the DNA pool, primers were designed to extend exons and 1 000 bp of upstream and downstream regulate regions. PCR products were tested and the objective strap were sequenced. The sequence peak maps were analyzed by DNAMAN and Chromas2 softwares, Sequenom Mass-array technology was used to genotype the detected SNP including the SNP obtained by previous GWAS. Haploview was used to construct haplotypes and analyze linkage disequilibrium of the polymorphic loci. Association analysis between the SNPs in RIPK2 gene and the growth traits were performed using SPSS 22.0 software. 【Result】 Four SNPs were detected. In exon 4 was a A5536G synonymous mutation rs01, there were three genotypes on this site and the frequencies of AA, AG and GG were 0.42,0.45 and 0.13 individually, the dominant allele was A with a frequency of 0.65. In exon 7 was a T8952C missen variation rs02 with three genotypes. The frequencies of TT, TC and CC genotypes were 0.82,0.16 and 0.02. T was the dominant allele and its frequency was 0.9. Rs05 was a T2836C mutation in exon 10 with only two genotypes TT and CC. The frequency of genotype TT was 0.25 as well as CC was 0.75. In the upstream regions a G5181C variation rs30 was detected with three genotypes GG, GC and CC. The frequency of heterozygous genotype GC was 0.50 and the other two genotypes CC, GG were 0.18 and 0.32 respectively. G was the dominant allele with its frequency was 0.58.The variation T4456C detected by GWAS was rs34 in this study and there were three genotypes. The frequency of TT genotype was 0.49, the frequency of TC was 0.37 and the frequency of CC was 0.14.T was the dominant allele with a frequency of 0.68. The χ² test indicated all the SNPs fit with Hardy-Weinberg equilibrium except rs34. Rs02 was in low polymorphic status (PIC＜0.25) and the other three SNPs were in moderate polymorphic status (0.25＜PIC＜0.5). Linkage disequilibrium analysis suggested that, rs01 and rs02 were strongly linked, the two sites constructed three haplotypes in the experiment population, AT was the dominant haplotype and the frequency was 0.645. Combining the genotypes of the two sites, we found 6 genotype combinations. AATT was the superior genotype and its frequency was 0.425. Single SNP correlation analysis revealed that: on rs01 site, individuals with genotype AA/AG genotype were significantly higher than those with GG genotype on four months body weight, chest girth and six months body height, chest girth (P＜0.05). On rs02 site, individuals with CC genotype were significantly higher on the four months weight, body height, chest girth and six months chest width (P＜0.05) than the individuals with TT and TC genotypes as well as individuals with CC genotype were extremely significantly different from individuals with TT and TC on the four months body length, six months body height and body length (P＜0.01). Individuals with GG genotype on rs30 site showed a significant difference with the individuals of the other two genotypes on the four months weight and chest girth (P＜0.05) and individuals with each genotype had a significant difference on the six months chest girth (P＜0.05). Individuals with each genotype of rs34 had significant difference on the four months weight (P＜0.05). Meanwhile, the combined genotype of rs01-rs02 correlation analysis revealed that: individuals with GGCC combined genotype was significantly different from individuals with the other five genotypes on the four months body weight, body height, body length, chest girth and six months body height, body length and chest width with (P＜0.05).【Conclusion】The results showed that the different SNPs and combined genotypes in RIPK2 gene had effect on Ujumqin sheep different growth traits with the SNPs being potentially valuable as genetic markers for economic traits in Ujumqin sheep.

Key words: RIPK2 gene, growth traits, polymorphisms, association, Ujumqin sheep

马晓萌，轩俊丽，王慧华，袁泽湖，吴明明，朱才业，刘瑞凿，魏彩虹，赵福平，杜立新，张莉. 乌珠穆沁绵羊RIPK2基因多态性与生长性状的关联[J]. 中国农业科学, 2016, 49(7): 1391-1407.

MA Xiao-meng, XUAN Jun-li,WANG Hui-hua,YUAN Ze-hu, WU Ming-ming, ZHU Cai-ye, LIU Rui-zao, WEI Cai-hong, ZHAO Fu-ping, DU Li-xin, ZHANG Li. Association of the RIPK2 Gene Genetic Variation with Ujumqin Sheep Growth Traits[J]. Scientia Agricultura Sinica, 2016, 49(7): 1391-1407.

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参考文献

[1] McCarthy J V, Ni J, Dixit V M.RIP2 is a novel NF-kappaB-activating and cell death-inducing kinase. The Journal of Biological Chemistry, 1998, 273(27): 16968-16975.

[2] Magalhaes J G, Lee J, Geddes K, Rubino S, Philpott D J, Girardin S E.Essential role of Rip2 in the modulation of innate and adaptive immunity triggered by Nod1 and Nod2 ligands. European Journal of Immunology, 2011, 41(5):1445-1455.

[3] Archer K A, Ader F, Kobayashi K S, Flavell R A, Roy C R.Cooperation between multiple microbial pattern recognition systems is important for host protection against the intracellular pathogen Legionella pneumophila. Infection and Immunity, 2010, 78(6):2477-2487.

[4] Aldhous M C, Soo K, Stark L A, Ulanicka A A, Easterbrook J E, Dunlop M G, Satsangi J.Cigarette smoke extract (CSE) delays NOD2 expression and affects NOD2/RIPK2 interactions in intestinal epithelial cells. PLoS One, 2011, 6(9):e24715.

[5] Wang X, Jiang W, Duan N, Qian Y, Zhou Q, Ye P, Jiang H, Bai Y, Zhang W, Wang W.NOD1, RIP2 and Caspase12 are potentially novel biomarkers for oral squamous cell carcinoma development and progression. International Journal of Clinical and Experimental Pathology, 2014, 7(4): 1677-1686.

[6] Taxman D J, Lei Y, Zhang S, Holley-Guthrie E, Offenbacher S, Ting J P.ASC-dependent RIP2 kinase regulates reduced PGE2 production in chronic periodontitis. Journal of Dental Research, 2012, 91(9): 877-882.

[7] Couturier-Maillard A, Secher T, Rehman A, Normand S, De Arcangelis A, Haesler R, Huot L, Grandjean T, Bressenot A, Delanoye-Crespin A, Gaillot O, Schreiber S, Lemoine Y, Ryffel B, Hot D, Nunez G, Chen G, Rosenstiel P, Chamaillard M.NOD2- mediated dysbiosis predisposes mice to transmissible colitis and colorectal cancer. The Journal of Clinical Investigation, 2013, 123(2): 700-711.

[8] Tigno-Aranjuez J T, Benderitter P, Rombouts F, Deroose F, Bai X, Mattioli B, Cominelli F, Pizarro T T, Hoflack J, Abbott D W.In vivo inhibition of RIPK2 kinase alleviates inflammatory disease. The Journal of Biological Chemistry, 2014, 289(43):29651-29664.

[9] Nachbur U, Stafford C A, Bankovacki A, Zhan Y, Lindqvist L M, Fiil B K, Khakham Y, Ko H J, Sandow J J, Falk H, Holien J K.A RIPK2 inhibitor delays NOD signalling events yet prevents inflammatory cytokine production. Nature Communications, 2015(6):6442.

[10] Minchenko D O, Davydov V V, Budreiko O A, Moliavko O S, Kulieshova D K, Tiazhka O V, Minchenko O H.The expression of CCN2, IQSEC, RSPO1, DNAJC15, RIPK2, IL13RA2, IRS1, and IRS2 genes in blood of obese boys with insulin resistance. Fiziolohichnyi Zhurnal, 2015, 61(1):10-18.

[11] Claire D'Andre H, Paul W, Shen X, Jia X, Zhang R, Sun L, Zhang X.Identification and characterization of genes that control fat deposition in chickens. Journal of Animal Science and Biotechnology, 2013, 4(1):43.

[12] Wang X A, Deng S, Jiang D, Zhang R, Zhang S, Zhong J, Yang L, Wang T, Hong S, Guo S, She Z, Zhang X D, Li H.CARD3 deficiency exacerbates diet-induced obesity, hepatosteatosis, and insulin resistance in male mice. Endocrinology, 2013, 154(2):685-697.

[13] Zhang L, Liu J, Zhao F, Ren H, Xu L, Lu J, Zhang S, Zhang X, Wei C, Lu G, Zheng Y, Du L.Genome-wide association studies for growth and meat production traits in sheep. PLoS One, 2013, 8(6):e66569.

[14] 李静. RIP2基因单核苷酸多态性及血清水平与系统性红斑狼疮的相关性研究[D]. 合肥: 安徽医科大学, 2012.

Li J.Association study of RIP2 gene polumorphisms,serum RIP2 level and systemic lupus erthematosus[D]. Hefei:Medical University of Anhui, 2012. (in Chinese)

[15] Thiebaut R, Douchin V, Jung C, Merlin F, Colombel J F, Lemann M, Almer S, Tysk C, O'Morain C, Gassull M, Finkel Y, Zouali H, Pascoe L, Hugot J P.RIP2 polymorphisms in inflammatory bowel diseases. Inflammatory Bowel Diseases, 2011, 17(4):1055.

[16] Nakashima K, Hirota T, Suzuki Y, Akahoshi M, Shimizu M, Jodo A, Doi S, Fujita K, Ebisawa M, Yoshihara S, Enomoto T, Shirakawa T, Kishi F, Nakamura Y, Tamari M.Association of the RIP2 gene with childhood atopic asthma. Allergology International : Official Journal of the Japanese Society of Allergology,2006, 55(1):77-83.

[17] Cavanagh C R, Jonas E, Hobbs M, Thomson P C, Tammen I, Raadsma H W.Mapping Quantitative Trait Loci (QTL) in sheep. III. QTL for carcass composition traits derived from CT scans and aligned with a meta-assembly for sheep and cattle carcass QTL. Genetics, Selection, Evolution, 2010, 42:36.

[18] Suzuki S, Wenyi Z, Hirai M, Hinokio Y, Suzuki C, Yamada T, Yoshizumi S, Suzuki M, Tanizawa Y, Matsutani A, Oka Y.Genetic variations at urotensin II and urotensin II receptor genes and risk of type 2 diabetes mellitus in Japanese. Peptides, 2004, 25(10): 1803-1808.

[19] Zheng Y, Devitt C, Liu J, Mei J, Skapek S X.A distant, cis-acting enhancer drives induction of Arf by Tgfbeta in the developing eye. Developmental Biology, 2013, 380(1):49-57.

[20] Ghiasvand N M, Rudolph D D, Mashayekhi M, Brzezinski J A T, Goldman D, Glaser T.Deletion of a remote enhancer near ATOH7 disrupts retinal neurogenesis, causing NCRNA disease. Nature Neuroscience, 2011, 14(5):578-586.

[21] Prud'homme B, Gompel N, Carroll S B. Emerging principles of regulatory evolution. Proceedings of the National Academy of Sciences of the United States of America, 2007, 104(Suppl 1): 8605-8612.

[22] Komar A A. Silent SNPs: impact on gene function and phenotype. Pharmacogenomics, 2007, 8(8):1075-1080.

[23] Yajima H, Motohashi N, Ono Y, Sato S, Ikeda K, Masuda S, Yada E, Kanesaki H, Miyagoe-Suzuki Y, Takeda S, Kawakami K.Six family genes control the proliferation and differentiation of muscle satellite cells. Experimental Cell Research, 2010, 316(17): 2932- 2944.

[24] Liu Y, Chu A, Chakroun I, Islam U, Blais A.Cooperation between myogenic regulatory factors and SIX family transcription factors is important for myoblast differentiation. Nucleic Acids Research, 2010, 38(20):6857-6871.

[25] 宋桃伟, 蔡惠芬, 罗卫星, 刘若余, 张依裕, 孙岩岩, 刘彬.两个贵州山羊品种GHSR和GHRL基因遗传变异及其与生长性状的关联性. 中国农业科学, 2015, 48(1):140-153.

Song T W, Cai H F, Luo W X, Liu R Y, Zhang Y Y, Sun Y Y, Liu B. Association of GHSR and GHRL gene genetic variation with growth traits in two guizhou goat breeds.Scientia Agricultural Sinica, 2015, 48(1):140-153. (in Chinese)

[26] Chang K C.Critical regulatory domains in intron 2 of a porcine sarcomeric myosin heavy chain gene. Journal of Muscle Research and Cell Motility, 2000, 21(5):451-461.

[27] Beauchamp N J, Daly M E, Makris M, Preston F E, Peake I R.A novel mutation in intron K of the PROS1 gene causes aberrant RNA splicing and is a common cause of protein S deficiency in a UK thrombophilia cohort. Thrombosis and Haemostasis, 1998, 79(6):1086-1091.

[28] Hirschey M D, Shimazu T, Goetzman E, Jing E, Schwer B, Lombard D B, Grueter C A, Harris C, Biddinger S, Ilkayeva O R, Stevens R D, Li Y, Saha A K, Ruderman N B, Bain J R, Newgard C B, Farese R V, Jr., Alt F W, Kahn C R, Verdin E.SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation. Nature, 2010, 464(7285):121-125.

[29] Orozco G, Hinks A, Eyre S, Ke X, Gibbons L J, Bowes J, Flynn E, Martin P, Wilson A G, Bax D E, Morgan A W, Emery P, Steer S, Hocking L, Reid D M, Wordsworth P, Harrison P, Thomson W, Barton A, Worthington J.Combined effects of three independent SNPs greatly increase the risk estimate for RA at 6q23. Human Molecular Genetics, 2009, 18(14):2693-2699.

[30] Horne B D, Camp N J.Principal component analysis for selection of optimal SNP-sets that capture intragenic genetic variation. Genetic Epidemiology, 2004, 26(1):11-21.

[31] 田万强. 牛AMPK家族7个基因SNP检测及其与生长和肉质性状的关联分析[D]. 杨凌：西北农林科技大学, 2013.

Tian W Q. Single nucleotide polymorphism of 7 genes of AMPK family aand their association with growth and meat quality traits in cattle[D]. Yangling: Northwest A & F University, 2013. (in Chinese)

[32] Howard T D, Whittaker P A, Zaiman A L, Koppelman G H, Xu J, Hanley M T, Meyers D A, Postma D S, Bleecker E R.Identification and association of polymorphisms in the interleukin-13 gene with asthma and atopy in a Dutch population. American Journal of Respiratory Cell and Molecular Biology, 2001, 25(3):377-384.