绵羊PDGF-D基因多态性与尾型的关联

doi:10.11843/j.issn.0366-6964.2019.04.002

摘要/Abstract

摘要：

旨在研究血小板源性生长因子-D（platelet-derived growth factor-D，PDGF-D）与绵羊尾型的关系，寻找可用的分子标记，同时对前期的研究结果进行验证。本研究在533只绵羊（湖羊、藏羊、杜泊×湖羊杂交羊）试验群体中，选取藏羊、湖羊两个品种各30个个体的血液样本，等量混合分别构建两个DNA混池，对PDGF-D基因的外显子及其上下游1 000 bp扩增，目的片段测序分析后，采用飞行质谱方法对检测到的SNP位点进行基因分型，Haploview4.1软件对突变位点进行连锁不平衡分析，使用SPSS19.0软件对PDGF-D基因SNP位点与尾型性状进行关联分析。结果显示，在PDGF-D基因及其上下游1 000 bp共找到6个突变位点，其中rs5、rs27、rs28与尾长、尾宽及尾周长均相关，rs6、rs19仅与尾长相关，rs13与尾宽、尾周长均显著相关（P<0.05）。连锁不平衡分析结果表明，rs6~rs13之间存在强连锁；组合基因型与尾型关联分析结果显示，CCGG型个体无论是在尾长、尾宽还是在尾周长上，其平均值均显著（P<0.05）或者极显著（P<0.01）低于其他组合基因型，但在群体中的数量较少。结果表明，PDGF-D基因对绵羊的尾型有着较为显著的影响，其SNP位点作为分子标记对于绵羊尾型选育工作有着一定的指导意义，可考虑将CCGG型作为筛选小尾绵羊的潜在分子标记，但是该基因型个体在群体中所占的比重较小，可适当增强人工选育以扩大数目。

Abstract:

We studied the possibility of the PDGF-D gene to be a novel candidate gene affecting the sheep tail traits, in order to verify the previous results, as well as to find new molecular markers related to the sheep tail traits. In the 533 sheep experiment group (Hu sheep, Tibetan sheep, Dorper sheep×Hu sheep), 30 DNA samples with a fixed concentration from Hu sheep and Tibetan sheep respectively were selected to construct the DNA pool, primers were designed to amplify exons and 1 000 bp of upstream and downstream regulatory regions. PCR products were tested and the target segments were sequenced. Sequenom Mass-array technology was used to genotype the detected SNPs. Haploview 4.1 was used to construct haplotypes and analyze linkage disequilibrium of the polymorphic loci. Association analysis between the SNPs of PDGF-D gene and the tail traits was performed using SPSS 19.0 software. The results showed that 6 SNPs were detected. rs5, rs27 and rs28 were significantly related to tail length, tail width and tail perimeter(P<0.05). rs6 and rs19 were only significantly involved in tail length(P<0.05). rs13 was significantly related to tail width and tail perimeter(P<0.05). The result of linkage disequilibrium analysis showed that there was close linkage from rs6 to rs13. Meanwhile, the association analysis of combined genotypes of rs6-rs13 with tail traits revealed that individuals with CCGG combined genotype were significantly lower than the individuals with other combined genotypes in tail length, tail width and tail perimeter (P<0.05 or P<0.01), but the number of individuals with CCGG was less in population. The results indicate that PDGF-D gene has significant effect on sheep tail traits, and the SNPs of PDGF-D gene are potentially valuable as genetic markers for breeding. The CCGG genotype can be considered as the potential molecular marker to breed small-tailed sheep. The amount of individuals with CCGG combined genotype should be expanded further.

中图分类号:

S826.2

李青, 王慧华, 卢曾奎, 金美林, 储明星, 魏彩虹. 绵羊PDGF-D基因多态性与尾型的关联[J]. 畜牧兽医学报, 2019, 50(4): 688-700.

LI Qing, WANG Huihua, LU Zengkui, JIN Meilin, CHU Mingxing, WEI Caihong. Association of the PDGF-D Gene Genetic Variation with Sheep Tail Traits[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(4): 688-700.

参考文献

[1] HAMADA T,UI-TEI K,IMAKI J,et al.Molecular cloning of SCDGF-B,a novel growth factor homologous to SCDGF/PDGF-C/fallotein[J].Biochem Biophs Res Commun,2001,280(3):733-737.
[2] LAROCHELLE W J,JEFFERS M,MCDONALD W F,et al.PDGF-D,a new protease-activated growth factor[J].Nat Cell Biol, 2001, 3(5):517-521.
[3] KURASAWA K,ARAI S,OWADA T,et al.Autoantibodies against platelet-derived growth factor receptor alpha in patients with systemic lupus erythematosus[J].Mod Rheumatol,2010,20(5):458-465.
[4] CHEN J H,YUAN W Z,WU L,et al.PDGF-D promotes cell growth,aggressiveness,angiogenesis and EMT transformation of colorectal cancer by activation of Notch1/Twist1 pathway[J].Oncotarget,2017,8(6):9961-9973.
[5] WANG Z W,KONG D J,BANERJEE S,et al.Down-regulation of platelet-derived growth factor-D inhibits cell growth and angiogenesis through inactivation of Notch-1 and nuclear factor-κB signaling[J].Cancer Res,2007,67(23):11377-11385.
[6] UUTELA M,WIRZENIUS M,PAAVONEN K,et al.PDGF-D induces macrophage recruitment,increased interstitial pressure,and blood vessel maturation during angiogenesis[J].Blood,2004,104(10):3198-3204.
[7] AHMAD A,WANG Z W,KONG D J,et al.RETRACTED ARTICLE:Platelet-derived growth factor-D contributes to aggressiveness of breast cancer cells by up-regulating Notch and NF-κB signaling pathways[J].Breast Cancer Res Trea, 2011, 126(1):15-25.
[8] BUHL E M,DJUDJAJ S,BABICKOVA J,et al.The role of PDGF-D in healthy and fibrotic kidneys[J].Kidney Int,2016, 89(4):848-861.
[9] BORKHAM-KAMPHORST E,VAN ROEYEN C R C,OSTENDORF T,et al.Pro-fibrogenic potential of PDGF-D in liver fibrosis[J].J Hepatol,2007,46(6):1064-1074.
[10] ZHAO T Q,ZHAO W Y,CHEN Y J,et al.Platelet-derived growth factor-D promotes fibrogenesis of cardiac fibroblasts[J].Am J Physiol:Heart Circ Physiol,2013,304(12):H1719-H1726.
[11] 张银辉,于晖,宋燕,等.血小板源性生长因子D抗体制备及其在人心脏纤维化组织中的表达[J].中国分子心脏病学杂志,2012,12(6):337-340.
ZHANG Y H,YU H,SONG Y,et al.PDGF-D promoted myocardial fibrosis byupregulation of collagen I[J].Molecular Cardiology of China,2012,12(6):337-340. (in Chinese)
[12] ARTEMENKO Y,GAGNON A,AUBIN D,et al.Anti-adipogenic effect of PDGF is reversed by PKC inhibition[J].J Cell Physiol,2005,204(2):646-653.
[13] HOLMSTRÖM T E,MATTSSON C L,FÄLTING J M,et al.Differential signalling pathways for EGF versus PDGF activation of Erk1/2 MAP kinase and cell proliferation in brown pre-adipocytes[J].Exp Cell Res,2008,314(19):3581-3592.
[14] VIRAKUL S,DALM V A S H,PARIDAENS D,et al.Platelet-derived growth factor-bb enhances adipogenesis in orbital fibroblasts[J].Invest Ophthalmol Vis Sci,2015,56(9):5457-5464.
[15] HYE KIM J,GYU PARK S,KIM W K,et al.Functional regulation of adipose-derived stem cells by PDGF-D[J].Stem Cells, 2015, 33(2):542-556.
[16] 赵辉,王威,张清润,等.高通量飞行时间质谱基因分型方法的研究[J].生物化学与生物物理进展,2005,32(7):667-672.
ZHAO H,WANG W,ZHANG Q R,et al.The study of high throughput MALDI-TOF genotyping assay[J].Progress in Biochemistry and Biophysics,2005,32(7):667-672.(in Chinese)
[17] BAI Y Y,CHEN J Z,SUN K,et al.A functional variant in promoter region of Platelet-derived Growth Factor-D is probably associated with intracerebral hemorrhage[J].J Neuroinflammation,2012,9:26.
[18] 高静,刘相飞,陈玉东.黄三角地区人群中PDGF-D基因多态性与冠心病的相关性[J].中国动脉硬化杂志,2016,24(9):944-948.
GAO J,LIU X F,CHEN Y D.Correlation of PDGF-D gene polymorphism with coronary artery disease in huangsanjiao population[J].Chinese Journal of Arteriosclerosis,2016,24(9):944-948.(in Chinese)
[19] 刘相飞,陈玉东,高静.血小板源性生长因子基因多态性与冠状动脉粥样斑块性质的相关性[J].实用医学杂志,2016,32(5):758-760.
LIU X F,CHEN Y D,GAO J.Correlation of PDGF-D gene polymorphism with coronary atherosclerotic plaque[J].The Journal of Practical Medicine,2016,32(5):758-760.(in Chinese)
[20] 杨志伟,王燕锋,朱大勇,等.血小板源性生长因子-D基因多态性与大肠癌遗传易感性关系的研究[J].医学研究杂志,2014,43(9):60-63.
YANG Z W,WANG Y F,ZHU D Y, et al.Study on Association of platelet-derived growth factor-d gene polymorphism with genetic susceptibility to colorectal cancer[J].Journal of Medical Research,2014,43(9):60-63. (in Chinese)
[21] 焦小丽,景炅婕,乔利英,等.绵羊Lpin2基因多态性及其与尾型和屠宰性状的关联分析[J].中国畜牧兽医,2017,44(4):1037-1045.
JIAO X L,JING J J,QIAO L Y,et al.Polymorphisms of Lpin2 gene and its association with tail type and slaughter traits in sheep[J].China Animal Husbandry & Veterinary Medicine,2017,44(4):1037-1045.(in Chinese)
[22] 高中元,林婄婄,袁亚男,等.PLIN基因多态性及其与绵羊尾形和屠宰性状的关联研究[J].山西农业大学学报:自然科学版,2012,32(2):158-164.
GAO Z Y,LIN P P,YUAN Y N,et al,Study on the polymorphism of PLIN gene and its association with tail and slaughter traits in sheep[J].Journal of Shanxi Agricultural University:Natural Science Edition, 2012,32(2):158-164.(in Chinese)
[23] 张静,景炅婕,张方,等.绵羊ANGPTL4基因的多态性及其与尾型和屠宰性状的关联研究[J].中国畜牧杂志,2016,52(5):12-18.
ZAHNG J,JING J J,ZHANG F,et al.Study on polymorphisms of ovine ANGPTL4 gene and the associations with tail type and slaughter traits[J].Chinese Journal of Animal Science,2016,52(5):12-18.(in Chinese)
[24] ZHU C Y,FAN H Y,YUAN Z H,et al.Genome-wide detection of CNVs in Chinese indigenous sheep with different types of tails using ovine high-density 600K SNP arrays[J].Sci Rep,2016,6:27822.
[25] WEI C H,WANG H H,LIU G,et al.Genome-wide analysis reveals population structure and selection in Chinese indigenous sheep breeds[J]. BMC Genomics,2015,16:194.
[26] 王慧华.中国地方绵羊群体结构分析及基因组选择痕迹挖掘[D].北京:中国农业科学院,2015.
WANG H H,Genome-wide analysis reveals population structure and selection in Chinese indigenous sheep breeds[D]. Beijing:Chinese Academy of Agricultural Sciences,2015. (in Chinese)
[27] 袁泽湖.利用选择信号分析方法筛选与绵羊尾型相关的基因[D].北京:中国农业科学院,2016.
YUAN Z H.Analysis of selection signatures identify genes associated with tail type in sheep[D].Beijing:Chinese Academy of Agricultural Sciences,2016.(in Chinese)
[28] 刘真,王慧华,刘瑞凿,等.不同尾型绵羊全基因组选择信号检测[J].畜牧兽医学报,2015,46(10):1721-1732.
LIU Z,WANG H H,LIU R Z,et al.Genome-wide detection of selection signatures of distinct tail types in sheep populations[J]. Chinese Journal of Animal and Veterinary Sciences,2015,46(10):1721-1732.(in Chinese)
[29] YANG J,LI W R,LV F H,et al.Whole-genome sequencing of native sheep provides insights into rapid adaptations to extreme environments[J].Mol Biol Evol,2016,33(10):2576-2592.
[30] ZHAO Y X,YANG J,LV F H,et al.Genomic reconstruction of the history of native sheep reveals the peopling patterns of nomads and the expansion of early pastoralism in East Asia[J].Mol BiolEvol,2017,34(9):2380-2395.
[31] MASTRANGELO S,MOIOLI B,AHBARA A,et al.Genome-wide scan of fat-tail sheep identifies signals of selection for fat deposition and adaptation[J].Anim Prod Sci,2018,doi:10.1071/AN17753.
[32] SUZUKI S,ZONG W Y,HIRAI M,et al.Genetic variations at urotensin Ⅱ and urotensin Ⅱ receptor genes and risk of type 2 diabetes mellitus in Japanese[J].Peptides,2004,25(10):1803-1808.
[33] 姜运良,李宁,杜立新,等.猪肌肉生长抑制素基因5'调控区T→A突变与生长性状的关系分析[J].遗传学报,2002,29(5):413-416.
JIANG Y L,LI N,DU L X,et al.Relationship of T→A mutation in the promoter region of myostatin gene with growth traits in swine[J].Journal of Genetics and Genomics,2002,29(5):413-416.(in Chinese)
[34] 余勐.新疆维吾尔妇女宫颈癌组织中HPV16上游调控区突变及其与宫颈癌发生关系的研究[D].乌鲁木齐:新疆大学,2006.
YU M.Studies on the relationship between the mutation of HPV16 URR from cervical carcinoma biopsies and the occurrence of cervical cancer in Xinjiang Uygur[D].Urumqi:Xinjiang University,2016.(in Chinese)
[35] 许艳丽.牛FSHβ基因上游调控区连锁突变对其转录的影响分析[D].长春:吉林大学,2011.
XU Y L.Effect of chain mutation of FSHβ gene upstream regulatory region on transcription in bovine[D].Changchun:Jilin University,2011.(in Chinese)
[36] BHATTACHARYYA N,BANERJEE D.Transcriptional regulatory sequences within the first intron of the chicken apolipoproteinAI (apoAI) gene[J].Gene,1999,234(2):371-380.
[37] CLEMENT J Q,WILKINSON M F.Rapid induction of nuclear transcripts and inhibition of intron decay in response to the polymerase Ⅱ inhibitor DRB[J].J Mol Biol,2000,299(5):1179-1191.
[38] KINCH L N,GRISHIN N V.Evolution of protein structures and functions[J].Curr Opin Struct Biol,2002,12(3):400-408.
[39] 王以婷,杨卫红,赵云龙,等.CYP3A4内含子10对CYP3A4基因表达的增强子作用[J].昆明医科大学学报,2017,38(1):13-17.
WANG Y T,YANG W H,ZHAO Y L,et al,The Enhancer role of CYP3A4 intron 10 in CYP3A4 gene expression[J].Journal of Kunming Medical University,2017,38(1):13-17.(in Chinese)
[40] 王金发.人雄激素芳香化酶基因内含子中启动子的研究[J].中山大学学报:自然科学版,1997,36(6):6-10.
WANG J F.The studies of the promoter in the intron Ⅱ of the human aromatase gene[J].Acta Scientiarum Naturalium Universitatis Sunyatseni,1997,36(6):6-10.(in Chinese)
[41] 谢晓玲,马思雨,吴希,等.两种新的F8内含子突变导致剪接异常的机制研究[J].诊断学理论与实践,2018,17(1):32-37.
XIE X L,MA S Y,WU X,et al.Molecular pathogenesis of two novel splice site mutations ofF8 in hemophilia A[J].Journal of Diagnostics Concepts & Practice,2018,17(1):32-37.(in Chinese)
[42] MIKOL D D,RONGNOPARUT P,ALLWARDT B A,et al.The oligodendrocyte-myelin glycoprotein of mouse:primary structure and gene structure[J].Genomics,1993,17(3):604-610.
[43] 胡守旺,梁希若,吴淑华.突变和修饰IFN-α2b基因的5'和3'端对其在E. coli表达的调控作用[J].免疫学杂志,2000,16(5):355-358.
HU S W,LIANG X R,WU S H.Mutagenesis and modifications of IFN-α 2b gene affect the expression level in E. coli[J].Immunological Journal,2000, 16(5):355-358.(in Chinese)
[44] 石统东,吴玉章,朱锡华.真核mRNA 3'非翻译区在基因表达中的作用[J].生物化学与生物物理进展,1998,25(3):195-196,253.
SHI T D,WU Y Z,ZHU X H.3' untranslated region of eukaryotic mRNA in gene regulation[J].Progress in Biochemistry and Biophysics,1998,25(3):195-196,253.(in Chinese)
[45] OROZCO G,HINKS A,EYRE S,et al.Combined effects of three independent SNPs greatly increase the risk estimate for RA at 6q23[J].Hum Mol Genet,2009,18(14):2693-2699.
[46] HORNE B D,CAMP N J.Principal component analysis for selection of optimal SNP-sets that capture intragenic genetic variation[J]. Genet Epidemiol, 2004, 26(1):11-21.