绵羊KISS1基因组织表达及其多态性与产羔数关联分析
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摘要
为研究KISS1基因在常年发情的小尾寒羊和季节性发情的草地型藏羊中的表达模式,以及KISS1基因多态性与绵羊繁殖之间的关系,实验采用qPCR技术对比分析KISS1基因在2个品种绵羊的10种繁殖相关组织中的表达差异,同时利用Sequenom MassARRAY~?SNP技术对常年发情绵羊(小尾寒羊、策勒黑羊和湖羊)和季节性发情绵羊(滩羊、苏尼特羊和草地型藏羊)KISS1基因2个SNPs位点多态性进行检测,并与小尾寒羊产羔数进行关联分析。qPCR结果显示:KISS1基因在小尾寒羊下丘脑、大脑、垂体和甲状腺中的表达量高于草原型藏羊(P<0.05);分型结果表明,g.1317523C>T位点基因型频率和等位基因频率在常年发情和季节性发情绵羊品种间差异均达到极显著水平(P<0.01);群体遗传学分析表明,g.1317523C>T位点在6个绵羊群体中均表现为中度多态(0.25T位点在小尾寒羊、滩羊、苏尼特羊和湖羊中均处于哈代温伯格平衡状态(P>0.05),g.1311578G>T位点在苏尼特羊和湖羊中处于哈代温伯格平衡状态(P>0.05);关联分析表明,这2个SNPs位点与小尾寒羊前3胎产羔数均无显著关联(P>0.05),但g.1311578G>T位点TT型各胎产羔数均大于GT和GG型。综上,KISS1基因与绵羊的季节性繁殖密切相关,并且g.1311578G>T位点对绵羊产羔性状有潜在调控作用。
In order to study the expression pattern of KISS1 gene in year-round estrous Small Tail Han sheep and seasonal estrous Prairie Tibetan sheep, and explore the relationship between the polymorphism and breeding in sheep. QPCR was performed to investigate the expression of KISS1 gene in 10 reproductive tissues at different breeds of sheep. Year-round estrous(Small Tail Han sheep, Cele black sheep and Hu sheep) and seasonal estrous(Tan sheep, Sunite sheep and Prairie Tibetan sheep) sheep breeds were selected and Sequenom MassARRAY~?SNP assay was applied to genotype two loci of KISS1 gene. Then the association was analyzed between KISS1 and litter size in Small Tail Han sheep. The results of gene expression showed that the KISS1 gene was higher in hypothalamus, brain, pituitary and thyroid of Small Tailed Han sheep than that in Prairie Tibetan sheep(P<0.05). From the result of genotyping, both genotype frequency and allele frequency were extremely different between year-round estrous and seasonal estrous sheep(P<0.01) at g.1317523C>T locus. Population genetic analysis showed that the g.1317523C>T locus had moderate polymorphism(0.25T locus of Small Tail Han sheep, Tan sheep, Sunite sheep and Hu Sheep was under Hardy-Weinberg equilibrium(P>0.05), and the g.1311578G>T locus of Sunite sheep and Hu sheep was under HardyWeinberg equilibrium(P>0.05). Association analysis indicated that the polymorphism of two SNPs had no correlation with the litter size in Small Tail Han sheep(P>0.05), while the litter size of TT was higher than GT and GG in the first three births at g.1317523C>T locus. Therefore, KISS1 gene is closely related to the seasonal reproduction of sheep, and the g.1311578G>T locus might provide a basis for litter size trait selection in sheep.
In order to study the expression pattern of KISS1 gene in year-round estrous Small Tail Han sheep and seasonal estrous Prairie Tibetan sheep, and explore the relationship between the polymorphism and breeding in sheep. QPCR was performed to investigate the expression of KISS1 gene in 10 reproductive tissues at different breeds of sheep. Year-round estrous(Small Tail Han sheep, Cele black sheep and Hu sheep) and seasonal estrous(Tan sheep, Sunite sheep and Prairie Tibetan sheep) sheep breeds were selected and Sequenom MassARRAY~?SNP assay was applied to genotype two loci of KISS1 gene. Then the association was analyzed between KISS1 and litter size in Small Tail Han sheep. The results of gene expression showed that the KISS1 gene was higher in hypothalamus, brain, pituitary and thyroid of Small Tailed Han sheep than that in Prairie Tibetan sheep(P<0.05). From the result of genotyping, both genotype frequency and allele frequency were extremely different between year-round estrous and seasonal estrous sheep(P<0.01) at g.1317523C>T locus. Population genetic analysis showed that the g.1317523C>T locus had moderate polymorphism(0.25
引文
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[16]黄冬维,储明星,狄冉,等.山羊繁殖季节性相关基因KiSS-1与RFRP的表达研究[J].畜牧兽医学报,2014,46(6):924-931.
[17]张伟,刘晓娜,邓双义,等.阿勒泰羊不同繁殖状态下丘脑Kiss1基因表达规律与其季节性繁殖的关联性分析[J].基因组学与应用生物学,2016,35(1):45-52.
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[19]甄婗,吕拥芬,李嫔.TTF1在雌鼠下丘脑的分布及其与KiSS1和GnRH表达的关系[J].上海交通大学学报(医学版),2018,38(6):598-604.
[20]郑杰,蒲思颖,刘芳田,等.山羊Kiss-1和GPR54基因的克隆及组织表达研究[J].畜牧与兽医,2017,49(8):10-15.
[21]刘靓,王婷,郑荣秀.中枢性性早熟与KISS1、MKRN3系统的关系研究进展[J].天津医科大学学报,2018,24(3):274-277.
[22]吴井生,王金玉,郭苹,等.猪KISS-1基因多态性分析[J].黑龙江畜牧兽医,2016(3):97-99.
[23]金慧慧.转猪FSHR和KISS1基因小鼠繁殖性能分析[D].重庆:西南大学,2015.
[24]An X P,Han P,Hou J X,et al.Molecular cloning and characterization of KISS1 promoter and effect of KISS1 gene mutations on litter size in the goat[J].Genet Mol Res,2013,12(4):4308-4316.
[25]Maitra A,Sharma R,Ahlawat S,et al.Association analysis of polymorphisms in caprine KiSS1 gene with reproductive traits[J].Anim Reprod Sci,2014,151(1-2):71-77.
[26]陈祥,李鹏程,孙振梅,等.黔北麻羊KISS1基因多态性及组织差异表达研究[J].农业生物技术学报,2018,26(7):1213-1218.
[2]West A,Vojta P J,Welch D R,et al.Chromosome localization and genomic structure of the KiSS-1 metastasis suppressor gene(KISS1)[J].Genomics,1998,54(1):145-148.
[3]Ohtaki T,Shintani Y,Honda S,et al.Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor[J].Nature,2001,411(6837):613-617.
[4]Papaoiconomou E,Msaouel P,Makri A,et al.The role of kisspeptin/GPR54 in the reproductive system[J].Vivo,2011,25(3):343-354.
[5]Caraty A,Smith J T,Lomet D,et al.Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes[J].Endocrinology,2007,148(11):5258.
[6]Tovar S,Vázquez M J,Navarro V M,et al.Effects of single or repeated intravenous administration of kisspeptin upon dynamic LH secretion in conscious male rats[J].Endocrinology,2006,147(6):2696.
[7]周梅,曹晓涵,贺小云,等.绵羊FGF7基因组织表达及其多态性与产羔数之间的关系[J].畜牧兽医学报,2018,49(3):525-533.
[8]李晓雨,贺小云,刘秋月,等.TAC1和PRLR基因在绵羊不同繁殖状态下的表达模式分析[J].畜牧兽医学报,2018,49(2):253-262.
[9]马晓萌,轩俊丽,王慧华,等.TXNRD1基因多态性与乌珠穆沁绵羊生长性状的关联分析[J].畜牧兽医学报,2016,47(5):909-921.
[10]Livak K J,Schmittgen T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCTmethod[J].Methods,2001,25(4):402-408.
[11]Schmittgen T D,Livak K J.Analyzing real-time PCR data by the comparative CT method[J].Nat Protoc,2008,3(6):1101-1108.
[12]田志龙,刘秋月,王翔宇,等.绵羊BMP15基因多态性及其与产羔数的关联分析[J].中国畜牧杂志,2018,54(7):23-26.
[13]Clarke I J,Caraty A.Kisspeptin and seasonality of reproduction[J].Adv Exp Med Biol,2013,784:411-430.
[14]Muir A I,Chamberlain L,Elshourbagy N A,et al.AXOR12,a novel human G protein-coupled receptor,activated by the peptide KiSS-1[J].J Biol Chem,2001,276(31):28969.
[15]臧猛,晁利刚,张志强,等.猪KISS-1基因克隆、序列分析[J].江西农业学报,2008,20(11):5-7.
[16]黄冬维,储明星,狄冉,等.山羊繁殖季节性相关基因KiSS-1与RFRP的表达研究[J].畜牧兽医学报,2014,46(6):924-931.
[17]张伟,刘晓娜,邓双义,等.阿勒泰羊不同繁殖状态下丘脑Kiss1基因表达规律与其季节性繁殖的关联性分析[J].基因组学与应用生物学,2016,35(1):45-52.
[18]王军,孙蕾,吕文发,等.KiSS-1/GPR54系统在不同发情周期母羊下丘脑中的表达规律[J].西北农林科技大学学报:自然科学版,2011,39(12):49-52.
[19]甄婗,吕拥芬,李嫔.TTF1在雌鼠下丘脑的分布及其与KiSS1和GnRH表达的关系[J].上海交通大学学报(医学版),2018,38(6):598-604.
[20]郑杰,蒲思颖,刘芳田,等.山羊Kiss-1和GPR54基因的克隆及组织表达研究[J].畜牧与兽医,2017,49(8):10-15.
[21]刘靓,王婷,郑荣秀.中枢性性早熟与KISS1、MKRN3系统的关系研究进展[J].天津医科大学学报,2018,24(3):274-277.
[22]吴井生,王金玉,郭苹,等.猪KISS-1基因多态性分析[J].黑龙江畜牧兽医,2016(3):97-99.
[23]金慧慧.转猪FSHR和KISS1基因小鼠繁殖性能分析[D].重庆:西南大学,2015.
[24]An X P,Han P,Hou J X,et al.Molecular cloning and characterization of KISS1 promoter and effect of KISS1 gene mutations on litter size in the goat[J].Genet Mol Res,2013,12(4):4308-4316.
[25]Maitra A,Sharma R,Ahlawat S,et al.Association analysis of polymorphisms in caprine KiSS1 gene with reproductive traits[J].Anim Reprod Sci,2014,151(1-2):71-77.
[26]陈祥,李鹏程,孙振梅,等.黔北麻羊KISS1基因多态性及组织差异表达研究[J].农业生物技术学报,2018,26(7):1213-1218.
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