小尾寒羊GnRH基因组织表达、多态性及其与产羔性状关联分析
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摘要
为揭示GnRH基因在小尾寒羊下丘脑-垂体-卵巢轴(Hypothalamic-pituitary-ovarian axis,HPOA)中的表达规律、多态性及其与产羔数的关系,深入了解其对小尾寒羊多羔的作用,采用实时荧光定量PCR技术对6只小尾寒羊(FecB++型单、多羔母羊各3只)的生殖组织及脑组织中GnRH基因的表达谱进行分析,同时采用Sequenom MassARRAY~?SNP技术对380只小尾寒羊和共380只的小尾寒羊、滩羊、苏尼特羊、策勒黑羊、湖羊和草原型藏羊GnRH基因2个单核苷酸多态性位点(Single nucleotide polymorphisms,SNPs)的进行多态性检测,并与小尾寒羊产羔数进行关联分析。结果显示,GnRH基因在下丘脑、卵巢和子宫组织均有表达,其中在下丘脑高表达。在小尾寒羊多羔群体中,GnRH基因在卵巢、大脑、下丘脑、垂体的表达均极显著高于单羔群体(P<0.01),暗示其可能参与了小尾寒羊多羔性状调控。分型发现GnRH基因2个位点基因型频率和等位基因频率在单、多羔品种间差异均不显著(P>0.05)。关联分析表明,GnRH基因的2个SNPs位点的多态性与小尾寒羊各胎产羔数差异均不显著(P>0.05);2个SNPs在所有绵羊品种中均表现为低度多态(PIC<0.25);卡方适合性检验表明,2个SNPs在各个绵羊品种中均处于Hardy-Weinberg平衡状态(P>0.05)。
This study was undertaken to evaluate the expression patterns and polymorphisms of GnRH gene in the hypothalamic-pituitary-ovarian axis(HPOA) and its association with litter size in Small Tail Han sheep.The real-time fluorescence quantitative PCR(RT-qPCR) method was used to detect the expression of the GnRH gene in reproductive tissues and brain tissues of six Small Tail Han sheep(Three polytocous and three monotocous ewes from the FecB++ genotype) in this study.380 Small Tail Han sheep and 380 in total for Small Tail Han sheep,Tan,Sunite,Cele Black,Hu and Prairie Tibetan sheep were selected and Sequenom MassARRAY~? SNP assay was applied to genotype 2 single nucleotide polymorphism sites(SNPs) of GnRH gene.Then the association was analyzed between GnRH and litter size in Small Tail Han sheep.The results showed that GnRH gene was expressed in hypothalamus,ovary and uterus tissues and among which,GnRH was highly expressed in hypothalamus.The expression of GnRH gene in ovary,hypothalamus and uterus of polytocous Small Tail Han ewes was extremely significant higher(P<0.01) than that of monotocous ewes,and the GnRH gene might be involved in the regulation of multiple lambs in Small Tail Han sheep.From genotyping,we found the genotype frequencies and allele frequencies of the 2 SNPs were not significantly different between uniparous and multiparous sheep(P>0.05),association analysis showed that these 2 SNPs in GnRH gene were not significantly correlated with litter size in Small Tail Han sheep(P>0.05).The population genetic analysis indicated that 2 SNPs showed low polymorphism in all sheep breeds(PIC<0.25),and the result of chi-square test showed that 2 SNPs were at Hardy-Weinberg equilibrium in most sheep breeds(P>0.05).
This study was undertaken to evaluate the expression patterns and polymorphisms of GnRH gene in the hypothalamic-pituitary-ovarian axis(HPOA) and its association with litter size in Small Tail Han sheep.The real-time fluorescence quantitative PCR(RT-qPCR) method was used to detect the expression of the GnRH gene in reproductive tissues and brain tissues of six Small Tail Han sheep(Three polytocous and three monotocous ewes from the FecB++ genotype) in this study.380 Small Tail Han sheep and 380 in total for Small Tail Han sheep,Tan,Sunite,Cele Black,Hu and Prairie Tibetan sheep were selected and Sequenom MassARRAY~? SNP assay was applied to genotype 2 single nucleotide polymorphism sites(SNPs) of GnRH gene.Then the association was analyzed between GnRH and litter size in Small Tail Han sheep.The results showed that GnRH gene was expressed in hypothalamus,ovary and uterus tissues and among which,GnRH was highly expressed in hypothalamus.The expression of GnRH gene in ovary,hypothalamus and uterus of polytocous Small Tail Han ewes was extremely significant higher(P<0.01) than that of monotocous ewes,and the GnRH gene might be involved in the regulation of multiple lambs in Small Tail Han sheep.From genotyping,we found the genotype frequencies and allele frequencies of the 2 SNPs were not significantly different between uniparous and multiparous sheep(P>0.05),association analysis showed that these 2 SNPs in GnRH gene were not significantly correlated with litter size in Small Tail Han sheep(P>0.05).The population genetic analysis indicated that 2 SNPs showed low polymorphism in all sheep breeds(PIC<0.25),and the result of chi-square test showed that 2 SNPs were at Hardy-Weinberg equilibrium in most sheep breeds(P>0.05).
引文
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[2] Cao C Y,Ding Y F,Kong X J,Feng G D,Xiang W,Chen L,Yang F,Zhang K,Chu M X,Wang P Q,Zhang B Y.Reproductive role of miRNA in the hypothalamic-pituitary axis[J].Molecular and Cellular Neuroscience,2018(88):130-137
[3] Mijiddorj T,Kanasaki H,Sukhbaatar U,Oride A,Hara T,Kyo S.Mutual regulation by GnRH and kisspeptin of their receptor expression and its impact on the gene expression of gonadotropin subunits[J].General and Comparative Endocrinology,2017(246):382-389
[4] 高广亮,王阳铭.GnRH基因的研究进展[J].畜禽业,2016 (6):48-50Gao G L,Wang Y M.Research progress of GnRH gene[J].Livestock and Poultry Industry,2016 (6):48-50 (in Chinese)
[5] Asa S L,Ezzat S.The pathogenesis of pituitary tumours[J].Nature Reviews Cancer,2002,2(11):836.
[6] 龚建文,黄峰,陶斌.浅析GnRH对提高母猪产仔数及存活数的作用[J].黑龙江动物繁殖,2011,19(3):49Gong J W,Huang F,Tao B.Analysis of the effect of GnRH on increasing litter size and survival number of sows[J].Heilongjiang Journal of Animal Reproduction,2011,19(3):49 (in Chinese)
[7] 丁赫,宫永胜,王军,吕文发.初情期启动过程中小尾寒羊下丘脑GnRH基因甲基化状态及表达量关系研究[J].中国畜牧杂志,2018,54(5):58-61Ding H,Gong Y S,Wang J,Lv W F.Relationship between methylation status and expression of GnRH gene in Small Tail Han sheep during onset of puberty[J].Chinese Journal of Animal Science,2018,54(5):58-61 (in Chinese)
[8] 李志惠,李德.榕江小香羊GnRH基因多态性及其与繁殖性状关联性分析[J].湖北农业科学,2016,55(8):2058-2061Li Z H,Li D.Association analysis on polymorphism of GnRH gene and reproduction traits in Rongjiang Small Xiang goat[J].Hubei Agricultural Sciences,2016,55(8):2058-2061 (in Chinese)
[9] 宋娜娜,钟金城,柴志欣,汪琦,何世明,吴锦波,蹇尚林,冉强,蒙欣,胡红春.三江黄牛全基因组数据分析[J].中国农业科学,2017,50(1):183-194Song N N,Zhong J C,Chai Z X,Wang Q,He S M,Wu J B,Jian S L,Ran Q,Meng X,Hu H C.The whole genome data analysis of Sanjiang cattle[J].Scientia Agricultura Sinica,2017,50(1):183-194 (in Chinese)
[10] 潘章源,贺小云,刘秋月,郭晓飞,曹晓涵,胡文萍,狄冉,王翔宇,储明星.全基因组测序(WGS)在畜禽群体进化和功能基因挖掘中的应用[J].农业生物技术学报,2016,24(12):1945-1954Pan Z Y,He X Y,Liu Q Y,Guo X F,Cao X H,Hu W P,Di R,Wang X Y,Chu M X.Application of whole genome sequencing (WGS) in population evolution and gene discovery of domestic animals[J].Journal of Agricultural Biotechnology,2016,24(12):1945-1954 (in Chinese)
[11] 汪文强,赵生国,马利青,郭继军,马月辉,赵倩君.动物基因组学重测序的应用研究进展[J].畜牧兽医学报,2016,47(10):1947-1953Wang W Q,Zhao S G,Ma L Q,Guo J J,Ma Y H,Zhao Q J.The research progress and application of resequencing based on animal genomics[J].Acta Veterinaria et Zootechnica Sinica,2016,47(10):1947-1953 (in Chinese)
[12] Ai H S,Fang X D,Yang B,Huang Z Y,Chen H,Mao L K,Zhang F,Zhang L L,Cui L L,He W M,Yang J,Yao X M,Zhou L S,Han L J,Li J J,Sun S L,Xie X H,Lai B X,Su Y,Lu Y,Yang H,Huang T,Deng W J,Nielsen R,Ren J,Huang L S.Adaptation and possible ancient interspecies introgression in pigs identified by whole-genome sequencing[J].Nature Genetics,2015,47(3):217-225
[13] 曾滔,赵福平,王光凯,吴明明,魏彩虹,张莉,李利,张红平,杜立新.基于群体分化指数FST的绵羊全基因组选择信号检测[J].畜牧兽医学报,2013,44(12):1891-1899Zeng T,Zhao F P,Wang G K,Wu M M,Wei C H,Zhang L,Li L,Zhang H P,Du L X.Genome-wide detection of selection signatures in sheep populations with use of population differentiation index FST[J].Acta Veterinaria et Zootechnica Sinica,2013,44(12):1891-1899 (in Chinese)
[14] 陈树林,毛海鹏,赵慧英,卿素珠,范光丽,张艳,李耀辉,周建平.青年奶山羊下丘脑GnRH神经元的分布特点[J].西北农林科技大学学报:自然科学版,2007,35(1):45-48+53Chen S L,Mao H P,Zhao H Y,Qing S Z,Fan G L,Zhang Y,Li Y H,Zhou J P.Distribution characteristics of GnRH neurons in hypothalamus of young dairy goats[J].Journal of Northwest A & F University:Natural Science Edition,2007,35(1):45-48+53 (in Chinese)
[15] 陈树林,林磊,蒋田园,雷治海,李文献,曾明华,张艳,范光丽,隋治国.妊娠期奶山羊下丘脑GnRH和OT免疫反应双标记细胞的分布[J].畜牧兽医学报,2007,38(9):966-971Chen S L,Lin L,Jiang T Y,Lei Z H,Li W X,Zeng M H Zhang Y,Fan G L,Sui Z G.Localization of double-labeled immunoreactive cells of gonadotropin releasing hormone and oxytocin in hypothalamus of dairy goat during pregnancy[J].Acta Veterinaria et Zootechnica Sinica,2007,38(9):966-971 (in Chinese)
[16] 宫永胜,苑志宇,张涛,吕文发,王军.初情期启动过程中MeCP2和GnRH在母羊下丘脑的分布定位[J].中国畜牧杂志,2017,53(12):53-55Gong Y S,Fan Z Y,Zhang T,Lv W F,Wang J.Distribution of MeCP2 and GnRH in the hypothalamus of ewes during initiation of puberty[J].Chinese Journal of Animal Science,2017,53(12):53-55 (in Chinese)
[17] 徐元青,王建林,宋国强,王凤玲,邵宝平.GnRH在妊娠期黄牛下丘脑-垂体-卵巢轴中的表达[J].畜牧兽医学报,2010,41(12):1649-1654Xu Y Q,Wang J L,Song G Q,Wang F L,Shao B P.Expression of GnRH in the hypothalamic-pituitary-ovarian axis of the pregnant cattle[J].Acta Veterinaria et Zootechnica Sinica,2010,41(12):1649-1654 (in Chinese)
[18] 徐元青,邵宝平,王建林.促性腺激素释放激素在妊娠牦牛下丘脑主要核团中的表达[J].畜牧与兽医,2016,48(2):82-84Xu Y Q,Shao B P,Wang J L.GnRH expression in hypothalamic nuclei of pregnant yaks[J].Animal Husbandry and Veterinary Medicine,2016,48(2):82-84 (in Chinese)
[19] Voigt C,Bennett N C.GnRH mRNA expression in the brain of cooperatively breeding female Damaraland mole-rats[J].Reproduction,2017,153(4):453-460
[20] 张克山,胡彦竞科,韩笑哲,高广亮,钟航,王启贵.鹅不同繁殖时期GnRH和GnIH基因表达和激素浓度变化分析[J].畜牧兽医学报,2016,47(8):1720-1726Zhang K S,Hu Y J K,Han X Z,Gao G L,Zhong H,Wang Q G.Analysis of the serum concentrations and mRNA expression levels of GnRH and GnIH in geese during different reproductive periods[J].Acta Veterinaria et Zootechnica Sinica,2016,47(8):1720-1726 (in Chinese)
[21] 刘岚,陈燕,李宁,王大伟,任飞,宋英,郭聪,刘晓辉.布氏田鼠GnRH基因克隆及不同组织和发育阶段的基因表达特征[J].四川动物,2017,36(5):498-506Liu L,Chen Y,Li N,Wang D W,Ren F,Song Y,Guo C,Liu X H.Molecular cloning,tissue distribution and expression of GnRH gene in different developmental stages of Lasiopodomys brandtii[J].Sichuan Journal of Zoology,2017,36(5):498-506 (in Chinese)
[22] Sun J R,Hui C C,Xia T J,Xu M,Deng D T,Pan F M,Wang Y M.Effect of hypothyroidism on the hypothalamic-pituitary-ovarian axis and reproductive function of pregnant rats[J].BMC Endocrine Disorders,2018,18(1):30
[2] Cao C Y,Ding Y F,Kong X J,Feng G D,Xiang W,Chen L,Yang F,Zhang K,Chu M X,Wang P Q,Zhang B Y.Reproductive role of miRNA in the hypothalamic-pituitary axis[J].Molecular and Cellular Neuroscience,2018(88):130-137
[3] Mijiddorj T,Kanasaki H,Sukhbaatar U,Oride A,Hara T,Kyo S.Mutual regulation by GnRH and kisspeptin of their receptor expression and its impact on the gene expression of gonadotropin subunits[J].General and Comparative Endocrinology,2017(246):382-389
[4] 高广亮,王阳铭.GnRH基因的研究进展[J].畜禽业,2016 (6):48-50Gao G L,Wang Y M.Research progress of GnRH gene[J].Livestock and Poultry Industry,2016 (6):48-50 (in Chinese)
[5] Asa S L,Ezzat S.The pathogenesis of pituitary tumours[J].Nature Reviews Cancer,2002,2(11):836.
[6] 龚建文,黄峰,陶斌.浅析GnRH对提高母猪产仔数及存活数的作用[J].黑龙江动物繁殖,2011,19(3):49Gong J W,Huang F,Tao B.Analysis of the effect of GnRH on increasing litter size and survival number of sows[J].Heilongjiang Journal of Animal Reproduction,2011,19(3):49 (in Chinese)
[7] 丁赫,宫永胜,王军,吕文发.初情期启动过程中小尾寒羊下丘脑GnRH基因甲基化状态及表达量关系研究[J].中国畜牧杂志,2018,54(5):58-61Ding H,Gong Y S,Wang J,Lv W F.Relationship between methylation status and expression of GnRH gene in Small Tail Han sheep during onset of puberty[J].Chinese Journal of Animal Science,2018,54(5):58-61 (in Chinese)
[8] 李志惠,李德.榕江小香羊GnRH基因多态性及其与繁殖性状关联性分析[J].湖北农业科学,2016,55(8):2058-2061Li Z H,Li D.Association analysis on polymorphism of GnRH gene and reproduction traits in Rongjiang Small Xiang goat[J].Hubei Agricultural Sciences,2016,55(8):2058-2061 (in Chinese)
[9] 宋娜娜,钟金城,柴志欣,汪琦,何世明,吴锦波,蹇尚林,冉强,蒙欣,胡红春.三江黄牛全基因组数据分析[J].中国农业科学,2017,50(1):183-194Song N N,Zhong J C,Chai Z X,Wang Q,He S M,Wu J B,Jian S L,Ran Q,Meng X,Hu H C.The whole genome data analysis of Sanjiang cattle[J].Scientia Agricultura Sinica,2017,50(1):183-194 (in Chinese)
[10] 潘章源,贺小云,刘秋月,郭晓飞,曹晓涵,胡文萍,狄冉,王翔宇,储明星.全基因组测序(WGS)在畜禽群体进化和功能基因挖掘中的应用[J].农业生物技术学报,2016,24(12):1945-1954Pan Z Y,He X Y,Liu Q Y,Guo X F,Cao X H,Hu W P,Di R,Wang X Y,Chu M X.Application of whole genome sequencing (WGS) in population evolution and gene discovery of domestic animals[J].Journal of Agricultural Biotechnology,2016,24(12):1945-1954 (in Chinese)
[11] 汪文强,赵生国,马利青,郭继军,马月辉,赵倩君.动物基因组学重测序的应用研究进展[J].畜牧兽医学报,2016,47(10):1947-1953Wang W Q,Zhao S G,Ma L Q,Guo J J,Ma Y H,Zhao Q J.The research progress and application of resequencing based on animal genomics[J].Acta Veterinaria et Zootechnica Sinica,2016,47(10):1947-1953 (in Chinese)
[12] Ai H S,Fang X D,Yang B,Huang Z Y,Chen H,Mao L K,Zhang F,Zhang L L,Cui L L,He W M,Yang J,Yao X M,Zhou L S,Han L J,Li J J,Sun S L,Xie X H,Lai B X,Su Y,Lu Y,Yang H,Huang T,Deng W J,Nielsen R,Ren J,Huang L S.Adaptation and possible ancient interspecies introgression in pigs identified by whole-genome sequencing[J].Nature Genetics,2015,47(3):217-225
[13] 曾滔,赵福平,王光凯,吴明明,魏彩虹,张莉,李利,张红平,杜立新.基于群体分化指数FST的绵羊全基因组选择信号检测[J].畜牧兽医学报,2013,44(12):1891-1899Zeng T,Zhao F P,Wang G K,Wu M M,Wei C H,Zhang L,Li L,Zhang H P,Du L X.Genome-wide detection of selection signatures in sheep populations with use of population differentiation index FST[J].Acta Veterinaria et Zootechnica Sinica,2013,44(12):1891-1899 (in Chinese)
[14] 陈树林,毛海鹏,赵慧英,卿素珠,范光丽,张艳,李耀辉,周建平.青年奶山羊下丘脑GnRH神经元的分布特点[J].西北农林科技大学学报:自然科学版,2007,35(1):45-48+53Chen S L,Mao H P,Zhao H Y,Qing S Z,Fan G L,Zhang Y,Li Y H,Zhou J P.Distribution characteristics of GnRH neurons in hypothalamus of young dairy goats[J].Journal of Northwest A & F University:Natural Science Edition,2007,35(1):45-48+53 (in Chinese)
[15] 陈树林,林磊,蒋田园,雷治海,李文献,曾明华,张艳,范光丽,隋治国.妊娠期奶山羊下丘脑GnRH和OT免疫反应双标记细胞的分布[J].畜牧兽医学报,2007,38(9):966-971Chen S L,Lin L,Jiang T Y,Lei Z H,Li W X,Zeng M H Zhang Y,Fan G L,Sui Z G.Localization of double-labeled immunoreactive cells of gonadotropin releasing hormone and oxytocin in hypothalamus of dairy goat during pregnancy[J].Acta Veterinaria et Zootechnica Sinica,2007,38(9):966-971 (in Chinese)
[16] 宫永胜,苑志宇,张涛,吕文发,王军.初情期启动过程中MeCP2和GnRH在母羊下丘脑的分布定位[J].中国畜牧杂志,2017,53(12):53-55Gong Y S,Fan Z Y,Zhang T,Lv W F,Wang J.Distribution of MeCP2 and GnRH in the hypothalamus of ewes during initiation of puberty[J].Chinese Journal of Animal Science,2017,53(12):53-55 (in Chinese)
[17] 徐元青,王建林,宋国强,王凤玲,邵宝平.GnRH在妊娠期黄牛下丘脑-垂体-卵巢轴中的表达[J].畜牧兽医学报,2010,41(12):1649-1654Xu Y Q,Wang J L,Song G Q,Wang F L,Shao B P.Expression of GnRH in the hypothalamic-pituitary-ovarian axis of the pregnant cattle[J].Acta Veterinaria et Zootechnica Sinica,2010,41(12):1649-1654 (in Chinese)
[18] 徐元青,邵宝平,王建林.促性腺激素释放激素在妊娠牦牛下丘脑主要核团中的表达[J].畜牧与兽医,2016,48(2):82-84Xu Y Q,Shao B P,Wang J L.GnRH expression in hypothalamic nuclei of pregnant yaks[J].Animal Husbandry and Veterinary Medicine,2016,48(2):82-84 (in Chinese)
[19] Voigt C,Bennett N C.GnRH mRNA expression in the brain of cooperatively breeding female Damaraland mole-rats[J].Reproduction,2017,153(4):453-460
[20] 张克山,胡彦竞科,韩笑哲,高广亮,钟航,王启贵.鹅不同繁殖时期GnRH和GnIH基因表达和激素浓度变化分析[J].畜牧兽医学报,2016,47(8):1720-1726Zhang K S,Hu Y J K,Han X Z,Gao G L,Zhong H,Wang Q G.Analysis of the serum concentrations and mRNA expression levels of GnRH and GnIH in geese during different reproductive periods[J].Acta Veterinaria et Zootechnica Sinica,2016,47(8):1720-1726 (in Chinese)
[21] 刘岚,陈燕,李宁,王大伟,任飞,宋英,郭聪,刘晓辉.布氏田鼠GnRH基因克隆及不同组织和发育阶段的基因表达特征[J].四川动物,2017,36(5):498-506Liu L,Chen Y,Li N,Wang D W,Ren F,Song Y,Guo C,Liu X H.Molecular cloning,tissue distribution and expression of GnRH gene in different developmental stages of Lasiopodomys brandtii[J].Sichuan Journal of Zoology,2017,36(5):498-506 (in Chinese)
[22] Sun J R,Hui C C,Xia T J,Xu M,Deng D T,Pan F M,Wang Y M.Effect of hypothyroidism on the hypothalamic-pituitary-ovarian axis and reproductive function of pregnant rats[J].BMC Endocrine Disorders,2018,18(1):30
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