小尾寒羊LHR基因组织表达、多态性及其与产羔性状的关联分析
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摘要
为了揭示LHR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产羔性状的作用。本研究采用实时荧光定量PCR技术对6只小尾寒羊(FecB++型单、多羔母羊各3只)的生殖及脑组织中LHR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380只其他品种绵羊(小尾寒羊、滩羊、苏尼特羊、策勒黑羊、湖羊和草原型藏羊)LHR基因7个单核苷酸多态性位点(single nucleotide polymorphisms, SNPs)的多态性进行检测,并与小尾寒羊产羔数进行关联分析。结果显示,LHR基因在小尾寒羊大脑、下丘脑和卵巢中均有表达,其中在卵巢高表达。LHR基因在小尾寒羊多羔群体卵巢、大脑和下丘脑的表达均极显著高于单羔群体(P<0.01)。分型发现LHR基因中,4个SNPs位点的等位基因频率和基因型频率在多羔和单羔品种间差异均达到极显著水平(P<0.01);7个SNPs在大多数绵羊品种中均表现为中度多态(0.250.05);关联分析表明,LHR基因有1个SNP多态性与小尾寒羊各胎产羔数显著相关(P<0.05),2个SNPs多态性与小尾寒羊各胎产羔数呈极显著相关(P<0.01)。本研究发现,LHR基因的3个SNPs位点的多态性与小尾寒羊产羔性状存在一定程度相关,暗示其可能参与小尾寒羊多羔性状调控。
This study was conducted to evaluate the expression patterns and polymorphisms of LHR gene in the hypothalamic-pituitary-ovarian axis(HPOA) and its association with litter size in Small Tail Han sheep, and further understand its effect on lambing traits of Small Tail Han sheep. The real-time fluorescence quantitative PCR(qPCR) method was used to detect the expression of the LHR gene in reproductive and brain tissues of 6 Small Tail Han sheep(3 polytocous and 3 monotocous ewes from the FecB ++ genotype) in this study. The 380 Small Tail Han sheep and total 380 sheep for Small Tail Han sheep, Tan, Sunite, Cele Black, Hu and Prairie Tibetan sheep were selected, and Sequenom MassARRAY~?SNP assay was applied to detect the polymorphism of 7 single nucleotide polymorphism sites(SNPs) of LHR gene. Then the association between LHR and litter size was analyzed in Small Tail Han sheep. The results showed that LHR gene was expressed in the brain, hypothalamus and ovary tissues, and highly expressed in ovary. The expression of LHR gene in ovary, brain and hypothalamus of polytocous Small Tail Han ewes was extremely significant higher than that of monotocous ewes(P<0.01). The genotype frequencies and allele frequencies of the 4 SNPs were extremely significantly different between monotocous and polytocous sheep breeds(P<0.01). Population genetic analysis indicated that 7 SNPs showed moderate polymorphism in most sheep breeds(0.250.05). Association analysis showed that the polymorphism of one SNP in the LHR gene were significantly correlated with litter size in Small Tail Han sheep(P<0.05), and the polymorphism of 2 SNPs were extremely significantly correlated with litter size in Small Tail Han sheep(P<0.01). The preliminary results of this study showed that there was a correlation between the polymorphisms of 3 SNPs of LHR gene and litter size in Small Tail Han sheep, and the LHR gene may be involved in the regulation of prolificacy in Small Tail Han sheep.
This study was conducted to evaluate the expression patterns and polymorphisms of LHR gene in the hypothalamic-pituitary-ovarian axis(HPOA) and its association with litter size in Small Tail Han sheep, and further understand its effect on lambing traits of Small Tail Han sheep. The real-time fluorescence quantitative PCR(qPCR) method was used to detect the expression of the LHR gene in reproductive and brain tissues of 6 Small Tail Han sheep(3 polytocous and 3 monotocous ewes from the FecB ++ genotype) in this study. The 380 Small Tail Han sheep and total 380 sheep for Small Tail Han sheep, Tan, Sunite, Cele Black, Hu and Prairie Tibetan sheep were selected, and Sequenom MassARRAY~?SNP assay was applied to detect the polymorphism of 7 single nucleotide polymorphism sites(SNPs) of LHR gene. Then the association between LHR and litter size was analyzed in Small Tail Han sheep. The results showed that LHR gene was expressed in the brain, hypothalamus and ovary tissues, and highly expressed in ovary. The expression of LHR gene in ovary, brain and hypothalamus of polytocous Small Tail Han ewes was extremely significant higher than that of monotocous ewes(P<0.01). The genotype frequencies and allele frequencies of the 4 SNPs were extremely significantly different between monotocous and polytocous sheep breeds(P<0.01). Population genetic analysis indicated that 7 SNPs showed moderate polymorphism in most sheep breeds(0.25
引文
[1] 程美玲,赵素梅,黄英,等.云岭黑山羊FSH基因表达量与其产羔数相关性研究[J].云南农业大学学报,2010,25(6):807-810.CHENG M L,ZHAO S M,HUANG Y,et al.Study on correlation between the expression level of FSH gene and lambing number in Yunling black goat[J].Journal of Yunnan Agricultural University,2010,25(6):807-810.(in Chinese)
[2] 顾祝荣,郭海燕,王强,等.湖羊GDF9基因多态性和产羔性能的关系[J].安徽农业科学,2018,46(6):87-89,185.GU Z R,GUO H Y,WANG Q,et al.The relationship between GDF9 gene polymorphism and lambing performance in the Hu sheep[J].Journal of Anhui Agricultural Sciences,2018,46(6):87-89,185.(in Chinese)
[3] 张军霞,乐祥鹏,王维民,等.绵羊KITLG基因的表达及其多态性与产羔性状关联分析[J].农业生物技术学报,2017,25(6):893-900.ZHANG J X,LE X P,WANG W M,et al.Expression and polymorphisms of KITLG gene and their association with litter size in sheep (Ovis aries)[J].Journal of Agricultural Biotechnology,2017,25(6):893-900.(in Chinese)
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[6] PATEL H,BHARTIYA D,PARTE S,et al.Follicle stimulating hormone modulates ovarian stem cells through alternately spliced receptor variant FSH-R3[J].J Ovarian Res,2013,6:52.
[7] ZHANG R,ZHANG S Z,ZHU X,et al.Follicle-stimulating hormone receptor (FSHR) in Chinese alligator,Alligator sinensis:Molecular characterization,tissue distribution and mRNA expression changes during the female reproductive cycle[J].Anim Reprod Sci,2015,156:40-50.
[8] 胡威,崔燕,潘阳阳,等.黄体生成素受体基因在牦牛发情周期不同阶段生殖系统中的表达模式[J].生物技术通报,2015,31(9):232-237.HU W,CUI Y,PAN Y Y,et al.Analysis of expression pattern of LHR gene in the reproductive system of yak during the estrous cycle[J].Biotechnology Bulletin,2015,31(9):232-237.(in Chinese)
[9] WOHLRES-VIANA S,ARASHIRO E K N,MACHADO M A,et al.Intrafollicular oestradiol production,expression of the LH receptor (LHR) gene and its isoforms,and early follicular deviation in Bos indicus[J].Reprod Fertil Dev, 2017,29(10):1958-1970.
[10] 狄冉,冯涛,储明星,等.山羊促黄体素受体基因(LHR)外显子11的PCR-SSCP分析[J].农业生物技术学报,2009,17(4):614-620.DI R,FENG T,CHU M X,et al.PCR-SSCP analysis on exon 11 of luteinizing hormone receptor (LHR) gene in goats[J].Journal of Agricultural Biotechnology,2009,17(4):614-620.(in Chinese)
[11] 吴井生,张跟喜,王金玉.小梅山猪LHR基因第11外显子PCR-RFLP多态性及其与产仔数的关联分析[J].中国兽医学报,2012,32(8):1239-1243.WU J S,ZHANG G X,WANG J Y.Polymorphism of PCR-RFLP in exon 11 of LHR gene and its relationship with litter size in Xiaomeishan pigs[J].Chinese Journal of Veterinary Science,2012,32(8):1239-1243.(in Chinese)
[12] 宋娜娜,钟金城,柴志欣,等.三江黄牛全基因组数据分析[J].中国农业科学,2017,50(1):183-194.SONG N N,ZHONG J C,CHAI Z X,et al.The whole genome data analysis of Sanjiang cattle[J].Scientia Agricultura Sinica,2017,50(1):183-194.(in Chinese)
[13] 潘章源,贺小云,刘秋月,等.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):1945-1954.PAN Z Y,HE X Y,LIU Q Y,et al.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)
[14] 汪文强,赵生国,马利青,等.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):1947-1953.WANG W Q,ZHAO S G,MA L Q,et al.The research progress and application of resequencing based on animal genomics[J].Acta Veterinaria et Zootechnica Sinica,2016,47(10):1947-1953.(in Chinese)
[15] AI H S,FANG X D,YANG B,et al.Adaptation and possible ancient interspecies introgression in pigs identified by whole-genome sequencing[J].Nat Genet,2015,47(3):217-225.
[16] 曾滔,赵福平,王光凯,等.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):1891-1899.ZENG T,ZHAO F P,WANG G K,et al.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)
[17] 何晶,耿仁德,计红,等.海兰褐蛋鸡卵巢与输卵管FSHR及LHR基因定量的研究[J].黑龙江八一农垦大学学报,2015,27(2):27-31,45.HE J,GENG R D,JI H,et al.Genetic quantitative research of FSHR and LHR in ovary and oviduct of Hy-line brown laying hens[J].Journal of Heilongjiang Bayi Agricultural University,2015,27(2):27-31,45.(in Chinese)
[18] THEMMEN A P N,HUHTANIEMI I T.Mutations of gonadotropins and gonadotropin receptors:elucidating the physiology and pathophysiology of pituitary-gonadal function[J]. Endocr Rev,2000,21(5):551-583.
[19] PAKARAINEN T, ZHANG F P, NURMIL,et al.Knockout of luteinizing hormone receptor abolishes the effects of follicle-stimulating hormone on preovulatory maturation and ovulation of mouse Graafian follicles[J]. Mol Endocrinol,2005,19(10):2591-2602.
[20] DE LOS REYES M,PALOMINO J,PARRAGUEZ V H,et al.Analysis of LH receptor in canine ovarian follicles throughout the estrous cycle[J].Theriogenology,2017,93:71-77.
[21] WEI S,SHEN X,GONG Z,et al.FSHR and LHR expression and signaling as well as maturation and apoptosis of cumulus-oocyte complexes following treatment with FSH receptor binding inhibitor in sheep[J].Cell Physiol Biochem,2017,43(2):660-669.
[22] CHEN H,CUI Y,YU S.Expression and localization of FSHR,GHR and LHR in different tissues and reproductive organs of female yaks[J].Folia Morphol,2018,77(2):301-309.
[23] HUO S D,CHEN S E,LONG R J,et al.Protein and mRNA expression of follicle-stimulating hormone receptor and luteinizing hormone receptor during the oestrus in the yak (Bos grunniens)[J].Reprod Domest Anim,2017,52(3):477-482.
[24] 胡威,陈虹,潘阳阳,等.LHR在牦牛肾和卵巢中表达情况的研究[J].中国兽医科学,2015,45(2):212-216.HU W,CHEN H,PAN Y Y,et al.Study on expression of luteinizing hormone receptor in the kidney and ovary of yak[J].Chinese Veterinary Science,2015,45(2):212-216.(in Chinese)
[25] 王利红,张伟,高勤学,等.湖羊促黄体素受体基因(LHR)外显子11多态性与繁殖性能相关性研究[J].安徽农业大学学报,2011,38(5):675-679.WANG L H,ZHANG W,GAO Q X,et al.The polymorphisms on exon 11 of luteinizing hormone receptor (LHR) gene and their correlation to production performance in Hu sheep[J].Journal of Anhui Agricultural University,2011,38(5):675-679.(in Chinese)
[26] 张善文.猪LHR和PGR基因的遗传变异及其与繁殖性状的相关性研究[D].长沙:湖南农业大学,2012.ZHANG S W.Genetic variation of porcine LHR and PGR gene and their association with reproductive traits[D].Changsha:Hunan Agricultural University,2012.(in Chinese)
[27] KATHIRAVAN R S,CHITRA R,MURALI N,et al.Monomorphism of luteinizing hormone receptor (LHR) gene in buffaloes by restriction fragment length polymorphism[J].J Entomol Zool Stud, 2018, 6(2):2010-2012.
[2] 顾祝荣,郭海燕,王强,等.湖羊GDF9基因多态性和产羔性能的关系[J].安徽农业科学,2018,46(6):87-89,185.GU Z R,GUO H Y,WANG Q,et al.The relationship between GDF9 gene polymorphism and lambing performance in the Hu sheep[J].Journal of Anhui Agricultural Sciences,2018,46(6):87-89,185.(in Chinese)
[3] 张军霞,乐祥鹏,王维民,等.绵羊KITLG基因的表达及其多态性与产羔性状关联分析[J].农业生物技术学报,2017,25(6):893-900.ZHANG J X,LE X P,WANG W M,et al.Expression and polymorphisms of KITLG gene and their association with litter size in sheep (Ovis aries)[J].Journal of Agricultural Biotechnology,2017,25(6):893-900.(in Chinese)
[4] MARSHALL J C,KELCH R P.Gonadotropin-releasing hormone:role of pulsatile secretion in the regulation of reproduction[J].N Engl J Med,1986,315(23):1459-1468.
[5] 孙明亮,马金成.FSHR与LHR的研究进展[J].黑龙江动物繁殖,2008,16(4):1-3.SUN M L,MA J C.Research progress of FSHR and LHR[J].Heilongjiang Journal of Animal Reproduction,2008,16(4):1-3.(in Chinese)
[6] PATEL H,BHARTIYA D,PARTE S,et al.Follicle stimulating hormone modulates ovarian stem cells through alternately spliced receptor variant FSH-R3[J].J Ovarian Res,2013,6:52.
[7] ZHANG R,ZHANG S Z,ZHU X,et al.Follicle-stimulating hormone receptor (FSHR) in Chinese alligator,Alligator sinensis:Molecular characterization,tissue distribution and mRNA expression changes during the female reproductive cycle[J].Anim Reprod Sci,2015,156:40-50.
[8] 胡威,崔燕,潘阳阳,等.黄体生成素受体基因在牦牛发情周期不同阶段生殖系统中的表达模式[J].生物技术通报,2015,31(9):232-237.HU W,CUI Y,PAN Y Y,et al.Analysis of expression pattern of LHR gene in the reproductive system of yak during the estrous cycle[J].Biotechnology Bulletin,2015,31(9):232-237.(in Chinese)
[9] WOHLRES-VIANA S,ARASHIRO E K N,MACHADO M A,et al.Intrafollicular oestradiol production,expression of the LH receptor (LHR) gene and its isoforms,and early follicular deviation in Bos indicus[J].Reprod Fertil Dev, 2017,29(10):1958-1970.
[10] 狄冉,冯涛,储明星,等.山羊促黄体素受体基因(LHR)外显子11的PCR-SSCP分析[J].农业生物技术学报,2009,17(4):614-620.DI R,FENG T,CHU M X,et al.PCR-SSCP analysis on exon 11 of luteinizing hormone receptor (LHR) gene in goats[J].Journal of Agricultural Biotechnology,2009,17(4):614-620.(in Chinese)
[11] 吴井生,张跟喜,王金玉.小梅山猪LHR基因第11外显子PCR-RFLP多态性及其与产仔数的关联分析[J].中国兽医学报,2012,32(8):1239-1243.WU J S,ZHANG G X,WANG J Y.Polymorphism of PCR-RFLP in exon 11 of LHR gene and its relationship with litter size in Xiaomeishan pigs[J].Chinese Journal of Veterinary Science,2012,32(8):1239-1243.(in Chinese)
[12] 宋娜娜,钟金城,柴志欣,等.三江黄牛全基因组数据分析[J].中国农业科学,2017,50(1):183-194.SONG N N,ZHONG J C,CHAI Z X,et al.The whole genome data analysis of Sanjiang cattle[J].Scientia Agricultura Sinica,2017,50(1):183-194.(in Chinese)
[13] 潘章源,贺小云,刘秋月,等.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):1945-1954.PAN Z Y,HE X Y,LIU Q Y,et al.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)
[14] 汪文强,赵生国,马利青,等.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):1947-1953.WANG W Q,ZHAO S G,MA L Q,et al.The research progress and application of resequencing based on animal genomics[J].Acta Veterinaria et Zootechnica Sinica,2016,47(10):1947-1953.(in Chinese)
[15] AI H S,FANG X D,YANG B,et al.Adaptation and possible ancient interspecies introgression in pigs identified by whole-genome sequencing[J].Nat Genet,2015,47(3):217-225.
[16] 曾滔,赵福平,王光凯,等.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):1891-1899.ZENG T,ZHAO F P,WANG G K,et al.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)
[17] 何晶,耿仁德,计红,等.海兰褐蛋鸡卵巢与输卵管FSHR及LHR基因定量的研究[J].黑龙江八一农垦大学学报,2015,27(2):27-31,45.HE J,GENG R D,JI H,et al.Genetic quantitative research of FSHR and LHR in ovary and oviduct of Hy-line brown laying hens[J].Journal of Heilongjiang Bayi Agricultural University,2015,27(2):27-31,45.(in Chinese)
[18] THEMMEN A P N,HUHTANIEMI I T.Mutations of gonadotropins and gonadotropin receptors:elucidating the physiology and pathophysiology of pituitary-gonadal function[J]. Endocr Rev,2000,21(5):551-583.
[19] PAKARAINEN T, ZHANG F P, NURMIL,et al.Knockout of luteinizing hormone receptor abolishes the effects of follicle-stimulating hormone on preovulatory maturation and ovulation of mouse Graafian follicles[J]. Mol Endocrinol,2005,19(10):2591-2602.
[20] DE LOS REYES M,PALOMINO J,PARRAGUEZ V H,et al.Analysis of LH receptor in canine ovarian follicles throughout the estrous cycle[J].Theriogenology,2017,93:71-77.
[21] WEI S,SHEN X,GONG Z,et al.FSHR and LHR expression and signaling as well as maturation and apoptosis of cumulus-oocyte complexes following treatment with FSH receptor binding inhibitor in sheep[J].Cell Physiol Biochem,2017,43(2):660-669.
[22] CHEN H,CUI Y,YU S.Expression and localization of FSHR,GHR and LHR in different tissues and reproductive organs of female yaks[J].Folia Morphol,2018,77(2):301-309.
[23] HUO S D,CHEN S E,LONG R J,et al.Protein and mRNA expression of follicle-stimulating hormone receptor and luteinizing hormone receptor during the oestrus in the yak (Bos grunniens)[J].Reprod Domest Anim,2017,52(3):477-482.
[24] 胡威,陈虹,潘阳阳,等.LHR在牦牛肾和卵巢中表达情况的研究[J].中国兽医科学,2015,45(2):212-216.HU W,CHEN H,PAN Y Y,et al.Study on expression of luteinizing hormone receptor in the kidney and ovary of yak[J].Chinese Veterinary Science,2015,45(2):212-216.(in Chinese)
[25] 王利红,张伟,高勤学,等.湖羊促黄体素受体基因(LHR)外显子11多态性与繁殖性能相关性研究[J].安徽农业大学学报,2011,38(5):675-679.WANG L H,ZHANG W,GAO Q X,et al.The polymorphisms on exon 11 of luteinizing hormone receptor (LHR) gene and their correlation to production performance in Hu sheep[J].Journal of Anhui Agricultural University,2011,38(5):675-679.(in Chinese)
[26] 张善文.猪LHR和PGR基因的遗传变异及其与繁殖性状的相关性研究[D].长沙:湖南农业大学,2012.ZHANG S W.Genetic variation of porcine LHR and PGR gene and their association with reproductive traits[D].Changsha:Hunan Agricultural University,2012.(in Chinese)
[27] KATHIRAVAN R S,CHITRA R,MURALI N,et al.Monomorphism of luteinizing hormone receptor (LHR) gene in buffaloes by restriction fragment length polymorphism[J].J Entomol Zool Stud, 2018, 6(2):2010-2012.