PER1基因组织表达及多态性与绵羊季节性繁殖的相关性研究
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摘要
绵羊(Ovis aries)的季节性繁殖限制了中国绵羊产业的发展,从分子水平上探究影响绵羊季节性繁殖的关键基因具有重要意义。为了探究钟基因1 (period 1 gene, PER1)基因表达水平及其多态性与绵羊季节性繁殖的相关性,本实验通过半定量反转录PCR技术(semi-quantitative reverse transcriptionpolymerase chain reaction, sqRT-PCR)和荧光定量PCR(quantitative real-time PCR, qPCR)技术研究PER1基因在季节性发情的苏尼特羊和常年发情的小尾寒羊不同组织(松果体,下丘脑,垂体,卵巢,输卵管和子宫)中的表达,同时采用Sequenom MassARRAY?SNP技术,对季节性发情绵羊品种(草原型藏羊,苏尼特羊,滩羊)和常年发情绵羊品种(小尾寒羊,湖羊,策勒黑羊)中PER1基因g.27342699G>A和g.27346502A>G的多态性进行检测,并与小尾寒羊产羔数进行关联分析。结果表明,PER1基因在不同发情性状绵羊所选的各个组织中均表达,其表达量在季节性发情的苏尼特羊中均高于常年发情的小尾寒羊,且在松果体、卵巢和输卵管中达到显著差异(P<0.05)。分型结果表明,PER1基因的两个多态位点g.27342699G>A和g.27346502A>G均存在3种基因型,但只有g.27342699G>A位点的等位基因频率在季节性发情和常年发情的绵羊品种中达到显著差异水平(P<0.05)。关联分析结果表明,PER1基因2个SNPs位点多态性与小尾寒羊各胎产羔数无显著关联。从以上结果可以看出,在重要的繁殖相关组织(松果体,卵巢和子宫)中,PER1基因在季节性繁殖绵羊品种中表达量均高于常年发情绵羊品种,初步推测PER1的高表达很可能与季节性发情调控有关,这将为研究季节性发情机制提供理论依据。
In China, seasonal reproduction limits both diversification and intensification of sheep(Ovis aries)production, thus it is necessary to explore the key genes that affect the seasonal reproduction of sheep at the molecular genetic level. In order to explore the association between tissue expression levels and polymorphisms of period 1 gene(PER1) with seasonal reproduction in sheep, semi-quantitative reverse transcription and polymerase chain reaction(sqRT-PCR) and quantitative real-time PCR(qPCR) were performed to investigate the expression of PER1 gene in different tissues including pineal gland,hypothalamus, pituitary, ovary, oviduct and uterus in Small Tail Han sheep and Sunite sheep respectively.Seasonal estrous breeds(Prairie Tibetan sheep, Sunite sheep and Tan sheep) and year-round estrous breeds(Small Tail Han sheep, Hu and Celeblack sheep) were selected, and Sequenom MassARRAY?SNP assay was applied to genotype 2 single nucleotide polymorphism sites(SNPs) of PER1 gene. Gene and genotype frequencies of those 2 sites were analyzed in sheep with different breeding traits. The results showed that PER1 expressed in all tissues selected. Expression levels of PER1 in Sunite sheep was higher than that of Small Tail Han sheep in all tested tissues, and there were significant differences in pineal gland, ovary, and uterus(P<0.05). There were three genotypes in both year-round estrous breeds and seasonal estrous breeds.The allele frequencies of the PER1 g. 27342699 G>A was significantly different between year-round estrous and seasonal estrous sheep(P<0.05). Association analysis indicated that there were no significant correlation between the polymorphisms of the 2 SNPs in PER1 gene and the litter size of each parity in Small Tail Han sheep. In conclusion, the expression of PER1 in Sunite sheep was higher than that of Small Tail Han sheep in the tissues relating with seasonal reproduction, which showed significant differences in pineal, ovary and uterus. Therefore, the higher level of PER1 gene was probably related to the seasonal estrus regulation, which will provide a theoretical basis for studying the seasonal reproduction mechanism of sheep.
In China, seasonal reproduction limits both diversification and intensification of sheep(Ovis aries)production, thus it is necessary to explore the key genes that affect the seasonal reproduction of sheep at the molecular genetic level. In order to explore the association between tissue expression levels and polymorphisms of period 1 gene(PER1) with seasonal reproduction in sheep, semi-quantitative reverse transcription and polymerase chain reaction(sqRT-PCR) and quantitative real-time PCR(qPCR) were performed to investigate the expression of PER1 gene in different tissues including pineal gland,hypothalamus, pituitary, ovary, oviduct and uterus in Small Tail Han sheep and Sunite sheep respectively.Seasonal estrous breeds(Prairie Tibetan sheep, Sunite sheep and Tan sheep) and year-round estrous breeds(Small Tail Han sheep, Hu and Celeblack sheep) were selected, and Sequenom MassARRAY?SNP assay was applied to genotype 2 single nucleotide polymorphism sites(SNPs) of PER1 gene. Gene and genotype frequencies of those 2 sites were analyzed in sheep with different breeding traits. The results showed that PER1 expressed in all tissues selected. Expression levels of PER1 in Sunite sheep was higher than that of Small Tail Han sheep in all tested tissues, and there were significant differences in pineal gland, ovary, and uterus(P<0.05). There were three genotypes in both year-round estrous breeds and seasonal estrous breeds.The allele frequencies of the PER1 g. 27342699 G>A was significantly different between year-round estrous and seasonal estrous sheep(P<0.05). Association analysis indicated that there were no significant correlation between the polymorphisms of the 2 SNPs in PER1 gene and the litter size of each parity in Small Tail Han sheep. In conclusion, the expression of PER1 in Sunite sheep was higher than that of Small Tail Han sheep in the tissues relating with seasonal reproduction, which showed significant differences in pineal, ovary and uterus. Therefore, the higher level of PER1 gene was probably related to the seasonal estrus regulation, which will provide a theoretical basis for studying the seasonal reproduction mechanism of sheep.
引文
黄冬维,储明星.2011.动物季节性繁殖分子调控机理研究进展[J].遗传,33(7):35-46.(Huang D W,Chu M X.2011.Research progress in molecular mechanism of animal seasonal reproduction[J].Hereditas,33(7):35-46.)
李瑞文,成姝婷,王正荣.2013.生物钟基因与哺乳动物生殖[J].生物学杂志,30(4):67-70.(Li R W,Cheng S T,Wang Z R.2013.Clock gene and mammal reproduction[J].Journal of Biology,30(4):67-70.)
刘玉凤.2015.我国羊肉供需及市场均衡模拟研究[D].硕士学位论文,导师:王明利,中国农业科学院.pp.9-10.(Liu Y F.2015.Study on the supply,demand and market equilibrium simulation of mutton in China[D].Thesis for M.S.,Supervisor:Wang M L,Chinese Academy of Agricultural Sciences.pp.9-10.)
魏志杰,曹斌云.2013.我国肉羊产业发展动态及主推技术[C]//2013中国羊业进展,115-121.(Wei Z J,Cao B Y.2013.Development trend and main technology of mutton sheep industry in China[C]//Progress of Sheep Industry in China in 2013,115-121.
周梅,曹晓涵,贺小云,等.2018a.绵羊FSTL1基因组织表达及其多态性与产羔数之间的关系[J].农业生物技术学报,26(05):801-810.(Zhou M,Cao X H,He X Y,et al.2018.Tissue expression and polymorphism of sheep(ovis aries)FSTL1 gene and their association with litter size[J].Journal of Agricultural Biotechnology,26(05):801-810.)
周梅,曹晓涵,贺小云,等.2018b.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,49(3):525-533.(Zhou M,Cao X H,He X Y,et al.2018.Expression and polymorphism of sheep FGF7 gene and their association with litter size[J].Chinese Journal of Animal and Veterinary Sciences,49(3):525-533.)
Albrecht U,Sun Z S,Eichele G,et al.1997.A differential response of two putative mammalian circadian regulators,mper1 and mper2,to light[J].Cell,91(7):1055-1064.
Andersson H,Johnston J D,Messager S,et al.2005.Photoperiod regulates clock gene rhythms in the ovine liver[J].General and Comparative Endocrinology,142(3):357-363.
Blaustein J D,Tetel M J,Ricciardi K H,et al.1994.Hypothalamic ovarian steroid hormone-sensitive neurons involved in female sexual behavior[J].Psychoneuroendocrinology,19(5-7):505-516.
Cao X,Wang X,Lu L,et al.2018.Expression and functional analysis of the BCL2-associated agonist of cell death(BAD)gene in the sheep ovary during the reproductive cycle[J].Front Endocrinol(Lausanne),9:1-13.
Chappell P E,White R S,Mellon P L.2003.Circadian gene expression regulates pulsatile gonadotropin-releasing hormone(GnRH)secretory patterns in the hypothalamic GnRH-secreting GT1-7 cell line[J].Journal Of Neuroscience,23(35):11202-11213.
Dardente H,Fustin J M,Hazlerigg D G.2009.Transcriptional feedback loops in the ovine circadian clock[J].Comparative Biochemistry And Physiology A-Molecular&Integrative Physiology,153(4):391-398.
Guo X,Wang X,Di R,et al.2018.Metabolic effects of FecBgene on follicular fluid and ovarian vein serum in sheep(Ovis aries)[J].International Journal of Molecular Sciences,19(2):1-14.
Hugues D,Wyse C A,Lincoln G A,et al.2016.Effects of photoperiod extension on clock gene and neuropeptide RNA expression in the SCN of the Soay Sheep[J].PLoSOne,11(7):4-5.
Johnson M H,Lim A,Fernando D,et al.2002.Circadian clockwork genes are expressed in the reproductive tract and conceptus of the early pregnant mouse[J].Reproductive Biomedicine Online,4(2):140-145.
Kennaway D J,Varcoe T J,Mau V J.2003.Rhythmic expression of clock and clock-controlled genes in the rat oviduct[J].Molecular Human Reproduction,9(9):503-507.
Konopka R J,Benzer S.1971.Clock mutants of Drosophila melanogaster[J].Proceedings of The National Academy of Sciences of the USA,68(9):2112-2116.
Lamont E W,James F O,Boivin D B,et al.2007.From circadian clock gene expression to pathologies[J].Sleep Medicine,8(6):547-556.
Miyake S,Sumi Y,Yan L,et al.2000.Phase-dependent responses of PER1 and PER2 genes to a light-stimulus in the suprachiasmatic nucleus of the rat[J].Neuroscience Letters,294(1):41-44.
Morgan P J,Ross A W,Graham E S,et al.2010.OPer1 is an early response gene under photoperiodic regulation in the ovine pars tuberalis[J].Journal of Neuroendocrinology,10(5):319-323.
Nakamura T J,Moriya T,Inoue S,et al.2005.Estrogen differentially regulates expression of Per1 and Per2 genes between central and peripheral clocks and between reproductive and nonreproductive tissues in female rats[J].Journal of Neuroscience Research,2(5):622-630.
Pan Z,Li S,Liu Q,et al.2018.Whole-genome sequences of89 Chinese sheep suggest role of RXFP2 in the development of unique horn phenotype as response to semi-feralization[J].GigaScience,7(4):1-15.
Pilorz V,Helfrich-F?rster C,Oster H.2018.The role of the circadian clock system in physiology[J].Pflügers Archiv-European Journal of Physiology,470(2):227-239.
Przekop F,Domański.1980.Abnormalities in the seasonal course of oestrous cycles in ewes after lesions of the suprachiasmatic area of the hypothalamus[J].Journal of Endocrinology,85(3):481-486.
Raghunand T R.1996.Hickory dickory dock[J].Resonance,1(4):76-81.
Ratajczak C K,Herzog E D,Muglia L J.2010.Clock gene expression in gravid uterus and extra-embryonic tissues during late gestation in the mouse[J].Reproduction,Fertility and Development,22(5):743-750.
Tang J,Hu W,Di R,et al.2018.Expression analysis of the prolific candidate genes,BMPR1B,BMP15,and GDF9in Small Tail Han ewes with three fecundity(FecB gene)genotypes[J].Animals,8(10):1-14.
Wharfe M D,Mark P J,Waddell B J.2011.Circadian variation in placental and hepatic clock genes in rat pregnancy[J].Endocrinology,152(9):3552-3560.
Wiggins G,Legge M.2016.Cyclic variation of cellular clock proteins in the mouse estrous ovary[J].Journal of Reproduction&Infertility,17(4):192-198.
Wood S H,Christian H C,Miedzinska K,et al.2015.Binary switching of calendar cells in the pituitary defines the phase of the circannual cycle in mammals[J].Current Biology,25(20):2651-2662.
Yang H,Lin S,Lei X,et al.2016.Identification and profiling of microRNAs from ovary of estrous Kazakh sheep induced by nutritional status in the anestrous season[J].Animal Reproduction Science,175:18-26.
Zhai M,Xie Y,Liang H,et al.2018.Comparative profiling of differentially expressed micro RNAs in estrous ovaries of Kazakh sheep in different seasons[J].Gene,664:181-191.
Zhang X L,Yan H L,Wang K,et al.2018.Goat CTNNB1:mRNA expression profile of alternative splicing in testis and association analysis with litter size[J].Gene,679:297-304.
Zheng Y T,Liu C,Li Y,et al.2019.Loss-of-function mutations with circadian rhythm regulator PER1/PER2 lead to premature ovarian insufficiency[J].Biology of Reproduction,100(4):1066-1072.
李瑞文,成姝婷,王正荣.2013.生物钟基因与哺乳动物生殖[J].生物学杂志,30(4):67-70.(Li R W,Cheng S T,Wang Z R.2013.Clock gene and mammal reproduction[J].Journal of Biology,30(4):67-70.)
刘玉凤.2015.我国羊肉供需及市场均衡模拟研究[D].硕士学位论文,导师:王明利,中国农业科学院.pp.9-10.(Liu Y F.2015.Study on the supply,demand and market equilibrium simulation of mutton in China[D].Thesis for M.S.,Supervisor:Wang M L,Chinese Academy of Agricultural Sciences.pp.9-10.)
魏志杰,曹斌云.2013.我国肉羊产业发展动态及主推技术[C]//2013中国羊业进展,115-121.(Wei Z J,Cao B Y.2013.Development trend and main technology of mutton sheep industry in China[C]//Progress of Sheep Industry in China in 2013,115-121.
周梅,曹晓涵,贺小云,等.2018a.绵羊FSTL1基因组织表达及其多态性与产羔数之间的关系[J].农业生物技术学报,26(05):801-810.(Zhou M,Cao X H,He X Y,et al.2018.Tissue expression and polymorphism of sheep(ovis aries)FSTL1 gene and their association with litter size[J].Journal of Agricultural Biotechnology,26(05):801-810.)
周梅,曹晓涵,贺小云,等.2018b.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,49(3):525-533.(Zhou M,Cao X H,He X Y,et al.2018.Expression and polymorphism of sheep FGF7 gene and their association with litter size[J].Chinese Journal of Animal and Veterinary Sciences,49(3):525-533.)
Albrecht U,Sun Z S,Eichele G,et al.1997.A differential response of two putative mammalian circadian regulators,mper1 and mper2,to light[J].Cell,91(7):1055-1064.
Andersson H,Johnston J D,Messager S,et al.2005.Photoperiod regulates clock gene rhythms in the ovine liver[J].General and Comparative Endocrinology,142(3):357-363.
Blaustein J D,Tetel M J,Ricciardi K H,et al.1994.Hypothalamic ovarian steroid hormone-sensitive neurons involved in female sexual behavior[J].Psychoneuroendocrinology,19(5-7):505-516.
Cao X,Wang X,Lu L,et al.2018.Expression and functional analysis of the BCL2-associated agonist of cell death(BAD)gene in the sheep ovary during the reproductive cycle[J].Front Endocrinol(Lausanne),9:1-13.
Chappell P E,White R S,Mellon P L.2003.Circadian gene expression regulates pulsatile gonadotropin-releasing hormone(GnRH)secretory patterns in the hypothalamic GnRH-secreting GT1-7 cell line[J].Journal Of Neuroscience,23(35):11202-11213.
Dardente H,Fustin J M,Hazlerigg D G.2009.Transcriptional feedback loops in the ovine circadian clock[J].Comparative Biochemistry And Physiology A-Molecular&Integrative Physiology,153(4):391-398.
Guo X,Wang X,Di R,et al.2018.Metabolic effects of FecBgene on follicular fluid and ovarian vein serum in sheep(Ovis aries)[J].International Journal of Molecular Sciences,19(2):1-14.
Hugues D,Wyse C A,Lincoln G A,et al.2016.Effects of photoperiod extension on clock gene and neuropeptide RNA expression in the SCN of the Soay Sheep[J].PLoSOne,11(7):4-5.
Johnson M H,Lim A,Fernando D,et al.2002.Circadian clockwork genes are expressed in the reproductive tract and conceptus of the early pregnant mouse[J].Reproductive Biomedicine Online,4(2):140-145.
Kennaway D J,Varcoe T J,Mau V J.2003.Rhythmic expression of clock and clock-controlled genes in the rat oviduct[J].Molecular Human Reproduction,9(9):503-507.
Konopka R J,Benzer S.1971.Clock mutants of Drosophila melanogaster[J].Proceedings of The National Academy of Sciences of the USA,68(9):2112-2116.
Lamont E W,James F O,Boivin D B,et al.2007.From circadian clock gene expression to pathologies[J].Sleep Medicine,8(6):547-556.
Miyake S,Sumi Y,Yan L,et al.2000.Phase-dependent responses of PER1 and PER2 genes to a light-stimulus in the suprachiasmatic nucleus of the rat[J].Neuroscience Letters,294(1):41-44.
Morgan P J,Ross A W,Graham E S,et al.2010.OPer1 is an early response gene under photoperiodic regulation in the ovine pars tuberalis[J].Journal of Neuroendocrinology,10(5):319-323.
Nakamura T J,Moriya T,Inoue S,et al.2005.Estrogen differentially regulates expression of Per1 and Per2 genes between central and peripheral clocks and between reproductive and nonreproductive tissues in female rats[J].Journal of Neuroscience Research,2(5):622-630.
Pan Z,Li S,Liu Q,et al.2018.Whole-genome sequences of89 Chinese sheep suggest role of RXFP2 in the development of unique horn phenotype as response to semi-feralization[J].GigaScience,7(4):1-15.
Pilorz V,Helfrich-F?rster C,Oster H.2018.The role of the circadian clock system in physiology[J].Pflügers Archiv-European Journal of Physiology,470(2):227-239.
Przekop F,Domański.1980.Abnormalities in the seasonal course of oestrous cycles in ewes after lesions of the suprachiasmatic area of the hypothalamus[J].Journal of Endocrinology,85(3):481-486.
Raghunand T R.1996.Hickory dickory dock[J].Resonance,1(4):76-81.
Ratajczak C K,Herzog E D,Muglia L J.2010.Clock gene expression in gravid uterus and extra-embryonic tissues during late gestation in the mouse[J].Reproduction,Fertility and Development,22(5):743-750.
Tang J,Hu W,Di R,et al.2018.Expression analysis of the prolific candidate genes,BMPR1B,BMP15,and GDF9in Small Tail Han ewes with three fecundity(FecB gene)genotypes[J].Animals,8(10):1-14.
Wharfe M D,Mark P J,Waddell B J.2011.Circadian variation in placental and hepatic clock genes in rat pregnancy[J].Endocrinology,152(9):3552-3560.
Wiggins G,Legge M.2016.Cyclic variation of cellular clock proteins in the mouse estrous ovary[J].Journal of Reproduction&Infertility,17(4):192-198.
Wood S H,Christian H C,Miedzinska K,et al.2015.Binary switching of calendar cells in the pituitary defines the phase of the circannual cycle in mammals[J].Current Biology,25(20):2651-2662.
Yang H,Lin S,Lei X,et al.2016.Identification and profiling of microRNAs from ovary of estrous Kazakh sheep induced by nutritional status in the anestrous season[J].Animal Reproduction Science,175:18-26.
Zhai M,Xie Y,Liang H,et al.2018.Comparative profiling of differentially expressed micro RNAs in estrous ovaries of Kazakh sheep in different seasons[J].Gene,664:181-191.
Zhang X L,Yan H L,Wang K,et al.2018.Goat CTNNB1:mRNA expression profile of alternative splicing in testis and association analysis with litter size[J].Gene,679:297-304.
Zheng Y T,Liu C,Li Y,et al.2019.Loss-of-function mutations with circadian rhythm regulator PER1/PER2 lead to premature ovarian insufficiency[J].Biology of Reproduction,100(4):1066-1072.
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