多浪羊Lin28A基因克隆及其在初情期启动过程中的表达研究
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摘要
旨在克隆绵羊Lin28A基因cDNA序列,探讨其在初情期启动过程中的表达规律和调控特性。本研究分别在初情期前(第1次发情前1周)、初情期、初情期后(第1次发情后1周)屠宰多浪羊各5只。采集下丘脑、垂体、卵巢组织,对其Lin28A基因cDNA进行克隆,利用DNAman 6.0软件对多浪羊Lin28A氨基酸与其他物种的Lin28A氨基酸序列进行同源性比较。采用Real-time PCR技术分析下丘脑、垂体、卵巢中Lin28A基因、let-7a、let-7b在初情期启动过程中表达的变化规律。结果表明,多浪羊Lin28AcDNA序列为3 581bp,包括2 963bp的3′UTR和618bp CDS;多浪羊Lin28A氨基酸与人、家鼠、牛和绵羊的同源性分别是87.56%、88.52%、92.68%和89.95%,与鸡、斑马鱼的同源性分别是76.10%、61.84%。在初情期前至初情期的过程中,下丘脑、卵巢中Lin28A表达显著降低(P<0.05),let-7a、let-7b的表达没有显著变化(P>0.05)。本研究结果对于揭示Lin28A基因在绵羊初情期启动中的作用及机制提供了科学依据。
The objectives of the present study were to clone the Lin28AcDNA of sheep,investigate its expression patterns and regulation properties during the onset of puberty in sheep.In this study,5Duolang sheep were slaughtered in the prepuberty(1week before the first estrus),puberty and postpuberty(1week after the first estrus),respectively,hypothalamus,pituitary and ovarian tissues were collected.The Lin28A cDNA of these tissues was cloned,the homology of the amino acids of Lin28Abetween Duolang sheep and other species were compared by DNAman6.0.The expression patterns of Lin28A,let-7aand let-7bin hypothalamus,pituitary and ovary during the puberal transition in Duolang sheep were detected by real-time PCR.The results showed that the fragment of Lin28AcDNA was 3 581bp which contained 2 963bp of 3′UTR and 618 bp of the coding region.The deduced protein sequence of the Lin28A coding region shared87.56%,88.52%,92.68%,89.95%,76.10% and 61.84% to that of human,house mouse,cattle,sheep,original chicken and zebrafish.The expression of Lin28A was significantly decreased in the hypothalamus and ovary from prepuberty to puberty (P<0.05),the expression levels of let-7aand let-7b did not change significantly(P>0.05).The results provided a foundation for revealing the function of Lin28A gene and its role in the onset of puberty in sheep.
The objectives of the present study were to clone the Lin28AcDNA of sheep,investigate its expression patterns and regulation properties during the onset of puberty in sheep.In this study,5Duolang sheep were slaughtered in the prepuberty(1week before the first estrus),puberty and postpuberty(1week after the first estrus),respectively,hypothalamus,pituitary and ovarian tissues were collected.The Lin28A cDNA of these tissues was cloned,the homology of the amino acids of Lin28Abetween Duolang sheep and other species were compared by DNAman6.0.The expression patterns of Lin28A,let-7aand let-7bin hypothalamus,pituitary and ovary during the puberal transition in Duolang sheep were detected by real-time PCR.The results showed that the fragment of Lin28AcDNA was 3 581bp which contained 2 963bp of 3′UTR and 618 bp of the coding region.The deduced protein sequence of the Lin28A coding region shared87.56%,88.52%,92.68%,89.95%,76.10% and 61.84% to that of human,house mouse,cattle,sheep,original chicken and zebrafish.The expression of Lin28A was significantly decreased in the hypothalamus and ovary from prepuberty to puberty (P<0.05),the expression levels of let-7aand let-7b did not change significantly(P>0.05).The results provided a foundation for revealing the function of Lin28A gene and its role in the onset of puberty in sheep.
引文
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[5]SHYH-CHANG N,DALEY G Q.Lin28:Primal regulator of growth and metabolism in stem cells[J].Cell Stem Cell,2013,12(4):395-406.
[6]THORNTON J E,GREGORY R I.How does Lin28let-7control development and disease?[J].Trends Cell Biol,2012,22(9):474-482.
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[10]SANGIAO-ALVARELLOS S,MANFREDI-LOZA-NO M,RUIZ-PINO F,et al.Testicular expression of the Lin28/let-7 system:Hormonal regulation and changes during postnatal maturation and after manipulations of puberty[J].Sci Rep,2015,5:15683.
[11]陈超,陈菊祥.Lin28基因功能的研究进展[J].第二军医大学学报,2011,32(2):206-208.CHEN C,CHEN J X.Function of Lin28:An advance[J].Academic Journal of Second Military Medical University,2011,32(2):206-208.(in Chinese)
[12]刘雪荣,田文洪,董小岩,等.过表达Lin28a/Lin28b对let-7家族活性的影响[J].病毒学报,2011,27(6):533-541.LIU X R,TIAN W H,DONG X Y,et al.Effects of Lin28aand Lin28bon let-7family activity[J].Chinese Journal of Virology,2011,27(6):533-541.(in Chinese)
[13]GAYTAN F,SANGIAO-ALVARELLOS S,MAN-FREDI-LOZANO M,et al.Distinct expression patterns predict differential roles of the miRNA-binding proteins,Lin28and Lin28b,in the mouse testis:Studies during postnatal development and in a model of hypogonadotropic hypogonadism[J].Endocrinology,2013,154(3):1321-1336.
[14]MOSS E G,LEE R C,AMBROS V.The cold shock domain protein LIN-28controls developmental timing in C.elegans and is regulated by the lin-4RNA[J].Cell,1997,88(5):637-646.
[15]ZHU H,SHAH S,SHYH-CHANG N,et al.Lin28a transgenic mice manifest size and puberty phenotypes identified in human genetic association studies[J].Nat Genet,2010,42(7):626-630.
[16]SANGIAO-ALVARELLOS S,MANFREDI-LOZA-NO M,RUIZ-PINO F,et al.Changes in hypothalamic expression of the Lin28/let-7system and related microRNAs during postnatal maturation and after experimental manipulations of puberty[J].Endocrinology,2013,154(2):942-955.
[17]GRIECO A,RZECZKOWSKA P,ALM C,et al.Investigation of peripubertal expression of Lin28aand Lin28bin C57BL/6female mice[J].Mol Cell Endocrinol,2013,365(2):241-248.
[18]侯文通.中国西北重要地方畜禽遗传资源[M].北京:中国农业出版社,2010.HOU W T.Important genetic resources of the native domestic animals in northwestern China[M].Beijing:China Agriculture Press,2010.(in Chinese)
[19]卢萍平,邢凤,刘博丹,等.Lin28B基因表达与绵羊初情期启动的关系研究[J].家畜生态学报,2014,35(9):14-17.LIU P P,XING F,LIU B D,et al.Lin28B gene expression and its role in the onset of puberty of sheep[J].Acta Ecologiae Animalis Domastici,2014,35(9):14-17.(in Chinese)
[20]DANTAS A,SIQUEIRA E R,FERNANDES S,et al.Influence of feeding differentiation on the age at onset of puberty in Brazilian Bergamasca dairy ewe lambs[J].Arq Bras Med Vet Zootec,2016,68(1):22-28.
[21]CAO G L,FENG T,CHU M X,et al.Subtraction suppressive hybridisation analysis of differentially expressed genes associated with puberty in the goat hypothalamus[J].Reprod Fertil Dev,2015,28(11):1781-1787,doi:10.1071/RD14434.
[22]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the method[J].Methods,2001,25(4):402-408.
[23]HEO I,JOO C,CHO J,et al.Lin28mediates the terminal uridylation of Let-7precursor microRNA[J].Mol Cell,2008,32(2):276-284.
[24]VISWANATHAN S R,DALEY G Q,GREGORY RI.Selective blockade of microRNA processing by Lin28[J].Science,2008,320(5872):97-100.
[25]OJEDA S R,DUBAY C,LOMNICZI A,et al.Gene networks and the neuroendocrine regulation of puberty[J].Mol Cell Endocrinol,2010,324(1-2):3-11.
[26]RYBAK A,FUCHS H,SMIRNOVA L,et al.A feedback loop comprising lin-28and let-7controls pre-let-7maturation during neural stem-cell commitment[J].Nat Cell Biol,2008,10(8):987-993.
[27]TSIALIKAS J,ROMER-SEIBERT J.LIN28:Roles and regulation in development and beyond[J].Development,2015,142(14):2397-2404.
[2]曹贵玲.SSH筛选济宁青山羊性早熟相关基因及KiSS-1、GPR54、Lin28B基因的研究[D].北京:中国农业科学院,2011.CAO G L.Screening of sexual precocity related genes in Jining grey goat using SSH and study on KiSS-1,GPR54and Lin28Bgenes[D].Beijing:Chinese Academy of Agricultural Sciences,2011.(in Chinese)
[3]KALTIALA-HEINO R,KOSUNEN E,RIMPELM.Pubertal timing,sexual behaviour and self-reported depression in middle adolescence[J].J Adolesc,2003,26(5):531-545.
[4]GOLUB M S,COLLMAN G W,FOSTER P M D,et al.Public health implications of altered puberty timing[J].Pediatrics,2008,121(S3):S218-S230.
[5]SHYH-CHANG N,DALEY G Q.Lin28:Primal regulator of growth and metabolism in stem cells[J].Cell Stem Cell,2013,12(4):395-406.
[6]THORNTON J E,GREGORY R I.How does Lin28let-7control development and disease?[J].Trends Cell Biol,2012,22(9):474-482.
[7]GUO Y Q,CHEN Y X,ITO H,et al.Identification and characterization of lin-28homolog B(LIN28B)in human hepatocellular carcinoma[J].Gene,2006,384:51-61.
[8]韩威,苏一军,朱云芬,等.文昌鸡性成熟启动前后下丘脑c-Myc/LIN28B/let-7a通路表达规律分析[J].畜牧兽医学报,2016,47(4):709-715.HAN W,SU Y J,ZHU Y F,et al.Analysis on changes of hypothalamic c-Myc/LIN28B/let-7apathway transcription during puberty onset in Wenchang chicken[J].Acta Veterinaria et Zootechnica Sinica,2016,47(4):709-715.(in Chinese)
[9]YOKOYAMA S,HASHIMOTO M,SHIMIZU H,et al.Dynamic gene expression of Lin-28during embryonic development in mouse and chicken[J].Gene Expr Patterns,2008,8(3):155-160.
[10]SANGIAO-ALVARELLOS S,MANFREDI-LOZA-NO M,RUIZ-PINO F,et al.Testicular expression of the Lin28/let-7 system:Hormonal regulation and changes during postnatal maturation and after manipulations of puberty[J].Sci Rep,2015,5:15683.
[11]陈超,陈菊祥.Lin28基因功能的研究进展[J].第二军医大学学报,2011,32(2):206-208.CHEN C,CHEN J X.Function of Lin28:An advance[J].Academic Journal of Second Military Medical University,2011,32(2):206-208.(in Chinese)
[12]刘雪荣,田文洪,董小岩,等.过表达Lin28a/Lin28b对let-7家族活性的影响[J].病毒学报,2011,27(6):533-541.LIU X R,TIAN W H,DONG X Y,et al.Effects of Lin28aand Lin28bon let-7family activity[J].Chinese Journal of Virology,2011,27(6):533-541.(in Chinese)
[13]GAYTAN F,SANGIAO-ALVARELLOS S,MAN-FREDI-LOZANO M,et al.Distinct expression patterns predict differential roles of the miRNA-binding proteins,Lin28and Lin28b,in the mouse testis:Studies during postnatal development and in a model of hypogonadotropic hypogonadism[J].Endocrinology,2013,154(3):1321-1336.
[14]MOSS E G,LEE R C,AMBROS V.The cold shock domain protein LIN-28controls developmental timing in C.elegans and is regulated by the lin-4RNA[J].Cell,1997,88(5):637-646.
[15]ZHU H,SHAH S,SHYH-CHANG N,et al.Lin28a transgenic mice manifest size and puberty phenotypes identified in human genetic association studies[J].Nat Genet,2010,42(7):626-630.
[16]SANGIAO-ALVARELLOS S,MANFREDI-LOZA-NO M,RUIZ-PINO F,et al.Changes in hypothalamic expression of the Lin28/let-7system and related microRNAs during postnatal maturation and after experimental manipulations of puberty[J].Endocrinology,2013,154(2):942-955.
[17]GRIECO A,RZECZKOWSKA P,ALM C,et al.Investigation of peripubertal expression of Lin28aand Lin28bin C57BL/6female mice[J].Mol Cell Endocrinol,2013,365(2):241-248.
[18]侯文通.中国西北重要地方畜禽遗传资源[M].北京:中国农业出版社,2010.HOU W T.Important genetic resources of the native domestic animals in northwestern China[M].Beijing:China Agriculture Press,2010.(in Chinese)
[19]卢萍平,邢凤,刘博丹,等.Lin28B基因表达与绵羊初情期启动的关系研究[J].家畜生态学报,2014,35(9):14-17.LIU P P,XING F,LIU B D,et al.Lin28B gene expression and its role in the onset of puberty of sheep[J].Acta Ecologiae Animalis Domastici,2014,35(9):14-17.(in Chinese)
[20]DANTAS A,SIQUEIRA E R,FERNANDES S,et al.Influence of feeding differentiation on the age at onset of puberty in Brazilian Bergamasca dairy ewe lambs[J].Arq Bras Med Vet Zootec,2016,68(1):22-28.
[21]CAO G L,FENG T,CHU M X,et al.Subtraction suppressive hybridisation analysis of differentially expressed genes associated with puberty in the goat hypothalamus[J].Reprod Fertil Dev,2015,28(11):1781-1787,doi:10.1071/RD14434.
[22]LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the method[J].Methods,2001,25(4):402-408.
[23]HEO I,JOO C,CHO J,et al.Lin28mediates the terminal uridylation of Let-7precursor microRNA[J].Mol Cell,2008,32(2):276-284.
[24]VISWANATHAN S R,DALEY G Q,GREGORY RI.Selective blockade of microRNA processing by Lin28[J].Science,2008,320(5872):97-100.
[25]OJEDA S R,DUBAY C,LOMNICZI A,et al.Gene networks and the neuroendocrine regulation of puberty[J].Mol Cell Endocrinol,2010,324(1-2):3-11.
[26]RYBAK A,FUCHS H,SMIRNOVA L,et al.A feedback loop comprising lin-28and let-7controls pre-let-7maturation during neural stem-cell commitment[J].Nat Cell Biol,2008,10(8):987-993.
[27]TSIALIKAS J,ROMER-SEIBERT J.LIN28:Roles and regulation in development and beyond[J].Development,2015,142(14):2397-2404.