策勒黑羊ERα基因克隆、生物信息学分析及卵巢组织表达研究

doi:10.16431/j.cnki.1671-7236.2022.05.009

摘要/Abstract

摘要： 【目的】克隆策勒黑羊雌激素受体α(estrogen receptor α，ERα)序列并进行生物信息学分析，同时测定策勒黑羊卵巢中ERα基因的表达情况。【方法】利用RT-PCR和TA克隆方法获得策勒黑羊ERα基因序列，并采用在线软件对其进行生物信息学分析。采用实时荧光定量PCR方法检测ERα基因在策勒黑羊卵泡期、黄体期和妊娠30 d母羊卵巢中的表达情况。【结果】成功克隆获得策勒黑羊ERα基因序列，长1 791 bp，编码596个氨基酸，与山羊和牛的相似性最高。生物信息学分析显示，ERα蛋白分子式为C₂₉₃₀H₄₆₀₀N₈₂₂O₈₆₂S₄₁，理论等电点(pI)为7.32，不稳定指数为50.33，为不稳定亲水性蛋白;存在28个O-糖基化位点和54个磷酸化位点，不存在N-糖基化位点;主要定位在细胞核中，不具备跨膜结构;二级结构主要为α-螺旋及无规则卷曲。实时荧光定量PCR检测显示，ERα基因在策勒黑羊卵泡期、黄体期和妊娠30 d的卵巢中均有表达，与卵泡期相比，黄体期ERα基因表达量极显著下降(P<0.01)，妊娠30 d ERα基因表达量极显著升高(P<0.01)。【结论】策勒黑羊ERα基因序列长1 791 bp，编码596个氨基酸，为亲水蛋白，二级结构以α-螺旋和无规则卷曲为主。ERα基因在策勒黑羊卵泡期、黄体期和妊娠30 d的卵巢中均有表达。本研究结果为进一步探究ERα基因在策勒黑羊卵巢卵泡发育、黄体形成和妊娠中发挥的作用提供参考。

关键词: 策勒黑羊; ERα基因; 克隆; 表达

Abstract: 【Objective】 The aim of this experiment was to clone the estrogen receptor α (ERα) gene sequence in Cele Black sheep,and bioinformatics analysis was carried out.At the same time,the expression of ERα gene in ovary of Cele Black sheep were measured.【Method】 The ERα gene sequence of Cele Black sheep was obtained by RT-PCR and TA cloning,and bioinformatics analysis was carried out by online softwares.The expression of ERα gene in ovary of Cele Black sheep at follicular phase,luteal phase and 30 days of pregnancy were detected by Real-time quantitative PCR.【Result】 The length of ERα gene sequence in Cele Black sheep was 1 791 bp,and encoded 596 amino acids,with the highest similarity with Capra hircus and Bos taurus.Bioinformatics analysis results showed that the molecular formula of the protein was C₂₉₃₀H₄₆₀₀N₈₂₂O₈₆₂S₄₁,the theoretical isoelectric point (pI) was 7.32,the instability index was 50.33,which was an unstable hydrophilic protein.There were 28 O-glycosylation sites and 54 phosphorylation sites,but no N-glycosylation sites.ERα protein was mainly located in nucleus and there was no transmembrane structure.The secondary structure of ERα protein was mainly alpha helix and random coil.Real-time quantitative PCR showed that ERα gene in Cele Black sheep was expressed in ovary at follicular phase,luteal phase and 30 days of pregnancy,compared with follicular phase,the expression of ERα gene in Cele Black sheep was decreased extremely significantly at luteal phase (P<0.01),and increased extremely significantly at 30 days of pregnancy (P<0.01).【Conclusion】 The length of ERα gene sequence in Cele Black sheep was 1 791 bp,and encoded 596 amino acids,which was a hydrophilic protein.The secondary structure of ERα protein was mainly alpha helix and random coil.ERα gene in Cele Black sheep was expressed in ovaries at follicular phase,luteal phase and 30 days of pregnancy.The results provided a basis for further exploration of ERα gene which played a role in ovarian follicle development,corpus luteum formation and pregnancy of Cele Black sheep.

Key words: Cele Black sheep; ERα gene; cloning; expression

中图分类号:

S823

张继虎, 米热妮萨, 王子渲, 李琳, 邢凤, 刘书东. 策勒黑羊ERα基因克隆、生物信息学分析及卵巢组织表达研究[J]. 中国畜牧兽医, 2022, 49(5): 1679-1687.

ZHANG Jihu, MI Renisa, WANG Zixuan, LI Lin, XING Feng, LIU Shudong. Cloning and Bioinformatics of ERα Gene and Its Expression of Ovary Tissue in Cele Black Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(5): 1679-1687.

参考文献

[1] EYSTER K M.The estrogen receptors:An overview from different perspectives[J].Methods in Molecular Biology,2016,1366:1-10.
[2] DUBEY R K,JACKSON E K.Potential vascular actions of 2-methoxyestradiol[J].Trends in Endocrinology and Metabolism,2009,20(8):374-379.
[3] DUBEY R K,TOFOVIC S P,JACKSON E K.Cardiovascular pharmacology of estradiol metabolites[J].Journal of Pharmacology and Experimental Therapeutics,2004,308(2):403-409.
[4] RUAN X,SEEGER H,WALLWIENER D,et al.The ratio of the estradiol metabolites 2-hydroxyestrone (2-OHE1) and 16α-hydroxyestrone (16-OHE1) may predict breast cancer risk in postmenopausal but not in premenopausal women:Two case-control studies[J].Archives of Gynecology and Obstetrics,2015,291(5):1141-1146.
[5] PATEL B,ELGUERO S,THAKORE S,et al.Role of nuclear progesterone receptor isoforms in uterine pathophysiology[J].Human Reproduction Update,2015,21(2):155-173.
[6] TSAI M J,O'MALLEY B W.Molecular mechanisms of action of steroid/thyroid receptor superfamily members[J].Annual Review of Biochemistry,1994,63(1):451-486.
[7] KRÓL M B,GALICKI M,GREŠNER P,et al.The ESR1 and GPX1 gene expression level in human malignant and non-malignant breast tissues[J].Acta Biochimica Polonica,2018,65(1):51-57.
[8] BREYER J,WIRTZ R M,LAIBLE M.ESR1,ERBB2,and Ki67 mRNA expression predicts stage and grade of non-muscle-invasive bladder carcinoma (NMIBC)[J].Virchows Archiv,2016,469(5):547-552.
[9] GOVERS L C,PHILLIPS T R,MATTISKE D M,et al.A critical role for estrogen signaling in penis development[J].FASEB Journal,2019,33(9):10383-10392.
[10] MIJIDDORJ T,KANASAKI H,SUKHBAATAR U,et al.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.
[11] DUBOIS E A,ZANDBERGEN M A,PEUTE J,et al.Evolutionary development of three gonadotropin-releasing hormone(GnRH) systems in vertebrates[J].Brain Research Bulletin,2002,57(3-4):413-418.
[12] SAKAGUCHI H,FUJIMOTO J,BAO L H,et al.Quantitative analysis of estrogen recept-proteins in rat ovary[J].Journal of Steroid Biochemistry and Molecular Biology,2005,94(7):83-91.
[13] SUNSHIN K,JUNG A P,KANG H J,et al.Epistasis between CYP19A1 and ESR1 polymorphisms is associated with premature ovarian failure[J].Fertility and Sterility,2011,95(1):353-356.
[14] PORTEOUS R,HERBISON A E.Genetic deletion of ESR1 in the mouse preoptic area disrupts the LH surge and estrous cyclicity[J].Endocrinology,2019,160(8):1821-1829.
[15] 李键,符梅,兰道亮,等.牦牛雌激素受体ERα和ERβ基因分子特征及组织表达研究[J].西南民族大学学报(自然科学版),2014,40(6):801-808. LI J,FU M,LAN D L,et al.Molecular characterization and tissue expression of estrogen receptor ERα and ERβ genes in yak[J].Journal of Southwest University for Nationalities (Natural Science Edition),2014,40(6):801-808.(in Chinese)
[16] TORA L,WHITE J,BROU C,et al.The human estrogen receptor has two independent nonacid transcriptional activation functions[J].Cell,1989,59(3):477-487.
[17] HIKER V J,THOMPSON E B.Structural dynamics,intrinsic disorder,and allostery in nuclear receptors as transcription factors[J].Journal of Biological Chemistry,2011,286(46):39675-39682.
[18] 张翠霞,丛李艳,雷岷,等.家兔雌激素受体1基因编码区序列分析[J].中国畜牧杂志,2017,53(10):29-33. ZHANG C X,CONG L Y,LEI M,et al.Sequence analysis of the coding region of Oryctolagus cuniculus estrogen receptor 1 gene[J].Chinese Journal of Animal Science,2017,53(10):29-33.(in Chinese)
[19] TZUKERMAN M T,ESTY A,SANTISO-MERE D,et al.Human estrogen receptor transactivational capacity is determined by both cellular and promoter context and mediated by two functionally distinct intramolecular regions[J].Molecular Endocrinology,1994,8(1):21-30.
[20] JUENGEL J L,HEATH D A,QUIRKE L D,et al.Oestrogen receptor alpha and beta,androgen receptor and progesterone receptor mRNA and protein localisation within the developing ovary and in small growing follicles of sheep[J].Reproduction,2006,131(1):81-92.
[21] 包莹莹,陈卫刚,何玉琴,等.甘加型藏羊发情周期血浆雌激素测定及HPO轴ERα的表达[J].西北农业学报,2021,30(4):481-489. BAO Y Y,CHEN W G,HE Y Q,et al.Determination of plasma estrogen and ERα in HPO axis expression in estrus cycle of Ganjia Tibetan sheep[J].Acta Agriculturae Boreali-Occidentalis Sinica,2021,30(4):481-489.(in Chinese)
[22] 王锋.动物繁殖学[M].北京:中国农业大学出版社,2012. WANG F.Animal Reproduction[M].Beijing:China Agricultural University Press,2012.(in Chinese)
[23] BERISHA B,PFAFFL M W,SCHAMS D.Expression of estrogen and progesterone receptors in the bovine ovary during estrous cycle and pregnancy[J].Endocrine,2002,17(3):207-214.