湖羊NR5A1基因全序列克隆和表达特征分析
|
摘要
以江苏特色绵羊品种——湖羊为研究对象,克隆湖羊NR5A1基因全序列,分析其序列特征,并对湖羊组织和大、小卵泡中NR5A1表达特征进行研究。结果表明,湖羊NR5A1基因全长19 016 bp,含6个外显子和5个内含子;编码区全长1 386 bp,编码461个氨基酸残基,包含经典的DNA结合区(DBD)和配体结合区(LBD)。RT-PCR结果显示NR5A1基因在湖羊组织中广泛表达,其中在卵巢中高表达,脾脏次之; qRT-PCR和Western blot结果显示在湖羊大卵泡中NR5A1 mRNA和蛋白质水平均显著高于小卵泡,表明NR5A1基因参与调控湖羊卵泡发育,并与繁殖性能有关。
Using Hu sheep( Jiangsu featured sheep breed) as the research object,NR5 A1 gene full sequences were cloned and the sequence characteristics were analyzed. The expression features of NR5 A1 gene in Hu sheep tissues and in large and small follicles were explored. The results showed that the complete sequence of ovine NR5 A1 was 19 016 bp in length and contained six exons and five introns. The coding region was 1 386 bp and encoded 461 amino acid residues,including the classical DNA binding domain( DBD) and ligand binding domain( LBD). RT-PCR results showed that NR5 A1gene was widely expressed in Hu sheep tissues,which was highly expressed in ovary,followed by kidney. qRT-PCR and western blot results showed that the expression of NR5 A1 in large follicles was significantly higher than that in small follicles,indicating that NR5 A1 gene was involved in regulating follicle development and speculating the reproductive performance of Hu sheep.
Using Hu sheep( Jiangsu featured sheep breed) as the research object,NR5 A1 gene full sequences were cloned and the sequence characteristics were analyzed. The expression features of NR5 A1 gene in Hu sheep tissues and in large and small follicles were explored. The results showed that the complete sequence of ovine NR5 A1 was 19 016 bp in length and contained six exons and five introns. The coding region was 1 386 bp and encoded 461 amino acid residues,including the classical DNA binding domain( DBD) and ligand binding domain( LBD). RT-PCR results showed that NR5 A1gene was widely expressed in Hu sheep tissues,which was highly expressed in ovary,followed by kidney. qRT-PCR and western blot results showed that the expression of NR5 A1 in large follicles was significantly higher than that in small follicles,indicating that NR5 A1 gene was involved in regulating follicle development and speculating the reproductive performance of Hu sheep.
引文
[1] IKEDA Y,LALA DS,LUO X,et al. Characterization of the mouseFTZ-F1 gene,which encodes a key regulator of steroid hydroxylasegene expression[J]. Molecular Endocrinology,1993,7(7):852-860.
[2] LIN L,ACHERMANN J C. Steroidogenic factor-1(SF-1,Ad4BP,NR5A1)and disorders of testis development[J]. Sexual Develop-ment,2008,2(4/5):200-209.
[3] LUO X,IKEDA Y,PARKER K L. A cell-specific nuclear recep-tor is essential for adrenal and gonadal development and sexual dif-ferentiation[J]. Cell,1994,77(4):481-490.
[4] TANTAWY S,LIN L,AKKURT I,et al. Testosterone productionduring puberty in two 46,XY patients with disorders of sex devel-opment and novel NR5A1(SF-1)mutations[J]. European Jour-nal of Endocrinology,2012,167(1):125-130.
[5] TAIEB J,GRYNBERG M,PIERRE A,et al. FSH and its secondmessenger c AMP stimulate the transcription of human anti-Müllerian hormone in cultured granulosa cells[J]. Molecular En-docrinology,2011,25(4):645-655.
[6] MATΜLIS C K,MAYO K E. The LIM domain protein FHL2 inter-acts with the NR5A family of nuclear receptors and CREB to acti-vate the inhibin-αsubunit gene in ovarian granulosa cells[J].Molecular Endocrinology,2012,26(8):1278-1290.
[7] MURAYAMA C,MIYAZAKI H,MIYAMOTO A,et al. Luteini-zing hormone(LH)regulates production of androstenedione andprogesterone via control of histone acetylation of St AR and CYP17promoters in ovarian theca cells[J]. Molecular Cellular Endocri-nology,2012,350(1):1-9.
[8] IMAMICHI Y,MIZUTANI T,JU Y,et al. Transcriptional regula-tion of human ferredoxin 1 in ovarian granulosa cells[J]. Molecu-lar Cellular Endocrinology,2013,370(1/2):1-10.
[9] LAW NC,WECK J,KYRISS B,et al. Lhcgr expression in granu-losa cells:roles for PKA-phosphorylatedβ-catenin,TCF3,andFOXO1[J]. Molecular Endocrinology,2013,27(8):1295-1310.
[10] YIN M,LM,YAO G,et al. Transactivation of microRNA-383by steroidogenic factor-1 promotes estradiol release from mouse o-varian granulosa cells by targeting RBMS1[J]. Molecular Endo-crinology,2012,26(7):1129-1143.
[11] FORTIN J,KUMAR V,ZHOU X,et al. NR5A2 regulates Lhband Fshb transcription in gonadotrope-like cells in vitro,but isdispensable for gonadotropin synthesis and fertility in vivo[J].PLoS ONE,2013,8(3):e59058
[12] SCHANG A L,QURAT B,SIMON V,et al. Mechanisms under-lying the tissue-specific and regulated activity of the Gnrhr promot-er in mammals[J]. Front Endocrinol(Lausanne),2012,3:162.
[13]李隐侠,张俊,钱勇,等.湖羊NR5A1基因SNPs筛选及其与产羔数的关联分析[J].江苏农业学报,2017,33(1):124-132.
[14] TAMURA K,PETERSON D,PETERSON N,et al. MEGA5:mo-lecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Mo-lecular Biology Evolution,2011,28(10):2731-2739.
[15] YAO Y,REHEMAN A,XU Y,et al. miR-125b contributes to o-varian granulosa cell apoptosis through targeting BMPR1B,a majorgene for sheep prolificacy[J]. Reproduction Science,2018. doi:10.1177/1933719118770544.
[16] OBA K,YANASE T,NOMURA M,et al. Structural characteriza-tion of human Ad4bp(SF-1)gene[J]. Biochemical BiophysicalResearch Communication,1996,226(1):261-267.
[17] YUMOTO F,NGUYEN P,SABLIN E P,et al. Structural basis ofcoactivation of liver receptor homolog-1 byβ-catenin[J]. Proceed-ings of the National Academy of Sciences of USA,2012,109(1):143-148.
[18] LI Y X,ZHANG J,QIAN Y,et al. Molecular characterization,expression,polymorphism of NR5A2 and its relationship with littersize in Hu sheep[J]. Genetics and Molecular Research,2015,14(4):12765-12775.
[19] MLYNARCZUK J,WROBEL M H,ZIOLKOWSKA A,et al. In-volvement of the orphan nuclear receptor SF-1 in the effect ofPCBs,DDT and DDE on the secretion of steroid hormones and ox-ytocin from bovine granulosa cells[J]. Animal Reproduction Sci-ence,2013,143(1-4):30-37.
[20] BUAAS F W,GARDINER J R,CLAYTON S,et al. In vivo evi-dence for the crucial role of SF1 in steroid-producing cells of thetestis,ovary and adrenal gland[J]. Development,2012,139(24):4561-4570.
[21] EL-KHAIRI R,MARTINEZ-AGUAYO A,FERRAZ-DE-SOUZAB,et al. Role of DAX-1(NR0B1)and steroidogenic factor-1(NR5A1)in human adrenal function[J]. Endocrine Develop-ment,2011,20:38-46.
[22] EHRLUND A,JONSSON P,VEDIN L L,et al. Knockdown ofSF-1 and RNF31 affects components of steroidogenesis,TGFβ,and Wnt/β-catenin signaling in adrenocortical carcinoma cells[J]. PLoS ONE,2013,7(3):e32080.
[23] FALENDER A E,LANZ R,MALENFANT D,et al. Differentialexpression of steroidogenic factor-1 and FTF/LRH-1 in the rodentovary[J]. Endocrinology,2013,144(8):3598-3610.
[24] JIN X,HAN C S,ZHANG X S,et al. Stem cell factor modulatesthe expression of steroidogenesis related proteins and FSHR duringovarian follicular development[J]. Fronters in Bioscience:a Jour-nal and Virtual Library,2005,10:1573-1580.
[25] YIN M,WANG X,YAO G,et al. Transactivation of micrornA-320 by microRNA-383 regulates granulosa cell functions by targe-ting E2F1 and SF-1 proteins[J]. The Journal of Biological Chem-istry,2014,289(26):18239-18257.
[26] WANG L,LI C,LI R,et al. MicroRNA-764-3p regulates 17β-es-tradiol synthesis of mouse ovarian granulosa cells by targeting ste-roidogenic factor-1[J]. In Vitro Cellular&Developmental BiologyAnimal,2016,52(3):365-373.
[27] PELUSI C,IKEDA Y,ZUBAIR M,et al. Impaired follicle devel-opment and infertility in female mice lacking steroidogenic factor 1in ovarian granulosa cells[J]. Biology of Reproduction,2008,79(6):1074-1083.
[2] LIN L,ACHERMANN J C. Steroidogenic factor-1(SF-1,Ad4BP,NR5A1)and disorders of testis development[J]. Sexual Develop-ment,2008,2(4/5):200-209.
[3] LUO X,IKEDA Y,PARKER K L. A cell-specific nuclear recep-tor is essential for adrenal and gonadal development and sexual dif-ferentiation[J]. Cell,1994,77(4):481-490.
[4] TANTAWY S,LIN L,AKKURT I,et al. Testosterone productionduring puberty in two 46,XY patients with disorders of sex devel-opment and novel NR5A1(SF-1)mutations[J]. European Jour-nal of Endocrinology,2012,167(1):125-130.
[5] TAIEB J,GRYNBERG M,PIERRE A,et al. FSH and its secondmessenger c AMP stimulate the transcription of human anti-Müllerian hormone in cultured granulosa cells[J]. Molecular En-docrinology,2011,25(4):645-655.
[6] MATΜLIS C K,MAYO K E. The LIM domain protein FHL2 inter-acts with the NR5A family of nuclear receptors and CREB to acti-vate the inhibin-αsubunit gene in ovarian granulosa cells[J].Molecular Endocrinology,2012,26(8):1278-1290.
[7] MURAYAMA C,MIYAZAKI H,MIYAMOTO A,et al. Luteini-zing hormone(LH)regulates production of androstenedione andprogesterone via control of histone acetylation of St AR and CYP17promoters in ovarian theca cells[J]. Molecular Cellular Endocri-nology,2012,350(1):1-9.
[8] IMAMICHI Y,MIZUTANI T,JU Y,et al. Transcriptional regula-tion of human ferredoxin 1 in ovarian granulosa cells[J]. Molecu-lar Cellular Endocrinology,2013,370(1/2):1-10.
[9] LAW NC,WECK J,KYRISS B,et al. Lhcgr expression in granu-losa cells:roles for PKA-phosphorylatedβ-catenin,TCF3,andFOXO1[J]. Molecular Endocrinology,2013,27(8):1295-1310.
[10] YIN M,LM,YAO G,et al. Transactivation of microRNA-383by steroidogenic factor-1 promotes estradiol release from mouse o-varian granulosa cells by targeting RBMS1[J]. Molecular Endo-crinology,2012,26(7):1129-1143.
[11] FORTIN J,KUMAR V,ZHOU X,et al. NR5A2 regulates Lhband Fshb transcription in gonadotrope-like cells in vitro,but isdispensable for gonadotropin synthesis and fertility in vivo[J].PLoS ONE,2013,8(3):e59058
[12] SCHANG A L,QURAT B,SIMON V,et al. Mechanisms under-lying the tissue-specific and regulated activity of the Gnrhr promot-er in mammals[J]. Front Endocrinol(Lausanne),2012,3:162.
[13]李隐侠,张俊,钱勇,等.湖羊NR5A1基因SNPs筛选及其与产羔数的关联分析[J].江苏农业学报,2017,33(1):124-132.
[14] TAMURA K,PETERSON D,PETERSON N,et al. MEGA5:mo-lecular evolutionary genetics analysis using maximum likelihood,evolutionary distance,and maximum parsimony methods[J]. Mo-lecular Biology Evolution,2011,28(10):2731-2739.
[15] YAO Y,REHEMAN A,XU Y,et al. miR-125b contributes to o-varian granulosa cell apoptosis through targeting BMPR1B,a majorgene for sheep prolificacy[J]. Reproduction Science,2018. doi:10.1177/1933719118770544.
[16] OBA K,YANASE T,NOMURA M,et al. Structural characteriza-tion of human Ad4bp(SF-1)gene[J]. Biochemical BiophysicalResearch Communication,1996,226(1):261-267.
[17] YUMOTO F,NGUYEN P,SABLIN E P,et al. Structural basis ofcoactivation of liver receptor homolog-1 byβ-catenin[J]. Proceed-ings of the National Academy of Sciences of USA,2012,109(1):143-148.
[18] LI Y X,ZHANG J,QIAN Y,et al. Molecular characterization,expression,polymorphism of NR5A2 and its relationship with littersize in Hu sheep[J]. Genetics and Molecular Research,2015,14(4):12765-12775.
[19] MLYNARCZUK J,WROBEL M H,ZIOLKOWSKA A,et al. In-volvement of the orphan nuclear receptor SF-1 in the effect ofPCBs,DDT and DDE on the secretion of steroid hormones and ox-ytocin from bovine granulosa cells[J]. Animal Reproduction Sci-ence,2013,143(1-4):30-37.
[20] BUAAS F W,GARDINER J R,CLAYTON S,et al. In vivo evi-dence for the crucial role of SF1 in steroid-producing cells of thetestis,ovary and adrenal gland[J]. Development,2012,139(24):4561-4570.
[21] EL-KHAIRI R,MARTINEZ-AGUAYO A,FERRAZ-DE-SOUZAB,et al. Role of DAX-1(NR0B1)and steroidogenic factor-1(NR5A1)in human adrenal function[J]. Endocrine Develop-ment,2011,20:38-46.
[22] EHRLUND A,JONSSON P,VEDIN L L,et al. Knockdown ofSF-1 and RNF31 affects components of steroidogenesis,TGFβ,and Wnt/β-catenin signaling in adrenocortical carcinoma cells[J]. PLoS ONE,2013,7(3):e32080.
[23] FALENDER A E,LANZ R,MALENFANT D,et al. Differentialexpression of steroidogenic factor-1 and FTF/LRH-1 in the rodentovary[J]. Endocrinology,2013,144(8):3598-3610.
[24] JIN X,HAN C S,ZHANG X S,et al. Stem cell factor modulatesthe expression of steroidogenesis related proteins and FSHR duringovarian follicular development[J]. Fronters in Bioscience:a Jour-nal and Virtual Library,2005,10:1573-1580.
[25] YIN M,WANG X,YAO G,et al. Transactivation of micrornA-320 by microRNA-383 regulates granulosa cell functions by targe-ting E2F1 and SF-1 proteins[J]. The Journal of Biological Chem-istry,2014,289(26):18239-18257.
[26] WANG L,LI C,LI R,et al. MicroRNA-764-3p regulates 17β-es-tradiol synthesis of mouse ovarian granulosa cells by targeting ste-roidogenic factor-1[J]. In Vitro Cellular&Developmental BiologyAnimal,2016,52(3):365-373.
[27] PELUSI C,IKEDA Y,ZUBAIR M,et al. Impaired follicle devel-opment and infertility in female mice lacking steroidogenic factor 1in ovarian granulosa cells[J]. Biology of Reproduction,2008,79(6):1074-1083.