水貂PRLR基因的克隆及生物信息学分析
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摘要
本研究旨在从水貂卵巢组织中克隆催乳素受体(prolactin receptor,PRLR)基因编码区全长序列,并进行生物学信息分析,为研究其结构和功能奠定基础。根据GenBank中登录的犬PRLR基因的mRNA预测序列(登录号:XM_025436332.1)设计1对引物,采集性成熟的健康水貂卵巢组织样本,提取RNA并反转录合成cDNA,以cDNA为模板PCR扩增PRLR基因,将其插入pEASY-Blunt Simple Vector中,筛选阳性克隆测序后进行生物信息学分析。结果显示,水貂PRLR基因编码区序列全长为1 878bp,编码了625个氨基酸,水貂PRLR基因核苷酸序列与海獭同源性最高,为97.7%。系统进化树分析也显示其和海獭亲缘关系最近。对水貂卵巢PRLR基因编码蛋白进行生物信息学分析表明,PRLR蛋白含有1个信号肽,1个细胞外区域,1个跨膜区和1个膜内区;水貂PRLR蛋白为单次跨膜蛋白,其N端的D1部分含有2对保守的二硫键(Cys36—Cys46和Cys75—Cys86),D2部分含有保守的WS-基序(WS-E-WS)。水貂PRLR蛋白具有和其他PRLR蛋白相似的膜外和膜内结构。本试验结果为进一步研究和揭示水貂PRLR蛋白的结构及功能奠定了基础。
The aim of this study was to clone the full-length coding sequence of prolactin receptor(PRLR)gene from mink ovary tissues and analyze its biological characteristics to lay a foundation for studying its structure and function.A pair of primers was designed based on the predicted mRNA sequence of the dog PRLRgene in GenBank(accession No.:XM_025436332.1),and the ovary tissue samples of sexually mature healthy mink were collected,and RNA was extracted and then was reverse transcribed to synthesize cDNA,and PRLRgene was amplified by PCR using cDNA as template.The PCR products then were inserted into the pEASY-Blunt Simple Vector.Bioinformatics analysis was carried out after positive clone sequencing.The results showed that the coding region of PRLRgene of mink was 1 878 bp in length and encoded 625 amino acids.The homology of nucleotide sequence of PRLRgene between mink and Enhydra lutris kenyoni wasthe highest which reached 97.7%.The phylogenetic tree analysis also showed the closest relationship with Enhydra lutris kenyoni.The bioinformatics analysis of the PRLR protein of mink ovary showed that it contained a signal peptide,an extracellular region,a transmembrane region and an intramembranous region.It was a single transmembrane protein,and the D1 domain at the N-terminal contained two pairs of conserved disulfide bonds(Cys36-Cys46 and Cys75-Cys86),the D2 domain contained a conserved WS-motif(WS-E-WS).The PRLR protein of mink had a similar structure to other PRLR proteins.In conclusion,the study provided basic experimental data and theoretical basis for the further study of the structure and function of PRLR protein.
The aim of this study was to clone the full-length coding sequence of prolactin receptor(PRLR)gene from mink ovary tissues and analyze its biological characteristics to lay a foundation for studying its structure and function.A pair of primers was designed based on the predicted mRNA sequence of the dog PRLRgene in GenBank(accession No.:XM_025436332.1),and the ovary tissue samples of sexually mature healthy mink were collected,and RNA was extracted and then was reverse transcribed to synthesize cDNA,and PRLRgene was amplified by PCR using cDNA as template.The PCR products then were inserted into the pEASY-Blunt Simple Vector.Bioinformatics analysis was carried out after positive clone sequencing.The results showed that the coding region of PRLRgene of mink was 1 878 bp in length and encoded 625 amino acids.The homology of nucleotide sequence of PRLRgene between mink and Enhydra lutris kenyoni wasthe highest which reached 97.7%.The phylogenetic tree analysis also showed the closest relationship with Enhydra lutris kenyoni.The bioinformatics analysis of the PRLR protein of mink ovary showed that it contained a signal peptide,an extracellular region,a transmembrane region and an intramembranous region.It was a single transmembrane protein,and the D1 domain at the N-terminal contained two pairs of conserved disulfide bonds(Cys36-Cys46 and Cys75-Cys86),the D2 domain contained a conserved WS-motif(WS-E-WS).The PRLR protein of mink had a similar structure to other PRLR proteins.In conclusion,the study provided basic experimental data and theoretical basis for the further study of the structure and function of PRLR protein.
引文
[1]RAVAULT J P,MARTINET L,BONNEFOND C,et al.Diurnal variations of plasma melatonin concentrations in pregnant or pseudopregnant mink(Mustela vison)maintained under different photoperiods[J].Journal of Pineal Research,1986,3(4):365-373.
[2]MURPHY B D,DIGREGORIO G B,DOUGLAS D A,et al.Interactions between melatonin and prolactin during gestation in mink(Mustela vison)[J].Journal of Reproduction and Fertility,1990,89(2):423-429.
[3]MURPHY B D,CONCANNON P W,TRAVIS H F,et al.Prolactin:The hypophyseal factor that terminates embryonic diapause in mink[J].Biology of Reproduction,1981,25(3):487-491.
[4]LOPES F L,DESMARAIS J A,MURPHY B D.Embryonic diapause and its regulation[J].Reproduction,2004,128(6):669-678.
[5]BINART N,BACHELOT A,BOUILLY J.Impact of prolactin receptor isoforms on reproduction[J].Trends in Endocrinology and Metabolism,2010,21(6):362-368.
[6]FREEMARK M.Ontogenesis of prolactin receptors in the human fetus:Roles in fetal development[J].Biochemical Society Transactions,2001,29(2):38-41.
[7]POSNER B I,KELLY P A,SHIU R P,et al.Studies of insulin,growth hormone and prolactin binding:Tissue distribution,species variation and characterization[J].Endocrinology,1974,95(2):521-531.
[8]GOFFIN V,KELLY P A.Prolactin and growth hormone receptors[J].Clinical Endocrinology,1996,45(3):247-255.
[9]张芬芬,王长法,黄金明,等.催乳素受体的研究进展[J].家畜生态学报,2009,30(3):6-9.ZHANG F F,WANG C F,HUANG J M,et al.Progress in the study on prolactin receptor[J].Acta Ecologiae Animalis Domastici,2009,30(3):6-9.(in Chinese)
[10]GOFFIN V,SHIVERICK K T,KELLY P A,et al.Sequence-function relationships within the expanding family of prolactin,growth hormone,placental lactogen,and related proteins in mammals[J].Endocrine Reviews,1996,17(4):385-410.
[11]DAMIANO J S,WASSERMAN E.Molecular pathways:Blockade of the PRLR signaling pathway as a novel antihormonal approach for the treatment of breast and prostate cancer[J].Clinical Cancer Research,2013,19(7):1644-1650.
[12]HAMMER A,DIAKONOVA M.Tyrosyl phosphorylated serine-threonine kinase PAK1is a novel regulator of prolactin-dependent breast cancer cell motility and invasion[J].Advances in Experimental Medicine and Biology,2015,846:97-137.
[13]DIAKONOVA M.Recent advances in prolactin research preface[J].Recent Advances in Prolactin Research,2015,846:8-9.
[14]GOFFIN V,BINART N,TOURAINE P,et al.Prolactin:The new biology of an old hormone[J].Annual Review of Physiology,2002,64:47-67.
[15]LIU X W,ROBINSON G W,WAGNER K U,et al.Stat5ais mandatory for adult mammary gland development and lactogenesis[J].Genes&Development,1997,11(2):179-186.
[16]PEZET A,FERRAG F,KELLY P A,et al.Tyrosine docking sites of the rat prolactin receptor required for association and activation of Stat5[J].Journal of Biological Chemistry,1997,272(40):25043-25050.
[17]CLEVENGER C V.Roles and regulation of stat family transcription factors in human breast cancer[J].American Journal of Pathology,2004,165(5):1449-1460.
[18]CLEVENGER C V,FURTH P A,HANKINSON S E,et al.The role of prolactin in mammary carcinoma[J].Endocrine Reviews,2003,24(1):1-27.
[19]BERLANGA J J,GUALILLO O,BUTEAU H,et al.Prolactin activates tyrosyl phosphorylation of insulin receptor substrate 1 and phosphatidylinositol-3-OHkinase[J].Journal of Biological Chemistry,1997,272(4):2050-2052.
[20]BOLE-FEYSOT C,GOFFIN V,EDERY M,et al.Prolactin(PRL)and its receptor:Actions,signal transduction pathways and phenotypes observed in PRL receptor knockout mice[J].Endocrine Reviews,1998,19(3):225-268.
[21]张宇飞,李晓霞,王士勇,等.北极狐及乌苏里貉抑制素α亚基基因克隆及生物信息学分析[J].中国畜牧兽医,2017,44(12):3391-3400.ZHANG Y F,LI X X,WANG S Y,et al.Cloning and bioinformatics analysis of inhibinα-subunit gene of arctic fox and Wusuli raccoon dog[J].China Animal Husbandry&Veterinary Medicine,2017,44(12):3391-3400.(in Chinese)
[22]FENELON J C,BANERJEE A,LEFEVRE P,et al.Polyamine-mediated effects of prolactin dictate emergence from mink obligate embryonic diapause[J].Biology of Reproduction,2016,95(1):6.
[23]FENELON J C,LEFEVRE P L,BANERJEE A,et al.Regulation of diapause in carnivores[J].Reproduction in Domestic Animals,2017,52(2):12-17.
[24]LIONGUE C,WARD A C.Evolution of classⅠcytokine receptors[J].BMC Evolutionary Biology,2007,7:120.
[25]HAXHOLM G W,NIKOLAJSEN L F,OLSEN J G,et al.Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes[J].Biochemical Journal,2015,468(3):495-506.
[26]DAGIL R,KNUDSEN M J,OLSEN J G,et al.The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation:Insight from structures of the prolactin receptor[J].Structure,2012,20(2):270-282.
[27]BUGGE K,PAPALEO E,HAXHOLM G W,et al.Acombined computational and structural model of the full-length human prolactin receptor[J].Nature Communications,2016,7:11578.
[2]MURPHY B D,DIGREGORIO G B,DOUGLAS D A,et al.Interactions between melatonin and prolactin during gestation in mink(Mustela vison)[J].Journal of Reproduction and Fertility,1990,89(2):423-429.
[3]MURPHY B D,CONCANNON P W,TRAVIS H F,et al.Prolactin:The hypophyseal factor that terminates embryonic diapause in mink[J].Biology of Reproduction,1981,25(3):487-491.
[4]LOPES F L,DESMARAIS J A,MURPHY B D.Embryonic diapause and its regulation[J].Reproduction,2004,128(6):669-678.
[5]BINART N,BACHELOT A,BOUILLY J.Impact of prolactin receptor isoforms on reproduction[J].Trends in Endocrinology and Metabolism,2010,21(6):362-368.
[6]FREEMARK M.Ontogenesis of prolactin receptors in the human fetus:Roles in fetal development[J].Biochemical Society Transactions,2001,29(2):38-41.
[7]POSNER B I,KELLY P A,SHIU R P,et al.Studies of insulin,growth hormone and prolactin binding:Tissue distribution,species variation and characterization[J].Endocrinology,1974,95(2):521-531.
[8]GOFFIN V,KELLY P A.Prolactin and growth hormone receptors[J].Clinical Endocrinology,1996,45(3):247-255.
[9]张芬芬,王长法,黄金明,等.催乳素受体的研究进展[J].家畜生态学报,2009,30(3):6-9.ZHANG F F,WANG C F,HUANG J M,et al.Progress in the study on prolactin receptor[J].Acta Ecologiae Animalis Domastici,2009,30(3):6-9.(in Chinese)
[10]GOFFIN V,SHIVERICK K T,KELLY P A,et al.Sequence-function relationships within the expanding family of prolactin,growth hormone,placental lactogen,and related proteins in mammals[J].Endocrine Reviews,1996,17(4):385-410.
[11]DAMIANO J S,WASSERMAN E.Molecular pathways:Blockade of the PRLR signaling pathway as a novel antihormonal approach for the treatment of breast and prostate cancer[J].Clinical Cancer Research,2013,19(7):1644-1650.
[12]HAMMER A,DIAKONOVA M.Tyrosyl phosphorylated serine-threonine kinase PAK1is a novel regulator of prolactin-dependent breast cancer cell motility and invasion[J].Advances in Experimental Medicine and Biology,2015,846:97-137.
[13]DIAKONOVA M.Recent advances in prolactin research preface[J].Recent Advances in Prolactin Research,2015,846:8-9.
[14]GOFFIN V,BINART N,TOURAINE P,et al.Prolactin:The new biology of an old hormone[J].Annual Review of Physiology,2002,64:47-67.
[15]LIU X W,ROBINSON G W,WAGNER K U,et al.Stat5ais mandatory for adult mammary gland development and lactogenesis[J].Genes&Development,1997,11(2):179-186.
[16]PEZET A,FERRAG F,KELLY P A,et al.Tyrosine docking sites of the rat prolactin receptor required for association and activation of Stat5[J].Journal of Biological Chemistry,1997,272(40):25043-25050.
[17]CLEVENGER C V.Roles and regulation of stat family transcription factors in human breast cancer[J].American Journal of Pathology,2004,165(5):1449-1460.
[18]CLEVENGER C V,FURTH P A,HANKINSON S E,et al.The role of prolactin in mammary carcinoma[J].Endocrine Reviews,2003,24(1):1-27.
[19]BERLANGA J J,GUALILLO O,BUTEAU H,et al.Prolactin activates tyrosyl phosphorylation of insulin receptor substrate 1 and phosphatidylinositol-3-OHkinase[J].Journal of Biological Chemistry,1997,272(4):2050-2052.
[20]BOLE-FEYSOT C,GOFFIN V,EDERY M,et al.Prolactin(PRL)and its receptor:Actions,signal transduction pathways and phenotypes observed in PRL receptor knockout mice[J].Endocrine Reviews,1998,19(3):225-268.
[21]张宇飞,李晓霞,王士勇,等.北极狐及乌苏里貉抑制素α亚基基因克隆及生物信息学分析[J].中国畜牧兽医,2017,44(12):3391-3400.ZHANG Y F,LI X X,WANG S Y,et al.Cloning and bioinformatics analysis of inhibinα-subunit gene of arctic fox and Wusuli raccoon dog[J].China Animal Husbandry&Veterinary Medicine,2017,44(12):3391-3400.(in Chinese)
[22]FENELON J C,BANERJEE A,LEFEVRE P,et al.Polyamine-mediated effects of prolactin dictate emergence from mink obligate embryonic diapause[J].Biology of Reproduction,2016,95(1):6.
[23]FENELON J C,LEFEVRE P L,BANERJEE A,et al.Regulation of diapause in carnivores[J].Reproduction in Domestic Animals,2017,52(2):12-17.
[24]LIONGUE C,WARD A C.Evolution of classⅠcytokine receptors[J].BMC Evolutionary Biology,2007,7:120.
[25]HAXHOLM G W,NIKOLAJSEN L F,OLSEN J G,et al.Intrinsically disordered cytoplasmic domains of two cytokine receptors mediate conserved interactions with membranes[J].Biochemical Journal,2015,468(3):495-506.
[26]DAGIL R,KNUDSEN M J,OLSEN J G,et al.The WSXWS motif in cytokine receptors is a molecular switch involved in receptor activation:Insight from structures of the prolactin receptor[J].Structure,2012,20(2):270-282.
[27]BUGGE K,PAPALEO E,HAXHOLM G W,et al.Acombined computational and structural model of the full-length human prolactin receptor[J].Nature Communications,2016,7:11578.
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