水牛PTHLH基因克隆分析及其真核表达载体构建
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摘要
为揭示甲状旁腺激素样激素(parathyroid hormone-like hormone,PTHLH)基因对水牛繁殖性能的影响,本研究对水牛PTHLH基因进行克隆,并对其核苷酸和氨基酸序列进行生物信息学分析。以牛PTHLH基因为种子序列(GenBank登录号:NM_001290949),应用CE Design软件设计引物序列,运用PCR扩增和测序技术获得水牛完整编码区序列,使用DNAMAN、ProtParam、SOPMA、PSORTⅡPrediction等在线软件分析PTHLH蛋白的一级结构、二级结构、三级结构与理化性质,并进行同源性比对分析及系统进化树构建。结果显示,试验克隆了水牛PTHLH基因完整编码区序列,该序列长为534bp,可编码177个氨基酸。水牛PTHLH基因编码区核苷酸序列与黄牛、猪、马、山羊、绵羊和骆驼的同源性分别为98.3%、90.4%、90.1%、98.1%、97.5%和89.2%,物种之间同源性较高,系统进化树分析结果与其亲缘关系远近一致,表明水牛PTHLH基因编码区在进化过程中比较保守。蛋白理化性质分析显示,水牛PTHLH蛋白分子式为C895H1451N271O266S2,分子质量为2 885u,半衰期为30h,理论等电点(pI)为10.00,水溶液在280nm处的消光系数为23 950,肽链N端为蛋氨酸(Met),不稳定系数为60.04,属于碱性不稳定蛋白;脂肪系数为72.15,总平均亲水性为-0.928,该蛋白属于不可溶性蛋白,亚细胞定位于细胞核、细胞质和线粒体。结构域预测结果显示,水牛PTHLH蛋白包含有1个PTH区域,同时还包含有1个低复杂度区域。二级结构分析显示,水牛PTHLH蛋白包含83个α-螺旋(46.89%)、17个延伸链(9.60%)、10个β-转角(5.66%)和67个无规则卷曲(37.85%),与三级结构预测结果相一致。试验构建了PTHLH基因真核表达载体pcDNA3.1-PTHLH,并通过电泳和测序验证了载体的准确性。PTHLH基因的成功克隆及其真核表达载体的成功构建为今后研究水牛PTHLH基因的功能和遗传特性提供了材料。
In order to reveal the effect of parathyroid hormone-like hormone (PTHLH) gene on the reproductive ability of buffalo,buffalo PTHLHgene was cloned,and its nucleotide and amino acid sequences were analyzed by bioinformatics.Primers were designed by CE Design according to the sequence of cattle PTHLHgene in GenBank (accession No.:NM_001290949).The coding region of buffalo PTHLH gene was cloned by PCR amplification and sequencing technology.The primary structure,secondary structure,tertiary structure and physicochemical properties of PTH-LH protein,and homology were analyzed,and phylogenetic tree of PTHLH was constructed by softwares such as DNAMAN,ProtParam,SOPMA and PSORTⅡ Prediction,et al.The results showed that PTHLHgene in buffalo was cloned,including a 534 bp length coding region and 177 amino acids.Compared with cattle,pigs,horses,goats,sheep and camels,the nucleotide sequence of gene coding region homology of PTHLH gene in buffalo were 98.3%,90.4%,90.1%,98.1%,97.5%and 89.2%,respectively.Thus,there was a high homology among different species,and the phyletic evolution was the same as their genetic relationship.The research indicated that PTHLH gene was conservative in the course of evolution.The physicochemical properties analysis result showed that the molecular formula of PTHLH protein was C895 H1451 N271 O266 S2,the molecular weight was 2 885 u,the half-life was 30 h,the theoretical electrical points (pI) was10.00,the extinction coefficient of water solution at 280 nm was 23 950,the peptide chain N-terminal was Met,the unstable coefficient was 60.04,thus PTHLH protein belong to the basic unstable protein.The fat coefficient of PTHLH protein was 72.15,and the total average hydrophilicity was-0.928,so it belonged to insoluble protein.The protein subpopulations were located in the nucleus,cytoplasm and mitochondria.The structural domain prediction results showed that buffalo PTHLH protein contained a PTH region and a low complexity region.The secondary structure analysis showed that buffalo PTHLH protein contained 83 alpha helix (46.89%),17 extended chain (9.60%),10 beta turn (5.66%) and 67 random coil (37.85%),and consistent with the tertiary structure prediction.In this study,the eukaryotic expression vector was constructed,and the accuracy of the carrier was verified by electrophoresis and sequencing.In summary,the successful cloning of PTHLH gene and the successful construction of its eukaryotic expression vector provide materials for the future study of the function and genetic characteristics of PTHLH gene in buffalo.
In order to reveal the effect of parathyroid hormone-like hormone (PTHLH) gene on the reproductive ability of buffalo,buffalo PTHLHgene was cloned,and its nucleotide and amino acid sequences were analyzed by bioinformatics.Primers were designed by CE Design according to the sequence of cattle PTHLHgene in GenBank (accession No.:NM_001290949).The coding region of buffalo PTHLH gene was cloned by PCR amplification and sequencing technology.The primary structure,secondary structure,tertiary structure and physicochemical properties of PTH-LH protein,and homology were analyzed,and phylogenetic tree of PTHLH was constructed by softwares such as DNAMAN,ProtParam,SOPMA and PSORTⅡ Prediction,et al.The results showed that PTHLHgene in buffalo was cloned,including a 534 bp length coding region and 177 amino acids.Compared with cattle,pigs,horses,goats,sheep and camels,the nucleotide sequence of gene coding region homology of PTHLH gene in buffalo were 98.3%,90.4%,90.1%,98.1%,97.5%and 89.2%,respectively.Thus,there was a high homology among different species,and the phyletic evolution was the same as their genetic relationship.The research indicated that PTHLH gene was conservative in the course of evolution.The physicochemical properties analysis result showed that the molecular formula of PTHLH protein was C895 H1451 N271 O266 S2,the molecular weight was 2 885 u,the half-life was 30 h,the theoretical electrical points (pI) was10.00,the extinction coefficient of water solution at 280 nm was 23 950,the peptide chain N-terminal was Met,the unstable coefficient was 60.04,thus PTHLH protein belong to the basic unstable protein.The fat coefficient of PTHLH protein was 72.15,and the total average hydrophilicity was-0.928,so it belonged to insoluble protein.The protein subpopulations were located in the nucleus,cytoplasm and mitochondria.The structural domain prediction results showed that buffalo PTHLH protein contained a PTH region and a low complexity region.The secondary structure analysis showed that buffalo PTHLH protein contained 83 alpha helix (46.89%),17 extended chain (9.60%),10 beta turn (5.66%) and 67 random coil (37.85%),and consistent with the tertiary structure prediction.In this study,the eukaryotic expression vector was constructed,and the accuracy of the carrier was verified by electrophoresis and sequencing.In summary,the successful cloning of PTHLH gene and the successful construction of its eukaryotic expression vector provide materials for the future study of the function and genetic characteristics of PTHLH gene in buffalo.
引文
[1]NEGLIA G,GASPARRINI B,VECCHIO D,et al.Long term effect of ovum pick-up in buffalo species[J].Reproduction in Domestic Animals,2011,123(3-4):180-186.
[2]WEIR E C,PHILBRICK W M,AMLING M,et al.Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(19):10240-10245.
[3]MIAO D,LI J,XUE Y,et al.Parathyroid hormonerelated peptide is required for increased trabecular bone volume in parathyroid hormone-null mice[J].Endocrinology,2004,145(8):3554.
[4]MARTIN T J.Osteoblast-derived PTHrP is a physiological regulator of bone formation[J].Journal of Clinical Investigation,2005,115(9):2322-2324.
[5]PINHEIRO P L,CARDOSO J C,GOMES A S,et al.Gene structure,transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chicken[J].BMC Evolutionary Biology,2010,10(1):373.
[6]CUTHBERTSON R M,KEMP B E,BARDEN J A.Structure study of osteostatin PTHrP[Thr107](107-139)[J].Biochimica et Biophysica Acta,1999,1432(1):64.
[7]ZHAO Q,BRAUER P R,XIAO L,et al.Expression of parathyroid hormone-related peptide(PthrP)and its receptor(PTH1R)during the histogenesis of cartilage and bone in the chicken mandibular process[J].Journal of Anatomy,2002,201(2):137.
[8]KLOPOCKI E,HENNIG B P,DATHE K,et al.Deletion and point mutations of PTHLH cause brachydactyly type E[J].American Journal of Human Genetics,2010,86(3):434-439.
[9]THIEDE M A,DAIFOTIS A G,WEIR E C,et al.Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in preterm rat myometrium[J].Proceedings of the National Academy of Sciences of the United States of America,1990,87(18):6969-6973.
[10]FERGUSON J E,SEANER R,BRUNS D E,et al.Expression of parathyroid hormone-related protein and its receptor in human umbilical cord:Evidence for aparacrine system involving umbilical vessels[J].American Journal of Obstetrics&Gynecology,1994,170(4):1024-1026.
[11]VASAVADA R C,CAVALIERE C,D’ERCOLE A J,et al.Overexpression of parathyroid hormone-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia,hyperinsulinemia,and hypoglycemia[J].Journal of Biological Chemistry,1996,271(2):1200-1208.
[12]WOJCIK S F,SCHANBACHER F L,MCCAULEY L K,et al.Cloning of bovine parathyroid hormone-related protein(PTHrP)cDNA and expression of PTHrPmRNA in the bovine mammary gland[J].Journal of Molecular Endocrinology,1998,20(2):271-280.
[13]AYA K,TANAKA H,ICHINOSE Y,et al.Expression of parathyroid hormone-related peptide messenger ribonucleic acid in developing kidney[J].Kidney International,1999,55(5):1696-1703.
[14]LUPARELLO C,SIRCHIA R,PUPELLO D.PTHrP[67-86]regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinaseplasminogen activator and MMP-1expression[J].Journal of Cell Science,2003,116(12):2421-2430.
[15]MASSFELDER T,DANN P,WU T L,et al.Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells:A critical role for nuclear targeting[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(25):13630-13635.
[16]AARTS M M,LEVY D,HE B,et al.Parathyroid hormone-related protein interacts with RNA[J].Journal of Biological Chemistry,1999,274(8):4832-4838.
[17]AARTS M M,RIX A,GUO J,et al.The nucleolar targeting signal(NTS)of parathyroid hormone related protein mediates endocytosis and nucleolar translocation[J].Journal of Bone&Mineral Research,1999,14(9):1493-1503.
[18]PACHE J C,BURTON D W,DEFTOS L J,et al.A carboxyl leucine-rich region of parathyroid hormone-related protein is critical for nuclear export[J].Endocrinology,2006,147(2):990-998.
[19]MONIZ C,BURTON P B,MALIK A N,et al.Parathyroid hormone-related peptide in normal human fetal development[J].Journal of Molecular Endocrinology,1990,5(3):259-266.
[20]SENIOR P V,HEATH D A,BECK F.Expression of parathyroid hormone-related protein mRNA in the rat before birth:Demonstration by hybridization histochemistry[J].Journal of Molecular Endocrinology,1991,6(3):281-290.
[21]郭晓莉.PTHLH在鸡卵泡发育中的功能、表达调控及其与产蛋性能的关系[D].泰安:山东农业大学,2017.GUO X L.Function and transcriptional regulation of parathyroid hormone-like hormone gene during follicle growth and its genetic effects on egg production traits in hens[D].Tai’an:Shandong Agricultural University,2017.(in Chinese)
[22]邓廷贤,庞春英,陈明棠,等.水牛信号转导子和转录激活子1基因电子克隆及序列分析[J].中国畜牧兽医,2014,41(8):9-13.DENG T X,PANG C Y,CHEN M T,et al.The electron cloning and sequence analysis of signal transduction and transcription activator 1genes in buffalo[J].China Animal Husbandry&Veterinary Medicine,2014,41(8):9-13.(in Chinese)
[23]马素珍,张方方,刘丹丹,等.人TRIM28基因及蛋白质的生物信息学分析[J].生物技术,2016,5:473-481.MA S Z,ZHANG F F,LIU D D,et al.Bioinformatics analysis of human TRIM28gene and protein[J].Biotechnology,2016,5:473-481.(in Chinese)
[24]孙伟,李达,苏锐,等.绵羊YAP1基因全长cDNA克隆及生物信息学分析[J].中国农业科学,2013,46(8):1725-1735.SUN W,LI D,SU R,et al.Full-length cDNA cloning and bioinformatics analysis of sheep YAP1gene[J].Chinese Agricultural Science,2013,46(8):1725-1735.(in Chinese)
[25]孙雪婧,杜晓华,杨孝朴,等.牦牛CYGB基因CDS区克隆与生物信息学分析[J].中国农业科学,2014,47(13):2690-2698.SUN X J,DU X H,YANG X P,et al.Cloning of CDSregion of yak CYGBgene and bioinformatics analysis[J].Chinese Agricultural Science,2014,47(13):2690-2698.(in Chinese)
[26]杨振宇.山羊PTHrP基因的克隆、原核表达和多克隆抗体制备[D].杨凌:西北农林科技大学,2010.YANG Z Y.Cloning,prokaryotic expression and polyclonal antibody preparation of goat PTHrP gene[D].Yangling:Northwest A&F Technology University,2010.(in Chinese)
[27]VAN DE STOLPE A,KARPERIEN M,LWIK C W,et al.Parathyroid hormone-related peptide as an endogenous inducer of parietal endoderm differentiation[J].Journal of Cell Biology,1993,120(1):235-243.
[28]WATSON P H,WESTHUSIN M E,WATSON A J.Expression of PTHrP and PTHR(PTH/PTHrP-r)mRNAs and polypeptides in bovine ovary and stimulation of bovine blastocyst development in vitro following PTHrP treatment during oocyte maturation[J].Anatomy&Embryology,2001,203(3):175-184.
[29]NOWAK R A,HAIMOVICI F,BIGGERS J D,et al.Transforming growth factor-beta stimulates mouse blastocyst outgrowth through a mechanism involving parathyroid hormone-related protein[J].Biology of Reproduction,1999,60(1):85-93.
[30]郭磊.甲状旁腺激素样激素(PTHLH)在小鼠卵母细胞成熟及着床前胚胎发育过程中的作用[D].北京:中国科学院,2011.GUO L.The role of parathyroid hormone-like hormone(PTHLH)during mouse oocytes maturation and preimplantation embryonis development[D].Beijing:Chinese Academy of Sciences,2011.(in Chinese)
[31]ERBACH G T,BIGGERS J D,MANNING P C,et al.Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching[J].Journal of Assisted Reproduction&Genetics,2013,30(8):1009-1015.
[2]WEIR E C,PHILBRICK W M,AMLING M,et al.Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(19):10240-10245.
[3]MIAO D,LI J,XUE Y,et al.Parathyroid hormonerelated peptide is required for increased trabecular bone volume in parathyroid hormone-null mice[J].Endocrinology,2004,145(8):3554.
[4]MARTIN T J.Osteoblast-derived PTHrP is a physiological regulator of bone formation[J].Journal of Clinical Investigation,2005,115(9):2322-2324.
[5]PINHEIRO P L,CARDOSO J C,GOMES A S,et al.Gene structure,transcripts and calciotropic effects of the PTH family of peptides in Xenopus and chicken[J].BMC Evolutionary Biology,2010,10(1):373.
[6]CUTHBERTSON R M,KEMP B E,BARDEN J A.Structure study of osteostatin PTHrP[Thr107](107-139)[J].Biochimica et Biophysica Acta,1999,1432(1):64.
[7]ZHAO Q,BRAUER P R,XIAO L,et al.Expression of parathyroid hormone-related peptide(PthrP)and its receptor(PTH1R)during the histogenesis of cartilage and bone in the chicken mandibular process[J].Journal of Anatomy,2002,201(2):137.
[8]KLOPOCKI E,HENNIG B P,DATHE K,et al.Deletion and point mutations of PTHLH cause brachydactyly type E[J].American Journal of Human Genetics,2010,86(3):434-439.
[9]THIEDE M A,DAIFOTIS A G,WEIR E C,et al.Intrauterine occupancy controls expression of the parathyroid hormone-related peptide gene in preterm rat myometrium[J].Proceedings of the National Academy of Sciences of the United States of America,1990,87(18):6969-6973.
[10]FERGUSON J E,SEANER R,BRUNS D E,et al.Expression of parathyroid hormone-related protein and its receptor in human umbilical cord:Evidence for aparacrine system involving umbilical vessels[J].American Journal of Obstetrics&Gynecology,1994,170(4):1024-1026.
[11]VASAVADA R C,CAVALIERE C,D’ERCOLE A J,et al.Overexpression of parathyroid hormone-related protein in the pancreatic islets of transgenic mice causes islet hyperplasia,hyperinsulinemia,and hypoglycemia[J].Journal of Biological Chemistry,1996,271(2):1200-1208.
[12]WOJCIK S F,SCHANBACHER F L,MCCAULEY L K,et al.Cloning of bovine parathyroid hormone-related protein(PTHrP)cDNA and expression of PTHrPmRNA in the bovine mammary gland[J].Journal of Molecular Endocrinology,1998,20(2):271-280.
[13]AYA K,TANAKA H,ICHINOSE Y,et al.Expression of parathyroid hormone-related peptide messenger ribonucleic acid in developing kidney[J].Kidney International,1999,55(5):1696-1703.
[14]LUPARELLO C,SIRCHIA R,PUPELLO D.PTHrP[67-86]regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinaseplasminogen activator and MMP-1expression[J].Journal of Cell Science,2003,116(12):2421-2430.
[15]MASSFELDER T,DANN P,WU T L,et al.Opposing mitogenic and anti-mitogenic actions of parathyroid hormone-related protein in vascular smooth muscle cells:A critical role for nuclear targeting[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(25):13630-13635.
[16]AARTS M M,LEVY D,HE B,et al.Parathyroid hormone-related protein interacts with RNA[J].Journal of Biological Chemistry,1999,274(8):4832-4838.
[17]AARTS M M,RIX A,GUO J,et al.The nucleolar targeting signal(NTS)of parathyroid hormone related protein mediates endocytosis and nucleolar translocation[J].Journal of Bone&Mineral Research,1999,14(9):1493-1503.
[18]PACHE J C,BURTON D W,DEFTOS L J,et al.A carboxyl leucine-rich region of parathyroid hormone-related protein is critical for nuclear export[J].Endocrinology,2006,147(2):990-998.
[19]MONIZ C,BURTON P B,MALIK A N,et al.Parathyroid hormone-related peptide in normal human fetal development[J].Journal of Molecular Endocrinology,1990,5(3):259-266.
[20]SENIOR P V,HEATH D A,BECK F.Expression of parathyroid hormone-related protein mRNA in the rat before birth:Demonstration by hybridization histochemistry[J].Journal of Molecular Endocrinology,1991,6(3):281-290.
[21]郭晓莉.PTHLH在鸡卵泡发育中的功能、表达调控及其与产蛋性能的关系[D].泰安:山东农业大学,2017.GUO X L.Function and transcriptional regulation of parathyroid hormone-like hormone gene during follicle growth and its genetic effects on egg production traits in hens[D].Tai’an:Shandong Agricultural University,2017.(in Chinese)
[22]邓廷贤,庞春英,陈明棠,等.水牛信号转导子和转录激活子1基因电子克隆及序列分析[J].中国畜牧兽医,2014,41(8):9-13.DENG T X,PANG C Y,CHEN M T,et al.The electron cloning and sequence analysis of signal transduction and transcription activator 1genes in buffalo[J].China Animal Husbandry&Veterinary Medicine,2014,41(8):9-13.(in Chinese)
[23]马素珍,张方方,刘丹丹,等.人TRIM28基因及蛋白质的生物信息学分析[J].生物技术,2016,5:473-481.MA S Z,ZHANG F F,LIU D D,et al.Bioinformatics analysis of human TRIM28gene and protein[J].Biotechnology,2016,5:473-481.(in Chinese)
[24]孙伟,李达,苏锐,等.绵羊YAP1基因全长cDNA克隆及生物信息学分析[J].中国农业科学,2013,46(8):1725-1735.SUN W,LI D,SU R,et al.Full-length cDNA cloning and bioinformatics analysis of sheep YAP1gene[J].Chinese Agricultural Science,2013,46(8):1725-1735.(in Chinese)
[25]孙雪婧,杜晓华,杨孝朴,等.牦牛CYGB基因CDS区克隆与生物信息学分析[J].中国农业科学,2014,47(13):2690-2698.SUN X J,DU X H,YANG X P,et al.Cloning of CDSregion of yak CYGBgene and bioinformatics analysis[J].Chinese Agricultural Science,2014,47(13):2690-2698.(in Chinese)
[26]杨振宇.山羊PTHrP基因的克隆、原核表达和多克隆抗体制备[D].杨凌:西北农林科技大学,2010.YANG Z Y.Cloning,prokaryotic expression and polyclonal antibody preparation of goat PTHrP gene[D].Yangling:Northwest A&F Technology University,2010.(in Chinese)
[27]VAN DE STOLPE A,KARPERIEN M,LWIK C W,et al.Parathyroid hormone-related peptide as an endogenous inducer of parietal endoderm differentiation[J].Journal of Cell Biology,1993,120(1):235-243.
[28]WATSON P H,WESTHUSIN M E,WATSON A J.Expression of PTHrP and PTHR(PTH/PTHrP-r)mRNAs and polypeptides in bovine ovary and stimulation of bovine blastocyst development in vitro following PTHrP treatment during oocyte maturation[J].Anatomy&Embryology,2001,203(3):175-184.
[29]NOWAK R A,HAIMOVICI F,BIGGERS J D,et al.Transforming growth factor-beta stimulates mouse blastocyst outgrowth through a mechanism involving parathyroid hormone-related protein[J].Biology of Reproduction,1999,60(1):85-93.
[30]郭磊.甲状旁腺激素样激素(PTHLH)在小鼠卵母细胞成熟及着床前胚胎发育过程中的作用[D].北京:中国科学院,2011.GUO L.The role of parathyroid hormone-like hormone(PTHLH)during mouse oocytes maturation and preimplantation embryonis development[D].Beijing:Chinese Academy of Sciences,2011.(in Chinese)
[31]ERBACH G T,BIGGERS J D,MANNING P C,et al.Localization of parathyroid hormone-related protein in the preimplantation mouse embryo is associated with events of blastocyst hatching[J].Journal of Assisted Reproduction&Genetics,2013,30(8):1009-1015.