版纳微型猪近交系STRA8基因克隆及功能生物信息学分析
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摘要
【目的】以版纳微型猪近交系(Banna mini-pig inbred line, BMI)为材料,初步研究STRA8基因的功能,为该基因在猪精子形成过程的作用研究奠定基础。【方法】以GenBank下载的猪STRA8基因序列JQ965783及多条猪EST序列为参考序列,设计特异性引物扩增版纳微型猪近交系(BMI) STRA8基因。对STRA8蛋白质进行理化特性和功能生物信息学分析,包括二级结构、蛋白功能域、疏水性、跨膜结构、信号肽、亚细胞定位、磷酸化位点以及蛋白质功能分析;搜索并下载其他物种的STRA8蛋白质序列构建多物种系统进化树。应用实时荧光定量PCR技术检测BMI STRA8在版纳微型猪近交系15个组织中的mRNA转录表达水平。【结果】获得了包含编码序列1 101 bp的全长序列1 316 bp,编码366个氨基酸,序列已提交GenBank,基因登录号KU705622,蛋白质分子量(Mw) 41.06 ku,等电点(pI) 4.52。STRA8二级结构中α螺旋含量最高,占52.19%;含有1个蛋白功能域;在氨基端有1个HLH结构域和1个卷曲螺旋区域,在羧基端有2个低复杂度区域;疏水性结果表明STRA8基因N末端、C末端均亲水;无跨膜结构,无信号肽序列;该蛋白质位于细胞核中的概率是56.5%;STRA8蛋白共有34个磷酸化位点;系统进化结果表明:BMI与羊、水牛的相似度最高。STRA8基因mRNA在检测的BMI 15个组织中呈现差异表达现象,睾丸中表达量最高。【结论】结果将为进一步研究STRA8基因在猪精子发生方面的作用及功能提供参考。
[Purpose]The aim of this study was to obtain the function of STRA8 gene in Banna mini-pig inbred line(BMI) and lay a foundation for further study of the gene in spermatogenesis role.[Method]Referring to STRA8 JQ965783 and EST sequences of pig downloaded from GenBank,we designed specific primers and amplified Banna mini-pig inbred line(BMI) STRA8 gene. The physicochemical properties and functional bioinformatics of STRA8 protein were analysed including the secondary structure, protein domain, hydrophobicity, transmembrane structure, signal peptide,subcellular localization, phosphorylation sites and protein function. The STRA8 amino acid sequences of other species were searched and downloaded for constructing phylogenetic trees. The mRNA transcription expression levels in BMI 15 tissues was detected by real-time quantitative PCR.[Result]The result obtained the BMI STRA8 full length sequence 1 316 bp, including coding sequence 1 101 bp,with a protein of 366 amino acids, which has been submitted to GenBank with mRNA accession number KU70562. The molecular weight(Mw) and the isoelectric point(pI) of this protein were 41.06 ku and 4.52, respectively. The most of secondary structure of STRA8 protein was alpha helix, which accounted for 52.19%. The BMI STRA8 protein contained a protein functional domain, an HLH structure domain and a curly spiral region in the amino terminal, two low-complexity regions in the carboxyl terminal. Hydrophobic analysis showed that the both N-terminus and C-terminus are hydrophilic without transmembrane structure and signal peptide sequence. The probability of the protein located in the nucleus is 56.5%. The STRA8 has 34 phosphorylation sites. Phylogenetic result demonstrated that BMI has the highest similarity with sheep and water buffalo. The mRNAs of STRA8 were differential expression in the BMI 15 tissues and there was the highest expression in the testes.[Conclusion]This study will provide a reference for studying STRA8 gene function in pig spermatogenesis.
[Purpose]The aim of this study was to obtain the function of STRA8 gene in Banna mini-pig inbred line(BMI) and lay a foundation for further study of the gene in spermatogenesis role.[Method]Referring to STRA8 JQ965783 and EST sequences of pig downloaded from GenBank,we designed specific primers and amplified Banna mini-pig inbred line(BMI) STRA8 gene. The physicochemical properties and functional bioinformatics of STRA8 protein were analysed including the secondary structure, protein domain, hydrophobicity, transmembrane structure, signal peptide,subcellular localization, phosphorylation sites and protein function. The STRA8 amino acid sequences of other species were searched and downloaded for constructing phylogenetic trees. The mRNA transcription expression levels in BMI 15 tissues was detected by real-time quantitative PCR.[Result]The result obtained the BMI STRA8 full length sequence 1 316 bp, including coding sequence 1 101 bp,with a protein of 366 amino acids, which has been submitted to GenBank with mRNA accession number KU70562. The molecular weight(Mw) and the isoelectric point(pI) of this protein were 41.06 ku and 4.52, respectively. The most of secondary structure of STRA8 protein was alpha helix, which accounted for 52.19%. The BMI STRA8 protein contained a protein functional domain, an HLH structure domain and a curly spiral region in the amino terminal, two low-complexity regions in the carboxyl terminal. Hydrophobic analysis showed that the both N-terminus and C-terminus are hydrophilic without transmembrane structure and signal peptide sequence. The probability of the protein located in the nucleus is 56.5%. The STRA8 has 34 phosphorylation sites. Phylogenetic result demonstrated that BMI has the highest similarity with sheep and water buffalo. The mRNAs of STRA8 were differential expression in the BMI 15 tissues and there was the highest expression in the testes.[Conclusion]This study will provide a reference for studying STRA8 gene function in pig spermatogenesis.
引文
[1]OULAD-ABDELGHANI M,BOUILLET P,DéCIMOD,et al.Characterization of a premeiotic germ cell-specific cytoplasmic protein encoded by STRA8,a novel retinoic acid-responsive gene[J].The Journal of Cell Biology,1996,135(2):469.DOI:10.1083/jcb.135.2.469.
[2]DONG R R,YANG S J,JIAO J,et al.Characterization of STRA8 in Southern catfish(Silurus meridionalis):evidence for its role in meiotic initiation[J].BMC Molecular Biology,2013,14:11.DOI:10.1186/1471-2199-14-11.
[3]ZHOU Q,NIE R,LI Y,et al.Expression of stimulated by retinoic acid gene 8(STRA8)in spermatogenic cells induced by retinoic acid:an in vivo study in vitamin A-sufficient postnatal murine testes[J].Biology of Reproduction,2008,79(1):35.DOI:10.1095/biolreprod.107.066795.
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[5]ZHANG Y N,ZUO Q S,LIU Z Y,et al.The induction effect of Am80 and TSA on ESC differentiation via regulation of STRA8 in chicken[J].PLoS One,2015,10(11):e0140262.DOI:10.1371/journal.pone.0140262.
[6]JIA W W,CHENG D,CHEN S,et al.Retinoic acid induces myoblasts transdifferentiation into premeiotic STRA8-positive cells[J].Cell biology International,2011,35(4):365.DOI:10.1042/CBI20100610.
[7]ZHOU Q,LI Y,NIE R,et al.Expression of stimulated by retinoic acid gene 8(STRA8)and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro[J].Biology of Reproduction,2008,78(3):537.DOI:10.1095/biolreprod.107.064337.
[8]CHEN W,JIA W W,WANG K,et al.Distinct roles for CBP and p300 on the RA-mediated expression of the meiosis commitment gene STRA8 in mouse embryonic stem cells[J].PLoS One,2013,8(6):e66076.DOI:10.1371/journal.pone.0066076.
[9]KRENTZ A D,MURPHY M W,SARVER A L,et al.DMRT1 promotes oogenesis by transcriptional activation of STRA8 in the mammalian fetal ovary[J].Developmental Biology,2011,356(1):63.DOI:10.1016/j.ydbio.2011.05.658.
[10]ENDO T,ROMER K A,ANDERSON E L,et al.Periodic retinoic acid-STRA8 signaling intersects with periodic germ-cell competencies to regulate spermatogenesis[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(18):E2347.DOI:10.1073/pnas.1505683112.
[11]HOGARTH C A,EVANOFF R,SNYDER E,et al.Suppression of STRA8 expression in the mouse gonad by WIN 18446[J].Biology of Reproduction,2011,84(5):957.DOI:10.1095/biolreprod.110.088575.
[12]WANG X Y,CHEN T F,SONG C Y,et al.Cloning and expression characteristics of the pig STRA8 gene[J].International Journal of Molecular Sciences,2014,5(7):12480.DOI:10.3390/ijms150712480.
[13]WANG P,HUO H L,WANG S Y,et al.Cloning,sequence characterization,and expression patterns of members of the porcine TSSK family[J].Genetics and Molecular Research,2015,14(4):14908.DOI:10.4238/2015.October.18.56.
[14]ZENG R,ZENG Y Z.Molecular cloning and characterization of SLA-DR genes in the 133-family of the Banna mini-pig inbred line[J].Animal Genetics,2005,36(3):267.DOI:10.1111/j.1365-2052.2005.01277.x.
[15]QIN C Y,WANG P,WANG S Y,et al.Complementary DNA cloning,sequence characterization and expression analyses of a novel gene MFN1 from Chinese Banna mini-pig inbred line(BMI)[J].Research Journal of Biotechnology,2014,9(5):1.
[16]HUO J L,WANG P,ZHAO Y,et al.Molecular cloning,mRNA expression and characterization of a novel FAIM1 gene from Chinese Banna mini-pig inbred line(BMI)[J].Journal of Animal and Veterinary Advances,2012,11(8):1080.DOI:10.3923/java.2012.1080.1086.
[17]YU P,ZHANG L,LI S,et al.Screening and analysis of porcine endogenous retrovirus in Chinese Banna minipig inbred line[J].Transplantation Proceedings,2004,36(8):2485.DOI:10.1016/j.transproceed.2004.07.055.
[18]张霞,曾日彬,霍金龙,等.版纳微型猪近交系RNF4基因克隆、多组织qPCR表达及功能生物信息学分析[J].东北农业大学学报,2017,48(3):24.DOI:10.3969/j.issn.1005-9369.2017.03.004.
[19]TANG F R,YAO X,ZHU H,et al.Expression pattern of Ngn3 in dairy goat testis and its function in promoting meiosis by upregulating STRA8[J].Cell Proliferation,2014,47(1):38.DOI:10.1111/cpr.12077.
[20]YAO X,TANG F R,YU M,et al.Expression profile of Nanos2 gene in dairy goat and its inhibitory effect on STRA8 during meiosis[J].Cell Proliferation,2014,47(5):396 .DOI:10.1111/cpr.12128.
[21]BAO J Q,MA H Y,SCHUSTER A,et al.Incomplete cre-mediated excision leads to phenotypic differences between STRA8-iCre;Mov10/1lox/lox and STRA8-iCre;Mov10/1lox/Δmice[J].Genesis,2013,51(7):481.DOI:10.1002/dvg.22389.
[22]DESIMIO M G,CAMPOLO F,DOLCI S,et al.SOHLH1 and SOHLH2 directly down-regulate stimulated by retinoic acid 8(STRA8)expression[J].Cell Cycle,2015,14(7):1036.DOI:10.1080/15384101.2015.1007721.
[23]ANDERSON E L,BALTUS A E,ROEPERS-GAJADI-EN H L,et al.STRA8 and its inducer,retinoic acid,regulate meiotic initiation in both spermatogenesis and oogenesis in mice[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(39):14976.DOI:10.1073/pnas.0807297105.
[24]韩为东,赵亚力,伍志强,等.FHL2通过相互作用抑制Id2的功能活性[J].中国生物化学与分子生物学报,2008,24(9):810.
[25]顾成磊,伍志强,孟元光,等.Id2通过非HLH结构域促进MCF-7细胞的侵袭性[J].现代肿瘤医学,2008,16(12):2043.DOI:10.3969/j.issn.1672-4992.2008.12.001.
[26]米美玲,杨蓓,徐斯凡,等.Stra8:生殖细胞有丝分裂转变为减数分裂前特异表达的基因[J].中华男科学杂志,2009,15(1):54.
[2]DONG R R,YANG S J,JIAO J,et al.Characterization of STRA8 in Southern catfish(Silurus meridionalis):evidence for its role in meiotic initiation[J].BMC Molecular Biology,2013,14:11.DOI:10.1186/1471-2199-14-11.
[3]ZHOU Q,NIE R,LI Y,et al.Expression of stimulated by retinoic acid gene 8(STRA8)in spermatogenic cells induced by retinoic acid:an in vivo study in vitamin A-sufficient postnatal murine testes[J].Biology of Reproduction,2008,79(1):35.DOI:10.1095/biolreprod.107.066795.
[4]MIYAMOTO T,SENGOKU K,TAKUMA N,et al.Isolation and expression analysis of the testis-specific gene,STRA8,stimulated by retinoic acid gene 8[J].Journal of Assisted Reproduction and Genetics,2002,19(11):531.DOI:10.1023/A:1020963919596.
[5]ZHANG Y N,ZUO Q S,LIU Z Y,et al.The induction effect of Am80 and TSA on ESC differentiation via regulation of STRA8 in chicken[J].PLoS One,2015,10(11):e0140262.DOI:10.1371/journal.pone.0140262.
[6]JIA W W,CHENG D,CHEN S,et al.Retinoic acid induces myoblasts transdifferentiation into premeiotic STRA8-positive cells[J].Cell biology International,2011,35(4):365.DOI:10.1042/CBI20100610.
[7]ZHOU Q,LI Y,NIE R,et al.Expression of stimulated by retinoic acid gene 8(STRA8)and maturation of murine gonocytes and spermatogonia induced by retinoic acid in vitro[J].Biology of Reproduction,2008,78(3):537.DOI:10.1095/biolreprod.107.064337.
[8]CHEN W,JIA W W,WANG K,et al.Distinct roles for CBP and p300 on the RA-mediated expression of the meiosis commitment gene STRA8 in mouse embryonic stem cells[J].PLoS One,2013,8(6):e66076.DOI:10.1371/journal.pone.0066076.
[9]KRENTZ A D,MURPHY M W,SARVER A L,et al.DMRT1 promotes oogenesis by transcriptional activation of STRA8 in the mammalian fetal ovary[J].Developmental Biology,2011,356(1):63.DOI:10.1016/j.ydbio.2011.05.658.
[10]ENDO T,ROMER K A,ANDERSON E L,et al.Periodic retinoic acid-STRA8 signaling intersects with periodic germ-cell competencies to regulate spermatogenesis[J].Proceedings of the National Academy of Sciences of the United States of America,2015,112(18):E2347.DOI:10.1073/pnas.1505683112.
[11]HOGARTH C A,EVANOFF R,SNYDER E,et al.Suppression of STRA8 expression in the mouse gonad by WIN 18446[J].Biology of Reproduction,2011,84(5):957.DOI:10.1095/biolreprod.110.088575.
[12]WANG X Y,CHEN T F,SONG C Y,et al.Cloning and expression characteristics of the pig STRA8 gene[J].International Journal of Molecular Sciences,2014,5(7):12480.DOI:10.3390/ijms150712480.
[13]WANG P,HUO H L,WANG S Y,et al.Cloning,sequence characterization,and expression patterns of members of the porcine TSSK family[J].Genetics and Molecular Research,2015,14(4):14908.DOI:10.4238/2015.October.18.56.
[14]ZENG R,ZENG Y Z.Molecular cloning and characterization of SLA-DR genes in the 133-family of the Banna mini-pig inbred line[J].Animal Genetics,2005,36(3):267.DOI:10.1111/j.1365-2052.2005.01277.x.
[15]QIN C Y,WANG P,WANG S Y,et al.Complementary DNA cloning,sequence characterization and expression analyses of a novel gene MFN1 from Chinese Banna mini-pig inbred line(BMI)[J].Research Journal of Biotechnology,2014,9(5):1.
[16]HUO J L,WANG P,ZHAO Y,et al.Molecular cloning,mRNA expression and characterization of a novel FAIM1 gene from Chinese Banna mini-pig inbred line(BMI)[J].Journal of Animal and Veterinary Advances,2012,11(8):1080.DOI:10.3923/java.2012.1080.1086.
[17]YU P,ZHANG L,LI S,et al.Screening and analysis of porcine endogenous retrovirus in Chinese Banna minipig inbred line[J].Transplantation Proceedings,2004,36(8):2485.DOI:10.1016/j.transproceed.2004.07.055.
[18]张霞,曾日彬,霍金龙,等.版纳微型猪近交系RNF4基因克隆、多组织qPCR表达及功能生物信息学分析[J].东北农业大学学报,2017,48(3):24.DOI:10.3969/j.issn.1005-9369.2017.03.004.
[19]TANG F R,YAO X,ZHU H,et al.Expression pattern of Ngn3 in dairy goat testis and its function in promoting meiosis by upregulating STRA8[J].Cell Proliferation,2014,47(1):38.DOI:10.1111/cpr.12077.
[20]YAO X,TANG F R,YU M,et al.Expression profile of Nanos2 gene in dairy goat and its inhibitory effect on STRA8 during meiosis[J].Cell Proliferation,2014,47(5):396 .DOI:10.1111/cpr.12128.
[21]BAO J Q,MA H Y,SCHUSTER A,et al.Incomplete cre-mediated excision leads to phenotypic differences between STRA8-iCre;Mov10/1lox/lox and STRA8-iCre;Mov10/1lox/Δmice[J].Genesis,2013,51(7):481.DOI:10.1002/dvg.22389.
[22]DESIMIO M G,CAMPOLO F,DOLCI S,et al.SOHLH1 and SOHLH2 directly down-regulate stimulated by retinoic acid 8(STRA8)expression[J].Cell Cycle,2015,14(7):1036.DOI:10.1080/15384101.2015.1007721.
[23]ANDERSON E L,BALTUS A E,ROEPERS-GAJADI-EN H L,et al.STRA8 and its inducer,retinoic acid,regulate meiotic initiation in both spermatogenesis and oogenesis in mice[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(39):14976.DOI:10.1073/pnas.0807297105.
[24]韩为东,赵亚力,伍志强,等.FHL2通过相互作用抑制Id2的功能活性[J].中国生物化学与分子生物学报,2008,24(9):810.
[25]顾成磊,伍志强,孟元光,等.Id2通过非HLH结构域促进MCF-7细胞的侵袭性[J].现代肿瘤医学,2008,16(12):2043.DOI:10.3969/j.issn.1672-4992.2008.12.001.
[26]米美玲,杨蓓,徐斯凡,等.Stra8:生殖细胞有丝分裂转变为减数分裂前特异表达的基因[J].中华男科学杂志,2009,15(1):54.