猪Sox9基因cDNA的全长克隆及功能预测
|
摘要
Sox基因是由众多富含HMG盒DNA绑定蛋白区域的基因家族,Sox9属于Sox家族成员之一,Sox9是与早期胚胎发育有关的基因,是与位于雄性动物Y染色体上的Sry相关的睾丸决定因子,可与DNA序列特异结合,在很多动物中都有表达,因此在进化上非常保守。在人类SOX9突变可引起CD综合征和性反转。在睾丸和卵巢中都可以检验到Sox9mRNA6勺转录本,暗示它在性腺发育的作用,显示它在猪的性腺中可能具有同样功能。对猪Sox9基因的克隆测序和初步分析,可以为研究猪Sox9基因的生物学功能和品种改良提供基础的基因信息,也为研究人的基因功能建立动物试验模型提供参考。
本研究采用电子克隆的方法,结合RT-PCR和RACE技术,获得了猪Sox9基因的cDNA全序列。利用生物信息学方法预测了该蛋白的结构和功能,并进行了同源序列多重比对和分子系统发生分析。利用实时定量PCR(Real-time quantitative PCR)技术分析了猪Sox9基因mRNA在不同组织的相对表达水平,具体内容如下:
1 Sox9基因的克隆测序
用人的Sox9基因的cDNA全序列作为电子探针,分别从NCBI的dbEST和UniGene数据库钓取了共12条猪的EST序列,将其输入DNAstar-SeqMan程序,最后拼接出1个重叠群(Contig1)序列。在此基础上,利用Primet Premier 5.0软件手动设计引物,借助RT-PCR和RACE技术一方面验证了电子克隆的正确性;另一方面扩增出ORF和cDNA全序列。
2 Sox9蛋白的生物信息学分析
借助借助生物信息学网络资源和软件,猪Sox9基因定位于12p13-p11,预测出完整的ORF编码框,位于第357-1886bp核苷酸之间,开放阅读框的长度为1530bp,编码509个氨基酸残基;通过对Sox9启动子的预测,可能性高于0.8的有10个启动子,从第187bp开始到第1541bp为CpG岛,长度为1355bp。CpG位点有151个;通过对蛋白质序列相似性检索,预测出猪Sox9蛋白不合有信号肽,不含有跨膜区,定位于细胞核中,在509个氨基酸中从第104到174位共71个氨基酸残基构成HMG盒,功能区主要是HMG盒。发现有HMG、DnaJ、GIT、HSF和Prim-Zn-Ribbon结构域。推测猪Sox9蛋白是一种核蛋白,有DNA结合能力,参与染色质修饰和基因转录调控。
3 Real-time quantitative PCR分析Sox9基因的组织表达谱
采用实时荧光定量PCR技术,按照双标准曲线法,分析了猪Sox9不同组织中的相对表达水平,结果表明:Sox9基因在猪的不同组织中广泛表达,但表达水平存在差异。在性腺和脑组织表达水平最高,而在脾、胸腺、淋巴组织表达水平最低,说明了Sox9基因在猪性腺、脑的发育以及软骨发生的功能与作用。
Sox9 is a member of the Sox family of transcription factors and contains a DNA-binding motif known as the high-mobility-group(HMG) domain.Sox9 is a related factor about early embryo development.It is an important male animal Sry-related testis-determining gene.The encoded proteins are capable of binding to DNA in a sequence-specific manner.To date,SOX9 has been identified in many different species, thereby testifying to its conserved role inevolution.It has been shown that mutations in the human SOX9 gene cause campomelic dysplasia(CD) and autosomal sex reversa.The detection of Sox9 mRNA in both the testis and ovary of human suggests that this gene might be involved in the gonad development of pig.The cDNA cloning and preliminary analysis of porcine Sox9 gene not only provides primary information for better understanding the biological functions of Sox9 in pig,but also for the establishment of animal model to investigate human gene funtion.
In this study,using in silico cloning approach combined with reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(5' RACE and 3' RACE),we cloned the full-length cDNA of Sox9.Bioinformatics methods are adopted to predict the structure and function of Sox9 protein,and also to construct a phylogenetic tree to reveal the evolutionary relationship of various species.To check related expression levels of Sox9 mRNA in various porcine tissues,the real-time PCR was performed.The contents are as followed:
1 cDNA cloning and sequencing of porcine Sox9 gene
The full-length cDNA sequence of human SOX9 gene served as an electronic probe, which was submitted to generate BLAST reciprocal best hits from dbEST and UniGene database,and about 12 EST sequences were retrieved.Using DNAstar-SeqMan program, these ESTs were assembled into one contigs.Based on the contig,the gene specific primers were designed by Primer Premier 5.0.We carried out not only to cheek the validity of in silico approach,but also to obtain ORF and the full-length sequence of porcine Sox9 cDNA by RT-PCR and RACE-PCR.
2 Bioinformatics analysis of Sox9 protein
Using bioinformatics network resources and relevant softwares,the full-length cDNA of porcine Sox9 gene was firstly acquired,which localized on 12p13-p11 of porcine chromosome,we predict that Sox9 cDNA has a 1530bp open reading frame(ORF) from the 357bp site to the 1886bp site which coding a 509-amino acid(aa) residues.We find it could had 10 promoters and there is a big CpG island which from 187bp to 1541bp about 1355bp length containing 151 CpG sites.It contains no signal peptide,typical hydrophobic regions or transmembrane region,but the nucleoprotein residing in nucleolus.There is a HMG box which as main functional region contained of 71-amino acid(aa) residues from 104aa to 174aa of proteide sequence.A conserved domain database alignment reveals that the protein contains several conserved domains,including HMG,DnaJ,GIT,HSF and Prim-Zn-Ribbon domain.We speculate Sox9 encoded proteins are capable of binding to DNA in a sequence-specific manner,participating in chromatin modification and transcriptional regulation.
3 Real-time PCR analysis of Sox9 mRNA tissue-specific expression profiles
According to the double standard curve method,real-time fluorescence quantitative PCR was performed to analyze the relative expression levels in various tissues.The results showed that Sox9 gene was expressed broadly in various tissues,but at very different levels. The strongest expression was observed in pituitary,cartilage and gonad,whereas the lowest was seen in spleen,thymus and lymph,suggesting that Sox9 protein plays important roles in porcine gonad,brain and chondrogenesis development function.
本研究采用电子克隆的方法,结合RT-PCR和RACE技术,获得了猪Sox9基因的cDNA全序列。利用生物信息学方法预测了该蛋白的结构和功能,并进行了同源序列多重比对和分子系统发生分析。利用实时定量PCR(Real-time quantitative PCR)技术分析了猪Sox9基因mRNA在不同组织的相对表达水平,具体内容如下:
1 Sox9基因的克隆测序
用人的Sox9基因的cDNA全序列作为电子探针,分别从NCBI的dbEST和UniGene数据库钓取了共12条猪的EST序列,将其输入DNAstar-SeqMan程序,最后拼接出1个重叠群(Contig1)序列。在此基础上,利用Primet Premier 5.0软件手动设计引物,借助RT-PCR和RACE技术一方面验证了电子克隆的正确性;另一方面扩增出ORF和cDNA全序列。
2 Sox9蛋白的生物信息学分析
借助借助生物信息学网络资源和软件,猪Sox9基因定位于12p13-p11,预测出完整的ORF编码框,位于第357-1886bp核苷酸之间,开放阅读框的长度为1530bp,编码509个氨基酸残基;通过对Sox9启动子的预测,可能性高于0.8的有10个启动子,从第187bp开始到第1541bp为CpG岛,长度为1355bp。CpG位点有151个;通过对蛋白质序列相似性检索,预测出猪Sox9蛋白不合有信号肽,不含有跨膜区,定位于细胞核中,在509个氨基酸中从第104到174位共71个氨基酸残基构成HMG盒,功能区主要是HMG盒。发现有HMG、DnaJ、GIT、HSF和Prim-Zn-Ribbon结构域。推测猪Sox9蛋白是一种核蛋白,有DNA结合能力,参与染色质修饰和基因转录调控。
3 Real-time quantitative PCR分析Sox9基因的组织表达谱
采用实时荧光定量PCR技术,按照双标准曲线法,分析了猪Sox9不同组织中的相对表达水平,结果表明:Sox9基因在猪的不同组织中广泛表达,但表达水平存在差异。在性腺和脑组织表达水平最高,而在脾、胸腺、淋巴组织表达水平最低,说明了Sox9基因在猪性腺、脑的发育以及软骨发生的功能与作用。
Sox9 is a member of the Sox family of transcription factors and contains a DNA-binding motif known as the high-mobility-group(HMG) domain.Sox9 is a related factor about early embryo development.It is an important male animal Sry-related testis-determining gene.The encoded proteins are capable of binding to DNA in a sequence-specific manner.To date,SOX9 has been identified in many different species, thereby testifying to its conserved role inevolution.It has been shown that mutations in the human SOX9 gene cause campomelic dysplasia(CD) and autosomal sex reversa.The detection of Sox9 mRNA in both the testis and ovary of human suggests that this gene might be involved in the gonad development of pig.The cDNA cloning and preliminary analysis of porcine Sox9 gene not only provides primary information for better understanding the biological functions of Sox9 in pig,but also for the establishment of animal model to investigate human gene funtion.
In this study,using in silico cloning approach combined with reverse transcription polymerase chain reaction(RT-PCR) and rapid amplification of cDNA ends(5' RACE and 3' RACE),we cloned the full-length cDNA of Sox9.Bioinformatics methods are adopted to predict the structure and function of Sox9 protein,and also to construct a phylogenetic tree to reveal the evolutionary relationship of various species.To check related expression levels of Sox9 mRNA in various porcine tissues,the real-time PCR was performed.The contents are as followed:
1 cDNA cloning and sequencing of porcine Sox9 gene
The full-length cDNA sequence of human SOX9 gene served as an electronic probe, which was submitted to generate BLAST reciprocal best hits from dbEST and UniGene database,and about 12 EST sequences were retrieved.Using DNAstar-SeqMan program, these ESTs were assembled into one contigs.Based on the contig,the gene specific primers were designed by Primer Premier 5.0.We carried out not only to cheek the validity of in silico approach,but also to obtain ORF and the full-length sequence of porcine Sox9 cDNA by RT-PCR and RACE-PCR.
2 Bioinformatics analysis of Sox9 protein
Using bioinformatics network resources and relevant softwares,the full-length cDNA of porcine Sox9 gene was firstly acquired,which localized on 12p13-p11 of porcine chromosome,we predict that Sox9 cDNA has a 1530bp open reading frame(ORF) from the 357bp site to the 1886bp site which coding a 509-amino acid(aa) residues.We find it could had 10 promoters and there is a big CpG island which from 187bp to 1541bp about 1355bp length containing 151 CpG sites.It contains no signal peptide,typical hydrophobic regions or transmembrane region,but the nucleoprotein residing in nucleolus.There is a HMG box which as main functional region contained of 71-amino acid(aa) residues from 104aa to 174aa of proteide sequence.A conserved domain database alignment reveals that the protein contains several conserved domains,including HMG,DnaJ,GIT,HSF and Prim-Zn-Ribbon domain.We speculate Sox9 encoded proteins are capable of binding to DNA in a sequence-specific manner,participating in chromatin modification and transcriptional regulation.
3 Real-time PCR analysis of Sox9 mRNA tissue-specific expression profiles
According to the double standard curve method,real-time fluorescence quantitative PCR was performed to analyze the relative expression levels in various tissues.The results showed that Sox9 gene was expressed broadly in various tissues,but at very different levels. The strongest expression was observed in pituitary,cartilage and gonad,whereas the lowest was seen in spleen,thymus and lymph,suggesting that Sox9 protein plays important roles in porcine gonad,brain and chondrogenesis development function.
引文
[1]杨增明,孙青原,夏国良.生殖生物学(M).科学出版社出版.2005,352-354.
[2]王建武,丁家桐,刘春玲等.哺育动物性别决定的基因调控[J].动物科学与动物医学,2003,4:26-58.
[3]Vincent RH,Michael JC,Ant hony A,The Molecular Action and Regulation of the Testis-Determining Factors,SRY(Sex-Determining Region on the Y Chromosome) and SOX9[SRY- Related High-Mobility Group(HMG) Box 9]Endocr[J].Rev,2003,24:466-487.
[4]Koopman P,Gubbay J,Vivian N,et aL Male development of chromosomally femal mice transgenic for Sry[J].Nature,1991,351(6322):117-121.
[5]Hanley NA,Hagan DM,Clement-Jones M,et al.SRY,SOX9,and DAX1 expression patterns during human sex determination and gonadal development[J].Mech Dev,2000,91:403-407.
[6]Margarit E,Dolors-Coil M.SRY gene transferred to the Iongarm of the X chromosome in a Y-positive XX true hermaphrodite[J].Anim Genet,2000,90:25-28.
[7]Lovell-Badge R.How does SRY act in mammalian sex determination[J].The 48 th NIBB Conference,Okazaki,2003,Japan.
[8]Koichiro N,Naoko H,Satoshi T,et al.DNA Methylation-mediated Control of Sry Gene Expression in Mouse Gonadal Development[J].J Biol Chem,2004,279:22306-22313.
[9]赵勇,王文波.Sox9基因与椎间盘退变[J].中华外科杂志,2005,43:544-545.
[10]Sive JI,Baird P,Jeziorsk M,et aL Expression of chondrocyte markers by cells of normal and degenerated intervertebral discs[J].Mol Pathol,2002,55:91-97.
[11]Argentaro A,Sim H,Kelly S,et al.A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia Pautosomal sex reversal[J].J Biol Chem,2003,278(36):33839-33847.
[12]Frojdman K,Harley VR,Pelliniemi LJ.Sox9 protein in rat sertoli cells is age and stage dependent[J].Histochem Cell Bio 1,2000,113(1):31-36.
[13]Wagner T,Wirth J,Meyer J,et al.Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene Sox9[J].Cell,1994,79:1111-1120.
[14]Kent J,Wheatley SC,Andrews JE,et al.A male-specific role for Sox9 in verterbrate sex determination[j].Development,1996,122:2813-2822.
[15]Koopman P.Sry,Sox9 and mammalian sex determination[J].EXS,2001,91:25-56.
[16]Morals da Silva S,Hacker A,Harley V,et al.Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds[J].Nat Genet 1996,14:62-68.
[17]李楠,王秀利,仇雪梅.SOX、DMRTT生别决定基因家族及其应用研究进展[J].生物技术通报,2005,3:5-9.
[18]Ellisen LW.Regulation ofgene expression by WT1 in development and turnorigenesis[J].Int J Hematol,2002,76(2):110-116.
[19]MacLaughlin DT,Donahoe PK.Sex determination and differentiation[J].N Engl J Med,2004,350(4):367-378.
[20]Anwar H,Grady F.Saunders:Role of Wilms Tumor 1(WT1) in the Transcriptional Regulation of the Mullerian-Inhibiting Substance Promoter[j].Biol Reproduct,2003,69(6):1808~1814.
[21]Matsuzawa-Watanabe Y,Inoue J,Semba K.Transcriptional activity of Testis-determining factor SRY is modulated by the Wilms'tumor 1 gene product,WT1[J].Oncogene,2003,22(39):7900-7904.
[22]Rey R,Lukas-Croisier C,Lasala C,et al.AMH/MIS:what we know already about the gene,the protein and its regulation[J].Mol Cell,Endocrinol,2003,211(1-2):21-31.
[23]Amiel J,Gigarel N,Benacki A.Prenatal diagnosis of respiratory chain deficiency by direct mutation screening[J].Prenat Diagn,2001,21(7):602--604.
[24]Niers L,vanden Heuvel L,Trijbels F,et al.Prerequisites and strategies for prenatal diagnosis of respiratory chain deficiency in chorionic villi[J].J Inherit Metab Dis,2003,26(7):647-658.
[25]Meeks JJ,Weiss J,Jameson JL.Daxl is required for testis determination[J].Nat Genet,2003,34(1):32-33.
[26]周林燕,张修月,王德寿.动物学研究,2004,25(1):81-88.
[27]Seibel P,Trappe J,Villani G,et al.Transfection of mitochondria:strategy towards a gene therapy of mitochondrial DNA diseases[J].NucleicAcids Res,1995,23(1):10-17.
[28]Klaus Kalthoff.Analysis of Biological Development[M].2001,2nd edition,。
[29]何巧平,孔祥清.WNT4基因在房间隔缺损患者心肌组织中的表达.南京医科大学学报[J].2005,25(11):810-812.
[30]Qin Y,Bishop CE.Sox9 is sufficient for functional testis development producing fertile male mice in the absence of Sry[J].Hum Mol Genet,2005,14(9):1221-1229.
[31]Jeays-Ward K,Hoyle C,Brennan J,et al.Endothelial and steroido-genic cell migration are regulated by WNT4 in the developing mammalian gonad[J].Development,2003,130(16):3663-3670.
[32]Anwar H,Grady F.Synergistic cooperation between the β-catenin signaling pathway and steroidogenic fatter 1 in the activation of the mullerian inhibiting substance type Ⅱ type receptor[J].Biol Chem,2003,278(29):26511-26516.
[33]Chardard D,Chesnel A,Goze C,et al.Pw Sox-1:the first member of the Sox gene family in Urodeles[J].Nucleic Acids Research,1993,21(15):3576.
[34]Wegner M.From head to toes:The multiple facets of Sox proteins[J].Nucleic Acids Res,1999,27(6):1409-1420.
[35]Foster JW et al.Sox-9 gene and protein and use in the regeneration of bone or cartilage[J].Nature,1994,372:525
[36]Sudbeck P,Schmitz ML,Baeuerle PA,et al.Sex reversal by loss of the C-terminal transactivation domain of human SOX9[J].Nat Genet,1996,13:230-232.
[37]Subdeck P,Scherer G..Two independent nuclear localization signals are present in the DNA-binding high-mobility group domains of SRY and SOX9[J].J Biol Chem,1997 OCT31,272(44):27848-27852.
[38]de Santa Barbara P,Moniot B,Poulat F,et al..Expression and subcellular localization of SF-1,SOX9,WT1 and AMH proteins during early human testicular development[J].Dev Dyn,2000,217(3):293-298.
[39]Argentaro A,Sim H,Kelly S,et al.A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal[J].J Biol Chem,2003,278(36):33839-33847.
[40]Morals da Silva S,Hacker A,Harley V,et aL SOX9 expression during gonadal development mplies a conserved role for the gene in testis differentiation in mammals and birds[J]. Nat Genet, 1996, 14:62-68.
[41] Jennifer A, Mersshll G Evolution of the mammalian Y chromosome and sex-determining genes[J].J Exp Zool, 1998,281:472-481.
[42] Pfeifer D, Poulat F, Holinski-Feder E, et al. The SOX8 gene is located within 700 kb of the tip of chromosome 16p and is deleted in a patient with ATR-16 syndrome[J]. Genomics, 2000, 63(1):108-116.
[43] Takada S, Koopman P. Regulation of Amh during sex determination in chickens: Sox gene expression in male and female gonads[J]. Cytogenet Genome Res, 2003, 101(3-4): 212-218.
[44] Schepers G, Wilson M, Wilhelm D, et al. SOX8 is expressed during testis differentiation in mice and synergizes with SF1 to activate the Amh promoter in vitro[J]. J Biol Chem, 2003,278(30):28101-28108.
[45] Akiyama H, Chaboissier MC, Martin JF, et al. The transcription facter Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and reqired for expression of Sox5 and Sox6[J]. Gene Dev, 2002, 16:2813-2828.
[46] Torres-M aldonado LC, Landa PA, et al. General & Comparative Endocrinology, 2002,129(1):20-26.
[47] JussiT IS. In vitro cartilage formation by human adult stem cells From bone marrow stroma defines the sequence of cellular and molecuLar events during chondrogenesis. PNAS, 2002, 99(7):4397-4402.
[48] Lefebvre V, Behringer RR, de Crombrugghe B.L-Sox5,Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway[J].Osteoarthritis and cartilage, 2001, S9A: s69-75.
[49] 赵勇,王文波.Sox9基因与椎间盘退变.中华外科杂志,2005, 43: 544-545.
[50] Sive JI, Baird P, Jeziorsk M, et al. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs[J]. Mol Pathol, 2002, 55(2):91-97.
[51] Lefebvre V, Huang W, Harley VR, et al... SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alphal(II) collagen gene[J].Mol. Cell. Biol, 1997, 17:2336-2346.
[52] Huang WD, Veronique Lefebvre XZ, de Crombrugghe B. Phosphorylation of SOX9 by Cyclic AMP-Dependent Protein Kinase A Enhances SOX9's Ability To Transactivate a Col2al Chondrocyte-Specific Enhancer[J]. Molecular and Cellular Biology, 2000,20(11): 4149-4158.
[53] Chen CW, Tsai YH, Deng WP, et al.Type I and II collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells[J]. J Orthop Res, 2005, 23(2): 446-453.
[54] Gruber HE, Norton HJ, Ingram JA, et al. The SOX9 transcription factor in the human disc: decreased immunolocalization with age and disc degeneration. Spine, 2005, 30(6): 625-630.
[55] Rozeman LB, Hameetman L, Cleton-Jansen AM, et al.Absence of IHH and retention of PTHrP signalling in enchondromas and central chondrosarcomas. J Pathol, 2005, 205(4): 476-482.
[56] Smits P, Li P, Mandel J, et al. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation[J]. Dev Cell, 2001, 1: 277-290.
[57] Lefebvre V & Smits P. Transcriptional control of chondrocytefate and differentiation[J]. Birth Defects Research, Part C:Embryo Today: Reviews, 2005, 75, 200-212.
[58] Ikeda T, Kawaguchi H, Kamekura S, et al. Distinct roles of Sox5, Sox6, and Sox9 in different stages of chondrogenic differentiation[J]. J Bone Miner Metab, 2005, 23: 337-340.
[59]Stolt CC,SchlierfA,Lommes P,et al.SoxD Proteins Influence Multiple Stages of Oligodendrocyte Development and Modulate SoxE Protein Function[J].Developmental Cell 2006,11:697-709.
[60]Yommerup N,Sehempp W,Meinecke P,et al.Assignment of an autosomal sex reversal locus (SRA 1) and eampomelic dysplasia(CMPD1).to 17q24.3-q25.1.Nat Genet,1993,4(2):170-174.
[61]Smith JM,Koopman PA.The ins and outs of transcriptional control:nucleocytoplasmie shuttling in development and disease[J].Trends Genet,2004,20(1):4-8.
[62]Lee YH,Aold Y,Hong CS,Saint-Germain N,et al.Early requirement of the transcriptional activator Sox9 for neural crest specification in Xenopus[J].Dev Biol,2004,275(1):93-103.
[63]Sottile V,Li M,Seotting PJ.Stem cell marker expression in the Bergmann glia population of the adult mouse brain[J].Brain Res,2006,1099(1):8-17.
[64]Montero JA,Giron B,Arrechedera H,et al.Expression of SoxS,Sox9 and Soxl0 in the developing valves and autonomic nerves of the embryonic heart[J].Mech Dev,2002,118(1-2):199-202.
[65]GoffSA,Ricke D,Lan TH,et al.A.draft sequence of the rice genome(Oryza sativa L.ssp.japonica)[J].Science,2002,296:92-100,
[66]Boguski MS,Tolstoshev CM,Bassett DE.Gene discovery in dbEST.Science 1994,265(5181):1993-1994
[67]Vitt U,Gietzen D,Stevens K,et al.Identification of can-didate disease genes by EST alignments,synteny,and expression and verification of Ensembl genes on rat chromosome lq43-54.Genome Res 2004,14(4):640-650
[68]Jongeneel CV.Searching.the expressed sequence tag(EST) databases:Panning for genes.Brieafings in Bioinformatics 2000,1:76-79.
[69]Schuler.Pieces of the puzzle:expressed sequence tags and the catalog of human genes.J Mol Med 1997,75(10):694-698
[70]Gasteiger E,Hoogland C.Protein Identification and Analysis Tools on the ExPASy Server,in Walker JM ed:The Proteomics Protocols Handbook.Humana Press 2005,571-607.
[71]Wilsker D,Patsialou A,Dallas PB,et al.ARID proteins:a diverse family of DNA binding proteins implicated in the control of cell growth,differentiation,and development.Cell Growth Differ 2002,13:95-106
[72]Bendtsen JD,Nielsen H,Von Heijne G.,Brunak S.Improved prediction of signal peptides:SignalP 3.0.J Mol Biol 2004,340:783-795
[73]Marchler-Baner A,Bryant SH.CD-Search:protein domain annotations on the fly.Nucleic Acids Res 2004,32:327-331
[74]常重杰,杜启艳,邵红伟.Sox基因家族研究的新进展.遗传,2002,24(4):470-476.
[75]Lefebvre V,Dumitriu B,M'endez AP,et al.Control of cell fate and differentiation by Sry-related high-mobility-group box(Sox) transcription factors.The International Journal of Biochemistry &Cell Biology,2007,39:2195-2214.
[76]杨坚,正常猪外周血淋巴细胞cDNA文库部分EST序列的电子克隆及初步实验验证,[硕士学位论文],广西大学硕士论文,2006.
[77]田兴国,猪肌肉组织EST的基因克隆与表达分析。[硕士学位论文],广州,华南农业大学,2004.
[78]Gasteiger E,Gattiker A.ExPasy:the proteomics server for in-depth protein knowledge and analysis.Nucleic Acids Res 2003,31:3784-3788.
[79]Moiler S,Croning MD,Apweiler R.Evaluation of methods for the prediction of membrane spanning regions.Bioinformatics 2001,17(7):646-653.
[80]Nakai K,Horton P.PSORT:a program for detecting the sorting signals of proteins and predicting their subcellular localization.Trends Biochem Sci 1999,24(1):34-35.
[81]袁红梅,张丽,张虹,黄永红.SOX9基因的研究进展.黑龙江农业科学,2007,(3):124-127.
[82]Vidal VP,Chaboissier MC,De Rooij DG,et al.Sox9 induces testis development in XX transgenic mice.Nature Genetic,2001,28:216-217.
[83]Erta P,Hawkins JR,Sinclair AH,et al.Genetic evidence equating SRY and the testis-determining factor.Nature,1990,348(6300):448-450.
[84]Clarkson MJ,Harley VR.Sex with two Sox on:SRY and SOX9 in testis development.Trend in Endocrinology & Metabolism,2002,13(3):106~111.
[85]张勇,陈淳,顾建新等.哺乳动物性别决定的研究进展.生物工程进展,2001,21(3):26-29.
[86]Foster JW,Granes JA.An SRY-related sequence on the Marsupial X Chromosome:Implications of the evolution of the mammalian testis-determining gene.Proc Natl Acad Sci USA,1994,91:1927-1931.
[87]朱必才,高建国,张子峰等.哺乳动物性别决定及其机制的研究.细胞生物学杂志,2002,24(5):282-286.
[88]Morais da Silva S,Hacker A,Harley V,et al.SOX9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds.Nat Genet,1996,14:62-68.
[89]Sudbeck P,Schmitz ML,Baeuerle PA,et al.Sex reversal by loss of the C-terminal transactivation domain of human SOX9.Nat Genet,1996,13:230-232.
[90]Wright EM,Snopek B,Koopan P.Seven new members of the Sox gene family expression during mouse development.Nucleic Acids Res,1993,21:774.
[90]Zanaria E,Muscatelli F,Bardoni B,et al.A novel and unusual member of the nuclear hormone receptor super family is responsible for X-linked adrenal hypoplasia congenital.Nature,1994,372(6507):635-641.
[92]Zhao Q,Eberspaecher H,Lefebvre V,et al.Parallel expression of Sox9 and Col2al in cells under going chondrogenesis.DevDyn,1997,209:377-386.
[93]Meyer J,S(u|¨)dbeck P,Held M,et aL Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal:lack of genotype/phenotype correlations.Human Molecular Genetics,1997,6:91-98.
[94]Kawakami Y,Rodriguez-Leon J,et al.The role of TGFbs and Sox9 during limb chondrogenesis.Current Opinion in Cell Biology 2006,18:723-729.
[2]王建武,丁家桐,刘春玲等.哺育动物性别决定的基因调控[J].动物科学与动物医学,2003,4:26-58.
[3]Vincent RH,Michael JC,Ant hony A,The Molecular Action and Regulation of the Testis-Determining Factors,SRY(Sex-Determining Region on the Y Chromosome) and SOX9[SRY- Related High-Mobility Group(HMG) Box 9]Endocr[J].Rev,2003,24:466-487.
[4]Koopman P,Gubbay J,Vivian N,et aL Male development of chromosomally femal mice transgenic for Sry[J].Nature,1991,351(6322):117-121.
[5]Hanley NA,Hagan DM,Clement-Jones M,et al.SRY,SOX9,and DAX1 expression patterns during human sex determination and gonadal development[J].Mech Dev,2000,91:403-407.
[6]Margarit E,Dolors-Coil M.SRY gene transferred to the Iongarm of the X chromosome in a Y-positive XX true hermaphrodite[J].Anim Genet,2000,90:25-28.
[7]Lovell-Badge R.How does SRY act in mammalian sex determination[J].The 48 th NIBB Conference,Okazaki,2003,Japan.
[8]Koichiro N,Naoko H,Satoshi T,et al.DNA Methylation-mediated Control of Sry Gene Expression in Mouse Gonadal Development[J].J Biol Chem,2004,279:22306-22313.
[9]赵勇,王文波.Sox9基因与椎间盘退变[J].中华外科杂志,2005,43:544-545.
[10]Sive JI,Baird P,Jeziorsk M,et aL Expression of chondrocyte markers by cells of normal and degenerated intervertebral discs[J].Mol Pathol,2002,55:91-97.
[11]Argentaro A,Sim H,Kelly S,et al.A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia Pautosomal sex reversal[J].J Biol Chem,2003,278(36):33839-33847.
[12]Frojdman K,Harley VR,Pelliniemi LJ.Sox9 protein in rat sertoli cells is age and stage dependent[J].Histochem Cell Bio 1,2000,113(1):31-36.
[13]Wagner T,Wirth J,Meyer J,et al.Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene Sox9[J].Cell,1994,79:1111-1120.
[14]Kent J,Wheatley SC,Andrews JE,et al.A male-specific role for Sox9 in verterbrate sex determination[j].Development,1996,122:2813-2822.
[15]Koopman P.Sry,Sox9 and mammalian sex determination[J].EXS,2001,91:25-56.
[16]Morals da Silva S,Hacker A,Harley V,et al.Sox9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds[J].Nat Genet 1996,14:62-68.
[17]李楠,王秀利,仇雪梅.SOX、DMRTT生别决定基因家族及其应用研究进展[J].生物技术通报,2005,3:5-9.
[18]Ellisen LW.Regulation ofgene expression by WT1 in development and turnorigenesis[J].Int J Hematol,2002,76(2):110-116.
[19]MacLaughlin DT,Donahoe PK.Sex determination and differentiation[J].N Engl J Med,2004,350(4):367-378.
[20]Anwar H,Grady F.Saunders:Role of Wilms Tumor 1(WT1) in the Transcriptional Regulation of the Mullerian-Inhibiting Substance Promoter[j].Biol Reproduct,2003,69(6):1808~1814.
[21]Matsuzawa-Watanabe Y,Inoue J,Semba K.Transcriptional activity of Testis-determining factor SRY is modulated by the Wilms'tumor 1 gene product,WT1[J].Oncogene,2003,22(39):7900-7904.
[22]Rey R,Lukas-Croisier C,Lasala C,et al.AMH/MIS:what we know already about the gene,the protein and its regulation[J].Mol Cell,Endocrinol,2003,211(1-2):21-31.
[23]Amiel J,Gigarel N,Benacki A.Prenatal diagnosis of respiratory chain deficiency by direct mutation screening[J].Prenat Diagn,2001,21(7):602--604.
[24]Niers L,vanden Heuvel L,Trijbels F,et al.Prerequisites and strategies for prenatal diagnosis of respiratory chain deficiency in chorionic villi[J].J Inherit Metab Dis,2003,26(7):647-658.
[25]Meeks JJ,Weiss J,Jameson JL.Daxl is required for testis determination[J].Nat Genet,2003,34(1):32-33.
[26]周林燕,张修月,王德寿.动物学研究,2004,25(1):81-88.
[27]Seibel P,Trappe J,Villani G,et al.Transfection of mitochondria:strategy towards a gene therapy of mitochondrial DNA diseases[J].NucleicAcids Res,1995,23(1):10-17.
[28]Klaus Kalthoff.Analysis of Biological Development[M].2001,2nd edition,。
[29]何巧平,孔祥清.WNT4基因在房间隔缺损患者心肌组织中的表达.南京医科大学学报[J].2005,25(11):810-812.
[30]Qin Y,Bishop CE.Sox9 is sufficient for functional testis development producing fertile male mice in the absence of Sry[J].Hum Mol Genet,2005,14(9):1221-1229.
[31]Jeays-Ward K,Hoyle C,Brennan J,et al.Endothelial and steroido-genic cell migration are regulated by WNT4 in the developing mammalian gonad[J].Development,2003,130(16):3663-3670.
[32]Anwar H,Grady F.Synergistic cooperation between the β-catenin signaling pathway and steroidogenic fatter 1 in the activation of the mullerian inhibiting substance type Ⅱ type receptor[J].Biol Chem,2003,278(29):26511-26516.
[33]Chardard D,Chesnel A,Goze C,et al.Pw Sox-1:the first member of the Sox gene family in Urodeles[J].Nucleic Acids Research,1993,21(15):3576.
[34]Wegner M.From head to toes:The multiple facets of Sox proteins[J].Nucleic Acids Res,1999,27(6):1409-1420.
[35]Foster JW et al.Sox-9 gene and protein and use in the regeneration of bone or cartilage[J].Nature,1994,372:525
[36]Sudbeck P,Schmitz ML,Baeuerle PA,et al.Sex reversal by loss of the C-terminal transactivation domain of human SOX9[J].Nat Genet,1996,13:230-232.
[37]Subdeck P,Scherer G..Two independent nuclear localization signals are present in the DNA-binding high-mobility group domains of SRY and SOX9[J].J Biol Chem,1997 OCT31,272(44):27848-27852.
[38]de Santa Barbara P,Moniot B,Poulat F,et al..Expression and subcellular localization of SF-1,SOX9,WT1 and AMH proteins during early human testicular development[J].Dev Dyn,2000,217(3):293-298.
[39]Argentaro A,Sim H,Kelly S,et al.A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal[J].J Biol Chem,2003,278(36):33839-33847.
[40]Morals da Silva S,Hacker A,Harley V,et aL SOX9 expression during gonadal development mplies a conserved role for the gene in testis differentiation in mammals and birds[J]. Nat Genet, 1996, 14:62-68.
[41] Jennifer A, Mersshll G Evolution of the mammalian Y chromosome and sex-determining genes[J].J Exp Zool, 1998,281:472-481.
[42] Pfeifer D, Poulat F, Holinski-Feder E, et al. The SOX8 gene is located within 700 kb of the tip of chromosome 16p and is deleted in a patient with ATR-16 syndrome[J]. Genomics, 2000, 63(1):108-116.
[43] Takada S, Koopman P. Regulation of Amh during sex determination in chickens: Sox gene expression in male and female gonads[J]. Cytogenet Genome Res, 2003, 101(3-4): 212-218.
[44] Schepers G, Wilson M, Wilhelm D, et al. SOX8 is expressed during testis differentiation in mice and synergizes with SF1 to activate the Amh promoter in vitro[J]. J Biol Chem, 2003,278(30):28101-28108.
[45] Akiyama H, Chaboissier MC, Martin JF, et al. The transcription facter Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and reqired for expression of Sox5 and Sox6[J]. Gene Dev, 2002, 16:2813-2828.
[46] Torres-M aldonado LC, Landa PA, et al. General & Comparative Endocrinology, 2002,129(1):20-26.
[47] JussiT IS. In vitro cartilage formation by human adult stem cells From bone marrow stroma defines the sequence of cellular and molecuLar events during chondrogenesis. PNAS, 2002, 99(7):4397-4402.
[48] Lefebvre V, Behringer RR, de Crombrugghe B.L-Sox5,Sox6 and Sox9 control essential steps of the chondrocyte differentiation pathway[J].Osteoarthritis and cartilage, 2001, S9A: s69-75.
[49] 赵勇,王文波.Sox9基因与椎间盘退变.中华外科杂志,2005, 43: 544-545.
[50] Sive JI, Baird P, Jeziorsk M, et al. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs[J]. Mol Pathol, 2002, 55(2):91-97.
[51] Lefebvre V, Huang W, Harley VR, et al... SOX9 is a potent activator of the chondrocyte-specific enhancer of the pro alphal(II) collagen gene[J].Mol. Cell. Biol, 1997, 17:2336-2346.
[52] Huang WD, Veronique Lefebvre XZ, de Crombrugghe B. Phosphorylation of SOX9 by Cyclic AMP-Dependent Protein Kinase A Enhances SOX9's Ability To Transactivate a Col2al Chondrocyte-Specific Enhancer[J]. Molecular and Cellular Biology, 2000,20(11): 4149-4158.
[53] Chen CW, Tsai YH, Deng WP, et al.Type I and II collagen regulation of chondrogenic differentiation by mesenchymal progenitor cells[J]. J Orthop Res, 2005, 23(2): 446-453.
[54] Gruber HE, Norton HJ, Ingram JA, et al. The SOX9 transcription factor in the human disc: decreased immunolocalization with age and disc degeneration. Spine, 2005, 30(6): 625-630.
[55] Rozeman LB, Hameetman L, Cleton-Jansen AM, et al.Absence of IHH and retention of PTHrP signalling in enchondromas and central chondrosarcomas. J Pathol, 2005, 205(4): 476-482.
[56] Smits P, Li P, Mandel J, et al. The transcription factors L-Sox5 and Sox6 are essential for cartilage formation[J]. Dev Cell, 2001, 1: 277-290.
[57] Lefebvre V & Smits P. Transcriptional control of chondrocytefate and differentiation[J]. Birth Defects Research, Part C:Embryo Today: Reviews, 2005, 75, 200-212.
[58] Ikeda T, Kawaguchi H, Kamekura S, et al. Distinct roles of Sox5, Sox6, and Sox9 in different stages of chondrogenic differentiation[J]. J Bone Miner Metab, 2005, 23: 337-340.
[59]Stolt CC,SchlierfA,Lommes P,et al.SoxD Proteins Influence Multiple Stages of Oligodendrocyte Development and Modulate SoxE Protein Function[J].Developmental Cell 2006,11:697-709.
[60]Yommerup N,Sehempp W,Meinecke P,et al.Assignment of an autosomal sex reversal locus (SRA 1) and eampomelic dysplasia(CMPD1).to 17q24.3-q25.1.Nat Genet,1993,4(2):170-174.
[61]Smith JM,Koopman PA.The ins and outs of transcriptional control:nucleocytoplasmie shuttling in development and disease[J].Trends Genet,2004,20(1):4-8.
[62]Lee YH,Aold Y,Hong CS,Saint-Germain N,et al.Early requirement of the transcriptional activator Sox9 for neural crest specification in Xenopus[J].Dev Biol,2004,275(1):93-103.
[63]Sottile V,Li M,Seotting PJ.Stem cell marker expression in the Bergmann glia population of the adult mouse brain[J].Brain Res,2006,1099(1):8-17.
[64]Montero JA,Giron B,Arrechedera H,et al.Expression of SoxS,Sox9 and Soxl0 in the developing valves and autonomic nerves of the embryonic heart[J].Mech Dev,2002,118(1-2):199-202.
[65]GoffSA,Ricke D,Lan TH,et al.A.draft sequence of the rice genome(Oryza sativa L.ssp.japonica)[J].Science,2002,296:92-100,
[66]Boguski MS,Tolstoshev CM,Bassett DE.Gene discovery in dbEST.Science 1994,265(5181):1993-1994
[67]Vitt U,Gietzen D,Stevens K,et al.Identification of can-didate disease genes by EST alignments,synteny,and expression and verification of Ensembl genes on rat chromosome lq43-54.Genome Res 2004,14(4):640-650
[68]Jongeneel CV.Searching.the expressed sequence tag(EST) databases:Panning for genes.Brieafings in Bioinformatics 2000,1:76-79.
[69]Schuler.Pieces of the puzzle:expressed sequence tags and the catalog of human genes.J Mol Med 1997,75(10):694-698
[70]Gasteiger E,Hoogland C.Protein Identification and Analysis Tools on the ExPASy Server,in Walker JM ed:The Proteomics Protocols Handbook.Humana Press 2005,571-607.
[71]Wilsker D,Patsialou A,Dallas PB,et al.ARID proteins:a diverse family of DNA binding proteins implicated in the control of cell growth,differentiation,and development.Cell Growth Differ 2002,13:95-106
[72]Bendtsen JD,Nielsen H,Von Heijne G.,Brunak S.Improved prediction of signal peptides:SignalP 3.0.J Mol Biol 2004,340:783-795
[73]Marchler-Baner A,Bryant SH.CD-Search:protein domain annotations on the fly.Nucleic Acids Res 2004,32:327-331
[74]常重杰,杜启艳,邵红伟.Sox基因家族研究的新进展.遗传,2002,24(4):470-476.
[75]Lefebvre V,Dumitriu B,M'endez AP,et al.Control of cell fate and differentiation by Sry-related high-mobility-group box(Sox) transcription factors.The International Journal of Biochemistry &Cell Biology,2007,39:2195-2214.
[76]杨坚,正常猪外周血淋巴细胞cDNA文库部分EST序列的电子克隆及初步实验验证,[硕士学位论文],广西大学硕士论文,2006.
[77]田兴国,猪肌肉组织EST的基因克隆与表达分析。[硕士学位论文],广州,华南农业大学,2004.
[78]Gasteiger E,Gattiker A.ExPasy:the proteomics server for in-depth protein knowledge and analysis.Nucleic Acids Res 2003,31:3784-3788.
[79]Moiler S,Croning MD,Apweiler R.Evaluation of methods for the prediction of membrane spanning regions.Bioinformatics 2001,17(7):646-653.
[80]Nakai K,Horton P.PSORT:a program for detecting the sorting signals of proteins and predicting their subcellular localization.Trends Biochem Sci 1999,24(1):34-35.
[81]袁红梅,张丽,张虹,黄永红.SOX9基因的研究进展.黑龙江农业科学,2007,(3):124-127.
[82]Vidal VP,Chaboissier MC,De Rooij DG,et al.Sox9 induces testis development in XX transgenic mice.Nature Genetic,2001,28:216-217.
[83]Erta P,Hawkins JR,Sinclair AH,et al.Genetic evidence equating SRY and the testis-determining factor.Nature,1990,348(6300):448-450.
[84]Clarkson MJ,Harley VR.Sex with two Sox on:SRY and SOX9 in testis development.Trend in Endocrinology & Metabolism,2002,13(3):106~111.
[85]张勇,陈淳,顾建新等.哺乳动物性别决定的研究进展.生物工程进展,2001,21(3):26-29.
[86]Foster JW,Granes JA.An SRY-related sequence on the Marsupial X Chromosome:Implications of the evolution of the mammalian testis-determining gene.Proc Natl Acad Sci USA,1994,91:1927-1931.
[87]朱必才,高建国,张子峰等.哺乳动物性别决定及其机制的研究.细胞生物学杂志,2002,24(5):282-286.
[88]Morais da Silva S,Hacker A,Harley V,et al.SOX9 expression during gonadal development implies a conserved role for the gene in testis differentiation in mammals and birds.Nat Genet,1996,14:62-68.
[89]Sudbeck P,Schmitz ML,Baeuerle PA,et al.Sex reversal by loss of the C-terminal transactivation domain of human SOX9.Nat Genet,1996,13:230-232.
[90]Wright EM,Snopek B,Koopan P.Seven new members of the Sox gene family expression during mouse development.Nucleic Acids Res,1993,21:774.
[90]Zanaria E,Muscatelli F,Bardoni B,et al.A novel and unusual member of the nuclear hormone receptor super family is responsible for X-linked adrenal hypoplasia congenital.Nature,1994,372(6507):635-641.
[92]Zhao Q,Eberspaecher H,Lefebvre V,et al.Parallel expression of Sox9 and Col2al in cells under going chondrogenesis.DevDyn,1997,209:377-386.
[93]Meyer J,S(u|¨)dbeck P,Held M,et aL Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal:lack of genotype/phenotype correlations.Human Molecular Genetics,1997,6:91-98.
[94]Kawakami Y,Rodriguez-Leon J,et al.The role of TGFbs and Sox9 during limb chondrogenesis.Current Opinion in Cell Biology 2006,18:723-729.