扬子鳄Sox 和DMRT基因的研究
摘要
本文由文献综述和研究报告两部分组成
一、文献综述
1、从Sox基因的发现、特点及功能三个方面概述Sox基因的研究现状。Sox基因含有一个能与DNA特异结合的高度保守HMG-box基序。在个体发育过程中，该基因广泛参与了动物的性别决定及多种组织器官的形成。
2、从DMRT基因的发现及功能等方面综述DMRT基因的研究进展。DMRT基因是一类新发现的含有DM保守盒的转录调控因子。该基因参与调节动物的性别决定与分化等过程。
3、综述了Sox基因与DMRT基因在性别决定中的相互作用关系。
二、研究报告
1、参照人SRY基因HMG-box保守区的序列，设计一对兼并引物，扩增了扬子鳄的Sox基因，并对扩增产物进行了克隆与测序。结果在雌雄中均筛选出8个不同的Sox基因，长度均为216bp，无性别特异性。其DNA序列和编码的氨基酸序列与人相应Sox基因的相似性分别为88%、88%、88%、89%、87%、93%、87%、93%和91%、91%、91%、96%、100%、98%、94%、96%。显示出进化上的高度保守性。
2、参照人DMRT1基因DM盒保守区的序列，设计一对特异引物，扩增了扬子鳄的DMRT1基因，并对扩增产物进行了SSCP分析和测序。结果显示：(1)扬子鳄DMRT1基因的扩增片段与人DMRT1基因扩增片段的大小相同，均为140bp；(2)雌雄扬子鳄DMRT1基因扩增片段大小虽然与人相同，但其单链迁移率与人有较大差异，而雌雄个体间无差异。(3)测序结果表明，雌雄扬子鳄DMRT1基因的序列一致，且其DNA序列和编码的氨基酸序列与人相应DMRT1基因的相似性分别为87%和95%。显示出高度的保守性。
本研究为探索扬子鳄的性别决定机制、Sox基因与DMRT基因的功能及其进化等提供了分子资料。
This paper consists of two parts,which are as follows:
PART ONE: reviews
1. The discovery,characteristic and function of Sox genes were discussed. Sox genes encode a group of proteins that carry a DNA-binding HMG domain. In various phages of embryonic development, Sox genes play a key role in sex-determining and in organism formation.
2. The findings and function of DMRT gene were reviewed. DMRT genes are a kind of transcription factor which possesses a common motif called the DM domain. DMRT genes are considered to be involved in sex determination and sex differentiation.
3. The interaction between Sox genes and DMRT genes in sex determining and sex differentiation was discussed.
PART TWO: studies
1. After a pair of degenerate primers based on the conservative region, HMG-box,of Human SRY gene were used, eight different fragments were amplified from both female and male Alligator sinensis whose length were all 216bp, then they were cloned by pUCm-T Vector and sequenced.The result indicated that they showed high homology to Human SOX genes, the identities to Human SOX genes in the DNA sequence and the amino acid sequence were 88%、88%、88%、89%、87%、93%、87%、93% and 91%、91%、91%、96%、100%、98%、94%、96%, respectively, which may be concluded that Sox genes were highly conservative in phylogenesis.
2. The DMRT1 gene of Alligator sinensis was amplified by using a pair of special primers which can amplify the conservative motif (DM domain) of human DMRT1 gene,and then it was sequenced. The length of amplification was 140bp in both male and female, which was consistent with that of human DMRT1 gene.The result of SSCP analysis showed that there were many differences in the DMRT1 gene sequence between Alligator sinensis and human,but no differences between male and female Alligator sinensis. The sequence analysis showed that the identities of Alligator sinensis DMRT1 gene to Human DMRT1 gene in the DNA sequence and the amino acid sequence were 87% and 95%, respectively. The results indicated that DMRT genes were highly conservative in phylogeny.
Our work provides molecular data for the study of sex-determining mechanism, the function of Sox genes and DMRT genes ,and the conservation of Sox genes' and DMRT genes' evolution.
一、文献综述
1、从Sox基因的发现、特点及功能三个方面概述Sox基因的研究现状。Sox基因含有一个能与DNA特异结合的高度保守HMG-box基序。在个体发育过程中，该基因广泛参与了动物的性别决定及多种组织器官的形成。
2、从DMRT基因的发现及功能等方面综述DMRT基因的研究进展。DMRT基因是一类新发现的含有DM保守盒的转录调控因子。该基因参与调节动物的性别决定与分化等过程。
3、综述了Sox基因与DMRT基因在性别决定中的相互作用关系。
二、研究报告
1、参照人SRY基因HMG-box保守区的序列，设计一对兼并引物，扩增了扬子鳄的Sox基因，并对扩增产物进行了克隆与测序。结果在雌雄中均筛选出8个不同的Sox基因，长度均为216bp，无性别特异性。其DNA序列和编码的氨基酸序列与人相应Sox基因的相似性分别为88%、88%、88%、89%、87%、93%、87%、93%和91%、91%、91%、96%、100%、98%、94%、96%。显示出进化上的高度保守性。
2、参照人DMRT1基因DM盒保守区的序列，设计一对特异引物，扩增了扬子鳄的DMRT1基因，并对扩增产物进行了SSCP分析和测序。结果显示：(1)扬子鳄DMRT1基因的扩增片段与人DMRT1基因扩增片段的大小相同，均为140bp；(2)雌雄扬子鳄DMRT1基因扩增片段大小虽然与人相同，但其单链迁移率与人有较大差异，而雌雄个体间无差异。(3)测序结果表明，雌雄扬子鳄DMRT1基因的序列一致，且其DNA序列和编码的氨基酸序列与人相应DMRT1基因的相似性分别为87%和95%。显示出高度的保守性。
本研究为探索扬子鳄的性别决定机制、Sox基因与DMRT基因的功能及其进化等提供了分子资料。
This paper consists of two parts,which are as follows:
PART ONE: reviews
1. The discovery,characteristic and function of Sox genes were discussed. Sox genes encode a group of proteins that carry a DNA-binding HMG domain. In various phages of embryonic development, Sox genes play a key role in sex-determining and in organism formation.
2. The findings and function of DMRT gene were reviewed. DMRT genes are a kind of transcription factor which possesses a common motif called the DM domain. DMRT genes are considered to be involved in sex determination and sex differentiation.
3. The interaction between Sox genes and DMRT genes in sex determining and sex differentiation was discussed.
PART TWO: studies
1. After a pair of degenerate primers based on the conservative region, HMG-box,of Human SRY gene were used, eight different fragments were amplified from both female and male Alligator sinensis whose length were all 216bp, then they were cloned by pUCm-T Vector and sequenced.The result indicated that they showed high homology to Human SOX genes, the identities to Human SOX genes in the DNA sequence and the amino acid sequence were 88%、88%、88%、89%、87%、93%、87%、93% and 91%、91%、91%、96%、100%、98%、94%、96%, respectively, which may be concluded that Sox genes were highly conservative in phylogenesis.
2. The DMRT1 gene of Alligator sinensis was amplified by using a pair of special primers which can amplify the conservative motif (DM domain) of human DMRT1 gene,and then it was sequenced. The length of amplification was 140bp in both male and female, which was consistent with that of human DMRT1 gene.The result of SSCP analysis showed that there were many differences in the DMRT1 gene sequence between Alligator sinensis and human,but no differences between male and female Alligator sinensis. The sequence analysis showed that the identities of Alligator sinensis DMRT1 gene to Human DMRT1 gene in the DNA sequence and the amino acid sequence were 87% and 95%, respectively. The results indicated that DMRT genes were highly conservative in phylogeny.
Our work provides molecular data for the study of sex-determining mechanism, the function of Sox genes and DMRT genes ,and the conservation of Sox genes' and DMRT genes' evolution.
引文
[1]Sinclair AH, Berta P, Palmer MS, et al.A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif[J]. Nature,1990,346:240-244.
[2]Gubbay J, collignon J, Koopman, P, et al.A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embry-onically expressed genes[J]. Nature,1990,346:245-250.
[3]Bergstorm D, Young M, Albecht K,et al. Related function of mouse Sox3, Sox9, and SRY HMG domains assayed by male sex determination[J]. Genesis, 2000, 28:111-124.
[4]Wilson M, Koopman P. Matching Sox: Partner proteins and Co-factors of the Sox family of transcriptional regulators[J]. Current opinion in Genetics & Development,2002,12:447-446.
[5]常重杰，杜启艳，邵红伟.Sox基因家族研究的新进展[J].遗传，2002,24(4):470-476。
[6]Berta P, Hawkins JR, Sinclair AH, et al. Genetic evidence equating SRY and the testis-determining factor[J]. Nature, 1990,348(6300):448-450.
[7]汪锐，程汉华，郭一清等.脊椎动物Sox基因家族的系统发生分析[J].遗传学报，2002,29(11):990-994。
[8]周荣家.参与发育的基因家族[J].遗传，2001，23(1):86-88.
[9]Wright Em, Smopek B, Koopman P. Seven new members of the Sox gene family expressed during mouse development[J]. Nucleic Acids Res,1993,21:744.
[10]Bowles J, Schepers G, Koopman P.Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators[J]. Dev Biol,2000,227(2):239-755.
[11]Yuan X, Lu M, Li T, et al. SRY interacts with and negatively regulates androgen receptor transcriptional activity[J]. J Biol Chem, 2001, 276:46647-46654.
[12]Koopman P, Gubbay J, Vivian N, et al. Male development of chromosomally femal mice transgenic for sry[J], Nature,1991,351(6322):117-121.
[13]Clarkson MJ, Harley VR. Sex with two SOX on: SRY and SOX9 in testis development[J]. Trend in Endocrinology & Metabolism,2002,13(3):106-111.
[14]张勇,陈淳,顾建新等.哺乳动物性别决定的研究进展[J].生物工程进展, 2001,21(3):26-29.
[15]Froster JW,Granes JA. An SRY-related sequence on the Marsupial X chromosome:Implications of the evolution of the mammalian testis-determining
gene[J].Proc Natl Acad Sci USA, 1994,91:1927-1931.
[16]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[J]. 细胞生物学杂志，2002,24(5):282-286.
[17]Michael W. From head to toes: the multiple facets of Sox proteins[J]. Nucleic Acids Res,1999,27(6): 1409-1420.
[18]Graves JA. Interactions between SRY and Sox genes in mammalian sex determination[J]. Bioessays,1998,20(3):264-269.
[19]Wachtel S.X-linked sex reversing genes[J]. Cytogene cell Gene, 1998, 80:222-225.
[20]Caple B. Sex in the 90's:SRY and the switch to the male pathway[J] Mammalian sex deter,1998,60:497-523.
[21]Jennifer A, Marsshll G. Evolution of the mammalian Y chromosomc and sex-determining genes[J]. J Exp zool,1998,281:472-481.
[22]Colvin JS, et al. Male-to female sex reversal in mice lacking fibroblast growth factor 9[J]. Cell,2000, 104:875-889.
[23]Moniot B, Berta P, Scherer, G, et al. Male specific expression suggests role of DMRT 1 in human sex determination[J]. Mech Dov,2000,91:323-325.
[24]Denny P, Swift S, Connor F, et al. An SRY-Related gene expressed during spermatogenesis in the mouse encodes a sequence-specific DNA binding protein[J]. EMBO Journal,1992,11:3705-3712.
[25]Kamaki Y, cheah KS, Kondoh H. Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealved by comparison of SOX1/2/3 and Sox9[J]. Mol Cell Biol,1999,19(1):107-120.
[26]Du Z, Cong H, Yao Z. Identification of putative downstream genes of oct-4 by suppression-subtractive hybridization[J]. Biochem Biophys Res Commun, 2001,282(3):701-706.
[27]Ambrosetti DC, Basilico C, Dailey L. Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites[J]. Mol cell Biol,1997,17(11):6321-6329.
[28]Wistow G, Sardarian L, Gan W, et al. The human gene for gamma-crystallin: alternative transcripts and expressed sequences from the first intron[J]. Mol Vis,2000,6:79-84.
[29]Meyer J,Südbeck P,Held M,Wsgner T,et al. Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal:lack of genotype/phenotype correlations[J].Human Molecular Genetics, 1997,6:91-98.
[30]Pevny LH, Lovell-badge R. Sox genes find their feet[J]. Current Opinion in Genetics & Developments,1997,7:338-344.
[31]赵寿元，乔守怡.现代遗传学[M].北京:高教出版社,2001,217-219.
[32]Yi W, Zarkower D. Similarity of DNA binding and transcriptional regulatin by Caenorhabditis elegans Mab-3 and Drosophila melanogaster DSX suggests conservation of sex determining mechanism[J]. Dev,1999, 126:873-881.
[33]任莉莉，程汉华，郭一清等.两栖类、爬行类和鸟类存在一个新的Dmrt基因家族[J].科学通报,2001,46(14):1187-1190。
[34]Kondo M, Froschauer A, Kitano A, et al.Molecular cloing and characterization of DMRT genes from the medaka Oryzias latipes and the platyfish Xiphophorus maculatus[J].Gene,2002, 295:213-222.
[35]Raymond CS, Murphy MW, O'Sullivan MG, et al. Dmrt1, a gene related to worm and fly sexual regulators, is required for mammaliam testis differentiation[J]. Genes Dev,2000,14:2587-2595.
[36]Ellegren H. Hens, cocks and avian sex determination:A quest for genes on Z or W[J]. EMBO reports,2001,2(3):192-196.
[37]Raymond CS, Kettlewell JR, Hirsch B, et al.Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development[J]. Dev Biol,1999,215:208-220.
[38]Kettlewell JR, Rymond CS, Zarkower D.Temperature-Dependant expression of turtle Dmrt1 prior to sexual differentiation[J].Genetics,2000,26(3): 174-178.
[39]Smith CA, McClive PJ, Western PS, et al. Conservation of a Sex-determining gene[J]. Nature, 1999,402:601-602.
[40]Kazuyuki S,Minoru T,Masahisa N.The Dmrt1 expression in sex-reversed gonad of amphibians[J].General & Comparative Endocrinology, 2002,127(3):232-242.
[41]Marchand O, Govoroun M, D'Cotta H, et al. DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss[J]. Biochimica et Biophysica Acta, 2000, 1493: 180-187.
[42]Smith CA, Smith MJ, Sinclair AH.gene expression during gonadogenesis in the chicken embryo[J]. Gene,1999,234: 395-402.
[2]Gubbay J, collignon J, Koopman, P, et al.A gene mapping to the sex-determining region of the mouse Y chromosome is a member of a novel family of embry-onically expressed genes[J]. Nature,1990,346:245-250.
[3]Bergstorm D, Young M, Albecht K,et al. Related function of mouse Sox3, Sox9, and SRY HMG domains assayed by male sex determination[J]. Genesis, 2000, 28:111-124.
[4]Wilson M, Koopman P. Matching Sox: Partner proteins and Co-factors of the Sox family of transcriptional regulators[J]. Current opinion in Genetics & Development,2002,12:447-446.
[5]常重杰，杜启艳，邵红伟.Sox基因家族研究的新进展[J].遗传，2002,24(4):470-476。
[6]Berta P, Hawkins JR, Sinclair AH, et al. Genetic evidence equating SRY and the testis-determining factor[J]. Nature, 1990,348(6300):448-450.
[7]汪锐，程汉华，郭一清等.脊椎动物Sox基因家族的系统发生分析[J].遗传学报，2002,29(11):990-994。
[8]周荣家.参与发育的基因家族[J].遗传，2001，23(1):86-88.
[9]Wright Em, Smopek B, Koopman P. Seven new members of the Sox gene family expressed during mouse development[J]. Nucleic Acids Res,1993,21:744.
[10]Bowles J, Schepers G, Koopman P.Phylogeny of the SOX family of developmental transcription factors based on sequence and structural indicators[J]. Dev Biol,2000,227(2):239-755.
[11]Yuan X, Lu M, Li T, et al. SRY interacts with and negatively regulates androgen receptor transcriptional activity[J]. J Biol Chem, 2001, 276:46647-46654.
[12]Koopman P, Gubbay J, Vivian N, et al. Male development of chromosomally femal mice transgenic for sry[J], Nature,1991,351(6322):117-121.
[13]Clarkson MJ, Harley VR. Sex with two SOX on: SRY and SOX9 in testis development[J]. Trend in Endocrinology & Metabolism,2002,13(3):106-111.
[14]张勇,陈淳,顾建新等.哺乳动物性别决定的研究进展[J].生物工程进展, 2001,21(3):26-29.
[15]Froster JW,Granes JA. An SRY-related sequence on the Marsupial X chromosome:Implications of the evolution of the mammalian testis-determining
gene[J].Proc Natl Acad Sci USA, 1994,91:1927-1931.
[16]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[J]. 细胞生物学杂志，2002,24(5):282-286.
[17]Michael W. From head to toes: the multiple facets of Sox proteins[J]. Nucleic Acids Res,1999,27(6): 1409-1420.
[18]Graves JA. Interactions between SRY and Sox genes in mammalian sex determination[J]. Bioessays,1998,20(3):264-269.
[19]Wachtel S.X-linked sex reversing genes[J]. Cytogene cell Gene, 1998, 80:222-225.
[20]Caple B. Sex in the 90's:SRY and the switch to the male pathway[J] Mammalian sex deter,1998,60:497-523.
[21]Jennifer A, Marsshll G. Evolution of the mammalian Y chromosomc and sex-determining genes[J]. J Exp zool,1998,281:472-481.
[22]Colvin JS, et al. Male-to female sex reversal in mice lacking fibroblast growth factor 9[J]. Cell,2000, 104:875-889.
[23]Moniot B, Berta P, Scherer, G, et al. Male specific expression suggests role of DMRT 1 in human sex determination[J]. Mech Dov,2000,91:323-325.
[24]Denny P, Swift S, Connor F, et al. An SRY-Related gene expressed during spermatogenesis in the mouse encodes a sequence-specific DNA binding protein[J]. EMBO Journal,1992,11:3705-3712.
[25]Kamaki Y, cheah KS, Kondoh H. Mechanism of regulatory target selection by the SOX high-mobility-group domain proteins as revealved by comparison of SOX1/2/3 and Sox9[J]. Mol Cell Biol,1999,19(1):107-120.
[26]Du Z, Cong H, Yao Z. Identification of putative downstream genes of oct-4 by suppression-subtractive hybridization[J]. Biochem Biophys Res Commun, 2001,282(3):701-706.
[27]Ambrosetti DC, Basilico C, Dailey L. Synergistic activation of the fibroblast growth factor 4 enhancer by Sox2 and Oct-3 depends on protein-protein interactions facilitated by a specific spatial arrangement of factor binding sites[J]. Mol cell Biol,1997,17(11):6321-6329.
[28]Wistow G, Sardarian L, Gan W, et al. The human gene for gamma-crystallin: alternative transcripts and expressed sequences from the first intron[J]. Mol Vis,2000,6:79-84.
[29]Meyer J,Südbeck P,Held M,Wsgner T,et al. Mutational analysis of the SOX9 gene in campomelic dysplasia and autosomal sex reversal:lack of genotype/phenotype correlations[J].Human Molecular Genetics, 1997,6:91-98.
[30]Pevny LH, Lovell-badge R. Sox genes find their feet[J]. Current Opinion in Genetics & Developments,1997,7:338-344.
[31]赵寿元，乔守怡.现代遗传学[M].北京:高教出版社,2001,217-219.
[32]Yi W, Zarkower D. Similarity of DNA binding and transcriptional regulatin by Caenorhabditis elegans Mab-3 and Drosophila melanogaster DSX suggests conservation of sex determining mechanism[J]. Dev,1999, 126:873-881.
[33]任莉莉，程汉华，郭一清等.两栖类、爬行类和鸟类存在一个新的Dmrt基因家族[J].科学通报,2001,46(14):1187-1190。
[34]Kondo M, Froschauer A, Kitano A, et al.Molecular cloing and characterization of DMRT genes from the medaka Oryzias latipes and the platyfish Xiphophorus maculatus[J].Gene,2002, 295:213-222.
[35]Raymond CS, Murphy MW, O'Sullivan MG, et al. Dmrt1, a gene related to worm and fly sexual regulators, is required for mammaliam testis differentiation[J]. Genes Dev,2000,14:2587-2595.
[36]Ellegren H. Hens, cocks and avian sex determination:A quest for genes on Z or W[J]. EMBO reports,2001,2(3):192-196.
[37]Raymond CS, Kettlewell JR, Hirsch B, et al.Expression of Dmrt1 in the genital ridge of mouse and chicken embryos suggests a role in vertebrate sexual development[J]. Dev Biol,1999,215:208-220.
[38]Kettlewell JR, Rymond CS, Zarkower D.Temperature-Dependant expression of turtle Dmrt1 prior to sexual differentiation[J].Genetics,2000,26(3): 174-178.
[39]Smith CA, McClive PJ, Western PS, et al. Conservation of a Sex-determining gene[J]. Nature, 1999,402:601-602.
[40]Kazuyuki S,Minoru T,Masahisa N.The Dmrt1 expression in sex-reversed gonad of amphibians[J].General & Comparative Endocrinology, 2002,127(3):232-242.
[41]Marchand O, Govoroun M, D'Cotta H, et al. DMRT1 expression during gonadal differentiation and spermatogenesis in the rainbow trout, Oncorhynchus mykiss[J]. Biochimica et Biophysica Acta, 2000, 1493: 180-187.
[42]Smith CA, Smith MJ, Sinclair AH.gene expression during gonadogenesis in the chicken embryo[J]. Gene,1999,234: 395-402.