河北小尾寒羊多脊柱调控基因Btg2和NR6A1的多态性分析
|
摘要
多脊椎现象是存在于河北小尾寒羊中的有益突变,多数突变都会引起绵羊脊柱的加长和体尺增加,从而显著提高产肉量。本论文采用PCR-RFLP、dCAPS、PCR-SSCP以及直接测序等技术首次从分子水平上对小尾寒羊的Btg2基因和NR6A1基因的多态性与多脊椎性状的关系进行研究,以期找到与小尾寒羊多脊椎突变相关的分子遗传标记,并为进一步标记辅助选择提高河北小尾寒羊的肉用性能,培育我国肉用绵羊品种奠定基础。
根据牛和猪的Btg2基因的DNA序列,选择保守区域设计引物,扩增绵羊Btg2基因的外显子3和3′UTR的部分序列,并对这些序列选取代表性个体进行测序,得到长度为1226bp的片段。全部片段测序结果经过同源性比对,共发现4个SNPs位点,突变位点依据拼接序列来命名分别为:A150G、C243T、A607G和T704C,其中有1个SNPs位于外显子,未引起氨基酸序列的改变。
应用PCR-RFLP及dCAPS技术,对Btg2基因的A150G、C243T和A607G突变位点分别设计引物进行PCR扩增和群体检测。独立性卡方检测结果表明150位点的不同表型个体基因型频率差异不显著(P>0.05);243位点的差异极显著(P<0.01),卡方检验再分割结果表明,基因型频率在T14L6与T14L7、T14L5表型之间差异极显著,T14L6与T13L7之间差异显著;607位点的差异呈显著水平(P<0.05),卡方检验再分割表明基因型频率在T14L6与T14L7中差异显著。结果表明243位点、607位点可能与多脊椎性状有关。经过独立性卡方检验,表明单体型在不同表型中差异显著(P<0.05),分析表明单体型GTA可能与多脊椎性状有关。
根据牛和猪的NR6A1基因的DNA及mRNA序列,设计8对引物,扩增绵羊的该基因的全部编码区序列。运用PCR-SSCP以及直接测序技术对扩增产物进行突变筛查,结果在外显子5的第25位碱基处上检测到1个T25G的单碱基突变,该突变未引起氨基酸序列的改变。对该位点进行群体检测分型,经独立性卡方检验后,发现基因型频率在5种不同多脊椎突变表型间差异达到显著水平(P<0.05),卡方检验再分割后发现基因型频率在T14L5与T14L6以及T14L5与T14L7中差异显著,表明该位点可能与多脊椎性状有关。
Multi-vertebra trait is a positive mutation existing in the Hebei Small Tail Han Sheep, most of which can result in lengthening of the spine, which increases the body size and mutton production significantly. PCR-RFLP, dCAPS, PCR-SSCP and sequencing were used to study the correlation between the polymorphism of Btg2 and NR6A1 gene, and the Multi-vertebra trait. The associated molecular markers were expected to be found, which will be used in Marker-Assisted Selection in order to improve meat performance of Hebei Small Tail Han Sheep, and will lay the foundation for the breeding of meat sheep.
The 1226bp fragment containing partial exon3 and partial 3′UTR was amplified using a pair of PCR primers designed based on the available nucleotide sequence of Btg2 gene from cattle and pig, and was sequenced in some representative individuals with distinct phenotypes. According to the homologous alignment of the sequenced results, 4 SNPs were detected (A150G, C243T, A607G and T704C), one of which was silent mutation existing in the exon.
The dCAPS primers were designed according to A150G, C243T and A607G, respectively, followed by population analysis via PCR-RFLP. The results of independence Chi-square test showed that genotype frequencies at Site 150 had no significant difference among different vertebral patterning sheep (P>0.05). At Site 243, the genotype frequencies had extremely significant difference among different vertebral patterning sheep (P<0.05), and the resegmentation of Chi-square test showed that the genotype frequencies were extremely significantly different (P<0.01) between T14L6 and T14L7, and between T14L6 and T14L5, and were significantly different (P<0.05) between T14L6 and T13L7. At site 607, the genotype frequencies were significantly different (P<0.05), and according to the resegmentation of Chi-square test, the genotype frequencies between T14L6 and T14L7 were significantly different. The results showed that Site 243 and Site 607 might be associated with multi-vertebra trait. According to independence Chi-square test, haplotypes showed significant difference among different phenotypic individuals, and the haplotype GTA might be related to multi-vertebra trait.
8 pairs of primers have been designed based on the available DNA and mRNA sequences of NR6A1 gene from cattle and pig, followed by amplification of the complete coding sequence (CDS) in Hebei Small Tail Han Sheep. Mutation screening has been carried out via PCR-SSCP and direct sequencing, which showed there was one silent mutation (T25G) at the 25th base in exon5. With respect to this site, the population genotyping and independence Chi-square test showed that the genotype frequencies were significantly different (P<0.05) among the five kinds of multi- vertebra phenotypes, and according to the resegmentation of Chi-square test, the genotype frequencies were significantly different (P<0.05) between T14L5 and T14L6, and between T14L5 and T14L7, showing this site might be related to the multi-vertebrae trait.
根据牛和猪的Btg2基因的DNA序列,选择保守区域设计引物,扩增绵羊Btg2基因的外显子3和3′UTR的部分序列,并对这些序列选取代表性个体进行测序,得到长度为1226bp的片段。全部片段测序结果经过同源性比对,共发现4个SNPs位点,突变位点依据拼接序列来命名分别为:A150G、C243T、A607G和T704C,其中有1个SNPs位于外显子,未引起氨基酸序列的改变。
应用PCR-RFLP及dCAPS技术,对Btg2基因的A150G、C243T和A607G突变位点分别设计引物进行PCR扩增和群体检测。独立性卡方检测结果表明150位点的不同表型个体基因型频率差异不显著(P>0.05);243位点的差异极显著(P<0.01),卡方检验再分割结果表明,基因型频率在T14L6与T14L7、T14L5表型之间差异极显著,T14L6与T13L7之间差异显著;607位点的差异呈显著水平(P<0.05),卡方检验再分割表明基因型频率在T14L6与T14L7中差异显著。结果表明243位点、607位点可能与多脊椎性状有关。经过独立性卡方检验,表明单体型在不同表型中差异显著(P<0.05),分析表明单体型GTA可能与多脊椎性状有关。
根据牛和猪的NR6A1基因的DNA及mRNA序列,设计8对引物,扩增绵羊的该基因的全部编码区序列。运用PCR-SSCP以及直接测序技术对扩增产物进行突变筛查,结果在外显子5的第25位碱基处上检测到1个T25G的单碱基突变,该突变未引起氨基酸序列的改变。对该位点进行群体检测分型,经独立性卡方检验后,发现基因型频率在5种不同多脊椎突变表型间差异达到显著水平(P<0.05),卡方检验再分割后发现基因型频率在T14L5与T14L6以及T14L5与T14L7中差异显著,表明该位点可能与多脊椎性状有关。
Multi-vertebra trait is a positive mutation existing in the Hebei Small Tail Han Sheep, most of which can result in lengthening of the spine, which increases the body size and mutton production significantly. PCR-RFLP, dCAPS, PCR-SSCP and sequencing were used to study the correlation between the polymorphism of Btg2 and NR6A1 gene, and the Multi-vertebra trait. The associated molecular markers were expected to be found, which will be used in Marker-Assisted Selection in order to improve meat performance of Hebei Small Tail Han Sheep, and will lay the foundation for the breeding of meat sheep.
The 1226bp fragment containing partial exon3 and partial 3′UTR was amplified using a pair of PCR primers designed based on the available nucleotide sequence of Btg2 gene from cattle and pig, and was sequenced in some representative individuals with distinct phenotypes. According to the homologous alignment of the sequenced results, 4 SNPs were detected (A150G, C243T, A607G and T704C), one of which was silent mutation existing in the exon.
The dCAPS primers were designed according to A150G, C243T and A607G, respectively, followed by population analysis via PCR-RFLP. The results of independence Chi-square test showed that genotype frequencies at Site 150 had no significant difference among different vertebral patterning sheep (P>0.05). At Site 243, the genotype frequencies had extremely significant difference among different vertebral patterning sheep (P<0.05), and the resegmentation of Chi-square test showed that the genotype frequencies were extremely significantly different (P<0.01) between T14L6 and T14L7, and between T14L6 and T14L5, and were significantly different (P<0.05) between T14L6 and T13L7. At site 607, the genotype frequencies were significantly different (P<0.05), and according to the resegmentation of Chi-square test, the genotype frequencies between T14L6 and T14L7 were significantly different. The results showed that Site 243 and Site 607 might be associated with multi-vertebra trait. According to independence Chi-square test, haplotypes showed significant difference among different phenotypic individuals, and the haplotype GTA might be related to multi-vertebra trait.
8 pairs of primers have been designed based on the available DNA and mRNA sequences of NR6A1 gene from cattle and pig, followed by amplification of the complete coding sequence (CDS) in Hebei Small Tail Han Sheep. Mutation screening has been carried out via PCR-SSCP and direct sequencing, which showed there was one silent mutation (T25G) at the 25th base in exon5. With respect to this site, the population genotyping and independence Chi-square test showed that the genotype frequencies were significantly different (P<0.05) among the five kinds of multi- vertebra phenotypes, and according to the resegmentation of Chi-square test, the genotype frequencies were significantly different (P<0.05) between T14L5 and T14L6, and between T14L5 and T14L7, showing this site might be related to the multi-vertebrae trait.
引文
[1]赵有璋.正确认识和利用小尾寒羊[J].中国草食动物,2003年专辑:1-6.
[2]路广计,王振来,郑文波,等.河北小尾寒羊饲养屠宰使用报告[J].河北畜牧兽医,2001,17(10):14.
[3]张立岭,斯琴毕力格.蒙古羊多脊椎基因的群体遗传学研究[J].遗传,1997,19 (增刊) : 67-69
[4]张立岭,斯琴毕力格,张世铨.多脊椎蒙古羊的胸腰椎长度对产肉性能的影响[J].中国畜牧杂志,1998,34(3):24-25.
[5]Venter, J.C., Adams, M.D., Myers, E.W, et al. The sequence of the human genome [J]. Science, 2001, 291:1304-1351.
[6]Lee, SJ, McPherson, AC. Regulation of myostatin activity and muscle growth [J]. Proc. Natl. Acad. Sci. 2000, 98: 9306-9311.
[7]Yeo C, Whitman M. Nodal signals to Smads through Criptodependent and Cripto-independent mechanisms [J]. Mol Cell. 2001, 7(5):949-957.
[8]Mathews LS, Vale WW. Expression cloning of an activin receptor, a predicted transmembrane serine kinase[J]. Cell, 1991, 65: 973-982.
[9]Feijen, A., Goumans, M J, van den Eijnden, et al. Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins [J]. Development, 1994, 120:3621-3637.
[10]Alexandra C McPherson, Ann M Lawler, Se-Jin Lee. Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11[J]. Nature genetics, 1999, 22 (3):260-264.
[11]Oh SP , Li E. The signaling pathway mediated by the type IIB activin receptor controls axial patterning and lateral asymmetry in the mouse [J]. Genes & Dev. 1997, 11: 1812-1826.
[12]Song J, Oh SP, Schrewe H, et al. The type II activin receptors are essential for egg cylinder growth, gastrulation, and rostral head development in mice[J]. Dev Biol, 1999, 213:157-169.
[13]Matzuk MM, Kumar TR., Bradley A, et al. Different phenotypes for mice deficient in either activins or activin receptor type II [J]. Nature, 1995, 374:356-360.
[14]Ethier JF, Houde A, Lussier JG,et al. Bovine activin receptor type II cDNA: cloning and tissue expression [J]. Mol Cell Endocrinol, 1994, 106(1-2):1-8.
[15]Ethier JF, Lussier JG, Silversides DW. Bovine activin receptor type IIB messenger ribonucleic acid displays alternative splicing involving a sequence homologous to Src-homology 3 domain binding sites [J]. Endocrinology, 1997, 138(6):2425-2434.
[16]Oh SP, Yeo CY, Lee Y, et al. Activin type IIA and IIB receptors mediate Gdf11 signaling in axial vertebral patterning [J]. Genes&Development, 2002, 16(21):2749-2754.
[17]Lee YJ, Hong KH, Yun J, et al. Generation of activin receptor type IIB isoform-specific hypomorphic alleles [J]. Genesis, 2006, 44(10):487-494.
[18]Awgulewitsch A, Utset MF, Hart CP, et al. Spatial restriction in expression of a mouse homoeobox locus within the central nervous system[J]. Nature, 320:328-335.
[19]Glavac D, Dean M.Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations [J]. Hum Mutate, 1993, 2:404-414.
[20]Zhao Y, Potter SS. Functional comparison of the Hoxa 4, Hoxa 10, and Hoxa 11 homeboxes[J]. Dev Biol, 2002, 244(1):21-36.
[21]Wellik DM, Capecchi MR. Hox10 and Hox11 genes are required to globally pattern the mammalian skeleton [J]. Science, 2003, 301(5631):363-367.
[22]Erselius JR, Goulding MD, Gruss P. Structure and expression pattern of the murine Hox3.2 gene [J]. Development, 1990, 110(2):629-642.
[23]Kwon Y, Shin J, Park HW, et al. Dynamic expression pattern of Hoxc8 during mouse early embryogenesis [J]. Nat Rec A Discov Mol Cell Evol Biol, 2005, 283(1):187-192.
[24]Van den Akker E, Fromental-Ramain C, De Graaff W, et al. Axial skeletal patterning in mice lacking all paralogous group 8 Hox genes [J]. Development, 2001, 128(10):1911-1921.
[25]Lei H, Wang H, Juan AH, et al. The identification of Hoxc8 target genes [J]. Proc Natl Acad Sci USA, 2005, 102(7):2420-2424.
[26]Juan AH, Lei H, Bhargava P, et al. Multiple roles of hoxc8 in skeletal development [J]. Ann N Y Acad Sci, 2006, 1068:87-94.
[27]Le Mouellic H, Lallemand Y, Brulet P. Homeosis in the mouse induced by a null mutation in the Hox-3.1 gene[J]. Cell, 1992, 69(2):251-264.
[28]Shashikant CS, Kim CB, Borbely MA, et al. Comparative studies on mammalian Hoxc8 early enhancer sequence reveal a baleen whale-specific deletion of a cis-acting element [J]. Proc Natl Acad Sci USA, 1998, 95(26):15446-15451.
[29]Juan AH, Rundle FH. Enhancer timing of Hox gene expression: deletion of the endogenous Hoxc8 early enhancer [J]. Development, 2003, 130(20):4823-4834.
[30]张立岭,吉尔嘎拉,周卫等.乌珠穆沁羊脊椎数变异的群体遗传特征研究[J].内蒙古畜牧科学,1996,4:1-3.
[31]张立岭,斯琴毕力格.动物脊柱的进化与蒙古羊脊椎变异间的关系[J].内蒙古农牧学院学报,1997,18(3):1-6.
[32]张立岭.乌珠穆沁羊Homeobox基因突变与椎骨数变异的研究[J].内蒙古农业大学学报,1996,17(3):29-33.
[33]张立岭,菊林花,杨丽君.蒙古羊胸椎数的亲本印记遗传研究[J].内蒙古农业大学学报, 2000,21(2):1-6.
[34]曹忠红.小尾寒羊多脊椎遗传现象的单核苷酸多态性分析[D].河北农业大学硕士学位论文,2004.
[35]刘建明.小尾寒羊多脊椎突变的分子标记研究[D].河北农业大学硕士学位论文,2006.
[36]Guardavaccaro D, Corrente G, Covone F, et al. Arrest of G1-S progression by the p53-inducible gene PC3 is Rb dependent and relies on the inhibition of cyclin D1 transcription[ J ]. Mol Cell Biol, 2000, 20 (5):1797 - 1815.
[37]Cyril D, Nicole F, Carole A, et al. The human BTG2 /TIS21 /PC3 gene: genomic structure,transcriptional regulation and evaluation as a candidate tumor suppressor gene [J]. Gene, 2002, 282 (12): 207– 214.
[38]Prév?t D, Voeltzel T, Birot AM, et al. The leukemia-associated protein Btg1 and the p53-regulated protein Btg2 interact with the homeoprotein Hoxb9 and enhance its transcriptional activation [J]. J Biol Chem, 2000, 275:147–153.
[39]Braat AK,Zandbergen MA,de Vries E, et al. Cloning and expression of the zebrafish germ cell nuclear factor [J]. Mol Reprod Dev, 1999, 53:369-75.
[40]Morasso MI, Grinberg A, Robinson G, et al. Placental failure in mice lacking the homebox gene Dlx3[J]. Proc Natl Acad Sci USA, 1999, 96:162-167.
[41]Katz D, Niederberger C, Slaugher GR, et al. Characterization of germ cell-specific expression of the orphan nuclear receptor,gem cell nuclear factor[J]. Endocrinology, 1997, 138:4364-4372.
[42]Lan ZJ, Chung AC, Xu X, et al. The Embryonic Function of Germ Cell Nuclear Factor Is Dependent on the DNA Binding Domain [J]. J Biol Chem, 2002, 52(477):50660-50667.
[43]Gu P, LeMenuet D, Chung AC, et al. Orphan Nuclear Recepor GCNF Is Required for the Repression of Pluripotenncy Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation [J]. Mol Cell Biol, 2005, 25(19):8507-8519.
[44]Noriko Sato, Mitsumasa Kondo, Ken-ichi Arai. The orphan nulear receptor GCNF recruits DNA methyltransferase for Otc-3/4 silencing [J]. BBRC, 2006, 344:845-851.
[45]Chung AC, Katz D, Pereira FA, et al. Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation [J]. Mol Cell Biol, 2001, 21(2):663-677.
[46]Mikawa S, Hayashi T, Nii M, et al.Two quantitative trait loci on Sus scrofa chromosomes 1 and 7 affecting the number of vertebrae [J]. J Anim Sci, 2005, 83(10):2247-2254.
[47]Mikawa S, Morozumi T, Shimanuki S, et al. Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1) [J]. Genome Res, 2007, 17(5):586-593.
[48]Wang D G,Fan J B,Siao C J,et al. Large-scale identification,mapping,and genotyping of single-nucleotide polymorphisms in the human genome[J]. Science, 1998, 280(5366):1077-1082.
[49]Syvanen A C. Accessing genetic variation: genotyping single nucleotide polymorphisms [J]. Nature Review Genetics, 2001, 2:930-942.
[50]Takeshita T,Morimoto K,Mao X,et al. Characterization of the three genotypes of low K maldehyde dehydrogenase in a Japanese population [J]. Human genetics, 1994, 94:217-223.
[51]Hayashi K. PCR-SSCP: A method for detection of mutations [J]. Genetic analysis,techniques and applications, 1992, 9(3):73-79.
[52]Spinardi L,Mazars R,Theillet C. Protocols for an improved detection of point mutation by SSCP [J]. Nucleic acids research, 1991, 19(14):4009.
[53]杨昭庆,洪坤学,褚嘉佑.单核苷酸多态性的研究进展[J].国外医学遗传学分册, 2000 , 23 (1) : 4- 8.
[54]Leabman MK, Huang CC, DeYoung J, et al . Natural variation in human embrane transporter genes reveals evolutionary and functional const raints [J]. Proc Natl Acad Sci USA, 2003, 100(8) :5896 -5901.
[55]Akey J, Jin L, Xiong M. Haplotypes vs single marker linkage disequilibrium tests: what do we gain?[J]. Eur J Hum Genet, 2001, 9(4):291- 300.
[56]Slager S L, Huang J, Vieland V J. Effect of allelic heterogeneity on the power of the transmission disequilibrium test [J]. Genet Epidemiol, 2000, 18(2):143- 156.
[57]Morris R W, Kaplan N L. On the advantage of haplotype analysis in the presence of multiple disease susceptibility alleles [J]. Genet Epidemiol, 2002, 23(3):221- 33.
[58]Chapman J M, Cooper J D. Detecting disease associations due to linkage disequilibrium using haplotype tags: a class of tests and the determinants of statistical power [J]. Hum Hered, 2003, 56(1- 3):18- 31.
[59]J.萨姆布鲁克,E.F弗里奇,T.曼尼阿蔕斯著,金冬雁、黎孟枫等译.分子克隆实验指南[M].北京:科学技术出版社,2002.
[60]Michaels SD, Amasino RM. A robust method for detecting single-nucleotide changes as polymorphic markers by PCR [J]. The Plant journal, for cell and molecular biology, 1998, 14(3): 381-385.
[61]唐时幸.单链构象多态性分析与应用[J].生物技术通讯, 1995,6(4):179-180.
[62]Sunnucks P, Wilson AC, Beheregaray LB, et al. SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology [J]. Mol Ecol, 2000, 9(11):1699-1710.
[63]Kukita Y, Tahira T, Sommer SS, et al. SSCP analysis of long DNA fragments in low pH gel [J]. Hum Mutat, 1997, 10(5):400-407.
[64]Fu Y, Kuhl DP, Pizzuti A, et al. Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox [J]. Cell, 1991, 67(6):1047-1058.
[65]Hayashi K. PCR-SSCP: A method for detection of mutations [J]. Genet Genet Anal Tech Appl, 1992, 9(3):73-79.
[66]刘佳健.猪PRKAR1A基因的PCR-RFLP和PCR-SSCP研究[D].武汉:华中农业大学硕士学位论文,1996.
[67]卢圣栋.《现代分子生物学实验技术》[M].北京:高等教育出版社, 1993.
[68]O'Connell CD, Tian J, Juhasz A, et al. Development of standard reference materials for diagnosis of p53 mutations: analysis by slab gel single strand conformation polymorphism [J]. Electrophoresis, 1998, 19(2):164-171.
[69]张学,孙开来. PCR-SSCP分析技术及其应用[J].国外医学遗传学分册, 1992, 5(5):225-231.
[70]姜运良,李宁.影响PCR-SSCP的因素分析[J].农业生物技术学报, 2000, 8(3):245-247.
[71]Clark AG. The role of haplotypes in candidate gene studies [J]. Genetic Epidemiology, 2004, 27(4): 321-333.
[72]焦传珍,王吉华,田志环.真核生物基因组中的非编码序列[J].生物学杂志, 2000,17(4):12-14.
[73]Park S, Lee YJ, Lee HJ, et al. B-cell translocation gene 2 (Btg2) regulates vertebral patterning by modulating bone morphogenetic protein/smad signaling [J]. Molecular and Cellular Biology, 2004, 24(23):10256-10262.
[2]路广计,王振来,郑文波,等.河北小尾寒羊饲养屠宰使用报告[J].河北畜牧兽医,2001,17(10):14.
[3]张立岭,斯琴毕力格.蒙古羊多脊椎基因的群体遗传学研究[J].遗传,1997,19 (增刊) : 67-69
[4]张立岭,斯琴毕力格,张世铨.多脊椎蒙古羊的胸腰椎长度对产肉性能的影响[J].中国畜牧杂志,1998,34(3):24-25.
[5]Venter, J.C., Adams, M.D., Myers, E.W, et al. The sequence of the human genome [J]. Science, 2001, 291:1304-1351.
[6]Lee, SJ, McPherson, AC. Regulation of myostatin activity and muscle growth [J]. Proc. Natl. Acad. Sci. 2000, 98: 9306-9311.
[7]Yeo C, Whitman M. Nodal signals to Smads through Criptodependent and Cripto-independent mechanisms [J]. Mol Cell. 2001, 7(5):949-957.
[8]Mathews LS, Vale WW. Expression cloning of an activin receptor, a predicted transmembrane serine kinase[J]. Cell, 1991, 65: 973-982.
[9]Feijen, A., Goumans, M J, van den Eijnden, et al. Expression of activin subunits, activin receptors and follistatin in postimplantation mouse embryos suggests specific developmental functions for different activins [J]. Development, 1994, 120:3621-3637.
[10]Alexandra C McPherson, Ann M Lawler, Se-Jin Lee. Regulation of anterior/posterior patterning of the axial skeleton by growth/differentiation factor 11[J]. Nature genetics, 1999, 22 (3):260-264.
[11]Oh SP , Li E. The signaling pathway mediated by the type IIB activin receptor controls axial patterning and lateral asymmetry in the mouse [J]. Genes & Dev. 1997, 11: 1812-1826.
[12]Song J, Oh SP, Schrewe H, et al. The type II activin receptors are essential for egg cylinder growth, gastrulation, and rostral head development in mice[J]. Dev Biol, 1999, 213:157-169.
[13]Matzuk MM, Kumar TR., Bradley A, et al. Different phenotypes for mice deficient in either activins or activin receptor type II [J]. Nature, 1995, 374:356-360.
[14]Ethier JF, Houde A, Lussier JG,et al. Bovine activin receptor type II cDNA: cloning and tissue expression [J]. Mol Cell Endocrinol, 1994, 106(1-2):1-8.
[15]Ethier JF, Lussier JG, Silversides DW. Bovine activin receptor type IIB messenger ribonucleic acid displays alternative splicing involving a sequence homologous to Src-homology 3 domain binding sites [J]. Endocrinology, 1997, 138(6):2425-2434.
[16]Oh SP, Yeo CY, Lee Y, et al. Activin type IIA and IIB receptors mediate Gdf11 signaling in axial vertebral patterning [J]. Genes&Development, 2002, 16(21):2749-2754.
[17]Lee YJ, Hong KH, Yun J, et al. Generation of activin receptor type IIB isoform-specific hypomorphic alleles [J]. Genesis, 2006, 44(10):487-494.
[18]Awgulewitsch A, Utset MF, Hart CP, et al. Spatial restriction in expression of a mouse homoeobox locus within the central nervous system[J]. Nature, 320:328-335.
[19]Glavac D, Dean M.Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations [J]. Hum Mutate, 1993, 2:404-414.
[20]Zhao Y, Potter SS. Functional comparison of the Hoxa 4, Hoxa 10, and Hoxa 11 homeboxes[J]. Dev Biol, 2002, 244(1):21-36.
[21]Wellik DM, Capecchi MR. Hox10 and Hox11 genes are required to globally pattern the mammalian skeleton [J]. Science, 2003, 301(5631):363-367.
[22]Erselius JR, Goulding MD, Gruss P. Structure and expression pattern of the murine Hox3.2 gene [J]. Development, 1990, 110(2):629-642.
[23]Kwon Y, Shin J, Park HW, et al. Dynamic expression pattern of Hoxc8 during mouse early embryogenesis [J]. Nat Rec A Discov Mol Cell Evol Biol, 2005, 283(1):187-192.
[24]Van den Akker E, Fromental-Ramain C, De Graaff W, et al. Axial skeletal patterning in mice lacking all paralogous group 8 Hox genes [J]. Development, 2001, 128(10):1911-1921.
[25]Lei H, Wang H, Juan AH, et al. The identification of Hoxc8 target genes [J]. Proc Natl Acad Sci USA, 2005, 102(7):2420-2424.
[26]Juan AH, Lei H, Bhargava P, et al. Multiple roles of hoxc8 in skeletal development [J]. Ann N Y Acad Sci, 2006, 1068:87-94.
[27]Le Mouellic H, Lallemand Y, Brulet P. Homeosis in the mouse induced by a null mutation in the Hox-3.1 gene[J]. Cell, 1992, 69(2):251-264.
[28]Shashikant CS, Kim CB, Borbely MA, et al. Comparative studies on mammalian Hoxc8 early enhancer sequence reveal a baleen whale-specific deletion of a cis-acting element [J]. Proc Natl Acad Sci USA, 1998, 95(26):15446-15451.
[29]Juan AH, Rundle FH. Enhancer timing of Hox gene expression: deletion of the endogenous Hoxc8 early enhancer [J]. Development, 2003, 130(20):4823-4834.
[30]张立岭,吉尔嘎拉,周卫等.乌珠穆沁羊脊椎数变异的群体遗传特征研究[J].内蒙古畜牧科学,1996,4:1-3.
[31]张立岭,斯琴毕力格.动物脊柱的进化与蒙古羊脊椎变异间的关系[J].内蒙古农牧学院学报,1997,18(3):1-6.
[32]张立岭.乌珠穆沁羊Homeobox基因突变与椎骨数变异的研究[J].内蒙古农业大学学报,1996,17(3):29-33.
[33]张立岭,菊林花,杨丽君.蒙古羊胸椎数的亲本印记遗传研究[J].内蒙古农业大学学报, 2000,21(2):1-6.
[34]曹忠红.小尾寒羊多脊椎遗传现象的单核苷酸多态性分析[D].河北农业大学硕士学位论文,2004.
[35]刘建明.小尾寒羊多脊椎突变的分子标记研究[D].河北农业大学硕士学位论文,2006.
[36]Guardavaccaro D, Corrente G, Covone F, et al. Arrest of G1-S progression by the p53-inducible gene PC3 is Rb dependent and relies on the inhibition of cyclin D1 transcription[ J ]. Mol Cell Biol, 2000, 20 (5):1797 - 1815.
[37]Cyril D, Nicole F, Carole A, et al. The human BTG2 /TIS21 /PC3 gene: genomic structure,transcriptional regulation and evaluation as a candidate tumor suppressor gene [J]. Gene, 2002, 282 (12): 207– 214.
[38]Prév?t D, Voeltzel T, Birot AM, et al. The leukemia-associated protein Btg1 and the p53-regulated protein Btg2 interact with the homeoprotein Hoxb9 and enhance its transcriptional activation [J]. J Biol Chem, 2000, 275:147–153.
[39]Braat AK,Zandbergen MA,de Vries E, et al. Cloning and expression of the zebrafish germ cell nuclear factor [J]. Mol Reprod Dev, 1999, 53:369-75.
[40]Morasso MI, Grinberg A, Robinson G, et al. Placental failure in mice lacking the homebox gene Dlx3[J]. Proc Natl Acad Sci USA, 1999, 96:162-167.
[41]Katz D, Niederberger C, Slaugher GR, et al. Characterization of germ cell-specific expression of the orphan nuclear receptor,gem cell nuclear factor[J]. Endocrinology, 1997, 138:4364-4372.
[42]Lan ZJ, Chung AC, Xu X, et al. The Embryonic Function of Germ Cell Nuclear Factor Is Dependent on the DNA Binding Domain [J]. J Biol Chem, 2002, 52(477):50660-50667.
[43]Gu P, LeMenuet D, Chung AC, et al. Orphan Nuclear Recepor GCNF Is Required for the Repression of Pluripotenncy Genes during Retinoic Acid-Induced Embryonic Stem Cell Differentiation [J]. Mol Cell Biol, 2005, 25(19):8507-8519.
[44]Noriko Sato, Mitsumasa Kondo, Ken-ichi Arai. The orphan nulear receptor GCNF recruits DNA methyltransferase for Otc-3/4 silencing [J]. BBRC, 2006, 344:845-851.
[45]Chung AC, Katz D, Pereira FA, et al. Loss of orphan receptor germ cell nuclear factor function results in ectopic development of the tail bud and a novel posterior truncation [J]. Mol Cell Biol, 2001, 21(2):663-677.
[46]Mikawa S, Hayashi T, Nii M, et al.Two quantitative trait loci on Sus scrofa chromosomes 1 and 7 affecting the number of vertebrae [J]. J Anim Sci, 2005, 83(10):2247-2254.
[47]Mikawa S, Morozumi T, Shimanuki S, et al. Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1) [J]. Genome Res, 2007, 17(5):586-593.
[48]Wang D G,Fan J B,Siao C J,et al. Large-scale identification,mapping,and genotyping of single-nucleotide polymorphisms in the human genome[J]. Science, 1998, 280(5366):1077-1082.
[49]Syvanen A C. Accessing genetic variation: genotyping single nucleotide polymorphisms [J]. Nature Review Genetics, 2001, 2:930-942.
[50]Takeshita T,Morimoto K,Mao X,et al. Characterization of the three genotypes of low K maldehyde dehydrogenase in a Japanese population [J]. Human genetics, 1994, 94:217-223.
[51]Hayashi K. PCR-SSCP: A method for detection of mutations [J]. Genetic analysis,techniques and applications, 1992, 9(3):73-79.
[52]Spinardi L,Mazars R,Theillet C. Protocols for an improved detection of point mutation by SSCP [J]. Nucleic acids research, 1991, 19(14):4009.
[53]杨昭庆,洪坤学,褚嘉佑.单核苷酸多态性的研究进展[J].国外医学遗传学分册, 2000 , 23 (1) : 4- 8.
[54]Leabman MK, Huang CC, DeYoung J, et al . Natural variation in human embrane transporter genes reveals evolutionary and functional const raints [J]. Proc Natl Acad Sci USA, 2003, 100(8) :5896 -5901.
[55]Akey J, Jin L, Xiong M. Haplotypes vs single marker linkage disequilibrium tests: what do we gain?[J]. Eur J Hum Genet, 2001, 9(4):291- 300.
[56]Slager S L, Huang J, Vieland V J. Effect of allelic heterogeneity on the power of the transmission disequilibrium test [J]. Genet Epidemiol, 2000, 18(2):143- 156.
[57]Morris R W, Kaplan N L. On the advantage of haplotype analysis in the presence of multiple disease susceptibility alleles [J]. Genet Epidemiol, 2002, 23(3):221- 33.
[58]Chapman J M, Cooper J D. Detecting disease associations due to linkage disequilibrium using haplotype tags: a class of tests and the determinants of statistical power [J]. Hum Hered, 2003, 56(1- 3):18- 31.
[59]J.萨姆布鲁克,E.F弗里奇,T.曼尼阿蔕斯著,金冬雁、黎孟枫等译.分子克隆实验指南[M].北京:科学技术出版社,2002.
[60]Michaels SD, Amasino RM. A robust method for detecting single-nucleotide changes as polymorphic markers by PCR [J]. The Plant journal, for cell and molecular biology, 1998, 14(3): 381-385.
[61]唐时幸.单链构象多态性分析与应用[J].生物技术通讯, 1995,6(4):179-180.
[62]Sunnucks P, Wilson AC, Beheregaray LB, et al. SSCP is not so difficult: the application and utility of single-stranded conformation polymorphism in evolutionary biology and molecular ecology [J]. Mol Ecol, 2000, 9(11):1699-1710.
[63]Kukita Y, Tahira T, Sommer SS, et al. SSCP analysis of long DNA fragments in low pH gel [J]. Hum Mutat, 1997, 10(5):400-407.
[64]Fu Y, Kuhl DP, Pizzuti A, et al. Variation of the CGG repeat at the fragile X site results in genetic instability: resolution of the Sherman paradox [J]. Cell, 1991, 67(6):1047-1058.
[65]Hayashi K. PCR-SSCP: A method for detection of mutations [J]. Genet Genet Anal Tech Appl, 1992, 9(3):73-79.
[66]刘佳健.猪PRKAR1A基因的PCR-RFLP和PCR-SSCP研究[D].武汉:华中农业大学硕士学位论文,1996.
[67]卢圣栋.《现代分子生物学实验技术》[M].北京:高等教育出版社, 1993.
[68]O'Connell CD, Tian J, Juhasz A, et al. Development of standard reference materials for diagnosis of p53 mutations: analysis by slab gel single strand conformation polymorphism [J]. Electrophoresis, 1998, 19(2):164-171.
[69]张学,孙开来. PCR-SSCP分析技术及其应用[J].国外医学遗传学分册, 1992, 5(5):225-231.
[70]姜运良,李宁.影响PCR-SSCP的因素分析[J].农业生物技术学报, 2000, 8(3):245-247.
[71]Clark AG. The role of haplotypes in candidate gene studies [J]. Genetic Epidemiology, 2004, 27(4): 321-333.
[72]焦传珍,王吉华,田志环.真核生物基因组中的非编码序列[J].生物学杂志, 2000,17(4):12-14.
[73]Park S, Lee YJ, Lee HJ, et al. B-cell translocation gene 2 (Btg2) regulates vertebral patterning by modulating bone morphogenetic protein/smad signaling [J]. Molecular and Cellular Biology, 2004, 24(23):10256-10262.