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山羊Agouti基因内含子3变异在不同毛色群体中分布研究
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摘要
本试验根据GenBank上已发表的绵羊(EU185099)Agouti基因序列进行设计引物,扩增山羊Agouti基因3’UTR区,测序结果表明该片断的长度为190bp。对山羊Agouti基因内含子3测序分析发现四个g.A4756C、g.C4971T、g.A5344G和g.AGG5124-5125变异位点,并对这四个变异位点进行分析。研究发现这4个突变位点共有6种单倍型,分别为ACAA(71.31%)、ATBG(13.11%)、CTBG(7.38%)、ATAA(4.92%)、ATAG(1.64%)、CTAG(0.82%)。在济宁青山羊(青灰色)、辽宁绒山羊(白色)、雷州黑山羊(黑色)山羊中单倍型几乎都为ACAA,但是在南江黄羊快长系(棕褐色)山羊中有不同的单倍型。因此,推测这些突变位点可能在山羊毛色形成过程中发挥一定作用。
     在山羊Agouti基因内含子3上g.A4756C位点发现了两个等位基因A和C,青灰色(济宁青山羊)、白色(辽宁绒山羊)和黑色(雷州黑山羊)为主的山羊品种中有较高频率的等位基因A。棕色山羊(南江黄羊快长系)A等位基因的频率为54.69%,其三种基因型分布比较平均。分析表明g.A4756C的C等位基因可能与控制山羊棕色表型的位点存在连锁关系,CC基因型为与棕色被毛相关的基因型。
     在山羊Agouti基因内含子区g.C4971T位点也发现了两个等位基因C和T,济宁青山羊(JN)中C等位基因的频率为98.33%;雷州黑山羊(LH)C等位基因的频率为93.33%,辽宁绒山羊(LR)中C等位基因的频率为100.00%,南江黄羊快长系(NK)中T等位基因的频率为100.00%,全部为TT基因型。分析表明g.C4971T的T等位基因可能与控制山羊棕色表型的位点存在连锁关系,TT基因型为与棕色被毛相关的基因型。
     在山羊Agouti基因内含子3上g.A5344G位点发现了两个等位基因A和G,其中A等位基因在济宁青山羊、雷州黑山羊和辽宁绒山羊中占优势,等位基因频率分别为:100.00%、96.67%、100.00%,但从基因型上看,济宁青山羊和辽宁绒山羊中全部为AA基因型,雷州黑山羊中AA基因型占绝对优势,只有南江黄羊快长系中有GG基因型并且占绝对优势。分析表明g.A5344G的G等位基因可能与控制山羊棕色表型的位点存在连锁关系,GG基因型为与棕色被毛相关的基因型。
     在山羊Agouti基因内含子3中g.AGG5124-5125插入位点发现了两个等位基因A(非插入AGG碱基)和B(插入AGG碱基)。其中A等位基因在济宁青山羊、雷州黑山羊和辽宁绒山羊中占优势,等位基因频率分别为:98.33%、90.00%、96.67%,但从基因型上看,济宁青山羊和辽宁绒山羊中全部为AA和AB基因型,雷州黑山羊中三种基因型都有,在这三个山羊品种中AA基因型占绝对优势,在南江黄羊快长系中BB(71.87%)基因型占绝对优势。由此我们推测g.AGG5124-5125插入位点的B等位基因可能与控制山羊棕色表型的位点存在连锁关系,BB基因型为与棕色被毛相关的基因型。
     经单倍型分析,发现ACAA(71.31%)、ATBG(13.11%)、CTBG(7.38%)、ATAA(4.92%)、ATAG(1.64%)、CTAG(0.82%)六种单倍型。在济宁青山羊(青灰色系)和辽宁绒山羊(白色系)两个品种中,几乎所有个体的单倍型为ACAA,CTBG的单倍型占少数,但是在雷州黑山羊(黑色系)和南江黄羊快长系(棕色系)的品种中有不同的单倍型,推测CTBG的单倍型可能与棕色表型相关。
     聚类结果表明4个地方山羊品种分为两类,第一类为具有棕色毛色表型位基因的南江黄羊快长系,第二类为具有黑色、灰色以及白色等位基因的济宁青山羊、辽宁绒山羊和雷州黑山羊。经过和毛色相关分析表明,在这4个地方山羊品种中,A4756C、g.C4971T、g.A5344G和g.AGG5124-5125这四个位点的突变和山羊的毛色有显著性的相关。本试验的聚类结果反映了4个不同地方山羊品种在Agouti基因A4756C、g.C4971T、g.A5344G和g.AGG5124-5125的群体间的遗传关系。
The primers are designed according to the sheep Agouti gene(EU185099) that has published in GenBank. The sequenced length of 3’UTR region of goat is 190bp. The g.A4756C、g.C4971T、g.A5344G and g.AGG5124-5125 variable sizes are found in the 3rd intron of goat Agouti gene, and then analyse the four variation sites. Research shows that they have six haplotypes that is ACAA(71.31%)、ATBG(13.11%)、CTBG(7.38%)、ATAA(4.92%)、ATAG(1.64%)and CTAG (0.82%) in the four variable sites. Almost all individuals of JN with grey coat color, LH with black coat color and LR with white coat color were haplotype ACAA, but NK with brown coat color had different haplotypes. It is infered that these variable sites may be play a certain role in color goat of the process of forming.
     A and C alleles of g.A4756C locus were found in the 3rd intron of goat Agouti gene, and the frequency of A allele is high in cinerous, white, and black variey. The frequency of A allele is 54.69% in Brown goats that the distribution of three genotype is hypodispersion. The C allele of g.A4756C may exist chain relation with sites that controlled the brown phenotype of goats, and CC genotype is related with brown hair by the analysis.
     The C and T allele of g.C4971T locus were found in intron region of Agouti gene. The frequency of C allele is 98.33% in JN, the frequency of C allele is 93.33% in LH, the frequency of C allele is 100% in LR and the NK, which the genotype is all TT. The T allele of g.C4971T may exist chain relation with sites that controlled the brown phenotype of goats, and TT genotype is related with brown hair by the analysis.
     The A and G allele of g.A5344G locus were found in the 3rd intron of Agouti gene. The A allele was dominated in JN、LH、LR, The genotype was AA in JN and LR, and AA genotype is absolute advantage in LH, on the contrary, the GG genotype was absolute advantage in the NK. The G allele of g.A5344G may exist chain relation with sites that controlled the brown phenotype of goats, and GG genotype is related with brown hair by the analysis.
     The A and B allele of g.AGG5124-5125 insert locus were found in the 3rd intron of Agouti gene, which the A was not inserted the AGG, but B was inserted by AGG. The A allele was dominated in JN, LH and LR, the frequency of A allele was 98.33%, 90.00% and 96.67% separately. The genotype was AA in JN, the genotype was AB in LR, and three of genotypes were existed in LH, the AA genotype was rich in these three breeds, on the contrary, the BB genotype (71.87%) was absolute advantage in the NK. So we conjectured that the B allele of g.A5344G may exist chain relation with sites that controlled the brown phenotype of goats, and BB genotype is related with brown hair by the analysis. After the single times type analysis,found six single times type that ACAA(71.31%)、ATBG(13.11%)、CTBG(7.38%)、ATAA(4.92%)、ATAG(1.64%)、CTAG(0.82%). Almost the JN and LR is ACAA, the single times type that ACAA is small proportion. But there are different single times type in LH and NK, single times type that CTBG could relate with brown phenotype.
     The result of cluster shows that the 4 local breeds of goat were divided into two classes, the one is the NK that allele has the brown hair phenotype; the other one is JN, LH and LR, which allele has black, grey and white hair phenotype. The relative analysis shows that the mutations of A4756C、g.C4971T、g.A5344G and g.AGG5124-5125 locus were significantly related with hair of goat in these 4 local breeds. The result of cluster can just reflect the heredity relation of these 4 local breed goat by the A4756C、g.C4971T、g.A5344G and g.AGG5124-5125 locus of Agouti gene, but not real heredity relation. We need to further study in order to the genetic relationship of 4 local goat breeds.
引文
[1]Kwon H Y, Bultman S I, Loffler C, et al. Molecular structure and chrom--osomal mapping of the human homolog of the Agouti gene [J]. Proc Natl Acad Sci USA,1994,91:9760-9764.
    [2]Voisey J,van Daal A.Agouti: from mouse to man,from skin to fat [J]. Pigment Cell Res.2002,15(l):10-18.
    [3]MICHEAL M, OLLMANN M, LYNN L, et al. Interaction of Agouti protein with the melanocortin 1 receptor in vitro and in vivo [J]. Gene and Development, 1998, 12: 316-330.
    [4]Prota G.Melanin and Melanogenesis[M].New York: Academic Press,1992.l-290.
    [5]刘甲斐,仇雪梅.黑色素及其相关基因的研究进展[J].生物技术通报,2007,4:55-58。
    [6]Kijas J M H et al. Melanocortin Receptor 1(MC1R) Mutation and Coat Color in Pigs [J].Ccnclica, 1998,150:1177-1185.
    [7]伍革民,彭光旭.动物黑色素研究进展[J].甘肃畜牧兽医,2005,1:39-41
    [8]Takeuchi S, Suzuki H, Hirose S, et al. Molecular cloning and sequence analysis of the chick melanocortin-1 receptor gene[J]. Biochimica et Biophysica Acta, 1996, 1306: 122-126.
    [9]常洪.山羊毛色遗传.国外畜牧科技[J],1995,22(5):23-26.
    [10]Searle A G.Comparative Genetics of Coat Colourin Mammals[M]. London : Logos Press , 1968.
    [11]Lush J L. Inheritance of horns waitles, and colorin grade Toggenburg goats[J]. Jour Hered, 1926,17:73-91.
    [12]Asdell, S. A., and F.A.E. Crewe. Inheritance of color, beard, tassels and horns in the goat[J].Jour. Hered , 1928 ,19:425-431.
    [13]Ollmann M M, Barsh G S. Down-regulation of melanocortin receptor signaling mediated by the amino terminus of Agouti protein in Xenopus melanophores[J]. Journal of Biological Chemistry, 1999, 274(22): 15837-15846.
    [14]Suzuki I, Tada A, Ollmann M M, et al. Agouti signaling protein inhibits melanogenesis and the response of human melanocytes toα-melanotropin[J]. Journal of Investigative Dermatology, 1997, 108(6): 838-842.
    [15]Argeson A C,Neison K K,Siracusa L D. Molecular basis of the pleiotropic phenotype of mice carrying the hypervariable yellow (Ahvy) mutation at the Agouti locus[J]. Genetics, 1996,142:557-567.
    [16]Bultman S J,Michaud E J,Woychik R P.Molecular characterization of the mouse Agouti locus[J]. Cell,1992,71:1195-1204.
    [17]Matsunaga N, Virador V, Santis C,et al. In situ localization of Agouti Signal Protein in murine skin hsing immunohistochemistry with an ASP-specific antibody[J]. Biochemical and Biophysical Research Communications, 2000,270:176-182.
    [18]Vrieling H,Duhl D M J,Millar S E,et al. Differences in dorsal and ventral pigmentation result fromregional expression of the mouse Agouti gene[J].Proc Natl Acad Sci USA,1994,91:5667-5671.
    [19]Bultman S J,Klebig M L,Michaud E J,et al. Molecular analysis of reverse mutations from nonAgouti (a) to black-and-tan (at) and white-bellied Agouti (Aw) reveals alternative forms of Agouti transcripts[J].Genes Dev,1994,8:481-490.
    [20]Joanne V,Neil F,Angela V D et al.A polumorphism study of the human Agouti gene and its association with MC1R pigment[J]. Cell Res, 2001,14:264-267.
    [21]Matsunaga N, Virador V, Sands C, et al.In situ localization of Agouti signal protein in murine skin using immunohistochemistry with an ASP- specific antibody[J]. Biochem Biophys Res Commun, 2000, 270:176-182.
    [22]Miltenberger, R J, Mynatt R L et al.An Agouti mutation lacking the basic domain induces yellow pigmentation but not obesity in transgenic mice[J]. Proc. Nat. Acad. Sci. 1999, 96:8579-8584.
    [23]Millar S E, Miller M W, Stevens M E et al. Exp2ssion and transgenic studies of the mouse Agouti gene provide insight into the mechanisms by which mamm- -alian coat color patterns a2 generated[J].Development, 1995, 121:3223-3232.
    [24]Kucera G T, Bortner D M, Rosenberg M P, Overexpression of an Agouti cDNA in the skin of transgenic mice recapitulates dominant coat color phenotypes of spontaneous mutants[J]. Development Biology,1996,173:162-173.
    [25]林大光.兔毛的遗传特性[J].中国养兔杂志,1984,(1):16.
    [26]Voisey J, Box N F, van Daal A.A polymorphism study of the hum an Agouti gene and its association with MC1R[J]. Pigment Cell Res, 2001, 14:264-267.
    [27]Kanetsky P A, Swoyer J, et al.A Polymorphism in the Agouti signaling protein gene is associated with human pigmentation[J]. Am. J. Hum. Genet. 2002, 70: 770-775.
    [28]Stefan R, Sead T, Denis M, et al.Mutations in the Agouti(ASIP), the extension (MC1R), and the brown (TYRP1) loci and their association to coat color phenotypes in horses (Equus caballus)[J].Mammalian Genome 2001,12:450-455.
    [29]Vage D I, Lu D, Klungland H, et a1.A non-epistatic interaction of Agouti and extension in the fox, Vulpes vulpes[J]. Nat Genet 1997 ,15 (3): 311-315.
    [30]Eizirik E, Yuhki N, Johnson W E, et al. Molecular genetics and evolution of melanism in the cat family[J]. Curr. Biol. 2003, 13:448-453.
    [31]Kijas J M, Tornsten A, Chowdhary B, et al.Porcine Agouti gene map position SSC17q21 [J].Chromosome Research,1998,6:243.
    [32]Kim K S, Mendez E A, Marklund S, et al.Rapid communication:Linkage map- -ing of the porcine Agouti gene[J].J.Anim.Sci.2000,78:1395-1396.
    [33]Wang Y, et al. Isulaliun, coloning and sequencing of porcine Agouti exon2 (PorAex2)[J].Pigment cell research, 1998,11:155-157.
    [34]Leeb T, et al. Canonic structure and nucleotide polymorphisms of the porcine Agouti signaling protein gene (ASIP)[J].Animal Genetics, 2000,31(5):335-336.
    [35]Michael G, Juliette M, Sylvain G. Widespread expression of the bovine Agouti gene results from atleast three alternative promoters[J].Pigment Cell Res.18:34-41.
    [36]Olison T A.The Genetics of Cattle[C].Wallingford Oxon:CAB Intemational,1999
    [37]赵静雯.山羊Agouti基因的遗传多样性研究(D).保定:河北农业大学硕士学位论文,2006.
    [38]唐春娟.山羊Agouti基因与毛色表型相关分析及其遗传变异的研究(D).保定:河北农业大学硕士学位论文,2008.
    [39]Li Xiang-long, Zhao Jing-wen, Tang Chun-juan, Zhou Rong-yan, Zheng Guiru, Li Lan-hui, Guo Xiu-li (2010) Sequencing of Part of the Goat Agouti Gene and SNP Identification. Biochemical Genetics 48 (1-2):152-156.
    [40]Weiwei Wu, Xianglong Li, Rongyan zhou, Lanhui Li, Huiqin zheng (2011 ) .Origin and differentiation of a special fragment from Capra hircus Agouti gene. African Journal of Biotechnology .Vol. 10(51):10373-10379
    [41]Tang Chun-Juan, Zhou Rong-Yan, Li Xiang-Long, Zhao Jing-Wen, Li Lan-Hui, Feng Fu-Jun, Li Dong-Feng, Wang Jian-Tao, Guo Xiu-Li, Kang Jing-Fen (2008) Variation of 423G>T in the Agouti gene exon 4 in indigenous Chinese goat breeds. Biochemical Genetics 46:770–780.
    [42]Tang Chun-Juan, Li Xiang-Long, Zhou Rong-Yan, Li Lan-Hui, Feng Fu-Jun, Li Dong-Feng, Wang Jian-Tao (2009) Study on genetic diversity of T128 del in Agouti gene intron 1 in Chinese main indigenous goat breeds. Acta Veterinarina et Zootechnica Sinaca 40 (4):320–326.
    [43]Vignal A, Milan D, SanCristobal M, et al. A review on SNP and other types of molecular markers and their use in animal genetics[J]. Genet. Se1. Evol, 2002(34): 275-305.
    [44]杜玮南,孙红霞,方福德.单核苷酸多态性的研究进展.国外医学(遗传学分册),2000(4):392-394.
    [45]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.
    [46]Syvanen A C. Accessing genetic variation: genotyping single nucleotide polymorphisms [J]. Nature Review Genetics, 2001, 2:930-942.
    [47]Hayashi K. PCR-SSCP: A method for detection of mutations [J]. Genetic analysis,techniques and applications, 1992, 9(3):73-79.
    [48]Spinardi L,Mazars R,Theillet C. Protocols for an improved detection of point mutation by SSCP [J]. Nucleic acids research, 1991, 19(14):4009.
    [49]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.
    [50]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.
    [51]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.
    [52]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.
    [53]Morris R W, Kaplan N L. On the advantage of haplotype analysis in the presence of multiple diseasesusceptibility alleles [J]. Genet Epidemiol, 2002, 23(3):221- 33.
    [54]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.
    [55]J.萨姆布鲁克,弗里奇E F,曼尼阿蔕斯T著,金冬雁、黎孟枫等译.分子克隆实验指南(第三版)[M].北京:科学技术出版社,2002.
    [56]Botstein D, White RL, Skolnick M and Davis RW (1980) Construction of a genetic liNJFage map in man using restriction fragment length polymorphisms. Am J Hum Genet 32:314-331.
    [57]Takezaki N, Nei M. Genetic distance and reconstruction of phylogenetic trees from microsatellite DNA[J].Genetics.1996, 144 :389-399.
    [58]Nei M, Tajima R, Tateno Y. Accuracy of estimated phylogenetic trees from moleculars data[J]. J. Mo1.Evo I .1983,19 : I53-170.
    [59]Thompson JD, et al.Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[J]. Nucleic Acids Res. 1994, 22(22): 4673-4680.
    [60]张成岗,贺福初.生物信息学方法与实践[M].北京:科学出版社, 2002.
    [61]Spinardi L, M azars R, Heillet C.Protocols for an improved detection of point mutations by SSCP[J]. Nucleic Acids Research, 1991,19(14):4009.
    [62]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.
    [63]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.
    [64]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.
    [65]韩立霞.河北小尾寒羊多脊椎调控基因Btg2和NR6A1的多态性分析[D].保定:河北农业大学硕士学位论文, 2009.
    [66]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.
    [67]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.
    [68]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.
    [69]张爱玲,马月辉,李宏滨,等.利用微卫星标记分析6个山羊品种遗传多样性[ J ].农业生物技术学报, 2006 , 14 ( 1 ):38 -44.
    [70]Stephens M, Scheet P (2005). Accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation. Am J Hum Genet 76: 449-462.
    [71]耿岩,杨章平,常洪等中国蒙系6个绵羊品种的遗传分化和基因流[J].扬州大学学报,2007,3:22-26.
    [72]Wright S. Evolution in Mendelian population [J]. Genetics, 1931, 16: 91-159.
    [73]WRIGHT S. Evolution and the genetics of populations. Variability within and among natural populat ions [M]. Chicago: Univer sity of Chicago Press, Vol. 4. 1978.
    [74]胡海芳,张华,孙捷,阳玉萍,王燕,谭国萍,向阳冰,陈俞(2011).新疆维、汉人群CD14基因单核苷酸多态性的连锁不平衡和单倍型分析.新疆医科大学学报. 34(7):682-686.
    [75]Gabriel SB, Schaffner SF, Nguyen H, et al (2002). The structure of haplotype blocks in the human genome [J]. Science, 296(5576): 2225 -2229.
    [76]Wall JD, Pritchard JK (2003). Haplotype blocks and linkage disequilibrium in the human genome [J]. Nat Rev Gen et, 4(8):587 - 597.
    [77]范瑞文.etal.哺乳动物毛色色素Agouti基因位点的研究进展[J]Progress in Veterinary Medicine动物医学进展,2004,25(3):59-61.
    [78]GIRARDOT M, GUIBERT S, LAFORET M P, et al. The insertion of a full-lengthBos TaurusLINE element is responsible for a transcriptional deregulation of the Normand Agoutigene[J].Pigment Cell Research, 2006, 19: 346-355.

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