KIT基因与荣昌猪毛色相关性研究
摘要
通常人们对白毛色猪肉较为偏爱。自中世纪以来，人们就开始对白色家猪进行选育，在选育过程中，通常将有杂色斑点的白猪淘汰。但由于毛色形成的原因极为复杂，隐性杂毛色等位基因较多，所以经过近100年的努力，人们仍无法将无斑点白猪的基因型固定下来。KIT基因对猪的白毛色的形成起起非常重要的作用，为了阐明荣昌猪白毛色形成的分子机制，本研究以荣昌猪(包括全白色和其他各种类型毛色)、杜洛克、大白和长白猪为实验动物，以编码肥大细胞生长因子受体的KIT基因为候选基因，通过聚合酶链式反应单链构象多态性分析(PCR-SSCP)和焦磷酸测序(pyrosequencing)，研究KIT基因的多态性和KIT基因的突变比例，分析KIT基因和荣昌猪毛色的相关性，为猪毛色遗传定向选育奠定基础。
结果：(1)通过PCR-SSCP方法检测了105头不同毛色类型的荣昌猪、30头杜洛克、40头长白和10头大白每一个体的KIT基因多态性。结果发现，相同品种中不同个体都得到相同的KIT基因条带，其中长白和大白有同样的4条带，而荣昌猪和杜洛克为相同的2条带。
(2)选择具有不同带型的样品，对其KIT基因17、18内含子进行双向测序，结果发现大白和长白猪17内含子第一核苷酸处发生了G→A的替换突变，18内含子内发生了4碱基的剪接突变，而荣昌猪和杜洛克上都没有发现这两种突变。
(3)从PCR-SSCP检测过的样品中，选择10头荣昌猪进行焦磷酸测序，获得不同毛色类型的荣昌猪KIT基因内含子17的第一核苷酸处G→A突变比例都为0％，再次证实荣昌猪不存在G→A的突变。
(4)对26头长白猪和4头大白猪的焦磷酸测序结果发现，白色猪种中不同个体G→A的突变比例差异较大，说明表型为全白的猪种不一定全为显性白基因型，很多个体中存在不同类型的杂合隐性基因。
(5)对杜洛克进行PCR—SSCP检测和焦磷酸测序结果都证明在其KIT基因的17、18内含子中均没有突变产生。
结论：(1)荣昌猪与长白、大白猪虽然在毛色上十分相似，但荣昌猪的白毛色可能不属于显性白毛色，推测它们的白色属于两种不同的毛色遗传体系，遗传本质上存在极大的差异。
(2)KIT基因的G→A的突变和AGTT缺失可能不是导致白毛色产生的唯一决定性突变，也许在KIT基因的其他区域还存在别的未被发现的决定性突变位点，这可能是导致荣昌猪白毛色形成的原因。另外，影响黑色素形成的基因较多，各基因间存在互作，这可能也是影响
White-coated meat was favored by consumers, and the selection for white coat-color domestic pigs was started since medieval times through which the pigment spots are usually eliminated from breeding herds. Unfortunately, breeders have not been able to completely fix the desired phenotype so far in spite of nearly 100-year strong selection due to the diversity of recessive alleles of pigment spots and the complexity of coat-color forming. With the speedy development of molecular biology and biotechnologies, researchers have been studying the genetic characters of coat color on the level of molecule. For the purpose of studying the polymorphisms of KIT gene and quantification of the ratio of wild-type/mutant nucleotide at the splice site and exploring the relationship between KIT gene and Rongchang pig coat color, this study was conducted to apply single strand conformation polymorphism analysis of polymearase chain reaction products (PCR-SSCP) and pyrosequencing in Rongchang pigs (including white and other coat color), Duroc, Landrace and Large White pigs, and chose KIT gene of mast/stem cell growth factor receptor (MGF) as candidate gene.
The results of polymorphisms of KIT gene showed that the genotypes of KIT gene were specific among the breeds. Electrophoresis patterns of PCR-SSCP for intron 17/18 of porcine KIT gene showed that the same breed detected individuals have the same strips of KIT gene; Landrace and Large White pig have an identical strips of KIT gene (4 strips) and Rongchang pig and Duroc have another identical strips of KIT gene(2 strips). KIT gene in pigs have different strips was sequenced. The result of sequencing KIT gene in a Rongchang pig showed that the mutations of KIT gene of Landrace and Large White pig (G→A and the deletion of AGTT) were not found in Rongchang Pig with the similar coat color, which was against that white coat color was controlled by traditional dominated white allele gene. Therefore, the conclusion was attained that the
结果：(1)通过PCR-SSCP方法检测了105头不同毛色类型的荣昌猪、30头杜洛克、40头长白和10头大白每一个体的KIT基因多态性。结果发现，相同品种中不同个体都得到相同的KIT基因条带，其中长白和大白有同样的4条带，而荣昌猪和杜洛克为相同的2条带。
(2)选择具有不同带型的样品，对其KIT基因17、18内含子进行双向测序，结果发现大白和长白猪17内含子第一核苷酸处发生了G→A的替换突变，18内含子内发生了4碱基的剪接突变，而荣昌猪和杜洛克上都没有发现这两种突变。
(3)从PCR-SSCP检测过的样品中，选择10头荣昌猪进行焦磷酸测序，获得不同毛色类型的荣昌猪KIT基因内含子17的第一核苷酸处G→A突变比例都为0％，再次证实荣昌猪不存在G→A的突变。
(4)对26头长白猪和4头大白猪的焦磷酸测序结果发现，白色猪种中不同个体G→A的突变比例差异较大，说明表型为全白的猪种不一定全为显性白基因型，很多个体中存在不同类型的杂合隐性基因。
(5)对杜洛克进行PCR—SSCP检测和焦磷酸测序结果都证明在其KIT基因的17、18内含子中均没有突变产生。
结论：(1)荣昌猪与长白、大白猪虽然在毛色上十分相似，但荣昌猪的白毛色可能不属于显性白毛色，推测它们的白色属于两种不同的毛色遗传体系，遗传本质上存在极大的差异。
(2)KIT基因的G→A的突变和AGTT缺失可能不是导致白毛色产生的唯一决定性突变，也许在KIT基因的其他区域还存在别的未被发现的决定性突变位点，这可能是导致荣昌猪白毛色形成的原因。另外，影响黑色素形成的基因较多，各基因间存在互作，这可能也是影响
White-coated meat was favored by consumers, and the selection for white coat-color domestic pigs was started since medieval times through which the pigment spots are usually eliminated from breeding herds. Unfortunately, breeders have not been able to completely fix the desired phenotype so far in spite of nearly 100-year strong selection due to the diversity of recessive alleles of pigment spots and the complexity of coat-color forming. With the speedy development of molecular biology and biotechnologies, researchers have been studying the genetic characters of coat color on the level of molecule. For the purpose of studying the polymorphisms of KIT gene and quantification of the ratio of wild-type/mutant nucleotide at the splice site and exploring the relationship between KIT gene and Rongchang pig coat color, this study was conducted to apply single strand conformation polymorphism analysis of polymearase chain reaction products (PCR-SSCP) and pyrosequencing in Rongchang pigs (including white and other coat color), Duroc, Landrace and Large White pigs, and chose KIT gene of mast/stem cell growth factor receptor (MGF) as candidate gene.
The results of polymorphisms of KIT gene showed that the genotypes of KIT gene were specific among the breeds. Electrophoresis patterns of PCR-SSCP for intron 17/18 of porcine KIT gene showed that the same breed detected individuals have the same strips of KIT gene; Landrace and Large White pig have an identical strips of KIT gene (4 strips) and Rongchang pig and Duroc have another identical strips of KIT gene(2 strips). KIT gene in pigs have different strips was sequenced. The result of sequencing KIT gene in a Rongchang pig showed that the mutations of KIT gene of Landrace and Large White pig (G→A and the deletion of AGTT) were not found in Rongchang Pig with the similar coat color, which was against that white coat color was controlled by traditional dominated white allele gene. Therefore, the conclusion was attained that the
引文
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[3] Hetzer HO. Inhertitance of coat color in swine. Ⅱ. Results of Landrace by Poland China crosses [J]. Journal of Heredity,, 1945b, 36: 187-192.
[4] Hetzer HO. Inhertitance of coat color in swine. Ⅲ. Results of Landrace by Berkshire crosses[J]. Journal of Heredity, 1945c, 36: 255-256.
[5] Hetzer HO. Inhertitance of coat color in swine. Ⅵ. Results of Yorkshire by Duroc-Jersey crosses [J]. Journal of Heredity, 1947, 38: 121-124.
[6] Hetzer HO. Inhertitance of coat color in swine. Ⅴ. Results of Landrace by Hampshire crosses[J]. Journal of Heredity, 1948, 39: 123-128.
[7] Hetzer HO. Effectiveness of selection for extension of black-spotting in Beltsille no 1 swinelnhertitance of coat color in swine.[J]. Journal of Heredity, 1954, 45: 215-223.
[8] Hetzer HO. Inhertitance of coat color in swine. Ⅴ. Results of Landrace by Duroc-Jersey crosses[J]. Journal of Heredity, 1946, 37: 217-224.
[9] 施启顺，马海明．猪的毛色遗传研究进展[J]．国外畜牧科技，2000，27(6)：29-33．
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[14] Celia JC, Francisco S, Takeshi K. A new enzymatic fuction in the melanogenic pathway. The 5,6-dihydroxyindole-2-carboxylic acid oxidase activity of tyrosinase-related protein-1 (TRP1).[J]. The Journal of biological chemistry, 1994, 269(27): 17993-18000.
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[16] Ollivier, Sellier P. Pig genetics: a reviw[J]. Annales de Genetique et de Selection Animale, 1982, 14: 481-544.
[17] Lauvergne JJ, Canope I. Etude de quelqwes variants colores du porc Creole de al Guadeloupe.[J]. Annales de Genetique et de Selection Animale, 1979, 11: 381-390.
[18] 张仲葛，李炳坦．中国猪品种志[M]．上海：上海科学技术出版社，1986．Pages．
[19] Kijas J. M. A,Tornsten BC, Andersson. L. Porcine Agouti gene map position SSC17q21[J]. Chromosome Research, 1998, 6: 243.
[20] Kim KS, Mendez EA, Marklund S. Rapid communication: Linkage mapping of the porcine Agouti gene[J]. JAnim Sci, 2000., 78: 1395-1396.
[21] Bultman SJ, Michaud EJ, Woychik RE Molecular characterization of the mouse Agouti locus[J]. Cell, 1992, 71: 1195-1204.[22] Bultman SJ, Russell LB, Gutierrez-Espeleta GA. Molecular characterization of a region of DNA associated with mutations at the Agouti locus in the mouse[J]. Proc Natl Acad Sci USA, 1991, 88: 8062-8066.
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