【作者】 李蓓；

【导师】 芒来；

【作者基本信息】 内蒙古农业大学 ， 动物遗传育种与繁殖， 2011， 博士

【摘要】 动物毛色基因研究是毛色遗传学研究的重要内容,毛色不仅是马品种和个体识别的重要依据,而且还可以作为某些疾病筛查的有力工具和手段。开展马毛色基因的遗传学研究不仅具有理论意义,更具有特殊的经济价值和人文意义。蒙古马是我国最古老的品种之一,其毛色种类繁多,是研究马毛色遗传的最佳模式动物。本研究以不同类群蒙古马为实验对象,首先对蒙古马MC1R基因、ASIP基因、TYRP1基因和STX17基因进行多态性检测,找出与毛色表型相关的基因位点,并对位点间的遗传效应进行了剖分;其次采用实时荧光定量PCR(QRT-PCR)的相对定量方法对蒙古马MC1R基因、ASIP基因、TYR基因、TYRP1基因、TYRP2基因和STX17基因在不同毛色皮肤、肝脏、垂体和肾脏组织中差异表达进行了研究;最后制备了MC1R蛋白和ASIP蛋白的特异性多克隆抗体,采用蛋白免疫印迹技术对蒙古马MC1R蛋白在不同毛色蒙古马皮肤和垂体组织的差异表达进行研究。通过以上研究,为蒙古马毛色遗传研究提供了重要的理论依据,并获得以下主要研究结果:(1)分别采用PCR-RFLP、PCR-AFLP和PCR-SSCP技术分别对蒙古马MC1R基因E位点、ASIP基因A位点和TYRP1基因B位点进行了遗传多态检测。研究结果表明MC1R基因在编码区第901bp处碱基发生C→T错义突变,该突变导致编码的氨基酸由丝氨酸突变为苯丙氨酸;ASIP基因在第二外显子2174-2184bp处发生碱基缺失突变;TYRP1基因在第二外显子189bp处碱基发生C→T错义突变,该突变导致编码的氨基酸由苏氨酸突变为蛋氨酸。(2)通过各多态位点与蒙古马毛色表型相关性分析得到,MC1R基因E位点的隐性纯合型与蒙古马栗色毛的形成具有较强的相关性,ASIP基因A位点的隐性纯合型与蒙古马的黑色毛具有较强的相关性,TYRP1基因B位点主要与毛色的深浅及有色毛的形成有关。(3)半巢式PCR扩增结果表明,在灰色蒙古马中均检测到了STX17基因第六内含子处4.6kb的重复片段,而在非灰色马中未检测到,证明STX17基因与蒙古马的灰色毛形成可能具有相关性。(4)采用NOIA模型对蒙古马MC1R基因E位点与ASIP基因A位点对皮肤反射系数影响的遗传效应和遗传方差进行了剖分。从剖分结果得到8个遗传效应组分均对蒙古马皮肤反射系数具有显著的影响;经过分析发现蒙古马MC1R基因与ASIP基因间不但具有显著的上位效应,并且具有拮抗作用,二者共同作用影响着毛色表型的形成;通过遗传方差剖分证明蒙古马的毛色性状主要由MC1R和ASIP基因的遗传效应所决定,环境效应影响不大。(5)采用QRT-PCR的2-△△Ct方法对蒙古马MC1R基因、ASIP基因、TYR基因、TYRP1基因、TYRP2基因及STX17基因在不同毛色皮肤、肝脏、垂体和肾脏组织进行了差异表达研究。结果表明各基因在所有组织中均有表达;对于皮肤组织,MC1R基因、TYR基因、TYRP1基因和TYRP2基因均在有色毛皮肤组织中的表达量较高,灰色毛皮肤组织中的表达量最低;ASIP基因在骝色毛皮肤组织中的表达量最高,STX17基因在灰色毛皮肤中表达量最高,二者均在黑色毛皮肤组织中的表达量最低。表明MC1R基因和酪氨酸酶家族基因是影响深色系毛色形成的主要候选基因,STX17基因是浅色系毛色形成的主效基因;ASIP基因与深色系毛色和浅色系毛色的形成均具有相关性。从各基因的差异表达情况可以看出ASIP基因与MC1R基因以及酪氨酸酶家族基因互为抑制。(6)采用多肽合成抗原及原核表达外源蛋白的方法制备了MC1R基因和ASIP基因的特异性多克隆抗体,并采用蛋白免疫印迹的方法对MC1R蛋白在不同毛色蒙古马皮肤和垂体组织中的表达量进行了半定量检测。结果显示MC1R蛋白在黑色毛皮肤组织中表达量最高,在灰色毛皮肤组织中表达量最低;在黑色毛垂体组织中表达量最高,在灰色垂体组织中表达量最低。更多还原

【Abstract】 Animal coat colour genes research is the important part of the colour genetics, the coat colour is not only the important basis to identification equine breeds and individuals, but also is the powerful means to disease screening. The research of the horse coat colour genes genetics not merely has theoretical significance, furthermore has special economical value and humanistic significance. The Mongolian Horse who has the various kinds of coat colour is one of the ancient indigenous horse breeds in our country and the ideal model animal to research about equine coat colour genetics. The research used five groups of Mongolian Horse as the experiment materials. First of all, the polymorphisms of MC1R gene, ASIP gene, TYRP1 gene and STX17 gene were detected and the interaction of related loci were analyzed; Secondly, differentially expression of MC1R gene, ASIP gene, TYR gene, TYRP1 gene, TYRP2 gene and STX17 gene in skin, liver, hypophysis, and kidney of different coat colour Mongolian Horse were researched by real-time fluorescence quantitative PCR(QRT-PCR); The specific polyclonal antibody of MC1R gene and ASIP gene were prepared, and the differentially expression level of MC1R in skin, hypophysis of different coat colour Mongolian Horse were detected by Western blotting. Given all the above researchs, important theoretical basis were provided for Mongolian Horse coat colour genetics, and the following mainly results were obtained:(1) The polymorphism of Mongolian Horse MC1R gene (locus E), ASIP gene (locus A) of and TYRP1 gene (locus B) were detected by PCR-RFLP, PCR-AFLP and PCR-SSCP respectively. The results showed that the MC1R gene had a C→T missense mutation in 901bp coding region and caused the Ser→Phe amino acid substitution. The ASIP gene had a deletion mutation in 2174-2184bp of exon 2. The TYRP1 gene had a C→T missense mutation C189T in 189bp of exon 2 and caused the Thr→Met amino acid substitution.(2) According to the analysis of correlation between polymorphisms with phenotype, the recessive homozygote in locus E of MC1R gene had a strong correlation with chestnut Mongolian Horse, the recessive homozygote in locus A of ASIP gene had a positive correlation with black Mongolian Horse, the locus B of TYRP1 gene mainly related with colour deep or light and coloured coat formation.(3) The results of semi-nested PCR showed that 4.6 kb duplication in intro 6 of STX17 gene were detected in gray Monglian Horse, but not found in non-gray. The results proved that the STX17 gene might correlate with the gray coat colour formation of Mongolian Horse.(4) The genetic effect and variance for skin reflectance of MC1R gene locus E and ASIP gene locus A of Mongolian Horse were analyzed by NOIA model. For the results of division, the 8 genetic effective componenets were all be found have significantly affect to skin reflectance of Mongolian Horse. The results showed that the MC1R gene and ASIP gene not noly had significantly epistatic effect, but also had antagonism effect, the Mongolian Horse coat colour phenotype generated by the effect of the two genes. The division of variance showed that the genetic effects were dominant factors to coat colour, the environmental effects were secondary factor.(5) The differential expression of MC1R gene, ASIP gene, TYR gene, TYRP1 gene, TYRP2 gene, STX17 gene in various coat colour skin, liver, hypophysis, and kidney of Mongolian Horse were analyzed by 2-△△Ct method of QRT-PCR. The results showed that the above mentioned genes were expressived in all the tissues. For the skin tissues, the expression level of MC1R gene, ASIP gene, TYR gene, TYRP1 gene, TYRP1 gene, TYRP2 gene was higher in coloured coat skin than in gray coat colour;The expression level of ASIP gene was highest in bay coat colour, the expression of STX17 gene was highest in gray coat colour, the expression of ASIP gene and STX17 gene were lowest in black colour. The results showed that the MC1R gene and tyrosinase-related protein family genes were dominant candidate gene to deep colour, the STX17 gene was dominant candidate gene to light colour, the ASIP gene had relationship between both of deep colour and light colour. The antagonism between ASIP gene with MC1R gene and tyrosinase-related protein family genes were found according to the differential expression of mentioned above genes.(6) The specific polyclonal antibody of MC1R gene and ASIP gene were prepared by synthesis of peptides and prokaryotic expression foreign protein for antigen, and the expression of MC1R protein in various coat colour skins and hypophysis of Mongolian Horse was semi-quantitative determinated by Western blotting. The results showed that the expression of MC1R protein was highest in black skin tissue,lowest in gray skin;highest in black hypophysis tissue,lowest in gray hypophysis.更多还原