The Study On Polymorphism Analysis And Tissue Expression Of Mongolian Horse Coat Colour Genes

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.