| The coat colour of Tibetan Sheep restricted the economic value of its wool, while the study of themolecular mechanism about the major gene and the mechanism for Tibetan Sheep coat color is stillblank. This test was carried out on the coat colour candidate genes of Tibetan sheep, Polymorphisms ofAgouti, MC1R, KIT, MITF gene were Analysised just Aimed to identify the gene mutation which wasassociated with the coat color, as well as filtered out those genes with difference expression in skintissue through Fluorescence quantitative PCR, using Western blot and immunohistochemical to analysisthe genes with difference expression, we had getten this results as follows:Firstly, we found35mutation sites within Agouti gene of Tibetan sheep, also detected Agouti gene withcopy number variation. Through association analysis of coat colour with the G.100-1045bp deletionmutation in exon2of Agouti gene and the copy number variation of Agouti gene, we had come to aconclution that neither pure white nor pure black coat sheep population was correlation with thismutation. MC1R coding region mutation was not found in this population, a new g.1360T﹥C mutationhad been detected in MITF and g.2340C﹥G had been detected in KIT, both this two mutation were incoding region. H-W equilibrium Detected the KIT g.2340C﹥G in pure black and pure white groupswas equilibrium,while MITF g.1360T﹥C was not equilibrium. The result Show that there may be nomutations associated with the coat color of the pigment candidate genes Agout, MC1R and KIT, MITFg.1360T>C site was in great selection pressure. The C allele is more conducive to generating purewhite coat phenotype.Secondly, there were no significant difference for the relative mRNA expression of Agouti and KITgenes between pure white and pure black coat skin tissue, also there was no significant in white andblack spots coat skin tissue of variegated individuals; MC1R relative mRNA expression of pure whitecoat individual was significantly higher than individuals with pure black coat, also white coat skin tissuewas significantly higher than black spot; MITF gene relative mRNA expression of pure black coatindividual was significantly higher than the individuals with pure white coat, yet black spots skin tissuewas significantly higher than white coat. The mRNA relative expression of Agouti and MITF in thesame coat color skin tissue of the same individual was no significant correlation. There was nosignificant correlation between Agouti gene and MITF gene mRNA expression in the same coat colorskin tissues of the same individual. The result of semi-quantitative analysis showed that MC1R gene andMITF-M subtypes only expression in the skin tissue. It is implied that high mRNA expression of MITFgene is conducive to the formation of black coat. High mRNA expression of MC1R gene was associatedwith the white coat.Thirdly, the expression of c-kit protein in the skin tissue of white coat and black spot for the variegatedTibetan sheep was basically the same, the expression of MITF protein in the skin tissue of black spotwas significantly higher than the white coat, the expression of c-kit protein in the Tibetan sheep skin tissue of pure white and pure black was basically the same, the expression of MITF protein in the skintissue of pure black was significantly higher than the pure white. MITF positive expression in skintissue mainly detected in the top layer of skin, hair follicle dermal papilla and outer root sheathdetection of hair follicle. The positive MITF staining colour of black coat skin tissue was deeper thanwhite coat. We get a conclution that the black hair of Tibetan sheep is associated with the highexpression of MITF protein in the dermal papilla and bulb of hair follicle, MITF plays an important rolefor the formation of wool color. |
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