| Coat color is an important characteristic to identify species and property, and also one of the important indices for varieties and animal breeding genetic uniformity. Among livestock and poultry, they have different coat color because exist, distribution and ratio differences of eumelanin and pheomelanin in melanocytes. In the research we focused on local swamp buffalo and LuChuan pigs as models to clone MC1R coding sequences by PCR, then analyzed expression pattern of the related genes(MC1R?KIT?MITF?TYR?TYRP1 DCT and EDNRB) of coat colour formation in different buffalo skin and black and white skin of LuChuan pigs by qRT-PCR, and detected expression level of MC1R? MTTF?TYR protein in different cattle and black and white skin of LuChuan pigs by western-blot. Moreover, skin texture of LuChuan pigs were observed by HE staining and characterizing the localization of TYR protein in skin of that by immunohistochemistry. The main research results are as follows:1. MC1R coding sequences of GuangXi local swamp buffalo and LuChuan pigs were cloned and analyzed. Sequence analysis indicated that MCIR genes of buffaloes and LuChuan pigs were 954bp and 1115bp in length, which encode 317 and 321 amino acids respectively. Cloning and sequencing MC1R of a Murrah buffalo, the white swamp buffalo and cattle in the same way, several basic groups substitution were found by multiple sequence alignment, and 476 C> T,618 C> G,930G>A,931 A > G, which causing Phe 159 Ser, Glu 310 Ala, Asp294 Ala, all of that are nonsynonymous.2. The melanogenesis related genes expressions profile and protein expression level in skin of different cattle were analyzed. We found gene expressions of melanogenesis related genes (MC1R?KIT?MITF?TYR? TYRP1?DCT and EDNRB) in different kinds of cattle by qRT-PCR. The results showed that expression of MC1R> KIT?MITF and TYR in the white swamp buffalo is lower than that of Murrah buffalo, swamp buffalo and cattle observably (P<0.05). In mRNA level, the expression of tyrosinase gene family (TYR?TYRP1?DCT) in Murrah buffalo is higher than that of swamp buffalo, the white swamp buffalo and cattle markedly. We hardly detected TYRP1 expression in the white swamp buffalo and cattle. Then we detected expression of MC1R?MITF?TYR in protein level. As a result, protein expression of MC1R?MITF and TYR in Murrah buffalo was lower than that of swamp buffalo. Protein expression of the selected genes in the white swamp buffalo was also lower than that of swamp buffalo, especially TYR gene protein band was very light, which was consistent with the previous step qRT-PCR.3. The melanogenesis related genes expressions profile and protein expression level in the black and white skin of LuChuan pigs were analyzed. We found gene expressions of melanogenesis related genes (MC1R, KIT?MITF?TYR?TYRP1?DCT and EDNRB) in the black and white skin of a LuChuan pig by qRT-PCR. The results displayed that the expression of TYR and KIT had not significant difference in mRNA level (P>0.05), whose relative expression was 0.8±0.05 and 1.38±0.04 respectively. However, the expression of other genes in black skin was higher than that of white skin and had significant difference (P<0.05).Relative expression of MC1R, TYRP1 and DCT in mRNA level in black skin was 13.83±1.67,12.38±1.45 and 7.73±0.43 respectively and was higher than that of white skin. HE staining.and the immunohistochemical results showed that TYR had positive staining in both black and white skin of LuChuan pigs and hair follicles were stained. We also detected that expression of TYR ptotein in both black and white skin of LuChuan pigs by western-blot and both expression level was close to each other. Combining previous results of qRT-PCR and immunohistochemical, all indicated that expression of TYR had not significant difference in mRNA and protein level. |
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