MC1R c.676A>G与山羊皮肤色素沉积的关系及其对黑色素合成的影响

Relationship between MC1R c.676A>G and goat skin pigmentation and its effect on melanin synthesis

为探讨山羊MC1R基因c.676A>G突变与山羊皮肤色素沉积的关系,及其对黑色素合成和cAMP信号通路下游色素相关基因表达的影响:一方面采用直接测序法检测酉州乌羊及其杂交后代群体(120个个体)MC1R基因c.676A>G的突变情况,用色差仪测量不同基因型个体的腹部皮肤色度值,分析c.676A>G突变与皮肤色素沉积的关系;另一方面,构建野生型(MC1R c.676A)和突变型(MC1R c.676G)真核表达载体,采用脂质体介导法转染到小鼠皮肤黑色素瘤细胞(B16-F10)中进行过表达,利用酶标仪检测各组细胞黑色素含量的差异,并通过实时荧光定量PCR方法检测各组细胞中cAMP信号通路下游色素相关基因(MITF、TYR、TYRP 1、DCT)的表达差异性。结果显示:酉州乌羊及其杂交后代群体中,c.676A>G位点均以GG基因型占优势,不同基因型群体的皮肤色度值差异不显著(P>0.05)。MC1R突变组(MC1R c.676G)细胞中的黑色素含量极显著(P<0.05)高于MC1R野生组(MC1R c.676A)。MITF、TYRP 1和DCT基因在MC1R突变组中的相对表达量极显著(P<0.01)高于野生组,TYR基因在MC1R突变组细胞中的相对表达量显著(P<0.05)高于野生组。综上,MC1R基因c.676A>G不直接影响山羊皮肤色素沉积,但MC1R c.676G能促进B16-F10细胞中黑色素的合成,也能促进cAMP信号通路下游MITF、TYR、TYRP 1和DCT等基因的表达,推测MC1R基因c.676A>G与黑色素合成密切相关。

In order to reveal the relationship between goat MC1R gene c.676A>G mutation and goat skin pigmentation,and the effect of mutation on melanin synthesis and the expression of pigment-related genes in the downstream of the cAMP signaling pathway:on the one hand,the direct sequencing was used to detect the MC1R c.676A>G mutation of Youzhou dark-skin goats and the hybrid offsprings(120 individuals),and the abdominal skin color of individuals of different genotypes was determined with the colorimeter,and the relationship between c.676A>G mutation and skin pigmentation was analyzed;on the other hand,the eukaryotic expression vectors of wild type(MC1R c.676A)and mutant type(MC1R c.676G)were constructed and transfected into B16-F10 cells for overexpression by liposome-mediated method,and the differences of melanin in each group were detected by the microplate reader and the expression differences of pigment-related genes in the downstream of the cAMP signaling pathway(MITF,TYR,TYRP1,DCT)in cells were detected by quantitative real-time PCR.The results showed that the c.676A>G site of Youzhou dark-skin goats and their hybrid offsprings was dominated by the GG genotype,and the skin color values of different genotype populations did not show significant difference(P>0.05).The melanin in mutant group cells(MC1R c.676G)showed significantly(P<0.01)higher value than that of the wild group cells(MC1R c.676A).The relative expression of MITF,TYRP 1 and DCT in the mutant group cells was significantly(P<0.01)higher than that of the wild group,and the relative expression of TYR in the mutant group was significantly(P<0.05)higher than that of the wild group.In summary,MC1R c.676A>G did not directly affect goat skin pigmentation.However,MC1R c.676G could promote the synthesis of melanin in B16-F10 cells and could increase the expression of MITF,TYR,TYRP 1 and DCT genes in the downstream of cAMP signaling pathway,indicating that MC1R c.676A>G was closely related to melanin synthesis.