The Effect Of Function For Melanocortin-1 Receptor Gene Regulated By MiR-324-3p From Melanocytes Of Alpaca

Melanocortin receptor 1 (melanocortin-1 receptor, MC1R) is located on surface of the grown up melanocytes, the gene is regarded as one of major and related genes.which regulated the synthesis and type transformation of animals melanin.The result of screening and prediction found that miR-324-3p may target MC1R gene and regulate the expression of the gene.To investigate the effect of function on melanocortin-1 receptor gene regulated by miR-324-3p, the research verified the targeting relationship between miR-324-3p and MC1R gene via dual luciferase reporter vector, transfected the overexpression vector of miR-324-3p into Alpaca melanocytes in vitro,measured the mRNAs expressive quantities by real-time quantitative PCR, measured the proteins expressive quantities by western blotting, as far as MC1R gene and its downstream genes,which related with coat color, measured melanin production by microplate reader. The exprimental methods and main results of this research are as follows:1. Application of qRT-PCR was to measure the expressive difference of miR-324-3p in melanocytes of white or brown coat color Alpaca. The results show that the expressive quantity of miR-324-3p in melanocytes of brown Alpaca is highly significantly than that of white Alpaca(P<0.01), and relatively expressive quantity in melanocytes of brown Alpaca is 1.64 times than the that of white Alpaca.2. Application of Dual luciferase reporter gene detection system was to measure the targeting relationship between miR-324-3p and MC1R gene, three experimental groups of 293T cells were a s follows:A group(pmirGLO-MC1R-3'UTR), B group(NC+pmirGLO-MC1R-3'UTR),C group(miR-32 4-3p+pmirGLO-MC1R-3'UTR). The results show that luciferase activity of C group is 0.66 times t han that of A group,and is 0.69 times than that of group B.3. Application of qRT-PCR was to measure the expressive quantities at transcriptional level of MC1R,Mitf,Tyr and Tyrp2 genes in three experimental groups, three experimental groups were as follows:treatment group (miR group, which transfected only miR-324-3p overexpression vectors), Negtive Control (NC group, which transfected only miR-NC vectors), control cells (KB group, which did not transfect vectors). The results show that expressive quantities at the transcriptional level of MCIR, Mitf, Tyr and Tyrp2 genes in miR group are respectively 0.194,0.473,0.550 and 0.920 times than that of NC group,expressive quantities at the transcriptional level of MC1R, Mitf and Tyr in miR group are significantly different with NC group(P<0.01), while expressive quantity at the transcriptional level of Tyrp2 in miR group is markedly different with NC group(P<0.05);Expressive quantities at the transcriptional level of MCIR, Mitf, Tyr and Tyrp2 genes in miR group are respectively 0.019,0.321,0.180 and 0.347 times than that of KB group, expressive quantities at the transcriptional level of MC1R, Mitf, Tyr and Tyrp2 from miR group are all significantly different with the KB group(P<0.01).4. Application of western blotting was to measure the expressive quantities at protein level of MC1R,Mitf,Tyr and Tyrp2 genes in three experimental groups.The results show that expressive quantities at the protein level of MC1R, Mitf, Tyr and Tyrp2 genes in miR group are respectively 0.815,0.389,0.903 and 0.808 times than that of NC group.and are respectively 0.811,0.382,0.889 and 0.733 times than that of KB group; Expressive quantities at protein level of MC1R, Mitf, Tyr and Tyrp2 from miR group are all significantly different with NC and KB group(P<0.01).5. Application of microplate reader was to measure the A475 absorbance values of melanin in three experimental groups, the ratio between A475 absorbance values of melanin on experimental groups and that of KB group represent the relative quantity of melanin. The results show that relative quantity of melanin in miR group is 0.498739±0.0112840, that of NC group is 0.878930±0.0202527, that of KB group is 1.000000±0.0342576, melanin relative quantity in miR group are all significantly than that of NC group and KB group(P<0.01).A conclusion is that miR-324-3p expressed in Alpaca melanocytes can target and down-regularte the expression of MCIR, and then down-regulate the expression of those genes as the downstream genes of MCIR and related to coat color, down-regulate ultimately synthesis quantity of melanin.The significance of this research is offering for reliable basis on function of miR-324-3p and MC1R gene in pigmentogenesis of Alpaca skin.