Study On The Relationship Between Mice Agouti Gene And Color Phenotype And Its Variation In Mice Populations

Mammalian coat color is a polygenic trait, as well as agouti gene is a major candidate gene about studying mammalian coat color. Agouti signaling protein are involved in a wide range of effects, such as coat color, diabetes, obesity, sexual behavior and cancer susceptibility and so on. In1994, the mouse Agouti signaling protein (ASP) was found that it has high affinity with Melanocortin-1-receptor (MC1R), after which it began the prologue of studying Agouti signaling protein pleiotropic mechanism.Firstly, in order to reveal the molecular mechanism of coat color difference in chromosome engineering mice, we detected mice coat color by color measurement spectrophotometer to make sure the exsitence of color differences, and found that the K/S values of eight strains respectively in the K/S value of C3H/He mice on both sides. According the K/S value of C3H/He mice, the eight strains of mice can be divided into two types, that is, light grey and deep grey. Secondly, DNA chip scanning confirmed a candidate chromosome fragment, which was located between rs13476817and rs13476889on chromosome2, where there was a Agouti gene related to coat color. Therefore, Agouti was chosen as the candidate gene for the following analysis.Based on sanger method, the sequences of candidate gene Agouti cDNA were obtained. The length of the open reading frame (ORF) of Agouti gene was396bp, which encodes131amino acids. The single nucleotide polymorphisms of the Agouti cDNA sequence was analyzed, resulting five SNPs:in light grey mice, there are four SNPs (A37G, G1O8A,G110A and A309G); in deep grey mice, there are two SNPs (A37G, C286G). Hence, we defined the mutations which occurs in light grey mice as type I, and defined the mutations which occurs in deep grey mice as type Ⅱ. Based on RT-PCR results, it was found that there were no significant differences in mRNA expression levels, indicating that the polymorphisms of Agouti cDNA sequence showed no effects on the expressions of Agouti gene. Manceau found that in the skin tissues of different coat colors, the spatial structure of Agouti signaling protein were different. According this, we prepare to analyse the space conformation of Agouti signaling protein. The N-glycosylation sites and cysteine-rich C-terminal region of the Agouti signaling protein are very important in keeping the intact biological functions of this protein, whereas the mutations in lysine/arginine-rich region seems no effects on the biological function of ASP. Bioinformatics analysis found that only the mutation (C286G) of type Ⅱ mutation occured in the cysteine-rich C-terminal, causing the arginine turns to glycine. The missense mutation of type Ⅱ Agouti signaling protein resulted that one β turn deletion and one y turn、one disulfide bond add up in the secondary structure of the mutant protein, as well as tertiary structure changed, leading to binding ability decreasing. And through the use of the color measurement spectrophotometer, We found that the color of type Ⅱ mutant mice was darker than wild type mice and type Ⅰ mutant mice. All of these are conforming to the argument which points out that mutations in the C-terminal of Agouti signaling protein will reduce its ability that inhibit a-MSH binding to MCIR receptors, resulting an increase in melanin/pheomelanin, and animal coat color mostly towards deeper color.In this study, a novel missense mutation is found in candidate Agouti gene. It plays critical role in receptor binding activity, reducing which may reflect on mice coat color changing from light gray to dark gray eventually.