Analyzing Coat Color Differences Between Red And Silver Fox (Vulpes Vulpes), Pigment Genes Cloning And Tentative Functional Investigation Of TYRP1

The silver fox has occurred as a mutation of the red fox and belongs therefore to the same species-Vulpes vulpes. These the two kinds of foxes have different coat color, and the molecular mechanism of coat color variation is unknown. Color is a major factor of fur price, and also the import economic trait, in addition to as to be the breeding goal. The study on the coat color variation provides the theoretical basis and technical support for breeding a new coat color type fox. It provides the basis for the molecular mechanism of coat color variation, and solid support for molecular breeding of the fox by mining genes and finding the signal pathway of coat color formation.In the present study, the red fox and silver fox were used. I carried out some experiments. First, the melanin component and content of fur were assayed in the red and silver fox. Meanwhile, karyotypes were analyzed in the two kinds. Second, different expression genes were screened from skin tissues of red and silver fox by RNA-seq. Third, differential expressed microRNAs were obtained by Small RNA-seq. and target genes of miRNAs also were predicted. Fourth, the CDS sequences of MC1 R, MITF and TYRP1 were cloned by the regular molecular experiments. Then structures and functions of these genes also were predicted by bioinformatics web and software. Fifth, the method of isolating and culture melanocytes from the fox skin tissues was established. Finally, a different expression gene by the RNA-seq information, that is TYRP1, was selected to construct the overexpression vector of PiggyBac. The TYRP1-PiggyBac vector was transfected into melanocytes and the function of TYRP1 in the process of melanin synthesis also was investigated. These experiments laid a technical foundation for investigating color genes and miRNAs function in the future.The main results and conclusions were summarized as follows:1. The skin biopsy showed the type of hair follicle in the fox skin is multiple hair follicles, and major parts are three hair follicles together. Moreover, the melanin content was assayed. The total melanin and eumelanin content from coat in the silver fox were higher than their in red fox with highly significant(p<0.01)., and the eumelanin content in the wood amd guard hair of silver fox also had a significant higher(p<0.05), in addition to the melanin content in other types of hairs between the two foxes have no significant difference.2. The karyotypes were no significant differences between red fox and silver fox, which both have 34 chromosomes(2n=32+X,Y), including several B type chromosomes(generally, 0-4), metacentric chromosome(12 pairs of autosomes and X sex chromosome) and submetacentric chromosome(4 pairs of autosomes and Y sex chromosome).3. From the RNA-seq, there are two differential expressed gene closely related to melanin formation. They were PTPRU( protein tyrosine phosphatase, receptor-type, U), has up-regulated expression and TYRP1, have down-regulated expression. The two genes played a crucial role in the coat color variation. Then, two highly different signal pathways, ECM-receptor interaction and Tyrosine metabolism were enriched.4. From the Small RNA sequencing, 23 differential expressed miRNAs were displayed, 16 miRNAs were up-regulated and 7 miRNAs were down-regulation. Of the most important, miR125 b and miR-145 have an important role in melanin synthesis. Moreover, 8 signal pathways were ensured to participating in the process of melanin synthesis. They are TGF-beta signaling pathway、Endocytosis、Lysosome、Cysteine and methionine metabolism、Tyrosine metabolism、Phenylalanine, tyrosine and tryptophan biosynthesis、Phenylalanine metabolism、Glutathione metabolism。5. The CDS sequence of MC1 R in the silver fox was 954 bp, and encoded a seven transmembrane protein. The protein has three glycosylation sites, three phosphorylation sites and G-protein coupled receptor signal sites. It has 6 general phosphorylation sites and special phosphorylation site in T-157. The CDS sequence of MITF in the silver fox was 1260 bp, and encoded a non-membrane binding protein. The protein has 9 phosphorylation sites, one Leucine zipper domain. It has 26 general phosphorylation sites and special phosphorylation site in T-38. The CDS sequence of TYRP1 in the silver fox was 1614 bp, and encoded a 2 transmembrane protein. The protein has 8 glycosylation sites, 17 Protein kinase phosphorylation sites, 2 growth factor structure domain sites and 2 Cu binding sites. It has 27 general phosphorylation sites and special phosphorylation site in T-128.6. The method of isolating and culture melanocytes from the fox skin tissues, including primary culture, cell cryopreserved and recovery, Purification culture and so on, was established.7. The overexpression vector of TYRP1-PiggyBac was constructed and transfected into melanocytes in vitro culture. The technology platform can be used to study the function of gene and miRNA in vitro in the future.