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    • br Introduction Coat colors are determined by both genetics

    2022-09-13


    Introduction Coat colors are determined by both genetics and environment, especially genetics. The phenotype of coat color of animals depends on two types of melanin, black to brown eumelanin and yellow to reddish brown pheomelanin produced in mammalian melanocyte (Ito et al., 2000; Ito and Wakamatsu 2008). The genetic basis for coat color is well understood in rodents (Slominski et al., 2004; Steingrímsson et al., 2006). Some common genes implicated in the regulation of coat color are also well documented in other species including sheep. For example, MC1R and ASIP loci are functionally linked to undesirable coat color phenotypes in sheep (Våge et al., 1999; Norris and Whan 2008), and TYRP1 is a strong positional candidate gene for color variation in Soay sheep (Gratten et al., 2007). In a previous study, we characterized the transcriptome profiles of sheep skins with white and black coat color, and identified differentially expressed genes (Fan et al., 2013) including known coat color genes (e.g., DCT, MATP, TYR and TYRP1). One of the differentially expressed genes is soluble guanylate cyclase (sGC), which showed significantly higher expression in white vs. black sheep skin. Guanylate cyclases are a family of enzymes that catalyze the conversion of GTP to cGMP. The family comprises both membrane-bound (particulate guanylate cyclase, pGC) and soluble (soluble guanylate cyclase, sGC) isoforms that are expressed in nearly all cell types (Fan et al., 2013). sGC is involved in many signal transduction pathways, most notably in the Pralatrexate australia and the nervous system (Denninger and Marletta 1999). The present study reports the characterization of sGC expression in sheep skins with white and black coat color, providing evidence to suggest that sGC might be related to coat color formation.
    Materials and methods
    Results and discussion From the skin transcriptome profile of sheep with different hair color, we found that sGC was expressed differently, which suggested that sGC may be related to the coat color (Fan et al., 2013). Here sGC was isolated and characterized and then anylyzed the expression to support the possibility. The full length of CDS is 1860bp, encoding a protein of 620 amino acids. The deduced amino acid sequence shares ∼99% sequence similarity with sGC proteins from other species including human, rat, pig, dog, cattle and sheep. The protein is predicted to contain several conserved functional domains including HNOB (Heme NO Binding), HNOBA (Heme NO Binding Associated), and CHD (Cyclase Homology Domain) (Fig. 1). Quantitative expression of sGC mRNA in white and black sheep skin was analyzed by real time PCR. As shown in Fig. 2, the expression of sGC mRNA is significantly higher (5 fold) in white than black sheep skin (P<0.01). Western blot analysis showed that the size of the sheep sGC protein is around 70kDa and that the expression of sGC protein in white sheep skin is higher compared to black sheep skin (Fig. 3A). Quantitative analysis showed that the average intensity of the sGC protein bands is ∼2.5 times stronger in white sheep skin than black sheep skin with significant difference (P<0.01) (Fig. 3B). As shown in Fig. 4A, the expression of sGC is significantly higher (7 fold) in cells transfected than untransfected (P<0.05). The expression of sGC protein in transfected cells is higher compared to untransfected cells(Fig. 4B). Quantitative analysis showed that the average intensity of the sGC protein bands is ∼2.7 times stronger in transfected cells than untransfected cells with significant difference (P<0.01) (Fig. 4C). The abundance of both mRNA and protein for MITF, TYR, TYRP1 and TYRP2 were examined in melanocytes transfected by the pmirGLO-sGC, compared to untransfected melanocytes. The over expression of pmirGLO-sGC in menlanocytes resulted in a decrease in the mRNA abundance for TYR, TYRP1 and TYRP2 (Fig. 5A). Western blot analysis showed that the protein expression of MITF, TYR, TYRP1 and TYRP2 was significantly reduced in melanocytes over-expressed by pmirGLO-sGC (Fig. 5B and C).