Molecular Cloning And Expression Analysis Of Pigmentation-related Genes, Sox10, Agouti, Tyrp1and Dct, In Oujiang Color Common Carp,Cyprinus Carpio Var. Color

Body color is special phenotypic traits in fish, which provides important valuesof biodiversity and genetics theory research. Body color variation of fish has beenused as a very important research topics in genetics，evolutionary biology anddevelopmental biology. Oujiang color common carp (Cyprinus carpio var. color) is aregional freshwater species distributing in the Oujiang River drainage in China. Thefish has five kinds of basic body color patterns, namely “whole red”,“whole red withbig black patch”,”whole red with scattered black spots”,“whole white” and “wholewhite with big black patch”, respectively. This fish can be used as a very good modeland material for studying body color inheritance in fish. In this study, based onprevious studies in our laboratory, Oujiang color common carp were used as researchmaterial of body color to conduct gene cloning and expression analysis of four genes(Sox10, Agouti, Tyrp1and Dct) in different skin types, which regulate pigmentation inmelanin synthesis pathway. The detailed contents are presented as follows:1. Cloning and expression analysis of Sox10geneThe full length of Sox10cDNA (2854bp) in Oujiang color common carp wascloned by using RACE techniques. The Sox10cDNA contained9bp5’ untranslatedregions (UTR),1375bp3’-UTR, and1446bp open reading frame (ORF) encoding481amino acids. The phylogenetic analyses based on amino acid sequence showedthat there were59%-94%similarities with some of previously reported other species.RT-PCR analysis revealed that the gene was highly level expressed in skin, muscle,eyes and swim bladder, weakly expressed in gill and heart and no expression werefound in the kidney and the liver. Real-time PCR showed that the expression of theSox10gene in white skin ("Whole white" body color) was significantly higher thanother color patterns (P<0.05), and the significantly lowest expression in black skin of “whole white with big black patch” color pattern (P<0.05), but there were nosignificant difference in expression level among the other color patterns (P＞0.05).The results primarily indicated that the Sox10gene is important in regulating bodycolor variation in Oujiang color common carp.2. Cloning and expression analysis of Agouti geneThe full length of Agouti cDNA (1278bp) in Oujiang color common carp wascloned by RACE techniques. The Agouti cDNA contained94bp5’-UTR,806bp3’-UTR, and378bp open reading frame (ORF) that encode125amino acids.Compared with other reported vertebrate Agouti sequences, the sequence identityranged from34%to90%. RT-PCR analysis revealed that the Agouti gene was highlyexpressed in skin and kidney, lowly expressed in muscle, eye, gill, swim bladder,heart and liver. Real-time PCR showed that the expression in “whole red with bigblack patch” red skin was significantly higher than the other patterns (P<0.01), andwas the significantly lowest in black skin of “whole white with big black patch” colorpattern (P<0.01), but were no significant differences among the other color patterns,such as black skin in “whole red with big black patch”, red skin in “whole red”, andwhite skin in “whole white”(P>0.05). The results primarily indicated that the Agoutigene is a key factor in regulating body color variation of Oujiang color common carp.3. Cloning and expression analysis of Tyrp1and Dct genesTwo pigmentation-related genes (Tyrp1and Dct) of Oujiang color common carpwere cloned by RACE techniques. The full-length cDNA of Tyrp1was1680bp with a87bp5-UTR, a1581bp open reading frame (ORF; translated to a protein with526amino acids), and a12bp3-UTR. The full-length cDNA of Dct was1712bp with a202bp5’-UTR, a1458bp open reading frame (translating to a485amino acids), anda52bp3’-UTR. RT-PCR analysis revealed that the expression of Tyrp1was higher inskin, muscle and eye than other tissue, including gill, swim bladder and heart. Theexpression was not observed in kidney and liver. The expression of Dct gene wasdetected in all studied tissues expect for liver, and the highest expression was found ineye. Real-time PCR showed that Tyrp1gene expression in black skins (black skin in“whole red with big black patch” and in “whole white with big black patch”) wassignificantly higher than those in other skins (P<0.01). Dct expression was thehighest in red skin in “whole red”, but was no significant differences among the otherskins (P>0.05). In the same tissues, it was found that the expressions of Tyrp1washigher than that of Dct.