The Observation Of Body Color Formation And Mitfa And Tyr Gene CDNA Cloning And Tissue Expression Study In Yellow River Carp (Cyprinus Carpio Haematopterus)

The Yellow River carp(Cyprinus carpio haematopterus)is one of the most important carp varieties in China and is known for its golden scales and yellow fins and tail.Same as Trachidermus fasciatus,Culter alburnus,Siniperca chuatsi,it is one of Chinses four famous freshwater fish.There are many researches on the fish body color and mainly focus on the model species such as zebrafish(Danio rerio)and medaka(Oryzias latipes),but the researches of the Yellow River carp body color are rarely reported.mitfa acts as one of the pioneer biomarkers of differentiation from neural crest cell to melanin,master regulator of melanocyte development,function and survival by modulating Tyrosinase family.Tyrosinase(Tyr)is the essential rate-limiting enzyme in melanogenesis,the expression and activity of Tyr determines the rate and yield of melanin.In this study,the Yellow River carp was selected as the experimental material,the key genes mitfa and tyr in melanin synthesis pathway were selected as the object of the study.Then full-length cDNA of mitfa and tyr were cloned,the expression level of two genes in different tissues of the Yellow River carp was detected by real time PCR,the process of body color formation in the Yellow River carp was observed,and two genes expression in different stages of body color formation was detected,to further investigate the roles of mitfa and tyr genes in fish body color formation.1.Molecular cloning,characterization and mRNA expression analysis of mitfa gene in Yellow River carpIn our study,the mitfa cDNA was first cloned from the Yellow River Carp(Cyprinus carpio haematopterus),then we obtained the full-length cDNA sequence of mitfa by rapid-amplification of cDNA ends(RACE)and examined the mRNA and protein expression levels of Tyr in different tissues using quantitative real-time PCR(qRT-PCR)and immunohistochemical methods.The results showed that mitfa cDNA from the yellow river carp was 1861 bp and contained an open reading frame of 1462 bp,coding for 486 amino acids,and the mitfa amino acid sequences from the Yellow river carp and Cyprinus carp were 95.2% identical.The results of real-time quantitative PCR showed that mitfa was expressed widely in adult tissues and the highest expression level was detected in muscle.Meanwhile,immunohistochemistry analysis again demonstrated not only mitfa protein was detected at the epidermis of black skin,but also at the epidermis and dermis of slivery skin without melanophores.2.Molecular cloning,expression pattern and location of tyrosinase gene in Yellow River carpTo study the relationship between tyr gene and the formation of body color in Cyprinus carpio haematopterus,we obtained the full-length cDNA sequence of tyr by RACE and examined the mRNA and protein expression levels of Tyr in different tissues using qRT-PCR and immunohistochemical methods.The full length cDNA of tyr was 1717 bp(Genbank ID: No.KY305667),including 41 bp at 5'-UTR,71 bp at 3'-UTR,and a 1605 bp open reading frame encoded a peptide of 535 amino acids.Sequence analysis exhibited that Tyr from Yellow River carp had the highest amino acid similarity with Cyprinidae,while has low similarity with mammalian and bird.And phylogenetic tree analysis indicated Yellow River carp has close relationship with Cyprinidae species like Cyprinus carpio Koi and Danio rerio.QRT-PCR results revealed that tyr mRNA was checked in all different tissues of Yellow River carp,among which the maximum level was detected in muscle,followed by black skin.But tyr was not detected in the slivery skin lacking of melanophores.The immunohistochemical positive signals were detected in the melanophores and mucous cells of black skin.In conclusion,the expression level of tyr is relevant to the abundance and location of melanophores,and could be associated with the body color formation of the yellow river carp.3.The expression of mitfa and tyr in process of the body color formation of the Yellow River carpWe observed and descripted the formation of body color and pigment cells of the Yellow River carp.The expression of mitfa and tyr gene in the body color formation of Yellow River carp were analyzed by the qRT-PCR.The results showed that the Yellow River carp had iridocyte,xanthophore and melanophore.And the observation of body color formation indicated that the back and abdomen of the Yellow River carp had melanophore in1 dph(days past hatch).2dph,the xanthophore appeared and the number of melanophore was increased gradually.31 dph ~ 41 dph,iridocyte and scales began to appear.51 dph body color formation.The results of qRT-PCR analysis showed that mitfa and tyr were expressed in the all body color formation stage of the Yellow River carp.The expression level of mitfa and tyr gene in the formation process was basically the same as the increase of melanocytes.