Molecular Cloning, Characterization And Tissue Distribution Of Megalobrama Amblycephala Melanocortin Receptors

The melanocortin (MC) peptides mediated their effects through melanocortin receptors. The melanocortin receptor family, with seven transmembrane alpha-helixs, is a member of the smallest G-protein-coupled receptor and of great importance in chordate physiological function. Researches have showed that there were five subtypes (MC1R, MC2R, MC3R, MC4R and MC5R) in mammalian and tetrapod, with rather diverse functions. Recent years, fish MCRs have gained much concern of a growing number of researchers. Different MCR subtypes have been cloned and studied in several fish species. The MCR of fish, the earliest vertebrates in evolution, is well conserved with mammalian in molecular characterization and physiology fuction, and it owns some special features of their own. It has reported that fish MCR was involved in the regulation of pigmentation, food intake and energy homeostasis, and also stress response.Megalobrama amblycephala, commonly known as the blunt snout bream or the Wuchang bream, has been one of the most important cultivated species of conventional freshwater fish in China for decades. Recently, the stress reponse of fish leads to many serious diease problems, causing significant economic loss. Therefor the study of fish MCR and the regulation of stress response will not only contribute to our understanding of the evolution of vertebrate MCR, but also provide new ideas to prevent and alleviate the harm of stress reponse, providing the basic information for fishery production.Here, the M. amblycephala MC2R and MC5R mRNA is cloned using PCR and rapid amplification of cDNA ends (RACE) methods. The sequences are analyzed by biology information technology. The gene tissue-specific expression pattern of 13 tissues are proceed by quantitive real-time PCR. Two reference genes are used to normalize the expression of MC2R and MC5R in tissues. MC2R responds to ACTH only and plays an essential role in stress response. The interaction of MC2R and ACTH results in the increasing of cAMP and cortisol production. Based on the known information above and the result of tissue-specific expression, this paper pays attention to MC2R expression and cortisol realease of recorvey from catching-stress in which fish were caught and kept out of water for 60 s. This paper also have an eye on the regulation of MC5R in M. amblycephala during this period.The results are as follows:1. The cloned M. amblycephala MC2R mRNA contains an 915 bp open reading frame (ORF), encoding 304 amino acids. The highest amino acid sequence indentity is shown with goldfish MC2R, nearly 87%. The results reveales that the MC2R mRNA is widely expressed in the 13 tested tissues. The MC2R is predominantly expressed in head kidney. The MC2R level in spleen is just a little lower than head kidney, testis and brain follows. The results also indicate that the actue stressor seems have no significant effect on MC2R expression of head kidney. While, after recovery for 4 h, the cortisol level is still upregulated, but the expression of MC2R is significant downregulated in spleen, brain and testis, compared to 1 h. Based on this study, this research aims to get a basic understanding of the MC2R regulation during the recovery from catching stress, and further to provide an important theoretical basis for the control after stressor exposure.2. The open reading frame (ORF) of cloned M. amblycephala MC5R mRNA is 990 bp, encoding 329 amino acids. Phylogenetic analysis shows that the M. amblycephala MC5R is clustered within the cyprinid fish. The result of evolution status is consistent with its taxonomic status. The tissue specific expression shows that M. amblycephala MC5R expressed in all the test tissues, and is found highest expression level in brain, eye and skin follows. The tissue expression is roughly in line with mammalines and the other fish species, and slight expression difference in some tissues. The competition binding potency order of M. amblycephala MC5R is NDP-MSH> α-MSH> β-MSH, which is conserved with human MC5R. The catching-stress experiment reveals that there’s an upregulation of MC5R gene in brain and eye within 1h after stress. While, the expression level in skin is significantly down-regulated. The expression of MC5R will all back to the level before stress after 24 h. In conclusion the results suggest that MC5R is participant in the regulation of stress reponse, and may also involvement in exocrine secretion, similar to the role of mammalian MC5R.