| Melanocortin receptor 3(Mc3r)and melanocortin receptor 4(Mc4r),play an important role in the regulation of energy conservation,belong to rhodopsin-like Family A G-protein-coupled receptors(GPCRs),which have 7 transmembrane domains and are highly expressed in the central nervous system.Melanocortin receptor accessory protein(Mrap2)is also highly expressed in the central nervous system and plays critical roles in energy balance by regulating the signaling pathways of Mc3 r and Mc4 r.This study takes topmouth culter as the research object.Topmouth culter,an important freshwater economic fish in china,has the characteristics of excellent taste,fast growth,high amino acid content and strong breeding performance.And topmouth culter is widely distributed in the rivers,reservoirs,lakes of china.In this experiment,we cloned the full-length sequences of the mc3 r,mc4r,mrap2 a and mrap2 b genes of topmouth culter and analyzed the differential expression of pomc,mc3 r,mc4r,mrap2 a and mrap2 b genes in different embryonic development stages,different body weights and the effect of fasting on their expressions in topmouth culter,we also explored the regulation of Mc3 r and Mc4 r expression by Mrap2.The results of this study were summarized as follows:1.We cloned the full-length sequences of the mc3 r,mc4r,mrap2 a,and mrap2 b genes in topmouth culter by PCR and RACE technology.The full-length sequence of mc3 r was 1487 bp including a 5’ untranslated region(UTR)of 280 bp,a 3’ UTR of 223 bp,and an ORF of 984 bp that encoded proteins of 327 amino acids.The full-length sequence of mc4 r was 1280 bp including a 5’ UTR of 84 bp,a 3’ UTR of 215 bp,and an ORF of 981 bp that encoded proteins of 326 amino acids.The full-length sequence of mrap2 a was 1719 bp including a 5’ UTR of 334 bp,a 3’ UTR of 731 bp,and an ORF of 654 bp that encoded proteins of 217 amino acids.The full-length sequence of mrap2 b was 1001 bp including a 5’UTR of 160 bp,a 3’ UTR of 247 bp,and an ORF of 594 bp that encoded proteins of 197 amino acids.And mc3 r and mc4 r both had 7 hydrophobic transmembrane domains,while mrap2 a and mrap2 b had only one transmembrane domain.2.RT-q PCR was used to detect the expressions of mc3 r,mc4r,mrap2 a,and mrap2 b genes in various tissues of topmouth culter.The results showed that the mc3 r,mc4r,mrap2 a and mrap2 b genes were all highly expressed in the central nervous system of the topmouth culter,especially in the hypothalamus,but the expression in peripheral tissues is different.The expression of mc3 r gene and mc4 r gene was generally lower in the peripheral tissues of female,but it was highly expressed in the gonads,liver and head kidney of male.The higher expression of mrap2 a was detected in the gill tissues of females,but its expression was lower in other peripheral tissues of males and females,and mrap2 b had higher expressions in the gonads of both males and females.It can be seen that mc3 r,mc4r,mrap2 a and mrap2 b were highly expressed in the central nervous system and can play an important role in the regulation of energy conservation through interaction.And the higher expression in some peripheral tissues indicates that mc3 r,mc4r,mrap2 a and mrap2 b may have other functions such as regulating reproductive development and immune response.3.The differential expression of pomc,mc3 r,mc4r,mrap2 a and mrap2 b genes in different embryonic development stages and different body weights of the topmouth culter were detected by RT-q PCR.The results of 1-5 days of embryonic development showed that the expression levels of the other four genes except mc3 r gene were increased with the development of the embryo.In the different body weights of the topmouth culter,the expression level of pomc,mc3 r,mc4r and mrap2 a genes were decreased except for the mrap2 b gene,but the expression level of the mrap2 b gene between 50 ± 3.25 g-500 ± 30.56 g was also decreased.It can be seen that mrap2 a and mrap2 b genes may play a more important role in regulating the MC3R/MC4 R signaling pathway during embryonic development,but the lower expression of pomc,mc3 r,and mc4 r may play a major role in the regulation of different body weights topmouth culter.4.The expressions of pomc,mc3 r,mc4r,mrap2 a and mrap2 b genes under starvation were detected by RT-q PCR.The results showed that the expressions of pomc,mc3 r,mc4r,mrap2 a and mrap2 b in the brain showed an upward trend during short-term starvation,while the expressions of pomc,mc3 r,mc4r,mrap2 a and mrap2 b in the brain showed an overall downward trend during long-term starvation.Therefore,we speculated that the mrap2 a and mrap2 b genes may play a more important role in the regulation of the Mc3r/Mc4 r signaling pathway to balance the energy changes caused by starvation during short-term starvation,while the lower expressions of pomc,mc3 r,and mc4 r genes may play a major role in the regulation of the Mc3r/Mc4 r signaling pathway to regulate energy conservation during long-term starvation.5.The influence of culter Mrap2 on the cell surface and total expressions level of Mc3 r and Mc4 r were performed by flow cytometry.The results showed that Mrap2 a decreased cell surface expression but didn’t affect on total expression of Mc3 r,however,Mrap2 b didn’t affect on both cell surface and total expression of Mc3 r.Mrap2a increased the cell surface and total expression of Mc4 r,but there were no significant differences among groups.Mrap2 b significantly increased cell surface and total expression of Mc4 r in 1:3 group compared with the 1:0 group.It can be seen that the regulation of Mc3 r and Mc4 r expression by Mrap2 is different.The above data will help us further revealing the relationship between the mrap2 a,mrap2b genes and the mc3 r,mc4r genes,and lay the foundation for further exploring the physiological functions of the mc3 r and mc4 r genes in topmouth culter.Topmouth culter as the parent of the hybrid line of BT(Megalobrama amblycephala,♀×Culter alburnus,♂)and TB(Culter alburnus,♀×Megalobrama amblycephala,♂)(our laboratory has been successfully cultivated the hybrid line)the basic research on the mc3 r,mc4r,mrap2 a and mrap2 b genes in topmouth culter will provide a theoretical basis for subsequent research in the BT and TB. |
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