团头鲂黑素皮质素3型受体基因克隆及表达分析

黑素皮质素3型受体(MC3R)系统与动物摄食行为以及能量调控密切相关。为丰富鱼类MC3R相关基础研究,探索鱼类摄食行为与能量调控机制,本研究克隆了团头鲂(Megalobrama amblycephala)mc3r基因,采用分子生物信息学和相对荧光定量PCR方法分别对氨基酸序列保守结构域、组织表达分布和禁食条件下的表达变化等开展分析研究。结果显示,团头鲂mc3r基因编码区全长984 bp,编码327个氨基酸,与现有报道的MC3R氨基酸序列高度相似,具有典型的7次跨膜结构域。组织表达图谱分析表明,mc3rmRNA在下丘脑、垂体、肝脏和卵巢有相对较高的表达。禁食试验结果显示,下丘脑和垂体中的转录变化与周期性摄食信号相关,其中下丘脑mc3r mRNA在禁食72 h和14 d的表达显著上调(P0.05),垂体mc3r mRNA在禁食72 h和禁食72 h后恢复投喂6 h表达量显著升高(P0.05),而肝脏中mc3r mRNA在禁食48 h和14 d的表达量显著上调(P0.05)。此外,对血液皮质醇和血糖的监测结果显示,两者均在禁食14 d有显著变化,其中皮质醇水平显著高于对照组(P0.05),而血糖水平显著低于对照组(P0.05)。综合本研究结果表明,MC3R在鲤科鱼类中具有高度相似的氨基酸序列和结构域;在组织中的转录表达变化与食物摄取有明显相关性,对调控鱼体摄食行为和能量代谢具有重要作用。

Molecular cloning, characterization, and expression analysis of Megalobrama amblycephala melanocortin receptor 3 during fasting

The melanocortin system consists of melanocortin peptides derived from the proopiomelanocortin gene, five melanocortin receptors, two endogenous antagonists, and two ancillary proteins. It plays a role in many important physiological functions of terrestrial animals. Studies on rodents have affirmed the role of melanocortin receptor 3 (mc3r) in feeding behavior and energy regulation. However, the function of mc3r in fish, including basic information, is not well understood. Thus, to determine the initial value of fish mc3r and assess the role of MC3R in fish feeding behavior and energy regulation, the full-length cDNA of Megalobrama amblycephala mc3r was cloned. The nucleotide sequence and deduced amino acid sequence were analyzed using bioinformatics methods. Blood glucose and cortisol levels were detected, and real-time quantitative PCR was employed to characterize the tissue expression profile and expression during fasting and refeeding. The results showed that M. amblycephala mc3r cDNA encodes a protein of 327 amino acids, which shares a high amino acid sequence identity with that of other animals and is characteristic with the typical seven transmembrane domain. Phylogenetic analyses showed that the mc3r cluster was within the MC3R clade of the Cyprinidae MC3Rs. The tissue expression analysis revealed that M. amblycephala mc3r was highly expressed in the hypothalamus, pituitary, liver, and ovary, whereas its expression was much lower in the other detected tissues. During fasting and refeeding, mc3r mRNA expression in the hypothalamus and pituitary showed a periodic variation with higher expression near the first feeding time every day both in the experimental and control groups. Furthermore, the expression of mc3r mRNA was significantly increased and was higher than that in the control group after 72 h and 14 d fasting in the hypothalamus (P < 0.05), whereas similar results occurred in the pituitary at 72 h fasting and 6 h after refeeding (P < 0.05), and in the liver at 48 h and 14 d after fasting (P < 0.05). In addition, an obvious change in blood glucose and cortisol was only observed with long term fasting (P < 0.05). The M. amblycephala mc3r is not only highly conservative in genetic information, but also in its tissue expression profile and physiological function during the evolutionary process, and may play an important role in feeding behavior and energy regulation.