Molecular cloning of AGRP and NPY and their expression analysis during fasting and refeeding in juvenile blunt snout bream (Megalobrama amblycephala)

Agouti-related protein (AGRP) and neuropeptide Y (NPY) are important factors involved in the regulation of feeding in both mammals and fish. In this study, the full-length cDNA sequences of AGRP and NPY genes of Megalobrama amblycephala were cloned by RACE technology for the first time, to investigate the expression patterns of AGRP and NPY in different tissues and different physiological conditions of fasting and refeeding in M. amblycephala by Quantitative Real-time PCR technology. Four hundred and eighty juvenile blunt snout breams with an average body weight of (5.30±0.01) g were randomly divided into 4 groups with 3 replicates in each group, i.e. the control group (the daily feeding experiment diet at 3% of the weight), the F/refeeding group (fasted from 1 d to 14 d, refed from 15 d to 28 d and the daily feeding experiment diet at 3% of the weight), the F/excessive refeeding group (fasted from 1 d to 14 d, refed from 15 d to 28 d and the daily feeding experiment diet at 8% of the weight), the fasted group (fasted for 28 d). The results showed as follows: ① The results of histological analysis showed that the epithelium columnar cells of intestine in the fasted group displayed obviously leukocyte infiltration in the submucosa. ② The full length cDNA of M. amblycephala AGRP and NPY are respectively 770 bp and 557 bp, consisting of a 5′ untranslated region of 77 bp and 101 bp, a 3′ untranslated region of 315 bp and 120 bp, and an open reading frame of 378 bp and 336 bp, encoding 125 amino acid and 111 amino acids. Based on similarity comparison, AGRP is relatively conservative and has the highest similarity to AGRP in Cyprinus carpio, and the phylogenetic tree analysis indicated that AGRP and NPY of M. amblycephala is associated with AGRP and NPY in other fishes, respectively. ③ Quantitative Real-time PCR analysis showed that AGRP and NPY were both expressed in all tested tissues with highest expression in brain and then liver and intestine. ④ There were significantly differences in AGRP and NPY mRNA of fish during fasting and refeeding. The expression of AGRP mRNA in the brain firstly increased and then decreased after refeeding, at 28 d, AGRP mRNA expression levels of brain tissue in F/refeeding group were higher than those in other groups, but AGRP mRNA expression levels of intestine tissue in F/excessive refeeding group were higher than those in other groups. Meanwhile, the expression of NPY mRNA showed a trend from decline to rise in the brain and intestine tissues, NPY mRNA expression of brain and intestine tissue in fasting group was higher than that of other three groups. In conclusion, this study showed that AGRP and NPY are involved in feeding regulation in M. amblycephala and have different expression patterns in brain and intestinal tissues.