Cloning And Expression Analysis Of Ghrelin,Neuropeptide Y And Cholecystokinin In Blunt Snout Bream, Megalobrama Amblycephala

Food intake play an important role on fish growth and production performance, feeding is regulated by a series of appetite-regulating peptides including appetite stimulators and inhibitors. Ghrelin, neuropeptide Y (NPY) and cholecystokinin (CCK) all have important roles in the regulation of feeding. In order to elucidate the role of ghrelin and CCK in the appetite regulation in blunt snout bream (Megalobrama amblycephala), we cloned the full-length cDNA sequences of ghrelin, NPY and CCK by gene cloning method, constructed the temporal and spatial expression profile, and assessed the effects of fasting and refeeding on the gene expression. The main conclusions are as follows:1. Cloning and sequence analysis of full-length cDNA of ghrelin, NPY and CCKThe full-length cDNA of blunt snout bream ghrelin was494bp (GenBank No. JQ301476), contained a59bp5’-untranslated region (5’-UTR), a123bp3’-UTR and a312bp open reading frame (ORF) encoding a preproghrelin with103amino acids (aa). Through the sequence analysis of the deduced amino acids of ghrelin, blunt snout bream ghrelin was exactly the same as that of grass carp, the similarity to Prenant’s schizothoracin, goldfish and zebrafish were92%,88%and71%, respectively.The full-length cDNA of blunt snout bream NPY was760bp (GenBank No. JQ301475), contained a65bp5’-UTR, a404bp3’-UTR and a291bp ORF encoding a preproghrelin with96amino acid (aa). Through the sequence analysis of the deduced amino acids of NPY, blunt snout bream NPY was exactly the same as that of grass carp and common carp. The similarity to other vertebrates were also high.The full-length cDNA of blunt snout bream CCK was770bp (GenBank No. JQ290110), contained a49bp5’-UTR, a309bp3’-UTR and a372bp ORF encoding a preproghrelin with123aa. Through the sequence analysis of the deduced amino acids of blunt snout bream CCK, the similarity to grass carp was98%and lower with other vertebrates.2. The temporal and spatial expression profile of ghrelin, NPY and CCK mRNAThe relative expression of ghrelin, NPY and CCK mRNA in embryonic development and post-hatching larvae stages, different adult tissues were analyzed by real-time quantitative PCR. During early development, ghrelin, NPY and CCK were all expressed throughout the embryonic and larval development stages, and the expression levels were higher in larval stages than in embryonic stages. During embryonic development, ghrelin mRNA expression levels were consistently low (P>0.05). However, NPY mRNA expression fluctuated, and showed a significant increasing at gastrula stage, then reduced at prophase of hatching. Levels of CCK mRNA expression showed a decreasing trend (P>0.05). During the early larval stage, ghrelin and NPY mRNA expressions were upregulated at1,3,5DAH, while CCK mRNA expression was reduced significantly at3DAH. The mRNA expression levels of three genes in larvae varied significantly until30DAH. Ghrelin, NPY and CCK were all expressed in all the tissues, Ghrelin mRNA was mainly expressed in the intestine while NPY and CCK mRNAs were mainly expressed in the brain. NPY mRNA was most strongly expressed in the hypothalamus, while CCK mRNA was most strongly expressed in the pituitary.3. Effects of starvation and refeeding on the expression of ghrelin, NPY and CCK mRNAUsing real-time quantitative PCR method detected the effects of starvation and refeeding on the expression of ghrelin, NPY and CCK mRNA in the brain and intestine of blunt snout bream. The results are showed as follows:in the brain, the ghrelin mRNA expression was upregulated during starvation, and was downregulated after refeeding; in the intestine, the ghrelin mRNA expression levels also increased significantly after starvation, and came up to the highest level after7days of starvation, after refeeding for7days, the ghrelin mRNA expression decreased to the same levels as the control group. In the brain, the NPY mRNA expression was upregulated during starvation, and decreased to the same levels as the control group after refeeding for4days. in the intestine, the NPY mRNA expression was not signnificantly changed (P>0.05). The CCK mRNA expression in the brain and intestine were all came up to the lowset level after4days of starvation, and increased to the same levels as the control group after refeeding for7days.