Cloning Of Neuropeptide Y CDNA And Its Expression Regulation By Dietary Protein In Rock Carp (Procypris Rabaudi (Tchang))

Neropeptide Y,a 36 amino acid,is widely distributed in both the central nervous system(CNS) and the peripheral nervous system(PNS),especially in telencephalon,preoptic area,optic tectal slices and hypothalamus.In hypothalamus,the NPY is concentrated in arcuate nucleus(ARC), paraventricular nucleus(PEVN),periventricular nucleus,suprachiasmatic nucleus(SCN),and optic chiasma postzone and so on.NPY is implicated in various physiological effects such as the stimulation of feeding,reproduction,cardiovascular function,stress,immune responses,fatness,sex reversal and so on.Moreover,NPY is involved in the regulation of circadian rhythms and sexual behavior in mammals as well as in fish..This study will help to evaluate effects NPY on food intake and growth in P.rabaudi,and provide a data for in sight into the neuroendocrine regulation mechanism in P.rabaudi.In the present study,we cloned the full length NPY by the reversetranscription-polymerase chain reaction(RT-PCR) and RACE methods from the rock carp P.rabaudi,deduced the potential amino acid sequence,and predicted and analysed the corresponding protein structure.Meanwhile, the changes of expression from brain tissue of P rabaudi by feeding the diet which contained different protein level(25%,30%,35%,40%,45%,50%) at the different stage time(first,second and fourth month) were determined.1.Primers designed based on multiple alignment analysis of other NPY genes cloned from Cyprinidae fishes,the full-length cDNA of P.rabaudi was amplificated by the RACE and RT-PCR technique using the total RNA isolated from the brain.Analyzed by bioinformation method,the gene was 699bp in length,consisting a 5′-untranslated region(5′UTR,10bp),the start codon(ATG),an open reading frame of 291 bp coding for a protein of 96 amino acids,the stop codon(TGA), 3′-untranslated region(3′UTR,398bp) containing a polyadenylation signal(AATAAA) and the poly A tail.Comparsion of the sequence homology between NPY cDNAs from various animals indicated that the cDNA fragment obtained in the study encodes the NPY protein.2.The rock carp NPY gene encoded a protein with 96 amino acids,the protein molecular weight was predicted 10892 D,and the isoelectric point was 5.72.Using on-line lookuping in SignalP,we found that the location of 1-28aa was the signal peptide of NPY.NPY contained a PAH (Pancreatic Hormone) domain which located from 29aa to 64aa and an intrinsic disorder domain which located 77aa to 95aa.Analysis of amino acid composition showed that the highest content of amino acids is leueine(10.42%),the second is alanine(9.38%),the third is glycine and threonine (7.29%),and the lowest is valine(1.04%).The proportions of acidic amino acids and basic amino acids were 13.5%and 9.38%,respectively.The protein contains four main secondary structures:α-helix,β-sheet,turn and random coil.Alpha helix was the dominant secondary structure andβ-sheet was the lowest.3.Blasting anligment in NCBI and clustering analysis by the software of MEGA4.0 suggested that the homogeneities were 98.3%,98.5%,97%and 97%compared with Cyprimius carpio,Spinibarbus sinensis,Carassius auratus and Danio rerio,which were highest homogeneity among these species.Furthermore,it had relatively low homology with other species such as orange-spotted grouper(Epinephelus coioides),european seabass(Dicentrarehus labrax),rainbow trout(Oncorhynchus mykiss),marbled electric ray(Torpedo marmorata) and channel catfish(Ictalurus punctatus)(88.8%,87.3%,88.1%,88.1%and 71.4%amino acid homology,respectively),but there is lowest homology when compared with Lampetra fluviatilis (only 67.9%).The evolution tree built from the NPY homology of P.rabaudi and other species was consistent with that of traditional taxonomic results.All of results suggested that Cyprinidae fishes NPYs were highly conservated.4.Three hundred of P.rabaudi juveniles were randomly allocated into six treatments with different dietary crude protein levels(control,40%;25%;30%;35%;45%;50%) respectively.The expression level of NPY in total brain tissue was quantified by RT-PCR at the different stage time(1, 2 and 4 month).Fish fed higher(45%and 50%) protein diets for 1 and 2 months exhibited significantly increased NPY gene expression in total brain compared to the control treatment(40%), however,fish fed a lower(25%,30%and 35%) protein diets after 1 and 2 months also exhibited significantly increased NPY gene expression in total brain.It was note that there were no significant difference between the fish fed 35%and 45%protein diets,which were significantly different comparing to the fish fed 25%,30%and 50%protein diets.NPY gene expression level were decreased in fish fed lower 25%and 30%protein diets for 4 months,and the two treatment showed significantly different compared to fish fed higher(35%,40%,45%and 50%) diets.The results suggest that NPY expression is influenced by protein intake in rock carp brain.