| Neropeptide Tyrosing(NPY)is a polypeptide containing 36 amino acid residues,is abundantly and 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 food intake,reproduction,cardiovascular function,reproduction,stress,immunologic response,immunologic response,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.In this study,a full length cDNA of grass carp NPY was cloned by RACE methods and analyzed by bioinformatics.At the same time,its potential amino acid sequence was deduced and the corresponding protein structure was predicted and analysed.Meanwhile,the change of expression from brain tissue of grass Carp in the different stage time(24,48,72,73,75 h)of starvation and in the 1h and 3h after refeed revealed the NPY's effect in regulation of food intake,and the results provided the scientific evidence for fish nutriology.1.Based on multiple alignment of a number of Cypr(?)n(?)dae NPY genes,the(?)ull-length cDNA of grass carp was amplificated by the RACE technique using the total RNA isolatedo from grass carp brain as template.The length of the cDNA is 765bp,which includes a 5'-untranslated region(5'UTR, 51bp),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,423bp)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 a NPY protein.2.The grass carp NPY pro-polypeptide of 96 amino acid residues includes 28 signal peptide amino acids.Its molecular weight(Mw)is 10987 and its isoelectnic point(pI)is.5.94.Analysis of amino acid composition showed that the highest content of amino acids is leucine(11.36%),the second highest content is alanine(9.38%),the third highest contents are glycine and threonine (7.29%),and the lowest content is valine(1.04%).The proportions of acidic amino acids and basic amino acids are 12.5%and 9.38%,respectively.The protein contains four main secondary structures:α-helix;β-sheet,turn and randona coil.Alpha helix is the dominant secondary structure andβ-sheet is the lowest.3.Analysis of phylogenetic tree showed that Cyprinidae fishes NPY proteins are highly homologous.The NPY proteins of grass carp and Cyprinus carpio have 96 amino acids and exhibit homology of 100%.Similarly,amino acid sequence of grass carp NPY protein also has high homology compared with other Cyprinidae fishes such as spinibarbus sinensis,goldfish and zebrafish(97.9%,93.8%,95.8%amino acid homology,respectively).Furthermore,it has relatively low homology with other species such as channel catfish,european seabass,rainbow trout,marbled electric ray and orange-spotted grouper(68.4%,68.4%,66.7%,61.5%and 65.3%amino acid homology,respectively),but there is lowest homology when compared with lampetra fluviatilis (only 41.1%).All of results suggested that Cyprinidae fishes NPYs are highly conservated in the evolutional process.4.After different periods(24h,48h,72h,73h and 75h)of feed deprivation and refeed at 73h or 75h after feed deprivation(73R,75R),the levels of NPY mRNA in the brain tissue were determined. The result showed that the expression levels of the NPY were increasing follow the time extension of feed deprivation(FD).The expression levels of NPY were highest in 72h FD fish(0h-72h),and continued for 1-3 h after 72h FD;whereas,refeeding reversed the increase of the NPY mRNA levels only in 1-3 h after refeeding(73R,75R),exhibiting a significant difference(P＜0.05)when compared with the levels of 73h and 75h FD.Taken together,these results further supported that NPY is a physiological transducer of brain involved in the regulation of daily appetite and feeding in grass carp.
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