| Neuropeptide Tyrosing is a polypeptide containing36amino acid residues, is abundantly and widely distributed in both the central nervous system(CNS) and the peripheral nervous system(PNS), specially in telencephalon, preoptic area, optic tectal. slices and hypothalamus. In hypothalamus, the NPY is concentrated in arcuate nucleus(ARC), paraventricular nucleus(PVN), suprachiasmatic nucleus(SCN), and optic chiasma postzone and so on, NPY is implicated in various physiological effects such as food intake, cardiovascular function, immunologic response, reproduction, fatness, sex reversal and so on.The southern catfish was used as the experimental subjects in this study. Using rapid amplification of cDNA end (RACE) cloning technology, we cloned the NPY full-length cDNA of southern catfish and analysed its nucleotide sequence. its potential amino acids sequence was deduced and its corresponding protein structure was predicted and analysed. Moreover, using quantitative qPCR, we identified the levels of NPY mRNA expression on brain, gill, kindey, stomach, liver, intestine, heart, oosphere stage, blastocyst, gastrula, neurula and before first-feeding stage. The results provided the scientific evidence for fish nutriology.1%In this study, a full length cDNA of southern catfish NPYwas cloned by RACE methods and analyzed by bio informatics. The southern catfish NPY cDNA nucleotide sequence is743bp in length, and contains a82bp of5’-untranslated region (5’-UTR), the start codon(ATG), a373bp of3’-untranslated region (3’-UTR) and a288bp of open reading frame(ORF) encoding a prepro-NPY with95amino acids, the stop codon(AGT).2、The deduced amino acid sequences contain a27-amino-acids signal peptide followed by the36amino acids of mature NPY, a glyeine-lysine-arginine processing site and a29amino acid C-terminal peptide of NPY(CPOP). Its molecular weight is10830.5and its isoelectnic point is6.54, and its molecular fomula is C487H754N130O136S7Analysis of amino acid composition showed that the highest content of amino acids is leucine(11.6%), the second highest content is alanine(8.4%), and the lowest content is hisine(1.1%). The proportions of acidic amino acids and basic amino acids are11.7%and10.6%, respectively. The protein contains four main secondary structures:a-helix(43.16%),β-turn(9.47%), extended strand(15.79%)and random coil(31.58%). Alpha helix is the dominant secondary structure and β-turn is the lowest.3%Analysis of phylogebetic tree showed that Siluriformes fishes are highly homologous. There is highest homology when compared with Channel catfish (79%) and Darkbarbel catfish(76%). Furthermore, it has relatively low homology with other saecies shch as godfishbarbode, Zebrafish, Common carp, Rock carp, Atlantic salmon, Chicken, Rhesus monkey and Human (69%,66%,66%,65%,65%,53%,57%,61%,61%amino acid homology, respectively). NPY of southern catfish is clustered closely with Channel catfish and Darkbarbel catfish.4、Using quantitative qPCR, we identified the levels of NPY mRNA expression on Tissues (brain, gill, stomach, intestine, kindey, liver and heart), ontogenetic process (oosphere stage, blastocyst, gastrula, neurula and before first-feeding stage). The result showed that in southern catfish, NPY mRNA was strongly expressed in the brain and much lower levels of expression were detected in heart, gill, intestine, kindey, stomach and liver. During embryonic development stages, the expression signals of NPY appeared in all stages, but the stage before first-feeding larva is significantly higher to other stage; It indicated that NPY might be involved in the embryonic of southern catfish. The expression of NPY mRNA leves increased with fasting day elongated and the highest levels appeared at fasting for7days, in the refeeding experiment, the expression of NPY mRNA leves of4hours after refeeding reduced rapidly compared to fasting for14days. Form that we conjecture that NPY regulating food intake in southern catfish. |
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