The CDNA Cloning And Characterization Analysis Of NPY Gene In Mystus Macropterus Bleeker

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), periventricul-ar 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 thythms and sexual behavior in mammals as well as in fish.In this study, cDNA of Mystus macropterus Bleeker NPY was cloned by de sig-ning gene-specific primer, 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 anaylzed. Meanwhile, real-time PCR was also conducted to detect the expression of NPY in tissues. Primary results are as follow: Full-length cDNA sequence of NPY from M. macropterus Bleeker.Based on multiple alignment of NPY from many kinds of fishes, two gene-specific primers were designed for amplification of middle segment, and then the 3'cDNA of NPYgenes was amplified by designing two special primers of 3'cDNA ends.The length of NPY is 686bp with 168bp middle segment and 566bp 3'cDNA.It includes 5'untranslated region, initiation codon(ATG), an open reading frame enconding the 88 amino acid, termination codon(TAA) and 287bp 3'untraslated region with poly(A) structure. It can be identified as the cDNA of NPY from M. macropterus Bleeder by homology analysis. Similarity analysis of amino acid sequence deduced by NPY cDNA of M. macropterus Bleeker.The complete sequence of the cDNA encoding M. macropterus Bleeder NPY peptide was identified to be 267bp in length, and encode a protein with 88 amino acids.The alignment result shows that the amino acids sequence of NPY from M. macropterus Bleeker share high similarity with other species.Comparision of the amino acid sequence of NPY from M. macropterus Bleeder to that of Ictalurus punctatus shows 84.9% similarity, and to that of other fishes such as Myxocyprinus asiaticus, Cyprinus carpio, Carassius auratus, Spinibarbus sinensis, Danio reri shows 67.0%,67.0%,67.0%,68.2%,68.2% similarity, and to that of other species such as Oryzias latipes, Homo sapiens, Mus musculus, Parallichthys olivaceus shows 50%,52.3%,53.4%,44.3% amino acid similarity. The phylogenetic tree analysis of NPY from M. macropterus BleekerThe phylogenetic tree based on NPY gene is congruent with their evolutionary relationships. M. macropterusBleeker belong to catfishes of pisces. homology NPY from cyclostomata, pisces, amphibia, aves and mammalia was aligned, then the phylogenetic tree analysis of NPY was conducted. The phylogenetic tree shows that fish of cypriniformes get together with catfishes, fish of salmoniformes, gadiformes, yprinodontiformes and perciformes get together.Meanwhile, compared to mammalia on distence, amphibia and aves is nearest, cycolstomata is nearer and pisces is far. Protein analysis and secondary predication of the deduced protein of NPY from M. macropterus BleekerThe predicted NPY protein of M. macropterus Bleeker has a molecular weight of 9933.3Da and a isoelectric point of 6.71, and its molecular formula is C439H690 N122_O131S5. Estimated half-life in reticular cells of mammalia is 30h, and instability parameter is 62.47.Therefore, it could be instable protein.The number of both full positively charged residues and full negatively charged residues is 10. Aliphatic index of the protein is 80.91and grand average of hydropathicity is -0.361. Analysis of amino acid composition showed that the highest content of amino acids is leucine(12.5%), but it does not contain Pyl and Sec.The protein contains three main secondary structures:a-helix,extended strand and random coil.Random coil is the dominant secondary stucture(56.82%), extended strand is the lowest(13.64%) andα-helix is in middle(29.55%).Tissue expression of NPY gene from M. macropterus Bleeker RT-PCR was conducted to detect the expression of NPY gene from M. macropterus Bleeker. The results show that NPY gene can be detected in brain, skin, gill, spiner,kidney,liver and intestine, but can not be detected in heart, stomach and muscle.This study encode NPY from Mystus macropterus(Bleeker) to provide a data for it's growth and breeding,and for researching biologic function of NPY.