| In this study,the open reading frame(ORF)sequences of genes including npy,orexin and ox2 r were obtained by using homologous cloning method in Seriola aureovittata.The tissue distribution characteristics of npy,orexin and ox2 r and the temporal expression patterns in gametes and during early life stages were determined,and the effects of the addititive of 5-HMF on the npy,orexin and ox2 r expression was investigated,the results provided basic data for revealing the physiological function of these three genes related with feeding fucntion.1.Cloning,phylogenetic analysis and spatial expression patterns of npy,orexin and ox2 r genes in Seriola aureovittataThe ORF of Seriola aureovittata npy was 300 nucleotides in length that encodeed a 99 amino acids(aa)precusor,with a calculated molecular mass and isoelectric point of 11.24 k Da and 5.02,respectively.The precursor protein was composed of a predicted signal peptide of 28-aa in size,36-aa mature peptide,a GKR protein proteolytic site and a 32-aa C terminus of unknown function.The ORF of the orexin gene was 450 bp in length and encoded a precusor of 149 aa,which included a signal peptide of 46 aa,a orexin A mature peptide of 43 aa,a orexin B mature peptide of 28 aa,26 peptides of unknown function at the C terminus and two GKR proteolysis sites.The predicted protein molecular weight and isoelectric point of interest were16.57 KDa and 9.46,respectively.The ORF of the ox2 r gene was 1269 bp in length and encoded receptor protein of 422 aa,7 transmembrane regions,4 N glycosylation sites(score>0.5),and 6 Ser/Thr phosphorylation sites(score>0.9).The predicted protein molecular weight and isoelectric point were 48.29 KDa and 8.97,respectively.Sequence analysis showed that Seriola aureovittata npy was highly conserved(>90%)with other teleosts npy.Similarly,and orexin(>70%)and ox2r(>80%)of Seriola aureovittata were also highly conserved with orexin and ox2 r of other teleosts.Phylogenetic analysis showed that in the Seriola aureovittata,these three genes were clustered into one branch with their conterparts in Seriola dumerili.The npy,orexin and ox2 r mRNA were significantly highly expressed mainly in the brain(P<0.05),followed by pituitary and stomach,with detectable minimal expression in other tissues.In addition,except for intestine and gonad,npy was found to have no significant difference in all other detected tissues of both sexes.Interestingly,the expression of orexin mRNA in the gonads varied significantly between female and male Seriola aureovittata(P<0.05),indicating a sexual dimorphism of the npy and orexin genes.2.Temporal expression patterns of npy,orexin and ox2 r genes during early life stages in Seriola aureovittataThe expression patterns npy,orexin and ox2 r mRNA were detected in gamets and all stages of embryo development in Seriola aureovittata.Results showed that the expression of the three genes was detected in both sperm and ovum,indicating that the three genes had parental inheritance properties.npy mRNA was evidently high expression in the early gastrula(P<0.05)and lower later,evidently upregulated in stage embryo encircling 2/3 of yolk sac and reached a maximal value in the hatching period(P<0.05).The expression of orexin mRNA were significantly upregulated from stage embryo encircling 2/3 of yolk sac and and then the expression was relatively stable.It decreased slightly at hatching.After embryo formation,the tail bud and heartbeat were formed successively,and the high expression patterns of npy,orexin and ox2 r mRNA after embryo formation suggested that it may participate in the regulation of organ formation and development during early embryonic development.The expression of ox2 r mRNA were significantly upregulated from stage embryo encircling 3/4 of yolk sac and and and reached a maximal value in the hatching period(P<0.05).In the larvae and juveniles,the relative expression level of npy mRNA increased at 5 dph and 7 dph(P<0.05).The expression level of npy mRNA was relatively lower at 10 dph-25 dph,then gradually increased from 30 dph,reached a peak at 60 dph(P<0.05).The expression trends of orexin and ox2 r mRNA were similar,with significant upregulation levels at 5 dph and 7 dph,then low expression levels,and upregulation levels at 60 dph(P<0.05).Upregulation of npy,orexin and ox2 r mRNA at 5 dph suggested their possible involvement in the switch from endogenous to exogenous nutrients in larvae.3.The expression characteristics of npy,orexin,and ox2 r genes to starvation-refeeding strategy in Seriola aureovittataThe effects of starvation-refeeding on the gene expression of npy,orexin,and ox2 r mRNA were examined in Seriola aureovittata.We found that starvation-refeeding induced similar expression trends of npy,orexin,and ox2 r mRNA in brain,pituitary,and stomach.Fasting induced increase of npy,orexin and ox2 r mRNA levels,refeeding normalized npy,orexin and ox2 r mRNA abundance following food deprivation.The pituitary npy mRNA showed more sensitive responses to fasting.Gene expression changes of orexin and ox2 r were similar but slightly different in these three tissues.At 21 d post starvation,orexin and ox2 r mRNA were significantly higher in the experimental group(P<0.05)and returned to normal levels after 7 d of refeeding.In pituitary,ox2 r mRNA was significantly upregulated after starvation for 14 d.4.The expression responses of npy,orexin,ox2 r genes to pentahydroxymethyl furfural(5-HMF)as additive in feed in Seriola aureovittataThe 5-HMF was as additive and added into feed of Seriola aureovittatawith different concentrations,FAW、WGR、SGR increased significantly(P<0.05).The relative expression levels of npy,orexin and ox2 r mRNA in the brain,pituitary and stomach were also detected.npy,orexin and ox2 r mRNA levels were increased in different tissues,but the degree of expression level was slightly different in different tissues at different concentrations.In addition,with different concentrations of 5-HMF,after feeding the Seriola aureovittata for 30 d,the activity of trypsin,pepsin,α-amylase and lipase were increased.Suggesting that 5-HMF promoted feeding digestion,consistent with the expression trend of feeding function genes. |
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