Molecular Cloning,Expression,Identification And Functional Study Of Neuropeptide F And Its Receptor In Sepiella Japonica

Sepiella japonica is one of the most economically important aquaculture species in the East China Sea,which belonging to the Mollusca and Cephalopoda.Neuropeptide Y(NPY)is the most effective oxygenating peptide in the brain of vertebrate,which participates in many physiological processes such as feeding,digestion,metabolism,etc.Neuropeptide F(NPF),as the homologue of NPY in invertebrates,is considered to perform similar functions.In this study,NPF and its receptor NPFR of S.japonica were used as targets to analyze the sequence characteristics,evolutionary status,tissue expression characteristics,tissue localization,subcellular localization in different development stages.Then,we also study the law of expression changes,individual anatomy,histology,and isoenzyme chemistry under feeding regulation(starvation for 5 days then refeeding for 3 days).The main results are as follows:1.The cloning and identification of the full-length cDNA sequence of NPF and its receptor NPFR from S.japonica showed that:the ORF(Open reading frame)of SjNPF is 240bp.Through the analysis and prediction of bioinformatics data,a signal peptide of 22 amino acids was found at the N-terminal of SjNPF.The tertiary structure prediction showed that SjNPF have an a helix and partial random crimps.The multiple amino acid sequence alignment and the phylogenetic tree indicated that SjNPF have the closest evolutionary relationship with Idiosepius paradoxus,with the homology of 82.72%,and clustered with other molluscs such as bivalve and gastropod.The ORF of SjNPFR is 1266bp encoding 421 amino acid residues,and no signal peptide was found.The multiple amino acid sequence alignment and the phylogenetic tree showed that SjNPFR has the closest evolutionary relationship with that of Sepia pharaonis,with homology as high as 99.52%,and is also grouped into one branch with bivalve and gastropod.2.In situ hybridization was used to map the mRNA levels of SjNPF and SjNPFR.For SjNPF,the signal density was the strongest at the intersection of the posterior basal lobes with vertical lobe and subvertical lobe in brain.While the signal density of SjNPFR was the strongest at the edge of the posterior pedal lobe and the palliovisceral lobe.Both of them were widely distributed in optic lobe.The subcellular localization technique was used for cellular localization.The results showed that SjNPF was located in the cytoplasm while SjNPFR was expressed on the cell membrane.3.qRT-PCR was applied to analyze spatio-temporal expression of male and female S.japonica at different development stages.The results showed that SjNPF was significantly expressed in digestive organs at the early development stages(stages Ⅰ-Ⅱ and stage Ⅲ)of male and female(p<0.05),while SjNPFR was significantly expressed in brain and optic lobe tissues in all stages of male and female(p<0.05)except male at stage VI.SjNPF and SjNPFR were significantly expressed in the accessory sexual gland(nidamental gland and spermatophore)of stage Ⅵ(p<0.05).4.Under feeding regulation,on the 3rd day,the vitality of S.japonica decreased and the hepatosomatic index of mature female S.japonica decreased significantly(p<0.05);on the 4th day,the body color turned white and and cannibalism came up;on the 5th day,the liver and intestine atrophied,and the intestinal structure was damaged.qRT-PCR analysis showed that the expression of SjNPF and SjNPFR increased in accordance with starvation in the experimental group and was significantly high on the 5th day(p<0.05),but decreased significantly on the 1st day of refeeding(p<0.05).The detection of pepsin enzyme activity showed that the protease activity of S.japonica decreased at first and then increased after refeeding(p<0.05).