Molecular Cloning,Expression And Function Analysis Of Neuropeptide FF-related Gene And Neuropeptide FF Receptors In Larimichthys Polyactis

Neuropeptide FF is an octapeptide belonging to the RFamide-related peptide family,which has regulatory functions in a variety of biological processes.In this paper,NPFF related gene(LpNPFF: Accession No.MT012894)and its two receptors(LpNPFFR1:Accession No.MT012894 and LpNPFFR2: Accession No.MT12896)were cloned from Larimichthys polyactis.Sequence analysis results showed that the LpNPFF gene cDNA of L.polyactis was 835 bp in length,and the open reading frame(ORF)encoded 127 amino acid residues,including a signal peptide with 21 amino acid residues and two mature PQRFamide peptides.The relative molecular weight(MW)of the protein was predicted to be 29.8 kDa and the isoelectric point(pI)to be 5.90.LpNPFFR1 and LpNPFFR2 belong to a typical family of G protein-coupled receptors,both of which have 7 transmembrane regions,and their ORF regions encode 427 and 444 amino acid residues,respectively.The results of multi-sequence homology and phylogenetic tree analysis showed that the LpNPFF,LpNPFFR1 and LpNPFFR2 showed the closest relationship with Larimichthys crocea.The results of RT-PCR and RT-qPCR showed that LpNPFF,LpNPFFR1 and LpNPFFR2 mRNAs were mainly expressed in the brain and the gonads of L.polyactis(stage ?).The localization and distribution of LpNPFF in L.polyactis was showed through in situ hybridization and immunohistochemistry.The results of in situ hybridization showed that a large number of LpNPFF mRNA positive cells were detected in optic tectum(OTec),torus lobe(TL),nodules pronucleus(NAT),lateral nodules(NLT),the hypothalamus(NDLI)of the midbrain and medulla oblongata(MO),while less positive cells were detected in telencephalon and cerebellum in L.polyactis.This may indicate that the LpNPFF transmits signals to different organizations in different ways.Immunohistochemical results showed that RFamid immunopositive signals were mainly visible in the surface cells of the brain and the connective tissue around the ovarian wall of L.polyactis.This study also investigated the effect of LpNPFF on the secretory activity of the protein in Chinese hamster ovary cells(CHO-K1)and the production of nitric oxide(NO)in macrophages(RAW264.7).The results of in vitro cells experiments showed that 0.01?M~10?M LpNPFF(1)and 0.01?M,0.1?M LpNPFF(2)had significant inhibitory effects on the total protein secretion activity of CHO-K1 cells(p<0.05).LcGnRH3 of 0.01?M ~100?M promoted the total protein secretion of CHO-K1 cells(p<0.05).When two RFamide mature peptides of LpNPFF and LcGnRH3 acted on cells together,LpNPFF(0.01?M ~100?M)inhibited the increased protein secretion caused by LcGnRH3 to some extent(p<0.05).LpNPFF(1)andLpNPFF(2)inhibited the production of NO in RAW264.7 cells at rest and LPS activation.In addition,the interaction between ligand and receptor was preliminarily verified by the NPFF receptor antagonist(RF9).RF9 had no effect on the NO of RAW264.7 macrophages.After blocking the NPFF receptor by RF9,the content of NO was not significantly different at PBS group and LPS group,which proved that LpNPFF inhibited the amount of NO secreted in cells.These results contribute to future studies of neuropeptide evolution or also provide new ideas for further exploration of the reproductive regulation of NPFF-related genes.