Molecular Cloning And Gene Expression Of Two Fmrfamide Receptors And Research On Potential Function Of Fmrfamide In Pharaoh Cuttlefish(Sepia Pharaonis)

The pharaoh cuttlefish(Sepia pharaonis)is an important marine economic cephalopod in China.Due to overfishing,habitat destruction,and habitat degradation,the pharaoh cuttlefish’s fishery resources have declined since the 1980 s and gradually become endangered species.The recovery and utilization of S.pharaonis resources depends on the relevant research on the reproductive characteristics of this species.However,at present,we still know little about the molecular mechanism of reproductive development of S.pharaonis.Neuropeptides are widely distributed in invertebrates and vertebrates and usually act as neurotransmitters,neuromodulators and neurohormones.The neuropeptide FMRFamide(FLP)is the most famous neuropeptide family,represented by the tetrapeptide FMRFamide(Phe-Met-Arg-Phe-NH2;FMRFa).Previous studies have demonstrated that FMRFamide and GnRH(Gonadotropin-releasing hormone)participate in the regulation of cephalopod reproductive development and the two receptors of FMRFamide play an important role in this process.Based on this,the cloning and sequence characterization of the two receptor genes of FMRFamide in S.pharaonis were firstly carried out in this paper,the distribution of the two receptors on the tissue specificity was studied,the mRNA of the two FMRFamide receptors and the precursor FMRFamide were localized,and finally,the effect of FMRFamide on the secretory activity of CHO-K1 cells was examined.The results were showed as follows:1.The localization and distribution of SpFMRFamide in S.pharaonis showed through in situ hybridization and immunohistochemistry.In situ hybridization experiments results showed that a large number of SpFMRFamide precursor mRNA positive cells were detected in the central medulla,marginal medulla and deep retina of the optic lobes of S.pharaonis.The distribution of mRNA of FMRFamide precursor gene in optic lobe tissue is basically consistent with the distribution of nucleus in optic lobe cells.The FMRFamide precursor gene can be observed at the basal lobe(bl)and vertical lobe(vl)and olfactory lobe(olf)of the supraesophageal mass.The results of immunohistochemical experiments showed that the FMRFamide immunoreactive area in the optic lobes of S.pharaonis had the following characteristics: FMRFamide immunological activity was detected in the reticulum of the deep retina of the optic lobes;The SpFMRFamide immunosignals are detected in the marginal medulla that extends in all directions through afferent nerve fibers;Dense immune responses are detected in the central medulla.There are also a large number of FMRFamide immune signals in the supraesophageal mass neurons.These immune response areas are mainly distributed in the nerve cells and nerve fibers between the vertical lobe,sub-vertical lobe,olfactory lobe,optic gland and basal lobes,and these rusts can be seen.The colored areas spread out along the connection of the vertical and base leaves.Experiments with neuropeptide stimulation cells have shown that FMRFamide and GnRH can inhibit cell protein secretion when they were combined to stimulate cells.2.The full-length cDNA sequence of G-protein receptor gene of FMRFamide in S.pharaonis was obtained by homologous cloning method and RACE technology and termed as SpFaGPCR.The cDNA of the FaGPCR gene of S.pharaonis is 1518 bp in length,including 225 bp 5’-UTR,36 bp 3’-UTR and 1257 bp ORF,encoding 418 amino acids.The protein’s relative molecular weight(MW)is predicted to be 49.3 kDa and its isoelectric point(pI)is 9.28.The protein was found to be a G protein-coupled receptor with 7transmembrane(TM)regions,containing 7 glycosylation sites and 35 phosphorylation sites.Homologous sequence alignment result shows that the amino acid sequence of SpFaGPCR has the closest genetic relationship with the FaGPCR sequence of Sepiella japonica(SjFaGPCR),and has a high similarity to the cephalopods and bivalves.Comparison shows that there are more conserved regions.Phylogenetics showed that the SpFaGPCR is grouped with FaGPCR of bivalve species,which has further relationship with vertebrates and arthropods.Tissue difference experiments showed that SpFaGPCR had higher expression in the optic lobe,brain and nidamental gland of S.pharaonis compared to other tissues.The results of in situ hybridization experiments showed that significant SpFaGPCR mRNA positive signals could be observed in the medulla area of??the optic lobe,the photoreceptor cells of the retina,and the outer layer of the valve leaflets of the nidamental gland.3.Homologous cloning and RACE technology were used to obtain the full-length cDNA sequence of the sodium channel receptor gene of FMRFamide in S.pharaonis and termed as SpFaNaC.The full-length cDNA of SpFaNaC gene is 2090 p,which includes265 bp 5’-UTR,46 bp 3’-UTR and 1799 bp ORF,encoding 593 amino acids.The protein’s MW is predicted to be 63.9 KDa and its pI is 12.5.It is predicted that SpFaNaC contains 6glycosylation sites and 70 phosphorylation sites.Prediction of the tertiary structure of SpFaNaC found that it contains 45% of the α-helical structure,12% of the extended chain and 43% of the random coil.Phylogeny shows that SpFaNaC and FaNaC of gastropods are grouped together,which is relatively far from bivalve.Tissue difference experiments showed that SpFaNaC had higher expression in the optic lobe and brain of S.pharaonis compared to other tissues.The results of in situ hybridization experiments showed that a positive signal could be detected in the deep retina of the optic lobe,the photoreceptor cell body of the retina,and the supraesophageal mass.