Molecular Cloning And Expression Analysis Of Sepiella Maindroni Neuropeptides Gene LFRFamide And GnRH

Neuropeptides LFRFamide and GnRH are two short peptides with multiple functions released by neuroendocrine cells.Sepiella maindroni is an important domestic economic species in East China Sea,and its genetical characterization decline and premature phenomena threat this species proliferation.In consideration of the multiple functions of LFRFamide and GnRH,both neuropeptides cDNA were cloned,and the tissue distributions and specific localization of mRNA in the brain were analysed in this paper.The potential functions of LFRFamide and GnRH in the cuttlefish reproduction regulation were supposed.The main results were as followed:1.The full-length sequence of LFRFamide was 841 bp,which contained an open reading frame(ORF)of 567 bp encoding 188 amino acid(aa)residues,a 5’-untranslated region(UTR)of 91 bp,and a 3’-UTR of 183 bp.The predicted protein of 188 aa contained a putative signal peptide of 22 aa and 4 different FLPs,one copy of hexapeptide NSLFRFa,three copy of hexapeptide GNLFRFa,one copy of pentapeptides TIFRFa,and one copy of heptapeptide PHTPFRFa.The predicted molecular weight(MW)was 21.5 kDa and estimated pI was 9.40.The C-terminus of each FLP had the conserved mature peptides ended in FRF.According to the phylogenetic tree,LFRFamide should belong to the LFRFamide subfamily.The transcript of LFRFamide was detected through quantitative real-time PCR method.In male,the transcript of LFRFamide was also significantly detected in brain.In female,the transcript of LFRFamide was significantly detected in brain,but low trace in nidamental gland and accessory nidamental gland.In situ hybridization performed in the cuttlefish brain was used to determine the cellular location of LFRFamide mRNA.The results showed that LFRFamide was transcripted in subpeduncle lobe(SPL),subvertical lobe(SVL),anterior basal lobe(ABL),posterior basal lobe(PBL)and superior frontal lobe(SFL)of supraoesophageal mass,magnocellular lobe(MAG),palliorisceral lobe(PVL),anterior chromatophore lobe(ACL),anterior pedal lobe(APL)and brachial lobe(BL)of subraoesophageal mass and optic lobe,suggesting its potential function in feeding,chromatophore regulation and reproduction.2.The full-length sequence of GnRH was 710 bp,which contained an ORF of 273 bp encoding 90 aa,a 5’-UTR of 85 bp,and a 3’-UTR of 352 bp.The predicted protein of 90 aa contained a putative signal peptide of 31 aa and a mature peptides of pQNYHFSNGWHPG and a GnRH-associated peptide of 44 aa.The predicted MW was 10.1 kDa and estimated pI was 7.71.The amino acid sequence had the conserved mature peptides.According to the phylogenetic tree,S.maindroni GnRH was in the same cluster with invertebrate GnRH.The transcript of GnRH was detected through quantitative real-time PCR method.In male,the transcript of GnRH was significantly detected in brain.In female,the transcript of GnRH was also significantly detected in brain,but low trace in ovary.In situ hybridization performed in the cuttlefish brain was used to determine the cellular location of GnRH mRNA.The results showed that GnRH was transcripted in SPL,SVL,ABL,PBL and inferior frontal lobe(IFL)of supraoesophageal mass,MAG,ACL,pedal lobe(PL),BL of subraoesophageal mass and optic lobes,suggesting its potential function in reproduction.This work was the first report about the full-length sequence of LFRFamide and GnRH in S.maindroni.The tissue distribution analysis and mRNA cellular location in the cuttlefish brain of the two neuropeptides genes were also performed.The preliminary study of S.maindroni neruopeptides LFRFamide and GnRH on reproduction regulation could provide theoretical basis for genetic resources conservation and artificial breeding.