虎斑乌贼(Sepia pharaonis)FMRFamide G蛋白偶联受体基因的克隆与表达定位研究

MOLECULAR IDENTIFICATION AND LOCALIZATION OF G-PROTEIN COUPLED RECEPTOR OF FMRFAMIDE IN PHARAOH CUTTLEFISH SEPIA PHARAONIS

以FMRFamide为典型代表的FMRFamide相关肽,是目前已知的最大的神经肽家族。FMRFamide广泛参与多种生理过程,包括摄食、心跳、渗透平衡、变态、防御和免疫等。采用同源克隆法与RACE技术获得了虎斑乌贼FMRFamide的G蛋白偶联受体基因cDNA全长序列(命名为SpFaGPCR,OL765295),SpFaGPCR cDNA全长1514 bp,包括222 bp的5′-非编码区(5′-UTR)、35 bp的3′-UTR以及1257 bp的开放阅读框(ORF),共编码418个氨基酸。预测相对分子量(MW)为49.8 kDa,等电点(pI)为9.76,具有7个跨膜结构域、糖基化位点7个、磷酸化位点36个。同源序列比对分析表明,SpFaGPCR与曼氏无针乌贼的FaGPCR氨基酸序列相似度最高,达98%。系统发育分析显示SpFaGPCR与双壳纲的FaGPCR聚为姐妹支。荧光定量结果显示SpFaGPCR在视叶、视腺、脑、缠卵腺中表达量相对较高(P<0.05)。进一步利用原位杂交技术检测到虎斑乌贼视叶的髓质区、视网膜的感光细胞和缠卵腺的瓣叶外层具有明显的阳性杂交信号。实验结果为进一步探讨FMRFamide通过FaGPCR的介导参与虎斑乌贼生理代谢功能奠定了前期基础。

Neuropeptides are one of active peptides that involved in the multiple physiological metabolic processes via interaction with corresponding receptors.FMRFamide-like peptides(FLPs)are the largest known neuropeptide family and the tetrapeptide FMRFamide is a typical representative involved in a wide range of physiological processes,including feeding,heart activity,osmotic balance,metamorphosis,defense,and immunity.In the study,the full-length cDNA sequence of G-protein coupled receptor of FMRFamide in Sepia pharaonis(designated SpFaGPCR,OL765295)was obtained by homologous cloning and rapid amplification of cDNA end(RACE)methods.The cDNA of SpFaGPCR is 1514 bp length,including 222 bp 5′-untranslated region(UTR),35 bp 3′-UTR,and 1257 bp opening reading frame(ORF),encoding 418 amino acid residues with 7 transmembrane(TM)regions.The predicted molecular weight(MW)is 49.8 kDa and theoretical isoelectric point(pI)is 9.76.Seven glycosylation sites and 36 phosphorylation sites were also predicted.Multi-sequence alignment revealed high homology of SpFaGPCR with SjFaGPCR from Sepiella japonica.Phylogenetic analysis showed that SpFaGPCR is clustered into a group with Bivalvia species.Quantitative real-time PCR(qRT-PCR)results showed that SpFaGPCR had relative higher expression level in the optic lobe,optic gland,brain,and nidamental gland(P<0.05).In situ hybridization(ISH)experiments showed that significant SpFaGPCR mRNA positive signals could be observed in the medulla area of optic lobe,the photoreceptor cells of retina,and the outer layer of the valve leaflets of the nidamental gland.These data provided solid data and theoretical supports for further study on the FaGPCR mediated role of FMRFamide in the physiological metabolism of S.pharaonis.