虎斑乌贼FMRFamide的表达定位及其Na^(+)通道受体基因的克隆鉴定、表达分析

FMRFAMIDE LOCALIZATION AND MOLECULAR CHARACTERIZATION,AND THE EXPRESSION OF ITS Na^(+)CHANNEL RECEPTOR IN SEPIA PHARAONIS

FMRFamide是神经肽家族的一类重要代表成员,参与无脊椎动物的多种生理调控过程。利用同源克隆和RACE技术获得了虎斑乌贼神经肽FMRFamide的Na^(+)通道受体(Sp FaNaC)的cDNA全长共2090 bp,开放阅读框(open reading frame,ORF)1782 bp,编码一个由593个氨基酸残基组成的多肽链,N端无信号肽,具有2个跨膜区;序列分析显示其与头足纲、腹足纲和双壳纲相似性较高,聚类为姐妹支。荧光定量PCR(qRT-PCR)检测到SpFaNaC主要分布在中枢神经系统的视叶、脑,其次是心脏和副缠卵腺。SpFaNaC原位杂交结果显示其阳性信号存在于视网膜感光细胞的细胞体、视叶深层视网膜的内颗粒细胞层和外细胞颗粒层、食道上神经团的各亚叶,尤以交界区最为集中。同时,原位杂交和免疫组化观察到神经肽FMRFamide主要分布在视叶的中央髓质区、边缘髓质区、内细胞颗粒层和外细胞颗粒层;食道上神经团的阳性信号较少,主要集中为亚叶交界处。实验结果为研究虎斑乌贼FMRFamide及Sp FaNaC的生理功能研究奠定一定的理论基础。

Many reports have showed that neuropeptides play pivotal regulatory roles in the neuroendocrine process.FMRFamide is an important representative member in neuropeptide family and involved in the physiological process.We cloned the full-length cDNA of FMRFamide Na^(+)channel receptor gene in Sepia pharaonis(designated SpFaNaC)with homologous amplification and RACE(rapid-amplification of cDNA ends).The cDNA of SpFaNaC is 2090 bp length,including a 1782 bp opening reading frame(ORF),encoding 593 amino acid residues with none a signal peptide at N-terminal and 2 transmembrane(TM)regions.Sequence analysis showed that SpFaNaC shared higher identity with cephalopods,gastropods,and bivalve species,and clustered as sister branches.Quantitative Real-time PCR(qRT-PCR)results showed SpFaNaC had relative higher expression level in the central nervous system optic lobe and brain followed by heart and accessory nidamental gland.In situ hybridization(ISH)results showed that SpFaNaC mRNA positive signals could be detected significantly in the inner cell granular layer and outer cell granular layer of the optic lobe,the photoreceptor cells of retina,the sub-lobes of supraoesophageal mass,and the positive signals are the most concentrated in the border area.In addition,ISH and immunohistochemical staining(IHC)results showed that FMRFamide mainly distributed in the central medullary area,peripheral medullary area,inner cell granular layer,and outer cell granular layer of the optic lobe.There were relatively few positive signals in the supraoesophageal mass,and the positive signals were most concentrated in the border area.This study provided a basis for studying the role of FMRFamide and its Na^(+)channel receptors in the physiological process of S.pharaonis.