曼氏无针乌贼神经肽sCAP基因克隆与组织表达定位

CLONING, EXPRESSION ANALYSIS AND LOCALIZATION OF NEUROPEPTIDE SCAP IN SEPIELLA JAPONICA

为研究曼氏无针乌贼(Sepiella japonica)小心激肽(small cardioactive peptides,sCAP)生理功能,通过RACE技术克隆曼氏无针乌贼sCAP基因(简称SjsCAP,GenBank登录号:MG779491),得到总长度为696 bp的cDNA序列,包括111 bp的5′非编码区(UTR)和324 bp的3′UTR,预测的开放阅读框(ORF)共261 bp,编码86个氨基酸,相对分子量(MW)为9.331 kD,等电点(pI)为8.52。信号肽以及跨膜区预测结果表明,sCAP中含有明显的信号肽序列和跨膜区结构。因此,推测该蛋白可能由细胞内分泌到细胞外发挥作用。亲水性分析显示该蛋白为亲水性蛋白。基于sCAP氨基酸序列进行的系统进化分析表明曼氏无针乌贼与商乌贼(Sepia officinalis)亲缘关系最近,相似性达到90%。通过荧光定量PCR (Quantitative real-time PCR,qRT-PCR)技术对SjsCAP基因在成熟雄性和雌性曼氏无针乌贼不同组织中的表达量进行分析,结果显示sCAP主要在视叶中显著表达,在脑中也有较高的表达量,其中雄性个体表达量显著高于雌性个体。原位杂交实验结果显示sCAP基因在曼氏无针乌贼的脑组织的视叶、食道上神经团的垂直叶、亚垂直、脑脚叶、背外侧叶和视腺均可以观察到明显的阳性杂交信号。sCAP基因的成功克隆以及组织表达定位分析为sCAP的亚细胞定位以及生物学功能研究奠定一定的基础,同时为曼氏无针乌贼的种质资源保护与开发提供一定的理论支持。

sCAP could stimulate heart movement and contraction of muscle and fallopian tube,enhance posterior bowel contraction and participate in biological molting.This study identified the sCAP gene (GenBank accession No.MG779491) from Sepiella japonica as SjsCAP.We cloned a 696 bp full-length cDNA sequence of SjsCAP gene by RTPCR and RACE techniques including 111 bp 5′ untranslated region (UTR),324 bp 3′UTR,and 261 bp open reading frame (ORF) encoding a protein with 86 amino acids.The molecular weight of the protein was 9.331 kD and its pI was 8.52.The prediction of signal peptides and transmembrane domain indicated that SjsCAP might play an important role in extracellular.The protein is hydrophilic.Phylogenetics analysis based on SjsCAP amino acid sequences demonstrated that S.japonica has the closest relationship with Sepia officinalis with a 90% similarity.Expression analysis of SjsCAP gene in male and female individuals showed that it mainly expressed in optic lobe and also has the high expression in the brain.The expression level in male was obviously higher than that of female cuttlefish.The results of in situ hybridization assay showed that significant positive hybridization signals were detected in the optic lobe of the brain tissue of S.japonica,the vertical lobe,subvertical lobe,peduncle lobe,dorsolateral lobe of the esophageal nerve mass,and optic gland.These results provide some theoretical basis for genetic resource conservation and development of S.japonica.