虎斑乌贼神经肽sCAP基因克隆与表达分析

Molecular Cloning and Expression Analysis of Neuropeptide sCAP in Sepia pharaonis

小心激肽(small cardioactive peptides,sCAP)是刺激心脏运动、牵引肌肉收缩、刺激输卵管收缩、增强后肠的收缩以及参与生物蜕皮的神经肽,开展虎斑乌贼sCAP及其生理功能研究具有重要意义,为其种质资源保护和开发奠定一定理论基础。虎斑乌贼sCAP基因(简称SpsCAP,Genbank登录号MN852577)cDNA全长666 bp,包括109 bp的5’-UTR和296 bp 3’-UTR,预测的开放阅读框(ORF)为261 bp,编码86个氨基酸,分子量(MW)为9.3 kD,等电点(pI)为8.52。预测的信号肽位于1号到20号氨基酸之间,该蛋白含有跨膜区结构,预测的二级结构含有33.72%的延伸链和66.28%的无规则卷曲,亲疏水性分析显示为亲水性蛋白。同源性氨基酸序列比对发现虎斑乌贼与曼氏无针乌贼的同一性最高,其次是商乌贼;构建系统进化树分析发现,虎斑乌贼sCAP基因的进化关系与曼氏无针乌贼和商乌贼最近,并且与软体动物聚为一支。组织表达分析显示,在虎斑乌贼所有组织中均检测到SpsCAP的mRNA,且视叶组织中表达量最高,其次是脑组织,显著高于其他组织(P<0.05);在雄性与雌性乌贼的视叶组织表达量存在显著性差异,且雄性乌贼的相对表达量是雌性乌贼的4倍。

A small cardioactive peptides(sCAP)gene was identified in a cuttlefish species called Sepia pharaonis and was designated as SpsCAP.The total length of the SpsCAP sequence was 666 bp while the ORF contained 261 bp encoding a protein of 86 amino acid residues with a predicted pI and MW of 8.52 and 9.3 kDa along with a 109 bp 5’-UTR and 296 bp 3’-UTR.Its signal peptide was located in amino acids ranging from 1 to 20,and the protein has a transmembrane structure.The analysis of the secondary structure of the protein revealed that it contains 33.72%extended strand and 66.28%random coil.Hydrophilicity analysis showed that it was a hydrophilic protein.The sCAP homologous amino acid sequence alignment showed that the identity was highest between the S.pharaonis and S.japonica,followed by S.officinalis.The phylogenetic tree analysis revealed that the evolutionary relationship of the sCAP gene in S.pharaonis was closest to those of S.japonica and S.officinalis,and it is grouped with mollusks.qRT-PCR results showed that sCAP gene expressed in all tested tissues,and the highest expression was found in optic lobe,followed by the brain,which has a significant difference with other tissues.There was also a significant difference in visual tissue between male and female cuttlefish,and the relative expression of male is 4 times of that in female.