于秋寒¹, 平洪领^1,2, 闫允君¹, 史会来², 吕振明¹, 杨静文¹
1.浙江海洋大学 海洋科学与技术学院, 国家海洋设施养殖工程与技术研究中心;2.浙江省海洋水产研究所

摘要:
采用cDNA末端快速扩增技术(rapid amplification of cDNA ends, RACE)克隆了刺参GPR54-like(Apostichopus japonicus, AjGPR54-like, 简称AjGPR54L)基因cDNA全长, 对AjGPR54L全长cDN 序列进行了生物信息学分析; 通过绿色荧光蛋白(GFP)融合表达, 对其在HEK293细胞中进行了亚细胞定位分析; 采用实时荧光定量PCR技术(qRT-PCR)检测AjGPR54L在组织中的表达。结果表明: 该基因全长1454 bp, 包含993 bp的开放阅读框(open reading frame, ORF), 编码330个氨基酸, 191 bp的5′非翻译区(UTR)和270 bp的3′-UTR. 预测蛋白质的相对分子质量为37.83 ku、等电点(pI)为9.81; 预测蛋白序列分析显示AjGPR54L具有GPCR家族蛋白典型的七次跨膜结构, 包含1个预测的N-连接糖基化位点、5个Asn-Xaa-Ser/Thr序列子和34个磷酸化位点, 无信号肽酶切位点; 与已鉴定的GPR54s氨基酸序列同源性分析表明, AjGPR54L与斑马鱼GPR54序列相似性最高, 为30.0%; 共聚焦显微镜检测显示AjGPR54L-EGFP可定位于细胞膜表面; 基因表达定量分析结果显示, AjGPR54L在刺参呼吸树、消化道、体壁、肌肉、神经环中均有表达分布, 且神经环中表达量显著高于其他组织。
关键词:刺参(Apostichopus japonicus)GPR54基因克隆亚细胞定位组织表达
DOI：10.11759/hykx20171220001
分类号:
基金项目:国家自然科学基金项目(41606150); 浙江省公益技术研究项目(2017C32074)

Molecular cloning and characterization of GPR54-like receptor in the sea cucumber Apostichopus japonicus
YU Qiu-han,PING Hong-ling,YAN Yun-jun,SHI Hui-lai,Lü Zhen-ming,YANG Jing-wen
Abstract:
In this study, a novel GPR54-like gene from Apostichopus japonicus was cloned using RACE technology, and then bioinformatics analysis was performed. The AjGPR54L gene was fused with the green fluorescent protein (GFP), and its subcellular localization in HEK293 cells was evaluated. The mRNA expression level in multiple tissues of the sea cucumber was also quantified. Results showed that the full-length cDNA of AjGPR54L was 1, 454 bp, which contained an open reading frame (ORF) of 993 bp, encoding 330 amino acid (aa) residues, a 5′-untranslated region (5′-UTR) of 191 bp, and a 3′-UTR of 270 bp. The molecular weight of the protein was predicted to be 37.83 ku, and the isoelectric point (pI) was 9.81. Sequence analysis of the predicted protein showed that AjGPR54L had seven transmembrane domains, containing one predicted N-linked glycosylation site, five Asn-Xaa-Ser/Thr sequences, and 34 phosphorylation sites, but no signal peptide cleavage site. The homology analysis of multiple sequence alignment revealed that AjGPR54L had the highest similarity to zebrafish GPR54 (30.0%). Confocal microscopy demonstrated the cell membrane localization characteristic of AjGPR54L-EGFP. AjGPR54L exhibited a ubiquitous expression in A. japonicus, and a significantly higher level of AjGPR54L transcription was detected in the nerve ring than in other tissues such as the respiratory tree, intestine, body wall, muscle, and ovary.
Key words:Apostichopus japonicusGPR54gene cloningsubcellular localizationtranscriptional expression





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