引用本文:	周林,李颖,吕振明,史会来,吴常文,迟长凤.曼氏无针乌贼(Sepiella japonica)精子表面蛋白17(Sp17)基因克隆以及组织表达特异性分析.海洋与湖沼,2017,48(5):1044-1051.

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*曼氏无针乌贼(Sepiella japonica)精子表面蛋白17(Sp17)基因克隆以及组织表达特异性分析*
周林¹, 李颖¹, 吕振明¹, 史会来², 吴常文¹, 迟长凤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技术研究中心海洋生物种质发掘与利用国家地方联合实验室舟山 316022;2.浙江省海洋水产研究所舟山 316021

摘要:

精子表面蛋白17（Sp17）是参与受精过程的关键分子。本研究采用RT-PCR以及RACE技术克隆得到了曼氏无针乌贼（Sepiella japonica）Sp17简称Sj-Sp17 cDNA的全长序列，序列全长1463bp，5'和3'非编码区分别为92bp和222bp，预测的开放阅读框（ORF）全长1149bp。编码的蛋白理论分子量为42.0548kDa，等电点4.66，是一种亲水性蛋白，不存在跨膜区以及信号肽序列，含有丰富的螺旋结构（54%）。同源氨基酸序列比对发现，与其他软体动物的相似性最高仅为44%，表明Sp17在进化中并不保守。基于Sp17氨基酸序列构建的系统进化分析表明，曼氏无针乌贼和加州双斑蛸（Octopus bimaculoides）进化关系最近。组织特异性分析发现Sp17在曼氏无针乌贼的生殖系统中均有较高的表达量，其中在精巢和储精囊中有显著表达。Sp17基因的成功克隆以及组织表达特异性分析对于深入研究其细胞定位以及生物学功能具有重要意义。

关键词: 曼氏无针乌贼 Sp17 cDNA 生物信息学 real-time PCR

DOI：10.11693/hyhz20170300060

分类号:

基金项目:科技部港澳台科技合作专项，2014DFT30120号；浙江省自然科学基金项目，LY15C190010号；舟山市科技专项，2016C41016号；浙江省科研院所专项，2015F50055号；浙江海洋大学"海洋科学"省重中之重学科开放课题，20160116号。

附件

MOLECULAR CLONING AND EXPRESSION ANALYSIS OF SPERM SURFACE PROTEIN 17 (SP₁7) GENE IN SEPIELLA JAPONICA

ZHOU Lin¹, LI Ying¹, LÜ Zhen-Ming¹, SHI Hui-Lai², WU Chang-Wen¹, CHI Chang-Feng¹

1.National Engineering Research Center of Marine Facilities Aquaculture, National and Provincial Joint Laboratory of Exploration and Utilization of Marine Aquatic Genetic Resources, School of Marine Science and Technology, Zhejiang Ocean University, Zhoushan 316022, China;2.Marine Fisheries Research Institute of Zhejiang, Zhoushan 316021, China

Abstract:

Sperm surface protein 17 (Sp17) is a key molecular in fertilization process. A 1463bp full-length cDNA of Sp17 gene from Sepiella japonica described as Sj-Sp17 was obtained with RT-PCR and rapid amplification of cDNA ends (RACE) techniques. Sp17 consists of a 92bp 5' untranslated region (UTR) and a 222bp 3' UTR. The predicted open reading frame (ORF) is 1149bp, the molecular weight of deduced protein is 42.0548kDa, and its pI is 4.66. Sj-Sp17 is hydrophilic, has no signal peptide sequence and transmembrane regions, but contains rich spiral structures (54%). The deduced amino acid sequence that is aligned with other species Sp17 shows low similarity (<44%), indicating that the structure of Sj-Sp17 is not conserved. Phylogenetic analysis based on Sp17 demonstrates that S. japonica is closely related with Octopus bimaculoides. Sj-Sp17 gene was mainly expressed in reproductive system by real-time PCR analysis, and had significant expression especially in testis and seminal vesicle. We believe that cloning and analysis on tissue-specific expression of Sj-Sp17 gene would be of great significance for understanding its cellular localization and biological function.

Key words: Sepiella japonica Sp17 cDNA bioinformatics real-time PCR