| 白斑狗鱼StAR基因克隆及其表达分析 |
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| 引用本文： | 张俊杰,李菁,赵瑞阳,古丽帕日·艾克拜.白斑狗鱼StAR基因克隆及其表达分析[J].南方农业学报,2021,52(11):3093-3101. |
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| 作者姓名： | 张俊杰 李菁 赵瑞阳 古丽帕日·艾克拜 |
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| 作者单位： | 新疆农业大学生命科学学院,乌鲁木齐 830052 |
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| 基金项目： | 国家自然科学基金项目（31660739）；新疆高校科研计划项目（XJEDU2016I201）；新疆农业大学大学生创新项目（201910758040） |
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| 摘 要： | 【目的】克隆白斑狗鱼（Esox lucius）类固醇激素合成急性调节蛋白（StAR）基因（ElStAR）并分析其组织表达差异，为开展StAR基因生物学功能研究及揭示白斑狗鱼性腺发育机制提供基础资料。【方法】根据GenBank已公布的白斑狗鱼基因组测序结果（NC_047581.1）设计特异性引物，采用RT-PCR克隆ElStAR基因cDNA序列，通过ClustalX、ExPASy、TargetP 1.1、TMHMM 2.0和SignalP 5.0等在线软件进行生物信息学分析，并采用实时荧光定量PCR进行ElStAR基因组织表达定量分析。【结果】ElStAR基因cDNA序列长度1485 bp，其开放阅读框（ORF）为864 bp，共编码287个氨基酸残基；ElStAR氨基酸序列与北极红点鲑StAR氨基酸序列的相似性最高（93.33%），与海鳟、条纹鲈鱼、金头鲷、大菱鲆、斑马鱼、半滑舌鳎的相似性均高于75.00%，而与小家鼠的相似性最低（59.15%）；基于StAR氨基酸序列相似性构建的系统发育进化树也显示白斑狗鱼与北极红点鲑和海鳟等鲑科鱼类处于同一分支。ElStAR蛋白相对分子量为32.23 kD，理论等电点（pI）为8.98，为不稳定的亲水性蛋白；具有START结构域，无信号肽及跨膜结构，符合线粒体靶向肽的基本特征。ElStAR蛋白二级结构由α-螺旋（占40.77%）、无规则卷曲（占35.89%）、延伸链（占18.12%）和β-折叠（占5.23%）组成，其三级结构是由α-螺旋配合多个β-折叠卷曲盘旋而成。ElStAR基因在白斑狗鱼精巢、卵巢、头肾、肝脏、肌肉及脑组织中均有不同程度的表达，且在精巢中的相对表达量极显著高于在卵巢中的相对表达量（P<0.01），呈明显的性别二态性表达模式。【结论】ElStAR基因编码蛋白结构和功能十分保守，具有典型的START结构域，对胆固醇的运输和调节起重要作用。ElStAR基因在白斑狗鱼精巢及卵巢中的表达存在极显著差异，可能在维持白斑狗鱼精巢的发育形成中发挥重要作用。
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| 关 键 词： | 白斑狗鱼 StAR基因 START结构域 胆固醇 性腺组织 |
| 收稿时间： | 2020-11-25 |
Cloning and expression analysis of StAR gene in Esox lucius |
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| ZHANG Jun-jie,LI Jing,ZHAO Rui-yang,Gulpari·Akbar.Cloning and expression analysis of StAR gene in Esox lucius[J].Journal of Southern Agriculture,2021,52(11):3093-3101. |
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| Authors: | ZHANG Jun-jie LI Jing ZHAO Rui-yang Gulpari·Akbar |
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| Institution: | College of Life Sciences, Xinjiang Agricultural University, Urumqi 830052, China |
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| Abstract: | 【Objective】 The aim of this study was to clone the steroidal hormone synthesis acute regulatory protein (StAR) gene(ElStAR) and analyze its expression in tissues, so as to provide basic data for studying the biological function of StAR gene and revealing gonadogenesis mechanism of Esox lucius.【Method】 Specific primers were designed based on the genomic sequence of E. lucius in GenBank(NC_047581.1), and the cDNA sequence of ElStAR gene was cloned by RT-PCR. Bioinformatics analysis was carried out by online softwares such as ClustalX, ExPASy, TargetP 1.1, TMHMM 2.0 and SignalP 5.0, and the tissue expression of ElStAR gene was analyzed by real-time fluorescence quantitative PCR.【Result】 The cDNA sequence of ElStAR gene was 1485 bp in length and its open reading frame(ORF) was 864 bp, and encoded 287 amino acids. Amino acid sequence of StAR had the highest similarity with Salvelinus alpinus(93.33%), higher than 75.00% with Salmo trutta, Morone saxatilis, Sparus aurata, Scophthalmus maximus, Danio rerio and Cynoglossus semilaevis, and the lowest similarity with Mus musculus(59.15%). The phylogenetic tree based on StAR amino acid sequence similarity also showed that pike was in the same branch as Salmonidae fish such as Salvelinus alpinus and Salmo trutta. ElStAR protein had a relative molecular weight of 32.23 kD and a theoretical isoelectric point(PI) of 8.98. It was an unstable hydrophilic protein. It had START domain, no signal peptide and transmembrane structure, which was consistent with the basic characteristics of mitochondrial targeting peptide. The secondary structure of ElStAR protein was composed of α- helix(40.77%), random coil(35.89%), extended chain(18.12%) and β- fold(5.23%), and its tertiary structure was composed of α- helix fit multiple β- fold with curling and circling. ElStAR gene was expressed in testis, ovary, head kidney, liver, muscle and brain of E. lucius. The relative expression in testis was extremely significantly higher than that in ovary(P<0.01), and showed the expression pattern of gender dimorphism.【Conclusion】 ElStAR gene encodes a protein with very conservative structure and function, with a typical START domain, and plays an important role in the transportation and regulation of cholesterol. There are extremely significant differences in the expression of ElStAR gene in testis and ovary of E. lucius, the gene may play an important supporting role in maintaining the development and formation of testis of pike. |
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