首页 >  广东海洋大学学报 >  尼罗罗非鱼marco基因克隆、亚细胞定位及表达分析

2022, 42(5): 27-37. doi: 10.3969/j.issn.1673-9159.2022.05.004

尼罗罗非鱼marco基因克隆、亚细胞定位及表达分析

1. 广东海洋大学水产学院;

2. 广东省水产动物病害防控与健康养殖重点实验室, 广东 湛江 524088;

2. 广东海洋大学深圳研究院;

4. 广东省水生动物健康评估工程技术研究中心;

5. 深圳市海水经济动物种苗健康评价公共技术服务平台, 广东 深圳 518120

收稿日期:2022-03-16
修回日期:2022-03-16

基金项目:   广东省现代农业产业体系项目(2019KJ141);广东省基础与应用基础研究基金项目(2021A1515110481) 

关键词: 尼罗罗非鱼 , marco , 无乳链球菌 , 清道夫受体 , 亚细胞定位 , 表达分析

Molecular Cloning, Subcellular Localization and Expression Analysis of marco Gene in Nile Tilapia(Oreochromis niloticus)

1. Fisheries College of Guangdong Ocean University;

2. Guangdong Provincial Key Laboratory of Aquatic Animal Disease Control and Healthy culture, Zhanjiang 524088, China;

2. Shenzhen Institute of Guangdong Ocean University;

4. Guangdong Provincial Engineering Research Center for Aquatic Animal Health Assessment;

5. Shenzhen Public Service Platform for Evaluation of Marine Economic Animal Seedings, Shenzhen 518120, China

Received Date:2022-03-16
Accepted Date:2022-03-16

Keywords: Oreochromis niloticus , marco , Streptococcus agalactiae , scavenger receptor , subcellular localization , expression analysis

【目的】研究尼罗罗非鱼(Oreochromis niloticus)胶原样结构巨噬细胞受体(macrophage receptor with collagenous structure,Marco)的结构特征和免疫功能,为揭示尼罗罗非鱼免疫应答机制提供理论基础。【方法】采用聚合酶链式反应从尼罗罗非鱼脾脏克隆marco基因序列,利用生物信息学和亚细胞定位等手段分析其结构特征,用实时荧光定量PCR(qRT-PCR)技术检测其表达情况。【结果】尼罗罗非鱼marco 基因(Onmarco)的开放阅读框(open reading frame,ORF)长1 071 bp,共编码356 个氨基酸,具有跨膜结构域、典型的胶原样结构域和C 端富半胱氨酸受体(scavenger receptor cystein-rich,SRCR)结构域;多序列比对和系统进化分析表明,Marco 在硬骨鱼类中具有较高的保守性;亚细胞定位显示,尼罗罗非鱼Marco(OnMarco)在HEK-293T细胞中为全细胞分布,主要在细胞膜上表达;qRT-PCR结果显示,Onmarco在健康尼罗罗非鱼各组织中均有表达,但在血液中的表达量最高;经灭活的无乳链球菌和聚肌胞苷酸(Poly I:C)刺激后,Onmarco 在罗非鱼肠道、头肾和脾脏中的表达量均呈时序性变化;用脂多糖(LPS)、Poly I:C 和灭活后的无乳链球菌刺激罗非鱼头肾白细胞,Onmarco 表达量均显著升高。【结论】Onmarco基因可能在鱼体抵御细菌和病毒入侵的免疫反应中发挥重要作用。

【Objective】To investigate the structural characteristics and immune function of macrophage receptor with collagenous structure (Marco) in Oreochromis niloticus, which will provide a theoretical basis for revealing the immune response mechanism in O.niloticus.【Method】The O.niloticus marco(Onmarco) was cloned from the spleen by polymerase chain reaction (PCR).Then, its structural characteristics were analyzed by bioinformatics and subcellular localization, and its expression level was detected by real-time quantitative PCR (qRT-PCR).【Results】 The results showed that the sequence of open reading frame (ORF) of Onmarco was 1 071 bp, which encoded 356 amino acids.Based on amino acids sequence, the structure of O.niloticus Marco (OnMarco) contained three domains: transmembrane domain, typical collagen domain and scavenger receptor cystein-rich (SRCR)domain.The results of multiple sequence alignment and phylogenetic analysis revealed that Marco is highly conserved in teleosts.In subcellular localization experiment, the OnMarco was expressed in the whole cell of HEK-293T cells, mainly seen on the cellular membrane.The result of qRT-PCR indicated that Onmarco was widely expressed in a variety of tissues of healthy fish with the highest expression in the blood.In the challenge experiments, fish were stimulated by inactivated Streptococcus agalactiae and Polyinosine-polycytidylic acid (Poly I:C), and the expression of Onmarco in the intestine, head kidney and spleen showed obvious temporal patterns of change.Besides, the expression of Onmarco in head kidney leukocytes increased significantly upon stimulation with lipopolysaccharide (LPS), Poly I:C and inactivated S.agalactiae.【Conclusion】These findings suggested that Onmarco plays an essential role in the immune response against bacterial and viral pathogens.

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尼罗罗非鱼marco基因克隆、亚细胞定位及表达分析