生物学杂志

生物学杂志

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池蝶蚌HsCTL的分子特征及表达分析

  

  1. 1. 南昌大学 生命科学学院， 南昌 330031； 2. 江西省水产动物资源与利用重点实验室， 南昌 330031；3. 江西省抚州市水产科学研究所，抚州 344000
  • 出版日期:2018-12-18 发布日期:2018-12-18
  • 通讯作者: 洪一江，教授，博导, 主要从事水产动物遗传育种学研究, E-mail：yijianghong2008@163.com
  • 作者简介:彭扣，讲师，博士, 主要从事水产动物分子免疫学研究, E-mail：pengkou@126.com
  • 基金资助:
    国家自然科学基金（31860737;31660337）；国家现代农业产业技术体系（CARS-49）；江西省自然科学基金（20151BAB204035）；江西省教育厅自然科学青年基金项目（GJJ14219）；江西省科技支撑项目（20121BBF60036）；江西省特种水产产业技术体系（JXARS-10）

Molecular characterization and expression analysis of the C-type lectin gene (HsCTL) from the freshwater pearl mussel, Hyriopsis schlegelii

  1. 1. School of Life Sciences, Nanchang University, Nanchang 330031; 2. Key Laboratory of Aquatic Animal Resources and Utilization of Jiangxi, Nanchang University, Jiangxi 330031；3. Fuzhou Fisheries Science Institute of Jiangxi, Fuzhou 344000, China
  • Online:2018-12-18 Published:2018-12-18

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摘要： C-型凝集素(C-type lectin, CTL)是存在动、植物及微生物中一类Ca2+依赖性的糖蛋白，能够识别和结合病原微生物表面的多糖物质，在机体免疫防御中起重要作用。为了解淡水育珠贝池蝶蚌（Hyriopsis schlegelii） CTL的分子特征及潜在作用，结合转录组高通量测序和末端快速扩增技术（Rapid amplification of cDNA ends, RACE）从池蝶蚌中鉴定了一条与其他物种CTL同源的cDNA序列，命名为HsCTL。该序列全长为1716 bp，包括5′ 端非编码区92 bp，3′ 端非编码区898 bp，开放阅读框长726 bp，编码241个氨基酸，预测蛋白质相对分子量为28 ku。其氨基酸序列存在一个由23个氨基酸残基组成的信号肽和一个糖配体识别结构域(carbohydrate recognition domain, CRD)，CRD结构域中仅包含1个QPD（Gln-Pro-Asp）基序。序列同源性及进化分析表明，HsCTL与斑节对虾 (Penaeus monodon) CTL同源性最高，达36%，而与目前GenBank公开的其他贝类CTLs的同源性为9.7%~18.1%。构建的系统进化树显示，HsCTL与斑节对虾和中国明对虾(Fenneropenaeus chinensiss)的同系物聚为一支，与三角帆蚌(Hyriopsis cumingii) perlucin和九孔鲍(Haliotis diversicolor) CTL聚为相邻支。通过实时荧光定量RT-PCR检测发现，HsCTL的mRNA广泛分布于池蝶蚌各个组织，其中在血细胞表达量最高，外套膜和肝胰腺次之。且进一步发现，注射脂多糖（lipopolysaccharide, LPS）后，HsCTL的mRNA表达量在血细胞和肝胰腺组织中显著增加。以上结果表明，HsCTL在池蝶蚌抵抗病原微生物的免疫防御反应中可能起到重要作用，结果为进一步研究HsCTL参与池蝶蚌先天免疫的分子机制打下基础。

关键词: 池蝶蚌, C-型凝集素, 克隆, 序列分析, 免疫应答, 实时荧光定量PCR

Abstract: C-type lectins (CTLs) are Ca2+-dependent carbohydrate-recognition proteins which are extensively present in plants, animals and microorganisms. CTLs were revealed to be able to recognize and bind the carbohydrate components on the cell surface of pathogenic microorganisms, and play crucial roles in innate immunity. To understand the molocular characterization of CTL and its potential function from a species of freshwater pearl mussel (Hyriopsis schlegelii), the cDNA sequence of one novel C-type lectin gene (named as HsCTL) in this study was cloned from H. Schlegelii for the first time by means of de novo transcriptome sequencing, combined with 5′ and 3′ rapid amplification of cDNA ends (RACE). The full-length cDNA consists of 1716 bp with one open reading frame (ORF) of 726 bp, encoding a polypeptide of 241 amino acids with 28 ku predicted molecular weight, one 5′-untranslated region (UTR) of 92 bp and one 3′-UTR of 898 bp. The HsCTL contained a typical signal peptide sequence with 23 amino acids and one carbohydrate recognition domain (CRD). The CRD of HsCTL was composed of 115 residues, including 6 conserved cysteine residues and a QPD (Gln-Pro-Asp) motif which has a predicted binding specificity for galactose-type sugarin immune reactions. BLASTP analysis showed that the HsCTL shared the highest identity (reaching 36%) with the ortholog in sugpo prawn (Penaeus monodon), and 9.7%-18.1% with other molluscon orthologs. The phylogenetic analysis showed that the HsCTL fell on the same branch with P. monodon and Fenneropenaeus chinensiss. And then they were clustered with Hyriopsis cumingii and Haliotis diversicolor in a big branch in the neighbor-joining tree. Tissue expression analysis showed that the HsCTL mRNA was expressed in hepatopancrea, hemocyte, mantle, gill, adductor muscle and gonad, with the highest expression in hemocyte and then in mantle, hepatopancreas, gill and adductor muscle, with the lowest expression in gonad. After challenge with lipopolysaccharide (LPS), HsCTL mRNA expression was upregulated significantly in the hemocytes and hepatopancreas. Our results implied that HsCTL plays an important role in the immune response in mussels and lays a foundation for studies on the H. schlegelii immune system.

Key words: Hyriopsis schlegelii, C-type lectin, cloning, sequence analysis, immune response, real-time PCR

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