引用本文:	李蒙,王金凤,黄骞,李才文.三疣梭子蟹含硒谷胱甘肽过氧化物酶基因克隆及其表达分析.海洋与湖沼,2019,50(1):149-158.

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三疣梭子蟹含硒谷胱甘肽过氧化物酶基因克隆及其表达分析
李蒙^1,2,3, 王金凤^1,2,3,4, 黄骞^1,2,3,4, 李才文^1,2,3,4
1.中国科学院海洋研究所海洋生态与环境科学重点实验室青岛 266071;2.青岛海洋科学与技术国家实验室海洋生态与环境科学功能实验室青岛 266237;3.中国科学院海洋大科学研究中心青岛 266071;4.中国科学院大学北京 100049

摘要:

为探究三疣梭子蟹的免疫机制以及谷胱甘肽过氧化物酶（GPx）在甲壳动物响应寄生虫感染免疫过程中的作用，本研究采用RACE方法从三疣梭子蟹中克隆获得硒半胱氨酸-谷胱甘肽过氧化物酶基因的全长cDNA序列，其cDNA序列全长为696bp，5'-UTR长度为102bp，3'-UTR长度为87bp，开放阅读框长度为507bp，编码168个氨基酸，其中含有一个典型的由蛋白石终止密码子（²²⁰TGA²²²）编码的硒半胱氨酸（⁴⁰U）。预测了该基因编码的氨基酸序列及其中的保守结构域，包括GPx家族签名序列（⁶⁴LAFPCNQF⁷¹）、活性位点序列（¹⁵²WNFEKF¹⁵⁷）以及与酶催化活性相关的氨基酸位点包括谷氨酰胺（⁷⁴Q）、精氨酸（⁹⁰R和¹⁶⁸R）和色氨酸（¹⁴²W）。相似性比对和系统发育分析结果表明，三疣梭子蟹硒半胱氨酸-谷胱甘肽过氧化物酶（SeGPx）与甲壳动物中的SeGPxs相似性较高，其中与拟穴青蟹SeGPx相似性最高，并与其在系统发育树中聚为一支。经血卵涡鞭虫侵染后（0-192h），SeGPx基因在三疣梭子蟹的血细胞、肝胰腺和鳃组织中的转录水平均发生显著性升高。该结果表明，SeGPx在三疣梭子蟹应对血卵涡鞭虫的免疫反应中发挥重要作用，可通过调控甲壳宿主体内被病原扰乱的氧化还原状态进而起到宿主组织保护作用。

关键词: 甲壳动物固有免疫寄生虫谷胱甘肽过氧化物酶基因分子克隆基因表达

DOI：10.11693/hyhz20180500128

分类号:

基金项目:国家自然科学基金面上项目，41676102号；支持“率先行动”中国博士后科学基金会与中国科学院联合资助优秀博士后项目，2016LH0034号；中国博士后科学基金第60批面上资助项目，2016M600561号。

附件

MOLECULAR CLONING AND EXPRESSION OF A SELENIUM-DEPENDENT GLUTATHIONE PEROXIDASE GENE FROM PORTUNUS TRITUBERCULATUS

LI Meng^1,2,3, WANG Jin-Feng^1,2,3,4, HUANG Qian^1,2,3,4, LI Cai-Wen^1,2,3,4

1.CAS Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.Marine Ecology and Environmental Science Laboratory, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China;3.Center for Ocean Mega-Science, Chinese Academy of Sciences, Qingdao 266071, China;4.University of Chinese Academy of Sciences, Beijing 100049, China

Abstract:

Glutathione peroxidase (GPx) is an essential antioxidant enzyme that associated with the immune defense of aerobic living organisms against invading pathogens. To investigate the potential role of GPx genes in crustacean immune response against parasitic infection, we isolated and characterized a novel selenium-dependent glutathione peroxidase gene (PtSeGPx) in Portunus trituberculatus. The open reading frame (ORF) encoded 168 amino acids, with a characteristic selenocysteine residue (⁴⁰U) encoded by an opal codon (²²⁰TGA²²²). The conserved domains including the GPx signature motif 2 (⁶⁴LAFPCNQF⁷¹) and the active site motif (¹⁵²WNFEKF¹⁵⁷), as well as the catalytically related amino acid residues consisting of Gln (⁷⁴Q), Arg (⁹⁰R), Trp (¹⁴²W), and Arg (¹⁶⁸R), were identified in the deduced PtSeGPx protein sequence. Amino acid comparison and phylogenetic analyses indicated that the PtSeGPx was closely related to and clustered with the crustacean SeGPxs. The PtSeGPx transcripts were extensively and induced significantly in multiple major tissues (e.g. hemocytes, hepatopancreas, and gills) of P. trituberculatus during its host immune response against the invasion of the parasitic dinoflagellate Hematodinium, implying its involvement in the crustacean innate immunity against parasitic infection.

Key words: crustacean innate immunity parasite glutathione peroxidase gene molecular cloning gene expression