凡纳滨对虾Nrf2在绿原酸诱导GPx基因转录的功能研究

Cultured shrimp is vulnerable to oxidative damage induced by different stresses. Antioxidases play key roles in animal oxidant stress. The results of previous indicate that the expression of GPx gene in shrimp was induced by chlorogenic acid. However, the pathway of the induction remains unknown. Transcription factor Nrf2 (NF-E2-Related factor 2) plays an important role in regulation of GPx gene in mammals. In this project, Litopenaeus vannamei was selected as the candidate animal. Nrf2 gene of L. vannamei was identified and characterized by cloning and bioinformatics technology. The distribution of Nrf2 in shrimp was confirmed by qRT-PCR and Western blot method. Then the involvement of Nrf2 in the regulation of GPx gene was clarified by the technique of Nrf2 gene silence in vivo and over expression of Nrf2 gene and GPx gene promoter recombined into luciferase report gene vector in vitro. Meanwhile, the roles of chlorogenic acid in the involvement of Nrf2 in the regulation of GPx gene were investigated by signal pathway blocking technique in primary hemolymph cell model of L. vannamei. The results of this project will deepen our understanding of the regulation of antioxidant gene transcription in aquatic crustaceans and provide a theoretic support for chlorogenic acid as an antioxidant for aquaculture purpose.

对虾容易遭受不同因子胁迫而诱发氧化损伤。抗氧化酶在机体抗氧化应激中起着重要作用。核转录因子Nrf2是调控哺乳动物抗氧化酶基因转录的关键分子。前期研究发现绿原酸明显诱导对虾GPx基因的表达，但是这种诱导作用的途径完全不清楚。鉴于此，本项目选用凡纳滨对虾作为研究对象，通过基因克隆和生物信息学技术鉴定目标基因Nrf2，明确其在对虾体内的分布规律；然后基于体内Nrf2基因沉默技术以及体外Nrf2过量表达及GPx启动子报告质粒转染技术阐明Nrf2参与对虾GPx基因转录的调控，同时以建立的凡纳滨对虾原代血细胞模型，运用信号阻断技术从细胞水平探讨绿原酸对Nrf2调控GPx基因转录的作用。本项目结果加深我们对水生甲壳动物抗氧化酶基因转录调控的了解，亦可为绿原酸作为渔用抗氧化剂提供了科学依据。

凡纳滨对虾Nrf2基因（LvNrf2）全长1830 bp，开放式阅读框729 bp，编码了242个氨基酸，预测分子量为27.83 kDa，pI为9.33。BLASTp比对分析显示LvNrf2编码的氨基酸序列与夏威夷明钩虾（Parhyale hawaiesis）CNC的相似性最高（87.60%），与斑马鱼（Danio rerio）Nrf2的相似性最低（38.12%）。CDD分析显示LvNrf2属于Cap′n′Colla （CNC）家族的碱性亮氨酸拉链蛋白，序列包含有CNC转录因子亮氨酸拉链结构域，功能是与DNA结合的二聚体化结构域。构建了原核表达载体，体外获得分子量约为35 kDa的纯化重组蛋白，获得了LvNrf2纯化的特异性多克隆抗体，Western blot结果显示LvNrf2蛋白在心脏、肌肉、肠道、胃和鳃中均表达，其中肌肉组织中该蛋白的表达量最高。荧光定量分析结果表明LvNrf2基因在血淋巴细胞、肝胰腺、胃、肠道和鳃组织中均有表达，在肠道组织中的表达量最高；饲料添加400 mg/kg绿原酸饲喂凡纳滨对虾56 d能显著诱导血淋巴细胞LvNrf2基因的表达水平，且低盐度和亚硝酸联合胁迫48 h后400 mg/kg绿原酸组对虾肝胰腺LvNrf2基因表达水平显著升高，有利于抵抗环境胁迫。dsLvNrf2注射凡纳滨对虾肌肉组织后，血淋巴细胞LvNrf2和谷胱甘肽过氧化物酶（GPx）基因的表达水平同时降低，说明LvNrf2有可能调控GPx基因表达，而MnSOD和CAT基因表达水平升高可能弥补GPx基因表达的下调。为了进一步研究LvNrf2与GPx之间的调控机制，采用Genome walking方法克隆获得了GPx启动子序列1524 bp，分析发现GPx-P含有丰富的腺嘌呤和胸腺嘧啶（A+T），含有典型的TATA序列。构建了LvNrf2-pCDNA3.1真核表达载体和GPx-P-pGL3荧光素酶报告基因载体，共转染至293T细胞中，结果表明LvNrf2与GPx-P-33段（-547至-433 bp）之间存在明显的相互作用效应。5 µg/mL绿原酸有利于增强LvNrf2和GPx-P启动子的相互作用。