郑佩华 汪蕾 张秀霞 李军涛 张泽龙 王冬梅 冼健安 王安利

摘要：【目的】明確凡纳滨对虾微粒体谷胱甘肽硫转移酶3（LvMGST3）在机体抗逆境胁迫与抵御病原体侵染过程中的功能作用，为有效提高对虾的抗胁迫能力和抗病力提供理论依据。【方法】采用RACE克隆LvMGST3基因cDNA序列，以EditSeq、ExPASy、SignalP 5.0、TMHMM 2.0、Clustal X和MEGA 6.0等在线软件进行生物信息学分析，再应用半定量PCR进行组织表达量分析。凡纳滨对虾分别进行氨氮胁迫（20.00 mg/L）及脂多糖（LPS，8 μg/g）刺激后，采用实时荧光定量PCR检测分析LvMGST3基因的表达响应情况。【结果】LvMGST3基因cDNA序列全长826 bp，包含85 bp的5'端非编码区（5'-UTR）、321 bp的3'端非编码区（3'-UTR）和420 bp的开放阅读框（ORF），在ploy（A）尾前端15个核苷酸处存在加尾信号AATAAA。LvMGST3基因编码139个氨基酸残基，包含MGST3亚家族共有的保守结构域FNCX1QRX2H，此序列为FNCYQRAH。LvMGST3蛋白分子量为15.53 kD，理论等电点（pI）为9.38，具有3个跨膜结构域，但不存在信号肽。LvMGST3氨基酸序列与软甲纲普通卷甲虫（Armadillidium vulgare）的MGST3氨基酸序列（RXG56258.1）相似性最高，为71.22%;基于MGST3氨基酸序列相似性构建的系统发育进化树显示，LvMGST3与节肢动物门和软体动物门的MGST3亲缘关系较近。LvMGST3基因在凡纳滨对虾肝胰腺、鳃组织和眼柄中的基础表达量较高;经氨氮胁迫后，肝胰腺中的LvMGST3基因相对表达量分别在胁迫6和24 h时极显著上调（P<0.01，下同），鳃组织中的LvMGST3基因相对表达量在胁迫12 h时显著上调（P<0.05，下同）、胁迫48 h时极显著上调;肝胰腺中的LvMGST3基因在LPS刺激前期表达受抑制，至刺激12 h时极显著上调，鳃组织中的LvMGST3基因分别在LPS刺激3和48 h时出现2个表达峰值，其相对表达量均极显著高于对照组，分别是对照组的4.39和6.45倍。【结论】LvMGST3基因具有典型的MGST3亚家族结构特征，主要在凡纳滨对虾的肝胰腺、鳃组织和眼柄中表达，且氨氮胁迫和LPS刺激可明显诱导凡纳滨对虾肝胰腺和鳃组织中LvMGST3基因的表达水平，即MGST3在对虾抗逆境胁迫及抵御病原菌感染的调控机制中发挥重要作用。

关键词： 凡纳滨对虾;谷胱甘肽硫转移酶3（MGST3）;基因克隆;氨氮胁迫;脂多糖（LPS）刺激

中图分类号： S945.49                                文献标志码： A 文章编号：2095-1191（2020）10-2311-10

Cloning and function analysis of microsomal glutathione transferase 3 gene（MGST3） from Litopenaeus vannamei

ZHENG Pei-hua1，2， WANG Lei1， ZHANG Xiu-xia2，3， LI Jun-tao2，3， ZHANG Ze-long2，3，WANG Dong-mei2，3， XIAN Jian-an2，3*， WANG An-li1*

（1School of Life Sciences， South China Normal University/Guangdong Provincial Key Laboratory for Healthy and Safe Aquaculture/Guangzhou Key Laboratory of Subtropical Biodiversity and Biomonitoring/Key Laboratory of Ecology and Environmental Science in Guangdong Higher Education， Guangzhou  510631， China; 2Institute of Tropical Biosciences and Biotechnology， Chinese Academy of Tropical Agricultural Sciences/Hainan Provincial Key Laboratory for Functional Components Research and Utilization of Marine Bio-resources， Haikou  571101， China; 3Hainan Institute of Tropical Agricultural Resources， Chinese Academy of Tropical Agricultural Sciences， Haikou  571101， China）

Abstract：【Objective】The purpose of this study was to clarify the function of the microsomal glutathione thitransfera-se 3（LvMGST3） in the process of resisting stress and pathogen infection， and to provide theoretical basis for effectively improving the stress resistance and disease resistance of Litopenaeus vannamei. 【Method】RACE was used to clone the cDNA sequence of LvMGST3 gene， and online softwares such as Editseq， Expasy， SignalP-5.0， TMHMM 2.0， Clustal X and MEGA 6.0 was used for bioinformation analysis， and then semi-quantitative PCR was used for tissue expression ana-lysis. Shrimp were stimulated by ammonia nitrogen stress（20.00 mg/L） and lipopolysaccharide（LPS， 8 μg/g） injection respectively， and the expression responses of LvMGST3 were analyzed by real-time fluorescence quantitative PCR. 【Result】LvMGST3 was 826 bp in length， including 85 bp 5' non-coding region（5'-UTR）， 321 bp 3'-UTR and 420 bp open reading frame（ORF）， and the polyadenylate signal AATAAA presented at 15 nucleotides in front of ploy（A）. LvMGST3 encoded 139 amino acids， including the unique structural sequence FNCX1QRX2H of MGST3 subfamily， this sequence was FNCYQRAH. The molecular weight of LvMGST3 protein was 15.53 kD， and the theoretical isoelectric point（pI） was 9.38. It had three transmembrane domains， but there was no signal peptide. The amino acid sequence of LvMGST3 had the highest similarity to MGST3 amino acid of Malacostraca Armadillidium vulgare（RXG56258.1）， which was 71.22%. Phylogenetic trees based on the similarity of MGST3 amino acid sequence showed that LvMGST3 was closely related to MGST3 of Arthropoda and Mollusca. The basal expression of LvMGST3 was high in hepatopancreas， gills and eyestalk. After ammonia-nitrogen stress， expressions of LvMGST3 in hepatopancreas were extremely significantly up-regulated at 6 h and 24 h of stress （P<0.01， the same below）， and expressions of LvMGST3 in gills were significantly up-regulated at 12 h of stress（P<0.05， the same below） and extremely significantly up-regulated at 48 h of stress. Under LPS stimulation， expression of LvMGST3 in hepatopancreas was inhibited in the early stage， and then it was extremely significantly up-regulated at 12 h. There were two expression peaks of LvMGST3 in gills at LPS stimulation for 3 h and 48 h， and their relative expression levels were significantly higher than those in control group， which were 4.39 times and 6.45 times of control， respectively. 【Conclusion】LvMGST3 has typical structural characteristics of MGST3 family， and is mainly expressed in hepatopancreas， gills and eyestalk. The expression levels of LvMGST3 in the hepatopancreas and gill of L. vannamei are significantly induced by ammonia nitrogen stress and LPS stimulation， indicating that LvMGST3 plays an important role in the defense mechanism of shrimp against environmental stress and pathogenic bacteria infection.

Key words： Litopenaeus vannamei; microsomal glutathione transferase 3（MGST3）; gene cloning; ammonia nitrogen stress; lipopolysaccharide（LPS）

Foundation item： Hainan Natural Science Foundation（319QN306）; Guangdong Natural Science Foundation （2017A0 30313194）; Basic Research Project of Chinese Academy of Tropical Agricultural Sciences（1630052019013，1630052016 011）

0 引言

【研究意義】凡纳滨对虾（Litopenaeus vannamei）又称南美白对虾，是节肢动物门（Arthropoda）甲壳纲（Crustacea）十足目（Decapoda）对虾科（Penaeidae）中的重要人工养殖物种之一。近年来，在市场需求量的刺激下，凡纳滨对虾养殖产业得到快速发展，但在高密度集约化养殖模式下，养殖水质恶化、病害频发等一系列严峻问题随之涌现，而造成虾类免疫功能受损及抗应激能力下降（麦康森等，2004;蒋葛等，2019）。……