三疣梭子蟹GST基因的cDNA克隆及表达分析

Cloning and expression analysis of the GST gene in Portunus trituberculatus

取健康三疣梭子蟹(Portunus trituberculatus)‘黄选1号’80日龄幼蟹,体重(32.66±6.27)g。通过RT-PCR及Smart-TM Race技术,克隆了三疣梭子蟹谷胱甘肽S-转移酶(glutathiones-transferase,简称GST)基因c DNA全长序列(Gen Bank登录号:KF781517)。GST基因全长1 010 bp,编码一个由216个氨基酸组成的多肽,预测理论等电点PI为5.294,分子量为24.59 k D。氨基酸序列中含有GST基因家族特有GST-N结构域和GST-C结构域。同源性及系统进化分析表明,三疣梭子蟹GST基因与中华绒螯蟹(Eriocheir sinesis)和脊尾白虾(Exopalaemon carinicauda)的同源性分别为78%和63%。实时荧光定量PCR结果表明,GST基因在肝胰腺、鳃、肌肉、血淋巴、心脏和眼柄中均有分布,在肝胰腺中表达量最高(P<0.05),其次是鳃(P<0.05),在心脏中表达最少。三疣梭子蟹肌肉注射低、中、高3个剂量组的磺胺嘧啶后,GST基因表达较对照组都有表达,均呈现先升高后降低再升高的趋势。同时,随着药物浓度的增加,低、中、高3个剂量组峰值出现的时间越晚,分别为6 h、24 h和48 h。本实验结果表明,磺胺嘧啶可以诱导三疣梭子蟹GST基因表达,GST基因可能参与三疣梭子蟹的药物代谢反应。

Drug residues from aquaculture have been associated with negative effects on human and environmental health. The in vivo metabolism of a drug is carried out by a number of enzymes and can influence the formation of drug residues or the drug effect. However, few studies have evaluated the effects of drugs used in aquaculture on drug metabolism enzymes in marine crustaceans. Glutathione S-transferases play an important role in drug metabolism. We isolated and characterized glutathione S-transferase gene in Portunus trituberculatus, a species that is widely cultured in the offshore waters of China. The c DNA encoding GST was first cloned using RT-PCR and Smart-Race. Analysis of the nucleotide sequence revealed that the full-length c DNA clone was 1 010 bp and encoded a protein of 216 amino acids, which had a predicted molecular weight of 24.59 k D with an estimated PI of 5.294. The protein was expected to possess the GST-N structure domain and GST-C structural domain, suggesting that it belonged to the GST subgroup. Comparison of amino acid sequences showed a similarity of more than 78% between GST in P. trituberculatus and Eriocheir sinensis. The phylogenetic analysis revealed that the GST in P. trituberculatus was in the same branch as that of E. sinensis. The level of GST gene expression in different tissues was analyzed by quantitative real-time PCR. GST was detected in all tested tissues of P. trituberculatus, including the hepatopancreas, gills, muscle, hemolymph, and eyestalk. Expression was highest in the hepatopancreas（P〈0.05） and lowest in the heart. GST expression was up-regulated in the hepatopancreas after intramuscular injection of sulfadiazine（P〈0.05）. This suggests that GST expression was induced by the exogenous drug and increased the capacity for detoxification of the organism. GST expression decreased with time and was significantly（P〈0.05） lower than controls between 12 and 18 h post injection. This suggests that the function of hepatopancreas cells was disrupted. However, expression of the GST gene increased significantly between 24 h and 48 h, suggesting that detoxification of hepatopancreas cells was restored. Our results suggest that GST may play a role in the drug-metabolic response of P. trituberculatus.