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2025年4月5日 星期六
农业生物技术学报  2021, Vol. 29 Issue (9): 1785-1794    DOI: 10.3969/j.issn.1674-7968.2021.09.013
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
丙烯酰胺对斑马鱼应激反应相关基因的研究
徐悦1,2, 梁利群2,*, 孙博2, 常玉梅2, 赵雪飞3
1 上海海洋大学 水产科学国家级实验教学示范中心,上海 201306;
2 中国水产科学研究院 黑龙江水产研究所,哈尔滨 150070;
3 东北林业大学 野生动物与自然保护地学院,哈尔滨 150040
Studies on Stress Response Related Genes Affected by Acrylamide in Zebrafish (Danio rerio)
XU Yue1,2, LIANG Li-Qun2,*, SUN Bo2, CHANG Yu-Mei2, ZHAO Xue-Fei3
1 National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, China;
2 Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China;
3 College of Wildlife and Protected Area, Northeast Forestry University, Harbin 150040, China
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摘要 丙烯酰胺(acrylamide, ACR)广泛用于土壤稳定化、水处理、工业产品和某些食品中,是重要的环境胁迫因子之一,其毒性正在引起全球关注。本研究以3日龄斑马鱼(Danio rerio)仔鱼和实验室自主培养的斑马鱼肠细胞(zebrafish intestine cells, ZFI)为研究对象,利用凝集素荧光标记技术和qRT-PCR对氧化应激反应代表基因谷胱甘肽硫还原酶(glutathione-disulfide reductase, GSR)、内质网应激反应代表基因X盒结合蛋白1 (X-box binding protein 1, xbp1u)、渗透压应激反应代表基因水通道蛋白4重组蛋白(recombinant aquaporin 4, aqp4)和应激信号通路结合蛋白钙/钙调蛋白依赖性蛋白激酶Ⅱ (calcium/calmodulin-dependent protein kinase Ⅱ γ2, CaMK2g2)进行表达检测。结果表明,凝集素点印迹显示,与0 mmol/L ACR耐受情况相比,12.5、25.0 mmol/L ACR耐受浓度下怀槐凝集素Ⅱ (maackia amurensis lectin Ⅱ, MAL-Ⅱ)、小麦胚芽凝集素(wheat germ agglutini, WGA)和花生凝集素(peanut agglutinin, PNA)的条带均在斑马鱼仔鱼及ZFI的45~60 kD中出现不同程度的变浅,其中ZFI在25.0 mmol/L耐受浓度下的凝集素条带改变更为明显。此外,在25.0 mmol/L ACR浓度下,ACR毒性对机体(18 h)和细胞(5 h)的损伤最大,qRT-PCR结果表明与上述应激反应相关的4个基因有所关联:对ZFI而言,aqp4xbp1u表达量极显著上调(P<0.01),CaMK2g2GSR表达量显著上调(P<0.05);对斑马鱼仔鱼而言,aqp4的表达量显著性升高(P<0.05),xbp1uGSR表达量极显著性上调(P<0.01),CaMK2g2表达量无显著性改变(P>0.05)。上述结果表明:ACR毒性对细胞造成损伤的同时兼具全身性,并且其毒性作用机制与非生物性胁迫应激响应机制高度相关。本研究揭示了鱼体对ACR胁迫应激响应机制,有利于保护鱼类等水生生物免受ACR毒性侵扰。
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徐悦
梁利群
孙博
常玉梅
赵雪飞
关键词 丙烯酰胺凝集素点印迹谷胱甘肽硫还原酶(GSR)X盒结合蛋白1 (xbp1u)水通道蛋白4重组蛋白(aqp4)钙离子/钙调蛋白依赖性蛋白激酶Ⅱγ2 (CaMK2g2)    
Abstract:Acrylamide (ACR), one of the critical environmental stressors, is widely used in soil stabilization, water treatment, industrial products, and certain foods, which toxicity was attracting global attention. This study focused on the toxic effects of ACR in non-neural organs such as the intestines. This study used 3-day-old zebrafish (Danio rerio) and laboratory-grown zebrafish intestine cells (ZFI) as experimental materials. Coagulant fluorescence markers and qRT-PCR were used to check some stress response genes, glutathione-disulfide reductase (GSR), X-box binding protein 1 (xbp1u), recombinant aquaporin 4 (aqp4), and calcium/calmodulin-dependent-protein (CaMK2g2) for expression testing. The 4 genes' functions were related to reaction mechanisms such as oxidative stress, endosurgen network stress, permeable pressure stress, and stress signal-transducing. The coagulation point imprinting results showed that the maackia amurensis lectin Ⅱ(MAL-Ⅱ), wheat germ agglutini (WGA), and peanut agglutinin (PNA) tricoagulant bars were at 12.5 and 25.0 mmol/L tolerances than the 0 mmol/L ACR tolerance. The bands showed varying degrees of shallow changes in zebrafish and ZFI in 45~60 kD, where the changes in the coagulant bands of ZFI at a tolerance concentration of 25.0 mmol/L were more pronounced. Besides, at a concentration of 25.0 mmol/L ACR, ACR toxicity caused the most significant damage to the body (18 h) and cells (5 h), and the above 4 genes associated with stress response showed that for ZFI, aqp4 and xbp1u showed very high significant expression (P<0.01), CaMK2g2 and GSR showed significant high expression in ZFI (P<0.05); For zebrafish, the expression of aqp4 increased significantly (P<0.05), xbp1u and GSR showed extremely high significant expression (P<0.01), and CaMK2g2 showed no significant change (P>0.05). The above results showed that ACR toxicity was systemic when it caused damage to cells, and the ACR toxicity mechanism was closely related to the non-biological stress response mechanism. Therefore, this study's significance lied in revealing the fish bodies' stress response mechanism to ACR stress. Multi-level interpretation of its toxicity mechanism would be conducive to protect fish and other aquatic organisms from ACR toxic intrusion to meet the growing aquatic food safety needs of China and the world's population.
Key wordsAcrylamide    Lectin blot    Glutathione-disulfide reductase (GSR)    X-box binding protein 1 (xbp1u)    Recombinant aquaporin 4 (aqp4)    Calcium/calmodulin-dependent protein kinase Ⅱγ2 (CaMK2g2)
收稿日期: 2020-12-11     
ZTFLH:  S917  
基金资助:国家重点研发计划“蓝色粮仓科技创新”(2019YFD0900405); 黑龙江省自然科学基金重点项目(ZD2018008)
通讯作者: * liangliqun@hrfri.ac.com   
引用本文:   
徐悦, 梁利群, 孙博, 常玉梅, 赵雪飞. 丙烯酰胺对斑马鱼应激反应相关基因的研究[J]. 农业生物技术学报, 2021, 29(9): 1785-1794.
XU Yue, LIANG Li-Qun, SUN Bo, CHANG Yu-Mei, ZHAO Xue-Fei. Studies on Stress Response Related Genes Affected by Acrylamide in Zebrafish (Danio rerio). 农业生物技术学报, 2021, 29(9): 1785-1794.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2021.09.013     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2021/V29/I9/1785
 
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