| 低温驯化下斑马鱼LINE1的表达检测 |
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| 作者姓名： | 陶筱帆 谢婷婷 李小霞 罗军涛 白雅静 韩兵社 张俊芳 |
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| 作者单位： | 上海海洋大学 水产种质资源发掘与利用教育部重点实验室 上海 201306;上海海洋大学 水产种质资源发掘与利用教育部重点实验室 上海 201306；上海海洋大学 水产科学国家级实验教学示范中心 上海 201306;上海海洋大学 水产种质资源发掘与利用教育部重点实验室 上海 201306；上海海洋大学 水产科学国家级实验教学示范中心 上海 201306；上海海洋大学 中国科学技术部海洋生物科学国际联合研究中心 上海 201306 |
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| 基金项目： | 国家自然科学基金项目(31372516,81770165);上海市教育委员会“东方学者”计划和上海市人才发展资金项目(201457)共同资助。 |
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| 摘 要： | 长散布核元件-1(Long spread nuclear element-1,LINE1)是跳跃基因。前期比较基因组研究发现,南极鱼经历漫长的低温适应进化后,与南极圈外的同亚目鱼类相比较,在基因水平上LINE1的扩增效率高达8~300倍,但LINE1的扩增与鱼类抵御寒冷之间的关系尚未明了。本实验对斑马鱼(Danio rerio)胚胎成纤维细胞ZF4进行了不同时间梯度的低温处理(18℃、5 d和18℃、30 d),同时对斑马鱼成鱼也进行了不同时间的低温处理(10℃,3 h、6 h、1 d、3 d、5 d)。采用RT-qPCR检测了LINE1的mRNA水平,并克隆了斑马鱼LINE1基因启动子区,利用Luciferase双荧光报告系统,在ZF4细胞中验证LINE15’UTR在低温压力下的生物活性。结果显示,短时间低温处理下,ZF4细胞中LINE1 mRNA水平有所降低,而在长时间低温处理中,LINE1的mRNA水平显著升高。在成鱼中,短时间低温处理下,LINE1 mRNA水平降低;长期低温处理下,LINE1 mRNA水平显著升高。在ZF4细胞中发现,LINE15’UTR具有生物活性。在低温处理(18℃,3 d)下,报告基因信号减弱,间接表明LINE1启动子活性减弱。研究结果表明,低温压力会影响LINE1在鱼类中的表达。本研究为进一步探究LINE1在鱼类适应低温环境中的作用机制奠定了基础。
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| 关 键 词： | LINE1 低温 斑马鱼 ZF4细胞 启动子 |
| 收稿时间： | 2019/2/26 0:00:00 |
| 修稿时间： | 2019/3/14 0:00:00 |
Expression of LINE1 in Zebrafish (Danio rerio) During Cold Acclimation |
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| Authors: | TAO Xiaofan XIE Tingting LI Xiaoxi LUO Juntao BAI Yajing HAN Bingshe ZHANG Junfang |
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| Institution: | Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306; National Demonstration Center for Experimental Fisheries Science Education, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306; National Demonstration Center for Experimental Fisheries Science Education, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306; International Research Center for Marine Biosciences, Shanghai Ocean University, Shanghai 201306 |
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| Abstract: | The long-spread nuclear element-1(LINE1)retrotransposon is a mobile element in genome.Previous comparative genomic studies found that Antarctic Notothenioid fish underwent a long low-temperature adaptation evolution,and compared with Notothenioid fish outside the Antarctic circle,LINE1 genes were duplicated by 8~300 fold.The link between this augmentation and the resistance of fish to cold is not known.In this study,zebrafish(Danio rerio)embryonic fibroblasts ZF4 were exposed to a low temperature(18℃)for 5 days and 30 days,and adult zebrafish were exposed to a low temperature(10℃)for 3 h,6 h,1 d,3 d,and 5 d.The mRNA expression of LINE1 was examined using RT-qPCR.The promoter regions of zebrafish LINE1 gene were cloned and the biological activity of LINE15’UTR at low temperature were verified in ZF4 cells by using the dual-luciferase reporter system.The following results were obtained:(1)In ZF4 cells,LINE1 mRNA expression was decreased by short-term low temperature treatment,but was significantly increased by long-term low temperature treatment.(2)In adult fish,LINE1 mRNA expression was decreased by short-term low temperature treatment,but was significantly increased in long-term low temperature treatment.(3)The LINE15’UTR was found to be biologically active in ZF4 cells.(4)It was found that during low temperature treatment(18℃,3 d),the reporter gene signal was weakened,which indirectly indicated that the LINE1 promoter activity was weakened.The results showed that low temperature stress affected LINE1 expression in fish,which presents a foundation for further study on the mechanism of action of LINE1 in fish adaptation to a low temperature environment. |
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| Keywords: | LINE1 Low temperature Danio rerio ZF4 cells Promoter |
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