文章摘要
低温驯化下斑马鱼LINE1的表达检测
Expression of LINE1 in zebrafish under cold acclimation
投稿时间:2019-02-26  修订日期:2019-03-14
DOI:
中文关键词: LINE1,低温,斑马鱼,ZF4细胞,启动子
英文关键词: LINE1, low temperature, Danio rerio, ZF4 cells, promoter.
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 支持项目; 上海市人才发展资金项目(201457)资助.
作者单位E-mail
陶筱帆 上海海洋大学 512099587@qq.com 
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中文摘要:
      长散布的核元素-1(LINE1)反转录转座子是跳跃基因。前期比较基因组研究发现,南极鱼经历了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,但逆转座子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℃,5d和18℃,30d),同时对斑马鱼成鱼也进行了不同时间长度的低温处理(10℃,3h、6h、1d、3d、5d)。使用RT-qPCR检验了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LINE1 5’UTR在低温(18℃,3d)压力下的生物活性。结果显示: (1) 在ZF4细胞中,短期低温处理LINE1 mRNA水平有所降低,而在长期低温处理中,LINE1的mRNA水平显著升高。(2) 在成鱼中,短期低温处理下LINE1 mRNA水平降低,长期低温处理LINE1 mRNA水平显著升高。(3) 在ZF4细胞中发现LINE1 5’UTR具有生物活性。(4) 发现在低温处理(18℃,3d)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,为进一步研究LINE1在鱼类适应低温环境中的作用机制奠定了基础。
英文摘要:
      The long-spread nuclear element-1 (LINE1) retrotransposon is a skip gene. Previous comparative genomic studies found that Antarctic fish experienced a long low-temperature adaptation evolution, compared with the Antarctic fish outside the Antarctic circle, the LINE1 amplification efficiency at the gene level is as high as 8 to 300 times, reversing the LINE1 The link between augmentation and the resistance of fish to cold is not known. In this experiment, zebrafish (Danio rerio) embryonic fibroblast ZF4 was treated with low temperature (18 ° C, 5 d and 18 ° C, 30 d) for different lengths of time, and the zebrafish adult fish were also subjected to low temperature for different lengths of time. Treatment (10 ° C, 3 h, 6 h, 1 d, 3 d, 5 d), mRNA levels of LINE1 were examined using RT-qPCR. The zebrafish LINE1 gene promoter region was cloned and the biological activity of LINE1 5'UTR under low temperature (18 ° C ,3d) pressure was verified in ZF4 cells using the Luciferase dual fluorescence reporter system. The results showed that: (1) In ZF4 cells, the LINE1 mRNA level was decreased in the short-term low temperature treatment, while the LINE1 mRNA level was significantly increased in the long-term low temperature treatment. (2) In adult fish, LINE1 mRNA levels were decreased under short-term low temperature treatment, and LINE1 mRNA levels were significantly increased in long-term low temperature treatment. (3) The LINE1 5'UTR was found to be biologically active in ZF4 cells. (4) It was found that under low temperature treatment (18 ° C, 3 d), the reporter gene signal was weakened, which indirectly indicated that the LINE1 promoter activity was weakened. The results show that low temperature stress will affect the expression of LINE1 in fish, which lays a foundation for further study on the mechanism of action of LINE1 in fish adaptation to low temperature environment.
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