低温驯化下斑马鱼LINE1的表达检测

Expression of LINE1 in Zebrafish (Danio rerio) During Cold Acclimation

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

中文摘要:

      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LINE1 5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，LINE1 5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。

英文摘要:

      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 LINE1 5'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 LINE1 5'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.