低温驯化下斑马鱼LINE1的表达检测

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

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。

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.