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赖氨酸特异性去甲基化酶4A基因(lysine demethylase 4A,kdm4A)编码的蛋白具有去甲基化酶活性,并且在基因表达调控中起重要作用。该基因在斑马鱼中有两种类型kdm4aa和kdm4ab。本实验室前期对18℃低温胁迫下斑马鱼(Danio rerio)胚胎成纤维样细胞(zebrafish embryos fibroblast like cell line,ZF4)的转录组数据分析发现kdm4aa表达量显著降低,但是该基因在鱼类低温胁迫下的作用仍不清楚。为了研究这两种基因在鱼类低温胁迫下的功能,本研究用EcoRⅠ和AgeⅠ酶切构建了pLKO.1-kdm4aa-shRNA、pLKO.1-kdm4ab-shRNA和pLKO.1-negative control (nc)三种重组质粒,并分别与慢病毒包装质粒pCMV-DR8.9.1、pCMV-VSVG共同转染至293T细胞中,经293T细胞包装的病毒,感染斑马鱼ZF4细胞,RT-qPCR检测表明ZF4细胞中kdm4aa和kdm4ab均成功被敲降。随后将细胞于28℃培养(Control)或经18℃低温处理1 d,使用台盼蓝染色法检测ZF4细胞存活率,并进一步检测了细胞内自噬相关基因beclin1和LC3的表达情况。结果显示,在18℃条件下,敲降kdm4aa的细胞与nc组细胞相比存活率显著上升(p<0.05),而敲降kdm4ab的细胞存活率与nc组相比无明显变化,证明敲降kdm4aa在低温胁迫下保护细胞。进一步的研究发现,敲降kdm4aa的细胞内自噬相关基因beclin1及LC3的表达量均显著上调,而敲降kdm4ab的细胞变化不显著,提示敲降kdm4aa在低温胁迫下对斑马鱼细胞的保护作用可能是通过上调自噬水平引起的。该研究为后期斑马鱼低温胁迫分子机制的研究提供新的思路。
Abstract：The protein encoded by the lysine demethylase 4 A(kdm4 A) has demethylase activity and plays an important role in gene expression regulation.This gene has two types of kdm4 aa and kdm4 ab in zebrafish.The transcriptome data of zebrafish embryos fibroblast like cell line(ZF4) at 18 ℃ under low temperature stress in the early stage of the laboratory showed that the expression of kdm4 aa was significantly decreased,but the gene was low in fish.The role under stress is still unclear.In order to study the function of these two genes under low temperature stress in fish,we constructed the pLKO.1-kdm4 aa-shRNA,pLKO.1-kdm4 ab-shRNA and pLKO.1-negative control(nc) by EcoR Ⅰ and Age Ⅰ digestion.Recombinant plasmids were transfected into 293 T cells together with lentiviral packaging plasmids pCMV-DR8.9.1 and pCMV-VSVG,respectively.The zebrafish ZF4 cells were infected with 293 T cell-packaged virus.RT-qPCR detection showed that kdm4 aa in ZF4 cells.And kdm4 ab were successfully knocked down.Subsequently,the cells were cultured at 28 ℃ or treated at 18 ℃ for 1 d.The survival rate of ZF4 cells was detected by trypan blue staining,and the expression of autophagy-related genes beclin1 and LC3 were further detected.The results showed that at 18 ℃,the survival rate of knockdown kdm4 aa cells was significantly higher than that of nc cells(p<0.05),while the cell survival rate of knockdown kdm4 ab was not significantly different from that of nc group,which proved that knockdown kdm4 aa protects cells under low temperature stress.Further studies showed that the expression levels of autophagy-related genes beclin1 and LC3 in knockdown kdm4 aa were significantly up-regulated,while the knockdown of kdm4 ab cells did not change significantly,suggesting that the protective effect of knockdown kdm4 aa on zebrafish cells under low temperature stress.May be caused by up-regulation of autophagy levels.This study provides a new idea for the study of the molecular mechanism of zebrafish low temperature stress.
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基本信息:
DOI：10.13417/j.gab.039.004467
中图分类号:S917.4
引用信息:
[1]刘玮,罗军涛,谢婷婷,等.kdm4A在斑马鱼细胞低温胁迫过程中的作用[J].基因组学与应用生物学,2020,39(10):4467-4474.DOI:10.13417/j.gab.039.004467.
基金信息:
国家自然科学基金项目(81770165);; 上海市教育委员会"东方学者"计划支持项目(31372516);; 上海市自然科学基金项目(13ZR1419500);; 上海市人才发展资金项目(201457)共同资助
2019-10-28
2019-10-28
2019-10-28