摘　要：之前研究表明在低温胁迫下斑马鱼多个组织中, circadian associated repressor of transcription a (ciarta, 也称为c1orf51)基因被广泛诱导表达。为了鉴定ciarta启动子中受低温响应的最敏调控区域, 克隆5个不同长度的截短片段(-1 992~+100 bp, -1 354~+100 bp, -881~+100 bp, -634~+100 bp和-247~+100 bp), 插入到荧光素酶报告载体pGL4.10中，随后将载体转染到斑马鱼来源的ZF4细胞中, 研究这5个截短片段低温诱导基因表达的能力。结果表明, 低温条件下, 5个截短片段都能显著诱导下游基因的表达, 其中-881~+100 bp截短片段表现出最高的启动子活性(达5.61±0.53倍)。通过对启动子中转录因子结合位点的预测结果进行分析发现，与-634~+100 bp截短片段相比, -881~+100 bp截短片段含有一个已知的低温调控元件(BCL6的结合位点), 进一步证实BCL6的结合位点具有低温诱导基因表达的能力。实验鉴定出的ciarta启动子低温诱导核心区域可以作为一个工具来诱导基因在低温下的表达, 为相关研究提供帮助。

Abstract: Previous study showed that the expression level of circadian associated repressor of transcription a (ciarta) gene, which is also known as c1orf51, is commonly induced in eight tissues of zebrafish under cold stress. To identify the core cold-regulatory region of ciarta promoter, five fragments of different lengths(-1 992~+100 bp, -1 354~+100 bp, -881~+100 bp, -634~+100 bp and -247~+100 bp) were cloned into pGL4.10, which were transfected into zebrafish-derived ZF4 cells. The cold-regulatory abilities of these 5 fragments were quantified by examining the expression changes of luciferase at a cold temperature. The results showed that all of the five fragments could significantly induce the gene expression at the cold temperature. Of these 5 fragments, one fragment (-881~+100 bp) showed the highest cold-induced ability and increased the downstream gene expression up to 5.61±0.53 fold. Compared with the fragment (-634~+100 bp), the fragment (-881~+100 bp) was found to contain a known cold-induced cis-element (BCL6 binding site). The finding verifies the cold-induced ability of BCL6 binding site. The cold-induced key promoter region of ciarta can be used as a tool to induce the expression of the gene at low temperature, which is helpful for the related research.