摘要： ATP6V0A4基因编码V-ATP酶的一个亚基，与H⁺转运相关，是遗传性远端肾小管性酸中毒的致病基因。在前期研究中发现，民猪在遭受冷应激后其背脂中ATP6V0A4基因的转录水平发生了显著上升（P<0.05）。为了分析ATP6V0A4基因在转录水平的调控机制，构建了一系列长度不同的含有该基因5'端启动子区域的双荧光素酶报告基因质粒，转染PK15细胞并检测双荧光素酶的相对活性。根据检测结果，利用重叠PCR技术定点靶序列，预判可能存在的转录调控因子。结果发现，该基因启动子区的-1 504~-1 bp片段具有转录活性，通过2轮启动子截短试验后判定-265~-1 bp区域内存在正调控元件，-581~-265 bp区域内存在负调控元件。定向缺失-205~-190 bp，-120~-110 bp小片段后，确定-205~-190 bp区域内存在调控该基因转录的正调控元件结合位点，通过在线软件预测后发现E2F3、SP2、EGR1、E2F6、CTCFL、E2F1、SP1和ETF可能是该基因的正调控转录因子。首次克隆了猪的ATP6V0A4基因的启动子区域并进行了转录因子结合位点的筛选与鉴定，为后续研究其在冷应激状态下的转录调控奠定了基础。

关键词: 猪, ATP6V0A4, 启动子, 核心区, 双荧光素酶报告系统

Abstract: The ATP6V0A4 gene encodes a subunit of V-ATPase, which is related to H⁺ transport and is the causative gene of hereditary distal renal tubular acidosis. In the previous study, our group found that the transcription level of the ATP6V0A4 gene in the back fat of Min pig was significantly increased after cold stress(P<0.05). In order to analyze the regulatory mechanism of the ATP6V0A4 gene at the transcriptional level, this study constructed a series of dual-luciferase reporter gene plasmids with different lengths containing the 5'-end promoter region of the gene, transfected PK15 cells and detected the relative dual-luciferase active. According to the test results, the overlap PCR technology was used to delete the target sequence to predict the possible transcription regulatory factors. The results showed that the-1 504﹣-1 bp fragment of the promoter region of the gene had transcriptional activity. After two rounds of promoter truncation tests, it was determined that there were positive regulatory elements in the-265﹣-1 bp region and negative regulatory elements in the-581﹣-265 bp. After targeted deletion of small fragments of-205﹣-190 bp and-120﹣-110 bp, it was determined that there were binding sites of positive regulatory elements that regulate the transcription of the gene in the region of-205﹣-190 bp. After prediction by online software, E2F3, SP2, EGR1, E2F6, CTCFL, E2F1, SP1 and ETF might be the positive regulatory transcription factors of this gene. The promoter region of the pig ATP6V0A4 gene was cloned for the first time and the transcription factor binding sites were screened and identified, which laid the foundation for the follow-up study of its transcriptional regulation under cold stress.

Key words: Pig, ATP6V0A4, Promoter, Core region, Dual-luciferase reporter system

中图分类号: 

• Q78

• S828