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

CLC Number: 

• Q78

• S828