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| Cloning and Transcriptional Activity Analysis of ACSL6 Gene Promoter in Dairy Goats (Capra hircus) |
| XIONG Tian-Tian, WU Yu, SHAO Yue-Xin, CHEN Chong, WANG Wan-Ting, CHENG Fei, LI Cong* |
| College of Animal Science and Technology, Northwest A&F University/Shaanxi Provincial Key Laboratory of Animal Genetics, Breeding and Reproduction, Yangling 712100, China |
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Abstract Acyl-CoA synthetase long chain family member 6 (ACSL6), a key enzyme in the uptake and transport of free fatty acids, is involved in the regulation of fatty acid metabolism during lactation in dairy animals. This study aimed to preliminarily investigate the transcriptional regulation mechanism of ACSL6 gene (Gene ID: XM_018050725.1) through promoter cloning, bioinformatics analysis and diluciferase activity detection in dairy goats (Capra hircus). The full-length sequence of the ACSL6 promoter was obtained by PCR amplification using the blood genomic DNA of Xinong Saanen dairy goats as a template. The sequence features of the binding sites and promoter regions with high prediction scores were screened using bioinformatics analysis software. The promoter deletion fragment vectors were cloned and constructed, which were co-transfected with the internal reference vectors into the mammary epithelial cells of dairy goats, and the promoter active regions were identified by dual luciferase activity assay. The results showed that the full-length sequence of the ACSL6 promoter was 2 108 bp. The multiple transcription factor binding sites were existed in the ACSL6 promoter. The two CpG islands with lengths of 101 and 460 bp were identified in the promoter sequences of -907~-807 bp and -513~-54 bp, respectively. The nine deletions obtained from the cloning were analysed by dual-luciferase activity, and it was found that the core region of the ACSL6 promoter was located at -170~-33 bp, and there might be negative regulatory elements upstream of the transcriptional start site at -2 004~-1 707 bp, -1 334~-957 bp, and -584~-271 bp. This study provides basic information to reveal the molecular mechanism of ACSL6 regulating milk fat metabolism in dairy goats, and provides a reference to improve the quality of goat milk at the molecular level.
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Received: 04 March 2024
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Corresponding Authors:
* congl@nwafu.edu.cn
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