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
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.
[1] 艾对, 张富新, 李延华, 等. 2015. 羊奶短中链脂肪酸与羊奶膻味关系的研究[J]. 食品工业科技, 36(06): 113-116, 121. (Ai D, Zhang F X, Li Y H, et al.2015. Study on the relationship between short and medium chain fatty acids and the odor of goat milk[J]. Food Industry Technology, 36(06): 113-116, 121.) [2] 程菲, 潘坛, 张飞, 等. 2021. 奶山羊ACSL6基因克隆及表达调控分析[J]. 中国畜牧兽医, 48(07): 2291-2301. (Chen F, Pan Y, Zhang F, et al.2021. Gene cloning and expression regulation analysis of ACSL6 in dairy goats[J]. Chinese Animal Husbandry and Veterinary Medicine, 48(07): 2291-2301.) [3] 赫秋亚, 罗军, 姚大为, 等. 2016. 奶山羊ELOVL6基因启动子的克隆及活性区域分析[J]. 农业生物技术学报, 24(06): 790-798. (He Q Y, Luo J, Yao D W, et al.2016. Cloning and active region analysis of the ELOVL6 gene promoter in dairy goats[J]. Journal of Agricultural Biotechnology, 24(06): 790-798.) [4] 吕丹薇. 2023. 山羊诱导乳腺上皮细胞泌乳相关miRNA筛选及调控网络建立[D]. 硕士学位论文, 广西大学,导师: 黄奔, 何仁春, pp. 63-78. (Lv D W.2023. Lactation-related miRNAs screening and regulatory networkestablishment in goat induced mammary epithelial cells[D]. Thesis for M.S., Guangxi University, Supervisor: Huang B, He R C, pp. 63-78.) [5] 任志敏. 2019. 羊奶中膻味成分的分析与不同处理方式对膻味的影响[D]. 硕士学位论文, 内蒙古农业大学,导师: 陈志军, pp. 1-14. (Ren Z M.2019. Analysis of the odor components in goat milk and the influence of different treatment methods on the odor[D]. Thesis for M.S., Inner Mongolia Agricultural University, Supervisor: Chen Z J, pp. 1-14.) [6] 宋书媛. 2020. ACSL6对奶牛乳腺上皮细胞乳脂合成的调控作用研究[D]. 硕士学位论文, 东北农业大学, 导师: 侯晓明, pp. 14-24. (Song S Y.2020. Regulation of ACSL6 on milk fat synthesis in mammary epithelial cells of dairy cows[D]. Thesis for M.S., Northeast Agricultural University, Supervisor: Hou X M, pp. 14-24.) [7] 王引泉, 郝丽霞, 石刚. 2010. 羊奶的营养与食疗特性[J]. 畜牧兽医杂志, 29(01): 66-67. (Wang Y Q, Hao L X, Shi G.2010. Nutrition and therapeutic characteristics of sheep's milk[J]. Journal of Animal Husbandry and Veterinary Medicine, 29(01): 66-67.) [8] 许会芬, 罗军, 李芳, 等. 2012. 山羊SREBP-1基因的超表达对脂肪酸代谢相关基因表达的影响[J]. 生物工程学报, 28(11): 1306-1316. (Xu H F, Luo J, Li F, et al., 2012. Effect of overexpression of the goat SREBP-1 gene on the expression of genes involved in fatty acid metabolism[J]. Journal of Bioengineering, 28(11): 1306-1316.) [9] 姚大为, 赵欣, 赵淑颖, 等. 2021. 奶山羊心脏型脂肪酸结合蛋白(FABP3)基因启动子的克隆及活性分析[J]. 中国饲料, 1(17): 10-15. (Yao D W, Zhao X, Zhao S Y, et al.2021. Cloning and activity analysis of the promoter of cardiac fatty acid-binding protein (FABP3) gene in dairy goats[J]. Chinese Feed, 1(17): 10-15.) [10] 藏赛鸽. 2022. 奶山羊BDH1基因对乳腺上皮细胞脂质代谢的影响研究[D]. 硕士学位论文, 西北农林科技大学, 导师: 罗军, 李聪, pp. 48-51. (Zang S G.2022. Effects of BDH1 gene on lipid metabolism of mammary epithelial cells in dairy goat[D]. Thesis for M.S., Northwest A&F University, Supervisor: Luo J, Li C, pp. 48-51.) [11] 钟瑜. 2011. LXRα基因RNA干扰和过表达对奶山羊乳腺上皮细胞脂肪酸代谢的影响[D]. 硕士学位论文, 西北农林科技大学, 导师: 罗军, pp. 43-52. (Zhong Y.2011. RNA interference and overexpression of LXRα gene on fatty acid metabolism in the mammary gland epithelial cell of dairy goat[D]. Thesis for M.S., Northwest A&F University, Supervisor: Luo J, pp. 43-52.) [12] Chandrasekaran P, Weiskirchen R.2024. The role of SCAP/SREBP as central regulators of lipid metabolism in hepatic steatosis[J]. International journal of molecular sciences, 25(2): 1109. [13] Fernandez R F, Kim S Q, Zhao Y, et al.2018. Acyl-CoA synthetase 6 enriches the neuroprotective omega-3 fatty acid DHA in the brain[J]. Proceedings of the National Academy of Sciences of the USA, 115(49):12525-12530. [14] Grevengoed T J, Klett E L, Coleman R A.2014. Acyl-CoA metabolism and partitioning[J]. Annual Review of Nutrition, 34(1): 1-30. [15] Hale B J, Fernandez R F, Kim S Q, et al.2019. Acyl-CoA synthetase 6 enriches seminiferous tubules with the ω-3 fatty acid docosahexaenoic acid and is required for male fertility in the mouse[J]. The Journal of Biological Chemistry, 294(39): 14394-14405. [16] Klose R J, Bird A P.2006. Genomic DNA methylation: The mark and its mediators[J]. Trends in Biochemical Sciences, 31(2): 89-97. [17] Lee E J, Kim H C, Cho Y Y, et al.2005. Alternative promotion of the mouse acyl-CoA synthetase 6 (mAcsl6) gene mediates the expression of multiple transcripts with 5′-end heterogeneity: Genetic organization of mAcsl6 variants[J]. Biochemical and Biophysical Research Communications, 327(1), 84-93. [18] Lee J Y, Kim A R, Jung Y H.et al.2018. Dissociation of systemic glucose homeostasis from triacylglyceride accumulation by reduced acsl6 expression in skeletal muscle[J]. Biotechnology and Bioprocess Engineering, 23(4): 465-472. [19] Liu D, Liu X, Wu Y, et al.2016. Cloning and transcriptional activity of the mouse omi/HtrA2 gene promoter[J]. International Journal of Molecular Sciences, 17(1): 119. [20] Lu S, Archer M C.2010. Sp1 coordinately regulates de novo lipogenesis and proliferation in cancer cells[J]. International Journal of Cancer, 126(2): 416-425. [21] Marszalek J R, Kitidis C, Dirusso C C, et al.2005. Long-chain acyl-CoA synthetase 6 preferentially promotes DHA metabolism[J]. Journal of Biological Chemistry, 280(11): 10817-10826. [22] Pawlak M, Lefebvre P, Staels B.2015. Molecular mechanism of PPARα action and its impact on lipid metabolism, inflammation and fibrosis in non-alcoholic fatty liver disease[J]. Journal of Hepatology, 62(3): 720-733. [23] Shibata M, Gulden F O, Sestan N.2015. From trans to cis: Ttranscriptional regulatory networks in neocortical development[J]. Trends in Genetics, 31(2): 77-87. [24] Stierwalt H D, Ehrlicher S E, Robinson M M,, et al.2020. Long-chain acyl-CoA synthetases relate to fat oxidation and storage in skeletal muscle of lean humans[J]. Medicine & Science in Sports & Exercise, 52(7S): 1007-1007. [25] Watkins P J, Jaborek J R, Teng F, et al.2021. Branched chain fatty acids in the flavour of sheep and goat milk and meat: A review[J]. Small Ruminant Research, 200:106398. [26] Williamson D L, Rideout T C.2017. Is ACSL6 at the crossroads of skeletal muscle lipid synthesis?[J]. The Journal of Physiology, 595(3): 619-620. [27] Wu N, Li J, Zheng Q, et al.2023. Characterization of the core region of grape VvHOS1 promoter activity and its upstream regulatory proteins[J]. Environmental and Experimental Botany, 207: 105199. [28] Xie D, He Z, Dong Y, et al.2020. Molecular cloning, characterization, and expression regulation of acyl-CoA synthetase 6 gene and promoter in common carp Cyprinus carpio[J]. International Journal of Molecular Sciences, 21(13): 4736.
