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| 中国荷斯坦奶牛乳脂中miR-2285f生物信息学分析及靶基因验证 |
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张迪1,2,刘佳敏1,2,禹保军1,2,李德生1,2,顾亚玲1,2,温万3,张娟1,2*
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| (1.宁夏大学 农学院, 银川 750021;2.宁夏回族自治区反刍动物分子细胞育种重点实验室, 银川 750021;3.宁夏畜牧工作站, 银川 750002) |
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| 摘要: |
| 旨为探索bta-miR-2285f和宿主基因TANK结合激酶1(TANK binding kinase 1，TBK1)的生物学功能，以及bta-miR-2285f与靶基因互作调节奶牛乳脂代谢的潜在机制，本研究通过生物信息学方法分析bta-miR-2285f在不同物种间的保守性，并通过上游序列CPG岛分析、启动子预测、进一步进行组织表达谱分析，预测靶基因并进行GO功能和KEGG通路富集分析。利用双荧光素酶报告试验验证bta-miR-2285f调控的下游靶基因钙结合蛋白39(Calcium binding protein 39，CAB39)，并在乳腺上皮细胞水平探究bta-miR-2285f的功能作用。结果表明:1)bta-miR-2285f是牛特异性miRNA，属于内含子miRNA；2)bta-miR-2285f与宿主基因TBK1上游序列2 kb处均不含有CPG岛。bta-miR-2285f在上游586 bp处为转录起始位点。宿主基因TBK1在上游105 bp处为转录起始位点；3)TargetScan和miRWalk预测miR-2285f有172个交集靶基因，主要参与新陈代谢、遗传信息传递、信号分析和细胞的相互作用，并且显著富集到mTOR信号通路、PI3K-AKT信号通路、ErbB信号通路、Hippo信号通路以及甘油酯代谢等乳脂代谢相关的信号通路并在STRING数据库中构建bta-miR-2285f靶基因之间的互作网络图；4)bta-miR-2285f在奶牛乳腺组织中的表达水平最高，与其他组织存在显著差异(P<0.05)；5)双荧光素酶报告基因试验证明bta-miR-2285f与基因CAB39的3’非翻译区(UTR)种子序列具有直接的靶向关系并且bta-miR-2285f mimic显著上调乳脂标志基因ELOVL6、SLC27A6、PPARG和FASN的表达，bta-miR-2285f inhibitor则具有相反的作用。综上，bta-miR-2285f-1可能通过反馈调节宿主基因TBK1从而参与乳脂代谢过程，并且bta-miR-2285f通过靶向基因CAB39进而参与奶牛乳脂代谢过程，为进一步探究bta-miR-2285f在中国荷斯坦奶牛乳脂代谢中的潜在机制提供基础。 |
| 关键词: 中国荷斯坦奶牛 miR-2285f 生物信息学 miRNA-mRNA互作 乳脂 |
| DOI：10.11841/j.issn.1007-4333.2023.07.10 |
| 投稿时间：2022-10-14 |
| 基金项目:宁夏自治区育种专项优质高产奶牛选育资助项目(2019NYYZ05)；宁夏回族自治区重点研发项目(2022BBF02017) |
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| Bioinformatics analysis and target gene identification of miR-2285f in Chinese Holstein dairy cows |
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ZHANG Di1,2,LIU Jiamin1,2,YU Baojun1,2,LI Desheng1,2,GU Yaling1,2,WEN Wan3,ZHANG Juan1,2*
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| (1.College of Agriculture, Ningxia University, Yinchuan 750021, China;2.Key Laboratory of Ruminant Molecular Cell Breeding, Ningxia Hui Autonomous Region, Yinchuan 750021, China;3.Animal Husbandry Extension Station, Yinchuan 750002, China) |
| Abstract: |
| To explore the biological functions of bta-miR-2285f and TBK1 (TANK binding kinase 1), and the potential mechanism of bta-miR-2285f interacting with its target gene in regulating milk fat metabolism in dairy cows, here, the conservation of bta-miR-2285f among different species was analyzed by bioinformatics methods, and the upstream sequence CPG island analysis, promoter prediction, and further tissue expression profiling were performed to predict the target gene, and GO function and KEGG pathway enrichment analysis were performed. The downstream target gene CAB39(calcium binding protein 39) regulated by bta-miR-2285f was verified by dual luciferase reporter assay, and the functional role of bta-miR-2285f was explored at the level of breast epithelial cells. The results showed that: 1)bta-miR-2285f was a bovine specific intronic miRNA; 2)bta-miR-2285f and host gene TBK1 did not contain CPG islands at the 2 kb upstream sequence. Bta-miR-2285f was the transcription start site at 586 bp upstream. The host gene TBK1 is the transcriptional initiation site at 105 bp upstream; 3)TargetScan and miRWalk predicted that miR-2285f had 172 intersection target genes, which were mainly involved in metabolism, genetic information transmission, signal analysis and cell interaction. The mTOR signaling pathway, PI3K-AKT signaling pathway, ErbB signaling pathway, Hippo signaling pathway and triglyceride metabolism were significantly enriched, and the interaction network between bta-miR-2285f target genes was constructed in STRING database; 4)The expression of bta-miR-2285f was the highest in mammary gland tissues, which was significantly different from other tissues(P<0. 05); 5)Dual luciferase reporter assay confirmed the direct targeting relationship between bta-miR-2285f and CAB39 3'UTR seed sequence and bta-miR-2285f mimic significantly up-regulated the expression of milk fat marker genes ELOVL6, SLC27A6, PPARG and FASN, while bta-miR-2285f inhibitor had the opposite effect. In conclusion, Bta-Mir-2285f-1 may participate in milk fat metabolism by feedback regulating host gene TBK1, and bta-miR-2285f participates in milk fat metabolism by targeting gene CAB39. This study provides a basis for further exploring the potential mechanism of bta-miR-2285f in milk fat metabolism in Chinese Holstein dairy cows. |
| Key words: Chinese Holstein dairy miR-2285f bioinformatics miRNA-mRNA interaction milk fat |
