文章摘要
施缘萍,朱文娇,张昌润,彭雅洁,乔 洁.smc5基因敲除斑马鱼肝脏转录组学分析[J].,2022,(23):4401-4406
smc5基因敲除斑马鱼肝脏转录组学分析
The Hepatic Transcriptome Analysis of smc5 Knockout Zebrafish Model
投稿时间:2022-04-23  修订日期:2022-05-18
DOI:10.13241/j.cnki.pmb.2022.23.001
中文关键词: CRISPR/Cas9  斑马鱼  smc5  代谢  转录组测序
英文关键词: CRISPR/Cas9  Zebrafish  smc5  Metabolism  RNA-seq
基金项目:国家自然科学基金项目(81873652);上海市自然科学基金项目(21ZR1438300)
作者单位E-mail
施缘萍 上海交通大学医学院附属第九人民医院内分泌科 上海 200011 sh1yuanping@126.com 
朱文娇 上海交通大学医学院附属第九人民医院内分泌科 上海 200011  
张昌润 上海交通大学医学院附属第九人民医院内分泌科 上海 200011  
彭雅洁 上海交通大学医学院附属第九人民医院内分泌科 上海 200011  
乔 洁 上海交通大学医学院附属第九人民医院内分泌科 上海 200011  
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中文摘要:
      摘要 目的:探讨smc5基因敲除对斑马鱼肝脏基因表达谱的影响,进一步明确smc5突变对斑马鱼代谢的影响。方法:用CRISPR/Cas9技术构建smc5基因敲除斑马鱼模型,取3个月的smc5-/-和野生型斑马鱼肝脏进行转录组测序,创建基因表达谱文库,观察smc5基因敲除后斑马鱼肝脏基因表达谱的变化,将筛选出的差异表达基因进行功能富集,并运用荧光定量PCR对KEGG通路中显著的差异表达基因进行验证。结果:成功构建出7号外显子上2碱基缺失造成移码突变的smc5基因敲除斑马鱼模型。RNA-seq发现smc5-/-斑马鱼的肝脏基因表达谱变化显著,包含p53的多个通路激活,如细胞周期和凋亡。糖酵解、脂肪酸降解与代谢、丙酮酸代谢等相关通路显著下调。荧光定量PCR结果与RNA-seq结果一致。结论:smc5基因敲除下调斑马鱼肝脏糖脂代谢。本研究结果为进一步研究SMC5基因在糖脂代谢调控中的潜在机制奠定基础。
英文摘要:
      ABSTRACT Objective: To investigate the hepatic transcriptomic changes and the effects on metabolism of smc5 gene knockout zebrafish. Methods: The smc5 knockout zebrafish model was constructed by CRISPR/Cas9 technology and total RNA of the liver from wildtype and smc5-/- zebrafish was extracted for RNA sequencing, and gene expression profile of smc5-/- zebrafish was established. To better understand the differential expressed genes (DEGs) in the smc5-/-, DEGs were subjected to the Kyoto encyclopedia of genes (KEGG). qRT-PCR analysis was used to validated a set of genes. Results: The smc5 knockout lines of zebrafish carrying 2 base deletion were successfully constructed, which is expected to cause a frame shift. Further bioinformatic analysis revealed that p53 related pathways are significantly enriched among the genes upregulated. The downregulated DEGs were mainly enriched in glycolysis/gluconeogenesis, fatty acid degradation and metabolism, pyruvate metabolism. qRT-PCR results also validated that a set of genes of metabolism was downregulated. Conclusion: smc5 deficiency affects hepatic metabolism in zebrafish. The results of this study lay a foundation for further research on the potential mechanism of SMC5 gene in the regulation of glucose and lipid metabolism.
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