利用CRISPR/Cas9技术研究保守非编码元件对斑马鱼血红蛋白生成的影响  

Impact of conserved non-coding element on zebrafish hemoglobin production using CRISPR/Cas9 technology

作　　者：曹瑞萌 徐逸程 产久林 许强华[1,2,3] 吴智超 胡鹏 CAO Ruimeng;XU Yicheng;CHAN Jiulin;XU Qianghua;WU Zhichao;HU Peng(College of Marine Living Resource Sciences and Management,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Sustainable Exploitation of Oceanic Fisheries Resources,Ministry of Education,Shanghai 201306,China;National Engineering Research Center for Oceanic Fisheries,Shanghai 201306,China;College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources,Ministry of Education,Shanghai Ocean University,Shanghai 201306,China)

机构地区：[1]上海海洋大学海洋生物资源与管理学院,上海201306 [2]大洋渔业资源可持续开发教育部重点实验室,上海201306 [3]国家远洋渔业工程技术研究中心,上海201306 [4]上海海洋大学水产与生命学院,上海201306 [5]上海海洋大学水产种质资源发掘与利用教育部重点实验室,上海201306

出　　处：《上海海洋大学学报》2025年第1期1-11,共11页Journal of Shanghai Ocean University

基　　金：国家自然科学基金青年科学基金(32200414)。

摘　　要：血红蛋白是高等生物血液循环系统中的关键组分,其功能障碍与贫血、心血管疾病等密切相关。南极冰鱼科(Channichthyidae)鱼类血液因缺乏功能性血红蛋白呈现白色,为研究血红蛋白的生物学功能提供了独特的模型。研究发现位于foxp1b基因上游的intergenic区域chr6:43629743-43629779可能作为关键因子参与鱼类血红蛋白生成,但其具体的调控功能尚不清楚。本研究通过CRISPR/Cas9基因编辑技术,以斑马鱼为模型构建了CNE敲除杂合突变体,并通过血红蛋白染色比较了野生型对照组与突变体斑马鱼的血红蛋白生成差异。结果表明,chr6:43629743-43629779的敲除显著降低了斑马鱼血红蛋白的生成。qRT-PCR分析结果表明,突变体斑马鱼foxp1b基因表达量显著低于野生型对照组。揭示了CNE通过调控下游基因表达从而调控血红蛋白生成的分子机制,提示CNE在血红蛋白生成中发挥了关键作用。Hemoglobin is a vital component of the circulatory system in higher organisms,and its dysfunction is closely associated with conditions such as anemia and cardiovascular diseases.Antarctic icefish,whose blood appears white due to the absence of functional hemoglobin,serve as a unique model for studying the biological functions of hemoglobin.We identified a conserved non-coding element(CNE)located in the intergenic region chr6:43629743-43629779,upstream of the foxp1b gene,which may play a key role in hemoglobin production in fish.However,its specific regulatory function remains unclear.In this study,we utilized CRISPR/Cas9 gene-editing technology to generate zebrafish heterozygous mutants with knockouts of this CNE.Hemoglobin staining was subsequently performed to compare hemoglobin production between the wild-type control group and the mutant zebrafish.The results demonstrated that the knockout of chr6:43629743-43629779 significantly reduced hemoglobin production in zebrafish.Additionally,qRT-PCR(Quantitative Reverse Transcription Polymerase Chain Reaction)analysis revealed that foxplb gene expression was significantly lower in the mutant zebrafish compared to the wild-type controls.This study indicates the molecular mechanism by which CNEs regulate hemoglobin production by modulating the expression of downstream genes.It highlights the critical role of conserved non-coding elements in hemoglobin synthesis and offers new insights into the regulation of this essential protein.

关 键 词：南极冰鱼 斑马鱼 血红蛋白 保守非编码元件 CRISPR/Cas9 

分 类 号：S917[农业科学—水产科学]