生物学杂志

生物学杂志 ›› 2023, Vol. 40 ›› Issue (3): 74-.doi: 10.3969/j.issn.2095-1736.2023.03.074

• 研究报告 • 上一篇    下一篇

hdac8基因敲除对斑马鱼运动能力的影响

罗贝贝1,2， 罗军涛1,2， 韩丽洁1,2， 韩兵社1,2， 张俊芳1,2   

  1. 1. 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；
    2. 上海海洋大学水产科学国家级实验教学示范中心， 上海 201306
  • 出版日期:2023-06-18 发布日期:2023-06-19
  • 通讯作者: 张俊芳，教授，研究方向为表观遗传学与环境适应，E-mail:jfzhang@shou.edu.cn
  • 基金资助:
    上海市科技兴农重点攻关项目\[NO.沪农科创字(2019)第1-2号\]； 国家自然科学基金面上项目(No.81770165)； 上海市教委水产一流学科建设项目

Effects of hdac8 gene knockout on locomotion capacity of zebrafish

LUO Beibei1,2, LUO Juntao1,2, HAN Lijie1,2, HAN Bingshe1,2, ZHANG Junfang1,2   

  1. 1. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education,
    Shanghai Ocean University, Shanghai 201306, China; 2. National Demonstration Center for Experimental Fisheries
    Science Education, Shanghai Ocean University, Shanghai 201306, China
  • Online:2023-06-18 Published:2023-06-19
  • About author:罗贝贝，硕士研究生，研究方向为表观遗传学，E-mail:1726470802@qq.com

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摘要： 为研究鱼类hdac8的生物学功能，以斑马鱼（Danio rerio）作为模式生物，利用CRISPR/Cas9基因编辑技术，针对hdac8基因的第3个外显子设计Cas9靶位点，并制备gRNA。将gRNA与Cas9蛋白显微注射至一细胞期的斑马鱼胚胎中。经过T7E1核酸内切酶酶切、测序和序列比对后确定hdac8敲除成功，进一步获得F2代缺失41个碱基对的hdac8的纯合突变体斑马鱼。RT-qPCR结果显示：与WT斑马鱼相比，hdac8-/-斑马鱼的hdac8mRNA表达量显著下调（P<0.0001）；而hdac1的mRNA的表达量补偿性增加（P<0.05），hdac3的mRNA表达水平无显著性差异。Western Blot显示hdac8-/-斑马鱼全鱼和肌肉组织中H3K27ac、H3K9me3、H3K4me3和H3K4me1的表达水平都没有发生显著变化。斑马鱼行为轨迹跟踪系统检测发现，与WT斑马鱼幼鱼相比，hdac8-/-斑马鱼幼鱼的最大加速度无明显差异，但运动总距离、平均速度、活动性均显著性降低（P<0.0001），说明敲除hdac8基因会对斑马鱼幼鱼的运动能力造成影响。

关键词: 组蛋白去乙酰化酶8, 斑马鱼, CRISPR/Cas9, 运动能力

Abstract: To explore the biological function of thehdac8gene in fish, zebrafish (Danio rerio) was used as a model organism, using CRISPR/Cas9 gene editing technology, the Cas9 target site was designed for the third exon of thehdac8gene, and gRNA was prepared. The gRNA and Cas9 protein were co-microinjected into zebrafish embryos at one-cell stage. After T7E1 endonuclease digestion, sequencing and sequence alignment, it was confirmed that thehdac8gene was successfully knocked out, and F2homozygous mutant zebrafish with a deletion of 41 base pairs in thehdac8gene were obtained. RT-qPCR showed that compared with that of wild-type zebrafish, the expression ofhdac8mRNA was significantly reduced (P<0.0001), while the expression ofhdac1mRNA was compensatively increased (P<0.05), no significant change was detected withhdac3mRNA expression. Western Blot results showed no significant change of H3K27ac, H3K9me3, H3K4me3 and H3K4me1 levels inhdac8-/- zebrafish and muscle tissue. Behavioral tests of wild-type andhdac8-/- zebrafish larvae using the Danio Vision trajectory tracking system were performed, compared with that of wild-type zebrafish larvae, the maximum acceleration ofhdac8-/- zebrafish larvae showed no significant difference, but the total distance, mean velocity, and mobility were all significantly reduced (P<0.000 1), those indicated that knocking out thehdac8gene affected the locomotion capacity of zebrafish larvae.

Key words: HDAC8, zebrafish, CRISPR/Cas9, locomotion capacity

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