生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (5): 12-.doi: 10.3969/j.issn.2095-1736.2021.05.012

• 研究报告 • 上一篇    下一篇

CRISPR/Cas9技术敲除hbae1.1基因对斑马鱼血红蛋白生成的影响

  

  1. 1.海洋生物科学国际联合研究中心（上海海洋大学）中国科学技术部，上海201306；
    2.水产种质资源发掘与利用教育部重点实验室（上海海洋大学），上海201306；
    3.水产科学国家级实验教学示范中心（上海海洋大学），上海201306
  • 出版日期:2021-10-18 发布日期:2021-10-20
  • 通讯作者: 陈良标，教授，主要从事动物进化与环境基因组学研究，E-mail: lbchen@ shou.edu.cn
  • 作者简介:孟　琳，硕士研究生，研究方向为分子生物学，E-mail: 237988835@qq.com
  • 基金资助:
    国家重点研发计划项目(2018YFD0900600)；上海市科委“一带一路”国际联合实验室项目（19590750500）；上海市教委研创新计划项目（2017-01-07-00-10-E00060）

The effect of hbae1.1 gene on hemoglobin production in zebrafish knocked out by CRISPR/Cas9

  1. 1.National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University,
    Shanghai 201306, China; 2. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of
    Education, Shanghai Ocean University, Shanghai 201306, China; 3. International Research Center for Marine
    Bioscience, Ministry of Science and Technology, Shanghai Ocean University, Shanghai 201306, China
  • Online:2021-10-18 Published:2021-10-20

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摘要： 利用CRISPR/Cas9技术敲除hbae1.1基因，获得缺失hbae1.1基因的纯合突变体，并研究缺失hbae1.1基因对斑马鱼血红蛋白生成的影响。根据斑马鱼hbae1.1基因设计敲除靶点，并制备相应gRNA，将gRNA与Cas9蛋白以一定比例混合后通过显微注射注入斑马鱼受精卵一细胞期的动物极。通过T7E1核酸内切酶酶切、测序和序列比对后检测到成功敲除了hbae1.1基因，并通过与WT交配和内交，成功获得F2代纯合突变体。将hbae1.1基因缺失的纯合突变体与野生型斑马鱼WT所产受精卵一起进行固蓝（o-Diansidine）染色，结果显示，与对照组WT相比，突变体的血红蛋白含量明显减少。研究表明，hbae1.1基因的缺失会对斑马鱼血红蛋白的生成造成一定影响。同时hbae1.1基因的研究为了解鱼类血红蛋白的发生发育提供一定基础。

关键词: 斑马鱼, 血红蛋白, hbae1.1基因, CRISPR /Cas9, 固蓝染色

Abstract: This study used the CRISPR/Cas9 technology to knock out the hbae1.1 gene and obtained the homozygous mutants of hbae1.1 gene. The effect of hbae1.1 gene deletion on hemoglobin production in zebrafish was investigated. The knockout target was designed according to zebrafish hbae1.1 gene, and the corresponding gRNA was prepared, gRNA and Cas9 protein were mixed in a certain proportion and injected into zebrafish fertilized egg at a cellular stage by microinjection. After T7E1 endonuclease digestion, sequencing and sequence comparison, it was detected that the hbae1.1 gene was successfully knocked out. By mating with wild type and internal crossover, the F2 homozygous mutant was successfully obtained. Comparing the o-Dianisidine staining of embryos produced by the homozygous mutant with hbae1.1 gene deletion and wild type, the results showed that the hemoglobin content of the mutant was significantly reduced compared with the wild type. Studies have shown that the deletion of hbae1.1 gene would have a certain effect on the production of zebrafish hemoglobin. At the same time, the study of hbae1.1 gene provided a certain basis for understanding the occurrence and development of fish hemoglobin.

Key words: zebrafish, hemoglobin, hbae1.1 gene, CRISPR/ Cas9, o-dianisidine

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