生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (5): 28-.doi: 10.3969/j.issn.2095-1736.2021.05.028

• 研究报告 • 上一篇    下一篇

CRISPR/Cas9构建斑马鱼igf2bp3突变体及后代雌雄性别分析

  

  1. 1．中国科学技术部 海洋生物科学国际联合研究中心（上海海洋大学）， 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，研究方向为发育分子生物学，E-mail: lbchen@shou.edu.cn
  • 作者简介:徐伟伟，硕士研究生，研究方向为分子生物学，E-mail: xww0200@163.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）

CRISPR/Cas9 construction of zebrafish igf2bp3 mutant and sex analysis of offspring

  1. 1. International Research Center for Marine Biosciences, Ministry of Science and Technology, 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. National Demonstration Center
    for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai 201306, Chinav
  • Online:2021-10-18 Published:2021-10-20

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摘要： 胰岛素样生长因子2 mRNA结合蛋白3（IGF2BP3）通过结合目标mRNA编码区调节mRNA转录和翻译,在细胞极化、运动、形态发生、新陈代谢、增殖及分化等过程中发挥重要作用。由于在哺乳动物中缺乏可用的突变体模型，研究利用CRISPR/Cas9技术构建igf2bp3基因斑马鱼突变体，通过将两个gRNA与Cas 9蛋白显微注射至斑马鱼胚胎的单细胞期，PCR及Sanger测序验证，成功构建缺失1513 bp的igf2bp3基因敲除品系。荧光定量PCR结果表明，igf2bp3基因在野生型成年斑马鱼各个组织中均有表达，但在生殖腺中表达量最高。突变体纯合子斑马鱼性别比例分析显示，雄鱼数目明显多于雌鱼，雌雄鱼比例接近1∶10，表明igf2bp3基因可能在斑马鱼性腺发育中发挥作用。实验结果为斑马鱼性别决定基因研究奠定基础，同时也为渔业养殖产业中单性育种提供新的思路。

关键词: 斑马鱼, igf2bp3基因, CRISPR/Cas9, 性别决定

Abstract: Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) regulates mRNA transcription and translation by binding to the target mRNA coding region, and it plays important roles in the processes of cell polarization, movement, morphogenesis, metabolism, proliferation and differentiation. Due to the lack of available mutant models in mammals, this study used CRISPR/Cas9 technology to construct igf2bp3 gene zebrafish mutants. By microinjecting two gRNA and Cas9 proteins into the single cell stage of zebrafish embryos, PCR and Sanger sequencing confirmed that the igf2bp3 gene knockout strain with a deletion of 1 513 bp was successfully constructed. Fluorescence quantitative PCR results showed that the igf2bp3 gene was expressed in all tissues examined of wild-type adult zebrafish, but the highest expression tissue was ovary, the number of males in homozygous zebrafish and wild-type zebrafish was significantly more than that of females, and the proportion of males and females was close to 0.1, indicating that the igf2bp3 gene might play a role in zebrafish gonad development. The result would lay a foundation for studying the sex-determining genes of zebrafish, and provided new ideas for obtaining fast-growing male fish in the fish farming industry.

Key words: zebrafish; igf2bp3, CRISPR/Cas9, sex determination

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