生物学杂志

生物学杂志 ›› 2021, Vol. 38 ›› Issue (3): 63-.doi: 10.3969/j.issn.2095-1736.2021.03.063

• 研究报告 • 上一篇    下一篇

青鳉脑型芳香化酶cyp19b基因的敲除和鉴定

  

  1. 1. 上海海洋大学 农业部淡水水产种质资源重点实验室, 上海 201306;
    2. 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;
    3. 上海海洋大学 水产科学国家实验教学示范中心, 上海 201306
  • 出版日期:2021-06-18 发布日期:2021-06-21
  • 通讯作者: 关桂君，教授，博士生导师，研究方向为鱼类性别调控和性别分化，E-mail:gjguan@shou.edu.cn
  • 作者简介:常宇阳，硕士研究生，研究方向为鱼类性别调控和性别分化，E-mail:cyy2013f@126.com
  • 基金资助:
    国家重点研发计划项目(2018YFD0900601)；国家自然科学基金项目(81771545)

Knockout and identification of brain type aromatase gene cyp19b in medaka

  1. 1. Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, 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, China
  • Online:2021-06-18 Published:2021-06-21

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摘要： 芳香化酶是调节雄激素向雌激素合成转换的关键酶。脑型芳香化酶(cyp19b)的表达在成鱼青鳉的脑中呈现显著雌雄性差异，在性腺中微弱表达且无明显性别差异。为探讨cyp19b基因的生理功能,采用CRISPR/Cas9系统靶向敲除cyp19b基因。 在青鳉cyp19b基因的第2和第4外显子上分别设计一个靶位点，通过显微注射gRNA和Cas9蛋白到一细胞期的受精卵进行cyp19b双靶点基因敲除。研究结果表明：成功获得两种可遗传的cyp19b突变等位基因型种鱼，并通过杂合子交配、基因组PCR及DNA测序分析，选育得到两种cyp19b敲除突变型纯合子品系。表型观察分析显示,两种突变型纯合子品系均无明显雌雄性偏差,胚胎均可以正常发育,成鱼均可以正常生殖，提示cyp19b基因的缺失可能不直接影响性腺的雌雄性分化。cyp19b基因如何影响性腺分化和发育，有待进一步深入探讨。

关键词: 青鳉, cyp19b基因, 基因敲除, CRISPR/Cas9

Abstract: Aromatase is the key enzyme for the synthesis and conversion of estrogens from androgens.Brain type aromatase(cyp19b) is predominantly expressed and displays a sexual dimorphism in adult brain, in contrast to a slight expression with no sexual dimorphism in adult gonad of medaka (Oryzias latipes). In order to explore the bio-function ofcyp19bgene in brain and gonad development, we used CRISPR/Cas9 system to targeted knockoutcyp19b. Two targeting sites were designed at exon 2 and exon 4, respectively.Microinjection of aliquots containing gRNAs and Cas9 protein was carried out into 1-cell fertilized eggs. Results showed that two heritablecyp19bmutant alleles were successfully obtained, and twocyp19bknockout mutant homozygous lines were selected through heterozygous mating, genomic PCR and DNA sequencing analysis. There was no significant male-or female-bias in both lines ofcyp19bknockout homozygotes. In both lines ofcyp19bknockout homozygotes, the embryo could develop normally and the adult could reproduce normally, suggesting that the loss ofcyp19bgene might not directly affect the sexual differentiation of gonads.

Key words: medaka;cyp19b, gene knockout, CRISPR/Cas9

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