遗传 ›› 2018, Vol. 40 ›› Issue (6): 478-487.doi: 10.16288/j.yczz.18-055

• 研究报告 • 上一篇    下一篇

果蝇睾丸基因敲除突变体的构建及表型分析

唐浚博1,曹浩伟1,许蕊1,张丹丹1,黄娟1,2()   

  1. 1. 南京医科大学基础医学院,南京 211166
    2. 南京医科大学生殖医学国家重点实验室,南京 211166
  • 收稿日期:2018-03-02 修回日期:2018-04-27 出版日期:2018-06-20 发布日期:2018-05-11
  • 通讯作者: 黄娟 E-mail:huangjuan@njmu.edu.cn
  • 作者简介:唐浚博,硕士研究生,专业方向：雄性生殖。E-mail: twg0312@hotmail.com
  • 基金资助:
    南京医科大学引进人才启动经费(2012RC04);生殖医学国家重点实验室开放课题资助(2012RC04)

Mutant generation of the testis genes and phenotype analyses in Drosophila

Junbo Tang1,Haowei Cao1,Rui Xu1,Dandan Zhang1,Juan Huang1,2()   

  1. 1. School of Basic Medical Sciences, Nanjing Medical University, Nanjing 211166, China
    2. State key laboratory of reproductive medicine,Nanjing Medical University, Nanjing 211166, China
  • Received:2018-03-02 Revised:2018-04-27 Online:2018-06-20 Published:2018-05-11
  • Contact: Huang Juan E-mail:huangjuan@njmu.edu.cn
  • Supported by:
    Supported by the Start-up Foundation from Nanjing Medical University(2012RC04);[Funding of State Key Laboratory of Reproductive Medicine(2012RC04)

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摘要：

生殖系统功能的正常维持是物种繁衍的基础,需要多基因协同作用,但其中许多基因的具体功能和作用机制并不清楚。本研究选取了果蝇(Drosophila melanogaster)中8个在睾丸中表达、功能未知且与人(Homo sapiens)和小鼠(Mus musculus)高度同源的基因(CG4161、CG11475、CG2921、CG10541、CG7276、CG3800、CG8117和CG16779),分析了它们在不同组织中的表达水平,并分别检测了它们在雄性生殖系统中的功能。在这8个基因中,前5个为睾丸优势表达基因,其余3个为全身性表达。首先,利用CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9)技术结合同源定向修复(homology-directed repair, HDR)在果蝇中对8个候选基因逐一进行敲除,建立了纯合的基因敲除突变品系;然后对这些品系的雄蝇进行了生育力测试及睾丸细胞学观察。结果显示,CG7276和CG3800基因敲除果蝇出现部分雄蝇不育且可育雄蝇后代数量较野生型显著下降。睾丸解剖观察显示CG7276和CG3800的功能缺失可导致雄蝇分别出现不同程度的精囊缩小及精原干细胞减少和细胞分布混乱;染色结果也提示CG7276和CG3800在精子的成熟过程中发挥一定作用。其他6个基因突变并未导致雄蝇育性变化或睾丸形态异常。这些突变体的获得及表型的初步分析为进一步研究基因功能及机制提供了良好的动物模型及基础。

关键词: 睾丸, CRISPR/Cas9, 果蝇, 基因敲除

Abstract:

Multiple genes work together to maintain the normal functions of the reproductive system. However, for many of these genes, little is known about their specific functions and mechanisms. In the present study, eight Drosophila genes, including CG4161, CG11475, CG2921, CG10541, CG7276, CG3800, CG8117 and CG16779, were selected for detailed studies based on their testis expression, undefined functions, and having highly homologous and conserved genes in humans (Homo sapiens) and mouse (Mus musculus). We analyzed their expression levels in different tissues, and determined their probably functions in male reproduction. The results showed that the first five genes were mainly expressed in testis, while other three showed ubiquitous expression in all tissues examined. Using the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) and homology-directed repair (HDR) strategies, we have systematically generated the mutants of these genes and studied their functions in male reproduction. Loss of function of CG7276 and CG3800 resulted in partial sterility and reduction of the offspring number, while other genes showed no significant impact on fertility. CG7276 -/- and CG3800 -/- mutants were partial sterile and showed various types of abnormities, including seminal vesicle atrophy, spermatogonial stem cell reduction and cellular distribution disorders. Results of DNA and F-actin staining also indicated that CG7276 and CG3800 could play important roles in spermiogenesis. The establishments of these mutants have provided means to unravel the functions and mechanisms of these genes in animal models.

Key words: testis, CRISPR/Cas9, drosophila, gene knockout