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植物研究 ›› 2019, Vol. 39 ›› Issue (6): 869-875.doi: 10.7525/j.issn.1673-5102.2019.06.009

• 研究报告 • 上一篇    下一篇

一种拟南芥基因高效编辑体系研究

欧阳乐军, 马铭赛, 陈梓洛, 黄嘉玲, 布梁灏, 陈自银, 李莉梅   

  1. 广东石油化工学院生物工程学院, 茂名 525000
  • 收稿日期:2018-04-28 出版日期:2019-11-05 发布日期:2019-11-16
  • 通讯作者: 李莉梅 E-mail:lilimeinh@163.com
  • 作者简介:欧阳乐军(1977-),男,博士,副教授,主要从事植物基因工程研究。
  • 基金资助:
    国家自然科学基金项目(31470677);广东省自然科学基金项目(2017A030307017);广东科技计划项目(2017A030303087);广东省教育厅重点项目(2018KZDXM047);广东省“扬帆计划”高层次人才项目

An Efficient Gene Editing Research System of Arabidopsis

OUYANG Le-Jun, MA Ming-Sai, CHEN Zi-Luo, HUANG Jia-Ling, BU Liang-Hao, CHEN Zi-Yin, LI Li-Mei   

  1. College of Biological and Food Engineering, Guangdong University of Petrochemical Technology, Maoming 5250000
  • Received:2018-04-28 Online:2019-11-05 Published:2019-11-16
  • Supported by:
    Supported by the National Natural Science Foundation of China(31470677); Natural Science Foundation of Guangdong(2017A030307017); Science and Technology Plan Project Foundation of Guangdong(2017A030303087); Provincial Department of Education Key Foundation of Guangdong(2018KZDXM047); "Sailing Plan" High-level Talent Foundation of Guangdong

摘要: CRISPR/Cas9基因编辑系统操作简单易行,无需引入外源基因,生物安全性高。但怎样快速筛选获得不含外源转化元件的基因编辑后代是一个关键技术问题。本研究创造性的将拟南芥种皮特异性启动子At2S3与荧光筛选标记基因mCherry组装进植物基因组定点编辑CRISPR载体pHDE中,以拟南芥as1为靶基因,构建一种通过荧光标记筛选、实现转化后代中Cas9 Free的基因高效编辑体系。结果表明,通过同源重组方法构建的带有筛选标记的CRISPR载体与设计相符,外源插入片段正确。挑选转化后种皮上带有红色荧光标记的阳性种子培育得到T1代植株,经PCR验证,成功获得as1定点敲除的纯合突变植株,纯合子比率达到40%;挑选T1代纯合突变上不带荧光的种子,培育得到的T2代植株中,PCR检测不到Cas9片段,实现了编辑后代的Cas9 Free。本研究构建的一种带有可视化筛选标记的基因高效编辑体系,成功实现编辑后代中无外源插入的Cas9等转化元件,生物安全性高,为基因组定点编辑技术在植物遗传资源改良中的高效利用提供了借鉴与参考。

关键词: CRISPR/Cas9, 基因编辑, 荧光筛选标记, as1

Abstract: The CRISPR/Cas9 gene-editing system is easy to handle and has a high gene editing efficiency. However, how to quickly screen and obtain gene editing descendants without exogenous transformation elements is a key technical problem. In this study, two pairs of single-guide RNAs were designed to simultaneously recognize two different sites in the one target gene encoding as1 in Arabidopsis. The CRISPR construct also carried a gene for a fluorescent selection marker. By fluorescence screening, a transgene-free progeny was easily obtained in the T2 population. T1 seeds with red fluorescence in their coat were selected to verify that the proportion of as1 knockout mutants traits were obvious. Seeds with no fluorescence were selected among the T1 generation and screened for homozygous mutations. In the T2 generation plants, the Cas9 fragment was not detected by polymerase chain reaction. Thus, a highly efficient DNA-editing construct, with a gene for a fluorescence screening marker, was successfully constructed, and the transgenic element was successfully eliminated from the progeny by selecting or avoiding seeds with the fluorescent marker. This system can be used as a plant genome site-specific editing tool and may be useful for improving plant genetic resources.

Key words: CRISPR/Cas9, gene editing, fluorescence screening, as1

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