浙江农业学报 ›› 2017, Vol. 29 ›› Issue (5): 685-693.DOI: 10.3969/j.issn.1004-1524.2017.05.01

• 作物科学 •    下一篇

利用CRISPR/Cas9技术靶向编辑水稻基因

原文霞1, 2, 王栩鸣2, 李冬月2, 3, 周洁2, 严成其1, 2, *, 陈剑平1, 2, *   

  1. 1.浙江师范大学 化学与生命科学学院,浙江 金华321004;
    2.浙江省农业科学院 病毒学与生物技术研究所,浙江省有害生物防控国家重点实验室培育基地,农业部植保生物技术重点实验室,浙江省植物病毒重点实验室,浙江 杭州310021;
    3.云南农业大学 植物保护学院,云南 昆明 650201
  • 收稿日期:2017-03-09 出版日期:2017-05-20 发布日期:2017-06-06
  • 通讯作者: 严成其,E-mail: yanchengqi@163.com;陈剑平,E-mail: jpchen2001@126.com
  • 作者简介:原文霞(1990-),女,山西汾阳人,硕士研究生,主要从事植物病理学研究。E-mail: yuanwenxia5@163.com
  • 基金资助:
    国家重点研发计划(2016YFD0100601,2016YFD0200804); 浙江省自然基金(2014C140001,2016C110017)

Application of the technology of CRISPR/Cas9 edit rice gene

YUAN Wenxia1, 2, WANG Xuming2, LI Dongyue2, 3, ZHOU Jie2, YAN Chengqi1, 2, *, CHEN Jianping1, 2, *   

  1. 1.College of Chemistry and Life Sciences, Zhejiang Normal University, Jinhua 321004, China;
    2.State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease Control, MOA Key Laboratory for Plant Protection and Biotechnology, Zhejiang Provincial Key Laboratory of Virology, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China;
    3.College of Plant Protection, Yunnan Agricultural University, Kunming 650201, China
  • Received:2017-03-09 Online:2017-05-20 Published:2017-06-06

摘要: 以现有水稻CRISPR/Cas9载体系统为起点,优化改造了相应的载体及其构建方法,实现了两步法构建基因敲除载体。利用新的载体和方法,针对水稻日本晴(Oryza sativa L.spp. Japonica cv. Nipponbare)D3基因的3个靶向位点分别构建了相应的CRISPR/Cas9载体,并通过农杆菌介导的遗传转化成功获得一系列转基因植株。T0代转基因植株靶位点测序结果显示,靶位点DDRC1包括5种突变类型,植株的突变率为35.48%;靶位点DDRC2包括5种突变类型,植株的突变率为25.00%;靶位点DDRC3包括1种突变类型,植株的突变率为20.00%。利用T1代纯合突变植株,进一步研究了所得纯合突变体的株高、分蘖数表型与CRISPR/Cas9编辑后基因型之间的关系,探讨了CRISPR/Cas9系统的基因编辑效率、位置效应和突变类型等特点,为进一步利用CRISPR/Cas9系统发展具有不同农艺性状的水稻种质材料提供了参考。

关键词: 水稻, CRISPR/Cas9, D3基因, 基因编辑

Abstract: In this study, the CRISPR/Cas9 vector system was optimized in order to construct gene editing constructors in two steps. With optimized CRISPR/Cas9 system, three genome editing constructors targeting different sites of D3 gene in rice (Oryza sativa L. spp. Japonica cv. Nipponbare) were generated, and the transgenic plants were successfully produced by Agrobacterium-mediated transformation. The sequencing results of T0 transgenic plants showed that the mutation rate of target site DDRC1 was 35.48%, and five mutation types were detected; the mutation rate of target site DDRC2 was 25.00%, and also with five mutation types; the mutation rate of target site DDRC3 was 20.00%, and only one mutation type was detected. The phenotype, height and tiller number, together with the genotypes of homozygous mutants were investigated, and the gene editing efficiency of the optimized CRISPR/Cas9 system along with the position effect and mutation patterns were also analyzed and discussed. In summary, the findings in this research provided important information for further developing and utilizing of rice germplasm on variety agronomic trait with the optimized CRISPR/Cas9 system.

Key words: rice, CRISPR/Cas9, D3 gene, gene editing

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