Subtropical Plant Science ›› 2022, Vol. 51 ›› Issue (3): 163-171.DOI: 10.3969/j.issn.1009-7791.2022.03.001

• Plant physiology, biochemistry and molecular biology •     Next Articles

Establishment of a High Efficient Gene Editing System for Nicotiana attenuata

LIANG Yong-xin, WU Jin-song*   

  1. (Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201,Yunnan China)
  • Received:2022-04-25 Accepted:2022-05-30 Online:2022-06-30 Published:2022-10-17
  • Contact: WU Jin-song

渐狭叶烟草高效基因编辑体系的建立

梁永鑫,吴劲松*   

  1. (中国科学院昆明植物研究所云南省野生资源植物研发重点实验室,云南 昆明 650201)
  • 通讯作者: 吴劲松
  • 基金资助:
    国家自然科学基金项目(31670262)

Abstract: Nicotiana attenuata is a model plant species for study of plant-insect and plant-pathogen interactions in the genus of Nicotiana. In order to provide a more powerful and efficient means to study gene function in N. attenuata, we explored and finally established the highest gene editing efficiency system for N. attenuata by using phytoene desaturase gene (PDS) as a target gene, pHSE401 as the multi-target gene editing vector, and hygromycin as the selecting marker. Finally, we obtained a gene editing efficiency of approximately 80% for the PDS gene, far exceeding the approximately 30% gene editing efficiency currently reported in N. attenuata. The gene editing efficiency was also validated by targeting the WRKY70 gene. After sequencing, it was found that the editing frequency of WRKY70 was 83%, with 50% large fragment deletion. Taken together, we successfully established and verified a gene editing system with the highest editing efficiency reported ever in N. attenuata, thus providing a solid foundation for future gene function research in N. attenuata.

Key words: Nicotiana attenuata, gene editing, CRISPR/Cas9

摘要: 渐狭叶烟草(Nicotiana attenuata)是烟草属植物中研究植物与昆虫、植物与病原菌互作的模式植物。本研究以八氢番茄红素脱氢酶基因(PDS)为靶标基因,建立一套以pHSE401为基因编辑载体,以潮霉素为抗性筛选标记的渐狭叶烟草高效基因编辑体系。利用该体系,获得PDS基因约80%的基因编辑效率,远远超过目前在渐狭叶烟草中报道的约30%的基因编辑效率。进一步使用WRKY70基因为靶标,对该体系对进行编辑效率验证,经测序发现WRKY70基因编辑材料中的基因编辑效率为83%,其中发生大片段缺失突变的频率为50%。因此,本研究成功建立了渐狭叶烟草高效基因编辑体系,为以后渐狭叶烟草的基因功能研究奠定基础。

关键词: 渐狭叶烟草, 基因编辑, CRISPR/Cas9

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