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植物学报 >

2023 , Vol. 58 >Issue 6: 905 - 916

DOI: https://doi.org/10.11983/CBB22250

技术方法

湖北地黄CRISPR/Cas9基因编辑体系的建立

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  • 1河南农业大学农学院, 郑州 450046
    2福建农林大学农学院, 福州 350002
† 共同第一作者

收稿日期: 2022-10-21

  录用日期: 2023-03-17

  网络出版日期: 2023-03-28

基金资助

国家自然科学基金(81473299);河南省高等学校重点科研项目(22A360009)

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Establishment of CRISPR/Cas9 Gene Editing System in Rehmannia henryi

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  • 1College of Agronomy, Henan Agricultural University, Zhengzhou 450046, China
    2College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, China
† These authors contributed equally to this paper

Received date: 2022-10-21

  Accepted date: 2023-03-17

  Online published: 2023-03-28

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摘要

湖北地黄(Rehmannia henryi)是一种具有重要价值的药用植物, 其基因编辑研究还未见报道。为建立湖北地黄基因编辑体系, 克隆了类胡萝卜素合成途径中八氢番茄红素脱氢酶(PDS)的编码基因, 并构建RhPDS1基因的CRISPR/Cas9载体, 以根癌农杆菌介导法转化湖北地黄基因组。结果表明, RhPDS1转录本含1 764 bp的开放阅读框(ORF), 其推测的氨基酸序列具有八氢番茄红素脱氢酶的典型结构域。RhPDS1基因在蕾、花和嫩叶中的相对表达量较高。利用基因编辑获得3个具有白化表型的再生株系, 白化苗分化率为3.7%。测序结果表明, 3个突变体属于2个基因编辑事件, 靶点序列分别为1 bp或/和5 bp碱基缺失, 造成移码突变。白化苗突变体的叶绿素和类胡萝卜素含量极显著低于野生型, RhPDS1基因的表达量也显著下降。综上, 该研究克隆了RhPDS1基因, 并利用CRISPR/Cas9技术实现了对RhPDS1基因的靶向敲除, 为湖北地黄的功能基因组学研究和野生驯化奠定了技术基础。

关键词： 湖北地黄; RhPDS1; CRISPR/Cas9; 基因敲除

本文引用格式

苗春妍, 李铭铭, 左鑫, 丁宁, 杜家方, 李娟, 张重义, 王丰青 . 湖北地黄CRISPR/Cas9基因编辑体系的建立[J]. 植物学报, 2023 , 58(6) : 905 -916 . DOI: 10.11983/CBB22250

Abstract

Rehmannia henryi is an important plant with great medicinal value, but no research of CRISPR/Cas9 has been done on this species. To establish the gene editing system of R. henryi, the gene encoding phytoene desaturase (PDS) in carotenoid biosynthesis was screened, and the CRISPR/Cas9 vector of RhPDS1 was constructed and transformed into R. henryi genome by Agrobacterium-mediated transformation method. The transcript of RhPDS1 with a 1 764 bp open reading frame (ORF) of RhPDS1 was obtained, the deduced amino acid sequence of RhPDS1 has the typical structural domains of phytoene desaturase. RhPDS1 showed higher expression levels in bud, flower and new leaf. Using CRISPR/Cas9 method, three regenerated shoots with albino phenotype were finally obtained, the differentiation rate of albino shoot was 3.7%. Sequencing analysis revealed that the three albino shoots belong to 2 editing events, in which deletion of 1 bp or (and) 5 bp occurred, respectively, which caused frame shift mutations. The contents of chlorophylls and carotenoids were significantly decreased in the albino mutants as compared to wild type, and the expression levels of RhPDS1 were also decreased in the albino mutants. Taken together, the RhPDS1 gene was cloned and knocked out by using CRISPR/Cas9 method, which laid down the foundations for functional genomics studies and de novo domestication of R. henryi.

Key words： Rehmannia henryi; RhPDS1; CRISPR/Cas9; gene knock out

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