Chinese Bulletin of Botany >

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

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

TECHNIQUES AND METHODS

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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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

Cite this article

Chunyan Miao, Mingming Li, Xin Zuo, Ning Ding, Jiafang Du, Juan Li, Zhongyi Zhang, Fengqing Wang . Establishment of CRISPR/Cas9 Gene Editing System in Rehmannia henryi[J]. Chinese Bulletin of Botany, 2023 , 58(6) : 905 -916 . DOI: 10.11983/CBB22250

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