Highly Efficient Gene Editing of Lignin Gene F5H in Switchgrass

doi:10.11983/CBB22240

Abstract

Abstract: Switchgrass (Panicum virgatum) is an important C₄ perennial lignocellulosic bioenergy and forage crop. In order to rapidly develop novel germplasm of switchgrass with high cell wall conversion rate, we cloned the ferulic acid 5-hydroxylase gene (PvF5H) from the heterotetraploid switchgrass cultivar Alamo. According to the PvF5H sequence, the editing target sgRNA was designed and used to construct CRISPR/Cas9-PvF5H editing vector. Finally, 59 positive transgenic switchgrass lines were generated by Agrobacterium tumefaciens mediated transformation. Sequencing analysis showed that the PvF5H was edited in most of the transgenic lines (94.9%), and the homozygous editing efficiency was 55.4%. Thus, we have successfully established a highly efficient gene editing system for switchgrass. It will facilitate manipulating target genes involved in cell wall quality and can be employed to breed novel switchgrass cultivars for production of biofuels and fodders in future.

Key words: bioenergy and forage grass, switchgrass, lignin, F5H, gene editing

Rui Qiu, Feng He, Rui Li, Yamei Wang, Sinian Xing, Yingping Cao, Yefei Liu, Xinyue Zhou, Yan Zhao, Chunxiang Fu. Highly Efficient Gene Editing of Lignin Gene F5H in Switchgrass[J]. Chinese Bulletin of Botany, 2023, 58(2): 298-307.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB22240

https://www.chinbullbotany.com/EN/Y2023/V58/I2/298

Figures/Tables 4

Figure 1 The basic molecular characterization of PvF5H in Panicum virgatum (A) Chromosomal position of F5H gene in P. virgatum (PvF5Ha: Pavir.9NG241700.1; PvF5Hb: Pavir.9KG138400.1); (B) F5H gene exons and introns distribution map of P. virgatum (blue boxes are exons, black thin lines are introns, thick black lines are 5' and 3' untranslated sequences); (C) Phylogenetic tree of F5H protein and gene conserved motif analysis in different species (P. virgatum: Pavir.9NG241700.1 (PvF5Ha), Pavir.9KG138400.1 (PvF5Hb); Oryza sativa: LOC_Os10g36848.1 (OsF5H); Zea mays: ZmPHB47.01G353500.1 (ZmF5H); Sorghum bicolor: Sobic.001G196300.1.p (SsF5H); Medicago truncatula: Medtr5g- 021390.1 (MtF5H); Arabidopsis thaliana: AT4G362201.1 (AtF5H). Different color of elements represent different nucleotide sequences in motif analysis)

Figure 2 Construction of PvF5H gene editing vector in Panicum virgatum (A) PvF5H1a and PvF5H1b gene sequence alignment (red box indicates target site); (B) Schematic diagram of F5H gene editing site in switchgrass (blue for exons, thin lines for introns); (C) Secondary structure prediction map of F5H gRNA target+gRNA skeleton in switchgrass; (D) F5H gene editing vector map (the expression of Cas9 was driven by maize Ubiquitin promoter and sgRNA by OsU3 promoter)

Figure 3 Generation and identification of CRISPR/Cas9_PvF5H transgenic switchgrass (A) Screening of switchgrass resistant calli grown on medium; (B) Calli turned green and produced shoots on differentiation medium after resistance screening; (C) Shoot enlongation; (D) Roots formation in rooting medium; (E) PCR analysis of transgenic switchgrass lines. No fragment band was amplified in the negative control (-, wild type switchgrass), while the same-sized band were amplified in all the resistant plants (lane 2 to 20) as in the positive control (+, CRISPR/Cas9_PvF5H plasmid). (A)-(D) Bars=1 cm

Figure 4 Gene editing methods and efficiency in switchgrass (A) Efficiency of F5H gene editing in switchgrass; (B) Gene editing patterns of PvF5H in switchgrass (I: Insertion; D: Delete; D&I: Delete+insertion)

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