CRISPR/Cas9-Mediated Editing of AGAMOUS-like Genes Results in a Late-Bolting Phenotype in Chinese Cabbage (Brassica rapa ssp. pekinensis)

Abstract

Due to the sudden change in temperature in spring, Chinese cabbage, a leafy vegetable cultivated for consumption, loses its commercial value due to the onset of bolting—the phenomenon of switching from vegetative to reproductive growth. In this study, we applied clustered regularly interspaced short palindromic repeats/(CRISPR)-associated system 9 (CRISPR/Cas9) technology to analyze AGAMOUS-like genes. We performed functional analysis of AGL19 and AGL24 genes related to bolting and flowering using CRISPR/Cas9-mediated Chinese cabbage transformation. Single-guide RNA (sgRNA) sequences were created with a low off-targeting probability to construct gene-editing vectors. Agrobacterium-mediated transformation was conducted, and tentative E0 AGL-edited lines were analyzed using molecular biotechnological methods. Two AGL19-edited lines with nucleotide sequence mutations in the target sequence of the AGL19 genes and four AGL24-edited lines with nucleotide sequence mutations in the target sequence of the AGL24 genes showed particularly late bolting compared to the inbred line ‘CT001.’ Generational progression using bud pollination obtained T-DNA-free E1 AGL-edited lines, which also showed late bolting. The loss of function of the AGL protein was caused by the occurrence of an indel mutation in the AGL19 and AGL24 genes, which results in an early stop codon. Furthermore, frameshift mutations led to structural changes and the introduction of an early stop codon in the AGL19 and AGL24 proteins. Our results indicate that CRISPR/Cas9-mediated editing of AGAMOUS-like genes results in a late-bolting phenotype and that CRISPR/Cas9 is a useful technology for analyzing gene function in Chinese cabbage (Brassica rapa ssp. pekinensis).

Keywords: AGL19 gene; AGL24 gene; Brassica rapa; CRISPR/Cas9; late bolting.

Figure 1

Gene structure analysis and construction of gene-editing vector. (A) Genomic structure of AGL and each homologous gene. Black box, exon regions; black line, intron regions; orange arrow, target site of single-guide RNAs (sgRNAs). (B) Schematic representation of the T-DNA in gene-editing vectors. LB, left border; T1, NOS terminator; Hyg^R, hygromycin resistance gene; P1, NOS promoter; P2, Arabidopsis U6 promoter; P3, 35S promoter; Cas9hc:NLS:HA, human-codon-optimized Cas9 with the nuclear localization signal and an HA epitope; T2, 35S terminator; RB, right border. Red box, sgRNA and sgRNA scaffold.

Figure 2

Selection of E₀ AGAMOUS-like (AGL)-edited lines by polymerase chain reaction (PCR) analysis. (A) PCR analysis with hyg^R and Cas9hc primer sets of E₀ AGL19-edited lines. (B) PCR analysis with hyg^R and Cas9hc primer sets of E₀ AGL24-edited lines. The 709 bp and 654 bp PCR amplicons are indicated with an arrow. P, positive control; M, 100 bp DNA ladder; N, negative control; numbering lane, tentative gene-edited lines.

Figure 3

Observation of bolting time and stem length of inbred line ‘CT001′ and E₀ AGL-edited lines. (A) Beginning stage of bolting. (B) Early stage of bolting. (C) Middle stage of bolting. Left, inbred line ‘CT001′; middle and right, E₀ AGAMOUS-like (AGL)-edited lines. (D) Stem length of inbred line ‘CT001′ and E₀ AGL-edited lines. Stem length gained from beginning to end of bolting in each line. The stem length was measured from the ground to the shoot apex.

Figure 4

Pod formation following bud pollination in ‘CT001′ inbred line and E₀ AGAMOUS-like (AGL)-edited lines. No difference in pod shape and seed formation was observed between inbred and E₀ AGL-edited lines.

Figure 5

Mutagenesis patterns induced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 in E₀ gene-edited lines. (A) Comparison of each nucleic and amino acid sequence of AGL19 genes (CT001_A03121400 and CT001_A08282630) in A1-2 and A1-9 lines. (B) Comparison of each nucleic and amino acid sequence of AGL24 genes (CT001_A03122450 and CT001_A01013460) in A2-1, A2-11, A2-16, and A2-22 lines. The underline indicates single-guide RNA (sgRNA), and the blue font indicates the protospacer adjacent motif (PAM) sequence; the red font represents the presence of sequence mutations and the resulting change in the amino acid sequence; red star, stop codon.

Figure 6

Analysis of T-DNA copy number and insertion position in E₀ AGAMOUS-like (AGL)-edited lines. (A) Southern hybridization analysis for identifying the copy number of T-DNA in the E₀ AGL-edited lines’ genome. Briefly, 30 μg of genomic DNA was digested with EcoRI, separated, and blotted onto Hybond N⁺ nylon membranes for hybridization with a [³²P]-labeled probe. The approximate DNA molecular size marker is indicated on the left. M, λ HindIII molecular ladder; N, negative control; lane, E₀ AGL-edited lines showing late bolting and sequence mutations. (B) A modified variable argument thermal asymmetric interlaced PCR (VA-TAIL PCR) analysis to confirm the flanking DNA sequence of T-DNA in the E₀ AGL-edited lines’ genome. Arbitrary degenerate (AD) primers and T-DNA border-specific primers were designed and amplified using 100 ng of genomic DNA as a template. M, 100 bp DNA ladder; lane, E₀ AGL-edited lines. (C) Schematic diagram of the T-DNA insertion site in the E₀ AGL-edited lines’ genome. T-DNA was inserted into the intergenic region of the genome of the E₀ AGL-edited lines.

Figure 7

Observation of the bolting and vegetative growth of the inbred line ‘CT001′ and the T-DNA-free E₁ AGL-edited lines. (A) Vegetative stage before bolting. (B) Beginning stage of bolting. (C) Early stage of bolting. (D) Middle stage of bolting. (E) End stage of bolting. Left, inbred line ‘CT001′; middle and right, T-DNA-free E₁ AGAMOUS-like (AGL)-edited lines.