Function Analysis of the PR55/ B Gene Related to Self-Incompatibility in Chinese Cabbage Using CRISPR/Cas9

Abstract

Chinese cabbage, a major crop in Korea, shows self-incompatibility (SI). SI is controlled by the type 2A serine/threonine protein phosphatases (PP2As). The PP2A gene is controlled by regulatory subunits that comprise a 36 kDa catalyst C subunit, a 65 kDa regulatory A subunit, and a variety of regulatory B subunits (50-70 kDa). Among them, the PP2A 55 kDa B regulatory subunit (PR55/B) gene located in the A05 chromosome has 13 exons spanning 2.9 kb, and two homologous genes, Bra018924 and Bra014296, were found to be present on the A06 and A08 chromosome, respectively. In this study, we performed a functional analysis of the PR55/B gene using clustered regularly interspaced short palindromic repeats/CRISPR-associated system 9 (CRISPR/Cas9)-mediated gene mutagenesis. CRISPR/Cas9 technology can be used to easily introduce mutations in the target gene. Tentative gene-edited lines were generated by the Agrobacterium-mediated transfer and were selected by PCR and Southern hybridization analysis. Furthermore, pods were confirmed to be formed in flower pollination (FP) as well as bud pollination (BP) in some gene-edited lines. Seed fertility of gene-edited lines indicated that the PR55/B gene plays a key role in SI. Finally, self-compatible T-DNA-free T₂ gene-edited plants and edited sequences of target genes were secured. The self-compatible Chinese cabbage developed in this study is expected to contribute to Chinese cabbage breeding.

Keywords: Brassica rapa; CRISPR/Cas9; PR55/B gene; self-incompatibility.

Figure 1

Vector structure and position of sgRNAs. (A) Detailed schematic representation of T-DNA. LB, left border; Hyg^R, hygromycin resistance gene; Cas9hc:NLS:HA, human-codon-optimized Cas9 with the nuclear localization signal and an HA epitope; T1, NOS terminator; T2, 35S terminator. RB, right border. Hyg^R, sgRNA, and Cas9hc:NLS:HA are under the control of NOS promoter (P1), Arabidopsis U6 promoter (P2), and 35S promoter (P3), respectively. (B) Summary of the genomic structure of the PR55/B gene and two homologous genes. Black box, exon regions; black line, intron regions; yellow bar, target site of sgRNA1; blue bar, target site of sgRNA3.

Figure 2

Selection of T₀ gene-edited lines by PCR analysis. (A) PCR analysis with hyg^R and Cas9hc primer sets of T₀ PT1 gene-edited lines. (B) PCR analysis with hyg^R and Cas9hc primer sets of T₀ PT3 gene-edited lines. The 709 bp and 654 bp expected PCR products are indicated with an arrow, respectively. P, positive control; M, 100 bp DNA ladder; N, negative control; Numbering lane, tentative gene-edited lines.

Figure 3

Self-compatibility analysis by FP and BP of inbred line ‘CT001′ and T₀ gene-edited Chinese cabbage lines. (A) Pods of inbred line ‘CT001′ using FP and BP. (B) Pods of T₀ gene-edited lines (PT1-2, -4, -8, and PT3-3) using FP and BP. (C) Seed formations of inbred line ‘CT001′ using FP and BP. (D) Seed formations of T₀ gene-edited lines (PT1-2, -4, -8, and PT3-3) using FP and BP.

Figure 4

Analysis of mutation patterns in T₁ gene-edited lines. (A) Confirmation of each nucleic and amino acid sequence of the PR55/B gene in PT1-2-2, -4-1, and -8-3. (B) Confirmation of each nucleic and amino acid sequence of the PR55/B gene, Bra018924, and Bra014296 in PT3-3-3, respectively. The underline indicates sgRNA, and the blue font indicates the PAM sequence; the red font represents the presence of indel mutations and the resulting change of amino acid sequence.

Figure 5

Southern hybridization analysis for identifying the copy number of T-DNA in the T₁ gene-edited lines genome. A total of 30 μg genomic DNA was digested with EcoRI, then separated on a 1.0% agarose gel and blotted onto a Hybond N⁺ nylon membrane for hybridization with a probed [³²P]-labeled 709 bp of hyg^R from a gene-editing vector. An approximate DNA molecular size marker is indicated on the left. M, λ HindIII molecular ladder; N, negative control; Lane, T₁ gene-edited lines showing self-compatible phenotype and sequence mutations.

Figure 6

Self-compatibility analysis by FP and BP of T₁ gene-edited Chinese cabbage lines. (A) Pods of inbred line ‘CT001′ using FP and BP. (B) Pods of T₁ gene-edited lines (PT1-2-2, -4-1, -8-3, and PT3-3-3) using FP and BP. (C) Seed formations of inbred line ‘CT001′ using FP and BP. (D) Seed formations of T₁ gene-edited lines (PT1-2-2, -4-1, -8-3, and PT3-3-3) using FP and BP.

Figure 7

Analysis of mutation patterns in T₂ gene-edited lines. (A) Confirmation of each nucleic and amino acid sequence of PR55/B gene, Bra018924 and Bra014296 in PT1-2-2-8, -4-1-4, and -8-3-15, respectively. (B) Confirmation of each nucleic and amino acid sequence of PR55/B gene, Bra018924, and Bra014296 in PT3-3-3-5. The underline indicates sgRNA, and the blue font indicates the PAM sequence; the red font represents the presence of indel mutations and the resulting change of amino acid sequence.