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Generation of novel bpm6 and dmr6 mutants with broad-spectrum resistance using a modified CRISPR/Cas9 system in Brassica oleracea
Wednesday, 2025/02/12 | 08:16:07
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Yulun Zhang, Jinhui Liu, Yingjie Li, Hongxue Ma, Jialei Ji, Yong Wang, Mu Zhuang, Limei Yang, Zhiyuan Fang, Jun Li, Chao Zhang, Liwang Liu, Marina Lebedeva, Vasiliy Taranov, Yangyong Zhang, Honghao Lv Journal of Integrative Plant Biology; First published: 28 January 2025 https://doi.org/10.1111/jipb.13842
Brassica oleracea is an important biennial herbaceous species in the Cruciferae family. With an estimated 3.77 million hectares planted worldwide, these cole crops, for instance, cabbage, broccoli, and cauliflower, constitute significant agricultural resources (Li et al., 2024).
In recent years, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has been extensively applied in major crops, including rice, wheat, and potato. This technology can be utilized to regulate or disrupt gene expression to develop high-quality, disease-resistant, or stress-tolerant crops (Gao, 2021). However, the current low regeneration efficiency of B. oleracea has led to a limited Agrobacterium-mediated transformation efficiency of less than 1.0%, consequently impacting CRISPR/Cas9 based gene editing efficiency, with values as low as 12.9% being observed, compared to 68% in rice (Li et al., 2021; Zhou et al., 2022). This study aimed to enhance the application of CRISPR/Cas9 technology in B. oleracea.
We observed that Cas9 from Streptococcus canis (ScCas9) requires a protospacer adjacent motif (PAM) sequence of 5'-NNG-3', after which any base can be selected. However, the editing efficiency is predicted to be greater when the base following the PAM sequence is a “T” (http://crispor.tefor.net/). To test whether a “T” following the PAM sequence for Streptococcus pyogenes Cas9 (SpCas9) can enhance the editing efficiency of CRISPR/Cas9 technology, we developed two separate expression vectors tailored for rice and cabbage to knock out the Phytoene Desaturase genes (OsPDS and BoPDS), leading to an albino phenotype in the edited plants. The sequences with a “T” immediately following the PAM sequence are designated “NGGT”, and others are referred to as “NGGN” (Figure 1A, B). The high-throughput sequencing and analysis (Pinello et al., 2016) results indicated that in rice line Nipponbare, compared with “NGGN,” “NGGT” exhibited an average increase in editing types of 28.6%, and the overall increase in editing efficiency was 13.8% (Figure 1C; Table S1). The results in cabbage inbred line M1-1 indicated that the plants edited with “‘NGGT” exhibited a greater degree of editing, representing a significant improvement in the editing efficiency from 20.4% to 68.7% (Figure 1D; Table S2). These findings suggest that selecting a 5'-NGGT-3' PAM sequence can enhance the diversity of editing types and improve the efficiency of CRISPR/Cas9 technology. In summary, the application of the optimized CRISPR/Cas9 technology to knock out the BoBPM6 and BoDMR6 genes resulted in the creation of new germplasms with broad-spectrum disease resistance. Our findings provide robust support for gene editing and disease resistance breeding techniques in B. oleracea.
See https://onlinelibrary.wiley.com/doi/10.1111/jipb.13842
Figure 1: Strategies employed for the updating and application of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) editing system (A) Schematic diagram of the CRISPR/Cas9 construct for PDS editing in rice. NGGN (N1-N3) and NGGT (T1-T3) represent the positions of the target sites. (B) Schematic diagram of the CRISPR/Cas9 construct for PDS editing in cabbage. NGGN (N1–N2) and NGGT (T1–T2) represent the positions of the target sites. (C) The top image shows rice in which the OsPDS gene was knocked out using “NGGN” and “NGGT,” and the unedited green plants are indicated in the red circle. The bottom diagrams display changes in rice editing types (blue line graph) and editing efficiency (yellow bar graph). (D) The top image shows that BoPDS was knocked out using “NGGN” and “NGGT”, with the “MOCK” group representing unedited cabbage plants. The bottom diagrams display changes in editing types (red line graph) and editing efficiency (blue bar graph). (E) Regeneration efficiency using GROWTH-REGULATING FACTOR (GRF)5–GRF-INTERACTING FACTOR (GIF)1–GRF5, GRF4–GIF1–GRF4, GRF5, and GIF1, and the blank control. (F and G) Diagram of BoDMR6 and BoBPM6 gene structures. Light pink and blue blocks represent the gene coding region; dark pink and blue blocks indicate intronic regions; red lines illustrate the single guide RNA (sgRNA) target region (target 1). The sequences of the wild-type (WT) and mutants are outlined below. The target sequence is underlined. The protospacer adjacent motif (PAM) sequence is highlighted in red, insertions in yellow, and deletions in blue. (H and I) Phenotype and disease index of black rot and clubroot in WT and knockout (bodmr6) plants. (J–L) Phenotype and disease index of Fusarium wilt, black rot, and clubroot in WT and knockout (bobpm6) plants. The cabbage sister lines M1-1 and M1-2 with slightly different genetic background were used for bodmr6 and bobpm6 knockout, respectively. For the disease inoculation tests, three knockout lines are selected for both bobpm6 and bodmr6, with 30 seedlings per line (10 seedlings for each of the three replicates). The whole inoculation experiment was repeated three times with similar results. **P < 0.01; ***P < 0.001; ****P < 0.0001 indicate significant differences determined by two-tailed Student's t-test. Scale bars, 10 mm.
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