CRISPR/Cas9-Mediated Editing of AGAMOUS-like Genes Results in a Late-Bolting Phenotype in Chinese Cabbage (Brassica rapa ssp. pekinensis)
Abstract
:1. Introduction
2. Results
2.1. Vector Construction and Agrobacterium-Mediated Transformation of Chinese Cabbage
2.2. Selection and Bolting Time Analysis of E0 AGL-Edited Chinese Cabbage Lines
2.3. Mutagenesis of AGL Induced by CRISPR/Cas9 in E0 AGL-Edited Lines
2.4. T-DNA Copy Number and Flanking Site Sequence Analysis of Late-Bolting E0 AGL-Edited Lines
2.5. Mutagenesis Inheritance Analysis in T-DNA-Free E1 AGL-Edited Lines
3. Discussion
4. Materials and Methods
4.1. sgRNA Design and Construction of Gene-Editing Vectors
4.2. Development of AGL-Edited Chinese Cabbage Lines and Selection Using PCR Analysis
4.3. Bolting Time Record and Stem Length Measurement of Selected AGL-Edited Lines
4.4. Identifying the Number of T-DNA Copies and Insertion Site
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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E0 Gene-Edited Line | E1 Gene-Edited Line | Chr. z | Target Gene | Mutation Type y |
---|---|---|---|---|
A1-2 | A1-2-5 | A03 | CT001_A03121400 | – |
A08 | CT001_A08282630 | – | ||
A1-9 | A1-9-2 | A03 | CT001_A03121400 | 1 bp In (T) |
A08 | CT001_A08282630 | – | ||
A2-1 | A2-1-2 | A03 | CT001_A03122450 | 60 bp Del |
A01 | CT001_A01013460 | 1 bp Del (T) | ||
A2-11 | A2-11-1 | A03 | CT001_A03122450 | 1 bp In (A) |
A01 | CT001_A01013460 | 1 bp In (C) | ||
A2-11-6 | A03 | CT001_A03122450 | 1 bp In (A) | |
A01 | CT001_A01013460 | 1 bp Tv, 7 bp Del | ||
A2-16 | A2-16-4 | A03 | CT001_A03122450 | 1 bp In (C) |
A01 | CT001_A01013460 | 6 bp Del | ||
A2-16-12 | A03 | CT001_A03122450 | 1 bp In (C) | |
A01 | CT001_A01013460 | 6 bp Del | ||
A2-22 | A2-22-5 | A03 | CT001_A03122450 | – |
A01 | CT001_A01013460 | 120 bp Del |
Reaction | Number of Cycles | Thermal Settings |
---|---|---|
Primary (AD-LSP1/RSP1) z | 1 | 95 °C 2 min |
5 | 94 °C 30 s, 72 °C 4 min | |
2 | 94 °C 30 s, 25 °C ramping to 72 °C in 4 min | |
15 | 94 °C 30 s, 72 °C 4 min 94 °C 20 s, 72 °C 4 min 94 °C 30 s, 44 °C 1 min, 72 °C 2 min 30 s | |
1 | 72 °C 5 min, 16 °C hold | |
Secondary (AD-LSP2/RSP2) | 1 | 95 °C 2 min |
5 | 94 °C 20 s, 72 °C 4 min | |
15 | 94 °C 30 s, 72 °C 4 min 94 °C 20 s, 72 °C 4 min 94 °C 30 s, 44 °C 1 min, 72 °C 2 min 30 s | |
5 | 94 °C 20 s, 44 °C 1 min, 72 °C 2 min 30 s | |
1 | 72 °C 5 min, 16 °C hold |
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Shin, Y.-H.; Lee, H.-M.; Park, Y.-D. CRISPR/Cas9-Mediated Editing of AGAMOUS-like Genes Results in a Late-Bolting Phenotype in Chinese Cabbage (Brassica rapa ssp. pekinensis). Int. J. Mol. Sci. 2022, 23, 15009. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232315009
Shin Y-H, Lee H-M, Park Y-D. CRISPR/Cas9-Mediated Editing of AGAMOUS-like Genes Results in a Late-Bolting Phenotype in Chinese Cabbage (Brassica rapa ssp. pekinensis). International Journal of Molecular Sciences. 2022; 23(23):15009. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232315009
Chicago/Turabian StyleShin, Yun-Hee, Hyun-Min Lee, and Young-Doo Park. 2022. "CRISPR/Cas9-Mediated Editing of AGAMOUS-like Genes Results in a Late-Bolting Phenotype in Chinese Cabbage (Brassica rapa ssp. pekinensis)" International Journal of Molecular Sciences 23, no. 23: 15009. https://0-doi-org.brum.beds.ac.uk/10.3390/ijms232315009