An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava
Abstract
:1. Introduction
2. Results
2.1. Acquisition of HbAN1-Overexpressing Transgenic Cassava
2.2. Evaluation of HbAN1-Overexpressing Genetic Transformation
2.3. Construction of AR-CRISPR/Cas9-gRNA Editing Vector and Genetic Transformation
2.4. Analysis of the Editing Effects of the MeCDD4 Gene
3. Discussion
4. Materials and Methods
4.1. Plant Materials
4.2. Construction of HbAN1 Expressing and AR-CRISPR/Cas9-MeCDD4-gRNA Editing Vectors
4.3. Cassava Genetic Transformation
4.4. Extraction and Measurement of Anthocyanins
4.5. Statistics of Cotyledons, Bud Regenerations and Seedlings of the Regenerated Plants
4.6. Hi-TOM Sequencing
4.7. qRT-PCR Validation
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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Line Name | Mutation Sequence | Reads Count | Reads Ratio | Mutation Type | Mutant Base |
---|---|---|---|---|---|
WT | GCTTCCCCCAACTGAGTGCGAGG | 68,606 | 83.04% | WT | |
meccd4-1 | GCTTCCCCCAA-----------TGCGAGG | 26,593 | 46.25% | 5D | CTGAG |
GCTTCCCCCAACTG------GCGAGG | 24,928 | 43.36% | 3D | AGT | |
meccd4-2 | GCTTCCCCCA--------TGCGAGG | 56,300 | 84.10% | 6D | ACTGAG |
meccd4-4 | GCTT------------------------------------------ | 33,361 | 49.97% | 26D | CCCCCAACTGAGTGCGAGGTGATTGA |
GCTTCC--------------------------CGAGG | 26,650 | 39.92% | 12D | CCCAACTGAGTG | |
meccd4-5 | GCTTCCCCCAACT----GTGCGAGG | 42,805 | 51.02% | 2D | GA |
GCTTCCCCCAACTGA----GCGAGG | 36,498 | 43.50% | 2D | GT | |
meccd4-6 | GCTTC---------------------AGT-TGAGG | 23,776 | 90.84% | 9D, 1D, SNP | CCCCAACTG,G,C->T |
meccd4-7 | GCTTCCCCCAA---------------CGAGG | 32,807 | 45.03% | 7D | CTGAGTG |
GCTTCCCCCAAC--------TGCGAGG | 30,596 | 41.99% | 4D | TGAG | |
meccd4-8 | GCTTCCCCCAACTGA----GCGAGG | 33,855 | 52.31% | 2D | GT |
GCTTCCCCCAACTG------GCGAGG | 13,453 | 20.78% | 3D | AGT | |
GCTTCCCCCAACT----GTGCGAGG | 3718 | 05.74% | 2D | GA | |
GCTTCCCCCAACTGAGT----GAGG | 3604 | 05.57% | 2D | GC | |
meccd4-9 | GCTTCCCCCA-------------TGCGAGG | 22,175 | 36.57% | 6D | ACTGAG |
GCTTCCCCCAACTGA----GCGAGG | 17,791 | 29.34% | 2D | GT | |
GCTTCCCCCAACT-------------GAGG | 14,399 | 23.75% | 6D | GAGTGC | |
meccd4-10 | GCTTCCCCCAACTGA--TGCGAGG | 25,004 | 45.79% | 1D | G |
GCTTCCCCCAAC---------TGCGAGG | 23,782 | 43.55% | 4D | TGAG | |
meccd4-12 | GCTTCCCCCA---------------GCGAGG | 32,546 | 44.66% | 7D | ACTGAGT |
GCTT-CCCCA----------------GCGAGG | 32,364 | 44.41% | 1D,7D | C,ACTGAGT |
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Zhen, X.-H.; Pan, R.-R.; Lu, X.-H.; Ge, Y.-J.; Li, R.-M.; Liu, J.; Wang, Y.-J.; Yi, K.-X.; Li, C.-X.; Guo, J.-C.; et al. An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava. Int. J. Mol. Sci. 2024, 25, 11808. https://doi.org/10.3390/ijms252111808
Zhen X-H, Pan R-R, Lu X-H, Ge Y-J, Li R-M, Liu J, Wang Y-J, Yi K-X, Li C-X, Guo J-C, et al. An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava. International Journal of Molecular Sciences. 2024; 25(21):11808. https://doi.org/10.3390/ijms252111808
Chicago/Turabian StyleZhen, Xing-Hou, Ran-Ran Pan, Xiao-Hua Lu, Yu-Jian Ge, Rui-Mei Li, Jiao Liu, Ya-Jie Wang, Ke-Xian Yi, Chun-Xia Li, Jian-Chun Guo, and et al. 2024. "An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava" International Journal of Molecular Sciences 25, no. 21: 11808. https://doi.org/10.3390/ijms252111808
APA StyleZhen, X.-H., Pan, R.-R., Lu, X.-H., Ge, Y.-J., Li, R.-M., Liu, J., Wang, Y.-J., Yi, K.-X., Li, C.-X., Guo, J.-C., Yao, Y., & Geng, M.-T. (2024). An Anthocyanin-Based Visual Reporter System for Genetic Transformation and Genome Editing in Cassava. International Journal of Molecular Sciences, 25(21), 11808. https://doi.org/10.3390/ijms252111808