Identification, Characterization, and Mutational Analysis of a Probable KEAP1 Ortholog in Rice (Oryza sativa L.)
Abstract
:1. Introduction
2. Results
2.1. Identification and Characterization of the KEAP1 Ortholog in Rice
2.2. Targeted Mutagenesis of 5′ UTR Downregulated OsKEAP1 Expression
2.3. Impact of OsKEAP1 Down-Regulation on Plant Growth and Development
2.4. Impact of OsKEAP1 Downregulation on Seed Germination and Sensitivity to Abscisic Acid (ABA)
2.5. Impact of OsKEAP1 Downregulation on Redox Regulation
3. Discussion
4. Materials and Methods
4.1. In Silico Identification and Characterization of KEAP1 Orthologs in Plants
4.2. Construction of CRISPR/Cas9 Vector
4.3. Identification and Development of OsKEAP1 Mutants
4.4. Real-Time Quantitative-PCR and Subcellular Localization
4.5. Agronomic and Seed Germination Tests of OsKEAP1 Mutants
4.6. Seedling Growth
4.7. Measurement of H2O2, MDA and Proline Content
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Name of Primers | Sequences (5′-3′) | Remarks |
---|---|---|
KEAP1-F | ggcaGGGTGCTACGAAACCCGC | OsKEAP1 sgRNA synthesize |
KEAP1-R | aaacGCGGGTTTCGTAGCACCC | |
Pk1-F | GGTTGATCGATGCTTGCTGC | For oskeap1 mutant identification and high resolution melting (HRM) analysis |
Pk1-R | CACCAATCGCGACCAAATCG | |
KPqpcr-F | CAAGCACTGGCCAGCTTAAT | q-PCR analysis |
KPqpcr-R | GATTAGCGCGAACAGGAGCA | |
qKp2-F | GCAGCCCGTCTATGATGAACTCTC | |
qKp2-R | AGAAACCCTCCCTTGGGTCATAC | |
qCATA-F | TCCCAGTGTGATGAGTCGTTGG | |
qCATA-R | TCTTCACATGCTTGGCTTCACG | |
qCATB-F | TCCTACTGGTCGCAGTGTGATG | |
qCATB-R | TTTCAGGTTGAGACGTGAAGCC | |
KpGFP F | gaattcATGGGTGCTGGAAAGAAGACTCA | Complementary DNA amplification |
KpGFP R | ggatccCAATGGCAACGGCGCATGC | |
HYG-F | AGAAGAAGATGTTGGCGACCT | Hygromycin marker primer |
HYG-R | GTCCTGCGGGTAAATAGCT |
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Liu, Y.-H.; Jiang, M.; Li, R.-Q.; Basnet, R.; Huang, J.-Z.; Song, S.-Y.; Shu, Q.-Y. Identification, Characterization, and Mutational Analysis of a Probable KEAP1 Ortholog in Rice (Oryza sativa L.). Plants 2020, 9, 1450. https://doi.org/10.3390/plants9111450
Liu Y-H, Jiang M, Li R-Q, Basnet R, Huang J-Z, Song S-Y, Shu Q-Y. Identification, Characterization, and Mutational Analysis of a Probable KEAP1 Ortholog in Rice (Oryza sativa L.). Plants. 2020; 9(11):1450. https://doi.org/10.3390/plants9111450
Chicago/Turabian StyleLiu, Yan-Hua, Meng Jiang, Rui-Qing Li, Rasbin Basnet, Jian-Zhong Huang, Shi-Yong Song, and Qing-Yao Shu. 2020. "Identification, Characterization, and Mutational Analysis of a Probable KEAP1 Ortholog in Rice (Oryza sativa L.)" Plants 9, no. 11: 1450. https://doi.org/10.3390/plants9111450