Stu-miR827-Targeted StWRKY48 Transcription Factor Negatively Regulates Drought Tolerance of Potato by Increasing Leaf Stomatal Density
Abstract
:1. Introduction
2. Results
2.1. Identification, Characterization, and Expression Analysis of stu-miR827-5p
2.2. Generation of Transgenic Potato with Repressed stu-miR827-5p Expression by STTM Approach
2.3. Suppression of microRNA827-5p Increases Stomatal Density
2.4. Suppression of microRNA827-5p Reduced Drought Tolerance
2.5. Suppression of microRNA827-5p Altered Drought-Relevant Physiological Indexes
2.6. Identification of stu-miR827-5p-Targeted StWRKY48 Gene
2.7. Subcellular Localization and Expression Pattern of StWRKY48
2.8. Knockdown of stu-miR827-5p Increases the Levels of StWRKY48
2.9. Suppression of microRNA827-5p Affects the Expression of Stomata Development-Related Genes
3. Discussion
3.1. Stu-miR827-Mediated StWRKY48 mRNA Cleavage In Vivo
3.2. Stu-miR827 Negatively Regulates Drought Tolerance in Potato
3.3. Stu-miR827 Positively Regulates Stomatal Density by Suppressing the Expression of StSDD1 and StTMM Genes
4. Methods
4.1. The Quantitative Real-Time PCR (qRT-PCR) Analysis
4.2. Construction of Silencing Plasmid of stu-miR827-5p
4.3. Transformation of Potato and Identification of Transgenic Lines
4.4. Analysis of Transgenic Potatoes’ Resistance to Drought Stress
4.5. Measurement of Reactive Oxygen Species (ROS), Proline, and Malonaldehyde (MDA) Levels and Antioxidant Enzyme Activity
4.6. Stomatal Density Measurement
4.7. Prediction and Validation of stu-miR827a-5p Target Genes
4.8. Subcellular Localization Assay
4.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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miRNA | stu-miR827 |
---|---|
MS | UUUGUUGAUGGUCAUCUAUUC |
Gene ID | MI0025949 |
PS | UUUGUUGAUGGUCAUCUAUUCAUCAUAUCAUUUGGCAUAGUUUUUGCAACCAUUAAUAUGCCAUGAUUAGAUGAACAUCAACAAACA |
LP (nt) | 87 |
Families | miR827 |
NM (nt) | 2 |
LM (nt) | 21 |
Side | 5 |
MFEs (kcal/mol) | −32.05 |
Chromosome | 12 |
GenBank | CP047558.1 |
miRNA | miRNA Sequence | Target Gene ID | Target Annotations |
---|---|---|---|
stu-miR827 | UUUGUUGAUGGUCAUCUAUUC | Soltu.DM.01G019140.1 | StWRKY48 |
Gene | Oligonucleotides Forward (F) and Reverse (R) | Amplicon Length (bp) |
---|---|---|
Stu-miR827 | F 5′-TGCCTGGCTCCCTGTATGCCA-3′ | - |
St18sRNA | R 5′-TTAGAGGAAGGAGAAGTCGTAACAA-3′ | - |
StWRKY48 | F 5′-TGCTACCACCAAGTTTCCA -3′ | 171 |
R 5′-CCGAAGAGTAAGAACCGAGG -3′ | ||
StSDD1 | F 5′-TGAAGGAGAGCCTGTCATAAG -3′ | 111 |
R 5′-GGCATAGGATGTAACCGATT -3′ | ||
StEPF | F 5′-AAGACAACATTGGCAAGGG -3′ | 169 |
R 5′-GCAACAGGACAAGTCTCAGC -3′ | ||
StTMM | F 5′-GAAATAATGGAGGTGAGCGT -3′ | 137 |
R 5′-GTTGCTAAACGATGCCTTG -3′ | ||
StSPCH | F 5′-GACCATCTATCTGTGCTACGC -3′ | 130 |
R 5′-TTGGCTTCTAAGGATTGGAG -3′ | ||
StMUTE | F 5′-CGAAAGAGCCTAAGCCCTA -3′ | 130 |
R 5′-AACATCAGCCACTGGAGAA -3′ | ||
StFAMA | F 5′-TGAGGAAGTTGAGAGCCAA -3′ | 118 |
R 5′-CCCTTTGAACATAGGAGCC -3′ | ||
StEF1α | F 5′- ATTGGAAACGGATATGCTCCA-3′ | 101 |
R 5′- TCCTTACCTGAACGCCTGTCA-3′ |
Target Gene | Genomic Locust | Outer Primer | Inner Primer | Size |
---|---|---|---|---|
StWRKY48 | Soltu.DM.01G019140.1 | CAGGCTATCAAATGCCGATGA | TGAGCAGAAAGGCAGCATTCAAGACAATC | 397 bp |
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Yang, J.; Zhang, N.; Bai, J.; Duan, X.; Zhang, L.; Liu, S.; Tang, X.; Jin, X.; Li, S.; Si, H. Stu-miR827-Targeted StWRKY48 Transcription Factor Negatively Regulates Drought Tolerance of Potato by Increasing Leaf Stomatal Density. Int. J. Mol. Sci. 2022, 23, 14805. https://doi.org/10.3390/ijms232314805
Yang J, Zhang N, Bai J, Duan X, Zhang L, Liu S, Tang X, Jin X, Li S, Si H. Stu-miR827-Targeted StWRKY48 Transcription Factor Negatively Regulates Drought Tolerance of Potato by Increasing Leaf Stomatal Density. International Journal of Molecular Sciences. 2022; 23(23):14805. https://doi.org/10.3390/ijms232314805
Chicago/Turabian StyleYang, Jiangwei, Ning Zhang, Jiangping Bai, Xiaoqin Duan, Luhe Zhang, Shengyan Liu, Xun Tang, Xin Jin, Shigui Li, and Huaijun Si. 2022. "Stu-miR827-Targeted StWRKY48 Transcription Factor Negatively Regulates Drought Tolerance of Potato by Increasing Leaf Stomatal Density" International Journal of Molecular Sciences 23, no. 23: 14805. https://doi.org/10.3390/ijms232314805