Abstract
Background
Cloning and characterizing the drought-inducible promoters is essential for their use in crop resistance's genetic improvement. Previous studies have shown that the TaNRX1-D gene participates in regulating the response of wheat to drought stress. However, its promoter has not yet been identified.
Objective
In this study, we aimed to characterize the promoter of the TaNRX1-D gene.
Methods
The promoter of TaNRX1-D (named P0, 2081 bp) was isolated from common wheat with several cis-acting elements that regulate in response to abiotic stresses and some core cis-acting elements. Functional verification of the promoter, eight 5′-deletion fragments of TaNRX1-D promoter, was fused to the β-glucuronidase (GUS) gene P0::GUS ~ P7::GUS and transformed into Arabidopsis, respectively. Agrobacterium-mediated GUS transient assay the P6a and P6b promoter regions in tobacco leaves under normal, osmotic or ABA stress.
Results
Activity analysis of the full-length promoter (P0) showed that the intensity of stronger β-glucuronidase (GUS) staining in the roots and leaves was obtained during the growth of transgenic Arabidopsis. P0::GUS displayed the GUS activity was much higher in the roots and leaves than in other parts of the transgenic plant under normal conditions, which was similarly within wheat. Analysis of the 5′-deletion fragments revealed that P0::GUS ~ P6::GUS responded well upon exposure to osmotic (polyethylene glycol-6000, PEG6000) and abscisic acid (ABA) stress treatments and expressed significantly higher GUS activity than the CaMV35S promoter (35S::GUS), while P7::GUS did not. GUS transient assay in tobacco leaves showed that the GUS activities of P6a and P6b were lower than P6 in the PEG6000 and ABA stresses.
Conclusion
The 193 bp (P6) segment was considered the core region of TaNRX1-D responding to PEG6000 or ABA treatment. GUS activity assay in transgenic Arabidopsis showed that this segment was sufficient for the PEG6000 or ABA stress response. The identified 193 bp promoter of TaNRX1-D in this study will help breed osmotic or ABA tolerant crops. The 36 bp segment between P6 and P6b (−193 to −157 bp) was considered the critical sequence for the TaNRX1-D gene responding to PEG6000 or ABA treatment.
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Abbreviations
- ABA:
-
Abscisic acid
- PEG 6000:
-
Polyethene glycol-6000
- GUS:
-
β-Glucuronidase
- CaMV35S:
-
Cauliflower mosaic virus 35S
- TRX:
-
Thioredoxin
- NRX:
-
Nucleotoreoxin
- PCR:
-
Polymerase chain reaction
- DMSO:
-
Dimethyl sulfoxide
- X-Gluc:
-
5-Bromo-4-chloro-3-indolyl-β-d-glucuronic acid cyclohexylammonium salt
- 4-MUG:
-
4-Methyl-umbelliferyl-β-d-glucuronide
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Acknowledgements
This study was sponsored by the National Natural Science Foundation of China (31671693), Key Research and Development Project of Shaanxi Province (2019ZDLNY04-05), The CAS "Light of West China" Program, The Funds for Local Science and Technology Development Guided by the Central Government (ZY20200089), Cooperation Project of Ningxia Academy of Agriculture and Forestry Science (DWX2019001)
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XZ, JX, YW, and DC provided fund support; XZ conceived the initial screening and research plans; JC and FW performed most of the experiments, analyzed the data, and wrote the initial draft; NL participated the manuscript modification and revisions; TS and MZ helped the data analysis, figures, and tables preparation. All authors approved the final manuscript.
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Cheng, J., Wei, F., Zhang, M. et al. Identification of a 193 bp promoter region of TaNRX1-D gene from common wheat that contributes to osmotic or ABA stress inducibility in transgenic Arabidopsis. Genes Genom 43, 1035–1048 (2021). https://doi.org/10.1007/s13258-021-01115-x
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DOI: https://doi.org/10.1007/s13258-021-01115-x