Masters Theses
Date of Award
5-2021
Degree Type
Thesis
Degree Name
Master of Science
Major
Plant Sciences
Major Professor
Charles Neal C Stewart, Jr.
Committee Members
Feng Chen, Tessa Burch-Smith, Reginald Millwood
Abstract
Drought can potentially affect the global rice supply. Recent climate modeling studies projected a more frequent and intense drought scenario, especially in top producing and exporting countries worldwide. Several drought-tolerant genes had been identified in recent years, and transgenic approaches had been employed to develop drought-tolerant rice. However, the constitutive expression of some drought-tolerant genes resulted in undesirable phenotypes and metabolic burden. The use of an inducible synthetic promoter is advantageous in preventing the possible pleiotropic effects from constitutively expressing a drought-tolerant gene. In this study, rationally designed synthetic promoters based on poplar (Populus trichocarpa) cis-motifs were tested for drought-stress inducible activity in rice. Three constructs (SD18-1, SD9-2, and negative control) were stably transformed in rice (Oryza sativa L. ssp. Japonica cv. ‘Taipei 309’) through Agrobacterium-mediated transformation. The two constructs SD18-1 and SD9-2 (synthetic promoter from drought-stress-inducible promoters), consisted of heptameric repeats of cis-motifs (motifs 18-1 and 9-2). The heptamerized cis-motifs were fused upstream of a core promoter, which consisted of the -46 35S promoter with the TMV Ω 5’ UTR leader sequence for transcriptional initiation, to express a green fluorescent TurboGFP gene. The negative control construct (herein referred to as Neg) contained the core promoter fused to the TurboGFP gene but had no heptameric repeats of any cis-motif. Synthetic promoter response to drought and salt-stress treatments was tested in two-month-old T1 transgenic rice in greenhouse conditions. Synthetic promoter activity was reported by acquiring the normalized green fluorescence values of the first three young and fully expanded leaves of the main tiller of the transgenic rice at 502 nm emission using 465 nm excitation through fluorescence spectroscopy. Drought-stress treatment was done by water-cessation for 15 days. The green fluorescence values were taken before and after 7-, 10-, and 15-days of treatment. Here, transgenic rice harboring SD18-1 and SD9-2 synthetic promoter constructs had statistically significant green fluorescence than the mock and the WT control (p<0.05) only after 15 days drought-stress treatment. The Neg transgenic rice had no statistically different green fluorescence intensity than the WT control (p>0.05) after 7-, 10-, and 15- days of drought-stress treatment. To determine the specificity of drought-stress inducible-response of the synthetic promoters in rice, SD18-1, SD9-2, and Neg transgenic rice were subjected to salt-stress treatment. Salt-stress treatment was done by applying 250 mM NaCl solution directly to the potting-mix for five days. The green fluorescence values were taken before and after 1-, 3-, and 5- days of treatment. Salt-stressed SD18-1, SD9-2, and Neg transgenic rice had statistically significant green fluorescence intensity than the mock control (p<0.05) after one and five days of salt-stress treatment. However, the green fluorescence intensities of the SD18-1, SD9-2, and Neg transgenic rice were statistically similar to the WT control (p>0.05). From the results, the transgenic rice harboring the SD18-1 and SD9-2 synthetic promoters only showed an inducible response to drought-stress treatment. The statistically no significant green fluorescence from the Neg transgenic rice versus the WT after the drought- and salt-stress treatments also showed that the core promoter was not driving TurboGFP gene expression in rice. Lastly, the rationally-designed synthetic promoters based on poplar cis-motifs demonstrating drought-stress inducible response in rice indicated that the SD18-1 and SD9-2 cis-motifs are likely highly conserved in both the dicot and monocot plant systems. Overall, this study showed two relatively short synthetic promoters with drought-stress-specific inducible response in rice.
Recommended Citation
Tagaloguin, Paolo Montano, "Drought-stress-inducible synthetic promoters in rice (Oryza sativa). " Master's Thesis, University of Tennessee, 2021.
https://trace.tennessee.edu/utk_gradthes/6214