밀의 Pseudo Response Regulator (PRR) 유전자군의 분자생물학적 특성 연구 = Molecular characterization of Pseudo Response Regulator (PRR) genes in Wheat (Triticum aestivum L. cv. Keumkang)

다국어 초록 (Multilingual Abstract)

Wheat (Triticum aestivum L.) is one of the most important staple crops cultivated worldwide, along with rice (Oryza sativa L.) and maize (Zea mays L.). However, global climate change and the resulting increase in both biotic and abiotic stresses have posed serious challenges to wheat cultivation, contributing to instability in global wheat supply. To overcome these challenges, genetic approaches to improve agronomic traits such as flowering time and stress tolerance have been intensively studied. Among these, genes belonging to the Pseudo-Response Regulator (PRR) family have emerged as key regulators of circadian rhythm and flowering in plants. The Photoperiod-1 (Ppd-1) gene, a member f the PRR family, has long been used as a marker in wheat breeding programs. However, recent evidence has shown that Ppd-1 does not always reliably correlate with flowering phenotype in diverse wheat germplasm, suggesting that additional PRR family members may also contribute to flowering regulation. In this study, I conducted a detailed analysis of the wheat PRR gene family (TaPRRs) to investigate their molecular features and possible functions in regulating the timing of flowering. Initially, I conducted sequence alignments and examined phylogenetic relationships using PRR genes from wheat, rice, maize, and Arabidopsis. This analysis showed that TaPRRs are preserved and organized into categories that match those found in different species. Looking at how TaPRRs are expressed over a 48-hour period showed patterns that change in a daily cycle, much like PRRs found in typical plants. Their highest levels of expression occur during the day, while levels drop at night. Experiments on where TaPRR proteins are located within cells, using GFP tags in wheat protoplasts, showed that they are mostly found in the nucleus, which aligns with their expected role in regulation. To explore how functions interact, yeast two-hybrid (Y2H) tests were conducted to see if the TaPRR proteins connect with wheat CONSTANS (TaCO1 and TaCO2) and Nuclear Factor Y (TaNFY) proteins, which are recognized companions of PRRs in Arabidopsis. The findings showed positive connections among various TaPRR proteins and the members of TaCO1, TaCO2, and TaNF-YC. Bimolecular fluorescence complementation (BiFC) tests gave more proof of these protein connections inside the nucleus, backing up their possible roles in controlling flowering processes. Taken together, these analyses offer important details of TaPRR gene family in wheat like their evolutionary traits and similarity to other PRR genes, expression patterns in 24 hour period, single locations in cells, and interactions to other central clock proteins and locations. These findings show that other TaPRRs, not only Ppd-1, may play a central role in regulating daily cycles, leading to flowering time control.

목차 (Table of Contents)

• Table of Contents i
• List of Figures iii
• List of Tables iv
• Abstract v
• 1. Introduction 1
• 2. Materials & Methods 5
• 2.1. Plant materials and growth conditions. 5
• 2.2. Gene cloning 5
• 2.3. Alignments and phylogenetic analysis. 6
• 2.4. Expression analysis for TaPRR genes 6
• 2.5. RNA expression analysis . 7
• 2.6. Yeast two-hybrid assay. 8
• 2.7. Subcellular localization assay. 10
• 2.8. Bimolecular Fluorescence Complementation assay 11
• 3. Results and Discussion 19
• 3.1. Phylogenetic tree and multiple sequence alignments of Arabidopsis, rice, corn, and wheat PRR gene family . 19
• 3.2. Diurnal expression patterns of TaPRR genes under long day (LD) and short day (SD) conditions 23
• 3.3. Subcellular localization patterns of TaPRR proteins . 26
• 3.4. Protein-protein interaction analysis between TaPRR and other wheat proteins (TaCO1, TaCO2, and TaNF-Y) 30
• 4. References 38
• Figure 1. Comparison of conserved domains and phylogenetic relationships of PRR proteins of Arabidopsis, rice, corn, and wheat. 21
• Figure 2. Experssion profiles of wheat PRR gene family under long day/short day conditions 25
• Figure 3. Subcellular localization of TaPRR proteins in wheat protoplast 28
• Figure 4. Interaction analysis between TaPRR and TaCO, TaNFY proteins using yeast two hybrid system 34
• Figure 5. Bimolecular Fluorescence Complementation assay between TaPRR and TaCO, TaNF-Y proteins 36
• Table 1. Primer lists used for TA Cloning 12
• Table 2. Primer lists used for RNA expression analysis 13
• Table 3. Primer lists used for yeast two-hybrid assay 14
• Table 4. Primer lists used for subcellular localization assay 16
• Table 5. Primer lists used for Bimolecular Fluorescence Complementation assay 17
• Table 6. Summary of subcellular localization patterns for TaPRR proteins 18