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研究生: 阮武保
Nguyen Vu Bao
論文名稱: 水稻OsCAF1蛋白質在熱逆境的特性分析
Characterization of OsCAF1 Protein Function in Rice Response to Thermal Stress
指導教授: 陸重安
Chung-An Lu
葉靖輝
Ching-Hui Yeh
口試委員:
學位類別: 博士
Doctor
系所名稱: 生醫理工學院 - 生命科學系
Department of Life Science
論文出版年: 2025
畢業學年度: 113
語文別: 英文
論文頁數: 135
中文關鍵詞: One keyword per line
外文關鍵詞: processing bodies (PBs), CCR4-associated factor 1 (CAF1), eukaryotic initiation factor 4AII (eIF4AII), DEAD-box ATP-dependent RNA helicase 8 (RH8)
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  • messenger RNA (mRNA) 降解的調節對基因表現控制至關重要,其中 CCR4-associated factor 1 (CAF1)蛋白在脫腺苷酸化過程中(deadenylation)發揮著關鍵作用。然而,水稻 CAF1(OsCAF1)蛋白在逆境下於細胞內位置及其功能意義仍未明確。在本研究中,我們探討了高溫逆境下 OsCAF1 蛋白於細胞內位置的改變及其在耐熱逆境中的作用。在正常條件下,OsCAF1A、OsCAF1G 與 OsCAF1H 分散於細胞質中,而 OsCAF1B 則定位於processing bodies (PBs)。高溫逆境促使 OsCAF1A、OsCAF1G 與 OsCAF1H 位移至PBs及stress granules,顯示其在逆境反應中調控 mRNA 穩定性及降解的作用。基因表現量(absolute RT-qPCR)結果顯示,OsCAF1A 在水稻幼苗中表現量最為豐富,且在高溫逆境下會被進一步誘導表現。功能性分析結果表明,在高溫逆境下,OsCAF1A 的大量表現可以減少細胞電解質洩漏以提高幼苗存活率,來增強植株耐熱性。相對地, 當OsCAF1A 發生突變時,則使得植株對高溫更為敏感。然而,在正常生長條件下,OsCAF1A 的過度表現會導致植株矮化及穀物產量減少。交互作用(BiFC)實驗結果證實,兩種 DEAD-box RNA 解旋酶-OseIF4AIIb 與 OsRH8-均為與 OsCAF1A 具有交互作用的蛋白質因子,對OsCAF1A於細胞內位置分佈具有截然相反的影響:在高溫逆境下,OseIF4AIIb 會限制 OsCAF1A 聚集於 PB,而 OsRH8 則促進OsCAF1A 聚集於 PB。這些發現強調了 OsCAF1 蛋白在水稻耐熱性中的重要作用,並展示了其在提高作物對氣候變遷適應能力方面的潛力


    The dynamic regulation of messenger RNA (mRNA) decay is crucial for gene expression control, with CCR4-associated factor 1 (CAF1) proteins playing a key role in deadenylation. However, the subcellular localization and functional sig-nificance of rice CAF1 (OsCAF1) protein under stress conditions remain largely uncharacterized. In this study, we investigated the re-localization of OsCAF1 proteins under high-temperature stress and their role in heat stress tolerance. Under normal conditions, OsCAF1A, OsCAF1G, and OsCAF1H were dispersed in the cytoplasm, whereas OsCAF1B localized to processing bodies (PBs). Heat stress triggered the re-localization of OsCAF1A, OsCAF1G, and OsCAF1H to PBs and stress granules, suggesting a role in stress-responsive mRNA regulation. Expression analysis revealed that OsCAF1A was the most abundantly expressed among OsCAF1 genes in rice seedlings, with its expression further induced by heat stress. Functional analysis demonstrated that OsCAF1A overexpression en-hanced heat tolerance by reducing electrolyte leakage and improving seedling survival, whereas OsCAF1A mutants exhibited increased sensitivity to heat stress. However, OsCAF1A overexpression lines also exhibited shorter plant height and reduced grain yield than wild type under normal growth conditions. Interaction studies identified OseIF4AIIb and OsRH8, two DEAD-box RNA helicases, as OsCAF1A-associated proteins with opposing effects on its re-localization: OseIF4AIIb restricted, whereas OsRH8 promoted OsCAF1A recruitment to PBs under heat stress. These findings highlight the crucial role of OsCAF1 proteins in rice thermotolerance and their potential for improving crop resilience to climate change.

    摘要 i Abstracts ii Acknowledgments iii List of Tables vii List of Figures viii List of Supplemental Data x Introduction 1 Materials and Methods 20 Results 29 1. OsCAF1A mRNA is highly expressed compared to other OsCAF1 genes in rice seedlings and further induced under high-temperature conditions 30 2. OsCAF1A enhances seed germination under elevated temperature 31 3. Functional characterization of OsCAF1A in the heat stress response of rice 33 4. OsCAF1A promotes rice seedling growth under sublethal high-temperature treatment 34 5. Transgenic rice overexpressing OsCAF1A displays defects at vegetative and reproductive stages 36 6. High temperature induces re-localization of OsCAF1 proteins in rice cells 37 7. Cycloheximide inhibits the assembly of heat-induced OsCAF1-containing cytoplasmic foci in rice protoplasts 39 8. OsCAF1 proteins localize to processing bodies and stress granules under high-temperature conditions 40 9. Functional conservation of OsCAF1A protein in OsCAF1A-GFP transgenic lines 42 10. Identification of OsCAF1A-interacting proteins and validation of their interactions 43 11. Subcellular localization of OseIF4AIIb and OsRH8 46 12. Interaction of OseIF4AIIb and OsRH8 with OsCCR4 and the central domain of OsNOT1 (OsMIF4G) in rice 48 13. Differential expression of OseIF4AIIb and OsRH8 in response to heat stress in rice seedlings 49 14. OseIF4AIIb and OsRH8 exert opposing effects on the high-temperature-induced localization of OsCAF1 proteins to processing bodies 50 Discussion 55 References 65 Supplemental data 105

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