臺灣博碩士論文加值系統

植物無法自由移動，因此在遭遇環境逆境時，需演化出相應的生存能力。為了探討阿拉伯芥中與熱逆境相關的基因，本實驗室先前利用正向遺傳學的策略，篩選出對熱敏感的突變株heat intolerance 4（hit4）。後續研究發現，HIT4蛋白質在常溫下位於細胞核內的染色質中心。而在植物受熱後，HIT4會在染色質中心消散前，由染色質中心轉移到核仁，且在熱逆境結束後，隨著染色質中心的重新形成而回到染色質中心。另一方面，hit4突變蛋白質雖然保有在高溫環境下轉移到核仁的能力，hit4突變株內的染色質中心卻不會因高溫而消散。據此推測，應有其他分子參與在HIT4媒介熱誘導染色質中心消散及植物耐熱能力的反應。為了找出這些潛在的分子，進而完整揭露HIT4在阿拉伯芥中的作用機制，本研究使用TurboID來標記HIT4附近的蛋白質，並以pull down assay與質譜分析（mass spectrometry），來辨認可能與HIT4有交互作用的蛋白質。隨後對這些候選蛋白質進行突變株的篩選，測試其對高溫逆境的耐受能力，並以雙分子螢光互補（BiFC）等實驗，對候選蛋白質進行分析。在候選蛋白質中，PUB49（PLANT U-BOX 49）在正常溫度下均勻散佈在整個細胞核中，但在遭遇熱逆境時，會先聚集到色質中心，並隨著時間推移，與HIT4一樣位移到核仁。BiFC實驗則顯示了兩者之間有交互作用。尤有甚者，pub49突變株也失去對高溫逆境的耐受能力。綜上結果，PUB49即為參與HIT4調節植物耐熱反應的分子之一。

Plants, unable to move freely, evolve corresponding survival abilities to cope with environmental adversities. To investigate genes related to heat stress in Arabidopsis, our laboratory previously utilized a forward genetic approach to screen for heat-sensitive mutant lines, identifying heat intolerance 4 (hit4). Subsequent studies revealed that the HIT4 protein is localized in the chromocenters of the nucleus at normal temperatures (23°C). Upon heat stress, HIT4 translocates from the chromocenters to the nucleolus before decondensing, and upon cessation of heat stress, it returns to the chromocenters as they reform. Conversely, the hit4 mutant protein, while retaining the ability to translocate to the nucleolus under high temperatures (37°C), does not allow the decondensation of chromocenters in mutant plant nuclei under heat stress. These results suggest the involvement of other molecules in HIT4-mediated heat-induced chromocenter decondensation and plant thermotolerance. To identify these potential molecules and fully elucidate the role of HIT4 in Arabidopsis, this study employed TurbolD to label proteins near HIT4 and utilized pull-down assays and mass spectrometry to identify proteins that may interact with HIT4. Subsequently, mutant strains of these candidate proteins were screened for thermal sensitivity, and candidate proteins were analyzed using techniques such as bimolecular fluorescence complementation (BiFC). Among the candidate proteins, PUB49 (PLANT U-BOX 49) was found to be uniformly distributed throughout the nucleus at normal temperatures but aggregates at the chromocenters upon heat stress, similar to HIT4, and relocates to the nucleolus over time, interacting with HIT4 as indicated by BiFC experiments. Importantly, pub49 mutant plants also lose their tolerance to high-temperature stress. These results collectively indicate that PUB49 is one of the molecules involved in regulating plant thermotolerance together with HIT4 in Arabidopsis.

摘要 I
Abstract II
誌謝 IV
總目錄 V
表目錄 VII
圖目錄 VIII
一、緒論 1
二、研究材料與方法 5
2-1 植物材料 5
2-1-1 植物品系 5
2-1-2 植物的培養 5
2-1-3 植物熱逆境（heat stress）與熱休克（heat shock）處理條件 6
2-2 質體的製備 6
2-2-1 細菌LB （Luria Broth）培養基製備 6
2-2-2 質體的轉殖、篩選與萃取 7
2-3 植物DNA萃取 9
2-4 建立基因轉殖植物 10
2-4-1 製備農桿菌勝任細胞 10
2-4-2 農桿菌電穿孔轉型 10
2-4-3 阿拉伯芥的農桿菌基因轉殖 10
2-5 阿拉伯芥原生植體的製備與轉殖 11
2-6 蛋白質膠體分析 12
2-6-1 製作SDS-PAGE與蛋白質的電泳分離 12
2-6-2 西方墨點法（western blot） 12
2-6-3 Coomassie Brilliant Blue染色 13
2-6-4 銀染（Silver stain） 13
2-7 Affinity Purification-Mass Spectroscopy (AP-MS) 14
2-8 反轉錄PCR（RT-PCR） 15
2-9 免疫共沉澱(Co-immunoprecipitation, Co-IP) 15
三、結果 17
3-1 尋找與HIT4交互作用的蛋白質 17
3-1-1 TbID-HIT4融合蛋白不會影響HIT4參與的耐熱反應 17
3-1-2 TbID-HIT4 pull down assay 18
3-2 分析候選基因與HIT4之間的關係 18
3-2-1 HEAT SHOCK PROTEIN (HSP) family 19
3-2-2 DEAD BOX RNA HELICASE 1 (DRH1) 19
3-2-3 CHR11與ARP4 20
3-2-4 NUCLEOLIN LIKE 1 (NUC1) 21
3-3 PUB49會參與HIT4誘導的耐熱性 21
3-3-1 PUB49在熱逆境下的位置與HIT4一致 22
3-3-2 PUB49會與HIT4進行交互作用 22
3-3-3 pub49突變株失去對高溫逆境的耐受能力 23
3-3-4 PUB49的突變不影響HIT4轉移到核仁的能力 23
四、討論 24
五、參考文獻 28

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