自噬作用在真核生物中是一種保守的細胞機制，參與植物營養缺乏的逆境反應。雖然植物細胞缺磷會造成自噬小體數量上升，但缺磷反應如何活化自噬作用相關(autophagy related, ATG)基因的轉錄調控路徑仍不明確。PHOSPHATE STARVATION RESPONSE 1 (PHR1)是調控植物缺磷反應基因群的核心轉錄因子，參與一系列的缺磷反應，包括促進磷酸吸收、維持細胞內磷酸恆定。已知PHR1或自噬作用基因的功能缺陷分別會影響細胞磷酸的恆定。我們進一步取得阿拉伯芥 phr1/atg5-1和phr1/atg7-3雙突變株並進行無機磷酸濃度的檢測。雙突變株的地上部磷酸濃度相較於phr1、atg5-1、atg7-3更低，意味著PHR1與自噬作用可能對於磷酸恆定有各自調控的路徑。實驗室前期的研究發現，阿拉伯芥幼苗中AtATG8家族成員AtATG8f 和AtATG8h會在缺磷時增加表現量，但在phr1突變株中，AtATG8f 和AtATG8h受到缺磷誘導的情形減少。為了找到缺磷下誘導AtATG8f 和AtATG8h基因表現的轉錄因子，我們以AtATG8f 和 AtATG8h 啟動子序列建立報告載體，利用酵母菌單雜合技術，在阿拉伯芥轉錄因子庫去篩選出有交互作用的轉錄因子。結果發現，AtPHR1沒有在陽性轉錄因子候選列表中。此外，我們根據RNA-seq數據中提供缺磷誘發的轉錄因子，從AtATG8f酵母菌單雜合結果的16個陽性轉錄因子中，選擇出AtTEM1、AtRAV1、At4G25210; AtATG8h 的11個陽性轉錄因子中選擇AtNAC032 、 AtNAC102、At4G25210，來進一步在植物細胞中驗證酵母菌單雜合結果。我們使用阿拉伯芥原生質體進行雙冷光素酶報告基因測定。結果表示，大量表現AtRAV1和AtTEM1轉錄因子會抑制 AtATG8f的表現。缺磷造成自噬作用的活化是否與AtTEM1、AtRAV1、AtNAC032 、 AtNAC102、At4G25210轉錄因子的調控有關仍須後續研究。

Autophagy is a conserved eukaryotic cellular process involved in stress responses. Although autophagosomes were found to increase in plant cells under phosphate (Pi) deficiency, the pathway of Pi deficiency-induced transcriptional and post-transcriptional regulation of autophagy-related (ATG) genes remains unclear. PHOSPHATE STARVATION RESPONSE 1 (PHR1) is a transcription factor that regulates plant transcriptional response to Pi starvation, including enhancement of Pi uptake and maintenance of cellular Pi homeostasis. Loss of function of PHR1 and core ATG genes led to alteration of Pi homeostasis. We further obtained the A. thaliana phr1/atg5-1 and phr1/atg7-3 double mutants and found the shoot Pi level of double mutants was lower than that of phr1, atg5-1 and atg7-3 single mutants, indicating that AtPHR1 and autophagy may independently regulate Pi homeostasis. In our previous study, the expression levels of the two AtATG8 family members, AtATG8f and AtATG8h, were upregulated in following Pi starvation. However, such upregulation is attenuated in the phr1 mutant. To find which transcription factor binds to AtATG8f and AtATG8h promoters, we generated the reporter constructs with the promoter sequences of AtATG8f and AtATG8h for yeast one-hybrid (Y1H) analysis with A. thaliana transcription factor library. AtPHR1 was not identified as a transcription factor candidate directly regulating the expression of AtATG8f and AtATG8h. Among the 16 positive transcription factor candidates that interact with the AtATG8f promoter, we selected 3 positive transcription factors with increased expression under Pi starvation based on the RNA-seq data: AtTEM1, AtRAV1,and At4G25210. Among the 11 positive transcription factor candidates that interact with the ATG8h promoter, AtNAC032, AtNAC102, and At4G25210 were selected. Using Arabidopsis protoplasts, we performed the dual-luciferase reporter assay to validate whether these transcription factors could regulate the promoter activity of AtATG8f and AtATG8h. Our results showed that overexpression of AtRAV1 and AtTEM1 inhibited the AtATG8f promoter. The involvement of AtTEM1, AtRAV1, AtNAC032, AtNAC102, and At4G25210 in Pi starvation-induced autophagy needs further study.

目錄
壹、 前言 1
一、 植物磷酸恆定之調控 1
二、 缺磷反應誘導自噬作用 4
三、 植物自噬作用ATG基因群的表現量調控 5
四、 研究動機 7
貳、 材料與方法 9
一、 阿拉伯芥與圓葉菸草種子表面消毒: 9
二、 阿拉伯芥與圓葉菸草生長條件 9
三、 基因轉質載體設計與製作 10
五、 阿拉伯芥原生質體表達系統 11
六、 雙分子互補綠螢光蛋白標定(split GFP-tagging) 11
七、 雙冷光素酶報告檢測 12
八、 圓葉菸草短暫表達系統 12
九、 阿拉伯芥收集與無機磷濃度檢測 13
十、 蛋白質含量測定 14
十一、 共軛焦顯微鏡觀察 14
參、 實驗結果 15
一、 測量phr1/atg5-1和phr1/atg7-3突變株的無機磷濃度 15
二、 atg1a-2、atg2-1、atg11-1 突變株的 AtPHT1;1/1;2/1;3蛋白表現量下降 15
三、 利用酵母單雜合篩選出結合到AtATG8f和AtATG8h啟動子上的轉錄因子 16
四、 利用split-GFP tagging確認RAV1、TEM1、NAC032、NAC102、At4G25210 在細胞核表現 17
五、 AtRAV1和AtTEM1表現能降低AtATG8f轉錄活性 18
六、 AtNAC102不是PHO2的下游目標 19
肆、 討論 21
一、 探討PHR1調控AtATG8f和AtATG8h的可能性 21
二、 探討AtRAV1和AtTEM1對AtATG8f啟動子活性的影響 22
三、 探討NAC102和NAC032與AtATG8h啟動子活性的影響 22
伍、 圖表 24
圖一、 atg突變株根部與地上部的無機磷酸濃度 24
圖二、 atg突變株的PHT1;1/1;2/1;3蛋白質表現量 25
圖三、 Y1H載體使用的AtATG8f 和AtATG8h啟動子序列示意圖 26
圖四、 Split GFP-tagging確認轉錄因子在細胞核的表達 27
圖五、 AtATG8f和AtATG8h啟動子雙冷光素酶報告檢測 28
圖六、 利用煙草短暫表達系統共同表現NAC102與PHO2 29
陸、 參考文獻 30
柒、 附錄 36
附圖一、 酵母菌單雜合使用的載體 36
附圖二、 攜有AtAtATG8f和AtAtATG8h啟動子報告基因的酵母菌進行3-AT 測試 37
附圖三、AtAtATG8f和AtAtATG8h啟動子報告基因進行Y1H的3-AT濃度梯度 38
附圖四、 酵母菌單雜合篩選出與AtATG8f和AtATG8h啟動子交互作用的轉錄因子 39
附圖五、AtATG8f啟動子的Y1H陽性結果中轉錄因子在缺磷下的mRNA表現變化 40
附圖六、AtATG8h啟動子的Y1H陽性結果中轉錄因子在缺磷下的mRNA表現變化 41
附表一、 建構質體所使用的引子 42
附表三、 實驗所使用的抗體 44

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