過去探討蛋白質交互作用時，常利用protein-fragment complement assay (PCA)、fluorescence resonance energy transfer (FRET)或是yeast two hybrid (Y2H)等方法。本論文探討新的PCA方法—三分子螢光互補 (tripartite split-GFP)技術偵測植物細胞內蛋白質交互作用之可行性。三分子螢光互補工具是將GFP拆解成GFP10、GFP11與GFP1-9OPT三個部分，透過蛋白質交互作用重新組裝成產生發螢光的GFP。此系統已被證明可應用在偵測大腸桿菌、酵母菌和哺乳類細胞中可溶性蛋白之間的交互作用。
先前的文獻指出磷酸運輸蛋白PHOSPHATE 1 (PHO1)和泛素接合酶PHOSPHATE 2 (PHO2)有交互作用。因此，我們選用這組蛋白質交互作用來探討tripartite split-GFP運用在植物細胞的可行性。實驗結果顯示，tripartite split-GFP系統可以偵測PHO1和PHO2在農桿菌感染的菸草細胞中產生蛋白質交互作用。PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR 1 (PHF1)也可以與PHO2、PHO1產生交互作用。除此之外，我們也發現PHF1 sandwich split-GFP在細胞質內產生螢光。Tail-anchored protein是在細胞質轉譯結束後才嵌至內質網或粒線體膜上的特殊蛋白，為單次穿膜，通常其 C端位於內質網內且較短。PHF1 與tail-anchored protein的條件相符。我們推測PHF1 sandwich在運往內質網之前，已在細胞質中進行螢光互補。因為PHO2與內質網上的 PHF1，以及和PHF1蛋白質序列相似的SEC12、SEC12-like皆產生交互作用，我們仍須探討PHO2是否易與在內質網上的許多蛋白有非專一性的交互作用。

In the past, many methods are developed to detect protein-protein interactions (PPIs), such as protein-fragment complement assay (PCA), fluorescence resonance energy transfer (FRET), and yeast two hybrid (Y2H). The usage of tripartite split-GFP has been applied in E. coli, mammalian cells, and Saccharomyces cerevisiae for detecting PPI of soluble proteins. In this study, we validated the use of this new method in the detection of membrane PPI in planta.
We chose the Arabidopsis phosphate homeostasis-related proteins, the phosphate transporter PHOSPHATE 1 (PHO1) and the ubiquitin conjugase PHOSPHATE 2 (PHO2), which were shown to interact in previous studies, to evaluate whether this tool can be used in planta. We demonstrated that the tripartite split-GFP is a reliable technique for detecting PPI of membrane proteins in agro-infiltrated tobacco leaves. Moreover, we found that PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1) can interact with PHO2 and PHO1. We also detected the signal of PHF1 sandwich split-GFP in the cytosol. The topology of PHF1 is predicted to have only one transmembrane domain with a short carboxyl-terminus. It is likely a tail-anchored protein translated in the cytosol and then inserted into the ER membranes. We proposed that the PHF1 sandwich split-GFP undergoes fluorescence complementation in the cytosol before trafficking to the endoplasmic reticulum (ER) membrane. We also found that PHO2 interacts with the guanine nucleotide-exchange factor SEC12 and SEC12-like proteins at the ER, which share amino acid sequence homology with PHF1. We need to study whether PHO2 is prone to have non-specific interactions with many ER membrane proteins.

目 錄
摘要 1
ABSTRACT 2
壹、 前言
一、 植物內維持磷酸恆定的蛋白質 3
二、 探討蛋白質交互作用之偵測方法 4
三、 農桿菌感染植物細胞 8
四、 菸草短暫表達蛋白質系統 9
五、 植物細胞誘導蛋白質表現系統 9
六、 研究動機 10
貳、 實驗結果
一、 PHO1、PHO2和PHF1的split-GFP三明治螢光蛋白表現 11
二、 PHO1和PHO2的寡聚物形成 11
三、 PHO1和PHO2在菸草短暫表達系統中的交互作用 12
四、 PHF1、SEC12和SEC12-like分別和PHO2、PHO1 13
和GONST1在菸草短暫表達系統中的交互作用
參、 討論
一、 PHO2與其他蛋白質交互作用之探討 15
二、 PHF1為 tail-anchored protein之探討 15
三、 GFP-SEC12-like在細胞內表現位置之探討 16
肆、 實驗方法
一、 菸草種子表面消毒 17
二、 植物種植與生長條件 17
三、 大腸桿菌和農桿菌之轉形 17
四、 菸草(N. benthamiana)短暫表達蛋白質 18
五、 共軛焦顯微鏡使用 18
伍、 圖表
圖一 、Split-GFP三明治螢光蛋白表現 19
圖二 、偵測PHO1和PHO2寡聚物形成 20
圖三 、菸草細胞中PHO1和PHO2交互作用位置 21
圖四 、PHO1、PHO2和PHF1之間的交互作用 22
圖五 、PHF1拓樸對於三分子螢光互補工具偵測蛋白質交互作用 23
的影響
圖六 、PHF1拓樸對於三分子螢光互補工具偵測蛋白質交互作用 24
之定量統計分析圖
圖七 、SEC12和SEC12-like綠螢光融合蛋白表現 25
圖八 、測試PHF1、SEC12和SEC12-like之非專一性蛋白交互 26
作用
表一 、PHF1拓樸對於三分子螢光互補工具偵測蛋白質的交互 27
作用之定量分析
陸、 附錄
圖一、 PHF1 拓撲預測 28
圖二、 PHF1親緣樹分析 29
表一、 PHF1、SEC12和SEC12-Like序列比對 30
表二、 三分子螢光互補工具相關之表現載體 31
表三、 引子與含切位蛋白質序列 32
柒、 參考文獻 34

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