植物細胞膜磷酸轉運蛋白屬於主要易化子超家族(Major Facilitator Superfamily)成員之一，幫助植物從土壤中吸收和轉運植物內不同組織或是器官之間的無機磷酸鹽。本研究探討阿拉伯芥磷酸轉運蛋白PHOSPHATE TRANSPORTER1;1 (AtPHT1;1) 的後轉譯調控機制。我們以先前對於PHT1;1動態模擬研究所提出的機械化學協同作用(mechanochemical coupling)作為基礎，分別進行六個AtPHT1;1點突變：V42C、I141S、W304H、F311Y、A445S 和G448S 。因為pho2突變株根部能大量吸收無機磷酸鹽並將其轉移至地上部，所以我們以其作為遺傳背景，希望通過AtPHT1;1點突變降低AtPHT1;1 D38和D308 的pKa來加速轉殖株轉運磷酸至細胞內。我們發現，AtPHT1;1(WT)/pho2 #6、#7、#12、#13轉殖株中的AtPHT1;1/2/3表現量超過pho2中的表現量，且 #6、#7、#12根部磷酸濃度比pho2高，可能是過度表現PHT1;1使得磷酸在根部累積。由於AtPHT1;1 F311Y以及A445S突變轉殖株也抑制PHO1表現而影響植物磷酸分佈，因此我們無法區分出大量表達PHT1;1(WT)和PHT1;1點突變對植物磷酸累積造成影響之差別。我們也探討位於內質網的磷酸轉運蛋白運輸輔助子(PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1, PHF1) 如何協助PHT1蛋白離開內質網。先前文獻指出，酵母菌伴護蛋白Shr1能協助同屬於主要易化子超家族的胺基酸通透酶GAP1 (General amino-acid permease)的氨基和羧基結構域重組，使其成為具有功能的轉運蛋白。我們推測AtPHF1也可能參與AtPHT1;1折疊或重組。我們將AtPHT1;1分成氨基和羧基結構域，根據三分子綠螢光互補原理使其與AtPHF1共同表現於菸草下表皮細胞，觀察AtPHF1是否參與AtPHT1;1的折疊或重組，使其靶向細胞膜。除此之外，我們也設計了三組含跨膜區的不同結構域AtPHF1變異體：分別是含跨膜區的胞質液域AtPHF1(N-TM)，僅含跨膜區的AtPHF1(TM)，含跨膜區的內質網內腔域AtPHF1(TM-C)，使其分別與AtPHT1;1共同表現，觀察交互作用。實驗結果顯示， 即使與AtPHF1共變現，AtPHT1;1的氨基和羧基結構域仍然滯留在內質網；以及AtPHF1(TM)就足以與AtPHT1;1在內質網上進行交互作用，但AtPHF1(N-TM)與AtPHT1;1交互作用型態為一種OsER(organized smooth ER)的螢光模式。

The plant plasma membrane-localized PHOSPHATE TRANSPORTER1 (PHT1) is one of the major facilitator superfamily (MFS) members, which helps plants absorb inorganic phosphate (Pi) from the soil and translocate Pi between different tissues or organs. In this study, we studied the post-translational regulatory mechanisms of the Arabidopsis thaliana PHT1;1 (AtPHT1;1). A previous study of AtPHT1;1 by dynamic simulation suggested mechanochemical coupling in AtPHT1;1 for Pi transport. We generated six point-mutations of AtPHT1;1, which surround the AtPHT1;1 D38 and D308 and could decrease the pKa of AtPHT1;1 D38 and D308: V42C, I141S, W304H, F311Y, A445S and G448S and thereby presumably enhancing Pi transport into plant cells. We used pho2, a Pi accumulator, as genetic background to introduce these AtPHT1;1 variants and measured Pi concentrations and AtPHT1;1/2/3 protein amounts in the transgenic plants. Our results showed that the expression of AtPHT1;1/2/3 in AtPHT1;1(WT)/pho2 #6, #7, #12 and #13 was more than that in pho2, and the root Pi concentrations of #6, #7 and #12 were increased relative to pho2. The increased expression of AtPHT1;1 can enhance Pi accumulation in the root. Because AtPHT1;1 F311Y and A445S-expressing transgenic plants exhibit suppression of PHO1 proteins and thus affect the Pi distribution within plants, we could not distinguish the effects of expression of PHT1;1(WT) and PHT1;1 point-mutations on Pi accumulation. Moreover, we studied the function of endoplasmic reticulum (ER)-localized PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR1 (PHF1), which assists the ER exit of AtPHT1;1. The yeast chaperone protein Shr1 was shown to assist the assembly of the amino and carboxyl domains of general amino-acid permease1 (GAP1), which was the MFS amino acid permease, into a functional transporter. We speculated that AtPHF1 may also participate in the folding or assembly of AtPHT1;1. We divided AtPHT1;1 into the amino and the carboxyl domains and used the tripartite split-GFP assay in agroinfiltrated tobacco to test whether AtPHF1 assists the folding and assembly of the AtPHT1;1 halves for plasma membrane targeting. In addition, we designed and expressed three AtPHF1 variants with different structural domains containing the transmembrane region: AtPHF1(N-TM), AtPHF1(TM), AtPHF1(TM-C), and tested the interaction between AtPHT1;1 and these AtPHF1 variants. The results showed that the amino and carboxyl domains of AtPHT1;1 retained in the ER in the presence of AtPHF1 and the transmembrane region of AtPHF1 is sufficient to interact with AtPHT1;1 in the ER, but AtPHF1 with both the transmembrane and cytoplasmic domains interacts with AtPHT1;1 in a OsER-like (organized smooth ER) pattern.

摘要……………………………………………………………………………………1
ABSTRACT…………………………………………………………………………3
壹、 前言
一、 高等植物內維持磷酸鹽恆定的機制和作用………………………………5
二、 磷酸轉運蛋白PHT1;1分子結構、轉運機制和動態模擬之現況………9
三、 協助膜蛋白合成，折疊，重組的伴護蛋白………………………………14
四、 研究動機…………………………………………………………………15
貳、 實驗方法
一、 圓葉菸草與阿拉伯芥種子表面消毒……………………………………16
二、 圓葉菸草與阿拉伯芥種植與生長條件…………………………………17
三、 大腸桿菌和農桿菌之質體轉形…………………………………………17
四、 轉殖基因表現載體設計與製作…………………………………………18
五、 建立阿拉伯芥轉殖株篩選流程…………………………………………19
六、 雙分子和三分子綠螢光互補技術………………………………………20
七、 圓葉菸草短暫表達系統…………………………………………………21
八、 共軛焦顯微鏡使用………………………………………………………21
九、 磷酸濃度測量……………………………………………………………21
十、 蛋白質含量測量…………………………………………………………22
參、 實驗結果
一、 觀察AtPHT1;1點突變轉殖株之性狀及測量其磷酸濃度……………23
二、 AtPHF1 表達對於AtPHT1;1氨基和羧基結構域交互作用之影響……27
三、 AtPHF1與其他磷酸鹽恆定相關蛋白交互作用之表現位置與形態……28
肆、 討論
一、 利用阿拉伯芥PHT1;1突變轉殖株探討機械化學協同作用參與磷酸
轉運………………………………………………………………………31
二、 利用菸草短暫表達系統來觀察AtPHT1;1 與AtPHF1之交互作用
(一) AtPHF1參與AtPHT1;1氨基和羧基結構域的重組之探討………36
(二) 利用AtPHF1的不同拓撲區與AtPHT1;1之交互作用來探討
AtPHF1功能…………………………………………………………36
伍、 圖表
圖一 、AtPHT1;1突變轉殖株T2幼苗磷酸濃度測量…………………………39
圖二 、AtPHT1;1突變轉殖株T2幼苗磷酸濃度之根冠比……………………40
圖三 、AtPHT1;1氨基和羧基結構域交互作用之表現位置與形態………41
圖四 、AtPHF1的不同拓撲區分別與AtPHT1;1交互作用之表現位置與形
態…………………………………………………………………………42
圖五 、磷酸鹽恆定相關蛋白交互作用之表現位置與形態…………………43
圖六 、AtPHT1;1突變轉殖株T2幼苗中PHT1;1/2/3和PHO1蛋白表現
量…………………………………………………………………………44
陸、 附錄
附圖一、AtPHT1;1突變轉殖株T2幼苗磷酸濃度測量補充………………45
附圖二、AtPHT1;1突變轉殖株T2幼苗中PHT1;1/2/3和PHO1蛋白表現
量之補充…………………………………………………………………46
附圖三、阿拉伯芥AtPHT1;1(WT) 突變轉殖株T1之性狀和磷酸濃度分
析…………………………………………………………………………47
附圖四、阿拉伯芥AtPHT1;1 (V42C) 突變轉殖株T1之性狀和磷酸濃度
分析………………………………………………………………………48
附圖五、阿拉伯芥AtPHT1;1 (I141S) 突變轉殖株T1之性狀和磷酸濃度
分析………………………………………………………………………49
附圖六、阿拉伯芥AtPHT1;1 (W304H) 突變轉殖株T1之性狀和磷酸濃度
分析………………………………………………………………………50
附圖七、阿拉伯芥AtPHT1;1 (F311Y) 突變轉殖株T1之性狀和磷酸濃度
分析………………………………………………………………………51
附圖八、阿拉伯芥AtPHT1;1 (A445S) 突變轉殖株T1之性狀和磷酸濃度
分析………………………………………………………………………52
附圖九、阿拉伯芥AtPHT1;1 (G448S) 突變轉殖株T1之性狀和磷酸濃度
分析………………………………………………………………………53
附表一、 AtPHT1;1特定胺基酸點突變影響D38和D308之pKa…………54
附表二、 阿拉伯芥AtPHT1;1(WT)突變轉殖株T1的篩選…………………55
附表三、 阿拉伯芥AtPHT1;1(V42C)突變轉殖株T1的篩選………………56
附表四、 阿拉伯芥AtPHT1;1(I141S)突變轉殖株T1的篩選………………57
附表五、 阿拉伯芥AtPHT1;1(W304H)突變轉殖株T1的篩選……………58
附表六、 阿拉伯芥AtPHT1;1(F311Y)突變轉殖株T1的篩選………………59
附表七、 阿拉伯芥AtPHT1;1(A445S)突變轉殖株T1的篩選………………60
附表八、 阿拉伯芥AtPHT1;1(G448S)突變轉殖株T1的篩選………………61
附表九、 雙分子和三分子螢光互補技術相關之表現載體列表……………62
附表十、 In-Fusion Cloning……………………………………………………63
附表十一、 使用引子列表……………………………………………………64
柒、 參考文獻………………………………………………………………65

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