摘要
三分子綠螢光互補技術 (tripartite split-GFP complementation assay) 是一種新的蛋白質交互作用工具，改良自傳統的雙分子螢光互補技術bimolecular fluorescence complementation (BiFC)。BiFC技術是將GFPN和GFPC兩個片段接在不同的兩個不同蛋白質上，易有干擾目標蛋白質的折疊、運送與功能等問題。三分子綠螢光互補技術改成GFP10 (GFP殘基194–212) 和GFP11 (GFP殘基213–233) 接在目標蛋白上，讓接在蛋白質上的片段縮小，從而改善上述的問題。我們欲探討此技術可否應用於植物細胞中，利用阿拉伯芥 (Arabidopsis thaliana) 中參與缺磷反應的核蛋白: phosphate starvation response 1 (PHR1) 和SPX domain-containing protein 1 (SPX1)進行測試。我們將GFP10 接到PHR1/SPX1的胺基端和GFP11接到羧基端，短暫表達於菸草的下表皮。在螢光顯微鏡觀察下可以發現，綠色螢光訊號出現在細胞核中，因此三分子綠螢光互補技術可以正確的表達PHR1/SPX1，我們欲了解此系統在植物細胞中偵測蛋白交互作用的情況，利用過去已知PHR1與SPX1會結合，分別將PHR1和SPX1的胺基端接上GFP10或羧基端接上GFP11，觀察之間交互作用情形。結果不僅顯示PHR1和SPX1有交互作用，也證實過去的推測PHR1在植物體內會形成二聚體以及新發現SPX1會形成二聚體或寡聚體。
我們進一步藉由表現阿拉伯芥核蛋白nitrogen limitation adaptation (NLA/ BAH1)，並觀察NLA與PHR1、SPX1交互作用的情形，來分析此系統的專一性。結果顯示，NLA與PHR1、SPX1都有交互作用。我們另外選擇PHR1的家族蛋白PHR1-like 3 (PHL3)，發現PHL3雖然與PHR1、SPX1都有交互作用，但在相同實驗條件下，SPX1和PHL3之間的螢光強度最強，其次是SPX1和PHR1，再來是PHR1二聚體，而PHL3和PHR1的組合結果最弱。
另一方面，我們以β-estradiol誘導GFP10-PHR1、SPX1-GFP11和GFP1–9表現於阿拉伯芥轉殖株。我們只有觀察到少數阿拉伯芥的幼苗根毛細胞核中有螢光，而且訊號的強度微弱，顯示三分子綠螢光互補技術在轉植株上並不如預期地成功。

ABSTRACT
Tripartite split-GFP complementation assay is a new protein-protein interaction technique improved from bimolecular fluorescence complementation (BiFC). Tripartite split GFP system is composed of a larger fragment, GFP1-9 (residues 1–193), and two shorter β-strands : GFP10 (residues 194–212) and GFP11 (residues 213–233). There are some disadvantages of BiFC because BiFC is based on bulky fragments that may increase the difficulty of protein folding and interfere with protein function. To test whether tripartite split-GFP is a useful tool in planta, we first chose two proteins involved in the phosphate starvation reponse: the phosphate starvation response 1 (PHR1) and the SPX domain-containing protein 1 (SPX1) proteins. The PHR1 and SPX1 sandwitch proteins (GFP10-PHR1-GFP11 and GFP10-SPX1-GFP11) were used for transient expression in tobacco leaves and localized in the nucleus. Next, we confirmed the previous results that PHR1 interacts with SPX1 using tripartite split-GFP system. This assay showed that PHR1 and SPX1 can form homo-dimer/oligomers itself.
We used the Arabidopsis nitrogen limitation adaptation (NLA/BAH1) to verify the specificity of the interaction between PHR1 and SPX1. Our results showed that NLA interacts with both PHR1 and SPX1. In addition, we chose the other PHR1 family protein PHR1-like 3 (PHL3) to test the interaction of PHL3 with PHR1 and SPX1 respectively. The signal strength between PHR1, SPX1 and PHL3 are different. From the strongest to the weakest is PHR1 and PHL3, PHR1 dimer, SPX1 and PHR1, and then the signal of SPX1 and PHL3 was the strongest.
Furthermore, we generated and obtained the Arabidopsis transgenic liens overexpessing GFP10-PHR1, SPX1-GFP11 and GFP1–9. Confonal analysis of these lines showed few signals in the root hair of seedlings, indicating that the tripartite spli-GFP system used in transgenic plants of Arabidopsis needs to be improved.

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