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.

目錄ii
摘要iv
ABSTRACTv
壹、前言1
第一節研究動機1
第二節三分子綠螢光互補技術1
第三節以阿拉伯芥作為分子研究的模式生物2
第四節農桿菌與植物轉殖株2
第五節植物的蛋白質表現系統2
第六節磷酸鹽與植物調控磷酸鹽的蛋白質3
貳、材料與方法5
參、結果9
第一節以菸草測試三分子綠螢光互補技術9
一、三分子綠螢光互補技術表達核蛋白9
二、利用三分子綠螢光系統偵測植物細胞中的蛋白質交互作用9
三、利用誘導系統表達三分子綠螢光系統9
四、利用其他核蛋白驗證三分子綠螢光系統中交互作用的專一性10
1Nitrogen limitation adaptation (NLA)10
2PHR1-like 3 (PHL3)11
第二節表現三分子綠螢光系統的阿拉伯芥轉殖株12
一、篩選表現GFP 1–9的轉殖株12
二、觀察轉殖株幼苗中的蛋白質交互作用12
肆、討論14
伍、圖表16
圖一：以三明治型式的綠螢光系統表達PHR1或SPX1於菸草16
圖二：以持續系統表達PHR1與SPX1的蛋白質交互作用組合於菸草17
圖三：以誘導系統表達PHR1與SPX1的蛋白質交互作用組合於菸草18
圖四：混和誘導系統和持續系統表達PHR1與SPX1的蛋白質交互作用組合於菸草19
圖五：以不同感染濃度與誘導濃度表達PHR1與SPX1的蛋白質交互作用於菸草20
圖六：以誘導系統表達NLA與PHR1、SPX1的蛋白質交互作用組合於菸草21
圖七：以誘導系統表達PHR1與PHL3的蛋白質交互作用組合於菸草22
圖八：以誘導系統表達SPX1與PHL3的蛋白質交互作用組合於菸草23
圖九：PHR1、SPX1、PHL3之間交互作用訊號的比較箱形圖24

表一：篩選阿拉伯芥T2轉殖株(UBQ10:XVE:GFP 1–9: Bar)25
圖十：以誘導系統表達GFP 1–9、GFP10-PHR1和SPX1-GFP11的阿拉伯芥轉植株幼苗26
陸、附錄27
附錄圖一：三分子綠螢光系統交互作用示意圖27
附錄圖二：XVE/OlexA 誘導系統示意圖28
附錄圖三：質體製作29
附錄圖四：Zeiss LSM 510正立共軛交顯微鏡結構圖30
附錄圖五：螢光顯微鏡拍攝畫面擷取圖31
附錄圖六：分析不同雷射強度與螢光強度32
附錄表一：引子序列表33
附錄表二：螢光強度換算後統計表34
柒、參考文獻35

柒、 參考文獻
1. Arabidopsis Genome Initiative. (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 408(6814), 796-815.
2. Benfey, P. N., Ren, L., & Chua, N. H. (1990). Tissue-specific expression from CaMV 35S enhancer subdomains in early stages of plant development. The Embo journal, 9(6), 1677-1684.
3. Borghi, L. (2010). Inducible gene expression systems for plants. Methods Molecular Biology, 655, 65-75.
4. Cabantous, S., Nguyen, H. B., Pedelacq, J. D., Koraichi, F., Chaudhary, A., Ganguly, K., . . . Waldo, G. S. (2013). A new protein-protein interaction sensor based on tripartite split-GFP association. Scientific Reports, 3, 2854.
5. Chung, M. H., Chen, M. K., & Pan, S. M. (2000). Floral spray transformation can efficiently generate Arabidopsis transgenic plants. Transgenic Research, 9(6), 471-476.
6. Clough, S. J., & Bent, A. F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal, 16(6), 735-743.
7. Dalrymple, M. A., McGeoch, D. J., Davison, A. J., & Preston, C. M. (1985). DNA sequence of the herpes simplex virus type 1 gene whose product is responsible for transcriptional activation of immediate early promoters. Nucleic Acids Research, 13(21), 7865-7879.
8. Duan, K., Yi, K., Dang, L., Huang, H., Wu, W., & Wu, P. (2008). Characterization of a sub-family of Arabidopsis genes with the SPX domain reveals their diverse functions in plant tolerance to phosphorus starvation. Plant Journal, 54(6), 965-975.
9. Gatz, C., & Lenk, I. (1998). Promoters that respond to chemical inducers. Trends in Plant Science, 3(9), 352-358.
10. Greene, G., Gilna, P., Waterfield, M., Baker, A., Hort, Y., & Shine, J. (1986). Sequence and expression of human estrogen receptor complementary DNA. Science, 231(4742), 1150-1154.
11. Hu, C. D., Chinenov, Y., & Kerppola, T. K. (2002). Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Molecular Cell, 9(4), 789-798.
12. Kant, S., Peng, M., & Rothstein, S. J. (2011). Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in arabidopsis. PLoS Genetics, 7(3), e1002021
13. Klopffleisch, K., Phan, N., Augustin, K., Bayne, R. S., Booker, K. S., Botella, J. R., . . . Jones, A. M. (2011). Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis. Molecular Systems Biology, 7, 532.
14. Lin, W.-Y., Huang, T.-K., & Chiou, T.-J. (2013). NITROGEN LIMITATION ADAPTATION, a target of microRNA827, mediates degradation of plasma membrane–localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis. The Plant Cell Online.
15. Lin, W. Y., Huang, T. K., & Chiou, T. J. (2013). Nitrogen limitation adaptation, a target of microRNA827, mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis. Plant Cell, 25(10), 4061-4074.
16. Neumann, G. (2015). The role of ethylene in plant adaptations for phosphate acquisition in soils–A review. Front in Plant Science, 6, 1224.
17. Nilsson, L., Muller, R., & Nielsen, T. H. (2007). Increased expression of the MYB-related transcription factor, PHR1, leads to enhanced phosphate uptake in Arabidopsis thaliana. Plant Cell Environ, 30(12), 1499-1512.
18. Norris, S. R., Meyer, S. E., & Callis, J. (1993). The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Molecular Biology, 21(5), 895-906.
19. Peng, M., Hannam, C., Gu, H., Bi, Y. M., & Rothstein, S. J. (2007). A mutation in NLA, which encodes a RING-type ubiquitin ligase, disrupts the adaptability of Arabidopsis to nitrogen limitation. Plant Journal, 50(2), 320-337.
20. Pitzschke, A., & Hirt, H. (2010). New insights into an old story: Agrobacterium-induced tumour formation in plants by plant transformation. Embo j, 29(6), 1021-1032.
21. Puga, M. I., Mateos, I., Charukesi, R., Wang, Z., Franco-Zorrilla, J. M., de Lorenzo, L., . . . Paz-Ares, J. (2014). SPX1 is a phosphate-dependent inhibitor of Phosphate Starvation Response 1 in Arabidopsis. Proceedings of the National Academy of Sciences, 111(41), 14947-14952.
22. Rajulu, C. (2014). Control of phosphate starvation responses in Arabidopsis thaliana: new regulators and regulatory interactions.
23. Rubio, V., Linhares, F., Solano, R., Martin, A. C., Iglesias, J., Leyva, A., & Paz-Ares, J. (2001). A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes and Development, 15(16), 2122-2133.
24. Schlucking, K., Edel, K. H., Koster, P., Drerup, M. M., Eckert, C., Steinhorst, L., . . . Kudla, J. (2013). A new beta-estradiol-inducible vector set that facilitates easy construction and efficient expression of transgenes reveals CBL3-dependent cytoplasm to tonoplast translocation of CIPK5. Molecular Plant, 6(6), 1814-1829.
25. Schubert, D., Lechtenberg, B., Forsbach, A., Gils, M., Bahadur, S., & Schmidt, R. (2004). Silencing in Arabidopsis T-DNA transformants: the predominant role of a gene-specific RNA sensing mechanism versus position effects. Plant Cell, 16(10), 2561-2572.
26. Stam, M., Mol, J. N. M., & Kooter, J. M. (1997). The silence of genes in transgenic plants. Annal of Botany, 79(1), 3-12.
27. Sun, L., Song, L., Zhang, Y., Zheng, Z., & Liu, D. (2016). Arabidopsis PHL2 and PHR1 Act Redundantly as the Key Components of the Central Regulatory System Controlling Transcriptional Responses to Phosphate Starvation. Plant Physiolgy, 170(1), 499-514.
28. Zhang, B., Rapolu, M., Liang, Z., Han, Z., Williams, P. G., & Su, W. W. (2015). A Dual-Intein Autoprocessing Domain that Directs Synchronized Protein Co-Expression in Both Prokaryotes and Eukaryotes. Scientific Reports, 5, 8541.
29. Zuo, J., Niu, Q. W., & Chua, N. H. (2000). Technical advance: An estrogen receptor-based transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant Journal, 24(2), 265-273.