1. Bachmair, A., Novatchkova, M., Potuschak, T., and Eisenhaber, F. (2001).
Ubiquitylation in plants: a post-genomic look at a post-translational modification.
TRENDS in Plant Science. 6, 463-470.
2. Bai, C., Sen, P., Hofmann, K., Ma, L., Goebl, M., Harper, J.W., and Elledge, S.J.
(1996). Skp1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box. Cell. 86: 263-274.
3. Bailey, T.L., and Gribskov, M. (1998). Methods and statistics for combining motif match scores. Journal of computational biology. 5: 211-221.
4. Brosché, M., Schuler, M.A., Kalbina, I., Connor, L., and Strid, Å. (2002). Gene regulation by low level UV-B radiation: identification by DNA array analysis.
Photochemical and photobiological sciences. 1: 656-664.
5. Burke, T.J., Callis, J., and Vierstra, R.D. (1988). Characterization of a TpolyubiquitinT gene from Arabidopsis thaliana. Molecular and general genetics. 213:435-443.
6. Callis,T J., Raasch, J.A., and Vierstra, R.D. (1990). TUbiquitin extension proteins of Arabidopsis thaliana. Structure, localization, and expression of their promoters in transgenic tobacco. Journal of biological chemistry. 265: 12486-12493.
7. Callis, J., Carpenter, T., Sun, C.W., and Vierstrat, R.D. (1995). Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia. Genetics. 139: 921-939.
8. Carter, C., Graham, R.A., and Thornburg, R.W. (1998). Arabidopsis thaliana contains a large family of germin-like proteins: characterization of cDNA and genomic sequences encoding 12 unique family members. Plant molecular biology. 38: 929-943.
9. Catic, A., and Ploegh, H.L. (2005). Ubiquitin - conserved protein or selfish gene?
Trends in biochemical sciences. 30: 600-604.
10. Chae, E., Tan, Q.K.G., Hill, T.A., and Irish, V.F. (2008). An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development. Development.
135: 1235-1245.
11. Chau, V., Tobias, J.W., Bachmair, A., Marriott, D., Ecker, D.J., Gonda, D.K., and Varshavsky, A. (1989). A multiubiquitin chain is confined to specific lysine in a targeted short-lived protein. Science. 243: 1576-1583.
12. Chomczynski, P., and Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Analytical biochemistry.
162: 156-159.
13. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. TThe plant journalT. 16:
735-743.
14. Crooks, G.E., Hon, G., Chandonia, J.M., and Brenner, S.E. (2004). WebLogo: a sequence logo generator. Genome research.14: 1188-1190.
15. Fan, W.H., and Dong, X.N. (2002). In vivo interaction between NPR1 and transcription factor TGA2 leads tosalicylic acid–mediated gene activation in Arabidopsis. The plant cell. 14:1377-1389.
16. Finley, D., Özkaynak, E., and Varshavsky, A. (1987). The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell. 48:
1035-1046.
17. Finley, D., and Chau, V. (1991). Ubiqitination. TAnnual review of cell biologyT. 7:
25-69.
18. Finley, D., Sadis, S., Monia, B.P., Boucher, P., Ecker, D.J., Crooke, S.T., and Chau, V. (1994). Inhibition of proteolysis and cell cycle progression in a
multiubiquitination- deficient yeast mutant. Molecular and cell biology. 14:
5501-5509.
19. Fisk, H.A., and Yaffe, M.P. (1999). A role for ubiquitination in mitochondrial inheritance in Saccharomyces cerevisiae. TJournal of cell biology.T 145: 1199-208.
20. Galan, J.M., and Haguenauer-Tsapis, R. (1997). Ubiquitin Lys63 is involved in ubiquitination and endocytosis of a yeast plasma membrane protein. The TEMBOT journal. 16: 5847-54.
21. Glickman, M.H., and Ciechanover, A. (2002). The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. TPhysiological reviewsT. 82: 373-428.
22. Goldstein, G., Scheid, M., Hammerling, U., Schlesinger, D.H., Niall, H.D., and Boyse, E.A. (1974). Isolation of a polypeptide that has lymphocyte- differentiating properties and is probably represented universally in living cells. TProceedings of the national academy of sciences of the United States of America.T 72: 11-15.
23. Gray, W.M., del Pozo, J.C., Walker, L., Hobbie, L., Risseeuw, E., Banks, T., Crosby, W.L., Yang, M., Ma, H., and Estelle, M. (1999). Identification of an SCF ubiquitin ligase complex required for auxin response in Arabidopsis thaliana. Genes and development. 13: 1678-1691.
24. Hershko, A., Heller, H., Elias, S., and Ciechanover, A. (1983). Components of ubiquitin-protein ligase system: resolution, affinity purification and role in protein breakdown. TThe journal of biological chemistry.T 258: 8206-8214.
25. Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984). ATP-dependent degradation of ubiquitin-protein conjugates. Proceedings of the national academy of sciences. 81: 1619-1623.
26. Hershko, A., and Ciechanover, A. (1992). The ubiquitin system. TAnnual review of clinical biochemistry.T 61: 761-807.
27. Hochstrasser, M. (2006). Lingering mysteries of Ubiquitin-Chain assembly. Cell. 124:
27-34.
28. Hoege, C., Pfander, B., Moldovan, G.L., Pyrowolakis, G., and Jentsch, S. (2002).
RAD6- dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature. 419: 135-41.
29. Hofmann, K., and Bucher, P. (1998). The PCI domain: a common theme in three multi-protein complexes. TTrends in Biochemical Sciences.T 23: 204-205.
30. Imaizumi, T., Tran, H.G., Swartz, T.E., Briggs, W.R., and Kay, S.A. (2003). FKF1 is essential for photoperiodic-specific light signalling in Arabidopsis. Nature. 426:
302-306.
31. TJefferson, R.A., TKavanagh, T.A., and Bevan, M.W. (T1987). GUS-fusions:
Tβ-glucuronidase as a sensitive and versatile fusion marker in higher plants. The EMBO journal. 6: 3901-3907.T
32. Lai, Z., Ma, W., Han, B., Liang, L., Zhang, Y., Hong, G., and Xue, Y. (2002). An F-box gene linked to the self-incompatibility (S) locus of Antirrhinum is expressed specifically in pollen and tapetum. Plant molecular biology. 50: 29-42.
33. Murashige, T., and Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. TPhysiologia plantarumT. 15: 473-497.
34. Ni, W., Xie, D., Hobbie, L., Feng, B., Zhao, D., Akkara, J., and Ma, H. (2004).
Regulation of flower development in Arabidopsis by SCF complexes. Plant physiology. 134: 1574-1585.
35. Norris, S.R., Meyer, S.E., and 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: 895-906.
36. Osterlund, M.T., Hardtke, C.S., Wei, N., and Deng, X.W. (2000). Targeted destabilization of HY5 during light-regulated development of Arabidopsis. Nature.
405: 462-466.
37. Özkaynak, E., Finley, D., and Varshavsky, A. (1984). The yeast ubiquitin gene:
head-to-tail repeats encoding a polyubiquitin precursor protein. Nature. 312: 663-666.
38. Patton, E.E., Willems, A.R., and Tyers, M. (1998). Combinatorial control in ubiquitin-dependent proteolysis: don't Skp the F-box hypothesis. Trends in genetics.
14: 236-243.
39. del Pozo, J.C., Timpte, C., Tan, S., Callis, J., and Estelle, M. (1998). The ubiquitin-related protein RUB1 and auxin response in Arabidopsis. TScience. 280:
1760-1763.T
40. Rose, A.B., Elfersi, T., Parra, G., and Korf, I. (2008). Promoter-proximal introns in Arabidopsis thaliana are enriched in dispersed signals that elevate gene expression.
The plant cell. 20: 543-551.
41. Schlessinger, D.H., Goldstein, G., and Niall, H.D. (1975). The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells. Biochemistry. 14: 2214-2218.
42. Schwechheimer, C., Serino, G., Callis, J., Crosby, W.L., Lyapina, S., Deshaies, R.J., Gray, W.M., Estelle, M., and Deng, X.W. (2001). Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFPTIR1P in mediating auxin response. TScience.
292: 1379- 1382.TT
43. Shanklin, J., Jabben, M., and Vierstra, R.D.T (1987).TTRed light-induced formation of ubiquitin-phytochrome conjugates: identification of possible intermediates of
phytochrome degradation.T The proceedings of the national academy of sciencesT. 84:
359-363.T
44. Sun, C.W., and Callis, J. (1997). Independnet modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes.
TThe plant journal.T 11: 1017-1027.
45. Tornero, P., Merritt, P., Sadanandom, A., Shirasu, K., Innes, R.W., and Dangl, J.L. (2002). RAR1 and NDR1 contribute quantitatively to disease resistance in
Arabidopsis, and their relative contributions are dependent on the R gene assayed. The
Plant Cell. 14: 1005-1015.
46. Xu, L., Liu, F., Lechner, E., Genschik, P., Crosby, W.L., Ma, H., Peng, W., Huang, D., and Xie, D. (2002). The SCFPCOI1P ubiquitin-ligase complexes are required for jasmonate response in Arabidopsis. The plant cell. 14: 1919-1935.
47. Yin, X.J., Volk, S., Ljung, K., Mehlmer, N., Dolezal, K., Ditengou, F., Hanano, S., Davis, S.J., Schmelzer, E., Sandberg, G., Teige, M., Palme, K., Pickart, C. and Bachmaira, A. (2007). Ubiquitin lysine 63 chain-forming ligases regulate apical dominance in Arabidopsis. The plant cell. 19: 1898-1911.
48. Zimmermann, P., Hirsch-Hoffmann, M., Hennig, L., and Gruissem, W. (2004).
GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant physiology. 136: 2621-2632.
表格與圖片
名稱 序列內容 目的
UBQ3 F2 gcctgcagGTCGGGCAACAAAAATCCTG 放大P,5,I序列 UBQ3 F3 GGATGGTCGCACACTTGCAG 進行RT-PCR實驗 UBQ3 R1 gcggatccCTGAAATAAAACAATAGAACAAGTAG 放大P,5,I序列 UBQ3 R2 gcggatccGAAAGAGAGAGTCGCGAGAG 放大P,5序列 UBQ3 R3 CGTTACAAGCCCAAACGAAGG 進行RT-PCR實驗 UBQ3 R4 gcggatccGAATTATTATCTTCCACACGAAG 放大P序列 UBQ4 F1 gcctgcagCTCGACATTTGCACTAATAAGC 放大P,5,I序列 UBQ4 F2 gcctgcagGCTTCTCTTAGTTTCATTGTTC 放大3,P,5,I序列 UBQ4 F3 CAGATATTTGTGAAGACCCTTAC 進行RT-PCR實驗 UBQ4 R1 gcggatccCTGCGAAGGAAGAGAGCAAG 放大P,5,I序列 UBQ4 R3 gcggatccCTTGAAAGAGAGAATCGTTCG 放大P,5序列 UBQ4 R4 GTTGAAACAGCAACCAAAGGTTC 進行RT-PCR實驗 UBQ4 R5 gcggatccGATAATACTTCGCTAGAAAATAG 放大P序列 UBQ10 F1 CTTCGTCAAGACTTTGACCG 進行RT-PCR實驗 UBQ10 R1 CTTCTTAAGCATAACAGAGACGAG 進行RT-PCR實驗 Actin7 F1 GGAAGCTCCTGGAATCCATG 進行RT-PCR實驗 Actin7 R1 GACAAGACACACTTAGAAGC 進行RT-PCR實驗 HSP70 F1 GATGCAATCGACCAAGCTATTG 進行RT-PCR實驗 HSP70 R1 CTTTCATAGGTCAGAGCGAG 進行RT-PCR實驗 表一 專一引子對序列資料
專一引子對序列資料包含名稱、序列內容與實驗目的。名稱欄內F 代表正向引 子，R 代表反向引子。序列內容中的大寫字母為目標基因序列，小寫字母為限 制酵素序列。實驗目的欄內P 代表啟動子，5 表示五端未轉譯區域，I 表示內插 子，3 表示三端未轉譯區域。
表二 UBQ3 與 UBQ4 轉殖株之 GUS 定量結果
利用偵測轉殖株所表現的GUS 活性，研究 UBQ3 與 UBQ4 的基因表現除了受啟 動子調控以外，內插子與五端未轉譯區域是否也有調控的能力。實驗使用14 日 齡之轉殖株，將根與葉片分開測定，各品系皆各使用4~6 株，野生型(Col.)則使 用3 株。而每株樣品皆重複測定 3 次後，計算 GUS 活性(GUS activity)的平均值 (mean)與標準誤差(stander error)，實驗結果單位為 pmol /min /μg protein。並以野 生型GUS 活性做基準，分析 UBQ3 與 UBQ4 轉植株 GUS 活性的表現(Fold change，轉殖株/野生型)。
Leaves Roots Name Gus activity
Mean ± Stander Error Fold change Name Gus activity
Mean ± Stander Error Fold change U3.1 24.07±1.87 80.2 U3.1 40.19±3.13 19.6 U3.2 0.65±0.05 2.1 U3.2 2.08±0.16 1.01 U3.3 0.4±0.02 1.3 U3.3 1.96±0.15 0.95 U4.1 4.67±0.69 15.5 U4.1 6.36±0.41 3.1 U4.4 22.43±3.18 74.7 U4.4 15.36±0.49 7.5 U4.5 7.78±1.74 25.9 U4.5 4.97±0.89 2.4 U4.2 30.45±2.2 101.5 U4.2 28.76±2.1 14.02 U4.3 15.81±0.76 52.7 U4.3 11.27±0.25 5.5 U4.6 14.61±2.53 48.7 U4.6 10.74±1.84 5.2 Col. 0.30±0.019 1 Col. 2.05±0.12 1
Promoter UBQ3 UBQ4 UBQ10 UBQ11 UBQ14
UBQ3 8 14 10 12
UBQ4 8 8 8
UBQ10 21 31
UBQ11 16
UBQ14
5’-UTR UBQ3 UBQ4 UBQ10 UBQ11 UBQ14
UBQ3 2 2 2 2
UBQ4 3 2 2
UBQ10 2 2
UBQ11 2
UBQ14
Intron UBQ3 UBQ4 UBQ10 UBQ11 UBQ14
UBQ3 4 4 4 4
UBQ4 6 6 6
UBQ10 6 6
UBQ11 6
UBQ14
表三 阿拉伯芥聚泛素基因序列之相似片段數目
五個聚泛素基因序列分為啟動子(Promoter)、五端未轉譯區域(5’-UTR)與內插子 (Intron)三部份作比對，後計算其相似序列的片段數目。啟動子相似片段的長度 自10~45 bp 不等，五端未轉譯區域為 11~49 bp，內插子為 11~40 bp。
位置 基因 序列資訊 4~30 bp UBQ3 tattttctgattctttttgtttttgat 1
4~30 bp UBQ4 cctgtttctcttattgttatttctatt
61~140 bp UBQ3 gattattgaatcttttgtataaattgcttttgacaatattgttcgtttcgtcaatccagcttctaaattttgtcctgatt 2
61~140 bp UBQ4 tgtatgatgtccttggatgattgaatcgggaaattattgattccgtttcatctatgcttttctcgattctttgatttat 175~251 bp UBQ3 taatttcttgcttgattgtgaaattaggattttcaaggacgatctattcaatttttgtgttttctttgttcgattc 3
170~246 bp UBQ4 actaagatctctcaattcgtagttcgtcaggtttttatgtttgtagtgatctttgttttgataagaagtaatatcg 306~376 bp UBQ3 ttctcttacggcttttgatttggtatatgttcgctgattggtttctacttgttctattgttttatttcag 4
316~386 bp UBQ4 gatttcttatcacagtttctagtttggttatttttgctgatcgattactacttgctctcttccttcgcag
表四 UBQ3與UBQ4內插子之相異序列
利用MEME軟體比對出UBQ3與UBQ4內插子序列相似處後，扣除其相似處，得 到四組內插子序列相異處，並將這四組序列的資訊與所在位置整理成表。
UBQ3 UBQ4
1~100 bp 18.95 20.81
101~200 bp 9.339 21.36 201~300 bp 25.303 11.033
301~ end 8.28 8.966
50~150 bp 9.4 17.897
150~250 bp 14.055 13.548 250~350 bp 20.386 11.529
表五 UBQ3與UBQ4內插子序列之強化訊息分析
已有研究證明許多基因的內插子序列含有增強基因表現的強化訊息，所以我們 利用IMEter分析UBQ3與UBQ4的內插子序列是否有強化訊息存在。取UBQ3與 UBQ4的內插子序列，分為七份，以每段長度約100 bp進行分析，並將所得之數 值整理為表。分析結果的數值越高者，表示序列帶有強化訊息的機率越高。在 UBQ3的內插子序列中，以第1~100 bp與200~350 bp的位置帶有強化訊息的機率 最高，而UBQ4的第100~200 bp內插子序列的機率最高。
Mutant
Mean ± Stander Error
Stimulus
Mean ± Stander Error
UBQ3 1.25±0.09 1.2±0.06
UBQ4 1.08±0.024 1.07±0.023
UBQ10 1.03±0.014 1.01±0.012
UBQ11 1.11±0.04 1.14±0.03
UBQ14 1.02±0.02 1.11±0.025
表六 聚泛素基因表現之微陣列資料分析
微陣列資料是聚泛素基因在不同突變株(mutant)中或接受環境刺激(stimulus)後 的表現，扣除對照組所得的比值(Ratio)。我們整理所得的數值並計算平均值 (mean)與標準誤(stander error)，並從結果分析聚泛素基因的表現。基因表現的分 數以1為標準值，當平均值越高或越低，代表此基因的表現量越容易受外在因素 的影響或其他基因的改變而有波動；標準誤越大，代表基因表現量的變異性越 大。在此分析中，所有聚泛素基因的表現量都相當穩定，但是聚泛素基因中UBQ3 的表現量最容易受到其他因素所影響；而UBQ10則是表現最穩定的聚泛素基因。
(Hochstrasser M., 2006) 圖一 泛素化作用機制
首先，泛素羧基端上的甘胺酸會和泛素激活酵素(E1)的半胱胺酸結合，促使泛 素活化。接著，泛素接合酵素(E2)，會取代E1與泛素結合，讓E1恢復原本的狀 態以再次進行活化泛素的工作。之後，泛素連接酶(E3)會和帶有泛素的E2接合，
幫助泛素鍵結到蛋白受質(S)上。而E3分為二種，ㄧ種是RING (Really Interesting New Gene) E3，另一種為HECT (Homologous to E6AP C-Terminus) E3。RING E3 只幫助E2和蛋白受質接合，泛素會直接從E2轉移到蛋白受質上；但若是和HECT E3作用，泛素會先從E2接到E3上後，再與蛋白受質接合，以進行泛素化作用。
圖二 阿拉伯芥聚泛素基因與泛素延伸基因之結構
阿拉伯芥中共包含五個聚泛素基因(UBQ3、UBQ4、UBQ10、UBQ11與UBQ14) 與五個泛素延伸基因(UBQ1、UBQ2、UBQ5、UBQ6與UBQ5/6 like)。(A)聚泛素 基因是重複2個以上的泛素序列所組成，聚泛素基因分類的特徵是各基因所生成 蛋白質之羧基端會帶有不同的胺基酸，UBQ3/UBQ4接有絲胺酸(serine, S)和苯基 丙胺酸(phenylalanine, F)，而另一類只接有苯基丙胺酸(phenylalanine, F)。(B)泛 素延伸基因是由一個泛素基因接著一個形成核醣蛋白(ribosome protein)的序列 所組成。這一類可以再分成二種，第一種是泛素序列接一個形成52個胺基酸的 核醣蛋白基因，稱為RPL40 (60S ribosomal protein L40)，而另一種則是形成ㄧ個 81個胺基酸核醣蛋白，稱為RPS27 (40S ribosomal protein S27)。
圖三 UBQ3啟動子連接不同組合五端未轉譯區域/內插子之構築序列
A:研究五端未轉譯區域/內插子是否會影響UBQ3基因表現所構築的三種不同質 體。U3.1為全長，U3.2為去除內插子序列，而U3.3則是只有啟動子序列之質體。
紅色箭頭為目標序列的引子。。B:各質體所篩選出之14日齡轉殖株，經由GUS 染色後的結果，其編號所代表的質體內容與A相同。U3.1-1/U3.1-2都是轉殖U3.1 序列的轉殖株，但因插於不同基因體(genome)位置中，所以加上編號區別，而 U3.2-1/U3.2-2、U3.3-1/U3.3-2也是相同原因。UBQ3在植物全株幾乎都有表現，
但去除內插子後，表現量有明顯的下降。再進一步去除五端未轉譯區域後，仍 維持與去除內插子相似的表現量。C:各轉殖株的花朵組織，經由GUS染色後的 結果，其編號所代表的質體內容與A相同。UBQ3會在萼片、花絲、花藥與柱頭 等部份表現。但去除內插子後，在花朵上的表現幾乎完全消失。且進一步去除 五端未轉譯區域後，仍維持與去除內插子相似的表現量。
圖四 UBQ4啟動子連接不同組合五端未轉譯區域/內插子之構築序列
A:研究五端未轉譯區域/內插子是否會影響UBQ4基因表現所構築的六種不同質 體。U4.1、U4.4與U4.5分別是全長、去除內插子與只有啟動子序列；U4.2、U4.3 與U4.6則是全長、去除內插子與只有啟動子序列外，另加上前方之GLP3基因的 三端未轉譯區域(3’UTR)。紅色箭頭為目標序列的引子。B:各質體所篩選出之14 日齡轉殖株，經由GUS染色後的結果，其編號所代表的質體內容與A相同。
U4.1-1/U4.1-2都是轉殖U4.1序列的轉殖株，但因插於不同基因體(genome)位置 中，所以加上編號區別，而U4.3-1/U4.3-2、U4.4-1/U4.4-2、U4.6-1/U4.6-2也是 相同原因。UBQ4的基因表現在營養組織中，無論是去除內插子或進一步去除五 端未轉譯區域皆無明顯的影響。C:各轉殖株的花朵組織，經由GUS染色後的結 果。從圖片結果可發現，UBQ4會在柱頭、花絲、萼片與成熟的花瓣等部分表現。
U4.1-1/U4.1-2都是轉殖U4.1序列的轉殖株，但因插於不同基因體(genome)位置 中，所以加上編號區別，而U4.3-1/U4.3-2、U4.4-1/U4.4-2、U4.6-1/U4.6-2也是 相同原因。UBQ4的基因表現在營養組織中，無論是去除內插子或進一步去除五 端未轉譯區域皆無明顯的影響。C:各轉殖株的花朵組織，經由GUS染色後的結 果。從圖片結果可發現，UBQ4會在柱頭、花絲、萼片與成熟的花瓣等部分表現。