綠豆低溫逆境基因篩選及泛素/蛋白解體途徑之研究

簡易檢索 / 詳目顯示

回結果列表

研究生：	王志強 Chih-Chiang Wang
論文名稱：	綠豆低溫逆境基因篩選及泛素/蛋白解體途徑之研究 The expression of Vigna radiata genes selected from temperature stress and the ubiquitin/26S proteasome pathway under low temperature
指導教授：	林彩雲 Tsai-Yun Lin
口試委員:
學位類別：	碩士 Master
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2005
畢業學年度：	93
語文別：	英文
論文頁數：	65
中文關鍵詞：	泛素、低溫逆境、微陣列晶片
外文關鍵詞：	ubiquitin, cold stress, microarray
相關次數：	點閱：2 下載：0
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

為探討綠豆低溫處理(4°C)所誘導表現基因，我們建立了1012個獨立的ESTs，含391個正常生長發芽三天的綠豆ESTs，和621個ESTs利用扣減法從發芽三天的綠豆分別來自低溫和高溫，利用cDNA 微陣列晶片技術去分析在低溫處理誘導的基因，從初步結果挑選了一些在低溫處理不同時間點被誘導的基因。本研究的重點在分析幾個有關泛素(ubiquitin)/蛋白解體(26S proteasome)途徑的基因，包括了泛素、泛素結合酶(ubiquitin conjugating enzyme)、泛素連接酶(ubiquitin ligase)、蛋白解體次單位(20S core protease、19S regulatory particle)，利用北方墨點法分析在不同溫度下處理的mRNA表現量，找到泛素結合基因在低溫處理下mRNA有明顯的表現量，目前在針對低溫下這途徑的研究相當的少，而這個結果建議只在低溫下這些泛素結合基因扮演終結蛋白質的角色。

To identify cold-inducible genes in mungbean, we prepared a mungbean cDNA set containing independent cDNAs combined with 391 cDNAs isolated from 3 day old seedlings and 621 cDNA from subtraction for cold- and heat-treatment. This study focused on some of the candidate genes obtained from our preliminary microarray data. We aimed to study the mungbean genes involved in the ubiqitin/26S proteosome pathway from our cold-subtracted library, including the ubiquitin, ubiquitin conjugating enzyme, RING type ubiquitin ligase, 20S core protease, and 19S regulatory particle. We confirmed those genes using Northern blot analysis and finally identified the cold-induced ubiquitin conjugation genes. These results suggest that the ubiquitin conjugation genes have roles during cold stress through the turnover of protein(s) via the ubiquitin/26S proteasome pathway, but not heat.

Abstract in Chinese••••••••••••••••••••••••••i
Abstract ••••••••••••••••••••••••••••••ii
Acknowledgement••••••••••••••••••••••••••iii
Abbreviations ••••••••••••••••••••••••••• iv
Table of Contents•••••••••••••••••••••••••• v
List of Figures & Tables••••••••••••••••••••••• vii

Introduction ••••••••••••••••••••••••••••1
Materials and Methods ••••••••••••••••••••••• 12
1. Plant growth and stress treatments•••••••••••••••••••  12
2. Subtraction library••••••••••••••••••••••••• 13
 2.1 Subtraction cDNA••••••••••••••••••••••••  13
 2.2 Ligation•••••••••••••••••••••••••••• 13
 2.3 Competent Cell Preparation•••••••••••••••••••• 13
 2.4 Bacterial Transformation••••••••••••••••••••••14
 2.5 Mini-preparation of Plasmid DNA••••••••••••••••••15
 2.6 Large number of Mini-preparation of Plasmid DNA•••••••••••15
 2.7 Restriction enzyme Digestion••••••••••••••••••••16
 2.8 Double-stranded DNA Sequencing••••••••••••••••••17
 2.9 EST library construction••••••••••••••••••••••18
3. Total cellular RNA extraction •••••••••••••••••••• 18
4. RT-PCR •••••••••••••••••••••••••••••19
5. Synthesis of DIG-labeled DNA Probes by PCR ••••••••••••• 20
6. Northern blotting ••••••••••••••••••••••••• 21
7. cDNA Microarry•••••••••••••••••••••••••• 23
 7.1 PCR Products Reaction for DNA Amplification••••••••••••23
 7.2 Preparation of Microarray•••••••••••••••••••• 24
 7.3 Hybridization••••••••••••••••••••••••• 24
 7.4 Scan•••••••••••••••••••••••••••••24
 7.5 Analysis••••••••••••••••••••••••••• 25

Results ••••••••••••••••••••••••••••••26
Discussion •••••••••••••••••••••••••••••33
Tables and Figures•••••••••••••••••••••••••• 40
References •••••••••••••••••••••••••••• 55





List of Figures & Tables

Table 1. The subtraction condition including different temperatures, germination stages, and time intervals•••••••••••••••••••••• 40

Table 2. ESTs of different function were involved up-regulation under cold stress•••••••••••••••••••••••••••••••• 45

Fig. 1 Functional categories of the NG1C library •••••••••••••••41

Fig. 2 Functional categories of the NG3C library •••••••••••••••42

Fig. 3 Functional categories of the NG3H library •••••••••••••••43

Fig. 4 The morphology of mungbean at different temperatures for different periods•••••••••••••••••••••••••••••••44

Fig. 5 Comparison of the amino acid sequences of the NG3C120, NG3C153, NG3C195, NG1C339, and NG3C024 to their Arabidopsis orthologues in the TAIR database •••••••••••••••••••••••••••46

Fig. 6. Northern blot analysis of the NG1C120 (polyubiquitin) transcripts at different time and different temperatures •••••••••••••••48

Fig. 7 RT-PCR analysis of NG1C120 (Polyubiquitin) transcripts at different time and different temperatures••••••••••••••••••••• 49

Fig. 8 Northern blot analysis of the NG3C153 (Ubiquitin-conjugating enzyme E2) transcripts at different time and different temperatures•••••• ••50

Fig. 9 Northern blot analysis of NG3C195 (RING E3) transcripts at different time and different temperatures •••••••••••••••••••••51

Fig. 10 RT-PCR analysis of NG3C195 (RING E3) transcripts at different time and different temperatures ••••••••••••••••••••••• 52

Fig. 11 Northern blot analysis of NG1C339 (20S CP) and NG3C024 (19S RP) expression at 4°C treatments••••••••••••••••••••• 53

Fig. 12 Overview for ubiquitin/26S proteasome at cold stress•••••••••54

                                

Azevedo, C. (2001) The U-box protein family in plants. Trends Plant Sci, 6, 354-358.

Bachmair, A., Novatchkova, M., Potuschak, T. and Eisenhaber, F. (2001) Ubiquitylation in plants: a post-genomic look at a posttranslational modification. Trends Plant Sci, 6, 463-470

Callis, J., Carpenter, T., Sun C.W. and Vierstra, R.D. (1995) Structure and evolution of genes encoding polyubiquitin and ubiquitin-like proteins in Arabidopsis thaliana ecotype Columbia. Genetics, 139, 921-939.

Callis, J. and Vierstra, R.D. (2000) Protein degradation in signaling. Curr. Opin. Plant Biol, 3, 381-386.

Capron, A., Okresz, L. and Genschik, P. (2003) First glance at the plant APC/C, a highly conserved ubiquitin-protein ligase. Trends Plant Sci, 8, 83-89.

Chang, S., Puryear, J. and Cairney, J. (1993) A simple and efficient method for isolating RNA from pine trees. Plant Mol. Biol, 11(2), 113-116.

Cheng, L., Watt, R. and Piper, P.W. (1994) Polyubiquitin gene expression contributes to oxidative stress resistance in respiratory yeast (Saccharomyces cerevisiae). Mol. Gen. Genet, 243, 358-362.

Chen, K.C., Lin, C.Y., Kuan, C.C., Sung, H.Y. and Chen, C.S. (2002) A novel defnsin encolded by a mungbean cDNA exhibits insecticidal activity against Bruchid. J. Agric. Food Chem, 50, 7258-7263.

Dahan, J., Etienne, P., Petitot, A.S., Houot, V., Blein J. P. and Suty, L. (2001) Cryptogein affects expression of alpha3, alpha6 and beta1 20S proteaome subunits encoding genes in tobacco. J. Exp. Bot, 52, 1947-1948.

Deshaies, R. J. (1999) SCF and Cullin/Ring H2-based ubiquitin ligases. Annu. Rev. Cell Dev. Biol, 15, 435-467.

Deyholos M. and Galbraith, D.W. (2001) High-density microarrays for gene expression analysis. Cytometry, 43, 229-238.

Downes B.P, Stupar R.M, Gingerich D.J, and Vierstra R.D. (2003) The HECT ubiquitinprotein ligase (UPL) family in Arabidopsis: UPL3 has a specific role in trichome development. Plant J, 35, 729-742

Finley, D., Ozkaynak, E. and Varshavsky, A. (1987) The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses. Cell, 48, 1035-1046.

Freemont, P. S. (2000) Ubiquitination: RING for destruction? Current Biology, 10, R84-R87.

Fu, H., Girod P.A., Doelling J.H., van Nocker, S., Hochstrasser, M., Finley, D. and Vierstra, R.D. (1999) Structure and functional analysis of the 26S proteasome subunits from plants. Mol. Biol. Rep, 26, 137-146.

Gagne J.M, Downes B.P, Shiu S.H., Durski A.M. and Vierstra R.D. (2002) The F-Box subunit of the SCF E3 complex is encoded by a diverse superfamily of genes in Arabidopsis. Proc. Natl. Acad. Sci. USA, 99, 11519-11524

Girke, T., Todd J., Ruuska, S., White J., Benning, C. and Ohlrogge, J. (2000) Microarray analysis of brassinosteroid-regulated genes of developing Arabidopsis seeds. Plant Physiol, 124, 1570-1581.

Girod, P.A., Carpenter, T.B., van Nocker, S., Sullivan, M.L. and Vierstra, R.D. (1993) Homologies of the essential ubiquitin conjugating enzymes UBC1, 4, and 5 in yeast are encoded by a multigene family in Arabidopsis thaliana. Plant J, 3, 545-552.

Glickman, M.H. (2000) Getting in and out of the proteasome. Semin. Cell Dev. Biol, 11, 149-158

Gray, W.M. and Kepinski, S. and Rouse, D. and Leyser, O. and Estelle, M.
Gray, W.M., Kepinski, S., Rouse, D., Leyser, O. and Estelle, M. (2001) Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Nature, 414, 271-276.

Hardtke, C.S., Okamoto, H. and Deng, X.W. (2002) Biochemical evidence for ubiquitin ligase activity of Arabidopsis COP1 interacting protein 8 (CIP8). Plant J, 30(4), 385-394.

Hatfield, P.M., Gosink, M.M., Carpenter, T.B. and Vierstra, R.D. (1997) The ubiquitinactivating enzyme (E1) gene family in Arabidopsis thaliana. Plant J, 11, 213-226.

Hellmann, H. and Estelle, M. (2002) Plant development: regulation by protein degradation. Science, 297, 793-797.

Hershko, A. and Ciechanover, A. (1998) The ubiquitin system. Annu. Rev. Biochem, 67, 425-479.

Huang, L., Kinnucan, E., Wang, G., Beaudenon, S., Howley, P.M., Huibregtse, J.M. and Payletich, N.P. (1999) Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade. Science, 286, 1321-1326.

Jackson, P.K., Eldridge, A.G., Freed, E., Furstenthal, L., Hsu, J.Y., Kaiser, B.K. and Reimann, J.D. (2000) The lore of the RINGs: substrate recognition and catalysis by ubiquitin ligases. Trends Cell Biol, 10, 429-439.

Jahngenhodge, J., Obin, M.S. and Gong, X., Shang, F., Nowell, T.R., Gong, J., Abasi, H., Blumberg, J. and Taylor, A. (1997) Regulation of Ubiquitin-conjugating Enzymes by Glutathione Following Oxidative Stress. J. Biol. Chem, 272, 28218-28226.

Joazeiro, C.A., Wing, S.S., Huang, H., Leverson, J.D. Hunter, T. and Liu, Y.C. (1999) The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science, 286, 309-312.

Jungmann, J., Reins, H.A., Schobert, C. and Jentsch, S. (1993) Resistance to cadmium mediated by ubiquitin-dependent proteolysis. Nature, 361, 369-371.

Kasuga, M., Miura, S., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2004) A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought- and low-temperature stress tolerance in tobacco by gene transfer. Plant Cell Physiol, 45(3), 346-350.

Kawasaki, S., Borchert, C., Deyholos, M., Wang, H., Brazille, S., Kawai, K., Galbraith, D. and Bohnert, H.J. (2001) Gene expression profiles during the initial phase of salt stress in rice. Plant Cell, 13, 889-905.

Kim, H. S. and Delaney, T. P. (2002) Arabidopsis SON1 is an F-box protein that regulates a novel induced defense response independent of both salicylic acid and systemic acquired resistance. Plant Cell, 14, 1469-1482.

Kosarev, P., Mayer, K.F. and Hardtke, C.S. (2002) Evaluation and classification of RING-finger domains encoded by the Arabidopsis genome. Genome Biol, 3, 1-12.

Kreps, J.A., Wu, Y., Chang, H.S., Zhu, T., Wang, X. and Harper, J.F. (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol, 130, 2129-2141.

Maruyama, K., Sakuma, Y., Kasuga, M. and Yamaguchi-Shinozaki, K. (2004) Identification of cold-inducible downstream genes of the Arabidopsis DREB1A/CBF3 transcriptional factor using two microarray systems. Plant J, 38, 982-993.

Muller-Taubenberger, A., Hagmann, J., Noegel A. and Gerisch, G. (1988) Ubiquitin gene expression in Dictyostelium is induced by heat and cold shock, cadmium, and inhibitors of protein synthesis. J. Cell Science, 90, 51-58.

Osterlund, M., Hardtke, C., Wei, N. and Deng, X.W. (2000) Targeted destabilization of HY5 during light regulated development of Arabidopsis. Nature, 405, 462-466.

Ozturk, Z.N., Talame, V., Deyholos, M., Michalowski, C.B., Galbraith, D.W., Gozukirmizi, N., Tuberosa, R. and Bohnert, H.J. (2002) Monitoring large scale changes in transcript abundance in drought- and salt-stressed barley. Plant Mol. Biol, 48, 551-573.

Pickart, C.M. (2000) Ubiquitin in chains. Trends Biochem. Sci, 25, 544-548.

Puhakainen, T., Hess, M. W., Makela, P., Svensson, J., Heino, P. and Palva, E. T. (2004) Overexpression of multiple dehydrin genes enhances tolerance to freezing stress in Arabidopsis. Plant Mol. Biol, 54, 743-753.

Rabbani, M.A., Maruyama, K., Abe, H.M., Khan, A., Katsura, K., Ito, Y. Yoshiwara, K., Seki, M., Shinozaki, K. and Yamaguchi-Shinozaki, K. (2003) Monitoring Expression Profiles of Rice Genes under Cold, Drought, and High-Salinity Stresses and Abscisic Acid Application Using cDNA Microarray and RNA Gel-Blot Analyses. Plant Physiol, 133(4), 1755-1767.

Reymond, P. Weber, H. Damond, M. and Farmer, E.E. (2000) Differential gene expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell, 12, 707-719.
.
Riechmann, J.L., Heard, J., Martin, G., Reuber, L., Jiang, C., Keddie, J., Adam, L., Pineda, O., Ratcliffe, O.J., Samaha, R.R., Creelman, R., Pilgrim, M., Broun, P., Zhang, J.Z., Ghandehari, D., Sherman, B.K. and Yu, G.. (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science, 290, 2105-2110.

Saurin, A. J., Borden, K. L., Boddy, M. N. and Freemont, P. S. (1996) Does this have a familiar RING? Trends Biochem Sci, 21(6), 208-214.

Schenk, P.M., Kazan, K., Wilson I., Anderson, J.P., Richmond, T., Somerville, S.C. and Manners, J.M. (2000) Coordinated plant defense responses in Arabidopsis revealed by microarray analysis. Proc. Natl. Acad. Sci. USA, 97, 11655-11660.

Seki, M., Narusaka, M., Abe, H., Kasuga, M., Yamaguchi-Shinozaki, K., Carninci, P., Hayashizaki, Y. and Shinozaki, K. (2001) Monitoring the expression pattern of 1300 Arabidopsis genes under drought and cold stresses using full-length cDNA microarray. Plant Cell, 13, 61-72.

Seki, M., Narusaka, M., Ishida, J. Nanjo, T. Fujita, M. Oono, Y. Kamiya, A. Nakajima, M. Enju, A. and Sakurai, T. (2002a) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J, 31, 279-292.

Seki, M., Ishida, J., Narusaka, M., Fujita, M., Nanjo, T., Umezawa, T., Kamiya, A., Nakajima, M., Enju, A. and Sakurai, T. (2002b) Monitoring the expression pattern of around 7, 000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray. Funct. Integr. Genomics, 2, 282-291.

Selitrennikoff, C.P. (2001) Antifungal proteins. Applied and environmental microbiology, 67(7), 2883-2894.

Seufert, W. and Jentsch, S. (1990) Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins. Embo J, 9, 543-550.

Shang, F., Gong, X. and Taylor, A. (1997) Age-related decline in ubiquitin conjugation in responses to oxidative stresses in the lens. J. Biol.Chem, 272, 23086-23093.

Shang, F. and Taylor, A. (1995) Oxidative stress and recovery from oxidative stress are associated with altered ubiquitin conjugating and proteolytic activities in bovine lens epithelial cells. J. Biol chem. J, 307, 297-303.

Shen, W.H., Parmentier, Y., Hellmann, H., Lechner, E., Dong, A., Masson, J., Granier, F., Lepiniec, L., Estelle, M. and Genschik, P. (2002) Null mutation of AtCUL1 causes arrest in early embryogenesis in Arabidopsis. Mol. Biol. Cell, 13, 1916-1928.

Shinozaki, K. and Yamaguchi-Shinozaki, K. (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr. Opin. Plant Biol, 26, 217-223.

Shiu, S.H. and Bleecker, A.B. (2001) Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases. Proc. Natl. Acad. Sci. USA, 98, 10763-10768.

Smalle, J. and Vierstra, R.D. 2004 The ubiquitin 26S proteasome proteolytic pathway. Annu. Rev. Plant Biol, 55, 555-590.

Somers, D.E., Schultz, T.F., Milnamow, M. and Kay, S.A. (2000) ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis. Cell, 101, 319-329.

Suzuki, G., Yanagawa, Y., Kwok, S.F., Matsui, M. and Deng, X.W. (2002) Arabidopsis COP10 is a ubiquitin-conjugating enzyme variant that acts together with COP1 and the COP9 signalosome in repressing photomorphogenesis. Genes Dev, 16, 554-559.

Takai, R., Matsuda, N., Nakano, A., Hasegawa, K. and Akimoto, C. (2002) El5, a rice N-acetylchitooligosaccharide elicitor-responsive RING-H2 finger protein, is a ubiquitin ligase which functions in vitro in cooperation with an elicitor-responsive ubiquitin-conjugating enzyme, OsUBC5b. Plant J, 30, 447-455.

Thomashow, M.F. (1999) Plant cold acclimation: Freezing tolerance genes and regulatory mechanisms. Annu. Rev. Plant Physiol. Plant Mol. Biol, 50, 571-599.

Treger, J.M., Heichman, K.A. and McEntee, K. (1988) Expression of the yeast UB14 gene increases in response to DNA-damaging agents and in meiosis. Mol. Cell Biol, 8, 1132-1136.

Vierstra, R.D. (2003) The ubiquitin/26S proteasome pathway, the complex last chapter in the life of many plant proteins. Trends in Plant Science, 8, 135-142.

Voges, D., Zwickl, P., and Baumeister, W. (1999) The 26S proteasome: a molecular machine designed for controlled proteolysis. Annu. Rev. Biochem, 68, 1015-1068.

von Arnim, A.G. and Deng, X.W. (1994) Light inactivation of Arabidopsis photomorphogenic COP1 involves a cell-specific regulation of its nucleo–cytoplasmic partitioning. Cell, 79, 1035-1045.

Wilkinson, K.D. (2000) Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome. Semin. Cell Dev. Biol, 11, 141-148.

Woo, H.R., Chung, K.M., Park, J.H., Oh, S.A., Ahn, T., Hong, S.H., Jang, S.K. and Nam, H.G. (2001) ORE9, an F-box protein that regulates leaf senescence in Arabidopsis. Plant Cell, 13, 1779-1790.

Wu, M.F. (2001) Expression profile analysis of Hsc70 of Vigna radiata. Master’s Dissertation, National Tsing Hua University, Hsinchu, Taiwan, R.O.C.

Yan, J., Wang, J., Li, Q., Hwang, J.R., Paterson, C. and Zhang, H. (2003) AtCHIP, a U-box-containing E3 ubiquitin ligase, plays a critical role in temperature stress tolerance in Arabidopsis. Plant Physiol, 132, 1-9.

Zhu, J.K. (2002) Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol, 53, 247-273.

全文公開日期本全文未授權公開 (校內網路)
全文公開日期本全文未授權公開 (校外網路)

簡易檢索 / 詳目顯示

相關論文