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研究生:蔡佩菁
研究生(外文):Pei-Ching Tsai
論文名稱:水稻OSCK1蛋白次細胞分佈及其啟動子活性分析
論文名稱(外文):Characterization of the Protein Subcellular Localization and Promoter Specificity of OSCK1 from Rice
指導教授:呂維茗
指導教授(外文):Wei-Ming Leu
學位類別:碩士
校院名稱:國立中興大學
系所名稱:農業生物科技學研究所
學門:農業科學學門
學類:農業技術學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:57
中文關鍵詞:水稻啟動子
外文關鍵詞:Ricepromoter
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Calcium-dependent calmodulin-independent protein kinase (CDPK) 為一種結鈣激活酵素,在植物中常以多基因家族型式存在,可能參予調控植物各種發育過程及生理反應。本實驗室藉由差異性表現基因選殖法,自水稻中篩選出一個水稻授粉前專一表現的基因,其序列與玉米花粉中專一表現之CDPK基因序列相似度極高,故命名為OSCK1 (Oryza sativa calcium-dependent calmoduin-independent protein kinase 1)。OSCK1蛋白除具有典型CDPK蛋白之特性外,其近N端點具有一個荳蔻酸化修飾訊息,可能使蛋白藉由荳蔻酸附著到細胞膜上;而蛋白近C端處則有一個典型bipartite的趨核訊號,可能可帶領蛋白進入細胞核中。本研究探討OSCK1蛋白在細胞內分布位置,希望藉以瞭解其生化特性與生理功能,利用大腸桿菌共同表現OSCK1蛋白與酵母菌的N-myristoyltransferase (NMT),結果顯示OSCK1蛋白可被荳蔻酸化,惟荳蔻酸化與否並未造成激活酵素活性差異,已進一步構築質體進行植物轉殖以探討OSCK1蛋白荳蔻酸化對其蛋白分布及功能之影響。以洋蔥表皮細胞暫時表現OSCK1-GUS融合蛋白的實驗顯示此蛋白並未特別集中於細胞核,但未排除OSCK1蛋白是否可以接受外源刺激而轉入核中。另一研究方向為探討OSCK1基因啟動子是否具有花粉專一性,故構築三個質體,分別帶有長度不等的OSCK1啟動子片段與GUS報告基因接合,以農桿菌轉殖法進行水稻轉殖,目前已得到多株轉基因植株,除經PCR方式確認其為轉殖株外,初步分析轉殖株之根、葉等營養器官,並未發現有GUS基因之表現,是否會在花粉中表現則有待研究。

The calcium-dependent calmodulin-independent protein kinases (CDPKs) are calcium-regulated protein kinases. The CDPKs are encoded by multigene family, although highly conserved in sequence, they often exhibit distinct expression patterns, implicating their specific roles involved in various developmental controls or physiological responses. By differential screening, we have isolated a CDPK gene, designated as OSCK1 (Oryza sativa calcium-dependent calmodulin-independent protein kinase 1), from rice. Database searching revealed that an myristoylation signal is located at the N-terminal end of OSCK1 and may be responsible for its membrane association. Moreover, a bipartite nucleus localization signal was found at the C-terminal of OSCK1. We aim to analyze the relevance of these two putative signals in directing the subcellular distribution and the biochemical activity of OSCK1. Our results showed that the OSCK1 was myristoylated when co-expressed with yeast N-myristoyl transferase (yNMT) in E. coli. However, no difference in kinase activity could be detected between proteins expressed with or without yNMT. Moreover, OSCK1-GUS fusion protein was not specifically accumulated in nucleus when transiently expressed in the epidermis of onion. To analyze the activity and specificity of OSCK1 promoter, we constructed three plasmids carrying OSCK1 promoter in various lengths fused with the GUS reporter gene. By transformation of rice, the preliminary results showed that no GUS activity could be detected in the vegetative tissues of transgenic plants.

目錄
中文摘要………………………………………………………….…………i
英文摘要………………………………………………………….…………ii
前言…………….……………………………………………………………1
材料與方法
一、少量質體DNA之萃取……………………………………………….7
二、DNA 片段之純化回收 .……………………………………………7
三、轉殖用穠桿菌之製備 .……………………………………………….8
四、穠桿菌之基因轉移 .…………………………………………………10
五、水稻基因組 DNA之萃取 ………………………………………….11
六、轉殖植物之初步確認 …..………………………………………….12
七、基因槍轉殖法 .………………………………………………………12
八、於大腸桿菌中表現 OSCK1不同區域的融合蛋白質 .…………..14
九、純化不同長度OSCK1之融合蛋白質 ……………………………..15
十、西方點墨法分析……………………………………………………...15
十一、激活酵素活性測試 ……………………………………………….16
結果
一、OSCK1啟動子活性分析
1. OSCK1啟動子活性分析載體之構築...…………………………….17
2.水稻之基因轉殖 ……………………………………………………18
3.水稻轉殖株之分析
(1)以聚合酶連鎖反應(PCR)分析基因是否轉入植株中.…………….18
(2)轉殖植株各部位組織GUS活性分析 ……………………..……...19
二、OSCK1蛋白次細胞分佈分析
1. 測試OSCK1蛋白C端趨核序列訊號...…………………………...20
2. 測試OSCK1蛋白N端荳蔻酸化序列訊號
(1)測試各OSCK1可溶性融合蛋白質之部分純化條件.……..………21
(2)測試OSCK1蛋白是否可被荳蔻酸化.……………………………...22
(3)測試OSCK1蛋白荳蔻酸化與否對蛋白活性的影響……………....23
(4)構築載體以於植物中表達野生型或荳蔻酸化訊號序列突變(G1A)
的OSCK蛋白..………………………………………………………23
討論
一、探討水稻基因轉殖之操作方法……………………………………...25
二、探討OSCK1基因花粉專一性啟動子序列特性分析……..……......26
三、探討OSCK1蛋白次細胞分佈狀況………………………………….27
參考文獻……………………………………………………………………..…28
附圖
圖一、OSCK1與其他植物CDPK的胺基酸序列比對………………33
圖二、OSCK1 啟動子分析質體之構築………………………….…....35
圖三、OSCK1啟動子活性分析質體構築之簡單示意圖 …………....36
圖四、CDPK-24質體構築流程……………….……….……………....37
圖五、CDPK-25質體構築流程………………………………….….…38
圖六、CDPK-26質體構築流程………………………………………...39
圖七、水稻癒合組織經農桿菌感染及植株情形……………………....40
圖八、OSCK1基因上引子位置示意圖及各引子之詳細序列..……….41
圖九、CDPK—25之轉殖水稻PCR分析……………………………….42
圖十、CDPK—26之轉殖水稻PCR分析…………………………..…...43
圖十一、含帶全長OSCK1 cDNA質體pCDPK-F 之構築流程….44
圖十二、表現OSCK1-GUS融合蛋白的暫存質體(transient plasmid)之
構築…………………………………………………………....45
圖十三、藉由大腸桿菌表現之各OSCK1融合蛋白質示意圖………..46
圖十四、以Ni-NTA進行可溶性OSCK1-17-1,OSCK1-18-1及OSCK1-20-1蛋白質之純化…………………………………..47
圖十五、大腸桿菌表現系統中OSCK1蛋白質荳蔻酸化情形………..48
圖十六、構築載體以於植物中表達野生型或G1A OSCK1蛋白……..49
表一、水稻之轉殖頻率……………………………………….…………………50
表二、轉殖植株各部位組織GUS活性測試…………………………………….51
附錄一、水稻組織培養所使用之培養基成份……………………………..…...52
附錄二、pCAMBIA1300 載體之基因圖譜………………………..…………...53
附錄三、pCAMBIA1301 載體之基因圖譜…………………………………….54

參考文獻
Bate, N. and D. Twell (1998). "Functional architecture of a late pollen promoter: pollen-specific transcription is developmentally regulated by multiple stage-specific and co-dependent activator elements." Plant Mol Biol 37(5): 859-69.
Bernaerts, M. and J. De Ley (1967). "Mechanism of the 3-ketolactose test for Agrobacterium." Arch Mikrobiol 56(1): 81-90.
Crossway, A., Oakes, J. V., Irvine, J. M., Ward, B., Knauf, V. C. and shewmarker,
C. K.( 1986) Intergration of forein DNA following microinjection of tobacco mesophyll protoplasts. Mol. Gen. Genet. 202, 179-185
Duronio, R. J., E. Jackson-Machelski, et al. (1990). "Protein N-myristoylation in Escherichia coli: reconstitution of a eukaryotic protein modification in bacteria." Proc Natl Acad Sci U S A 87(4): 1506-10.
Estruch, J. J., S. Kadwell, et al. (1994). "Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase." Proc Natl Acad Sci U S A 91(19): 8837-41.
Fromm, M. E., L. P. Taylor, et al. (1986). "Stable transformation of maize after gene transfer by electroporation." Nature 319(6056): 791-3.
Gallusci, P., F. Salamini, et al. (1994). "Differences in cell type-specific expression of the gene Opaque 2 in maize and transgenic tobacco." Mol Gen Genet 244(4): 391-400.
Grosset, J., R. Alary, et al. (1997). "Characterization of a barley gene coding for an alpha-amylase inhibitor subunit (CMd protein) and analysis of its promoter in transgenic tobacco plants and in maize kernels by microprojectile bombardment." Plant Mol Biol 34(2): 331-8.
Hamilton, D. A., Y. H. Schwarz, et al. (1998). "A monocot pollen-specific promoter contains separable pollen-specific and quantitative elements." Plant Mol Biol 38(4): 663-9.
Hansen, G., A. Das, et al. (1994). "Constitutive expression of the virulence genes improves the efficiency of plant transformation by Agrobacterium." Proc Natl Acad Sci U S A 91(16): 7603-7.
Harmon, A. C., B. C. Yoo, et al. (1994). "Pseudosubstrate inhibition of CDPK, a protein kinase with a calmodulin-like domain." Biochemistry 33(23):
7278-87.
Hellens, R., P. Mullineaux, et al. (2000). "Technical Focus:a guide to Agrobacterium binary Ti vectors." Trends Plant Sci 5(10): 446-51.
Hiei, Y., T. Komari, et al. (1997). "Transformation of rice mediated by Agrobacterium tumefaciens." Plant Mol Biol 35(1-2): 205-18.
Hiei, Y., S. Ohta, et al. (1994). "Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA." Plant J 6(2): 271-82.
Hong, Y., M. Takano, et al. (1996). "Expression of three members of the calcium-dependent protein kinase gene family in Arabidopsis thaliana." Plant Mol Biol 30(6): 1259-75.
Hooykaas-Van Slogteren, G. M., P. J. Hooykaas, et al. (1992). "Expression of Ti plasmid genes in monocotyledonous plants infected with Agrobacterium tumefaciens. 1984." Biotechnology 24: 382-3.
Kawasaki T., Hayashida N., Baba T., Shinocaki K., and Shimada H. (1993). The gene encoding a calcium-dependent protein kinase located near the sbeI gene encoding starch branching enzyme I is specifically expressed in developing rice seeds. Gene 129: 183-189.
Klein, T. M., Wolf, E. D., Wu, R. and Sanford, J. C. (1987a) High-velocity microprojectiles for delivering nucleic acids into living cell. Nature, 327, 70-73.
Klein, T. M., Harper, E .C., Svab, Z., and Sanford, J. C., Fromm, M. E. and Krens, F. A., Moleendijk, L., Wullens, G. J. and Schiperoort, R.A.(1987b) In vitro transformation of plant protoplast with Ti-plasmid DNA. Nature, 296, 72-74.
Martin, M. L. and L. Busconi (2000). "Membrane localization of a rice calcium-dependent protein kinase (CDPK) is mediated by myristoylation and palmitoylation." Plant J 24(4): 429-35.
Roberts, D., and Harmon, A. (1992). Calcium-Modulated Proteins: Targets of Intracellular Calcium Signals in Higher Plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 43, 375-414.
Slogtern, H. V., Hooykaas, P. J. J. and Schilperoort, R. A.(1984)Expression of Ti-plasmid genes in monocotyledonous plants infected with Agrobacterium tumeffaciens. Nature, 311, 763-764.
Takezawa D., Patil S ., Bhatia A., and Poovaiah B.W. (1996). Calcium-dependent protein kinase gene in corn roots. J. Plant physiol. 149:329-335.
Thomashow, M. F., J. E. Karlinsey, et al. (1987). "Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment." J Bacteriol 169(7): 3209-16.
Toki, S. (1997) Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Mol. Biol. Rep. 15, 16-21
Vasil, I. K. (1996) Milestones in crop biotechnology-transgenic cassava and Agrobacterium-mediated transformation of maize. Nature Biotechnology, 14, 702-703.
Varagona, M. J., R. J. Schmidt, et al. (1992). "Nuclear localization signal(s) required for nuclear targeting of the maize regulatory protein Opaque-2." Plant Cell 4(10): 1213-27.
Vitart, V., J. Christodoulou, et al. (2000). "Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase." Biochemistry 39(14): 4004-11.
Vitart, V. V., J. Christodoulou, et al. (2000). "Intramolecular activation of a Ca(2+)-dependent protein kinase is disrupted by insertions in the tether that connects the calmodulin-like domain to the kinase." Biochemistry
39(39): 12102.
Weterings, K., J. Schrauwen, et al. (1995). "Functional dissection of the promoter of the pollen-specific gene NTP303 reveals a novel pollen-specific, and conserved cis-regulatory element." Plant J 8(1): 55-63.
Willing RP, Bashe D, Mascharenhas JP: An analysis of the quantity and diversity of messenger RNAs from pollen and shoots of Zea mays. Theor Appl Genet 75:751-753 (1998)
Yalovsky S, Rodr Guez-Concepcion M, Gruissem W. Lipid modifications of proteins - slipping in and out of membranes. : Trends Plant Sci 1999 Nov;4(11):439-445
Zhao, Y., B. Kappes, et al. (1993). "Gene structure and expression of an unusual protein kinase from Plasmodium falciparum homologous at its carboxyl terminus with the EF hand calcium-binding proteins." J Biol Chem 268(6): 4347-54.

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