跳到主要內容

簡易檢索 / 詳目顯示

研究生: 吳怡蓁
Yi-Chen Wu
論文名稱: 阿拉伯芥蛋白激酶AtYak1之功能探討
The studies of AtYak1, a novel DYRK family member in Arabidopsis thaliana
指導教授: 吳素幸
Shu-Hsing Wu
吳少傑
Shaw-Jye Wu
口試委員:
學位類別: 碩士
Master
系所名稱: 生醫理工學院 - 生命科學系
Department of Life Science
畢業學年度: 92
語文別: 中文
論文頁數: 89
中文關鍵詞: 光訊息傳導蛋白激酶
外文關鍵詞: AtYak1, light signal transduction, DYRK protein kinase
相關次數: 點閱:11下載:0
分享至:
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
  • 植物對於周遭自然環境狀態的改變非常敏感,因此必須藉由不同的
    反應機制來適應環境的變動;其中,光對植物的影響最為重要,它不僅
    是能量的來源,同時也是植物生長與發育過程中不可或缺的因子之一。
    植物一方面透過光接受器接收或偵測外在光源,另一方面則在光訊息傳
    遞路徑過程中藉由許多基因的表現與調節,進而對光產生多重層次的詮
    釋及反應。經由DNA 微矩陣技術,在光照對阿拉伯芥基因表現影響的
    初步研究中,鑑定到許多在光訊息傳遞中受調控的基因;在這群基因
    中,先針對一個具有WD-repeat motif 的蛋白質做測試,由於此基因的
    表現量隨光照時間增加而增加,而於暗處理時,又明顯造成此基因表現
    量降低,因此顯示其受光調節,故將此基因命名為LWD1。如同其他具
    有WD-repeat motif 的蛋白質,WD-repeat motif 提供了穩定各個蛋白質
    間交互作用的平台,故假設LWD1 也藉由和其他不同蛋白質間的交互
    作用,進而調節植物體內一些訊號的傳遞。利用酵母菌雙雜合技術可發
    現, AtYak1 是其中一個表現與LWD1 有交互作用之蛋白質。AtYak1
    也是第一個在植物中找到的DYRKs (dual specificity Yak1-related
    protein kinase)。之前研究顯示DYRKs 可調節細胞的生長與發育,為了
    更進一步了解AtYak1 是如何在光訊息路徑中調控植物生長和發育,本
    論文將針對AtYak1 的生物功能來作探討,透過AtYak1 在植物細胞中表
    現的位置、不同組織之表現量及突變株性狀之觀察與分析,進而推測其
    生理功能及意義。結果顯示,當AtYak1 與DNA binding domain 融合單
    獨送入酵母菌時即啟動β-galactosidase 活性,因此我們推測AtYak1 可
    能具有轉錄作用活化者的功能。我們也可發現AtYak1 分佈於細胞質及
    細胞核內,由此我們認為在植物中之AtYak1 可能也和其他真核細胞之
    DYRKs 功能類似,而且會依據外在環境因素或細胞本身之生理狀態而
    存在細胞中不同的位置以執行不同之功能。在觀察AtYak1 在組織表現
    量方面,結果顯示,其於葉片、花、果實、根部均有表現,其中以根部
    表現量最多。另外,藉由突變株的觀察,我們發現AtYak1 突變株在長/
    短日照中生長葉片的型態和發育均和野生型不同,植株也較小,尤其以
    短日照最為明顯,故推測AtYak1 可能也參與在阿拉伯芥葉片型態生長和發育路徑中


    Light provides an important environmental cue regulating plant growth
    and development. Light perception is mediated by the action of several
    photoreceptors. The signal transduction and light responses in higher plants
    involve the regulation of differential gene expression. Considerable amount of
    efforts have been put in the studies of light-mediated signaling pathway in
    plants. DNA microarray technology serves as a powerful approach in addition
    to the biochemical and genetic methods in identifying novel molecular
    components in light signaling pathway. After analyzing microarray, we
    choose a gene encoding WD-containing protein, termed LWD1, which is
    clearly up-regulated in response to light treatment and is dramatically
    down-regulated in dark-treated Arabidopsis leaves. Similar to other
    WD-containing proteins, LWD1 could function in interacting or coordinating
    multiple protein partners responsible for proper signal transduction. Via yeast
    two-hybrid, preliminary data reveal strong protein-protein interaction between
    LWD1 and AtYak1. AtYak1 is the first DYRK(dual specificity Yak1-related
    protein kinase)family member identified in plants and exists as a single copy
    gene in Arabidopsis. In this thesis, I will report the initial efforts in
    characterizing the biological properties and functions of AtYak1. Results
    suggest that AtYak1 is a putative transcriptional activator. Subcellular
    localization of AtYak1 is analyzed by generating a AtYak1-GFP translational
    fusion protein for protoplast transient assay. AtYak1-GFP is observed both in
    the cytoplasm and in the nucleus. Real time RT-PCR was employed to study
    the expression pattern of AtYAK1 in various tissues of Arabidopsis, including
    rosette leaves, flowers, silique, and roots. Results show that AtYak1 is
    expressed ubiquitously but predominantly in roots. Furthermore, to gain more
    insights on the biological functions of AtYak1, we obtained AtYak1 T-DNA
    insertion lines and transgenic Arabidopsis overexpressing AtYak1. Phenotype
    comparison between wild type and AtYak1 mutant Arabidopsis indicate that
    AtYak1 may involve in leaf differentiation and development.

    中文摘要·······································································································i 英文摘要······································································································ii 目錄············································································································ iii 圖表目錄······································································································v 縮寫對照表·································································································vi 前言··············································································································1 一、植物光訊息傳導(light signal transduction)及光型態形成 ( photomorphogenesis) ··········································································· 1 二、阿拉伯芥受紅光/遠紅光調控之基因························································ 2 三、LWD1(light-regulated WD repeat 1)基因·············································· 5 1.LWD1 基因簡介······················································································· 5 2.WD-repeat 蛋白質結構與功能································································ 6 四、AtYak1(Arabidopsis thaliana Yak1-related protein kinase)基因··········· 8 1. AtYak1 同源基因與結構的簡介····························································· 8 2. AtYak1 同源基因的表現與功能····························································· 9 五、研究目的····································································································· 11 材料與方法····································································································12 一、酵母菌雙雜合反應····················································································· 12 1). pACT2(AD)-LWD1 及pAS2(BD)-AtYak1 質體之建構······················ 12 2). 酵母菌細胞轉型··················································································· 16 3). β-galactosidase 活性分析······································································· 18 二、AtYak1 基因功能研究分析········································································ 19 1). AtYak1 基因在細胞中的表現位置························································ 19 2). AtYak1 基因在各組織之基因表現分析················································ 23 iv 三、AtYak1 突變株之篩選、性狀觀察與分析················································ 27 1). 阿拉伯芥突變株種植與生長······························································· 27 2). 阿拉伯芥AtYak1 T-DNA 嵌入突變株之篩選···································· 27 3). T-DNA 嵌入數目之確認-南方墨漬法·················································· 29 4). 大量表現AtYak1 基因阿拉伯芥轉殖株之獲得·································· 37 5). 大量表現AtYak1 轉殖株與AtYak1 T-DNA 插入 突變株之基因表現··············································································· 41 結果與討論····································································································42 一、LWD1 與AtYak1 交互作用之確認··························································· 42 二、AtYak1 基因功能推測················································································ 44 1). AtYak1 在細胞中位置的定位(subcellular localization) ·························· 44 2). AtYak1 於植物組織之表現量分析(expression pattern)·························· 45 三、AtYak1 突變株之性狀觀察與分析···························································· 46 1). 阿拉伯芥AtYak1 T-DNA 嵌入突變株同型合子的獲得···························· 46 2). 阿拉伯芥突變株中T-DNA 嵌入數目之確認············································· 47 3). 利用RT-PCR 對AtYak1 於突變株之基因表現分析·································· 48 4). T-DNA 嵌入AtYak1 基因突變株表型的觀察············································· 49 5). 利用Real time RT-PCR 確認AtYak1 基因於轉殖株中大量表現·············· 50 未來展望········································································································54 參考文獻········································································································55 表····················································································································62 圖····················································································································63 附圖················································································································79 附錄················································································································84

    Altafaj, X., Dierssen, M., Baamonde, C., Marti, E., Visa, J., Guimera, J.,
    Oset, M., Gonzalez, J.R., Florez, J., Fillat, C., and Estivill, X. (2001).
    Neurodevelopmental delay, motor abnormalities and cognitive deficits in
    transgenic mice overexpressing Dyrk1A (minibrain), a murine model of
    Down''s syndrome. Hum. Mol. Genet. 10, 1915-1923.
    Ballesteros, M.L., Bolle, C., Lois, L.M., Moore, J.M., Vielle-Calzada, J.-P.,
    Grossniklaus, U., and Chua, N.-H. (2001). LAF1, a MYB transcription
    activator for phytochrome A signaling. Genes Dev. 15, 2613-2625.
    Becker, W., and Joost, H.G. (1999). Structural and functional characteristics
    of Dyrk, a novel subfamily of protein kinases with dual specificity. Prog
    Nucleic Acid Res Mol Biol 62, 1-17.
    Bhalerao, R.P., Salchert, K., Bako, L., Okresz, L., Szabados, L.,
    Muranaka, T., Machida, Y., Schell, J., and Koncz, C. (1999). Regulatory
    interaction of PRL1 WD protein with Arabidopsis SNF1-like protein kinases.
    Proc Natl Acad Sci U S A 96, 5322-5327.
    Briggs, W.R., and Olney, M.A. (2001). Photoreceptors in Plant
    Photomorphogenesis to Date. Five Phytochromes, Two Cryptochromes, One
    Phototropin, and One Superchrome. Plant Physiol 125, 85-88.
    Bouquin, T., Meier, C., Foster, R., Nielsen, M. E., and Mundy, J. (2001).
    Control of specific gene expression by gibberellin and brassinosteroid. Plnat
    Physiol 127, 450-458.
    Leyser, H.M.O., Lincoln, C.A., Timpte, C., Lammer, D., Turner, J., and
    Estelle, M. (1993) Arabidopsis auxin-resistance gene AXR1 encoded to
    ubiquitin-activating enzyme E1. Nature 364, 161-164.
    Campbell, L.E., and Proud, C.G. (2002). Differing substrate specificities of
    members of the DYRK family of arginine-directed protein kinases. FEBS Lett
    510, 31-36.
    - 56 -
    Choi, G., Yi, H., Lee, J., Kwon, Y.K., Soh, M.S., Shin, B., Luka, Z., Hahn,
    T.R., and Song, P.S. (1999). Phytochrome signalling is mediated through
    nucleoside diphosphate kinase 2. Nature 401, 610-613.
    D. Li., and R. Roberts. (2001). Human genome and disease: review,
    WD-repeat proteins: structure characteristics, biological function, and their
    involvement in human diseases. Cellular and molecular life sciences 58,
    2085-2097.
    de Vetten, N., Quattrocchio, F., Mol, J., and Koes, R. (1997). The an11
    locus controlling flower pigmentation in petunia encodes a novel WD-repeat
    protein conserved in yeast, plants, and animals. Genes Dev 11, 1422-1434.
    Fankhauser, C., Yeh, K.C., Lagarias, J.C., Zhang, H., Elich, T.D., and
    Chory, J. (1999a). PKS1, a substrate phosphorylated by phytochrome that
    modulates light signaling in Arabidopsis. Science 284, 1539-1541.
    Fankhauser, C., Yeh, K.-C., Clark, J., Lagarias, Zhang, H., Elich, T.D.,
    and Chory, J. (1999b). PKS1, a Substrate Phosphorylated by Phytochrome
    That Modulates Light Signaling in Arabidopsis. Science 284, 1539-1541.
    Fields, S. (1993)The two-hybrid system to detect protein-protein interactions.
    METHODS: A Companion to Meth. Enzymol 5, 116-24.
    Flick, J.S. and Johnston, M.(1990)Two systems of glucose repression of
    the GAL1 promoter in Saccharomyces cerevisiae. Mol.Cell. Biol 10, 4757-69.
    Garrett, S., and Broach, J. (1989). Loss of Ras activity in Saccharomyces
    cerevisiae is suppressed by disruptions of a new kinase gene, YAKI, whose
    product may act downstream of the cAMP-dependent protein kinase. Genes
    Dev. 3, 1336-1348.
    Himpel, S., Panzer, P., Eirmbter, K., Czajkowska, H., Sayed, M.,
    Packman, L.C., Blundell, T., Kentrup, H., Grotzinger, J., Joost, H.G., and
    Becker, W. (2001). Identification of the autophosphorylation sites and
    characterization of their effects in the protein kinase DYRK1A. Biochem J
    359, 497-505.
    Ho, Y., Gruhler, A., Heilbut, A., Bader, G.D., Moore, L., Adams, S.-L.,
    - 57 -
    Millar, A., Taylor, P., Bennett, K., Boutilier, K., Yang, L., Wolting, C.,
    Donaldson, I., Schandorff, S., Shewnarane, J., Vo, M., Taggart, J.,
    Goudreault, M., Muskat, B., Alfarano, C., Dewar, D., Lin, Z.,
    Michalickova, K., Willems, A.R., Sassi, H., Nielsen, P.A., Rasmussen, K.J.,
    Andersen, J.R., Johansen, L.E., Hansen, L.H., Jespersen, H.,
    Podtelejnikov, A., Nielsen, E., Crawford, J., Poulsen, V., Sorensen, B.D.,
    Matthiesen, J., Hendrickson, R.C., Gleeson, F., Pawson, T., Moran, M.F.,
    Durocher, D., Mann, M., Hogue, C.W.V., Figeys, D., and Tyers, M. (2002).
    Systematic identification of protein complexes in Saccharomyces cerevisiae
    by mass spectrometry. Nature 415, 180-183.
    Hoecker, U., and Quail, P.H. (2001). The Phytochrome A-specific Signaling
    Intermediate SPA1 Interacts Directly with COP1, a Constitutive Repressor of
    Light Signaling in Arabidopsis. J. Biol. Chem. 276, 38173-38178.
    Hoecker, U., Tepperman, J.M., and Quail, P.H. (1999). SPA1, a WD-repeat
    protein specific to phytochrome A signal transduction. Science 284, 496-499.
    Hsieh, H.-L., Okamoto, H., Wang, M., Ang, L.-H., Matsui, M., Goodman,
    H., and Deng, X.W. (2000). FIN219, an auxin-regulated gene, defines a link
    between phytochrome A and the downstream regulator COP1 in light control
    of Arabidopsis development. Genes Dev 14, 1958-1970.
    Hudson, M.E. (2000). The genetics of phytochrome signalling in Arabidopsis.
    Seminars in Cell & Developmental Biology 11, 475-483.
    Ito, H., Fukada, Y., Murata, K. and Kimura, A.(1983)Transformation of
    intact yeast cells treated with alkali cations. J. Bacterial. 153, 163-8.
    Kim, J., Yi, H., Choi, G., Shin, B., and Song, P.S. (2003). Functional
    characterization of phytochrome interacting factor 3 in
    phytochrome-mediated light signal transduction. Plant Cell 15, 2399-2407.
    Kircher, S., Kozma-Bognar, L., Kim, L., Adam, E., Harter, K., Schafer, E.,
    and Nagy, F. (1999). Light Quality  ependent Nuclear Import of the Plant
    Photoreceptors Phytochrome A and B. Plant Cell 11, 1445-1456.
    Leder, S., Czajkowska, H., Maenz, B., De Graaf, K., Barthel, A., Joost,
    H.G., and Becker, W. (2003). Alternative splicing variants of dual specificity
    - 58 -
    tyrosine phosphorylated and regulated kinase 1B exhibit distinct patterns of
    expression and functional properties. Biochem J 372, 881-888.
    Li, K., Zhao, S., Karur, V., and Wojchowski, D.M. (2002). DYRK3
    activation, engagement of protein kinase A/cAMP response element-binding
    protein, and modulation of progenitor cell survival. J Biol Chem 277,
    47052-47060.
    Lin J.-F, Wu S.-H (2004) Molecular events in senescing Arabidopsis leaves. Plant J.
    In press.
    Mao, J., Maye, P., Kogerman, P., Tejedor, F.J., Toftgard, R., Xie, W., Wu,
    G., and Wu, D. (2002). Regulation of Gli1 transcriptional activity in the
    nucleus by Dyrk1. In J Biol Chem, 277, 35156-35161.
    Moriya, H., Shimizu-Yoshida, Y., Omori, A., Iwashita, S., Katoh, M., and
    Sakai, A. (2001). Yak1p, a DYRK family kinase, translocates to the nucleus
    and phosphorylates yeast Pop2p in response to a glucose signal. Genes Dev
    15, 1217-1228.
    Ni, M., Tepperman, J.M., and Quail, P.H. (1998). PIF3, a
    phytochrome-interacting factor necessary for normal photoinduced signal
    transduction, is a novel basic helix-loop-helix protein. Cell 95, 657-667.
    Oda, A., Fujiwara, S., Kamada, H., Coupland, G., and Mizoguchi, T.
    (2004). Antisense suppression of the Arabidopsis PIF3 gene does not affect
    circadian rhythms but causes early flowering and increases FT expression.
    FEBS Lett, 259-264.
    Osterlund, M.T., Hardtke, C.S., Wei, N., and Deng, X.W. (2000b).
    Targeted destabilization of HY5 during light-regulated development of
    Arabidopsis. Nature 405, 462-466.
    Payne, C.T., Zhang, F., and Lloyd, A.M. (2000). GL3 Encodes a bHLH
    Protein That Regulates Trichome Development in Arabidopsis Through
    Interaction With GL1 and TTG1. Genetics 156, 1349-1362.
    Quail, P.H. (2002). Photosensory perception and signalling in plant cells:
    - 59 -
    new paradigms? Current Opinion in Cell Biology 14, 180-188.
    Quail, P.H., Boylan, M.T., Parks, B.M., Short, T.W., Xu, Y., and Wagner,
    D. (1995). Phytochromes: photosensory perception and signal transduction.
    Science 268, 675-680.
    Sakamoto, K., and Nagatani, A. (1996). Nuclear localization activity of
    phytochrome B. Plant J 10, 859-868.
    Schafer, E., and Bowle, C. (2002). Phytochrome-mediated photoperception
    and signal transduction in higher plants. EMBO Rep 3, 1042-1048.
    Sheen, J., Chiu, W. L., Niwa, Y., Zeng, W., Hirano, T. and Kobayashi, H.
    (1996). Engineered GFP as a vital reporter in plants. Current Biology 6,
    325-330
    Sherman,F.,G.R. Fink and J.B. Hicks.(1986)Methods in yeast genetics: A
    Laboratory Manual(Cold Spring Harbor, New York: Cold Spring Harbor
    Laboratory Press).
    Skurat, A.V., and Dietrich, A.D. (2004). Phosphorylation of Ser640 in
    muscle glycogen synthase by DYRK family protein kinases. J Biol Chem 279,
    2490-2498.
    Smith, H. (2000). Phytochromes-The Enigma Solved? Semin Cell Dev Biol
    11, 445-447.
    Smith, T.F., Gaitatzes, C., Saxena, K., and Neer, E.J. (1999). The WD
    repeat: a common architecture for diverse functions. Trends Biochem Sci 24,
    181-185.
    Sompornpailin, K., Makita, Y., Yamazaki, M., and Saito, K. (2002). A
    WD-repeat-containing putative regulatory protein in anthocyanin biosynthesis
    in Perilla frutescens. Plant Mol Biol 50, 485-495.
    Souza, G., Lu, S., and Kuspa, A. (1998). YakA, a protein kinase required for
    the transition from growth to development in Dictyostelium. Development
    125, 2291-2302.
    - 60 -
    Sullivan, J.A., and Deng, X.W. (2003). From seed to seed: the role of
    photoreceptors in Arabidopsis development. Dev Biol 260, 289-297.
    Takase, T., Nakazawa, M., Ishikawa, A., Manabe, K., and Matsui, M.
    (2003). DFL2, a New Member of the Arabidopsis GH3 Gene Family, is
    Involved in Red Light-Specific Hypocotyl Elongation. In Plant Cell Physiol
    44, 1071-1080.
    Tepperman, J.M., Zhu, T., Chang, H.S., Wang, X., and Quail, P.H. (2001).
    Multiple transcription-factor genes are early targets of phytochrome A
    signaling. Proc Natl Acad Sci U S A 98, 9437-9442.
    Ulm, R., Baumann, A., Oravecz, A., Mate, Z., Adam, E., Oakeley, E.J.,
    Schafer, E., and Nagy, F. (2004). Genome-wide analysis of gene expression
    reveals function of the bZIP transcription factor HY5 in the UV-B response of
    Arabidopsis. Proc Natl Acad Sci U S A 101, 1397-1402.
    van der Voorn, L., and Ploegh, H. L. (1992). The WD-40 repeat. FEBS Lett
    307, 131-134.
    Van Nocker, S., and Ludwig, P. (2003). The WD-repeat protein superfamily
    in Arabidopsis: conservation and divergence in structure and function. BMC
    Genomics 4, 50.
    von Groote-Bidlingmaier, F., Schmoll, D., Orth, H.M., Joost, H.G., Becker,
    W., and Barthel, A. (2003). DYRK1 is a co-activator of FKHR
    (FOXO1a)-dependent glucose-6-phosphatase gene expression. Biochem
    Biophys Res Commun 300, 764-769.
    Yeh, K. C., and Lagarias, J.C. (1998). Eukaryotic phytochromes:
    light-regulated serine/threonine protein kinases with histidine kinase ancestry.
    Proc Natl Acad Sci U S A 95, 13976-13981.
    Yeh, K. C., Wu, S. H., Murphy, J.T., and Lagarias, J.C. (1997). A
    Cyanobacterial Phytochrome Two-Component Light Sensory System. Science
    277, 1505-1508.
    Zhao, L., Nakazawa, M., Takase, T., Manabe, K., Kobayashi, M., Seki, M.,
    - 61 -
    Shinozaki, K., and Matsui, M. (2004). Overexpression of LSH1, a member
    of an uncharacterised gene family, causes enhanced light regulation of
    seedling development. In Plant J 37, 694-706.

    QR CODE
    :::