Title page for 91224016


[Back to Results | New Search]

Student Number 91224016
Author Ju-Yu Kuo(郭如玉)
Author's Email Address s1224016@cc.ncu.edu.tw
Statistics This thesis had been viewed 2481 times. Download 11 times.
Department Life Science
Year 2004
Semester 1
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Characterization of AtNPR1 transgenic tomato and study of defense mechanism
Date of Defense 2004-09-29
Page Count 86
Keyword
  • AtNPR1 transgenic tomato
  • cDNA microarray
  • defense resistance mechanism
  • Abstract Abstract
    In nature, crop plants constantly encounter various biotic and abiotic stresses, which can severely affect agricultural productivity. Pathogens, particularly, are limiting factors reducing crops quality and quantity seriously. Genetic engineering of disease-resistance through transferal of plant defense-related genes into crop is a valuable disease-control approach. Among the defense genes used to genetically engineer  systemic acquired resistance in plants, Arabidopsis NPR1 (nonexpresser of PR genes) is of particular interest for its being a central regulator of plant defense responses. The exploration of empolying Arabidopsis NPR1 (AtNPR1) gene for genetics engineering disease-resistance in tomato plants have led to the production of transgenic tomato lines conferring broad-spectrum disease-resistance, especially to vascular and leaf pathogens. In this work, the possible resistance mechanism employed by a selected AtNPR1 transgenic line and its interactions with Ralstonia solanacerarum were further studied. The results showed that some PR (pathogenesis-related) genes were constitutively expressed at a high level in the leaf tissues of the transgenic plants and thus may account for the enhanced resistances to the leaf pathogens. By further employing cDNA microarray approach, using customized tomato cDNA microarray, a group of putative AtNPR1 overexpression-induced genes were identified and a model for defense mechanism is proposed. By cross-referencing these data with the microarray data obtained from other abiotic stress studies, possible cross-links between biotic stress and heavy metal stress response were revealed. Evaluation of the horticulture traits of the transgenic tomato showed that the transgenic plants responded similarity, as did the wild type plants under drought and salinity stress. Furthermore, because none of the transgenic lines generated in the genetic background of CL5915 tomato cultivar conferred enhanced resistance to virus diseases, a tomato cultivars, CLN2116B, which carries the resistance trait to tomato yellow leaf curl virus was used as the background cultivar for transformation with AtNPR1. New CLN2116B transgenic plants were selected and characterized. Molecular analyzes revealed that expression of some PR genes was constitutively enhanced in these transgenic plants, suggesting a potential enhanced of disease-resistance in these transgenic plants.
    Table of Content 目錄
    中文摘要…………………………………………………………………
    英文摘要…………………………………………………………………
    目錄………………………………………………………………………
    圖目錄……………………………………………………………………
    表目錄……………………………………………………………………
    附錄………………………………………………………………………
    縮寫與全名對照表………………………………………………………
    第一章 緒論……………………………………………………………..
    一、番茄簡介……………………………………………………………
    二、番茄病害……………………………………………………………12
    三、植物抗病機制………………………………………………………
    四、植物基因轉殖於抗病育種上之應用………………………………
    五、NPR1在植物系統性誘導抗病機制中所扮演之角色…………….
    六、DNA微陣列應用於植物病害研究………………………………..
    七、研究動機……………………………………………………………
    第二章 材料與方法……………………………………………………..
    一、實驗材料……………………………………………………………
    (一) 植物材料………………………………………………………
    (二) 番茄的種植及逆境條件的處理………………….……………
    1. 生物性逆境的處理條件……………………………………..
    2. 非生物性逆境的處理條件…………………………………..
    二、實驗方法……………………………………………………………
    (一)DIG標記探針之製作……………………………………….…
    (二)總核醣核酸之純化及電泳分析……………………………….
    (三)北方點墨法…………………………………………………….
    (四)反轉錄聚合酶連鎖反應………………………………….……
    (五)cDNA微陣列技術……………………………………….…….
    1. cDNA探針之製備………………………………………..….
    2. 雙色螢光基因微陣列雜交反應…………………………….
    3. 資料分析…………………………………………………….
    (六)基因組去氧核醣核酸之純化及電泳分析…………….………
    (七)南方墨點法…………………………………………….………
    (八)番茄CLN2116B品系之基因轉殖…………………….………
    第三章 結果…………………………………………………………
    一、AtNPR1轉殖番茄品系抗病機制之探討…………………….…….
    (一)轉殖基因及防禦相關基因在轉殖品系之表現………….……
    (二)轉殖品系中可能受AtNPR1或青枯病菌誘導之基因….…….
    二、AtNPR1轉殖番茄品系對非生物性逆境之耐受性比較…………..
    三、番茄CLN2116B抗病品系之培育………………………………...
    第四章 討論…………………………………………………………..….
    第五章 建議………………………………………………………………
    參考文獻…………………………………………………………………

    圖目錄
    圖一、 以北方點墨法分析 AtNPR1基因在轉殖植物中的表現…….
    圖二、 以北方點墨法分析 PR1b1基因在轉殖植物中的表現………
    圖三、 以北方點墨法分析 PR1p6基因在轉殖植物中的表現………
    圖四、 以北方點墨法分析β-1, 3-glucanase基因在轉殖植物中
    的表現…………………………………………………………..
    圖五、 以北方點墨法分析acidic chitinase 9基因在轉殖植物中
    的表現…………………………………………………………..
    圖六、 在AtNPR1轉殖品系可能受誘導表現之基因……………..
    圖七、 以青枯病菌處理AtNPR1轉殖品系後可能受誘導表現之基因……………………………………………………………
    圖八、 轉殖品系可能受AtNPR1誘導表現之基因來源分配圖……….
    圖九、 維恩圖表示可能共同受生物性及非生物性逆境誘導之
    基因……………………………………………………………..
    圖十、 確認微陣列實驗所得基因之表現……………………………..
    圖十一、未轉殖與轉殖品系對非生物性逆境處理之耐受性比較…….
    圖十二、番茄CLN2116B轉殖品系之篩選…………………………….
    圖十三、以分子生物技術分析CLN2116B轉殖品系基因表現……….
    圖十四、以北方點墨法分析在CLN2116B轉殖品系中防禦基因
    之表現…………………………………………………………..
    圖十五、AtNPR1轉殖品系可能被誘導之防禦機制……………………

    表目錄
    表一、番茄病害防禦微陣列中可能受AtNPR1誘導之基因……….….
    表二、IBS-Tom微陣列中處理青枯病菌後受AtNPR1誘導之
    基因………………………………………………………………
    表三、AtNPR1轉殖品系中共同受青枯病菌與其他非生物性
    逆境誘導之基因………………………………………………….

    附錄
    附錄一、pNPR1之建構圖……………………………………………….
    附錄二、常用之細菌培養基配方……………………………………….
    附錄三、實驗使用之引子序列………………………………………….
    附錄四、改良式 Hoagland水耕培養液……………………………….
    附錄五、番茄基因轉殖所需培養基配方………………………………..
    附錄六、AtNPR1轉殖品系之抗病測試結果……………………………
    Reference 參考文獻
    柯勇. (1996). 植物病理學. 國立中興大學教材.
    Adams, M.D., Soares, M.B., Kerlavage, A.R., Fields, C., and Venter, J.C. (1993). Rapid cDNA sequencing (expressed sequence tags) from a directionally cloned human infant brain cDNA library. Nat Genet 4, 373-380.
    Alexander, D., Goodman, R.M., Gut-Rella, M., Glascock, C., Weymann, K., Friedrich, L., Maddox, D., Ahl-Goy, P., Luntz, T., Ward, E., and et al. (1993). Increased tolerance to two oomycete pathogens in transgenic tobacco expressing pathogenesis-related protein 1a. Proc Natl Acad Sci U S A 90, 7327-7331.
    Cao, H., Li, X., and Dong, X. (1998). Generation of broad-spectrum disease resistance by overexpression of an essential regulatory gene in systemic acquired resistance. Proc Natl Acad Sci U S A 95, 6531-6536.
    Cao, H., Bowling, S.A., Gordon, A.S., and Dong, X. (1994). Characterization of an Arabidopsis Mutant That Is Nonresponsive to Inducers of Systemic Acquired Resistance. Plant Cell 6, 1583-1592.
    Cao, H., Glazebrook, J., Clarke, J.D., Volko, S., and Dong, X. (1997). The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88, 57-63.
    Chen, Z., Silva, H., and Klessig, D.F. (1993). Active oxygen species in the induction of plant systemic acquired resistance by salicylic acid. Science 262, 1883-1886.
    Chern, M.S., Fitzgerald, H.A., Yadav, R.C., Canlas, P.E., Dong, X., and Ronald, P.C. (2001). Evidence for a disease-resistance pathway in rice similar to the NPR1-mediated signaling pathway in Arabidopsis. Plant J 27, 101-113.
    Delaney, T.P., Friedrich, L., and Ryals, J.A. (1995). Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance. Proc Natl Acad Sci U S A 92, 6602-6606.
    Dong, J., Chen, C., and Chen, Z. (2003). Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Mol Biol 51, 21-37.
    Dong, X. (1998). SA, JA, ethylene, and disease resistance in plants. Curr Opin Plant Biol 1, 316-323.
    Dowd, C., Wilson, I.W., and McFadden, H. (2004). Gene expression profile changes in cotton root and hypocotyl tissues in response to infection with Fusarium oxysporum f. sp. vasinfectum. Mol Plant Microbe Interact 17, 654-667.
    Durrant, W.E., and Dong, X. (2004). Systemic acquired resistance. Annu Rev Phytopathol 42, 185-209.
    Felton, G.W., Korth, K.L., Bi, J.L., Wesley, S.V., Huhman, D.V., Mathews, M.C., Murphy, J.B., Lamb, C., and Dixon, R.A. (1999). Inverse relationship between systemic resistance of plants to microorganisms and to insect herbivory. Curr Biol 9, 317-320.
    Fitzgerald, H.A., Chern, M.S., Navarre, R., and Ronald, P.C. (2004). Overexpression of (At)NPR1 in rice leads to a BTH- and environment-induced lesion-mimic/cell death phenotype. Mol Plant Microbe Interact 17, 140-151.
    Genin, S., and Boucher, C. (2002). Ralstonia solanacerum: secrets of a major pathogen unveiled by analysis of its genome. Molecular Plant Pathology 3, 111-118.
    Genin, S., and Boucher, C. (2004). Lessons learned from the genome analysis of ralstonia solanacearum. Annu Rev Phytopathol 42, 107-134.
    Guisset, J.-L., and Vries, G.M.P.-D. (2000). The Redox States and Circadian Rhythms. Kluwer Academic Publishers.
    Jach, G., Gornhardt, B., Mundy, J., Logemann, J., Pinsdorf, E., Leah, R., Schell, J., and Maas, C. (1995). Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco. Plant J 8, 97-109.
    JF, W., and CH, L. (2004). Colonization capacuty of Ralstonia solanacearum tomato strains differing in aggressiveness on tomato and weeds. in:allen C, Prior P and Hayward AC(eds), Bacterial Wilt: The disease and the Ralstonia solanacearum Species Complex. APS Press, St Paul (in press).
    Jongedijk, E., Tigelaar, H., S.C., J., and Melchers, L.S. (1995). Synergistic activity of chitinase and B-1,3-glucanases enhances fungal resistance in transgenic tomato plants. Euphytica 85, 173-180.
    Kasprzewska, A. (2003). Plant chitinases--regulation and function. Cell Mol Biol Lett 8, 809-824.
    Kinkema, M., Fan, W., and Dong, X. (2000). Nuclear localization of NPR1 is required for activation of PR gene expression. Plant Cell 12, 2339-2350.
    Lam, E., Kato, N., and Lawton, M. (2001). Programmed cell death, mitochondria and the plant hypersensitive response. Nature 411, 848-853.
    Li, L., and Steffens, J.C. (2002). Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215, 239-247.
    Li, Y., Li, T., Liu, S., Qiu, M., Han, Z., Jiang, Z., Li, R., Ying, K., Xie, Y., and Mao, Y. (2004). Systematic comparison of the fidelity of aRNA, mRNA and T-RNA on gene expression profiling using cDNA microarray. J Biotechnol 107, 19-28.
    Lin., W.-C., Lu., C.-F., Wu., J.-W., Cheng., M.-L., Lin., Y.-M., Black., L., Green., S.K., Wang., J.-F., and Cheng, C.-P. (2004). Transgenic tomato plants expression the Arabidopsis NPR1 gene confer enhanced resistance to a spectrum of fungal and bacterial diseases. Transgenic research (in press).
    Liu, D., Raghothama, K.G., Hasegawa, P.M., and Bressan, R.A. (1994). Osmotin overexpression in potato delays development of disease symptoms. Proc Natl Acad Sci U S A 91, 1888-1892.
    Lu, R., Malcuit, I., Moffett, P., Ruiz, M.T., Peart, J., Wu, A.J., Rathjen, J.P., Bendahmane, A., Day, L., and Baulcombe, D.C. (2003). High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance. Embo J 22, 5690-5699.
    Maleck, K., Levine, A., Eulgem, T., Morgan, A., Schmid, J., Lawton, K.A., Dangl, J.L., and Dietrich, R.A. (2000). The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nat Genet 26, 403-410.
    Mengiste, T., Chen, X., Salmeron, J., and Dietrich, R. (2003). The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis. Plant Cell 15, 2551-2565.
    Mithofer, A., Schulze, B., and Boland, W. (2004). Biotic and heavy metal stress response in plants: evidence for common signals. FEBS Lett 566, 1-5.
    Mou, Z., Fan, W., and Dong, X. (2003). Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes. Cell 113, 935-944.
    Pieterse, C.M.J., Ton, J., and Loon, L.C.V. (2001). Cross-talk between plant defense signalling pathways:boost or burden. AgBiotechNet 3, 1-8.
    Ryals, J., Lawton, K.A., Delaney, T.P., Friedrich, L., Kessmann, H., Neuenschwander, U., Uknes, S., Vernooij, B., and Weymann, K. (1995). Signal transduction in systemic acquired resistance. Proc Natl Acad Sci U S A 92, 4202-4205.
    Ryals, J., Weymann, K., Lawton, K., Friedrich, L., Ellis, D., Steiner, H.Y., Johnson, J., Delaney, T.P., Jesse, T., Vos, P., and Uknes, S. (1997). The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor I kappa B. Plant Cell 9, 425-439.
    Ryals, J.A., Neuenschwander, U.H., Willits, M.G., Molina, A., Steiner, H.Y., and Hunt, M.D. (1996). Systemic Acquired Resistance. Plant Cell 8, 1809-1819.
    Schaller, F., Biesgen, C., Mussig, C., Altmann, T., and Weiler, E.W. (2000). 12-Oxophytodienoate reductase 3 (OPR3) is the isoenzyme involved in jasmonate biosynthesis. Planta 210, 979-984.
    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 U S A 97, 11655-11660.
    Schweizer, P., Christoffel, A., and Dudler, R. (1999). Transient expression of members of the germin-like gene family in epidermal cells of wheat confers disease resistance. Plant J 20, 541-552.
    Tornero, P., Gadea, J., Conejero, V., and Vera, P. (1997). Two PR-1 genes from tomato are differentially regulated and reveal a novel mode of expression for a pathogenesis-related gene during the hypersensitive response and development. Mol Plant Microbe Interact 10, 624-634.
    Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S., Dincher, S., Chandler, D., Slusarenko, A., Ward, E., and Ryals, J. (1992). Acquired resistance in Arabidopsis. Plant Cell 4, 645-656.
    van Wees, S.C., de Swart, E.A., van Pelt, J.A., van Loon, L.C., and Pieterse, C.M. (2000). Enhancement of induced disease resistance by simultaneous activation of salicylate- and jasmonate-dependent defense pathways in Arabidopsis thaliana. Proc Natl Acad Sci U S A 97, 8711-8716.
    Verhagen, B.W., Glazebrook, J., Zhu, T., Chang, H.S., van Loon, L.C., and Pieterse, C.M. (2004). The transcriptome of rhizobacteria-induced systemic resistance in arabidopsis. Mol Plant Microbe Interact 17, 895-908.
    Vierheilig, H., Alt, M., and Boller, T. (1995). Colonization of transgenic tobacco constitutively expression pathogenesis-related proteins by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae. Applied and Enviroment Microbiology, 3031-3034.
    Wan., J., Dunnung., F.M., and F.Bent, A. (2002). Probing plant-pathogen interactions and downstream defense signaling using DNA microarays. Funct Integr Genomucs 2, 259-273.
    Wang, J.F., Olivier, J., Thoquet, P., Mangin, B., Sauviac, L., and Grimsley, N.H. (2000). Resistance of tomato line Hawaii7996 to Ralstonia solanacearum Pss4 in Taiwan is controlled mainly by a major strain-specific locus. Mol Plant Microbe Interact 13, 6-13.
    Wang, W., Vinocur, B., Shoseyov, O., and Altman, A. (2004). Role of plant heat-shock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci 9, 244-252.
    Whitham, S., Dinesh-Kumar, S.P., Choi, D., Hehl, R., Corr, C., and Baker, B. (1994). The product of the tobacco mosaic virus resistance gene N: similarity to toll and the interleukin-1 receptor. Cell 78, 1101-1115.
    Wong, C.E., Carson, R.A., and Carr, J.P. (2002). Chemically induced virus resistance in Arabidopsis thaliana is independent of pathogenesis-related protein expression and the NPR1 gene. Mol Plant Microbe Interact 15, 75-81.
    Y.H.Lee, I.S.Yoon, S.C.Suh, and H.I.Kim. (2002). Enhanced disease resistance in transgenic cabbage and tobacco expressing a glucose oxidase gene from Aspergillus niger. Plant Cell Rep 20.
    Yu, D., Chen, C., and Chen, Z. (2001). Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13, 1527-1540.
    Zhou, J.M., Trifa, Y., Silva, H., Pontier, D., Lam, E., Shah, J., and Klessig, D.F. (2000). NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid. Mol Plant Microbe Interact 13, 191-202.
    Advisor
  • Ming-Tsair Chan(詹明才)
  • Shir-Ly Huang(黃雪莉)
  • Files
  • 91224016.pdf
  • disapprove authorization
    Date of Submission 2004-10-05

    [Back to Results | New Search]


    Browse | Search All Available ETDs

    If you have dissertation-related questions, please contact with the NCU library extension service section.
    Our service phone is (03)422-7151 Ext. 57407,E-mail is also welcomed.