跳到主要內容

臺灣博碩士論文加值系統

(44.192.22.242) 您好!臺灣時間:2021/08/03 18:21
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:戴宏光
研究生(外文):Hung-Kuang Tai
論文名稱:建構單次可誘導轉位子以中止篩選標記基因之功能
論文名稱(外文):Construction of an inducible transposable element to terminate selectable marker gene
指導教授:常玉強
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農藝學研究所
學門:農業科學學門
學類:一般農業學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:67
中文關鍵詞:嘉磷塞篩選標記基因轉位子EPSPS轉位
外文關鍵詞:glyphosateselectable marker genestransposable elementEPSPS
相關次數:
  • 被引用被引用:0
  • 點閱點閱:153
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
使用篩選標記基因(selectable marker genes)可以快速、有效率的篩選出轉殖株。然而在獲得轉殖株後,篩選標記基因就無特殊用途,並有生物安全疑慮。本論文目的是建構單次可誘導轉位子以終止篩選標記之功能。將轉位酶基因上游構築可誘導啓動子,且轉位子之5’構築於轉位子顯子 (exon) C和D間,而將轉位子之3’構築在修飾之水稻EPSPS ( 5-enolpyruvylshikimate-3-phosphate synthase ) 顯子1和2之間,完成之構築命名為Cpt5ne3。透過誘導劑水楊酸處理,企圖使轉位子轉位 (transposition) 將兩端及其內部分轉位酶基因和部分修飾之EPSPS基因片段切離,達到終止轉位酶基因和修飾之EPSPS基因之功能。將Cpt5ne3以農桿菌法轉形至水稻、菸草和阿拉伯芥。結果共獲得4個水稻癒傷組織轉殖系、33個菸草轉殖系(共55株菸草轉殖株)以及26個阿拉伯芥轉殖系(共210株阿拉伯芥轉殖株)。透過南方墨點分析,阿拉伯芥T-DNA之拷貝數為1~3之間,符合農桿菌低拷貝數預期。以RT-PCR分析三種物種之轉位酶基因均正確表現,但目前尚未發現有轉位現象發生;修飾之EPSPS基因在三物種中均正確表現,以glyphosate 進行抗性測試,水稻轉殖癒傷組織能抗10 mM glyphosate持續增生癒傷組織﹔而阿拉伯芥轉殖株能抗1000 ppm glyphosate,並可正常結種子,此結果可作為未來以glyphosate篩選轉殖株之建議濃度。另外本研究顯示將轉位子5’構築在轉位酶基因隱子(intron)和將轉位子3’構築在修飾之EPSPS基因隱子中均不影響基因轉錄,提供日後將轉位子5’及轉位子3’構築在基因之不同隱子中是否會影響基因正確表現之參考。
Selectable marker genes (SMGs) are often necessary during the process of plant transformation, but unnecessary once transgenic plants have been obtained. Besides, SMGs is involved in biosafety. Therefore, in this work, an inducible transposable element was constructed to terminate the selectable marker gene:modified EPSPS gene. Firstly, the 3’ end of the Ac was inserted in the first intron of the modified rice EPSPS gene, which triggered by the nos promoter. Secondly, The 3’ end of the Ac was inserted in the fourth intron of the inducible Ac transposase gene trigged by PR-1a promoter. Thus, this inducible transposable element contains not only the exon E of Ac transposase gene but also the first exon of the modified EPSPS gene and its promoter. This construct, which termed as Cpt5ne3, was introduced into rice, tobacco, and arabidopsis. The modified EPSPS gene can be transcribed normally in all three species and allow the transgenic rice and arabidopsis to be Glyphosate-resistant. Also, the transposase gene is transcribed correctly. Therefore, it is expected that transposition event can be induced by salicylic acid
目錄……………………………………………………………………………………I
圖表目錄、附錄目錄…………………………………………………………………II
縮寫字對照表……………………………………………………………………… III



中文摘要………………………………………………………………………………1
英文摘要………………………………………………………………………………2
前言……………………………………………………………………………………3
材料與方法 …………………………………………………………………………13
結果 …………………………………………………………………………………30
討論 …………………………………………………………………………………36
未來展望 …………………………………………………………………………40
引用文獻 ……………………………………………………………………………41
李冠德 (2005) 可誘導轉位子單次移除篩選標記基因之研究
謝嘉珊 (2004) 構築含無啟動子報導基因之可誘導轉位子
Aarts M.G., Dirkse W.G., W. J. Stiekema and A. Pereira (1993) Transposon tagging of a male sterility gene in Arabidopsis. Naure. 363(6431): 715-717.
Balcells L., Sundberg E., Coupland G. (1994) A heat-shock promoter fusion to the Ac transposase gene drives inducible transposition of a Ds element during Arabidopsis embryo development. Plant Journal. 5(5): 755-764.
Bradshaw L.D., Padgette S.R., Kimball S.L., Wells BH (1997) Perspectives on glyphosate resistance. Weed Technology 11: 189–198.
Brotherton J. E., Jeschke M. R., Tranel P. J. and Widholm J. M. (2006) Identification of Arabidopsis thaliana Variants with Differential Glyphosate Response. Plant Physiology. In press
Cajacob C.A., Feng P.C., Heck G.R., Murtaza F.A., Sammons R.D., Padgette S.R. (2004) Engineering resistance to herbicides, in Handbook of biotechnology, ed by Christou P and Klee H, Chapter 1, John Wiley&Sons, Chichester, UK, pp 353-372.
Charng Y.C., Pfitzner A. J. P., Pfitzner U. M., Charng C.K.F., Chen C.M., Tu J., Kuo T.T. (2000) Construction of an inducible transposon, INAc, to develop a gene tagging system in higher plants. Molecular Breeding 6: 353-367.
Charng Y.C., Hsieh C.S., (2004) Construction of an inducible transposon with promoterless reporter gene. Crop, Environment and Bioinformatics 1: 89-101.
Chen L., Marmey P., Taylor N.J., Brizard J.P., Espinoza, C., D’Cruz P., Huet H., Zhang S., de Kochko A., Beachy R.N., Fauquet C.M., (1998) Expression and inheritance of multiple transgens in rice plants. Nature. Biotechnology 16:1060–1064.
Cobb A.H., Kirkwood R.C. (2000) Herbicides and their Mechanisms of Action. CRC Press, Boca Raton, FL.
Coppoolse E.R., Vroomen M.J., Roelofs D., Smit J., van Gennip J., Hersmus B.J.M., Nijkamp H.J.J., van Kaaren M.J.J. (2003) Cre recombinase expression can result in phenotypic aberrations in plant. Plant Molecular Biology 51:263-279.
Cotsaftis O., Sallaud C., Breitler J.C., Meynard D., Greco R., Pereira A., Guiderdoni E., (2002) Transposon-mediated generation of T-DNA- and marker-free rice plants expressing a Bt endotoxin gene. Molecular Breeding. 10:165–180.
Daley M., Knauf V.C., Summerfelt K.R., Turner J.C. (1998) Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants. Plant Cell Report 17:489-496.
Dill G. M. (2005) Glyphosate-resistant Crops: History, Status and Future. Pest Management Science. 61:219-224.
Ebinuma H., Sugita K., Matsunaga E., Yamakado M., (1997) Selection of marker-free transgenic plants using the isopentyl transferase gene. Proceeding of the National Academic of Sciences U.S.A. 94, 2117–2121.
Enoki H., Izawa T., Kawahara M., Komatsu M., Koh S., Kyozuka J., Shimamoto K. (1999) Ac as a tool for the functional genomics of rice. Plant Journal 19(5): 605-613.
Firek S., Martin D. J., Roberts M. R., Sturgess F., Scott R., Draper J. (1996)Gametophyte-specific transposition of the maize Ds element in transgenic tobacco. Plant J. 10:569-578.
Fitzmaurice W. P., Nguyen L. V., Wernsman E. A., Thompson W. F. and Conkling M. A. (1999) Transposon Tagging of the Sulfur Gene of Tobacco Using Engineered Maize Ac/Ds Elements. Genetics 153:1919-1928.
Goldsbrough A.P., Lastrella C.N., Yoder J.I., (1993) Transposition mediated re-positioning and subsequent elimination of marker genes from transgenic tomato. Biotechnology 11:1286-1292.
Greco R., Ouwerkerk P. B. F., Kam R. J. d., Sallaud C., Favalli C., Colombo L., Guiderdoni E., Meijer A. H., Hoge J. H. C., Pereira A. (2003) Transpositional behaviour of an Ac/Ds system for reverse genetics in rice. Theoretical and Applied Genetics 108(1): 10-24.
Greco R., Ouwerkerk P. B., Taal A. J., Favalli C., Beguiristain T., Puigdomenech P., Colombo L., Hoge J. H., Pereira A. (2001) Early and multiple Ac transpositions in rice suitable for efficient insertional mutagenesis. Plant Molecular Biology 46(2): 215-227.
Hajdukiewicz P.T. (2001) Multiple pathways for Cre/lox-mediated recombination in plastids. Plant Journal 27:161-170.
Haring M. A., Rommens C. M., Nijkamp H. J., Hille J. (1991) The use of transgenic plants to understand transposition mechanisms and to develop transposon tagging strategies. Plant Molecular Biology. 16(3): 449-461.
Howe A. R., Gasser C. S., Brown S. M., Padgette S.R., Hart J., Parker G.B., Fromm M.E., Armstrong C.L. (2002) Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants. Molecular Breeding 10:153-164.
Hu T., Metz S., Chay C., Zhou H. P., Biest N., Chen G., Cheng M., Feng X., Radionenko M., Lu F., Fry J. (2003) Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Reports 21:1010-1019.
Ipek A. and Simon P. (2002) Developing an Ac/Ds Based Two-Element Transposon Tagging System in Carrot. Plant & Animal Genome Meeting.
Jander G., Baerson S.R., Hudak J.A., Gonzalez K.A., Gruys K.J., Last R.L., (2003) Ethylmethanesulfonate saturation mutagenesis in Arabidopsis to determine frequency of herbicide resistance. Plant Physiology 131:139-146.
Jones D. A., Thomas C. M., Hammond-Kosack K. E., Balint-Kurti P. J., Jones J. D. (1994) Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging. Science 266(5186): 789-793.
Kim S.R., Lee J., Jun S.H., Park S., Kang H.G., Kwon S., An G. (2003) Trangene structures in T-DNA-inserted rice plants. 52: 761-773
Kishore G.M. and Shah D.M. (1988) Amino acid biosynthesis inhibitors as herbicides. Annu. Review Biochemistry 57:627-663.
Klimyuk, V. I. and Jones J. D. G. (1997) AtDMC1, the Arabidopsis homologue of the yeast DMC1 gene: characterization, transposon-induced allelic variation and meiosis-associated expression. Plant Journal 11(1): 1-14.
Kohli, A., Xiong J., Greco R., Christou P., Pereira A. (2001) Tagged Transcriptome Display (TTD) in indica rice using Ac transposition. Molecular Genetics and Genomics 266(1): 1-11.
Komari T., Hoeo Y., Saito Y., Murai N., Kumashiro T. (1996) Vectors carrying two separate T-DNAs for co-transformation for higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant Journal 10:165-174.
Koprek T., Rangel S., McElroy D., Louwerse J. D., Williams-Carrier R. E., Lemaux, P. G. (2001) Transposon-Mediated Single-Copy Gene Delivery Leads to Increased Transgene Expression Stability in Barley. Plant Physiology. 125:1354-1362.
Liu Y.G., Mitsukawa N., Oosumi T., Whittier R. F. (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant Journal 8: 457-463.
Lyznik L.A., Rao K.V., Hodges T.K. (1996) FLP-mediated recombination of FRT sites in the maize genome. Nucleic Acids Research 24:3784-3789.
Matthews P.R., Waterhouse P.M., Thornton S., Fieg S.J., Gubler F., Jacobsen J.V., (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent ‘twin T-DNAs’ on a standard Agrobacterium transformation vector. Molecular Breeding 7:195–202.
McClean P., (1998) http://www.ndsu.edu/instruct/mcclean/plsc731/transgenic/transgenic3.htm
McCormac A.C., Elliott M.C., Chen D.F. (1998) A simple method for the production of highly competent cells of Agrobacterium for transformation via electroporation. Molecular Biotechnology 9:155-159.
Meissner R., Chague V., Zhu Q., Emmanuel E., Elkind Y., Levy A. A. (2000) A High Throughput System for Transposon Tagging and Promoter Trapping in Tomato. The plant journal. 22(3):265-274.
Miki B. and McHugh S. (2004). "Selectable marker genes in transgenic plants: applications, alternatives and biosafety." Journal of Biotechnology 107(3): 193-232.
Miller M., Tagliani L., Wang N., Berka B., Bidney D., Zhao Z.Y., (2002) High efficiency transgene segregation in co-transformed maize plants using an Agrobacterium tumifaciens 2 T-DNA binary system. Transgenic Reseasrch 11:381–396.
Morita R., Hattori Y., Yokoi S., Takase H., Minami M., Hiratsuka K., Toriyama K. (2003) Assessment of utility of meiosis-associated promoters of lily for induction of germinal Ds transposition in transgenic rice. Plant Cell Physiology 44(6): 637-642.
Muller-Neumann M., Yoder J. I., Starlinger P., (1984) The DNA sequence of the transposable element of Ac of Zea mays L. Molecular Genetics Genomics 198: 19-24.
Nakagawa Y., Machida C., Machida Y., Toriyama K. (2000) Frequency and pattern of transposition of the maize transposable element Ds in transgenic rice plants. Plant Cell Physiology 41(6): 733-742.
Nishal B., Tantikanjana T., Sundaresean V. (2005) An Inducible Targeted Tagging System for Localized Saturation Mutagenesis in Arabidopsis. Plant Physiology 137:3-12.
Ochman H., Gerber A. S., Hartl D. L. (1988). Genetic applications of an inverse polymerase chain reaction. Genetics 120: 621-623.
Padgette S.R., Re D.B., Barry G.F., Eichholtz D.E., Delannay X., Fuchs R.L., Kishore G.M., Fraley R.T. (1996) New weed control opportunities: development of soybeans with a Roundup Ready gene. In S Duke, ed, Herbicide Resistant Crops: Agricultural, Economic, Environmental, Regulatory, and Technological Aspects. CRC Press, Boca Raton, FL, pp53–84.
Padgette S.R., Re D.B., Gasser C.S., Eichholtz D.A., Frazier R.B., Hironaka C.M., Levine E.B., Shah D.M., Fraley R.T., Kishore G.M. (1991) Site-directed mutagenesis of a conserved region of the 5-enolpyruvylshikimate-3-phosphate synthase active site. Journal Biology. Chemistry 266: 22364–22369.
Perez-Jones A., Park K-W, Polge N., Colquhoun J., Mallory-Smith C. A. (2007) Investigating the Mechanisms of Glyphosate Resistance in Lolium multiflorum. Planta. In press
Podevin N., Buck S. D., Wilde C. D., Depicker A. (2006) Insights into recognition of the T-DNA border repeats as termination sites for T-strand synthesis by Agrobacterium tumefaciens. Transgenic Research 15:557-571.
Sambrook J., Fritsch E. F., Maniatis T. (1989) Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Schönbrunn E., Eschenburg S., Shuttleworth W.A., Schloss J.V., Amrhein N., Evans J.N.S., Kabsch W. (2001) Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail. Proceeding of the National Academic of Sciences U.S.A. 98: 1376–1380.
Sha Y., Li S., Pei Z., Luo L., Tian Y., He C., (2004) Generation and flanking sequence analysis of a rice T-DNA tagged population. Theoretical and Applied Genetics 108:306-314.
Shah D.M., Horsch R.B., Klee H.J., Kishore G.M., Winter J.A., Tumer N.E., Hironka C.M., Sanders P.R., Gasser C.S., Ayken S., Siegel N.R., Rogers S.G., Fraley R.T. (1986) Engineering herbicide tolerance in transgenic plant. Science 233:478-481.
Sheu J. J., Yu T. S., Yu S. M. (1996) Carbohydrate starvation stimulates differential expression of rice alpha-amylase genes that is modulates through complicated transcriptional and posttranscriptional processes. Journal Biology Chemistry 271:26998-7004.
Southern E. M. (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal Moecular. Biology 98:503-17.
Sugita K., Kasahara E., Matsunaga E., Ebinuma H. (2000) A transformation vector for the production of marker-free transgenic plants containing a single copy transgene at high frequency. Plant Journal 22:461-469.
Sugita K., Matsunaga E., Ebinuma H. (1999) Effective selection system for generating marker-free transgenic plants independent of sexual crossing. Plant Cell Report 18:941-947.
Swinburne J., Balcells L., Scofield S. R., Jones J. D. G.,Coupland, G. (1992) Elevated Levels of Activator Transposase mRNA Are Associated with High Frequencies of Dissociation Excision in Arabidopsis. The Plant Cell 4:583-595.
Thomas C.M., Jones J.D.G., (2007) Molecular analysis of Agrobacterium T-DNA integration in tomato reveals a role for left border sequence homology in most integration events Molecular Genetics Genomics In press
Tissier A. F., Marillonnet S., Klimyuk V., Patel K., Torres M.A., Murphy G., Jones J.D. (1999) Multiple independent defective suppressor-mutator transposon insertions in Arabidopsis: a tool for functional genomics. Plant Cell 11: 1841-1852.
Triglia T.,Peterson M.G.,Kemp D.J. (1988). A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Research 16: 186.
Upadhyaya N. M., Zhou X.R., Zhu Q.H., Ramm K., Wu L., Eamens A., Sivakumar R., Kato T., Yun D.W., Santhoshkumar C., Narayanan K.K., Peacock J.W, Dennis E.S. (2002) An iAc/Ds gene and enhancer trapping system for insertional mutagenesis in rice. Functional Plant Biology 29(5): 547-559.
Wang J.X., Zhao F.Y., Xu P. (2006) Use of aroA-M1 as a selectable marker for Brassica napus transformation. Crop Science 46:706-711.
Wei C.F., Kunze R. (2000) Transposition of maize Ac/Ds transposable elements in the yeast Saccharomyces cerevisiae. Nature Genetics 26: 187–190
Widholm J.M., Chinnala A.R., Ryu J.H., Song H.S., Egett T., Brotherton J.E. (2001) Glyphosate selection of gene amplification in suspension cultures if three plant species. Physiology Plant 112:540-545.
Wu L, Nagano H, Yoshida K, Kawasaki S, Kishima Y, Sano Y (2001) Mapping of the sequences expressed in the rice callus onto a BAC contig: identification of a genomic segment carrying closely linked genes encoding EPSP synthase and rps20. Unpublished
Yan Y., An C., Li L., Gu J., Tan G., Chen Z. (2003) T-linker-speciAc ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends. Nucleic Acids Research 31(12): e68, 7 pp.
Yuan C.I., Chiang M.Y., Chen Y.M. (2002) Triple mechanisms of glyphosate-resistance in a naturally occurring glyphosate-resistant plant Dicliptera chinensis. Plant Science 163: 543-554.
Zhou H., Arrowsmith J.W., Fromm M.E., Hironaka C.M., Taylor M.L., Rodriguez D., Pajeau M.E., Brown S.M., Santino C.G., Fry J.E. (1995) Glyphosate-tolerant CP4 and GOX genes as a selectable marker in wheat transformation Plant Cell Report 15:159-163.
Zhou M., Xu H., Wei X., Ye Z., Wei L., Gong W., Wang Y., Zhu Z. (2006) Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy. Plant Physiology 140:184-195.
Zubco E., Scutt C., Meyer P., (2000) Intrachromosomal recombination between attP regions as a tool to remove selectable marker genes from tobacco transgenes. Nature Biotechnology 18:442–445.
Zuo J., Niu Q.W., Moller S.G., Chua N.H., (2001) Chemical-regulated, site-specific DNA excision in transgenic plants. Nature Biotechnology 19:157–161.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top