Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36276
標題: 以農桿菌浸潤菸草之暫時性轉殖分析評估T-DNA傳送過程之輔助蛋白的促轉效果
Examination Effect of Accessory Protein in T-DNA transferring by Transient Agroinfiltration of Tobacco
作者: 林怡君
Lin, Yi-Jyun
關鍵字: Agrobacteria-mediated plant transformation
農桿菌轉殖
出版社: 生物科技學研究所
引用: Anand, A., Krichevsky, A., Schornack, S., Lahaye, T., Tzfira, T., Tang, Y., Citovsky, V., and Mysore, K.S. (2007). Arabidopsis VIRE2 INTERACTING PROTEIN2 is required for Agrobacterium T-DNA integration in plants. Plant Cell 19, 1695-1708. Carrington, J.C., Kasschau, K.D., and Johansen, L.K. (2001). Activation and suppression of RNA silencing by plant viruses. Virology 281, 1-5. Christie, P.J., Atmakuri, K., Krishnamoorthy, V., Jakubowski, S., and Cascales, E. (2005). Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu Rev Microbiol 59, 451-485. Dafny-Yelin, M., Levy, A., and Tzfira, T. (2008). The ongoing saga of Agrobacterium-host interactions. Trends Plant Sci 13, 102-105. Ding, Z.Y., Atmakuri, K., and Christie, P.J. (2003). The outs and ins of bacterial type IV secretion substrates. Trends Microbiol 11, 527-535. Friesner, J., and Britt, A.B. (2003). Ku80- and DNA ligase IV-deficient plants are sensitive to ionizing radiation and defective in T-DNA integration. Plant J 34, 427-440. Fromm, M., Taylor, L., and Walbot, V. (1985). Expression of genes transferred into monocot and dicot plant cells by electroporation. Proc Natl Acad Sci U S A 82, 5824-5828. Gelvin, S.B., Tenea, G.N., Spantzel, J., Lee, L.Y., Zhu, Y.M., Lin, K., and Johnson, S.J. (2009). Overexpression of Several Arabidopsis Histone Genes Increases Agrobacterium-Mediated Transformation and Transgene Expression in Plants. Plant Cell 21, 3350-3367. Griesbach, R.J. (1987). Chromosome-mediated transformation via microinjection. Plant Science 50, 69-77. Hwang, H.H., and Gelvin, S.B. (2004). Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation. Plant Cell 16, 3148-3167. Jin, S.G., Prusti, R.K., Roitsch, T., Ankenbauer, R.G., and Nester, E.W. (1990). Phosphorylation of the VirG protein of Agrobacterium tumefaciens by the autophosphorylated VirA protein: essential role in biological activity of VirG. J Bacteriol 172, 4945-4950. Klein, T.M., Wolf, E.D., Wu, R., and Sanford, J.C. (1987). High-velocity microprojectiles for delivering nucleic acids into living cells. Nature 327, 70-73. Lacroix, B., and Citovsky, V. (2009). Agrobacterium aiming for the host chromatin: Host and bacterial proteins involved in interactions between T-DNA and plant nucleosomes. Commun Integr Biol 2, 42-45. Lacroix, B., Vaidya, M., Tzfira, T., and Citovsky, V. (2005a). The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation. Embo Journal 24, 428-437. Lacroix, B., Vaidya, M., Tzfira, T., and Citovsky, V. (2005b). The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation. EMBO J 24, 428-437. Levy, A., Dafny-Yelin, M., and Tzfira, T. (2008). Attacking the defenders: plant viruses fight back. Trends Microbiol 16, 194-197. Li, J., Krichevsky, A., Vaidya, M., Tzfira, T., and Citovsky, V. (2005a). Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium. Proc Natl Acad Sci U S A 102, 5733-5738. Li, J.X., Vaidya, M., White, C., Vainstein, A., Citovsky, V., and Tzfira, T. (2005b). Involvement of KU80 in T-DNA integration in plant cells. P Natl Acad Sci USA 102, 19231-19236. Luo, Z.X., and Wa, R. (1988). A simple method for the transformation of rice via pollen-tube pathway. plant Mol. Biol. Report 6, 165-174. McCormac, A.C., Fowler, M.R., Chen, D.F., and Elliott, M.C. (2001). Efficient co-transformation of Nicotiana tabacum by two independent T-DNAs, the effect of T-DNA size and implications for genetic separation. Transgenic Res 10, 143-155. Mimori, T., and Hardin, J.A. (1986). Mechanism of interaction between Ku protein and DNA. J Biol Chem 261, 10375-10379. Mitsuhara, I., Ugaki, M., Hirochika, H., Ohshima, M., Murakami, T., Gotoh, Y., Katayose, Y., Nakamura, S., Honkura, R., Nishimiya, S., Ueno, K., Mochizuki, A., Tanimoto, H., Tsugawa, H., Otsuki, Y., and Ohashi, Y. (1996). Efficient promoter cassettes for enhanced expression of foreign genes in dicotyledonous and monocotyledonous plants. Plant Cell Physiol 37, 49-59. Mysore, K.S., Nam, J., and Gelvin, S.B. (2000). An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration. Proc Natl Acad Sci U S A 97, 948-953. Negrutiu, I., Shillito, R., Potrykus, I., Biasini, G., and Sala, F. (1987). Hybrid genes in the analysis of transformation conditions. I. Setting up a simple method for direct gene transfer in plant protoplasts. Plant Mol Biol 8, 363-373. Schrammeijer, B., Risseeuw, E., Pansegrau, W., Regensburg-Tuink, T.J., Crosby, W.L., and Hooykaas, P.J. (2001). Interaction of the virulence protein VirF of Agrobacterium tumefaciens with plant homologs of the yeast Skp1 protein. Curr Biol 11, 258-262. Schulte-Uentrop, L., El-Awady, R.A., Schliecker, L., Willers, H., and Dahm-Daphi, J. (2008). Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks. Nucleic Acids Res 36, 2561-2569. Sheng, J.S., and Citovsky, V. (1996). Agrobacterium plant cell DNA transport: Have virulence proteins, will travel. Plant Cell 8, 1699-1710. Sparkes, I., Tolley, N., Aller, I., Svozil, J., Osterrieder, A., Botchway, S., Mueller, C., Frigerio, L., and Hawes, C. (2010). Five Arabidopsis reticulon isoforms share endoplasmic reticulum location, topology, and membrane-shaping properties. Plant Cell 22, 1333-1343. Tenea, G.N., Spantzel, J., Lee, L.Y., Zhu, Y.M., Lin, K., Johnson, S.J., and Gelvin, S.B. (2009). Overexpression of Several Arabidopsis Histone Genes Increases Agrobacterium-Mediated Transformation and Transgene Expression in Plants. Plant Cell 21, 3350-3367. Tzfira, T., and Citovsky, V. (2002). Partners-in-infection: host proteins involved in the transformation of plant cells by Agrobacterium. Trends Cell Biol 12, 121-129. Tzfira, T., Vaidya, M., and Citovsky, V. (2004). Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature 431, 87-92. van der Fits, L., Deakin, E.A., Hoge, J.H., and Memelink, J. (2000). The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43, 495-502. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003a). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33, 949-956. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003b). An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J 33, 949-956. Zhu, J., Oger, P.M., Schrammeijer, B., Hooykaas, P.J.J., Farrand, S.K., and Winans, S.C. (2000). The bases of crown gall tumorigenesis. Journal of Bacteriology 182, 3885-3895.
摘要: 農桿菌轉殖為常見的植物基因轉殖方法,在農桿菌傳送T-DNA至植物細胞的過程中需要許多輔助蛋白協助,除了來自農桿菌的蛋白,如VirE2、VirE3等,來自植物的BTI1/BTI2/BTI3/RAB8、VIP1、VIP2、Ku70/Ku80、H2A等蛋白也是必要的。曾有文獻指出,經基因轉殖而大量表現輔助蛋白的擬南芥,其T1世代再度被轉殖的效率大幅提升,由此推測輔助蛋白可能有提升轉殖效率的功能。 實驗室將可轉譯出輔助蛋白的水稻同源基因(實驗室將之命名為enhance transformation gene,簡稱為ET基因或促轉基因)構築於植物表現載體上,使用共轉(co-transformation)策略測試促轉基因在轉殖當代的作用。所謂共轉策略也就是將一帶有促轉基因的農桿菌與另一帶有報導基因(mGFP5)的農桿菌混合後進行轉殖,本論文以此方法測試菸草短暫性轉殖效率是否可因促轉基因存在而提升。 實驗結果發現,一些促轉基因能有效提升轉殖率,而將啟動子更換為2X35S之後,促轉效果也跟著提升。由於促轉基因在農桿菌轉殖過程中分別扮演不同角色,本實驗進一步混合不同功能的促轉基因,結果產生協同作用的效果,其中以VirE2+VIP1+H2A的組合,促轉效率最高。此外,為了證明轉殖率提升的結果確實來自促轉蛋白的幫助,我們將促轉基因的長度縮短,構築出突變版本進行轉殖測試,結果顯示,促轉基因其蛋白讀碼被破壞後,促轉效果即消失。 另一方面,我們使用阿拉伯芥花序為材料,以floral dip的方式對其進行永久性轉殖,初步測試促轉基因的效果,結果顯示BTI3提升轉殖效率約2.2倍。此外,把輔助蛋白VirG原生型或其突變型(N54D)質體轉入農桿菌EHA105中,使過度表現VirG蛋白,結果發現使用來自農桿菌種C58的VirG 基因可增加EHA105的致毒性,而來自農桿菌種LBA4404者則否。
For the Agrobacterium-mediated plant transformation, the long journey for T-DNA transferring from bacteria till the final destination not only requires bacterial proteins, such as VirE2, VirE3, etc., but also is aided by various plant factors, including BTI1/BTI2/BTI3/Rab8, VIP1, VIP2, Ku70/Ku80, H2A, etc. Many reports had demonstrated that T1 progenies of transgenic Arabidopsis overproducing various accessory proteins exhibited higher transformation efficiency. Gene orthologs encoding the accessory proteins were isolated from rice and designated as “ET genes” for purpose of “enhance transformation”. Co-transformation strategy, infiltrate one Agrobacterium carries an ET gene and the other contains the mGFP5 reporter gene, was employed to evaluate transient transformation efficiency in tobacco. Several ET genes were already identified to be effective. In this study, ET gene expressed by 2X35S promoter mostly exerted better results than the previous constructs. Synergistic effects were observed when several ET genes were combined, with the best effect observed for mixture of VirE2, VIP1 and H2A genes. Moreover, to demonstrate that enhancement of transformation is caused by expression of ET proteins, truncated ET genes were generated. As a result, disrupt open reading frame of ET gene concomitantly diminish its ET effect. A preliminary test using floral dip of Arabidopsis revealed that BTI3 enhance permanent transformation frequency to be about ~2.2 fold. Besides, Agrobacterium EHA105 was transformed with plasmids to overproduce VirG proteins in its wildtype or constitutively active (N54D) version. VirG gene from C58, but not from LBA4404, was found to increase the “virulence” of EHA105.
URI: http://hdl.handle.net/11455/36276
其他識別: U0005-0908201114373900
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0908201114373900
Appears in Collections:生物科技學研究所

文件中的檔案:

取得全文請前往華藝線上圖書館



Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.