Please use this identifier to cite or link to this item:
標題: 以表現輔助蛋白提升農桿菌的植物轉殖效率
Increase Agrobacteria-mediated plant transformation efficiency by expression of accessory proteins
作者: 林家誠
Lin, Jia-Cheng
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: 周光宇、翁堅、龔蓁蓁、曾以申、楊曉霞、沈慰芳、王自芬、陶金洲,農業分子育種-授粉後外源DNA導入植物的技術,中國科學 第21期,第1-6頁(1988)。 詹明才、張新雄。1991。農桿菌轉殖系統之影響因素。科學農業 39:249-255。 Anzai, H., Ishii Y., Shichinohe M., Katsumata K., Nojiri C., Morikawa H.,and Tanaka M. (1996). Transformation of Phalaenopsis by particle bombardment. Plant Tissue Cult. Lett 13, 265-271. 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. Armstrong, C.L., Petersen, W.L., Buchholz, W.G., Bowen, B.A., and Sulc, S.L. (1990). Factors Affecting Peg-Mediated Stable Transformation of Maize Protoplasts. Plant Cell Rep 9, 335-339. Bates, G.W. (1999). Plant transformation via protoplast electroporation. Methods Mol Biol 111, 359-366. Boyko, A., Matsuoka, A., and Kovalchuk, I. (2009). High frequency Agrobacterium tumefaciens-mediated plant transformation induced by ammonium nitrate. Plant Cell Rep 28, 737-757. Carrington, J.C., Kasschau, K.D., and Johansen, L.K. (2001). Activation and suppression of RNA silencing by plant viruses. Virology 281, 1-5. Chen, W.S., Chiu, C.C., Liu, H.Y., Lee, T.L., Cheng, J.T., Lin, C.C., Wu, Y.J., and Chang, H.Y. (1998). Gene transfer via pollen-tube pathway for anti-fusarium wilt in watermelon. Biochem Mol Biol Int 46, 1201-1209. Cheng, M., Fry, J.E., Pang, S.Z., Zhou, H.P., Hironaka, C.M., Duncan, D.R., Conner, T.W., and Wan, Y.C. (1997). Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115, 971-980. 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. Christou, P. (1992). Genetic-Transformation of Crop Plants Using Microprojectile Bombardment. Plant J 2, 275-281. Clough, S.J., and Bent, A.F. (1998). Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16, 735-743. 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. Doty, S.L., Yu, M.C., Lundin, J.I., Heath, J.D., and Nester, E.W. (1996). Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens. J Bacteriol 178, 961-970. Erikson, O., Hertzberg, M., and Nasholm, T. (2004). A conditional marker gene allowing both positive and negative selection in plants. Nat Biotechnol 22, 455-458. 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.P., and Walbot, V. (1985). Expression of Genes Transferred into Monocot and Dicot Plant-Cells by Electroporation. Proc Natl Acad Sci USA 82, 5824-5828. Gordon-Kamm, W., Dilkes, B.P., Lowe, K., Hoerster, G., Sun, X.F., Ross, M., Church, L., Bunde, C., Farrell, J., Hill, P., Maddock, S., Snyder, J., Sykes, L., Li, Z.S., Woo, Y.M., Bidney, D., and Larkins, B.A. (2002). Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway. Proc Natl Acad Sci USA 99, 11975-11980. Grange, W., Duckely, M., Husale, S., Jacob, S., Engel, A., and Hegner, M. (2008). VirE2: A unique ssDNA-compacting molecular machine. Plos Biol 6, 343-351. Hiei, Y., Komari, T., and Kubo, T. (1997). Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol 35, 205-218. Hu, C.Y., and Wang, L.Z. (1999). In planta soybean transformation technologies developed in China: Procedure, confirmation and field performance. In Vitro Cell Dev-Pl 35, 417-420. 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. (2005). The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation. Embo Journal 24, 428-437. Lazzeri, P.A., Brettschneider, R., Luhrs, R., and Lorz, H. (1991). Stable Transformation of Barley Via Peg-Induced Direct DNA Uptake into Protoplasts. Theor Appl Genet 81, 437-444. 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. Proc Natl Acad Sci USA 102, 19231-19236. Li, S.H., Kuoh, C.S., Chen, Y.H., Chen, H.H., and Chen, W.H. (2005). Osmotic sucrose enhancement of single-cell embryogenesis and transformation efficiency in Oncidium. Plant Cell Tiss Org 81, 183-192. Marion, J., Bach, L., Bellec, Y., Meyer, C., Gissot, L., and Faure, J.D. (2008). Systematic analysis of protein subcellular localization and interaction using high-throughput transient transformation of Arabidopsis seedlings. Plant J 56, 169-179. Mu, H.M., Liu, S.J., Zhou, W.J., Wen, Y.X., Zhang, W.J., and Wei, R.X. (1999). Transformation of wheat with insecticide gene of arrowhead proteinase inhibitor by pollen tube pathway and analysis of transgenic plants In Process Citation. Yi Chuan Xue Bao 26, 634-642. 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. Neuhaus, G., Spangenberg, G., Scheid, O.M., and Schweiger, H.G. (1987). Transgenic Rapeseed Plants Obtained by the Microinjection of DNA into Microspore-Derived Embryoids. Theor Appl Genet 75, 30-36. Park, J., Lee, Y.K., Kang, B.K., and Chung, W.I. (2004). Co-transformation using a negative selectable marker gene for the production of selectable marker gene-free transgenic plants. Theor Appl Genet 109, 1562-1567. Rachmawati, D., Hosaka, T., Inoue, E., and Anza, H. (2004). Agrobacterium-mediated transformation of Javanica rice cv. Rojolele. Biosci Biotechnol Biochem 68, 1193-1200. Sato, S., Newell, C., Kolacz, K., Tredo, L., Finer, J., and Hinchee, M. (1993). Stable Transformation Via Particle Bombardment in 2 Different Soybean Regeneration Systems. Plant Cell Rep 12, 408-413. 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. Stachel, S.E., Messens, E., Vanmontagu, M., and Zambryski, P. (1985). Identification of the Signal Molecules Produced by Wounded Plant-Cells That Activate T-DNA Transfer in Agrobacterium-Tumefaciens. Nature 318, 624-629. 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. Tjokrokusumo, D., Heinrich, T., Wylie, S., Potter, R., and McComb, J. (2000). Vacuum infiltration of Petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation. Plant Cell Rep 19, 792-797. 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. (2001). VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity. EMBO J 20, 3596-3607. Tzfira, T., Vaidya, M., and Citovsky, V. (2004). Involvement of targeted proteolysis in plant genetic transformation by Agrobacterium. Nature 431, 87-92. Vain, P., Mcmullen, M.D., and Finer, J.J. (1993a). Osmotic Treatment Enhances Particle Bombardment-Mediated Transient and Stable Transformation of Maize. Plant Cell Rep 12, 84-88. Vain, P., Keen, N., Murillo, J., Rathus, C., Nemes, C., and Finer, J.J. (1993b). Development of the Particle Inflow Gun. Plant Cell Tiss Org 33, 237-246. Vanwert, S.L., and Saunders, J.A. (1992). Reduction of Nuclease Activity Released from Germinating Pollen under Conditions Used for Pollen Electrotransformation. Plant Sci 84, 11-16. Voinnet, O., Rivas, S., Mestre, P., and Baulcombe, D. (2003). 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. Ward, D.V., and Zambryski, P.C. (2001). The six functions of Agrobacterium VirE2. Proc Natl Acad Sci USA 98, 385-386. Yuan, Z.C., Edlind, M.P., Liu, P., Saenkham, P., Banta, L.M., Wise, A.A., Ronzone, E., Binns, A.N., Kerr, K., and Nester, E.W. (2007). The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium. Proc Natl Acad Sci U S A 104, 11790-11795. Zambryski, P., Tempe, J., and Schell, J. (1989). Transfer and Function of T-DNA Genes from Agrobacterium Ti-Plasmid and Ri-Plasmid in Plants. Cell 56, 193-201. 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.
農桿菌常被廣泛應用於植物基因轉殖技術中,因其操作簡易,可使目標基因單一且完整的插入染色體組中,並且不易引發基因靜默 (gene silencing)反應,因此,近30年來以此法進行轉殖成功的植物超過百種,然而仍有許多植物難以進行基因轉殖,其中包括蘭花、百合等重要花卉作物,故如何提升植物轉殖效率仍舊是極為重要的課題。農桿菌轉殖法中的T-DNA傳送過程需要許多輔助蛋白 (accessory protein)參與,包括來自農桿菌或植物的蛋白質,例如BTI、VIP1、Ku80、H2A等。許多文獻曾指出經基因轉殖使大量表現輔助蛋白之擬南芥,其T1世代植株被再度轉殖的效率大幅提升,暗示輔助蛋白可能扮演提升轉殖效率的角色。

由於上述研究材料均僅侷限於雙子葉之模式植物-擬南芥,因此本實驗選用可轉譯出輔助蛋白的水稻同源基因,稱之為促轉基因 (enhance transformation genes,簡稱為ET gene),構築於植物表現載體上,分別對文心蘭與擬南芥進行轉殖實驗,希望在單、雙子葉植物中均測試促轉基因的效果,並於T0世代(即野生株)即進行評估,以達到利用促轉基因提升目標基因轉殖效率的實用目的。實際作法上乃利用分別帶有促轉基因或GUS報導基因的兩種農桿菌,進行共同轉殖 (co-transformation),數日後藉由偵測短暫性表現的GUS蛋白活性研判促轉基因是否可提升轉殖效率。循此想法,首先需測試雙農桿菌的共轉殖效率,以分別帶有紅、綠螢光蛋白的兩株農桿菌共同感染文心蘭,其後製成原生質體 (protoplast)觀察,發現農桿菌共同轉殖單一細胞的效率高達90%。故以空植物表現載體為控制組,以促轉基因為實驗組,分別與帶有GUS報導基因的農桿菌進行共同感染,發現GUS報導基因於文心蘭中的表現活性極低,難以用於進行定量評估,故將改以強啟動子或病毒的抑制基因 (viral suppressor gene)提升GUS報導基因的基礎表現量,以真正評估促轉基因於文心蘭轉殖的效果。另一方面,擬南芥的測試顯示共轉殖ET2、ET6與ET9促轉基因可提升暫時性GUS基因的表現量 (p< 0.05,Student’s t-test),暗示T-DNA被傳送入核的頻率上升。然而,於永久性轉殖實驗,發現ET7可提升2.5倍的轉殖效率(p< 0.05,Student’s t-test),經聚合酶鏈鎖反應及南方墨點法,不論促轉基因同時插入染色體的頻率或GUS報導基因的嵌入複本數均與控制組無太大差異。暗示ET7促轉基因可提升GUS報導基因永久性轉殖效率,但不會提升嵌入的複本數。

Agrobacteria-mediated transformation of higher plants is a well-known and powerful tool for T-DNA delivery to plant cells. The method results in mostly single or low-copy integration of full-length transgene, and is less likely to trigger gene silencing than gene-gun mediated method. Various technical modifications, either on Agrobacteria or plant side, were employed to improve the transformation efficiency ever since its invention on 1983. However, many plant species or cultivars are still granted as “recalcitrant” that barely give rise to regenerable and non-chimeric transgenic lines. Therefore, it is still critical to improve efficiency of this technique.
The long journey for T-DNA transferring till the final destination not only requires bacteria proteins but also is aided by various plant factors, including BTI, VIP1, Ku80, H2A, etc. Interestingly, T1 progenies of transgenic Arabidopsis overproducing several accessory proteins were found to exhibit higher transformation efficiency, suggesting roles of the accessory proteins in “enhance transformation”, designed as “ET genes” in this study. To test if this condition could be employed at the T0 stage, so to enhance delivery of target gene directly, we use co-transformation strategy with two T-DNA harbored in two Agrobacteria. One T-DNA carries simply the ET gene ortholog from rice and the other T-DNA contains GUS reporter gene together with hygromycin selection marker. Oncidium and Arabidopsis were chosen as the organisms to test.
To evaluate the efficiency of Agrobacteria-mediated co-transformation in Oncidium, protoplast was prepared after transformation and observed for frequency of co-presence of GFP and RFP proteins within single cell. Surprisingly, the co-transformation efficiency reached ~90%, indicates a feasibility of our strategy. Unfortunately, the expression level of GUS protein in protocorm-like bodies (PLBs) of Oncidium was too low for quantitative assays, even strong promoter and anti-silencing viral suppressors were employed for boosting gene expressions. On the other hand, using Arabidopsis root segments as materials did identify three ET genes, ET2, ET6 and ET9, in enhancement of transient transformations to a significant level (p<0.05 in Student's t-test). Moreover, floral-dip method for producing transgenic Arabidopsis was improved by the co-presence of ET7 to 2.5-fold. The co-integration frequency of ET gene and copy number of transgene were not particularly elevated in the transgenic lines examined, suggesting a usage of ET7 to increase the transformation frequency without a deleterious effect.
其他識別: U0005-1808201018173600
Appears in Collections:生物科技學研究所

Show full item record

Google ScholarTM


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