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標題: 利用菸草暫時性轉殖系統評估共同表現之輔助蛋白對T-DNA傳送效率之影響
Evaluation of Co-expressed Accessory Proteins in the Enhancement of T-DNA Transfer Efficiency Using Agroinfiltration Analyses on N. benthamiana
作者: 戴于政
Dai, Yu-Cheng
關鍵字: N. benthamiana
transient co-expression
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, Mysore KS. (2007) Arabidopsis VIRE2 INTERACTING PROTEIN2 Is Required for Agrobacterium T-DNA Integration in Plants. Plant Cell. 19(5):1695-708. Christie PJ, Atmakuri K, Krishnamoorthy V, Jakubowski S, Cascales E. (2005) Biogenesis, architecture, and function of bacterial type IV secretion systems. Annu Rev Microbiol. 59:451-85. Dafny-Yelin M, Levy A, Tzfira T. (2008) The ongoing saga of Agrobacterium-host interactions. Trends Plant Sci. 13(3):102-5. Ding Z, Atmakuri K, Christie PJ. (2003) The outs and ins of bacterial type IV secretion substrates. Trends Microbiol. 11(11):527-35. Djamei A, Pitzschke A, Nakagami H, Rajh I, Hirt H. (2007) Trojan Horse Strategy in Agrobacterium Transformation: Abusing MAPK Defense Signaling. Science. 318(5849):453-6. Dumas F, Duckely M, Pelczar P, Van Gelder P, Hohn B. (2001) An Agrobacterium VirE2 channel for transferred-DNA transport into plant cells. Proc Natl Acad Sci U S A. 98(2):485-90. Friesner J., Britt AB. (2003) Ku80- and DNA ligase IV-deficient plants are sensitive to ionizing radiation and defective in T-DNA integration. Plant J. 34(4):427-40 Grange W, Duckely M, Husale S, Jacob S, Engel A, Hegner M. (2008) VirE2: A Unique ssDNA-Compacting Molecular Machine. PLoS Biol. 6(2):e44. Hwang HH, Gelvin SB. (2004) Plant Proteins That Interact with VirB2, the Agrobacterium tumefaciens Pilin Protein, Mediate Plant Transformation. Plant Cell. 16(11):3148-67. Lacroix B, Vaidya M, Tzfira T, Citovsky V. (2005) The VirE3 protein of Agrobacterium mimics a host cell function required for plant genetic transformation. EMBO J. 24(2):428-37. Levy A, Dafny-Yelin M, Tzfira T. (2008) Attacking the defenders: plant viruses fight back. Trends Microbiol. 16(5):194-7. Li J, Krichevsky A, Vaidya M, Tzfira T, 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(16):5733-8. Li J, Vaidya M, White C, Vainstein A, Citovsky V, Tzfira T. (2005b) Involvement of KU80 in T-DNA integration in plant cells. Proc Natl Acad Sci U S A. 102(52):19231-6. Marillonnet S, Thoeringer C, Kandzia R, Klimyuk V, Gleba Y. (2005) Systemic Agrobacterium tumefaciens-mediated transfection of viral replicons for efficient transient expression in plants. Nat Biotechnol. 23(6):718-23 McCormac AC, Fowler MR, Chen DF, Elliott MC. (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(2): 143-55. Mysore KS, Nam J, Gelvin SB. (2000) An Arabidopsis histone H2A mutant is deficient in Agrobacterium T-DNA integration. Proc Natl Acad Sci U S A. 97(2):948-53. Sainsbury F, Lomonossoff GP. (2008) Extremely High-Level and Rapid Transient Protein Production in Plants without the Use of Viral Replication. Plant Physiol. 148(3):1212-8. Schulte-Uentrop L, El-Awady RA, Schliecker L, Willers H, 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(8):2561-9 Tzfira T, Vaidya M, Citovsky V. (2002) Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis nuclear protein VIP1. Proc Natl Acad Sci U S A. 99(16):10435-40. Voinnet O, Rivas S, Mestre P, 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(5):949-56. Yi H, Mysore KS, Gelvin SB. (2002)Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation. Plant J. 32(3):285-98. Yi H, Sardesai N, Fujinuma T, Chan CW, Veena, Gelvin SB. (2006) Constitutive Expression Exposes Functional Redundancy between the Arabidopsis Histone H2A Gene HTA1 and Other H2A Gene Family Members. Plant Cell. 18(7):1575-89. Yuan ZC, Edlind MP, Liu P, Saenkham P, Banta LM, Wise AA, Ronzone E, Binns AN, Kerr K, Nester EW. (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(28):11790-5. Zambryski P, Tempe J, Schell J. (1989) Transfer and function of T-DNA genes from Agrobacterium Ti and Ri plasmids in plants. Cell. 56(2):193-201. Zhu J, Oger PM, Schrammeijer B, Hooykaas PJ, Farrand SK, Winans SC. (2000) The Bases of Crown Gall Tumorigenesis. J Bacteriol. 182(14):3885-95.
摘要: 在植物轉殖技術中,農桿菌(Agrobacterium tumefaciens)是最常用的工具之一。農桿菌感染植物後,Ti質體中的Transfer DNA (T-DNA)會轉移至植物細胞內,並嵌入染色體上。T-DNA的傳送過程需要許多輔助蛋白參與,包括來自農桿菌或植物的蛋白質。T-DNA在細菌中的傳送是藉由細菌本身合成的virulence (Vir)蛋白協助,而當T-DNA進入植物細胞時,一些Vir蛋白也會透過通道進入植物細胞,繼續協助T-DNA的傳送,例如:VirE2、VirE3與VirF等蛋白。除此之外,尚需許多植物本身所合成的輔助蛋白參與,例如BTI、RAB8、VIP1、H2A與KU80蛋白等。研究指出,在輔助蛋白大量表現的阿拉伯芥轉殖株中(即T1世代植株),其基因轉殖效率上升。根據此些已被報導具有促進轉殖效率的輔助蛋白資訊,我們自水稻選殖出其同源基因,並命名為ET gene (enhance transformation gene)。本實驗希望在異源系統中測試水稻ET gene的作用,所以選擇菸草作為實驗對象。將這些水稻基因分別構築於表現載體,實驗組取分別攜有mGFP5基因與ET 基因的農桿菌;控制組則取分別攜有mGFP5基因與空載體的農桿菌,均以等比例混合後,利用農桿菌注入法(agroinfiltration)分別感染以中央葉脈為界的左、右兩側葉片。在感染三天後剪下葉片,比較葉片左右兩側的螢光表現,並以軟體計算量化,發現相對於空載體控制組,其中三個ET基因之感染區域有較強的螢光表現。此結果暗示水稻ET基因的表現可提升菸草細胞中T-DNA的傳送效率,造成暫時性表現的增加。未來或許可利用此暫時性共表現ET基因的策略提昇難轉殖植物的永久轉殖效率。
Agrobacterium-mediated transformation of higher plants is a well-known and powerful tool for transgene delivery to plant cells. The use of plants as expression systems for valuable recombinant proteins often involves transfer and integration of a transferred DNA, known as T-DNA, from Agrobacterium into the plant genome. In the bacteria side, various virulent proteins are involved in the assembly and export of T-DNA. After entering of T-DNA into plant cells, various accessory proteins including VirE2, VirE3, VirF, etc. imported from Agrobacterium and BTI, RAB8, VIP1, H2A, KU80, etc. provided by plant cells, are required for T-DNA's long journey from the cell peripheral into nucleus for chromosome integration. Previous reports indicated that the transformation efficiency of T1 transgenic Arabidopsis, in which certain accessory proteins were overproduced, could be greatly promoted. Based on the sequence information, their orthologous versions were isolated from rice and named as ET (enhance transformation) genes. To evaluate that can the transformation efficiency be increased by rice ET genes in a heterologous system, Agroinfiltration of N. benthamiana was chosen to use. Two cultures of A. tumefaciens containing ET and mGFP5 genes, respectively, were mixed and co-infiltrated into the young leaves of N. benthamiana. Three days after inoculation, leaves were cut and fluorescent images were recorded. Quantification of GFP expressions revealed that three ET genes did enhance fluorescent signals to a significant level, as comparing with control experiment by using an empty vector. This result demonstrated that the transiently expressed rice ET proteins can facilitate T-DNA transferring into plant nucleus. Such a transient co-expression strategy may be used to promote the permanent transformation frequency of recalcitrant plants.
其他識別: U0005-1908200913193800
Appears in Collections:生物科技學研究所



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