Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/90048
標題: Increase efficiency of Agrobacterium-mediated plant transformation
提升農桿菌的植物轉殖效率之研究
作者: 鄭淞尹
Sung-Yin Cheng
關鍵字: 無;NO
引用: 林怡君. (2011). 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. In 生物科技學研究所 (台中市: 中興大學) 林家誠. (2010). 以表現輔助蛋白提升農桿菌的植物轉殖效率. In 生物科技學研究所 (台中市: 中興大學) 蔡靜琪.(2013).表現輔助蛋白提升農桿菌之水稻轉殖效率與水稻突變株 rolts之性狀分析. In 生物科技學研究所 (台中市: 中興大學) 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. 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. Hiei, Y., Ohta, S., Komari, T., and Kumashiro, T. (1994). Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant journal : for cell and molecular biology 6, 271-282. Hwang, H.H., and Gelvin, S.B. (2004). Plant proteins that interact with VirB2, the Agrobacterium tumefaciens pilin protein, mediate plant transformation. The 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. Journal of bacteriology 172, 4945-4950. Lacroix, B., and Citovsky, V. (2011). Extracellular VirB5 enhances T-DNA transfer from Agrobacterium to the host plant. PloS one 6, e25578. 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. The EMBO journal 24, 428-437. Li, J.X., Vaidya, M., White, C., Vainstein, A., Citovsky, V., and Tzfira, T. (2005). Involvement of KU80 in T-DNA integration in plant cells. P Natl Acad Sci USA 102, 19231-19236. 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. Sheng, J.S., and Citovsky, V. (1996). Agrobacterium plant cell DNA transport: Have virulence proteins, will travel. Plant Cell 8, 1699-1710. 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. Tsuda, K., Qi, Y., Nguyen le, V., Bethke, G., Tsuda, Y., Glazebrook, J., and Katagiri, F.(2012). An efficient Agrobacterium-mediated transient transformation of Arabidopsis. The Plant journal : for cell and molecular biology 69, 713-719. 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. (2002). Increasing plant susceptibility to Agrobacterium infection by overexpression of the Arabidopsis nuclear protein VIP1. Proceedings of the National Academy of Sciences of the United States of America 99, 10435-10440. Yi, H., Mysore, K.S., and Gelvin, S.B. (2002). Expression of the Arabidopsis histone H2A-1 gene correlates with susceptibility to Agrobacterium transformation. The Plant journal : for cell and molecular biology 32, 285-298. 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.
摘要: 
植物基因轉殖技術中,農桿菌轉殖系統常被廣泛的應用,因為其操作簡便而且可以使目標基因單一且完整的插入染色體組中,不易引發基因靜默反應(gene silencing)。因此,有超過數百種植物用此方法轉殖成功,但是還有許多植物在基因轉殖上有很大的困難,所以如何提升植物轉殖效率是極為重要的課題。農桿菌感染植物後,T-DNA 傳送至植物細胞的過程中需要許多輔助蛋白的協助,有來自農桿菌的蛋白,例如:VirE2、VirE3 與 VirF 等,也有來自植物的蛋白,例如:BTI、VIP1、KU80 與 H2A 等。H2A 在前人的研究當中,發現和目標 T-DNA 一起插入植物基因體的比率很高,有 85。故本實驗欲研究 H2A 共轉殖之機制:將水稻 H2A 點突變,使之無法轉譯成完整蛋白且 DNA 序列改變不大,命名為 H2Am,以此做法可得知 H2A 高共轉殖率之機制是 DNA 特性導致此現象,還是 H2A 之蛋白質會幫助 H2A 插入植物的基因體中。實驗結果發現,pH2A 及 pH2A 之點突變版本皆與空載體相近,並沒有高共轉效率之現象,,分別為 18及 20 和前人所做出 H2A 同時嵌入植物基因體的頻率 85有很大的差異。

另外,本實驗將也將水稻 H2A 和目標 T-DNA 之農桿菌一起感染阿拉伯芥,發現水稻 H2A 在阿拉伯芥當中,並沒有高的共轉殖效率。故本實驗測試阿拉伯芥的 H2A,在阿拉伯芥當中是否也有高共轉殖效率之現象,在此實驗中,發現阿拉伯芥之 H2A 基因在阿拉伯芥中並沒有高共轉殖之現象。

另外,AvrPto 是來自植物病原菌 Pseudomonas syringae,此蛋白會干擾植物免疫受體,抑制植物免疫反應,在擬南芥中表現 AvrPto,可提升暫時性轉殖效率;而 VirB5 蛋白是組成細菌 T 線毛的成員之一 它會移動到細菌表面並和植物表面,接觸,是一個農桿菌感染早期幫助農桿菌和植物接觸的角色。本實驗使用暫時性轉殖分析系統評估 AvrPto 及外加 VirB5 蛋白的功效 評估的方式為利用帶有 GUS,報導基因的農桿菌以及帶有欲測試基因的農桿菌共同感染植物後,在 T-DNA 尚未插入植物染色體之前 以 MUG assay 分析 GUS 活性 和控制組比較得知AvrPto,。
之轉殖效率提升約為 1.3 倍;而在外加 VirB5 蛋白之水稻暫時性轉殖效率並沒有明顯的提升。

Agrobacterium-mediated T-DNA transfer is well-known to be a powerful plant transformation tool. This method mostly confers single or low-copy integration of full-length gene, and leads to less problems in gene-silencing. But, a lot of important crops are still 'recalcitrant' to transformation. Thus, it is still important to improve the plant transformation efficiency using Agrobacterium-mediated method. The long journey for T-DNA transferring from bacteria till the final destination requires bacterial proteins and various plant factors. As co-transformation of T-DNAs carrying GUS/hyg and rice H2A genes, respectively, often cause co-integration of the H2A gene in absence of selection marker, we were interested to investigate its mechanism. A single nucleotide change leads to nonsense mutation (H2Am) was constructed. The high co-integration frequency of H2A gene will be clarified to be caused by DNA
itself or the encoded protein. In this study, the H2A and H2Am both have no high co-integration frequency, this result is different with previous studies.

In addition, we found the rice H2A is not significant high co-integration frequency in Arabidopsis. So, we will test Arabidopsis H2A Group I can enhance co-integration frequency in Arabidopsis or not. In these experiment the Arabidopsis H2A is not significant high co-integration frequency.

Moreover, AvrPto is an effector protein that suppresses plant immunity. Expression of AvrPto in Arabidopsis dramatically enhances its transient transformation efficiency mediated by Agrobacterium. VirB5 is an essential virulence (Vir) protein and represents a minor component of the T-pilus. Besides, VirB5 is translocated to the physical interface between bacterial and plant cell surface, suggests an additional role of VirB5 involving early interactions between Agrobacterium and the host cell. Using co transformation of T-DNAs carrying GUS/hyg and AvrPto genes, respectively, transient transformation efficiency of GUS was found to be ~1.3-fold higher than control experiment. Other experiments we examine that addition of purified VirB5
proteins when infected rice callus. we found the VirB5 proteins is not significant high transformation rate in rice.
URI: http://hdl.handle.net/11455/90048
Rights: 同意授權瀏覽/列印電子全文服務,2015-07-15起公開。
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