臺灣博碩士論文加值系統

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.

壹、前言.................................................1
貳、前人研究.............................................3
一、各種植物基因轉殖技術.................................3
二、農桿菌轉殖植物細胞的機制.............................4
三、農桿菌轉殖過程中影響轉殖效率的相關蛋白...............6
四、RNA靜默之病毒抑制子 (viral suppressor)...............8
參、材料與方法..........................................11
一、 試驗材料...........................................11
(一) 植物材料.......................................11
(二) 質體構築.......................................11
二、 試驗方法...........................................12
(一) 瓊脂凝膠(Agarose)膠片之配製....................12
(二) 質體構築方法...................................13
(三) 大腸桿菌及農桿菌質體DNA之萃取..................14
(四) 大腸桿菌及農桿菌勝任細胞的製備及轉形作用.......15
(五) 阿拉伯芥基因轉殖與篩選轉基因植物...............16
(六) 菸草基因轉殖與數據分析.........................17
(七) 西方墨點法分析.................................19
(八) 以Ni-NTA純化蛋白質.............................20
肆、結果................................................21
一、測試促轉基因是否提升阿拉伯芥花序之永久性轉殖效率....21
二、修飾載體期望提升促轉基因的表現及促轉效率............21
三、測試來自農桿菌的促轉基因VirG在模擬磷酸化之後是否促進植物
轉殖................................................23
四、混合不同促轉基因測試其是否有協同作用(synergistic effect)
的現象..............................................23
五、破壞促轉基因以確認促轉基因蛋白的效果................25
六、偵測促轉基因蛋白的表現..............................25
伍、討論................................................27
一、探討促轉基因對阿拉伯芥永久性轉殖之影響..............27
二、探討促轉基因對菸草短暫性轉殖之影響..................27
三、探討不同VirG基因對菸草短暫性轉殖之影響..............27
四、探討促轉基因混合施打對菸草短暫性轉殖之影響..........28
五、評估促轉基因蛋白的表現..............................28
陸、參考文獻............................................30

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