根毛農桿菌對菸草之多基因轉形研究

Agrobacterium rhizogenes-mediated genes transformation of Nicotiana tabacum Wisconsin 38

蘇敬岳(Ching-Yeuh Su)

指導教授 ： 李昆達

國立臺灣大學/生命科學院/微生物與生化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.01582

摘要

本論文在進行根毛農桿菌對菸草之多重基因共轉形之探討。菸草威斯康新 38 號被選為農桿菌感染研究用材料。本研究選用GFP與GUS兩個報導蛋白質之基因進行菸草之多基因共轉形。本研究在設計上有兩個策略，策略一為一個雙元質體攜帶一個報導基因。亦即：單一菌體同時攜帶雙元質體 (2BV)，或兩質體分別位於兩農桿菌來同時感染菸草 (2AR)。策略二為，一個雙元質體內含有雙報導基因：我們建構了兩個質體，分別是兩個報導基因位於同一個 T-DNA 區域中 (pCGG-1TD)，或兩個基因位於二個不同的 T-DNA 區域中 (pCGG-2TD)。四類欲用來探討菸草多基因轉形策略的農桿菌已建構完成，並已分別對菸草進行感染。2AR 及 2BV 二組的誘導率分別為 81％ 及75％，而帶有雙基因的雙元質體組 (pCGG-1TD 與pCGG-2TD) 誘導率很低。報導基因表現方面，2BV 及 pCGG-1TD 兩組已經以螢光顯微鏡及 b-葡萄糖酸苷酶染色技術證實該兩組可同時表現綠色螢光蛋白及 b-葡萄糖酸苷酶。2BV 組是目前較好的共轉形及共表現策略。在本篇研究中，如果各個策略皆可順利將基因轉形至植物中，我們將可同時運用兩種以上的策略來對植物進行多基因的轉形，以得到可大量生產植物次級代謝物的毛狀根。

關鍵字

根毛農桿菌 ； 菸草 ； 毛狀根 ； 綠色螢光蛋白質 ； b-葡萄糖酸苷酶

並列摘要

Transformation of multiple genes into tobacco by Agrobacterium rhizogenes-mediated method was performed in this study. Nicotiana tabacum Wisconsin 38 was selected as the material for hairy root induction. Two reporter genes, gfp from pCAMBIA 1302 and gus from pCAMBIA 1201, were used for co-transformation study. Two strategies were developed. The first one, two reporter genes were carried by two respective plasmids. And then, transformation was conducted either by one transformed A. rhizogenes with two binary vectors (2BV), or conducted by co-cultured at the same time with two separate transformed A. rhizogenes which harboring one reporter gene (2AR). The second one was, constructing the two reporter genes in one binary vector. Thus, two reporter genes located in one T-DNA region or separated in two T-DNA regions were constructed, and they were named pCGG-1TD and pCGG-2TD, respectively. The above plasmids were transformed into A rhizogenes, and then inoculated tobacco leaf discs. The root inducing rate of 2AR and 2BV were 81 % and 75 %, respectively. On the other hand, those induced by pCGG-1TD and pCGG-2TD caused less hairy roots. Regarding expression of reporter genes, in 2BV and pCGG-1TD groups, we have observed GFP expression by fluorescence microscopy and GUS expression by tissue staining. Nowadays, the construction of 2BV was the better strategy to gain co-transformation and co-expression hairy roots. In this study, if all strategies can succeed to transform multiple genes into plants, we can integrate those strategies and transform multi-genes into plants at the same time to induce hairy roots for secondary metabolite production.

並列關鍵字

Agrobacterium rhizogenes ； tobacco ； hairy roots ； GFP ； GUS

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