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題 名 | Construction of the Binary Vector with Bi-Selectable Markers for Generating Marker-Free Transgenic Plants=生產無篩選標示轉基因植物的多功能轉殖載體之開發應用 |
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作 者 | 林靜宜; 古新梅; 陳志威; 葉錫東; 詹富智; | 書刊名 | Botanical Studies |
卷 期 | 52:3 2011.07[民100.07] |
頁 次 | 頁239-248 |
分類號 | 434.28 |
關鍵詞 | 共轉型法; 無篩選標示; 選擇性標示基因; 轉基因植物; Co-transformation; Marker gene-free; Transgenic plant; |
語 文 | 英文(English) |
中文摘要 | 植物轉殖通常需要利用抗生素或殺草劑的抗性基因作為選擇性標示基因以區分其中少數轉殖成功的植物。然而存在轉基因植物中的選擇性標示基因之安全性近來已成為大眾關注的話題,因此自轉基因植物中移除選擇性標示基因乃為當務之急。本研究中,我們以共轉型法發展了一套生產無篩選標示轉基因植物的多功能轉殖載體,pGA2TNH,並且可適用更多的植物種類,尤其是對kanamycin具有天然耐/抗性的作物。我們利用單載體攜帶兩組T-DNA為策略,並於其中一組T-DNA中構築兩種不同的選擇性標示基因,其一為hpt而另一為nptII基因。為了作為對照,我們也構築了兩組載體pGANP-CP1/pBin19與pGA2T-CP1,藉以比較三者生產無篩選標示基因轉基因植物的效率。前者為單一菌株內兩個獨立的質體各自攜帶有目標基因或標示基因的T-DNA,後者為同一質體中同時帶有目標基因及標示基因的T-DNA。經pGA2TNH系統再生之轉基因菸草的共轉型效率為50%,其效率和其他兩組系統相當。至於兩組T-DNA 於子代的分離現象亦符合預期,於單一標示基因套數的轉基因植物中,移除標示基因的比例可達17.5%,而pGA2T-CP與pGANP-CP1/pBin19則分別為18.6%與24.1%。結果證實我們所發展之系統確實可行且能有效率地移除選擇性標示基因,進一步可提供簡便及實用性兼具的工具應用至更為廣泛的物種上生產無篩選標示轉基因植物,且有助於提升大眾對轉基因作物的接受度。 |
英文摘要 | Plant transformation typically involves antibiotic or herbicide resistance genes as selection markers to identify the transformed plants. However, there have been public concerns over the safety of the marker genes that remain in transgenic plants. It is therefore desirable to remove marker genes prior to the release of transgenic plants. In this study, we used the co-transformation strategy to develop a binary vector with bi-selectable markers, pGA2TNH. Such strategy enables the generation of marker-free transgenic plants and increases utilization of additional plant species, especially crops with natural resistance or tolerance to kanamycin. One plasmid carrying two separated T-DNAs was adopted in order to construct multiple selectable markers in one of the T-DNAs. Markers used were the hpt gene for hygromycin resistance and the nptII gene for kanamycin resistance. pGANP-CP1/pBin19 and pGA2T-CP1 were constructed to evaluate and compare their efficacy in generating marker-free transgenic plants. The former consisted of two individual plasmids carrying separate T-DNA of the target and marker genes, while the latter contained a single plasmid carrying two T-DNAs for the target and marker genes. In pGA2TNH system, the co-transformation frequency of the R0 transgenic Nicotiana benthamiana plants with both selection markers and target gene was 50%, which was as efficient as the other two systems. As expected, segregation of the two T-DNAs was observed in progeny. In one marker gene copy of transgenic plants, the elimination of marker gene was found at a ratio of 17.5% in pGA2TNH system, and 18.6% and 24.1% in pGA2T-CP and pGANP-CP1/pBin19, respectively. This demonstrated that the binary vectors we constructed were efficient and feasible in eliminating marker genes. It also provides a practical and simple tool for generating marker-free transgenic crops, which will have a significant impact on their acceptance by the public. |
本系統中英文摘要資訊取自各篇刊載內容。