- 學位論文
建構單次可誘導轉位子以中止篩選標記基因之功能
Construction of an inducible transposable element to terminate selectable marker gene
摘要
使用篩選標記基因(selectable marker genes)可以快速、有效率的篩選出轉殖株。然而在獲得轉殖株後，篩選標記基因就無特殊用途，並有生物安全疑慮。本論文目的是建構單次可誘導轉位子以終止篩選標記之功能。將轉位酶基因上游構築可誘導啓動子，且轉位子之5’構築於轉位子顯子 (exon) C和D間，而將轉位子之3’構築在修飾之水稻EPSPS ( 5-enolpyruvylshikimate-3-phosphate synthase ) 顯子1和2之間，完成之構築命名為Cpt5ne3。透過誘導劑水楊酸處理，企圖使轉位子轉位 (transposition) 將兩端及其內部分轉位酶基因和部分修飾之EPSPS基因片段切離，達到終止轉位酶基因和修飾之EPSPS基因之功能。將Cpt5ne3以農桿菌法轉形至水稻、菸草和阿拉伯芥。結果共獲得4個水稻癒傷組織轉殖系、33個菸草轉殖系(共55株菸草轉殖株)以及26個阿拉伯芥轉殖系(共210株阿拉伯芥轉殖株)。透過南方墨點分析，阿拉伯芥T-DNA之拷貝數為1~3之間，符合農桿菌低拷貝數預期。以RT-PCR分析三種物種之轉位酶基因均正確表現，但目前尚未發現有轉位現象發生；修飾之EPSPS基因在三物種中均正確表現，以glyphosate 進行抗性測試，水稻轉殖癒傷組織能抗10 mM glyphosate持續增生癒傷組織﹔而阿拉伯芥轉殖株能抗1000 ppm glyphosate，並可正常結種子，此結果可作為未來以glyphosate篩選轉殖株之建議濃度。另外本研究顯示將轉位子5’構築在轉位酶基因隱子(intron)和將轉位子3’構築在修飾之EPSPS基因隱子中均不影響基因轉錄，提供日後將轉位子5’及轉位子3’構築在基因之不同隱子中是否會影響基因正確表現之參考。
並列摘要
Selectable marker genes (SMGs) are often necessary during the process of plant transformation, but unnecessary once transgenic plants have been obtained. Besides, SMGs is involved in biosafety. Therefore, in this work, an inducible transposable element was constructed to terminate the selectable marker gene：modified EPSPS gene. Firstly, the 3’ end of the Ac was inserted in the first intron of the modified rice EPSPS gene, which triggered by the nos promoter. Secondly, The 3’ end of the Ac was inserted in the fourth intron of the inducible Ac transposase gene trigged by PR-1a promoter. Thus, this inducible transposable element contains not only the exon E of Ac transposase gene but also the first exon of the modified EPSPS gene and its promoter. This construct, which termed as Cpt5ne3, was introduced into rice, tobacco, and arabidopsis. The modified EPSPS gene can be transcribed normally in all three species and allow the transgenic rice and arabidopsis to be Glyphosate-resistant. Also, the transposase gene is transcribed correctly. Therefore, it is expected that transposition event can be induced by salicylic acid