Investigation of inducible transposon to remove selection marker

轉位子可分為兩部分：轉位酶基因及受轉位酶作用之兩端點。本研究之目的及原理為將可誘導轉位子的端點構築在目標基因的隱子（intron）中，目標基因經由剪接作用（splicing），插入隱子的端點不影響基因正常表現；當目標基因表現後，以誘導劑驅動可誘導轉位子表現，使其轉位至染色體的其他位置，帶走部份目標基因，使該基因不完整，以達到移除目標基因的目的。 本研究實例以EPSPS ( 5-enolpyruvylshikimate-3-phosphate synthase )作為轉基因篩選標記基因，水稻內生EPSPS基因經二點突變（將胺基酸序列位置168之Glycine突變為Alanine；位置211之Glycine突變為Aspartic acid），使其由對嘉磷塞（glyphosate）敏感型轉為抵抗型，而成為篩選標記基因。將 Ac 轉位子的 5’端插入修飾之 EPSPS 基因的第一個隱子，並以 CaMV 35S promoter作為啟動子；另將PR-1a啟動子（pathogenesis-related protein 1a, 可受水楊酸誘導表現）與 Ac 轉位酶融合，構築於 Ac的兩端點之間。完成上述構築後，以農桿菌法將此構築轉殖至水稻癒傷組織，於添加嘉磷塞之篩選培養基，在野生型癒傷組織的致死濃度下（5 mM），篩選出轉殖系後，證明修飾之EPSPS基因可作為篩選標記。其後，將癒傷組織移至含誘導劑水楊酸的培養基，誘導轉位，在T0世代的癒傷組織中，經 PCR 分析，的確觀察有轉位事件（transposition event），即包含轉位子本身，CaMV 35S 啟動子與修飾之 EPSPS 基因的第一個顯子（exon）同時隨之轉位，修飾之 EPSPS 基因的基因序列不再完整。據此，癒傷組織發育成完整植株後，可在後代取得完全轉位的轉殖株，達到篩選標記基因移除的目的。The maize transposable element Ac can move to new location within genome. In this work, an Ac–based inducible transposon was constructed to develop a one-time gene expression system. Firstly, the 5’ end of the Ac was inserted in the first intron of the modified rice EPSPS gene, which triggered by the CaMV35S promoter. The inducible transposon was completed by locating the PR-1a promoter-transposase fusion and the CaMV35S promoter was between the 5’ and 3’ ends. Thus, this inducible transposon contains not only the PR-TPase fusion but also the first exon of the modified EPSPS gene and its promoter. This construct, which termed as KCEH, was introduced into rice. The modified EPSPS gene can be transcribed normally and allow the transgenic rice to be Glyphosate-resistant. After the Glyphosate screening, the transgenic rice plants were treated with salicylic acid for the excision of the inducible transposon. As a result of this, the modified EPSPS gene was truncated by losing its first exon and the 35S promoter. Emphasis has been placed on the theoretical and practical aspects of the approach that may be relevant to its application to other gene expression regulatory system. It provides a means to investigate a one-time gene expression system.目錄……………………………………………………………………………I 圖表目錄、附錄目錄…………………………………………………………II 中文摘要………………………………………………………………………1 英文摘要………………………………………………………………………2 前言……………………………………………………………………………3 前人研究………………………………………………………………………4 材料與方法………………………………………………………………….12 結果………………………………………………………………………….30 討論………………………………………………………………………….37 未來展望…………………………………………………………………….42 引用文獻…………………………………………………………………….4