應用轉位子系統去除轉殖蝴蝶蘭篩選標誌基因之研究

Abstract

篩選標誌基因之表達有助於分離出轉殖細胞，一旦確立轉殖事件後，篩選標誌基因便不具有其他的用途，若持續添加抗生素或殺草劑等篩選試劑，則易降低轉殖細胞增殖及分化的能力。另外，轉殖植株之抗藥性基因可能影響生態環境的平衡，易引發大眾對其安全性的疑慮。本研究利用可受水楊酸誘導之菸草PR-1a 啟動子與轉位酶 (transposase) 基因融合，應用於四種轉位子 (transposon) 系統，以 3 mM 水楊酸處理轉殖蝴蝶蘭癒傷組織及菸草葉圓片5天，接著抽取基因組 DNA 進行聚合酶連鎖反應 (polymerase chain reaction, PCR)，將 PCR 所得到的產物定序分析，結果顯示轉位事件 (transposition) 發生後，產生四種轉位類型，除了預期的左邊界 (left border) 及右邊界 (right border)，也保留了35 - 128 bp之部分質體序列。本研究進一步針對篩選標誌基因轉位後是否再插入其它位置進行探討，以南方氏雜交法進行分析，轉殖 pGcET 和 pGEnT 植株只雜合到轉位後之片段，並未獲得篩選標誌基因片段，顯示轉位作用已達到篩選標誌基因移除之目標。

Expression of selectable marker genes allows scientists to identify and isolate the transgenic cells. Once the transgenic plant has been generated, marker gene generally no longer serves an essential purpose. Furthermore, the propagation and development of transgenic cells will be retarded if the antibiotic or herbicide remains in the growth medium. Besides, it is concerned by publics about biosafety of antibiotic and herbicide resistance genes in transgenic plants. We have developed four different constructs under the control of the promoter PR-1afor inducible transposon systems as marker-free strategy and theses constructs were transformed into Phalaenopsis calli and tobacco plants. Transgenic plants were treated with 3 mM salicylic acid for 5 days to eliminate marker genes. Four patterns of transposition have been identified base on sequence analysis. Residual DNA sequences of 35 - 128 bp were fused as footprints after excision of the transposon. Southern analysis confirmed further that the herbicide resistance geneEPSPS,had been removed together with the transposition.