本研究的目的為建立能讓橙黃壺菌BL10穩定表現外源基因的方法，大致的作法，是製作15種以neo或 Sh ble為selection marker gene的環狀或線狀片匣，搭配電破及後續的agar plate成長篩選，計算 agar plate上所形成的BL10 colony個數來比較不同DNA constructs的轉殖效率，並以PCR及qRT-PCR的方法，驗證外來基因能否在BL10細胞內穩定的存在及表現。研究結果顯示，所有片匣中，以含有P1啟動子及酵母菌維持序列CEN6-ARSH4-HIS3 (CAH) 片段的環狀片匣轉殖BL10的效率最高，同時可以讓繼代培養4代後的轉殖株仍能穩定的表現外源基因，此可能和P1啟動子具有較強的活性，同時CAH片段能讓外源基因以episome的游離態、而非插入染色體的形式存在於BL10的細胞核內有關。後續將進一步確認外源基因是否真以episome的形式存在。綜而言之，本研究得到穩定表現外源基因的stable clone，並藉由構築表現片匣提升轉殖效率。

Our objective in this study was to establish a protocol by which to achieve the stable expression of foreign genes in the Aurantiochytrium strain BL10. To confirm the presence and expression of foreign genes in BL10 cells, we constructed 15 linear or circular expression cassettes, which were then transformed into BL10 via electroporation and then selected on antibiotic agar plates. The cassette with the P1 promoter and yeast-maintain-sequence, CEN6-ARSH4-HIS3 (CAH), presented the highest transformation efficiency, the transformants of which stably expressed the foreign gene after being subcultured four times. This may be attributable to the pronounced activity of the P1 promoter and CAH sequence, which makes it possible for the foreign gene to be maintained as an episome rather than undergoing integration into the chromosome. Subsequent confirmation will confirm whether the foreign gene is maintained as episome.

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陳筠方，利用基因靜默法改善橙黃壺菌BL10品系之脂肪酸組成，國立成功大學生物科技研究所碩士論文，2015。

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