本研究旨在建立BL10的insertion及episome基因轉殖法。前者是在原有線狀constructs基礎上進行簡化，並更換啟動子及 (或) selectable marker，製作出8種分別以neo及aphVIII為selectable marker的線狀constructs後，搭配電穿孔法及抗生素抗性篩選，進行轉殖效率的評估。後者則是使用含有酵母菌維持序列CEN6-ARSH4-HIS3 (CAH) 片段的環狀construct進行基因轉殖後，對所得轉殖株進行stability maintenance assay，分別培養於有無抗生素的環境中，觀察環狀轉殖株是否會在無篩選壓力條件下喪失抗性，藉以觀測episome的存在與否。
實驗結果表明，(1) 利用線狀constructs能夠成功轉殖並篩選到stable clones，且能夠通過PCR偵測到外源基因訊號。含aphVIII的線狀constructs轉殖效率優於含neo者，其中又以含BL10 ubiquitin啟動子之UAG1效率最高，說明 aphVIII更適合作為selectable marker被應用於BL10的基因轉殖中。(2) 將來自線狀construct E2NG1及環狀construct pP2NG-CAH-18S轉殖的stable clones經過2代的無抗生素培養後，部分環狀轉殖株維持率相較於線狀轉殖株顯著低下，說明外源基因可能以episome形式游離存在於環狀轉殖株中，推測CAH序列或有助於環狀construct形成episome。本研究為日後BL10之基因工程以及基因功能相關研究提供優良基礎。

This study sought to establish methods for the transformation of BL10, including genome insertion and episome maintenance. The original linear constructs were optimized for the former by replacing the promoter and/or selectable marker. Eight linear constructs with neo or aphVIII as the selectable marker were constructed. Transformation efficiency was evaluated after electroporation and antibiotic resistance screening. As for episome maintenance, a circular construct containing yeast-maintenance-sequence CEN6-ARSH4-HIS3 (CAH) element was delivered to BL10 via electroporation. A stability maintenance experiment was performed on the transformants, involving cultivation with or without antibiotics. The presence of episome was detected by measuring the difference in resistance maintenance rates between transformants derived from linear or circular constructions. Our results revealed that stable clones could be obtained via transformation using linear constructs, and foreign gene signals could be detected by PCR. Linear constructs containing aphVIII presented higher efficiency than those containing neo, and UAG1 containing BL10 ubiquitin promoter was the most efficient. This indicates that aphVIII as a selectable marker, is well suited to the transformation of BL10. We also determined that after two generations of cultivation without selection, the maintenance rate of some circular construct strains was significantly lower than that of linear construct strains, which suggests that the circular construct may exist as an episome in corresponding transformants, due to the contribution of the CAH fragment. This study provides a basis for future genetic engineering and gene function studies of BL10.

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翁伯瑋，橙黃壺菌 L-BL10 品系之基因轉殖平台建立，國立成功大學生物科技研究所碩士論文，2014。

梁又方，建立可靠的方案以達成橙黃壺菌品系 BL10 之穩定基因轉殖，國立成功大學生物科技與產業科學系碩士論文，2018。

莊凱荃，培養條件對 Aurantiochytrium sp BL10 之 DHA 產量及脂肪酸組成的影響，國立成功大學生物科技研究所碩士論文，2011。

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