橙黃壺菌BL10品系 (Aurantiochytrium mangrovei strain BL10) 為真核微藻，會出現不具細胞壁的阿米巴細胞世代，相較於其他具有細胞壁的真核生物更容易送入外源基因，進而表現。故本研究目的，在於建立BL10基因轉殖的方法。首先，藉由細胞存活率的分析，排除容易造成細胞死亡的DNA傳送法，接著利用PCR及Cy5螢光標定的方法，確認外源基因是否能有效送入細胞內。而後，利用real-time PCR方法分析送入細胞後外源基因的表現量，比較不同DNA傳送法及啟動子對於外源基因表現的影響。研究結果顯示，在以sorbitol清洗藻細胞後，搭配適當的電場條件進行電穿孔，亦或是利用微脂體進行轉染，其細胞存活率均超過九成。而電穿孔法送入外源DNA至細胞的總量雖然稍低於微脂體的攜帶，然卻能使外源基因 (rfp) 相較控制組之表現量提高4倍 (p<0.05)。分析三種來自BL10基因的啟動子活性：延長因子 (EF1-α)、以及兩種不同長度之甘油醛-3-磷酸脫氫酶 (GAPDH) 啟動子中，其以長度較長的GAPDH啟動子的活性最強，相較控制組之表現量提高16倍 (p<0.05)。接續並以同源重組設計抗生素篩選平台，以利於日後以BL10為轉殖平台的應用性試驗，而這些研究成果有助於後續stable clone的建立。

Aurantiochytrium mangrovei strain, BL10 has fast growth rate and unique life cycle that is amoeboid cells which characterized with lacking cell wall. It is easier to transmit exogenous gene into BL10 which compared with having cell wall organism. The purpose of this study was to establish genetic transformation in BL10. So, we used electroporation and liposome transfection of DNA delivery system to transmit exogenous genes. The results showed that DNA delivery system had significantly reduction of mortality rate in electrical pulse conditions (sorbitol, 5 kV/cm) and liposome (final concentration: 13 μg/mL). Both of DNA delivery system can transmit exogenous gene, it was detected by PCR and Cy5 labeling methods. Moreover, electroporation caused higher gene expression than liposome transfection. However, we also changed promoter of gene expression cassette for increasing expression. Finally, we successfully detected the cassette of random insertion with GAPDH promoter had significant up-regulation by electroporation.

王嘉慧，藉由RNA干擾、蛋白合成抑制及突變株篩選改善橙黃壺菌 L-BL10品系脂肪酸組成，國立成功大學生物科技研究所碩士論文，2013。

吳哲安，橙黃壺菌L-BL10品系之聚酮合成酶分析，國立成功大學生物科技研究所碩士論文，2014。

周思丞，探討橙黃壺菌BL10品系阿米巴細胞形成與遷移的機制，國立成功大學生物科技研究所碩士論文，2013。

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。

莊凱筌，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。

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。

劉致寧，橙黃壺菌L-BL10品系之聚酮合成酶基因序列與表現分析，國立成功大學生物科技研究所碩士論文，2013。

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