橙黃壺菌L-BL10 品系 (Aurantiochytrium sp. strain L-BL10) 為真核 微藻，其具有培養成本低廉、成長快速、以及具有無細胞壁世代 (阿米巴 細胞) 的優點，加上已完成初步的基因體分析，因此具有成為新一代真核 表現平台的潛力。
本研究的目的，即為建立L-BL10 外源性基因表現之轉 殖平台。首先，藉由L-BL10 的基因體資料庫預測基因序列之密碼子使用 偏性；同時取得延長因子的啟動子、基因及終止子序列，以作為起始及 終止外源基因轉錄的元件；其次，分別選用綠色螢光蛋白基因以及遺傳 黴素抗性基因-紐奧黴素磷酸轉移酶II 基因，作為基因片匣中的報導基因 或篩選標誌；最後進行細胞轉化測試，搭配微脂粒或電穿孔法並測試以 不同微脂粒包覆核酸之比例或脈衝條件等。 本研究共統計16788 個密碼子使用偏性，其中UUA、CUA、AUA 以及 CGA 在對應於同胺基酸的密碼子中，其使用率小於1 %。在核酸構 築已完成延長因子啟動子、外源基因以及延長因子終止子等三個片段連 接成的基因片匣。最後，以電穿孔法及微脂粒法皆能成功將綠色螢光蛋 白基因組序列送入細胞中且能偵測到外源基因之mRNA，此外能在微脂 粒化轉細胞中觀察到具有綠色螢光訊號之藻細胞。

Aurantiochytrium sp. strain L-BL10 is a eukaryotic microalgae that incurs lower culture costs, displays higher growth, and has lower rates of cell wall generation (amoeba cells) than other eukaryotes currently being used to express foreign proteins. The objective of this study was thus to construct a transgenic platform for exogenous gene expression in L-BL10. For this, we first designed nucleic acid constructs. L-BL10 genome databases were accessed during platform design to predict and correct codon usage bias within the gene sequences. We obtained housekeeping genes, which included: (1) the promoter and terminator sequences of the elongation factor 1-α (EF-1α) and (2) the sequences of 18s ribosomal DNA. Next, we selected green fluorescent protein (GFP) gene, red fluorescent protein (RFP) gene, and neomycin phosphotransferase II (NPT II) gene. Those genes replaced sequences of the elongation factor, thereby serving as the reporter genes or selection markers in gene cassettes. For these, we coated the nucleic acids with different proportions of Lipofectamine or performed electroporation under different pulsed conditions. From the 16 predicted gene sequences of L-BL10, we recorded 16,788 instances of codon usage bias, among which the usage rates of UUA, CUA, AUA, and CGA in corresponding codons of the same amino acid were less than 1%. We had already completed gene cassettes during nucleic acid construction. Finally, we successfully transferred GFP gene sequences into cells using electroporation and Lipofectamine, and we detected the mRNA of exogenous genes. In addition, we observed green fluorescent signals in the algal cells transformed using Lipofectamine.

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。

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。

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。

蘇昱銘，來自北台灣之海洋異營性微藻分離株: Aurantiochytrium sp. Strain BL10之生物特性研究，國立成功大學生物科技研究所碩士論文，2012。

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