本研究旨在開發一種適用於蛞形橙黃壺菌（Aurantiochytrium limacinum）的外染色體自主複製質體系統 pUNG2-PIT24，並對其進行特性驗證。研究重點是建立一種非整合型、穩定且具自我剔除潛力的質體系統，以克服現有轉殖技術在穩定性與效率上的限制。pUNG2-PIT24 質體引入了來三角褐指藻 （Phaeodactylum tricornutum）的 centromere-like 序列，以支持質體的外染色體維持，從而實現穩定的基因表達。
研究結果顯示，pUNG2-PIT24 在蛞形橙黃壺菌 BL10 品系中能夠穩定維持外染色體形式，並在去除篩選壓力後展現出自我剔除潛力，分裂效率約97.7 %。此外，該質體具有支持雙重 expression cassette 的潛力。通過電穿孔條件優化和 G418 篩選濃度的調整，成功提高了轉殖效率。本研究開發的質體系統為橙黃壺菌提供了一種穩定的基因操作平台，對於未來的功能性基因驗證、基因編輯以及代謝工程應用具有重要的技術價值。

This study aims to develop a plasmid system, pUNG2-PIT24, for extrachromosomal replication in Aurantiochytrium limacinum and to validate its characteristics. The focus of the research is to establish a non-integrative, stable, and self-eliminating plasmid system to address the challenges of stability and efficiency in existing transformation technologies. The pUNG2-PIT24 plasmid incorporates centromere-like sequences from Phaeodactylum tricornutum to support extrachromosomal maintenance, enabling stable gene expression and gene editing. The results show that pUNG2-PIT24 can stably maintain an extrachromosomal form in Aurantiochytrium limacinum BL10 and exhibits self-elimination potential upon removal of selective pressure, with a segregation efficiency of approximately 97.7 %. Additionally, the plasmid has the potential to support dual expression cassettes. By optimizing electroporation conditions and adjusting G418 selection concentrations, the transformation efficiency was successfully improved, and high-efficiency plasmid screening was achieved. The plasmid system developed in this study provides a stable genetic manipulation platform for Aurantiochytrium and holds significant technological value for future functional gene validation, gene editing, and metabolic engineering applications.

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研究所碩士論文，2018。
張宜婷，橙黃壺菌BL10基因轉殖平台的改良，國立成功大學生物科技研究所碩士論文，2021。
黃大祐，以UV致變及基因編輯法提高橙黃壺菌 BL10 的DHA產量，國立成功大學生物科技研究所碩士論文，2023。
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