在過去的研究中，已知Aurantiochytrium limacinum 品系BL10會透過轉錄、轉錄後及轉譯後層級的多層次調控，影響 polyunsaturated fatty acid synthase (Pfa) 蛋白的活性，來調節高度不飽和脂肪酸的生合成，同時已確認缺氮是誘發 pfa genes表現的重要因子，然而，尚有：1. 缺氮，出現第一波pfa genes上調後，為何還會有另一波的pfa genes上調? 2.無法確定Pfa1L及1S的比例變化究竟是只和泛素化有關，還是也和基因表現量的差異有關? 3. 無法確定三個pfa genes，乃至於其他油脂生合成重要基因在不同生長階段的轉錄起始點偏好 (影響轉錄後調控)。也因此，本研究的目的，即在釐清前述問題。首先，由培養期間培養基內總磷含量變化的分析，及與pfa基因表現量變化的比對，結果顯示磷濃度的波動與 pfa 基因表現上調的時序並未呈現明顯相關性。接著，由高專一性引子對的設計，搭配即時PCR方法，所確認出pfa1L和1S基因表現變化十分一致的結果，判定Pfa1L與1S蛋白質異構體比例的變化和轉錄調控無關。最後，發現mRNA樣品的完整度攸關能否成功利用RNA-ligation mediated RACE的方法標定出pfa，乃至於其他參與油脂生合成重要基因的轉錄起始點。後續將以RNA-sequencing的方法分析，由此確認前述基因在不同生長階段的轉錄起始點變化。

Previous studies have shown that Aurantiochytrium limacinum strain BL10 regulates the biosynthesis of highly unsaturated fatty acids (HUFAs) through multilayered control of polyunsaturated fatty acid synthase (Pfa) proteins at the transcriptional, post-transcriptional, and post-translational levels. Nitrogen limitation has been identified as a major factor inducing pfa gene expression. However, several important questions remain unresolved. First, it is unclear why a second wave of pfa gene upregulation occurs after the initial response to nitrogen depletion. Second, although the ratio between the Pfa1L and Pfa1S protein isoforms is known to change, it remains uncertain whether this shift is solely due to ubiquitination or also influenced by differences in gene expression. Third, the transcription starts site (TSS) preferences of the three pfa genes and other lipid biosynthesis-related genes at different growth stages have not been characterized, leaving possible post-transcriptional regulatory mechanisms unexplored. To address these questions, this study first examined changes in total phosphorus concentration in the culture medium during cultivation and compared them with pfa gene expression patterns. The findings indicate that the timing of pfa gene upregulation does not show a clear correlation with phosphorus levels. Next, the use of highly specific primer sets, and quantitative real-time PCR revealed that the expression of pfa1L and pfa1S is highly consistent, indicating that the ratio of their protein isoforms is not regulated at the transcriptional level. Finally, it was found that RNA integrity is critical for successful identification of transcription start sites using RNA-ligation mediated RACE. RNA sequencing of RNA-ligated samples will be conducted to determine TSS variation of pfa and other lipid biosynthesis genes at different growth stages.

吳玥慶. 在缺氮條件下，橙黃壺菌BL10之多元不飽和脂肪酸合成酶基因的轉錄後及轉譯後調控反應，國立成功大學生物科技與產業科學系碩士論文，2023。
郭智宇. 橙黃壺菌BL10之多元不飽和脂肪酸合成酶的偵測與分析，國立成功大學生物科技與產業科學系碩士論文，2021。
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