橙黃壺菌BL10 (Aurantiochytrium sp. strain BL10) 已知可以利用一種含有三個催化次單元 (catalytic subunits)：AmPFA1, 2, 3的酵素來製造22碳6烯酸DHA，然相關於AmPFA對DHA生合成的調控仍不清楚。本研究目的，即為開發一套可用以偵測AmPFA的方法，確認BL10內AmPFA的含量變化及形式，藉以推估BL10如何調控DHA的生合成。首先完成4種小鼠多株抗體的製備，由此挑選出夠專一辨識AmPFA1的A1，及同時辨識AmPFA2及AmPFA3的B2，在分析不同生長階段的BL10時，發現三種次單元會在細胞停止分裂、對應基因表現量最高時達到最高產量，隨後觀察到DHA產量的顯著增加。後續由免疫共沉澱實驗發現AmPFA1有兩個isomer，其中較小的AmPFA1-S相對於較大的AmPFA1-L的比例會隨著培養時間的增加而遞減。進一步以質譜分析，發現AmPFA1-S有ubiquitination的修飾。上述結果說明BL10內DHA合成除了會受AmPFA基因轉錄後修飾的調控，也可能和AmPFA1-S的轉譯後修飾有關。

Researchers have established that Aurantiochytrium sp. strain BL10 produces DHA using an enzyme that comprises three catalytic subunits: AmPFA 1, 2, and 3. However, the precise mechanism by which AmPFA regulates DHA biosynthesis remains unknown. This research developed a method by which to detect changes in the content and form of AmPFA in BL10. We first created mouse polyclonal antibodies and selected A1 for the specific recognition of AmPFA1 and B2 for the simultaneous recognition of AmPFA2 and AmPFA3. Analysis of AmPFA protein levels in various BL10 growth stages revealed that the production of three catalytic subunits peaked after the overexpression of the three genes and prior to DHA synthesis. Immunoprecipitation experiments revealed that AmPFA1 possesses two isomers. Subsequent LC-MS/MS analysis revealed that AmPFA1-S had undergone ubiquitination modification. Taken together, these results indicate that DHA synthesis in BL10 is regulated by the post-transcriptional regulation of AmPFA as well as the post-translational modification of AmPFA1-S.

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