橙黃壺菌L-BL10以二十二碳六烯酸 (Docosahexaenoic acid, C22:6ω3, DHA) 為主要不飽和脂肪酸情形異於其他真核生物。過去研究經由脂肪酸合成酶抑制劑淺藍菌素的添加，推知棕櫚酸並非L-BL10合成DHA的前驅物。分析L-BL10的基因體後，發現有類似裂殖壺菌 (Schizochytrium sp.) 中參與高度不飽和脂肪酸合成的聚酮合成酶 (Polyketide synthase, PKS) 基因存在，且DHA的累積出現在該基因表現量增加後，推測PKS與DHA的合成有關。然而目前對於PKS蛋白質產物培養後不同時間的變化以及是否存在其餘小分子調控其活性仍不清楚。因此，本研究目的在於製備出PKS抗體，探討PKS蛋白質產物變化。首先篩選PKS抗原決定位，以模板重複聚合酶連鎖反應與轉接端鏈聚合酶連鎖反應製造線性陣列抗原，藉由兔子及小鼠製備抗體。後續以LC-MS/MS與西方墨點法鑑定PKS身份及分析蛋白質產物變化。首先在鑑定結果，PKS A、B與C分別比對到L-BL10數據庫中18%、58%、24%的胺基酸片段，初步證實確實含有PKS，並在培養不同時間後觀察到PKS B與PKS C在12小時開始產生訊號，20-28小時訊號為最強，與基因表現趨勢符合，綜合過去研究資料，推知L-BL10面臨氮源缺乏會誘發PKS產生，合成DHA抵禦環境中營養源的變化。

Unlike other eukaryotes, which primarily possess unsaturated fatty acids, Aurantiochytrium sp. strain L-BL10 is characterized by docosahexaenic acid (DHA). Previous researchers have found that incubating L-BL10 with cerulenin failed to decrease the amount of DHA, indicating that palmatic acid is not the precursor of DHA. After analysis of the draft genome of L-BL10 revealed a number of partial genes resembling segments of the polyketide synthase (pks) gene, which is responsible for synthesizing DHA in Schizochytrium sp. During L-BL10 cultivation, DHA were observed accumulating soon after the up-regulation of pks genes. Those findings suggest that PKS enzyme involved in the synthesis of DHA in L-BL10. However, the amount of protein products produced in the various stages of DHA synthesis remains unclear. This study prepared a PKS antibody to elucidate the changes in polyketide synthase protein product. We designed epitopes and then used template-repeated polymerase chain reaction (TR-PCR) and adapter polymerase chain reaction (AD-PCR) to create linear array epitopes (LAE). And produce antibody by rabbit and mice. Finally, we used western blot and LC-MS/MS to identify and analyze PKS. LC-MS/MS results revealed that PKS A, B, and C comprised 18%, 58% and 24% coverage, respectively, compared with the L-BL10 polyketide synthase database. Western blot showed that PKS B and C produced strong signals at 20-28 hours. Finally, we suggested that L-BL10 use PKS to produce DHA. It is able to withstand stress in nitrogen deficient.

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