Aurantiochytrium sp. strain L-BL10為一富含22碳6烯酸 (docosahexaenoic acid, DHA) 的海洋原生生物，其DHA含量可達乾重的18 %，然也連帶產生高量的棕櫚酸 (palmitic acid, PA)，PA為一種有引起心血管疾病疑慮的長鏈飽和脂肪酸，對於藻油產品的健康訴求將產生負面影響。添加cerulenin，一種fatty acid synthase (FAS) 抑制劑的作法，可有效降低L-BL10細胞中PA的含量，然因其為高單價之抗生素並無法實際應用在工業級量產中。
因此，本研究的目的在於評估利用RNA干擾、FAS蛋白質生產抑制及突變株篩選的方式，達到降低PA產量的目的。在RNA干擾部分，首先針對L-BL10的FAS基因進行定序，再利用其序列設計real-time PCR及合成dsRNA之引子。接著利用real-time PCR分析FAS基因的表現趨勢，並挑選適合之處理時間點後，處理由T7 RiboMAX™ Express RNAi System合成之dsRNA，期望可有效抑制FAS基因表現，進而降低PA之產量。在FAS蛋白質產量抑制部分，則是於上述適合之處理時間點處理morpholino以抑制蛋白質產量。而在突變株篩選的部分，首先檢測致變劑MNNG (N-methyl-N'-nitro-N-nitrosoguanidine) 的施用劑量後，再利用GC/MS分析突變株之油質組成，挑選出PA產量較低的突變株。
在RNA干擾實驗部分目前已完成L-BL10之FAS 定序，長度為13242 bp，屬於type Ia FAS中的bacteral FAS，其基因表現量在細胞之對數成長後期，也就是PA大量累積前8~10個小時會開始大量表現。進一步也確立將dsRNA送入藻細胞之條件，但在送入fas dsRNA測試中，發現並無法有效抑制fas之表現，推測可能本次設計之dsRNA片段無法激起RNA干擾反應，抑或是L-BL10並不具有RNA干擾之機制。於FAS蛋白質抑制測試中，發現其轉染效率僅0.2 %，需進一步針對轉染法進行修改以供抑制測試。而在突變株篩選的實驗中，在處理0.1 mg/ml MNNG的情況下，細胞存活率為1.35 %，選取本劑量進行致變處理，由此所獲得的150株品系，初步篩選之中發現有5株PA比率明顯較低，進一步確認後發現其中以編號M48之突變株下降比率最高，證明致變劑處理的方式確實有機會降低L-BL10的PA含量。

Aurantiochytrium sp. L-BL10, an oleaginous microalgal strain rich in DHA, is a promising candidate for the production of vegetative DHA. The sole concern limiting the use of L-BL10 is the co-existence of palmitic acid (PA), an undesirable fatty acid which counteracts the human health effects of DHA. In the past, PA production was reduced through treatment with cerulenin, an inhibitor of fatty acid synthase. Unfortunately, cerulenin is an antibiotic that can negatively affect food safety.
The purpose of this research was to reduce PA production in L-BL10 through RNA interference (RNAi) of the fatty acid synthase gene (fas), inhibits the synthesis of fatty acid synthase (FAS) proteins and mutagenesis. In this study, we first sequenced fas and then designed real-time PCR primers in order to analyze gene expression patterns over various durations following incubation. These results provided a foundation for the following investigation. After ascertaining the appropriate timing, we conducted RNAi analysis on fas dsRNA and treated this gene with morpholino in order to inhibit FAS protein synthesis. For mutagenesis, we first determined the correct dose of MNNG mutagen and then treated L-BL10 with MNNG to obtain mutant lines. The fatty acid composition of the mutant lines was analyzed using GC/MS.
fas sequencing was completed to a length of 13257 bps. fas expression was up-regulated after 16 hrs of incubation, representing the late logarithmic phase of cell growth. At this point, dsRNA was combined with Lipofetamine to induce RNA interference; however, these treatments did not significantly inhibit FAS gene expression. Regarding the inhibition of protein synthesis, the efficiency of transfection was found to be only 0.2 %. In order to facilitate inhibition tests, it would be necessary to modify the conditions of transfection. Regarding mutagenesis, a concentration of 0.68 mM MNNG caused a 1.35 % reduction in cell survival rate. Under these conditions, we selected 150 lines of mutant strains, 5 of which were identified as having low PA accumulation compared with the original line. In the mutant line M48, the reduced PA ratio was as high as 8.38 %. These results suggest that mutagenesis has the potential to reduce PA production.

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