以分析基因表現與代謝物產量之關聯性探討牛樟芝 Coenzyme Q3 類緣物生合成之候選基因

Exploring the Candidate Genes for the Biosynthesis of Coenzyme Q3 Analogues in Antrodia cinnamomea using Correlation Analysis between Gene Expression and Metabolite Contents

周家因(Chia-Yin Chou)

指導教授 ： 許輔

國立臺灣大學/生物資源暨農學院/園藝暨景觀學系/碩士(2020年)

https://doi.org/10.6342/NTU202001779

摘要

泛醌 (coenzyme Q3, CoQ3) 類緣物 (analogues) 為牛樟芝 (Antrodia cinnamomea) 中一類具生理活性的化合物，antroquinonol (AQ) 與 4-acetylantroquinonol B (4-AAQB) 兩種泛醌類緣物之生合成途徑，已被證實與泛醌的生合成途徑相似。因此，參與泛醌生合成途徑的 COQ 蛋白可能具有調節泛醌類緣物產量之潛力。本研究欲分析參與 CoQ 生合成之酵素基因表現量與泛醌類緣物產量之間的關聯性，找出對其產量具有關鍵影響之基因。為獲得牛樟芝 COQ 蛋白之核苷酸序列，本研究以次世代定序進行牛樟芝轉錄體分析，並於全新組裝 (de novo assembly) 後得到 48,653 條鄰接序列 (contig)，可對應到 8,034 個特定基因 (unigene)，其中共有 15 個特定基因註解到 COQ 蛋白，挑選其中 8 個可能編碼 COQ 蛋白的特定基因，包含 COQ2 異戊烯基轉移酶 (polyprenyltransferase)、COQ3 O-甲基轉移酶 (O-methyltransferase)、COQ5 C-甲基轉移酶 (C-methyltransferase)、COQ6 單加氧酶 (monooxygenase)、COQ7 單加氧酶 (monooxygenase)，於後續實驗中分析其基因表現量。將牛樟芝液態培養 7、10、13、16、19、22、25、28 天後，收集菌絲體，將其乙醇萃取液以液相層析串聯質譜儀 (liquid chromatography-tandem mass spectrometry, LC-MS/MS) 進行泛醌類緣物定量，並以即時聚合酶連鎖反應 (real-time polymerase chain reaction) 分析 8 個 COQ 蛋白之基因表現量。關聯性分析結果顯示，特定基因 AC2588 (COQ2-4) 與泛醌類緣物產量有較高的相關性，具有調節泛醌類緣物產量的潛力，其轉譯後之蛋白可能為聚異戊二烯轉移酶 (EC 2.5.1.39)。本研究根據轉錄體定序結果，提出 AC2588 蛋白質編碼序列 (protein-coding sequence) 與對應之胺基酸序列。未來可就本研究提出之序列，進一步探討其蛋白質功能與酵素催化活性，以期後續能應用於提升牛樟芝泛醌類緣物產量。

關鍵字

牛樟芝 ； 泛醌類緣物 ； antroquinonol ； 4-acetylantroquinonol B ； 聚異戊二烯轉移酶

並列摘要

Coenzyme Q3 (CoQ3) analogues are a group of bioactive compounds in Antrodia cinnamomea. Most of CoQ3 analogues exhibited excellent anti-cancer effects, especially antroquinonol (AQ) and 4-acetylantroquinonol B (4-AAQB). Recently, AQ and 4-AAQB were proved to have similar biosynthetic pathways with that of CoQ. Therefore, COQ proteins involved in the biosynthesis of CoQ might have the potential in regulating the yields of CoQ3 analogues in A. cinnamomea. To obtain the nucleotide sequences of COQ proteins from A. cinnamomea, a transcriptome database containing 48,653 contigs corresponding to 8,034 unigenes was established. There were fifteen unigenes annotated to COQ proteins, and eight of them annotated to COQ2 (polyprenyltransferase), COQ3 (O-methyltransferase), COQ5 (C-methyltransferase), COQ6 (monooxygenase), and COQ7 (monooxygenase) were selected for subsequent analysis. The mycelium of A. cinnamomea sampled at eight time points during submerged fermentation was collected. Quantification of CoQ3 analogues from mycelium was carried out using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Gene expression levels of selected COQ proteins were analyzed by real-time polymerase chain reaction. After correlation analysis between gene expression profile and the contents of CoQ3 analogues, unigene AC2588 (COQ2-4) that might encode a polyprenyltransferase (EC 2.5.1.39) was found the most correlated to the contents of CoQ3 analogues and might be potential to regulate the yields of CoQ3 analogues in A. cinnamomea. The open-reading frame and deduced amino acid sequence for COQ2-4 were proposed in this study. It was recommended that further studies could be performed to investigate the protein function and catalytic activity of COQ2-4.

並列關鍵字

Antrodia cinnamomea ； CoQ3 analogues ； antroquinonol ； 4-acetylantroquinonol B ； polyprenyltransferase

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