真菌的二次代謝調控 (secondary metabolism) 主要是用來調節生長及發育，同時也會因為環境的差異產生不同的二次代謝物。麴菌屬參與二次代謝調控機轉的基因多以基因叢集 (gene cluster) 的方式存在，催化酵素主要是以非還原性聚酮合成酶 (nonreducing polyketide synthase, NRPKS) 參與代謝路徑，但對於催化後所得到的代謝物種類、功能及活性尚不明確。本實驗材料選用的蛹蟲草或稱北蟲草 (Cordyceps militaris)，可寄生昆蟲幼蟲或蛹體產生功能性成分如蟲草素、蟲草酸、核苷、蟲草多醣等，在人體醫療保健方面廣泛應用。由於蛹蟲草本身可產生多樣的二次代謝物、具有應用價值，但對於代謝物生物活性有許多仍是未知，為闡明非還原性聚酮合成酶基因功能與分析二次代謝物，實驗利用融合聚合酶鏈鎖反應 (fusion polymerase chain reaction, fusion PCR) 將擴增出的標的基因結合篩選標記等基因片段，轉形進入構巢麴菌宿主進行異源表達，使可轉譯酵素並催化產生二次代謝物。以高效能液相層析法分析產生的二次代謝物，藉以推論蛹蟲草非還原性聚酮合成酶的功能，同時分析所催化而生產的二次代謝物。目前已擴增出兩段具有養份代謝篩選標記之蛹蟲草NRPKS基因片段，轉形進入構巢麴菌宿主，挑選出7個轉形株。以PCR確認轉形株染色體是否嵌入標的基因，結果顯示擴增出約3.8 kb之DNA片段。將轉形株進行高效能液相層析法分析後，結果顯示轉形株有別於宿主及蛹蟲草的訊號出現，推測標的基因有嵌入宿主內，並催化產生二次代謝物；但是這些訊號所代表的二次代謝物種類與是否具有生物活性則有待釐清。 Fungal secondary metabolism is very important in fungal growth and development. The production of secondary metabolites is relied on the changes of environment. In Aspergillus, gene cluster is involved in the regulation of secondary metabolism, and the major products of the gene cluster are belong to non-reducing polyketide synthases (NRPKSs). However, it is not very clear about the function and activity of the secondary metabolites catalyzed by NRPKSs. Cordyceps militaris is a useful herbal medicine grew on some insect larvae or pupae. Cordyceps militaris contains many kinds of functional components such as cordycepin, cordycepic acid, nucleosides, and Cordyceps polysaccharide. We assume that the secondary metabolic pathway is also existed in C. militaris. In order to clarify the function of NRPKSs in the regulation of secondary metabolism and the secondary metabolites in C. militaris, fusion PCR technique was used to the recombination of selective markers , alcA promoter, and target gene. Heterologous expression was performed in Aspergillus nidulans. In this study, the use of fusion PCR completed which NRPKS fusion gene, and seven of transformants were obtained. The variety of secondary metabolites was analyzed by high-performance liquid chromatography. The results indicated that some secondary metabolites were produced in transformants. This study showed a great potential for developing the application of fungal secondary metabolites and studying the function of NRPKS genes.
