探討鮑魚菇轉形株菌絲體、子實體與擔孢子之異源基因表現

Abstract

菇類分子農場係以菇類為生物反應器，生產醫藥用蛋白質或工業用酵素，具備安全性高、操作簡單及成本低廉等優勢，為近年來新興之生物技術應用。過去研究藉由農桿菌媒介轉形法成功進行多種菇類轉形，亦發展不同分生策略提升異源蛋白質表現量。前人發現可表現綠色螢光蛋白質之金針菇轉形株，其綠色螢光有聚集於蕈褶之現象，推測異源基因可能集中於蕈褶或是擔孢子中大量表現，因此本研究欲探討菇類轉形系統是否具有表現部位差異，以提供未來發展口服疫苗食用部位之參考。使用前人建立經序列刪減之金針菇甘油醛-3-磷酸脫氫酶 (glyceraldehyde-3-phosphate dehydrogenase, gpd) 啟動子 (gpd-d1) 表現報導基因綠色螢光蛋白質，而篩選標誌採用金針菇來源之萎鏽靈抗性基因 (carboxin resistance gene, cbr)。探討異源基因表現部位之差異，分為菌絲體以及子實體之蕈柄、蕈傘、擔孢子等四個部位，並同時探討鮑魚菇表現金針菇來源之啟動子與篩選標誌之可行性。為方便擔孢子分析，選用可大量產生擔孢子之鮑魚菇 (Pleurotus ostreatus) 做為表現宿主。結果顯示於篩選培養基可順利篩選出鮑魚菇轉形株，並確認目標基因嵌入至宿主染色體 DNA 與綠色螢光蛋白質表現，證明金針菇來源之 gpd-d1 啟動子與篩選標誌可於鮑魚菇中表現，拓展金針菇萎鏽靈篩選標誌跨物種之應用；另外不同部位異源基因分析結果發現，蕈柄與菌絲體總可溶性蛋白質中之綠色螢光蛋白質表現量最高，而訊息 RNA 則以擔孢子表現量較高。不同部位異源蛋白質表現量分析結果，可提供未來菇類分子農場發展之參考。

Mushroom molecular pharming, an emerging biotechnology application using mushrooms as bioreactors to produce pharmaceutical proteins or industrial enzymes, exhibits advantages of being safe, easy in manipulation, and less expense. Agrobacterium-mediated transformation has been established and the heterologous gene expression has also been enhanced by various molecular biology strategies in our laboratory. The previous study shows that the major green fluorescence was found on the pileus of Flammulina velutipes enhanced green fluorescent protein (eGFP) transformants fruiting body, indicating the major heterologous gene expression might occur in pileus or basidiospores. In order to analyze the basdiospores, eGFP gene was transformed into Pleurotus ostreatus using the vector which contains partially deleted F. velutipes glyceraldehyde-3-phosphate dehydrogenase promoter d1 (gpd-d1) and carboxin resistance gene. The expression of eGFP between mycelia, pileus, stipes and basdiospores of the transformants was determined and compared. The heterologous genes could be driven by F. velutipes gpd-d1 in P. ostreatus. The high protein expression level was found both in mycelia and stipes, while the high mRNA expression was found in basdiospores. The results suggested the carboxin resistance selection marker of F. velutipes can be applied in other mushrooms and the distribution of heterologous gene expression between various tissues need to be considered in development of mushroom molecular pharming.