以農桿菌媒介轉形法進行金針菇表達系統之研究

Abstract

分子農場 (molecular farming) 泛指透過植物來生產具醫藥等級、工業應用或保健食品之重組蛋白質或酵素。除了植物以外，真菌也是一個極具發展潛力之表達系統，近二十年來已有許多利用酵母菌以及絲狀真菌生產重組蛋白質之例子；然而以菇類為主的擔子菌表達系統始終缺乏有效且穩定的轉形方法。 本研究成功建立金針菇之農桿菌媒介轉形系統。農桿菌媒介轉形策略是利用本身所帶的 Ti (tumor inducing) 質體將外來 DNA 藉由感染的過程轉入金針菇染色體，具有高穩定度及低染色體 DNA 嵌入處等優點。以洋菇與金針菇之甘油醛-3-磷酸脫氫脢啟動子，GUS (b-glucuronidase) (uidA)、green fluorescent protein (gfp) 為報導基因，hygromycin phosphotransferase (hph) 為篩選標記建構表現載體，再以金針菇的菌絲體、菌絲塊及子實體蕈褶組織為材料，與帶有表現載體之農桿菌株 LBA4404、AGL-1 進行共培養 3 ~ 6 天，皆能夠順利得到轉形株。篩選標記 hph 基因之轉形效率：菌絲體每毫升混合菌液可獲得 2 ~ 3 個轉形株；菌絲塊以及子實體蕈褶組織之轉形效率各為 15 ~ 30% 及 5 ~ 15%。經由南方氏雜合分析可確定目標基因確實插入金針菇染色體 DNA 中，且異源基因嵌入金針菇染色體為隨機的方式，嵌入位置數目為 1 ~ 2 個。使用金針菇與洋菇之甘油醛-3-磷酸脫氫脢啟動子及其第一個內含子，皆能於金針菇中表現 EGFP。經過酵素免疫分析定量結果顯示，洋菇與金針菇啟動子所表現之 EGFP 最高濃度分別為可溶性蛋白質的 0.0089% 及 0.00035%，兩者表現量相差了 25 倍。綜合以上結果可知在金針菇表達系統中，洋菇異源啟動子所啟動之 egfp 表現量高於同源啟動子，顯示洋菇啟動子較適合作為金針菇表現 egfp 基因之啟動子。

Molecular farming is the production of pharmaceutically important and industrially valuable proteins or enzymes in plants and mushrooms. Research in the past two decades has developed heterologous protein expression in yeasts and filamentous fungi. However, the lack of an efficient and stable transformation system in filamentous fungi is one of the major problems for recombinant protein production in mushrooms. In this study, the expression system of Flammulina velutipes using Agrobacterium tumefaciens-mediated transformation (ATMT) was developed. A. tumefaciens is able to transfer a portion of the resident Ti- plasmid, the T-DNA (transferred DNA), into F. velutipes nuclear genome during infection. The advantages of ATMT over other transformation methods, include high stability of heterologous gene expression and almost single-copy integrated DNA. Three expression plasmids were constructed harboring Escherichia coli hygromycin B phosphotransferase (hph) as the selectable marker, and GUS (β-glucuronidase) (uidA) or enhanced green fluorescent protein (egfp) as reporter genes under control of the glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter from Agaricus bisporus or F. velutipes. A. tumefaciens LBA4404 and AGL-1 carrying these expression plasmids were cocultivated with vegetative mycelia, modified mycelial pellets (MMP) and gill tissues of F. velutipes for 3 ~ 6 days, respectively. The transformation efficiency of hph gene varied 2 ~ 3 transformants per mL mycelia suspensions, 15 ~ 30% with MMP and 5 ~ 15% with gill tissues. Southern-blot analysis showed that the hph gene was integrated into the genome randomly and predominantly with one insertion site. The homologous and heterologous A. bisporus gpd promoters with their first intron were successful in egfp expression in F. velutipes. The amount of egfp expressed by A. bisporus promoter constituted 0.0089% of total soluble protein (TSB), about 25 times more than that using F. v. promoter with 0.00035% of TSB. This study showed that heterologous A. bisporus gpd promoter was more efficient for egfp expression in F. velutipes than homologous gpd promoter.