利用農桿菌媒介轉形法研究美白菇表達系統

Abstract

分子農場可生產具有高價值的醫藥保健用重組蛋白質或工業應用之酵素。建立菇類的基因轉形系統，有助於分子育種和分子農場的發展。本研究使用農桿菌媒介轉形法，經由感染宿主的過程，將 Ti (tumor-inducing) 質體上的目標基因 T-DNA 隨機鑲嵌入宿主染色體，可使目標基因較穩定且有利於後續蛋白質之分析。實驗中建構帶有同源美白菇甘油醛-3-磷酸脫氫酶 (GPD) 啟動子、篩選標記潮黴素抗藥性基因 (hygromycin phosphotransferase, hph) 和報導子綠色螢光蛋白質 (enhanced green fluorescent protein, egfp) 基因的表現載體，將帶有此表現載體之農桿菌與轉形材料美白菇菌絲、菌絲塊進行共培養，皆可順利得到表現 EGFP 之轉形株。在潮黴素選擇性培養基上得到表現 EGFP 之轉形株。篩選標記 hph 轉形效率：菌絲塊具有較佳的轉形效率，轉形效率約 30-40 %，而菌絲體每毫克可獲得 1 個轉形株。帶有異源洋菇、金針菇甘油醛-3-磷酸脫氫酶 (GPD) 啟動子之農桿菌皆可順利感染美白菇，在潮黴素選擇性培養基上得到表現 EGFP 之轉形株。在酵素免疫分析進行 EGFP定量後，美白菇同源啟動子所測得最高 EGFP 含量為每克菌粉乾重中達 27.4 ng，高於洋菇和金針菇啟動子所測得之 EGFP 含量，顯示美白菇啟動子較適合作為美白菇表現 egfp 之啟動子。在研究中選殖出琥珀酸去氫酶 (succinate dehydrogenase, sdi1) 基因序列，進行特定序列的點突變後，利用農桿菌媒介轉形法將此質體鑲嵌入美白菇染色體，可得到具有同源性篩選標記萎銹靈 (carboxin) 之轉形株。

The molecular farming may produce pharmaceutically valuable protein or enzymes of the industrial application. The establishment of gene transformation contribute to develop molecular breeding and molecular farming. In this study, the expression system using Agrobacterium tumefaciens-mediated transformation (ATMT) was developed. A. tumefaciens inserts the T-DNA which is a portion of Ti-plasmid into the genome DNA randomly during infecting host. It could make the target gene more stable and enhance protein analysis. The expression plasmids were constructed to harbor homologous glyceraldehyde-3-phosphate (gpd) promoter from Hypsizygus marmoreus variant (Hmv), Escherichia coli hygromycin B phosphotransferase gene (hph) as the selectable marker and enhanced green fluorescent protein gene (egfp) as reporter gene. A. tumefaciens carrying these plasmids were cocultivated with vegetative mycelia, modified mycelia pellets (MMP) of Hmv for 3~6 days, respectively. The transformation efficiency was 1 transformant per mg dry weight of mycelia and about 30~40% with MMP. The heterologous gpd promoters from Agaricus bisporous and Flammulina velutipes expressed egfp gene successfully in Hmv. The western blotting analysis revealed the EGFP was expressed indeed. From the ELISA quantification analysis, the egfp expressed by Hmv promoter could reach to 27.4 ng per gram dry weight of mycelia, which was higher than by A. bisporus and F. velutipes. This study show the homologous Hmv gpd promoter was more efficient for egfp expression in Hmv than heterologous promoter. In this study, we cloned the succinate dehydrogenase gene (sdi1) from Hmv and introduced a point mutation that confers carboxin resistance into it. After transfer the recombinant plasmid into Hmv by ATMT, we could get the transformants which confers homologous selectable resistance.