利用農桿菌媒介重複轉形法提升美白菇異源蛋白質表現量

Abstract

分子農場(molecular farming)泛指利用轉基因植物生產具有高價值的重組蛋白質，如醫藥用蛋白質。菇類於分子農場之應用近年來也受到重視，建立完善之菇類異源表達系統將有助於分子農場的發展。過去研究已成功應用於多種菇類進行異源基因表現。農桿菌媒介轉形法(Agrobacterium tumefaciens-mediated transformation, ATMT)具有外源基因穩定性高的優點，然而缺點是異源蛋白質表現量偏低。本研究使用美白菇(Hypsizygus marumoreus)為表達宿主，進行農桿菌媒介重複轉形，期望能提升轉形株異源蛋白質表現量。本研究先以帶有萎銹靈抗性基因(carboxin resistance gene , cbxr)為篩選標記與綠色螢光蛋白質基因(enhanced green fluorescent protein , egfp)為報導基因的農桿菌進行第一次轉形，接著再對該轉形株以相同之農桿菌進行第二次轉形以取得單一抗藥性之二次轉形株；或是使用另一種帶有潮黴素抗性基因(hygromycin phosphotransferase, hph)為篩選標記與綠色螢光蛋白質基因的農桿菌進行第二次轉形，以取得雙重抗藥性之二次轉形株，篩選過後比較一次轉形株母體與二次轉形株的綠色螢光蛋白質表現量。結果顯示單一抗藥性之二次轉形株其綠色螢光蛋白質最高為每克總可溶性蛋白質中含有319.58 ng，百分比為3.19*10-5，係一次轉形株母體的4.25倍，另外雙重抗藥性之二次轉形株其綠色螢光蛋白質最高為每克總可溶性蛋白質中含有418.83 ng，百分比為4.18*10-5，係一次轉形株母體的5.5倍，顯示農桿菌媒介重複轉形法可以提升異源蛋白質表現量。

Mushroom molecular farming recently attracts tremendous attention because of its application pontentials and the advantages over plant molecular farming. Agrobacterium tumefaciens-mediated transformation (ATMT) is commonly used in mushroom transformation but its application was limited due to the low heterologous gene expression. In this study, Hypsizygus marumoreus was chosen as the expression host and was transformed by multiple ATMT in order to enhance heterologous protein expression. For the first step of multiple ATMT, A. tumefaciens harboring p0390-Cbx-Hiegfp, a Ti-plasmid contains carboxin resistance gene (cbxr) and enhanced green fluorescent protein (egfp), was used for transformation. The single transformants were re-transformed by A. tumefaciens harboring p0390-AH-Aiegfp, which contains hygromycin phosphotransferase (hph) and egfp, or re-transformed by A. tumefaciens harboring p0390-Cbx-Hiegfp. Finally, EGFP was analyzed by ELISA and the copy number of egfp was determined by real-time PCR. This study demonstrated that the heterologous gene expression was enhanced by multiple ATMT in H. marumoreus. The highest EGFP production in two-vector double transformants was 418.83 ng/g TSP (total soluble protein), which increased up to 5.5 folds in comparison with its parental single transformant. In one-vector double transformants, the highest EGFP production was 319.58 ng/g TSP, which raised to 4.25 folds and the transgene copy number accordingly increased, too. Multiple ATMT described in this study provides a new approach in improvement of the heterologous gene expression.