金針菇琥珀酸脫氫酶次單元C基因選殖及不同萎鏽靈抗性基因比較

Abstract

菇類分子農場 (mushroom molecular pharming) 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、製程簡單和成本低廉等優勢，為現今生物產業應用之新趨勢。過去研究以農桿菌媒介轉形法成功建立菇類轉形平台，應用於多種菇類上。前人為提升轉基因菇類的生物安全性，將琥珀酸脫氫酶 (succinate dehydrogenase, Sdh) 之次單元B (SdhB) 上胺基酸單一點突變成為萎鏽靈抗性基因cbrB，建立以金針菇為材料來源之同源性篩選系統。然而後續應用卻發現此抗性篩選系統並不如預期穩定。因此，本篇研究欲發展以次單元C (SdhC) 為基礎之萎鏽靈抗性基因cbrC，並比較帶有兩段抗性基因之轉形株的琥珀酸脫氫酶活性，找出活性較高之萎鏽靈抗性篩選標誌。實驗結果證實帶有cbrC 之金針菇轉形株確實可生長在萎鏽靈篩選培養基上，顯示 cbrC 確實具有萎鏽靈抗性能力，而內含子的存在可幫助金針菇表現cbrC。本篇研究建立菇類琥珀酸脫氫酶活性測試系統，抽取轉形株粒線體進行活性分析，實驗結果發現在含有萎鏽靈抗生素之環境下，轉形株之酵素活性皆比野生株高，而帶有 cbrC 的轉形株活性較帶有 cbrB的轉形株活性高，顯示菇類同源性篩選系統以 SdhC 為較佳的篩選標誌。而未來希望能結合兩段抗性基因以提高此同源篩選系統之為萎鏽靈抗性能力。

Mushroom molecular pharming, a novel and promising industry that use mushrooms as bioreactors to produce pharmaceutical proteins or industrial enzymes, possesses lots of advantages such as safety, easy manipulation and low cost. In our laboratory, we have been successfully established Agrobacterium-mediated transformation in mushroom system. For biosafety of transgenic mushrooms, we also developed homologous selectable system via a single point mutation of succinate dehygrogenase subunit B (SdhB) to make transformants against harmless agent, carboxin. However, previous study show the instability of transformants after several generations of subcultures. In order to improve the efficiency of selection and stability of transformants, we would like to clone another carboxin-binding site of succinate dehydrogenase to enhance the selectable resistance against carboxin. In this study, succinate dehygrogenase subunit C (SdhC) was selected as the target sequence of novel selectable marker. We successfully cloned carboxin-resistance gene (cbrC) based on the SdhC, and found out that gpd-d1 promoter with the first intron improved the expression of cbrC. The activity of succinate dehydrogenase was assaged to compare these two selectable system, SdhB (cbrB) and SdhC (cbrC). The results suggested that carboxin-resistance gene (cbrC) based on the SdhC is a better carboxin resistance selection marker than that based on SdhB. We demonstrated that carboxin-resistance gene (cbrC) based on the SdhC is a new homologous selectable marker, and its seleciotn ability was measured by the succinate dehydrogenase activity in mushrooms. This allows us to develop a dual carboxin-resistant selectable system to improve the stability of transgenic mushrooms.