应用alc基因开关系统在糙皮侧耳中表达绿色荧光蛋白

Expression of Green Fluorescent Protein Controlled by alc-gene Switch in Pleurotusostreatus

为探明担子菌糙皮侧耳(Pleurotus ostreatus)中是否存在与构巢曲霉相似的乙醇代谢调控机制,及能否在糙皮侧耳中应用构巢曲霉alc基因开关系统调控表达外源基因,从糙皮侧耳PC15v2.0基因组数据库中电子克隆得到其假拟乙醇脱氢酶基因(alcA)及其上游序列,该基因上游1 317bp序列中含有1个正向和2个反向调控因子AlcR蛋白的特异性结合位点序列。将构建的含有alcA基因启动子控制绿色荧光蛋白基因表达的质粒pEGFP-C1-PalcA转入糙皮侧耳天达300中,转化子在含有葡萄糖的培养基中没有绿色荧光蛋白表达,在含有乙醇的培养基中则有绿色荧光蛋白表达。综合上述,糙皮侧耳的乙醇代谢调控机制与构巢曲霉相似,仅用构巢曲霉的alcA基因启动子替代目的基因的启动子即可在糙皮侧耳中有效调控目的基因的表达。

To explore if there is a similar ethanol metabolic regulation mechanism to Aspergillus nidulans in Pleurotus ostreatus,the hypothetical alcohol dehydrogenase gene （alcA） and its upstream sequences was cloned by electronic cloning technology from P.ostreatus PC15 v2.0 genome database.There were one forward and two reverse AlcR specific binding sites in the upstream region （1 317 bp） of alcA.To explore if ethanol-inducible alc-gene switch from A.nidulans can be used in P.ostreatus,the promoter of alcA（PalcA） was inserted in plasmid pEGFP-C1 and fused with the green fluorescent protein gene （GFP）,generating the plasmid pEGFP-C1-PalcA.The plasmid pEGFP-C1-PalcA was transformed into P.ostreatus TD300.The GFP genes in the transformants expressed when the transformants were cultured in medium containing alcohol,but did not express when the transformants were cultured in the medium containing glucose.Overall,the ethanol metabolic regulation mechanism in P.ostreatus is similar to A.nidulans,and replacing the promoter of target gene by PalcA can effectively regulate the target gene expression in P.ostreatus.