玉米核糖体失活蛋白基因z108在烟草原生质体中的转化及其表达  

Transformation and Expression of Maize Ribosome-inactivating Protein Gene z108 in Tobacco Protoplasts

作　　者：周颖[1] 姜国勇[1] 

机构地区：[1]青岛农业大学植物基因工程研究所,青岛266109

出　　处：《生物技术通报》2010年第4期99-102,共4页Biotechnology Bulletin

基　　金：国家自然科学基金(30771440);青岛市科技攻关计划

摘　　要：利用与根癌农杆菌共培养的方法将玉米核糖体失活蛋白基因z108及其融合基因GUS导入烟草叶肉原生质体细胞。试验结果表明,烟草叶片在含有1.0%纤维素酶和0.5%离析酶的裂解液中,以0.5M甘露醇、5000.0mg/LCaCl2为渗透压调节剂,25℃保温12-14h,可获得纯化的原生质体细胞;纯化的叶肉原生质体细胞与根癌农杆菌菌株pCam1301/91-108共培养30min,经25mg/L潮霉素筛选,转化率可达17.84%。转化体细胞经X-Gluc染色、PCR和RT-PCR检测证明玉米核糖体失活蛋白基因z108已整合到烟草原生质体细胞的核基因组中并获得表达。In this study,maize ribosome-inactivating protein gene z108 and its fusion gene GUS were introduced into tobacco mesophyll protoplasts mediated by A.tumefaciens.The results showed that,through the treatments using enzyme-regent mixture of 1.0% cellulase,0.5% macerozyme,0.5 mol/L mannitol and 5 000.0 mg/L CaCl2 incubated at 25℃ for 12-14 h,high purified tobacco protoplasts were obtained.The protoplasts were co-cultured with A.tumefaciens pCam1301/ 91-108 for 30min and then screened under 25 mg/L hygromycin.The transformation ratio could reach to 17.84%.Furthermore,by X-Gluc Staining,PCR detecting and RT-PCR analysis,it confirmed that z108 had been integrated into tobacco genome of protoplasts and expressed in vivo.

关 键 词：玉米核糖体失活蛋白 烟草原生质体 转化 

分 类 号：S572[农业科学—烟草工业]