根癌农杆菌介导的双孢蘑菇转基因体系的建立  

Establishment of A High-Efficiency Agrobacterium-mediated Transformation System in Agaricus bisporus

作　　者：乔燕楠 林星雨 高雅 闫静[2] 张岚岚[1] 李南羿[1] QIAO Yannan;LIN Xingyu;GAO Ya;YAN Jing;ZHANG Lanlan;LI Nanyi(College of Agricultural and Food Science,Zhejiang Agricultural and Forestry University,Hangzhou 311300,Zhejiang,China;Hangzhou Academy of Agricultural Sciences,Hangzhou 310024,Zhejiang,China)

机构地区：[1]浙江农林大学农业与食品科学学院,浙江杭州311300 [2]杭州市农业科学研究院,浙江杭州310024

出　　处：《食用菌学报》2021年第1期48-54,共7页Acta Edulis Fungi

基　　金：国家自然科学基金(31201668);浙江省“十三五”食用菌新品种选育重大科技专项子项目(2016C02057-4)。

摘　　要：构建了gpd启动子驱动的双孢蘑菇(Agaricus bisporus)双元表达载体,研究了受体材料、农杆菌浓度、侵染时间、乙酰丁香酮(acetosyringone,AS)浓度和共培养时间对转化效率的影响。结果表明:不同受体的转化效率有较大的差异;以直径2~4 cm的菌丝球为受体,当农杆菌菌液D600为0.2,侵染时间30 min,共培养AS浓度为200μmol·L^-1,共培养1 d时,获得抗性转化子的百分率最高,为56.8%。拟转化子的PCR鉴定和GUS(β-glucuronidase,GUS)染色验证外源基因已整合到双孢蘑菇基因组并得到稳定表达。A binary expression vector driven by the gpd promoter was constructed for Agrobacterium-mediated transformation of Agaricus bisporus.Key factors involved in the transformation,including recipient tissue,bacteria concentration,infection time,acestosyringone(AS)concentration,and co-culture time were studied.The results showed that recipient tissue had a significant effect on transformation efficiency.The highest transformation efficiency reached 56.8%by infecting mycelial aggregates of 2-4 cm in diameter with Agrobacterium at D6000.2 for 30 min,followed by co-culture with 200μmol·L-1AS for 1 d.PCR and GUS staining results showed that the reporter gene was integrated into the A.bisporus genome and expressed stably.

关 键 词：双孢蘑菇 农杆菌 转化 gpd启动子 GUS染色 

分 类 号：S646.11[农业科学—蔬菜学]