农杆菌介导南瓜遗传转化体系的建立

Establishment of Cucurbita moschata Genetic Transformation System by Agrobacterium tumefaciens Transfection

以南瓜金辉一号(Cucurbita moschata' Jinhui1')为实验材料,利用根癌农杆菌(Agrobacterium tumefaciens)介导转化南瓜子叶节,研究了预培养时间、侵染时间、乙酰丁香酮(AS)浓度和共培养时间,抗生素羧苄青霉素(Carb)、头孢霉素(Cef)以及筛选剂卡那霉素(Kan)等因素对离体不定芽的影响,建立了南瓜最适遗传转化体系。结果表明:外植体预培养0天,侵染时间30分钟,AS浓度为100mg·L-1,共培养5天可获得最高遗传转化效率;最适除菌剂为Cef,其最适浓度为500mg·L-1;最适Kan筛选浓度为100mg·L-1;在MS培养基上培养抗性芽生根,经PCR和Southern blot检测,证明为转基因植株。

Establishing a transformation system is fundamental to transgenic study.We developed an efficient Agrobacterium tumefaciens-mediated transformation and screening system for Cucurbita moschata ＇Jinhui 1＇.The leaves from tissue culture were excellent explants for transformation.To construct a genetic transformation system,factors affecting the transformation frequency included pre-culture time of explants,infection time,and concentration of acetosyringone（AS）.Additionally,co-culture time and the concentration of antibiotics（Carb,Cefotaxime and Kanamycin）,which inhibit the growth of Agrobacterium,were also examined in the adventitious bud.The data were analyzed by DPS software,and a suitable genetic transformation system was established.Pre-culturation for 0 days,infection for 30 min,AS at 100 mg·L^-1 and co-culture for 5 days produced the highest transformation efficiency.The medium with Cefotaxime（500 mg·L^-1） and Kanamycin（100 mg·L^-1） was optimal for selecting transgenic plant cultivation-resistant shoots on MS for eventual root induction.PCR and Southern blot results showed the Kanamycin-resistant exogenous gene had been integrated into the genome of Cucurbita moschata.