农杆菌介导的烟草瞬时表达影响因素研究

通过探究根癌农杆菌(Agrobacterium tumefaciens)介导的烟草瞬时表达体系的影响因素,确定烟草瞬时表达的最佳条件,为目的基因的功能研究提供技术支持。利用马铃薯晚疫病抗性基因RB和致病疫霉菌效应基因Avrblb 1作为报告基因,采用农杆菌介导的注射渗透法,摸索农杆菌菌悬液OD600值和菌系(遗传背景)、烟草品种及其生长时期等因素,确定烟草高效的基因瞬时表达条件。研究结果表明:介导烟草基因瞬时表达的根癌农杆菌菌悬液适宜OD600值为0.3~0.8;在菌悬液OD600值为0.1及以上时,AGL1、GV3101和C58C1介导的报告基因瞬时表达效率相似,均引发较强的叶片坏死反应;在农杆菌菌悬液OD600值小于0.1时,AGL1介导的瞬时表达效率最高。测试的烟草品种都具备较高的瞬时表达效率,苗龄6~8周的烟草适合分析。通过测试农杆菌菌悬液OD600值和菌系(遗传背景)、烟草品种及其生长时期等因素,确定本氏烟和普通烟均可实现基因高效瞬时表达的条件。

Optimization of Agrobacterium-Mediated Transient Assay of Gene Expression in Nicotiana tobacum

By analyses of factors important for Agrobacterium tumefaciens-mediated gene expression in tobacco (Nicotiana tobacum),this study aims at determining the optimal conditions for transient expression of target genes.The potato late blight resistant gene RB and the cognate Phytophthora infestans effector gene AvrblbQX(Y15]1 were used as reporter genes to determine the effects of bacterial OD₆₀₀,strains,tobacco genotypes and development stages on the efficacies of A.tumefaciens-mediated transient gene expression.In the range of bacterial OD₆₀₀ of 0.3-0.8,reasonable gene expression levels were achieved for all three examined strains AGL1,GV3101 and C58C1,as determined by enabling RB/Avrblb1-mediated rapid necrosis,which was achieved even at lower OD₆₀₀ (0.1 and above).Strain AGL1 was more efficient at low OD₆₀₀ (0.1 and lower).All six examined tobacco cultivars and lines displayed high-efficiency transient gene expression.Tobacco plants of 6-8 weeks were suitable for agroinfiltration assays.Through consideration of bacterial OD₆₀₀,strains (genetic background),tobacco genotypes and growth stages,optimized conditions for A.tumefaciens-mediated high-efficiency transient gene expressions were determined for N.benthamiana and N.tobacum.