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采用Streptomyces hygroscopicus(放线菌)源的BAR基因,通过微粒子轰炸遗传转化三个属兰花:长萼兰(Brassia)、卡特兰(Cattleopya)和五唇蝶兰(Doritaensis)的原球茎状体。采用bialaphos(一种除草剂)完成转化细胞的选择。在含1mg/l bialaphos选择培养基上增殖的原球茎状体切碎后转入含3mg/l上述除草剂的选择培养基上。在两个月的间隔期中此种选择重复两次。假定转化的五唇蝶兰的小植株在不含bialaphos中再生,长萼兰和卡特兰则在含3mg/l bialaphos中再生。通过PCR和Southern印迹分析证实了转化植株中存在BAR基因。通过Northern印迹分析证实了BAR基因的转录。通过直接将bialaphos涂于叶片所进行的抗性分析证实了所有三种兰花的小植株BAR基因的功能性表达。
Three genera of orchids were genetically transformed by microparticle bombardment using the BAR gene from the Streptomyces hygroscopicus source: prothallomes of the genus Brassia, Cattleopya and Doritaensis. Bialaphos (a herbicide) is used to complete the selection of transformed cells. Protocorms multiplied on bialaphos selection medium containing 1 mg / l were chopped and transferred to selection medium containing 3 mg / l of the above herbicide. This choice is repeated twice during the two-month interval. It is assumed that the transformed plantlets of the five-leaved Phalaenopsis are regenerated in the absence of bialaphos, while the long-euphorbiae and Cattleran are regenerated in 3 mg / l bialaphos. The presence of the BAR gene in the transformed plants was confirmed by PCR and Southern blot analysis. BAR gene transcription was confirmed by Northern blot analysis. Resistance analysis by direct application of bialaphos to the leaves confirmed the functional expression of the BAR genes of the plantlet of all three orchids.