番茄遗传转化体系的建立及LC和GFP标记基因的表达

The Expression of Lc and GFP Marker Gene in Transformation of Tomato

本研究以小型番茄(miniature Lycopersicon esculentum Mill.)Micro-Tom为试材,建立其以子叶、下胚轴为外植体的高效、稳定再生体系及遗传转化体系。结果表明,以子叶、下胚轴为外植体时,诱导愈伤组织和芽分化的适宜培养基均为MS+6-BA2.0mg/L+IAA0.2mg/L,芽分化率均可高达92%。两种外植体适宜的生根培养基均为MS+NAA0.1mg/L。同时,通过根癌农杆菌介导法,建立了Micro-Tom的遗传转化体系。而乙酰丁香酮能大大提高根癌农杆菌转化番茄的效率,当AS=180μm/L时,芽分化率为52.8%;OD600=0.14是转化的最佳菌液浓度;子叶外植体与农杆菌共培养后,经过诱导,根的再生,获得了抗性苗,利用绿色荧光蛋白(GFP)检测和整株表型为紫色(Lc)基因的表达,初步证明外源基因已经整合到Micro-Tom中,为番茄激活标签体系的建立奠定了基础。

Micro-Tom （miniature Lycopersicon esculenturn Mill.） was used as the material, to establish the high efficient and stable plant regeneration by cotyledon and hypocotyl. The results showed that with cotyledon and hypocotyl as explants, the best callus initiation and shooting medium was MS＋6-BA 2.0mg/ L＋IAA 0.2mg/L. The best rooting medium for both the explants was MS＋NAA 0.1mg/L. Meanwhile established genetic transformation system of Micro-Tom though Agrobactefium tumefaciens, and gained purple mutant. The results showed that AS could improve the transformation rate. when AS is 180μm/L, the differentiation rate was 52.8%, OD600=0.14 was the best infect concentration; Kan resistant plant obtained from the infection of explants with Agrobacterium. Detection of green fluorescent protein under microscope and the express of Lc gene firstly confirmed that transgene was integrated into Micro-Tom.