1、根癌农杆菌介导烟草GUS基因转化Agrobacterium mediated GUS gene transformation of tobacco,根癌农杆菌(Agrobacterium tumefaciens)介导转化法,A. tumefaciens is a gram-negative soil bacterium which naturally transforms plant cells, resulting in crown gall (cancer) tumors. Tumor formation is the result of the transfer, integration
2、 and expression of genes on a specific segment of tumefaciens plasmid DNA called the T-DNA (transferred DNA). The T-DNA resides on a large plasmid called Ti (tumor inducing) found in A. tumefaciens.,Ti-Plasmid,Ti质粒可分为4个区：1、T-DNA区 (transferred-DNA regions) 转移DNA2、Vir区 (virulence region) 激活T-DNA转移3、Co
3、n区 (regions encoding conjugations) 转移编码区，调控Ti质粒在农杆菌之间的转移。4、Ori区 (origin of replication) 调控Ti质粒的自我复制。,Triparential mating,Agrobacterium-mediated plant transformation,GUS检测,荧光检测,GFP,luciferase,Chances in lab ,本周实验安排：烟草叶片组织培养与芽的再生1. MS培养基的配制 ( 2-3h )1）母液配制2）烟草愈伤组织诱导与芽再生培养基 MS + 2,4 D + 6-BAP（请同时准备下周要用的培养基）2. 培养基高压灭菌 ( 2-3h )3. 叶片外植体接种 ( 2-3h),下周实验安排：1. 烟草叶片组织与根癌农杆菌共培养感染（2-3h）共培养（2-3d）瞬时表达检测2. GUS基因瞬时表达的组织化学检测（3-4h）,
