Agrobacterium –mediated GUS gene transformation of tobacco 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 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DNA 2、Vir区 (virulence region) 激活T-DNA转移 3、Con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 luciferase GFP
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）