plantmolecularbiology-生物探索-探索生物科技價值的新

1、根癌農(nóng)桿菌介導(dǎo)煙草GUS基因轉(zhuǎn)化Agrobacterium –mediated GUS gene transformation of tobacco,根癌農(nóng)桿菌(Agrobacterium tumefaciens)介導(dǎo)轉(zhuǎn)化法,A. tumefaciens is a gram-negative soil bacterium which naturally transforms plant cells, resulting in cr

2、own 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 resid

3、es on a large plasmid called Ti (tumor inducing) found in A. tumefaciens.,Ti-Plasmid,Ti質(zhì)?？煞譃?個區(qū)：1、T-DNA區(qū) (transferred-DNA regions) 轉(zhuǎn)移DNA2、Vir區(qū) (virulence region) 激活T-DNA轉(zhuǎn)移3、Con區(qū) (regions encoding conju

4、gations) 轉(zhuǎn)移編碼區(qū)，調(diào)控Ti質(zhì)粒在農(nóng)桿菌之間的轉(zhuǎn)移。4、Ori區(qū) (origin of replication) 調(diào)控Ti質(zhì)粒的自我復(fù)制。,Triparential mating,Agrobacterium-mediated plant transformation,,GUS檢測,熒光檢測,GFP,luciferase,Chances in lab ……,本周實驗安排：煙草葉片組織培養(yǎng)與芽的

5、再生1. MS培養(yǎng)基的配制 ( 2-3h )1）母液配制2）煙草愈傷組織誘導(dǎo)與芽再生培養(yǎng)基 MS + 2,4 D + 6-BAP（請同時準備下周要用的培養(yǎng)基）2. 培養(yǎng)基高壓滅菌 ( 2-3h )3. 葉片外植體接種 ( 2-3h),下周實驗安排：1. 煙草葉片組織與根癌農(nóng)桿菌共培養(yǎng)感染（2-3h）→共培養(yǎng)（2-3d）→瞬時表達檢測2. GUS基因瞬時表達的組織化學(xué)檢測（3