Plant Expression System. M olecular B iology Lab oratory Oh Chang Jae. Overview of methods for plant transformation. A. Delivery of DNA into a single plant cell → transient assay B. Integration of DNA into the plant cell genome
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Plant Expression System
Molecular Biology Laboratory
Oh Chang Jae
Overview of methods for plant transformation
A. Delivery of DNA into a single plant cell→ transient assay
B. Integration of DNA into the plant cell genome
C. Regenerationof the transformed cell into a whole plant
In planta transformation with Agrobacterium
(Bechtold et al. 1993 )
Floral dipping method
(Bent et al., 1998)
- crown gall disease
- Ti plasmid : T-DNA + vir genes
( > 200 Kbp )
… cause disease primarily on dicots.
However, they can even infect gymnosperms,
not infectious on monocots, although transfer of the T-DNA into monocot can occur.
1. Tumor formation caused by the elevation of auxin and cytokinin levels by genes on a natural T-DNA
2. Opines synthesis
T-DNA used for scientific experimentation have usually been deleted of iaaM, iaaH, ipt and opine synthesis genes. → “ disarmed”
T-DNA transfer as mediated by vir gene functions
Plant transformation vector
Binary vector system
- Basic cloning vector (plant expression vector)
- plant transformation vector
Cointegrate vector system
- Cointegrate vector
- Disarmed Ti plasmid
General Vector composition
: Ti-plasmid right border (RB) + MCS + positive selectable marker + Ti-plasmid left border (LB)
Constitutve : 35S CaMV (Cauliflower mosaic virus)
Inducible : heat shock, glucocorticoid hormones, alcohol etc.
A plant promoter will often work in many different plant species, but yeast or human or bacterial promoter do not function.
Derived from CaMV, nopaline synthase gene etc.
√Common plant selectable marker : antibiotics , herbicides
Schematic diagram of binary vector
Basic cloning vector (pRT101)
P35S : CaMV 35S Promoter
pACaMV :CaMV polyadenylation site
ColE1 replication origin
bla ; Amp resistance gene
Plant transformation vector (pBIN20)