Plant Expression System

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Plant Expression System
Molecular Biology Laboratory Oh Chang Jae
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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. Regeneration of the
transformed cell into a whole plant
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In planta transformation with Agrobacterium
Vacuum infiltration (Bechtold et al. 1993
) Floral dipping method (Bent et al.,
1998) Co-cultivation
with Agrobacterium
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Agrobacterium tumefaciens
- crown gall disease
- Ti plasmid T-DNA vir genes
( gt 200 Kbp )
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Host Range 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.
Pathogenesis
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
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T-DNA transfer as mediated by vir gene functions
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Plant transformation vector
Binary vector system - Basic cloning vector
(plant expression vector) - plant
transformation vector
Cointegrate vector system - Cointegrate
vector - Disarmed Ti plasmid
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General Vector composition
v T-DNA cassette
Ti-plasmid right border (RB) MCS positive
selectable marker Ti-plasmid left border (LB)
v Promoter
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.
v Terminator
Derived from CaMV, nopaline synthase gene etc.
v Common plant selectable marker antibiotics ,
herbicides
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Schematic diagram of binary vector
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Basic cloning vector (pRT101)
P35S CaMV 35S Promoter
pACaMV CaMV polyadenylation site
ColE1 replication origin
bla Amp resistance gene
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(No Transcript)
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Plant transformation vector (pBIN20)
Kanamycine Resistance
NOS promoter
NOS terminator
Border