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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

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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. Regenerationof 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

( > 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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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

√T-DNA cassette

: Ti-plasmid right border (RB) + MCS + positive selectable marker + Ti-plasmid left border (LB)

√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.

√Terminator

Derived from CaMV, nopaline synthase gene etc.

√Common plant selectable marker : antibiotics , herbicides

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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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Plant transformation vector (pBIN20)

Kanamycine Resistance

NOS promoter

NOS terminator

Border

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