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Plant Expression System

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

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  1. Plant Expression System Molecular Biology Laboratory Oh Chang Jae

  2. 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

  3. In planta transformation with Agrobacterium Vacuum infiltration (Bechtold et al. 1993 ) Floral dipping method (Bent et al., 1998) Co-cultivation with Agrobacterium

  4. Agrobacterium tumefaciens - crown gall disease - Ti plasmid : T-DNA + vir genes ( > 200 Kbp )

  5. 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”

  6. T-DNA transfer as mediated by vir gene functions

  7. Plant transformation vector Binary vector system - Basic cloning vector (plant expression vector) - plant transformation vector Cointegrate vector system - Cointegrate vector - Disarmed Ti plasmid

  8. 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

  9. Schematic diagram of binary vector

  10. Basic cloning vector (pRT101) P35S : CaMV 35S Promoter pACaMV :CaMV polyadenylation site ColE1 replication origin bla ; Amp resistance gene

  11. Plant transformation vector (pBIN20) Kanamycine Resistance NOS promoter NOS terminator Border

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