HAU16008-02-2 - The Mechanism of Plant Transformation...

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1 The Mechanism of Plant Transformation Lecture 2 October 28, 2008 Core Concepts : Broad host range plasmid Chimeric genes Selectable marker genes Scorable marker genes Disarmed Agrobacterium strains Cointegrating vectors Binary vectors Multiplication/amplification of DNA molecules. Plasmid vectors -Narrow host range vectors -Broad host range vectors Cloning and Genetic selection -Cloning DNA fragments -Transformation of bacteria -Antibiotic selection Plasmids are autonomous replicating units capable of replicating in a limited (narrow host range) number of bacteria or broad (broad host range) range of different gram -ve bacteria by virtue of their replication regions. They also have well defined maps and many single restriction enzyme sites
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2 Antibiotic resistance genes found on many plasmids encode resistance to antibiotics. Antibiotics have a selective cellular target and can either kill (bacteriocidal) or stop the growth (bacteriostatic) of bacteria. Chimeric genes are functional genes that have been constructed by joining segments from genes obtained from different sources. Chimeric genes. Basic anatomy of a gene -Structural regions -5' and 3' regulatory regions Disarmed vectors are Ti-plasmids that contain modified T-DNA regions. The tumor forming genes (oncogenes; i.e. those responsible for auxin and cytokinin biosynthesis) have been removed.
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3 A binary system is one in which the T- DNA and vir -region are on different plasmids in the same bacteria. In a cointegrating system the plasmid containing the T-DNA region is integrated into the plasmid (Ti or Disarmed-Ti) containing the vir -region via homologous recombination (due to a region of homology between the Ti and plasmid containing the T-DNA). Binary Cointegrating Agrobacterium vectors. Disarmed Ti plasmid -Cointegrating vector -Binary vector Genetic selection is the process of selecting preferentially for those cells that have been transformed by the incoming T-DNA. A selective advantage can be conferred upon the transformed cells through the introduction of genes encoding antibiotic resistance or resistance to some metabolic inhibitor such as a herbicide for example. In the presence of the antibiotic or herbicide, the untransformed cells die whereas the transformed cells grow and multiply. Nutrient selection (mannitol utilization) Marker/foreign genes for selection/screening Chimeric reporter genes Selectable Antibiotic resistance(APH3'II, HPH) Herbicide tolerance (BAR) Nutrient selection (MTL) Scorable/reporter Tumor/root Opine synthesis (NOS, OCS) Enzymatic (GUS, LUC) Non-enzymatic, non-destructive (GFP) Marker Free Transformation Excision of selectable markers (cre-lox) Regeneration stimulating genes Green Fluorscent Protein (GFP) CAT A scorable marker gene allows the visual detection of transformed cel s, tissues or plants through the expression of genes normally not found within the host plant's genome without necessarily imposing any form of selection. These genes usually encode an enzyme/protein that is readily detectable itself (GFP) or through the use of chromogenic, fluorogenic, photon emitting or radioactive substrates (GUS, LUC, LAC, CAT etc).
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HAU16008-02-2 - The Mechanism of Plant Transformation...

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