Note
Studied by 6 people(2)Genetic Engineering of Plants
Transgenic Plants
Reasons for development:
Improve agricultural, horticultural, or ornamental value.
Act as bioreactors.
Study gene action during growth.
Genetically determined traits that can be introduced:
Insecticidal activity, disease resistance.
Delay of senescence, environmental stress tolerance.
Altered flower pigmentation, improved nutritional quality, extended shelf life.
Production of therapeutic agents, polymers, antibody fragments.
Expression of viral antigenic determinants for edible vaccines.
Reduces plant breeding program durations.
Agrobacterium tumefaciens and Ti Plasmid
Gram-negative soil phytopathogen.
Transforms dicotyledonous plants by transferring T-DNA.
T-DNA contains genes for:
Plant hormone biosynthesis (auxin, cytokinin).
Opine synthesis.
Infection Mechanism:
Attachment to wound site.
Production of cellulose fibrils.
Induction of vir genes by plant phenolic compounds.
Transfer and integration of T-DNA into plant genome.
Ti Plasmid Components
vir genes: Essential for T-DNA transfer and integration.
T-DNA region: Bordered by right (RB) and left (LB) borders; contains genes for hormone and opine biosynthesis.
ori: Origin of replication.
Opine catabolism genes.
T-DNA Genes
Genes for auxin synthesis:
iaaM: tryptophan-2-monooxygenase (tryptophan to indole 3-acetamide).
iaaH: indole 3-acetamide hydrolase (indole 3-acetamide to indole acetic acid).
tmr/ipt: isopentenyltransferase (formation of cytokinins).
Opines
Unique condensation products (amino acid + keto acid or amino acid + sugar).
Octopine: arginine + pyruvic acid.
Nopaline: arginine + α-ketoglutaraldehyde.
Agropine: bicyclic sugar derivative of glutamic acid.
Ti Plasmid-Derived Vector Systems
Limitations of native Ti plasmid:
Phytohormone production, opine synthesis, large size, incompatibility with E. coli.
Modified Ti plasmids include:
Selectable marker gene (e.g., neomycin phosphotransferase for kanamycin resistance).
ori for replication in E. coli and A. tumefaciens.
Right border (RB) sequence.
Multiple cloning site.
Binary and Cointegrate Vector Systems
Binary Vector:
Shuttle vector active in both E. coli and A. tumefaciens.
A. tumefaciens contains a disarmed Ti plasmid with vir genes but lacking functional T-DNA.
Cointegrate Vector:
Contains plant selectable marker gene, target gene, RB, E. coli ori, and bacterial selectable marker gene.
Disarmed Ti plasmid lacks tumor-producing genes and RB of T-DNA.
Physical Methods of Gene Transfer
Microprojectile Bombardment (Biolistics):
Gold or tungsten particles coated with DNA.
Accelerated to high speeds using a particle gun.
Helium used as propelling force.
Extent of penetration controlled by gas pressure, distance, or particle size.
Chloroplast Engineering
Chloroplasts contain DNA and proteins encoded by both nuclear and chloroplast DNA.
High copy number of chloroplasts per cell and DNA copies per chloroplast.
Methods for introducing recombinant proteins:
Fusion gene with segments directing protein transport into chloroplast.
Direct insertion of recombinant gene into chloroplast DNA.
Secretion of Foreign Proteins
Production of therapeutics, antibody fragments, antibodies, and biopolymers.
Rhizosecretion: Secretion of foreign proteins through roots into hydroponic culture medium.
Oleosins
Oil body proteins in plant seeds.
Hydrophobic proteins embedded in oil droplets.
N- and C-terminal regions are hydrophilic.
Fusion with water-soluble proteins possible.
Cleavable linker used for recombinant protein recovery.
Stable accumulation in seeds.
Rhizosecretion Experiments
Testing secretion of proteins like xylanase, GFP, and human placental secreted alkaline phosphatase.
Efficient secretion requires a signal peptide upstream of the gene.
35S and mas2' promoters direct synthesis in root tissues.
35S promoter can be used for protein recovery from guttation fluid.
Glycosylation of Proteins
Plant genomes can be modified for glycosylation patterns similar to mammalian cells.
Transient Gene Expression
Rapid production of recombinant proteins without integration into plant DNA.
Genes exist stably in cytoplasm under strong expression signals (e.g., viral promoters).
High yield, rapid production, but not inherited.
Removing Marker Genes from Transgenic Plants
Concerns about toxicity, allergenicity, and transfer of antibiotic resistance.
Methods:
Introduce marker gene and gene of interest on separate plasmids, then separate by breeding.
Removing Marker Genes – Using A Transposase
Marker gene cloned between two plant transposable elements (Ds elements).
Transposase excises DNA between Ds elements.
Selectable marker moved to another chromosomal site and can be removed by breeding.
Removing Marker Genes – Using A Recombinase
Useful for woody, vegetatively propagated, or sterile plants.
Flank selectable marker with specific DNA sequences for removal from the genome.
Removing Marker Genes from Chloroplasts
Selectable bacterial gene (e.g., aadA) flanked by direct repeat sequences.
Gene excised by homologous recombination in the absence of selective pressure.
Totipotency in Transgenic Plants:
Totipotency is the ability of a single plant cell to differentiate and regenerate into a whole plant. This is crucial for generating transgenic plants because:
Transformation often occurs at the single-cell level (e.g., using Agrobacterium).
The transformed cell must be capable of developing into a complete, fertile plant.
Techniques like tissue culture rely on totipotency to regenerate plants from transformed cells or tissues.
Reasons for Generating Transgenic Plants:
Improve agricultural value (e.g., insect resistance).
Act as bioreactors (e.g., production of therapeutic agents).
Study gene action during growth and development.
Key Elements in Agrobacterium-Mediated Transformation:
Agrobacterium tumefaciens: A Gram-negative soil bacterium that naturally transforms dicotyledonous plants. It transfers a portion of its Ti (tumor-inducing) plasmid into the plant cell's genome.
Ti Plasmid: A large plasmid in A. tumefaciens that contains genes essential for the transformation process.
T-DNA (Transfer DNA): The region of the Ti plasmid that is transferred to the plant cell's genome. It contains genes for plant hormone biosynthesis (auxin, cytokinin) and opine synthesis.
Binary Vector: A shuttle vector active in both E. coli and A. tumefaciens. A. tumefaciens contains a disarmed Ti plasmid with vir genes but lacking functional T-DNA.
Cointegrate Vector: Contains plant selectable marker gene, target gene, RB, E. coli ori, and bacterial selectable marker gene. Disarmed Ti plasmid lacks tumor-producing genes and RB of T-DNA.
Disarmed Ti Plasmid: A modified Ti plasmid that has had its tumor-producing genes removed, making it safe for creating transgenic plants. It retains the necessary components for T-DNA transfer and integration.
vir (Virulence) Genes: Genes on the Ti plasmid essential for T-DNA transfer and integration into the plant genome. They are induced by plant phenolic compounds.
Opine: Unique condensation products of amino acids and keto acids or sugars, synthesized by enzymes encoded by T-DNA genes. They serve as a carbon and nitrogen source for Agrobacterium.
Auxin: A plant hormone that, when overproduced due to T-DNA genes (iaaM, iaaH), causes cell proliferation and tumor formation.
Cytokinin: Another plant hormone, overproduction of which (due to tmr/ipt gene) contributes to tumor formation in infected plants.
Opine Catabolism Genes: Genes that allow Agrobacterium to utilize opines as a nutrient source.
Microprojectile Bombardment (Biolistics):
A physical method of gene transfer where DNA-coated gold or tungsten particles are accelerated into plant cells using a particle gun.
Helium is used as the propelling force.
The extent of penetration is controlled by gas pressure, distance, and particle size.
Chloroplast Engineering:
Foreign DNA can be targeted to the chloroplast through:
Fusion genes with segments directing protein transport into the chloroplast.
Direct insertion of the recombinant gene into chloroplast DNA.
Reasons for targeting:
High copy number of chloroplasts per cell and DNA copies per chloroplast, leading to high levels of protein expression.
Rhizosecretion and Transient Gene Expression:
Rhizosecretion: The secretion of foreign proteins through roots into a hydroponic culture medium.
Transient Gene Expression: Rapid production of recombinant proteins without integration into plant DNA, where genes exist stably in the cytoplasm under strong expression signals (e.g., viral promoters).
ZMAP Example: High-yield, rapid production in tobacco plants, but not inherited.
Methods for Removing Marker Genes:
Transfect Plants with Marker and Gene of Interest Followed by Selective Breeding:
Separate marker gene and gene of interest by breeding.
Advantage: Simple.
Disadvantage: Time-consuming.
Use of a Transposase Followed by Selective Breeding:
Marker gene cloned between two plant transposable elements (Ds elements).
Transposase excises DNA between Ds elements.
Advantage: Can remove marker genes.
Disadvantage: Requires additional genetic elements.
Use of a Recombinase:
Flank selectable marker with specific DNA sequences for removal from the genome.
Advantage: Useful for woody, vegetatively propagated, or sterile plants.
Disadvantage: Requires specific DNA sequences and recombinase enzyme
Definitions:
Agrobacterium tumefaciens:
Gram-negative soil bacterium.
Naturally transforms dicotyledonous plants.
Transfers a portion of its Ti plasmid into the plant cell's genome.
Ti Plasmid:
A large plasmid in A. tumefaciens.
Contains genes essential for the transformation process.
vir (Virulence) Genes:
Genes on the Ti plasmid.
Essential for T-DNA transfer and integration into the plant genome.
Induced by plant phenolic compounds.
T-DNA (Transfer DNA):
The region of the Ti plasmid that is transferred to the plant cell's genome.
Contains genes for plant hormone biosynthesis (auxin, cytokinin) and opine synthesis.
Auxin:
A plant hormone.
Overproduction due to T-DNA genes (iaaM, iaaH) causes cell proliferation and tumor formation.
Cytokinin:
Another plant hormone.
Overproduction (due to tmr/ipt gene) contributes to tumor formation in infected plants.
Opine:
Unique condensation products of amino acids and keto acids or sugars.
Synthesized by enzymes encoded by T-DNA genes.
Serve as a carbon and nitrogen source for Agrobacterium.
Binary Vector:
A shuttle vector.
Active in both E. coli and A. tumefaciens.
A. tumefaciens contains a disarmed Ti plasmid with vir genes but lacking functional T-DNA.
Disarmed Ti Plasmid:
A modified Ti plasmid.
Tumor-producing genes have been removed, making it safe for creating transgenic plants.
Retains the necessary components for T-DNA transfer and integration.
Cointegrate Vector:
Contains plant selectable marker gene, target gene, RB, E. coli ori, and bacterial selectable marker gene.
Disarmed Ti plasmid lacks tumor-producing genes and RB of T-DNA.
Microprojectile Bombardment (Biolistics):
A physical method of gene transfer.
DNA-coated gold or tungsten particles are accelerated into plant cells using a particle gun.
Chloroplast:
Foreign DNA can be targeted to the chloroplast through:
Fusion genes with segments directing protein transport into the chloroplast.
Direct insertion of the recombinant gene into chloroplast DNA.
Rhizosecretion:
The secretion of foreign proteins