Lecture 7 Flashcards
Planta Transformation
What is a Ti Plasmid?
A large (100-200 kb) plasmid found in Agrobacterium tumefaciens that enables the bacterium to transfer genes into plant cells, leading to tumor formation (galls).
What are the different regions of a Ti Plasmid?
- T-DNA (Transferred DNA)
- Vir (Virulence) Genes
- Opine Catabolism (Noc) Genes
- Ori (Origin of Replication)
What is the T-DNA region responsible for?
Contains genes responsible for tumor formation and opine biosynthesis.
What are oncogenes?
Genes that induce uncontrolled cell proliferation (gall formation).
What do opine synthesis genes encode?
Enzymes that produce rare amino acids (e.g., nopaline or octopine), serving as nutrients for Agrobacterium.
What are direct repeats in the context of T-DNA?
23 base pair sequences flanking T-DNA, essential for transfer.
What are Vir genes?
Essential for transferring T-DNA into plant cells; located outside the T-DNA region.
What activates Vir genes?
Phenolic compounds secreted by wounded plants, such as acetosyringone.
What is acetosyringone?
A phenolic compound released by wounded plants that activates Agrobacterium Vir genes.
What role do opine catabolism (Noc) genes play?
Enable Agrobacterium to metabolize opines as a nutrient source.
What is the function of the Ori region in a Ti Plasmid?
Allows plasmid replication in Agrobacterium.
Which part of the Ti Plasmid is necessary for plant transformation?
Vir genes are necessary for transferring T-DNA into the plant genome.
What do opines serve as for Agrobacterium?
A unique carbon and nitrogen source.
What is a shuttle vector?
A plasmid capable of replication in multiple host organisms.
What are the requirements for a shuttle vector?
- One origin of replication functional in E. coli (e.g., ColE1 ori)
- One origin of replication functional in Agrobacterium (e.g., pVS1 ori)
What is a gene of interest?
A gene that is integrated into the plant genome via T-DNA insertion.
How is T-DNA integrated into the plant genome?
Via illegitimate recombination.
What is a gene gun?
A physical method to introduce foreign DNA into plant cells.
How does the gene gun process work?
- DNA is coated onto gold or tungsten particles
- Particles are propelled using helium pressure
- DNA is delivered into the target cell
What is hydroponic culture?
A method of growing plants in nutrient solutions without soil.
What is rhizosecretion?
The secretion of proteins or small molecules into the surrounding medium via roots.
What are the advantages of using chloroplasts for foreign gene insertion?
- High gene expression levels
- Non-Mendelian inheritance
- Lower risk of transgene contamination in wild plants
Is chloroplast DNA circular or linear?
Circular.
How would you remove a selectable marker from the nuclear genome?
Using Cre-LoxP recombination or transposon-based strategies.
What selectable marker would you use for chloroplast transformation to avoid removal?
Betaine aldehyde dehydrogenase (BADH) gene.
