L13- Genetic Engineering Flashcards
What is Grown Gall disease caused by?
A naturally occurring soil bacteria- Agrobacterium
Where does it usually affect?
Near the base of the shoot at wound sites.
Why is Agrobacterium able to transform plants?
It can inject the T-DNA from a plasmid into the plant cell, which is incorporated into the plant genome.
How does the plant trigger Agrobacterium infection?
The plant is wounded, the wound releases a compound into the soil- “Acetysyringone”. The Vir A/G on the bacteria’s membranes detects the syringone.
How is the infection process initiated?
After the Vir A/G has detected the syringone, the bacteria moves by chemotaxis towards the wound site. Vir D + B make a bridge, physically joining the agrobacterium to the plant.
What is the plant’s defence response to the bacterium?
The plant’s plasma membrane has receptors for flagellum. When flagella are detected, transcription factors in the cytoplasm get phosphorylated. They then go into the nucleus and activate the DNA to transcribe defence proteins.
What is this transcription factor? When flagella are detected by the plant these transcirption factors get phosphorylated..
VIP1. Gets phosphorylated to VIP1-P
What does VirF do?
It contains everything needed to transport itself into the nucleus. Once in the nucleus it suppresses host defence by degrading VIP. VIP can no longer activate defence proteins being made.
How does T-DNA get into the nucleus?
By binding onto VIP, because it’s good at getting into the nucleus.
When T- DNA is integrated into the plant’s DNA, what is made?
- Auxin and cytokinin are made- which promote tumour formation in the infected plant cells.
- Opines are made- which are amino acids that the bacteria can metabolize but the plant can’t
What does the engineered T-DNA look like?
Instead of one of the genes for hormones and opines in the normal wild type t dna. Now there’s an antibiotic resistance gene instead.
How do you engineer the T- DNA region?
- Remove the genes for the hormones and opine biosynthesis.
- Replace with whatever gene you want.
- > only the region between the T-DNA borders gets transferred.
In the binary vector systems- you can put the T-DNA region on a small plasmid instead. (binary vector). What is needed?
All plasmids need on origin of replication. (and the wanted genes)
What’s the 1st step of plant transformation?
Co-cultivation. Mimick wounding by cutting discs from a leaf and putting them in an agar plate. With the agrobacterium carrying the binary vector. With the wanted gene and antibiotic resistance.
What’s the 2nd step of plant transformation?
Selection. Need to be able to select the transfered cells from untransformed. Usually have selectable marker, e.g. antibiotic resistance. Put the discs into selective environment. See which have uptaken the binary vector.
