Plant Genome Manipulation Flashcards
What is Genetic Transformation?
Introduction of genes into genomes by means other than gamete fusion or somatic cells
How does transformation differ from sexual reproduction?
DNA can be introduced from unrelated sources, and can introduce single genes other than whole genomes
What is transient transformation?
DNA has not been integrated into the genome and so may be lost when cell divides
DNA integrated into genome will be passed down by meiosis and mitosis
What is crown gall disease?
Tumour like growth often caused by insects at the crown of the plant (where root meets stem)
Caused by soil bacterium Agrobacterium tumefaciens which detects and infects wounded cells (common at crown where bending occurs)
What causes tumour production?
Agrobacterium alters the host cells by introducing factors into them
Tumours become independent of bacteria presence after long enough (4 or so days?)
High levels of IAA (Auxin) and CK (cytokinin) in gall tissues suggest that the cells have acquired the capacity for autonomous growth by permanent activation of growth substance synthesising systems
What is the difference between culturing normal plant tissue and Gall tissue?
Normal plant tissue need exogenous factors to live indefinitely in medium
Gall tissue does not
What unusual metabolites are present in the gall tissue of Jerusalem artichoke
Octopine and Nopaline
What metabolites are found in all gall tissues?
Opines (Arginine derivatives)
Different opines depending on agrobacterium strain
What is the effect of opine production on the plant cell?
Opines can’t be metabolised by the plant only the bacterium
Locks away nutrition from the plant cells for bacterial nutrition
What causes the Agrobacterium to be virulent?
The tumour inducing (Ti) plasmid
This plasmid is absent from avirulent strains (cannot form tumours)
Transferring the plasmid to Avr strain transfers virulence
Ti plasmid is present in DNA from tumour cells
What is the structure of the Ti plasmid?
T-DNA (transfer DNA)
T-DNA is flanked by 2 25bp repeat border sequences
Virulence genes which are required for T-DNA movement into plant cell
What induces Vir gene expression?
Small plant derived molecules such as Acetosyringone
Acetosyringone likely perceived by VirA protein encoded on Ti plasmid
VirA and VirG induce other vir genes in response to plant signals
How is the T-DNA transferred?
By a multi-subunit type IV secretion system (secretion through a protein complex that traverses the cell envelope) made up of different vir gene products
What does opine production induce in the Agrobacterium?
Expression of genes required for opine uptake and catabolism (permeate and hydrolase enzymes)
Why is tumour formation beneficial for the bacterium?
More cells producing opines?
How does the T-DNA region produce tumours?
Contains genes for auxin and cytokinin production
Helps to induce tissue growth and tumours
What industrial use does Agrobacterium have?
As a vector for plant transformation
Modify Ti plasmid to make it non-oncogenic and non opine producing
Regenerate transgenic plant from transformed cells to produce full plant
How can the altered plasmid be put into the Agrobacterium?
Electroporation
Conjugation