13. transforming plants Flashcards
1
Q
What is the meaning of genetic engineering/recombinant dna technology/genetic modification/manipulation (GM)? 1
A
- involves the direct manipulation of an organism’s genes
2
Q
What is a genetically modified organism (MGO)? 1
A
- an organism whose genetic material has been altered using genetic engineering tehchniques
3
Q
What does transgenic mean? 1
A
- Genes from one organism in another
4
Q
What does cisgenic mean? 2
A
- Swapping order of genes to change behaviour
2. debate over whether this is really gm
5
Q
Whnat is crown gall disease? 5
A
- found on a wide variety of plant species eg. rosebush
- caused by naturally occurring soil bacteria, agrobacterium, which genetically engineer the plant to grow nodules
- several related species, but agrobacterium tumefaciens most widely studied
- usually affects near the base of shoot at wound sites. some proliferate root growth
- natural crop pathogen, generally of little economic importance
6
Q
How does an Agrobacterium infection result in crown gall disease? 6
A
- has plasmid with vir genes (virulence genes) and t-dna region, the part that’s transferred
- something of a host-pathogen arms race, pathogens break down defenses, host evolved to resist them. up to 10% of genes suppress defenses
- Plant wounds secrete a molecule called acetosyringone to the soil
- agrobacteria use this to move towards wounded plants by chemotaxes
- VirA/G is always transcribed by the bacterium
- A typeT pilus forms a connection between host and bacterium so genes can be passed through to host cell
7
Q
How do plants respond to an agrobacterium infection? 5
A
- flagellin in type t pilus is highly conserved
- the host plant has receptors for it
- binding triggers a phosphorylation cascade, where a transcription factor in the cytoplasm is phosphorylated
- the transcription factor enters the nucleus and transcription of defense genes begins
- these can kill the bacterium
8
Q
What is the role of VirF in crown gall disease? 5
A
- virf is a virulence protein that contains everything needed to transport the disease into the nucleus
- a single stranded copy of the disease plasmid is made inside the host
- this binds to vip1 plant protein, which moved into nucleus
- once in the nucleus, virf degrades the VIP1, which is needed to transcribe the defense genes
- the plasmid is then integrated into the genome by an inknown mehcanism
9
Q
What happens in crown gall disease after T-DNA integration? 5
A
- t-dna makes plant produce auxin and cytokinin
- these cause production of galls for bacteria to live in
- opines are formed, which the plants can’t use - an unusualy form of amino acids
- bacteria use them for themselves to grow
- in wild type t-dna, anything between the boarders of LB (left border) and RB (right boarder), incl genes for production of hormones and opines, will be transcribed
10
Q
Hoe can we exploit the t-dna region? 3
A
- the genes promoting tumours are not useful, so we remove hormone and opine genes
- replace with a selective marker, usually antibiotic resistance
- also add gene for whatever it is we want plant to do
11
Q
What are binary vector systems? 5
A
- in the lab, we use several useful plasmids, with useful restriction sites that occur once and about 3000base pairs
- the t-dna plasmid is 2000kbp, much too big for easy manipulation
- we need the origin of replication and virulence genes transporting to a helper pplasmid of 150kbp
- after this, we transfer the origin replication, along wtih selectable marker and desired genes, to a 10kb binary vector
- much easier to handle
12
Q
What is co-cultivation? 3
A
- we minoc wounding by cutting discs out of leaves
- these are placed in an agar plate
- agrobacterium carrying the binary vector are added
13
Q
How do we select which cells have been transformed when genetically engineering plants? 6
A
- transformation is inefficient, begin with billions and end up with transforming 1-2
- need to be able to select one transformed cell from many untransformed
- selectable markers used eg. kanamycin, an antibiotic with no medical use
- eg. basta-herbicide with bacterial targets that are conserved in chloroplats
- the bacteria with the antibiotic resistant marker gene survive, and the others don’t
- genes used inlc. neomycin phophototransferase or phosphinothriain acetly (np22 or bar PAT)
14
Q
How do we regenerate a plant from one transformed cell? 5
A
- many plant cells are totipotent
- can manipulate plant cell division using endogenous plant growth regulators
- a higher cytokinin to auxin ratio can turn a callous to a plant structure
- under the correct conditions, cells are able to irganise themselves to form the shoot meristem
- once a shoot has formed, rooting is easy eg. use of cuttings
15
Q
What is arabidopsis? 3
A
- ovules in flowers are the targets of agrobacterium transformation using flower dipping methods/method in planta
- when the flower is dipped, the tiny agrobacterium genome is sucked into intracellular spaces
- agrobacterium then affects germline and we’re not sure how
