lecture 13 Flashcards
non bacterial ways of transforming plants (cont.) research applications of transgenic plants
1
Q
What are plastids?
A
- Plastids are a major organelle in plant and algal cells
- they are semi-autonomous with their own genetic material
- plastids are capable of differentiating into various types
- some store large amounts of starch (amyloplasts)
- others are photosynthetic (chloroplasts)
2
Q
Where is the second genome of animals cells found? How is this important?
A
- in the mitochondria
- it means that you could also transform the mitochondria
3
Q
How many genomes do plants have?
A
3:
- nuclear genome
- mitochondrial genome
- plastid genome (e.g. chloroplast)
4
Q
Why bother putting genes into a plastid?
A
- plastids are maternally inherited
- some proteins can be toxic in the cytoplasm
- proteins/polymers can accumulate to high levels (it’s what they usually do)
- integration is by homologous recombination
5
Q
What is the plastid genome?
A
- Ds DNA 120-220 kb circular and linear forms
- up to 10,000 copies per cell
- ~120 genes
- 20-30 kb inverted repeats (IRa and IRb) separate the large and small single copy regions (LSC and SSC)
6
Q
What experiment was performed with unicellular alga?
A
- Chlamydomonas has a single large chloroplast
- using biolistics, a non-photosynthetic Chlamydomonas mutant was transformed with an ATPase subunit gene
- a high frequency of direct homologous replacement events in the chloroplast was found but there was no integration of plasmid sequences
7
Q
What is homologous recombination in plastids?
A
- complete replacement of the target gene with the replacement gene
- the only way you can integrate DNA in a plastid
8
Q
What is the standard design of transformation construct?
A
- the standard design of a transformation construct has two regions of plastid DNA surrounding the gene of interest and a spectinomycin resistance gene (aadA)
- don’t need the shoulder regions
9
Q
Can flowering plant plastids be transformed?
A
- yes, flowering plant plastids can also be transformed
10
Q
How do you get the transformation construct into the cell?
A
- put it onto the beads
- load them into the gun
- fire up the gun e.g. helium gun
- accelerates disc which hits stopping plate and small pellets come through and into the tissue
11
Q
What is heteroplasmy?
A
- a plant cell can have have hundreds of chloroplasts
- for transformation to occur, a DNA-coated particle has to enter a chloroplast
- in a typical experiment, only one or two chloroplasts will have such particles
- this results in a situation called “heteroplasmy”
- this is the presence of two or more different plastid genomes in a cell
12
Q
What is the problem of heteroplasmy?
A
- heteroplasmic situations are genetically unstable and usually resolve spontaneously (‘homoplasmy’)
- this sorting-out is due to random genome segregation upon plastid division, as well as random plastid segregation upon cell division
- 50-100 plastids/cell, 50-100 genomes/plastid
- primary event changes a single plastid genome
- cell/organelle division under spectinomycin selection — ptDNA is heteroplasmic
- Regenerated plant reselected on spectinomycin — ptDNA is homoplasmic
- go through rounds of selection with spectinomycin until you eventually have a homoplasmic plant
- can use GFP reporter
13
Q
What is a biotechnology application of the Bt toxin?
A
- Dipel
- Bt toxins are insecticidal proteins from the Gram positive bacteria Bacillus thuringiensis
- proteins found as crystal inclusions in sporulated bacteria
- potent toxins of specific classes of insects
- Lepidoptera - butterflies and moths
- Hymenoptera - ants, bees, wasps, etc.
- Coleoptera - beetles and weevils
- Early biotech interest in expressing Bt toxins in plants
14
Q
What were some expression problems with the Bt toxin?
A
- Agrobacterium-mediated transformation used to introduce Bt toxins into plants
- Poor levels of insect resistance because of low protein expression
- Re-engineering the Bt gene increased insect resistance to acceptable levels
- Protein expression increased from barely detectable (WT protein) to <100 ng per 50µg total protein (0.2%)
- preferences for G+C in plants - increased numbers in re-engineered version - changing codons without changing amino acid (redundancy)
15
Q
How were these problems with the Bt toxin overcome?
A
- McBride et al (1995) expressed WT Bt toxin in plastids
- Bt toxin represented 3-5% of total leaf protein
- High levels of insect resistance and no adverse effects on plant growth
- Plastid transformation attracts considerable attention for high level production of foreign proteins
16
Q
How can we use viruses to deliver genes?
A
- viral nucleic acids are infective, can cross cell walls, and can spread from cell-to-cell
- most plant viruses have ssRNA genomes that serve as mRNA without further transcription
17
Q
What is potato virus X?
A
- potato virus X (PVX) is a widespread virus, with symptoms ranging from mild leaf mottling to a severe mosaic
- however symptoms are often not visible
- the ssRNA genome is 6.4kb long and encodes an RNA-dependent RNA polymerase (RdRp), a coat protein (CP) and 3 movement proteins (MP1-3)
18
Q
How do we take advantage of PVX ?
A
- cDNA inserts can be placed between MP3 and CP genes
- in vitro transcription of the DNA from a bacterial promoter produces infective RNA, which can be rubbed onto leaves with a little ground glass
- this leads to transient but systemic expression of the cDNA-encoded protein
- size limitations (1-2kb)
why not incorporate PVX into a T-DNA?
- can put the entire virus inside a T-DNA
- enters into the nucleus
- using Agrobacterium to deliver viral T-DNAs increases the number of infected cells and improves the yield of protein
- you want to get translation to occur and harvest the plant before its natural defences occur
19
Q
How can plants act as factories for recombinant protein production?
A
- Depending on the protein, yields of up to 0.5-5g recombinant protein per kg leaf biomass have been achieved
a) growing plants and bacteria
b) agroinfiltration
c) plant incubation
d) biomass harvest
e) extraction
f) protein purification
20
Q
Why use plants as protein factories?
A
- technology in hand
- simple, inexpensive means of producing complex proteins
- elimates the risk of contamination with animal pathogens (animal ethics)
- heat stable environment
21
Q
What are some Agrobacterium-mediated transformation research application?
A
- what Agrobacterium can do
- T-DNA ‘plug-n-play’
- promoter trapping
- GAL4 trapping
- cytokinins and root development
22
Q
What can Agrobacterium do?
A
- transfer T-DNA to region to a eukaryotic cell
- get T-DNA into the nucleus
- T-DNA genes may be expressed without integration (transient transformation)
- T-DNA may be integrated randomly into genome (stable transformation)
23
Q
What is in the plant molecular biologists’ toolkit?
A
Using Agrobacterium you can:
- add new functions to an organism (e.g. herbicide resistance to soybeans)
- remove an existing function (e.g. FlavrSavr tomato)
- express proteins transiently in mature organs (e.g. leaf)
24
Q
What does a “typical” gene have?
A
- three modular parts
- each has its own separate and largely independent function
- promoter: regulates the expression of a nearby gene
- coding region: will be expressed if near a promoter
- 3’ UTR
- Non-Coding DNA isn’t expressed
25
Q
What is T-DNA plug-n-play?
A
- right and left border
- if you want to add a function: promoter driving function of a coding region both of which are inserted in T-DNA, also a stop sequences (3’ UTR)
- left and right border + promoter –> random integration so if it lands near something it will change the way it is expressed
- coding region + stop signal = if it happens to land near promoter plant will express coding region according to that promoter
- adding NC DNA may inactivate something
26
Q
What is promoter trapping?
A
- randomly insert a promoterless Kan^r gene into a plant genome and screen for kanamycin resistant calli
- has to be inserted in cells undergoing division
- to be expressed, the promoterless Kan^r gene must insert near a promoter that is activate in calli
- region around where there is active transcription occurring is a good site for T-DNAs to insert into them - conformation of the T-DNA allows that to happen better than the conformation of DNA in genes that are inactive
- T-DNAs seem to preferentially insert next to genes that are being transcribed (evidenced by the fact that inserting a fully functional gene vs a promoterless gene has little impact on the number of cells that are transformed, [5 vs 10 in 10,000])
