Midterm 2 - Notes 6 (Part 7) Flashcards
What are plant transformation goals?
All cells of a plant should contain the new construct
What is a problem in plant tranformation?
Only identical cells are being transformed to received the T-DNA
What is plant transformation?
Delivery of DNA to a single plant cell
- integration of DNA into the genome
- conversion of transformed cell into a whole plant
What are all plant cells?
Totipotent
Totipotent
Can regenerate complete plants when provided with nutrients and growth hormones
What do reporter gene constructs follow?
The organ, tissue and cell type of specific expression of YGI
What do reporter gene constructs isolate?
The promoter of YGI
What are 2 examples of a reporter gene?
- GUS
2. LUC
What does GUS encode?
A beta-glucuronidase that cleaves a colourless substrate (X-Gluc) to produce a blue precipitate
What does LUC encode?
A firefly enzyme (luciferase) that oxidizes luciferin and thereby emits light (luminescence)
What does YGI reporter gene fusion constructs follow?
The sub-cellular localization of YGI
What are the 5 steps involving YGI-reporter gene fusion constructs?
- Follow the sub-cellular localization of YGI
- Isolate the open reading frame of YGI
- Fuse it to a reporter gene
- GFP - Fuse construct to a constitutive promoter
- Visualize fluorescence in vivo using confocal laser microscopy
What results would you get?
Would get both expression, where it is normally expressed in sub cellular expression
What can over expressed constructs identify?
GOF and LOF
Can cDAN of YGI come from any organism?
Yes
What kind of marker is knock down constructs?
Always a selective marker between the LR and the RB
- located towards the IB
Where does T-DNA degration start?
At the RB and the LB is integrated last
- if you get selection marker present you can be sure the construct of integration is first and the selection marker follows that
What is the knock down constructs used for?
To abolish/reduce expression of YGI
What are the 5 steps in knock down constructs (anti-sense)?
- Isolate outer reading frame of YGI
- Clone it in inverse orientation in from of a promoter (anti-sense)
- Or clone parts of it twice in reverse orientation, separated by an intron (RNAi)
- Transform the plant of origin to YFG
- When transcribed, the transgene produces an anti-sense mRNA (complementary to the endogenous sense-mRNA from YGI)
What are 3 promoter choices for knock down constructs?
- Constitutive
- cauliflower - Inducible promoter
- YGI promoter
What are 3 promoter choices for over expression constructs?
- Constitutive
- cauliflower - Inducible promoter
- Tissue/cell style promoters
What is an example of transgenic plants used in food production?
Herbicide resistant plants
What are herbicides normally?
Non selective
- can kill both weed and crop plant
What do herbicides target?
Biological processes that is not important in other organisms groups
What are herbicides specific to?
Killing plants and not animals
When are herbicides usually applied?
Before crop plants germinate
What are 4 possibilities to engineer herbicide resistant plants?
- Inhibition of uptake
- Add protein that inactivates herbicide
- Over production target protein
- produce way more of the target protein so even though its being killed off there is still enough to function - Replace target protein with resistant variety **
What is an example of transgenic plants use in herbicide resistance?
Glyphosate resistant plants - roundup ready
What pathway is present in plants, bacteria and some fungi, but not animals?
Shikimate pathway
Why is shikimate pathway specific to plants?
Because the pathway does not exist in animals
- this is the reason the aromatic amino acids are essential when taking up our food
What does glyphosphate resistant plants lead to?
Biosynthesis of essential aromatic amino acids
What does glyphosate bind to?
Binds to the active site of ESPS synthase and inactivates it
What is the mutated ESPS synthase version from E.Coli insensitive to?
Glyphosate
- but still active
- does not inhibit the version of the enzyme
- allows them to easily create transgenic plants
