Midterm 2 - Notes 6 (Part 7)

Question 1

What are plant transformation goals?

Answer 1

All cells of a plant should contain the new construct

Question 2

What is a problem in plant tranformation?

Answer 2

Only identical cells are being transformed to received the T-DNA

Question 3

What is plant transformation?

Answer 3

Delivery of DNA to a single plant cell

• integration of DNA into the genome
• conversion of transformed cell into a whole plant

Question 4

What are all plant cells?

Answer 4

Totipotent

Question 5

Totipotent

Answer 5

Can regenerate complete plants when provided with nutrients and growth hormones

Question 6

What do reporter gene constructs follow?

Answer 6

The organ, tissue and cell type of specific expression of YGI

Question 7

What do reporter gene constructs isolate?

Answer 7

The promoter of YGI

Question 8

What are 2 examples of a reporter gene?

Answer 8

1. GUS

2. LUC

Question 9

What does GUS encode?

Answer 9

A beta-glucuronidase that cleaves a colourless substrate (X-Gluc) to produce a blue precipitate

Question 10

What does LUC encode?

Answer 10

A firefly enzyme (luciferase) that oxidizes luciferin and thereby emits light (luminescence)

Question 11

What does YGI reporter gene fusion constructs follow?

Answer 11

The sub-cellular localization of YGI

Question 12

What are the 5 steps involving YGI-reporter gene fusion constructs?

Answer 12

1. Follow the sub-cellular localization of YGI
2. Isolate the open reading frame of YGI
3. Fuse it to a reporter gene
   - GFP
4. Fuse construct to a constitutive promoter
5. Visualize fluorescence in vivo using confocal laser microscopy

Question 13

What results would you get?

Answer 13

Would get both expression, where it is normally expressed in sub cellular expression

Question 14

What can over expressed constructs identify?

Answer 14

GOF and LOF

Question 15

Can cDAN of YGI come from any organism?

Answer 15

Yes

Question 16

What kind of marker is knock down constructs?

Answer 16

Always a selective marker between the LR and the RB

- located towards the IB

Question 17

Where does T-DNA degration start?

Answer 17

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

Question 18

What is the knock down constructs used for?

Answer 18

To abolish/reduce expression of YGI

Question 19

What are the 5 steps in knock down constructs (anti-sense)?

Answer 19

1. Isolate outer reading frame of YGI
2. Clone it in inverse orientation in from of a promoter (anti-sense)
3. Or clone parts of it twice in reverse orientation, separated by an intron (RNAi)
4. Transform the plant of origin to YFG
5. When transcribed, the transgene produces an anti-sense mRNA (complementary to the endogenous sense-mRNA from YGI)

Question 20

What are 3 promoter choices for knock down constructs?

Answer 20

1. Constitutive
   - cauliflower
2. Inducible promoter
3. YGI promoter

Question 21

What are 3 promoter choices for over expression constructs?

Answer 21

1. Constitutive
   - cauliflower
2. Inducible promoter
3. Tissue/cell style promoters

Question 22

What is an example of transgenic plants used in food production?

Answer 22

Herbicide resistant plants

Question 23

What are herbicides normally?

Answer 23

Non selective

- can kill both weed and crop plant

Question 24

What do herbicides target?

Answer 24

Biological processes that is not important in other organisms groups

Question 25

What are herbicides specific to?

Answer 25

Killing plants and not animals

Question 26

When are herbicides usually applied?

Answer 26

Before crop plants germinate

Question 27

What are 4 possibilities to engineer herbicide resistant plants?

Answer 27

1. Inhibition of uptake 2. Add protein that inactivates herbicide 3. Over production target protein - produce way more of the target protein so even though its being killed off there is still enough to function 4. Replace target protein with resistant variety **

Question 28

What is an example of transgenic plants use in herbicide resistance?

Answer 28

Glyphosate resistant plants - roundup ready

Question 29

What pathway is present in plants, bacteria and some fungi, but not animals?

Answer 29

Shikimate pathway

Question 30

Why is shikimate pathway specific to plants?

Answer 30

Because the pathway does not exist in animals | - this is the reason the aromatic amino acids are essential when taking up our food

Question 31

What does glyphosphate resistant plants lead to?

Answer 31

Biosynthesis of essential aromatic amino acids

Question 32

What does glyphosate bind to?

Answer 32

Binds to the active site of ESPS synthase and inactivates it

Question 33

What is the mutated ESPS synthase version from E.Coli insensitive to?

Answer 33

Glyphosate - but still active - does not inhibit the version of the enzyme - allows them to easily create transgenic plants