Nodulation Prac Flashcards
1
Q
visualisation
A
GFP-labelling
2
Q
Describe nodulation
A
- on Pisum sativum by Rhizobium leguminosarum single cell (competes)
- Pisum sativum releases flavonoids
- nod gene membrane-bound product detects flavonoids; activates nod operon
3
Q
nod operon
A
- synthesises lipo-oligosachharide
- detected by plant Lysm receptors
- initiates Sym
4
Q
Sym =
A
symbiosis pathway
5
Q
describe nodulation differentiation
A
- N-fixing bacteroids induce nif and fix expression: nitrogenase, metabolic genes
- N2 fixed to ammonia
- 10^9 pop
6
Q
Questions:
A
1) are different strains better at colonising?
2) do plants exhibit partner choice (how do plants respond to non-N2-fixing strains)
7
Q
Protocol:
A
1) compare morphologies of wet-preparation nodule- and lab- rhizobia
2) compare nodule size and abundance of wt strain
8
Q
Protocol specifics
A
1) crush 2-3 nodules of wt-infected w 100microlitres sterile water
2) transfer 20microlitres to compound microscopic slide + coverslip
3) view at 10x, 40x, and 100x (with immersion oil)
4) scientific drawing
9
Q
Competition for nodulation
A
- two strains: 3841 and UPM791
- inoculate under sterile conditions at different ratios (1:1, 1:10, 10:1)
- grow for 4 weeks
- add X-glu
- heat-treat at 70 degrees
- X-gal
- plot relative nodule occupancy against relative inoculation
10
Q
3841
A
- heat-stable b-galactosidase gene (celB) integrates into chromosome
- X-galactose = blue
11
Q
UPM791
A
- glucuronidase (gusA) gene integrated into chromosomes
- X-glucuronide
- stains pink
12
Q
Investigating partner choice
A
- based on N2 fixing efficiency
- 2 strains: 3481 wt, 3481 deltanifH
- inoculate at ratios 1:0, 1:1, 0:1
- count and plot nodule occupancy wrt inoculation ratio and conduct analysis of abundance, size and distribution
13
Q
3481deltanifH
A
no fixation; gusA marks pink
