Nodulation Prac Flashcards

1
Q

visualisation

A

GFP-labelling

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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
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3
Q

nod operon

A
  • synthesises lipo-oligosachharide
  • detected by plant Lysm receptors
  • initiates Sym
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4
Q

Sym =

A

symbiosis pathway

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5
Q

describe nodulation differentiation

A
  • N-fixing bacteroids induce nif and fix expression: nitrogenase, metabolic genes
  • N2 fixed to ammonia
  • 10^9 pop
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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)

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7
Q

Protocol:

A

1) compare morphologies of wet-preparation nodule- and lab- rhizobia
2) compare nodule size and abundance of wt strain

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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

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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
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10
Q

3841

A
  • heat-stable b-galactosidase gene (celB) integrates into chromosome
    • X-galactose = blue
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11
Q

UPM791

A
  • glucuronidase (gusA) gene integrated into chromosomes
  • X-glucuronide
  • stains pink
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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
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13
Q

3481deltanifH

A

no fixation; gusA marks pink

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