L6 - (l4 ang) RNA interference Flashcards
RNAi Definition
The introduction of double-stranded RNA into a cell to inhibit the expression of a gene
Also known as RNA silencing, inhibitory RNA, and RNA inactivation
Why Use RNAi for Pest Control?
• RNAi has the potential to be a very targeted method of crop protection vs invertebrate pests and pathogens
Success of RNAi technology depends on?
However success of the technology will depend on effective and acceptable delivery mechanisms
-> Need to demonstrate there are no/low “off- target” and “non-target” effects
Give an example of a study which targeted Detoxification Enzymes via RNAi?
- Targeting detoxification enzymes: Mao et al. 2007,
• The cotton bollworm’s antidote against one of the cotton plant’s natural insecticides (gossypol) is detoxified by a cytochrome P450 monooxygenase which is induced by gossypol itself, and appears to neutralize it
- Silencing of the gene encoding this protein by RNAi exposes the insect to the full force of the insecticide
- Larvae fed plant material expressing dsRNA specific to CYP6AE14 show lower transcript levels and retarded growth
Give an example of a study which targeted key cellular processes via RNAi?
Baum et al. 2007
Silence Key Genes in Insects
- Targeted a key metabolic enzyme V-ATPase
- Transgenic maize plants expressing a V-ATPase targeting dsRNA are protected from corn rootworm feeding damage
What results are wanted with dsRNA studies
dose-dependent reaction.
Increase in dsRNA doseage causes an increase in mrotality
The majority of RNAi methods attack what in the insect? Targeting what would be more specific?
CNS
iron channels in synapses esp NA
highly conserved across animal kingdom tho tho so little species specificity
Ca+ ion channels less conserved - greater species specificity
Most studies on RNAi attack what species?
Coleoptera (very sensitive to RNAi)
Lepidoptera - v hard
To test targetting Ca ion what was tested?
Tribolium castaneum (red flour beetle) Sodium channel para type (DmNav1) gene
Effects of dsRNA targeted to TcNav on development and survival: via injection?
- Significantly reduces larval survival and adult emergence in a dose-dependent manner
- 80% reduction in adult emergence 2 weeks post injection
TcNav gene expression post injection:
• Larvae:
• Pupae:
DW too much
• Larvae:
- Dose dependant reduction in TcNav gene expression
- Up to 60% knock down of gene expression at 160 ng/larva at 48 h
• Pupae:
- 30% knock down of gene expression at 160 ng/early-stage pupa at 48 h
- 42% knock down of gene expression at 160 ng/late-stage pupa at 48 h
TcNAV dsRNA feeding effects:
• Induced RNAi response in larvae
Dose dependant gene knockdown
Lethal phenotype
TcNAV Adult Emergence
• RNAi response propagated through pupation for both delivery methods
Overal what did the study using TcNAV dsRNA achieve?
- Provides proof of concept that a dsRNA-based biopesticide targeting Nav channels could be effective
- Highly specific – no homology to 744 insect Nav (MegaBLAST) held by NCBI
What dsRNA was used to target protein channels?
results showed?
what was done in regards to biosafety?
dsRNA Targetting sk
(Small conductance calcium-activated potassium channel (SK))
• Dose-dependent larval mortality in Tribolium castaneum (red flour beetle)
Biosafety: test effects on adult honey bees
Results:
• No significant effects on adult honey bees
• No significant effects on gene expression
