EXOGENOUS APPLICATION OF RNAS TO DISCOVER GENE FUNCTION Flashcards
When can RNA silencing occur?
Even after gene silencing!
How is gene silencing achieved?
By a stable or transient expression of genetic constructs that produce hpRNA
hpRNA
hairpin RNA
How do we discover gene expression?
By inhibiting the gene expression at a post transcriptional level
Reverse genetics
A way to find out gene function; it is based on phenotypical changes that can occur when we over or underexposes a gene
Plants own PTGS
It helps the plant to protect against viruses
How to we use a Plants own PTGS?
- we let DCL splice the dsRNA into sRNA and miRNA (21-24 of them)
- We add Argonaute containing silencing effector complexes
- it guides a sequence specific mRNA cleavage
- the silencing pathway starts
How does exogenous applied dsRNA enter a plant cell?
It enters through wounded or abraded surfaces -> moves away from the initial point of application
What is the best cell type to silence something? Endogenous ones or transgenes?
Transgenes!
How does a silencing signal move inside a plant?
Via vascular tissues from “the root to the shoot”
Vascular tissues
Vascular tissue is comprised of the xylem and the phloem, the main transport systems of plants.
Stable transformation
Used for the stable introduction of a gene into a plant- meaning the gene will be fully integrated in the host genome, so it is expressed continuously, and will also be expressed in later generations of the plant.
How do we use a stable transformation to silence RNA
We use siRNA generated constructs that activate the RNA silencing pathway
What is the problem with choosing stable transformations too silence RNAi?
It is labour intensive and time consuming
Genetic engineering via biolistic method
regeneration and selection of stable trans genetic plants; molecular analysis of the plants trans genetic state;
phenotypical analysis of plant generations
Agrobacteric method
By adding a silencing signal to the agrobacterium it gets spread in the plant organism
Agrobacterium
A pest that causes tumors in plants. It attacks the plant via genetic code
Transient transformation
Is short lived and won’t be expressed over multiple rounds of transformation
Is RNAi influenced by position effects in the genome when using transient transformation?
Nah miss gurl 🤗🙇🏻♀️🙇🏻♀️
What doesn’t RNAi when using transient transformation require?
Selection of a specific plant tissue 🧈🕴🏻
For what is RNAi using transient transformation applied for?
Producing a high amount of foreign proteins and gene silencing in PTGS
How can RNAi using transient transformation be obtained?
By infiltration of cells via Agrobacteria
How do molecules move from cell to cell in a plant organism?
Mostly via “plasmodesmata” or the vasculate
Vasculate
specialized conducting system that includes xylem and phloem
Plasmodesmata
Plasmodesmata (singular: plasmodesma) are microscopic channels which traverse the cell walls of plant cells
Cross kingdom definition
between interacting organisms
What can move cross kingdom in a plant?
sRNA
Does an exogenous application work better with endogenous genes or with trans genes?
With transgenes
How does dsRNA application work on a molecular level?
It gets processed by a dicer in the cell;
It produces siRNAs that are homologous to different positions of the target mRNA;
the RNAi complex recognises and cleaves the mRNA
