7/29/2019 Mutants and Their Uses - Pick Kuen
1/21
Mutants: Their Uses
Pick Kuen Chan
7/29/2019 Mutants and Their Uses - Pick Kuen
2/21
What is a Mutation? permanent change in the DNA sequence
Range from single DNA base to a largesegment of a chromosome
7/29/2019 Mutants and Their Uses - Pick Kuen
3/21
What Causes DNA Mutations? DNA damage from environmental agents
- ultraviolet light (sunshine)
- nuclear radiation
- certain chemicals
Mistakes that occur when a cell copies itsDNA in preparation for cell division
7/29/2019 Mutants and Their Uses - Pick Kuen
4/21
Types of Mutations Point mutations
single nucleotide base changes in a gene's DNA
sequence- Missense mutations
- Nonsense mutations
- Silent mutations
Insertion and deletion mutationsadd or remove one or more DNA bases. Insertion and
deletion mutations cause frameshift mutations
7/29/2019 Mutants and Their Uses - Pick Kuen
5/21
The Central Dogma of Biology
Transcription
Translation
DNA
RNA
Protein
7/29/2019 Mutants and Their Uses - Pick Kuen
6/21
Genome to Gene Function
Genome sequence
Gene discovery
Gene functional annotation In silico prediction
Gene expression Protein-protein interaction
Mutant analysis
7/29/2019 Mutants and Their Uses - Pick Kuen
7/21
Mutant Analysis Known as forward genetics
Phenotype-centric process
Reliable means to assign gene function
Enormous effort to identify each generesponsible for a particular phenotype
7/29/2019 Mutants and Their Uses - Pick Kuen
8/21
Mutant Analysis
Reverse genetics
Prediction of function based on DNA
sequence
Required large collections gene-indexed
mutation
7/29/2019 Mutants and Their Uses - Pick Kuen
9/21
Mutant Analysis
Forward Genetics
Select a biological process
Generate highly redundant
mutant populations
Screen for mutants with adesired phenotype
Map and clone the generesponsible for the phenotype
Reverse Genetics
Select a gene or genes ofinterest
Generate highly redundantmutant populations
Develop and conductsequence-based mutantscreens
Analyse the phenotype ofthe mutants
Alfonso and Ecker, 2006
7/29/2019 Mutants and Their Uses - Pick Kuen
10/21
Generation of Mutant Population Chemical agents
Ethyl methanesulphonate (EMS) andnitrosomethylurea (NMU)
Physical agents Fast neutrons, X-ray and carbon ions
Biological agents Transposons and transferred-DNA (T-DNA)
7/29/2019 Mutants and Their Uses - Pick Kuen
11/21
EMS Mutagenesis Efficient mutagens in plant
- Soybean, pea, Lotus japonicus,Medicago truncatula
Generate a broad range of DNA alteration
- Point mutation
Unbiased distribution of mutation
7/29/2019 Mutants and Their Uses - Pick Kuen
12/21
Wild type nts1007
EMS
Supernodulation mutant
7/29/2019 Mutants and Their Uses - Pick Kuen
13/21
Ethylene insensitive mutants
Wild type
Skl
Penmetsa et al., 2003
7/29/2019 Mutants and Their Uses - Pick Kuen
14/21
Fast Neutron Mutagenesis Intermediate efficiency
Produce double stranded DNA breaks thatare then repaired
DNA alteration
- Big chromosomal insertions, deletionsand rearrangement
7/29/2019 Mutants and Their Uses - Pick Kuen
15/21
Examples
wildtype
ga1-3mutant
transgenicga1-3
Sun et al., 1992
FN37 wild type
Men et al., 2002
7/29/2019 Mutants and Their Uses - Pick Kuen
16/21
Biological agents DNA or RNA molecules as mutagen
Introduction of engineered DNA/RNAmolecule into living cell.
- Agrobacterium tumefaciens as shuttle
- Plant-associated bacteria
- DNA/RNA viruses
- Particle bombardment
7/29/2019 Mutants and Their Uses - Pick Kuen
17/21
Application in Plant Transformatiom
Insertional mutagenesis
Ectopic expression
Gene silencing
Homologous recombination
7/29/2019 Mutants and Their Uses - Pick Kuen
18/21
Criteria for T-DNA Selectable marker
- Herbicide resistance gene, antibioticresistance gene
Reporter genes
- GFP, -glucuronidase (GUS), luciferase
Gene/s of interest
7/29/2019 Mutants and Their Uses - Pick Kuen
19/21
Schematic Diagram on
Transformation
7/29/2019 Mutants and Their Uses - Pick Kuen
20/21
Examples
Ottenschlger et al., 2003
7/29/2019 Mutants and Their Uses - Pick Kuen
21/21
Workshop on Mycorhizal