GM plants and animals II – GM Food Flashcards
Roundup-Ready soybeans
epsps (5-enolpyruvylshikimate-3-phosphate synthase) gene in plants is responsible for aromatic amino acid synthesis.
Glyphosate inhibits epsps gene
Epsps enzyme from Agrobacterium is resistant to glyphosate
Inserted into soybean chloroplasts using biolistics
Selected on glyphosate
Most GM plants have been created using one of two approaches:
the biolistic method or Agrobacterium tumefaciens - mediated transformation technology.
The biolistic method is a
physical method of introducing DNA into cells. Particles of heavy metals such as gold are coated with the DNA that will transform the cells; these particles are then fired at high speed into plant cells in vitro, using a device called a gene gun. Cells that survive the bombardment may take up the DNA-coated particles, and the DNA may migrate into the cell nucleus and integrate into a plant chromosome. Plants that grow from the bombarded cells are then selected for the desired phenotype
Golden rice 1
Created by expressing two transgenes, one from a bacterium and one from daffodil, in rice kernels.
Expressed low levels of b-carotene (±20% of daily requirement)
Golden rice 2
Swopped psy gene from daffodil for maize ortholog.
20X higher expression levels of b-carotene.
Currently in several field trials.
To use the Ti plasmid as a transformation vector, scientists remove the T-DNA segment and replace it with cloned DNA of the genes to be introduced into the plant cells. In order to have the newly introduced gene expressed in the plant, the gene must be cloned next to an appropriate promoter sequence that will direct transcription in the required plant tissue. For example, the beta-carotene pathway genes introduced into Golden Rice were cloned next to a promoter that directs transcription of the genes in the rice endosperm.
Agrobacterium tumefaciens is a
pathogen of woody dicot plants.
Agrobacterium tumefaciens Contains a
Ti-plasmid (tumor-inducing) that can effect natural horizontal gene transfer.
How does Agrobacterium tumefaciens work
Gene of interest (GOI) and marker gene inserted (with appropriate regulatory elements) in T-DNA region of Ti-plasmid.
Virulence genes mobilise T-DNA into nucleus of host cell.
Higher transformation rate than Biolistics
Random integration and low copy number
Negative (HygR) vs. positive selection (PMI)
negative selection involves use of a marker gene such as the hygromycin-resistance gene - plant cells that express the gene can survive on synthetic culture medium that contains hygromycin.
example of positive selection involves the use of a selectable marker gene such as that encoding phosphomannose isomerase (PMI) - Plant cells that express the pmi gene can survive on synthetic culture medium that contains only mannose as a carbon source.
Biolistics (biological ballistics)
is an example of direct DNA transfer.
How does Biolistics (biological ballistics) work?
Plasmid DNA containing GOI, marker gene, regulatory elements is precipitated onto heavy metal (gold or tungsten) microprojectiles.
Accelerated at high speed (high pressure Helium gas) into host cells.
Microprojectiles lodge in the nucleus, and may be incorporated into the genome during normal cell replication.
Random integration and high copy number.
Genome-editing allows for:
Different types of modifications to be made
Much faster methods
Much cheaper methods
Process vs. product-based trigger for regulation.
Meganucleases; ZFNs; TALENs and CRISPR/Cas
Why is genome editing not considered genetically modified (GM) by regulatory agencies in many countries.
Precise nucleotide or single-gene mutations or deletions without introducing foreign DNA into the organism.
Genome editing - the CRISPR/Cas immune system
Its natural!
