BIO2015: Lecture 9

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  • Created by: LMoney
  • Created on: 17-05-14 07:10
why alter plants using GM- what advantages can it confer?
1) Plant productivity 2) Nutritional value 3) New & modified proteins 4) Metabolic Pathway regulation 5) Agronomic traits 6) Novel traits 7) Phytoremeditation 8) Pharmaceutical compounds
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what is stable transformation?
Transgene & novel genetic characteristics maintained during life of culture or plant, transgene is usually (not always) integrated into host genome
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what is transient expression?
expression of transgene detected in first few days after introduction into cells, subsequent decline in gene activity indicates expression results from non-integrated DNA
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what is integrative transformation?
transgene covalently integrated into genome of host cell- in fertile plants- transgene inherited by next generation
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what is nuclear transformation?
gene transfer into nuclear genome of host cell- confirmed by cellular fractionation, eukaryotic type expression or mendelian inheritance
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what is organellar transformation?
Gene transfer into plasmid or mitochondrial genome of host cell, as confirmed by cellular fractionation, prokaryotic-type expression or menndelian inheritance
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what is episomal transformation?
viral genomes or 'mini-chromosomes' which replicate independently from host genome
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what is Polyethylene glycol (PEG)-mediated Protoplast fusion?
treating plant tissue with enzymes that digest the cell wall, releasing protoplasts
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In microprecipitation, what 2 pieces of DNA are first dissolved in phosphate buffer?
1) DNA of gene to be put into cells 2) selectable marker gene
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In microprecipitation, what is then added slowly to the DNA?
calcium chloride solution, very fine precipitate (calcium phosphate) forms with DNA adsorbed to it
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what happens next in micro precipitation?
solution incubated briefly w/ cells in tissue culture plates, after rinsing, cells cultured further- then selection for marker gene is applied
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how is the gene usually integrated In micro precipitation?
as tandem arrays
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what are liposomes?
lipid-bilayer bounded vesicles, can be formed by hydrating lipids in aqueous solutions
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in liposome fusion, what happens if DNA is present in the liposome solution?
DNA becomes incorporated into liposomes
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what kind of cells do liposomes interact with?
wall-less cells- liposomal contents are transferred to inside of cell
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what have been implicated as mechanisms of liposome fusion?
membrane fusion and endocytosis have both been implicated as mechanisms
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genes present in the transferred DNA can be expressed how?
transiently- transferred DNA may also integrate into chromosomes and cell lines containing integrated gene may be selected
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how does Agrobacterium make plants produce the food it needs to live off?
By inserting small piece of its own DNA into genome of the plant- scientists use this to make genetically modified plants
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which Agrobacterium plasmid gives the bacterium the capacity to transfer part of the plasmid (T-DNA) to a plant?
The Ti Plasmid
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wounds sites of plants attract agrobacterium through release of which compounds?
Phenolic compounds
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which chromosomal and which plasmid genes are needed for successful infection?q
Chromosomal genes ChvA and ChvB- for binding to plant cell, bacterial genes in operons vitR-virH needed for DNA transfer
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The T-DNA contains genes which only function where?
in the plant cell
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what does this T-DNA cause?
formation of galls and production of nutrients utilised by Agrobacterium
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T-DNA in vectors used for plant genetic engineering have what removed?
Agrobacterium genes removed- cannot cause gall formation and nutrient production- Agrobacterium genes replaced by marker gene & gene to be expressed in transgenic plants
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what is B-glucuronidase (GUS) used for?
reporter gene assays
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where is B-glucuronidase (GUS) taken from?
E.coli
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how is GUS localised?
histochemical localisation of enzyme activity
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what are tissues incubated in to ascertain whether the GUS marker has been incorporated?
5-bromo-4-chloro-3-indonyl glucuronide (X-gluc)
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hydrolysis by GUS produces a precipitate of what colour?
blue
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what is GFP?
another reporter gene assay- stands for green fluorescent protein
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where is GFP gene taken from?
Aequoria victoria- a jellyfish- mutant forms available, fluoresce at different wavelengths
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what happens in a binary vector system?
T-DNA and vir genes are separated onto 2 different plasmids
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which organism is the binary vector manipulate in first before transfer to agrobacterium?
E. coli
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the binary vector is transferred from E.coli to E.coli to A.tumefaciens- it is therefore...?
A triparental mating
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what is used to obtain the desired transformant?
antibiotic selection- because the selectable marker gene functions in plant cells that have taken up T-DNA- makes them resistant to antibiotic
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how is plant tissue infected?
plant tissue is usually co-cultivated with Agrobacterium- infects at cut edges- gives rise to transformed cells- these are selected by antibiotic and develop into callus in tissue culture
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what is a callus?
actively dividing, non-organised tissue of undifferentiated (sometimes differentiated) cells, often develop from point of injury (wounding)
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what is organogenesis?
development of organs (roots, buds, shoots, flowers, etc) from unusual points of origin including a callus
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which growth regulators (hormones/phytohormones) are used in plant medium?
1) auxin 2) cytokinins
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what do auxins do?
group of plant hormones (natural and synthetic) which induce cell elongation, or in some cases cell division. Often induce adventitious roots and inhibit adventitious shoots
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what do cytokinis do?
group of plant hormones (natural and synthetic) which induce cell division and often induce adventitious shoots and inhibit adventitious root formation
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what happens if there are equal amounts of auxin & cytokinin?
callus formation
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if there is more cytokinin than auxin?
shoot formation
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if there is more auxin than cytokinin?
root formation
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what is subculturing?
transplanting of cell tissues and organs etc from one expended nutrient medium to another fresh one
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what is micropropagation?
vegetative propagation of plants in vitro
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what is totipotency?
each cell possesses potential to develop into another plant
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cous
COUS
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Card 2

Front

what is stable transformation?

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Transgene & novel genetic characteristics maintained during life of culture or plant, transgene is usually (not always) integrated into host genome

Card 3

Front

what is transient expression?

Back

Preview of the front of card 3

Card 4

Front

what is integrative transformation?

Back

Preview of the front of card 4

Card 5

Front

what is nuclear transformation?

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Preview of the front of card 5
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