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Making transgenic plants
1.Identify and clone DNA sequence encoding desired protein into
suitable vector = DNA molecule that allows sequence to be
propagated in chosen host
• create a recombinant DNA molecule
Making transgenic plants
1) create recombinant DNA
2) transform recombinant
molecules into suitable host
Making transgenic plants
1) create recombinant DNA
2) transform recombinant
molecules into suitable host
3) identify hosts which have taken
up your recombinant molecules
Making transgenic plants
1) create recombinant DNA
2) transform recombinant molecules into suitable host
3) identify hosts which have taken up your recombinant molecules
4) Confirm they contain the recombinant DNA
Making transgenic plants
1.Identify and clone DNA sequence encoding desired protein into
suitable vector = DNA molecule with:
• Origin of replication that functions in chosen host
• “Selectable marker” = gene encoding protein allowing
selection of hosts that
have taken up the
recombinant molecule
• Cloning site =
dispensable region
where foreign DNA
can be inserted
Making transgenic plants
1.Identify and clone DNA sequence encoding desired protein into
suitable vector = DNA molecule with:
• Origin of replication that functions in chosen host
• “Selectable marker” = gene encoding protein allowing
selection of hosts that
have taken up the
recombinant molecule
• Cloning site =
dispensable region
where foreign DNA
can be inserted
Making transgenic plants
1.Identify and clone DNA sequence encoding desired protein into
suitable vector
2.Vectors for plant transformation add promoters, terminators and
selectable markers that work in plant cells
Making transgenic plants
1.Digest DNA and vector with same restriction enzyme
•Assume have identified the DNA sequence to clone, eg by PCR
Making transgenic plants
1.Digest DNA and vector with same restriction enzyme
2.Allow them to anneal, then seal nicked backbone with DNA ligase
Making transgenic plants
1.Digest DNA and vector with same restriction enzyme
2.Allow them to anneal, then seal nicked backbone with DNA ligase
3.Transform into bacteria
Making transgenic plants
1.Digest DNA and vector with same restriction enzyme
2.Allow them to anneal, then seal nicked backbone with DNA ligase
3.Transform into bacteria
4.Extract plasmid
Making transgenic plants
1.Digest DNA and vector with same restriction enzyme
2.Allow them to anneal, then seal nicked backbone with DNA ligase
3.Transform into bacteria
4.Extract plasmid
5.Directly add to plants or transfer to Agrobacterium tumefasciens
Making transgenic plants
1.Digest DNA and vector with same restriction enzyme
2.Allow them to anneal, then seal nicked backbone with DNA ligase
3.Transform into bacteria
4.Extract plasmid
5.Directly add to plants or transfer to Agrobacterium tumefasciens
6.Select transgenics
Agrobacterium tumefasciens (Rhizobium radiobacter)
Gram-negative pathogenic soil bacterium of Rhizobiaceae (same
family as Rhizobium symbionts)
Agrobacterium tumefasciens (Rhizobium radiobacter)
Gram-negative pathogenic soil bacterium of Rhizobiaceae (same
family as Rhizobium symbionts)
Causes crown galls in over 140 dicot plant spp.
Agrobacterium tumefasciens (Rhizobium radiobacter)
Gram-negative pathogenic soil bacterium of Rhizobiaceae (same
family as Rhizobium symbionts)
Causes crown galls in over 140 plant spp.
Contains 206,000 bp Tumor-inducing (Ti) plasmid
Agrobacterium tumefasciens (Rhizobium radiobacter)
Contains 2006,000 bp Tumor-inducing (Ti) plasmid
When infects host transfers T-DNA (from left to right border of Ti
plasmid) that inserts into host chromosome
Agrobacterium tumefasciens (Rhizobium radiobacter)
Contains 2006,000 bp Tumor-inducing (Ti) plasmid
When infects host transfers T-DNA (from left to right border of Ti
plasmid) that inserts into host chromosome
Process resembles
conjugation
Agrobacterium tumefasciens (Rhizobium radiobacter)
When infects host transfers T-DNA (from left to right border of Ti
plasmid) that inserts into host chromosome
Process resembles conjugation
T-DNA contains “oncogenic genes” that cause overproduction of
auxin and cytokinin
Agrobacterium tumefasciens (Rhizobium radiobacter)
When infects host transfers T-DNA (from left to right border of Ti
plasmid) that inserts into host chromosome
Process resembles conjugation
T-DNA contains “oncogenic genes” that cause overproduction of
auxin and cytokinin: make transformed cells form tumors
Agrobacterium tumefasciens (Rhizobium radiobacter)
T-DNA contains “oncogenic genes” that cause overproduction of
auxin and cytokinin: cause transformed cells to form tumors
Also have gene forcing cell to make opines: funny amino acids that
only Agro can use
Agrobacterium tumefasciens (Rhizobium radiobacter)
T-DNA contains “oncogenic genes” that cause overproduction of
auxin and cytokinin: cause transformed cells to form tumors
Also have gene forcing cell to make opines: funny amino acids that
only Agro can use: convert host into factory feeding Agro!
Agrobacterium tumefasciens (Rhizobium radiobacter)
T-DNA contains “oncogenic genes” that cause overproduction of
auxin and cytokinin: cause transformed cells to form tumors
Also have gene forcing cell to make opines: funny amino acids that
only Agro can use: convert host into factory feeding Agro!
Plant mol biologists have “disarmed” the Ti plasmid by removing
“oncogenic genes” (remember Ti plasmid is 206,000 bp!)
Agrobacterium tumefasciens (Rhizobium radiobacter)
Plant mol biologists have “disarmed” the Ti plasmid by removing
“oncogenic genes” (remember Ti plasmid is 206,000 bp!)
Added genes for plant and bacterial selectable markers
Origins that work in E. coli and in Agrobacterium
Promoter and terminator that work in plants
Agrobacterium tumefasciens (Rhizobium radiobacter)
1.Clone your gene into an E. coli plasmid
Agrobacterium tumefasciens (Rhizobium radiobacter)
1.Clone your gene into an E. coli plasmid
2.Add plant promoters and terminators
Agrobacterium tumefasciens (Rhizobium radiobacter)
1.Clone your gene into an E. coli plasmid
2.Add plant promoters and terminators
3.Transfer cassette into a disarmed (AKA binary) Ti plasmid
between left and right border and transform into E. coli
Agrobacterium tumefasciens (Rhizobium radiobacter)
1.Clone your gene into an E. coli plasmid
2.Add plant promoters and terminators
3.Transfer cassette into a disarmed (AKA binary) Ti plasmid
between left and right border and transform into E. coli
4.Verify plasmid, then transform into Agrobacterium
Agrobacterium tumefasciens (Rhizobium radiobacter)
1.Clone your gene into an E. coli plasmid
2.Add plant promoters and terminators
3.Transfer cassette into a disarmed (AKA binary) Ti plasmid
between left and right border and transform into E. coli
4.Verify plasmid, then transform into Agrobacterium
5.Infect plants with this Agrobacterium: will transfer T-DNA
carrying your gene into new host
Agrobacterium tumefasciens (Rhizobium radiobacter)
•Infect plants with this Agrobacterium: will transfer T-DNA carrying
your gene into new host
•Select transgenic plants containing your new gene
Lipid metabolism
Unique aspects in plants
Make fatty acids by
same reactions, but in
plastids with a prokaryotic
fatty acid synthase
12 proteins, cf one
multifunctional
protein
Lipid metabolism
Make fatty acids in plastids with a prokaryotic FAS
• 12 proteins, instead of one multifunctional protein
• Assemble some lipids in CP, others in ER
• Acetyl-CoA carboxylase is also prokaryotic = 4 subunits, except
in grasses (profoxydim & other grass herbicides inhibit ACCase)
Lipid metabolism
“16:3 plants” assemble lipids in cp using FA-ACP = prokaryotic
pathway (“primitive”)
“18:3 plants” export FA, assemble lipids in ER using FA-CoA =
eukaryotic pathway (“advanced”)
Substrates for most desaturases
are lipids, not FA!
Lipid metabolism
Chloroplasts have lots of galactolipids: sugar linked directly to
diacylglycerol : saves PO4
A) MGDG (Monogalactosyl diacylglycerol) 50% cp
B) DGDG (Digalactosyl diacylglycerol) 28% cp
C) SQDG ( Sulphoquinovosyldiacylglycerol) 16% cp
Lipid metabolism
Chloroplasts have lots of galactolipids: sugar linked directly to
diacylglycerol : saves PO4
A) MGDG (Monogalactosyl diacylglycerol) 50% cp
B) DGDG (Digalactosyl diacylglycerol) 28% cp
C) SQDG ( Sulphoquinovosyldiacylglycerol) 16% cp
• Very unsaturated!
• Makes membranes
very fluid
Lipid metabolism
Oleosomes: oil-storing organelles with only outer leaflet
• Put oils between the leaflets as they are made
• Add oleosin proteins to outside: curve the membrane
• Oils often have unusual fatty acids
Lipid metabolism
Biological roles
• Plasma membrane lipids help
survive freezing
• Unacclimated cells vesiculate as
they lose water & pop when it returns
• Acclimated cells shrivel & reswell
Lipid metabolism
log respiration (nmol O2/min/mg protein)
Biological roles
• Plasma membrane lipids help survive freezing
• Mito lipid composition may also influence chilling sensitivity
• CS plants (eg bananas) are damaged at 10˚ C
• Mito show defects at <10˚ C not seen in other plants
3.0
B) SB-H mitochondria
A) fr2 x fr1 mitochondria
34°
2.8
2.6
18°
2.4
35°
25°
24°
2.2
2.0
16°
Control
17:0
18:1
Control
17:0
18:1
1.8
3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.15
3.60 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60
1/T (°K x 1000)
1/T (°K x 1000)
Lipid metabolism
CS plants (eg bananas) are damaged at 10˚ C
• Mito show defects at <10˚ C not seen in other plants
• Membrane lipids show phase changes at these T
Lipid metabolism
CS plants (eg bananas) are damaged at 10˚ C
• Mito show defects at <10˚ C not seen in other plants
• Membrane lipids show phase changes at these T
• Blamed on saturated PG
log respiration (nmol O2/min/mg protein)
Lipid metabolism
Biological (& commercial) roles
• Plasma membrane lipids help survive freezing
• Mito lipid composition influences chilling sensitivity
• Mito show defects at <10˚ C not seen in other plants
•
unsaturated FA did not fix CS, but saturated FA made it
worse: reason for GM desaturases
3.0
B) SB-H mitochondria
A) fr2 x fr1 mitochondria
34°
2.8
2.6
18°
2.4
35°
25°
24°
2.2
2.0
16°
Control
17:0
18:1
Control
17:0
18:1
1.8
3.15 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.15
3.60 3.20 3.25 3.30 3.35 3.40 3.45 3.50 3.55 3.60
1/T (°K x 1000)
1/T (°K x 1000)
Lipid metabolism
Other commercial aspects
• Yield and quality (especially unsaturation) of seed oil is very
important:12 million tons/year
• Want more double bonds, especially w-3, for health
• Want less double bonds for shelf life and taste
• Each double bond increases p(oxidation) 40x
• Have GM oils with more & less double bonds
Lipid metabolism
Other commercial aspects
• Yield and quality of seed oil is very important:12 million tons/yr
• Also have markets for many specialized oils
Lipid metabolism
Other commercial aspects
• Yield and quality of seed oil is very important
• Also have markets for many specialized oils
• Have genetically-engineered many crops to alter seed oils or
produce specific fats
Lipid metabolism
Biofuels are now very fashionable
• Biodiesel = fatty acid methyl esters
• Trans-esterify oils to make them volatile