Making transgenic plants 1.Identify and clone DNA sequence encoding desired protein into suitable vector = DNA molecule that allows sequence to be propagated.

Making transgenic plants1.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 plants1) create recombinant DNA2) 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 plants1.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 plants1.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 plants1.Identify and clone DNA sequence encoding desired protein into suitable vector2.Vectors for plant transformation add promoters, terminators and selectable markers that work in plant cells

Making transgenic plants1.Digest DNA and vector with same restriction enzyme•Assume have identified the DNA sequence to clone, eg by PCR

Making transgenic plants1.Digest DNA and vector with same restriction enzyme2.Allow them to anneal, then seal nicked backbone with DNA ligase

Making transgenic plants1.Digest DNA and vector with same restriction enzyme2.Allow them to anneal, then seal nicked backbone with DNA ligase3.Transform into bacteria

Making transgenic plants1.Digest DNA and vector with same restriction enzyme2.Allow them to anneal, then seal nicked backbone with DNA ligase3.Transform into bacteria4.Extract plasmid

Making transgenic plants1.Digest DNA and vector with same restriction enzyme2.Allow them to anneal, then seal nicked backbone with DNA ligase3.Transform into bacteria4.Extract plasmid5.Directly add to plants or transfer to Agrobacterium tumefasciens

Making transgenic plants1.Digest DNA and vector with same restriction enzyme2.Allow them to anneal, then seal nicked backbone with DNA ligase3.Transform into bacteria4.Extract plasmid5.Directly add to plants or transfer to Agrobacterium tumefasciens6.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) plasmidWhen 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) plasmidWhen infects host transfers T-DNA (from left to right border of Ti plasmid) that inserts into host chromosomeProcess resemblesconjugation

Agrobacterium tumefasciens (Rhizobium radiobacter)When infects host transfers T-DNA (from left to right border of Ti plasmid) that inserts into host chromosomeProcess resembles conjugationT-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 chromosomeProcess resembles conjugationT-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 tumorsAlso 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 tumorsAlso 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 tumorsAlso 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 markersOrigins that work in E. coli and in AgrobacteriumPromoter 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 plasmid2.Add plant promoters and terminators

Agrobacterium tumefasciens (Rhizobium radiobacter)1.Clone your gene into an E. coli plasmid2.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 plasmid2.Add plant promoters and terminators 3.Transfer cassette into a disarmed (AKA binary) Ti plasmid between left and right border and transform into E. coli4.Verify plasmid, then transform into Agrobacterium

Agrobacterium tumefasciens (Rhizobium radiobacter)1.Clone your gene into an E. coli plasmid2.Add plant promoters and terminators 3.Transfer cassette into a disarmed (AKA binary) Ti plasmid between left and right border and transform into E. coli4.Verify plasmid, then transform into Agrobacterium5.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 metabolismUnique aspects in plantsMake fatty acids by same reactions, but in plastids with a prokaryoticfatty acid synthase12 proteins, cf one multifunctional protein

Lipid metabolismMake 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 metabolismChloroplasts have lots of galactolipids: sugar linked directly to

diacylglycerol : saves PO4

A) MGDG (Monogalactosyl diacylglycerol) 50% cpB) DGDG (Digalactosyl diacylglycerol) 28% cpC) SQDG ( Sulphoquinovosyldiacylglycerol) 16% cp

Lipid metabolismChloroplasts have lots of galactolipids: sugar linked directly to

diacylglycerol : saves PO4

A) MGDG (Monogalactosyl diacylglycerol) 50% cpB) DGDG (Digalactosyl diacylglycerol) 28% cpC) SQDG ( Sulphoquinovosyldiacylglycerol) 16% cp• Very unsaturated!• Makes membranes very fluid

Lipid metabolismOleosomes: 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 metabolismBiological roles• Plasma membrane lipids helpsurvive freezing• Unacclimated cells vesiculate as they lose water & pop when it returns• Acclimated cells shrivel & reswell

Lipid metabolismBiological 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

Lipid metabolismCS 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 metabolismCS 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

Lipid metabolismBiological (& 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

Lipid metabolismOther 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 metabolismOther commercial aspects• Yield and quality of seed oil is very important:12 million tons/yr• Also have markets for many specialized oils

Lipid metabolismOther 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 metabolismBiofuels are now very fashionable• Biodiesel = fatty acid methyl esters• Trans-esterify oils to make them volatile