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

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  • Making transgenic plantsIdentify and clone DNA sequence encoding desired protein into suitable vector = DNA molecule that allows sequence to be propagated in chosen hostcreate a recombinant DNA molecule

  • Making transgenic plants1) create recombinant DNA2) transform recombinant molecules into suitable host

  • Making transgenic plants1) create recombinant DNA2) transform recombinant molecules into suitable host3) identify hosts which have taken up your recombinant molecules

  • Making transgenic plants1) create recombinant DNA2) transform recombinant molecules into suitable host3) identify hosts which have taken up your recombinant molecules4) Confirm they contain the recombinant DNA

  • Making transgenic plantsIdentify and clone DNA sequence encoding desired protein into suitable vector = DNA molecule with:Origin of replication that functions in chosen hostSelectable marker = gene encoding protein allowing selection of hosts that have taken up the recombinant moleculeCloning site = dispensable region where foreign DNA can be inserted

  • Making transgenic plantsIdentify and clone DNA sequence encoding desired protein into suitable vector = DNA molecule with:Origin of replication that functions in chosen hostSelectable marker = gene encoding protein allowing selection of hosts that have taken up the recombinant moleculeCloning site = dispensable region where foreign DNA can be inserted

  • Making transgenic plantsIdentify and clone DNA sequence encoding desired protein into suitable vectorVectors for plant transformation add promoters, terminators and selectable markers that work in plant cells

  • Making transgenic plantsDigest DNA and vector with same restriction enzymeAssume have identified the DNA sequence to clone, eg by PCR

  • Making transgenic plantsDigest DNA and vector with same restriction enzymeAllow them to anneal, then seal nicked backbone with DNA ligase

  • Making transgenic plantsDigest DNA and vector with same restriction enzymeAllow them to anneal, then seal nicked backbone with DNA ligaseTransform into bacteria

  • Making transgenic plantsDigest DNA and vector with same restriction enzymeAllow them to anneal, then seal nicked backbone with DNA ligaseTransform into bacteriaExtract plasmid

  • Making transgenic plantsDigest DNA and vector with same restriction enzymeAllow them to anneal, then seal nicked backbone with DNA ligaseTransform into bacteriaExtract plasmidDirectly add to plants or transfer to Agrobacterium tumefasciens

  • Making transgenic plantsDigest DNA and vector with same restriction enzymeAllow them to anneal, then seal nicked backbone with DNA ligaseTransform into bacteriaExtract plasmidDirectly add to plants or transfer to Agrobacterium tumefasciensSelect 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)Clone your gene into an E. coli plasmid

  • Agrobacterium tumefasciens (Rhizobium radiobacter)Clone your gene into an E. coli plasmidAdd plant promoters and terminators

  • Agrobacterium tumefasciens (Rhizobium radiobacter)Clone your gene into an E. coli plasmidAdd plant promoters and terminators Transfer cassette into a disarmed (AKA binary) Ti plasmid between left and right border and transform into E. coli

  • Agrobacterium tumefasciens (Rhizobium radiobacter)Clone your gene into an E. coli plasmidAdd plant promoters and terminators Transfer cassette into a disarmed (AKA binary) Ti plasmid between left and right border and transform into E. coliVerify plasmid, then transform into Agrobacterium

  • Agrobacterium tumefasciens (Rhizobium radiobacter)Clone your gene into an E. coli plasmidAdd plant promoters and terminators Transfer cassette into a disarmed (AKA binary) Ti plasmid between left and right border and transform into E. coliVerify plasmid, then transform into AgrobacteriumInfect 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 hostSelect 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 FAS12 proteins, instead of one multifunctional proteinAssemble some lipids in CP, others in ERAcetyl-CoA carboxylase is also prokaryotic = 4 subunits, except in grasses (profoxydim & other grass herbicides inhibit ACCase)

  • Lipid metabolism16:3 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 PO4A) 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 PO4A) MGDG (Monogalactosyl diacylglycerol) 50% cpB) DGDG (Digalactosyl diacylglycerol) 28% cpC) SQDG ( Sulphoquinovosyldiacylglycerol) 16% cpVery unsaturated!Makes membranes very fluid

  • Lipid metabolismOleosomes: oil-storing organelles with only outer leaflet Put oils between the leaflets as they are madeAdd oleosin proteins to outside: curve the membraneOils often have unusual fatty acids

  • Lipid metabolismBiological rolesPlasma membrane lipids helpsurvive freezingUnacclimated cells vesiculate as they lose water & pop when it returnsAcclimated cells shrivel & reswell

  • Lipid metabolismBiological rolesPlasma membrane lipids help survive freezingMito lipid composition may also influence chilling sensitivityCS plants (eg bananas) are damaged at 10 CMito show defects at
  • Lipid metabolismCS plants (eg bananas) are damaged at 10 CMito show defects at
  • Lipid metabolismCS plants (eg bananas) are damaged at 10 CMito show defects at
  • Lipid metabolismBiological (& commercial) rolesPlasma membrane lipids help survive freezingMito lipid composition influences chilling sensitivityMito show defects at
  • Lipid metabolismOther commercial aspectsYield and quality (especially unsaturation) of seed oil is very important:12 million tons/yearWant more double bonds, especially w-3, for healthWant less double bonds for shelf life and tasteEach double bond increases p(oxidation) 40xHave GM oils with more & less double bonds

  • Lipid metabolismOther co