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Making transgenic plants

Making transgenic plants 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

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Making transgenic plants

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  1. Making transgenic plants • 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

  2. Making transgenic plants 1) create recombinant DNA 2) transform recombinant molecules into suitable host

  3. Making transgenic plants 1) create recombinant DNA 2) transform recombinant molecules into suitable host 3) identifyhostswhich have taken up your recombinant molecules

  4. Making transgenic plants 1) create recombinant DNA 2) transform recombinant molecules into suitable host 3) identifyhostswhich have taken up your recombinant molecules 4) Confirm they contain the recombinant DNA

  5. Making transgenic plants • 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

  6. Making transgenic plants • 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

  7. Making transgenic plants • Identify and clone DNA sequence encoding desired protein into suitable vector • Vectors for plant transformation add promoters, terminators and selectable markers that work in plant cells

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

  9. Making transgenic plants • Digest DNA and vector with same restriction enzyme • Allow them to anneal, then seal nicked backbone with DNA ligase

  10. Making transgenic plants • Digest DNA and vector with same restriction enzyme • Allow them to anneal, then seal nicked backbone with DNA ligase • Transform into bacteria

  11. Making transgenic plants • Digest DNA and vector with same restriction enzyme • Allow them to anneal, then seal nicked backbone with DNA ligase • Transform into bacteria • Extract plasmid

  12. Making transgenic plants • Digest DNA and vector with same restriction enzyme • Allow them to anneal, then seal nicked backbone with DNA ligase • Transform into bacteria • Extract plasmid • Directly add to plants or transfer to Agrobacterium tumefasciens

  13. Making transgenic plants • Digest DNA and vector with same restriction enzyme • Allow them to anneal, then seal nicked backbone with DNA ligase • Transform into bacteria • Extract plasmid • Directly add to plants or transfer to Agrobacterium tumefasciens • Select transgenics

  14. Agrobacterium tumefasciens (Rhizobium radiobacter) Gram-negative pathogenic soil bacterium of Rhizobiaceae (same family as Rhizobium symbionts)

  15. 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.

  16. 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

  17. 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

  18. 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

  19. 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

  20. 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

  21. 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

  22. 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!

  23. 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!)

  24. 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

  25. Agrobacterium tumefasciens (Rhizobium radiobacter) Clone your gene into an E. coli plasmid

  26. Agrobacterium tumefasciens (Rhizobium radiobacter) Clone your gene into an E. coli plasmid Add plant promoters and terminators

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

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

  29. Agrobacterium tumefasciens (Rhizobium radiobacter) Clone your gene into an E. coli plasmid Add plant promoters and terminators Transfer cassette into a disarmed (AKA binary) Ti plasmid between left and right border and transform into E. coli Verify plasmid, then transform into Agrobacterium Infect plants with this Agrobacterium: will transfer T-DNA carrying your gene into new host

  30. 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

  31. 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

  32. 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)

  33. 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!

  34. 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

  35. 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

  36. 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

  37. 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

  38. Lipid metabolism 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

  39. 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

  40. 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

  41. 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

  42. 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

  43. 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

  44. 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

  45. Lipid metabolism Biofuels are now very fashionable • Biodiesel = fatty acid methyl esters • Trans-esterify oils to make them volatile

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