Bio 226: Cell and Molecular Biology

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) identify hosts which have taken
up your recombinant molecules
4
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
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
• 163 plants assemble lipids in cp using FA-ACP
  prokaryotic pathway (primitive)
• 183 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 lt10 C not seen in other
  plants

39

• Lipid metabolism
• CS plants (eg bananas) are damaged at 10 C
• Mito show defects at lt10 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 lt10 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 lt10 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 important12 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
  important12 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