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Lecture 25 The future of transgenic
plantsChapter 16
Neal Stewart
Discussion questions• What is the main dichotomy between innovation and caution (or
risk, or the perception of risk)?
• What is real-time PCR and why is it better than regular PCR?
• Describe site-specific recombination and how it could lead to greater precision in plant transformation.
• How might site-specific recombination enhance biosafety?
• What are zinc-finger nucleases, and how might they alter the future of plant biotechnology?
• How do feelings and trust influence plant biotechnology?
• What are key issues in future applications in bioenergy?
Real-time PCR or Quantitative PCR
• Real-time PCR uses fluorescence as an output for DNA amplification in real-time.
• The amount of starting template DNA (or cDNA for RNA measurement (real-time RT-PCR) is correlated with the Ct number.
• More DNA = lower Ct; Ct is the cycle number when a threshold amount of DNA is produced.
http://www.youtube.com/watch?v=QVeVIM1yRMU
http://www.rt-pcr.com/
Problems in plant biotechnology:might be addressed with new
technologies
• Agrobacterium- and especially biolistics-mediated transformation are imprecise
• Transgenic plants are regulated because they are transgenic
• Gene flow (hybridization and introgression) remains to be a major issue in regulation.
The case of “Terminator” technologyAKA Technology Protection System
AKA Gene Use Restriction Technology
http://cls.casa.colostate.edu/TransgenicCrops/terminator.html
Promoter Toxin geneRecombinase gene
Recombinase protein
Promoter Toxin gene
Blocking DNA
Recombinase gene
1. A recombinase gene is under the control of an ethanol inducible promoter. In this case no ethanol is applied. Result– toxin gene is not expressed since blocker DNA remains in place and seeds can germinate.
2.
1.
Toxin protein
2. Ethanol is applied and turns on expression of recombinase gene. The recombinase acts to remove the blocking DNA from the toxin gene. Result– toxin gene is expressed and kills embryo in seeds so they cannot germinate.
Stewart 2004, Genetically Modified Planet Fig 5.2
Ethanol-inducible promoter
Figure 16.1
41
3 2
1 2 3 4
1 2 3 4
1
23
4
1 2
3 4
1 4
3 2
A.
B.
C.
(BB’)
(PP’)
(BP’) (PB’)
(BB’)(PP’)
(PB’)(BP’)
(BB’)
(PP’)
(BP’)
(PB’)
Figure 16.1 Recombination between recombination sites (arrowheads) leading to (A) deletion (excision of circular molecule 2,3 from molecule 1,2,3,4; or integration (insertion of molecule 2,3 into molecule 1,4; (B) inversion (of DNA segment 2,3 flanked by recombination sites of opposite orientation) or (C) translocation (of DNA of different molecules). Some recombination systems use recombination sites that differ in sequence generally known as attB, attP, attL and attR, here shown as BB’, PP’, BP’ and PB’, respectively. In these systems, recombination between attL and attR requires an excisionase protein in addition to an integrase protein.
This figure is slightly different from the one in the book—correct.
Figure 16.2
LY038cordapA
cordapA nptII
cross in cre gene
segregate away cre gene
Figure 16. 2 Renessen’s high lysine corn line LY038 used site-specific recombination to remove the transformation selectable marker, the kanamycin resistance gene nptII, after stable incorporation of cordapA that directs high lysine production in seed. Cre recombinase, introduced from hybridization with a cre transgenic plant, excised the nptII marker flanked by directly oriented lox recombination sites. The cre gene was subsequently segregated away in the following generation.
Site-specific recombinase-mediated transgene excision
Transgene Cre loxPloxP
Cre Transgene loxPloxP
loxPlo
Figure 16.3
trait nptII
Recombinase gene induced by developmental cues
rec inducible
Figure 16.3 Recombination sites that flank the entire transgenic locus permits removal of transgenic DNA upon induced expression of a recombinase gene. For instance, if the recombinase gene is placed under the control of sperm-specific or fruit-specific promoters, the excision of transgenic DNA may help reduce the outcross of transgenes, or minimize the production of transgene-encoded proteins needed elsewhere in the plant but not in the edible portions of food.
Site-specific recombinase-mediated transgene excision in pollen
RS
LBRBPollen genome
Pollen-specific promoter LAT52 activates recombinase in polle
excision
LAT52 pro Recombinase NOS ter RS GUS- NPTII35S pro 35S terRS
LAT52 pro Recombinase NOS terRS
LB
RS GUS-NPTII35S pro 35S ter
RBPollen genome
Luo et al. 2007 Plant Biotechnol J 5:263
GM-gene-deletor system(Luo et al. 2007 Plant Biotechnol J 5:263)
Cre-loxP/FRT vector
No recombinase vector
Fused recombination sites increase efficiency of excision
Luo et al. 2007 Plant Biotechnol J 5:263
Hudson et al 2001 Mol Ecol Notes 1:321
GFP marker for field trials
RB
LAT52 pro Recombinase NOS terRS Bar NOS ter
LB
RSNOS pro GFPLAT59 pro 35S ter
RB
LAT52 pro NOS terRS Bar NOS ter
LB
RSNOS pro GFPLAT59 pro 35S ter
• Cre recombinase with loxP recognition sites• ParA recombinase with MRS recognition sites• CinH recombinase with RS2 recognition sites• Cre recombinase with fused loxP-FRT recognition sites• No recombinase with loxP recognition sites
Zinc finger nucleases
www.bmb.psu.edu, www.wpclipart.com, www.faculty.ucr.edu
ZFNs in gene therapy
Nature 435:577
Double-strand break by zinc finger nuclease
Promoter activates ZFN
5’-TTCTTCCCCGAATTCGGGGAAGAA-3’
ZFN recognition sites
Promoter Zinc finger Nuclease Ter 3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’
ZFN cutting sites
Plant genome
Promoter Zinc finger Nuclease Ter 5’-TTCTTCCCCG
3’-AAGAAGGGGCTTAA
ZFN recognition sites
Double-strand Break
GCCCCT TCT T-5’
AATTCGGGGAAGAA-3’Plant genome
Double-strand break occurs between ZFN recognition sites
Zinc finger nuclease-mediated transgene excision in pollen
Pollen genome
Pollen-specific promoter LAT52 activates ZFN in pollen
excision
LBRBPollen genomeR R
LAT52 pro ZFN NOS ter NPTII35S pro 35S terR R
LAT52 pro ZFN NOS ter NPTII35S pro 35S ter
RB LB
R R R R
Excision sites
ZFN constructs
5’-TTCTTCCCCGAATTCGGGGAAGAA-3’
3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’
QQR ZFN recognition sites
LAT52 pro QQR ZFN Ter 35S pro GUS::NPTII Ter 5’-TTCTTCCCCGAATTCGGGGAAGAA-3’
3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’
QQR ZFN recognition sites
RB LB
5’-TTCTTCCCCGAATTCGGGGAAGAA-3’
3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’
QQR ZFN recognition sites
LAT52 pro Ter 35S pro GUS::NPTII Ter 5’-TTCTTCCCCGAATTCGGGGAAGAA-3’
3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’
QQR ZFN recognition sites
RB LB
• ZFN domain under the control of pollen specific promoter LAT52• ZFN recognition sites • GUS and NPTII fusion under the control of 35SLloyd et al. 2005 PNAS 102:2232
Figure 16.4
1 2 3 4
2’ 3’
1 2’ 3 4
1 2 3 4
CTCCCTGTC GCCACTCTC
Figure 16.4 A possible approach for homologous gene replacement in plants. Example shows replacement of gene 2 by gene 2’, mediated by two heterologous zinc finger nucleases, each binding a unique 9 bp sequence separated by a spacer of ~6 bp. Each zinc finger (triangle) recognizes a 3-nucleotide sequence. Cleavage at the spacer DNA promotes DNA repair and a higher rate of homologous recombination.
Last questions
• Is food too emotionally hot to be addressed by biotechnology? Where on earth?
• What is the scientist’s role here?
• What about non-food plant biotechnology such as bioenergy?
1996 - 1998
40
48
46
51
35
36
32
34
45
52
38
21
24
27
44
20
39
22
45
33
22
31
30
22
21
44
29
44
10
15
0 20 40 60 80 100
United Kingdom
Switzerland
Sweden
Netherlands
Italy
Germany
France
Austria
United States
Canada
Percent Response
False (Correct) Don't Know True
“Ordinary tomatoes do not contain genes, while genetically modified ones do”
Slide courtesy of Tom Hoban
People in different countries have varied knowledge about the facts of genetics and biotechnology.
American consumers’ trust in biotechnology information sources
4
6
7
11
16
20
20
28
32
41
43
48
52
52
54
61
66
59
51
46
52
46
41
37
30
19
14
12
17
13
0% 20% 40% 60% 80% 100%
Activist Groups
Chefs
Food Manufacturers
Biotechnology Companies
TV News Reporters
Farmers
Registered Dietitians
University Scientists
Food and Drug Administration
American Medical Association
A Lot Some None Slide courtesy of Tom Hoban
7
9
4
8
9
16
17
20
6
6
4
7
7
21
14
26
0 5 10 15 20 25 30
None (Spontaneous)
Don't Know
Mass Media
Government
Universities
Medical Profession
Environmental Groups
Consumer Organizations
Percent Response
1999
1996
Source of information trusted most to tell the truth about biotechnology(includes all European countries)
Slide courtesy of Tom Hoban
Path to cellulosic ethanol
Whole Genome Microarrays
Yesterday
TodayTomorrow
Accele
rated
Domestica
tion
MetabolicProfiling
Carbon allocation
ConventionalForestry
Short rotation hardwoods
High yield wood crops
Bioenergy and plant genomics:Expanding the nation’s renewable
energy resources
Brian Davison ORNL
Cell wall structure
Nature Reviews Molecular Cell Biology 2, 33-39 (2001)
Dixon and Chen 2007 Nature Biotechnology 25: 759-761
Dixon and Chen 2007 Nature Biotechnology 25: 759-761
Biomass/bioenergy crops
• Should not be food crops• Should not interfere with food production• Must be sustainable• Will probably require biotechnology for
better yield and cell wall digestion• Major biosafety issue with transgenic
switchgrass will be gene flow• An opportunity to do it right from the
beginning