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Lecture 25 The future of transgenic plants Chapter 16 Neal Stewart

Lecture 25 The future of transgenic plants Chapter 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?

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Lecture 25 The future of transgenic plants Chapter 16 Neal Stewart

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  1. Lecture 25 The future of transgenic plantsChapter 16Neal Stewart

  2. 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?

  3. 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.

  4. http://www.rt-pcr.com/ http://www.youtube.com/watch?v=QVeVIM1yRMU

  5. 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.

  6. The case of “Terminator” technologyAKA Technology Protection SystemAKA Gene Use Restriction Technology http://cls.casa.colostate.edu/TransgenicCrops/terminator.html

  7. 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. 1. Ethanol-inducible promoter Blocking DNA Toxin gene Recombinase gene Promoter 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. Recombinase protein 2. Promoter Recombinase gene Toxin gene Toxin protein Stewart 2004, Genetically Modified Planet Fig 5.2

  8. Figure 16.1 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. (BP’) (PB’) A. 1 2 3 4 3 2 (PP’) 1 4 (BB’) (PP’) B. (BB’) 1 2 3 4 1 3 2 4 (BP’) (PB’) (BB’) C. (BP’) 1 2 1 4 3 4 3 2 (PP’) (PB’) This figure is slightly different from the one in the book—correct.

  9. Figure 16.2 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 markerflanked by directly oriented lox recombination sites. The cre gene was subsequently segregated away in the following generation. cordapA nptII cross in cre gene segregate away cre gene cordapA LY038

  10. Transgene Cre Cre loxP loxP loxP loxP loxP lo Site-specific recombinase-mediated transgene excision Transgene

  11. Figure 16.3 trait nptII rec inducible Recombinase gene induced by developmental cues 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.

  12. LB LB Pollen genome GUS-NPTII GUS- NPTII 35S ter 35S pro 35S pro Recombinase LAT52 pro LAT52 pro NOS ter RB RB Pollen-specific promoter LAT52 activates recombinase in polle excision RS RS 35S ter Recombinase NOS ter RS RS Pollen genome RS Site-specific recombinase-mediated transgene excision in pollen Luo et al. 2007 Plant Biotechnol J 5:263

  13. GM-gene-deletor system(Luo et al. 2007 Plant Biotechnol J 5:263) No recombinase vector Cre-loxP/FRT vector

  14. Fused recombination sites increase efficiency of excision Luo et al. 2007 Plant Biotechnol J 5:263

  15. Hudson et al 2001 Mol Ecol Notes 1:321

  16. LB LB RB RB Bar GFP GFP NOS ter 35S ter LAT59 pro LAT59 pro NOS pro Recombinase LAT52 pro LAT52 pro NOS ter Bar 35S ter NOS pro NOS ter NOS ter RS RS RS RS GFP marker for field trials • 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

  17. Zinc finger nucleases www.bmb.psu.edu, www.wpclipart.com, www.faculty.ucr.edu

  18. ZFNs in gene therapy Nature 435:577

  19. Promoter Promoter Ter Ter ZFN cutting sites Plant genome Plant genome Zinc finger Nuclease ZFN recognition sites Promoter activates ZFN Double-strand break occurs between ZFN recognition sites Double-strand Break 5’-TTCTTCCCCGAATTCGGGGAAGAA-3’ 5’-TTCTTCCCCG 3’-AAGAAGGGGCTTAA GCCCCT TCT T-5’ AATTCGGGGAAGAA-3’ 3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’ Zinc finger Nuclease ZFN recognition sites Double-strand break by zinc finger nuclease

  20. Excision sites LB LB Pollen genome NPTII 35S ter ZFN 35S pro LAT52 pro LAT52 pro NOS ter RB RB excision Pollen-specific promoter LAT52 activates ZFN in pollen NPTII ZFN 35S ter 35S pro NOS ter Pollen genome R R R R R R R R Zinc finger nuclease-mediated transgene excision in pollen

  21. LB QQR ZFN 35S pro LAT52 pro 35S pro LAT52 pro Ter Ter Ter Ter RB 5’-TTCTTCCCCGAATTCGGGGAAGAA-3’ 3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’ 5’-TTCTTCCCCGAATTCGGGGAAGAA-3’ 5’-TTCTTCCCCGAATTCGGGGAAGAA-3’ 3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’ 3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’ 5’-TTCTTCCCCGAATTCGGGGAAGAA-3’ 3’-AAGAAGGGGCTTAAGCCCCTTCTT-5’ QQR ZFN recognition sites QQR ZFN recognition sites GUS::NPTII QQR ZFN recognition sites LB RB GUS::NPTII QQR ZFN recognition sites ZFN constructs • ZFN domain under the control of pollen specific promoter LAT52 • ZFN recognition sites • GUS and NPTII fusion under the control of 35S Lloyd et al. 2005 PNAS 102:2232

  22. Figure 16.4 CTCCCTGTC GCCACTCTC 1 2 3 4 2’ 3’ 1 2 3 4 1 2’ 4 3 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.

  23. 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?

  24. “Ordinary tomatoes do not contain genes, while genetically modified ones do” 1996 - 1998 People in different countries have varied knowledge about the facts of genetics and biotechnology. Slide courtesy of Tom Hoban

  25. American consumers’ trust in biotechnology information sources Slide courtesy of Tom Hoban

  26. Source of information trusted most to tell the truth about biotechnology(includes all European countries) Slide courtesy of Tom Hoban

  27. Path to cellulosic ethanol

  28. Bioenergy and plant genomics:Expanding the nation’s renewable energy resources Whole Genome Microarrays Tomorrow Carbon allocation Today Short rotation hardwoods High yield wood crops Accelerated Domestication Conventional Forestry Yesterday Metabolic Profiling Brian Davison ORNL

  29. Cell wall structure Nature Reviews Molecular Cell Biology2, 33-39 (2001)

  30. Dixon and Chen 2007 Nature Biotechnology 25: 759-761

  31. Dixon and Chen 2007 Nature Biotechnology 25: 759-761

  32. 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

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