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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

Lecture 25 The future of transgenic plantsChapter 16Neal Stewart


Discussion questions
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 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.rt-pcr.com/

http://www.youtube.com/watch?v=QVeVIM1yRMU


Problems in plant biotechnology might be addressed with new technologies
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 SystemAKA Gene Use Restriction Technology

http://cls.casa.colostate.edu/TransgenicCrops/terminator.html


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


Figure 16.1 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.

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.


Figure 16.2 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.

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


Transgene 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.

Cre

Cre

loxP

loxP

loxP

loxP

loxP

lo

Site-specific recombinase-mediated transgene excision

Transgene


Figure 16.3 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.

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.


Site specific recombinase mediated transgene excision in pollen

LB 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.

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


Gm gene deletor system luo et al 2007 plant biotechnol j 5 263
GM-gene-deletor system 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.(Luo et al. 2007 Plant Biotechnol J 5:263)

No recombinase vector

Cre-loxP/FRT vector


Fused recombination sites increase efficiency of excision
Fused recombination sites increase efficiency of excision 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.

Luo et al. 2007 Plant Biotechnol J 5:263


Hudson et al 2001 Mol Ecol Notes 1:321 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.


Gfp marker for field trials

LB 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.

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


Zinc finger nucleases
Zinc finger nucleases 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.

www.bmb.psu.edu, www.wpclipart.com, www.faculty.ucr.edu


Zfns in gene therapy
ZFNs in gene therapy 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.

Nature 435:577


Promoter 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.

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


Excision sites 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.

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


Zfn constructs

LB 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.

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


Figure 16.4 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.

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.


Last questions
Last questions 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.

  • 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?


“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



Source of information trusted most to sourcestell the truth about biotechnology(includes all European countries)

Slide courtesy of Tom Hoban



Bioenergy and plant genomics expanding the nation s renewable energy resources
Bioenergy and plant genomics: sourcesExpanding 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


Cell wall structure
Cell wall structure sources

Nature Reviews Molecular Cell Biology2, 33-39 (2001)




Biomass bioenergy crops
Biomass/bioenergy crops sources

  • 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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