ppt - Ntu.edu.tw

Methods to produce marker‐free
transgenic plants
Darbani, B., Eimanifar, A., Stewart, C. N., and Camargo, W.
Biotechnol. J. 2: 83‐90, 2007.
演講者: 林玉儒
指導老師: 黃慶璨 教授
演講日期: 2010/03/15
The purpose of transgenic plant
• World Bank Report:
– If distributed evenly, the world’s food supply can feed 6.4 billion (2350 calories/person); is even distribution a viable option?
– Cultivation lands will drop from 0.26 hectare /person in 1997 to 0.15 in 2050.
Applications of transgenic plant
(Hain et al., 1997)
Molecular breeding
Molecular farming
Processes of transgenic plant technology
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=cell&part=A1360&rendertype=figure&id=A1427
gene transfer into the plant genome
Target
Marker
Gene transfer into the plant genome
• Agrobacterium
• Biolistic
Selection
Regeneration
Current selectable marker genes
• A gene identified cells that have been transfomed or transfected with a vector
(Miki and McHugh, 2004)
• Positive selection
– Antibiotics
– Herbicides
Toxicity
– Carbon supply
– Phytohormone precursor
Antibiotic resistance genes
(Sakthivel et al., 2008)
P
Herbicide resistance genes
(Sakthivel et al., 2008)
Ac
(Franz et al., 1990)
Potential pitfalls of resistant selection methods
• Public concerns
Horizontal gene transfer
biological hazards
jblaque.livejournal.com/tag/environment
– Widespread in ecosystem
– Food safety
http://scrink.com/blog/wellness/uploaded_images/antibiotic-resistance-798176.jpg
http://www.isaaa.org/kc/CBTNews/press_release/images/briefs34/figures/acres/figure%20cover_acres.jpg
Methods to produce resistant marker‐free
transgenic plants
• Strategies
– Non‐antibiotic or herbicide resistance gene
– Excise or segregate resistant marker gene
•
•
•
•
Co‐transformation
Site‐specific recombination
Intrachromosomal recombination
Transposon
Markers not based on antibiotic or herbicide resistance
• Selection
– Nutrients
– Morphologies
(Miki and McHugh, 2004)
Analysis of mannose selection used for transformation of sugar beet (Okkels et al., 1998)
• PMI: phosphomannose isomerase
Mannose‐6‐phosphate
fructose‐6‐phosphate
Methods to produce resistant marker‐free
transgenic plants
• Strategies
– Non‐antibiotic or herbicide resistance gene
– Excise or segregate resistant marker gene
•
•
•
•
Co‐transformation
Site‐specific recombination
Intrachromosomal recombination
Transposon
Co‐transformation
• cannot be used for vegetative propagated plants
(Komamine et al., 2001)
Negative selection
• Types – Inhibition of plant growth
– Convert non‐toxic substrate to toxic WT= wild type Z= target gene
M= positive select marker M= negative select marker offspring
http://www.gmo-safety.eu/imagescontent/gentransfer/35p_grafik.gif
Co‐transformation using a negative selectable marker gene for the production of selectable marker gene‐
free transgenic plants (Chung et al., 2004)
negative select
Positive select
+
codA
GUS
• npt II: Kanamycin r
• codA: Cytosine deamidase
5‐fluorocytosine (5‐FC):non‐toxic
5‐fluorouracil (5‐FU): toxic
P‐DNA
• Backbone‐free transgenic plant
• Using plant DNA (P‐DNA)
– T‐DNA border‐like sequence
– Lacking open reading frame
– High A/T content
(Swords et al., 2004)
Site‐specific recombination‐mediated marker deletion
• Systems
– Cre/loxp: Bacteriophage P1
(Komamine et al., 2001)
– FLP/FRT: Saccharomyces cerevisiae
– R/RS: Zygosaccharomyces rouxii
Site‐specific recombination‐mediated marker deletion
• Mechanism:
RBE: recombinase binding element
Core sequence
(Hare and Chua, 2002)
(Hare and Chua, 2002)
Excision
Cre/loxp‐mediated marker gene excision in transgenic maize (Zea mays L.) plants
(Gilbertson et al., 2003)
• HSP70: Heat shock‐inducible promoter protein
• pHSP17.5E: Heat shock‐inducible promoter
• NptII: Kanamycinr
Callus
Light
Shoots
UV
Light
UV
GFP+
Control
42oC
Control
42oC
42oC
GFP L
Contol 42oC
GFP ‐
Control
Intrachromosomal recombination system
• Non‐recombinase
• Spontaneous excision
– attp: attachment P region of bacteriophage λ
Marker gene
(Meyer et al., 2002)
Intrachromosomal recombination between attP
regions as a tool to remove selectable marker genes from tobacco transgenes (Meyer et al., 2000)
• npt II – Positive selection
– Kanamycinr
• tms 2
– Negative selection
– Mechanism
• Naphthalene acetamide
NAM NAA
• [Auxin]
• Root development
+ tms2
‐ tms2
Transposon‐based marker methods
Komamine et al., 2001
Transposon
• Discovery:
• –Application of produced marker‐free transgenic plant 1940s
– Barbara McClintock
– Controlling element
• Ac (Activator)
• Ds (Disassociation) Induced Ac excision
(Yamakado et al., 1997)
(Comfort, 2001)
http://personal.agron.ntu.edu.tw/bocharng/images/%E8%B7%B3%E4%B8%8D%E8%B7%B3%E
6%9C%89%E9%97%9C%E4%BF%82.pdf
An inducible transposon system to terminate the function of a selectable marker in transgenic rice
(Tu et al., 2008)
Marker (epsps)
Transposon
SA‐induced transposed
Excision
• epsps:
–Glyphosate tolerance
–5‐enolpyruvylshikimate‐ 3‐phosphate synthase
• PR‐1a –Salicylic acid (SA) induced promoter
Un‐transposed
Conclusion
Selectable marker
Producing marker-free
unit.aist.go.jp/rigb/gf‐pmt/research_e.html forum.pchome.com.tw/content/69/21582
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