lecture 6.plant transformation - ubc...
TRANSCRIPT
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Plant transformationObjectives:
1. What is plant transformation?
2. What is Agrobacterium? How and why does it transform plant cells?
3. How is Agrobacterium used as a tool in molecular genetics?
References:
Hooykaas and Schilperoort. 1992. Agrobacterium and plant genetic engineering. Plant Molecular Biology 19: 15-38.
Westhoff et al. Molecular Plant Development:from gene to plant. Chapter 7, 236-243.
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Plant transformation
• Transient – no incorporation of exogenous DNA into the genome
• Stable – incorporation of introduced exogenous DNA into the genome
Introduction of exogenous DNA into a plant cell
Transformation of multicellular organisms:
• Cannot directly transform every cell - Transformation involves one cell which then regenerates an entire organism
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Agrobacterium tumefaciens:a natural tool for plant transformation
Soil gram positive bacterium
Agrobacterium tumefaciens attached to plant tissue
Martha Hawes
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Agrobacterium tumefaciens:a natural tool for plant transformation
• Causes Crown Gall disease - tumors (galls) form at base of stem in many dicotyledonous plants (dicots)
Photographs supplied by Sharon von Broembsen, Oklahoma State University
• production of tumors is caused by the transfer of bacterial DNA to the plant, which integrates into theplant genome
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Agrobacterium tumefaciens:a natural tool for plant transformation
• Genes involved in crown gall disease are not present on the chromosome of A. tumefaciens but on a large plasmid, called the Ti (tumor-inducing) plasmid.
Ti
A. tumefaciens
Circular chromosome
virgenes
LB
RBT-DNA
Ti plasmid~ 120 kbp
ori
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A. tumefaciens T-DNA Structure
LB and RB – 25 bp direct repeatsNos - nopaline synthase – opine biosynthetic gene*Shi - shoot inducing - 2 genes for auxin synthesis*Roi - root inducing - gene for cytokinin synthesis*
Shi NosShi Roi
*have eukaryotic promoters – these genes are not expressed in Agrobacterium!!!
RBLB
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T-DNA transfer into plants
•T-DNA transfer process is activated when Agrobacterium gets in contact with damaged plant tissue
• T-DNA is nicked at the RB, T-DNA gets replicated to the LB and moved intothe plant cell – these processes are catalyzed by products of vir genes
http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF
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•T-DNA is inserted into plant nuclear genome at random sites.
• Transformed cell starts proliferating upon DNA integration resulting in tumor formation. Why?
• Transformed cells make opines = N and C-rich nutrients (amino acidderivatives) for bacterium (“Genetic colonization”)
T-DNA transfer into plants
http://www.plantsci.cam.ac.uk/Haseloff/SITEGRAPHICS/Agrotrans.GIF
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Agrobacterium tumefaciens as a tool for genetic engineering
Problems: tumor, size of the Ti plasmid
How can we engineer Agrobacterium to make it useful for genetic engineering?
• Delete auxin, cytokinin and opine genes
• Retain vir genes, LB&RB, ori
• Ti plasmid is huge (~120 kb) – need to make it smaller
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Binary vector
LB
T-DNARB
Agrobacterium tumefaciens as a tool for genetic engineering
• vir genes and T-DNA can be on separate plasmids
• only left and right borders (LB & RB) are required for T-DNA to be transferred
virgenes
Ti plasmid
ori(Agrobacterium)
ori(E.coli)
Selectable marker
(Bacteria)
Selectable marker(Plants)
Selectable marker
(Bacteria)
Cloning site for plant genes
ori(Agrobacterium)
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Steps in plant transformation
1. Propagate binary vector in E. coli
3. Re-introduce engineered binary vector into E. coli to amplify
4. Isolate engineered binary vector from E. coli and introduce into Agrobacterium already containing a modified (smaller) Ti plasmid with vir genes
5. Infect plant tissue with engineered Agrobacterium(T-DNA containing the gene of interest gets inserted into a plant cell genome at random sites)
2. Isolate binary vector from E.coli and engineer (introduce a gene of interest)
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• In each cell T-DNA gets integrated at a different site in the genome
Plant transformation
• Consequences of the insertion: - Foreign DNA is inserted- Insertional mutagenesis (does not kill the cell – the organism is diploid!)
• Each cell is hemizygous for the insertion – only one of the homologous chromosomes gets the insertion
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Problem:
We want to transform the whole organism, not one cell!!!
Plant transformation
This is done by:
• Transforming plant cells in culture, selecting transformed cells and regenerating the entire plant from the transformed cell (eg. tobacco)
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Plant transformation
Solanum chacoense
http://en.wikipedia.org/wiki/File:Transformation_with_Agrobacterium.JPG
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Problem:
We want to transform the whole organism, not one cell!!!
Plant transformation
This is done by:
• Transforming plant cells in culture, selecting transformed cells and regenerating the entire plant from the transformed cell (eg. tobacco)
• In planta transformation of Arabidopsis- Dip flowering plants into Agrobacterium suspension
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Plant transformation
In planta transformation of Arabidopsis(Floral dip method)
Systemic infection in Arabidopsis is accomplished by transformation of female gametes!
plbio.life.ku.dk
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Problem:
We want to transform the whole organism, not one cell!!!
Plant transformation
This is done by:
• Transforming plant cells in culture, selecting transformed cells and regenerating the entire plant from the transformed cell (eg. tobacco)
• In planta transformation of Arabidopsis- Dip flowering plants into Agrobacterium suspension- Harvest seed and select for transformants – (they are
hemizygous!)
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Binary vector
LB
T-DNARB
Agrobacterium tumefaciens as a tool for genetic engineering
• vir genes and T-DNA can be on separate plasmids
• only left and right borders (LB & RB) are required for T-DNA to be transferred
virgenes
Ti plasmid
ori(Agrobacterium)
ori(E.coli)
Selectable marker
(Bacteria)
Selectable marker(Plants)
Selectable marker
(Bacteria)
Cloning site for plant genes
ori(Agrobacterium)
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Selection of transformants
http://krauthammerlab.med.yale.edu/imagefinder/
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Screening for transformants
DsRed selection using green light excitation
http://www.isb.vt.edu/articles/jan0803.htmhttps://www.emsdiasum.com/microscopy/technical/datasheet/sfa-2.aspx