Download Biotechnology and Genetic Engineering

Chapter 18-Genetic Engineering of
Plants: Methodology
• Plant transformation with the Ti plasmid of
Agrobacterium tumefaciens
• Ti plasmid derived vector systems
• Physical methods of transferring genes to plants
• Microprojectile bombardment
• Use of reporter genes in transformed plant cells
• Manipulation of gene expression in plants
• Production of marker-free transgenic plants
Why genetically engineer plants?
• To improve the agricultural, horticultural or
ornamental value of a crop plant
• To serve as a living bioreactor for the production of
economically important proteins or metabolites
• To provide a renewable source of energy
• To provide a powerful means for studying the action
of genes (and gene products) during development
and other biological processes
Plant transformation with the Ti plasmid of
Agrobacterium tumefaciens
• A. tumefaciens is a gram-negative soil bacterium
which naturally transforms plant cells, resulting in
crown gall (cancer) tumors
• Tumor formation is the result of the transfer,
integration and expression of genes on a specific
segment of A. tumefaciens plasmid DNA called the
T-DNA (transferred DNA)
• The T-DNA resides on a large plasmid called the Ti
(tumor inducing) plasmid found in A. tumefaciens
The Ti plasmid of Agrobacterium tumafaciens and the
transfer of its T-DNA to the plant nuclear genome
Fig. 18.3 The Ti plasmid of Agrobacterium tumafaciens and
its T-DNA region containing eukaryotic genes for auxin,
cytokinin, and opine production.
Fig. 28-27
Crown Gall on
Tobacco
Fig. 18.1 Infection of a
plant with A. tumefaciens and
formation of crown galls
Fig. 17.3 Ti plasmid:
structure & function
The infection process:
1. Wounded plant cell releases phenolics and nutrients.
2. Phenolics and nutrients cause chemotaxic response of A. tumefaciens
3. Attachment of the bacteria to the plant cell.
4. Certain phenolics (e.g., acetosyringone, hydroxyacetosyringone) induce vir
gene transcription and allow for T-DNA transfer and integration into plant
chromosomal DNA.
5. Transcription and translation of the T-DNA in the plant cell to produce opines
(food) and tumors (housing) for the bacteria.
6. The opine permease/catabolism genes on the Ti plasmid allow A. tumefaciens
to use opines as a C, H, O, and N source.
Fig. 18.4 The right and left borders of the T-DNA of the Ti
plasmid are 25 bp direct “repeats” important for
mobilization of the T-DNA by vir gene products
Right
Left
5’-TGNCAGGATATATNNNNNNGTNANN-3’
5’-TGGCAGGATATATNNNNNTGTAAAN-3’
Fig. 18.7 The binary Ti plasmid system involves using a small
T-DNA plasmid (shown below) and a disarmed (i.e., no TDNA) Ti plasmid in A. tumefaciens
Clone YFG (your favorite gene) or
the target gene in the small T-DNA
plasmid in E. coli, isolate the plasmid
and use it to transform the disarmed
A. tumefaciens as shown.
(disarmed)
Plant genetic engineering with
the binary Ti plasmid system
Transgenic
plant
Table 18.1 Plant cell DNA-delivery methods
Method
Comment
Ti plasmid-mediated gene
transfer *
Excellent and highly effective, but
limited to dicots
Microprojectile bombardment * Easy and effective; used with a
wide range of plants
Viral vectors
Not very effective
Direct gene transfer into plant
protoplasts
Only certain protoplasts can be
regenerated into whole plants
Microinjetion
Tedious and slow
Electroporation *
Limited to protoplasts that can be
regenerated into whole plants
Liposome fusion
Limited to protoplasts that can be
regenerated into whole plants
* Most commonly used methods
Fig. 18.10
Microprojectile
bombardment or
biolistic-mediated
DNA transfection
equipment
(a) lab version
(b) portable version
When the helium pressure
builds to a certain point, the
plastic rupture disk bursts, and
the released gas accelerates the
flying disk* with the DNAcoated gold particles on its
lower side. The gold particles
pass the stopping screen, which
holds back the flying disk, and
penetrate the cells of the plant.
*
Table 18.5 Some plant cell reporter and
selectable marker gene systems
Enzyme activity
Selectable
marker
Reporter
gene
Neomycin phosphotransferase (kanr)
Yes
Yes
Hygromycin phosphotransferase (hygr)
Yes
Yes
Nopaline synthase
No
Yes
Octopine synthase
No
Yes
b-glucuronidase (GUS)
No
Yes
Firefly luciferase
No
Yes
b-galactosidase
No
Yes
Bromoxynil nitrilase
Yes
No
Green fluorescent protein (GFP)
No
Yes
Reporter Genes
• For how reporter genes work, see:
http://bcs.whfreeman.com/lodish5e/pages/bcsmain.asp?v=category&s=00010&n=15000&i=15010.01&o=|00510|00610|00520|00530|005
40|00560|00570|00590|00600|00700|00710|00010|00020|00030|00040|00050|01000|
02000|03000|04000|05000|06000|07000|08000|09000|10000|11000|12000|13000|140
00|15000|16000|17000|18000|19000|20000|21000|22000|23000|99000|&ns=1322
• GFP Researchers Win Nobel Prize (October 8, 2008)
Osamu Shimomura, Martin Chalfie, and Roger Tsien won the Nobel Prize in chemistry
for their work on green flourescent protein, a tool that has become ubiquitous in
modern biology as a tag and molecular highlighter, vastly improving our ability to
understand what goes on inside cells.
• Perhaps you may even want to see a 10 minute YouTube video on GFP; if so please
see http://www.youtube.com/watch?v=Sl2PRHGpYuU
Manipulation of gene expression in plants
• Strong, constitutive promoters (35S Cauliflower mosaic virus
promoter or 35S CaMV or 35S)
• Organ and tissue specific promoter (e.g., the leaf-specific
promoter for the small subunit of the photosynthetic enzyme
ribulosebisphosphate carboxylase or rbc)
• Promoterless reporter gene constructs to find new organ- and
tissue-specific promoter (see Fig. 18.15)
• Inducible promoters
• Secretion of transgene products by inclusion of a signal peptide
sequence between a root promoter and YFG and growing the
transgenic plant hydroponically (YFG product will be secreted)
Fig. 18.26 Marker genes may be a safety issue, so it is best to
remove them—here is one strategy
LB
Recombinase Selectable
gene
marker
Recombinase recognition
sequence
Target gene
RB