Biotechnology and Genetic Engineering-PBIO 450/550

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12 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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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. 18.1 Infection of a

plant with
A. tumefaciens

and
formation of crown galls

Fig. 28
-
27

Crown Gall on

Tobacco

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 5’
-
TGNCAGGATATATNNNNNNGTNANN
-
3’

Left 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 T
-
DNA) Ti plasmid in
A. tumefaciens

Plant genetic engineering with
the binary Ti plasmid system

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.

Transgenic

plant

(disarmed)

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 DNA
-
coated 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

(
kan
r
)

Yes

Yes

Hygromycin

phosphotransferase

(
hyg
r
)

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/bcs
-
main.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

RB

Recombinase

gene

Selectable

marker

Target gene

Recombinase recognition

sequence