cloning and expression vector in plants

CLONING AND EXPRESSION
VECTOR IN PLANTS
PRESENTED BY –
ALEX MATHEW
ROLL NO -3
M.Sc. Biotechnology

CLONING VECTOR
• A cloning vector is a small piece
of DNA, taken from a virus, a
plasmid, that can be stably
maintained in an organism, and
into which a foreign DNA
fragment can be inserted for
cloning purposes.
• FEATURES -
 Origin of replication.
 Cloning site.
 Selectable marker.
 Reporter gene.
EXPRESSION VECTOR
• The expression vector is usually
a plasmid or virus designed
for protein expression in cells.
• FEATURES-
Expression vectors must have
strong promoter, a strong
termination codon, adjustment of
the distance between the promoter
and the cloned gene, and the
insertion of a transcription
termination sequence and
a portable translation initiation
sequence

Vectors used In plants
 Plasmids
 Viruses
 Bacteriophages
 Cosmids

Plasmid
• Extra chromosomal DNA molecules.
•Self-replicating.
•Circular & Double stranded.
•Short sequence of DNA.
• Found in prokaryotes.

Agrobacterium-mediated transformation
 Gram negative bacteria.
 Found in soil.
 Causes crown-gall disease.
 Ability to introduce DNA into plant.
Contains
- Ti-plasmid.
- Ri-plasmid

Recombinant Ti-plasmid
 Place target gene in T-DNA region.
 Recombinant T-DNA introduced into plants

The discovery that the vir genes do not need to be in the
same plasmid with a T-DNA region to lead its transfer and
insertion into the plant genome led to the construction of a
system for plant transformation where the T-DNA region and
the vir region are on separate plasmids.
The subsequent gene transfer in to plants is obtained by co-
integrative vectors. Co-integration of the two plasmids is
achieved with in Agrobacterium by homologous
recombination.
Co-integrative vector Binary vector
A binary vector consists of a pair of plasmids of which
one contain vir region and other contains disarmed T-
DNA sequence with right and left border sequences.
The plasmid contain disarmed T-DNA are called micro-Ti or
mini-Ti

Binary vector strategy Cointegrative strategy

Viral vectors
“Viruses which are used to carry genetic material into cells” are called viral
vectors.
Viruses are used in two ways
• Virus directly inserted into plant
• Virus indirectly inserted (bacteria)
1.Cauliflower mosaic virus based vectors.
2.Cowpea mosaic virus
3.Bean pod mottle virus (BPMV)
4.TMV based vectors.
5.Potato virus X (PVX)
6.Bean yellow dwarf virus
7.Bacteriophage Lambda Vectors

Cauliflower mosaic virus
 DNA virus
 Infectious when simply rubbed on leaves
 Mechanical and aphid mediated transmission
 Up to 106 copies per cell within 3-4 weeks of infection in plant.
 Small insertions (10-30 bp) in various sites abolished infectivity
 The largest insert is 256-531 bp
 CaMV genome can be inserted into Ti vector.

BACTERIOPHAGE VECTOR
 Cloning Vector that uses a Bacteriophage as a means for making and storing exact copies
of segments of DNA.
 The bacteriophages used for cloning are the phage λ and M13 phage.
 1000 times more efficient than plasmid vectors
 Clone DNA fragments in range of 10,000 - 20,000 bps

• Cosmids are plasmids that incorporate a segment of
bacteriophage λ DNA that has the cohesive end site (cos)
which contains elements required for packaging DNA into λ
particles.
• It is normally used to clone large DNA fragments between 25
and 45 Kb.
• They can replicate as plasmids if they have a suitable origin of
replication.
• They can also be packaged in phage capsids, which allows the
foreign genes to be transferred into cells by transduction.
Advantages :
• High transformation efficiency.
• The cosmid vector can carry up to 45 kb whereas plasmid and
Lambda phage vectors are limited to 25 kb.
Cosmid

cloning and expression vector in plants

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cloning and expression vector in plants