This document discusses cloning and expression vectors used in plants. It describes cloning vectors as small DNA molecules that can stably maintain foreign DNA for cloning purposes. Expression vectors are designed for protein expression and must contain strong promoters and terminators. Common vectors used in plants include plasmids, viruses, bacteriophages, and cosmids. Agrobacterium-mediated transformation is described as a method for introducing DNA into plants using Ti or Ri plasmids. Viral vectors like CaMV can also be used to directly or indirectly insert genes into plants.

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

2. 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

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

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

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

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

9. 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

10. Binary vector strategy Cointegrative strategy

11. 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

12. 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.

13. 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

14. • 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