cloning and expression vector in plants

cloning and expression vector in 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.

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

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

7. 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

8. Binary vector strategy Cointegrative strategy

9. 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) Cauliflower mosaic virus based vectors. Cowpea mosaic virus Bean pod mottle virus (BPMV) TMV based vectors. Potato virus X (PVX) Bean yellow dwarf virus Bacteriophage Lambda Vectors 

10. Cauliflower mosaic virus DNA virus Infectious when simply rubbed on leaves Mechanical and aphid mediated transmission Up to 10 6 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.

11. 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

12. 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