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Presentation on:
Plant transformation vectors and their types
Presented to:Dr. Hamid Manzoor
Presented by:Hina Mushtaq …………... BSBT-02Zahra Naz ……………….. BSBT-04
07th May, 2015
Institute of Molecular Biology & Biotechnology, Bahauddin Zakariya
University, Multan, Pakistan.
Contents
Plant transformation Vectors Types of vectors Plant transformation vectors Plasmids Viruses Bacteriophages
– Advantages – Disadvantages
Plant Transformation
”Transformation is the genetic alteration of
a cell resulting from the direct uptake and incorporation of exogenous
genetic material from its surroundings.”
or“Integration of gene into genome by
means other than fusion of gametes”file:///G:/agri%20biotech/NPTEL%20__%20Biotechn
ology%20-%20Plant%20Biotechnology.html
Steps of Plant
Transformation
Plant Transformation
Methods1.Indirect method or vectored
methods
oAgro bacterium-mediated transformation.
oVirus mediated.
2.Direct method.
o Protoplast electroporation.
o Protoplast polyethylene glycol
method.
o Gene gun method.
Vector
“A DNA molecule used as a vehicle to
carry foreign genetic material into
another cell.”
Types Of Vector:
-Plasmids. -Viral vectors.
-Cosmids. -Artificial
chromosome.file:///G:/agri%20biotech/PLANT%20TRANSFORMATION%20VECTORS%20_%20What%20Is%20The%20Biotechnology.html
Characteristics of vectors
Origin of replication
Self-replicating
Bacterial selectable markers
Gene constructs of interest
file:///G:/agri%20biotech/PLANT%20TRANSFORMATION%20VECTORS%20_%20What%20Is%20The%20Biotechnology.html
Vector classification
Cloning vectors“Small piece of DNA into which a foreign DNA fragment is inserted
for cloning purposes.”
Expression vectors“Also known as an
expression construct, is usually a plasmid or virus designed for protein expression in cells.”
file:///G:/agri%20biotech/PLANT%20TRANSFORMATION%20VECTORS%20_%20What%20Is%20The%20Biotechnology.html
In plants
Plasmids Viruses Bacteriophag
es Cosmids
Plant Physiol. 2007 Dec; 145(4): 1118–1128.doi: 10.1104/pp.107.106104PMCID: PMC2151730Focus Issue on Vector Systems for Plant Research and BiotechnologyDelivery of Multiple Transgenes to Plant CellsMery Dafny-Yelin and Tzvi Tzfira
Plasmid
• Extra chromosomal DNA
molecules.
• Self-replicating.
• Circular & Double stranded.
• Short sequence of DNA.
• Found in prokaryotes.
file:///G:/agri%20biotech/NPTEL%20__%20Biotechnology%20-%20Plant%20Biotechnology.html
Classification
Classification of plasmidso Fertility plasmid
e.g. F plasmid of E. colio Col plasmid
e.g. ColE1 of E. colio Resistance plasmid
e.g. RP4 in Pseudomonaso Degradative plasmid
e.g. TOL of P. putidao Virulence plasmid
e.g. Ti plasmids of A. tumefaciens
file:///C:/Users/%20/Documents/d.html
Based on the origin or source of plasmids
Two major classes :i) Natural plasmids:
They occur naturally in prokaryotes Example: ColE1.
ii) Artificial plasmids: They are constructed in-vitro by re-combining selected segments of two or more plasmids.
Example: pBR322.
file:///C:/Users/%20/Documents/d.html
Nomenclature of Plasmid
pBR322
p Plasmid
B Boliver
R Rodriguez
322 Number given to distinguish
http://blog.addgene.org/plasmids-101-how-to-name-your-plasmid-in-3-easy-steps
Advantages
Occur naturally in bacteriaHave different restriction sites.Replicate completely independent of bacteriaGenes are easily inserted into plasmidsEasily transformed into bacteria
https://answers.yahoo.com/question/index?qid=20061217124130AAsvKqk
Disadvantages
Cannot accept large fragments Sizes range from 10-20 kb Standard methods of transformation
are inefficient
https://answers.yahoo.com/question/index?qid=20061217124130AAsvKqk
Agrobacterium-mediated
transformation Gram negative bacteria.
Found in soil.
Causes crown-gall disease.
Ability to introduce DNA into plant.
Contains
- Ti-plasmid.
- Ri-plasmid
Curr Top Microbiol Immunol. 2014;375:155-92. doi: 10.1007/82_2013_352. Plant viral vectors for delivery by Agrobacterium. Gleba YY, Tusé D, Giritch A
Agrobacterium tumefaciens
Recombinant Ti-plasmid Place target gene in T-DNA region.
Recombinant T-DNA introduced into
plants
http://www.csun.edu/~hcbio027/biotechnology/lec2/PL/pl.htm
Plant genetic engineering using T-DNA vector
Method of screening
White-Blue screening
Colonies with recombinant plasmid are white Colonies with non-recombinant plasmids are blue.
For example: pUC19Resistance to ampicilline.
Contains portion of the lacZ which codes for beta-galactosidase.
Viral vectors
“Viruses which are used as gizmo by molecular biologists to
carry genetic material into cells” are called viral vectors.
Viral vectors are non-integrative as compared to bacterial vectors
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613721/
Examples
1.Cauliflower mosaic virus based vectors.
2.Cowpea mosaic virus3.Bean pod mottle virus (BPMV)4.TMV based vectors.5.Potato virus X (PVX) 6.Bean yellow dwarf virus7.Bacteriophage Lambda Vectors
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613721/
Characteristics of viral vectors
Safety Low toxicity Stability Cell type specificity
http://www.ncbi.nlm.nih.gov/pubmed/12828943
Viruses are used in two ways
–Virus directly inserted into plant
–Virus indirectly inserted (bacteria)
http://www.ncbi.nlm.nih.gov/pubmed/12828943
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.
Use of viral vectors for vaccine production in plants, M Carmen Cañizares, Liz Nicholson and George P Lomonossoff. John Innes Centre, Norwich, UK
Small insertions (10-30 bp) in various sites abolished infectivityThe largest insert is 256-531 bpCaMV genome can be inserted into Ti vector
transcription
nucleus
35S RNA
19S RNA
translation
Reverse transcription
uncoating
Gene IV
Gene V
Gene III/IV
assembly
Inclusion body(gene VI)
Gene I
CaMV activity in plant cell
Bacteriophage Lambda Vectors
Viruses that can infect bacteria 1000 times more efficient than
plasmid vectors Clone DNA fragments inrange of 10,000 - 20,000 bps
Bacteriophage. 2012 Oct 1; 2(4): 215–224.Soil-based systemic delivery and phyllosphere in vivo propagation of bacteriophagesTwo possible strategies for improving bacteriophage persistence for plant disease controlFanny B. Iriarte, Aleksa Obradović, Mine H. Wernsing, Lee E. Jackson, Botond Balogh, Jason A. Hong, M. Timur Momol, Jeffrey B. Jones, and Gary E. Vallad
Steps
Advantages
Fast processing ,low cost, high yield
Good at targeting and entering cells
Mostly target specific types of cells
Used as virus-induced gene silencing (VIGS) in reverse genetic studies
2003 Aug;30(4):296-303.Virus-induced gene silencing in plants.Lu R, Martin-Hernandez AM, Peart JR, Malcuit I, Baulcombe DC.
Express proteins in plants for the - Study of gene function- Production of vaccines - Study of metabolic engineering
- Analysis of plant-microbe interactions
Hum Vaccin. 2011 Mar;7(3):331-8. Epub 2011 Mar 1.Geminiviral vectors based on bean yellow dwarf virus for production of vaccine antigens and monoclonal antibodies in plants.Chen Q, He J, Phoolcharoen W, Mason HS
Disadvantages
Worst effects to plants by–Producing severe disease–Giving undesired products–Affecting the plant adversely
(due to highest mutation rate)
http://plantsciences.utk.edu/stewart.htm
Cosmid
Derived from bacteriophage & plasmid
Cohesive sites + plasmid = cosmid
Less used for plant transformation
Carry DNA fragments of about 40 kb
E.g. US 8298819 B2
J Virol. 2000 Oct; 74(19): 8972–8979.PMCID: PMC102093Generation of Mutant Murine Cytomegalovirus Strains from Overlapping Cosmid and Plasmid ClonesMariam E. Ehsani, Tshge W. Abraha ,Cecile Netherland-Snell,Niklaus Mueller, Meghan M. Taylor, and Barry Holwerda
Cohesive ends or sticky ends
A single-stranded end of a linear duplex DNA molecule which can form hydrogen-bond with a complementary single-strand base sequence from the end of the same or another DNA molecule
http://www.dnalc.org/view/15019-Cohesive-sticky-ends-and-their-significance-in-genetic-engineering-Paul-Berg.html