Vector mediated gene transfer methods for transgenesis in Plants
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- 1. VECTOR MEDIATED GENE TRANSFER METHODS FOR TRANSGENESIS IN
PLANTS - Akshay More, M.Sc. Biotechnology-1., Modern College,
Ganeshkhind, Pune.
- 2. OUTLINE Vector mediated gene transfers: A. Bacteria. B.
Viruses. C. Transposable Genetic Elements. D. Other Possible
Vectors.
- 3. A) Bacteria: Agrobacterium (Natures little genetic engineer)
- Genus include: A. tumefaciens, A. rhizogenes, A. radiobacter, A.
rubi. - Gram Negative, Soil Bacteria. - Infect plant and causes
cancerous growth in tissue. - A. rubi has narrow host range as
compared to others. - cancerous growth is because of plasmid.
Agrobacterium tumefaciens: -Almost all strains have Ti Plasmid Ti
Plasmid- Large ds circular DNA, size- 150-230 kbp., M.W.-(120-160
MD). Denaturation temp. is above 37 C , loses its tumorigenic
property. Has region encoding unusual amino acids like opine. Has
region for synthesis of opines in its catabolism. Has sequence for
conjunctive transfer of plasmid. Has sequence for virulence (T-DNA)
and replication.
- 4. General Structure of Ti Plasmid
- 5. Types: a) Octopine: -Synthesized from Arginine. -Molecular
formula is C9H18N4O4 -e.g. pTi-B6, pTi-ACH b) Nopaline: -Molecular
formula is C9H16N4O6 e.g. pTi- C58
- 6. OVERVIEW OF INFECTION PROCESS Source:
http://www.nature.com/nature/journal/v433/n7026/images/433585a-f2.2.jpg
- 7. Gene transfer through Ti plasmid: Desired foreign gene is
isolated An intermediate vector plasmid (pBR322) is constructed
using Col E1 plasmid Ti plasmid is isolated from Agrobacterium
& T-DNA is separated. T-DNA +pBR 322 = Shuttle Vector (has
strong promoter, replicates both in E. coli &Agrobacterium
T-DNA is then cut with restriction enzymes and then prepared
foreign DNA is inserted into T-DNA. Chimeric DNA is then inserted
into E. coli (Competent cell preparation). Transformed E.coli +
Agrobacterium, incubation, bacterial conjugation, transfer of
plasmid to Agrobacterium.
- 8. In Agrobacterium, chimeric DNA undergoes homologous
recombination with Ti Plasmid, Agrobacterium transformed. Selective
screening, and selected transformed bacteria used for transgenesis.
Methods for obtaining Agrobacterium transformed plant tissue: 1)
Co-cultivation- protoplasts +transformed Agrobacterium, plants
cells grown as callus. 2) Leaf-disk method- Leaf disk +transformed
Agrobacterium, plants cells grown as callus. 3)
Agroinfiltrationforcing Agrobacterium with transgenes into
leaves
- 9. Results of gene transfer plants- 1) Strain of Salmonella
grows vigorously in media having glyophosphate. So gene is isolated
from Salmonella and insert into plant Arabidopsis through
Agrobacterium. Resulting plant show resistance to glyophosphate. 2)
Nif- genes are transferred to plants for nitrogen fixation, but has
not given beneficial results. 3) Kemp (1981) transferred gene for
phaseolin synthesis from beans to the cells of sunflower. The
transformed cells produced mRNA of phaseolin in them. 4) The United
States Department of Agriculture transferred a gene for a storage
protein of maize into the cells of Sunflower. Transformed cells
produced desired storage protein of maize in them.
- 10. B) Virus Mediated Gene Transfer: -Viral genome doesnt
integrate into plant genome. -Vectors are episomal vectors,
therefore they have high copy number per cell. -The gene product is
very rapidly accumulated. -Viral genome sequences are excellent
source of promoters, enhancers and other components useful for
designing gene vectors. -Virus is systemically spread in the plant
body. -Generally have wide host range. Most Notables: 1)
Caulimovirus based vectors. 2) Gemini virus based vectors. 1)CaMV
based vectors.
- 11. CHARACTERISTICS: -dS DNA virus. Packaged as nucleosome.
-Mechanical and aphid mediated transmission -Virion DNA alone or
cloned CaMV DNA is infectious when simply rubbed on leaves -Up to
106 copies per cell. 3-4 weeks for systemic infection through
plant.
- 12. Two regions of the CaMV genomeopen reading frames (ORFs) II
and VIIdo not seem to be essential for infection, as both can be
either deleted or expanded by small inserts of foreign DNA. Results
of gene transfer plants- Brisson et al. (1984) transferred CaMV
vector containing the bacterial dihydrofolate reductase gene, into
turnip plant cells gives resistance to methotrexate. 2) Gemini
virus based vectors
- 13. CHARACTERISTICS: - Genome size is 2.6- 3.0 kb. - ssDNA, can
cause diseases to cereals, cassava, tomato, Digitaria. - Has
geminated morphology, hence name. - Capsids are icosahedral,
dimensions= 10-20*20 nm. - Most have insect mediated transmission.
No mechanical transmission. - Replicated by rolling circle
mechanism.
- 14. *GENE TRANSFER STRATEGY: pWDV2 V2 deletion vector Results
of gene transfer plants- Use of vectors based on the gemini tomato
golden mosaic virus (TGMV) to introduce the neomycin
phosphotransferase (neo gene) into cereals, cassava plants.
{*Source: Nature 334, 179 - 182 (14 July 1988)}
- 15. C. TRANSPOSABLE GENETIC ELEMENTS. - Structurally and
genetically discrete segments of DNA capable of moving from one
position in genome to other. - First discovered by Barbara
McClintok (1940) in Zea mays, jumping genes. - Insertion of
transposon interfere with expression of that gene(translation). -
E.g. Ac-Ds elements in maize, Insertion Elements, Tn Family, Maize
Elements, Ty elements in Yeast, Copia Elements.
- 16. Cis and Trans Transposon Mediated Gene Delivery Cis and
Trans transposon mediated gene delivery. GOI, gene of interest;
5TR, 5 terminal repeat; 3TR, 3terminal repeat; the yellow and beige
arrows indicate promoters to drive gene expression.
- 17. *Copia Elements- -Found in Drosophila. -e.g. FB (Fold
Back), P elements. P elements- - Variable size, largest element is
1907 bp long having 31 long terminal inverted repeats. - Foreign
DNA can be inserted into transposase element to develop recombinant
P element. - Recombinant P element then microinjected into
fertilized egg along with normal P element. Result of gene
transfer- - Gierl et al (1989) prepared recombinant P element by
inserting DNA fragment of rosy gene. - Recombinant P element then
inserted into plasmid along with normal P element. Both plasmids
were co-injected into Drosophila embryos. - 50 % progeny were rosy
progeny.
- 18. D) Other Possible Vectors 1) Maize Mitochondrial Elements:
- Studies on Cytoplasmic Male Sterility have reveal several of
these elements. - Unlike Transposons, they replicate autonomously
in mitochondria and integrate into mitochondrial genome. - Size
around 1-4 kb. - Can be act as vector for gene transfer. 2) Nuclear
Genomic Components: - Constructed from yeast chromosomal DNA,
integrate into host by homologous recombination. - E.g. YAC, YIC. -
High success in mammals. - Plants lack homologous recombination
system, so limited success.
- 19. 3) Viroids : - Smallest and simplest pathogenic agents. -
Small, 300-400 bases long, circular, single stranded having naked
RNA. - These non-protein coding viroids replicate in host using
hosts replicative enzyme probably RNA Pol II. - They are
mechanically transmissible, able to move throughout the sap and
infect other parts of plant. - They are certainly associated with
nucleus and may replicate in it. - Can be act as vector for gene
transfer.
- 20. References: 1) R. C. Dubey, A textbook of
Biotechnology,2006, first edition, S. Chand and Company Ltd.
Chapter 5. 2) Sunandan Saha and Matthew H. Wilson, chapter 11, Gene
Therapy - Tools and Potential Applications, Intech,
http://dx.doi.org/10.5772/52527. 3) Kjemtrup S, Sampson KS, Peele
C, Nguyen LV, Conkling MA, Thompson WF and Robertson D (1998). Gene
silencing from plant DNA carried by a geminivirus. The Plant
Journal, 14, 91-100. 4) Darbani B., Eimanifar A., Stewart C. N.,
and Camargo W., Methods to produce markerfree transgenic plants,
Biotechnology Journal. Vol. 2,2007, 8390. 5) S. Ignacimuthu,sii,
Plant Biotechnology, 2001, Oxford and IBH Publishing Co. Pvt.
Ltd.
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