gene cloning in agri

1. GENE CLONING IN
AGRICULTURE
Jannat Iftikhar
B11-16
6th semester
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2. Contents
• Gene addition
• Gene subtraction
• Problem with genetically modified crops
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3. Agriculture: World ‘s oldest biotechnology
Human have constantly searched for improved varieties
of their crop plants.
Better nutritional qualities, higher yields.
Gene cloning provides a new dimension to crop breeding.
Enable directed changes to be made to genotype of a
plant.
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4. Two general strategies
 Gene addition:
Cloning is used to alter characteristics of a
plant by providing it by one or more new genes.
 Gene subtraction:
Gene engineering techniques are used to
inactivate one or more of plant’s existing genes.
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5. Gene addition approach
 Gene addition:
Use of cloning techniques to introduce into a plant
one or more new genes coding for a useful characteristic
that plant lacks.
 A good example:
Development of plants that resist insect attack by
synthesizing insecticides coded by cloned genes.
 A number of projects are being carried out around the
world.
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6. Plants that make their own insecticides
 Most conventional insecticides e.g. Pyrethroids &
organophosphates.
 Relative non-specific poisons that kill a broad spectrum
of insects.
 High toxicity-some also have potentially harmful side
effects for other members of local biosphere.
 Exacerbate need to apply them to plants’ surfaces by
spraying.
 Subsequent movement of them in ecosystem cannot be
controlled.
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7. The δ-endotoxins of Bacillus thuringiensis
 Intracellular crystalline bodies contain an insecticidal
protein (δ-endotoxins).
 Highly poisonous to insects.
 More toxic than organophosphates (80,000X).
 Relatively selective.
 Different strains of bacterium synthesizing proteins
effective against larvae of different groups of insects.
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8. Mode of action of δ-endotoxins
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9. Cloning a δ-endotoxin gene in maize
A major pest: European corn borer (Ostrinia nubilialis).
1st attempt at countering this pest by engineering maize
plants was made in 1993, with CryǀA version of protein.
Cry protein is 1155 amino acid in length.
Toxic activity residing in 29-607 amino acids.
Rather than isolating the natural gene, a shortened
version containing the first 648 codons was made by
artificial gene synthesis.
Introduction into the maize embryos.
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10. Procedure used to obtain GM maize
plants expressing an artificial δ- endotoxin
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11. Cloning a δ-endotoxin gene in maize
• Immunological test
• Amount of δ-endotoxin varies from about 250ng to
1750ng.
• Difference due to positional effect.
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12. Cloning δ-endotoxin genes in chloroplasts
 Tobacco: CryIIA(a2) gene
 A broader toxicity spectrum: two-winged fly as well as
Lepidopterans.
 Fig. CryIIA(a2) operon, One advantage: chloroplasts like
bacteria is able to express all genes in an operon.
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13. Countering insect resistance to δ-
endotoxin crop
Crops synthesizing δ-endotoxin might become ineffective
after a few seasons.
Resistance among insect populations.
 Various strategies have been proposed to prevent the
development of o-endotoxin resistant insects.
1st, to develop crops expressing both the CryI and CryII
genes.
2nd, to engineer toxin production in such a way that
synthesis occurs only in those parts of the plant that need
protection.
3rd, to mix GM plants with non-GM ones.
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14. Herbicide resistant crops
Most important transgenic plants:
Those have been engineered to withstand herbicide
glyphosate.
Widely used by farmers & horticulturists.
Environmentally friendly: non-toxic to insects & animals; a
short residence time in soils; breaking down over a period
of a few days into harmless products.
Glyphosate kills all plants (both weeds & crops).
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15. ‘Roundup ready crops’
1st engineered crop for glyphosate, by Monsanto Co.
called ‘roundup ready’.
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16. A new generation of glyphosate resistant
crops
Recently a few report
 Organisms can actively degrade glyphosate.
Relatively common among genus Bacillus.
Possess an enzyme: glyphosate N- acetyltransferase
(GAT).
Detoxify glyphosate is by adding an acetyl group.
Most active detoxifier: a strain of Bacillus licheniformis.
Rates are too low to be of value if transferred to a GM
crop.
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17. Multigene shuffling; a type of directed
evolution
Bacterium possesses 3 related genes.
Take parts of each member of a multigene family &
reassembling these parts to create new gene variants.
Most active genes are identified.
Clone all variants in E. Coli & assay recombinant
colonies for GAT activity.
As substrates for next round of shuffling.
11 rounds: a gene specifies a GAT with 10,000X activity.
GM maize: 6X in glyphosate tolerance.
without any reduction in productivity of plant.
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19. Other gene addition projects
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20. Gene subtraction
Misnomer
modification does not involve actual removal of a gene,
merely its inactivation.
Several strategies
Most successful, antisense technology
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21. Antisense technology
The gene to be clone is ligated into the vector in reverse
orientation.
When the cloned gene is transcribed, the RNA that is
synthesized is the reverse compliment of the mRNA,
sometimes abbreviated to asRNA.
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22. Antisense RNA & engineering of fruit
ripening in tomato
GM tomato by antisense technology.
Fruit ripening process is slowed down.
Leave fruits on plant until they ripen to stage where flavor
has fully developed.
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23. Timescale for development of a fruit
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24. Using antisense RNA to inactivate
polygalacturonase gene
Partial inactivation of polygalacturonase
730 bp restriction fragment
Orientation was reversed
Cauliflower mosaic virus promoter
Plant poly(A) signal
Ti plasmid pBIN19
Agrobacterium tumefaciens
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25. Using antisense RNA to inactivate
polygalacturonase gene
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26. Using antisense RNA to inactivate
ethylene synthesis
Ethylene: a gas, acts as a hormone
Switch on gene involved in later stage of tomato ripening
2nd way: delaying plant ripening
Engineer plant: not synthesize ethylene
Unable to complete ripening process
Artificial ripening
Spraying tomatoes with ethylene
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27. Other gene subtraction projects
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28. Problems with genetically modified plants
Safety concerns with selectable markers
The terminator technology
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29. The terminator technology
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30. Conclusion
Gene cloning has revolutionized the agricultural
practices.
With the help of gene cloning we can introduce desired
characteristers into the plants.
There are many aspect yet to be discovered.
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