GENE CLONING IN

AGRICULTURE

Jannat Iftikhar

B11-16

6th semester

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Contents

• Gene addition

• Gene subtraction

• Problem with genetically modified crops

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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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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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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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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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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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Mode of action of δ-endotoxins

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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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Procedure used to obtain GM maize

plants expressing an artificial δ- endotoxin

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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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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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Countering insect resistance to δ-

endotoxin cropCrops 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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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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‘Roundup ready crops’

1st engineered crop for glyphosate, by Monsanto Co.

called ‘roundup ready’.

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A new generation of glyphosate resistant

cropsRecently 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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Multigene shuffling; a type of directed

evolutionBacterium 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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Other gene addition projects

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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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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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Antisense RNA & engineering of fruit

ripening in tomatoGM 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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Timescale for development of a fruit

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Using antisense RNA to inactivate

polygalacturonase genePartial 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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Using antisense RNA to inactivate

polygalacturonase gene

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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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Other gene subtraction projects

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Problems with genetically modified plants

Safety concerns with selectable markers

The terminator technology

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The terminator technology

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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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