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Gene cloning in agriculture

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gene cloning in agri

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Gene cloning in agriculture

  1. 1. GENE CLONING IN AGRICULTURE Jannat Iftikhar B11-16 6th semester 1
  2. 2. Contents • Gene addition • Gene subtraction • Problem with genetically modified crops 2
  3. 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. 3
  4. 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. 4
  5. 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. 5
  6. 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. 6
  7. 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. 7
  8. 8. Mode of action of δ-endotoxins 8
  9. 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. 9
  10. 10. Procedure used to obtain GM maize plants expressing an artificial δ- endotoxin 10
  11. 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. 11
  12. 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. 12
  13. 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. 13
  14. 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). 14
  15. 15. ‘Roundup ready crops’ 1st engineered crop for glyphosate, by Monsanto Co. called ‘roundup ready’. 15
  16. 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. 16
  17. 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. 17
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  19. 19. Other gene addition projects 19
  20. 20. Gene subtraction Misnomer modification does not involve actual removal of a gene, merely its inactivation. Several strategies Most successful, antisense technology 20
  21. 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. 21
  22. 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. 22
  23. 23. Timescale for development of a fruit 23
  24. 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 24
  25. 25. Using antisense RNA to inactivate polygalacturonase gene 25
  26. 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 26
  27. 27. Other gene subtraction projects 27
  28. 28. Problems with genetically modified plants Safety concerns with selectable markers The terminator technology 28
  29. 29. The terminator technology 29
  30. 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. 30
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