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Markers and reporter genes

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Markers and reporter genes

  1. 1. Marker and Reporter genes used for plant transformation Deepak Raj Pant Central Department of Botany Tribhuvan University, Kirtipur 1
  2. 2. Genetic Manipulation of plants Techniques of introducing DNA into plant cells: 10 hrs Marker and reporter genes used for plant transformation, Model plants and their Role in genetic manipulation, Indirect transformation: Genetic transformation of plant tissues with the use of Agrobacterium, Ti-plasmid and mechanism of T-DNA transfer (different protein involved and their rolr, vir region and other genes involved), Ti plasmid derived plant vector systems; binary and cointegrative vectors transformation process, regeneration of the transformed lines, Plant Viruses as biological vectors. Direct gene transfer methods in plants (Microprojectile bombardment, Electroporation; polyethylene glycol (PEG)- mediated gene transformation, Silica carbom fibres whiskers). Transformation of protoplasts with naked DNA Genetic modified plant and their Application 5 hrs Genetic engineering for plant improvement: Development of Pest resistance, herbicide resistance, resistance against viruses, improving stress tolerance, Protoplast fusion and its implication, Importance of GM plants 2
  3. 3. Genetic Manipulation of plants • Manipulation of genome of an organism • Can be done by traditional method (breeding) or by using the techniques of molecular cloning and genetic transformation (i.e., by genetic engineering) • Genetic engineering is used for genetic manipulation due to its advantages 3
  4. 4. Genetic Manipulation of plants Genetic engineering is advantageous because • Changes can be manipulated in lab and introduced into target organisms/tissues/ cells without affecting other genes • It overcomes the requirements for sexual compatibility • It enables us to study the gene function by inactivation of gene, altered expression of gene, ectopic expression of genes, altering the proteins, etc 4
  5. 5. Genetic Manipulation of plants What we need? • A fully functional gene with all its regulatory sequences • A competent target (Embryogenic competence; competence for transgene uptake) • Proven method of delivery (biological, chemical, or physical) • Method of verifying the progress of transformation and selection of transformants (i.e., reporter and selectable marker genes) 5
  6. 6. Selectable markers • The selectable markers are the genes which enable the selection/recovery of transformed cells/tissues from untransformed tissues in the growth media containing selecting agent (usually an antibiotic or a herbicide) • These genes provide selective advantage for the transformants (which occur at very low frequency) during the course of selection, i.e., the growth of cells/tissues in media containing the toxic substance (selecting agent) 6
  7. 7. Selectable markers • The selectable markers usually encode for a protein that detoxifies the compound used for selection of transformed cells • Some of the selectable markers also involve the genes that are mutated and produce target proteins that are insensitive to toxic substances (usually antibiotics or herbicides) used for selection • Some encode for proteins that convert a non-metabolite into a metabolite 7
  8. 8. Antibiotics Resistance as Selectable markers 8 Selectable marker gene(s) Resistance against Uses Neomycin phosphotranferase II (npt II) G418 sulphate, Kanamycin, Neomycin, etc Dicots/ monocots Hygromycin phosphotransferase (hpt or aphIV) Hygromycin B Dicots/monocots Aminoglycoside -3 adenyltransferase (aad A) or streptomycin resistance gene (spt) Streptomycin/ Spectinomycin dicots Gentamicin 3-N- acetyl transferase (gat) Gentamicin Dicots Dihydrofolate reductase (dfr) Methotrexate Dicots Unknown gene (ble) of Tn5 transposon Bleomycin
  9. 9. Herbicide Resistance as Selectable markers 9 Selectable marker gene(s) Resistance against Uses EPSPS/ gox from Achromobacter sp. Glyphosate/ Roundup Dicots/monocots Bialaphos (Basta) resistance (bar) or phosphinothricin acectyl transferase (pat) Glufosinate/ PPT/Basta/ Dicots/monocots Chlorosulfuron resistance (csr) Sulfonylurea (chlorosulfuron) Dicots/monocots Bromoxynil nitrilase (bxn) Bromoxynil (Buxtril) Dicots/monocots Mutant of Acetolactate synthase (als) Imidazolinones (Imazethapyr) Dicots Unknown monoxygenases Auxins like 2,4-D, 2,4,5-T Dicots
  10. 10. Selectable Markers from AA synthesis pathway 10 Selectable marker gene(s) Resistance against Dihydropicolinate synthase (dhps) from bacteria S-aminoethyl L-cysteine (AEC), a toxic analogue of L-Lysine Aspartate Kinase (AK) from bacteria Lys and Thr (These normally inhibit AK and cause starvation for methionine) Tryptophan Decarboxylase (TD) from Catharanthrus roseus 4-methyl tryptophan (a toxic analogue of tryptophan) Other selectable Markers Selectable marker gene(s) Resistance against Phosphomannose isomerase (pmi) from E. coli Mannose; can utilise mannose as substrate; convert mannose 6-p to fructose 6-P Xylose isomerase (xyl A) from Streptomyces rubiginosus Xylose; conversion of D-Xylose to D-xylulose Ipt, BADH, rol A , etc can also be used Cytokinin autotrophic growth, osmoprotectant, hairy roots
  11. 11. 11 Negative Selectable markers Selectable marker gene(s) Killed by Nitrate reductase gene (nia) Chlorate on medium containing NH4 + Cytosine deaminase (codA) 5-fluorocytosine, a non toxic compound, due to its conversion into 5-fluorouracil (toxic) • Negative selectable markers are not much useful in routine transformation • These can be used in transposon tagging to select the non-mutant populations
  12. 12. Reporter genes • The reporter genes encode for the products (scorable markers) that can be easily scored/quantified in transformed tissues by simple assays • These reporter genes also help detect/screen the transformation events during gene transfer experiments • These markers encode for products that are themselves fluorescent/ coloured or give fluorescence/ colour reactions upon treatment with other substrates 12
  13. 13. Reporter genes 13 Reporter gene Enzyme/potein Substrate Assay gfp gfp none Fluorescence observation Gus or uidA* Β-Glucuronidase X- Gluc Histochemical, photometric, fluorometric Lux/ luc Bacteria/ Firefly Luciferase D- Luciferin, ATP Fluorometric phoA Alkaline phosphatase Phosphate Fluorescece measurement; histochemical cat Chloramphenicol acetyl transferase 14C- chloramphnicol, Acetyl CoA Acetyl chloramphenicol detection by chromatography/ autoradiography lacZ Β-Galactosidase IPTG and X-Gal Colour formation (blue/ white) Npt II Neomycin phospho- transferase Kanamycin and 32P- ATP Detection of radioactivity in tissues (in situ)
  14. 14. Green Fluorescent protein (gfp) 14 • From Jellyfish Aquoria victoria, glows in blue light 395 nm giving green fluorescence (510 nm) • Can be detected in vivo (non destructively) by using fluorescence microscope • Different variants like EGFP, Red GFP, EYFP, etc available • Does not require any substrate, can be detected directly
  15. 15. Gus gene (uid A) 15 • From Escherichia coli • Codes for the β glucuronidase which breaks x-gluc (5 bromo-4 chloro-3 indol β D glucuronide) into blue colour; can be used for histochemical analysis of gene expression • Converts 4MUG (4- methylumbelliferyl-β- D glucuronide) into a fluorescent compound 4MU (Methyl Umbelliferone); can be used for quantification by fluorescent measurement • Can also be quantified spectro- photometrically by using p-nitrophenyl galactoside as substrate Gus expression in young leaves
  16. 16. Gus gene (uid A) 16
  17. 17. Luciferase gene luc or lux 17 • From firefly (luc) or bacteria (lux) • Converts luciferin into oxyluciferin in the presence of oxygen and ATP • Oxyluciferin is highly unstable, singlet excited compound which emits fluorescence upon relaxation to ground state
  18. 18. Alkaline phosphatase pho A 18 • From Bacteria E. coli • Cleaves compounds containing phosphate group like o- nitrophenly phosphate, x-phos (5 bromo-4 chloro-3 indolyl phosphate) or 4MUP (4-methyl umbelliferyl phosphate) • Can be used for colorimetric or histochemical detection (with o-nitrophenyl phosphate or X-phos) or by fluorescence measurement (with 4-MUP) • Forms yellow nitrophenol with o-nitrophenyl phosphate • Gives dark blue coloured precipitate with X-phos in the same manner as does β-glucuronidase with x-gluc
  19. 19. • Chawla H.S. 2009. Introduction to Plant Tissue Culture. Third Edition. Oxford and IBH • Guerineau, F. 1996. Tools for expressing foreign genes in plants. In: Jones, H. (ed), Plant gene transfer and expression protocols. Humana press. Pp 1-32 • Gupta, P.K. 2009. Biotechnology and Genomics. Rastogi Publications, pp 442-444 • Slater, A., Scott, N.W. and Fowler, M.R. 2008. Plant Biotechnology, the genetic manipulation of plants. Oxford University Press.pp 86-91 References 19