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Plastids transformation
- 1. Plastid Transformation Presented by: Baljeet Kaur L-2017-A-109-M
- 2. • Plastids • Chloroplast structure and Genome • Chloroplast transformation • Why Plastid transformation is preferred over nuclear transformation??? • Traits of Interest and limitations • Markers elimination systems • Conclusion Content
- 3. Membrane – bound organelles present in Plants, Algae, Several taxa of marine mollusks and in some Protistans Site of manufacture and storage of various chemical compounds Semiautonomous in nature Show Maternal inheritance Having double stranded circular DNA Plastids
- 4. Origin of Plastids Chan and Bhattacharya (2010) J Nature Education 3(9): 84
- 5. Muroplast Rhodoplast Proplastid/ Eoplast Amyloplast Elaioplast Proteinoplast Chromoplast Chloroplast Etioplast Types of Plastids Robert R(2006) Structure and Functions of Plastids, pp 3-26 Gerontoplast
- 6. Plastids development cycle Lieber et al (2017) Frontiers in Plant science 8: 23-27
- 7. Chloroplast Ribosome Plastoglobule Internal membrane Inter membrane Space External membrane Granum Chloroplast DNA Stroma Thylakoid Lamella Lumen Thylakoid membrne
- 8. Plastome Oleiniczak et al (2016) Planta 244: 517-527 Plastid and cpDNA copy are variable in cells
- 9. Plastid Transformation Homologous recombination between the transformation vector and the plastid genome, resulting in integration of the foreign gene(s) of interest at a predictable, predetermined site in the Plastid genome
- 10. Earlier Reports Year Host DNA delivery Approach Selection 1988 Chlamydomonas reinhardtii 1st stable plastid transformation Biolistic Homologous targeting Photosynthetic competence 1990 Nicotiana tabacum Biolistic Homologous targeting Spectinomycin (rrn16) 1993 Nicotiana tabacum PEG Homologous targeting Spectinomycin, Kanamycin
- 11. Chloroplast transformation requirements Chloroplast specific vector DNA delivery Methods Homologous recombination Efficient selection of transplastome
- 12. Choice of vector Maliga (2002) Plant Biology 5:164-172 There are no universal vector for chloroplast transformation
- 13. Vector designing single gene expression multiple genes expression Ahmad et al (2016) J Experimental Botany 67: 5945-5960
- 14. Commonly used Promoters, Un- translated regions and Insertion sites Promoter 5’-UTRs 3’UTRs Popular insertion sites PrrnA T7G10 rps16 trnI – trnA PpsbA ggagg rbcL rbcL –accD PrbcL rbcL petD rp132- trnL atpB psbA petA – psbJ psbA 3’-rps12/7 – trnV cry2a trn16/V – 3’-rps12/7 trnfM – trnG trnN – trnR atpB – rbcL rps7 – ndhB Ycf3 - trnS Ahmad et al (2016) J Experimental Botany 67: 5945-5960
- 15. DNA delivery Methods Plastid vector DNA is coated on to tungsten or gold micro projectiles (0.4- 1um in diameter), which are then delivered at high velocity through cell wall & membrane and then through double plastid membrane Heifetz (2000) Biochimie 82:655-666 Biolistic Transformation
- 16. PEG (Polyethylene Glycol) mediated transformation Isolation of Protoplast by degrading cell wall enzyme Plant Protoplast treated with PEG along with Ca2+ , followed by introduction of naked DNA into system Plasma membrane is destabilized by PEG & Ca2+ action and become permeable to naked DNA, which enter into plastids and integrate into genome Now PEG & cation are removed from system and transformed plastids are transferred to media for further regeneration and selection Heifetz (2000) Biochimie 82:655-666
- 17. Microinjection mediated transformation Syringe Mitochondria Chloroplast Nucleus Nuclear genome Knoblauch et al (1999) Nature Biotech 17
- 19. Selectable Markers Marker Selection organism Photosynthesis atpB, psaA/B, petB Photoautotrophy Chlamydomonas petA, rpoA, rbcL Photoautotrophy Tabacco Antibiotic resistance rrnS Spectinomycin- Streptomycin Chlamydomonas, Tabacco, Tomato rrnL Erythromycin Chlamydomonas aadA Spectinomycin- Streptomycin Commonly used for most of the crops nptII Kanamycin Tabacco, Cotton
- 20. Marker Selection organism Herbicide resistance psbA Metribuzin Chlamydomonas Bar Phosphinothricin Tobacco EPSPS Glyphosate Tobacco Metabolism BADH Betaine aldehyde Tobacco Negative selectable marker codA 5- fluorocytosine Tobacco Day and Clermont (2011) Plant Biotech J 9:540-553
- 21. Selection of Transplastomics Bock(2001) J Mol Biol 321:425-438
- 22. Transformation Selection Subcloning Selection Subcloning Heteroplasmic Homoplasmic Selection propagation Selection propagation Selection propagation Heteroplasmic Heteroplasmic Chimeric Homoplasmic Non- chimeric Day and Clermont (2011) Plant Biotech J 9:540-553
- 23. Confirmation of Transplastomics Using PCR primers In- Vitro screening Southern Blot Analysis Northern Blotting Western Blotting
- 24. Gene containment Why Plastid Transformation is preferred over Nuclear transformation ?? Mogensen (1996) Am J Bot 83: 383-404
- 25. Mode of Integration and Position effect NHEJ HR HR Donor DNA template Premature stop codon Indel mutation Homologous sequence A Homologous sequence B Promotor mg goi Plastid DNA Transformation Vector Homologous sequence A Homologous sequence B DNA DSB
- 26. Gene Silencing Cerutti (2003) Trends in genetics 9:1 NH2 NH2 NH2 NH2 NH2 NH2 NH2 Ribosome Promotor RNA polymerase 5’ 5’ 5’ Sence transgene Inverted repeat transgene DNA methylation Aberrant RNA Repressive chromatin structure dsRNA dsRNA SiRNA SiRNA RNA synthesis RNA synthesis Dicer Activated RISC Nucleus Cytoplasm RNA degradation RNA degradation Activated RISC like? Short or long dsRNA Primer independent RdRP? Primer dependent RdRP?
- 27. Transgene copy number and Level of Gene expression genes
- 28. Toxicity of Foreign Protein Toxic proteins accumulating within cytosol might results in serious pleiotropic effects Adverse effect of transgenic proteins can be minimized by chloroplast compartmentalizati on Epigenetic effects Homogeneity at ploidy level Epigenetic effects are not observed Lines are Homoplasmic. Homoplasmy achieved through repetitive selection and regeneration Epigenetic effects are observed Either hetrozygous or homozygous. Homozygosity achieved either by selfing or crossing Nucleus transformation Chloroplast transformation Ahmad et al (2016) J Experimental Botany 67: 5945-5960
- 29. Gene Site of Integration Efficiency of expression Enhanced traits Crop References cry9Aa2 trnI/trnA 10% TSP Resistance to potato tuber moth Tobacco Chakarbarti et al (2005) RC1011 trnI/trnA >17% TSP Resistance to TMV Tobacco Lee et al (2012) TCY Prs14/trnG NR Cold tolerance Tobacco Lu et al (2013) y-TMT trnI/trnA >7.7% TSP Increased salt tolerance Tomato Jin et al (2014) Spo, cys, chi trnI/trnA 5.9% TSP Biotic and Abiotic stress Tobacco Chen et al (2014) Traits of Interest Resistance to Biotic and Abiotic stress
- 30. Production of Vaccine antigens and Biopharmaceuticals Traits Gene Expression Host plant References Dengue virus DENV 0.8-1.6% TSP Tobacco Oey et al (2009) Hepatitis virus coat protein Cp 0.1% TSP Tobacco Madesis et al (2010) Polio virus VP1 4-5% TSP Tobacco Lakshmi et al (2013) HIV gp120 16ug g-1FW Tobacco Morgenfeld et al (2014) Tuberculosis antigen CTB-ESAT6 0.75% TSP Lettuce Gorantala et al (2014) CTB-tb72F 7.5% TSP Tobacco Gorantala et al (2014) Human proinsulin Pins-protein A 0.2% TSP Tobacco Yarbakht et al (2015)
- 31. Production of Biomaterial/Enzymes Enzymes/ Biomaterials Gene Crop Yield Reference Polyhydroxybutyr ate Phb Tobacco 18.8% TSP Peterson and Bock (2011) Endo- gluconase celB Tobacco 60% TSP Madanala et al (2015) Xylanase Xyn Tobacco 35% TSP Madanala et al (2015) Cellulases cel6B Tobacco 5-40% TSP Espinosa et al (2015) Elastin derived polymer Ep121 Tobacco Not detected Castigalia et al (2016)
- 32. Metabolic Engineering Trait Genes Target site Crop References Beta carotein Lycopene beta cyclase gene trnfM- trnG Tomato Apel and Bock (2009) Vitamin E TCY, TMT trnfM- trnG Tomato Lu et al (2013) Astaxanthin BKT, BHY rbcL- accD Tomato Harada et al (2014) Rubisco efficiency L8 A S8 t atpB- accD Tobacco Whitney et al (2015) Artemisinin production Nine genes were incorporated trnfM- trnG Tobacco Fuentus et al (2016)
- 33. Phytoremedation Mercuric ion reductase (merA) Organgomercurial lyase (merB) Both helps to improve the capacity accumulate more mercury without harmful effect Production of biofuels Production of biofuel from lingocellulosic material is limited. E.g. Xylanase 10 expressed in tobacco chloroplast for to maximize the conversion of methyl glucuronoxylan to fermentable sugars Adem et al (2017) Plant Methods 13:30
- 34. Limitations of Chloroplast Transformation Narrow range of Transformable plant species Poor expression of transgenes in non- green plastids Transgene containment in the chloroplast genome is not absolute Limited availability of inducible gene expression system Absence of Glycosylation Stability of protein Ahmad et al (2016) J Experimental Botany 67: 5945-5960
- 35. trnfM trnG Patp goi aadA Prrn TpsbA Trps 16 Sac I NcoI XbaI Eco RI Spel Spel
- 37. Why and How??
- 38. Reasons to remove Markers Potential metabolic burden imposed by high levels of marker gene expression Spread of antibiotic resistance genes from plant to bacteria then to pathogenic bacteria to create ‘Super bugs’ resistance to antibiotics Unintended consequences of protein product of antibiotic resistance genes on metabolism of plant, their toxicity and Allergenicity Day and Clermont (2011) Plant Biotech J 9:540-553
- 39. aadA Direct DNA repeats Alignment Lost Recombination Marker Free Recombination between direct repeats results in excision Day and Clermont (2011) Plant Biotech J 9:540-553 Markers Elimination Systems aadA free chloroplast DNA (herbicide resistance) aadA and bar free chloroplast DNA 418 bp 3’UTR 174 bp promotor gusA gusA aadA bar bar rbc L rbc L rbc L accD accD accD
- 40. Isolation of marker free plants using transient co- integration of the maker gene
- 41. Cre- Lox site- specific recombinase Maliga (2002) Plant Biology 5:164-172
- 42. Chloroplast Nucleus Int goi mg mg goi TP-ptDNA TP-ptDNA Marker gene excision by phage Int/att site- specific recombinase Lutz and Maliga (2007) Plant Biotech 18:107-114
- 43. Conclusion • Plastids has pivotal role in life- sustaining • Biolistic and PEG treatment is most common techniques used in chloroplast transformation • Absence of gene silencing & position effect, high expression, uni-parental inheritance and site directed integration of gene are important characteristics of chloroplast transformation • Int/att and Cre/loxP are most efficient methods for making marker free transplastomes
- 44. Although the technology to obtain marker free transplastomic plants is available, no transplastomic crops are yet grown commercially Thank You