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Chloroplast transformation

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Presented by- MD JAKIR HOSSAIN
Doctoral Research Scholar
Department of Agricultural Genetic Engineering ,
Faculty of Agricultural Sciences and Technologies,
Nigde Omer Halisdemir University, Turkey
E. Mail- mjakirbotru@gmail.com

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Chloroplast transformation

  1. 1. Chloroplast Transformation and its Applications to Plant Improvement MD JAKIR HOSSAIN Doctoral Student Department of Agricultural Genetic Engineering , Faculty of Agricultural Sciences and Technologies, Nigde Omer Halisdemir University, Turkey Presented By
  2. 2. What is Chloroplast ?  Chloroplast is a plastid containing chlorophyll and other pigments occurring in plants and eukaryotic algae and which possess their own genome or plastome, besides nuclear genome that carry out photosynthesis. Fig- Diagrammatic Presentation of Chloroplast
  3. 3.  Genetic materials in plants is distributed into nucleus, plastids and mitochondria.  There are up to 300 plastids in one plant cell.  In most angiosperm plant species (80%) plastids are strictly maternally inherited. What is Chloroplast ?
  4. 4. Organization of Chloroplast Genome - Size ~120–150 (30-201) kb that encode ~120 genes, responsible for gene expression, photosynthesis and metabolism  Example- 107 kb in Cathaya argyrophylla , 218 kb in Pelargonium - 1,000-10,000 copies of Genomes in a single cell - In case of Arabidopsis thaliana nucleus encode about 2100 chloroplast proteins and the whole chloroplast genome encodes for 117 proteins.
  5. 5. Organization of Chloroplast Genome  Circular double-stranded DNA through construction of -  complete genome maps,  a large single copy (LSC),  a small single copy (SSC), and duplication of a large (~25 kb) Inverted region (IRs)  The number of copies of plastomes per leaf cell- -1000 to 1700 in Arabidopsis thaliana and - up to 50,000 in Triticum sp.
  6. 6. Why Chloroplast is a Unique Transformation tool ?  Sources of Enormous Advantages -sequestration of carbon, production of starch, and evolution of oxygen, synthesis of amino acids, fatty acids, and pigments, and key aspects of sulfur and nitrogen metabolism  Have diverse functions • Chromoplasts– for pigment synthesis and storage • Gerontoplasts – control dismantling of photosynthetic apparatus during senescence • Leucoplasts –– monoterpene synthesis  An attractive alternative to nuclear gene transformation -High protein levels, the feasibility of expressing multiple proteins from polycistronic mRNAs, and gene containment through the lack of pollen transmission.
  7. 7.  Precursor for Photosynthesis Getaway to high-throughput genome sequencing - more than 230 photosynthetic organisms including 130 higher plants.  An excellent tool for phylogenetic and evolutionary studies -Endosymbiosis -cyanobacterial cell - engulfed by heterotrophic eukaryote -Chloroplast organelle -evolved from photosynthetic bacteria  Storage Compartment for Biosynthetic Pathways. Why Chloroplast is a Unique Transformation tools ?
  8. 8. Why Chloroplast is a Unique Transformation tools ? Protein accumulator - soluble proteins and intrinsic membrane proteins.  Cellular location for valuable recombinant products  Own genetic systems and genomes, high copy number, transcription translation machinery.  Plastid posses prokaryotic gene expression machinery.
  9. 9. Why Plastid transformation is Preferred? • High level of transgene expression and protein accumulation • The possibility of co-expressing several transgenes in operons • The precise transgene integration by homologous recombination . • The feasibility of expressing multiple proteins from polycistronic mRNAs • Regeneration of crop plants with higher resistance to biotic and abiotic stresses and molecular pharming.
  10. 10. • Absence of epigenetic effects • Uni-parental inheritance is commercially favored • Easy transgene stacking in operons • Foreign protein accumulation of up to > 40% of TSP, 70% in Tobacco • Absence of position effects due to lack of a compact chromatin structure • Efficient transgene integration by homologous Why Plastid transformation is Preferred?
  11. 11. Plastid Transformation- Superior than Nuclear Transformation Chloroplast transformation Nuclear transformation Reduced gene dispersal - maternal inheritance Extensive Gene dispersal -parental nature Higher expression and accumulation of foreign proteins Lower expression and accumulation of foreign proteins Efficient multiple gene expression multiple gene expression is very poor Single promoter for expression of multi-subunit complex protein Several promoters for xpression of respective subunits Simultaneous expression of several genes - prokaryotic gene expression system Do not have prokaryotic expression system can’t undergo simultaneous expression of several genes Homologous recombination- avoids position effects and gene silencing Random integration- presents position effects and gene silencing Transgenes is much higher; up to 18%. Transgenes expression of total proteins-0.5 - 3%
  12. 12. Traits Nuclear genome Plastid genome Chromosomes Two copies of each of many chromosomes the number of chromosomes per diploid cell is species -specific ~60 copies of a single circular chromosome per plastid Genes per chromosome Could be thousands ~120–150 Arrangement and operons transcription of genes Each gene is separate (individually transcribed ) Many genes are in ( transcribed together) Comparison of the nuclear and plastid genome
  13. 13. Milestones of Chloroplast engineering Year Milestones DNA Delivery Approach Selection 1988 1st stable plastid transformation in Chlamydomonas reinhardtii Biolistic Homologous Targeting Photosynthetic Competence 1990 1st stable plastid transformation in Nicotiano tabacum Biolistic Homologous Targeting Spectinomycin 1993 1st high level foreign protein expression in Nicotiano tabacum PEG Homologous Targeting Spectinomycin kanamycin 1995 New agronomic traits: Bacillus thuringiensis Marker gene elimination: Co- transformation Biolistic Homologous Targeting Spectinomycin 1998 1st stable plastid transformation in Arabidopsis thaliana Biolistic Homologous Targeting Spectinomycin 1999 1st stable plastid transformation in Solanum tuberosum and 1st stable plastid transformation in Oryza sativa Biolistic Homologous Targeting Spectinomycin
  14. 14. Milestones of Chloroplast engineering Year Milestones DNA Delivery Approach Selection 2000 1st human protein expression in Nicotiano tabacum Biolistic Homologous Targeting Photosynthetic Competence 2001 1st stforeign protein in Lycopersicon esculentum (tomato), Marker gene elimination: CRE-lox, New agronomic traits: glyphosate tolerance and PPT resistance Biolistic Homologous Targeting Spectinomycin 2002 1st stable plastid transformation in Porphyridium sp. PEG Homologous Targeting Spectinomycin 2003 Chlamydomonas reinhardtii: Foot-and-mouth disease virus VP1 protein expression, 1st stable plastid transformation in Brassicacea (oil seeds) and Phytoremediation: Murkery Biolistic Homologous Targeting Spectinomycin 2004 1st stable plastid transformation in Gossypium hirsutum (cotton), 1st stable plastid transformation in Glycin max (soybean), PHB polymer expression in Linum usitatissimum L. (flax) Biolistic Homologous Targeting Spectinomycin Aph-A6 npt 2
  15. 15. Chloroplast transformation requires 1. A chloroplast specific expression vector 2. A method for DNA delivery 3. An efficient selection for the transplastome
  16. 16. key conditions to achieve plastid transformation Generally, three key conditions have to be full-filled to achieve plastid transformation: (1) A robust method of DNA delivery into the chloroplast (2) The presence of active homologous recombination machinery in the plastid, and (3) The availability of highly efficient selection and regeneration protocols for transplastomic cells
  17. 17. Steps in chloroplast genetic engineering • Aseptic growth of plant on MS medium • Biolistic particle treated with vector and other chemicals • Injection of recombinant DNA Plasmid into chloroplast using Gene gun or other methods. • After 2 days, leaves cut into section and transferred to medium containing an antibiotic ,for recombinant selection.
  18. 18. Steps in chloroplast genetic engineering  Green calli formed on the bleached leaf are sub-cultured on the same medium Calli formed shoots  These shoots were rooted on MS medium to obtain plants, express the desired protein.
  19. 19. Presently, both Biolistic and PEG (polyethylene Glycol) treatment of protoplasts have been used to DNA delivery  The first one consists in bombarding of tissue or cells with DNA coated particles.  The second method treats isolated protoplasts with PEG.  Micro injection is also used (femto syringe) METHODS USED IN GENE DELIVERY INTO PLASTIDS
  20. 20. METHODS USED IN GENE DELIVERY INTO PLASTIDS Table 1- Chloroplast transformation methods and selection conditions for different plant species Plant spieces Methods Selection Expressed genes Chlamydomonas reinhardtii Bombardment photosynthetic competence atpB Nicotiana tabacum (tobacco) Bombardment Spectinomycin rrn16 Nicotiana tabacum (tobacco) PEG Spectinomycin rrn16 Nicotiana tabacum (tobacco) Bombardment Kanamycin nptII Nicotiana tabacum (tobacco) Bombardment Spectinomycin uidA Nicotiana tabacum (tobacco) Bombardment Spectinomycin Bar & aadA, Bt Arabidopsis thaliana Bombardment Spectinomycin aadA Solanum tuberosum (potato) Bombardment Spectinomycin aadA & gfp Rice Bombardment Spectinomycin aadA & gfp Lycopersicon esculentum (tomato) Bombardment Spectinomycin aadA Brassica napus (oilseed rape) Bombardment Spectinomycin aadA & cry1Aa10 Gossypium hirsutum (Cotton) Bombardment Kanamycin aphA-6 Glycine max (Soybean) Bombardment Spectinomycin aadA
  21. 21. Fig. Steps in volves in Plant plastid engineering by Gene gun
  22. 22. BIOLISTIC METHODS OF GENE DELIVERY Advantages Simple operation and high efficiency makes it a favorable No need to obtain protoplast as the intact cell wall can be penetrated. This device offers to place DNA or RNA exactly where it is needed into any organism. Disadvantages The transformation efficiency may be lower than Agrobacterium- mediated transformation. Associated cell damage can occur.  The target tissue should have regeneration capacity.
  23. 23. PEG METHODS OF GENE DELIVERY  PEG-mediated transformation of plastids requires enzymatically removing the cell wall to obtain protoplasts, then exposing the protoplasts to purified DNA in the presence of PEG.  The protoplasts first shrink in the presence of PEG, then lyse due to disintegration of the cell membrane. Removing PEG before the membrane is irreversibly damaged reverses the process.  Treatment of freshly isolated protoplasts with PEG allows permeabilization of the plasma membrane and facilitates uptake of DNA.
  24. 24. PEG METHODS OF GENE DELIVERY Plasmid DNA passes the plastid membranes and reaches the stroma where it integrates into the plastome as during biolistic transformation.  A relatively small number of species have been transformed using this approach, mainly because it requires efficient isolation, culture and regeneration of protoplasts, a tedious and technically demanding in vitro technology.
  25. 25. PEG METHODS OF GENE DELIVERY Advantages  A large number of protoplasts can be simultaneously transformed.  This can be successfully used for a wide range of plant species with adequate modifications. Disadvantages  The DNA is susceptible for degradation and rearrangement.  Random integration of foreign DNA into genome may result in undesirable traits.  Regeneration of plants from transformed protoplasts is a difficult task.
  26. 26. Plant species Gene introduced Nicotiana tabacum rrn16, nptII, uidA, hST, cry, cry9Aa2, Bar & aadA, rbcL, DXR, gfp, Cor 15a-FAD7, Delta(9) desaturase , AsA2, PhaG & PhaC Solanum tuberosum aadA & gfp Oryza sativa aadA & gfp Solanum lycopersicon aadA, Lyc Brassica napus aadA & cry1Aa10, aadA Daucus carota dehydrogenase (badh) Gossypium hirsutum aphA-6 Glycine max aadA Lactuca sativa gfp Brassica oleracea gus & aadA Lettuce gfp Brassica oleracea aadA & uidA Beta vulgaris aadA & uidA Solanum melongena aadA Arabidopsis thaliana pre-Tic40-His Zea mays ManA A List of Some Transplastomic Plants that has Engineered for Various Agronomic Traits
  27. 27. Vector Design for Chloroplast Transformation Fig. Basic design of a typical vector for transforming the plastid genome P- Promoter and direction of transcription, T- Terminators, White circles- UTRs, The thin dotted lines with arrows indicate homologous recombination.
  28. 28. Vector Design for Chloroplast Transformation Selectable Marker genes-  Spectinomycin resistance- The most efficient and routinely used  16S rRNA (rrn16) gene- Initially used and selected by spectinomycin resistance with low efficiency.  aadA (aminoglycoside 3′ adenylyltransferase) gene- Dominant marker gene that confers resistance to streptomycin and spectinomycin by inactivation of antibiotics.  Plastid expressed GFP (green fluorescent protein)- a visual marker for identification of plastid transformants at the early stage of selection and shoot regeneration.  The npt II- Transformation efficiency was low, i.e. about one transplastomic line per 25 bombarded samples
  29. 29. Vector Design for Chloroplast Transformation Selectable Marker genes-  neo gene-yielded 34 kanamycin resistant clones out of Bombardment of 25 leaves  The bacterial bar gene, encoding phosphinothricin acetyltransferase (PAT)- it was not good enough  Betaine aldehyde dehydrogenase (BADH) gene- efficiency was 25- fold higher with betaine aldehyde (BA) selection than with spectinomycin in tobacco
  30. 30. Insertion sites-  Insertion of foreign DNA in intergenic regions of the plastid genome had been accomplished at 16 sites, most commonly used insertion sites are - trnV-3'rps12 ,trnI-trnA and trnfM-trnG  The trnV-3'rps12 and trnI-trnA sites- located in the 25 kb inverted repeat (IR) region of ptDNA and a gene inserted into these sites would be rapidly copied into two copies in the IR region. Vector Design for Chloroplast Transformation
  31. 31. Insertion sites-  The trnfM-trnG site- Located in the large single copy region of the ptDNA, and the gene inserted between trnfM and trnG should have only one copy per ptDNA.  The pPRV series vectors- Targeting insertions at the trnV-3'rps12 intergenic region, the most commonly used vectors in tobacco and yield high levels of protein expression  The trnI and trnA genes- These two tRNAs are located between the small (rrn16) and large (rrn23) rRNA subunit genes and the operon is transcribed from promoters upstream of rrn16. Vector Design for Chloroplast Transformation
  32. 32. Vector Design for Chloroplast Transformation Regulatory sequences  The level of gene expression in plastids is predominately determined by regulatory sequences such as promoter as well as 5′ UTR elements .  Strong promoter is required to ensure high mRNA level for high- level of protein accumulation e.g. rRNA operon (rrn) promoter (Prrn).  Most commonly used promoter is CaMV 35S promoter of cauliflower mosaic virus which drives high level of transgene expression in dicots.
  33. 33. Vector Design for Chloroplast Transformation Reporter genes used in plastids  GUS (β-glucuronidase), chloramphenicol acetyl transferase (CAT), and GFP(Green Fluorescent Protien)  The enzymatic activity of GUS can be visualized by histochemical staining  GFP is a visual marker that allows direct imaging of the fluorescent gene product in living cells. GFP has been used to detect transient gene expression. GFP has also been fused with AadA and used as a bi-functional visual and selectable marker
  34. 34. How Chloroplasts are Transformed ? Fig- Sorting ptDNA at the organelle and cellular
  35. 35. How Chloroplasts are Transformed ?
  36. 36. Selection of Transplastomic  Common selection marker used for plastid transformation is the bacterial spectinomycin resistance gene aadA (3´aminoglycoside- adenyltransferase).  Transplastomic clones are identified as green shoots on spectinomycin medium.  Spectinomycin inhibits greening and shoots regeneration of wild type.  After integration, Homoplastomic cells obtained by several rounds of cell division and organelle segregation.
  37. 37. Confirmation of transgene integration into chloroplast genome  Integration of transgenes into the chloroplast genome can be confirmed by PCR using internal primers, first primer anneals to the flanking sequence and second primer anneals to the transgene region.  An expected size of PCR product was amplified and this confirmed integration of the transgenes in different cell cultures of plant  Integration of the transgenes into plastid genome can be investigated by Southern blot analysis.
  38. 38.  Genomic DNA from transformed and untransformed cultures Can be digested with appropriate restriction enzymes, transferred to nitrocellulose membrane and probed with P32- radio-label .  Transformed chloroplast genomic DNA digested with restriction enzymes yielded an expected 3.3 kb size hybridizing fragment. Confirmation of transgene integration into chloroplast genome
  39. 39. Applications of chloroplast Transformation to Plant Improvement  The expression of foreign genes in chloroplasts offers several advantages over their expression in the nucleus: 1. Improvement of Agronomic traits - Biotic stresses or Insect and Diseases resistance - Abiotic stresses or Drought and Salinity tolerance 2. Production of biopharmaceuticals and vaccines in plants 3. Metabolic pathway engineering 4. Research on RNA editing 5. Phytoremediation 6. Production of Industrial enzymes and Biofuels
  40. 40. Agronomic trait development through Chloroplast Transformation Enhanced traits Site of integration Regulatory sequences Transgenes Efficiency of expression Resistance to Phthorimaea operculella trnI/trnA Prrn/ggagg/rbcL Bt cry9Aa2 ~10% of TSP Tolerance to high temperature stress rbcL/accD Prrn/rbcL 3′ panD > 4-fold β-alanine Drought tolerance trnI/trnA Prrn/ggagg/psbA tps1 >169-fold transcript Resistance to herbicide rbcL/rbcL psbA/psbA/3′rbL Hppd 5% TSP Salt tolerance trnI/trnA Prrn/T7 10/rps16 Badh 93–101 μM g−1 FW Cold-stress tolerance prs14/trnG Prrn/T7 g10/TrbcL HTP, TCY, TMT NR Resistance against rice blast fungus trnI/trnA Prrn/Trps16 MSI-99 89.75 μg g−1 FW Broad-spectrum resistance against viral/bacterial/ phloem-feeding insects trnI/trnA 5′psbA/3′psbA Pta 7.1–9.2% TSP Resistance against whitefly and aphid trnI/trnA 5′psbA/3′psbA Bgl-1 >160-fold enzyme
  41. 41. Production of biopharmaceuticals and vaccines in plants  Protein drugs made by plant chloroplasts overcome most of these challenges like expensive fermentation systems, prohibitively expensive purification from host proteins, the need for refrigerated storage and transport.  E7 HPV type 16 protein is an attractive candidate for anticancer vaccine development in Tobacco.  Plastid transformation systems became successful in the oral delivery of vaccine antigens against cholera, tetanus, anthrax, plague, and canine parvovirus.  Above 7.6% Protein accumulation . Example- OspA
  42. 42. Phytoremadiation  Phytoremediation is a safe and cost-effective system for cleaning up contaminated environments using plants.  Two bacterial genes encoding two enzymes, mercuric ion reductase (merA) and organomercurial lyase (merB), were expressed as an operon in transgenic tobacco chloroplasts.  Phytoremediation of toxic mercury was achieved by engineering of tobacco chloroplast with metallothionein enzyme.
  43. 43. Production of industrial enzymes and biomaterials  To produced the highest level of the poly (p-hydroxybenzoic acid (pHBA) polymer (25% dry weight) in normal healthy plants poly hydroxy butyrate (PHB) was designed using an operon extension strategy  To date, the highest levels of PHB have been achieved in plastids due to the high flux of the PHB pathway substrate acetyl-CoA through this organelle during fatty acid biosynthesis
  44. 44. Metabolic Pathway Engineering  Plastid- ‘biosynthetic centre of the plant cell’  The most complex metabolic pathway- synthesis of the bioplastic polyhydroxybutyrate (PHB) , cause male sterility (b-ketothiolase expression ) and severe growth retardation  1st in Tobacco, Recent- in tomato to alter carotenoid biosynthesis towards producing fruits with elevated contents of β-carotene.  Successful example of engineering a nutritionally important biochemical pathway in non-green plastids by transforming the chloroplast genome.

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