Chloroplast genomics and biotechnology

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Chloroplast genomics and biotechnology

  1. 1. Chloroplast Genomics and Biotechnology Submitted by: Nidhi Singh
  2. 2. Outline • Introduction • History • Genome organization • Advantages • Different Transformation system • Molecular biology of Chloroplast Transformation • Chloroplast functional genomics by reverse genetics • Transgenic Chloroplast in Biotechnology • Challenges • Case study • Perspectives 10/12/2010 2
  3. 3. INTRODUCTION • Chloroplast word derived from Greek word : chloros – green and plast – organelle or cell. • One of the form of a plastid such as Amyloplast for storing starch, Elaioplast for fat storage, Chromoplast for pigment synthesis and storage. • Derived from proplastid. • Present in plant cell and capture light energy from sun to produce free energy via photosynthesis. 10/12/2010 3(R .Bock, 2001)
  4. 4. • Transplastomic technologies allow precise targeted integration of trait genes into chloroplasts without marker genes. • Industrial and therapeutic proteins expressed in chloroplasts accumulate to extraordinarily high levels providing an attractive production platform for manufacture of high-value products for industry and health ,which is both sustainable and carbon-neutral. • Maternal inheritance of chloroplast genes prevents the pollen-mediated spread of transgenes providing a natural form of gene containment for the next generation of Biotech crops . 10/12/2010 4
  5. 5. 10/12/2010 Chloroplast evolution Source:cps.ci.cambridge.ma.us 5
  6. 6. 10/12/2010 Plastid differentiation Source: biofortified.org 6
  7. 7. History • Theory of chloroplast inheritance, 1909, by Erwin baur, in Pelargonium and Mirabilis. • Presence of unique DNA in chloroplast, 1963, by Ruth sagar and Masahiro R. Ishida , in Chlamydomonas. • Circular DNA molecule in chloroplast,1971, by Manning et al., in Euglena. • Physical map of chloroplast,1976, by Jhon R. Bedbrook & Lawrence Bogorad, in Maize. • Physical map of Tobacco chloroplast DNA,1980, by Jurgenson & Bourque. 10/12/2010 (Masahiro Sugiura, 2003) 7
  8. 8. • First chloroplast transformation, 1988, by Boynton and Gillham , in Chlamydomonas. • First chloroplast transformation in higher plants, 1990, by Pal Maliga & coworkers, in Tobacco. 10/12/2010 (Pal Maliga, 2004) 8
  9. 9. Genome organization • Gene content highly conserved. • Semiautonomous organelles. • Highly polyploid genome. • Circular double stranded DNA. • Prokarotically organized. • Most plant genome have two identical copies of 20 to 30 kb inverted repeats separating a large single copy and small copy region. 10/12/2010 (Adrian C. Barbrook et al., 2010) 9
  10. 10. • Chloroplast DNA constitute 10 to 20 percent of total cellular DNA content. • Genome size ranges from Acetabularia spp. with 1.5Mbp size and Ostreoccocus tauri having 86 genes only. • Nucleomorph, feature of some complex chloroplast having retention of eukaryotic nucleus and located between the inner and outer chloroplast membrane. (Adrian C. Barbrook et al., 2010) 10/12/2010 10
  11. 11. 10/12/2010 Rice chloroplast DNA Source: shigen.nig.ac.jp 11
  12. 12. (Adapted from: TRENDS in Biotechnology, May 2005 Vol.23 No.5)10/12/2010 12
  13. 13. Different Transformation system • Direct DNA delivery methods such as particle gun or Biolistic method. • Microinjection techniques. • Embryogenesis. • Organogenesis via protoplast or leaves. 10/12/2010 13 (Dheeraj Verma et al., 2007)
  14. 14. 10/12/2010 14(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
  15. 15. 10/12/2010 15(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143,)
  16. 16. e 10/12/2010 16 (Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
  17. 17. Molecular biology of Chloroplast Transformation • Stable transformation system depends on integration of the transforming DNA into the plastid genome by homologous recombination. • Sequence to be introduced into the plastid genome must flanked on both side by region of homology with the chloroplast genome. • Primary transplastomic event results hetroplasmic cells. • Hetroplasmy is unstable so it will resolve into homoplasmy . 10/12/2010 17 (Ralph Bock, 2001)
  18. 18. 10/12/2010 18 (Adapted from : Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
  19. 19. Analysis of DNA isolated from putative transplastomic shoots 10/12/2010 19 PCR analysis : Lane 1: untransformed plant Lanes 2 to 4: transformed lines lane 1kb+: DNA marker Lanes 5 to 7: transformed lines PCR products of 3P/3M primers PCR product with 5P/2M primers chloroplast genome is probed with a radiolabeled flank fragment Sothern blot analysis: Lane 1: Untransformed plant lanes 2 and 4: homoplasmic transplastomic plant lane 3: heteroplasmic transplastomic plan. (Adapted from : Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
  20. 20. Chloroplast functional genomics by reverse genetics • Reverse genetics: study from genotype to phenotype. • Used for functional characterization of chloroplast genome. • Approaches used: 1. Knock out analysis 2. Site directed mutagenesis • Model system 1. Chlamydomonas reinhardtii 2. Tobacco 10/12/2010 20 (Ralph Bock, 2001) (Adapted from: PLASTID TRANSFORMATION IN HIGHER PLANTS, 2004)
  21. 21. Strategy 10/12/2010 21 (Ralph Bock, 2001)
  22. 22. Arabidopsis Chloroplast 2010 Project • Mutation of gene At1g10310 • A: At1g10310 gene model and two insertion alleles, Rectangles: exons , black rectangle: 5’ & 3’ UTR, Solid lines: introns & intergenic regions, Gray triangle : T-DNA insertion. • B: At1g10310 steady-state mRNA levels in wild-type Columbia (WT Col), a line derived from a wild-type segregant plant (WT Segregant), SALK_125505C (T-DNA), and RIKEN mutant line 15-1699-1 (Ds). • C: Seed fatty acid composition of a line derived from a wild-type segregant plant (WT Segregant), the Columbia ecotype (WT Col), and the two mutant lines, T-DNA and Ds. • Differences between the mutant and the wild type of greater than 15% . 10/12/2010 22(Adapted from: Plant Physiology, February 2010, Vol. 152, pp. 529–540)
  23. 23. Transgenic Chloroplast in Biotechnology 10/12/2010 23
  24. 24. 10/12/2010 24 (Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
  25. 25. 10/12/2010 25 (Adapted from : Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
  26. 26. Challenges • Chloroplast transformation has been accomplished in relatively few species. • Unavailability of the genome sequence. • The chloroplast transformation vectors utilize homologous flanking regions for recombination and insertion of foreign genes. • species-specific chloroplast vectors for transformation of grasses • Its transformation is a tissue culture-dependent process, a better understanding of DNA delivery, selection, regeneration, and progression toward homoplasmy is essential to achieve it in different taxonomic groups 10/12/2010 26
  27. 27. Case study 10/12/2010 27
  28. 28. 10/12/2010 28
  29. 29. Introduction • Rapeseed (Brassica napus L.) is one of the most important oil-producing crops worldwide. • Foreign genes escape through pollen from transgenic rapeseed plants to other closely related species under complicated ecological environment. • Natural crosses occur among B. napus, B. rapa and B. Juncea. • Chloroplast genes inheriting in a strictly maternal fashion, minimizes the possibility of out crossing of transgenes. 10/12/2010 29
  30. 30. Materials and methods 10/12/2010 30
  31. 31. 10/12/2010 31
  32. 32. • Construct rapeseed chloroplast transformation vector pCL308 targets the expression cassette into the trnI-trnA region of the rapeseed chloroplast genome. • Transform the chloroplast by bombardment of cotyledons, two days after bombardment cut it into 0.3x 0.3 cm explants & 80 explants were placed per Petri dish on B5 medium for callus induction. • After 6 to 8 week of culture, spectinomycin resistant calli started to grow. • Shoot producing spectinomycin resistant calli were transferred to a shoot regenerating medium. • Regenerated roots were transferred in to rooting medium. • Transplastomic rapeseed resistant to spectinomycin were selected for transferring onto B5 medium. 10/12/2010 32
  33. 33. Optimization of the culture system for efficient regeneration of rapeseed 10/12/2010 33
  34. 34. 10/12/2010 34 Construction of rapeseed chloroplast transformation vector PCR analysis of transgene integration into the rapeseed chloroplast genome
  35. 35. 10/12/2010 35 Analysis of the chloroplast vector cassette in transformed rapeseed by Southern blot hybridization Northern blot analysis of aadA mRNA in the transplastomic plants
  36. 36. Inheritance of the transplastomic aadA gene 10/12/2010 36
  37. 37. Upshot • A callus induction and regeneration system derived from cotyledonary tissues of elite rapeseed cultivars was developed and successfully used for rapeseed chloroplast transformation. • This chloroplast transformation method may serve as the basis of a method to investigate further integration of genes into the chloroplast genome of rapeseed to breed cultivars with improved agronomic characters. 10/12/2010 37
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  43. 43. 10/12/2010 43
  44. 44. Perspectives • Important task for the coming years will be implementing plastid transformation in the major crops. • Identifying bottlenecks in the recalcitrant species and combining suitable tissue culture systems with efficient molecular tools. • Exploitation of gene maintenance mechanisms in chloroplasts is expected to lead to improvements in transplastomic technologies and the design of transplastomic crops. • Now reached a more mature phase when it is expected to make a broader impact through agricultural and industrial applications. • Biotechnological applications of this new and exciting area of science are underpinned by fundamental research on the genes present in chloroplasts. 10/12/2010 44
  45. 45. DISCUSSION 10/12/2010 45
  46. 46. 10/12/2010 46

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