chloroplast genomics and biotechnology
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chloroplast genomics and biotechnologyTRANSCRIPT
Chloroplast Genomics and Biotechnology
Submitted by: Nidhi Singh
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
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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)
• 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 .
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Chloroplast evolution Source:cps.ci.cambridge.ma.us
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Plastid differentiationSource: biofortified.org 6
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
• First chloroplast transformation, 1988, by Boynton and Gillham , in
Chlamydomonas.
• First chloroplast transformation in higher plants, 1990, by Pal Maliga &
coworkers, in Tobacco.
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(Pal Maliga, 2004)
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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
• 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
10/12/2010Rice chloroplast DNASource: shigen.nig.ac.jp 11
(Adapted from: TRENDS in Biotechnology, May 2005 Vol.23 No.5) 10/12/2010 12
Different Transformation system• Direct DNA delivery methods such as particle gun or Biolistic method.
• Microinjection techniques.
• Embryogenesis.
• Organogenesis via protoplast or leaves.
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(Dheeraj Verma et al., 2007)
10/12/2010 14(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
10/12/2010 15(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143,)
e
10/12/2010 16(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
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 .
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(Ralph Bock, 2001)
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(Adapted from : Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
Analysis of DNA isolated from putative transplastomic shoots
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PCR analysis :Lane 1: untransformed plantLanes 2 to 4: transformed lineslane 1kb+: DNA markerLanes 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)
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)
Strategy
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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)
Transgenic Chloroplast in Biotechnology
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(Adapted from: Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
10/12/2010 25(Adapted from : Plant Physiology, December 2007, Vol. 145, pp. 1129–1143)
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
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Case study
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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.
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Materials and methods
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• 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.
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Optimization of the culture system for efficientregeneration of rapeseed
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Construction of rapeseed chloroplast transformationvector
PCR analysis of transgene integration into the rapeseedchloroplast genome
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Analysis of the chloroplast vector cassette in transformedrapeseed by Southern blot hybridization
Northern blot analysis of aadA mRNA in the transplastomicplants
Inheritance of the transplastomic aadA gene
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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.
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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.
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DISCUSSION
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