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.

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• 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.

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• 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

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(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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