Chloroplast transformation · plastome - DNA is attached to thylakoid membrane (nucleoid) - 15...
Transcript of Chloroplast transformation · plastome - DNA is attached to thylakoid membrane (nucleoid) - 15...
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Chloroplast transformation
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Plastids contain DNA – plastome
- Maternal inheritance
(advantage for biotechnological application)
(Mirabilis japonica, Correns 1909)
- 100 x 100 plastoms/cell
- Prokaryotic origin
- gene transfer
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Gene expression in plastids is procaryotic
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Inheritance in plastids
- Pelargonium:
- biparental
- maternal (most of the angisperms)
- paternal (gymnosperms, Sequoia, Pinus)
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plastome
- DNA is attached to thylakoid membrane (nucleoid)
- 15 nucleoids/plastid, 10 DNA molecules/nucleotid (polyploid)
- circular DNA
- 130 bis 160 kb
- inverse duplication
- small and large single copy region
- loss of inverse duplication e.g. conifers, Papilionaceae
Epiphagus
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The plastome of the holoparasiteEpifagus virginiana issubstantially reduced
Model system for plastidgenetics
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Plastomes of land plants
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Genes of the plastome
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Gene expression in plastids requires pro- and eukaryotic elements
Operons und Introns
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Most of the promoters are procaryotic – but not all of them
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Plastids contain two RNA-polymerases
- Epiphagus: lost the genes for RNA-polymerases, but still contain white plastids
- nuclear-encoded RNA-polymerase - plastid-encoded RNA-polymerase
- phage type - bacterial type- one subunit - ~13 subunits, nuclear- and plastid-encoded
- sigma factors (bacteria-like) - Expression of early genes - Expression of late genes
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psbB operon: complex processing steps
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psbB operon- multiple promotors, multiple transcription start sites
- both strands encode genes
- polycistronic transcripts
- primary transcript is large and unstable
- RNA codes for independent proteins
- transcript ripening, oligocistronic transcripts
- monocistronic transcripts
- specific endonucleases
- Exonucleases: processing of 3´-ends
- hair pin loops stabilizes RNA
- secondary structures prevent degradation
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Editing change plastid transcripts –Hydrolytic deamination of cytidine to uridine
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Most of the transcripts are stable
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Many genes from plastids were transferred to thenucleus
- DNA fragment
- as RNA after reverse transcription (e.g. as edited transcripts)
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Chloroplasts transformation
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Most of the transformationprotocols use protoplasts
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Regeneration is similar to nucleartransformation
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Pt transformation works in monocots - rice
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Problem: generation of homoplastomic lines
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Plastid transformationvectors are based on
homologousrecombination
eventsI
Different from nucleartransformation
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Plastid transformationvectors are based on
homologousrecombination events
II
Careful choice of insertion site
Requirement of flanking sequence for
recombination
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aad as selection marker- use of two selectable markers
- aad: procaryotic expression
- neomycin: eukaryoticexpression
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Reciprocal crosses show maternalinheritance (here: resistance gene)
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Advantages of plastidtransformation I
• Huge production of proteins• Maternal inheritance• Application of eatable plastids:
– Chromoplasts from tomato (no denaturation)– Amyloplasts from potato (boiling)
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Advantages of plastidtransformation II
• Chloroplast gene expression is mainlyregulated posttranscriptionally
• mRNA is present, although proteinsdo not accumulate (photosynthesisgenes in tomato and potato)
• Transformation of chloroplasts, expression in etio- or amyloplasts
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Expression can be >500-fold higherthan in the nucleus
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Gene inactivation identifies the role of plastidencoded proteins: inactivation of plastid RNA
polymerase
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Tomato and potato are crucialplants for plastid transformation
Higher carotenoid level