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biology and biotechnology of plastome engineering, presented at Shahid Beheshti University, Tehran, IRAN
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بسم اهللا الرحمن الرحيم
Chloroplast Genome
Engineering
Biology and Biotechnology
Seyed Javad DavarpanahFaculty of Bioscience, Shahid Beheshti University
June 19, 2010
A 50-290 kb double stranded circular molecule
A pair of 20-30 kb inverted repeat (IR) sequence
Prokaryotic protein synthesis machinery
100 chloroplasts per mesophyll cell and 100 genome copies per chloroplast (100 x 100 = 10,000 genome copies per cell)
Chloroplast genetic system
Chloroplast Genome Structure
Typical Chloroplast Genome Exception
Euglena
Pea
Higher plants plastome structure
Minicircle Structure in Dinoflagellates
Typical coding minicircles
Circular DNA molecules ranging insize between 2.2-3.8 kb
Around 14 genes –Mostly one gene in a minicircle (one gene - one circle or two genes - one circle )
250-500 bp non-coding core region
Gene (s) always in the same orientationregarding the core region
Core region may function as replication origin or promoter
Other Minicircles:Empty, Chimeric minicircles, Jumbled minicircles and Microcircles
Nuclear transformation
Biosafetyrisk of gene flow to the environmentsuperweed productionpollen poisoning for non-target insects
Stability of expression of transgenetransgene silencing (TGS) and (PTGS)
Expression level of foreign genes is higher than nuclear transformation; 5–80 (Chlamydomonas) or 500–10,000 (Nicotiana) DNA copies per cell
Multiple genes can be introduced as an operon
No risk of transgene escape – environmentally friendly
No position effect
No transgene silencing
Sequestration of foreign proteins in the organelle
Chloroplast transformation
Advantages of transplastomic plants
Transgenic pollen toxic to non-target insects of 60 major crop plants, only 11 have no wild relatives
No gene escape to WT (exceptions being alfalfa and possibly rice and pea => No WT insensitiveness to herbicides
Introgression of WT genes to transplastomic is in general in unusual
introgression of the common weed Raphanus raphanistruminto Brassica napus (oilseed rape) occurred at higher rates than the reciprocal cross of Brassica napus pollen into Raphanus raphanistrum.
• Transformation of plastids has already been achieved for tobacco , Arabidopsis, soybean , cotton, lettuce, cauliflower, poplar and potato
• The cereal crops rice, maize and wheat continue to be recalcitrant
• plastid-mediated molecular pharming will lead to the biofabrication of a range of biopolymers and pharmaceutical proteins
Stable transplastomic plants
Plastid transformed plants
Basics of Chloroplast Transformation
Chloroplast Transgenic Production Homologous Recombination Homoplasmy Process
Chloroplast transformation techniques
Biolistic delivery systemsPolyethylene glycol (PEG) treatment of protoplast• For unknown reasons, the technique has a lower success rate
than biolistic bombardment• long selection times required after initial DNA delivery• technically demanding and requires specialized tissue culture
skillsFemtoinjection technique: injection of DNA material into
chloroplasts using syringes with extremely narrow tipsAgrobacterium-mediated plastid transformation:• Two preliminary and thus far unconfirmed reports
Particle Delivery System
Advantages and disadvantages of biolistic method
Relatively high efficiency
Technical simplicity
Potential for mechanical shearing of large plasmidsduring particle preparation or delivery
Chemical attack by tungsten (a reactive transition metal) which can promote modifications or cleavage of DNA
Advantages of femtoinjection technique
Cells survive the injection
Transformed cell can be spotted easily
Cellular context remains intact
• The fate of the inserted gene or gene products to be followed.
Galinstan Expansion Femtosyringe (GEF)
Ex: Phormidium laminosum, bla gene: spectinomycingfp gene under the control of a chloroplast rRNA promoter
Chl autofluorescence GFP fluorescence overlay of both channels
marginal mesophyll cells of tobacco leaf
Reporter gene strategies
• Gene coding for the green fluorescent protein (GFP)• Resistance genes against lethal agents (e.g. spectinomycin and
streptomycin)• disadvantage of resistance marker genes: transformed cells must be
traced by stringent methods• Vectors carrying the bacterial gene aphA-6, coding for an
aminoglycoside phosphotransferase that detoxifies kanamycin or amikacin
• FLARE-S system, the aminoglycoside 3′′ adenyltransferase (aadAgene), which confers resistance against spectomycin and streptomycin,is translationally fused to the gfp gene of Aequoreavictoria
• In the case of an optical marker like GFP, difficulties arise with the regeneration of a plant from a single GFP-expressing cell
Reporter gene strategy: genetic contamination problems
Reasons to produce marker-free transplastomic plants
Potential metabolic burden imposed by high levels of marker gene expressionhomoplastomic state :the marker gene product 5% to 18% of the total cellular soluble protein
Shortage of primary plastid selective markersonly genes that confer resistance to spectinomycin and streptomycin (aadA) or kanamycin (neo or kan and aphA-6
Opposition to having any unnecessary DNA in transgenic crops, especially antibiotic resistance genes
Approaches for production of marker free transplastomic plants
Homology-based excision via directly repeated sequences
Excision by phage site-specific recombinanses
Transient co-integration of the marker gene
Cotransformation-segregation approach
Homology-based excision of Marker gene via directly repeated sequences
Recognition sequence of site-specific recombinanse
Marker gene excision by phage site-specific recombinanses
Marker gene excision by phage site-specific recombinanses
1-transplastomics carry marker gene flanked by two directly oriented recombinase target sites
2-removal of marker gene by introduction of a gene encoding a plastid-targeted recombinase in the plant nucleus
• recombinases (Cre and Int)• absence of homology between the attB and attP sites
and the absence of pseudo-att sites in ptDNA=> Intbetter than Cre
Transient cointegration of the marker gene to obtain marker-free plants
Cotransformation-segregation
New marker genes applying RNA editing in plastids
conversion of specific C nucleotides to U in plastids
Mediated by a nuclear encoded complex
Some plastid genes (e.g., psbL, ndhD, rpl2) the start codon is
encoded as ACG and must be edited to AUG
=>constructing new selectable marker gene only expressible
when integrated into the plastome
System Overall cost
Production timescale
scale-up capacity
Product quality
Glycosylation Contamination risks
Storage cost
Bacteria Low Short High Low None Endotoxins Moderate
Yeast Medium Medium High Medium Incorrect Low risk Moderate
Mammalian cell culture
High Long Very low Very high
Correct Viruses, prions and oncogenic DNA
Expensive
Transgenic animals
High Very long Low Very high
Correct Viruses, prions and oncogenic DNA
Expensive
Plant cell cultures
Medium Medium Medium High Minor differences
Low risk Moderate
Transgenic plants
Very low Long Very high
High Minor differences
Low risk Inexpensive
Comparison of Systems for Production of Heterologous Protein
Heterologous genes expressed stably in plastids of tobacco
Production of various protein classes
• expression of genes coding for insecticidal proteins or allowing for herbicide resistance
Bacillus thuringiensis (Bt) toxin: the gene (cry1A) coding for the Bt toxin Cry1A(c)
cry2Aa2 Bt geneNucleus: suboptimal production of toxin=> toxin
resistanceChloroplast:100% mortality of resistant insects 20-30 fold higher Bt prototoxin production
Oxyfluorfen resistance
• plastomic insertion of the Bacillus subtilis gene encoding protoporphyrinogen oxidase (protox)
• a diphenyl herbicide resistant
• higher degree of oxyfluorfen resistance
Glyphosate resistance
• EPSPS: a nuclear encoded, plastid targeted enzyme
• Integration of the petunia EPSPS (5-enol-pyruvyl shikimate-3-phosphate synthase) gene into the tobacco plastome
• Overproduction of EPSPS
• Glyphosate resistance
• production of a human somatropin in a soluble biologically active form
• biodegradable protein-based polymers in tobacco• introduce into plants a set of bacterial genes for the
biosynthesis of polyhydroxyalkanoates (PHAs)• PHAs: a class of biodegradable polymers • fermentative production has proven too costly for large-scale
production • Targeting of PHA biosynthetic genes from Ralstonia eutropha• Proteins involved in the metabolic pathways of plastidsRubisco, Reaction Center proteinsrbcL of Synechococcus: mRNA production but no protein or
enzyme activity
Engineering of plastid metabolism
Site-directed mutagenesis of Rubiscoo deletion of rbcL, replacement with chimeric plastid targeted LSUo rbcL replacement with cyanobacterial
homologues: no translation Plastid reverse genetics• function of several chloroplastic open reading frames (ORFs)ycf1,ycf2,ycf9 transplastomics: all lines heteroplasmicycf9 ORF: stabilisation of LHCycf6: involved in construction of cyt b6f complex• functioning of plastidic RNAfunctioning of plastidic RNA endonuclease• chloroplast structure and physiology only partly suffered from knocking
out plastid-encoded RNA polymerase
Requirements for widespread application of chloroplast engineering
the number of plant species to which plastome technology is applicable needs to be increased considerably
the success rate of gene insertion into the plastome has to be increased
the screening protocols must be simplified and become applicable to a large range of plant species
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