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Chloroplast genome engineering


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biology and biotechnology of plastome engineering, presented at Shahid Beheshti University, Tehran, IRAN

biology and biotechnology of plastome engineering, presented at Shahid Beheshti University, Tehran, IRAN

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  • 1. ‫ﺑﺴﻢ ﺍﷲ ﺍﻟﺮﺣﻤﻦ‬ ‫ﺍﻟﺮﺣﻴﻢ‬
  • 2. Chloroplast Genome Engineering Biology and Biotechnology Seyed Javad Davarpanah Faculty of Bioscience, Shahid Beheshti University June 19, 2010
  • 3. Chloroplast genetic system  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)
  • 4. Chloroplast Genome Structure Euglena Pea Typical Chloroplast Genome Exception
  • 5. Higher plants plastome structure
  • 6. Minicircle Structure in Dinoflagellates Circular DNA molecules ranging in size 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 orientation Typical coding minicircles regarding the core region Core region may function as replication origin or promoter Other Minicircles: Empty, Chimeric minicircles, Jumbled minicircles and Microcircles
  • 7. Nuclear transformation  Biosafety risk of gene flow to the environment superweed production pollen poisoning for non-target insects  Stability of expression of transgene transgene silencing (TGS) and (PTGS)
  • 8. Chloroplast transformation  Expression level of foreign genes is higher than nuclear transformation; 5–80 (Chlamydomonas) or 500–10,000 (N icotiana) 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
  • 9. 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 raphanistrum into Brassica napus (oilseed rape) occurred at higher rates than the reciprocal cross of Brassica napus pollen into Raphanus raphanistrum.
  • 10. Stable transplastomic plants • 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
  • 11. Plastid transformed plants
  • 12. Basics of Chloroplast Transformation Chloroplast Transgenic Production Homologous Recombination Homoplasmy Process
  • 13. Chloroplast transformation techniques Biolistic delivery systems Polyethylene 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 skills Femtoinjection technique: injection of DNA material into chloroplasts using syringes with extremely narrow tips Agrobacterium-mediated plastid transformation: • Two preliminary and thus far unconfirmed reports
  • 14. Particle Delivery System
  • 15. Advantages and disadvantages of biolistic method  Relatively high efficiency  Technical simplicity  Potential for mechanical shearing of large plasmids during particle preparation or delivery  Chemical attack by tungsten (a reactive transition metal) which can promote modifications or cleavage of DNA
  • 16. 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.
  • 17. Galinstan Expansion Femtosyringe (GEF) Chl autofluorescence GFP fluorescence overlay of both channels marginal mesophyll cells of tobacco leaf Ex: Phormidium laminosum, bla gene: spectinomycin gfp gene under the control of a chloroplast rRNA promoter
  • 18. 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 (aadA gene), which confers resistance against spectomycin and streptomycin,is translationally fused to the gfp gene of Aequorea victoria • 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
  • 19. Reasons to produce marker-free transplastomic plants  Potential metabolic burden imposed by high levels of marker gene expression homoplastomic state :the marker gene product 5% to 18% of the total cellular soluble protein  Shortage of primary plastid selective markers only 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
  • 20. 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
  • 21. Homology-based excision of Marker gene via directly repeated sequences Recognition sequence of site-specific recombinanse
  • 22. Marker gene excision by phage site- specific recombinanses
  • 23. 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=> Int better than Cre
  • 24. Transient cointegration of the marker gene to obtain marker-free plants
  • 25. Cotransformation-segregation
  • 26. 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
  • 27. Comparison of Systems for Production of Heterologous Protein System Overall Production scale-up Product Glycosylation Contamination Storage cost cost timescale capacity quality risks Bacteria Low Short High Low None Endotoxins Moderate Yeast Medium Medium High Medium Incorrect Low risk Moderate Mammalian High Long Very low Very Correct Viruses, prions Expensive cell culture high and oncogenic DNA Transgenic High Very long Low Very Correct Viruses, prions Expensive animals high and oncogenic DNA Plant cell Medium Medium Medium High Minor Low risk Moderate cultures differences Transgenic Very low Long Very High Minor Low risk Inexpensive plants high differences
  • 28. Heterologous genes expressed stably in plastids of tobacco
  • 29. 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 gene Nucleus: suboptimal production of toxin=> toxin resistance Chloroplast:100% mortality of resistant insects 20-30 fold higher Bt prototoxin production
  • 30. Oxyfluorfen resistance • plastomic insertion of the Bacillus subtilis gene encoding protoporphyrinogen oxidase (protox) • a diphenyl herbicide resistant • higher degree of oxyfluorfen resistance
  • 31. 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
  • 32. • 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 plastids Rubisco, Reaction Center proteins rbcL of Synechococcus: mRNA production but no protein or enzyme activity
  • 33. Engineering of plastid metabolism  Site-directed mutagenesis of Rubisco o deletion of rbcL, replacement with chimeric plastid targeted LSU o rbcL replacement with cyanobacterial homologues: no translation  Plastid reverse genetics • function of several chloroplastic open reading frames (ORFs) ycf1,ycf2,ycf9 transplastomics: all lines heteroplasmic ycf9 ORF: stabilisation of LHC ycf6: involved in construction of cyt b6f complex • functioning of plastidic RNA functioning of plastidic RNA endonuclease • chloroplast structure and physiology only partly suffered from knocking out plastid-encoded RNA polymerase
  • 34. 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
  • 35. Thanks for your patience