CHLOROPLAST TRANSFORMATION

Ekatpure SachinMSc. Agril. Plant Biotech

• photosynthetic chloroplasts, starch-storing amyloplasts, colorful chromoplasts of fruit

• Site of photosynthesis, the biosynthesis of amino acids, fatty acids, vitamins, etc.

• the consequence of an endosymbiotic event between a eukaryotic host cell and an ancestor of the cyanobacteria

• have their own genetic systems, and their own genomes

Plastid (1)

• 1 to 900 chloroplasts per plant cell• ~10,000 cpDNA per cell• CpDNA is packed into discrete structures called chloroplast nucleoids• genome size :

- 30kb – 201 kb- variat ion in length mainly due to presence of inverted

repeat (IR)- General ly 100-250 genes : gene expression,

photosynthesis, metabolism

Plastid (2)

IR IR

SSC

LSC

• The risk of transgene escape natural containment because of lack of pollen transmission

• Low expression level high levels of transgene expression because of the high copy number of the plastomes foreign protein accumulation of up to > 30% of TSP

• gene silencing absence of posit ion effects due to lack of a compact chromatin structure and efficient transgene integration by homologous recombination

Why genetically engineer chloroplasts?

Science, 1999; p. 886

• codon usage virtual ly any bacterial gene can be inserted

Production of polyhydroxybutyrate by polycistronic expression

• diff iculty of gene stacking multiple transgene expression due to polycistronic mRNA transcription

. Method for DNA delivery

Biolistics DNA delivery PEG-mediated transformation Agrobacterium transformation Microinjection

• biological + ball ist ic• Using high-pressure He gas as propellant• Tungsten or gold particles• Leaves, cotyledons, or cultured cells

Biolist ics DNA delivery

PEG-mediated transformation

Galistan expansion femtosyringe

• This is a novel approach involves the microinjection of DNA into chloroplast (Knoblauch et al., 1999) and is not widely used.  • The heat-induced expansion of a l iquid metal, gal istan, within a glass syringe forces the transformation plasmid DNA through a capi l lary t ip with a diameter of approximately 0.1 mm.

• expose the protoplasts to purif ied DNA in the presence of PEG• regeneration step required

Agrobacterium-mediated transformation • unsuccessful as yet

transformation of the chloroplast 1988 : - putting the foreign genes into chloroplast genome

Late 1990 : several biotech companies have initiated major programmes on chloroplast transformation

1998 : Chloroplast transformation has been touted as a means of “containing”

transgenes; that is, preventing them from transferring to non-GM crops or wild relatives through pollen, and hence preventing the creation of transgenic herbicide tolerant weeds. The theory is that chloroplasts are inherited exclusively through the female line.

Milestone of chloroplast transformation

Year Milestone DNA delivery

Approach Selection Reference

1988 Chlamydomonas reinhardtii1st stable plastid transformation

Biolistic Homologous targeting

Photosynthetic competence

Boynton & Gillham (Science, 240)

1990 Nicotiana tabacum1st stable plastid transformation

Biolistic Homologous targeting

Spectinomycin (rrn16)

Svab et al (PNAS, 87)

1993 Nicotiana tabacum1st high level foreign protein (2.5% GUS)

PEG Homologous targeting

Spectinomycin Kanamycin

Golds et al (Biotech. 11)O’Neill et al (Plant J. 3)

1995 Nicotiana tabacumNew agronomic trait: B. thruingiensisMarker gene elimination: co-transformation

Biolistic Homologous targeting

Spectinomycin McBride et al (Biotech. 13)Carrer and Maliga (Biotech. 13)

1998 Arabidopsis thaliana1st stable plastid transformation

Biolistic Homologous targeting

Spectinomycin Sikdar et al (Plant Cell Rep. 18)

1999 Solanum tuberosum (potato)1st stable plastid transformationOryza sativa (rice)1st stable plastid transformation

Biolistic Homologous targeting

Spectinomycin Sidorov et al (Plant J. 19)Khan and Maliga (Nat. Biotech. 17)

Year Milestone DNA delivery

Approach Selection Reference

2000 Nicotiano tabacum1st human protein expression

Biolistic Homologous targeting

Spectinomycin Staub et al (Nat. Biotech. 18)

2001 Lycopersicon esculentum (tomato)1st foreign protein in fruitMarker gene elimination: CRE-lox New agronomic traits: glyphosate tolerance and PPT resistance

Biolistic Homologous targeting

Spectinomycin Ruf et al(Nat. Biotech. 19)Corneille et al (Plant J. 19)Ye et al (Plant J. 25)Lutz et al (Plant Physiol. 125)

2002 Porphyridium sp.1st stable plastid transformation

Biolistic Homologous targeting

Spectinomycin Lapidot et al (Plant Physiol. 129)

2003 Chlamydomonas reinhardtii : Foot-and-mouth disease virus VP1 protein expressionBrassicacea (oil seeds)1st stable plastid transformationPhytoremediation: Mercury

Biolistic Homologous targeting

Spectinomycin Sun et al (Biotechnol Lett. 25)Skarjinskaia et al (Transgenic Res. 12)Ruiz et al (Plant Physiol. 132)

2004 Gossypium hirsutum (cotton)1st stable plastid transformationGlycin max (soybean)1st stable plastid transformationLinum usitat issimum L. (f lax):PHB polymer expression

Biolistic Homologous targeting

aph A-6 npt I ISpectinomycin

Kumar et al (PMB. 56)Dufourmantel et al (PMB. 55)Wrobel et al (J. Biotech. 107)

1. A chloroplast specif ic expression vector.

2. A method for DNA delivery through a double membrane of the chloroplast.

3. An efficient selection for the transplastome.

Chloroplast transformation requires:

Annu. Rev. Plant Bio. (2004) P. Maliga

Sort ing ptDNA at the organelle and cellular levels

How are chloroplasts transformed?

• Leaf discs are bombarded with plasmid constructs containing a selectable antibiotic resistance marker physically linked to the gene of interest, flanked by DNA for inserting into the correct site of the chloroplast genome. The antibiotic resistance marker most frequently used is the aadA gene encoding resistance for spectinomycin and streptomycin, driven by the promoter of the chloroplast encoded 16S rRNA gene.

• this transformation procedure applied to tobacco, Arabidopsis or oil seed rape, generates plants in which all the chloroplast genomes are uniformly transformed (a condition referred to as homoplasmic), despite the fact that tobacco leaf cells may contain 100 chloroplasts, each containing 100 copies of the chloroplast genome.

• Transformation of the chloroplast genome by bombarding tobacco leaves with microprojectiles coated with DNA. Following bombardment, leaf discs are placed onto antibiotic-containing medium (panel A). Transgenic plants are regenerated from the transformed tissue that is able to develop green chloroplasts (panel B)

CHLOROPLAST TRANSFORMATION

ChloroplastDNA

DNA of interest

Chloroplast DNA

Tsung Jeng (Kent) Chen

March 19, 2009

kentchen@interchange.ubc.ca

Gene Gun Homologous Recombination

Into chloroplast

Pros • Much better chance of localizing transgene compared

to when transgenes are in nuclear genome– Maternal inheritance of chloroplasts: no transgene in

pollens

Cons• Not suitable for all crops

– Some crops have biparental or paternal inheritance of chloroplasts • Will need to scren lines to ensure paternal

transmission of chloroplasts does not occur

References

Maliga, P. 2004. Plastid Transformation in Higher

Plants. Annual Review Plant Biology 55: 289-313.

1. A chloroplast specif ic expression vector• Depends on the integration of the foreign DNA into the chloroplast genome by homologous recombination.

• > 400 bp of homologous sequence on each side of the construct is generally used to obtain chloroplast transformants at a reasonable frequency.

• Chloroplast-specif ic promoters and termination signals.

• transcribed as operons, which allows more than two ORFs to transcribe under the same promoter.

• the selectable marker and the gene of interest are placed between the promoter and the terminator which are f lanked by the 5’ and 3’ untranslated regions.

Maliga, 2002

Prrn

PpsbAPrbcL

ggagg

psbA

rbcL

atpB

Bt

aadA

NptII

AphA-6Badh

psbA

rbcL

petD

rps16

P 5’ SM 3’UTR P 5’ GOI 3’UTRLTR RTR

LTR/RTR LTR/RTRtrnI/ trnA 3’rps12/trnV

trnN/trnR Ych3/trnS

trnG/trnfM rbcL/accD

trnH/pbA ndhB/rps7

rrn16/trnI 5’rps12/clpP

trnV/rrn16 petD/rpoA

rpl32/trnL petA/psbJ

Intergenic regions

• do not have special propert ies• may derive from any part of plastid genome• 14 intergenic regions

rrnP psbA T 3’16S 3’

pCtVG04

aadA

RBS

GFP trn Atrn I

trnI rrnP aadARBS

GFP psbA T 3’ RclpP S. PEPC HCE T trnA16S 3’

pCtVG04-SyPEPC

• Another advantage of chloroplast transformation is that foreign genes can be over-expressed, due to the high gene copy number, up to 100 000 compared with single-copy nuclear genes. And there does not seem to be gene-silencing and other instability that plague nuclear transformation. The gene product is retained inside the chloroplasts or can in principle be targeted to a specific compartment in the

chloroplast.

• Chloroplast transformation involves homologous recombination

• In practice, the inserted transgene has short DNA sequence tails added at each end, the tails are homologous to sequences on the chloroplast target gene, which thus initiate homologous recombination. Once the transgene is inserted into the chloroplast chromosome, the target gene is disrupted. The disruption of the target gene is expected to alter the growth and metabolism of the plant

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