plastome engineering

By: Kanchan Rawat

Plastids are cellular organelles with circular double

stranded DNA.

Various forms of plastids are Amyloplast for storing

starch, Elaioplast for fat storage, Chromoplast for

pigment synthesis and storage and Choloroplast for

photosynthesis.

Chloroplast origin is prokaryotic.

~900 chloroplasts per

plant cell

Each cell contain ~10,000

identical copies of each

plastid gene.

CpDNA is packed into

discrete structures called

chloroplast nucleoids.

Genome size :

120-160 kb

Each plastid cell contain

120 genes.

Risk of transgene escape: chloroplast genome is maternallyinherited so it provides gene containment thus reduces theescape of transgene.

Expression level: Higher level and multiple transgeneexpression due to polycistronic mRNA.

Homologous recombination: It minimizes the insertion ofunnecessary DNA that accompanies in nuclear genometransformation.

Gene silencing is absent.

Disulphide bond formation and folding human proteinsresults in high level production of proteins.

Nuclear genome Chloroplast genome

Gene silencing results in

decrease or elimination of

transgene expression.

Gene silencing is absent.

Paternal transgene inheritance

results in outcrossing among

crops and weeds.

Maternal gene inheritance in

most crop plants results in

natural gene containment.

Highly variable gene expression. Uniform gene expression.

Each transgene is independently

inserted and transcribed into a

monocistronic mRNA.

Genes transcribed into

polycistronic RNA so that

multiple transgenes can be

introduced and expressed in a

single transformation event.

2 successful methods include biolistics and

polyethylene glycol mediated transfer.

Biolistic DNA delivery is used when the targets in

plastids are intact tissue.

Polyethylene glycol treatment is used for DNA

introduction into protoplasts.

Biolistics is preferred as it is less time-consuming and

demanding.

The plastid genome segments that are included in the vector are

marked as the left (LTR) and right targeting regions (RTR). A

selectable marker gene and gene of interest is inserted in vector.

HSA is synthesized in the liver and functions as a

carrier protein for many exogenous and endogenous

metabolites and drugs.

It accounts for 60% of the total protein in blood

serum.

It is the most widely used intravenous protein in a

number of human therapies.

It is highly susceptible to proteolytic degradation in

recombinant systems and is expensive to purify.

Very low expression levels of HSA were attained

(0.02% tsp) via nuclear transformation.

The annual world need of HSA exceeds 500 tons.

Only source of HSA is blood so there is chance of

transmitting pathogenic viruses.

In addition, good recombinant systems are still not

available for many human proteins that are expensive

to purify or highly susceptible to proteolytic

degradation.

Integration of transgene cassettes into the chloroplast genome.

HSA is driven in all cassettes by the Prrn promoter upstream of the

aadA gene for spectinomycin resistance with additional promoters

and control elements.

Southern blot analysis.

b)Probe P1 and P2 used for southern blotting.

c)Lane 1:untransformed DNA; 2,3 :DNA from plants transformed with

pLDAsdHSA; 4,5: DNA from plants transformed with pLDApsbAHSA.

d)Plants for the first (T0) and second (T1) generation were analysed.

2,4: T0 generation. 3,5: T1 generation.

Analysis of HSA accumulation in transgenic chloroplasts.

(a) ELISA of HSA accumulation in leaves at different stages of development.

(b) Study after different hours of illumination. Samples of leaves were collected from

potted plants transformed with pLDApsbAHSA after the 8-h dark period or at

indicated hours in the light.

Colorimetric immunoblot detection of tobacco protein extracts

from mature leaves.

Total protein extracts were loaded in the gel. 1)pure HSA; 2: mw

marker; 3,5: untransformed plant extract; 4: pLDAsdHSA plant

extract; 6: pLDApsbAHSA plant extract.

HSA accumulation into inclusion bodies.

(a–d) EM of immunogold labelled tissues from untransformed (a)

and transformed mature leaves with the chloroplast vector

pLDApsbAHSA (b–d)

HSA extraction from inclusion bodies.

(a) SDS-PAGE gel showing 1: pure HSA; 2: marker; 3,4:soluble

fraction obtained after centrifugation of pLDApsbAHSA transformed

plant extract, 5: HSA after solubilization from the pellet; 6: proteins

from untransformed plant.

Immunoblot detection of protein extracts.

1: pure HSA; 2: HSA from a plant transformed with pLDApsbAHSA

during the solubilization process, showing mono, di and trimeric

forms; 3: proteins from an untransformed; 4: same HSA from lane 2

but in a more advanced stage of solubilization; 5: completely

monomerized HSA after the end of the solubilization treatment.

Plant T1 phenotypes

1,2: untransformed plants; 3: plant transformed with pLDAsdHSA;

4:plant transformed with pLDApsbAHSA.

500 folds higher concentration of HSA was observedthan usual concentration.

11.1% of tsp of HSA was observed as compared to0.02% observed in nuclear transformation.

Inclusion bodies facilitated purification of HSA .

Regulatory elements eg. psbA 5’UTR served as amodel system for enhancing expression of foreignproteins.

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