chloroplast genetic engineering

Download
Share  Add to  Flag Embed
Views:
 
Category: Education
     
 
License:   All Rights Reserved

Presentation Description

No description available.

Comments

 

By: shef2004 (7 month(s) ago)

sir pls send me this informative slide at my email id sstinurocks88@gmail.com

 

By: shef2004 (7 month(s) ago)

sir pls send me this informative slide at my email id sstinurocks88@gmail.com

By: nishat420 (8 month(s) ago)

plz Provide your Email id so that i can send it directly to you....

 
By: vidyalatha.kolli (2 month(s) ago)

hii sir can u provide the above ppt, i am intrested to do project in the chloroplast genetic eng strea ur ppt will help me alot plz can u send me my mail id is vidyalatha.kolli@gmail.com

 
 

By: vmurali1 (11 month(s) ago)

very informative

 

By: ashirin_muni (17 month(s) ago)

very nice presentation

Presentation Transcript

Slide 1: 

Chloroplast Genetic Engineering: Recent Advances and Future Perspectives Sandeep Yadav 2009BS125M

Slide 2: 

Introduction of Plastid (Chloroplast) Why genetically engineer chloroplasts? Milestone of chloroplast transformation How are chloroplasts transformed? Applications for chloroplast genetic engineering Limitations and future directions of chloroplast genetic engineering Contents

Slide 3: 

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

Slide 4: 

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 - variation in length mainly due to presence of inverted repeat (IR) - Generally 100-250 genes : photosynthesis, metabolism Plastid (2)

Slide 7: 

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 position effects due to lack of a compact chromatin structure and efficient transgene integration by homologous recombination Advantages : Science, 1999; p. 886

Slide 8: 

codon usage virtually any bacterial gene can be inserted Production of polyhydroxybutyrate by polycistronic expression difficulty of gene stacking multiple transgene expression due to polycistronic mRNA transcription

Slide 9: 

Milestone of chloroplast transformation

Slide 11: 

Henry Daniell et al 2002 Chloroplast transformation

Slide 12: 

Annu. Rev. Plant Bio. (2004) P. Maliga Sorting ptDNA at the organelle and cellular levels Chloroplast transformation

Slide 13: 

A chloroplast specific expression vector. A method for DNA delivery through a double membrane of the chloroplast. 3. An efficient selection for the transplastome. Chloroplast transformation requires:

Slide 14: 

1. A chloroplast specific 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-specific 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 flanked by the 5’ and 3’ untranslated regions.

Slide 15: 

Benjamin Meyers et al 2010

Slide 16: 

Intergenic regions do not have special properties may derive from any part of plastid genome 14 intergenic regions

Slide 17: 

2. Method for DNA delivery Biolistics DNA delivery PEG-mediated transformation Agrobacterium transformation Microinjection biological + ballistic Using high-pressure He gas as propellant Tungsten or gold particles Leaves, cotyledons, or cultured cells Biolistics DNA delivery

Slide 18: 

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. expose the protoplasts to purified DNA in the presence of PEG regeneration step required Agrobacterium-mediated transformation unsuccessful as yet

Selectable marker genes : 

Selectable marker genes The systems to select the transformed cells, tissues or organisms from the non-transformed ones are indispensable and selectable marker genes are vital to the plant transformation process. Marker genes enable the transformed cells to survive on medium containing the selective agent, while non-transformed cells and tissues die. Antibiotics (kanamycin or hygromycin) and herbicide (phosphinothricin, PPT) are mostly used. Selectable marker genes can be divided into several categories depending on whether they confer positive or negative selection and whether selection is conditional or non-conditional in the presence of external substrates.

Slide 20: 

3. Efficient selections positive selection - aadA : encodes aminoglycoside 3’-adenyltransferase (AAD), confers resistance to aminoglycoside type antibiotics such as spectinomycin and streptomycin. neomycin phosphotransferase (nptII) gene : confers kanamycin resistance. betaine aldehyde dehydrogenase (BADH) gene : produces and enzyme that converts toxic betaine aldehyde to non-toxic glycine betaine. genes that confer resistance to the herbicides phosphinothricin or glyphosate or to the antibiotic hygromycin.

Slide 21: 

negative selection codA - encodes cytosine deaminase (CD) - catalyzes deamination of cytosine to uracil - be utilized to identify seedlings on 5-fluorocytosine-medium from which codA was removed by the CRE-loxP site-specific recombinase reporter genes GFP, GUS

Marker gene elimination : 

Marker gene elimination Cre/lox site-specific recombination. CRE is a phage site-specific recombinase that excises any sequences between two directly oriented 34 bp lox-sites. Nuclear Cre is introduced by Agrobacterium transformation or by pollination, and is subsequently removed by segregation in the seed progeny. Pal Maliga 2002

Slide 23: 

Application: Biotechnology Improvement of agronomic traits Green factory to produce recombinant proteins

Slide 24: 

Foreign gene expression in chloroplasts of higher plants

Slide 26: 

Therapeutic proteins Daniell et al., TIB. 23 (2005)

Application of chloroplast engineering : 

Application of chloroplast engineering Abiotic stresses The unsaturation level of fatty acids (FA) in plant lipids has several implications for the stress tolerance of higher plants as well as for their nutritional value and industrial utilisation. ∆9 desaturase gene, an important gene in lipid metabolic pathways, was transformed into tobacco chloroplast. The transplastomic plants demonstrated the feasibility of using plastid transformation to engineer lipid component in both vegetative and reproductive tissues for increasing cold tolerance. (Craig et al.,2008).

Slide 30: 

Agronomic traits Daniell et al., TIB. 23 (2005)

Research Paper : 

Research Paper Establishment of a Gene Expression System in Rice Chloroplast and Obtainment of PPT-Resistant Rice Plants LI Yi-nü et al. Agricultural Sciences in China 2009, 8(6): 643-651

Slide 34: 

Plasmid pREF, showing the two homologous fragments, ndhF and trnL, in the wild-type rice chloroplast genome. B. Plasmid pRB, showing the integration of the bar gene expression cassette between ndhF and trnL fragments of pREF. Gene bar is expressed under the control of 16S rRNA gene promoter Prrn (16S) and psbA terminator sequence (psbA 3´). The sites of primers (P7 and P8) for PCR assay of transplastomic plants are also shown in the maps Construction of chloroplast transformation vector and localization of foreign gene bar in the vector

Bombardment of gold-coated DNA microprojectiles on rice calli : 

Bombardment of gold-coated DNA microprojectiles on rice calli The parameters set for bombardment in their study were: 28 inches Hg vacuum, 9 cm target distance, 1100 psi helium pressure, and 1.0 μm gold particle diameter.

Slide 36: 

Results

Slide 40: 

Limitations and future directions Improve the efficiency and range of transformable plants - highly efficient only in tobacco - inadequate tissue culture and regeneration protocols of crop plants - Arabidopsis: 1 transplatomic line per 40 – 151 bombardments and infertile - potato: 1 line per 25 bombardments - tomato: 1 line per 10 bombardments - soybean: 1 line per 984 bombardments of embryogenic suspension cultures - rice: highly heteroplasmic and transgene is not carried over to the next generation - maize, barley, wheat, sunflower, corn 2. Expression of transgenes in plastids other than the chloroplast - expression foreign genes in the amyloplasts, leucoplasts, etioblasts, and chromoplast - development of transformation techniques - development of appropriate regulatory sequences functioning in non-green plastids

Slide 41: 

To date, over forty transgenes have been stably integrated and expressed via the tobacco chloroplast genome to confer important agronomic traits, as well as to produce industrially valuable biomaterials and therapeutic proteins. The high expression of recombinant proteins within plastid engineered systems offers a cost effective solution for using plants as a bioreactor. The marker gene elimination systems facilitate the bio-safety of the plastid transformation. Removal of the plastid marker gene will facilitate public acceptance of the new transplastomic crops. The plastid transformation offered a good platform of foreign gene expression in high plants. However, this is only the first step. This technology hasn’t resulted in any product commercialization because problems in the protein purification and the expression level control still need to be solved. Plastid transformation is now routinely carried out only in tobacco while the efficiency of transformation in other plants is still too low. More experiments will be undertaken to move this technology toward practical utilization.

Slide 42: 

Improved biohydrogen production with an expression of codon-optimized hemH and lba genes in the chloroplast of Chlamydomonas reinhardtii

Future perspectives : 

Future perspectives Genetic engineering to enhance mercury phytoremediation Oscar N Ruiz and Henry Daniell Current Opinion in Biotechnology 2009, 20:213–219

Slide 44: 

Mercury is usually released in the metal or ionic form, accumulating in sediments where it becomes methylated by anaerobic sulfate-reducing bacteria to produce methyl mercury, a highly toxic organomercurial compound. Inorganic mercury forms are usually less harmful than organic forms, partly because they bind strongly to soil components that reduce their availability and absorption. On the contrary, organomercurials are highly toxic because of their hydrophobicity, which facilitates their movement across cell membrane and accumulation in membrane bound organelles, inhibiting essential oxidative and photosynthetic pathways. In plants, ionic mercury tends to affect the plasma membrane where it damages membrane transporters such as aquaporins, leading to nutrient and water disruption. Organomercurials rapidly localize to plastids where they accumulate and disrupt important metabolic functions

MerAB engineered via the chloroplast genome : 

MerAB engineered via the chloroplast genome By using chloroplast transformation, multigenetic pathways can be developed in a single transformation event and plastids have retained the genetic machinery from bacterial ancestor, their genomes can transcribe and translate operons. There is no need for codon optimization of bacterial genes for their expression in plastids. Limitation: Unlike transformation of the nuclear genome of different crop species using the same transformation vectors, species-specific vectors are necessary for chloroplast transformation. The native bacterial merA and merB genes were integrated into the tobacco chloroplast genome. The transgenic plants were resistant to very high concentrations of PMA, up to 400 mM. Mercury, especially in the organic form, is targeted to the chloroplast. Mercuric ion reductase functions better in chloroplasts because of the abundance of NADPH.

Slide 46: 

This shows that organic-Hg is transported more efficiently than inorganic forms. The transgenic plants were shown to volatilize Hg0 efficiently, independent of the form of Hg in the soil, confirming that both mercuric ion reductase and organomercurial lyase were active in transgenic chloroplasts. The mer operon has three known membrane transporter genes involved in the process of translocating Hg2+ into the cell: merC, merP and merT. To take advantage of these transport mechanisms, transgenic plants should be modified with the merA gene as well; this should provide resistance against higher levels of transported Hg. Alternatively, the merC can be coupled to a chelator gene like polyphosphate kinase ( ppk) or metallothionein (mt), to develop transgenic plants that could accumulate Hg.

Slide 47: 

First report of the expression of a membrane protein via the chloroplast genome. Overexpression of the membrane protein resulted in massive proliferation of the inner envelope membrane (up to 19 layers in electron micrograph) and this opens the possibility of engineering mercury transporters in plant cells. (Singh ND et al 2008)

Slide 49: 

One public concern regarding the use of the merAB system is the release of Hg0 into the atmosphere. Therefore, an alternative approach would be the sequestration of ionic mercury inside the cell by binding it to a chelating molecule. The bacterial ppk gene coding for polyphosphate kinase synthesized polyphosphates, which are negatively charged; the long phosphate polymers reduced cytotoxicity of heavy metals by chelation

Selectable marker genes commonly used in cereal transformation : 

Selectable marker genes commonly used in cereal transformation