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Transplastomics in Vegetable Crops.pptx
- 1. Student J.DELINCE 2015603402 Transplastomics in Vegetable Crops Chairman Dr. T. ARUMUGAM Professor and Head, Dept. of Vegetable Crops Dr. S. GANESHRAM Professor and Head, Department of Plant Genetic Resources Dr. V. PREMALAKSHMI Assistant Professor, Department of Vegetable Crops Members
- 2. Introduction Plastids and Plastomes Milestones in Plastid engineering Methods of gene transfer Selection Validation Case studies Drawbacks
- 3. Transplastomics • Plastome – Genome of the plastids • Transplastomics – transfer of gene into the genome of plastids • A technology for biological containment of genes (Bock and Khan, 2004)
- 4. Plastids and Plastomes (ptDNA) • Major organelle of plant and algal cells • Site of manufacture and storage of important chemical compounds • Replicates autonomously of the cell • Thought to have been originated from endosymbiotic bacteria • Plastid genes show maternal inheritance • Has circular, dsDNA with size of 120 – 160kb • Present in numbers upto 10,000 copies per plant cell • It is tetra partite and consists of 1. Large single copy region (LSC) 2. Small single copy region (SSC) 3. 2 inverted repeats (IRs) (Herrmann and Possingham, 1980)
- 5. Aspinall et al., 2006
- 6. Why Plastids? • High yield of target protein, • Removing the risk of outcrossing with weeds, • lack of silencing mechanisms, • Absence of epigenetic effects • Ability to engineer the entire metabolic pathways rather than single gene traits. (Łojewska et al, 2016)
- 7. Milestones Year Milestone DNA delivery Approach Selection Reference 1988 Chlamydomonas reinhardtii 1st stable plastid transformation Biolistic Homologous targeting Photosynthetic competence Boynton & Gillham , 1988 1990 Nicotiana tabacum 1st stable plastid transformation Biolistic Spectinomycin (rrn16) Svab et al., 1990 1993 Nicotiana tabacum 1st high level foreign protein (2.5% GUS) PEG Spectinomycin Kanamycin Golds et al., 1993 1999 Solanum tuberosum (potato) Biolistic Spectinomycin Sidorov et al., 1999 2001 Lycopersicon esculentum (tomato) 1st foreign protein in fruitMarker gene elimination: CRE- lox Biolistic Spectinomycin Ruf et al., 2001 2003 Brassicacea (oil seeds) Phytoremediatio n: Mercury Biolistic Spectinomycin Ruiz et al., 2003 Rigano et al., 2012
- 8. Year Milestone DNA delivery Approach Selection Reference 2004 Linum usitatissimum L. (flax): PHB polymer expression Daucus carota Abiotic stress tolerance Biolistic Homologous targeting aph A-6 npt II Spectinomycin Wrobel et al., 2004 Kumar, Dhingra et al, 2004 2007 Brassica oleracea var. capitata (Cabbage) Biolistics Spectinomycin and streptomycin Liu et al., 2004 2008 Lycopersicon esculentum (tomato) HIV antigens Biolisic Spectinomycin Zhou, Badillo‐Corona et al, 2008 2009 Lycopersicon esculentum (tomato) Enhanced β-carotene Biolisic Spectinomycin Apel and Bock, 2009 2010 Lettuce Fructose- 1,6/sedoheptulose-1, 7-bisphosphatase Biolistics Spectinomycin Ichikawa et al., 2010 2013 Lettuce hG-CSF Biolistics Spectinomycin Tabar, Habashi et al.,2013
- 9. Homologous recombination • Genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA • Chloroplast expression has flanking regions of genes present in the plastome (Day and Goldschmidt‐Clermont, 2011)
- 10. Method of Chloroplast transformation (Haider et al., 2010)
- 11. Designing the expression vectors Navaneeth et al , 2012
- 12. Methods of transferring gene • Biolistics and polyethylene glycol-mediated transfer • Biolistics is preferred as it is less time-consuming and demanding • Integration of foreign DNA into plastid genome occurs via homologous recombination
- 13. Biolistics • Direct gene transfer methods • Expression vectors are coated with gold nanoparticles (carriers) • And they are delivered to the plant sample at high speeds • Costlier method but with quicker transformation No of plates Rupture disc Distance (cm) Independen t events Events per plate Efficiency 30 650psi 6 0 0 0 24 9 1 1/24 4.2 26 12 0 0 0 36 900psi 6 0 0 0 34 9 1 1/34 2.9 36 12 0 0 0 38 1100psi 6 0 0 0 34 9 3 1/10 6.7 30 12 4 1/7.5 13.3 ( Dhingra et al, 2004) Biolistics in Carrot calli
- 14. Polyethylene Gylcol (PEG) Method • Lettuce seeds were sterilized and sown on MS medium with 2% sucrose • Shoot tips from leaves obtained were transferred to MS medium with 3% sucrose • The leaves were cut into pieces and incubated in PG solution, followed by enzyme solution consisting of 1% cellulase and 25% macerozyme • Protoplast suspension was filtered through nylon mesh (McCabe et al, 2005)
- 15. Contd.., • Protoplasts were collected at surface after centrifugation at 70g for 8min • 10µl transforming DNA and 0.6ml PEG solution was added to protoplast suspension and incubated at 25ºC for 10min • Protoplasts were mixed with 1:1 solution of B5 and 2% agarose to a density of 3.6 X 104 protoplasts per ml • Needs more standardization of procedures but with less equipments.
- 16. Homoplasmy and Heteroplasmy • Heteroplasmy refers to the presence of more than one type of organelle genome • In transplastomics, after bombardment we get some pt DNA transformed some may not. • After repeated cycles of multiplication and selection we reach homoplasmy. • Homoplasmic plastids are more stable than Heteroplasmic plastids. (Day and Goldschmidt‐Clermont, 2011)
- 18. Selection and Validation • Selection by presence of antibiotic resistance gene or herbicide tolerance gene. • aadA gene - aminoglycoside-3″-adenylyltransferases –resistance to streptomycin and Spectinomycin • Npt II – Neomycin phosphotransferase – Neomycin Validation: • Gene – PCR • Northern blotting • Western blotting
- 19. Plastid Expressed Betaine Aldehyde Dehydrogenase gene in carrot cultured cells, root, and leaves, confers salt tolerance • Glycine Betaine – osmoprotectant • Over expression of betaine by manipulation of badh gene. • Insertion at 16S/trnI- trnA/23S region of chloroplast • Transformation through particle bombardment • Selection in spectinomycin containing media • Regeneration through somatic embryogenesis (Dhingra et al, 2004)
- 20. Visual Selection & PCR validation A: Transformed B: Non- transformed C & D : Heteroplasmic calli Lane 1 – ladder Lane 2 – untransformed Lane 3 – 9 : Transformed lines
- 21. T – Transformed U - Untransformed
- 22. Enhancement of Carotenoid Biosynthesis in Transplastomic Tomatoes by Induced Lycopene- to-Provitamin A Conversion • Lycopene β- Cyclase genes from Erwinia herbicola and daffodil (Narcissus pseudonarcissus). • Raised till homoplasmy condition along with antibiotic selection • Screened with herbicide CPTA (2-(4- chlorophenylthio)- trimethylamine). (Apel and Bock, 2009)
- 24. Enzyme activity assay using herbicide CPTA
- 26. Human Granulocyte Colony-Stimulating Factor (hG-CSF)Expression in Plastids of Lactuca sativa • hG – CSF – a valuable biopharmaceutical for cancer treatment and research • hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator by replacing the GUS gene • Vector was coated with gold nanoparticles and transformed using biolistics method (Habashi et al, 2013)
- 27. Verification by PCR Lane M – 1Kb plus ladder Lanes 1 -5 – Transplastomic plants Lane w – Wild type
- 28. Immunoblot analysis Immunoblot analysis 1- 4 transgenic lines W –Wilf type
- 29. Generation of transplastomic lettuce with enhanced growth and high yield • The lettuce chloroplasts were transformed with genes of cyanobacterial fbp/sbp encoding fructose-1,6-/sedoheptulose-1,7- bisphosphatase (FBP/SBPase) • pLD6 transformation vector is used. • Screened with antibiotic spectinomycin • Increased photosynthetic ability (Tamoi et al, 2010)
- 32. Expression of a Bacillus thuringiensis toxin (cry1Ab) gene in cabbage (Brassica oleracea L. var. capitata L.) chloroplasts confers high insecticidal efficacy against Plutella xylostella • K–Y cross and Summer Summit are the varieties used • Cry 1Ab gene is transformed into the chloroplast genome • Usage pASCC201 chloroplast vector. (Lin et al, 2008)
- 33. Cry 1Ab expression Cabbage Cultivars Bombarded leaf samples Antibiotic resistant calli Southern positive PCR positive Northern positive K –Y Cross 100 8 8 5 4 Summer Summit 100 8 8 5 5 Regeneration and genetic analysis of chloroplast-transformed cabbage
- 34. % insect damage Cultivar Insect death (%) K – Y Cross WT – 1 0 A1 67 A3 100 A5 93 A6 91 Summer Summit WT 2 0 B2 67 B4 80 B6 100 B7 100 B9 67
- 35. Transformation of Solanum tuberosum plastids allows high expression levels of β-glucuronidase both in leaves and microtubers developed in vitro • Vector - pBSW-utr • Trangenes –uidA and aadA • Transformed between genes 16S and trnI. • Transformation done on S. tuberosum cv. Desiree. • β-glucuronidase (GUS) accumulation levels up to 41% of TSP in mature leaves (Lentz et al, 2012)
- 37. High-level expression of human immunodeficiency virus antigens from the tobacco and tomato plastid genomes • HIV contains three important retroviral genes gag, env and pol. • Also some regulatory and accessory proteins (Tat, Rev, Vpu, Vpr, Vif and Nef). • Gag encodes for structural components (p24 –conical core) and an important target for T-cell mediated immunity • P24 is key component of vaccine • In tomato plastid it is expressed in the plastome by selecting good combination of gene cassete with p24-Nef gene in the tobacco earlier (Badillo‐Corona et al, 2008)
- 40. Improvement of vitamin E quality and quantity in tobacco and lettuce by chloroplast genetic engineering • Lactuca sativa L. cv Green Wave • The Tocopherol synthesis pathway is engineered by introducing the gene for Toc cyclase (TC) and c-Toc methyltransferase (c-TMT) • The TC gene inserted in pTTC gene cassette • The TMT gene inserted in pTMT gene cassette • Both TC & TMT gene inserted as operon in pTTC –TMT gene cassette (Tanaka et al, 2013)
- 41. Tocopherol synthesis RT – PCR Analysis
- 43. Drawbacks • Difficult to deliver foreign DNA through double membrane • Repeated number of selection is required to reach homoplasmy • Not all plant species have plastome sequenced • Monocots are recalcitrant to the plastid transformation ( Scotti et al., 2012)
- 44. Thank you