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SUCCESS STORIES OF VITAMINE & AMINO ACIDS ENHANCEMENT IN TRANSGENIC CROPS
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SUCCESS STORIES OF VITAMINE & AMINO ACIDS ENHANCEMENT IN TRANSGENIC CROPS

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  • 1. SUCCESS STORIES OFV ITAMINE & AMINO ACIDS ENHANCEMENT IN TRANSGENIC CROPS
  • 2. What is a Transgenic Crop? Transgenic indicates that a transfer of genes has occurred using recombinant DNA technology. Generally a transgenic crop contains one or more genes that have been inserted artificially either from an unrelated plant or from different species altogether.
  • 3. How are Transgenic CropsMade?• In order to make a transgenic crop, there are five main steps:- 1. Extracting DNA. 2. Cloning a gene of interest. 3. Designing the gene for plant infiltration. 4. Transformation. 5. Breeding.
  • 4. STEPS INVOVED IN TRANSGENICS:-
  • 5. VITAMINES VITAMINE S
  • 6. VITAMIN E•Vitam in E is a group of eight hyd rophobic com pound s(known as vitam ers), the m ost potent of which is a-tocopherol. Vitam in E is obtained m ainly from seed s.•Function:- prevent the oxidation and polymerization ofunsaturated fatty acids in body.•Deficiency:- •general wasting •kid ney d egeneration •infertility•The levels of vitam in E activity can be increased either byincreasing the total am ount of vitam in E or by shifting them etabolic flux toward s a-tocopherol
  • 7. In plants, tocopherol synthesis requires input from twometabolic pathways :-1.T shikimae pahw ygener t homogentisic acid, w f ms t aomaic r oft he t t a aes hich or he r t ing ocopher , ol 2.T side cha is der ed fom phytyldiphosphate, apr oft met l yhr olphosphae(M pahw y he in iv r oduct he hyer t it t EP) t a.•These precursors are joined together by homogentisic acidprenyltransferase(HPT) to form the intermediate 2-methyl-6-phytylbenzoquinol(MPBQ) .•M Qis t substae f t o enzy t PB he r t or w mes, ocopher cy a a M Qmet lr nsf a ol cl se nd PB hyta er se. Tocopherol cyclase form d -tocopherol MPBQ methyltransferase forms 2,3-dimethyl-5-phytylbenzoquinol.T a ion oft he ct ocopher cy a on 2,3 hy-5 phyybenzoquinol ol cl se -dimet l t lproduces g-t ocopher . B h g-t ol nd ocopher ae substaes f g-tocopherol ol ot ocopher a d-t ol r r t ormethyltransferase pr oducing a- a b-t nd ocopher , r iv y ol espect el .
  • 8. shikimate pathway MEP pathway Homoginistic acid Phytyldiphospate HPT 2 methyle-6-phytyle bezoquinol {MPBQ} MPBQ Tocopherol Methyle Cyclase transferase 2,3 dimethyle 5-d-tocopherol phytylebenzoquinol Tocopherol TMT Cyclase TMT a & b-tocopherol g-tocopgerol Tocopherol synthesis
  • 9. •Shintani and Della-Penna expressed the Arabid opsisgenes encod ing g-tocopherol m ethyltransferase (g-TM T)in Arabid opsis seed s, resulting in a fund am ental shift of g/d-tocopherol a/b-tocopherol this showed that nutritional enhancem ent in plants was possible without altering total vitam in E levels.•The expression of Arabid opsis hom ogentisic acidprenyltransferase (H PT) prod uced twice the level ofvitam in E found in norm al seed s.
  • 10. In case of soybean• The Arabid opsis genes encod ing 2-methyl-6- phytylbenzoquinol (MPBQ) methyltransferase and g -TMT were used .• Transgenic soybeans showed a significant elevation in the total am ount of vitam in E activity (fivefold greater than that of wild -type plants), which was attributable m ainly from its norm al 1 0% of total vitam in E to over 95% .
  • 11. VITAMIN AVitam in A d eficiency is prevalent in the d eveloping worldand is probably responsible for the d eaths of two m illionchild ren annually.• Deficiency :- blind ness•H um ans can synthesize vitam in A if provid ed with theprecursor m olecule b -carotene (provitam in A), a pigm entfound in m any plants but not in cereal grains.•Therefore, a strategy was d evised to introd uce the correctm etabolic steps into rice end osperm to facilitate b -carotene synthesis.
  • 12. GOLDEN RICE•Professor Ingo Potrykus, Dr. PeterBeyer & other European scientists in august 1999.•At Swiss Federal Institute ofTechnology & University of Freiburgin Germany.•Produced by combining geneticmaterial from:- •daffodils, •peas, and •Japonica rice.
  • 13. Donor DNA Plasmid vector Selectable antibiotic resistance marker Donor DNA cut with EcoRI Donor DNA fragments Vector cut with EcoRI Add DNA ligase Plasmids Tetracycline-resistant Bacterial colony from transformed cell Introduce into E. coliRecombinant DNA Transformed cell
  • 14. PLANT GENE TRANSFER VIA AGROBACTERIUM The bacterium that causes crown gall disease in plants has a natural vector for transformation of desirable traits from one plant to another. T-DNA
  • 15. Agrobacterium tumefaciens A specific gene is chromosomal “cut out” of the DNA Plasmid DNA is donor DNA using cut open with the same enzyme. an enzyme. plasmid DNA New gene is When the plant cell inserted into divides, each daughter the plasmid. cell receives the newPlasmid is transformed gene, giving the wholeinto Agrobacterium. plant a new trait. When mixed with plant The new gene is transferred cells, Agrobacterium into the chromosomal DNA duplicates the plasmid. of the plant cell.
  • 16. Cloned Gene in Vector DNA Molecule Biolistic bombardment Transformation of (gene gun) AgrobacteriumProtoplast transformation Agrobacterium-mediatedfollowed by cell wall transformation of plantregeneration cell Migration and integration of gene into nucleus Plant cells Regeneration of grown in genetically tissue culture modified plant from tissue culture
  • 17. BIOSYNTHESIS OF b-CAROTENE• Joining of two geranylgeranyldiphosphate(GGDP) molecules toform the precursor phytoene.•The conversion of phytoene intob-carotene requires threeadditional enzyme activities: •phytoene desaturase •b-carotene desaturase •lycopene b-cyclase.•Cereal grains, such as rice,accumulate GGDP but lack thesubsequent enzymes in thepathway, so the genes for all threeenzymes are required to form b-carotene.
  • 18. •This has led to sim ilar progress in other crops,includ ing, m ost recently, “yellow potato’, ‘orangecauliflower’, carrots with enhanced b-carotene inthe taproot and tom atoes with the b-carotenem etabolic pathway transferred to the plastid s.•A recently d eveloped potato variety containing thephytoene synthase, phytoene d esaturase andlycopene b-cyclase from E rwinia herbicolacontained 1 1 4 mg carotenoid s per gram of d ryweight and 47 mg b-carotene per gram of d ryweight.
  • 19. FOLATES• Folate is a B-group vitamin critical for normal cellular function and division.• Deficiency:- • megaloblastic anaemia • cardiovascular disease • cancers and • cognitive decline • spina bifida and anencephaly
  • 20. •Folate is produced from multistep process from:- • pteridine synthesized in cytosol •glutamate moieties •p-aminobenzoate(PABA) synthesized in plastid•These moieties are then transported to the mitochondria,where they condense to form dihydropteroate and areconjugated to glutamate.•Rice plants transgenic for wheat 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase/7,8-dihydropteroatesynthase(HPPK/DHPS) which operates at a central point in theproduction pathway, gives elevated folate levels.
  • 21. The folate production pathway. PABA is synthesized fromchorismate in the chloroplast, pterin is synthesized in the cytoplasm.These are transported into the mitochondria where the two arecondensed and the product glutamated.
  • 22. PROCEDURE•Wheat HPPK/DHPS cDNA was isolated.•It was cloned into Sma1-Sac1 digested bombardment vectorpUbi.gfp.nos. so as to replace the green fluorescent protein(gfp)fragment with HPPK/DHPS.•Introduced the gene under the control of the maize ubiquitinpromoter into the Australian rice variety Jarrah of Oryza Sativavia particle bombardment using the Biolistic PDC-1000/He system.•Transgenic plants were selected for by growth on hygromycinmedia, and further subject to PCR for the presence of the HPPK/DHPS gene.
  • 23. AMINO ACIDS
  • 24. IntroductionThe nutritional quality of cereals and legumes has been improved by using biotechnological methods. Two genetic engineering approaches have been used to improve the seed protein quality.• First case:- A transgene (e.g. gene for protein containing sulphur rich amino acids) was introduced into pea plant (which is deficient in methionine and cysteine, but rich in lysine) under the control of seed-specific promoter.Second case:- The endogenous genes are modified so as to increase the essential amino acids like lysine in the seed proteins of cereals.These transgenic routes have helped to improve the essential amino acids contents in the seed storage proteins of a number of crop plants. E.g. overproduction of lysine by de-regulation.
  • 25. Enhancement of Mithionine & cysteine in pea• It is based on INTODUCTION OF TRANSGENE APPROACH. •a new gene encoding for storage protein rich in deficient amino acid is intoduced into crop to correct its amino acid deficiency.•vicilin seed storage protein of pea(Pisum sativum) have 7%lysine is deficient in the sulfur-containing amino acidsmethionine and cysteine.•Sunflower seeds protein, sunflower albumin 8(SFA8) has23% mithionine+cysteine content.
  • 26. •Gene cod ing for SFA8 is isolated and fused with vicilin geneprom otor.•Transferred the viciline gene prom otor-SFA gene construct into pea. Viciline promotor-SFA8 gene construct •This has enhanced the level of sulphur containing am ino acid s upto 40%
  • 27. Enhancement of Lysin in corn•Corn has become the most productive major crop.•Deficiency:- lysineStrategies for lysine genetic engineering in corn:-Supressing α-zein production:-•Natural maize opaque mutants have nutritionally poor corn proteinknown as α-zein.•RNAi mechanism has been used to specifically suppress α-zeinproduction in transgenic corn, resulting in a doubling of the lysinecontent of corn grain from 2400 ppm to 4800 ppm2.•α-zeins comprise roughly 40% of the total kernel protein, but containalmost no lysine. By reducing α-zeins, other lysine-containing kernelproteins were comparatively increased, raising the lysine content incorn protein from 2.8% to 5.4%.•
  • 28. RNAi mechanism to supress the α-zein production
  • 29. genetically modify Lysine metabolic pathway•lysine, along with methionine, threonine, and isoleucine, isderived from aspartate.•dihydrodipicolinate synthase (DHDPS) catalyzes the firstcommitted step of lysine biosynthesis.•A bifunctional enzyme, lysine-ketoglutaratereductase/saccharopine dehydrogenase (LKR/SDH), is responsiblefor lysine catabolism.•The free lysine level in plant cells is thought to be regulated by:- • lysine feedback inhibition of DHDPS and • feed-forward activation of LKR/SDH.
  • 30. To Enhance the levels of lysine:-•Activated the expression of a lysine feedback-insensitive DHDPS from Corynebacteriumglutamicum, CordapA.•suppressed the enzyme LKR/SDH•To further enhance the accumulation of free lysine incorn, we recently developed transgenic corn lines thatcombine CordapA expression and LKR/SDHsuppression7, by using a novel bifunctional transgenecassette.
  • 31. •An inverted repeat sequence corresponding to partialLKR/SDH cDNA was inserted into the intron of anexpression cassette containing CordapA as the codingregion.•Principle:- the expression of this transgene shouldgenerate an intron-derived, double-stranded RNAagainst LKR/SDH and an mRNA encoding CordapA.
  • 32. Lysin enhancement in sorghum•Sorghum is one of the m ain staples of the world ’s poorest an m ost food -insecure people.•It have low nutritional quality because of low lysine content.•Genetically enhancing the nutritional quality of grain sorghum by theintrod uction of genes encod ing:-•Methionine-rich maize beta-zein•L ysine-rich barley chymotrypsin inhibitor C I-2 proteins.TRA NSG E NIC STRA TE GIE S 1 . Transgenic sorghum plants were prod uced via A grobacterium- mediated transformation using im m ature zygotic em bryos as explant. 2. by particle bombardment O f im m ature inflorescences and shoot
  • 33. •Dihydropicolinate synthase, the first enzyme of the lysine-specific pathway•A functional gene which codes for a feedback insensitivedihydropicolinate synthase, was introduced into the genome ofsorghum with the goal of producing transgenic sorghum plantswith increased lysine contentRE QIURE ME NTS:- •Two transformable sorghum genotypes •five A frican sorghum genotypes which are highly regenerable and transform able. •The plant expression vectors containing:- 1 . The reporter gene uid A (GU S), 2. The selectable m arker genes bar or hpt II, 3. The lysine-rich C I-2 gene und er control of the gam m a-zein prom oter.
  • 34. •Four constructs were prepared for particlebombardment-mediated transformation of grainsorghum:- •One construct containing the wild type CI-2 gene driven by the maize gamma-zein (γ-zein) promoter •Three constructs were prepared containing the genetically engineered CI-2 gene, with additional lysine substitutions in a reactive loop or hairpin region, driven by the maize gamma-zein (γ-zein) promoter.•Two constructs were prepared containing themethionine-rich beta-zein gene or fusion protein genedriven by gamma-zein promoter, respectively
  • 35. Vector is introd uced into the sorghum genom e via A grobacterium-mediated transformation of selected sorghum genotypes.
  • 36. Enhancing protein quality in amaranthus albuminpotatoes‘Increased nutritive value of transgenic potato by expressing a nonallergenic seed albumin gene from Amaranthus hypochondriacus’• Potato is the fourth most abundant global crop and used for food,animal feed and production of starch and alcohol• Limited in lysine, tyrosine, methionine and cysteine•Transformed potato with seed albumin from Amaranthushypochondriacus .• Expression in tuber 5-10 fold higher with GBSS promoter than with 35Spromoter•Total protein content also increased (35-45%)
  • 37. • A gene that encod es a seed -specific protein, am aranth seed album in (Am A1 ) from Amaranthus hypochond riacus• The Am A1 protein has great potential as a d onor proteinfor the following reasons:- (i) It is a well-balanced protein in term s of am ino acid com position and even better than the values recom m end ed by the World H ealthO rganization for a nutritionally rich protein; (ii) It is a nonallergenic protein in its purified form (iii) It is encod ed by a single gene and thus would facilitate gene transfer into target plants with less d ifficulty.
  • 38. • The expression plasm id pSB8 was constructed by using AmA1 cod ing sequence along with 1 02 bp of 39 AmA1 untranslated region under the control of CaMV 35S promoter in pBI121and pSB8G, wherein 35S prom oter was replaced by GBSS prom oter 2 alternative pSB8 constructs. p35S CaMV AmA1 Nos 3’ Promoters constitutive or tuber-specific pGBSS AmA1 Nos 3’ pSB8G