Transcript Problem
Golden Rice Kameron Starr Ingo Potrykus Problem • Many developing countries suffer from Vitamin A deficiency due to the predominant consumption of rice. -Rice lacks Vitamin A and pro-vitamin A in the edible endosperm. • This deficiency leads to blindness, especially in children Problem • Many developing countries suffer from Vitamin A deficiency due to the predominant consumption of rice. -Rice lacks Vitamin A and pro-vitamin A in the edible endosperm. • This deficiency leads to blindness, especially in children Solution • Genetically engineer rice to produce pro-vitamin A, β-carotene. Carotenoid • organic pigment (red/orange/yellow color) Chlorophyll dominating Carotenoid dominating The Biosynthesis Pathway Enzymes involved 1. IPP/DMAPP-Isomerase 2. GGPP-Synthase 3. Phytoene synthase (psy) 4. phytoene desaturase 5. ζ-carotene desaturase 6. lycopene –cyclase (lcy) Catalyze introduction of two double bonds each (shown on next slide) bacterial phytoene desaturase (crtI) Catalyze introduction of all 4 double bonds Where did the foreign genes come from? Daffodil (psy), (lcy) Erwinia uredovora (crtI) The Biosynthesis Pathway Ti plasmid-mediated gene transfer using Agrobacterium tumafaciens Recall: Agrobacterium: Plant pathogen that delivers DNA to plants. • Can be used in genetic engineering - Delete the phytohormone genes - Add cloning site for YFG Single Transformation • Initially this plasmid was used to insure that lycopene (red) was being synthesized. • Contains genes for two enzymes - Phytoene-Synthase (psy) - Bacterial Phytoene Desaturase (crtI) Co-Transformation LB Gt1p psy nos! 35sp tp crtI nos! RB • These plasmid were used to complete the synthesis of β-Carotene (Yellow) • pZPsC is very similar to pB19hpc - provides machinery for the synthesis of Lycopene • pZLcyH was added to introduce Lycopene β-Cyclase which should complete the pathway SURPRISE! • To the researcher’s surprise, the single transformation produced β-carotene without the accumulation of lycopene. - α,β-cyclase may be constitutively expressed in endosperm - formation of lycopene turns these genes on • This makes transformation much more simple. Only two genes must be added. • Pb19hpc was used and modified in the following two ways - PMI selection replaced hygromycin - nptII selectable marker removed Modified pB19hpc Results • The transgene was inherited by offspring with a 3:1 Segregation, (Golden:WT) • 1.6 μg carotenoid/g dry rice endosperm - A child would need 3,330g(7.3lbs)/day - Usually eat a max of 120g/day • Proof of concept Results • Seed content analyzed by high performance liquid chromatography (HPLC) • Co-transformants much more variable • Lutein and zeaxanthin also synthesized\ -other carotenoids contributing to eye health A. B. C. D. Control seeds Single transformant Co-tranformant Co-transformant Golden Rice 2 • Psy was found to be the limiting factor - hypothesis based on the lack of lycopene accumulation • Psy genes from various other plants were used in an attempt to increase carotenoid synthesis. These include Maize, Pepper, and Tomato • Maize yielded the best results Results • The transgene was inherited by offspring with a 3:1 Segregation, (Golden:WT) • 37 μg carotenoid/g dry rice endosperm - A child would need 144g/day - compares to the max of 120g/day • Still no lycopene accumulation, further optimization possible Controversy • People are reluctant to consume this due to its genetic alteration • Farmers fear this could contaminate WT rice • Riots and “rice picking’s” have occurred