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EPSPS and Crops Developed by Dr. M. Zapiola for CSS/Hort 431 Glyphosate Herbicide • Glyphosate inhibits EPSPS enzyme - impeding the synthesis of aromatic amino acids CO2 EPSPS H2 O aromatic amino acids Proteins aromatic amino acids Proteins NH3 CO2 H2O NH3 glyphosate EPSPS Commercial RR crops "Two key elements needed for the development of commercially viable glyphosate-tolerant crops are: – a resistant target enzyme – sufficient expression of that enzyme within the transgenic plant” Heck et al. 2005. Development and Characterization of a CP4 EPSPS-Based, Glyphosate-Tolerant Corn Event: Crop Sci. 45:329-339 (2005). Roundup Ready Crops • When incorporated into the genome of RRsusceptible plants, the Roundup ReadyTM gene (CP4 EPSPS) confers resistance to glyphosate CO2 H2O NH3 glyphosate EPSPS aromatic amino acids CP4 EPSPS Proteins Gene Construct promoter (leading sequence) resistant EPSPS terminator • The gene is introduced into the plant nuclear genome via different mechanisms • The trait is then incorporated to elite varieties by backcrossing Gene Construct: RR soybean • LOCUS AB209952.1 • Size 2457 bp • SOURCE Glycine max • FEATURES – CaMV35S – Chloroplast transit peptide – CP4EPSPS – Nos 3’ terminator – Repeated fragment of cp4epsps truncate TGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAG ATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGA CGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGACAAG CTGACTCTAGCAGATCTTTCAAGAATGGCACAAATTAACAACATGGCACAAGGGATACAAACCCTTAATCCCAATTCCAATTTCCATAAACC CCAAGTTCCTAAATCTTCAAGTTTTCTTGTTTTTGGATCTAAAAAACTGAAAAATTCAGCAAATTCTATGTTGGTTTTGAAAAAAGATTCAATT TTTATGCAAAAGTTTTGTTCCTTTAGGATTTCAGCATCAGTGGCTACAGCCTGCATGCTTCACGGTGCAAGCAGCCGGCCCGCAACCGCCCG CAAATCCTCTGGCCTTTCCGGAACCGTCCGCATTCCCGGCGACAAGTCGATCTCCCACCGGTCCTTCATGTTCGGCGGTCTCGCGAGCGGTG AAACGCGCATCACCGGCCTTCTGGAAGGCGAGGACGTCATCAATACGGGCAAGGCCATGCAGGCCATGGGCGCCAGGATCCGTAAGGAA GGCGACACCTGGATCATCGATGGCGTCGGCAATGGCGGCCTCCTGGCGCCTGAGGCGCCGCTCGATTTCGGCAATGCCGCCACGGGCTGC CGCCTGACCATGGGCCTCGTCGGGGTCTACGATTTCGACAGCACCTTCATCGGCGACGCCTCGCTCACAAAGCGCCCGATGGGCCGCGTGT TGAACCCGCTGCGCGAAATGGGCGTGCAGGTGAAATCGGAAGACGGTGACCGTCTTCCCGTTACCTTGCGCGGGCCGAAGACGCCGACGC CGATCACCTACCGCGTGCCGATGGCCTCCGCACAGGTGAAGTCCGCCGTGCTGCTCGCCGGCCTCAACACGCCCGGCATCACGACGGTCAT CGAGCCGATCATGACGTGCGATCATACGGAAAAGATGCTGCAGGGCTTTGGCGCCAACCTTACCGTCGAGACGGATGCGGACGGCGTGC GCACCATCCGCCTGGAAGGCCGCGGCAAGCTCACCGGCCAAGTCATCGACGTGCCGGGCGACCCGTCCTCGACGGCCTTCCCGCTGGTTG CGGCCCTGCTTGTTCCGGGCTCCGACGTCACCATCCTCAACGTGCTGATGAACCCCACCCGCACCGGCCTCATCCTGACGCTGCAGGAAATG GGCGCCGACATCGAAGTCATCAACCTGCGCCTTGCCGGCGGCGAAGACGTGGCGGACCTGCGCGTTCGCTCCTCCACGCTGAAGGGCGTC ACGGTGCCGGAAGACCGCGCGCCTCCGATGATCGACGAATATCCGATTCTCGCTGTCGCCGCCGCCTTCGCGGAAGGGGCGACCGTGATG AACGGTCTGGAAGAACTCCGCGTCAAGGAAAGCGACCGCCTCTCGGCCGTCGCCAATGGCCTCAAGCTCAATGGCGTGGATTGCGATGAG GGCGAGACGTCGCTCGTCGTGCGTGGCCGCCCTGACGGCAAGGGGCTCGGCAACGCCTCGGGCGCCGCCGTCGCCACCCATCTCGATCAC CGCATCGCCATGAGCTTCCTCGTCATGGGCCTCGTGTCGGAAAACCCTGTCACGGTGGACGATGCCACGATGATCGCCACGAGCTTCCCGG AGTTCATGGACCTGATGGCCGGGCTGGGCGCGAAGATCGAACTCTCCGATACGAAGGCTGCCTGATGAGCTCGAATTCGAGCTCGGTACC GGATCCAATTCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAATTTCT GTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTATACAT TTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGGATCGATC CCCCACCGGTCCTTCATGTTCGGCGGTCTCGCGAGCGGTGAAACGCGCATCACCGGCCTTCTGGAAGGCGAGGACGTCATCAATACGGGC AAGGCCATGCAGGCCATGGGCGCCAGGATCCGTAAGGAAGGCGACACCTGGATCATCGATGGCGTCGGCAATGGCGGCCTCCTGGCGCC TGAGGCGCCGCTCGATTTCGGCAATGCCGCCACGGGCTGCCGCCTGACCATGGGCCTCGTCGGGGTCTACGATTTCAAGCGCATCATGCTG GGAA Gene bank gi|62318478|dbj|AB209952.1|[62318478] PCR Primer Design Name tm Product size CaM_1F 60 117 CaM_1R 60 CaM_2F 60 CaM_2R 60 CTP_1F 60 CTP_1R 60 CP4_F 60 CP4_R 60 Nos_F 60 Nos_R 58 121 150 286 216 TGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAG ATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTG ACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAGAGGACACGCTGACAA GCTGACTCTAGCAGATCTTTCAAGAATGGCACAAATTAACAACATGGCACAAGGGATACAAACCCTTAATCCCAATTCCAATTTCCATAAAC CCCAAGTTCCTAAATCTTCAAGTTTTCTTGTTTTTGGATCTAAAAAACTGAAAAATTCAGCAAATTCTATGTTGGTTTTGAAAAAAGATTCAA TTTTTATGCAAAAGTTTTGTTCCTTTAGGATTTCAGCATCAGTGGCTACAGCCTGCATGCTTCACGGTGCAAGCAGCCGGCCCGCAACCGCC CGCAAATCCTCTGGCCTTTCCGGAACCGTCCGCATTCCCGGCGACAAGTCGATCTCCCACCGGTCCTTCATGTTCGGCGGTCTCGCGAGCGG TGAAACGCGCATCACCGGCCTTCTGGAAGGCGAGGACGTCATCAATACGGGCAAGGCCATGCAGGCCATGGGCGCCAGGATCCGTAAGG AAGGCGACACCTGGATCATCGATGGCGTCGGCAATGGCGGCCTCCTGGCGCCTGAGGCGCCGCTCGATTTCGGCAATGCCGCCACGGGCT GCCGCCTGACCATGGGCCTCGTCGGGGTCTACGATTTCGACAGCACCTTCATCGGCGACGCCTCGCTCACAAAGCGCCCGATGGGCCGCGT GTTGAACCCGCTGCGCGAAATGGGCGTGCAGGTGAAATCGGAAGACGGTGACCGTCTTCCCGTTACCTTGCGCGGGCCGAAGACGCCGA CGCCGATCACCTACCGCGTGCCGATGGCCTCCGCACAGGTGAAGTCCGCCGTGCTGCTCGCCGGCCTCAACACGCCCGGCATCACGACGG TCATCGAGCCGATCATGACGTGCGATCATACGGAAAAGATGCTGCAGGGCTTTGGCGCCAACCTTACCGTCGAGACGGATGCGGACGGCG TGCGCACCATCCGCCTGGAAGGCCGCGGCAAGCTCACCGGCCAAGTCATCGACGTGCCGGGCGACCCGTCCTCGACGGCCTTCCCGCTGG TTGCGGCCCTGCTTGTTCCGGGCTCCGACGTCACCATCCTCAACGTGCTGATGAACCCCACCCGCACCGGCCTCATCCTGACGCTGCAGGAA ATGGGCGCCGACATCGAAGTCATCAACCTGCGCCTTGCCGGCGGCGAAGACGTGGCGGACCTGCGCGTTCGCTCCTCCACGCTGAAGGGC GTCACGGTGCCGGAAGACCGCGCGCCTCCGATGATCGACGAATATCCGATTCTCGCTGTCGCCGCCGCCTTCGCGGAAGGGGCGACCGTG ATGAACGGTCTGGAAGAACTCCGCGTCAAGGAAAGCGACCGCCTCTCGGCCGTCGCCAATGGCCTCAAGCTCAATGGCGTGGATTGCGAT GAGGGCGAGACGTCGCTCGTCGTGCGTGGCCGCCCTGACGGCAAGGGGCTCGGCAACGCCTCGGGCGCCGCCGTCGCCACCCATCTCGA TCACCGCATCGCCATGAGCTTCCTCGTCATGGGCCTCGTGTCGGAAAACCCTGTCACGGTGGACGATGCCACGATGATCGCCACGAGCTTC CCGGAGTTCATGGACCTGATGGCCGGGCTGGGCGCGAAGATCGAACTCTCCGATACGAAGGCTGCCTGATGAGCTCGAATTCGAGCTCGG TACCGGATCCAATTCCCGATCGTTCAAACATTTGGCAATAAAGTTTCTTAAGATTGAATCCTGTTGCCGGTCTTGCGATGATTATCATATAAT TTCTGTTGAATTACGTTAAGCATGTAATAATTAACATGTAATGCATGACGTTATTTATGAGATGGGTTTTTATGATTAGAGTCCCGCAATTAT ACATTTAATACGCGATAGAAAACAAAATATAGCGCGCAAACTAGGATAAATTATCGCGCGCGGTGTCATCTATGTTACTAGATCGGGGATC GATCCCCCACCGGTCCTTCATGTTCGGCGGTCTCGCGAGCGGTGAAACGCGCATCACCGGCCTTCTGGAAGGCGAGGACGTCATCAATAC GGGCAAGGCCATGCAGGCCATGGGCGCCAGGATCCGTAAGGAAGGCGACACCTGGATCATCGATGGCGTCGGCAATGGCGGCCTCCTGG CGCCTGAGGCGCCGCTCGATTTCGGCAATGCCGCCACGGGCTGCCGCCTGACCATGGGCCTCGTCGGGGTCTACGATTTCAAGCGCATCAT GCTGGGAA Scanning 96 Plants
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