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Supplemental figure 1 AB Supplementary Figure 1. Alternative postulation of intramolecular recombination to release the subgenomic circles in the transplastome.

Published byHarold Malone Modified over 8 years ago

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Presentation on theme: "Supplemental figure 1 AB Supplementary Figure 1. Alternative postulation of intramolecular recombination to release the subgenomic circles in the transplastome."— Presentation transcript:

1 Supplemental figure 1 AB Supplementary Figure 1. Alternative postulation of intramolecular recombination to release the subgenomic circles in the transplastome of the transplastomic plants. (A) Generation of 21 kb and 139 kb subgenomic circles via intramolecular recombination between psbA terminators. (B) Generation of 2.8, 18.2, 139, and 154 kb subgenomic circles by intramolecular recombination between rrn promoters and psbA terminators from initial transplastome

2 AGCTAGCCTAGGCTCGAGAAGCTTGTCGAC GAATTC AGAT ATAGGGGCAGTTGACAATTG AATCCGATTTTGACCATTATTTTCATATCCGTAATAGTGCGAAAAGAAGGCCCGGCTCCAAGTTGTTCAAGAATAGT GGCGTTGAGTTTCTCGACCCTTTGACTTAGGATTAGTCAGT TCTATTTCTCGATGGGGCGGGGAAGGGATATAACTCAGCGGTAGAGTGTCACCTTGACGTGGTGGAAGTCATCAGTTCG AGCCTGATTATCCCTA AGCCCAATGTGAGTTTTTCT agttggatttgctcccccgccgtcgttcaatgagaatggataagaggctcgtgggattgacgtgagggggcagggatggctatatttctgggagcgaactccgggcgaatacgaag cgcttggatacagttgtagggagggattt ATGGCAGAAGCGGTGATCGCCGAAGTATCGACTCAACTATCAGAGGTAGTTGGCGTCATCGAGCGCCATCTCGAACCGACGTTGCTGGCCGTACATTTGT ACGGCTCCGCAGTGGATGGCGGCCTGAAGCCACACAGTGATATTGATTTGCTGGTTACGGTGACCGTAAGGCTTGATGAAACAACGCGGCGAGCTTTGATCAACGACCTTTTGGAAACTTC GGCTTCCCCTGGAGAGAGCGAGATTCTCCGCGCTGTAGAAGTCACCATTGTTGTGCACGACGACATCATTCCGTGGCGTTATCCAGCTAAGCGCGAACTGCAATTTGGAGAATGGCAGCGC AATGACATTCTTGCAGGTATCTTCGAGCCAGCCACGATCGACATTGATCTGGCTATCTTGCTGACAAAGCAAGAGAACATAGCGTTGCCTTGGTAGGTCCAGCGGCGGAGGAACTCTTTGAT CCGGTTCCTGAACAGGATCTATTTGAGGCGCTAANTGAAACCTTANCGCTATGGAACTCGCCGCCCGACTGGGCTGGCGATGACCNAATGTAATGCTNACGTTGTCCCCATTG------------------- ----------------------------------------------CCGACTTCTNCAGAGCGCCTGCCNGGGGGATACGGCNGGAGGGNCCNTCTCTTTCCAGGACGCCGGNACTNCAGACACGTGCGGAGTCCAGGTTGAGG AGACCCCCTCGTCACCAGGATCGAGCTTAAGGGNATCGATTTCCAGGAGGACGGAACATCCTCGGCCACAAGTTGGAATACAACTACAACTCCCACAACGTATACATCACGGCAGACAAAC AAAAGAATGGAATCAAAGCTAACTTCAAAATTAGACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAA CCATTACCTGTCCACACAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATGGTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAACTATACAAATA AGATATCTAGAGCGATCCTGGCCTAGTCTATAGGAGGTTTTGAAAAGAAAGGAGCAATAATCATTTTCTTGTTCTATCAAGAGGGTGCTATTGCTCCTTTCTTTTTTTCTTTTTATTTATTTACTA GTATTTTACTTACATAGACTTTTTTGTTTACATTATAGAAAAAGAAGGAGAGGTTATTTTCTTGCATTTATTCATGATTGAGTATTCTATTTTGATTTTGTATTTGTTTAAAATTGTAGAAATAGAAC TTGTTTCTCTTCTTGCTAATGTTACTATATCTTTTTGATTTTTTTTCCAAAAAAAAAATCAAATTTTGACTTCTTCTTATCTCTTATCTTTGAATATCTCTTATCTTTGAAATAATAATATCATTGAAAT AAGAAAGAAGAGCTATATTCGA ACTTGAATCTTTTGTTTTCTAATTTAAATAATGTAAAAACGGAATGTAAGTAGGCGAGGGGGCGGATGTAGCCAAGTGGATCAAGGCAGTGGATTGTGA ATCCACCATGCGCGGGTTCAATTCCCGT ATCACGAATTCTGGATCCGATACGAACGCGTCGCAGCTGCG A) GNCNGGACACCTACACGCATCTTCCAGACCAAGAACTACGAAGAATCGCCCCTTTCATTCTGGGGTGACGGAGGGATCGTACCATTCGAGCCGTTTTTTTCTTGACTCGAAATGGGAGCAGG TTTGAAAAAGGATCTTAGAGTGTCTAGGGTTGGGCCAGGAGGGTCTCTTAACGCCTTCTTTTTTCTTCTCATCGGAGTTATTTCACAAAGACTTGCCAGGGTAAGGAAGAAGGGGGGAACAA GCACACTTGGAGAGCGCAGTACAACGGAGAGTTGTATGCTGCGTTCGGGAAGGATGAATCGCTCCCGAAAAGGAATCTATTGATTCTCTCCCAATTGGTTGGACCGTAGGTGCGATGATTTA CTTCACGGGCGAGGTCTCTGGTTCAAGTCCAGGATGGCCCA GGATCCCGAATTGGGATCGGTACCCAAAGCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTGGGATTGA CGTGAGGGGGCAGGGATGGCTATATTTCTGGGAGCGAACTCCGGGCGAATATGAAGCGCATGGATACAAGTT ATGCCTTGGAATGAAAGACAATTCCNAATCCGCTTTGTCTACGAACAA GGAAGCTATAAGTAATGCAACTATGAATCTCATGGAGAGTTCGATCCTGGCTCAGGATGAACGCTGGCGGCATGCTTAACACATGCAAGTCGGACGGGAAGTGGTGTTTCCAGTGGCGGAC GGGTGAGTAACGCGTAAGAACCTGCCCTTGGGAGGGGAACAACAGCTGGAAACGGCTGCTAATACCCCGTAGGCTGAGGAGCAAAAGGAGGAATCCCCCCGAGGAGGGGCTCGCGTCTG ATTAGCTAGTTGGGTGAGGCAATANCTTACCAAGGCGATGATCANTANCTGGTCCNANAGGATGATCANCCNCNCTGGGACTGAAACCCGNCCCAAACTCCNTACGGGAGGCANCANTGGG GAATTTTCCCNATGGGCNAAANCCTGACGGAACATTNCCCCNNGNAGNAAAAGNCCCCNGGTCCTNAACTTTTTTNCCCGNAAAAAACANGACNGNNTCNGGGAAAAAANATCNGNTAANNT TNNNCCNCNNCCCNGNAAAANAANNANCNANC B) CGNTNCANCTCCGGCCGCCATGGCGGCCGCGGGAATTCGATT AATTGGTTGGACCGTAGGTGCGATGATTTACTTCACGGGCGAGGTCTCTGGTTCAAGTCCAGGATGGCCCA GGATCCCGAATTGGGATCGGTA CCCAAAGCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTGGGATTGACGTGAGGGGGCAGGGATGGCTATATTTCTGGGAGCGAACTCCGGGCGAATATGAAGCGCATG GATACAAGTT ATGCCTTGGAATGAAAGACAATTCCGAATCCGCTTTGTCTACGAACAAGGAAGCTATAAGTAATGCAACTATGAATCTCATGGAGAGTTCGATCCTGGCTCAGGATGAACGCT GGCGGCATGCTTAACACATGCAAGTCGGACGGGAAGTGGTGTTTCCAGTGGCGGACGGGTGAGTAACGCGTAAGAACCTGCCCTTGGGAGGGGAACAACAGCTGGAAACGGCTGCTAATA ----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- CCCGGGCCTTGTACACACCGCCCGTCACACTATGGGAGCTGGCCATGCCCGAAGTCGTTACCTTAACCGCAAGGAGGGGGATGCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAA CAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCTTTTCAGGGAGAGCTAATGCTTGTTGGGGTATTTTGGTTTGACACTGCTTCACACCCCCAAAAAANAAGAAGGGAGCTACGTC TGAGTTAAACTTGGAGATGGAAGTCTTCTTTCCTTTCTCGACGGTGAAGTAAGACCAAGCTCATGAGCTTATTATCCTAGGTCGGAACAAGTTGATAGGACCCCCTTTTTTACGTCCCCATGTT CCCCCCGTGTGGCGACATGGGGGCGAAAAAAGGAAAGAGAGGGATGGGGTTTCTCTCGCTTTTGGCATAGCGGGCCCCCAGTGGGAGGCTCGCACGACGGGCTATTAGCTCAGTGGTAG AGCGCGCCCCTGATAATTGCGTCGTTGTGCCTGGGCTGTGAGGGCTCTCAGCCACATGGATAGTTCAATGTGCTCATCGGCGCCTGACCCTGAGATGTGGATCATCCAAGGCACATTAGCA TGGCGTACTCCTCCTGTTCGAACCGGGGTTTGAAACCAAACTCCTCCTCAGGAGGATAGATGGGGCGATTCGGGTGAGATCCAATGTAGATCCAACTTTCGATTCACTCGTGGGATCCGGG CGGTCCGGGGGGGACCACCACGGCTCCTCTCTTCCCGAGAATCCATACATCCCTTATCAGTGTATGGACAGCTATCTCTCGAGCACAGGTTTAGCAATGGGAAAATAAAATGGAGCACCTAA CAACGCATCTTCACAGACCAAGAACTGCGAGATCGCCCCTTTCATTCTGGGGTGACGGAGGGATCGTACCATTCGAGCCGTTTTTTTCTTGACTCGAAATGGGAGCAGGTTTGAAAAAGGAT CTTAGAGTGTCTAGGGTTGGGCCAGGAGGGTCTCTTAACGCCTTCTTTTTTCTTCTCATCGGAGTTATTTCACAAAGACTTGCCAGGGTAAGGAAGAAGGGGGGAACAAGCACACTTGGAGA GCGCAGTACAACGGAGAGTTGTATGCTGCGTTCGGGAAGGATGAATCGCTCCCGAAAAGGAATCTATTGATTCTCTCCCAATTGGTTGGACCGTAGGTGCGATGATTTACTTCACGGG AATCACT AGTGAATTCGCGGCCGCCTGCAGGTCGACCATATGGGAGAGCTCCCACGCGTGAGNNG C)

3 Suplementary Figure 2. Seuqence analysis confirming 18.2 kb and 2.8 kb subgenomic circles. Sequence of cloned 2.5 kb PCR product using 70B and H, confirming the existence of 18.2 kb. The sequence does not have the 16S rRNA and trnI genes between the trnV gene and the rrn promoter of transgene unit. Small capital, pSTBlue-1 vector sequence. Bold letters with underline, primer sequences of 70B and H (Table 1) Bold red with italic, trnV gene. Bold black with lower case, vector driven rrn promoter sequence. Red capital, aadA gene. Green capital, gfp gene. Blue capital, vector driven psbA terminator sequence. Bold green capital, trnH sequence derived from plastome. Sequencing was conducted using T7 and SP6 primers which bound on pSTBLUE-1 vector. B) Direct sequencing using trnIF3 primer for 2 kb PCR product using trnIF4 and trnIR7 confirming the existence of 2.8 kb circle. The sequence compose of only two genes of trnI and 16S rRNA sequences with vector driven rrn promoter. Red capital, trnI gene. Blue capital, 16S rRNA gene. Bold black, vector driven rrn promoter sequence. C) Sequence of cloned 2.8 kb PCR product using trnIF4 and trnIR7 confirming the existence of 2.8 kb circle. The sequence compose of only two genes of trnI and 16S rRNA sequences with vector driven rrn promoter. Small capital, pGEM T-easy vector sequence. Red letters with underline, primer sequences of trnIF4 and trnIR7 (Table 1). Red capital, trnI gene. Blue capital, 16S rRNA gene. Bold black, vector driven rrn promoter sequence. Black capital, spacer region between two genes. Sequencing was conducted using T7, SP6, Trrn-16F17 (AAGTCCGCCGTCAAATCCCA) and Trrn-IR8 (ACCCCATCCCTCTCTTTCCT) primers

4 WT C 1 2 3 6 12 Suplementary Figure 3. PCR analysis using trnIF4 and trnIR7 primers with inward orientation to confirm 2.8 kb subgenomic circle in laccase transplastomic plants. WT, wild-type plant; C, transplastomic plant (T 3 generation) generated by pCTVG vector; 1- 12, independent transplastomic plants (T 1 generation) generated by pCTVL carring aadA and laccase (from Pleurotus ostreatus) transgenes PrrnrbsTpsbA pCTVL 16S -3' trnItrnA aadA Laccase

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