Supplementary Materials Figure S1
A * 20 * 40 * 60 * 80 * Ciclev10020814m : ATGGGTTCAACCAGTTCAGAAACTCAAATAAGTCCAGCCCAAGGCTCGGATGAAGAGGCAAACCTCTTGGCCATGCAATTAACCAGTGCCTCAGT : 95 CrOMT2 : ATGGGTTCAACCAGTTCAGAAACTCAAATAAGTCCAGCCCAAGGCTCGGATGAAGAGGCAAACCTCTTGGCCATGCAATTAACCAGTGCCTCAGT : 95 HM641694.1 : ATGGGTTCAACCAGTTCAGAAACTCAAATAAGTCCAGCCCAAGGCTCGGATGAAGAGGCAAACCTCTTGGCCATGCAATTAACCAGTGCCTCAGT : 95 ATGGGTTCAACCAGTTCAGAAACTCAAATAAGTCCAGCCCAAGGCTCGGATGAAGAGGCAAACCTCTTGGCCATGCAATTAACCAGTGCCTCAGT
100 * 120 * 140 * 160 * 180 * Ciclev10020814m : CTTGCCTATGGTTCTCAAATCAGCCATTGAGCTTGATCTTTTAGAGATCATCGCTAAAGCTGGGCCAGATGCTTTCATGTCTCCAAAAGACATAG : 190 CrOMT2 : CTTGCCTATGGTTCTCAAATCAGCCATTGAGCTTGATCTTTTAGAGATCATCGCTAAAGCTGGGCCAGATGCTTTCATGTCTCCAAAAGACATAG : 190 HM641694.1 : CTTGCCTATGGTTCTCAAATCAGCCATTGAGCTTGATCTTTTAGAGATCATCGCTAAAGCTGGGCCCGATGCTTTCATGTCTCCAAAAGACATAG : 190 CTTGCCTATGGTTCTCAAATCAGCCATTGAGCTTGATCTTTTAGAGATCATCGCTAAAGCTGGGCCaGATGCTTTCATGTCTCCAAAAGACATAG
200 * 220 * 240 * 260 * 280 Ciclev10020814m : CTTCTCAGCTGCCCACAAAGAACCCAGATGCCCATATCGTGCTTGATCGTATATTGCGCCTTCTGGCGAGCTATTCAGTCCTTAATTGCTCTTTG : 285 CrOMT2 : CTTCTCAGCTGCCCACAAAGAACCCAGATGCCCATATCGTGCTTGATCGTATATTGCGCCTTCTGGCGAGCTATTCAGTCCTTAATTGCTCTTTG : 285 HM641694.1 : CTTCTCAGCTGCCCACAAAGAACCCAGATGCCCATATCGTGCTTGATCGTATATTGCGCCTTCTGGCGAGCTATTCAGTCCTTAATTGCTCTTTG : 285 CTTCTCAGCTGCCCACAAAGAACCCAGATGCCCATATCGTGCTTGATCGTATATTGCGCCTTCTGGCGAGCTATTCAGTCCTTAATTGCTCTTTG
* 300 * 320 * 340 * 360 * 380 Ciclev10020814m : CGCAATCTCCCCGACGGCAAAGTTGAGAGGCTTTATGGCCTTGCCCCCGTTTGTAAATTCCTCACTAAAAATGAAGATGGTGTTACACTTTCCGA : 380 CrOMT2 : CGCAATCTCCCCGACGGCAAAGTTGAGAGGCTTTATGGCCTTGCCCCCGTTTGTAAATTCCTCACTAAAAATGAAGATGGTGTTACACTTTCCGA : 380 HM641694.1 : CGCAATCTCCCCGACGGCAAAGTTGAGAGGCTTTATGGCCTTGCCCCCGTTTGTAAATTCCTCACTAAAAATGAAGATGGTGTTACACTTTCCGA : 380 CGCAATCTCCCCGACGGCAAAGTTGAGAGGCTTTATGGCCTTGCCCCCGTTTGTAAATTCCTCACTAAAAATGAAGATGGTGTTACACTTTCCGA
* 400 * 420 * 440 * 460 * Ciclev10020814m : TCTTTGTCTCATGAACCAAGACAAGGTTCTCATGGAGAGCTGGTACTACTTAAAAGATGCAGTGCTTGAAGGTGGCATTCCATTTAACAAGGCCC : 475 CrOMT2 : TCTTTGTCTCATGAACCAAGACAAGGTTCTCATGGAGAGCTGGTACTACTTAAAAGATGCAGTGCTTGAAGGTGGCATTCCATTTAACAAGGCCT : 475 HM641694.1 : TCTTTGTCTCATGAACCAAGACAAGGTTCTCATGGAGAGCTGGTACTACTTAAAAGATGCAGTGCTTGAAGGTGGCATTCCATTTAACAAGGCCT : 475 TCTTTGTCTCATGAACCAAGACAAGGTTCTCATGGAGAGCTGGTACTACTTAAAAGATGCAGTGCTTGAAGGTGGCATTCCATTTAACAAGGCCt
480 * 500 * 520 * 540 * 560 * Ciclev10020814m : ATGGGATGAATGCATTCGATTACCATGGCAAAGATCTAAGATTCAACAAGATTTTCAACAATGGAATGTCTTCTCATTCTACCATTACCATGAAG : 570 CrOMT2 : ATGGGATGAATGCATTCGATTACCATGGCAAAGATCTAAGATTCAACAAGATTTTCAACAATGGAATGTCTTCTCATTCTACCATTACCATGAAG : 570 HM641694.1 : ATGGGATGAATGCATTCGATTACCATGGCAAAGATCTAAGATTCAACAAGATTTTCAACAATGGAATGTCTTCTCATTCTACCATTACCATGAAG : 570 ATGGGATGAATGCATTCGATTACCATGGCAAAGATCTAAGATTCAACAAGATTTTCAACAATGGAATGTCTTCTCATTCTACCATTACCATGAAG
580 * 600 * 620 * 640 * 660 Ciclev10020814m : AAAATTCTTGAAAATTACAAAGGGTTTGAAGGCCTCAACTCAGTTGTCGACGTTGGTGGTGGAATTGGAGCCACACTTAACATGATTATCTCCAA : 665 CrOMT2 : AAAATTCTTGAAAATTACAAAGGGTTTGAAGGCCTCAACTCAGTTGTCGACGTTGGTGGTGGAATTGGAGCCACACTTAACATGATTATCTCCAA : 665 HM641694.1 : AAAATTCTTGAAAATTACAAAGGGTTTGAAGGCCTCAACTCAGTTGTCGACGTTGGTGGTGGAATTGGAGCCACACTTAACATGATTATCTCCAA : 665 AAAATTCTTGAAAATTACAAAGGGTTTGAAGGCCTCAACTCAGTTGTCGACGTTGGTGGTGGAATTGGAGCCACACTTAACATGATTATCTCCAA
* 680 * 700 * 720 * 740 * 760 Ciclev10020814m : GTATCCATCGATTAAAGGCATCAACTTTGATTTGCCACATGTGTCGAG------CATGTTAGGGGGAGACATGT : 733 CrOMT2 : GTATCCATCGATTAAAGGCATCAACTTTGATTTGCCACATGTTATTCAGGATGCTCCAGCTTTTCCTGGTGTCGAGCATGTTGGGGGAGACATGT : 760 HM641694.1 : GTATCCATCCATTAAAGGCATTAACTTTGATTTGCCACATGTTATTCAGGATGCTCCAGCTTTTCCTGGTGTCGAGCATGTTGGGGGAGACATGT : 760 GTATCCATCgATTAAAGGCATcAACTTTGATTTGCCACATGTtattcaggatgctccagcttttcctggtgtcgagcatgTtGGGGGAGACATGT
* 780 * 800 * 820 * 840 * Ciclev10020814m : TTGTTAGTGTTCCAAAGGGAGATGCCATTTTTATCAAGTGGATATGTCATGATTGGAGTGATGAGCACTGCGTGAAATTCTTGAAGAACTGCTAT : 828 CrOMT2 : TTGTTAGTGTTCCAAAGGGAGATGCCATTTTTATCAAGTGGATATGTCATGATTGGAGTGATGAGCACTGCGTGAAATTCTTGAAGAACTGCTAT : 855 HM641694.1 : TTGTTAGTGTTCCAAAGGGAGATGCCATTTTTATCAAGTGGATATGTCATGATTGGAGTGATGAGCACTGCGTGAAATTCTTGAAGAACTGCTAT : 855 TTGTTAGTGTTCCAAAGGGAGATGCCATTTTTATCAAGTGGATATGTCATGATTGGAGTGATGAGCACTGCGTGAAATTCTTGAAGAACTGCTAT
860 * 880 * 900 * 920 * 940 *
1 Ciclev10020814m : GAAGCACTCCCAGTAAATGGGAAAGTCATTGTTGCTGAATCTATCCTCCCAGTAACCCCGGACACAAGCCTCGCATCCAAAGTAGTCATCCATGT : 923 CrOMT2 : GAAGCACTCCCAGTAAATGGGAAAGTCATTGTTGCTGAATCTATCCTCCCAGTAACCCCGGACACAAGCCTCGCATCCAAAGTAGTCATCCATGT : 950 HM641694.1 : GAAGCACTCCCAGTAAATGGGAAAGTCATTGTTGCTGAATCTATCCTCCCAGTAACCCCGGACACAAGCCTTGCATCCAAAGTAGTCATCCATGT : 950 GAAGCACTCCCAGTAAATGGGAAAGTCATTGTTGCTGAATCTATCCTCCCAGTAACCCCGGACACAAGCCTcGCATCCAAAGTAGTCATCCATGT
960 * 980 * 1000 * 1020 * 1040 Ciclev10020814m : CGACTGCATCATGTTGGCTCATAACCCGGGTGGCAAAGAGAGGACTGAACAAGAGTTCAGAGCATTGGCTAAGGCTGCTGGATTCCAAGGTTTCC : 1018 CrOMT2 : CGACTGCATCATGTTGGCTCATAACCCGGGTGGCAAAGAGAGGACTGAACAAGAGTTCAGAGCATTGGCTAAGGCTGCTGGATTCCAAGGTTTCC : 1045 HM641694.1 : CGACTGCATCATGTTGGCTCATAACCCGGGTGGCAAAGAGAGGACTGAACAAGAGTTCAGAGCATTGGCTAAGGCTGCTGGATTCCAAGGTTTCC : 1045 CGACTGCATCATGTTGGCTCATAACCCGGGTGGCAAAGAGAGGACTGAACAAGAGTTCAGAGCATTGGCTAAGGCTGCTGGATTCCAAGGTTTCC
* 1060 * 1080 * 1100 Ciclev10020814m : AAGTTGTGAGCTCTGCTTTTAATACTTACATTATGGAATTTCTCAAGAGTGCTTGA : 1074 CrOMT2 : AAGTTGTGAGCTCTGCTTTTAATACTTACATTATGGAATTTCTCAAGAGTGCTTGA : 1101 HM641694.1 : AAGTTGTGAGCTCTGCTTTTAATACTTACATTATGGAATTTCTCAAGAGTGCTTGA : 1101 AAGTTGTGAGCTCTGCTTTTAATACTTACATTATGGAATTTCTCAAGAGTGCTTGA
B * 20 * 40 * 60 * 80 * Ciclev10020814m : MGSTSSETQISPAQGSDEEANLLAMQLTSASVLPMVLKSAIELDLLEIIAKAGPDAFMSPKDIASQLPTKNPDAHIVLDRILRLLASYSVLNCSL : 95 CrOMT2 : MGSTSSETQISPAQGSDEEANLLAMQLTSASVLPMVLKSAIELDLLEIIAKAGPDAFMSPKDIASQLPTKNPDAHIVLDRILRLLASYSVLNCSL : 95 ADK97702.1 : MGSTSSETQISPAQGSDEEANLLAMQLTSASVLPMVLKSAIELDLLEIIAKAGPDAFMSPKDIASQLPTKNPDAHIVLDRILRLLASYSVLNCSL : 95 MGSTSSETQISPAQGSDEEANLLAMQLTSASVLPMVLKSAIELDLLEIIAKAGPDAFMSPKDIASQLPTKNPDAHIVLDRILRLLASYSVLNCSL
100 * 120 * 140 * 160 * 180 * Ciclev10020814m : RNLPDGKVERLYGLAPVCKFLTKNEDGVTLSDLCLMNQDKVLMESWYYLKDAVLEGGIPFNKAHGMNAFDYHGKDLRFNKIFNNGMSSHSTITMK : 190 CrOMT2 : RNLPDGKVERLYGLAPVCKFLTKNEDGVTLSDLCLMNQDKVLMESWYYLKDAVLEGGIPFNKAYGMNAFDYHGKDLRFNKIFNNGMSSHSTITMK : 190 ADK97702.1 : RNLPDGKVERLYGLAPVCKFLTKNEDGVTLSDLCLMNQDKVLMESWYYLKDAVLEGGIPFNKAYGMNAFDYHGKDLRFNKIFNNGMSSHSTITMK : 190 RNLPDGKVERLYGLAPVCKFLTKNEDGVTLSDLCLMNQDKVLMESWYYLKDAVLEGGIPFNKAyGMNAFDYHGKDLRFNKIFNNGMSSHSTITMK
200 * 220 * 240 * 260 * 280 Ciclev10020814m : KILENYKGFEGLNSVVDVGGGIGATLNMIISKYPSIKGINFDLPHVS------SMLGGDMFVSVPKGDAIFIKWICHDWSDEHCVKFLKNCY : 276 CrOMT2 : KILENYKGFEGLNSVVDVGGGIGATLNMIISKYPSIKGINFDLPHVIQDAPAFPGVEHVGGDMFVSVPKGDAIFIKWICHDWSDEHCVKFLKNCY : 285 ADK97702.1 : KILENYKGFEGLNSVVDVGGGIGATLNMIISKYPSIKGINFDLPHVIQDAPAFPGVEHVGGDMFVSVPKGDAIFIKWICHDWSDEHCVKFLKNCY : 285 KILENYKGFEGLNSVVDVGGGIGATLNMIISKYPSIKGINFDLPHViqdapafpgveh6GGDMFVSVPKGDAIFIKWICHDWSDEHCVKFLKNCY
* 300 * 320 * 340 * 360 Ciclev10020814m : EALPVNGKVIVAESILPVTPDTSLASKVVIHVDCIMLAHNPGGKERTEQEFRALAKAAGFQGFQVVSSAFNTYIMEFLKSA : 357 CrOMT2 : EALPVNGKVIVAESILPVTPDTSLASKVVIHVDCIMLAHNPGGKERTEQEFRALAKAAGFQGFQVVSSAFNTYIMEFLKSA : 366 ADK97702.1 : EALPVNGKVIVAESILPVTPDTSLASKVVIHVDCIMLAHNPGGKERTEQEFRALAKAAGFQGFQVVSSAFNTYIMEFLKSA : 366 EALPVNGKVIVAESILPVTPDTSLASKVVIHVDCIMLAHNPGGKERTEQEFRALAKAAGFQGFQVVSSAFNTYIMEFLKSA
C Percent identity matrix for DNA
Ciclev10020814m CrOMT2 HM641694.1 Ciclev10020814m 100.00 98.79 98.42 CrOMT2 100.00 99.64 HM641694.1 100.00
Percent identity matrix for protein
Ciclev10020814m CrOMT2 ADK97702.1 Ciclev10020814m 100.00 98.60 98.60 CrOMT2 100.00 100.00 ADK97702.1 100.00
2 Figure S1 Sequences alignment of Citrus reticulata O-methyltransferase 2 (CrOMT2) and two paralogous genes from different citrus species at DNA and protein levels.
Complete CDs (A) and the deduced amino acid sequences (B) of CrOMT2 and the two paralogous genes were aligned using Clustal2.1 and analyzed by GeneDoc, including Ciclev10020814m from
Citrus clementina, CrOMT2 from Citrus reticulata, and a putative caffeic acid OMT from Citrus aurantium (GenBank accession: HM641694.1 for complete CDs and ADK97702.1 for the deduced protein). The percent identity matrixes of the aligned sequences created by Clustal2.1 are shown (C).
3 Figure S2
Figure S2 Biochemical characterization of the recombinant CrOMT2.
(A) SDS-PAGE profiles of the recombinant CrOMT2. Lanes: 1, the molecular weight of standard proteins; 2, unpurified fractions after induction; 3, purified enzyme.
(B) The dependency of the enzyme on pH and temperature.
Enzyme activity was measured with luteolin as the substrate, and the corresponding product catalyzed by CrOMT2 was analyzed using HPLC. The data are shown as means ± SE (n = 3).
4 Figure S3 A E chrysoeriol standard syringetin standard
B F tricin standard quercetin 3,3'-dimethyl ether standard
C G baicalein 7-methyl ether standard isokaempferide standard
D H isorhamnetin standard homoeriodictyol standard
Figure S3 MS/MS spectra of the authentic standards corresponding to the methylated products catalyzed by CrOMT2. Chrysoeriol (A), tricin (B), baicalein 7-methyl ether (C), isorhamnetin (D), syringetin (E), quercetin 3,3'-dimethyl ether (F), isokaempferide (G), homoeriodictyol (H). (A, F, H) were operated in negative ions mode and the rest were in positive ions mode.
5 Figure S4
Figure S4 HPLC analysis of dihydroflavonols and caffeic acid methylation by recombinant CrOMT2.
HPLC chromatograms of enzyme reactions using caffeic acid (A), dihydroquercetin (B), dihydromyricetin (C) and dihydrokaempferol (D) are shown. The methylated product of caffeic acid produced by the recombinant CrOMT2 were inferred by comparison of the retention times with corresponding standards. For each dihydroflavonols substrate, a single peak generated by CrOMT2 was indicated. Caf, caffeic acid; Fer, ferulic acid; Dhq, dihydroquercetin; Dhm, dihydromyricetin;
Dhk, dihydrokaempferol.
6 Figure S5 CaOMT1 1 M Á ...... Á L Á ...... F A Á MQL A Á SAS V Á LPM VLK ÄÅÆ S AIE ÄÅÅÆ L D L Á L EII ÄÅÆ R AtOMT1 1 M Â GSTAETQL.T.P.VQ...... V..T...DDE...... A Â A L Â ...... F A Â MQL A Â SAS V Â LPM ALK Ç S ALE Ç L D L Â L EIM Ç A ShMOMT1 1 M Â ALSMDNI...... V...... I..S...NEE.....EICM Â M Â ...... K A Â MHLPCGLYLNM Â Â VLK Ç A AIE Ç L D L Â F EII Ç A ShMOMT2 1 M Â ASNNNC...... AYELIE...... A Â E A Â ...... QSWDY Â I Â LSY L Â RPSC Ç IK C AI Ç QL G I Â P DIL Ç H ShMOMT3 1 M Â ALSMDNI...... V...... I..S...NEE.....EIYM Â M Â ...... K A Â MHIPCGLYLNM Â Â VLK Ç A AIE Ç L D L Â F EII Ç A SlMOMT4 1 M Â ALSMDNI...... V...... I..S...NEE.....EIYM Â M Â ...... K A Â MHIPCGLYLNM Â Â VLR Ç A AIE Ç L D L Â F EII Ç A TaOMT2 1 M Â GSIA...... A...... G..A...DED...... A Â C M Â ...... Y A Â LQL V Â SSS I Â LPMT Ç LK N AIE Ç L G L Â L E Ç T L M TaOMT1 1 M Â GSTAADMA...... A...... S..A...DEE...... A Â C M Â ...... Y A Â LQL V Â SSS I Â LPMT Ç LK N AIE Ç L G L Â L E Ç T L V OsOMT1 1 M Â GSTAADMA...... A...... A..A...DEE...... A Â C M Â ...... Y A Â LQL A Â SSS I Â LPMT Ç LK N AIE Ç L G L Â L E Ç T L Q CrOMT2▲ 1 M Â ELQSS...... EIRN...... A Â Q A Â ...... HFFTQ Â V Â FSFTSMSS Â Ç LK C AV Ç QL G I Â P DAI Ç H CrCOMT1 1 M Â GSANPDNK.N....S...... M..T.KEEEE...... A Â C L Â ...... S A Â MRL A Â SAS V Â LPM VLK Ç S AIE Ç L D L Â L ELI Ç K CrOMT6 1 M Â DLQTA...... EFRE...... A Â Q A Â ...... K I Â WSQ A Â FSF A Â NCA ALK Ç C AV Ç KL G I Â A DAI Ç D ObFOMT1 1 M Â GRDEEA...... A...... A Â Q A Â ...... E A Â WNHGFGF Â I Â KTS VIK Ç T AIE Ç L E I Â P DIL Ç H ObFOMT2 1 M Â GRDEEA...... A...... A Â R A Â ...... E A Â WNHGFGF Â I Â KTS VIK Ç T AIE Ç L E I Â P DIL Ç H ObFOMT3 1 M Â AVDKEV...... Q...... L Â H A Â ...... Q A Â WEH A Â LSY I Â NST AL Ç SA AVE Ç L E I Â P DIL Ç E ObFOMT4 1 M Â AVDKEV...... E...... L Â H A Â ...... Q A Â WDH A Â LSY I Â TPT AL Ç SA AVE Ç L E I Â P DIL Ç E ObFOMT5 1 M Â VADEEA...... Q...... L Â H A Â ...... Q A Â WDH A Â LSY I Â KPT AL Ç SA AVE Ç L E I Â P DIL Ç E ObFOMT6 1 M Â AVDKEV...... Q...... L Â H A Â ...... Q A Â WDH A Â LSY I Â KPT AL Ç DA AVE Ç L E I Â P DIL Ç E ObF8OMT-1 1 M Â PSSSGV...... DSTQELLD...... A Â Q A Â ...... H I Â WNH I Â FNH I Â NSMT Ç LK W AV Ç QL G I Â P DII Ç H MpOMT3 1 M Â EASFENGR.K.RSSS...... S..S.S.EEE...... S A Â FS...... F Â A Â MEL A Â AGS V Â LPM VIK Ç S AID Ç L N L Â L ELI Ç K MpOMT1A 1 M Â APEEDS...... L...... A Â L A Â ...... E A Â WNHGFGF Â I Â KTS IVK Ç T AVE Ç L E I Â P DIL Ç E MpOMT1B 1 M Â APKEDS...... L...... A Â L A Â ...... E A Â WNHGFGF Â I Â KTS IVK Ç T AVE Ç L G I Â P DIL Ç E MpOMT4 1 M Â VADEEV...... R...... V Â R A Â ...... E A Â WNN A Â FGY I Â KPT AV Ç AT AVE Ç L G L Â P DIL Ç E MpOMT2 1 M Â ALPNGI...... SSKQELLE...... A Â Q A Â ...... H V Â WNH I Â YSY I Â NSMS Ç LK C AI Ç QL G I Â P DAI Ç H CdFOMT5 1 M Â GSIVD...... GER.....DQSF Â A Â ...... Y A Â SQL V Â MGT V Â LPM AI Ç QA V Ç Y EL G I Â F EIL Ç D ZmOMT1 1 M Â GSTAGDVA...... A...... V..V...DEE...... A Â C M Â ...... Y A Â MQL A Â SSS I Â LPMT Ç LK N AIE Ç L G L Â L EVL Ç Q Hv7OMT 1 M Â QDTSSTQH.KSL.PNNIIEMDMVTSMPLEANSNGQILQ...... A Â E A Â ...... E L Â FCHSFGY Â L Â KSM AL Ç QS VV Ç KL R I Â P DVL Ç H HvOMT1 1 M Â GSIA...... A...... G..A...DED...... A Â C M Â ...... Y A Â LQL V Â SSS I Â LPMT Ç LK N AIE Ç L G L Â L E Ç T L M MsCOMT1 1 M Â GSTGETQI.T.P.TH...... I..S...DEE...... A Â N L Â ...... F A Â MQL A Â SAS V Â LPM ILK Ç S ALE Ç L D L Â L EII Ç A CrOMT2 1 M Â GSTSSETQIS.P.AQ...... G..S...DEE...... A Â N L Â ...... L A Â MQLTSAS Â V Â LPM VLK Ç S AIE Ç L D L Â L EII Ç A SaDMPM 1 M Ã APTEATRG.G.PADP...... A..P...APEAHRGGHTEH Ã A Ã DPAEHAAQFGAQER I Ã LTL V Ã WGY I Ã SSE IL ÉÈ DL A ÉÉÈ TR L D L Ã P DLM ÉÈ G consensus>70 M...... e...... a.q.....i....lk.aieL...#...
CaOMT1 31 GQ...... DT..C MS ÄÆ PT EI ÄÆ ASHLPTT.NPDAP.A.M V Á DRILR ÄÅÅÆ LL ÄÆ SCYS V Á VTCSVRSVD...... D...QRV.. Y Á GL Á AP V Á CK Á YL Á TKN AtOMT1 50 KNG...... S..P MS Ç PT EI Ç ASKLPTK.NPEAP.V.M L Â DRILR Ç LL Ç TSYS V Â LTCSNRKLS...... GDG.VERI.. Y Â GL Â GP V Â CK Â YL Â TKN ShMOMT1 48 KST...... TQ.K LS Ç SY EI Ç ASQIPTK.NPNASSL.V L Â ERILR Ç FLA Ç SQS L Â LTCNITKND...... DGNVHTT.. Y Â NL Â TP L Â SQS Â L Â ISD ShMOMT2 45 KNA....D..PI... MS Ç LS DL Ç IAALPNL.NPSKT.T.F I Â PI ÇLMR VLV Ç DFG L Â FNYHQ...... QQGDG.. Y Â SL Â TT V Â GR Â LL Â VEN ShMOMT3 48 KST...... TQ.K LS Ç SY EI Ç ASQIPTK.NPNASSL.V L Â ERILR Ç FLA Ç SQSFLTCNITKND...... DGIVHTS.. Â Y Â NL Â TP L Â SQS Â L Â ISD SlMOMT4 48 KST...... TQ.K LS Ç SY EI Ç ASQIPTK.NPNASSL.V L Â ERILR Ç FLA Ç SQSFLTCNITKND...... DGNVHTS.. Â Y Â NL Â TP L Â SQS Â L Â ISD TaOMT2 43 AAG....G..KF..LTPAEV Ç ÇA AKLPSAA.NPEAP.D.M V Â DRMLR Ç LLA Ç SYN V Â VSCRTEDGK...... DGRLSRR.. Y Â GA Â AP V Â CK Â YL Â TPN TaOMT1 47 AAG....G..KL..LTPAEV Ç ÇA AKLPSTA.NPAAA.D.M V Â DRMLR Ç LLA Ç SYN V Â VSCTMEEGK...... DGRLSRR.. Y Â RA Â AP V Â CK Â FL Â TPN OsOMT1 47 SAAVAGGGG.KAALLTPAEV Ç ÇA DKLPSKA.NPAAA.D.M V Â DRMLR Ç LLA Ç SYN V Â VRCEMEEGA...... DGKLSRR.. Y Â AA Â AP V Â CK Â WL Â TPN CrOMT2▲ 42 SHG....K...P... M Ç ALS DL Ç TNSLPI..NPSKA.P.Y I Â YRLMR Ç ILV Ç AAGYFSE...... EEKNV.. Â Y Â SL Â TPFT Â R Â LL Â LKN CrCOMT1 50 KSG....P..GA..Y VS Ç PS EL Ç AAQLPTQ.NPDAP.V.M L Â DRILR Ç LLA Ç SYS V Â LNCTLKDLP...... DGGIERL.. Y Â SL Â AP V Â CK Â FL Â TKN CrOMT6 42 NHD....K..KA... LT Ç LS EL Ç TEELSI..KPSKS.P.F L Â QRLMR Ç QLV Ç NAGFFTEAKQLRDDNKD.....GRTTTA.. Â Y Â AL Â TP V Â SR Â LL Â LKN ObFOMT1 40 NQG....G...P... LS Ç LSA ÇL SSAVGV..P...P.D.R L Â HRIMR Ç FLA Ç HHG V Â SKKTASP...... P.GESDYY.. Y Â AETA Â V Â SR Â SL Â TKD ObFOMT2 40 NHG....A...P... LS Ç LSA ÇL SSAVGV..P...P.D.R L Â HRIMR Ç FL Ç THHG V Â SKKTASP...... P.GESDYY.. Y Â AETA Â V Â SR Â SL Â TKD ObFOMT3 40 DHG....G...L... MS Ç LS EL Ç SAASGC..P...R.E.P L Â YRLMR Ç FLI Ç FHG I Â FTKSD...... DC.. Y Â AQSP Â L Â SR Â LFTRE Â ObFOMT4 40 DHG....G...L... MS Ç LS EL Ç SAASGC..P...R.E.P L Â YRLMR Ç FLI Ç FHG I Â FTKSN...... DC.. Y Â AQSP Â L Â SR Â VFTRE Â ObFOMT5 40 NHG....G...P... MT Ç LS EL Ç SAASGC..P...R.E.P L Â YRLMR Ç FLI Ç FHG I Â FTKSD...... DC.. Y Â AQSP Â L Â SR Â LFTTE Â ObFOMT6 40 DHG....G...P... MS Ç LS EL Ç SAATGC..P...R.E.P L Â YRLMR Ç FLI Ç FHG I Â FTKSD...... DC.. Y Â AQSP Â L Â SR Â LFTRE Â ObF8OMT-1 47 KHD....K...P... MT Ç LSQ ÇL ADAIPI..NRAKS.D.A L Â HRIMR Ç ILV Ç HSKFFDRVRT...... LP....NEEEA.. Â Y Â CL Â TR A Â SR Â LL Â LRD MpOMT3 53 RGG....E...E..G AS Ç AY EL Ç AAQINAE.NPKAA.AEM I Â DRIL Ç QL LA Ç AHS V Â LTCRVETPP...... SRR..RR.. Y Â SL Â AA V Â CK Â FL Â TRD MpOMT1A 40 SRG....A...P... VS Ç IP EL Ç ATAVDC..S...A.D.R I Â YRVMR Ç FLA Ç YHG I Â FKRTKPPPES...... T.EGGSVY.. Y Â AQTP Â V Â SR Â RL Â TRE MpOMT1B 40 SRG....A...P... VS Ç IP EL Ç AAAVDC..S...A.D.R L Â YRVMR Ç FLA Ç YHG I Â FKRTEPPPES...... T.GGGSVY.. Y Â AQTL Â V Â SR Â RL Â TRE MpOMT4 40 NHD....G...P... MS Ç LL EL Ç SAATDC..P...A.E.P L Â HRLMR Ç FLV Ç FHG I Â FKKTAKPPLS...... NEAVY.. Y Â ARTA Â L Â SR Â LFTRD Â MpOMT2 47 KHG....N...P... IT Ç LSQ ÇL ADALNI..NKAKS.H.G L Â FRLMR Ç ILV Ç HSGFFDKVKV.K..VKVEGEDEEEEEDA.. Â Y Â SL Â TP A Â SR Â LL Â LRS CdFOMT5 43 KVG....P..GAK.. L Ç CAS DI Ç AAQLLTK.NKDAP.M.M L Â DRILR Ç LLA Ç SYS V Â VECSL..DA...... SG.ARRL.. Y Â SL Â NS V Â SK Â YYVPN Â ZmOMT1 47 K....EAGGGKAA.L A Ç PEEV ÇV ARMPAAPGDPAAAAA.M V Â DRMLR Ç LLA Ç SYD V Â VRC.QMEDR...... DGRYERR.. Y Â SA Â AP V Â CK Â WL Â TPN Hv7OMT 69 RYG....G...A... AS Ç LP EL Ç LSTVPI..HPNKL.P.Y L Â PRLMK Ç MLA Ç AAG I Â FTAEDVPATV...... GDGEPTTL.. Y Â HL Â NA V Â SR Â LL Â VDD HvOMT1 43 SAG....G..KF..LTPAEV Ç ÇA AKLPSTA.NPEAP.D.M V Â DRMLR Ç LLA Ç SYK V Â VSCRTEESK...... DGRLSRR.. Y Â GA Â AP V Â CK Â YL Â TPN MsCOMT1 50 KAG....P..GA..Q IS Ç PI EI Ç ASQLPTT.NPDAP.V.M L Â DRMLR Ç LLA Ç CYI I Â LTCSVRTQQ...... DGKVQRL.. Y Â GL Â AT V Â AK Â YL Â VKN CrOMT2 51 KAG....P..DA..F MS Ç PK DI Ç ASQLPTK.NPDAH.I.V L Â DRILR Ç LLA Ç SYS V Â LNCSLRNLP...... DGKVERL.. Y Â GL Â AP V Â CK Â FL Â TKN SaDMPM 71 TEE...... R..A A È ..A EL È AASLDT.....DP.V.ATL Ã RLLR ÉÉÈ AF ÈA ALG L Ã A...... EETGAGR F Ã RL Ã TP A Ã GH Ã RL Ã ..R consensus>70 ...... e...... r.$r.l....v...... %...... l..n
CaOMT1 95 QD...GVS I Á AALC ÄÅÆ LM NQDK V Á LMES.W.YHLK D Á A V Á .....L.DGGI.P.F.NK A Á Y G Á MSS F Á EYHGT D Á PR.. F Á .NK V ÄÅÆF N RG M Á SDHST I Á T.MKK AtOMT1 117 ED...GVS I Â AALC Ç LM NQDK V Â LMES.W.YHLK D Â A I Â .....L.DGGI.P.F.NK A Â Y G Â MSA F Â EYHGT D Â PR.. F Â .NK V ÇF N NG M Â SNHST I Â T.MKK ShMOMT1 117 KD...GTS I Â APF LLL Ç ATDP V Â GVHA.C.FHLK D Â A I Â .....L.EGEI.P.F.NK A Â H G Â VHA F Â EYHGK D Â SR..M.NG Â L ÇF N KA M Â QNLTC I Â E.MKR ShMOMT2 107 HH..F..GNRSFF Â Ç L FAQHP V Â VLNT.A.ASVG D Â W L Â ...... KDDLRTA.F.ET A Â D G Â KSH W Â DYCGA D Â PE.. F Â .NG V ÇF N DA M Â AGDSR L Â M.SNL ShMOMT3 117 KD...GSS I Â APF LLL Ç ATDP V Â AVNS.W.FHFK D Â A I Â .....L.EGEI.P.F.NK A Â H G Â VHA F Â EYHGK D Â SR..M.NG Â L ÇF N RA M Â QN.VTCTEMKR Â SlMOMT4 117 KD...GSS L Â APF LLL Ç HSES V Â VVNS.C.FLLK D Â A I Â .....L.QGEV.P.F.NK A Â Y G Â MNA F Â EYTKK D Â SR..M.NG Â L ÇF N KA M Â QNVTC I Â E.MKK TaOMT2 112 ED...GVS M Â SAL ALM Ç NQDK V Â LMES.W.YYLK D Â A V Â .....L.DGGI.P.F.NK A Â Y G Â MSA F Â EYHGT D Â PR.. F Â .NR V ÇF N EG M Â KNHSI I Â I.TKK TaOMT1 116 ED...GVS M Â AAL ALM Ç NQDK V Â LMES.W.YYLK D Â A V Â .....L.DGGI.P.F.NK A Â Y G Â MSA F Â EYHGT D Â PR.. F Â .NR V ÇF N EG M Â KNHSI I Â I.TKK OsOMT1 123 ED...GVS M Â AAL ALM Ç NQDK V Â LMES.W.YYLK D Â A V Â .....L.DGGI.P.F.NK A Â Y G Â MTA F Â EYHGT D Â AR.. F Â .NR V ÇF N EG M Â KNHSV I Â I.TKK CrOMT2▲ 100 DP..L..NSISM Â VL Ç GVNQI A Â ELKA.W.NAMS E Â WF...... QNEDLTA.F.ET Â A Â H G Â KNF W Â DFGAE D Â K... Y Â .GKN ÇF DGV M Â AADSI L Â V.SKM CrCOMT1 119 ED...GVS M Â AAL LLM Ç NQDK V Â LMES.W.YHLK D Â A V Â .....L.EGGI.P.F.NK A Â Y G Â MTA F Â EYHGK D Â PR.. F Â .NK V ÇF N QG M Â SNHST I Â I.MKK CrOMT6 112 EQWNL..RGI.. Â VL Ç TMLDP A Â ELKA.W.SVLN D Â WF...... KNDDPTA.F.QT Â A Â HEKNY Â W Â DYTAENTQ..H.CQ Â Â I ÇF EDA M Â ANDSV L Â V.SKL ObFOMT1 101 N...... L Â GPF VLL Ç QGA.Q.RGP.SACITAQG Â Â L Â ...... KSRERPG.V.EE L Â GSDPL Â Y Â ....E D Â PI.. F Â TEK V ÇF RDA M Â TCHAR V Â T.TSA ObFOMT2 101 N...... L Â GAF VLL Ç QGA.Q.RGP.SACITAQG Â Â L Â ...... KSRERPG.V.EE L Â GSDPL Â Y Â ....E D Â PI.. F Â TKM V ÇF RDA M Â ACHAR L Â T.TSA ObFOMT3 94 N...... L Â GPY MLM Ç QATP V Â TRSP.A.GLSG E Â A L Â ...... KTGTSLY.L.KS I Â R G Â EDS W Â ....S D Â PAYG Y Â HMK A ÇF T NA M Â IAHAR L Â T.AAA ObFOMT4 94 N...... L Â GPY MLM Ç QATP V Â TRSP.A.GLSG E Â A L Â ...... KTGTPLY.L.KS I Â R G Â EDS W Â ....N D Â PAYG F Â HMR A ÇF T NG M Â AAHAR L Â T.AAA ObFOMT5 94 N...... L Â GPY MLM Ç QATP V Â TRCP.T.GLSG E Â A L Â ...... KTGTSLY.L.KS I Â R G Â EDS W Â ....S D Â PAYG Y Â HMK A ÇF T NA M Â TAHAR L Â T.AAA ObFOMT6 94 N...... L Â GLY MLM Ç QATP A Â TRSP.A.GLSG E Â A L Â ...... KTGTPLY.L.KS I Â R G Â EDS W Â ....N D Â PAYG F Â HMR A ÇF T NA M Â TAHSR L Â T.AAA ObF8OMT-1 110 EP..L..S L Â TPF ALA Ç VLDEDLMGT.F.HCVP Â E Â WF...... GNECPSP.L.EFKHEKSIREFAENNQR.. Â Â Â Â Â W Â .SL L ÇF N EG M Â ANDAR L Â V.GSI MpOMT3 120 ED...GAS L Â APLS Ç LL VQDR V Â FMEP.W.YHLK D Â V I Â .....V.EGGV.A.F.ER A Â Y G Â VHA F Â EYHAK D Â PK.. F Â .NK I ÇF N QA M Â HNQSI I Â F.MKR MpOMT1A 104 N...... L Â GPF VLL Ç QGT. M Â .REP.SGCVTA E Â T L Â ...... RTSKRPG.VVNENESDHL Â Â Y Â ....E D Â PV.. F Â SMK V ÇF RDA M Â ASHAR M Â T.TAA MpOMT1B 104 N...... L Â GPF VLL Ç QGT. M Â .REP.SGCVTA E Â T L Â ...... RMSKRPG.LVDDNDSDRL Â Â Y Â ....E D Â PV.. F Â SMK V ÇF RDA M Â ASHAR M Â T.TAA MpOMT4 102 E...... L Â GDF MLL Ç QTGP L Â SQHP.A.GLTASS Â L Â ...... RTGKPQF.I.RS V Â N G Â EDS W Â ....T D Â PVNG Y Â HMK V ÇF S DA M Â AAHARET.TAA Â MpOMT2 118 EP..L..S V Â APF ALA Ç MSDP V Â YTET.W.HHLS E Â WF...... RNDAVAA.F.DTKY Â Â G Â MTFPEYAVA Â D Â DR..L.NV Â L ÇF N EA M Â ACDAGFV.NSI Â CdFOMT5 109 KD...GVL L Â GPL L Ç Q M NQDK V Â LLES.W.SQLK D Â A I Â .....L.EGGI.P.F.NR A Â H G Â VHI F Â EYAGL D Â PK.. F Â .NKR ÇF N TA M Â YNYTS L Â V.LSN ZmOMT1 120 ED...GVS M Â AAL ALM Ç NQDK V Â LMES.W.YYLK D Â A V Â .....L.DGGI.P.F.NK A Â Y G Â MTA F Â EYHGT D Â SR.. F Â .NR V ÇF N EG M Â KNHSV I Â I.TKK Hv7OMT 137 ASVNGGAS M Â SPC VLL Ç GTVP L Â FLGA.S.LKLH E Â W L Â QSEEQATTETP.F.M.LAH. Â G Â GTL Y Â GIGGR D Â SE.. F Â .NT V ÇF N KA M Â GASSEFV.AAL Â HvOMT1 112 ED...GVS M Â SAL ALM Ç NQDK V Â LMES.W.YYLK D Â A V Â .....L.DGGI.P.F.NK A Â Y G Â MSA F Â EYHGT D Â PR.. F Â .NR V ÇF N EG M Â KNHSI I Â I.TKK MsCOMT1 119 ED...GVS I Â SALN Ç LM NQDK V Â LMES.W.YHLK D Â A V Â .....L.DGGI.P.F.NK A Â Y G Â MTA F Â EYHGT D Â PR.. F Â .NK V ÇF N KG M Â SDHST I Â T.MKK CrOMT2 120 ED...GVT L Â SDLC Ç LM NQDK V Â LMES.W.YYLK D Â A V Â .....L.EGGI.P.F.NK A Â Y G Â MNA F Â DYHGK D Â LR.. F Â .NK I ÇF N NG M Â SSHST I Â T.MKK SaDMPM 122 TD...VPDSLHAF Ã ÉÈ V ..RQG M Ã GVFRQA.WSHF D Ã HS.....I.RTGE.PAF.DQ Ã V Ã F G Ã TDF F Ã SYLSERPE..L.SGT Ã Ã ÉÈF T SS M Ã REATRTM.STA Ã consensus>70 e...... lm...... d...... n...g...... d....f....F.d.M......
7 CaOMT1 167 V ÄÆ FQA. Y Á ..QG F Á Q GL ÄÆ T SLV ÄÅÅÅÅÅÆ D VGG GT G ÄÆ A T L Á TM IL ÄÆ SK Y ÄÆP T IR ÄÅÅÆ C I N F ÄÅÅÆD L P H VI ÄÆ ED A Á PEY.P GIE ÄÅÆ ...... H VG ÄÅÅÅÅÆ G DMF V SV ÄÅÆ P K.G DAI ÄÅÅÅÅÅÅÅÅÅ F MKWI CH AtOMT1 189 IL Ç ET. Y Â ..KG F Â E GL Ç T SLV Ç D VGG GI G Ç A T L Â KM IV Ç SK Y ÇP N LKGI Ç N F ÇD L P H VI Ç ED A Â PSH.P GIE Ç ...... H VG Ç G DMF V SV Ç P K.G DAI F MKWI CH ShMOMT1 189 IV Ç EC. Y Â ..NG F Â Q GV Ç KE Ç II D VGG GL G Ç I S L Â AS II Ç SK Y ÇP N IKGI Ç N F ÇD L P H VI Ç KD A Â PTY.E GIE Ç ...... H VG Ç G DMW D SI Ç P Q.G ELIILK A VL H ShMOMT2 179 LI Ç SD.CCAGV Â F Â E GL Ç T SLV Ç D IGG GT G Ç A V A Â MA IA Ç GA F ÇP S LK Ç C I VL ÇD L P H VI Ç ADRKGS.GN Â Ç LE ...... F V Ç AG S MF DK Ç IP H.AN AILLKWIL H ShMOMT3 189 IV Ç EC. Y Â ..NG F Â Q GV Ç KE Ç II D VGG GL G Ç I S L Â AT II Ç SK Y ÇP N IKGI Ç N F ÇD L P H VI Ç KD A Â PTY.E GIE Ç ...... H VG Ç G DMF K SV Ç P Q.R ELILLK A IL H SlMOMT4 189 IV Ç EC. Y Â ..NG F Â E GV Ç KET Ç ID VGG GL G Ç I S L Â AS II Ç SK Y ÇP N IKGI Ç N F ÇD L P H VI Ç KD A Â PTY.E GIE Ç ...... H VG Ç G DM LK SV Ç P Q.G ELIILK E IL H TaOMT2 184 LL Ç ES. Y Â ..KG F Â E GL Ç G TLV Ç D VGG GV G Ç A T V Â AA I Ç TAH Y ÇP T IKGI Ç N F ÇD L P H VI Ç SE A Â PPF.P GV Ç T...... H VG Ç G DMF QK Ç VP S.G DAILMKWIL H TaOMT1 188 LL Ç EV. Y Â ..KG F Â E GL Ç G TIV Ç D VGG GV G Ç A T V Â GA I Ç TAA Y ÇP A IKGI Ç N F ÇD L P H VI Ç SE A Â QPF.P GV Ç T...... H VG Ç G DMF QK Ç VP S.G DAILMKWIL H OsOMT1 195 LL Ç DL. Y Â ..TG F Â DA Ç A S TVV Ç D VGG GV G Ç A T V Â AA VV Ç SRH ÇP H IRGI Ç N Y ÇD L P H VI Ç SE A Â PPF.P GVE Ç ...... H VG Ç G DMF A SV Ç P RGG DAILMKWIL H CrOMT2▲ 171 LI Ç PE. F Â N.YL F Â E GL Ç D SLV Ç D VGG GT G Ç TI A Â KA IA Ç KS F ÇP D LK Ç CTV F ÇD L P H VV Ç AN L Â EST.EN Ç LE ...... F VG Ç G DMF EK Ç IP S.AN AILLKWIL H CrCOMT1 191 IL Ç EI. Y Â ..QG F Â Q GL Ç K TVV Ç D VGG GT G Ç A T L Â NM IV Ç SK Y ÇP S IKGI Ç N F ÇD L P H VI Ç ED A Â PSY.P GVD Ç ...... H VG Ç G DMF V SV Ç P K.G DAI F MKWI CH CrOMT6 184 LV Ç TE. Y Â K.FL F Â E GL Ç T SLI Ç D LGG ST G Ç TI A Â KA LA Ç KS F ÇP N LK Ç CTV F ÇD L P H VV Ç AN L Â EST.KN Ç LE ...... F VG Ç G DMF EK Ç LP P.SN AILLKWIL H ObFOMT1 166 VI Ç EN. Y Â G.EG F Â R GV Ç G SLV Ç D VGG SY G Ç M T L Â GM LV Ç EA F ÇP W IRGI Ç C Y ÇD L P P VV Ç AK A Â KPL.H GVE Ç ...... F V Ç AG S MF E SV Ç P K.A DVIML M FVL H ObFOMT2 166 VI Ç EN. Y Â G.EG F Â R GV Ç G SLV Ç D VGG SY G Ç M T L Â GM LV Ç EA F ÇP W IRGI Ç C Y ÇD L P Q VV Ç AK A Â KPL.H GVE Ç ...... F V Ç AG S MF E SV Ç P E.A DVVML M FVL H ObFOMT3 162 IV Ç SN. Y Â P.AA F Â D GL Ç R SVV Ç D VGG RH G Ç TA I Â GR LV Ç EA F ÇP W VRGI Ç A F ÇD L P E IV Ç AD A Â PPR.K GVD Ç ...... F VG Ç G DMF E SV Ç P K.A DAVML M WIL H ObFOMT4 162 IV Ç TN. Y Â P.TA F Â N GV Ç R SVV Ç D VGG RH G Ç M A I Â GK LV Ç EA F ÇP W VRGI Ç A F ÇD L P E VV Ç AD A Â PPR.K GVD Ç ...... F VG Ç G DMF E SL Ç P K.A DAVML M WVL H ObFOMT5 162 IV Ç RN. Y Â P.AA F Â D GV Ç Q SVV Ç D VG SRH G Ç TA I Â GK LV Ç EA F ÇP W VRGI Ç A F ÇD L P E IV Ç AD A Â PPR.K GVD Ç ...... F VG Ç G DMF E SV Ç P K.A DAVML M WIL H ObFOMT6 162 IV Ç TN. Y Â P.TA F Â D GV Ç R SVV Ç D VGG RH G Ç M A I Â GR LV Ç EA F ÇP W VRGI Ç T F ÇD L P E IV Ç AD A Â PPL.K GVD Ç ...... F VG Ç G DMF K SV Ç P K.A DAVML M WIL H ObF8OMT-1 182 LA Ç KE.SR.KV Â F Â E GL Ç E TMV Ç D VGG GT G Ç M VSKA Â IV Ç DA F ÇP G MKGI Ç VL ÇD L P Y VV Ç SG L Â KGS.GN Ç L R...... Y VG Ç G DMF H SV Ç P P.A DAV F LKWIL H MpOMT3 192 IL Ç EI. Y Â ..KG F Â E GV Ç K SLV Ç D VGG GT G Ç A SSKM Â IV Ç SK Y ÇP L IK Ç A I N F ÇD L P H VI Ç QD A Â SPH.PE Ç VE ...... H VG Ç G DMF V SV Ç P K.A DAI F LKWI CH MpOMT1A 170 VI Ç EN. Y Â G.EG F Â Q GV Ç G SLV Ç D VGG SY G Ç M A L Â SM LV Ç KA F ÇP W LRGI Ç C F ÇD L P E VV Ç AR A Â SPL.K GVE Ç ...... F VG Ç G T MF E SI Ç P K.A DVVML M FVL H MpOMT1B 170 VI Ç EN. Y Â G.EG F Â L GV Ç G SLV Ç D VGG SY G Ç M A L Â SM LV Ç KA F ÇP W LRGI Ç C F ÇD L P E VV Ç AR A Â SPL.K GVE Ç ...... F V Ç AG S MF E SI Ç P K.A DVVML M FVL H MpOMT4 170 IV Ç RY.CP.AA Â F Â E GI Ç G TVV Ç D VGG RH G Ç V A L Â EK LV Ç AA F ÇP W VRGI Ç S F ÇD L P E IV Ç AK A Â PPR.P GIE Ç ...... F VG Ç G SF F E SV Ç P K.G DLVLL M WIL H MpOMT2 190 L Ç TTE.CR.EI Â F Â D GL Ç E SMV Ç D VGG GT G Ç A T A Â KG IA Ç AA F ÇP G M Ç ECTVL ÇD L P N VV Ç GG L Â KGS.EN Ç L S...... F V Ç SG DMF DF Ç IP H.A DAI F MKFIL H CdFOMT5 181 IL Ç ES. Y Â ..KG F Â DN Ç I K QLV Ç D VGG SL G Ç V T L Â QA I Ç TTK Y ÇP Y IKGI Ç N F ÇD Q P H VI Ç DH A Â PPH.PR Ç IE ...... H VG Ç G DMF Q SV Ç P K.G DAIIMK S VL H ZmOMT1 192 LL Ç DF. Y Â ..TG F Â E GV Ç S TLV Ç D VGG GV G Ç A T L Â HA I Ç TSRH ÇP H I Ç S GV N F ÇD L P H VI Ç SE A Â PPF.P GV Ç R...... H VG Ç G DMF A SV Ç P .AG DAILMKWIL H Hv7OMT 217 AV Ç RE.CR.DV Â F Â A GI Ç K SLV Ç D V A G GN G Ç TT A Â RT IA Ç EA F ÇP Y VK Ç CSVL ÇD L P Q VI Ç QG I Â SSH.GT Ç VE ...... F V Ç AG DM MEF Ç VP P.A EAVLLKYVL H HvOMT1 184 LL Ç ES. Y Â ..KG F Â E GL Ç G TLV Ç D VGG GV G Ç A T V Â GA II Ç AR Y ÇP A VKGI Ç N F ÇD L P H VI Ç SE A Â PAF.P GV Ç T...... H IG Ç G DMF QK Ç VP S.G DAILMKWIL H MsCOMT1 191 IL Ç ET. Y Â ..TG F Â E GL Ç K SLV Ç D VGG GT G Ç A V I Â NT IV Ç SK Y ÇP T IKGI Ç N F ÇD L P H VI Ç ED A Â PSY.P GVE Ç ...... H VG Ç G DMF V SI Ç P K.A DAV F MKWI CH CrOMT2 192 IL Ç EN. Y Â ..KG F Â E GL Ç N SVV Ç D VGG GI G Ç A T L Â NM II Ç SK Y ÇP S IKGI Ç N F ÇD L P H VI Ç QD A Â PAF.P GVE Ç ...... H VG Ç G DMF V SV Ç P K.G DAI F IKWI CH SaDMPM 194 LA È KEEE..YD Ã F Ã SSYG È TVV ÉÉÉÉÉÈ D IGG AD G È SL L Ã AA VL È SAH ÈP G V ÉÉÈ E GV V F ÉÉÈD S P EG È A RD A Ã AATLDA ÉÈ A GVGERGRVET ÉÉÉÉÈ G D F F TR ÉÈ VP GGG DL ÉÉÉÉÉÉÉÉÉ Y VLK S IL H consensus>70 .....y....Feg....!Dvgg..G...... fP.i.gi.fDlP.vi.da.....gve...... vgGdmf.svP...#ai.lk.!lH
CaOMT1 246 DWS ÅÅÅÅÆ D E H CL ÄÆ K LLK ÄÅÆ N C Á Y DALP ÄÅÅÆ N.....N GKVILAE ÄÅÅÅÅÅÆ C. IL ÄÆ PEV P Á . D Á .SSLA.TKGVVH I ÄÅÆD V IT ÄÅÆ VA HNPG GKE ÄÅÅÅÆ R T EK EF ÄÆ EA LA ÄÆ KA A ÄÅÆGF QGFQ. V Á AtOMT1 268 DWS Ç D E H CV Ç KF Ç LK N C Â Y E Ç S LP E.....D GKVILAE Ç C. IL Ç PET P Â . D Â .SSLS.TKQVVH V ÇD CI MLA Ç HNPG GKE Ç R T EK EF Ç EA LA Ç KAS ÇGF KGIK. V Â ShMOMT1 268 SLD Ç D E D CV Ç K ILK Ç N C Â WR Ç ALP N.....D GKVVVIE Ç Q. I Ç QPKY P Â . E Â .TNLL.SKRSFSF ÇD I S MMI Ç MFHG GKE Ç R T KQQ Ç F ED LA Ç KQ A ÇGF TYIK. V Â ShMOMT2 260 N Ç W DD E D CV Ç K LLK Ç K C Â K E Ç S I SSR..ENG GKVIIID Ç M. IM Ç EDNY.N.NKQL.VQSQHL Â Â M ÇD L I M Ç R I T.YAS Ç KE R T EK EW Ç EK L Ç FLE A ÇGF SGYK. I Â ShMOMT3 268 DW Ç DD E Y CV Ç K ILK Ç N C Â WR Ç ALP K.....D GKVVVIE Ç Q. M Ç QPEY P Â . E Â .TNLI.SKNSSS V ÇD M L MM Ç TMLDG GKE Ç R T KQQ Ç F ED LA Ç KQ A ÇGF TVFK. I Â SlMOMT4 268 N Ç W DD E D CV Ç K ILK Ç N C Â WR Ç ALP N.....D GKVVVIE Ç Q. I Ç QPEY P Â . E Â .TNLL.SKHLFA L ÇD I S MMI Ç MFHG GKE Ç R T KQQ Ç F ED LA Ç KQ A ÇGF TSIK. V Â TaOMT2 263 DWS Ç D E H CA Ç T LLK Ç N C Â Y DALP Ç A.....H GKVVLVE Ç C. IL Ç PVN P Â . E Â .ATPK.AQGVFH V ÇD M I MLA Ç HNPG GRE Ç R YER EF Ç EA LA Ç KG A ÇGF AAMK.T Â TaOMT1 267 DWS Ç D E H CA Ç T LLK Ç N C Â Y DALP Ç A.....H GKVVLVE Ç C. IL Ç PVN P Â . E Â .ATPK.AQGVFH V ÇD M I MLA Ç HNPG GRE Ç R YER EF Ç EA LA Ç KG A ÇGF KAIK.T Â OsOMT1 275 DWS Ç D E H CA Ç R LLK Ç N C Â Y DALP Ç E.....H GKVVVVE Ç C. VL Ç PESS. Â D Â .ATAR.EQGVFH V ÇD M I MLA Ç HNPG GKE Ç R YER EF Ç RE LA Ç RA A ÇGF TGFK. A Â CrOMT2▲ 251 DW Ç KD E E CV Ç K VLK Ç M C Â RK Ç AIP EK..EKG GKVILIE Ç TV LM Ç DSKKH Â E Â .NEEA.VKAQISS ÇD I D MMV Ç F.FTA Ç KE R T EE EW Ç AT L Ç FRE A ÇGF SGYK. I Â CrCOMT1 270 DWS Ç D AH CL Ç KF Ç LK N C Â H EALP Ç E.....N GKVILAE Ç C. LL Ç PEA P Â . D Â .STLS.TQNTVH V ÇD V I MLA Ç HNPG GKE Ç R T EK EF Ç EA LA Ç KG A ÇGF RGFIK V Â CrOMT6 264 DWN Ç D E D CV Ç K ILK Ç N C Â KK Ç AI QEK..GNG GKVIIID Ç T. VV Ç YSQK.N.EKEL.VDLQIS Â Â M ÇD M A MVI Ç N.FAA Ç KE R T EE EW Ç EH LI Ç RE A ÇGF SGHK. I Â ObFOMT1 246 N Ç WS D NE CI Ç D ILK Ç R C Â K EAIP Ç A....ET GRLMIID Ç A. II Ç DED G Â . E Â .GDEF.AGARLG L ÇD V T MMA Ç VTYE GKE Ç R T HR EW Ç AY IL Ç TE A ÇGF RKYV. V Â ObFOMT2 246 N Ç WS D NE CI Ç D ILK Ç R C Â K EAIP Ç R....ET GKVMIID Ç A. II Ç EED G Â . E Â .GDEF.AEARLG L ÇD V T MMA Ç VTFE GKE Ç R T HR EW Ç AF IL Ç KE A ÇGF RKYV. V Â ObFOMT3 242 DWS Ç D D K CI Ç E ILK Ç K C Â K EAIP Ç A....ST GKVMIVD Ç A. II Ç NED G Â . E Â .GDEF.SGARLS L ÇD M I MLA Ç VMAQ GKE Ç R T YK EW Ç VH LL Ç NE A ÇGF SKHT. V Â ObFOMT4 242 DWS Ç D D K CI Ç E ILK Ç K C Â K EAIP Ç T....ST GKVMIVD Ç A. II Ç NEE G Â . E Â .GDEF.SGARLS L ÇD M T MMA Ç MTTQ GKE Ç R S YK EW Ç VH LL Ç NE A ÇGF SKHT. V Â ObFOMT5 242 DWS Ç D D K CI Ç E ILK Ç K C Â K EAIP Ç A....NI GKVMIVD Ç A. II Ç NED G Â . E Â .GDEF.SGTRLS L ÇD M I MLA Ç VMAQ GKE Ç R T YK EW Ç VH LL Ç NE A ÇGF SKHT. I Â ObFOMT6 242 DWS Ç D D K CI Ç E ILK Ç K C Â K EAIP Ç T....ST GKVMIVD Ç A. II Ç NEE G Â . E Â .GDEF.SGARLS L ÇD M I MMA Ç TTTQ GKE Ç R S YK EW Ç VH LL Ç NK A ÇGF SKHS. V Â ObF8OMT-1 262 N Ç WS D D E CI Ç K IL Ç EK C Â K EAI Ç TTSKNMKG GKVIIVD Ç M. IL Ç GYEK.Q.QDEA.VETQLFF Â Â ÇD M M MM Ç TT.LT GKE Ç R T EQ EW Ç AK I Ç FFA A ÇGF KTYK. I Â MpOMT3 271 DWS Ç D E H C Ç RK LLK Ç N C Â Y DAI Ç LG.....N GKVIIAE Ç S.T Ç L PED P Â .N.SGPD.TIHAIRG Â ÇD V I ML Ç TVNPG GKE Ç R T EK EF Ç RT LA Ç LQ A ÇGF KRLVK V Â MpOMT1A 250 N Ç WS D E E CV Ç E ILK Ç R C Â K DAV Ç SK....DK GKVIIID Ç A. VI Ç DED G Â . D Â .GDEF.TGARLG L ÇD V T MMA Ç TMFE GRE Ç R T YV EW Ç AR II Ç NE A ÇGF RRHV. V Â MpOMT1B 250 N Ç WS D E E CV Ç E ILK Ç R C Â K DAVP Ç K....NK GKVIIID Ç A. VI Ç DED G Â .N.GDEF.TGARLG Â L ÇD V T MMA Ç NMFE GRE Ç R T YV EW Ç AH II Ç NE A ÇGF RRHV. V Â MpOMT4 250 DWS Ç D E S CI Ç E IMK Ç K C Â K EAIP Ç T....S. GKVMIVD Ç A. IV Ç DED G Â . E Â .GDDF.AGARLS L ÇD L I MMA Ç VLAR GKE Ç R T YR EW Ç EY LL Ç RE A ÇGF TKFV. V Â MpOMT2 270 DWN Ç D E E CV Ç K ILK Ç K C Â K EAI Ç SRSN.NSCR Ç KIILVE I. VM Ç EDEK. Â E Â .THEA.TETKLFF ÇD M Q MLA Ç I.IT GKE Ç R S EK EW Ç GK L Ç FFD A ÇGF TNYK. I Â CdFOMT5 260 DW Ç DD E H CL Ç K LLK Ç N C Â YKS Ç IP E.....D GKVIVVE Ç S. ML Ç PEV P Â .N.TSIE.SKSNSH Â L ÇD V L MMI Ç QSPG GKE Ç R T RH EF Ç MT LA Ç TG A ÇGF GGIS.C Â ZmOMT1 271 DWS Ç D AH CA Ç T LLK Ç N C Â Y DALP Ç E.....N GKVIVVE Ç C. VL Ç PVNT. Â E Â .ATPK.AQGVFH V ÇD M I MLA Ç HNPG GKE Ç R YER EF Ç RE LA Ç KG A ÇGF SGFK. A Â Hv7OMT 297 N Ç WS D QD CV Ç K IL Ç TR C Â R EAI Ç SHG..EKA GKVIIID Ç T. VV Ç GSPS.Q...QI.LESQVT Â Â M ÇD L S MMM Ç .LFN GK Ç VR EEQN Ç W HK I Ç FLE A ÇGF SHYK. I Â HvOMT1 263 DWS Ç D E H CA Ç T LLK Ç N C Â Y DALP Ç A.....H GKVVLVE Ç C. IL Ç PVN P Â . E Â .ATPE.VQGVFH V ÇD M I MLA Ç HNPG GRE Ç R YER EF Ç EA LA Ç KG A ÇGF AAMK.T Â MsCOMT1 270 DWS Ç D E H CL Ç KF Ç LK N C Â Y EALP Ç D.....N GKVIVAE Ç C. IL Ç PVA P Â . D Â .SSLA.TKGVVH I ÇD V I MLA Ç HNPG GKE Ç R T QK EF Ç ED LA Ç KG A ÇGF QGFK. V Â CrOMT2 271 DWS Ç D E H CV Ç KF Ç LK N C Â Y EALP Ç V.....N GKVIVAE Ç S. IL Ç PVT P Â . D Â .TSLA.SKVVIH V ÇD CI MLA Ç HNPG GKE Ç R T EQ EF Ç RA LA Ç KA A ÇGF QGFQ. V Â SaDMPM 282 DWS ÉÉÉÉÈ D ARS È A D ILR ÉÈ TVRA Ã ÉÉÈ AMP A.....HA ÉÉÉÉÉÈ RLLVVE V. LL È PDTV. Ã D Ã SSA.HPLGY..LS ÉÈD L Y MLV ÉÈ NM.G GRE ÉÉÉÈ R S ER D È LRS LL È SDT ÉÈGF RTTR. V Ã consensus>70 dwsDe.c...$k.c.ea.p...... gkviiv#..i...... #...... D..ml.....gkeR...#...l...aGF.....v
CaOMT1 326 FCNAFN.T..Y IIE ÄÅÆ FSKQICN AtOMT1 348 VCDAFG.V..N LIE Ç LLKKL.. ShMOMT1 348 VARAYY.S..W LIE Ç LYKY... ShMOMT2 342 ITSLGL.R..S LIE Ç IYP.... ShMOMT3 348 VARAYY.C..W VIE Ç LYK.... SlMOMT4 348 MARAYY.Y..W VIE Ç FYKY... TaOMT2 343 TYIYAN.A..W AIE Ç FTK.... TaOMT1 347 TYIYAN.A..F AIE Ç FTK.... OsOMT1 355 TYIYAN.A..W AIE Ç FTK.... CrOMT2▲ 335 FPMIDF.R..SP ÇIE VYP.... CrCOMT1 351 .CCAYN.S..W IME Ç LLK.... CrOMT6 346 FPMYDF.R..S IIE Ç VYP.... ObFOMT1 327 NNIKAL.E..S LIE Ç AYP.... ObFOMT2 327 KNIKAL.E..S LIE Ç AYP.... ObFOMT3 323 KNIKSI.E..S VIE Ç AYP.... ObFOMT4 323 KNIKTI.E..F VIE Ç AYP.... ObFOMT5 323 KNIKAM.E..F VIE Ç AYP.... ObFOMT6 323 KNIKTI.E..F VID Ç AYP.... ObF8OMT-1 346 YPLLGL.R..S LIE Ç VFP.... MpOMT3 352 .CAAFH.T..C IME Ç CHK.... MpOMT1A 331 KNIKTL.E..S VIE Ç AYP.... MpOMT1B 331 KNIKTL.E..S VIE Ç AYP.... MpOMT4 330 KNINTV.E..F VIE Ç AYP.... MpOMT2 353 TRVLGL.R..S VIE Ç VFP.... CdFOMT5 340 EL.AIGSL..W VME Ç FY.K... ZmOMT1 351 TYIYAN.A..W AIE Ç FIK.... Hv7OMT 377 HNVLGM.R..S LIE Ç VQP.... HvOMT1 343 TYIYAN.A..W AIE Ç FTK.... MsCOMT1 350 HCNAFN.T..Y IME Ç FLKKV.. CrOMT2 351 VSSAFN.T..Y IME Ç FLKSA.. SaDMPM 360 RTPPGL.TPFS LIE ÉÈ AAPV... consensus>70 ...... i#......
8 Figure S5 Sequence alignment of CrOMT2 and other caffeic acid O-methyltransferases (COMTs) from plants.
Protein sequences were aligned via T-Coffee (Expresso) using structural information and analyzed using ESpript 3.0. Residues that are important for dimer formation, substrate specificity, SAM binding and catalysis are shown [29], and are indicated with asterisks in green, blue, red and black, respectively. To avoid ambiguity, CrOMT2 from Catharanthus roseus was marked with a triangle.
9 Figure S6
Figure S6 Cell viability after treated with flavonoids substrates or methylated products of CrOMT2 in vitro. For each cell line, bars marked by different letters within each index are significantly different
(p < 0.05) following Tukey’s test.
10 Table S1 Percent identity of AtOMT1 against OMTs in Citrus clementina.
Query Subject Percentage of Expect value accesion.version accession.version identical matches AtOMT1 Ciclev10020814m 73.35 0 AtOMT1 Ciclev10024037m 72.53 0 AtOMT1 Ciclev10005230m 70.67 0 AtOMT1 Ciclev10020761m 70.20 0 AtOMT1 Ciclev10001661m 59.34 1.25E-147 AtOMT1 Ciclev10031952m 57.40 6.45E-141 AtOMT1 Ciclev10018226m 56.79 2.81E-142 AtOMT1 Ciclev10031953m 56.50 6.40E-138 AtOMT1 Ciclev10017683m 56.39 1.41E-142 AtOMT1 Ciclev10031951m 56.19 5.49E-138 AtOMT1 Ciclev10015685m 56.11 3.10E-142 AtOMT1 Ciclev10020875m 56.03 1.97E-142 AtOMT1 Ciclev10031949m 55.89 5.32E-137 AtOMT1 Ciclev10021145m 55.51 7.62E-94 AtOMT1 Ciclev10001555m 55.14 5.37E-154 AtOMT1 Ciclev10032454m 55.07 1.45E-85 AtOMT1 Ciclev10026361m 54.74 9.03E-81 AtOMT1 Ciclev10013542m 54.60 1.06E-127 AtOMT1 Ciclev10020880m 54.60 1.80E-140 AtOMT1 Ciclev10021670m 54.58 9.74E-90 AtOMT1 Ciclev10026359m 54.31 1.45E-80 AtOMT1 Ciclev10017649m 54.10 2.49E-131 AtOMT1 Ciclev10021888m 54.01 9.69E-86 AtOMT1 Ciclev10021678m 53.79 2.35E-90 AtOMT1 Ciclev10032453m 53.74 1.15E-82 AtOMT1 Ciclev10020874m 53.74 1.53E-138 AtOMT1 Ciclev10032027m 53.47 1.03E-124 AtOMT1 Ciclev10012042m 53.43 2.42E-137 AtOMT1 Ciclev10021321m 53.25 2.98E-79 AtOMT1 Ciclev10021326m 53.25 2.98E-79 AtOMT1 Ciclev10023568m 52.87 9.58E-137 AtOMT1 Ciclev10020870m 52.87 7.54E-136 AtOMT1 Ciclev10020873m 52.87 7.54E-136 AtOMT1 Ciclev10032452m 52.86 3.64E-82 AtOMT1 Ciclev10023994m 52.59 2.50E-128 AtOMT1 Ciclev10032460m 52.42 1.19E-80 AtOMT1 Ciclev10020782m 51.62 3.91E-130 AtOMT1 Ciclev10010860m 51.29 3.91E-81 AtOMT1 Ciclev10020818m 50.82 1.05E-130 AtOMT1 Ciclev10020821m 50.29 5.51E-120 AtOMT1 Ciclev10020377m 50.14 6.29E-122 AtOMT1 Ciclev10020820m 50.00 9.12E-121
11 Query Subject Percentage of Expect value accesion.version accession.version identical matches AtOMT1 Ciclev10005251m 49.45 8.43E-127 AtOMT1 Ciclev10020828m 49.13 1.75E-119 AtOMT1 Ciclev10023965m 46.49 5.23E-114 AtOMT1 Ciclev10003737m 46.24 2.02E-81 AtOMT1 Ciclev10003323m 43.15 1.69E-87 AtOMT1 Ciclev10012087m 42.07 4.53E-89 AtOMT1 Ciclev10005309m 40.85 2.85E-84 AtOMT1 Ciclev10006704m 39.94 4.03E-78
12 Table S2 Primers used for amplification. Gene Forward primer (5' to 3') Reverse primer (5' to 3') CrOMT2-CDs ATGGGTTCAACCAGTTCAGAAAC TCAAGCACTCTTGAGAAATTCC CrOMT2-PET ATAGGATCCATGGGTTCAACCAG GAAGGACTCGAGAGCACTCTTG TTCAGAAAC AGAAATTCC Underlined sequences represent restriction enzyme sites
13 Table S3 UniProt entries of COMT proteins used in the phylogenetic tree. Protein names UniProt entry Organism AtOMT1 Q9FK25 Arabidopsis thaliana (Mouse-ear cress) CrOMT2 Q8GSN1 Catharanthus roseus (Madagascar periwinkle) (Vinca rosea) CrCOMT1 Q8W013 Catharanthus roseus (Madagascar periwinkle) (Vinca rosea) CrOMT6 Q6VCW3 Catharanthus roseus (Madagascar periwinkle) (Vinca rosea) CaOMT1 P59049 Chrysosplenium americanum (Golden saxifrage) CdFOMT5 A0A125T1T5 Citrus depressa HvOMT1 Q43771 Hordeum vulgare (Barley) Hv7OMT A5YTR4 Hordeum vulgare subsp. vulgare (Domesticated barley) MsCOMT1 P28002 Medicago sativa (Alfalfa) MpOMT3 Q6VMV9 Mentha piperita (Peppermint) (Mentha aquatica x Mentha spicata) MpOMT1A Q6VMW2 Mentha piperita (Peppermint) (Mentha aquatica x Mentha spicata) MpOMT1B Q6VMW1 Mentha piperita (Peppermint) (Mentha aquatica x Mentha spicata) MpOMT4 Q6VMV8 Mentha piperita (Peppermint) (Mentha aquatica x Mentha spicata) MpOMT2 Q6VMW0 Mentha piperita (Peppermint) (Mentha aquatica x Mentha spicata) ObFOMT1 K0I977 Ocimum basilicum (Sweet basil) ObFOMT2 K0II72 Ocimum basilicum (Sweet basil) ObFOMT3 K0I7Q2 Ocimum basilicum (Sweet basil) ObFOMT4 K0I210 Ocimum basilicum (Sweet basil) ObFOMT5 K0ICR0 Ocimum basilicum (Sweet basil) ObFOMT6 K0I986 Ocimum basilicum (Sweet basil) ObF8OMT-1 S5DWK8 Ocimum basilicum (Sweet basil) OsOMT1 Q6ZD89 Oryza sativa subsp. japonica (Rice) ShMOMT1 F2YTN4 Solanum habrochaites (Wild tomato) (Lycopersicon hirsutum) ShMOMT2 F2YTN5 Solanum habrochaites (Wild tomato) (Lycopersicon hirsutum) ShMOMT3 M9Z1G5 Solanum habrochaites (Wild tomato) (Lycopersicon hirsutum) SlMOMT4 A0A088MF62 Solanum lycopersicum (Tomato) (Lycopersicon esculentum) TaOMT2 Q38J50 Triticum aestivum (Wheat) TaOMT1 Q84N28 Triticum aestivum (Wheat) ZmOMT1 Q6VWG3 Zea mays (Maize) SaDMPM P42712 Streptomyces alboniger
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