Presentation on theme: "Supplementary Figure 1. A phylogram displaying the relationship of the homologs to other MYB proteins. The data indicates that OsMYB80 and TaMYB80 are."— Presentation transcript:
Supplementary Figure 1. A phylogram displaying the relationship of the homologs to other MYB proteins. The data indicates that OsMYB80 and TaMYB80 are closely related to GhMYB80, AtMYB80 and BnMYB80 than other MYB proteins. The tree distances in the phylogram are included in brackets. The phylogram was generated using VectorNTI software (Invitrogen) and peptide sequences obtained from TAIR and TIGR databases. AtMYB80 (0.0321) BnMYB80 (0.0308) GhMYB80 (0.1860) OsMYB80 (0.0838) TaMYB80 (0.0791) AtMYB35 (0.3191) AtMYB4 (0.1944) AtMYB32 (0.1934) AtMYB41 (0.2087) AtMYB74 (0.1371) AtMYB102 (0.1546) Os03g18480 (0.3100) Os02g42870 (0.2969) Os08g37970 (0.2996) Os04g38740 (0.3109) Os12g07640 (0.2380)
Supplementary Figure 2. Promoter sequence alignment of Arabidopsis, Canola, Rice and Wheat MYB80. The promoter sequences are immediately upstream of the ATG translational start site. The alignment was performed using MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/). Blue represents high and purple represents moderate similarity, respectively. A highly conserved region is underlined in black.
Supplementary Figure 3. The AtMYB80 promoter contains a conserved region immediately upstream of the transcript start site. PLACE analysis identified four motifs which are conserved amongst the four species within this region. The motifs are underlined and coloured as follows, TGAC, W-BOX; GTGA and TCAC, GTGANTG10; ACCTACC, MYB4 ( A / T ACC MYB-core). The purple underlined sequence (CGTTGAATTCTTTA) is present in all four promoters with a highly conserved DOFCOREZM binding site ( A / T AAAG) (orange). Motif analysis was performed using cis-PLACE (http://www.dna.affrc.go.jp/PLACE/). GGAGTTGACCA.....TGGGTGACCTACCGG....ATCACA.....GTAGCGTTGAATTCTTTA -300bp
CTGCTCCTATATATGTACAGAATGTCGTCATATTATTGCCATGATACTTGGAACGGTGAAAGGAACCTGCTCTTGAATG ATGTATAAATCAGAGAGAGAAAAAAAGACTGCTCAGTACTGTGTTTATCTATCAGCTTCGTAGAGCTCGGTATGCATC GATTAATTGGTTTCAGGTCGCGTTGGCATTGAGCATGCGTAAGCCATGTATCTGCGTGGTTGATTTTACTGAAATGGTG GGAAAAGAACATGATTCAGGGTGTTCTTGGAACTGGCAGCCGCTGGCCTTTGTGTTCTTGATGATAAGGATAGGGGAA TAAAATAGCATCCTCATATCAAGAGTGTAATCAACTAATCATAGTGCTGCAGCCCATAGAAATATGCATTCCCCTTGCT TGTAAAGATCCTACTAGGATAAAACTGTGTGCAGAAATGAATCGTTTATCTTTGACAGTTTTTTTTTTCTTTTTGAGTCT ATCCTCCTGAACCTAAGGCTGTAAATTTGTTCGGACAAAGGATCATTACTCATAGCCTGCTATGTTCACAAGGTTTTAT ATTACTGAATAGTGGTGTTTCATCAAGCAGCATTAGTGAATAGTTGCAGTGGCAGAGTGGTGCAGGGCGTCAGTGAGC GAGATGGCGAGGCGAGAACGCGAGCGGCGCCGGGAGCGCGCGGGGCTTGGACCGTGGCCGTGAGGGGGGTTGACCCG GCGCTGCGCGGGTGGGCAACCTGTCCCGCCCCCCCGGGGGGAGGCTCACGCGTTGCGCGGGCGTTGAATTCTTTATGC CGCCCATGCCGCCTTCGCCCCCCGTGCGCGCGCGCGCGCGCCCACTCGCCGGCGCGGCAGCGCGCGCGCGCGCGCGCG GGCGGTCGCGAGGCCGCGAGCCTATTTATAACACGCGGCGCGGCCGTGTGAGCGCCGCACGCACGCAGCAGCAGCGG AATCTCGACGGCGGCGACATCATG Supplementary Figure 4. Sequence analysis of the OsMYB80 promoter deletion constructs. The promoter deletions occurred at -168 bp (highlighted in grey) for the construct pOS-168 while pOS-328 occurred at -328 bp and also included the conserved region (highlighted in green). The putative TATA box is bold and underlined (black), a putative Initiator (Inr) motif is bold and underlined (blue), upstream motifs are highlighted red (DOFCOREZM) and yellow (GTGANTG10). Motif analysis was performed using cis-PLACE (http://www.dna.affrc.go.jp/PLACE/).
Supplementary Figure 5. The conserved amino acid sequence downstream of the MYB domain forms two α-helical structures (underlined). The sequence analysis was performed using NPS@: Network Protein Sequence Analysis (http://npsa-pbil.ibcp.fr/cgi- bin/npsa_automat.pl?page=/ NPSA/npsa_server.html). IDPVTHKPFSHLMAEITTTLNPPQVSHLAEAALGCFKDEMLHLLTKKRV
PrimerPurpose (V = vector construction)Sequence 5’ to 3’ ADTaMYB80 Genomic walk (GW) Adapter primerGTAATACGATCACTATAGGGC NADTaMYB80 GW Nested adapter primerACTATAGGGCACGCGTGGT Ta+245RTaMYB80 GW gene specific RTCTTCCCGCAGCGCTGCAGCCCTGTCA Ta+225RTaMYB80 GW nested gene specific RCTGTCACGCGCGGCAACCTCGTTCAGC Ta+138RTaMYB80 GW nested gene specific RACTCACCGGCGTTCTTGGGGATGAGGC OsP-1021FOsMYB80 pro:GUS & complementation F VCACAAGCTTGTCAGAGGGTGGTGGCACTCT OsP-ATGOsMYB80 pro:GUS R VTTGGATCCCATGATGTCGCCGCCGTCGAGATTC OsP-168ROsMYB80 pro:GUS -168 truncation R VGTTGGATCCCATGGGCGGCATAAAGAATTCAAC OsP-328ROsMYB80 pro:GUS -328 truncation R VGTTGGATCCCTCGCTCACTGACGCCCTGCACCA TaP-FTaMYB80 pro:GUS F VCACCGGCCCCTGCAAAACTCCCGCA TaP-RTaMYB80 pro:GUS F VGTCGGCGCGTCCGCCTCCGAGGC OTi2+344FOs/TaMYB80 intron spanning RT-PCR FTCGTCGGCAACAGGTGGTCGGTGATCG OTx3+816ROs/TaMYB80 3 rd exon RT-PCR RCTTGATGCGCTCGATGGTGTCGTC TaBtubFWheat β-tubulin RT-PCR FAGGTCTCCACCTCTGTTGTT TaBtubRWheat β-tubulin RT-PCR RAGCCATACAACAGTGTCCTG AtPF-BamAtMYB80 promoter complementation F VATATCGGATCCAAGCCTTAGATAACTATTACAGAGA AtPR-H3AtMYB80 promoter complementation R VTTAAAAGCTTTTCTTCTTTCTTTCTTTCTAG TaATG-FTaMYB80 gene complementation F VATAAAAGCTTATGGGCCGGATCCCTTGCTGCGA TaCode-RTaMYB80 gene complementation R VATTAACTAGTTTAGTCACACATGTTATTCGT OsATG-FOsMYB80 gene complementation F VTATTAAGCTTATGGGGCGGGTGCCGTGCTGCGA OsCode-ROsMYB80 gene complementation R VATTAACTAGTCTACACCATGTGATTCGTGAG TaPF-BamTaMYB80 promoter complementation F VATATGGATCCGGCCCCTGCAAAACTCCCGCA TaPR-H3TaMYB80 promoter complementation R VAATAAAGCTTGTCGGCGCGTCCGCCTCCGAGG OsPR-H3OsMYB80 promoter complementation R VATATAAGCTTGATGTCGCCGCCGTCGAGATTCCG BnPFBnMYB80 promoter complementation F VAATTGGATCCCGTGGGGGTCCTGTAATTG BnCRBnMYB80 gene complementation & RT-PCR RAATTAAGCTTTCAAACCACATGATTAATGAG Bnx2+FBnMYB80 2 nd exon RT-PCR FGTGGACAAACTATTTGCGTCCTGACC BnEAR-RBnMYB80:EAR fusion R V ACTAGTTTACGCAAATCCTAGTCTGAGCTCGAGGTCCAA ATCTAAACCAACCACATGATTAATGAGATC AtBnx2FAt/BnMYB80 2 nd exon RT-PCR FGTCAAGTTTCACTCTGTTCTTGGT At80-RAtMYB80 RT-PCR RTCAAACCATATGATTGATGAGATCA EAR-REAR RT-PCR R, used in all EAR-related exp.ATCCTAGTCTGAGCTCGAGGTC β-Tub F2β-tubulin RT-PCR FGGACACTACACTGAAGGTGCTGAG β-Tub R3β-tubulin RT-PCR RGGCTCTGTATTGCTGTGATCCACG BnAct7-FBnActin 7 RT-qPCR FTCCCTCAGCACTTTCCAACAG BnAct7-RBnActin 7 RT-qPCR RACACTCACCACCACGACCCAG qBn80FBnMYB80 RT-qPCR FTGGCAGAGATAACCACTACACTC qBn80R BnMYB80 RT-qPCR RGTGATCCAAAATGAATCAAACCAC qEAR-RBnMYB80:EAR RT-qPCR RTCCTAGTCTGAGCTCGAGGTCC R2R3+44RTruncated MYB80:EAR fusion R V TTAATAAGCTTACGCAAATCCTAGTCTGAGCTCGAGGTCC AAATCTAAACCCTTGTGGGTCACCGGATCTATT R2R3-RTruncated MYB80:EAR fusion R V TTAATAAGCTTACGCAAATCCTAGTCTGAGCTCGAGGTCC AAATCTAAACCAACACGTTTCTTGGTGAGCAAG At+46FAtMYB80 +46bp RT-PCR FCAATGGACTCCTGAAGAAGACAAC Supplemental Table 1. Primers used in this article are listed below in the order that they were used according to the figures presented.
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