Post on 08-Mar-2018
ACT3
MYB1
bHLH1
28 !
26!
28 !
Fig. S1 Expression analyses of two transcription factor genes in CrV and CrW.
CrV CrW Cycle !no.
Additional file 2: Figure S1
RT-PCR analysis of TfMYB1 and TfbHLH1 and the β-actin gene. TfMYB1 and TfbHLH1 are suggested to be involved in anthocyanin biosynthesis in torenia. Gene names and PCR cycles are shown to the left and right of each panel, respectively.
B
! ! !I V V S R R R L P P G P T G F P !CrV ⋅⋅⋅⋅ATCGTCGTCTCCCGCAGACGGTTACCCCCCGGCCCAACGGGCTTCCCC⋅⋅⋅⋅ CrV, CrW ⋅⋅⋅⋅ATCGTCGTCTCCCGCAGACGGTTACCCCC-GGCCCAACGGGCTTCCCC⋅⋅⋅⋅
! !I V V S R R R L P P A Q R A S P
ΔC!
exon1!TfF3′5′H
A
! N L F A A G T D T T S !CrV ⋅⋅⋅tttttctttctgcagAACCTTTTTGCTGCGGGAACGGACACGACGTCG⋅⋅⋅!CrV,CrW ⋅⋅⋅tttttctttctgcagAACCTTTT------------GGACACGACGTCG⋅⋅⋅ !
! ! N L L D T T S
exon3!TfF3′H
Δ12bp
exon2!
exon1!
Fig. S2 Schematic structure and mutated sequences of ThF3′H and ThF3′5′H genes.
exon1!
500bp Additional file 2: Figure S2
(A) The ThF3′H gene consists of three exons and two introns. The deduced amino acid sequence corresponding to the 12-bp deletion in CrW is shown. Small letters indicate intron sequence, and an acceptor site (ag) is boxed. (B) The ThF3′5′H gene consists of two exons and one intron. The deduced amino acid sequence corresponding to the cytosine deletion in CrW is shown.
! ! ...GCACGAACCAAGTCATTCATTTAATGATGTGAGAAAATGTCATACTTTAATTGATATTTTGGAATACATGTGGAGAAATTAGCTCATGAACTTGGCTAGTTGGAGCTGAAA! ! TSD!
!ACTGGTTGCATGGTTTTGTTTCTGGTTGTGTGAAGAATCGTCGATGCTTATGAGAAGAAAATGCAGTGGAAGAGTAAAGAGTTATAAGTGTGTTATCACTTATAAAGTTGC!!TGTTTACTATATCACGTTTTCTTTGAGAGCTGCACATGGGAACGGTTAATGAGCAGTTATTAAGCTGAAGCAAGATTGTGTGTTCTGTTAGTTCGGTTTCAAGTAGCAGTT!!TTGCAGATCTCATAACTTGGTGTGTAGTTAGCCAATTTAAGCGATTTAAATTGCGTTGAAAAGCTCACATTACATACAACACATTGTAACTCATCATACTCAATAAAATTC!!TCTCTTCGTCCCGTGGAGTAGGGCTAAAGTTAGCGCCGAACCACGTTAAATCTGTTTTCTTCTGAGACTTTTCTGATTTTCTGTTCTTATTCAATCAAGTTTCTTTCGATC!!CAAACGCGTAGTAAACATATCACAAGACTTGTTAACTTTAATCTTGAGCATCTAAGAGCAAGAATTCTCAACAAGTGGTGCGGTGAGCGTGGATAACTCGAAGATGGCTGC!
PBS ! M A A!!GAGGTTCGAAGTTGAAAAGTTCACGGGTGATAATGACTTCGGTTTGTGGAAAATGAAGATGAGGGCGTTGCTTACCCAACAAGGGCTGATAGAGGTGTTGATGGTGGAGGA!
!R F E V E K F T G D N D F G L W K M K M R A L L T Q Q G L I E V L M V E D!!TCCACCGGCCACAGTGGTGGCTGGGACTGCTCCGACGGGCCAAGAAGATGCCGCTGCTGCAGCTGTAAATGCACAAGCTGCGGCAGAGAAGAAAATCTTGGATTCGAAGGC!
!P P A T V V A G T A P T G Q E D A A A A A V N A Q A A A E K K I L D S K A!!GCATTCAGTGATCATTCTGAGTTTGGGAGATCGAGTCTTGCGTCAAGTCTCTCATGAATCAACCGCTCTTGGTCTGTGGAAGAAATTGGAAGAGCTTTACATGACAAAGTC!
!H S V I I L S L G D R V L R Q V S H E S T A L G L W K K L E E L Y M T K S!!TCTTGCCAATCGACTTTATCTCAAGCAAGCGCTCTATTCGTTCAAGATGATTGAAGAAAAGGCCATTGATGAGCAGATGGATCAGTTTATCAAGCTGATTTTGGATCTTGA!
!L A N R L Y L K Q A L Y S F K M I E E K A I D E Q M D Q F I K L I L D L E!!AAACATCGAAGTGAAGATAGAGGATGAGGATCAAGCCTTGCTCTTGGTGTGTGCTTTGCCTCGGAGTTACAACACATTCAAGGACACGTTGTTGTATGGCCGAGAGACTTT!
!N I E V K I E D E D Q A L L L V C A L P R S Y N T F K D T L L Y G R E T L!!GACTTTGAAAGAGGTGCAAGCCGCGTTGAAGTCGAAACAGCTGAACACGAGGATTGATAACAAGGCAGTGGGATCTACATCCGAGGCTCTTTATGTTAAAGGGAAAGGTGA!
!T L K E V Q A A L K S K Q L N T R I D N K A V G S T S E A L Y V K G K G E!!AGAGAAGAAAACACACAAAGAAAGAAAGAACAAGTCCAAAAAGAAGGTTAAATGTTTCTATTGCGATGAGGAGGGTCATATGTGCAAGAATTGTCCCAAGAAGGAGAGAGA!
!E K K T H K E R K N K S K K K V K C F Y C D E E G H M C K N C P K K E R D!!CAAGGGCAAGAAAGTGGAGCAAGGCGAGGCTGCAATGGCTTGTGAAAGTTATGAGTCGGCTGACGTCTTGGCCGTGACTCATGAAGATCAAGATGTGACCAAATCTGAAAA!
!K G K K V E Q G E A A M A C E S Y E S A D V L A V T H E D Q D V T K S E K!!ATCTGGTAAATGGCTACTTGATTCCGCTTCTTCTTTTCATGTCACATGTGTTAAGTCTTGGATTAAAGACTTTAAGGGTTGTGATGGTTGTCTGGTCTCTGTTGGGGAAGA!
!S G K W L L D S A S S F H V T C V K S W I K D F K G C D G C L V S V G E E!!AAAGCAATACAAAATTCTGGGCTTTGGAACGGTTAAAATAAGGCTTAAAACCGGTGGAGTAAGAATTTTGAGAAATGTGAAGTTCATTCCGGATTTGGGTAGAAACCTTAT!
!K Q Y K I L G F G T V K I R L K T G G V R I L R N V K F I P D L G R N L I!!TTCTGTAGGGTTGTTGGATGTTCAAGGCTTTAAGTGTGTTGCTGGAAATGGTGTGATGAAGGTTTTCAAAGGGTCTAAGGTGATTATGAGTGGTACTTTGCAGAAAAACAG!
!S V G L L D V Q G F K C V A G N G V M K V F K G S K V I M S G T L Q K N R!!AACTTATCATGTTACAGGATCTGAAACAGTGGTGAACTGGGCTGGTTTGGGTGCTCGTAAAATCTGCTGTGGTGATTCTGGGAGTGAGAGGTTTGTTCAGGGTAATAGTAT!
!T Y H V T G S E T V V N W A G L G A R K I C C G D S G S E R F V Q G N S I!!CCTATGTGGTGATCATGATAAGGCCGGGTTTGAGCAGGTTACACAAGGGGAGATCACAGATCAGAGGGCTCAAAGTGAGGGATCTGACACTTTATTGTCTGAGGTGGAGCA!
!L C G D H D K A G F E Q V T Q G E I T D Q R A Q S E G S D T L L S E V E Q!!GATTTGTGTGAGAGTTGGTAGAGTTCTTTGTCCAGTTTCTGGGAAAGATTTGAATTATCTGAGGAATGGGGATATTCCTGAGGCTGATTGTGGTGGCCAACTACAAGGATC!
!I C V R V G R V L C P V S G K D L N Y L R N G D I P E A D C G G Q L Q G S!!GAATCTGCAAGTGACAAAGGAAGTGGGACTTATCAAGTTTGAAAGGCTGAAAAATGTGATTTCTGAGTCGTGTTTGGTGGTGGATAACTGTGGTGCTTCGAGTTTTCCTGG!
!N L Q V T K E V G L I K F E R L K N V I S E S C L V V D N C G A S S F P G!!GGCTGCTGAAGATCAAACTCAGTTTAATCTGCAGAATGGGAGTTGGTTTACAGCTGAGGTGGAGGAAAATGAAGCAAATGGTCAGTTTTATGAATGCTCAAAATCTGATGA!
!A A E D Q T Q F N L Q N G S W F T A E V E E N E A N G Q F Y E C S K S D D!!TGGCAGCAAGCTTGGAACAACAGTGGGCAAGTAGGTACCTTTGCGAGGTTGTAGCAGGCTGTGTGGGTGGATTTTGAGGAGGCTGTGGGATGCTTCGGGTTCACATCATAT!
!G S K L G T T V G K *!!GGTACTTCAATGTTCTGGGGATTTAACTCAGGTGGAGTTAAAGAGGAATCCACAAATTAAGCTCAAAGTACTCGGTTTGCGCATATTTGGTACAGAGTTTCTGGTTTGTTT!
!!TTGGGGGCTAAAACTTGGTTTTCTGGGCTGTTAACAAGTTGTTCTTGTGCTGAGAAAGGTTGTTAGCTGTGAGGTAACCAGAGTTGTGGATGTAGAACTGAAGTTTGGACT!!TGATGTGATAGTATTTGAGGTTGATCGAGTGAATACAAGTTCGCTGAAAAATTCTCAAATTTGTGTTCGTTTGGCTGGTTTCTGGGGCGTGTTCTGGATCGTTCTTGAGGG!!AATTCAGCTGGATGTTGTTTGCTTGGGAAAGTAGGCATGGTGGACTGAAGAGGGAAGTGGAGAGTTAATGAGCTAAGGTGGAGATTTGTGGAGAAATTAGCTCATGAACTT!
! PPT!!GGCTAGTTGGAGCTGAAAACTGGTTGCATGGTTTTGTTTCTGGTTGTGTGAAGAATCGTCGATGCTTATGAGAAGAAAATGCAGTGGAAGAGTAAAGAGTTATAAGTGTGT!!TATCACTTATAAAGTTGCTGTTTACTATATCACGTTTTCTTTGAGAGCTGCACATGGGAACGGTTAATGAGCAGTTATTAAGCTGAAGCAAGATTGTGTGTTCTGTTAGTT!!CGGTTTCAAGTAGCAGTTTTGCAGATCTCATAACTTGGTGTGTAGTTAGCCAATTTAAGCGATTTAAATTGCGTTGAAAAGCTCACATTACATACAACACATTGTAACTCA!!TCATACTCAATAAAATTCTCTCTTCGTCCCGTGGAGTAGGGCTAAAGTTAGCGCCGAACCACGTTAAATCTGTTTTCTTCTGAGACTTTTCTGATTTTCTGTTCTTATTCA!!ATCAAGTTTCTTTCGATCCAAACGCGTAGTAAACATATCACAAGACTTGTTAACTTTAATCTTGAGCATCTAAGAGCAAGAATTCTCAACAATACAATTAATAGGTACTCT...!
! TSD!Fig. S3 Sequence of the 5′-upstream region of F3H in CrW. The figure shows 5 bp of the target site duplication (TSD; blue letters), 560 bp of long terminal repeats (LTRs; red letters), the primer binding site (PBS; green letters), and the polypurine tract (PPT; brown letters). The amino acid sequence deduced from the open reading frame (ORF) is indicated by black letters.
Additional file 2: Figure S3
Fig. S4 Insertion of the solo-LTR in 5′-upstream region of F3H in CrW.
3.6k ATG TfF3H
-249
TGTAT TGTAT
500bp
LTR 560bp TSD TSD
ACGT containing element ! (CACGT)
P recognition element ! (CCT/AACC)
Binding sequence of vertebrate MYB protein ! (C/TAACT/GG)
+1
Additional file 2: Figure S4
A putative solo-LTR of 560 bp was identified in F3H promoter of CrW. It was flanked by 5-bp target site duplications (TSD) and the position was identical to the TORE1 insertion site.
H E X H E X CrW CrV
H E X H E X CrW CrV
Fig. S5 Southern blot analysis of F3′H and F3′5′H in CrV and CrW.
10 6
4
3
1.5
2.5
2
1
A B
Additional file 2: Figure S5
Total genomic DNAs were digested with Hind III (H), Eco RI (E), and Xba I (X) and transferred to nylon membranes as described in Methods. Membranes were probed with DIG-labeled sequences of F3′H (A) and F3′5′H (B). DNA marker sizes (kbp) are shown.
//
E H H X X E E E H
TORE1
Xba I
Eco RI
Hin dIII
10.5 kb
3.0 kb
4.5 kb 3.7 kb 4.7 kb
//
E H H X X
4.5 kb 4.9 kb
Xba I
Eco RI
Hin dIII
5.2 kb
7.0 kb
Fig. S6 Schematic diagram of the genomic structure of the F3H gene in CrV and CrW.
Probe
Probe
CrV
CrW
Additional file 2: Figure S6
Sites of restriction enzymes Xba I, Eco RI, and Hind III used in the Southern blot analysis are shown. Restriction sites at the 3′-proximal region were deduced from the results of Figure 4. Lengths following complete digestion are shown in blue, green, and red for Xba I, Eco RI and Hind III, respectively. The F3H probe used is shown in orange.
Fig. S7 HPLC analysis of flower petal anthocyanidins in GtF3H-overexpressing transgenic CrW.
1.75
1.93
2.36 2.63
2.75
12.15
14.00
14.45
16.32
16.72
17.03
17.28
18.81
19.56
21.55
21.85
22.56
24.71
0 2 4 6 8 10 12 14 16 18 20 22 24
保持時間(min)
0.00
0.05
0.10
0.15
0.20
0.25
信号強度(AU)
1.67
1.93
2.33 2.63
2.73
10.21
11.85
12.12 13.92
24.73
0 2 4 6 8 10 12 14 16 18 20 22 24
保持時間(min)
0.00
0.05
0.10
0.15
0.20
0.25
信号強度(AU)
Dp
Cy
Pg
1.76
1.95
2.35 2.64
2.76
13.96
18.13
21.87
24.65
0 2 4 6 8 10 12 14 16 18 20 22 24
保持時間(min)
0.00
0.05
0.10
0.15
0.20
0.25
信号強度(AU)
Pg
CrW
Standard
No. 4
Sign
al in
tens
ity (A
U)
Sign
al in
tens
ity (A
U)
Sign
al in
tens
ity (A
U)
Retention time
Additional file 2: Figure S7
After acid hydrolysis of petal extracts, the hydrolysates containing anthocyanidins were subjected to HPLC analysis. Dp, Cy, and Pg correspond to delphinidin, cyanidin, and pelargonidin, respectively.
Fig. S8 Example of pigment recovery in a CrW petal. (A) Whole flower. (B) Magnification of boxed red area in A.
A B
Additional file 2: Figure S8