Supplemental Figure 1.
A
AtBG1 transgenic lines
(Kb)
S
#1
#2
#23
#24 #36
#61 #63 NT
0.5
bar
(499bp)
0.5
AtBG1
(500bp)
1.0
Actin
(1046bp)
B
HindIII
EcoRI
HindIII
EcoRI
EcoRI
NT #1 #2 #3 #4 #14 NT #1 #2 #3 #4 #14 #23 #24 #25 #36 #23 #24 #25 #36 #2 #3 #63
(Kb)
6
3
Supplemental Fig. 1. Genomic PCR and Southern blot analyses of the AtBG1 transgenic
lines.
(A) Genomic PCR analysis of putative transgenic lines. The coding regions of the AtBG1
and bar transgenes were amplified by PCR from genomic DNA using the following sets of
oligonucleotide primers: 5′-TTGGATCACATTTAACGAGCC-3′ (forward) and 5′CAACTCGGACTCTTAGGATCAG-3′
(reverse)
for
AtBG1,
and
5′CTACCATGAGCCCAGAACGACG-3′
(forward)
and
5′CTGCCAGAAACCCACGTCATGCCAGTTC-3′ (reverse) for bar. The actin gene of
creeping bentgrass was also amplified using the same template and the primers 5′AACTGGGACGACATGGAGAAGATA-3′
(forward)
and
5′CGTCAGGGAGCTCGTAGTTCTTC-3′ (reverse), and then run as a loading control of
genomic DNA. The bar and AtBG1 was successfully amplified in all putative transgenic
lines. S, Standard DNA markers; NT, Non-transgenic control plant.
(B) Southern blot analysis of transgenic lines. The genomic DNA was digested with either
HindIII or EcoRI, and Southern blot hybridizations were carried out with [32P] dCTPlabeled bar probe. The results showed that several independent lines of AtBG1 transgenic
plants were produced .
Supplemental Figure 2.
NT
#1
#2
#23
#24
Supplemental Fig. 2. Apparent phenotypes of representative AtBG1 transgenic plants.
Fully-grown AtBG1 transgenic plants and non-transgenic control plants (NT) were shown.
Supplemental Figure 3.
TATTATCATT
GCAAGTGATA
CCATTGCCAA
TTCTTTTTAT
AGCTGGTATG
GTAAAGTATG
GTTTTTCCTT
CCAAAAGATC
ACCCCTGGTG
AGCTCAGCGC
TGCTTGACAG
CGGACGTTTT
CTGGATTTTG
ACATACTAAG
BAR
CGAGTGACGA
AAATATTCAG
CATCCAACAT
GAAAGAAAAT
GGCAGGCCAG
AGCTGAATGG
GACACATCTC
AACTACATAC
AAATGCTAAC
AGCAAATTTG
GACAGCCATT
TAATGTACTG
GTTTTAGGAA
GGTTTCTTAT
35S
CGTCCACACC
TTGGTTGCAA
GAAGGAGTTA
AAGAAAAGGG
ATAGCCAATA
CCGAAGCTCT
CGGAAGACAG
CTATATGTTT
TAGGAGTAGA
AACAAGACGC
CCACAGCGAG
AATTAACGCC
TTAGAAATTT
ATGCTCAACA
AtBG1
AGAAAAGAAA
CAGGAAAACT
ATTGAGATCT
AGATAACGAA
GCTTTGTCTG
TGTCATGGAT
ATCCGTTCCA
GACAAGGAAA
GTAGAATAAT
GAGGGGTAGC
TGACGAGATC
GAATTAATTC
TATTGATAGA
CATGAGCG
ATTTTAGTTG
TTCAAGTTGC
GAGTCAATTC
GGACGCAATT
CCCGTCGTAT
GTGTATGCAT
AAAACCCTGA
ATTCCCGCAA
TCAATAAACT
ACAAGCACAA
GACAACTTAA
GGGGGATCTG
AGTATTTTAC
Ubiquitin-P
35S
CCACGTGTGA
AAAGTTTCTT
TGAATTACGT
TTTTTATGAT
GCGCAAACTA
TCTAAACAAT
ATTCACCTGT
TTAGATATAT
TACTGTGCAG
AGAGAGAACA
AGATATGCAT
TCTCCTCGTA
ATTACAGGTG
AAGATTGAAT
TAAGCATGTA
TAGAGTCCCG
GGATAAATTA
GCATGTTCAC
TTTTTACTAC
TTGAACACTA
CTCACAGTAT
AAAAGTTGTC
GTCCTCATTC
ATATTATTAG
CGACCATAGT
ACGGTTGCAC
TCAATCGTAT
ATTCATGGCA
AGAATTTCTG
GCACGCCCCT
TGTAGTAAGT
AATTCACGCT
GATTGTAAAC
TCCCTTGTAC
TAACACATTG
GATTTTAGTA
AAATACAAAT
35S poly A tail
Intron-GUS
GCAGGG
ACCAGTTCGA
CCTGTTGCCG
ATAATTAACA
CAATTATACA
TCGCGCGCGG
CTATCATTGA
TTTATCCGTC
CTCATATTGA
ACAGCCACAT
AATACTAAAA
TCATAATTTT
CTGAGGTGAT
NOS terminator
AGGCAAACAA
ATTTCCCCGA
GTCTTGCGAT
TGTAATGCAT
TTTAATACGC
TGTCATCTAT
AAGAATATCG
CTATAAAGGA
CATCTTTACA
ATACCAAGTT
GGAAAAAAAC
TTCTGTCCGT
GCTAGCCACC
TCGTTCAAAC
GATTATCATA
GACGTTATTT
GATAGAAAAC
GTTACTAGAT
AATGTGAATT
TGTCATAAAT
TTTACAGATA
TGTCTCTCAC
ATTGTCTGCC
AGCTTCTCAT
ACCACCACCA
ATTTGGCAAT
TAATTTCTGT
ATGAGATGGG
AAAATATAGC
GATGCTAGCA
ATGCATAGGT
TTGTATAAAC
TGCAATCAAG
AAAAATATAG
AATGCCAGTC
CCCGTGAACA
Supplemental Fig 3. Determination of the T-DNA insertion site in AtBG1 #23 transgenic
line by I-PCR.
Inverse PCR (I-PCR) of transgenic plants were performed as previously reported (Ochman
et al., 1988) with some modifications. Total genomic DNA was isolated from the leaves of
greenhouse-grown plants and restriction digests were carried out using 2 μg of genomic
DNA. For circularization, restriction fragments were self-ligated overnight and 50 ng of
circularized DNA were used for the first round I-PCR. The DNA products from the first
round PCR reactions were used for the second round PCR and the PCR products were
fractionated on a 0.8% agarose gel. The purified PCR products were cloned into pGEM-T
vector (Promega) and sequencing analysis was performed. The flanking sequences of TDNA were analysed using BLAST in NCBI site, and the insertion of T-DNA was found to
be a non-coding region. Non-coding region was shown in Black, and CaMV 35S polyA
and NOS terminator of T-DNA region in the #23 transgenic plant were shown in red and
blue, respectively. The T-DNA region of the binary vector plasmid pCAMBIA3301
harbouring AtBG1 was shown in the middle of sequence.
Ochman H, Gerber AS, Hartl DL (1988) Genetic applications of an inverse polymerase
chain reaction. Genetics 120:621-623
Supplemental Figure 4.
TCTGCGTCGC
AGGAATGCCG
TTGGCCGTTA
AGCTCCACGA
GAGATATATG
ATACAGTCAC
CCGGTGATAT
GAACGAAGAA
GACGTTTTTA
GGATTTTGGT
ATACTAAGGG
CGTCGATCAC
CCATTGCACC
TGTGTGGGTG
ATCTGTGAAA
CATCCTTATC
ATTAATTATT
GGTTGCAAAT
TATTGTGGTG
ATGTACTGAA
TTTAGGAATT
TTTCTTATAT
BAR
35S
AGCTCGCATG
AAACTACAAG
AAAACCTGCT
AGAATACATA
TAATGTCTGA
GCACATGGGC
ATTAATTGGA
TAAACAAATT
TTAACGCCGA
AGAAATCTTA
GCTCAACACA
AtBG1
TTCATCTTGT
AGTGTGAGAG
ACCCTGGTTG
TGCATGGATC
ACTCTCAAGT
AGCTGAGTTA
TGCAGACAGA
GACGCTTAGA
ATTAATTCGG
TTGATAGAAG
TGAGCGAATC
GGGTCATAAG GAAGCACTGC
ACCTTAGGCT GTTAGGCAGG
CTCTTATATA TGCACGGCGA
GATATGACAA TATGCCAATC
CAATATAAAT TTGGATTAAA
GCATTCGACC ATGCATAGCT
TAACCATCAC GAGTTTAGTG
CAACTTAATA ACACATTGCG
GGGATCTGGA TTTTAGTACT
TATTTTACAA ATACAAATAC
GAATTC
35S poly A tail
Ubiquitin-P
35S
Intron-GUS
CGATGATTAT
GCATGACGTT
ACGCGATAGA
CTATGTTACT
ATCGAATGTG
AGGATGTCAT
TACATTTACA
AGTTTGTCTC
AAACATTGTC
CCGTAGCTTC
GTGTATACCT
CATATAATTT
ATTTATGAGA
AAACAAAATA
AGATGATGCT
AATTATGCAT
AAATTTGTAT
GATATGCAAT
TCACAAAAAT
TGCCAATGCC
TCATCCCGTG
GTCAAAGTAA
NOS terminator
ACTAGTGATT
ACGTTAAGCA
TGATTAGAGT
ACTAGGATAA
CAATGCATGT
CTGTTTTTTA
ATATTTGAAC
GCAGCTCACA
AACAAAAAGT
GCATGTCCTC
CGTAATATTA
GAATCCTGTT
TGTAATAATT
CCCGCAATTA
ATTATCGCGC
TCACCTATCA
CTACTTTATC
ACTACTCATA
GTATACAGCC
TGTCAATACT
ATTCTCATAA
TTAGCTGAGG
GCCGGTCTTG
AACATGTAAT
TACATTTAAT
GCGGTGTCAT
TTGAAAGAAT
CGTCCTATAA
TTGACATCTT
ACATATACCA
AAAAGGAAAA
TTTTTTCTGT
TTATTACCCG
CTGTTGAATT
TGGGTTTTTA
TAGCGCGCAA
AGCATCTAAA
AGGTATTCAC
AAATTTAGAT
CAAGTACTGT
ATAGAGAGAG
AGTCAGATAT
AACATCTCCT
GATAAAATCG
Supplemental Fig. 4. Determination of T-DNA insertion site in AtBG1 #2 transgenic line
by I-PCR.
Inverse PCR on #2 transgenic plant was performed using the same methods described in
Supplemental Figure 3. The T-DNA was integrated into a non-coding region. The noncoding region was shown in Black, and CaMV 35S polyA and NOS terminator of T-DNA
region in the #2 transgenic plant were shown in red and blue, respectively. The T-DNA
region of the binary vector plasmid pCAMBIA3301 harbouring AtBG1 was shown in the
middle of sequence.