Supplement Experimental Procedures
Southern blot
Genomic DNA from Tulipa gesneriana was prepared by the CTAB method as
described by Murray et al. (1980). Samples (10 μg) of DNA were digested with or
EcoRI or HindIII or XbaI overnight at 37 °C. Digested DNA was then separated on to a
TAE/1.0% agarose gel for 6.5 h at 25 V. After depurination by soaking the gel in 0.25
M HCl, denaturation in 0.5 M NaOH/1.5 M NaCl, and neutralization by 0.5 M Tris-HCl
pH 7/1.5 M NaCl, DNA was transferred to a nylon filter (Nylon membranes, positively
charged; Roche, http://www.roche-applied-science.com/) with 20x SSC. Membrane was
hybridized with DIG-labeled DNA probe corresponding to the 3’ region of the cording
region of TgVit1 using PCR DIG Probe Synthesis Kit (Roche) and the following
primers
(5’-GTTTGAGCTTGGATTGGAG-3’
and
5’-TCACACAGATTGAACAGCC-3’). TgVit1 probe was hybridized in DIG Easy Hyb
(Roche) overnight at 42 °C. The membrane was washed in 0.5x SSC/0.1% SDS at
65 °C, twice for 15 min. Immunological detection was performed using the DIG-High
Prime DNA Labeling and Detection Starter Kit II (Roche) according to the instruction
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manuals. The Luminescence signal was exposed to X-ray film (RX-U; FUJIFILM,
http://www.fujifilm.com/) for 1 h at 25 °C.
cDNA cloning
Total RNA was extracted from epidermal tissues of purple petal segments of T.
gesneriana using a Plant RNA Isolation Reagent (Invitrogen, Carisbad, CA, USA).
Partial fragments of the TgYSL3 cDNA were amplified by PCR using primers (Ysl-F;
5’-TGGAGCTTCTTCCAGTGGTT-3’
and
Ysl-R;
5’-GTCTTGAAGTCGTGCATCA-3’) that were designed based on A. thaliana and O.
sativa YSL sequences. The PCR-amplified fragments were cloned into pGEM®-T Easy
(Promega) and sequenced using a MegaBACE 500 system (GE Healthcare). The
5’-UTR of TgYSL3 was isolated by 5’-RACE (Invitrogen) using the following
gene-specific
primers
(GSP1,
5’-TCCGACATACGTCAGACTAAATCAAAGAGATG-3’,
5’-CCACACACATCTCCTCCAGAGTAAAACCAC-3’).
The
and
full-length
GSP2,
TgYSL3
cDNA was synthesized from total RNA using a GeneRacerTM Kit (Invitrogen),
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according
to
the
manufacturer’s
instructions,
and
gene
specific-primers
(5’-ATGACCAGATCTAACCCCTCTCATT-3’ and GeneRacerTM 3’ primer).
Quantitative PCR analysis
Total RNA was isolated from various tissues using the Plant RNA Isolation Reagent
(Invitrogen), according to the manufacturer’s instructions. Isolated RNA was treated
with DNase I (TaKaRa, Otsu, Japan), and 500 ng of total RNA was subjected to
first-strand cDNA synthesis using a PrimeScript RT reagent Kit (TaKaRa). Quantitative
PCR was carried out using 2 μl of dilute (1:10) cDNA and an ABI PRISM 7300 system
(Applied Biosystems) with SYBR Premix Ex Taq II (TaKaRa). The following primers
were
used:
TgYsl-F
(5’-TCTTGGTTTCTGCAATGCTTATG-3’),
TgYsl-R
(5’-CCGTAGTTGTAGGCCATGTTCA-3’).
References
Murray, M.G. and Thompson, W.F. (1980) Rapid isolation of high molecular weight
plant DNA. Nucleic Acids Res. 8, 4321-4325.
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