Abstract. The cDNA clone, TAN1, encoding anthocyanidin synthase (ANS) was isolated by screening of a ..... structure and role in plant metabolism. Annu. Rev.
331 Note
Plant Biotechnology, 17(4),331-335 (2000)
cDNA Cloning and Gene Expression of Anthocyanidin Synthase from Torenia fournieri Jun-ichiro
NAKAJIMA1, Yoshikazu TANAKA2, Mami YAMAZAKI1 and Kazuki SAITO1*
1Laboratory of Molecular Biology and Biotechnology , Research Center of Medicinal Resources, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan 2Institute for Fundamental Research , Suntory Ltd., Wakayama-dai 1-1-1, Shimamoto, Mishima, Osaka 618-8503, Japan * Corresponding author E-mail address:k Received 17 July 2000; accepted4 September2000 Abstract The cDNA clone, TAN1, encoding anthocyanidin synthase (ANS) was isolated by screening of a cDNA library constructed from flower petals of Torenia fournieri cv. Summer Wave Blue using a heterologous probe. Nucleotide sequence analysis revealed that it contains an open reading frame encoding a polypeptide of 376 amino acids. The postulated amino acid sequence shows 48% to 72% identities with those of previously reported ANSs. Southern blot analysis suggested that there is a single copy of ANS gene in the genome of T. fournieri. Northern blot analysis indicated that ANS gene is expressed in flower bud but not in leaf or mature flower. The
nucleotide
accession
number
Anthocyanins, olite
in
cDNAs
the
pathway
have
been
(Dooner
and
Holton 1992).
leading the
metabolites, cyanin al., reaction
we
Perilla
their
TAN1
a
Torenia
fournieri,
from
from
mercial
1991;
color
an
In
our
cDNA
plant
(Suzuki
fournieri
cv. of
constructed
from probe
isolated
enzyme study,
from
we
the
with
with
ANS
and
also
et
a suitable
engineering
of
was Blue
cDNA
from
the
of
of
and
60-80%
pletely cies,
Luca,
exper-
1996;
flower
308)
petals Perilla
isolated
from
(Suntory library
Ltd.) (Stratagene)
using frutescens
the
hetero-
conserved which
of
(Britsch 1994;
(Saito
(Lukacin
by
2000),
is
indicated
tree of
acid
also (Fig. divot the
Sequence
1993;
residues
as
The the
2B),
presence
plant at
Carolis
and
Prescott
and
site
of
et
From ANS
phylogenetic distinct
of
2
from
Lukacin
ANSs.
Torenia
The
De John,
(306-
binding
1997; in
the oxy-
Arg-Xaa-Ser
Britsch,
the
spe-
iron
3ƒÀ-hydroxylase
conserved
ANSs.
com-
soluble
specific
flavanone and
(Fig.
are
with
De
1997;
with identity
several
bind
at
TAN1
48-72%
His-296
The
(TAA)
of
and
et al.,
1). confirmed
ATG.
from
al.,
1993).
in
codon
iron-containing
et
reported
stop
amino
to
the
Matsuda
Prescott, motif,
acids
amino (Fig.
first
ANSs
thought
center
376
was
indicated in
in
are
open
sequence
Asp-242
petunia
group
the acid
of
contained
colon
in-frame
similarity
T.
genetic
ATG
ANSs
His-240,
genases
com-
an
amino
reported
catalytic
encoding
from
of
those
first
of
upstream
bp
sequence
and
polypeptide
the
presence
-oxoglutarate
Wave
flower
42,130-Da
have
press).
II
a
long,
1128
DSQ-2000L,
nucleotide
by
forming
from
of
a
1348
was method
(model
The
of
2A).
proteins is
was
analysis
termination
sequencer
frame
ANS
flowers
T. fournieri
TAN1,
a. ăZAP
(Saito
chain
Japan).
reading
by
sequence
dideoxy
automatic
comparison
antho-
of
using
18-bp
colored
in
paper
compared
in
Summer
step
present
genetic
et al.,
of
mechanism
ANS
the
leucoantho-
formation
recombinant
plant
ANS cDNA,
screening
from
species.
for
databases
Nucleotide by
authenticity
Gerats,
catalyzes
previous
encoding
1993;
and
(ANS)
the
the
species
Forkmann,
important
the
and
plant
Martin
the
In
model
The
for
thus
ornamental
imental
various
anthocyanidin
by
various
described,
1995;
frutescens.
isolated
logous
and
been
and
1999). out
Shimadzu,
studied
catalyzed
in GenBank/EMBL/DDBJ
al.,
most
step
formation. 1999),
carried
for
synthase
and
et
(Heller
cloned
to
metab-
pathway
have
first
are
responsible
Anthocyanidin
cyanidin,
species,
Enzymes
Cornish,
reaction
deposited
biosynthetic
Robbins,
and
has been
formed
secondary
plant
1993).
of
in this paper
AB044091.
most
flavonoid
Forkmann, steps
reported
representative
found
through
sequence
al.,
phylo-
falls
into
tree difference
also
a
332
Fig. 1.
Nucleotide indicates
between
the
sequence
in-flame
sequences
of
and deduced
monocotyledoneae
and
dicotyledoneae. For DNA
Southern from
leaves
described gested which
with do
a
T,
cut
agarose
2.5ƒÊg
fournieri
(Murray
several not
DNAs 0.8%
analysis, of
method
digested on
blot
et
different within
were
the separated
gel,
of
transferred
acid
sequence
al.,
1980)
was
the di-
of Torenia
ANS.
Then
electrophoresis onto
Hybond-
the
dCTP
using
(Takara
with
of
Shuzo,
a
ANS
Primer and
Biotech). in
0.1•~SSPE,
Hybridization BAS-2000
at
cDNA
Japan)
washed 10min.
and
probe
Random
(Pharmacia
finally •Ž for
(Amersham) cDNA
region
Column
enzymes,
cDNAs. by
by
N+ membrane 32P -labeled coding
genomic
isolated
restriction ANS
amino
Asterisk
stop colon.
hybridized 60•Ž
was
As
a
labeled
DNA
0.1%
Analyzer
a
probe,
by
32P-
Labeling
purified The
signals Image
.
with
Kit
by
NAP5
membrane SDS were
was at
60
detected (Fuji
Film,
333
(A)
(B)
Fig. 2.
Multiple alignment (A) and phylogenetic tree (B) of deduced amino acid sequences of ANSs from several plant species. (Continued on back page)
334
Multiple
alignment
(Thomopson
et al.,
similar
amino
show Asterisks
His
2000).
to
Torenia
grape,
vinifera
apple,
number
tree
incana
batatas
(Min
(Shiokawa
rice,
Oryza
and
et al.,
sativa
(A)
snapdragon,
1994);
forsythia,
1993);
carrot,
Callistephus
Forkmann,
accession
number
accession
then
hybrida
(Weiss
et
al.,
1993);
x
the
same
Zea
ern
blot
of
ANS
gene
from
Torenia
Genomic
DNA
leaves
were
HindIII
and
(2.5ƒÊg)
digested PstI,
on
(0.8%),
transferred
as
the
from
with probe.
petals
leaves (1.2%),
ing
agarose
gel
buds,
TAN1
to the
mature
cDNA RNA
on a
probe. (ANS) (rRNA)
and coding
(10ƒÊg)
electrophoresed
with
ribosomal
RNA
or for
al.,
bud
kb)
and
also
As was
probed RNA,
shown
in
detected
no
evident
signal
in
mature
flower
observed
of
ribosomal
1985).
(1.4
South-
loading
was for
by
for
equivalent
DNA
et
performed
condition
membrane
rice
flower
was
same
verify
the
membrane.
Fig.
only of
in
mRNA
petal
and
This
expression
timing
of
pattern
coloring
is
an
probe rice a load
in
well
petal
of
consistent
with
fournieri.
The
T
developmentally
ANS
gene
was
regulated
reported
(Rosati
for et al.,
expression
the
flower
of
of
Forsythia
1999).
then region
Acknowledgement
isolated flower
This
and
agarose
membrane, The
the
To
was
Hybond-N+
membrane
transcript
similar
EcoRV,
an
TAN1
1990);
from
a membrane,
Total
flower
transferred
hybridized labeled
(B) of
were
onto 32P-labeled
the
blot,
x intermedia hybridized
et al.,
fournieri.
EcoRI,
separated
Ipomoea
(B)
isolated
with
a
under
(Takaiwa
of
the (A)
commonstock,
(Messen
onto
the
ANS
leaf. analyses
al.,
accession
1997);
mays
analysis.
expression blot
et accession
sweetpotato,
32P-labeled
petals northern
Seitz,
Forkmann,
al.,
of
on
3B,
and
(Rosati
and
and et
probe
pRR217
(A)
intermedia
transferred
RNA
blot
1987). 1991);
AF026058);
with
Southern
et
Nei,
Y07955).
(B)
3.
and
al.,
Hybridization
Fig.
Lukacin
et
(Hirner
maize,
from
1997;
(Martin
(Pelletier
AB023786);
number
coordination.
(Saitou
carota
number
shows
Arrowheads
majus
(Henkel
thaliana
shading
Britsch,
Forsythia
chinensis
Arabidopsis
program
3ƒÀ-hydroxylase
and method
Daucus
gray
ferrous-ion
Antirrhinum
et al.,
W
alignment.
flavanone
Petunia
clustal
and
best for
neighbor-joining
petunia,
accession
(Reddy,
required in
by
acids
for
(Lukacin
1999);
(Davies,
arabidopsis,
as
al.,
chinaaster,
AF015885);
Matthiola
et
(Sparvoli
sp.
AF184273);
number
study);
(Saito
amino
conserved
by
out
sequences
2-oxoglutarate
generated
(this
L.
Malus
the
motif of
carried
identical
in
postulated
site
was
was
indicates gaps
residues
a binding
frutescens
Vitis
1999);
shading indicate
Asp
fournieri
Perilla
sequences
Arg-Xaa-Ser
be
Phylogenetic
torenia, perilla,
and the
assumed
acid
Black
Dashes
indicate
petunia,
amino
1994).
acids.
conserved
al.,
of
gel
and
then
Aid
work for
was
supported
Scientific
Education,
Science,
by
from
a
grant
in
Research
part
from
Sports
and
Research
by the
Grants-inMinistry
Culture,
for
the
of
Japan;
Future
and
Program
was 32pDNA
encod-
control.
(96I00302) tion
of
from
the
Science,
Japan
Society
for
the
Promo-
Japan.
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