cDNA Cloning and Gene Expression of Anthocyanidin Synthase from

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Abstract. The cDNA clone, TAN1, encoding anthocyanidin synthase (ANS) was isolated by screening of a ..... structure and role in plant metabolism. Annu. Rev.
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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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