Báo cáo Y học: Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica, involved in biosynthesis of bryonolic acid ppt

Molecular cloning and characterization of isomultiflorenol synthase, a
new triterpene synthase from
Luffa cylindrica
, involved in biosynthesis
of bryonolic acid
Hiroaki Hayashi
1
, Pengyu Huang
1
, Kenichiro Inoue
1
, Noboru Hiraoka
2
, Yasumasa Ikeshiro
2
, Kazufumi Yazaki
3
,
Shigeo Tanaka
4
, Tetsuo Kushiro
5
, Masaaki Shibuya
5
and Yutaka Ebizuka
5
1
Gifu Pharmaceutical University, Japan;
2
Niigata College of Pharmacy, Japan;
3

Graduate School of Biostudies, Kyoto University, Japan;
4
Graduate School of Pharmaceutical Sciences, Kyoto University, Japan;
5
Graduate School of Pharmaceutical Sciences, University of
Tokyo, Japan
An oxidosqualene cyclase cDNA, LcIMS1, was isolated
from cultured cells of Luffa cylindrica Roem. by
heterologous hybridization with cDNA of Glycyrrhiza
glabra b-amyrin synthase. Expression of LcIMS1 in yeast
lacking endogenous oxidosqualene cyclase activity resulted
in the accumulation of isomultiflorenol, a triterpene. This is
consistent with LcIMS1 encoding isomultiflorenol synthase,
an oxidosqualene cyclase involved in bryonolic acid
biosynthesis in cultured Luffa cells. The deduced amino-
acid sequence of LcIMS1 shows relatively low identity with
other triterpene synthases, suggesting that isomultiflorenol
synthase should be classified into a new group of triterpene
synthases. The levels of isomultiflorenol synthase and
cycloartenol synthase mRNAs, which were measured with
gene-specific probes, correlated with the accumulation of
bryonolic acid and phytosterols over a growth cycle of the
Luffa cell cultures. Isomultiflorenol synthase mRNA was
low during the early stages of cell growth and accumulated
to relatively high levels in the late stages. Induction of this
mRNA preceded accumulation of bryonolic acid. In
contrast, cycloartenol synthase mRNA accumulated in the
early stages of the culture cycle, whereas phytosterols
accumulated at the same relative rate throughout the whole
growth cycle. These results suggest independent regulation

of these two genes and of the accumulation of bryonolic acid
and phytosterols.
Keywords: bryonolic acid biosynthesis; isomultiflorenol
synthase; Luffa cylindrica; triterpene synthase.
Higher plants have the capacity to accumulate a wide range
of chemically diverse triterpenes in addition to sterols [1].
Sterols and triterpenes share a common biosynthetic
intermediate, 2,3-oxidosqualene, which is cyclized by
various oxidosqualene cyclases (OSCs) to give polycyclic
skeletons. OSCs are situated at the putative branch point
capable of channeling biosynthesis toward sterols or various
triterpenes in higher plants [2]. In contrast, lanosterol
synthase, the only OSC found in animals, plays a crucial role
in cholesterol biosynthesis. As part of a continuing study on
the evolution and reaction mechanism of OSCs in higher
plants, cDNAs for b-amyrin synthase (EC 5.4.99 ) [3–6],
lupeol synthase (EC 5.4.99 ) [7,8], multifunctional triter-
pene synthase (EC 5.4.99 ) [5,9,10], and cycloartenol
synthase (EC 5.4.99.8) [3,11– 14] were cloned, and
functionally characterized. However, more than 90 different
triterpene skeletal types have been found in nature [15],
suggesting the existence of additional OSCs. As part of an
effort to understand the molecular mechanism of the
triterpene cyclization reactions, as well as the molecular
evolution of these proteins in higher plants, we have a
continuing program to isolate as many of the different
triterpene synthases as possible.
Cell suspension cultures of Luffa cylindrica Roem.
(Cucurbitaceae) are capable of producing a large amount of
bryonolic acid (D:C-friedoolean-8-en-3b-ol-29-oic acid)

[16,17], a friedooleanane-type pentacyclic triterpene, which
exhibits antiallergic activity against cutaneous anaphylaxis
and contact dermatitis [17,18]. Bryonolic acid was first
isolated from the roots of Bryonia dioica (Cucurbitaceae)
[19], and was later shown to accumulate exclusively in the
roots and cultured cells of various cucurbitaceous plants
[20]. The biosynthetic pathway for bryonolic acid was
elucidated by tracer and enzymological experiments using
cultured watermelon cells [21]. 2,3-Oxidosqualene, the
common precursor derived from mevalonic acid, is cyclized
by an OSC to form isomultiflorenol, the pentacyclic
triterpene intermediate, which is then converted into
bryonolic acid. Hence, the OSC isomultiflorenol synthase
is a potential regulatory enzyme controlling the biosynthesis
of the bryonolic acid skeleton (Fig. 1).
We have been studying the regulation of bryonolic acid
biosynthesis in L. cylindrica as a means of investigating
Correspondence to H. Hayashi, Gifu Pharmaceutical University, 5-6-1
Mitahora-higashi, Gifu 502-8585, Japan. Fax: þ 81 58 237 5979,
Tel.: þ 81 58 237 3931, E-mail:
Enzymes: isomultiflorenol synthase (EC 5.4.99 ); b-amyrin synthase
(EC 5.4.99 ); lupeol synthase (EC 5.4.99 ); cycloartenol synthase
(EC 5.4.99.8).
Note: The nucleotide sequence reported in this paper has been deposited
in the DDBJ/EMBL/GenBank under the accession number AB058643.
(Received 6 June 2001, revised 8 October 2001, accepted 9 October
2001)
Abbreviations: DIG, digoxigenin; LC-APCIMS, liquid
chromatography-atmospheric pressure chemical ionization mass
spectrometry; OSC, oxidosqualene cyclase.

Eur. J. Biochem. 268, 6311–6317 (2001) q FEBS 2001
how triterpenoid formation is controlled relative to the sterol
biosynthesis pathway, which is necessary for cell growth. So
far, two OSC cDNAs, LcCAS1 [13] and LcOSC2 [22], have
been isolated from cultured Luffa cells by heterologous
hyridization with pea cycloartenol synthase cDNA. LcCAS1
encodes a functional cycloartenol synthase involved in
sterol biosynthesis [13], whereas expression of LcOSC2 in
yeast does not result in any triterpene accumulation [22].
The relatively low identity of the deduced LcOSC2 protein
with other pentacyclic triterpene synthases suggested that
LcOSC2 did not encode isomultiflorenol synthase [22].
Furthermore, as the reaction mechanism proposed for
triterpene formation by isomultiflorenol synthase is more
similar to that by b-amyrin synthase than to that by
cycloartenol synthase, an isomultiflorenol synthase is
expected to share greater sequence similarity with b-amyrin
synthase. In this study, we used the Glycyrrhiza glabra
GgbAS1 b-amyrin synthase cDNA [6] to isolate cDNA for a
new OSC family member from the Luffa cDNA library by
heterologous hybridization. Functional expression of this
cDNA in yeast indicated that the newly obtained cDNA
coded for the isomultiflorenol synthase involved in
bryonolic acid biosynthesis. Furthermore, the pattern of
expression of isomultiflorenol synthase was compared with
that of the cycloartenol synthase responsible for sterol
biosynthesis in cultured Luffa cells, which showed
sophisticated regulation of triterpenoid biosynthesis.
MATERIALS AND METHODS
Chemicals

Authentic isomultiflorenol and bryonolic acid were kindly
provided by W. Kamisako (Mukogawa Women’s University,
Nishinomiya, Japan). Other chemicals were purchased from
Wako Pure Chemicals (Osaka, Japan) and Nakalai Tesque
(Kyoto, Japan). Customized oligonucleotide primers were
synthesized by Amersham Pharmacia Biotech.
Plant material and culture conditions
Cell suspension cultures of Luffa [16] were maintained in a
300-mL Erlenmeyer flask containing 60 mL Linsmaier and
Skoog medium [23], supplemented with 100 n
M 1-naphthal-
eneacetic acid in the dark at 25 8C, and subcultured at
intervals of 4 weeks. For the time course experiments, the
cells (1 g fresh weight) were cultured in a 100-mL
Erlenmeyer flask containing 30 mL of the above medium.
Harvested cultured cells were frozen with liquid nitrogen
and stored at 2 80 8C.
Construction of the
L. cylindrica
cDNA library
Total RNA was prepared from 10-day-old-cultured Luffa
cells by guanidine thiocyanate/hot phenol extraction [24].
Poly(A)-rich RNAwas purified by an mRNA purification kit
(Pharmacia), and a Luffa cDNA library was constructed
using a lZAP-cDNA synthesis kit (Stratagene).
Cloning of a new triterpene synthase cDNA from
L. cylindrica
A 1227-bp digoxigenin (DIG)-labeled DNA probe, corre-
sponding to amino-acid residue numbers 353–729 of
G. glabra GgbAS1 b-amyrin synthase [6], was prepared by

PCR using GgbAS1 as a template, Taq DNA polymerase
(Takara Shuzo, Kyoto, Japan), the primers 5
0
-GAAGCATA
TCCACTATGAAGATGA-3
0
and 5
0
-TGAATACTCCCGTG
ATTTCCTGTTG-3
0
, and DIG–dNTP mixture (Roche
Diagnostics), according to the manufacturer’s manual. The
cDNA library prepared from cultured Luffa cells was
screened with the DIG-labeled probe under conditions of
low stringency as previously reported [14]. The hybridized
DIG-labeled probe was detected using a DIG-nucleic acid
detection kit (Roche Diagnostics), according to the
manufacturer’s manual. One positive clone was subcloned
into pBluescript SK(–) (Stratagene) by in vivo excision,
and was sequenced in both strands by the dideoxy chain-
termination method using a model 373A DNA sequencer
(PE Biosystems). Nucleotide and amino-acid sequences
were analysed by Genetyx-Mac software (Software
Development, Tokyo, Japan). This clone was designated
LcIMS1.
Fig. 1. Pathways of bryonolic acid biosynthesis
in Luffa cylindrica.
6312 H. Hayashi et al.(Eur. J. Biochem. 268) q FEBS 2001
Functional expression in yeast mutant GIL77

To construct an expression plasmid for yeast, Kpn I and
Xba I sites were introduced into the 5
0
and 3
0
-termini of the
deduced ORF of LcIMS1 cDNA by PCR (25 cycles with
40 s at 94 8C, 40 s at 50 8C and 2 min at 72 8C) with Pfu
DNA polymerase (Stratagene) used as the DNA polymerase.
Two primers of 5
0
-CTGGTACCGATTGAGTTGAGGTG
ATTG-3
0
(Kpn I site underlined) and 5
0
-CCTCTAGAGTA
AAAGTCTCCAATC-3
0
(Xba I site underlined) were used to
modify the cDNA. The PCR product was digested with
Kpn I and XbaI, and ligated to the sites of pYES2
(Invitrogen), to obtain pYES2-LcIMS1 in which the ORF
of the cDNA was ligated to the GAL1 promoter in the sense
orientation. The nucleotide sequence of the inserted DNA
was confirmed by sequencing. The ERG7-deficient yeast
strain GIL77 [3] was transformed with pYES2-LcIMS1 by
the lithium acetate method [25]. The protocols for the
culture condition, induction by galactose, and preparation of
triterpene mono-alcohol fraction have been described

previously [3]. After purification by preparative TLC, the
triterpene mono-alcohol fraction was analyzed by liquid
chromatography–atmospheric pressure chemical ionization
mass spectrometry (LC-APCIMS). LC-APCIMS was
performed with an LCQ (Thermo Quest, Tokyo, Japan)
under the following HPLC conditions: column, SUPER-
ODS (diameter 4.6 mm, length 200 mm; Tosoh); solvent
system, 95% acetonitrile aq.; flow rate, 1 mL·min
21
;
column temperature, 40 8C; detection, UV 202 nm;
retention time for isomultiflorenol, 21.7 min
Isomultiflorenol: MS m/z 409 [M þ H–H
2
O]
þ
, MS/MS
(precursor ion at m/z 409) 313 (20%), 299 (41%), 245
(45%), 231 (100%), 217 (75%), 191 (56%).
Northern-blot analysis
The DIG-labeled RNA probes were prepared from
Bam HI-digested LcCAS1 (probe length 1.3 kb) [13]
and Bam HI-digested LcIMS1 (probe length 1.9 kb) using
T7 RNA polymerase and DIG RNA Labeling Mix (Roche
Diagnostics), according to the manufacturer’s manual. The
two DIG-labeled RNA probes specifically hybridized to the
respective cDNA under conditions of high stringency.
Cultured Luffa cells frozen by liquid nitrogen were
homogenized in a mortar to give total RNA by an Extract-
A-Plant RNA isolation kit (Clontech). Five micrograms of

total RNA (per lane) were separated on a 1% agarose gel
containing formaldehyde, and blotted to a positively
charged nylon membrane (Roche Diagnostics). The
membrane was hybridized with the DIG-labeled RNA
probe as previously reported [14]. The hybridized
membrane was washed twice for 5 min at room temperature
with 2 £ NaCl/Cit containing 0.1% SDS, and then twice for
10 min at 65 8C with 0.2 £ NaCl/Cit containing 0.1% SDS.
The hybridized DIG probe was detected using the DIG
Nucleic Acid Detection Kit according to the manufacturer’s
manual.
Quantitative analysis of bryonolic acid
Freeze-dried powdered samples (50 mg) were extracted
with ethyl acetate (2 mL twice, 60 8C for 1 h), and
cholesterol (1 mg) was added to the extract as an internal
standard. The dried sample was dissolved in pyridine
(50 mL), and an aliquot (10 mL) of the pyridine solution was
treated with N,O-bis(trimethylsilyl)acetamide (10 mL) at
50 8C for 1 h. An aliquot (1 mL) of the solution was applied
to GC for the quantitative analysis of bryonolic acid. The
rest of the pyridine solution was saponified with 6% (w/v)
KOH in methanol (1 mL) at 90 8C for 1 h to hydrolyze steryl
esters. Sterols were then extracted with n-hexane (3 mL).
The dried extract was dissolved in a mixture of pyridine
(40 mL) and N,O-bis(trimethylsilyl)acetamide (40 mL), and
incubated at 50 8C for 1 h. An aliquot (1 mL) of the solution
was analyzed by GC for total sterols. GC analysis was
Fig. 2. LC-APCIMS analysis of the triterpene mono-alcohol
fraction from GIL77 transformed with pYES2-LcIMS1. (A)
HPLC profile of the triterpene mono-alcohol fraction of the

pYES2-LcIMS1 transformant; (B) MS/MS (precursor ion at m/z 409)
fragmentation patterns of the peak at 21.7 min of the pYES2-LcIMS1
transformant; (C) MS/MS (precursor ion at m/z 409) fragmentation
patterns of authentic isomultiflorenol.
q FEBS 2001 Cloning of isomultiflorenol synthase (Eur. J. Biochem. 268) 6313
performed under the following conditions: column, Ultra
Alloy
þ
-17 capillary column (15 m £ 0.5 mm internal
diameter; film thickness 1 mL; Frontier Laboratory Ltd,
Koriyama, Japan); column temperature, 200–300 8C
(20 8C·min
21
); injector and detector temperature, 320 8C;
carrier gas, He 3.5 mL·min
21
. The contents of bryonolic
acid and total sterols were calculated from the peak area of
their trimethylsilylated products relative to that of the
internal standard. Sterol content was the total content of
stigmasta-7,22-dien-3b-ol, stigmasta-7,25-dien-3b-ol, and
stigmasta-7,22,25-trien-3b-ol, which are D
7
-phytosterols
characteristic of cucurbitaceous plants [21].
RESULTS
Cloning of a new OSC cDNA from
L. cylindrica
A lZAP cDNA library constructed from 10-day-old
cultured Luffa cells was screened using a b-amyrin synthase

cDNA from G. glabra as a heterologous hybridization probe
under low-stringent conditions. One positive clone was
isolated from < 200 000 plaques of the cDNA library, from
which the phagemid pBluescript SK(–) containing the
cDNA insert was recovered by in vivo excision. This cDNA
was 2551 nucleotides in length, and contained an ORF of
2277 nucleotides. A stop codon was present 9 bp upstream
of the initial ATG codon in the frame. The cDNA sequence
was divergent from those of LcCAS1 [13] and LcOSC2
[22], previously isolated from this plant species. Translation
of the coding region yields a putative polypeptide of 759
amino-acid residues with a predicted molecular mass of
87.7 kDa. The QW motif, which occurs repeatedly in the
sequence of all known OSCs [26], and the DCTAE motif,
corresponding to an active site of the OSC family [27], were
observed in the deduced amino-acid sequence. We
presumed therefore that the new OSC cDNA was a
candidate for isomultiflorenol synthase of L. cylindrica.
Functional expression of the new OSC cDNA in the
ERG7-deficient yeast mutant GIL77
To elucidate the function of the new OSC cDNA, its ORF
was inserted into a yeast expression plasmid with a
galactose-inducible GAL1 promoter. The completed plas-
mid, designated pYES2-LcIMS1, was introduced into an
ERG7-deficient yeast mutant GIL77 [3], which lacks a
functional lanosterol synthase gene and produces no
detectable endogenous lanosterol or other triterpene mono-
alcohols. The transformed yeast was treated with galactose
to induce expression of the recombinant protein, and was
extracted with hexane. The extract of pYES2-LcIMS1

transformant gave a spot with an R
f
value corresponding to
triterpene mono-alcohol on preparative silica gel TLC (data
not shown), which was absent from the control pYES2
transformant [3]. This spot corresponding to triterpene
mono-alcohol was extracted and further analyzed by
LC-APCIMS. As shown in Fig. 2, a major peak at
21.7 min was observed in the HPLC profile. This main
product was identified as isomultiflorenol by comparing
its retention time and MS/MS (precursor ion at m/z
409) fragmentation patterns with those of authentic
isomultiflorenol. As the control pYES2 transformant
produced no triterpene mono-alcohol [3], accumulation of
Fig. 3. Multiple alignment of deduced amino-acid sequences of isomultiflorenol synthase (LcIMS1), cycloartenol synthase (LcCAS1), and
putative oxidosqualene cyclase (LcOSC2). Hyphens were inserted to maximize homology. Amino-acid residues identical in two out of the three
protein sequences are boxed. The DCTAE motif is marked by a double underline.
6314 H. Hayashi et al.(Eur. J. Biochem. 268) q FEBS 2001
isomultiflorenol in the pYES2-IMS1 transformant indicates
that the new OSC cDNA, designated LcIMS1, encodes
isomultiflorenol synthase, the key enzyme in bryonolic acid
biosynthesis in L. cylindrica. In addition to isomultiflorenol,
a minor compound at 18.9 min, which, like isomultiflorenol,
gave an ion at m/z 409, was also observed on LC-APCIMS
analysis (Fig. 2). This compound, which seems to be a side
product of LcIMS1 isomultiflorenol synthase, could not be
identified.
Sequence comparison
Figure 3 shows the multiple alignments of deduced amino-
acid sequences of three OSCs from L. cylindrica. The

sequence of the Luffa LcIMS1 isomultiflorenol synthase
exhibits 67–66%, 61–57%, 61%, 51% and 50% identities
with those of b-amyrin synthases [3–6], lupeol synthases
[7,8], Pisum PSM multifunctional triterpene synthase [5],
Luffa LcCAS1 cycloartenol synthase [13], and Luffa
LcOSC2 putative OSC [22], respectively. To clarify the
evolutionary relationship of isomultiflorenol synthase
among plant OSCs, the phylogenetic tree was constructed
from the deduced amino-acid sequences of the LcIMS1 and
other OSCs so far cloned from higher plants (Fig. 4). The
relatively low identities (67–57%) of the LcIMS1 protein
with other triterpene synthases suggest that isomultiflorenol
synthase belongs to a new class of triterpene synthases in
higher plants.
Differential expression of OSC mRNAs in the cultured cells
of
L. cylindrica
Isomultiflorenol synthase and cycloartenol synthase are
situated at a putative branch point for biosynthesis of
bryonolic acid and sterol, respectively (Fig. 1). Therefore, it
was of interest to compare the expression patterns of
isomultiflorenol synthase and cycloartenol synthase mRNAs
in cultured Luffa cells over a culture growth cycle. Northern-
blot analyses were performed with gene-specific DIG-
labeled RNA probes for the two OSCs: isomultiflorenol
synthase and cycloartenol synthase (LcCAS1) [13], so far
cloned from Luffa. Figure 5 shows the time course of cell
Fig. 4. Phylogenetic tree constructed from the deduced amino-acid
sequences of LcIMS1 cDNA and other plant OSCs. The phylogenetic
tree was constructed by the unweighted pair group method using

arithmetric averages [29] with Genetyx-Mac software (Software
Development, Japan). The GenBank database accession numbers used
in this analysis are as follows, AB009030 (Panax PNY), AB014057
(Panax PNY2), AB037203 (Glycyrrhiza GgbAS1), AB034802 (Pisum
PSY), AB034803 (Pisum PSM), U49919 (Arabidopsis LUP1),
AC002986 (Arabidopsis MFS [9,10]), AB058643 (Luffa LcIMS1),
AB025343 (Olea OEW), AB025345 (Taraxacum TRW), Y15366
(Medicago MtN18), AB009031 (Panax PNZ), AB025346 (Taraxacum
TRV), AB033335 (Luffa LcOSC2), AB025968 (Glycyrrhiza GgCAS1),
D89619 (Pisum PSX), AB025344 (Olea OEX), AB009029 (Panax
PNX), U02555 (Arabidopsis CAS1), AB033334 (Luffa LcCAS1) and
AB025353 (Allium AMX). bAS, b-amyrin synthase; CAS, cycloartenol
synthase; LUS, lupeol synthase; MFS, multifunctional triterpene
synthase; IMS, isomultiflorenol synthase.
Fig. 5. Time course of bryonolic acid accumulation and levels of
isomultiflorenol synthase and cycloartenol synthase mRNA in
cultured Luffa cells. (A) Time course of accumulation of bryonolic
acid and total sterol in cultured Luffa cells. Mean of three replicates. Bar
indicates SD. (B) Time course of levels of isomultiflorenol synthase and
cycloartenol synthase mRNA in cultured Luffa cells at different stages
of growth. Total RNA (5 mg) was separated on 1% agarose gel
containing formaldehyde, blotted to a positively charged nylon
membrane, and then hybridized with the DIG-labeled RNA probe of
LcIMS1 or LcCAS1. Ethidium bromide staining of the gel before
transfer is shown below.
q FEBS 2001 Cloning of isomultiflorenol synthase (Eur. J. Biochem. 268) 6315
growth, accumulation of bryonolic acid and total sterol, and
mRNA levels of isomultiflorenol synthase and cycloartenol
synthase in the cultured Luffa cells. Although the total sterol
level increased during all growth phases except the

stationary phase, the bryonolic acid level increased during
the late growth phase only [16,17]. The level of
isomultiflorenol synthase mRNA was high at days 16 and
20 and low at days 4 and 8. The induction of this mRNA
preceded the accumulation of bryonolic acid. In contrast, the
level of cycloartenol synthase mRNAwas high at days 4 and
8 and decreased at later growth stages. Similar results were
also obtained in the repeated time course experiment (data
not shown). These results suggest independent regulation of
these two genes and of the accumulation of bryonolic acid
and phytosterols. It is noteworthy that the changes in their
mRNA levels correlated with the accumulation of the
respective end products.
DISCUSSION
In this study, a new OSC cDNA (LcIMS1) was cloned from
cultured Luffa cells by heterologous hybridization with that
of G. glabra (licorice) b-amyrin synthase. We tried to
express the new OSC in the yeast mutant GIL77 [3], an
ergosterol auxotroph lacking a functional lanosterol
synthase gene, which lacks endogenous triterpene mono-
alcohol and accumulates oxidosqualene inside the cells. As
the triterpene synthase activity in a transformed yeast was
too low to identify cyclization products, we sought to detect
cyclization products in the yeast transformed with the
LcIMS1 clone. Expression of LcIMS1 in the mutant yeast
resulted in the accumulation of isomultiflorenol. This result
is consistent with LcIMS1 encoding isomultiflorenol
synthase, an OSC involved in bryonolic acid biosynthesis
in cultured Luffa cells.
Despite the use of licorice b-amyrin synthase cDNA as

the hybridization probe, a cDNA for b-amyrin synthase,
which produce b-amyrin as a main triterpene, was not
obtained. The major triterpenoid in culured Luffa cells is
bryonolic acid, a friedooleanane-type triterpenoid, and
oleanane-type triterpenenoids derived from b-amyrin have
not been isolated from cultured Luffa cells. As oleanane-
type triterpene saponins, together with dammarane-type
triterpene saponins, were isolated from the aerial parts of
Luffa [28], additional OSC genes for b-amyrin synthase may
be obtained if a cDNA library prepared from green tissues or
a genomic library is screened.
Although many cDNAs of OSCs have been cloned from
higher plants, there are few reports [6,14] on their gene
expression in the context of the physiological regulation of
sterol and triterpenoid biosynthesis. Molecular cloning of
the isomultiflorenol synthase gene will provide a useful tool
not only for elucidating bryonolic acid biosynthesis in
cultured Luffa cells, but also for studying the regulation of
the branch point in triterpenoid biosynthesis to supply
diverse triterpene molecules in plant cells. Further
experiments are under way to confirm the regulation of
the isomultiflorenol synthase gene.
ACKNOWLEDGEMENT
The authors are grateful to Dr W. Kamisako (Professor Emeritus,
Mukogawa Women’s University) for the gift of authentic isomulti-
florenol and bryonolic acid.
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