Expression of D6, D5 desaturase and GLELO elongase genes from Mortierella alpina for production of arachidonic acid in soybean [Glycine max (L.) Merrill] seeds

Abstract

We report the production of arachidonic acid (ARA) in seeds of soybean [Glycine max (L.) Merrill] by seed-specific expression of the genes encoding Δ6 desaturase, fatty acid elongase, and Δ5 desaturase from a filamentous fungus, Mortierella alpina 1S-4, as well as the down-regulation of an endogenous gene of soybean, Δ15 desaturase. Several fatty acids, γ-linolenic acid (GLA), eicosa-8, 11-dienoic acid (EDA), dihomo-γ-linolenic acid (DGLA), and ARA, which the host plant does not produce, accumulated up to 11.0 and 8.4% of the total fatty acids in the transgenic histodifferentiated somatic embryos and the transgenic mature seeds, respectively. The ability to synthesize these novel fatty acids was inherited to their progenies. These results indicate that metabolic engineering of the fatty acid biosynthetic pathway to produce desired very long-chain polyunsaturated fatty acids in oilseed crops is feasible.

Full text

Expression of D6, D5 desaturase and GLELO elongase genes
from Mortierella alpina for production of arachidonic
acid in soybean [Glycine max (L.) Merrill] seeds
Ren Chen
a,1,
*, Keisuke Matsui
a,1
, Miyuki Ogawa
a
, Mieko Oe
a
, Misa Ochiai
a
,
Hiroshi Kawashima
b
, Eiji Sakuradani
c
, Sakayu Shimizu
c
, Masao Ishimoto
d
,
Makoto Hayashi
e
, Yoshikatsu Murooka
e
, Yoshikazu Tanaka
a
a
Institute for Advanced Technology, Suntory Limited, 1-1-1, Wakayamadai, Shimamoto-cho,
Osaka 618-8503, Japan
b
Institute for Health Food, Suntory Limited, Osaka, Japan
c
Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan
d
Department of Low-Temperature Sciences, National Agricultural Research Center for Hokkaido Region,
Hokkaido, Japan
e
Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan
Received 9 June 2005; received in revised form 25 September 2005; accepted 27 September 2005
Available online 7 October 2005
Abstract
We report the production of arachidonic acid (ARA) in seeds of soybean [Glycine max (L.) Merrill] by seed-specific expression of the genes
encoding D6 desaturase, fatty acid elongase, and D5 desaturase from a filamentous fungus, Mortierella alpina 1S-4, as well as the down-regulation
of an endogenous gene of soybean, D15 desaturase. Several fatty acids, g-linolenic acid (GLA), eicosa-8, 11-dienoic acid (EDA), dihomo-g-
linolenic acid (DGLA), and ARA, which the host plant does not produce, accumulated up to 11.0 and 8.4% of the total fatty acids in the transgenic
histodifferentiated somatic embryos and the transgenic mature seeds, respectively. The ability to synthesize these novel fatty acids was inheritedto
their progenies. These results indicate that metabolic engineering of the fatty acid biosynthetic pathway to produce desired very long-chain
polyunsaturated fatty acids in oilseed crops is feasible.
#2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Gene transformation; Fatty-acid desaturase and elongase; Mortierella alpina; Very long-chain polyunsaturated fatty acids; Arachidonic acid; Soybean
1. Introduction
Very long-chain polyunsaturated fatty acids (VLC-PUFAs),
such as arachidonic acid (ARA, C20:4, n-6), eicosapentaenoic
acid (EPA, C20:5, n-3), and docosahexaenoic acid (DHA,
C22:6, n-3), play important roles as structural components of
membrane phospholipids and as precursors of eicosanoids of
signaling molecules, including prostaglandins, thromboxanes,
and leukotrien [1,2]. They are important in both the medical and
pharmaceutical fields, being involved in the human inflamma-
tory response, reproductive function, immune response, blood
pressure regulation, cholesterol metabolism, and infant retinal
and brain development [3,4]. Although these fatty acids are
either directly available through a diet including fish and meats
or synthesized from linoleic acid (LA, C18:2) and a-linolenic
acid (ALA, C18:3) by the intake of green vegetables and some
plant oils [5], their synthesis is limited in infants and the elderly
[6,7]. Furthermore, dietary changes over the last decades have
resulted in an insufficient or poor balance in the ingestion of
these essential fatty acids [8]. In order to circumvent this deficit,
VLC-PUFAs are currently produced from fish or cultivated
microorganisms, but their high production cost and diminishing
feedstock limit their supply and usage. To obtain a more
suitable source for the large-scale production of these desired
VLC-PUFAs, the modification of the fatty acid biosynthetic
www.elsevier.com/locate/plantsci
Plant Science 170 (2006) 399–406
* Corresponding author. Tel.: +81 75 962 8807; fax: +81 75 962 8262.
E-mail address: chenren@agr.kyushu-u.ac.jp (R. Chen).
1
These authors contributed equally to this work.
0168-9452/$ – see front matter #2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.plantsci.2005.09.006

pathway in higher plants (especially in oilseed crops) by
genetic manipulation would be the best alternative [9–11].
The fatty acid biosynthesis pathway leading to ARA is shown
in Fig. 1. Most higher plants terminate the fatty acid synthesis at
the chain length of C18, and, thus, all of their chloroplastic fatty
acids are of the C16 and C18 varieties. In other words, the
principal saturated fatty acids are palmitic (PA, C16:0) and
stearic (SA, C18:0), and the principal unsaturated fatty acids are
oleic (OA, C18:1), LA, and ALA. In marked contrast with this
property in higher plants, in animal cells and some microorgan-
isms, various desaturases with appropriate elongases are
responsible for the desaturation and elongation of unsaturated
fatty acids to form VLC-PUFAs derived from LA and ALA. For
example, for the conversion of the polyunsaturated C18 fatty
acids into ARA or EPA, three consecutive enzymatic steps are
formally required: D6 desaturation, D6 elongation, and D5
desaturation (Fig. 1). These findings suggest that the isolation
and transformation of the genes encoding these enzymes into
plant cells should lead to the synthesis of VLC-PUFAs in plants.
During the last few years, many of the genes that are
responsible for the biosynthesis of PUFAs have been cloned
from various organisms, including fungi, algae, mosses, plants,
nematodes, and mammals [12]. The possibility of the
heterologous reconstitution of this pathway has been demon-
strated in yeast, Aspergillus oryzae, and some model plants,
such as tobacco [13] and Arabidopsis thaliana [14]. In 2004,
three different reports by Qi et al. [15], Abbadi et al. [16] and
Kinney et al. [17] described the production of VLC-PUFA in
leaves of Arabidopsis (ARA and EPA), seeds of linseed (ARA
and EPA) and somatic embryos [ARA, EPA and docosapen-
taenoic acid (DPA, C22:5, n-3)] or seeds (ARA, EPA and DHA)
of soybean by expressing various heterologous genes. These
successes have led to the long-term goal of producing such fatty
acids in transgenic oilseed crops [11,16].
Soybean, one of the most important crops in the world,
which accounts for 56% of the global oilseed production, is the
most suitable species as a host plant to produce desired VLC-
PUFAs by fatty-acid metabolic engineering. This is because
soybean accumulates large amounts of fatty acids in seeds that
can be used as substrates for the modification of VLC-PUFA
synthesis, is capable of symbiotic nitrogen fixation and can thus
grow well even in infertile soil, and is a self-pollinated plant,
whereby gene dispersal by pollen can be avoided. We report
here the seed-specific expression of D6, D5 desaturase, and
GLELO elongase genes isolated from M. alpina 1S-4 and the
down-regulation of an endogenous D15 desaturase gene in
soybean to produce VLC-PUFAs, such as ARA, which leads to
a new ecological and economical way to produce VLC-PUFAs.
M. alpina is used to produce ARA for human consumption and
may be a good gene source for the consumption.
2. Materials and methods
2.1. Vector construction
A soybean seed-specific promoter, the a0subunit of the b-
conglycinin promoter [18], and a terminator of the manopine
synthase gene (mas) [19] were used in the expression cassettes
of all the fatty acid-modifying enzymes. A cauliflower mosaic
virus (CaMV) 35S promoter and a nopaline synthase (nos)
terminator were used in the expression cassettes [20] of
hygromycin phosphotransferase (HPT) as a selective marker.
The genes of D5, D6 desaturase, and GLELO elongase isolated
from M. alpina 1S-4 were amplified by PCR using KOD+
polymerase (Toyobo). The PCR products were ligated to
subcloning vectors, which have suitable restriction sites. The
cDNA of the D15 desaturase gene (accession no. P48625) was
synthesized by RT-PCR with the total RNA extracted from
immature soybean seed as a template and then subcloned to a
pCR II vector using the TOPO Cloning Kit (Invitrogen). The
5+591 bp (filled-in BamHI and XhoI sites) and
5+791 bp (filled-in SacI and XhoI sites) fragments
spanning from the start codon of the D15 desaturase gene
were subjected to PCR and ligated at the XhoI site, generating
an inverted repeat structure of the dsRNA transcript [RNA
interference (RNAi) structure]. This structure was then cloned
into the BamHI and SacI sites under the control of the b-
conglycinin promoter and nos terminator.
The pUCAP [21], which has two 8-base pair recognition sites
of AscIandPac I, was modified by inserting another two 8-base
pair recognition sites of SgfIandFseI as pUCSAPF. The
expression cassettes of D5 desaturase, D6 desaturase, GLELO
elongase, dsD15 desaturase, and HPT were subcloned to the
corresponding sites of pUCSAPF, generating pSPB1877 (Fig. 2).
2.2. Tissue culture, transformation, and regeneration
The somatic embryos of Jack, a soybean cultivar with high
embryogenic capacity, were induced as described by Bailey
et al. [22] and proliferated in an FN Lite liquid medium [23].
The proliferated embryos were transformed with pSPB1877
using the Biolistic PDS-1000/He Particle Delivery System (Bio-
Rad) in accordance with the method of El-Shemy et al. [24].
R. Chen et al. / Plant Science 170 (2006) 399–406400
Fig. 1. Simplified scheme of the plant fatty acid biosynthesis pathway and
modification by M. alpina fatty acid desaturases and elongase. PA, palmitic
acid; SA, stearic acid; OA, oleic acid; OTA, 6,9-octaecadienoic acid; EDA,
eicosa-8, 11-dienoic acid; MA, mead acid; LA, linoleic acid; GLA, g-linolenic
acid; DGLA, dihomo-g-linolenic acid; ARA, arachidonic acid; ALA, a-lino-
lenic acid; SDA, stearidonic acid; ESA, 8,11,14,17-eicosatetraenoic acid; EPA,
eicosapentaenoic acid; D9, D12, D15, D5, and D6, D9, D12, D15, D5, and D6
desaturases; GLELO, GLELO elongase.

Following bombardment, the embryos were selected in an FN
Lite medium supplemented with 15, 30, and 45 mg/mL
hygromycin B (Nacalai Tesque) for 1 month, 1 month, and 2
weeks, respectively. Surviving green embryos (hygromycin-
resistant somatic embryos) were proliferated and maintained in
an FN Lite medium supplemented with 15 mg/mL hygromycin
B; some of them were transferred to an FNL0S3S3GM medium
[25] for histodifferentiation for 1 month. The histodifferentiated
somatic embryos (T0 embryos) were desiccated and germinated
following the method of Bailey et al. [22]. The transgenic plants
(T0 primary transgenic plants) and their first progenies (T1
progenies) were cultivated in a greenhouse until they flowered
and produced seeds (T1 and T2 seeds).
2.3. DNA and RNA analysis
Total DNA was isolated from the T0 somatic embryos using
the DNeasy Kit (Qiagen) according to the instructions of the
manufacturer. For genomic PCR, three pairs of oligonucleo-
tides were synthesized as forward and reverse primers
according to their sequences to amplify the D6, D5 desaturase,
and GLELO elongase genes by PCR with the isolated 200 ng of
the total DNA as a template.
Total RNA was isolated from the PCR-positive transgenic T0
embryo lines using the RNeasy Kit (Qiagen) and was treated with
the RNase Free DNase (Qiagen) to digest the contaminated
DNA. RT-PCR was conducted using the SuperScript First-Strand
Synthesis System to synthesize the first-strand cDNA libraries.
These templates were used for the amplification of the D6, D5
desaturase, and GLELO elongase gene transcripts using the same
primer sets as those used in genomic PCR.
Southern blot hybridization was conducted to confirm the
stable integration transgenes; 50 mg of total DNA from the
leaves of T0 transgenic plantlet lines or their T1 progenies was
completely digested with EcoRV (Toyobo), separated by
electrophoresis in 0.8% agarose gel, and then transferred onto a
Hybond
+
membrane (Amersham). The filter was hybridized
with a full-length cDNA fragment probe of D6, D5 desaturase,
or GLELO elongase gene labeled using the Gene Images
Detection Module CDP-STAR labeling system (Amersham)
under the conditions specified by the manufacturer.
2.4. Fatty acid extraction and analysis
The fatty acid compositions of whole lipids from the T0
embryos and mature seeds of the T1 and T2 were extracted
according to the method described [26]. The lipids were
derivatized to fatty acid methyl esters (FAMEs) for gas
chromatography (GC, HP-6800, Hewlett Packard) analysis.
The fatty acid compositions were identified by comparison with
the retention times of the FAME standards and GC–mass-
spectrometry (GC–MS, HP-5973, Hewlett Packard). The
relative percentages of the fatty acids were estimated from
the peak areas.
3. Results
3.1. Regeneration of transgenic soybean plants and
molecular analysis
In order to produce ARA by the stepwise modification of LA
in soybean, it is necessary to express three genes, D6, D5
desaturase, and GLELO elongase genes (Fig. 1). LA is also a
substrate for an endogenous D15 desaturase gene in soybean to
produce ALA. Therefore, to facilitate ARA production, a
reduction of ALA content by the down-regulation of the
endogenous D15 desaturase gene is also required. The pSPB1877
expression vector, which harbors the three structural genes for
ARA (D5, D6 desaturase, and GLELO elongase derived from M.
alpina 1S-4) under the control of a seed-specific soybean b-
conglycinin promoter, the soybean endogenous D15 desaturase
gene with an RNAi structure, and a selective HPT marker gene,
was introduced into soybean somaticembryos via bombardment-
mediated transformation. Nine hygromycin-resistant somatic
embryo lines were obtained after selection in a medium
supplemented with increasing concentrations of hygromycin
B from 15–45 mg/mL. Genomic PCR analysis revealed that
Lines 1, 2, 4, 8, and 9 contained the genes of D6, D5 desaturase,
and GLELO elongase, while the others contained only one or two
of them (Table 1). RT-PCR analysis showed that Lines 1, 4, 8, and
9 contained the transcripts of the three genes (Table 1 ). Lines 4
and 8 were deleted from further analyses and regeneration
because they did not have altered fatty acid compositions.
RT-PCR analysis of Lines 1 and 9 also revealed that the
transcript level of the D15 desaturase gene was dramatically
decreased (Fig. 3), indicating that RNAi suppression of the
gene had been successful.
To analyze the integration patterns of transgenes, leaves of
T0 primary transgenic plant Line 1 and its T1 progenies were
assessed by Southern analysis using the DNA probes of D6, D5
desaturase, and GLELO elongase. The result showed that the
copy numbers varied depending on the genes (Fig. 4); each
probe hybridized more than three DNA fragments in the blot,
while only two fragments (indicated with two arrows in Fig. 4)
R. Chen et al. / Plant Science 170 (2006) 399–406 401
Fig. 2. Diagrammatic representation of pSPB1877. 35S, CaMV 35S promoter; nos, terminator of the nopaline synthase; Con, a’ subunit of the b-conglycinin
promoter; mas, terminator of the manopine synthase gene; ds D15, inverted repeat structure of the dsRNA transcript of the D15 desaturase gene; GLELO, GLELO
elongase gene; D6, D6 desaturase gene; D5, D5 desaturase gene; HPT, hygromycin phosphotransferase gene.

were detected in the same size among the three probes. There
were not significantly different bands between the T0 and T1.
The results demonstrate that at least two copies of plasmid
DNA containing the full length of D6, D5 desaturase, and
GLELO elongase genes were integrated into the transgenic
soybean genome and stably inherited to its progenies.
3.2. Fatty acid analysis of transgenic somatic embryos and
mature seeds
Since the fatty acid composition of the soybean somatic
embryo has been shown to predict that of seeds regenerated
from somatic embryos [27], we analyzed the fatty acid
composition of transgenic lines at the somatic embryo stage.
The results (Table 1) showed that Lines 1 and 9 in the T0
embryo stage had altered fatty acid compositions from that of
the host and several new peaks. These peaks were subjected to
GC–MS analysis of the dimethyl disulfide derivatives of their
methyl esters as g-linolenic acid (GLA, C18:3, n-6), eicosa-8,
11-dienoic acid (EDA, C20:2, n-9), dihomo-g-linolenic acid
(DGLA, C20:3, n-6), and ARA and accounted for 2.8, 4.4, 1.7,
and 2.1% of the total fatty acids in Line 1, respectively. Line 9
also had similar but smaller-extent fatty acid changes in fatty
acid composition.
For further study, the T0 embryos of Line 1 were regenerated
to plants and cultivated in a greenhouse until they flowered and
produced mature seeds (T1 seeds); then, T1 seeds were sown to
obtain next-generation seeds (T2 seeds). The four kinds of
altered fatty acids accounted for 2.5, 4.0, 1.1, and 0.8% of the
total fatty acids in T1 seeds as well as 1.6, 2.1, 0.5, and 0.5% on
average in T2 seeds (Table 2). Although the proportions of the
four kinds of fatty acids in the T2 seeds were only half of those
found in the T1 seeds, the phenotype of altered fatty acids was
R. Chen et al. / Plant Science 170 (2006) 399–406402
Table 2
Fatty acid composition of total lipids from the histodifferentiated somatic embryo, mature seed of transgenic soybean Line 1, and its progenies
Fatty acid composition (%) T0 embryo T1 seed T2 seed
Host Line 1 Host Line 1 Line 1–1 Line 1–2 Line 1–3 Line 1–4
PA 16:0 10.7 13.3 11.4 10.2 12.6 13.1 13.8 12.5
SA 18:0 3.8 6.4 4.3 4.8 5.8 6.3 6.1 7.6
OA 18:1 (n-9) 20.3 14.0 15.2 19.2 17.3 14.7 15. 8 20.1
LA 18:2 (n-6) 56.3 44.0 57.9 51.2 55.2 55.2 53.3 50.1
ALA 18:3a(n-3) 7.6 6.5 9.3 1.9 2.5 3.3 3.1 2.8
GLA 18:3g(n-6) 0.0 2.8 0.0 2.5 1.2 1.6 1.7 1.7
EDA 20:2 (n-9) 0.0 4.4 0.0 4.0 2.1 2.2 2.3 1.9
DGLA 20:3 (n-6) 0.0 1.7 0.0 1.1 0.4 0.5 0.6 0.4
ARA 20:4 (n-6) 0.0 2.1 0.0 0.8 0.4 0.5 0.5 0.5
Others 1.3 4.8 1.9 4.3 2.5 2.6 2.8 2.4
Total 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0
Table 1
PCR, RT-PCR, and fatty acid analysis of transgenic soybean lines at the
histodifferentiated somatic embryo stage
Transgenic line
1 23456789
PCR
D6 Desaturase + + + + + + + + +
D5 Desaturase + + ++++
GLELO elongase + + + ++
RT-PCR
D6 Desaturase + ++++++
D5 Desaturase + + ++++
GLELO elongase + + + +++
D15 Desaturase 
Fatty acid (%)
GLA 2.8 0.8
EDA 4.4 0.0
DGLA 1.7 0.7
ARA 2.1 0.7
+, positive; , negative; blank space, not performed yet.
Fig. 4. Southern blot hybridization of DNA from the leaves of transgenic
soybean Line 1 and its progeny. M, molecular standards of the DNA marker;
Lanes 1, 2, and 3, DNA from the leaves of Line 1 hybridized with D6 desaturase,
GLELO elongase, and D5 desaturase gene as a probe; Lanes 4, 5, and 6, DNA
from the leaves of Line 1 progeny hybridized with D6 desaturase, GLELO
elongase, and D5 desaturase gene as a probe.
Fig. 3. RT-PCR analysis of the D15 desaturase gene mRNA transcript level in
the transgenic histodifferentiated somatic embryos of soybean. M, molecular
standards of the DNA marker; Lanes 1 and 2, Lines 1 and 9; Lanes 3 and 4,
untransformed lines as negative controls.

evidently inherited to the next generation. There were also
some trace peaks in the transgenic seeds that the host did not
present, but they were too small in amount to be identified
properly and are shown together under the category ‘‘others’’ in
Table 2.
A decrease in the ALA content was observed, especially in
T1 seeds, from 9.3 to 1.9% (Table 2). It is consistent with the
decrease in the transcript level of the D15 desaturase gene found
in RT-PCR (Fig. 3). These results illustrate that D15 desaturase
inhibition may be achieved through an RNAi-mediated
approach.
4. Discussion
In the present work, we sought to modify plant fatty acids by
the expression of these VLC-PUFA-associated genes in legume
crop plants for actual industrial production and to optimize the
conditions on gene expression promoter and gene competition
for increasing the yields. We successfully produced VLC-
PUFAs such as GLA, EDA, DGLA, and ARA in soybean by
expressing D6, D5 desaturase, and GLELO elongase genes
from M. alpina 1S-4, which is used industrially to produce
ARA. Our result indicates that soybean can functionally
express foreign genes and that its fatty acid synthesis pathway
can be successfully modified. The GLA is suggested to be
produced from the desaturation of LA, which is most abundant
in host cells, by the introduction of D6 desaturase. GLA was
elongated by GLELO elongase and sequentially desaturated by
D5 desaturase, resulting in the production of the corresponding
DGLA and ARA. In fact, D6 desaturase also introduced a
double bond at position 9 of endogenous substrate OA,
resulting in the production of the corresponding EDA in the
transgenic soybean histodifferentiated embryos and seeds.
However, the activity of the n-9 biosynthetic pathway was
interrupted after GLELO elongation (Table 2). This may be due
to substrate preference, in other words, to the fact that D5
desaturase of M. alpina was specifically active on both the n-6
and n-3 fatty acid substrates [28].
There are two ways to synthesize ARA and EPA. We
adopted the conventional pathway to synthesize ARA, which
starts with the D6 desaturation of LA or ALA (Fig. 1). Abbadi
et al. [16] also adopted this pathway to obtain transgenic linseed
producing ARA or EPA by expressing the D5 and D6
desaturases from Phaeodactylum tricornutum together with
the D6 elongase from Physcomitrella patens. On the other hand,
ARA and EPA can also be synthesized via an alternative D8
pathway, through which LA and ALA are first elongated by a
D9-specific elongase and then desaturated by D8 desaturase. Qi
et al. [15] achieved up to 22.5% C20 PUFAs and 6.1% ARA
accumulation in Arabidopsis leaves by expressing the genes of
D9 elongase from Isochrysis galbana,D8 desaturase from
Euglena gracilis, and D5 desaturase from M. alpina with
repeated transformation. The former pathway is the major and
most active aerobic route, which operates in most eukaryotic
organisms, including M. alpina [29],P. tricornutum, and P.
patens [30]. The favorite substrates of D6 desaturase are LA
and ALA [31], while those of GLELO are GLA and stearidonic
acid (SDA, C18:4, n-3) [28] and that of D5 desaturase is DGLA
[31]. The latter pathway is the intermediate of the conventional
route in rat or human testis as well as glioma and breast cancer
cell lines and appears to lack D6 desaturase activity [12]. It has
been found that the D8 and D5 desaturases probably require
lipid-linked substrates. The elongated products had to be
incorporated into phospholipids before the subsequent desa-
turation steps. Detailed analyses of leaf lipids of transgenic
Arabidopsis have confirmed that LA and ALA were efficiently
elongated, followed by incorporation into all major extra-
plastidial phospholipids and subsequent desaturation resulting
C20-PUFA accumulated in high level, but the seed oil of
transgenic Arabidopsis constitutively expressing the D9
elongase alone had only 5% C20-LCPUFA, highlighting that
there are additional hurdles still to overcome in seeds [32].
Which pathway is the most appropriate for reconstitution in
legume crop remains to be determined.
LA can be sequentially desaturated to ALA by D15
desaturase in plants (Fig. 1). Since D6 desaturase shows no
major preference for n-6 or n-3 substrates, it acts on both LA
and ALA as a substrate with very similar levels of conversion
rates for both fatty acids [12]. Low amount of LA will rate-
limited n-6 fatty acid synthesis. In our previous study,
transgenic tobacco plants, which constitutively expressed the
same three genes, accumulated only small amounts of GLA and
DGLA but not any ARA in the leaves (data not shown).
However, the leaf of transgenic tobacco was able to synthesize
ARA when excessive amounts of GLA were exogenously
supplied (data not shown). The results indicate that D6
desaturation may not be efficient enough (for discussion see
below) or that LA was mostly desaturated to ALA and too low
to support high levels of ARA biosynthesis (Fig. 1). In the latter
case, endogenous D15 desaturase gene expression should be
suppressed. In our present study, RNAi inhibition of D15
desaturation resulted in lower D15 desaturase transcripts and
thus sharply decreased the ALA content in transgenic lines
(from 9.3 to 1.9% in T1 seeds, Table 2), illustrating that D15
desaturase suppression was effectively achieved by transcribing
an inverted repeat RNAi structure of the D15 desaturase gene.
In this way, LA could be maintained in a constant amount as the
substrate for the synthesis of ARA.
Although the CaMV 35S promoter is used most frequently
to regulate heterologous genes in transgenic plants, as in the
studies by Qi et al. [15], Sayanova et al. [13] and Zou et al. [33],
it is an unspecific strong and constitutive promoter [20].
Expression of a borage D6 desaturase under the control of the
35S promoter in tobacco and flax resulted in a high-level
accumulation of GLA in vegetative tissue (more than 20% of
the total fatty acids), but the level in seeds was low (less than
2% of the total fatty acids) [34,35]. Replacement of the 35S
with a strong Brassica napus seed-specific napin promoter
increased the accumulation of GLA (3–9%) in mature seeds of
the transgenic B. juncea [35], and the expression of D6
desaturase (along with D12 desaturase from M. alpina or from
Pythium irregulare) resulted in the production of up to 40% of
GLA in seeds of B. napus [36,37] or B. juncea [38]. Moreover,
high levels of no-native unusual fatty acids (VLC-FAs or VLC-
R. Chen et al. / Plant Science 170 (2006) 399–406 403

PUFAs) expression in vegetative parts of the plant may have
some detrimental effects on the growth of the transgenic plant,
dramatic morphological abnormalities, and even death [39].It
is possible that higher unusual fatty acids accumulation in seeds
could not be achieved using the 35S promoter [40]. To avoid
such site-specific effect of gene function, we placed the fatty
acid genes under the control of a soybean seed-specific
promoter, the a0subunit of the b-conglycinin promoter. b-
Conglycinin, one of the major seed-storage proteins in soybean
seed (embryonic axis, cotyledons, and endosperm), is only
expressed during the mid-late stages of embryogeny [18]. The
expression of genes under the b-conglycinin promoter has
shown that the transcript level in immature seeds is much higher
(20–100 times) than that in leaves and roots in petunia [18],
soybean [41], and tobacco plants [42]. The specific soybean
seed promoter reduces the potential risks of the accumulated
VLC-PUFAs, causing some disadvantageous effects on the
transgenic plant during the growth stage. Our results showed
that transgenic soybean and their progenies grew and produced
seeds normally.
ARA has recently been shown to play pivotal roles in infant
brain development, inflammatory responses, blood pressure
regulation, blood clotting, and cell signalling [43].Since
soybean is more important in the agricultural and food
industrial foreground than model plants such as tobacco or
Arabidopsis, the results presented here are useful for the
production of ARA in crop plant seeds economically and in
large-scale. Legume plants include many economically
important crop plants that are utilized in human foodstuffs,
herbal medicines, oil materials and as animal forages, our
resultsinsoybeancanalsobeusedtoobtaininsightsforthe
metabolic engineering of fatty acids and the inheritability of
the engineered fatty acids in legumes. We previously expressed
the same D6 fatty acid desaturase gene in a model legume,
Lotus japonicus,andinVigna angularis,whichisan
economically important and widely grown legume crop in
Asia. As a result, GLAwas synthesized and accumulated more
than 13.75 and 2.65% of the total fatty acids in the leaves and
seeds of transgenic plants, respectively. The ability to
synthesize GLA was found to be inherited to their progenies
[44].
The content of ARA in transgenic soybean seed is still low in
this study. This is most likely due to the unsatisfactory D6
desaturase and D6 elongase activities derived from M. alpina in
soybean. Although the soybean host cell contained more than
50% LA, less than 3% GLA (2.8, 2.5, 1.6% in T0 embryos, T1
and T2 seeds, respectively. Table 2) were desadurated, which
were considerably lower than those of previous studies
expressing D6 desaturase genes in yeast from borage and P.
irrgulare [35,38],inB. juncea from borage [35,38],inA. oryzae
from M. alpina [45], in tobacco, linseed from borage and P.
tricornutum [16,34,35] or in the same soybean plant from S.
diclina and M. alpina [17]. Furthermore, it was recently found
in reconstituted yeast that D6 desaturase mainly used the LA in
the sn-2 position of phosphatidylcholine (PC), while D6
elongase required GLA in the acyl-CoA pool. Therefore, an
inefficient transfer of the D6 desaturated products from PC into
the acyl-CoA pool most probably restricted the yield of ARA
[11,15]. This finding can explain the reason why the transgenic
linseed plants contained high proportions of D6-C18-PUFAs
(30–40% GLA + SDA), but only 1.6% EPA and 2.7% ARA
[16].
On the other hand, expression of a D6 desaturase (either from
the oomycete fungus Saprolegnia diclina or M. alpina), a D6
elongase and a D5 desaturase from M. alpina in soybean somatic
embryos resulted in the production of 19.6% EPA [17]. Although
the reasons for the higher yields obtained in soybean than in
linseed are not clear, it is thought that the most significant
difference between the results from linseed and soybean is the
extent of D6 elongation: the moss elongase was very active that
could be the ancillary by acyl-CoA:lysophosphatidylcholine
acyltransferase activity [11]. Disappointingly, also in soybean,
the totals of EDA + DGLA + ARA in the present study were only
8.2, 5.9 and 3.1% in T0 embryos, T1 and T2 seeds (Table 2),
respectively. Such difference could be interpreted as follows: we
used the same promoter (a0subunit of the b-conglycinin
promoter) for four expression cassettes (D6 desaturase, GLELO,
D5 desaturase and dsD15 desaturase genes) in pSPB1877 while
Kinney et al. [17] used different seed-specific promoters in each
cassette. Existence of repetitive sequences, such as multi-copy
interactions, frequently causes gene silencing in transgenic
plants. Such gene silencing is often promoted by DNA
methylation of transgene sequence especially in its promoter
region [46]. It is likely that silencing by genomic methylation
might occur in our transgenic soybean to some extent. As
mentioned above, the contents of EDA + DGLA + ARA
decreased gradually by generation (8.2, 5.9, and 3.1% in T0
embryos, T1 and T2 seeds, respectively). The decrease may
suggest that the DNA methylation in the promoter region
accumulated through the generations.
We expect that more sophisticated gene expression systems,
gene sources other than M. alpina, and/or the achievement of
more transgenic soybean lines may overcome this obstacle. We
have also obtained a transgenic soybean embryo line
transformed by another vector containing the same three
genes, which accumulated more than 18.8% C20 PUFAs and
10.7% ARA, but the line was confirmed to be sterile
(unpublished data). Similar results were also found in A.
thaliana [47] and B. napus [33], namely, that high proportions
of seed VLC-FAs or VLC-PUFAs had significantly decreased
seed size and showed poor germination. This indicates that
exorbitant high level of novel fatty acids may negatively affect
the agronomic characters and that accumulation of a moderate
content of ARA in transgenic soybean seed is crucial. Although
high homogeneity of the fatty acids to be used for chemical
purposes is required, the proportions of VLC-PUFAs in
transgenic oils can be rather low (in the order of a few
percent) for direct consumption as food or for medical and
pharmaceutical usage [16].
Acknowledgement
This work was supported by the New Energy and Industrial
Technology Development Organization (NEDO), Japan.
R. Chen et al. / Plant Science 170 (2006) 399–406404

References
[1] D.B. Jump, The biochemistry of n-3 polyunsaturated fatty acids, J. Biol.
Chem. 277 (2002) 8755–8758.
[2] A.A. Spector, Essentiality of fatty acids, Lipids 34 (1999) S1–S3.
[3] D.F. Horrobin, Nutritional and medical importance of gamma-linolenic
acid, Prog. Lipid Res. 31 (1992) 163–194.
[4] P. Benatti, G. Peluso, R. Nicolai, M. Calvani, Polyunsaturated fatty acids:
biochemical, nutritional and epigenetic properties, J. Am. Coll. Nutr. 23
(2004) 281–302.
[5] H. Okuyama, T. Kobayashi, S. Watanabe, Dietary fatty acids – the N-6/N-
3 balance and chronic elderly diseases. Excess linoleic acid and relative
N-3 deficiency syndrome seen in Japan, Prog. Lipid Res. 35 (1996) 409–
457.
[6] P. Willatts, J.S. Forsyth, M.K. DiModugno, S. Vama, M. Colvin, Effect of
long-chain polyunsaturated fatty acids in infant formula on problem
solving at 10 months of age, Lancet 352 (1998) 688–691.
[7] R. Perichon, J.M. Bourre, Effects of ageing on peroxysomatic breakdown
of polyunsaturated fatty acids—what are the health implications, Ocl-
Oleagineux Corps Gras Lipides 3 (1996) 85–89.
[8] A.P. Simopoulos, The importance of the ratio of omega-6/omega-3
essential fatty acids, Biomed. Phar. 56 (2002) 365–379.
[9] H. Drexler, P. Spiekermann, A. Meyer, F. Domergue, T. Zank, P. Sperling,
A. Abbadi, E. Heinz, Matabolic engineering of fatty acid for breeding of
new oilseed crops: strategies, problems, and first results, J. Plant Physiol.
160 (2003) 779–802.
[10] S.P. Singh, X. Zhou, Q. Liu, S. Stymne, A.G. Green, Metabolic engi-
neering of new fatty acids in plants, Curr. Opin. Plant Biol. 8 (2005) 197–
203.
[11] F. Domergue, A. Abbadi, E. Heinz, Relief for fish stocks: oceanic fatty
acids in transgenic oilseeds, Trends Plant Sci. 10 (3) (2005) 112–116.
[12] O.V. Sayanova, J.A. Napier, Eicosapentaenoic acid: biosynthetic routes
and the potential for synthesis in transgenic plants, Phytochemistry 65
(2004) 147–158.
[13] O.V. Sayanova, M.A. Smith, P. Lapinskas, A.K. Stobart, G. Dobson, W.W.
Christie, P.R. Shewry, J.A. Napier, Expression of a borage desaturase
cDNA containing an N-terminal cytochrome b5 domain results in the
accumulation of high levels of D6-desaturated fatty acids in transgenic
tobacco, Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 4211–4216.
[14] P. Broun, C. Somerville, Accumulation of ricinoleic, lesquerolic, and
densipolic acids in seeds of transgenic Arabidopsis plants that express a
fatty acyl hydroxylase cDNA from castor bean, Plant Physiol. 113 (1997)
933–942.
[15] B. Qi, T. Fraser, S. Mugford, G. Dobson, O. Sayanova, J. Butler, J.A.
Napier, A.K. Stobart, M. Lazarus, Production of very long-chain poly-
unsaturated omega-3 and omega-6 fatty acids in plants, Nat. Biotechnol.
22 (2004) 739–745.
[16] A. Abbadi, F. Domergue, J. Bauer, J.A. Napier, R. Welti, U. Za
¨hringer, P.
Cirpus, E. Heinz, Biosynthesis of very-long-chain polyunsaturated fatty
acids in transgenic oilseeds: constraints on their accumulation, Plant Cell
16 (2004) 2734–2748.
[17] A.J. Kinney, E.B. Cahoon, H.G. Damude, W.D. Hitz, Z. Liu, C.W. Kolar.
Production of very long chain polyunsaturated fatty acids in oilseed plants,
WO 2004/071467 A2, 2004.
[18] Z. Chen, M.A. Schuler, R.N. Beachy, Functional analysis of regulatory
elements in a plant embryo-specific gene, Proc. Natl. Acad. Sci. U.S.A. 83
(1986) 8560–8564.
[19] V. Sundaresan, P. Springer, T. Volpe, S. Haward, J.D. Jones, C. Dean, H.
Ma, R. Martienssen, Patterns of gene action in plant development revealed
by enhancer trap and gene trap transposable elements, Gene Dev. 9 (1995)
1797–1810.
[20] P.N. Benfey, N.H. Chua, The cauliflower mosaic virus 35S promoter:
combinational regulation of transcription in plants, Science 250 (1990)
959–966.
[21] F.A. van Engelen, J.W. Molthoff, A.J. Conner, J.P. Nap, A. Pereira, W.J.
Stiekema, pBINPLUS: an improved plant transformation vector based on
pBIN19, Transgenic Res. 4 (1995) 288–290.
[22] M.A. Bailey, H.R. Boerma, W.A. Parrott, Genotype effects on proliferative
embryogenesis and plant regeneration of soybean, In Vitro Cell Dev. Biol.
29 (1993) 102–108.
[23] J.J. Finer, A. Nagasawa, Development of an embryogenic suspension
culture of soybean (Glycine max Merrill), Plant Cell Tissue Organ Cul. 15
(1988) 125–136.
[24] H.A. El-Shemy, M. Khalafalla, K. Wakasa, M. Ishimoto, Reproducible
transformation in two grain legumes—soybean and azuki bean—using
different systems, Cell Mol. Biol. Lett. 7 (2002) 709–719.
[25] V.M. Samoylov, D.M. Tucker, W.A. Parrott, A liquid medium-based
protocol for rapid regeneration from embryogenic soybean cultures, Plant
Cell Rep. 18 (1998) 49–54.
[26] M. Yamada, Experimentation of Plant Lipid Metabolism, Japan Scientific
Societies Press, Tokyo, 1989, pp. 3–70.
[27] A.J. Kinney, Development of genetically engineered soybean oils for food
applications, J. Food Lipids 3 (1996) 273–292.
[28] J.M. Parker-Barnes, T. Das, E. Bobik, A.E. Leonard, J.M. Thurmond, L.
Chaung, Y. Huang, P. Mukerji, Identification and characterization of an
enzyme involved in the elongation of n-6 and n-3 polyunsaturated fatty
acids, Proc. Natl. Acad. Sci. U.S.A. 97 (2000) 8284–8289.
[29] Y. Huang, S. Chaudhary, J.M. Thurmond, E.G. Bobik Jr., L. Yuan, G.M.
Chan, S.J. Kirchner, P. Mukerji, D.S. Knutzon, Cloning of delta 12- and
delta 6-desaturases from Mortierella alpina and recombinant production
of gamma-linolenic acid in Saccharomyces cerevisiae, Lipids 34 (1999)
649–659.
[30] T. Girke, H. Schmidt, U. Zahringer, R. Reski, E. Heinz, Identification of a
novel delta 6-acyl-group desaturase by targeted gene disruption in Phys-
comitrella patens, Plant J. 15 (1998) 39–48.
[31] F. Domergue, J. Lerchl, U. Za
¨hringer, E. Heinz, Cloning and functional
characterization of Phaeodactylum tricornutum front-end desaturases
involved in eicosapentaenoic acid biosynthesis, Eur. J. Biochem. 269
(2002) 4105–4113.
[32] T.C. Fraser, B. Qi, S. Elhussein, S. Chatrattanakunchai, A.K. Stobart, C.M.
Lazarus, Expression of the Isochrysis C18-D9 polyunsaturated fatty acid
specific elongase component alters Arabidopsis glycerolipid profiles,
Plant Physiol. 135 (2004) 859–866.
[33] J. Zou, V. Katavic, E.M. Giblin, D.L. Barton, S.L. MacKenzie, W.A.
Keller, X. Hu, D.C. Taylora, Modification of seed oil content and acyl
composition in the Brassicaceae by expression of a yeast sn-2 acyltrans-
ferase gene, Plant Cell 9 (1997) 909–923.
[34] O.V. Sayanova, G.M. Davies, M.A. Smith, G. Griffiths, A.K. Stobart, P.R.
Shewry, J.A. Napier, Accumulation of D6-unsaturated fatty acids in
transgenic tobacco plants expressing a D6-desaturase from Borago offi-
cinalis, J. Exp. Bot. 50 (1999) 1647–1652.
[35] X. Qiu, H.P. Hong, N. Datla, S.L. MacKenzie, D.C. Tayler, T.L. Thomas,
Expression of borage D6-desaturase in Saccharomyces cerevisiae and
oilseed crops, Can. J. Bot. 80 (2002) 42–49.
[36] D.S. Knutzon, J.M. Thurmond, Y. Huang, S. Chaudhary, E.G. Bobik Jr.,
G.M. Chan, S.J. Kirchner, P. Mukerji, Identification of D5-desaturase from
Mortierella alpina by heterologous expression in Baker’s yeast and
canola, J. Biol. Chem. 273 (1998) 29360–29366.
[37] J. Liu, S. DeMichele, M. Bergana, E. Bobik, C. Hastilow, L. Chuang, P.
Mukerji, Y. Huang, Characterization of oil exhibiting high gamma-lino-
lenic acid from a genetically transformed canola strain, J. Am. Oil Chem.
Soc. 78 (2001) 489–493.
[38] H. Hong, N. Datla, D.W. Reed, P.S. Covello, S.L. MacKenzie, X. Qiu,
High-level production of g-linolenic acid in Brassica juncea using a D6
desaturase from Pythium irregulare, Plant Physiol. 129 (2002) 354–362.
[39] A.A. Millar, M. Wrischer, L. Kunst, Accumulation of very long-chain
fatty acids in membrane glycerolipids is associated with dramatic altera-
tions in plant morphology, Plant Cell 10 (1998) 1889–1902.
[40] A.A. Millar, L. Kunst, Very long-chain fatty acid biosynthesis is controlled
through the expression and specificity of the condensing enzyme, Plant J.
12 (1997) 121–131.
[41] A.J. Kinney, R. Jung, E.M. Herman, Cosuppression of the asubunits of b-
conglycinin in transgenic soybean seeds induces the formation of endo-
plasmic reticulum-derived protein bodies, Plant Cell 13 (2001) 1165–
1178.
R. Chen et al. / Plant Science 170 (2006) 399–406 405

[42] S. Chamberland, N. Daigle,F. Bernier, The legumin boxes andthe 30part of a
soybean beta-conglycinin promoter are involved in seed gene expression in
transgenic tobacco plants, Plant Mol. Biol. 19 (1992) 937–949.
[43] J.E. Kinsella, B. Lokesh, S. Broughton, J. Whelan, Dietary polyunsatu-
rated fatty acids and eicosanoids: potential effects on the modulaton of
inflammatory and immune cells: an overview, Nutrition 6 (1990) 24–44.
[44] R. Chen, S. Tsuda, K. Matsui, M. Fukuchi-Mizutani, M. Ochiai, S.
Shimizu, E. Sakuradani, T. Aoki, R. Imaizumi, S. Ayabe, Y. Tanaka,
Production of g-linolenic acid in Lotus japonicus and Vigna angularis by
expression of the D6-fatty-acid desaturase gene isolated from Mortierella
alpine, Plant Sci. 169 (2005) 599–605.
[45] E. Sakuradani, M. Kobayashi, S. Shimizu, D6-Fatty acid desaturase from
an arachidonic acid-producing Mortierella fungus: gene cloning and its
heterologous expression in a fungus, Aspergillus, Gene 238 (1999) 445–
453.
[46] M. Fagard, H. Vaucheret, (Trans)gene silencing in plants: how many
mechanisms? Annu. Rev. Plant Physiol. Plant Mol. Biol. 51 (2000) 167–
194.
[47] S. Singh, S. Thomaeus, M. Lee, S. Stymne, A. Green, Transgenic
expression of a D12-epoxygenase gene in Arabidopsis seeds inhibits
accumulation of linoleic acid, Planta 212 (2001) 872–879.
R. Chen et al. / Plant Science 170 (2006) 399–406406