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Molecular cloning and characterisation of an acyl carrier
protein thioesterase gene (CocoFatB1) expressed in the
endosperm of coconut (Cocos nucifera) and its heterologous
expression in Nicotiana tabacum to engineer the accumulation
of different fatty acids
Yijun Yuan
A,B
, Yinhua Chen
B
, Shan Yan
A
, Yuanxue Liang
A
, Yusheng Zheng
A,C
and Li Dongdong
A,C
A
Department of Biotechnology, Hainan University, Haikou, Hainan 570228, China.
B
Hainan Key Laboratory for Sustainable Utilisation of Tropical Bioresource, Hainan University, Haikou,
Hainan 570228, China.
C
Corresponding authors. Emails: liddfym@hotmail.com; hainanzyh@yahoo.com.cn
Abstract. Coconut (Cocos nucifera L.) contains large amounts of medium chain fatty acids, which mostly recognise acyl-
acyl carrier protein (ACP) thioesterases that hydrolyse acyl-ACP into free fatty acids to terminate acyl chain elongation
during fatty acid biosynthesis. A full-length cDNA of an acyl-ACP thioesterase, designated CocoFatB1, was isolated from
cDNA libraries prepared from coconut endosperm during fruit development. The gene contained an open reading frame of
1254 bp, encoding a 417-amino acid protein. The amino acid sequence of the CocoFatB1 protein showed 100% and 95%
sequence similarity to CnFatB1 and oil palm (Elaeis guineensis Jacq.) acyl-ACP thioesterases, respectively. Real-time
fluorescent quantitative PCR analysis indicated that the CocoFatB1 transcript was most abundant in the endosperm from 8-
month-old coconuts; the leaves and endosperm from 15-month-old coconuts had ~80% and ~10% of this level. The
CocoFatB1 coding region was overexpressed in tobacco (Nicotiana tabacum L.) under the control of the seed-specific napin
promoter following Agrobacterium tumefaciens-mediated transformation. CocoFatB1 transcript expression varied 20-fold
between different transgenic plants, with 21 plants exhibiting detectable levels of CocoFatB1 expression. Analysis of the
fatty acid composition of transgenic tobacco seeds showed that the levels of myristic acid (14 : 0), palmitic acid (16 : 0) and
stearic acid (18 : 0) were increased by 25%, 34% and 17%, respectively, compared with untransformed plants. These results
indicated that CocoFatB1 acts specifically on 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP, and can increase medium chain
saturated fatty acids. The gene may valuable for engineering fatty acid metabolism in crop improvement programmes.
Additional keywords: medium chain fatty acids, myristic acid, palmitic acid, stearic acid, transgenic tobacco.
Received 6 March 2013, accepted 31 May 2013, published online 8 July 2013
Introduction
Coconut (Cocos nucifera L.), a member of the monocotyledonous
family Aracaceae (Palmaceace), is an important oil-yielding
plantation crop that is of considerable economic and social
importance in the tropics (Rivera et al.1999; Samsudeen et al.
2006). The endosperm tissue of coconut stores a substantial
amount of oil, which has been used extensively for human
consumption and other purposes all over the world. In
physicochemical terms, coconut oil differs from other vegetable
oils in that itis rich in saturated oil (93%), with a high percentage
of medium chain fatty acids (MCFA) (60%), especially lauric
acid (12 : 0) (50%) (Ceniza et al.1991; Bhatnagar et al.
2009). Compared with long-chain triacylglycerols, medium-
chain triacylglycerols that contain caprylic acid (8 : 0), capric
acid (10 : 0), lauric acid (12 : 0) and myristic acid (14 : 0) are
more soluble in water and have a lower melting point, enabling
them to be absorbed and metabolised faster (Jeukendrup and Sarah
2004; Beermann et al.2007). In addition, the unique antiviral,
antibacterial and antiprotozoal properties of medium-chain
triacylglycerols have found applications in the food industry
(Enig 1998), and MCT oils have been used therapeutically since
the 1950s. The use of MCT oils as part of a ketogenic diet to treat
epilepsy, premature infants and patients infected with human
immunodeficiency virus, as well as to prevent fat malabsorption
in cystic fibrosis patients,is widely accepted (Ramírez et al.2001;
Beermann et al.2007). The potential industrial and medical
applications of uncommon seed oils have resulted in rapid
advances in efforts to bioengineer their accumulation.
CSIRO PUBLISHING
Functional Plant Biology,2014, 41,80–86
http://dx.doi.org/10.1071/FP13050
Journal compilation CSIRO 2014 www.publish.csiro.au/journals/fpb
The mechanisms by which coconut endosperm accumulates
unusual fatty acids are still unknown. Fatty acid biosynthesis in
higher plants occurs predominantly in the plastids by a de novo
iterative ‘polymerisation’process, which is commonly primed
with the acetyl moiety from acetyl-CoA and proceeds via iterative
chain extension through reaction with malonyl- acyl carrier
protein (ACP). The synthesis of 16- and 18-carbon (C16 and
C18) fatty acids is terminated by the acyl-ACP thioesterase,
which catalyses acyl-ACP thioester bond hydrolysis, the
terminal reaction of fatty acid biosynthesis that releases a free
fatty acid and ACP (Voelker 1996; Jing et al.2011). Therefore,
the specificities of thioesterases largely determine the chain
lengths of most plant fatty acids (Stumpf 1987).
Acyl-ACP thioesterases are nuclear-encoded, plastid-targeted
globular proteins (Voelker et al.1992). Based on amino acid
sequence alignments, these enzymes have been functionally
characterised and classified into two general families, termed
FatA and FatB (Jones et al.1995). All FatAs are orthologous in
different species, with the highest activities towards oleoyl–ACP
(18 : 1
~9
-ACP) (Hitr and Yadav 1992; Sánchez-García et al.
2010). In contrast with the high level of conservation in the
specificity of FatAs, FatBs primarily hydrolyse saturated acyl-
ACPs with chain lengths that contain between 8 and 18 carbons
(Voelker and Davies 1994; Jones et al.1995; Jha et al.2010). The
first FatB gene was isolated from the developing seeds of
California Bay Tree (Umbellularia californica), and the strong
preference of the enzyme for 12 : 0-ACP was verified in
Arabidopsis thaliana (L.) Heynh. (Davies et al.1991; Voelker
et al.1992). This work demonstrated, for the first time, the role of
FatB in determing the chain lengths of fatty acids, which spurred
efforts to isolate similar enzymes from other plant species with
unusual fatty acid phenotypes. Such enzymes included the
MCFA-specific thioesterases from Cuphea (Dehesh et al.
1996; Leonard et al.1997), American elm (Ulmus americana
L.; Voelker et al.1997) and coconut (Jing et al.2011). Because of
the potential applications of special seed oils, several studies have
focussed on engineering plant thioesterases with medium-chain-
specificities. Three acyl-ACP thioesterases (CnFatB1
(JF338903), CnFatB2 (JF338904), CnFatB3 (JF338905)) from
coconut have been isolated and characterised (Jing et al.2011).
However, their in vivo activities and substrate specificities were
only shown in Escherichia coli, with no function analyses
performed using plants. In the present work, a full-length
cDNA of an acyl-ACP thioesterase (CocoFatB1: JX275886)
was isolated from cDNA libraries prepared from coconut
endosperm during fruit development (Li and Fan 2009). The
CocoFatB1 gene was heterologously expressed in transgenic
tobacco (Nicotiana tabacum L.) under the control of the seed-
specific napin promoter (Kridl et al.1991; Zheng et al.2007).
Our results provide new insights into the function of CocoFatB1
and how it might be used to impact on the composition of plant
oils.
Materials and methods
Plant materials
Coconut (Cocos nucifera L.) fruits (i.e. coconuts), including
immature coconuts (8 months old) and ripe coconuts
(15 months old), and coconut leaves were obtained from the
Coconut Research Institute, Chinese Agricultural Academy of
Tropical Crops, Hainan, China. The fruits and leaves were
harvested at random, and endosperm tissues were physically
isolated and immediately frozen in liquid nitrogen to form
three sample pools. The samples were then analysed in
duplicate for RNA concentration, cDNA synthesis, gene
amplification and differential expression analysis. Escherichia
coli strain DH5a(Clontech Palo Alto, CA, USA), which was
grown in Lysogeny broth (LB) medium (Sangon, Shanghai,
China) supplemented with 50 mg L
–1
ampicilin or kanamycin
at 37C,was used for bacterial cloning. The pCAMBIA1300S
vector was donated by Yongjun Lin (Professor of Huazhong
Agricultural University). All chemicals, endonucleases and other
required enzymes were obtained from Sigma (St. Louis, MO,
USA) or TaKaRa (Dalian, China), unless otherwise stated.
RNA extraction and cDNA library construction
Total RNA from the pulp of coconuts was extracted using the
cetyltrimethylammonium bromide-based methods described by
Li and Fan (2007). The quantity and quality of isolated total RNA
was examined using spectrophotometry and gel electrophoresis,
respectively. Construction of the cDNA library prepared from
RNA isolated from coconut endosperm have been reported
previously (Li and Fan 2009).
Cloning of the CocoFatB1 gene and bioinformatic analysis
EST sequence information was obtained for 1000 clones that were
randomly selected from the cDNA library. All clones were
sequenced by Oebiotech Co. (Shanghai, China). A homology
search was conducted based on BLAST searches using the
National Center for Biotechnology Information BLAST
server (http://www.ncbi.nlm.nih.gov/BLAST). Among the EST
sequences obtained from the library, a partial clone was
discovered as being 91% identical to the palmitoyl-acyl carrier
protein thioesterase gene from oil palm (GenBank accession:
AF147879.2). Complete sequencing of the clone was performed
using the primers M13+ (50-GTAAAACGACGGCCAGT-30)
and M13–(50-AACAGCTATGACCATGTTCA-30) followed
by primer-walking until complete overlapping sequence data
were obtained from both sides.
Fluorescence quantitative reverse transcription–PCR
analysis
Total RNA from coconut leaves and endosperm tissues were
isolated separately from immature coconuts (8 months old) and
ripe coconuts (15 months old). First-strand cDNA was
synthesised from 2 mg of total RNA using the TIANScript
OneStep RT-PCR Kit (TIANGEN, Beijing, China). Reverse
transcription was performed at 42C for 60 min, with a final
denaturation at 70C for 15 min. The cDNA was then subjected to
real-time fluorescent quantitative reverse transcription–PCR
(RT-PCR) using standard methods (Marone et al.2001). The
RT-PCR primers for CocoFatB1 were designed using the Primer3
program based on the cDNA sequence. The b-actin gene was used
as an internal control for expression. The primers used in this
study were:
RTActin-F:5
0-TTACTCTGAAATACCCCATTGAGC-30,
RTActin-R:5
0-CTCTCTGTTAGCCTTGGGGTTG-30,
Function of the ACP thioesterase gene in coconut Functional Plant Biology 81
RTFatBF:5
0-ACACTTCTTGATTGGAAACCACG-30, and
RTFatBR:5
0-GCGTTTCTATAGAAGCCGTCC-30.
The RT-PCR amplification step was performed using the
SYBR Premix Ex Taq II (TaKaRa) and a RT-PCR detector
(TaKaRa Smart Cycler II system) by using the SYBR Green I
chimeric fluorescence method according to the manufacturer’s
instructions. Expression was quantified in terms of comparative
threshold cycle (Ct) using the 2
–~~Ct
method, and the results
were expressed as the binary logarithm of the relative quantity of
the transcript used to normalise gene expression (Livak and
Schmittgen 2001). Reactions were performed in triplicate,
including the ‘no template’and ‘no reverse transcriptase’
controls, and were monitored using an Applied Biosystems
(Foster City, CA, USA) 7500 RT-PCR instrumentation
outfitted with SDS software ver. 1.3.1 (Applied Biosystems).
Construction of expression vectors
The CocoFatB1 coding sequence was cloned from cDNA
prepared from coconut endosperm, and KpnI and BamHI sites
were added at the 50and 30ends, respectively. The primers used
for the PCR amplification were: FatBF 50-TATGGTACCATGG
TTGCTTCAGTTGCCGCTT-30(forward) and FatBR 50-TAT
GGATCCTCAAGCACTTCCAGCTGAAGTGG-30(reverse).
The conditions for PCR amplification were 94C for 3 min, 30
cycles of 94C for 45 s, 47C for 45 s, 72C for 1 min and
extension at 72C for 8 min. The PCR product was cloned into
trhe pMD18-T vector (TaKaRa) and the recombinant plasmid
was transformed into the Escherichia coli strain DH5a.To
generate the plant overexpression construct, the coding region
of CocoFatB1 was subcloned into the binary pCAMBIA1300S
vector (KpnIorBamHI site) under the control of the seed-specific
napin promoter (Kridl et al.1991; Fig. 1).
Transformation, selection and regeneration
of Nicotiana tabacum
The CocoFatB1 gene in the pCAMBIA1300S expression vector
was transformed into Agrobacterium tumefaciens LBA4404/
EHA105 by electroporation, as described by Hoekema et al.
(1983). Young leaves from wild-type tobacco (Nicotiana
tabacum L.) were cut into small square discs (0.5 0.5 cm
2
)
and immersed in 10diluted cultures of Agrobacterium
tumefaciens for 15 min. The leaf discs were first transferred to
sterile filter paper to remove excess Agrobacterium, and
then transferred onto solidified cocultivation MS medium
(Sigma; Murashige and Skoog 1962). After cocultivation,
the transformants were selected on MS medium containing
500 mg L
–1
carbenicillin (Sigma) and 10 mg L
–1
hygromycin B
(Sigma). Regenerated tobacco plants propagated in vitro
were transferred to soil and grown to maturity in a greenhouse
in a 16-h-light ;8-h-darkness photoperiod with a PPFD of
200–900 mmol m
–2
s
–1
. Positive transformants were identified
by PCR screening of genomic DNA. Primary transformants
were self-fertilised and the seeds were collected.
Analysis of CocoFatB1 expression in transgenic tobacco
using fluorescence quantitative RT-PCR
To detect the introduced transgene with PCR in the putative
regenerated transgenic plants, DNA samples were extracted from
leaf tissue using the DNAeasy Mini-kit (Qiagen, Hilden,
Germany) following the manufacturer’s instructions. The
FatBF–FatBR primer pair was used for PCR. The PCR
conditions were identical to those described above, and the
PCR products were then electrophoresed on a 1.0% (w/v)
agarose gel and visualised under ultraviolet light. Positive
transformants that yielded a single PCR product (~1200 bp)
were selected for future investigation.
Total RNA from each of the mature tobacco seeds was isolated
as described above. First-strand cDNA synthesis and fluorescent
quantitative RT-PCR were carried out as described above
using the RTFatB-F (50-GTAGCCAAACCCACCTCT-30) and
RTFatB-R (50-TTTCAGCCCAACCTTCG-30) primers, which
were used to detect the expression level of CocoFatB1 in the
seeds of transgenic plants. Transcripts of the 18S gene, which was
used as an internal control for expression, were amplified
using the RT18S-F (50-GCAACAAACCCCGACTTCT-30) and
RT18S-R (50-GCGATCCGTCGAGTTATCAT-30) primers.
Fatty acid methyl ester analysis by GC
Total lipids were extracted in triplicate using dichloromethane :
methanol (2 :1) from mature seeds from single plantlets of the
transgenic and wild-type tobacco plants. The fatty acid methyl
esters were recovered using N-hexane. Analysis of fatty acid
methyl esters was performed using GC, with methyl
heptadecanoate (17 : 0) (Sigma) as an internal standard. All
GC analysis was performed using a HP5890 GC instrument
equipped with a BPX-70 (30 m 0.25 mm) chromatography
column (SGE, Melbourne, Vic., Australia). The initial column
temperature (90C) was held for 10 min and then raised at 4C
min
–1
until it reached 240C, after which it was held at this
temperature for another 10 min (Beermann et al.2007; Chi et al.
2011).
RB
CaMV35S promoter
CaMV35S poly A
LB
Hygromycin (R)
Nos poly A Napin promoter lacZ alpha
CocoFatB1
Fig. 1. The T-DNA region of the construct used to transform Nicotiana tabacum plants. Sequences of
functional importance are the left border (LB), the right border (RB), cauliflower mosaic virus 35S
(CaMV35S) promoter and polyA addition sequences and the nopaline synthase (Nos) polyA addition
sequence. The seed-specific expression cassette consists of a napin promoter fragment and CocoFatB1 from
coconut.
82 Functional Plant Biology Y. Yuan et al.
Statistical analysis
All experiments were performed in triplicate, and the data
provided are means s.d. Intergroup comparisons between the
two tested groups were performed using a paired t-test, using
Statview ver. 6.0 software (SAS Institute, Cary, NC, USA) and
Microsoft Office Excel ver. 2007 (Microsoft Corporation,
Richmond, WA, USA). A P-value of <0.05 was regarded as
indicating a statistically significant difference.
Results
Complementary DNA cloning and conservation analysis
of the CocoFatB1 sequence
The full-length cDNA sequence (1858 bp) of an acyl-ACP
thioesterase was isolated (termed CocoFatB1; GenBank
accession: JX275886) from total RNA from the coconut
endosperm using EST sequences and RT-PCR. Sequence
analysis revealed that the CocoFatB1 sequence was
homologous to other acyl-ACP thioesterases and the predicted
protein has similar properties to previously identified orthologues
and is homologous across its entire length. CocoFatB1 has 73%
amino acid identity with the rice enzyme (Os06 g0143400).
Expression of CocoFatB1 in different tissues
and at different development stages
To reveal the expression of CocoFatB1 genes during the
development of coconut pulp, fluorescence quantitative RT-
PCR was used to analyse CocoFatB1 expression during two
different developmental stages: 8-month-old and 15-month-old
coconut fruits, with the abundance of b-actin transcripts
providing an internal control. The CocoFatB1 transcript was
most abundant in the endosperm from 8-month-old coconuts,
whereas the leaves and endosperm from 15-month-old coconuts
had ~80% and ~10% of this level.
Generation of transgenic tobacco plants expressing the
CocoFatB1 gene under the control of the napin promoter
To further determine the function of CocoFatB1 and to establish
whether its expression can change the fatty acid profile in
plants, we investigated the effects of CocoFatB1 expression in
transgenic tobacco. Analysis of the transgenic plants’genomic
DNA using PCR indicated the presence of the CocoFatB1 coding
sequence in the tobacco genome. Following transformation and
selection in the presence of hygromycin, 32 independent
transgenic plants were obtained. CocoFatB1 transcript
expression varied 20-fold between different transgenic plants,
with 21 plants exhibiting detectable levels of CocoFatB1
expression (Fig. 2). Four transformant lines (7, 8, 9 and 2) that
showed different levels of CocoFatB1 transcript were selected for
further analysis.
Analysis of fatty acid composition
As an acyl-ACP thioesterase, CocoFatB1 was expected to
increase the medium chain saturated fatty acid composition
of tissues in which it is expressed. To further confirm its
function in vivo, the fatty acid composition of transgenic and
untransformed tobacco plants were determined and compared.
Expression of transgenic CocoFatB1 in tobacco seed increased
levels of myristic acid (14 : 0), palmitic acid (16: 0) and stearic
acid (18 : 0) by 10.2%, 3.9%, 8.4% and 4.3% in Plants 7, 8, 11 and
15 respectively. Meanwhile, there was no obvious difference in
the levels of other fatty acids when the seeds of transgenic and
untransformed tobacco plants were compared (Table 1).
Discussion
Plant fatty acids are synthesised in the stroma of the plastids of
both leaves and developing seeds (Weaire and Kekwick 1975;
25.00
20.00
15.00
10.00
5.00
0.00
1234567 89
Different CocoFatB1 trans
g
eni plants
10 11 12 13 14 15 16 17 18 19 20 21
Expression level (fold)
Fig. 2. Expression levels of the CocoFatB1 gene in CocoFatB1 transgenic
plants. Expression is quantitated as the fold increase compared with plants
expressing the lowest CocoFatB1.
Table 1. Comparison of the fatty acid composition of untransformed and transgenic tobacco seed oil
Mature seeds from single plants were used for the analysis and extracted in triplicate. Data are means from three measurements, with s.d. Asterisks
indicate statistically significant differences compared with the control (Student’sttest: *, P<0.05; **, P<0.01). 14 : 0, myristic acid; 14 : 1,
myristoleic acid; 16 : 0, palmitic acid; 16 : 1, palmitoleic acid; 18 : 0, stearic acid; 18 :1, oleic acid; 18 : 2, linoleic acid; 18 :3, linolenic acid
Fatty Untransformed CocoFatB1 transgenic line
acid tobacco 7 8 9 2
14 : 0 0.08 ± 0.001 0.10 ± 0.002** 0.11 ± 0.008** 0.06 ± 0.006** 0.18 ± 0.005**
16 : 0 8.98 ± 0.003 18.15 ± 0.010** 12.32 ± 0.010** 16.89 ± 0.136** 12.06 ± 0.033**
16 : 1 0.12 ± 0.007 0.10 ± 0.096** 0.09 ± 0.003** 0.13 ± 0.004 0.24 ± 0.008**
18 : 0 2.85 ± 0.013 3.84 ± 0.0048** 3.37 ± 0.108** 3.36 ± 0.148** 3.98 ± 0.005**
18 : 1 12.53 ± 0.025 12.40 ± 0.125 10.9 ± 0.010** 11.89 ± 0.231* 11.16 ± 0.011**
18 : 2 69.97 ± 0.066 62.53 ± 0.007** 68.20 ± 0.055** 64.77 ± 0.004** 67.66 ± 0.012**
18 : 3 0.95 ± 0.005 1.22 ± 0.008** 1.32 ± 0.043** 1.08 ± 0.070* 1.90 ± 0.075**
Function of the ACP thioesterase gene in coconut Functional Plant Biology 83
Ohlrogge et al.1979). Accordingly, CocoFatB1 transcripts are
detected not only in the endosperm, but also in the leaves of
coconut plants. Thioesterases play a pivotal role in fatty acid
synthesis owing to their role in catalysing the terminal reaction of
fatty acid biosynthesis, which regulates the fatty acid composition
of storage lipids, especially in plant seeds (Brown et al.2010; Jing
et al.2011). The expression of thioesterase genes displayed the
highest levels in expanding tissues that are typically very active in
lipid biosynthesis, such as developing seed endosperm and young
expanding leaves (Oo and Stumpf 1979; Sánchez-García et al.
2010).
To confirm the activity and substrate specificity of CocoFatB1
in plants, we analysed the effects of its expression in transgenic
tobacco. This result indicated that CocoFatB1 showed specificity
towards 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP. Compared with
the results of function analysis of CnFatB1,CocoFatB1 is specific
not only towards 14 : 0-ACP and 16 : 0-ACP, which have been
demonstrated in E. coli by Jing et al.(2011), but also showed
specificity to 18 : 0-ACP in plant. These results are similar to those
previously reported for FatB thioesterases from other plants,
such as Elaeis guineensis (Othman et al.2000), Jatropha
curcas L. (Wu et al.2009), Cuphea hookeriana (Jones et al.
1995), Diploknema (Madhuca) butyracea (Jha et al.2006) and
Indian mustard (Brassica juncea L. Czern.; Jha et al.2010). All
of these enzymes displayed a high level of activity towards
16 : 0-ACP; BjFatBs from B. juncea were also specificto
18 : 0-ACP. Moreover, like other FatB thioesterases, which
preferably hydrolyse acyl-ACPs with saturated fatty acid
chains (Jones et al.1995), CocoFatB1 showed a preference for
saturated acyl-ACPs, especially palmitoyl-ACP.
However, compared with some FatBs from other plants that
contain rich MCFAs, CocoFatB1 still displayed some unique
characteristics. The FatBs from U. californica (Pollard et al.
1991; Voelker et al.1992), A. thaliana (Dormann et al.1995),
U. americana (Voelker et al.1997) and nutmeg (Myristica
fragrans; Voelker et al.1997) are specific for 12 : 0-ACP and
play a critical role in MCFA production. Unlike these FatBs,
CocoFatB1 shows a preference for 14 : 0-ACP, 16 : 0-ACP and
18 : 0-ACP. Recently, CnFatB3 has been demonstrated to be
specific for 12 : 0-ACP, 14 : 0-ACP and 14 : 1-ACP in E. coli
(Jing et al.2011), which may make a great contribution to
fatty acid profiles containing abundant MCFAs in coconut
endosperm. More functional analysis is needed to confirm the
characterisation of CnFatB3, especially in plants. Meanwhile,
some crucial enzymes involved in fatty acid synthesis may also
play an important role in determining the lengths of fatty acid
chains.
Although the specificities of thioesterases determine the
chain length of most plant fatty acids to a large extent, the
action of specificb-ketoacyl-ACP synthases (KAS) and acyl-
ACP acyltransferases shift the synthesis of fatty acids towards
molecules with shorter chains (Davies et al.1995; Leonard et al.
1998). Of three known classes of plant KAS enzymes, only
KASI elongates substrates from 4 : 0-ACP to 14 : 0-ACP
(Shimakata and Stumpf 1983). Seeds transformed with
CwKASA and CwFatB2 thioesterases in comparison with the
seeds transformed with thioesterases only had greatly increased
concentrations of 10 : 0 (capric acid) and 12 : 0 (lauric acid).
Coexpression of CwKASA with California bay FatB1 in
transgenic canola (Brassica napus L.) increased amounts of
12 :0 fatty acids when compared with expression of the FatB1
only (Leonard et al.1998). Additionally, expression of the
LPAAT gene of coconut endosperm in E. coli and canola
indicates that the enzyme displays a marked preference for the
transfer of medium-chain CoAs to 12 : 0-lysophosphatidic acid
relative to unsaturated long-chain substrates(Wiberg et al.
1997). Co-expressed with thioesterase, lysophosphatidyl
acyltransferase (LPAAT) expression generated triacylglycerol
(TAGs) with high levels of lauric acid (12 : 0) at sn-2 (Davies et al.
1995; Knutzon et al.1999; Wiberg et al.2000).
The aim of this study was to explore why coconut endosperm
is so rich in MCFAs and to identify one of the genes responsible
for this phenotype. The CocoFatB1 gene we have isolated and
characterised was highly effective in redirecting plant fatty acid
synthases to palmitate and myristate production, and appears to be
specific to 14 : 0-ACP, 16 : 0-ACP and 18 : 0-ACP. The ectopic
expression of C. nucifera CocoFatB1 in N. tobaccum increased
the levels of saturated acids, including myristic acid, palmitic acid
and stearic acid, to provide a fatty acid profile distinct from that of
other oil crops that produce large amounts of MCFAs, such as
U. californica (Pollard et al.1991; Voelker et al.1992). Based on
the previous studies, more detailed research about the actions of
the thioesterases, KAS and LPAAT from coconut endosperm is
needed to confirm the function of these crucial enzymes in fatty
acid synthesis. Co-expression of special thioesterases with KASI
or LPAAT in plants should enable the effects of the various
enzymes on fatty acid composition to be separated. The ability
to engineer the accumulation of these MCFAs, especially lauric
acid (12 : 0), will be beneficial in efforts to improve crops by
engineering fatty acid metabolism.
Acknowledgements
YY and YC contributed equally to the work. This research was supported
by the National Natural Science Foundation of China (No.: 31160171,
31060259 and 31260193) and Youth Foundation of Hainan University
(No.: QNJJ1001).
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