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Philippine Journal of Science
142 (1): 39-44, June 2013
ISSN 0031 - 7683
Date Received: ?? Feb 20??
A Proposed Regulatory Mechanism of Ontogenetically
Expressed DITAA-Containing Coconut Transcripts
Marni E. Cueno* and Rita P. Laude
Institute of Biological Sciences, College of Arts and Sciences (CAS),
University of the Philippines Los Baños (UPLB), College, Laguna 4031
Accumulation of medium-chain oils into the coconut endosperm has been shown to follow
a temporal pattern of fatty acid gene expression. The mechanism by which these genes are
regulated, however, has not been explored. Using 3’ RACE, we identified two coconut DITAAcontaining transcripts (DCTs), appropriately labeled DCT1 and DCT2, and found both
transcripts in the 5-6 and 6-7 month old (mo) coconut endosperms following an ontogenetic
pattern. Comparison of both amplified transcripts from the 5-6 and 6-7 mo endosperms show
100% homology between DCT2 transcripts. Interestingly, 52 nt downstream of the DCT1
sequences prior to the poly(A) tail are missing in the DCT1-6mo transcript. Visual inspection
for potential motifs shows the presence of CCGCC-like (DCT1-5mo and DCT1-6mo) and
TGTG-like (DCT1-6mo only) motifs. Both motifs have been published to be known regulatory
motifs in other species suggesting a probable similar function in coconut.
Key Words: 3’ RACE, coconut endosperm, Cocos nucifera, ontogenetic expression, regulation
INTRODUCTION
Coconut (Cocos nucifera L.) has often been considered
the “tree of life” due to its several end products used in
food, structure materials, and decorative items (Banzon
and Velasco 1982). Coconut is the major export crop
of the Philippines due to its oil (Harries 1994) and, at
the moment, it is either exported as copra or as virgin
coconut oil. Coconut oil is predominantly composed
of lauric acids and the coconut’s lauric oil content
commands a premium price in world markets (Banzon
and Velasco 1982; Harries 1994), wherein, coconut (from
the Philippines) and palm kernel (from Malaysia) are
the major sources of lauric acid-rich vegetable oils with
48.2% and 50.9%, respectively (Banzon and Velasco
1982). Lauric oils represent about 6% (4.4 million metric
tons) of the 72.7 million tons of the world’s oil and fats
production, and 5.5% of the world’s total fats and oils
consumption (Harries 1994).
*Corresponding author: [email protected]
It was previously suggested from the work of Villalobos
et al. (2001) that coconut fatty acid synthesis follows an
ontogenetic pattern of expression, however, it has never
been proven to date. Moreover, as the solid endosperm
develops in the coconut drupe, medium-chain fatty acids
concurrently start to accumulate in the solid endosperm
(Banzon and Velasco 1982; Harries 1994) further
suggesting ontogenetic gene expression which coincides
with the hypothesis made by Villalobos et al. (2001).
This would suggest a temporal pattern of gene regulation
among coconut genes involved in fatty acid synthesis.
The untranslated region (UTR) is well established to be
the region for potential post-transcriptional regulatory
pathways that control mRNA localisation, stability, and
translation efficiency (Pesole et al. 2000; Rothnie 1996).
In particular, the 3’ UTR plays an important role in many
aspects of RNA function and metabolism, particularly
in mRNA translation and turnover (Barreau et al. 2006;
Jackson and Standart 1990; Rothnie 1996; Sachs and
Whale 1993). In addition, the 3’ UTR was found to be
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Philippine Journal of Science
Vol. 142 No. 1, June 2013
involved in post-transcriptional mRNA processing and
gene expression regulation (Rothnie 1996; Pesole et al.
2000).
In this paper, we designed primers based on the DITAA
(Asp-Ile-Thr-Ala-Ala) motif which is part of the highly
conserved region of ketoacyl carrier protein synthase
(KAS) as indicated in a previously published work
(Tai and Jaworski 1993). We looked at the 3’end of
two coconut DITAA-containing transcripts (DCT). We
initially established that DCT1 (DCT isoform 1) and
DCT2 (DCT isoform 2) are ontogenetically transcribed
and, through sequence alignment, found 52 nucleotides
(nt) missing upstream of the poly(A) tail in DCT1
obtained from the 5 month old (mo) coconut endosperm.
Interestingly, sequence inspection upstream of the
missing 52 nt fragment reveals the presence of a known
regulatory motif which would suggest its involvement in
coconut gene regulation.
MATERIALS AND METHODS
mRNA extraction
Coconut endosperm tissues ages 4-5, 5-6, and 6-7 mo were
collected from the age-verified drupes of the Laguna Tall
coconut variety obtained from the Philippine Coconut
Authority-Davao. Coconut endosperm was freshly
obtained for mRNA isolation. The Micro-FastTrack
2.0 mRNA Isolation Kit (Invitrogen, USA) was used to
isolate pure mRNA from coconut endosperm following
the manufacturer’s instructions with some modifications.
All chemicals and tubes were provided by the kit.
Briefly, equal amounts of pulverized coconut endosperm
samples were treated with lysis buffer and incubated at
45°C for 20 min before centrifugation. The supernatant
was then transferred to a new tube and NaCl concentration
adjusted to 0.5M concentration. Remaining DNA was
sheared by quickly passing the lysate 5 times through a 1
mL pipette tip. Cell lysate was added to a tube of oligo(dT)
cellulose and allowed to swell before placing the tube
horizontally in a platform and incubated for 20 min at
room temperature. Oligo(dT) cellulose was resuspended in
binding buffer and centrifuged again after centrifugation.
This process was repeated until the buffer was no longer
cloudy. The resin was then resuspended in binding
buffer, transferred to a spin-column then centrifuged.
After repeating this step thrice, low salt wash buffer was
added to resuspend the resin before centrifugation. The
spin-column was then placed into a new tube and 100 µL
of elution buffer was added and mixed into the cellulose
bed before centrifugation. This step was repeated twice.
The mRNA was precipitated from the solution using
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Cueno and Laude: Regulation of Coconut Transcripts
glycogen carrier, sodium acetate, and ethanol placed
in -80°C overnight. The mRNA was pelleted through
centrifugation and resuspended in 10 µL elution buffer
for use in this study.
GC-rich cDNA synthesis and cloning of DITAAcontaining transcripts (DCTs)
The designed forward primer (5’-GAY ATH ACN GCN
GCN-3’) used for the study was partly based on the highly
conserved region of KAS (Tai and Jaworski 1993). The 3’
RACE (Random Amplification of cDNA Ends) procedure
was carried out using the 3’ RACE Kit (Invitrogen, USA)
following the manufacturer’s instructions for GC-rich
transcripts in order to avoid reverse transcribing truncated
cDNAs. A combined Hot-start Touchdown PCR condition
performed consists of an initial denaturation at 95°C for 5
min and 85°C for 3 min during which Taq polymerase is
added. This was then followed by 5 cycles of 94°C for 1
min, 55°C for 1 min, and 72°C for 2min; another 5 cycles
of 94°C for 1 min, 53C for 1 min, and 72°C for 2min; and
lastly consists of 25 cycles of 94°C for 1 min, 50°C for 1
min, and 72°C for 2min. A final 10min extension at 72°C
was performed before proceeding to cloning.
Gel electrophoresis was used to visually detect the
presence of amplified PCR products among the varying
ages of coconut endosperm used as cDNA template. The
detected PCR bands were immediately eluted and used in
the TOPO TA Cloning® Kit (Invitrogen, USA) following
manufacturer’s instructions. At least five randomly
selected clones from each eluted product were purified
for sequencing.
Sequencing and analysis
Purified plasmid DNA was commercially sequenced
by the Division of the Biomolecular Research Facility,
University of New Castle, Callaghen, Australia. Sequence
comparison and visual inspection was performed using
the Vector NTI Suite software.
RESULTS AND DISCUSSION
Ontogenetic expression of DCTs
Medium-chain fatty acid accumulation concurrently
occurs with coconut endosperm thickening (Banzon and
Velasco 1982; Harries 1994). To readily establish the
DCT ontogenetic expression in the coconut endosperm,
3’ RACE was performed. As shown in Figure 1, DCTs
are present in the 5-6 and 6-7 mo coconut endosperms
with two bands detected. No band was detected in the
4-5 mo. old endosperms. We classified the two distinct
bands detected as DCT1 (~700bp) and DCT2 (~500bp).
Philippine Journal of Science
Vol. 142 No. 1, June 2013
Cueno and Laude: Regulation of Coconut Transcripts
Figure 1. Ontogenetic pattern of DITAA-containing transcripts observed in varying ages of coconut endosperm.
Hot-start Touchdown RACE-PCR was performed using the three ages of coconut endosperm as
template: 4-5, 5-6 and 6-7 months old. A partial DITAAC-based oligonucleotide primer was used
for the 3’RACE procedure.
Considering our previous work (Cueno et al. 2010)
where we established that the coconut enolase gene can
be detected in the 4-5, 5-6, 6-7, and 7-8 mo coconut
endosperms, the absence of PCR bands in the 4-5 mo
coconut endosperm and the presence of PCR bands in
the 5-6 and 6-7 mo coconut endosperms would imply that
ontogenetic gene expression occurs in the coconut which
is in agreement with a previous work (Villalobos et al.
2001). The forward primer used was partly based on the
DITAAC amino acid region which is a highly conserved
motif in the KAS enzyme (Tai et al. 1994 and Slabaugh
et al. 1995). Among the KAS isoforms, only KASIII is
involved in fatty acid biosynthesis (Tai and Jaworski
1993). Considering the ontogenetic pattern observed
among the DCTs (Figure 1) and the results obtained by
Villalobos et al. (2001), it would then suggest that the
amplified DCTs represent a putative coconut KASIII.
Sequence profiling of the DCT 3’ends
To determine the cause of this discrepancy, we looked
into the DCT sequence profiles. All sequences obtained
showed high sequence similarities within each eluted
product (templates from coconut endosperm tissues ages
5-6 and 6-7 mo), thus, representative sequences were used
for further analyses. Independent alignments, as seen in
Figure 2, clearly show the high homology shared between
DCT1-5mo and DCT1-6mo; and DCT2-5mo and DCT26mo. Visual inspection of the first 15 nt of DCT2 show
the forward primer binding site confirming successful
PCR amplification. Interestingly, the first 18 nt of DCT2
translates into the DITAAC amino acid region (Table 1)
which we earlier mentioned to be highly conserved among
KAS enzymes. It is also worth mentioning that positions
16-18 nt of DCT2 translates to Cys, a known active-site
in the DITAAC amino acid region (Siggaard-Andersen
1993). Considering the designed primer used in this study
was partly based on the highly conserved amino acid
sequence of KAS and the presence of Cys immediately
after our DITAA-based primer, taken together this would
further suggest that the sequenced DCTs represent a
putative coconut KASIII enzyme.
Noticeably, DCT2 transcripts have 100% homology,
whereas, there is a major difference in sequence homology
between DCT1-5mo and DCT1-6mo. In particular, low
homology is observed at the start and end of both DCT1
sequences (Figure 2A). It is also worth mentioning that
the primer binding site can be seen in DCT2 sequences
(Figure 2B) but is not observed in DCT1 sequences
(Figure 2A) possibly since the effective sequencing
length is ~300 nt and DCT1 transcripts were observed
to have ~700 nt length (Figure 1). This suggests that the
DCT1 transcript sequences obtained were partial 3’ end
sequences (mostly from the 3’UTR) which would then
explain the observed low homology in the start sequence.
The 3’UTR always have a longer nucleotide sequence
compared to the 5’UTR (Pesole et al. 2000) owing to
the various control mechanisms present (Beelman and
Parker 1995; Mazumder et al. 2003), especially in plants
(Mazumder et al. 2003; Rothnie 1996). Considering both
DCT1 transcripts were collected from two different stages
41
Figure 2. Pairwise sequence alignment of DITAA-containing transcripts (DCTs). DCT1 and DCT2 sequences from 5- and 6- month old coconut endosperms were aligned using the
Vector nti Suite software. (A) DCT1 alignment; (B) DCT2 alignment.
Philippine Journal of Science
Vol. 142 No. 1, June 2013
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Cueno and Laude: Regulation of Coconut Transcripts
Cueno and Laude: Regulation of Coconut Transcripts
Philippine Journal of Science
Vol. 142 No. 1, June 2013
Table 1. DCT sequence profiles
DCT1
5 mo.*
Amino acid region
DITAAC
DCT1
6 mo.*
Not seen in sequence
DCT2
5 mo.*
DCT2
6 mo.*
1-18
1-18
Proposed function
Conserved region identified
only in KAS enzymes
Identified motifs
TAAT
CCGCC-like
TGTG-like
228-231
235-239
240-243
216-219
247-251
Not observed
235-238
Not observed
Not observed
Missing nucleotide sequence
240-291
Not observed
Not observed
polyadenylation site
Regulatory motif1
Regulatory motif2
*All sequence positions are indicated as nucleotides (nt)
1
JSNP database as cited by Tsuge et al. (2005)
2
Iengar and Joshi (2009)
of the coconut endosperm and, as seen in Figure 1, visible
differences in terms of PCR band intensity were observed,
we did expect sequence differences even in the 3’UTR.
The major sequence difference between DCT1-5mo and
DCT1-6mo transcripts is the 52 nt missing from DCT16mo transcripts (Table 1 and Figure 2A).
Visual inspection for potential coconut regulatory motifs
To offer an explanation why 52 nt was missing in the DCT16mo transcript, we looked for potential coconut motifs near
the poly(A) tail attributable to this phenomena. As shown
in Table 1, we identified a putative polyadenylation site
and two potential coconut motifs that have been previously
identified as regulatory motifs. The CCGCC-like motif
can be found in both DCT1 transcripts and, interestingly,
prior to the missing 52 nt (Figure 2A) while the TGTG-like
motif can only be found immediately after the CCGCClike motif and is part of the missing 52 nt where it is found
to be the starting sequence (Figure 2B). For purposes of
providing an explanation to why 52 nt was missing in
the DCT1-6mo transcript, we attribute this event to the
two potential coconut motifs we identified. Furthermore,
the obvious difference in band intensity between DCT15mo and DCT1-6mo (Figure 1) would seem to suggest
that regulation of the DCT1-5mo transcript is somehow
ascribable to the 52 nt deletion.
The CCGCC motif has been previously reported to be a
deletion-insertion site as seen in the JSNP database (as cited
by Tsuge et al. 2005). The TGTG motif has been reported
to function in genes related to cytoplasmic translation,
DNA replication, proteasomes, mitochondria, organellar
translation, merozoite invasion and actin myosin motors in
Plasmodium falciparum (Iengar and Joshi 2009). Though
the TGTG motif has not yet been identified in plants, when
we consider the positions where both our proposed coconut
motifs (CCGCC-like and TGTG-like) are found with
respect to the missing 52 nt (Figure 2A), the positioning
of both proposed coconut motifs favor a regulatory role
consistent with previously published works.
SUMMARY AND CONCLUSION
We established that coconut DCTs are ontogenetic
expressed. In addition, we noticed a difference in band
intensity between DCT1-5mo and DCT1-6mo, and
proposed a possible explanation for this phenomenon
through sequence profiling. Moreover, we identified 52 nt
missing prior to the poly(A) tail and attributed this to our
two proposed coconut regulatory motifs, namely CCGCClike and TGTG-like motifs. In addition, we hypothesize
that regulation of the DCT1-5mo transcript is associated to
the 52 nt deletion possibly attributable to the two proposed
coconut regulatory motifs. We emphasize, however, that
the conclusions we derived from our results are mainly
theoretical and requires further confirmation.
ACKNOWLEDGEMENTS
This study was funded by the University of the Philippines
Los Baños-PCARRD-DOST project entitled: Cloning of
Important Genes from Coconut. We would also like to
thank the Biochemistry Laboratory of the Institute of Plant
Breeding, UPLB for their invaluable help and permission
to use most of their equipment.
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