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Abstract |
Mesocarp-specific
Metallothionien-Like Gene Promoter For Genetic Engineering Of Oil
Palm
Primers from within the coding region were used to capture the 5'
regulatory sequence of the mesocarpspecific metallothionein-like gene,
MT3-A, via PCR-based genome walking. The amplified 1040 bp genomic
fragment was cloned and sequenced. The sequence of the genomic clone
showed total homology with the MT3-A cDNA sequence within their overlapping
regions. Rapid amplification of 5’-cDNA ends (5’-RACE)
was used to determine the full length cDNA sequence and the putative
transcription site of the gene. The adenine residue at the 5’-end
of the RACE product was chosen as the likely transcription start site.
The 986 bp promoter region upstream of the adenine contains putative
regulatory elements including a TATA box, an ethylene responsive element
in reverse orientation and two I-boxes. Functional analysis of the
MT3-A promoter was performed using a transient assay system. Transient
expression of ß-glucuronidase (GUS) examined using qualitative
histochemical GUS assay can be detected in both oil palm mesocarp
and leaf tissue slices bombarded with the pBI221 transformation vector
which contains the GUS reporter gene under the control of the constitutive
cauliflower mosaic virus (CaMV) 35S promoter. However, when the CaMV-35S
promoter was replaced with MT3-A promoter in the transformation vector
and used for bombardment, transient expression of GUS was detected
in the oil palm mesocarp slices only and not in the leaf tissue. This
suggests that the MT3-A promoter can be used to target specific gene
expression into oil palm mesocarp tissues.
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Isolation
and High Resolution Microscopy of Native Bacterial Polyhydroxybutyrate
Inclusions
Polyhydroxybutyrate (PHB) is a carbon reserve in some bacteria and
under nutrient-limiting conditions, accumulates intracellularly in
the form of inclusion bodies. These inclusions contain proteins, and
the PHB within the inclusions exists in an amorphous state. In this
study, a procedure to recover native PHB inclusions was developed,
and the isolated inclusions characterized using 13C NMR, western blotting,
atomic force microscopy and fluorescence microscopy. High resolution
images of native PHB granules were obtained and the surface features
and dimensions of the granules analysed. The size, morphology and
composition of the PHB inclusions are important parameters for analysing
inclusions formed within PHB-producing oil palm to support commercial
objectives. The methods reported in this work support the development
of analytical techniques for transgenic bioplastic-producing oil palm
samples.
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Light-Harvesting
Chlorophyll A/B Binding Protein (LHCB) Prometer for Targeting Specific
Expression in Oil Palm Leaves
Oil palm leaves have the potential to be manipulated as a green factory
to produce novel metabolites. In order to direct the specific expression
of transgenes into the leaf tissue, a leaf-specific promoter is required.
In these studies, efforts were carried out to isolate a leaf-specific
gene and its regulatory sequence from the oil palm. Rapid amplification
of 5’-cDNA Ends (5’-RACE) resulted in isolation of a 962
bp full length sequence of the light-harvesting chlorophyll a/b binding
protein (LHCB). The amino acid sequence of this gene exhibited more
than 88% homology with the LHCB1 gene isolated from monocots and dicots.
Characterization of this transcript using Northern blot analysis revealed
LHCB to be abundantly expressed in oil palm green leaf tissues, but
not in the non-photosynthetic tissues such as kernel, mesocarp, germinated
seedlings and inflorescences. Results from Southern blot analysis confirmed
that at least four copies of the LHCB gene are present in the oil palm
genome. The promoter region of LHCB was then obtained through genome
walking approach. Based on the sequence analysis, it was found that
the LHCB promoter lacks a TATA-box. Initiation of transcription can
therefore be replaced by an initiator element (Inr) located at positions
-1 to -7. Furthermore, a few putative cis-acting elements responsive
to light, wounding, abscisic acid and heat-shock were also found in
the distal and proximal regions of the LHCB promoter.
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Non-radioactive
Assay for Acetyl-CoA Carboxylase Activity
Acetyl-CoA carboxylase is a key enzyme in oil biosynthesis and is critical
for the oil deposition pathway. This biotinylated enzyme catalyzes the
first committed step in fatty acid biosynthesis, the ligation of a carbon
to acetyl-CoA to form malonyl-CoA. The acetyl-CoA carboxylase holoenzyme
has four distinct protein domains: biotin carboxylase, biotin carboxylase
carrier protein, and the alpha and beta transcarboxylase domains. Biotin
carboxylase catalyzes the addition of carbon dioxide to biotin carboxylase
carrier protein, while the alpha and beta subunits of carboxyltransferase
ligate the activated CO2 to acetyl-CoA. The canonical assays for monitoring
the activity of acetyl-CoA carboxylase rely on incorporation of radiolabelled
acetyl-CoA. In this work, we describe the development of a discontinuous,
non-radioactive spectrophotometric assay for acetyl-CoA carboxylase
activity. Permeabilized Corynebacterium glutamicum cells were added
to an assay mixture containing acetyl-CoA, bicarbonate, magnesium and
ATP, and aliquots were removed at set time points and stopped by the
addition of trifluoroacetic acid. The level of acetyl-CoA remaining
in each aliquot was determined via a citrate synthase assay, in which
the formation of the yellow compound dithiobisnitrobenzoic acidthiophenolate
was followed spectrophotometrically at 412 nm.
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Construction
of PHB and PHBV Transformation Vectors for Bioplastics Production in
Oil Palm
The construction of
transformation vectors carrying bioplastic biosynthetic genes driven
by constitutive and oil palm mesocarp-specific promoters was completed.
Four planned transformation vectors were produced. The poly-3-hydroxybutyrate
(PHB) producing constructs carried the phbA, phbB and phbC genes, while
the polyhyroxybutyrate-co-valerate (PHBV) producing constructs carried
the bktB, phbB, phbC and tdcB genes. Each of these genes was fused with
the transit peptide (Tp) of the oil palm acyl-carrier-protein (ACP)
for targeting into the plastids of plant cells. All vectors carry the
phosphinothricin acetyltransferase gene (bar) driven by an ubiquitin
promoter as plant selectable marker. The matrix attachment region from
tobacco (RB7MAR) was also included for stabilization of the transgene
expression and to minimize the gene silencing due to positional effects.
All constructs were verified by restriction analysis, polymerase chain
reaction (PCR) and DNA sequencing.
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Reagents
for Generation and Analysis of Bioplastic Producing Plants
Polyhydroxyalkanoates
(PHAs) are a class of biodegradable polyoxoesters synthesized from acetyl-CoA
that naturally accumulate as intracellular granules in a diverse range
of bacteria. Biosynthesis of the simplest PHA (PHB, poly-3-hydroxybutyrate)
can be accomplished though the action of three enzymes, beta-ketothiolase,
acetoacetyl-CoA reductase and PHA polymerase. We constructed plasmids
that contain genetic elements for the production of PHB, or the closely
related copolymer PHBV (poly-3-hydroxybutyrate-co-3-hydroxyvalerate),
in oil palm, which produces abundant levels of acetyl-CoA. These bacterial
PHA biosynthetic genes have been engineered to include plant plastid
targeting signals, in order to direct biosynthesis of the polymer inside
the plastids. In addition, we generated antibodies for the detection
of PHA biosynthetic enzymes. The plasmids and antibodies reported in
this work should be suitable as tools and reagents for the construction
and analysis of PHA-producing oil palm.
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Quantitative
Analysis of Flux Control Over Lipid Biosynthesis in Oil Palm (Elaeis
guineensis) Mesocarp
Plant storage oils
are of major commercial importance, yet our understanding of the regulation
and control of their synthesis is poor. The control of lipid biosynthesis
fluxes in the mesocarp of oil palm (Elaeis guineensis) was studied
using modular (top-down) metabolic control analysis (TDCA). This allowed
us to determine the relative contribution of two groups of reactions,
fatty acid formation (Block A) and lipid assembly (Block B), to the
control structure of overall pathway to triacylglycerol. The pathway
was manipulated in two ways. Single manipulation involved the addition
of oleate which inhibited fatty acid formation in Block A and stimulated
lipid assembly in Block B. In double manipulation experiments, cerulenin
was used in inhibition of Block A and Block B was inhibited by bromooctanoate.
Single manipulation-TDCA revealed that the group flux control coefficient
for fatty acid synthesis was 0.65 and 0.35 for lipid assembly. Double
manipulation- TDCA has a value of 0.6 for fatty acid synthesis and
0.4 for lipid assembly. Taken together, these data showed that under
our experimental conditions, about 60%-65% of the total metabolic
flux control lay in the fatty acid synthesis group of reactions. Nevertheless
because both parts of the lipid biosynthesis pathway exert significant
flux control, we suggest strongly that manipulation of single enzyme
will not affect the product yield appreciably.
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Transformation
of PHB and PHBV Genes Driven by Maize Ubiquitin Promoter into Oil Palm
for the Production of Biodegrabable Plastics
Three bacterial genes coding
for the enzymes, 3-ketothiolase (bktB), acetoacetyl-CoA reductase (phaB)
and PHB synthase (phaC), required for the synthesis of PHB from acetyl-CoA
in bacteria were transformed into oil palm embryogenic calli. For the
production of copolymer polyhydroxybutyrate–co-valerate (PHBV),
the threonine dehydratase (tdcB) gene from Escherichia coli was also
transformed into oil palm embryogenic calli for producing propionyl-CoA,
the substrate for hydroxyvalerate. These genes were under the control
of the maize ubiquitin promoter. Currently, many transformed embryogenic
lines resistant to the herbicide Basta have already been produced. These
transformed calli were later regenerated to produce a few hundred plantlets
which are now growing in a biosafety screenhouse. Molecular analyses
have demonstrated stable integration of the transgenes in their genome.
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Gene
Discovery via Expressed Sequence Tags from Oil Palm (Elaeis guineensis
Jacq.) Mesocarp
Expressed sequence tags (ESTs)
have been used for many applications such as to reveal gene expression
patterns, gene regulation and sequence diversity. A total of 1011 unique
transcripts corresponding to 1463 genes have been identified from the
ESTs generated from 17-week oil palm mesocarp cDNA library. This approach
was found to be successful in the discovery of new and important genes
expressed in the mesocarp tissue which are associated with the various
cellular processes of the tissue. It was observed that 12.9% of the
total genes expressed in the 17-week mesocarp cDNA library can be categorized
under metabolism. This is in agreement with the function of the tissue
which is involved in many biochemical processes including amino acid
and fatty acid metabolism. Most importantly are the discoveries of genes
playing important roles in the fatty acid and wax biosynthesis pathway
such as acetyl-CoA carboxylase, stearoyl-ACP desaturase, acyl carrier
protein (ACP), lysophosphatidic acid acyltransferase, ?6-palmitoyl-ACP
desaturase and lipase. These genes can serve as targets for genetic
manipulation where such endeavours have been extensively carried out
in other plants such as Brassica napus and Olea europaea to help increase
the economic value of the oil. Genes and protein associated with ethylene
synthesis and signal transduction pathway were also identified from
the 17-week mesocarp ESTs. Dot blot analysis was carried out to help
in identifying potential tissuespecific genes, which can lead to the
isolation of the tissue-specific promoters for manipulation of the mesocarp
tissue. This is in particular to direct accumulation of transgenic products,
such as new specialty oils and value-added products like pharmaceuticals
and nutraceuticals to the mesocarp.
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Isolation
and Utilization of Acetyl-CoA CArboxylase from Oil Palm (Elaeis
guineensis Jacq.) Mesocarp
One of the targets of the
MPOB oil palm genetic engineering programme is to synthesize biodegradable
plastics. Biodegradable plastics were first discovered in bacterial
systems. Polyhydroxybutyrate (PHB), the most common biodegradable plastic,
is synthesized from acetyl-CoA by the sequential action of the following
three enzymes: ß-ketothiolase (phbA or bktB), acetoacetyl-CoA
reductase (phbB) and PHB synthase (phbC). Acetyl-CoA is also the main
substrate for fatty acid synthesis, where acetyl-CoA carboxylase (ACCase)
catalyses the conversion of acetyl-CoA to malonyl-CoA, the building
block for fatty acid synthesis. Down-regulating ACCase could divert
the central metabolite acetyl-CoA to higher value products such as PHB.
In this study, efforts were made to isolate both the multifunctional
form of ACCase and biotin carboxylase (BC), a component protein of the
multisubunit form of ACCase. Initially, reverse transcriptase polymerase
chain reaction (RT-PCR) using degenerate primers designed based on the
conserved region of plant biotin carboxylase gene was used to amplify
a partial length of the oil palm cDNA. This was then used for further
isolation of the full length cDNA by random amplification of cDNA ends
(RACE), followed by end-to-end PCR. The RT-PCR was similary used to
isolate a partial length multifunctional ACCase employing degenerate
primers designed based on conserved regions of plant ACCase. After confirmation
through sequencing and cross-reference with gene bank, the partial length
cDNA of multifunctional ACCase was incorporated in an intervention strategy,
where the cDNA wasadded in the antisense orientation into existing PHB
and PHBV transformation vectors driven by an oil palm mesocarp specific
(MSP1) promoter. It was envisaged that by down-regulating the activity
of ACCase, fatty acid biosynthesis activity will be reduced and thus
the acetyl-CoA pool diverted to production of PHB and PHBV. The resulting
vectors were later transformed into oil palm embryogenic calli using
the BiolisticsTM approach. After selection on medium containing the
herbicide Basta, resistant colonies were isolated and are currently
undergoing regeneration into full plants.
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Carotenoid
Profiles and Preliminary Investigation on Carotenoid Biosynthesis in
the Oil Palm (Elaeis guineensis Jacq.) Mesocarp
The changes in carotenoid
content and profile were studied in oil palm (E. guineensis) mesocarp
at various stages of development. Spectrophotometric analysis showed
that chlorophyll synthesis predominates in the young fruits but it then
shifts strongly to carotenoids as the fruit ripens. The major carotenoids
in the oil from ripe fruits are a- and ß-carotenes. A very wide
variation in total carotenoid content was observed in various E. guineensis
genotypes. High performance liquid chromatography (HPLC) analysis using
both C18 and C30 stationary phases showed the C30 phase to be superior
in separating the cis- and trans- isomers of both a- and ß-carotenes
and resolving other components. Lycopene was not detected in at all.
Incorporation studies were carried out with various 14C-labelled substrates
- acetate, glyceraldehyde-3- phosphate (G3P), isopentenyl, pyrophosphate
(IPP), mevalonic acid (MVA) and pyruvate. IPP was the most incorporated
showing it to be a major intermediate in carotenoid synthesis in the
oil palm. There was also considerable incorporation of acetate, IPP
and MVA into a- and ß-carotenes. G3P and pyruvate were not incorporated
into a- and ß-carotenes suggesting that carotenoid synthesis in
the oil palm follows the acetate/ mevalonate pathway.
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Activity
Studies, Gene Characterization and Manipulation of ß-Ketothiolase
of Oil Palm (Elaeis guineensis Jacq.) Mesocarp
The enzyme ß-ketothiolase
plays a major role in isoprenoid metabolism as well as in polyhydroxybutyrate
(PHB) biosynthesis. PHB is a member of the polyhydroxyalkanoate (PHA)
family of polymers with potential as biodegradable replacements for
the current petrochemical plastics used. A coupled assay system for
ß-ketothiolase activity in oil palm mesocarp crude extracts was
designed and optimized. The highest levels of ß-ketothiolase specific
activity were seen in oil palm fruits 8 to 11 weeks after anthesis (WAA)
with ripe fruits showing lower activity. A cDNA coding for oil palm
(Elaeis guineensis Jacq.) ß-ketothiolase was isolated through
RT-PCR and RACE techniques. The longest reading frame encoded a protein
of 415 amino acids with a predicted relative molecular weight of 43
217 Da, and considerable similarities to the gene/enzyme in other plant
thiolases and, to a lesser extent, prokaryotic thiolases. There is no
evidence for the presence of a signal peptide, suggesting that the ß-ketothiolase
cDNA encodes a cytosolic protein. Genomic DNA gel blot analysis suggested
a small family of ß-ketothiolase isogenes. Northern analysis revealed
that ß-ketothiolase mRNA transcripts are present in higher quantities
in the riper (13, 17, 20 WAA) than younger fruits at 6, 8 and 11 WAA,
contradicting the biochemical activity profile. This discrepancy may
be caused by interfering substances in the oil palm crude extract such
as lipids or competition for substrates by other enzymes. Other explanatory
factors include genotype dependency, thiolase mRNA transcripts of the
same size and post-translational modification. The expression profile
obtained in the Northern analysis is in agreement with that of sterol
and carotenoid accumulation during fruit ripening. Sequence analysis
with biocomputing tools showed that ß-ketothiolase cDNA is relatively
lowly expressed in oil palm mesocarp throughout its development. The
gene was used in an intervention strategy to substitute for bacterial
ß-ketothiolase by redesigning the PHB transformation vector driven
by oil palm mesocarp-specific promoter (MSP1), for synthesizing biodegradable
plastics in oil palm. The new vector, designated pMS35, was later transformed
into oil palm embryogenic calli using the biolistics approach. Currently,
Basta-resistant embryogenic calli have been obtained and are undergoing
proliferation and regeneration.
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