Cloning, characterization and expression of sesquiterpene synthase 1 and delta guaiene synthase 1 genes associated with 'Gaharu" formation in aquilaria malaccensis Lam
'Gaharu' is a valuable fragrant resin produced by several Aquilaria spp., many of which are considered threatened. The product is highly recognized for its vast medicinal values as a digestive, sedative and antiemetic drug and also popular as perfumes and incenses in the Middle East, So...
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| Format: | Thesis |
| Language: | English |
| Published: |
2015
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/91275/1/FH%202015%201%20-%20IR.pdf |
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| Summary: | 'Gaharu' is a valuable fragrant resin produced by several Aquilaria spp., many of which
are considered threatened. The product is highly recognized for its vast medicinal
values as a digestive, sedative and antiemetic drug and also popular as perfumes and
incenses in the Middle East, South Asia, Japan and China. Additionally, 'gaharu'
sculpturing for interior decoration is another important of its value which generates an
income in Asia. Phytochemical studies revealed that sesquiterpenes are one of the main
components in gaharu. Therefore, in this study, two genes, sesquiterpene synthase 1
and o-guaiene synthase 1 in the terpenoid biosynthesis pathway have been successfully
cloned from A. malaccensis. These encoding genes were successfully isolated by rapid
amplification of cDNA ends. They were designated as SesTPSI and GuaiSI,
respectively. In the course of obtaining high quality RNA for gene cloning experiments,
callus was successfully induced from leaf tissues of in vitro A. malaccensis on
Murashige & Skoog medium supplemented with 2.2 11M6-Benzylaminopurine (BAP)
and 1.1 11Mnaphthaleneacetic acid (NAA). Cell line A2 and A4 developed into healthy
calli with cremish yellow color and the increased in diameter measurement. SesTPSI
and GuaiSI were cloned from callus RNA, using specific primers derived from
transcriptomic data, in a reverse transcription-PCR reaction. The results, the full-length
cDNA sequence of SesTPSI was 1632 bp encoding for 544 amino acids, while GuaiSI
was 1644 bp encoding for 547 amino acids. Sequence alignment analysis showed that
SesTPSI shared between 99% to 100% identity with sesquiterpene synthase from
Aquilaria sinensis while GuaiSI shared between 95% to 99% identity with o-guaiene
synthases from Aquilaria crassna and A. sinensis. The genes were functionally
characterized in a time-course wounding experiment using 3-year-old living trees. Two
types of wood samples were collected: 1) from wounded area (S 1) and, 2) from 5 cm below the wounded area (S2). SesTPSl was highly expressed after 6 hours post
wounding for both SI and S2, at a level 3-to 6-folds higher than that of control (0 h).
The expression of SesTPSl was downregulated between 8 h to 24 h after which it
climbed to between I-to 2-fold at 48 h. The pattern of expression of GuaiSl was not
very different when compared to SesTPSl. GuaiSl was drastically induced after just 2 h
of wounding to 2.8-folds. The expression levels fell back to lower levels (I-fold of
control) but increased to 2-folds at 24 h. The expression patterns of SesTPSl and
GuaiSl prevealed that they both responded similarly to wounding. In conclusion, fulllength
SesTPSl and GuaiSl genes were successfully cloned from A. malaccensis. This
is the first report on sesquiterpene genes from this species. It can be deduced that
wounding triggers genes in the sesquiterpene synthesis pathway and that might lead to
'gaharu' formation. |
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