Isolation and expression of hydroxyphenylpyruvate reductase (HPPR) in Orthosiphon aristatus
Orthosiphon aristatus or locally known as ‘Misai Kucing’ is a medicinal herb that contains rosmarinic acid. Rosmarinic acid is a natural plant constituent that can be found in Lamiaceae herbs and has been proven to have antioxidant, antiviral and antibacterial properties. In the biosynthetic path...
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| Format: | Thesis |
| Language: | English |
| Published: |
2014
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| Subjects: | |
| Online Access: | http://ir.unimas.my/id/eprint/8351/1/Zuliza%20%28full%29.pdf |
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| Summary: | Orthosiphon aristatus or locally known as ‘Misai Kucing’ is a medicinal herb that
contains rosmarinic acid. Rosmarinic acid is a natural plant constituent that can be
found in Lamiaceae herbs and has been proven to have antioxidant, antiviral and
antibacterial properties. In the biosynthetic pathway, hydroxyphenylpyruvate
reductase (HPPR) is one of the enzymes in production of rosmarinic acid. It is
responsible in reducing 4-hydroxyphenylpyruvate to 4-hydroxyphenyllactate in
dependence of NAD(P)H. In this study, partial length of the HPPR gene cDNA was
isolated. Reverse transcription-polymerase chain reaction (RT-PCR) and rapid
amplification of cDNA ends (RACE) technique were used in the experiment. Primers
were designed based on HPPR gene from other species obtained from the NCBI
database. The internal fragment nucleotide was obtained and the size of the sequence
is 583 bp. The 3’ and 5’ end of the HPPR gene was successfully isolated. Sequencing
result shows that the 3’ and 5’ end band contains 842 bp and 791 bp nucleotides
respectively. BLAST search revealed that the 3’ and 5’ end band nucleotide sequence
of O. aristatus HPPR had sequence similarities to HPPR genes from other plant
species (86 to 89% homology). In silico analysis was performed where the 3’ end
and 5’ end fragments were merged. The open reading frame (ORF) contains 924 bp
which is equivalent to a putative amino acid of 307 long. In this study, it is shown
that the deduced protein sequence contains a D-isomer specific 2-hydroxyacid
dehydrogenases NAD-binding signature and a putative catalytic domain. These
domains are typical domains for the family of D-isomer-specific 2-hydroxyacid
dehydrogenases. UV light is an abiotic stimulus that has been shown to induce
accumulation of rosmarinic acid in plants. In the UV analysis study, it is shown that
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UV treatment on O. aristatus plant increase expression of HPPR. The amplification
product intensity is lowest in control plant and is highest in plants exposed to UV for
60 minutes. |
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