Isolation and expression of hydroxyphenylpyruvate reductase (HPPR) in Orthosiphon aristatus

Zuliza, binti Ahmad (2014) Isolation and expression of hydroxyphenylpyruvate reductase (HPPR) in Orthosiphon aristatus. Masters thesis, Universiti Malaysia Sarawak, (UNIMAS).

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Abstract

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 iii 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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