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|Universiti Kebangsaan Malaysia, Malaysia|
|Keynote: J Plant Pathol Microbiol|
|Statement of the Problem: Persicaria minor (synonym Polygonum minus) produces a broad range of secondary metabolites such as sesquiterpenes that contribute towards the unique aroma of this plant. In an effort to understand the biosynthesis of these compounds, candidate genes involved in the sesquiterpene biosynthetic pathway have been identified from an expressed sequences tags (ESTs) collection. The purpose of this study is to characterize a gene which was initially identified as an-alpha farnesene synthase gene from the EST studies. Methodology & Theoretical Orientation: The full length cDNAs from P. minus was isolated and cloned into Escherichia coli, Lactococcus lactis and Arabidopsis thaliana following standard protocols. Enzymatic assay for the recombinant sesquiterpene synthase was performed using farnesyl diphosphate as substrate and the products from the enzymatic assays were analyzed using gas chromatography-mass spectrometry (GC-MS). Findings: The full length sequence of P. minor sesquiterpene synthase cDNA clone (PmSTS) was 2035 bp in size and was expressed in E. coli as a ~65 kDa soluble protein whereas in L. lactis, the size of the recombinant protein was ~63kDa. For enzyme activity assay, the major product of the recombinant PmSTS in E. coli was α-farnesene whilst for L. lactis recombinant protein, the major product was 5-sesquiphellandrene with beta-farnesene as a minor product. Subsequent expression in A. thaliana also produced transgenic lines with increased α-sesquiphellandrene production. Finally, new expression in E. coli produced a recombinant PmSTS that released 5-sesquiphellandrene as a major product and α-farnesene as a minor product, similar to the L. lactis recombinant protein. Conclusion & Significance: The identity of a plant sesquiterpene synthase has been confirmed as a 5-sesquiphellandrene synthase. The correct identity of the gene was finally achieved due to the updated version of the mass spectral library used in identifying the products from GC-MS.|
Roohaida Othman received her PhD in Biochemistry from University of Southampton, and joined Universiti Kebangsaan Malaysia as a Lecturer in 1995. Her research interest is focused on understanding the molecular mechanisms underlying the biosynthesis of commercially important metabolites in plants and algae. Her research group has employed tools of molecular biology, biochemistry and physiology as well as genomics and transgenics technology platforms to study the enzymes involved in these pathways. They have also developed protocols for higher plant and algae RNA extraction methods and overexpression of recombinant proteins in bacterial systems. She has been Editor-in-Chief for the Journal of Tropical Plant Physiology since 2010 and has been reviewer for several journals including International Journal of Food Properties.
Email: [email protected]
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