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Transcriptome profiling, and the cloning and characterization of a monoterpene synthase from the seeds of Coriandrum sativum L.

University of British Columbia

Plant terpenes are a large and diverse class of naturally-derived compounds, valuable in the medicinal, perfume and culinary industries. The seeds of Coriandrum sativum (coriander) produce essential oil (EO) rich in monoterpenes, volatile C₁₀ terpenes. In this study the coriander seeds were viewed under a scanning electron microscope and stomata as well as vittae structures were observed on the seed surface and in cross-section, respectively. The EO of C. sativum seeds was extracted and qualitatively and quantitatively analyzed using gas chromatography-mass spectrometry. The EO terpene content findings agreed with previous literature, with linalool being the most abundant monoterpene in coriander EO. The transcriptome of coriander seeds, at three developmental stages (early, mid and late) was sequenced via Illumina technology. Analysis of the differential transcript abundance of select terpene biosynthetic genes between these stages revealed that two terpene production pathways are constitutively active in the seeds, with slight upregulation in the mid-developmental stage. All the genes involved with active photosynthesis and fatty acid biosynthesis and metabolism were also identified from the coriander transcript library. To validate the usability of the transcriptome sequence data, a terpene synthase candidate gene, CsγTRPS, encoding a 611 amino acid protein was expressed in bacteria and the recombinant protein purified by Ni-NTA affinity chromatography. Enzymatic assays with geranyl diphosphate (GPP), the precursor to monoterpenes, revealed that this 65.86 kDa recombinant protein catalyzed the conversion of GPP to γ-terpinene, with apparent Vmax, Km, and kcat values of 2.2 ± 0.2 pkat/mg, 66 ± 13 µM and 1.476 × 10-⁴ s-¹, respectively. Knowledge gained from these experiments will facilitate future studies concerning essential and fatty acid oil production in coriander. They will also enable efforts to improve the EO of coriander through metabolic engineering or plant breeding.

Text

2014-01-02

Attribution 2.5 Canada

http://hdl.handle.net/2429/45688

Biochemistry and Molecular Biology

University of British Columbia

UBCO

http://creativecommons.org/licenses/by/2.5/ca/