Molecular cloning and characterization of monoterpene synthases from Litsea cubeba (Lour.) Persoon

Abstract

Mountain pepper (Litsea cubeba (Lour.) Persoon) is an evergreen tree in the Lauraceae family and has attracted attention for the antioxidant and antibacterial effects of its essential oils. The dominant components of the essential oil of L. cubeba are monoterpenes. To investigate the biosynthesis of monoterpenes that have bioactivity in L. cubeba, three cDNAs corresponding to monoterpene synthase genes were isolated, and the corresponding enzymes were functionally characterized in vitro. The genes obtained were named LcTPS1, LcTPS2, and LcTPS3. Transcript levels for LcTPS1 were most prominent in the leaf tissues, and levels of LcTPS2 and LcTPS3 were more evident in fruit tissues than in leaf tissues. Phylogenetic analyses indicated that LcTPS1, LcTPS2, and LcTPS3 all belong to TPS-b subfamily of angiosperm monoterpene synthases. For each genes, the corresponding enzymes were expressed and functionally characterized. LcTPS1 converted geranyl diphosphate to trans-ocimene, and LcTPS2 converted geranyl diphosphate to α-thujene, while LcTPS3 is a mutifunctional enzyme that converted geranyl diphosphate to α-thujene and (+)-sabinene, respectively.

Transcriptome Sequencing and Expression Analysis of Terpenoid Biosynthesis Genes in Litsea cubeba

“…The number of terpene synthases identified from L. cubeba , ten monoterpene synthases and four sesquiterpene synthases, and the three functionally characterized monoterpene synthase gene sequences have been deposited in the NCBI GenBank database and functionally characterized [11]. At present, a relatively large number of TPSs have been found in plants with genomic sequences.…”

“…Despite the medicinal and economic importance of L. cubeba , little genomic information is available for this non-model genus, and only 40 nucleotide sequences have been deposited in the NCBI GenBank database (as of the 22 nd of May 2013). To date, three genes involved in monoterpene synthases have been functionally characterized [11], showing the potential for genomic research on this plant. However, a lack of sequence data has limited extensive and intensive research.…”

Identification, Functional Characterization, and Evolution of Terpene Synthases from a Basal Dicot

“…3), in which the majority of angiospermous monoterpene synthases reside (Chen et al, 2011). The TPS sequences most similar to LnTPS1 are those of a-thujene synthase from L. cubeba (Lauraceae; Chang and Chu, 2011), with 92% identity, and a-terpineol synthase from M. grandiflora (Lee and Chappell, 2008), with 57% identity.…”

“…Previous studies in the basal dicots have identified a few TPS genes encoding monoterpene synthases in Litsea cubeba (Lauraceae; Chang and Chu, 2011) and monoterpene and sesquiterpene synthases in Magnolia grandiflora (Lee and Chappell, 2008). These studies have shown that the monoterpene synthases fell into clade b, to which most monoterpenes from the eudicot species belong, suggesting that clade b diverged from the rest of the TPS gene family at the base of the angiosperms.…”

Cloning and functional characterization of a monoterpene synthase gene from Eleutherococcus trifoliatus

“…Functional characterization of EtLIM: The activity of EtLIM was measured in vitro GC/MS analysis as described by Chang and Chu (2011) with slight modifications. The purified protein (20 μg) was added to monoterpene synthase buffer [25 mM HEPES (pH 7.2), 100 mM KCl, 10 mM MgCl 2 , 10% (v/v) glycerol, 5 mM DTT] with 30 μM GPP (Sigma).…”