Genomic characterization, molecular cloning and expression analysis of two terpene synthases from Thymus caespititius (Lamiaceae)
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The identification, isolation and functional characterization of two genes encoding two monoterpene synthases—γ-terpinene synthase (Tctps2) and α-terpineol synthase (Tctps5)—from three chemically distinct Thymus caespititius (Lamiaceae) genotypes were performed. Genomic exon–intron structure was also determined for both terpene synthase genes, revealing an organization with seven exons and six introns. The cDNA of Tctps2 was 2,308 bp long and had an open reading frame of 1,794 bp encoding for a protein with 598 amino acids. Tctps5 was longer, mainly due to intron sequences, and presented high intraspecific variability on the plants analyzed. It encoded for a protein of 602 amino acids from an open reading frame of 1,806 bp comprising a total of 2,507 bp genomic sequence. The amino acid sequence of these two active Tctps genes shared 74 % pairwise identity, ranging between 42 and 94 % similarity with about 50 known terpene synthases of other Lamiaceae species. Gene expression revealed a multi-product Tctps2 and Tctps5 enzymes, producing γ-terpinene and α-terpineol as major components, respectively. These enzymatic results were consistent with the monoterpene profile present in T. caespititius field plants, suggesting a transcriptional regulation in leaves. Herewith reported for the first time for this species, these two newly characterized Tctps genes improve the understanding of the molecular mechanisms of reaction responsible for terpene biosynthesis and chemical diversity found in T. caespititius.
KeywordsGene expression Genomic characterization Terpene synthase α-Terpineol γ-Terpinene Thymus
Inter-simple sequence repeat
Random amplification of polymorphic DNA
Solid phase micro extraction
T. caespititius terpene synthase 2 gene (γ-terpinene synthase)
T. caespititius terpene synthase 5 gene (α-terpineol synthase)
This study was partially funded by the Fundação para a Ciência e Tecnologia (FCT) under Pest/OE/EQB/LA0023/2011 and research contract PTDC/AGR-GPL/101334/2008. The funding from CRUP/DAAD, Ref. A11/12 supported the collaboration with AG Pharmaceutical Biotechnology group from Martin Luther University Halle-Wittenberg. The authors are grateful to Prof. Dr. K. Bahcevandziev (Escola Superior Agrária de Coimbra) for the maintenance of T. caespititius plants; Eng. V. Lopes (Banco Português de Germoplasma Vegetal) for T. caespititius samples from mainland Portugal; Prof. Dr. L. Domingues and to C. Oliveira (Universidade do Minho) for all the help, teaching and availability in the beginning of work lab with cloning and functional expression of Tctps; Prof. Dr. M. Oliveira and to M. Rodrigues (Instituto de Tecnologia Quimica e Biológica) for their availability and all the help with Gateway system.
Conflict of interest
The authors declare that they have no conflict of interest.
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