Domingues, Lucília

Lima, Ana Sofia

Oliveira, Carla Cristina Marques de

Trindade, Helena

Universidade do Minho: RepositoriUM

Microbiotec11 | Functional Genomics and Systems and Synthetic Biology Posters (PS8)  418 PS8: 2 Isolation and molecular cloning of γ-terpinene synthase gene from Thymus caespititius Ana Sofia Lima1Faculty of Sciences of University of Lisbon, Plant Biology Department, Institute for Biotechnology and Bioengineering, Centre of Plant Biotechnology, C2, Lisbon, Portugal; 2IBB-Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Braga, Portugal 1, Carla Oliveira2, Lucilia Domingues2, Helena Trindade1 Thymus caespititius Brot., commonly known as ‘tormentelo’ or ‘erva-úrsula’, is a Lamiaceae aromatic specie endemic of the NW Iberian Peninsula, and of the Azores and Madeira archipelagos characterized for high essential oil chemical variability [1, 2]. As part of an ongoing effort to isolate genes involved in scent production on different chemotypes, genomic characterization of exon and intron numbers, sizes and placement, of a putative gene encoding a monoterpene synthase, γ-terpinene synthase (TcTPS2), was performed on chemically distinct T. caespititius accessions collected at Azorean islands and in the Mainland Portugal. In Origanum vulgare TPS2 is responsible for the first step of the ‘cymyl’-pathway, giving rise to phenolic terpene isomers thymol and carvacrol and related compounds [3]. Being these terpenes the main components in two of the chemotypes of T. caespititius, the present work aims at showing the expression of TcTPS2 in Thymus. T. caespititius mRNA was isolated from aerial parts collected during the flowering stage and a homology based RT-PCR strategy was used to clone the TcTPS2 gene. One cDNA clone (TcTPS2-D1) was chosen to perform the heterologous expression in Escherichia coli for further characterization. A BLASTP search on GenBank revealed 27 to 93% of similarity of the cloned TcTPS2 gene to other known terpene synthases genes from different members of other Lamiaceae species. Full-length His•Tag TcTPS2-D1 cDNA was ligated to the vector pET-29a(+) for protein expression. Recombinant TPS2 was detected in E. coli cultures by SDS-PAGE with the predicted molecular weight (67 kDa). The best soluble protein production was obtained for cultures induced with 0.2mM of isopropyl-1-thio-b-d-galactopyranoside (IPTG) for 19h at 20ºC in a rotary shaker. Scale-up protein production is in progress, and further purification as well as enzymatic assays will be performed. Herewith reported for the first time for the genus Thymus, these cloning and expression approaches will contribute to elucidate the function of these TPS genes. 1. Figueiredo A.C. et al. (2010) Nat. Prod. Commun. 5: 1465-1476, 2. Figueiredo A.C. et al. (2008). Cur. Pharm. Design 14: 3120-3140, 3. Crocoll C. et al. (2010). Plant Mol. Biol. 73: 587-603. Acknowledgments: This study was partially funded by the Fundação para a Ciência e Tecnologia (FCT) under research contract PTDC/AGR-GPL/101334/2008.   

Isolation and molecular cloning of γ-terpinene synthase gene from thymus caespititius

http://repositorium.sdum.uminho.pt/bitstream/1822/17015/1/Pages%20from%20Microbiotec11-6.pdf

Isolation and molecular cloning of γ-terpinene synthase gene from thymus caespititius

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Universidade do Minho: RepositoriUM