| Summary: | 碩士 === 國立成功大學 === 生物科技研究所碩博士班 === 95 === Many plants emit volatile compounds as a means of attracting pollinators or defense function. The volatile components mainly consist of terpenoid, phenylpropanoid, benzenoids, and fatty acid derivatives. TPS are key enzymes in terpene biosynthesis pathway. Many TPS genes have been isolated and characterized in dicotyledon and in monocotyledon. However, so far no TPS have been documented in orchids. Phalaenopsis spp. has been developed as an important cash plant in Taiwan ornamental industry. Therefore, I aimed to isolate and functionally characterize TPS gene, and compare the TPS transcripts from both scentless P. equestris and scented P. bellina.
First, floral volatiles emitted form both scentless P. equestris and scented P. bellina were compared. Although the contents of terpene in scented P. bellina were remarkable and mainly composed of monoterpenes (Hsiao et al., 2006), the contents of fatty acid derivatives were similar in both scentless P. equestirs and scented P. bellina. A full-length cDNA encoding sesquiterpene synthase was cloned and sequenced from the EST database of P. equestris flower buds, and thus named as PeTPS. It harbored an ORF cDNA of 1,647 bp and can be translated to a protein of 549 amino acids. An ORF cDNA of terpene synthase (PbTPS) from P. bellina was also cloned using PCR with a pair of primers derived from PeTPS sequence. Alignments for PeTPS and PbTPS deduced amino acid sequences showed a high identity (96.1 %) containing the conserved DDIYD motif involved in the binding of a divalent metal cofactor and an R(H/Q)X8W motif. In addition, no any transit peptides were predicted, suggesting that its subcellular localization was in the cytoplasm. Phylogenetic analysis of plant terpene synthases showed that the PeTPS and PbTPS were classified in the TPSa subfamily, which consists of sesquiterpene synthase and diterpene synthase. The PeTPS gene was a single-copy gene as examined by using Southern blot analysis. Temporal expression of PeTPS transcript was expressed from the first stage of flower buds to day 10 post-anthesis of P. equestris, with a maximal expression on the blooming day. However, the maximal expression of PbTPS was detected at the bud stages of P. bellina. Spatially, PeTPS was highly expressed in lip and pedicle and to a small amount in the vegetative tissues and all other floral organs. The PeTPS protein was expressed by using in vitro system of the RTS 100 Wheat Germ CECF kit. An expected protein size of 65 kDa encoded by PeTPS was detected by using western blot analysis. The recombinant PeTPS protein catalyzed the formation of three monoterpenes, including cis-citral, geraniol and trans-citral, when GDP was added as the substrate in the in vitro functional assay and analyzed by GC-MS. However, little or no sesquiterpenes or diterpenes were produced when FDP and GGDP were added as substrates, respectively. We concluded that the PeTPS was a monoterpene synthase even though its amino acid sequence was similar to sesquiterpene synthase. PeTPS and PbTPS are the first isolated terpene synthase genes in orchids. In future transgenic plants ectopic expressing PeTPS can be generated to emit aromatic volatiles, including cis-citral, geraniol and trans-citral. Therefore, scentless plants species could be changed to scented plant species by introducing the PeTPS or PbTPS gene.
|
|---|
