# Functional Analysis of Two Terpene Synthase Genes Isolated from the Flowers of _Hosta_ 'So Sweet'
## 3 Results  
### Phylogenetic Analysis of TPS Genes from H. 'So Sweet'  
The RNA of _Hosta_ flower was reverse-transcribed into cDNA and analyzed by PCR. Hs_TPS1_ (1758 bp) and Hs_TPS2_ (1770 bp) encode 586 and 590 amino acids, respectively, with molecular weights of 67.48 and 68.1 kDa. The Hs_TPS1_ full-length sequence shared 56.79% amino acid identity with the (R)-linalool synthase from _Magnolia champaca_ (ART66980.1). In addition, Hs_TPS1_ had 56.69% and 56.50% homology with trans-ocimene synthase from _Litsea cubba_ (AEJ91554.1) and _Cinamoun micranthum f. kanehirae_ (RWR 88332.1). The homology of Hs_TPS1_ with monoterpene synthase from a _Lilium_ hybrid cultivar (QBP79170.1) was 52.90%. The Hs_TPS2_ full-length sequence shared 63.06% and 61.75% identity with the linaloal synthase from a _Freesia_ hybrid cultivar (AQMS05913.1) and 54.02% identity with the myrcene synthase from _Lilium_ sp. BT-2016 (AMT81307.1). Hs_TPS1_ and Hs_TPS2_ shared 44.46% identity with each other (Figure 1). Terpenoid synthases are usually categorized into seven branches, named TPS-a, TPS-b, TPS-c, TPS-d, TPS-e/f, and TPS-g [6]. The following motifs have already been reported to be implicated in catalysis by TPS: an anarginine-enriched region, often appearing as an RRx8W motif; a specific linaloal synthase region, often appearing as (M/L)L(S/Q/N) L(F/Y)EAS; asparticacid-enriched and glutamic acid-enriched regions, appearing as DDXXD and NSE/DTE motifs [37; 38]. The RRx8W motif is characteristic of the N-terminal domain of monoterpene synthases, as well as the TPS-b subfamily in angiosperms and the TPS-d subfamily in gymnosperms [39; 40; 41]. It has also been demonstrated to be isomerized in the first step from geranyl diphosphate to linalalyl diphosphate [42; 43]. These motifs may be involved in the linaloal synthase activity of Hs_TPS1_ and 2. The LSYEA(S/A) motif of Hs_TPS_ was located in a highly similar region to that seen in other m_TPS_s, considered to be an active site peptide. The highly conserved DDXXD motif is generally recognized as a binding site for the metal ion-chelated diphosphate ester substrate [44]. In contrast, NSE/DTE motifs are less conserved, and they are known to be combined with divalent metal ion cofactors in enzyme activity [37; 45]; they were present as the DDL(G/A)TS(E/T)AE motif in the Hs_TPS_ amino-acid sequences. In general, monoterpenes tend to cluster by genus rather than function [37]. Thirty-five different m_TPS_s from twenty-seven different species and two Hs_TPS_s were aligned in this study, and a phylogenetic tree was constructed (Figure 2). It is worth mentioning that most of the reported linaloal/nerolidol synthases belong to the TPS-g group. This is a subfamily closely related to the TPS-b group, which comprises angiosperm acyclic terpene synthases. Both TPSs were clustered into the TPS-b group, close to the TPS-g group.  
Figure 1: Protein sequence alignment of the two HsTPS was constructed using ClustalW and was edited using Escript 3.0. The three conserved motifs, RRx8W, DDxD, and DDxxTxxxE, characteristic of terpene synthases, were overlined in gray.  
Figure 2: Results of phylogenetic analysis of terpene synthase proteins from \(H\). ‘So Sweet’ (Hs_TPS1_,Hs_TPS2_) and other plants, done using 1000 replications for bootstrapping by MEGA 6 program. Circle at the branches of the tree shows bootstrap values.