## 2 Results  
### Screening and Isolation of Sesquiterpene Synthase Gene from P. minus  
Two new candidates of sesquiterpene synthase genes, _Pm_STPS1 (comp62410_co_seq6) and _Pm_STPS2 (comp47018_c0_seq1), were successfully identified through the sequence analysis of _P. minus_ transcriptome [40]. The 1098 bp _Pm_STPS1 transcript contained an open reading frame (ORF) of 1047 bp, encoding 348 amino acids with a calculated molecular mass of 40.9 kDa and an isoelectric point (pI) of 6. 15 (Figure S1). The ORF of _Pm_STPS1 started from the nucleotide position at 25 and ended at position 1071. The deduced amino acid sequence of _Pm_STPS1 (GenBankank accession no. MG921605) showed no signal peptide. A ProtParam analysis of the predicted amino acid sequence of _Pm_STPS1 revealed 47 negatively charged residues (Asp and Glu) and 52 positively charged residues (Arg and Lys), which represented the aliphatic index of this protein. This was a positive factor for the increased thermostability of the globular protein. The second sesquiterpene synthase transcript, _Pm_STPS2 (GenBank accession No. MG921606), was 1974 bp long and had an ORF of 1695, which encoded a polypeptide of 564 amino acids (Figure S2). The ORF of _Pm_STPS2 started from the nucleotide position at 97 and ended at position 1798. The calculated molecular mass of the mature protein was approximately 65.96 kDa, with a predicted pI of 5.75. A ProtParam analysis of the predicted amino acid sequence of _Pm_STPS2 identified 83 negatively charged residues (Asp and Glu) and 70 positively charged residues (Arg and Lys). Based on the BLASTx analysis, the predicted amino acid sequences of _Pm_STPS1 (Table 1) and _Pm_STPS2 (Table 2) had the closest hit to dimenol synthase from _Persicaria hydropiper_, with a 96% and 46% identity, respectively. The predicted amino acid sequence of _Pm_STPS2 was consistent with those of other sesquiterpene synthases encoding proteins of 550-580 amino acids, with molecular weights of 60-70 kDa. Conversely, the length of _Pm_STPS1, with only 348 amino acids, was much shorter than that of the other sesquiterpene synthases. Therefore, only _Pm_STPS2 met the range of other reported plant terpene synthases [21; 41; 42; 43; 44; 45]. The presence of the conserved domains in the _Pm_STPS1 and _Pm_STPS2 proteins was consistent with and similar to that of the other terpene synthase features. The terpene synthase family N-terminal domain (PF01397) and the synthase family C-terminal metal-binding domain (PF03936) contained highly conserved aspartate-rich motifs (DDoxD), which were essential for enzyme-substrate binding and catalytic function. The first aspartate-rich motif played a role in the determination of the chain length for the resulting premyl pyrophosphate. Based on the multiple sequence alignment of _Pm_STPS1 (Figure 1), several conserved motifs that were found in typical terpene synthases were identified, including the DDoxD (residue 100-104) and NSE/DTE (residue 245-253) motifs. The DDoxD and NSE/DTE motifs flanked the entrance of the active site. In addition to these motifs, there was a highly conserved arginine-rich RxR motif, which was involved in the complexing of the diphosphate group, after the ionisation of FPP [18; 39]. The RxR motif was located at 45 amino acids, upstream of the first DDoxD motif. For _Pm_STPS2, the conserved arginine-rich (RxR) region at amino acid position 278-281 was conserved in all of the terpene synthases [19]. Moreover, the aspartate-rich motif of DDoxD, which might have been the Mg\({}^{2+}\) binding site, was located at position 314-318 of the amino acid sequence. Another metal binding motif, the NSE/DTE motif, was detected at amino acid position 461-467 (Figure 1). The less conserved motif NSE/DTE, apparently evolved from a second motif that was conserved in premyl transferase. In general, these motifs were located on the opposite sides of the active site [6; 7]. The metal binding residues appeared as NSE in most microbial and fungal cyclases and as DTE in most plant cyclases.