## 2 Results and Discussion
### Identification of Gene Encoding 3S-Linalool Synthase from Grape Berries
\begin{table}
\begin{tabular}{c c c c c} \hline \begin{tabular}{c} **GenBank** \\ **Number** \\ \end{tabular} & \begin{tabular}{c} **Gene ID (12\(\times\))** \\ \end{tabular} & \begin{tabular}{c} _VvTPS Gene_ \\ **Model**\({}^{\dagger}\) \\ \end{tabular} & \begin{tabular}{c} **Cloned Genes** \\ \end{tabular} & \begin{tabular}{c} **Note**\({}^{\ddagger}\) \\ \end{tabular} \\ \hline \begin{tabular}{c} XM002270140.1 \\ \end{tabular} & GSVIVT01005272001 & _VvTPS56_ & \begin{tabular}{c} _VvRiLinNer_ (JQ062931, \\ this study) _VvPNLinNer2_ \\ (HM807392) _VvCSLinNer_ \\ (HM807393) \\ \end{tabular} & Full \\ \
\begin{tabular}{c} XM002270071.1 \\ \end{tabular} & GSVIVT01005271001 & _VvTPS65_ & No gene has been cloned & Stop in \\
\begin{tabular}{c} XM002266813.1 \\ \end{tabular} & GSVIVT01005221001 & _VvTPS54_ & _VvPNLinNer1_(HM807391) & Full \\ \hline \end{tabular} \({}^{\dagger}\) and \({}^{\ddagger}\), The “_VvTPS_ Gene Model” and “Note” are annotated by [PERSON] _et al._[14].  
\end{table} 
Table 1: Annotation of the three terpenids synthase (_VvTPS_) genes. **Figure 1.** Phylogenetic tree illustrating the VvRiLinNer as a member of terpene synthase (TPS)-g subfamily. The unrooted neighbor-joining tree was created using ClustalX and visualised in TreeView. Division into subfamilies is based on cluster analysis ([PERSON] _et al._ 1998 [11]; [PERSON] 2003 [12]; [PERSON] _et al._, 2011 [10]). Only a subset of the known plant TPSs is shown. Sequence abbreviations: AaLis, _Actinidia argute_ linalool synthase (ADD81294); AgBis, _Abies grandis_ (_E_)-a-bisabolene synthase; AgMys, _Abies grandis_, myrcene synthase (AAB71084); AgPis, _Abies grandis_, pinene synthase (AAB71085); AmLinNer2, _Antirrhinum majus_ linalool/nerolidol synthase (ABR24418); AmMys, _Antirrhinum majus_myrcene synthase (AAO41726); AtLis, _Arabidopsis thaliana_ linalool synthase (AAO85533); CiGer, _Cichorium intybus_ germacrene A synthase (AAM21658); CmCds, _Cucurbita maxima_ copalyl diphosphate synthase (AAD04292); FaNes1, _Fragaria \(\times\) ananassa_ nerolidol synthase1 (CAD57083); MaLis, _Mentha aquatic_ linalool synthase (AAL99381); MpFas, _Mentha \(\times\) piperta_ (_E_)-\(\beta\)-farnesene synthase (AAB95209); PaBis, _Picea abies_ (_E_)-\(\alpha\)-bisabolene synthase (AAS47689); PaLis, _Picea abies_ (_-_)-linalool synthase (AAS47693); PcLis, _Perilla citriodora_ linalool synthase (AAX16075); SlCds, _Solanum bycopersicum_ copalyl diphosphate synthase (BAA84918); SmDiTPS, _Selaginella moellendorffii_ a bifunctionalditerpene synthase (AEK75338); StVes, _Solanum tuberosum_ vetispiradiene synthase (BAA82092); VvGerD, _Vitis vinifera_ (_-_)-germacrene D synthase (AAS66357); VvGwbOcs, _Vitis vinifera_ L. Gewurztraminer (_E_)-beta-ocimene synthase (ADR74204); VvRiLinNer, _Vitis vinifera_ L. [PERSON] linalool/nerolidol synthase (JQ062931); VvTer, _Vitis vinifera_ (_-_)-\(\alpha\)-terpineol synthase (AAS79352); VvVal, _Vitis vinifera_ (_+_)-valencene synthase (AAS66358). To investigate the catalytic properties of the VvRiLinNer protein, we expressed _VvRiLinNer_ in _E. coil_ cells and purified the recombinant protein (Figure 3A). _In vitro_ reaction analysis showed that VvRiLinNer could convert GPP into (3\(S\))-linalool and convert FPP into (_E_)-nerolidol (Figure 3B,C). This means that VvRiLinNer is a bi-functional enzyme _in vitro_, which is similar to VvPNLinNer2 and VvCSLinNer [14]. In addition, we also detected trace levels of \(\beta\)-myrccene, (_E_)-\(\beta\)-ocimene, (_Z_)-\(\beta\)-ocimene and alloocimene in the GPP-containing reaction system, as well as trace levels of \(\alpha\)-bisabolol, \(\alpha\)-farnesene and \(\beta\)-farnesene in the FPP-containing reaction system (Figure 3B,C). No monotepene or sesquiterpene was detected in two controls. Figure 2: Alignment of the amino acid sequence of VvRiLinNer with other VvUPS. The alignment of VvRiLinNer predicted amino acid sequence with other VvUPS was performed using Align X (Vector NTI Advance 11, Invitrogen). VvTer (GenBank No.