## 2 Results and Discussion  
### Identification of Gene Encoding 3S-Linalool Synthase from Grape Berries  
According to the report of [PERSON] _et al._[14], a total of 32 gene models were annotated as linalool synthase genes or bifunctional linalool/nerolidol synthase in 12\(\times\) grape genome based on nucleotide sequence similarity. Eighteen gene models out of the above 32 genes were considered to be pseudo genes that might be generated by mutation like nucleotide deletion in the progress of evolution. Only seven full-length cDNA fragments corresponding to six putative gene models (TPS54, 56-58, 61, 63) were screened out from grape cultivars Pinot Noir and Cabernet Sauvignon (PN and CS) cDNA libraries. These fragments coded for VvLinNer, and most of them were from the mixed PN template extracted from stems, berries, flowers and leaves. Here it should be noticed that VvPNLNG11-4 (corresponding to TPS 57, TPS 63, TPS 58 and TPS 61) all lacked a transit signal peptide at their _N_-terminal. It is suggested that they might only catalyze the generation of nerolidol rather than linalool _in vivo_ since sesquiterpenes like nerolidol are synthesized in cytoplasm and monoterenes like linalool are synthesized in plastid. In the present study, we had been trying to get full-length cDNAs of the TPS54 and TPS56 gene models from \"Riesling\" berries, but only _VvRiLinNer_, corresponding to TPS56 was successfully obtained from young \"Riesling\" berries (Table 1). This _VvRiLinNer_ sequence (accession numbers: JQ062931)contained an open reading frame (ORF) of 1734 base pairs for a predicted protein of 577 amino acids, a molecular weight of 66.1 kDa and a calculated isoelectric point of 6.22 (conducted by NAMAN6.0). [PERSON] _et al._[14] previously cloned _VvPNLinNer2_ (HM807392 in Genbank) from _Vitis vinifera_ Pinot Noir and _VvCSLinNer_ (HM807393 in Genbank) from _Vitis vinifera_ Carbernet Sauvignon based on the designated gene _VvTPS56_. Here, the _VvRiLinNer_ fragment obtained shared very high similarity in ORF with _VvPNLinNer2_ (99.9%) and _VvCSLinNer_ (99.1%). Accordingly, it is suggested that _VvRiLinNer_, _VvCSLinNer_and _VvPNLinNer2_ were considered as allele genes of _VvTPS56_ (GSVIVT01005272001) distributing in different grape cultivars. However, the length of _VvPNLinNer2_ from \"Pinot Noir\" was 1839 bp, which had an additional 105 nucleotides in its 5'-terminus compared with the sequences of _VvRiLinNer_ and _VvCSLinNer_. Several primers were designed on the basis of GSVIVT01005272001 nucleotide sequence to clone this putative 105-nucleotide sequence from _Vitis vinifera_ Riesling berries, but no PCR product was attained. Interestingly, the localization of the VvRiLinNer was _in silico_ predicted using three different databases (TargetP, PSORT and ChloroP). The results all showed that this enzyme was present in chloroplast, which is totally the same as VvPNLinNer2 with an additional 105 bp sequence. This phenomenon is difficult to explain at present. Here it was worthwhile to note that the VvRiLinNer exhibited only approximately 55% and 54% amino acid homology with _Antirrhinum majus_ nerolidol/linalol synthases [15], approximately 48% with _Arabidopsis thaliana_ lanolol synthase [18], as well as about 55% with _Actinidia arguta_ lanolol synthase [23]. Based on putative amino acid sequences, the VvRiLinNer was grouped into the TPS-g subfamily (Figure 1), which lacks the RRxsW motif in the _N_-terminal domain, a key feature of all monoterene synthases of the TPS-b and TPS-d groups [11,24]. In the _C_-terminal domain, the VvRiLinNer contained two highly-conserved aspartate-rich motifs, DDxxD and NSE/DTE that are functional in the metal-dependent ionization of prenyl diphosphate substrates (Figure 2). The VvRiLinNer also showed the same feature sequences as VvPNLinNer2 and VvCSLinNer (Figure 2). This homology analysis strongly supports this view that the VvRiLinNer performs almost the same biological function as VvPNLinNer2 and VvCSLinNer in grape berry.