## 3 Discussion  
_Scutellaria_ is a useful plant genus with a broad global distribution. The transcriptome data generated in this study give us an overview of terpenoid biosynthesis in _S. burbata_. Thirty-three genes encode all known enzymes involved in terpenoid backbone biosynthesis and 14 terpene synthase genes were identified. Three diterpene synthase genes were further cloned from the root and functionally identified. Consistent with their close phylogenetic relationship with known diterpene synthases from Lamiaceae, _Sb_TPS8 and _Sb_TPS12 were found to produce miltiradiene, and _Sb_TPS9 would be involved in gibberellin biosynthetic pathway in _S. burbata_. Results of this study showed that besides the well-known _neo_-clerodane diterpenoids, normal-CPP mediated abietane diterpenoid biosynthesis also exists in _S. burbata_. The size of most gene families in _S. burbata_ are similar to the other 48 Lamiaceae species [12]. Some families have a much smaller number of genes, including HMGR in the MVA pathway, DXS in the MEP pathway, and TPS genes in the TPS-a and TPS-b subfamilies that are responsible for mono- and sesquiterpene biosynthesis [36]. In Lamiaceae species, the HMGR gene family in _Hyssopus officinalis_ (11), _Pogostemon cablin_ (12), _Prunella vulgaris_ (15), and _Thymus vulgaris_ (10) are even larger than those in _Gossypium raimondii_, which was identified to have the largest HMGR gene family (nine genes) in a survey including sequences from 14 land plants [42]. The DXS gene family is also highly expanded from three to 21 in _Pogostemon cablin_[12]. The TPS gene family expansion is correlated with mono- and sesquiterpene diversity [12], thus, the contraction of TPS-a and TPS-b clade genes together with HMGR in the MVA pathway may coincide with the lower chemical diversity of those compounds in _S. burbata_. HMGR and DXS have been reported as bottleneck enzymes of the MVA and MEP pathways, respectively [43]. DXS is the first step in the MEP pathway, which catalyzes pyruvate and glyceraldehyde 3-phosphate in the formation of deoxyxylulose 5-phosphate. Multiple studies have shown that DXS plays a central role in modulating the production of MEP-derived precursors to plastidial isoprenoids such as carotenoids, chlorophylls [44; 45; 46], and tanshinones in _Sa. Miltiorrhiza_[47; 48; 49]. HMGR catalyzes the conversion of HMG-CoA to MVA, which is widely used as the metabolic engineering target for improving terpenoid production [49; 50; 51]. The hierarchical clustering of gene expression demonstrated that genes in the MVA and MEP pathways should have different biological roles. Most of the genes in the DXP pathway (five of the seven enzymes) and MVA pathway (four of the six enzymes) had highest expression in the root (group II, Figure 3B). This group also included the complete enzymes from IPP to the diterpene synthase _Sb_TPS8 and _Sb_TPS12 (Figure 3), thus illustrating root is the most metabolically active tissue in _S. burbata_ for abietane diterpenoid biosynthesis. In group III, two of the DXS genes, DXS1 and DXS 5, had higher expression level in aerial parts than in root and flower. In particular, DXS1 was closely clustered with _Sb_TPS11, while HMGR1 and GGPPS1 were closely clustered with _Sb_TPS14, thus these genes provide further direction for modulating the bioactive _neo_-clerodane or abietane diterponoids with genetic engineering. _Neo_-clerodane diterponoids are signature metabolites of _Scutellaria_ species. Based on the specific expression level of _Sb_TPS11, we presumed that _Sb_TPS11 will function as a kolavenyl diphosphate synthase, which is the first step toward _neo_-clerodane diterponoids. The close relationship of _Sb_TPS11 with the identified CLPP synthase from _Salvia divinorum_[31; 33] and _Vitex agnus-castus_[34] in Lamiaceae further supports this hypothesis. However, _Sb_TPS11 showed no significant phylogenetic relationship with the known CLPP synthases (_Tw_TPS10 and _Tw_TPS14) of _Tripterygium wilfordii_[39].