# Cloning and Characterization of Two Iridoid Synthase Homologs from _Swertia Mussotii_
## 1 Introduction  
_Swertia mussotii_ Franch belongs to the Gentianaceae family and grows on the Qinghai Tibetan Plateau at an altitude of greater than 3800 m [1; 2]. The entire _S. mussotii_ plant is used in Tibetan medicine known as Zang-Yin-Chen in Chinese. Zang-Yin-Chen has been widely used to treat diseases, such as liver disease and blood disease [3]. To date, a diverse array of pharmaceutically active compounds have been isolated from the _S. mussotii_ plant [4], including xanthones and their derivatives, flavonoids, terpenoids, iridoids and secoriridoid glycosides, such as swertiamarin and genitopicoside.  
Genitopicoside has been assessed to confirm its anti-inflammatory, analgesic, and anti-hepatitis effects [5; 6], whereas swertiamarin has been reported to have anti-hepatitis [7], anti-cancer [8], anti-diabetics [9] and anti-arthritic [10] activities. These results undoubtedly demonstrate the importance of _S. mussotii_ as a medicinal plant. Therefore, biological research concerning secondarymetabolism in _S. mussotii_ should be strengthened. The biosynthesis of swertiamarin and related compounds in _Swertia chirayita_ has been investigated and some of the enzymes involved in the biosynthetic pathway have been reported [11].  
At present, only one gene involved in swertiamarin biosynthesis has been characterized in _S. mussotii_[2]. To characterize other genes involved in this biosynthetic pathway in this important medicinal plant, we obtained the transcriptome in leaves of _S. mussotii_ using RNA-sequencing.  
Figure 1: Schematic representation of the proposed in secoiridoid pathway. GPPS, geranyl diphosphate synthase; GES, geraniol synthase; GSH, geraniol 8-hydroxylase; 8-HGO, 8-hydroxygeraniol oxidoreductase; IS, iridoid synthase; IO, iridoid oxidase; 7-DLGT, 7-deoxyloganetic acid glucosyltransferase; 7-DLH, 7-deoxyloganic acid hydroxylase; LAMT, loganic acid O-methyltransferasease; SLS, secologanin synthase. Genes in bold letters were previously cloned in _S. mussotii_; the gene in red was identified in this study.  
Based on the RNA-seq data and sequence similarity comparison with genes of _Catharanthus roseus_, we proposed assessing the secoriidoid biosynthesis pathway in _S. mussotii_, in which swertiamarin and gentiopicroside are processed via a classical MVA/MEP route of terpene biosynthesis followed by the secoriidoid pathway (Figure 1) [12]. Iridoid biosynthesis is initiated from geranyl diphosphate (GPP), a condensation product of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). Secoriidoids are derived from iridoids via the opening of a cyclopentane ring. In this study, we were particularly interested in characterization of the gene(s) encoding iridoid synthase (IS) because the enzyme catalyses the crucial step in the biosynthesis of iridoids [13]. IS belongs to the plant progesterone 5\(\beta\)-reductase (P5\(\beta\)R) family and uses the linear monoterpene 8-oxogeranial as substrate, probably couples an initial NAD(P)H-dependent reduction step with a subsequent cyclization step via a Michael reaction [14]. Recently, it was reported that iridoid synthase activity is common among the plant progesterone 5\(\beta\)-reductases and two (CrIS and CrP5\(\beta\)R4) of six _C. roseus_ P5\(\beta\)R genes may participate in secoriidoid biosynthesis [15]. In our RNA-seq data, only three P5\(\beta\)R gene homologs were recognized. We cloned the full-length cDNA of two genes, SmIS1 and SmIS2. After hetero expression in _E. coli_, we compared the structure by homology modelling and catalytic properties. We also compared the expression of these two genes in different organs.