## Results
### Identification of OSCs in the Transcriptome of E. Tirucalli  
It is well known that the cyclisation of 2,3-oxidosqualene catalyzed by OSCs is a critical branching point for sterol and triterpenoid biosynthesis [32]. In the present study, seven putative _OSCs_ (termed as _EtOSCs_) were identified in the _[PERSON]_ transcriptome (Table S4). These seven EIOSCs were subjected to the phylogenetic analysis together with many previously well-characterized OSCs. As shown in Figure 5, EtOSC1, EtOSC4, and EtOSC7 are clustered in the same sub-clade with multifunctional triterpene synthases, including AtLUP1 and AtLUP2, which cyclize 2,3-oxidosqualene to mainly yield luped with some minor by-products. EtOSC2 shows the 82% amino acid identity with a cycloartenol synthase (CAS) AtCAS1 from _A. thaliana_, indicating that EtOSC2 is a CAS in _[PERSON]_. EtOSC3, previously named as EtAS [21], is a \(\beta\)-amyrin synthase. EtOSC5 and EtOSC6 show relatively closer relationship with AtPEN3 (At5g36150) from _A. thaliana_. It has been reported that the cyclisation of 2,3-oxidosqualene by AtPEN3 predominantly produces intracalla-7,24-dien-3-ol with  
Figure 4: Expression analysis of the unigenes in the steps to euphol and \(\beta\)-sitosterol across the four _[PERSON]. [PERSON]_ tissues. (**A**) Proposed biosynthetic pathways for euphol and \(\beta\)-sitosterol; (**B**) Heatmap analysis showing the expression patterns of DEGs in different tissues. The transcript abundances of relevant genes were analyzed by the FPKM method. The green color indicates the low transcriptional level, and the red color indicates the high transcriptional level. In the figure: ACAT, acetyl-CoA acetyltransferase; HMGS, hydroxymethylglutaryl-CoA synthase; HMGR, hydroxymethylglutaryl-CoA reductase; MK, mevalonate kinase; PMK, phosphomevalonate kinase; MVD, mevalonate diphosphate decarboxylase; DXPS, 1-deoxy-D-xylulose-5-phosphate synthase; DXR, 1-deoxy-D-xylulose 5-phosphate reductio isomerase; MCT, 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase; CMK, 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase; MECPS, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase; HDS, (E)-4-hydroxy-3-methylbut-2-enyl-diphosphate synthase; HDR, 4-hydroxy-3-methylbut-2-enyl diphosphate reductase; IDI, isopentenyl-diphosphate delta-isomerase; GPPS, geranyl diphosphate synthase; FPPS, farnesyl diphosphate synthase; SQS, squalene synthase; SE, squalene epoxidase; and OSC, oxidosqualene cyclase. several minor side products (i.e., 6% of triucallol) [33; 34]. Both euphol and triucallol are the isomers of triucalla-7,24-dien-3-ol. As shown in Figure 4B, EtOSC5 and EtOSC6 were highly expressed in latex compared to the other tissues. These data tempted us to speculate that EtOSC5 and EtOSC6 might be the candidate OSC enzymes for euphol synthesis in _E. triucalli._  
Figure 5: Phylogenetic analysis of the putative OSCs identified in the _E. triucalli_ transcriptome. The OSCs protein sequences were aligned by ClustalX software and the phylogenetic tree was constructed by the neighbor-joining method using MEGA ver. 5.0 software with 1000 bootstraps. In the figure: LjLAS: BAE95410.1, _Lotus japonicus_; PgPNZ1: BAA33462.1, _Panax ginseng_; AtLAS1: NP_001327789.1, _A. thaliana_; NcLAS: JAU32215.1, _Noccasea caerulescens_; ThLAS: XP_010520561.1, _Tarenayu hasslerianu_; RsCAS: BAF73929.1, _Rhizophora stylosa_; AmCAS1: AAG44096.1, _Abies magnifica_; BpBPX2: BAB83086.1, _Betula platphylulla_; LjOSC5: BAE53431.1, _L. japonica_; GsCAS1: BAA76902.1, _Glycyrrhiza glabra_; AtCAS1: AAC04931.1, _A. thaliana_; NcMS: BAF35580.1, _Kandelia candel_; BpBPY: BAB83088.1, _B. platphylulla_; GubAS: ACV21067.1, _Glycyrrhiza valensis_; AeAS: ADK12003.1, _Aralia elata_; EtOSC: BAE43643.1, _E. triucalli_; EtAS: BAE43642.1, _E. triucalli_; DhBAS: KZV14753.1, _Dorocceras hygrometric_; VhBS: ABK76265.1, _Vacaria hispanica_; PsBSY: BAA97558.1, _Pisum sativum_; AabAS: ACA13386.1, _Artemisia annua_; EsBAS: APZ88354.1, _Eleutherococcus sentiscous_; GgbAS1: Q9 MB42.1, _G. glabra_; LjAMY1: BAE53429.1, _L.