Minning And Identification Of OSCs In Gynostemma Pentaphyllun Based On Analysis Of Transcriptome

Gynostemma(Cucurbitaceae)is a genus of perennial creeping herbs.Gypenosides(triterpenoid saponins),the main effective components of G.pentaphyllum,are mainly distributed in leaves.More than 160 glycosides were found in G.pentaphyllum.According to modern pharmacology,gypenosides have many pharmacological activities including anticancer function,antianxiety function,antidiabetics,anti-atherosclerosis,blood fat-reducing and immunoprotection.Gypenosides draw a lot of public attentions as their structural similarities to glycosides found in Panax ginseng.OSCs play an important role in the synthesis of triterpenoids backbone.In this study,we have cloned and identified OSCs from G.pentaphyllum and lay a solid foundation for elucidating gypenosides biosynthesis pathway and its regulations.The plant signaling compound MeJA can induce the expression of FPS and SS,which belonging to the gypenosides upstream biosynthesis pathway.Therefore,we speculated that the OSCs also induced by MeJA in G.pentaphyllum.Based on this speculation,we analyzed the transcriptional levels of the G.pentaphyllum handled with MeJA by Illumina HiSeqTM 2000.After sequencing,we obtained 202 million raw reads and assembled into 82,073 unigenes.In GO analysis,45 genes possibly involved in secondary metabolic process and 2,015 genes possibly involved in metabolic regulation.In KEGG analysis,291 genes possibly involved in terpenoid metabolism,in which 82 genes are related to the terpene backbone biosynthesis and 42 genes are related to the synthesis of sesquiterpenes and triterpenoids.Based on annotation and homology alignment,five OSCs named GpOSCl-5 were screened and several highly conserved motifs,including DCTAE,MWCHCR and QW,were found in these candidate OSCs.According to real-time PCR,GpOSC1 shows high expression in leaves and GpOSC2-5 show high expression in stems.GpOSC1 and GpOSC4 were downregulated by MeJA.GpOSC2,GpOSC3 and GpOSC5 were also suppressed in preliminary stage,but gradually increase until 24h.According to phylogenetic analysis,GpOSC1 clustered with β-Amyrin;GpOSC2 clustered with cycloartenol synthase and shared 87.8%similarity with identified CAS in Cucurbita pepo;GpOSC3 clustered with lanosterol synthase;GpOSC4 clustered with cucurbitadienol synthase and shared more than 80%similarity with identified CbQ;GpOSC5 clustered with isomultiflorenol synthase and shared 82.3%similarity with identified IMS in Luffa aegyptiaca.For identifying the function of candidate OSCs,GpOSC1-5 were cloned into the yeast expression vector pYES2 and heterologously expressed in Saccharomyces cerevisiae strain GIL77(gal2 hem3-6 erg7 ura3-167)lacking lanosterol synthase activity.After induction with galactose,the cells were collected,refluxed with 20%KOH/50%ethanol and extracted with the same volume of n-hexane.Reaction products were analyzed by GC-MS and the results showed that GpOSCl can cyclize 2,3-oxidosqualene into two compounds(product 1 and 2)and GpOSC4 can cyclized 2,3-oxidosqualene into one compound(product 3).According to the analysis results,these three compounds’retention time show distinct difference with authentic dammarenediol Ⅱ,lupeol,β-amyrin,cucurbitadienol and betulin.The structures of these three products will be further studied by NMR.In this study,we identified the function of candidate OSCs based on the combination of RNA-seq and heterologous expression.This work not only provided useful components for gypenosides metabolic engineering and synthetic biology,but also provided theoretical support for molecular breeding of G.pentaphyllum.