Identification And Functional Characterization Of Genes Involved In Terpenoid Biosynthesis In Scutellaria Barbata And Prunella Vulgaris

Scutellaria barbata and Prunella vulgaris belong to the family Lamiaceae.S.barbatais an important medicinal plant widely used in Asian countries such as China,South Korea and India.The neo-clerodane-type diterpenoids are the main active ingredients in S.barbata and has good cytotoxicity against various cancer cells.P.vulgaris is the most widely distributed species in the Lamiaceae family and is a commonly used Chinese herbal medicine and herbal preparations.Triterpenoids are the main active constituents of P,vulgaris.In this thesis,Illumina sequencing and analysis were carried out on different tissues of S.barbata and P.vulgaris,and the genes in the biosynthesis pathway of terpenoids were identified systematically,and some diterpene synthase genes were functionally identified.These results gave a comprehensive understanding of the terpenoid biosynthesis in S.barbata and P.vulgaris,and lay a foundation for future studies on the functional characterization of candidate genes involved in the biosynthesis of pharmacologically active terpenoids in S.barbata and P.vulgaris.The main results are as follows:1.Transcriptome sequencing and assembly of different tissues of S.barbata and P.vulgaris.Extracting RNA from three tissues of B.sylvestris(BZLR),stem and leaf(BZLS),and flower(BZLF);Prunella vulgaris root(XKCR),stem and leaf(XKCS),flower spike(XKCF),and immature seeds(XKCZ)RNA of four tissues.Each organization repeats twice.121,958,564 clean RNA-sequence reads from S.barbata were assembled into 88,980 transcripts for a total of 52,211 unigenes.The average length of unigenes is 1370 nt,and the length of N50 is 2144 nt.121,809,359 clean RNA-sequence reads of P.vulgaris were assembled into 108,672 transcripts for a total of 57,985 unigenes.Unigene has an average length of 1,120 nt and an N50 of 1,843 nt.By searching common public databases such as Swiss-Prot,TrEMBL,Pfam(HMM),Gene Ontology and KEGG,there were 36,659 unigenes(70.23%)in S.barbata and 33,323 unigenes(54.47%)in P.vulgaris were annotated.2.Using NCBI public database and known genesfrom Salvia miltiorrhiza and Arabidopsis thaliana to identify genes involved in the biosynthesis of terpenoid backbone in S.barbata and P.vulgaris.Noted 71 unigenes and 53 unigenes,respectively.A total of 33 genes(32 with full-length cDNA)and 35 genes(35 with full-length cDNA were identified to be involved in the biosynthesis of terpenoid backbone in S.barbata and P.vulgaris.Compared with the whole genome identification of backbone genesin Sa.miltiorrhiza,we identified all the 17 gene families involved in the biosynthesis of terpenoid backbone in S.barbata and P.vulgaris,including AACT,HMGS,HMGR,MVK,PMK,MDD genes in the MVA pathway;DXS,DXR,MCT,CMK,MDS,HDS,HDR,IDI,IPK genes in the MEP pathway;prenyltransferase FPPS,GGPPS,GPPS.SSU gene.Ten gene families were found to be conserved among the three species,including AACT,HMGS,MK,PMK,MDD,DXS,DXR,MCT,CMK and MDS.The other seven gene families show a certain degree of expansion and contraction.For example,the HMGR gene family has 4 genes in P.vulgaris and Sa.miltiorrhiza,while only 3 genes in S.barbata.DXS has 5 genes in S.barbata and Sa.miltiorrhiza,while 6 genes in P.vulgaris.The IDI,FPPS,HDR and GGPPS gene families are also slightly different in the three speicies.3.14 and 17terpenoid synthase genes were identified in S.barbata and P.vulgaris Cluster analysis showed that one and two genes in S.barbata and P.vulgaris belonged to the TPS-a gene family,respectively,and it was speculated to participate in the biosynthesis of monoterpenes.There are 5 and 9 genes belonging to the TPS-b gene family,respectively,which are presumed to be involved in the biosynthesis of sesquiterpene in S.barbata and P.vulgaris.There are 8 and 6 diterpene synthase genes in S.barbata and P.vulgaris.Since P.vulgaris is rich in triterpenoids,the triterpenoid synthesis-related genes in P.vulgaris were identified.A total of 9 genes including 2 squalene synthase,2 squalene monooxygenase,and 5 oxidized squalene cyclasewere identified.The OSC gene family is responsible for the diversified skeleton formation of triterpenoids.Cluster analysis showed that PvOSC2,PvOSC3 in P.vulgaris and SbOSC2 in S.barbata were grouped with the dammarenediol ? synthase from Panax ginseng,which was involved in the ginsenosides biosynthesis in P.ginseng.Thus their catalytic function deserves further exploration.To farther reveal the possible biological functions of the above-mentioned terpenoid biosynthesis-related genes and their relationship,we performed cluster analysis of FPKM values based on log-2 transformation on the expression profile data of S.barbata and P.vulgaris.The results showed that the diterpene synthase genes TPS8,TPS2 and HMGR3 of S.barbata were mainly expressed in roots and flowers.The genes in the DXP pathway show high levels of expression in different tissues,which is consistent with the recognition that diterpenoids are mainly synthesized by the DXP pathway.The DXS1 and diterpene synthase gene TPS 11 are specifically expressed in the stems and leaves of the aerial part.Therefore,this gene may be involved in the biosynthesis of the neo-clerodane-type diterpenoids in S.barbata.Both monoterpene and sesquiterpene synthases have lower expression levels in different tissues.Therefore,the diterpenoid biosynthetic pathway plays an important role in S.barbata.It was found that the genes highly expressed in P.vulgaris were mainly derived from MVA pathways,such as HMGR1,HMGR3,IDI1,AACT1,FPPS1,and HMGS and triterpene synthase-related genes SQS1,OSC2 and OSC3,which are also mainly derived from MVA pathway.It is consistent with reports that P.vulgaris is rich in triterpenoids.4.Cloning and in vitro functional identification of diterpene synthase gene in S.barbata and P.vulgaris Three and two diterpene synthase genes were cloned from the root cDNAs of S.barbata and P.vulgaris,respectively,and their sequences were consistent with the transcriptome assembly sequence.The cDNA lengths of SbTPS8,SbTPS9 and SbTPS12 were 2793 bp,2690 bp and 3102 bp,the protein lengths were 803aa,810aa and 594aa,respectively.The CDNA lengths of PvTPS14 and PvTPS16 are 2403 bp and 1785 bp,the protein lengths are 800 aa and 593 aa,respectively.SbTPS8,SbTPS9 and PvTPS14 have a conserved DxDD domainwhich was known for the Class ? diterpene synthase.SbTPS12 and PvTPS16 have a conserved DDxxD domain which was belonged to the Class ? diterpene synthases.A systematic phylogenetic tree was established with 52 known functional diterpene synthases from Lamiaceae.It showed that SbTPS9 and PvTPS15 were grouped together with diterpene synthase involved in ent-CPP formation.SbTPS8 and PvTPS14 are clustered with specialized CPP/LDPP synthase in the Lamiaceae family.Further prokaryotic expression in E.coli revealed that the recombinant proteins of SbTPS8,SbTPS9 and SbTPS12 were well soluble,and the target protein after purification was consistent with the prediction.PvTPS14 and PvTPS16 have low levels of soluble proteins under the same conditions.No soluble protein was obtained despite the optimized induction temperature and purification conditions.The purified recombinant SbTPS8 and SbTPS9 individually reacted with the known enzyme of SmKSL1 from Sa.miltiorrhiza and AtKS from A.thaliana.SbTPS8 in combination with SmKSL1 formed miltiradiene,which was identified by comparison to the product of combination of normal-CPP synthase(SmCPSl)and miltiradiene synthase(SmKSLl)from Sa.miltiorrhiza.No product was observed when combining SbTPS8 and AtKS(specific to ent-CPP),showing that SbTPS8 was a normal-copalyl diphosphate synthase.The similar combination of SbTPS9 with AtKS and SmKSLl showed that SbTPS9 only produced ent-kaurene when combined with AtKS;thus SbTPS9 was found to be an ent-copalyl diphosphate synthase.Further,SbTPS12 was found to produce miltiradiene when combined with SbTPS8,thus SbTPS12 was identified to be a miltiradiene synthase in S.barbata.In vitro assay of PvTPS14 and PvTPS16 showed no significant stable activity.5.Class I diterpene synthase from different origins were found to have extreme promiscuity.Thus,to probe the substrate promiscuity of SbTPS12 and PvTPS16,a previously reported modular metabolic engineering system was utilized.This system enables facile co-expression of CPSs and KSLs in E.coli with high production.We first verified the system by coexpression the sclareol synthase(SsSS)from Salvia sclarea with 12 class ? diterpene synthases,and the results showed that SsSS can react with 9 diterpene synthaseswith the high production which was consistent with the literature,while the product is low when coexpressed with CLPP(12)and ent-CLPP(13).The special resistance of the terpentedienyl diphosphate(9)vector was not suitalbe for high-throughput operation.Thus,eleven class II diTPS were assembled into the metabolic engineering system for coexpression with SbTPS12 and PvTPS16.It showed that SbTPS12 co-expressed with copalyl diphosphate synthase produced the same product of miltiradiene with the in vitro assay.PvTPS16 also produced miltiradiene when co-expressed with copalyl diphosphate synthase,which was accordance with close phylogenetic relationship with other miltiradiene synthase.SbTPS 12 can also react with ent-kolavenyl diphosphate(12)to produce ent-kaurene.PvTPS16 react with endo-CPP(6)and 8a-hydroxy-CPP(7)to produce 13R-(+)-manoyl oxide and miltiradiene individually.It is indicated that SbTPS12 and PvTPS16 have different substrate promiscuityIn summary,this study used Illumina sequencing,digital expression profiling,E.coli in vitro enzymatic identification and E.coli engineering system to systematically identify and analyze the genes involved in terpenoid biosynthesis related genes in S.barbata and P.vulgaris.The high level expression of the DXP pathway gene,the expansion of the diterpene synthase gene and the different expression patterns in the root and aerial parts of S.barbata suggest that the diterpenoid biosynthesis pathway is the dominant pathway of S.barbata.However,the MVA pathway of P,vulgaris and a series of enzyme genes of triterpenoid biosynthesis showed higher expression in different tissues,suggesting that the triterpenoid biosynthesis pathway is the dominant pathway in P.vulgaris.The identification of four diterpene synthase functions and the study of substrate promiscuity clarified the function of the related genes and their catalytic reaction characteristics.These results led us to a comprehensive understanding of the terpenoid biosynthesis of S.barbata and P.vulgaris?and illustrate the molecular mechenism of the chemical diversity of terpenoids in S.barbata and P.vulgaris,and further provide useful information for enhancing the production of bioactive neo-clerodane diterpenoids or triterpeoids through genetic engineering in S.barbata and P.vulgaris.