Cloning And Functional Identification Of Diterpene Synthase Genes From Three Species Of Aconitum

Aconitum plants are perennial or annual herbs in Ranunculaceae,with about 400 species distributed in the world and more than 200 species in China,which includes a variety of traditional Chinese medicine,such as:Aconitum carmichaelii and so on.This genus is rich in diterpenoid alkaloids,which play a significant role in analgesia,anti-inflammation,anti-tumor,anti-arrhythmia and so on.Since most of the diterpenoid alkaloids are toxic,their clinical application is greatly limited.Thus,the analysis of the biosynthesis pathway of these components in plants will clarify the molecular mechanism of toxicity formation of Aconitum herbs from the source,and provide an important basis for the subsequent selection or cultivation of low-toxic or non-toxic varieties by means of molecular marker-assisted breeding and genetic engineering,and also for the safety and sustainable utilization of Aconitum herbs.Previous study systematically identified the function of terpene synthase gene family in A.carmichaelii.To fully understand the function of terpene synthase genes of this genus,this study systematically compared and studied terpene biosynthesis related genes of the other three Aconitum species,A.stylosum,A.pendulum and A.gymnandrum.,and identifying the function of diterpene synthase gene family.The transcriptome information of the above three plants was obtained by the combination of Pacbio Iso-Seq full-length transcriptome and Illumina RNA-Seq.Through bioinformatics analysis,the upstream genes and terpene synthase genes involved in terpenoid skeleton biosynthesis in the three plants were deeply excavated,and compared with the related gene families of A.carmichaelii,the gene families of contraction and expansion in different plants were identified.Subsequently,16 diterpene synthase genes(diTPSs)were cloned for functional identification,and 15 enzymes were identified to have the function of diTPSs,including copalyl-diphosphate synthase(CPS)and kaurene synthase-like(KSL).AsCPS3 and AgCPS5 were the first cloned ent-8,13-CPP synthase.Functional characterization of KSL synthase from three species further confirmed that its product ent-kaurene and ent-atiserene were the precursor of diterpene alkaloids.This study laid a solid foundation for the functional differentiation of diterpene synthase and the biosynthesis of diterpene alkaloids in Aconitum plants.The main conclusions are as follows:1.Transcriptome analysis and identification of genes related to terpene biosynthesis in three species.In order to fully obtain full-length transcripts and gene expression information,Iso-Seq and RNA-Seq were conducted for A.stylosum、A.gymnandrum and A.pendulum respectively.A.stylosum was grown for one month in an artificial climate chamber after seed germination,and A.gymnandrum was grown on MS solid medium for one month after seed disinfection.The total RNA of the aerial parts(stems and leaves)and roots of A.stylosum and A.gymnandrum were extracted,and the Iso-Seq was conducted after equally mixing,meantime the aerial parts and roots were analyzed for RNA-seq.Previous study has already conducted the RNA-Seq of the field collected A.pendulum in the three parts of root,leaf and flower.The ent-CPP synthase was found to be mainly expressed in the root,and aconitine and 2-hydroxyaconitine were detected as the main components So we only carried out Iso-Seq on the root of A.pendulum which was grown for one month in an artificial climate chamber this time.Totally,the raw data of 31.66 Gb,20.53 Gb and 91.50 Gb were obtained from the following three species A.stylosum、A.pendulum and A.gymnandrum,respectively.After CCS extraction,FLNCs construction,second-generation short sequencing error correction,horizontal cluster analysis and de-redundant sequence,20556,15400 and 111975 fulllength transcripts of unigenes were obtained,respectively,with an average length of 2342 bp,1938 bp and 1511 bp.Gene annotation and homologous sequence alignment were used to mine the genes involving in terpene synthesis in the three species.The results revealed that 30、39、39 candidate genes in A.stylosum、A.pendulum A.gymnandrum.Compared with the published transcriptome data of A.carmichaelii,it was found that A.carmichaelii had the largest number of genes involved in terpene synthesis,followed by A.gymnandrum,A.pendulum and A.stylosum.Using the terpene biosynthesis gene families of A.carmichaelii as reference,among the 20 identified gene families,we found that 3 gene families were very conservative in four species and had consistent number of genes.While 8 gene families experienced some contraction in the other three Aconitum plants.For example,there were 11 diterpene synthase genes in A.carmichael,7,7 and 4 genes in A.gymnandrum,A.pendulum and A.stylosum,respectively.The other 11 gene families experienced some contraction and expansion in four species.The distribution of these genes in different Aconitum lays a foundation for the following study of the biosynthesis of special compounds in specific species.To further predict the function of the above genes,we used RNA-seq data to systematically analyze the expression profiles of terpene biosynthesis related genes in our four species of Aconitum.It was found that the diterpene biosynthesis pathway was dominant in the above four plants,which may be related to the rich diterpene alkaloids in them.MEP(Methylerythritol phosphate)pathway and candidate diterpene synthase genes AsCPS2,AsCPS3,AsKSLl and AsKSL2 were predominantly expressed in aboveground parts of A.stylosum.MEP pathway and candidate diterpene synthase genes AgCPS1,AgCPS2,AgCPS3,AgCPS5 and AgKSL1 were predominantly expressed in aboveground parts of A.gymnandrum,and the genes of MEP pathway were mainly expressed in leaves of A.pendulum.MEP pathway and diterpene synthase genes in were predominantly expressed in aboveground and root of A.carmichael.It has been reported that nine diterpene synthase genes in A.carmichael are active,among which AcKSL2-1 and AcKSL2-2 genes with high expression level in roots are considered to be key enzyme genes involved in diterpenoid alkaloid biosynthesis.Therefore,the candidate diterpene synthase genes highly expressed in the aboveground parts or roots of the above three plants are likely to be involved in the biosynthesis of diterpene alkaloids in various species.2.Cloning and bioinformatics analysis of diterpene synthase geneSixteen candidate diterpene synthase genes obtained by above full-long transcriptome analysis were cloned.The results showed that 13 diterpene synthase genes were cloned successfully,except AsKSL2,ApCPS1-2 and AgCPS5.When cloning the transcript_HQ_zcw_trans cript13343/f3p0/2492,two transcripts were obtained,named AsCPS1-1 and AsCPS1-2,respectively.When cloning the transcript_HQ_zcw_transcript1 2480/f2p0/257,three transcripts were obtained,named AsKSL1-1,AsKSL1-3,AsKSL1-4.Finally,16 diterpene synthase genes were totally cloned,including 7 diterpene synthase genes from A.stylosum,named AsCPS1-1,AsCPS1-2,AsCPS2,AsCPS3,AsKSL1-1,AsKSLl-3 and AsKSL1-4,3 from A.pendulum,named ApCPS1,ApCPS2 and ApKSL1,6 from A.gymnandrum,named AgCPS1,AgCPS2,AgCPS3,AgCPS4,AgCPS5 and AgKSL1.Bioinformatics analysis of the sixteen diterpene synthase genes demonstrated that the length ranged from 2340 to 2409bp and the amino acid length were 789-802.Online ExPASy analysis revealed that the molecular weight of the candidate genes were between 89.4 and 91.97 kDa,and the theoretical isoelectric point of the candidate genes were 5.3-5.99.According to the conserved domain,AsCPS1-1,AsCPS1-2,AsCPS2,AsCPS3,ApCPS1,ApCPS2,AgCPS1,AgCPS2,AgCPS3,AgCPS4 and AgCPS5 all contained DXDD domain,as Class Ⅱ diterpene synthase.AsKSLl-1,AsKSL1-3,AsKSL1-4,ApKSL1 and AgKSL1 all contained DDXXD,as Class Ⅰ diterpene synthase.To further predict their possible biochemical functions,a phylogenetic tree was constructed with the above diterpene synthase with conserved domain and 77 identified diterpene synthase The results showed that AsCPS3 and AgCPS5 formed a separate branch between monocotyledon and dicotyledonous Class Ⅱ diterpene synthase,which suggested that they were relatively primitive.The other CPSs synthase clustered with the identified A.carmichael CPS synthases into a separate group,that formed a sister group with CPS mainly involved in gibberellin biosynthesis in dicotyledons,suggesting that they may also catalyze geranylgeranyl diphosphate(GGPP)to produce ent-copalyl diphosphate(ent-CPP).The KSL synthases in the three plants were also clustered into a separate group with KSL in A.carmichael,suggesting that it may also participate in the diterpene biosynthesis pathway based on ent-CPP.And we also found that diterpene synthase genes in Aconitum plants were different from those in Labiatae and form separate branches,indicating that the functional differentiation of diterpene synthase in Aconitum plants mostly occurs in this genus,which is probably related to recent genome replication and polyploidy of Aconitum.3.Function identification 16 diterpene synthase through in vitro and in vivo assay.Through homologous recombination,16 diterpene synthase genes were constructed into prokaryotic expression vector pET32a and transformed into E.coli Transetta(DE3).The expression was induced by isopropyl-β-D-thiogalactoside(final concentration was 0.4mM).After ultrasonic fragmentation,the total protein was extracted and purified by His Trap HP purification column,the purified protein was used for enzymatic reaction in vitro,and the enzymatic product was identified by GCMS,thus identifying the biochemical function of the protein.Diterpene precursor GGPP and 11 purified proteins of class Ⅱ diterpene synthase were used for enzymatic reaction in vitro.CPP was dephosphorylated by CIAP so that it could be captured by GC-MS.The results showed that ten CPSs were active(except AgCPS3).Eight CPSs,AsCPS1-1,AsCPS1-2,AsCPS2,ApCPS1,ApCPS2,AgCPS1,AgCPS2 and AgCPS4 all had a single peak 1 in 16.7min.The retention time and mass spectrometry were consistent with the dephosphorized products of ZmCPS2 in wheat.Whereas,the product peak 2 of AsCPS3 and AgCPS5 were 12.01 min.It was found that the mass spectrometry consistent with 8,13-CPP the product of PvCPS3 from Panicum virgatum.Subsequently,since the phylogenetic tree predicts the above 8 CPSs as entCPP synthase,each CPSs was co-catalyzed with AtKS(specific reaction with ent-CPP)to determine the configuration of the product.The results showed that all the eight CPSs with GGPP reaction products reacted with AtKS to produce ent-kaurene.The retention time and mass spectrometry of the eight co-catalytic products were the same as those of the of ZmCPS2 and AtKS,indicating that their products are all the ent-CPP.We also found that AsCPS3 and AgCPS5 can also react with AtKS to produce ent-kaurene,indicating that their products are ent-8,13-CPP.Since the CPS products of A.carmichael and the above three kinds of plants are mainly ent-CPP,the five cloned KSL synthases were co-expressed with ZmCPS2 to identify their functions in vitro.According to the modular metabolic system of diterpene synthase in E.coli reported by Reuben J.Peters team,two-step diterpene synthase reaction was completed in it,and then the function of KSL was identified.Using the above two systems to identified KSLs,the results show that AsKSL1-3 can catalyze ent-CPP to form ent-kaurene,ApKSLl and AgKSLl can catalyze ent-CPP to form entatiserene.However,AsKSL1-1 and AsKSL1-4 only detected ent-kaurene in the metabolic engineering system of E.coli.The above results suggest that we should try different systems such as tobacco,yeast,E.coli and other systems for functional identification.Because of the extreme promiscuity of KSLs in Labiatae,we continue to use E.coli metabolic engineering system to co-express the above ApKSL1 and AgKSL1 with 11 different CPS to observe the promiscuity of KSLs in Aconitum plants.The results show that both KSLs in addition to being able to react with ent-CPP,they can also react with class Ⅱ diTPSs which can produce 8β-hydroxy-ent-CPP and ent-kolavenyl diphosphate.Both of them produced ent-atiserene and manoyl oxide,when they reacted with 8β-hydroxy-ent-CPP.Similarly,both of them produced ent-atiserene when they reacted with ent-kolavenyl diphosphate,but,only ApKSL1 produced manoyl oxide.Based on the above results,AcKSL2-1(ent-atiserene synthase)in A.carmichael were co-expressed with class II diterpene synthases producing 8β-hydroxy-ent-CPP and ent-kolavenyl diphosphate,respectively.We found that when AcKSL2-1 reacts with 8βhydroxy-ent-CPP,like ApKSL1 and AgKSLl,ent-atiserene and manoyl oxide were produced.The coexpression of AcKSL2-1 with class Ⅱ diterpene synthase producing ent-kolavenyl diphosphate was the same as that of AgKSLl,but only ent-atiserene was produced.It can be noted that although the KSL synthase of A.carmichael has substrate promiscuity,the main products of its reaction with different substrates are only entatiserene and manoyl oxide,which is completely different from the product spectrum of KSLs synthase in Labiatae,and the specific mechanism needs to be further studied.In this study,the full-length transcriptome of A.gymnandrum,A.pendulum and A.stylosum were sequenced and compared with the genes related to the terpenoid synthesis skeleton in A.carmichael.Sixteen diterpenoid synthase genes were identified and the substrate promiscuity of KSLs of Aconitum was preliminarily explored.These results provide important basic data and reference for the future study of the functional differentiation of diterpene synthase genes of Aconitum and the subsequent analysis of diterpenoid alkaloid(DAs)biosynthesis pathway.