Studies On The Biosynthesis And Regulation Of Diterpenoids,Taxol And Tanshinones

Diterpenoids and their derivatives are important sources of drugs,among which Taxol and tanshinones are representative compounds.Taxol,as a tetracyclic diterpenoid compound,has a significant effect on the treatment of ovarian cancer,breast cancer,etc.Currently,it is one of the highest-selling botanicals;Tanshinones,belonging to abietane-type diterpene quinones,are one of the main bioactive ingredients in Salvia miltiorrhiza Bunge.and have good clinical effects on the treatment of cardiovascular and cerebrovascular diseases.In this work,we explored the biosynthesis of Taxol and tanshinones and their regulation at transcriptional or post transcriptional level with high-throughput sequencing technologies and so on.To understand the biosynthesis of Taxol and its regulation,we sequenced the transcriptomes from the roots,stems,leaves of Taxus cuspidata Sieb.et Zucc.with Illumina and Pacbio sequencing platforms,respectively.A total of 58,602 unigenes were acquired and,among them,13,636 unigenes underwent AS events.RT-PCR results confirmed that a tissue-preferential alternative splicing(AS)pattern existed for some genes.We found a total of 139 cytochrome P450s(CYP450s),31 BAHD acyltransferases(ACTs)and 1940 transcription factors(TFs).Among all the CYP450s,79 CYP450s were discovered for the first time in T.cuspidata,and 66 CYP450s were gymnosperm-specific.Based on motif,evolution and coexpression analysis,we found that 9 CYP450s and 7 BAHD ACTs were potential leading candidates for Taxol biosynthesis and 6 TFs were possibly involved in the regulation of this process.A system was setup for full-length transcriptome analysis of species without reference genome and discovery of genes related to secondary metabolism,thus laying the foundation for the study on the secondary metabolism and its post-transcriptional regulation,such as AS,using high-throughput sequencing technologies.Our previous work on S.miltiorrhiza genome sequencing suggested that the genes for tanshinone biosynthesis were possibly clustered together in the genome.By constructing and screening the S.miltiorrhiza BAC library and sequencing the selected BAC clones with Illumina and Pacbio platforms,we obtained two tanshinone biosynthetic gene clusters with lengths of 374.44kb and 464.46kb respectively.Both of gene clusters contain 7 CYP450s,3 terpene synthases,2 TFs and 1 F-box protein genes.CYP450s,TFs and F-box protein genes mostly have tandem duplication,but some genes have different copy numbers in the two gene clusters.These two gene clusters contain the identified genes for the signature enzymes CPS1,CPS2,KSL1 and genes for the tailing enz-ymes CYP76AH1 and CYP76AH3,which provide good evidences for the existence of the tanshinone gene cluster.Four candidates and three identified CYP450s were cloned and heterologously expressed in Saccharomyces cerevisiae.The catalytic activities of these 7 enzymes were tested with 21 tanshinone-type compounds,and it was confirmed that CYP71D410 had catalytic activities on ferruginol,neocryptotanshinone Ⅱ and 20-deoxocarnosol.NMR results showed that CYP71D410 hydroxylated the C-3 position of ferruginol to produce 8,11,13-abietatriene-3,12-diol.In addition,the function of CYP71D410 in S.miltiorrhiza was further confirmed by RNAi experiment in the hairy root system.The identification of tanshinone biosynthetic gene clusters will not only greatly contribute to the elucidation of tanshinone biosynthetic pathway,but also further deepen our understanding on the basic biological principle of plant gene clusters.Abscisic acid(ABA),as a plant hormone,could increase the accumulation of secondary metabolites in various plants.To investigate effects of the abscisic acid on the production of bioactive components in S.miltiorrhiza,we sequenced the root and leaf transcriptomes before and after treatment with ABA using novel nanopore PromethION technology.A total of 63,213,822 clean reads were generated,and after removing redundancy,58,487 transcripts were obtained.After treatment with exogenous ABA,approximately 50.0%unigenes were differentially expressed.Among them,expression levels of genes related to anthocyanin synthesis and glycosyl transfer were significantly up-regulated,and expression levels of genes involved in flavonoid synthesis and methyl transfer were significantly down-regulated.Approximately 61.0%of the genes underwent AS events in S.miltiorrhiza and intron retention(IR)was the main type.After treatment with ABA,the number of AS events generally showed a decreasing trend,which revealed that ABA extensively affected the AS pattern in S.miltiorrhiza.Among the five types of AS events,IR was most affected by ABA.The AS events in leaves were more affected by ABA than in roots,which suggested the response of AS events to ABA was tissue-preferential.After treatment with ABA,the number of splicing isomers of several genes involved in ABA signaling pathway increased significantly,which showed that AS may be involved in the regulation of ABA signal transduction.Most of genes involved in salvianolic acid biosynthesis were up-regulated by ABA,so we inferred that the exogenous ABA could promote the salvianolic acids biosynthesis in S.miltiorrhiza,while the genes for tanshinone biosynthesis can’t be induced by ABA.Our work suggested that the production of secondary metabolites can be regulated by ABA at transcriptional and post-transcriptional level.By elucidating Taxol and tanshinone biosynthetic pathways,we can provide valuable components for their study on synthetic biology and lay the foundation for the production of Taxol,tanshinones and their intermediates by constructing "cell factory",thus greatly benefiting the production and R&D of the related drugs.In addition,the understanding on the regulatory mechanism underlying the biosynthesis of Taxol and tanshinones will provide functional molecular markers for molecular breezing and accelerate the selection process of fine varieties,thus ensuring the sustainable development of the Traditional Chinese Medicine industry.