Cloning And Functional Study Of The Terpene Synthase Genes From Aconitum Carmichaelii

Aconitum carmichaelii is the most representative medicinal herb in Aconitum.It has been widely used to treat rheumatism,rheumatoid arthritis,ankylosing spondylitis,lumbar muscle strain,and nerves.Pain and other diseases for two thousand years.It has made great contributions to human health.The diterpene alkaloids(DAs)are its main medicinal components in Aconitum,which have anti-inflammatory,analgesic,anti-arrhythmic and other biological activities.At present,the acquisition of diterpene alkaloids mainly depends on the processing of Raw herbs.With the increasing demand for A.carmichaelii in clinical and the traditional Chinese medicine industry,it may face the sustainable development problem.Parsing the biosynthetic pathway of diterpenoids alkaloids is a necessary precondition for adjusting the content of diterpene alkaloids and obtaining diterpene alkaloids in A.carmichaelii by the metabolic engineering,which is of great significance to the sustainable utilization of aconitum resources.In this study,we applied a combination of Pacbio full-length transcriptome sequencing and 13 tissue RNA sequencing to provide the full-length transcriptome of A.carmichaelii(N50 2446bp).We got Nineteen TPSs and five alternative splicing isoforms by gene annotation,homology alignment and PCR cloning.Twelve diterpene synthase and two sesquiterpene synthase were identified by prokaryotic expression and diterpene metabolism engineering system.Seven of the TPS-c subfamily genes reacted with geranyl geranyl pyrophosphate(GGPP),the common substrate of diterpene,to produce the intermediate ent-copalyl diphosphate(ent-CPP),and five Ac KSLs separately reacted with ent-CPP to produce ent-kaurene,ent-atiserene,and ent-13-epi-sandaracopimaradie,among which Ac KSL3-2 is the first identified diterpene synthase gene that catalyses the production of ent-13-epi-sandaracopimaradie,and this compound has not been reported in Aconitum.In combination with the function of enzyme,the expression profile of terpenoid synthase genes in 13 tissues and the content analysis of DAs,we eliminated the possibility that other terpenoid synthases were involved in DAS biosynthesis and validated that ent-CPP is the sole precursor to all DAs biosynthesis,with Ac KSL1,Ac KSL2 s and Ac KSL3-1 responsible for C20 atisine and napelline type DAs biosynthesis,respectively.Atisine may be an important precursor for the formation of C19-diterpene alkaloids.We parsed the first two steps of the biosynthetic pathway of diterpenoids alkaloids of A.carmichaelii after GGPP.The high quality full-length transcriptome and the functional analysis of terpenoid synthase gene family laid a solid foundation for the further study of the C19-DAs biosynthetic pathway and took an important step to increase the content of diterpene alkaloids in Aconitum and the industrial production of the important active ingredients such as aconitine,which is of far-reaching significance to the sustainable development of Chinese medicine resources.