Cloning And Functional Analysis Of Genes Involved In The Biosynthesis Of Sesquiterpene Lactones In Xanthium Strumarium L.

As a large class of secondary metabolites,sesquiterpene lactones(STLs)have various pharmaceutical activities,holding promising for drug development.The identification of genes involved in the formation of STLs would help understand how these STLs form in plants and would provide gene components for metabolic engineering them in microorganisms.To date,knowledge regarding the biosynthesis of STLs is limited.Based on their carbocyclic skeletons,STLs are classified as germacranolide,guaianolide,xanthanolide,pseudo-guaianolide and eudesmanolide.Xanthium strumarium L.,a traditional Chinese herb,is rich of xanthanolide and guaianolide,making this plant an idea platform to investigate the biosynthesis of STLs.In this study,we have constructed a comprehensive transcriptome database of X.strumarium and have identified a series of candidate genes putatively involved in STL biosynthesis.Using in vitro biochemical characterizations,several genes coding for sesquiterpene synthases and cytochrome P450s have been functionally confirmed to be involved in the pathways to xanthanolide in X.strumarium.Basically,the data obtained from this study are summarized as follows:1,Transcriptome databases from young leaves and isolated glandular trichomes of X.strumarium have been constructed.For this purpose,two X.strumarium cultivars,Hubei-and Zunyi-cultivars were used.Purified glandular trichomes were detached from the young leaves of the Hubei-cultivar,and total RNA from the purified trichomes were isolated.Using IlluminaHiSeq 2000 sequencing platform,we are the first to successfully construct the X.strumarium glandular trichome transcriptome.As a comparison,the transcriptome of the young leaves of both cultivars was also established.A total of 14.2 G data was produced and about 157 million clean reads were generated which assembled into 91,861 unigenes,among which 59,858 unigenes were annotated with public databases.2,Screening candidate genes in sesquiterpene lactone biosynthesis pathway in X.strumarium:In X.strumarium transcriptome database,11 sesquiterpene synthases have been identified.Moreover,genes encoding tailing enzymes in the modification steps to STLs have also been found,which include 70 cytochromes P450,30 alcohol dehydrogenases,20 aldehyde dehydrogenases,27 acetyltransferases,8 epoxide hydrolase unigenes,4 oxide cyclase and 116 transcription factors.3 The identification and function analysis of sesquiterpene synthases in X.strumarium:In this study,the cDNAs coding for three sesquiterpene synthases(designated as XsTPS1,XsTPS2 and XsTPS3)were isolated based on the X.strumarium transcriptome.XsTPSl,XsTPS2 and XsTPS3 were revealed to have the primary activities forming germacrene D,guaia-4,6-diene and germacrene A,respectively,by either ectopic expression in yeast cells or the purified recombinant protein based in vitro assays.Quantitative real-time polymerase chain reactions and metabolite profiling analysis in different plant tissues revealed that they are relevant to the biosynthesis of the targeted sesquiterpenes in vivo.In particular,we have found that XsTPS3 is the most likely sesquiterpene synthase to be involved in xanthanolide biosynthesis in X.strumarium.4 The identification and funcrtion analysis of X.strumarium glandular trichome specifically expressed genes for cytochrome P450s:Germacrene A acid(GAA)is a central precursor of diverse STLs.The hydroxylations of GAA at C6 or C8 will lead to the formation of C12-6 and C12-8-STLs,respectively.Interestingly,one glandular highly expressed CYP450 candidate called XsCYP3 in this study was found to have efficient conversion activity on GAA,producing an unknown product.This new product has been purified and the analysis of its structure is being undertaken by NMR analysis.