Metabolome And Transcriptome Analyses Reveal The Types Of Alkaloids And Their Biosynthetic Pathways In Dendrobium Officinale

Dendrobium officinale Kimura et Migo is a medicinal plant of Dendrobium genus in Orchidaceae.Previous studies suggested that terpenoid indole alkaloids(TIAs)are the main alkaloid type of D.officinale.However,only one of the TIAs,quinine,was found in D.officinale.Therefore,clarifying whether D.officinale contains TIAs or other types of alkaloids is of great significance for the analysis of their biosynthetic pathways and metabolic regulation mechanism.In this study,D.officinale protocorm-like bodies(PLBs)were used as materials,and treated with TIAs precursors(tryptophan and secologanin)and methyl jasmonate(MeJA).Alkaloids and their biosynthesis-related genes of D.officinale were identified through metabolome and transcriptome.The differences of metabolites and genes which related to alkaloids,jasmonic acid(JA)and JA signal transduction pathways were also analyzed after TIAs precursors and MeJA treatment.And then combined with genomic data,to identify DoCYP450s involved in TIAs biosynthesis.The aim of this study is to clarify the alkaloid types and biosynthesis pathways of D.officinale,and to explore the molecular mechanism of TIAs precursors and MeJA for regulating their biosynthesis from the metabolism,transcription and whole genome levels.The main results are as follows:1.16 alkaloids were identified from D.officinale PLBs by using LC-MS/MS technology.Among them,sempervirine,carapanaubine and glycoperine were TIAs.The remaining 13 alkaloids were tropane,monoterpene,diterpene,pyrrolizidine,isoquinoline,quinolinidine and piperidine alkaloids.2.The total alkaloid content of D.officinale PLBs increased significantly treated with TIAs precursors and MeJA after 36 days.The results of metabolomics analysis showed that the content of 29 metabolites changed significantly under this treatment.Among them,carapanaubine(TIA)and its synthetic precursor tryptophan,xanthoplanine(isoquinoline alkaloid),carvone and rishitin(terpenes)exhibted significantly increased levels,indicating that the biosynthesis of these metabolites was positively regulated by TIAs precursors and MeJA.In contrast,the levels of fructose,glucose and galactose(sugars),aspartic acid and glutamic acid(amino acids)sharply decreased.Sugars and amino acids were negatively correlated with changes in the content of carapanaubine,xanthoplanine,carvone or rishitin.Such phenomena reflects that TIAs precursors and MeJA treatment can lead to the metabolic flow from primary metabolism(sugars and amino acids)to secondary metabolic processes such as TIAs,isoquinoline alkaloids and terpenes in D.officinale PLBs.3.Transcriptome sequencing results of D.officinale PLBs showed that 125 unigenes were involved in the biosynthesis of TIAs,isoquinoline alkaloids and tropane alkaloids.5883 differentially expressed genes(DEGs)were obtained by comparing the PLBs transcriptome which grew normally to 4 h and 24 h with that of TIAs precursors and MeJA treatment.35 DEGs were related to alkaloid biosynthesis,31 DEGs were involved in JA biosynthesis and its signal transduction pathway,and most of their expression levels were up-regulated.It is suggested that TIAs precursors and MeJA can regulate the expression level of genes related to alkaloid biosynthesis pathway,and mediate alkaloid biosynthesis through endogenous JA and its signal transduction pathway.The transcription factors,which belong to b HLH,AP2/ERF,WRKY,MYB and b ZIP family,had a strong co-expression relationship with the DEGs related to alkaloid biosynthesis pathway.It is concluded that these transcription factors can regulate the expression of genes related to the alkaloid biosynthesis pathway and promote the accumulation of alkaloids in D.officinale PLBs.4.93 DoCYP450 family members of D.officinale were analyzed by standard naming,classification and bioinformatics.Among them,DoCYP76AB4 was highly expressed in flower,leaf,stem and root tip,and its transcription level could be improved by TIAs precursors and MeJA treatment.Protein tertiary structures showed that DoCYP76AB4 had a similar structure to the G10 H with known function.These results showed that DoCYP76AB4 is DoG10 H.In addition,MeJA,SA and ABA could positively regulate the expression level of DoCYP76AB4,suggesting that they could regulate the biosynthesis of TIAs in D.officinale.