| Nelumbo nucifera belongs to Nymphaeaceae,Nelumbo,has a high economic value in actual production practice.A large number of metabolites,including alkaloids,steroids,flavonoids,triterpenoids,glycosides and polyphenols,have been extensively detected in Nelumbo nucifera and are tightly related to their pharmacological activity,such as anti-ischaemia,antioxidant,anticancer,antiviral,as well as anti-obesity.As the most abundant natural metabolites in plants,terpenoid compounds have a variety of important biological functions.However,genes related to terpenoids and their synthetic pathways have been little studied in Nelumbo nucifera.A total of six parts of Nelumbo nucifera,seeds(S),young leaves(YL),mature leaves(ML),white flowers(WF),yellow flowers(YF)and red flowers(RF)were selected as experimental materials for metabolomic and transcriptomic analyses.Co-expression networks of genes related to metabolite and terpenoid synthesis were constructed to screen key genes.Key genes were cloned and bioinformatically analyzed.The vitro bacterial color assay was used to identification of the enzymatic activity of NnGGPPS.Transgenic tobacco was constructed to characterize the biological function of the NnGGPPS1 gene.The main conclusions are as follows.(1)21 terpenoids,including 2 monoterpenes,3 sesquiterpenes,4 diterpenes and 12 triterpenes,were detected in 6 sets of samples by extensive targeted metabolomics analysis,and the terpenoid content varied in different samples.A total of 126.95 GB of clean data was generated by transcriptome analysis and assembled into 29,630 Unigenes in this study.Functional annotation analysis resulted in the screening of 127 Unigenes associated with terpenoid synthesis.(2)The co-expression network of terpenoids and terpenoid synthesis-related genes was constructed,which showed that HMGR and DXS,as rate-limiting enzymes in the MVA and MEP pathways,were highly correlated with a variety of terpenoids.In addition,GGPPS1,GGPPS3,GGPPS4 and the synthesis of several terpenoids such as Hesperic acid,catalpol methyl triacetate,betulinic acid and 12,13-dihydroursolic acid were closely related.Furthermore,multiple metabolites were associated with each terpene synthase gene.(3)A total of four genes encoding geranylgeranyl pyrophosphate synthases,named NnGGPPS1,NnGGPPS2,NnGGPPS3 and NnGGPPS4,were obtained by functional annotation analysis of the transcriptome,encoding amino acid sequences ranging in length from 320 to 366 and protein molecular weights from 34.986 to 39.821 KD.Phylogenetic analysis showed that NnGGPPPS1 and NnGGPPS2 belong to the same evolutionary branch,GGPPS.LSU,NnGGPPS3 and NnGGPPS4 belong to two different evolutionary branches,GGPPS.SSUII and GGPPS.SSUⅠ.Conserved sequence motif analyzed of four NnGGPPS proteins showed that NnGGPPS1 and NnGGPPS2 and the GGPPS of other species have similar motif composition,indicating that the GGPPS.LSU of NnGGPPS1 and NnGGPPS2 and the rest of species may have similar functions.The amino acid sequence comparison results showed that NnGGPPS1 and NnGGPPS2 conform to the characteristics of GGPPS.LSU,containing a CxxxC(x indicates an arbitrary amino acid,same below)structural domain,and two aspartate-rich structural domains-(first aspartate rich motif,FARM)DDxxxxD and(second aspartate rich motif,SARM)DDxxD.NnGGPPS3 is GGPPS.SSUII,containing two CxxxC and one DDxxxxD structural domains with DDxxD mutated to DDxxE.NnGGPPS4 conforms to the sequence characteristics of GGPPS.SSUⅠ,which lacks the FARM and SARM domains and contains only two CxxxC domains.(4)The co-expression network of NnGGPPS1 and NnGGPPS2 with genes related to the synthesis of MEP pathway,chlorophyll,carotenoids,sterols and plastid quinones was constructed.The results showed that NnGGPPS1 was highly correlated with some MEP pathway genes,carotenoid synthesis,gibberellin synthesis and chlorophyll synthesis compared to NnGGPPS2,indicating that NnGGPPS1 plays an important role in the synthesis of carotenoids and chlorophyll.The NnGGPPS genes were cloned and the pMAL-c5x-NnGGPPS prokaryotic expression vector were constructed,and the protein encoded by NnGGPPS1 gene was shown to have enzymatic activity by bacterial color in vitro experiments.(5)OE#NnGGPPS1 transgenic tobacco was constructed to investigate the biological function of NnGGPPS1 gene.Phenotypic analysis of transgenic tobacco showed that overexpression of NnGGPPS1 could promote the growth of tobacco,resulting in increased leaf area,taller plants and increased biomass in transgenic tobacco.The carotenoid and chlorophyll contents of the transgenic tobacco were measured and found to be significantly higher in chlorophyll,α-carotene,β-carotene,octahydrolycopene and zeaxanthin compared to wild-type tobacco.Chlorophyll fluorescence assay showed that overexpression of NnGGPPS1 enhanced the photosynthetic efficiency of transgenic tobacco.The expression patterns of MEP pathway-related genes,carotenoid and chlorophyll synthesis-related genes were analyzed by qPCR,and the results showed that the expression of these genes were higher in transgenic tobacco than in wild-type tobacco. |
