Transcriptomics Analysis And Targeted Metabolic Profiling For Pathway Elucidation And Identification Of A Geraniol Synthase And Three Iridoid Synthases Involved In Iridoid Biosynthesis From Gardenia Jasminoides

1 ObjectiveGardenia jasminoides Ellis,an evergreen shrub of the family Rubiaceae widely existing in tropical and subtropical regions,known as ’shanzhi’,’baichan’ and’huangzhizi’ in Chinese herbal name,is a traditional Chinese medicine.The main medicinal part of G.jasminoides is the dried fruit,and its main constituents are iridoid glycosides,such as gardenoside and geniposide.Recently,a large number of reports have proved that it has pharmacological activities,such as anti-inflammatory,antitumor,treatment of diabetes,liver protection,nervous system and cardiovascular system protection,etc.Through transcriptome sequencing and bioinformatics analysis of G.jasminoides,our research group predicted the biosynthesis pathway of iridoid by refering to published research reports and characterized the function of GjGES(Gardenia jasminoides Geraniol Synthase)and GjIS,GjIS2 and GjIS4(Gardenia jasminoides Iridoid Synthase),to provide a theory for synthesis biology of iridoids glycosides.2 Method2.1 Profiling of iridoid metabolites in different tissues of G.jasminoidesThe iridoid glycosides of G.jasminoides were extracted by 50%methanol using ultrasonic and detected by UPLC-MS.Then compared with seven standard substances to analyze their metabolic differences.2.2 Transcriptome sequencing of G.jasminoides and identifying genes involved in the biosynthesis of iridoids in G.jasminoidesTotal RNA of lower leaves,top leaves,flowers,and fruits(three biological replicates for each tissue;12 RNA samples in total)were isolated and used for cDNA library construction and transcriptome sequencing.Then transcriptome data was assembbled and were annotated against public databases(NR,KEGG,SwissProt,UniProt,KOG).In addition to functional annotation,putative genes involved in the biosynthesis of iridoid were further screened by NCBI BLAST and iridoid ring scaffold pathway of G.jasminoides was profiled by refering to known pathways.2.3 Differential gene expression analysisFPKM values from transcriptome squencing and RT-qPCR(real-time quantitative PCR)experiments were performed for analysis of expression abundance of GjGES(G.jasminoides Geraniol Synthase)and GjIS,GjIS2 and GjIS4(G.jasminoides Iridoid Synthase)in different tissues.2.4 Cloning,heterologous expressions and functional identification of two enzymesPrimers were designed to amplify the open reading frames of the two key enzyme genes screened by transcriptome data,and subcloned into the p-ENTR-D/Topo vector for sequencing.Using the cloning vector as template,the target gene was subcloned into the prokaryotic expression vector pMAL-c5X(MBP tag).Then the recombinant plasmid was transformed into Rosetta for prokaryotic expression,and the protein was purified by the MBP tag protein purification column.Enzyme assays were performed in vitro,and the product was analyzed by GC-MS.2.5 Sequence analysis of Geraniol Synthase and Iridoid SynthaseAmino acid sequences of known geraniol synthase and iridoid synthase were downloaded from NCBI and ExPASy.Phylogenetic analysis was performed using the tool MEGA and iTOL.Multiple amino acid sequence alignments were performed by DNAMAN.Genomic DNA of G.jasminoides was extracted,and subclone into pLB vector for sequencing.After squencing,introns and exons were analyzed by Snapgene software.2.6 Functional differences of two iridoid synthaseThe change of NADPH in the reaction was taken as the detection object,and Catharanthus roseus Iridoid Synthase(CrIS)was used as the positive control.The change of NADPH was detected with an enzyme-labeled instrument,and the detection wavelength is 340nm,then kinetic parameters was calculated.3 Results3.1 Profiling of iridoid metabolites in different tissues of G.jasminoidesGeniposide and Gardenoside were highly detected in fruits and fruits.Geniposidic acid was highly accumulated in top leaves and lower leaves.3.2 Transcriptome sequencing of G.jasminoides and identifying genes involved in the biosynthesis of iridoids in G.JasminoidesA total of 256,328 unigenes were obtained by transcriptome sequencing.Among 192,074(74.93%)uingenes were annotated in at least one public database(Nr,KEGG,SwissProt,UniProt,KOG).Almost all putative genes of the iridoid biosynthesis pathway can be found in the transcriptome data,including the MVA,MEP pathways and iridoid ring scafflod pathway.3.3 Expression levels of two key enzymes in the biosynthesis pathway of iridoidAccording to RPKM and RT-qPCR data analysis,it was found that GjGES was highly expressed in flowers and fruits.Combined with the metabolization results of G.jasminoides,indicating that GjGES is a key in the iridoid biosynthesis pathway.GjIS is highly expressed in flowers and fruits,and GjIS2 and GjIS4 are highly expressed in top leaves and lower leaves.Combined with the detection of G.jasminoides metabolites,it is more likely that GjIS is the vital gene.3.4 Cloning and functional analysis of two key enzyme genesGjGES,GjIS,GjIS2 and GjIS4 were constructed into the prokaryotic expression vector pMAL-c5X,and the purified proteins were obtained through protein induction and purification.GjGES catalyzed Geranyl pyrophosphate(GPP)to Geraniol.GjIS,GjIS2 and GjIS4 have the functions of catalysis of Cis-trans-nepetalactol from 8Oxogeranial and Progestrone to 5-beta-prognane-3,20-dione.3.5 Sequence analysis of geraniol synthase(Gj GES)and terpene cyclinase(GjIS,GjIS2,GjIS4)GjGES has seven exons and six introns,provides reference for the study of sequence splicing.GjIS,GjIS2 and GjIS4 has fewer introns,both only have two exons,one intron.3.6 Enzyme kinetics of iridoid synthaseThe kcat/Km of GjIS2 and GjIS4 were higher.Moreover,GjIS4 was close to CrIS(positive control).The enzyme activity of GjIS was too low to detect,and the product can only be dectected on GC-MS.4 ConclusionThis study mainly focuses on different tissue parts of Gardenia jasminoides,such as top leaves,lower leaves,flowers,fruits.Using the transcriptome squence and chemical constituent research,we provide the iridoid biosynthetic pathway of a large number of putative genes(including upstream MVA and MEP pathway that offer carbon flow,and iridoid ring scaffold pathway).The first branch step and cyclization step in the biosynthetic pathway of iridoid,Geraniol Synthase and Iridoid Synthase,has been cloning and functional characterted,elucidate the biosynthetic pathway of G.jasminoides medicinal composition and provide a theoretical and technical support study of synthesis iridoid glycoside in the future.