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Transcriptome analysis and targeted metabolic profiling for pathway elucidation and identification of a geraniol synthase involved in iridoid biosynthesis from Gardenia …

Industrial Crops and Products. 2019;

PengYeaShuangchengLiangaXiaominWangaLixinDuanbFengyangJiang-YanaJinfenYangaRuotingZhanaDongmingMaa

Products/Services Used	Details	Operation
Proteins, Expression, Isolation and Analysis	Target proteins were individually eluted with?elution?buffer (20?mM?maltose?in binding buffer), and 1-mL fractions were collected. For each GES homolog, 15-μL aliquots of every other fraction were analyzed using SDS-PAGE with gradient (10% acrylamide) gels, and proteins were visualized through an eStain?L1 protein?staining system (Genscript, USA). Fractions containing each MBP-tagged GES (molecular mass of approximately 109?kDa) were combined and dialyzed in PD-10 desalting columns (GE Healthcare) following the manufacturer’s instructions. For the removal of the MBP tag, Factor Xa (BioLabs, China) was used to cut the MBP tag in buffer (20?mM?HEPES, 500?mM NaCl, 2?mM CaCL2, and 50% glycerol, pH 8.0). The reaction mixture was incubated at 23?°C for 6?h. The protein concentration was determined using the Bradford method, with?bovine serum albumin?used as the standard.	Get A Quote

摘要

Iridoid compounds have been reported to be accumulated in the dried fruits of?Gardenia jasminoides?(Rubiaceae), which are used in traditional Chinese medicine. These compounds exhibit obvious pharmacological activities, including effective protective effects on the liver and therapeutic efficacy for cardiovascular diseases. However, the biosynthetic pathway of iridoid remains uninvestigated. In this study, 12 transcriptomes from four tissues (lower leaves, top leaves, flowers, and fruits) were analyzed to characterize the accumulation of active constituents and to identify the corresponding genes involved in iridoid biosynthesis. Almost all genes in the mevalonate (MVA) pathway and the 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway were found in our transcriptome database; these pathways comprise upstream pathways of iridoid compounds (monoterpene derivatives), most of which are abundant in flowers and fruits. Geraniol synthase (GES), which is involved in the first branch step in the iridoid biosynthetic pathway, was cloned, and quantitative real-time PCR (qRT-PCR) revealed that GjGES showed a similar expression pattern to that in the transcriptome in terms of reads per kilobase per million (RPKM) mapped reads. The recombinant GjGES protein efficiently converted geranyl diphosphate (GPP) to geraniol. Subsequently, the metabolic profiling of seven iridoids and iridoid glycosides, namely geniposide, gardenoside, genipin, geniposidic acid, genipin-1-β-gentiobioside, shanzhiside, and shanzhiside methyl ester, revealed that these compounds were highly accumulated in fruits. Consequently, the metabolites and speculated unigenes involved in iridoid metabolic pathways enabled the identification of glucosyltransferase, hydroxylase, and O-methyltransferase candidate genes responsible for the biosynthesis of iridoid and iridoid glycosides. The results of the present study can serve as a reference for the functional characterization of enzyme-coding genes and are beneficial for the engineering of biosynthetic pathways of iridoids and iridoid glycosides in the future.