Screening And Analysis Of2,3,5,4’-tetrahydroxy Stilbene-2-O-β-D-glucoside Biosynthesis Related Transcripts From Fallopia Multiflora

Fallopia multiflora (F.multiflora) is a traditional Chinese medicinal herb, which has beenwidely used for thousands of years. There are many active components beneficial to humanhealth in F.multiflora, such as flavonoids, anthraquinone etc.. One of the most important is2,3,5,4’-tetrahydroxy stilbene-2-O--D-glucoside (THSG), which belongs to stilbene. It wasbeen set as the main standard physiochemical identification of F.multiflora by ChinesePharmacopoeia, stipulates the content of THSG should not less than1%(dry weight) inofficinal F.multiflora. Pharmacodynamic study confirmed THSG has good applicationprospects on antihyperlipidemics, anti-oxidative, anti-infammatory and endothelial-protectiveactivities. In previous studies, the biosynthesis of stilbene backbone is classified in“Stilbenoid, diarylheptanoid and gingerol biosynthesis”(KEGG pathway：map00945). Studiesshow stilbene biosynthesis is derived from phenylalanine biosynthesis via the generalphenylpropanoid pathway. Trihydroxystibene resveratrol and tetrahydroxy piceatannol weresynthesized via thus way. Hitherto, as a four-hydroxy-stilbene glycoside derivatives, thebiosynthesis pathway of THSG is still unclear.Pursuing the objective to identify novel genes involved in THSG biosynthesis in ourstudy, we screened genes by Suppression subtractive hybridization (SSH) firstly. Andfull-length sequences were obtained by using3’5’RACE. Meanwhile, we performed the firstglobal analysis of F.multiflora transcriptomes using Illumina HiSeq2000. Furthermore, wecompared the gene expression profiles of four different F.multiflora tissues (root, stem andleaf) using the Digital Gene Expression (DGE) system. Through the analysis of gene functionand metabolism of differentially expressed genes, we conclude that THSG is synthesized fromtyrosine initiation via p-coumaric acyl coenzyme A. Specific contents and results are asfollows:⑴F.multiflora plants was gathered from13cities (counties). HPLC was used to analyze theTHSG contents. Results indicated that there was a significant difference (P-value <0.001) ofTHSG content in F.multiflora root from different origins. Detection showed root samples fromGuilin City have the most abundant of THSG, with a content of6.37%. Chongqing City,Xinyu County and Tianlin County’s samples have lowest level of THSG, with a content less than0.1%. Significant differences were found even within a same place. Eg. Guilin samples,the lowest is1.46%, the highest is6.37%. There was a significant difference of THSG contentin different tissues too, stems are less than roots, but more than leaves.⑵F.multiflora plants from Guilin and Chongqing were selected to perform SSH in3groups:I, leaves of Guilin’s F.multiflora as the tester, root of Guilin’s F.multiflora as thedriver; II, Guilin root as the tester, Chongqing root as the driver; III, Chongqing root asthe tester, Guilin root as the driver. Screened genes were connected to pMD19T-vector andthen sequenced. Guilin leaves tester group obtained5differential expressed genes,Guilin roots tester group obtained4differences genes, Chongqing roots tester group obtained7differential expressed genes (4of which are the same with the Guilin leaves tester group).By comparison to NCBI and KEGG database, in which, YCTS1and CTS1may be encodedcatalase involved in secondary metabolism, tryptophan metabolism, glyoxylate anddicarboxylate metabolism. The activity of tryptophan metabolism in Guilin leaves tester andChongqing root tester suggests that the low content of THSG in this two samples may be dueto the vigorousness of the pathway from chorismate to tryptophan, and competitive inhibitionof chorismate to phenylalanine metabolism. GTS1isolated from Guilin root tester may beinvolved in metabolic pathways and photosynthesis in the form ofphotosystem II oxygen-evolving to enhancer protein2.⑶There are4genes fragments obtained by SSH with no significant similarity in the NCBIdatabases. We pursue the full-length of the4genes fragments by rapid amplification of cDNAends(RACE), in order to obtain sequence information more. We got1full-length sequences ofGE and CF fragments separately,3possible full-length sequence of GW,2possible full-length sequence of CA. Finding open reading frame by NCBI ORF Finder showsthere are complete reading frame information in all7full-length sequence.⑷we performed de novo transcriptome assembly of F.multiflora roots from Deqing andChongqing samples using the Illumina RNA-seq system.65,653unigenes wereobtained. Compared to Nr, Nt, SwissProt, KEGG, COG, GO database, a total of73.38%genes annotated results returned. Pathway assignment showed,204genes took part instilbenoid, diarylheptanoid and gingerol biosynthesis (PATH: ko00945). And391genes wereinvolved in phenylpropanoid biosynthesis, which is closed to stilbenoid biosynthesis.116genes were involved in the biosynthesis of phenylalanine and tyrosine.55genes had polyketide synthase activity.⑸We performed DGE profiling analysis of F.multiflora roots(Dr), leaves(Dl) and stems(Ds)from Deqing sample and roots(Cr) from Chongqing sample. All tags were mapped totranscriptome reference database.17,884(Cr)，16,197(Dr)，14,643(Dl)，16,457(Ds) genes weremapped in transcriptome reference database by unambiguous tags.Clustering analysis ofdifferential gene expression pattern, GO functional enrichment analysis and pathwayenrichment analysis revealed16genes were involved in stilbenoid, diarylheptanoid andgingerol biosynthesis,29genes were involved in the phenylpropanoid biosynthesis,17geneswere participated in the phenylalanine metabolism.⑹Based on the DGE profiling analysis,21genes may be related to THSG biosynthesis weredetected by RT-PCR. Comprehensive analysis, we think THSG is synthesized byp-coumaricacyl coenzyme A via tyrosine pathway.We screened putative genes involved in the biosynthesis of2,3,5,4’-tetrahydroxystilbene-2-O-β-D-glucoside comprehensively by SSH, transcriptome sequencing and DGEprofiling analysis. We conclude that THSG is synthesized from tyrosine initiation, and list thegenes may be involved in THSG biosynthesis. This paper pointed out the direction for THSGbiosynthesis research, and provides a solid platform for researches on other secondarymetabolites of F.multiflora and tissue development, germplasm transformation.