Study On UPLC/Q-TOF MS Metabolomics And DGE Transcriptomics Technologyin Triterpenoid Saponins Biosynthetic Pathwayof Polygala Tenuifolia

RATIONALES:Triterpenoid saponins is one of the most important components of Polygala medicinal secondary metabolites, which as potential active substance has been shown to have calm, expectorant, fall blood sugar, and other pharmacological activities. Improve the content of Radix Polygala Tenuigenin can influence the efficacy of medicines Polygala. Improve the contente of herbs saponin in Polygala, mainly depends on the keygenes for saponin biosynthetic pathway.Currently, controling the secondary metabolites of polygala through the gene regulation survive the following questions:(1) Domestic and foreign scholars study Polygala focused largely on pharmacological activity, chemical composition and cultivation techniques, but molecular research is relatively small, and Polygala related gene database is lacking. (2) Triterpenoid saponins synthesis pathway has its own laws to each species.The triterpenoid saponins synthesis pathway of P. tenuifolia is not yet clear. (3) Research Group has use second-generation high-throughput Illumina sequencing technology studiedPolygala root tissue, but the technology can only be measured in all the genes Polygala herbs, and can not provide their exact content.OBJECTIVE:1. To rich genetic resources of P. tenuifolia. based on Digital expression profiling high-throughput sequencing technologyl.2. To find differentially expressed function genes in different developmental stages of P. tenuifolia through the correlation of polygala triterpenoid saponins peak intensity and triterpenoid saponins biosynthesis functional genea expression. It would be helpful for future regulation onjisaponin content through the bioengineering.METHOD:1. Performing the transcriptome sequencing via DGE high-throughput sequencing technology, and get a lot genetic information of P. tenuifolia. by bioinformatics research.2. Based UPLC/Q-TOF MS metabolomics technologyto identify and analyze the content of Secondary metabolites in P. Tenuifolia.different developmental stages.3. Based on previous DGE transcriptome database, using qRT-PCR technique to screen P. tenuifolia. reference gene. Using 2-△△CT method to analyze which involved in saponins biosynthetic pathway of different developmental stages of P. tenuifolia. Then combined saponin biosynthesis genes expression with saponin peak intensity based on the correlation UPLC/Q-TOF MS to elucidate the molecular mechanisms resulting in econdary metabolism differences in different developmental stagesof the P. Tenuifolia.RESULTS:1. In this study, DGE transcriptome sequencing data of P. tenuifolia. were assembled and statisticed.The statisticalresults obtained 39,625 Unigenes, total length of 54,594,810 bp. The unigenes were performedfunctional alignment and annotation via comparison on the Nr, Go database, Ko database, COG database and Swissprot protein database.2. In the study,3 DGE libraries at different development stages of P. Tenuifolia were constructed. Approximately 12,187 Unigenes may be involved in 419 metabolic or signalling pathways.. Compared the DGE library at 6Y with 9Y,9Y with 11Y, and 6Y with 11Y, there were 1,143,520, and 1,625 genes expressed differentially.3. Through the identified compounds based on UPLC/Q-TOF MS,24 compounds were identified, including 2xanthone,6 sucrose esters, 1 Ooligosaccharide multiesters and 6 triterpenoid saponins substances.Polygalasaponin XXVIII, Sibiricoses A5, Tenuifoliside A, Tenuifoliose K, and Tenuifoliose E can impact the active substances at different developmental stages Polygala.4. Using 2-△△CT method to analyze21 genesinvolved in saponins biosynthetic pathway of different developmental stages of P. tenuifolia. Then combined saponin biosynthesis genes expression with saponin peak intensity based on the correlation UPLC/Q-TOF MS to elucidate the molecular mechanisms resulting in econdary metabolism differences in different developmental stagesof the P. tenuifolia. The results showed that the correlation between thesaponin peak intensity and the expression of YP716B1, CYP98A3, CYP86B1, CYP94A1, UGT83Alwas highest in different developmental stages of P. Tenuifolia.Conclusion:In this study, theDGE transcriptome sequencing data were assembled and bioinformatics were analyzed of P. tenuifolia. GAPDH gene was screened as reference gene in different tissues of P. tenuifolia.Combined UPLC/Q-TOF MS metabolomics technology with qRT-PCR gene expression technology, research the expression patternof cytochrome P450 and enzyme glycosyltransferase genes in saponin biosynthesis withits content. From the correlation saponin biosynthesis enzyme genes expression with saponin content to explain the molecular mechanism causing saponin content differences inP. Tenuifolia.Ultimately six genes were selected to play a key role in the synthesis of Polygala triterpenoid saponins.