Analysis Of Fagopyrum Cymosum Resources And The Mechanism Of Buckwheat Bioactive Compounds Biosynthesis By Multi-Omics Analysis

Fagopyrum cymousum is a perennial dicotyledonous plant of Polygonaceae family,Fagopyrum mill.It is a kind of precious plant originated in China can be used as medicine and forage.Currently,the research on the investigation and analysis of Wild F.cymosum resources and the metabolic regulation mechanism of bioactive components in China is still very rare.So,In this study,the germplasm resources of F.cymosum in Southwest China were collected,and phylogenetic analysis was carried out by means of molecular markers to identify their kinship,for providing material support for variety selection and improvement;using the analysis technology of HPLC-Q-TOF-MS,metabonomics analysis were carried out for the secondary metabolites research of F.cymosum,and identifying different compounds,which provides reference for identification and evaluation of active components in F.cymosum;Carring out transcriptome research and analyzing key genes regulated by jasmonate,screening the transcription factors closely related to quality traits,analyzing the key genes regulated by jasmonic acid,exploring the expression rules of key enzyme genes of flavonoids bioactivesubstances,establishing the hairy root genetic transformation system,optimizing the key parameters;The genetic transformation system of F.cymosum hairy root was established,and the mechanism of MYB transcription factor regulating the metabolism of flavonoids in F.cymosum was also studied by using hairy root system as material,which explores the laboratory micro fermentation model about a new way for the extraction of Flavonoids from Fagopyrum tataricum,and having guiding significance for the comprehensive utilization of flavonoids in hairy roots in industrial production as well.The above studies laid a foundation for the in-depth and systematic analysis of the medicinal value of F.cymosum,conducive to the preparetion of F.cymosum drugs and health products as well as F.cymosum industry development.The detailed results of this thesis are as follows:1.Muritiple germplasm resources of F.cymosum were collected in Yunan,Sichuan,Guizhou and Chongqing,4 provience in Southwest China,and 15 represtive materia were seleted for further study.The molecular markers ITS and mat K were used to analysis the relationship of F.cymosum and other species in Fagopyurm mill;The F.cymosum consensus haplotype sequence of ITS and mat K were obtained,and phylogentic trees were built.the locations in different phylogenetic tree of F.cymosum were different,which indicated F.cymosum could be a mixed species.What's more,the phylogenetic relationship of F.cymosum was closer to F.tatarium rather than F.esculentum.2.Based on the platform of HPLC-Q-TOF-MS,496 compounds were identified by metabonomics analysis of alcohol extracts from the main root,the lateral root of F.cymosum.and the main root of F.tatarium.Among these compouns,there were 102 phenolic acids,34 esters,59 alcohols,24 flavonoids and its intermediates.This research provides a reference for the analysis of metabolic components,quality identification and the establishment of quality control standards of F.cymosum and its processed products.3.By using RNA SEQ transcriptome high-throughput sequencing method,a large number of differentially expressed genes related to response to jasmonate were found between the JAs treated group and control.A total of 12 buckwheat samples were sequenced to produce 70.2 GB of original data.By comparing the reference genome and gene function annotation,an average of 45983804 clean reads were obtained,with an average annotation gene coverage of 95.5%,which provided candidate genes for further study of jasmonate pathway in F.cymosum.Through KEGG annotation,we predicated that the bHLH family maybe the biggest group of transcription factorswhich has been found,NAC,B3 and MYB next.4.The hairy root transformation system of F.cymosum was established based on Agrobacterium rhizogenes A4,the optimal induce condition for A4 was 0.6~0.8OD,10 min for infection and 48 h for co-culture.5.TrMYB4 gene from purple V-marking white clover was transferred to study its effect on flavonoids metabolism of F.cymosum hairy root.The results showed that the TrMYB4 gene could significantly induce the accumulation of flavonoids in hairy roots,when compared with the control group,the accumulation of total flavonoids and rutin in transgenic hairy roots was about 1 time and 5 times higher,indicating a great potential for application.