Analysis Of Diversity Of The Fagopyrum Cymosum In Yunnan And Preliminary Study On The Biosynthesis Mechanism Of Flavonoids

Buckwheat is an important food-drug homologous crop.As the original center of buckwheat,China has abundant variety of buckwheat resources.Fagopyrum cymosum is the most widely distributed wild buckwheat species.The roots of F.cymosum containing a lot of bioactive components including rutin,quercetin,epicatechin and other flavonoids,which have important medicinal value.As one of the original centers of buckwheat,the F.cymosum resources in Yunnan are extremely rich.A With the large-scale collecting wild F.cymosumhas caused serious damage to its habitat,and the germplasm resourceshas been greatly reduced and near extinction.Therefore,the investigation of F.cymosum resources in Yunnan plays an important role in the protection of F.cymosum resources and the maintenance of ecological balance.Using the hairy root system to analyze the molecular regulation mechanism of flavonoids biosynthesis in F.cymosum has important theoretical significance for the metabolic engineering of bioactive substances in F.cymosum.More than ten counties in 6 cities including Shangri-La,Lijiang,Dali,were involved in the investigation.At the same time,a high-frequency genetic transformation system of F.cymosum was successfully induced by Agrobacterium rhizogenes A4,which laid a foundation for screening metabolic mutants and transgenic studies of F.cymosum,and created an ideal research system of the molecular mechanism of flavonoids synthesis in F.cymosum.The main results of this study are as follows:1.Further clarify the basic situation of the distribution of F.cymosum resources in Yunnan Province,It is mainly distributed in the central Yunnan and northwestern areas(Shangri-La Hutiaoxia,Shuanglang Town of Dali City,Guandu District of Kunming,etc.).After further observation and recording of the growth status and morphological characteristics of F.cymosum,The height of the F.cymosum plants in the Hutiaoxia area is generally high.The blocky stems of the F.cymosum in the Tonghai County road are spherical.At the same time,15 wild buckwheat resources were collected from different regions.The content of total flavonoids,rutin in the stems were measured by spectrophotometry and high performance liquid chromatography(HPLC).The total flavonoid content is between 0.440 and 3.428 mg/g,and the rutin content is between 0.084 and 1.069 mg/g.There was a large differences in the content of total flavonoids and rutin of F.cymosum stems collected in different regions were detected.Genetic diversity and genetic relationship of different F.cymosum resources were studied using ITS(Internal Transcribed Spacer)and matK molecular markers,The results show that the alfalfa is located at an altitude of 2000 m.The F.cymosum is clustered into one branch,and the elevation below 2000 m is a cluster.2.Fagopyrum esculentum var.was found in the coastal area of the Jinsha River in Yunnan.Morphological observation showed that the F.esculentum var.were similar to the F.cymosum in the morphology of flowers,fruits and leaves,while similar to the cultivated F.esculentum in root morphology.By karyotype analysis,the chromosome type of F.cymosum was 2 n = 2 X = 16 = 15 m + 1 sm,and the chromosome types of F.esculentum var.and F.esculentum were 2 n = 2 X = 16 = 16 m.The result of FISH showed that the F.esculentum var.is closely related to the F.esculentum.After sequencing the chloroplast genome of the F.esculentum var.and comparing with,the chloroplast genomes of the existing Fagopyrum cymosum,F.luojishanense,Fagopyrum esculentum and Fagopyrum tartary phylogenetic trees were established.The results showed that the relationship between the F.esculentum var.and the F.esculentum was the closest,followed by the F.cymosum,and then the phylogenetic tree was established using ITS,matK,psbA-trnH molecular markers to verify this result.In the electrophoretograms based on SSR molecular markers,the bands of Fa.esculentum var.relatives showed co-dominance with F.cymosum and F.esculentum.In conclusion,we speculated that the F.esculentum var.was a transitional species between F.cymosum and F.esculentum,and it was produced by hybridization under natural conditions.3.The hairy root system of F.cymosum was established using Agrobacterium rhizogenes A4.The foreign genes TrMYB4 and TrMYB44 were introduced into the hairy roots of F.cymosum,and the expression levels of key enzyme genes(phenylalanine ammonia-lyasegene:PAL?chalcone synthas gene:CHS?flavonol synthase gene:FLS)in the secondary metabolites of F.cymosum were up-regulated,and the content of rutin and quercetin were alsoincreased.