| Fagopyrum tataricum is a combination of nutrition,health care and medical treatment and known as "the king of five cereals",which has a long history of medicine in China.F.tataricum is rich in a variety of nutrients,especially the flavonoids,rutin,quercetin,and phenols in the seeds.They have various functions such as lowering blood sugar,lowering blood lipids,lowering blood pressure and anti-oxidation,anti-aging,anti-cancer,and improving the microcirculation of blood vessels.The related researches mainly focus on the separation,purification and analysis of the flavonoids from F.tataricum,and there is no in-depth study on the regulation mechanism of flavonoid metabolism.The active ingredient of F.tataricum is an important product in the metabolic pathway of flavonoids,and its molecular mechanism is not very clear.MYB families are widely involved in the secondary metabolic pathways of flavonoids,In this study,MYB regulatory genes in flavonoid metabolism pathway were extracted,and the expression analysis and functional verification were performed based on the data of the transcriptome and homologous comparison.In order to reveal the molecular mechanism of MYB transcription factor and flavonoid synthesis pathway in secondary metabolism regulation,it provides a theoretical basis for the clarification of the synthesis mechanism of flavonoids.The results of this study are as follows:1.Bioinformatics analysis of MYB transcription factor family.In this study,162 protein sequences containing MYB domains were identified by combining bioinformatics with the genome and transcriptome database of F.tataricum.152 R2R3-MYB subfamily,5 3R-myb subfamily and 5 4R-myb subfamily were collected.The number of R2R3-MYB families is further divided into 25 subgroups(S1-S25)and 40 Unclassified subgroups.The related MYB function studies showed that the transcription factor related to the biosynthesis pathway of flavonoids were mainly concentrated in S4,S5,S6 and S7 subgroups.2.Screening,cloning and analysis of flavonoid anabolic regulatory genes.Firstly,the MYB genes related to flavonoid anabolism were selected by homologous analogy,RNA-seq and photoinduced treatment.Then the candidate gene was cloned and verified.Finally,5 genes were cloned:FtMYB45,FtMYB64,FtMYB102,FtMYB116,FMYB162.A real-time quantitative PCR analysis was performed on the expression of MYB genes in various tissues and organs during the development of F.tataricum,and the expression model of MYB gene was confirmed as the expression of tissue differences.3.Functional verification of flavonoid synthetic metabolic regulatory genes.Using Gateway clone system,5 target genes were constructed to pK7WG2D terminal expression vector.Then transferred it to the source plant F.tataricum and the model plant Nicotiana tabacum L.We successfully obtained 3 transgenic F.tataricum hairy root strains and 2 transgenic tobacco plants.Finally,molecular and chemical detection of transgenic materials was carried out.Real-time fluorescence quantitative PCR test rsults showed that the relative expression of target gene in transgenic materials was significantly increased.The detection results of flavonoids in Genetically modified materials showed that the contents of rutin,quercetin,catechin and epicatechin were raised to a certain extent.The molecular and chemical test results showed positive correlation trend.It is concluded that FtMYB102 and FtMYB 116 play a positive role in the metabolism of flavonoids.FtMYB 102 had a positive effect on the metabolism pathway of flavonols,and FtMYB116 had a positive effect on procyanidins and flavonol metabolism pathways. |
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