Cloning And Functional Analysis Of R2R3 Type MYB S4 Transcription Factor Gene Family In Fagopyrum Tataricum

Known as "the king of the five grains",tartary buckwheat(Fagopyrum tataricum)contains rich flavonoids,such as rutin,with balanced amino acid composition,It is a medicinal and edible plant with both nutritional and healthcare functions,and like blood fat-lowering,blood giucose-lowering,blood pressure-lowering,anti-oxidation,anti-aging,anti-cancer,anti-cancer,and microcirculation-improving.At present,researches on Tartary buckwheat mainly focus on its chemical composition,protein extraction and separation,but no further studies have been conducted on the regulation mechanism of flavonoids such as rutin,quercetin,catechin and flavonoid metabolic pathways contained in Tartary buckwheat.Transcription factors(TFs)play important roles in plant growth and development by regulating gene expressions.MYB family:contains the MYB domain,is one of the largest transcription factor families in plant,this family plays roles in plant development and flavonoid biosynthesis of secondary metabolism.And can be divided into four subfamilies:1R-MYB,R2R3-MYB,3R-MYB,and 4R-MYB.In this study,the transcription factors of Tartary buckwheat R2R3-MYB S4 subgroup were cloned by homologous gene cloning with reference to Tartary buckwheat transcriptome data,and the changes in their expression were studied,so as to speculate their functions.In order to reveal the mechanism of action between the secondary metabolites of flavonoids and target genes.It is believed that with the further research,new MYB transcription factors and regulatory mechanisms in Tartary buckwheat will be discovered one after another,and the metabolic regulatory network of rutin and other flavonoids will become clearer,which will provide strong scientific support for the molecular breeding and quality improvement of Tartary buckwheat.The results of this study are as follows:1.Bioinformatics analysis of MYB transcription factor family.According to the genome and transcriptome of Tartary buckwheat reported in literatures,a total of 162 transcription factors of MYB family have been found,including 152 R2R3-MYB transcription factors,5 3R-MYB transcription factors and 5 4R-MYB transcription factors.R2R3-myb transcription factor is the largest and can be divided into 25 subgroups(SG).The functional study of MYB protein showed that R2R3-MYB S4 subfamily played a role in flavonoid secondary metabolism.2.Cloning and analysis transcription factors of Tartary buckwheat R2R3-MYB S4 subgroup.In this study,the genome database of Tartary buckwheat was analyzed through phylogenetic tree,and the transcription factors of Tartary buckwheat R2R3-MYB S4 subgroup were obtained,which were cloned and molecular verified.Finally,three genes were cloned:FtMYB45,FtMYB63 and FtMYB67.Tartary buckwheat was induced by ultraviolet light,real-time quantitative PCR was used to detect changes in the gene expression of the target gene,and the content of flavonoids was detected by LC-MS,and transcriptome sequencing was performed.It was found that the expression levels of FtMYB45,FtMYB63 and FtMYB67 genes were decreased,flavonoid biosynthesis genes were up-regulated and flavonoid compounds content was increased.It was speculated that UVB could reduce the expression levels of transcription factors of Tartary buckwheat S4 subgroup,so that flavonoid compounds could be accumulated in large quantities to resist UVB stress.3.Functional analysis of Tartary buckwheat R2R3-MYB S4 subgroup transcription factors.By Gateway cloning technology,the target gene was constructed into pK7WG2D expression vector,and was successfully transferred into Tartary buckwheat.Three Tartary buckwheat hairy root transgenic lines were obtained,and molecular and chemical tests were conducted on the transgenic materials.The molecular detection results showed that the relative expression of the target gene in the transgenic materials was significantly up-regulated,and the chemical detection showed that the content of flavonoids rutin,catechin and epicatechin was decreased.The results of molecular and chemical detection showed a negative correlation,FtMYB45,FtMYB63 and FtMYB67 were preliminarily determined to play a negative regulatory role in the flavonoid metabolic pathway.And through the yeast one-hybrid experiment,the corresponding vector was constructed,and it was found that FtMYB45 and FtMYB63 interacted with the CHI promoter of the flavonoid metabolic pathway and participate in the regulation of flavonoids.