The Mining And Functional Study Of FtMYB17,the Key Gene Regulating Pericarp Color Of Tartary Buckwheat

Tartary buckwheat is a dicotyledonous plant of the Polygonaceae genus,known as the "king of grains".In recent years,Tartary buckwheat and its processed products have become more and more popular among consumers because of its high nutritional,edible,medicinal and economic values.Pericarpl color is an important agronomic trait of Tartary buckwheat,which mainly depends on the type and content of flavonoids in the peel.Flavonoids was the key factor to determine the nutritional value of Tartary buckwheat.Therefore,the study on the relationship between pericarp color and the molecular mechanism related to flavonoids metabolism is very important to guide the breeding of Tartary buckwheat.In this study,554 Tartary buckwheat germplasm resources from different places were used as materials.Then,using the existing single nucleotide polymorphism(SNP)data,genome wide association study(GWAS)was performed on the phenotype of seed pericarp color and genome re-sequencing data.Finally,the function of candidate genes was studied.The main results are as follows:1.Firstly,the pericarp color of 554 Tartary buckwheat germplasm was identified.The results showed that there were 8 main peel colors in these germplasm: black,dark brown,brown,light brown,black-and-white variegated,dark gray,gray and light gray.And,there were 27,35,140,76,31,90,89 and 66 materials with different colors respectively.2.Genome wide association analysis in pericarp color of Tartary buckwheat showed five related SNPs loci,which were located on chromosomes 1,2,3,5 and 7 respectively.According to the results of haplotype analysis and expression analysis in different materials,one R2R3-MYB transcription factor FtMYB17 on chromosome 2 was selected for further study.Haplotype analysis showed that there was an SNP site at 1365 bp in upstream of the start codon(ATG)of the gene.There are three haplotypes: Hap 1(A/A),Hap.2(A/T),Hap.3(T/T).We speculate that hap 1(A/A)is related to the darker color of Tartary buckwheat.The expression analysis of the gene in different materials showed that the expression of the gene in Tartary buckwheat with black pericarp was significantly higher than that in light colored materials.3.The total length of CDS of FtMYB17 was 630 bp,encoding 209 amino acids.The molecular weight of the protein was 24.26 KDa and the theoretical isoelectric point was 5.39.Phylogenetic tree analysis showed that the gene was highly similar to At TT2 in Arabidopsis.Transcriptional activation activity analysis showed that the C-terminal of FtMYB17 had transcriptional activation activity.4.After overexpression of FtMYB17 in hairy roots,the content of total flavonoids in transgenic hairy roots increased.q RT-PCR results showed that overexpression of FtMYB17 promoted the expression of key enzyme genes in flavonoids synthesis(Ft PAL,Ft C4 H,Ft4CL,Ft CHS,Ft CHI,Ft F3 H,Ft F3’H,Ft DFR,Ft ANR and Ft LAR).The HPLC results showed that the contents of epicatechin,catechin,cyanidin,rutoside,dihydroquercetin and naringin in hairy roots overexpressing FtMYB17 gene increased.In the overexpression lines in Arabidopsis,the expression of key flavonoid synthesis enzyme genes(At PAL,At C4 H,At4CL,At CHS,At CHI,At F3 H,At F3’H,At FLS,At DFR,At ANR and At ANS)were significantly up-regulated.In addition,compared with wild-type Arabidopsis,the leaf of transgenic lines in Arabidopsis showed dark purple,indicating that the FtMYB17 can promote the accumulation of anthocyanin,a flavonoid derivative.The above results suggest that FtMYB17 may be involved in the flavonoid synthesis pathway of Tartary buckwheat.5.Yeast two hybrid experiment showed that FtMYB17 could interact with Ftb HLH2,Ft Pin G0505498300,Ft Pin G0505916000,Ft Pin G0505915500 and Ft Pin G0505811400.Therefore,we speculate that FtMYB17 may cooperate with these proteins to regulate the flavonoid synthesis of Tartary buckwheat.Although its specific regulatory mechanism needs to be further studied.To sum up,this study located a MYB transcription factor(FtMYB17)that regulates Tartary buckwheat peel color,which can promote the synthesis of related flavonoids by up regulating the expression of key genes,so as to regulate tartary buckwheat peel color.The results are of great significance for further understanding the mechanism of peel color of Tartary buckwheat and accelerating buckwheat molecular breeding.