Preliminary Analysis The Regulation Mechanism Of Tartary Buckwheat Secondary Metabolism Under Water Stress

Tartary buckwheat is a dual-purpose usage crop in medicine and grain with high stress resistance,which is rich in flavonoids with high biological activity.Flavonoid secondary metabolites in plants have important physiological functions such as scavenging oxygen free radicals,improving peroxidase activity and anti-oxidation.Moderate drought stress can increase the synthesis of plant secondary metabolites.Analysis of the changes in the content and composition of tartary buckwheat flavonoids under water stress and its regulation mechanism is of great value to reveal the relationship between tartary buckwheat stress resistance and the accumulation of flavonoids.In this study,Jiujiang tartary buckwheat was used as the material to analyze the changes of total flavonoid content under water stress,and analyze the basic characteristics of transcription factors that may participate in the regulation of flavonoid metabolism under drought stress.Tobacco and Arabidopsis overexpression systems were used to analyze the regulation of transcription factors on the accumulation of secondary metabolites,and yeast two-hybrid technology was used to analyze the proteins interacting,in order to lay the foundation for the analysis of the regulation mechanism of the secondary metabolism of tartary buckwheat under water stress.The following results were obtained:?1?After the water stress treatment of tartary buckwheat,under the condition that the soil relative water content was 30%-40%of the field water holding capacity?severe drought?,the content of flavonoids in tartary buckwheat leaves increased significantly;The expression level of FtbHLH1?Ft WRKY22 and Ft WRKY53 and the flavonoid content showed a similar trend.These transcription factors are likely to participate in the regulation of the accumulation of secondary metabolites in tartary buckwheat under stress conditions.?2?By combining the Plant TFDB database and tartary buckwheat transcriptome database,we predict and analyze the downstream regulatory genes of tartary buckwheat FtbHLH1,Ft WRKY22 and Ft WRKY53.The downstream target genes of FtbHLH1 involved in flavonoid metabolic pathways including TT8,CHS,ANS,FLS1,etc.The downstream regulatory genes of Ft WRKY22 and Ft WRKY53 involved in the flavonoid metabolic pathway including DFR and UFGT.Bioinformatics analysis shows that FtbHLH1 belongs to the bHLH family IIIf subgroup in evolution,and members of this subgroup can form transcription activation complexes with other regulatory factors to regulate the secondary metabolic pathway of flavonoids in plants.?3?The q RT-PCR analysis under stress and hormone treatments indicated that FtbHLH1may be an important transcription factor that widely responds to abiotic stress in tartary buckwheat,and Ft WRKY22 and Ft WRKY53 genes may be involved in JA-mediated signaling pathways.?4?Constructing plant overexpression recombinant plasmids p CAMBIA3301-FtbHLH1-e GFP,p CAMBIA3301-Ft WRKY22-e GFP,p CAMBIA3301-Ft WRKY53-e GFP,which were transformed into plant through Agrobacterium,and obtained the FtbHLH1,Ft WRKY22overexpressed T₃generation homozygous Arabidopsis lines by flower dipping method,seed germination experiment,root development experiment and flavonoid metabolism pathway related genes q RT-PCR analysis indicate that the seed germination rate of FtbHLH1overexpressing lines under mannitol treatment was higher than that of wild-type plants,and the root elongation was significantly higher than that of WT plants.The expression of flavonoid metabolism pathway-related genes was significantly up-regulated.The seed germination rate of Ft WRKY22 overexpressing lines under mannitol treatment was lower than that of WT plants,and the root elongation was significantly lower than that of WT plants.The expression of genes related to flavonoid metabolism pathway was significantly up-regulated.Ft WRKY22 and Ft WRKY53 overexpression tobacco T₁generation plants were obtained by leaf disc method,and the flavonoid content of T₁generation plants was measured.The results showed that the flavonoid content of Ft WRKY22 overexpression lines decreased.?5?Ft WRKY22 interacting proteins were screened by Y2H as Ft CSN5B,and verification analysis showed that Ft WRKY22 could interact with Ft CSN5B,and Ft CSN5B could interact with Ft VTC1.However,bimolecular fluorescence complementary verification showed that Ft WRKY22 could not interact with Ft CSN5B,but Ft CSN5B could interact with Ft VTC1.