Study On Fagopyrum Tataricum HY5 Regulating The Biosynthesis Of Flavonoids Under Blue Light Induction

As a kind of quasi-grain,Fagopyrum tataricum is a medicinal and edible plant that integrates edible,medicinal and medical care.It is called "the king of grains" and has a long history of cultivation in China.Tartary buckwheat is rich in nutrients.It is also known as the "Sanjiang Food" and because it contains a variety of flavonoids,such as rutin,quercetin,catechins and other substances."Health food for humans in the 21st century",tartary buckwheat is also the only quasi-grain containing rutin.The active components of tartary buckwheat are important products in the flavonoid metabolism pathway.Related research mainly focuses on the separation,purification and analysis of flavonoids of tartary buckwheat.This study comprehensively utilizes the theories of genome,transcriptome,metabolome,bioinformatics,and various molecular biology techniques,start-ing from the regulation mechanism of flavonoid metabolism pathway,and studying the effect of bZIP family HY5 transcription factors on regulating tartary buckwheat flavonoids under blue light treatment.The main research results of the molecular mechanism of compound synthesis are as follows:1.Analysis of metabolome data of tartary buckwheat seedlings under dark and blue light conditionsIn this study,the flavonoid metabolomics detection platform was established by LC-QQQ-MS to detect the content of flavonoids in 4d raw buckwheat seedlings after 48h dark and 48h blue light treatment.Compared with the blue light treatment and dark treatment,the content of naringin in the 4d raw buckwheat seedlings was increased by 3 times and the content of kaumferol was increased by 2.3 times,the content of rutin increased by 0.8 times,the content of quercitrin increased by 0.8 times,the content of isoquercitrin increased by 0.7 times,the content of catechin increased by 0.6 times,the content of epicatechin increased by 0.4 times,indicating that blue light promoted the synthesis of flavonoids in bitter buckwheat seedlings.2.Bioinformatics analysis of tartary buckwheat HY5 transcription factor and identification of its expression pattern under blue light.In this study,we identified the promoter of FtHY5 and the key enzyme gene of flavonoid metabolism pathway by using the genomic database and bioinformatics methods,and analyzed its sequence characteristics,conserved motif,gene structure,evolutionary characteristics and homeopathic elements.In this study,two FtHY5 transcription factors were identified,named FtHY5-1 and FtHY5-2,respectively.Both of them contained three conserved domains,namely,the VPE/DΦG motif and bZIP domain,which interact with the phosphorylation site of casein kinase Ⅱ and COP1 protein.By analyzing the transcriptome data of Tartary Buckwheat seedlings under blue light,it was found that the gene expressions of FtHY5-1 and FtHY5-2 were increased,and the gene expressions of key enzymes of flavonoids metabolism pathway FtF3H,FtF3’H,FtFLS1,FtCHI and FtCHS2 were also increased,and the qPCR experiment was conducted to verify the results.Consistent with trends in transcriptome data.With the support of the above data,two Fthy5-1 and Fthy5-2 genes and the promoters of key enzyme genes FtF3H,FtF3’H,FtFLS1,FtCHI and FtCHS2 were cloned by PCR.3.FtHY5 interacts with the promoters of key enzyme genes in the flavonoid metabolism pathway of tartary buckwheatIn this study,two target genes were successfully constructed on the pGREENⅡ 62-SK expression vector using enzyme digestion and ligation technology,and the promoters of key enzyme genes in the tartary buckwheat flavonoid metabolism pathway were constructed on the reporter gene pGREENⅡ0800-LUC vector,based on the transient expression of tobacco.The dual luciferase experiment under the system proved that FtHY5-1 and FtHY5-2 interacted with the promoters of FtF3H,FtF3’H,FtFLS2,FtCHI,and FtCHS2;The two target genes FtHY5-1 and FtHY5-2 were successfully constructed on the pB42AD expression vector using restriction enzyme digestion and ligation technology,and the promoters of the key enzyme genes of the tartary buckwheat flavonoid metabolism pathway were constructed on the reporter gene p Laczi vector through yeast one-hybridization The experiment verified that FtHY5-1 interacted with the FtCHI promoter,which further demonstrated that HY5 played a positive regulatory role in the metabolic pathway of tartary buckwheat flavone.4.Interaction between FtHY5 and other transcription factors that regulate flavonoid metabolismIn this study,two target genes were successfully constructed on pCAMBI-A1300-Nluc expression vector using restriction enzyme digestion and ligat-ion technology,and MYB transcription factor,which has been reported to regulate the biosynthesis of tartary buckwheat flavonoids,was constructed on pCAMBIA1300-Cluc vector.Based on tobacco transient The dual luciferase complementation experiment under the expression system proved that FtHY5-1-FtMYB7 and FtHY5-2-FtMYB7 proteins interact,indicating that FtHY5 protein can also recruit other transcription factors when regulating the biosynthesis of tartary buckwheat flavonoids.5.Construction of FtHY5 transcription factor transgenic systemIn this experiment,Gateway gene recombination technology was used to construct overexpression vectors,and two overexpression vectors,pK7WG2D-FtHY5-1 and pK7WG2D-FtHY5-2,were obtained.Using heat shock freezing and thawing method,an empty pK7WG2 D vector plasmid and the obtained two recombinant plasmid vectors were transformed into Agrobacter-ium rhizogenes strain ACCC10060 strain,and 3 strains were obtained:ACCC10060-pK7WG2D-CK,ACCC10060-pK7WG2D-FtHY5-1And ACCC10060-pK7WG2D-FtHY5-2.Subsequently,3 strains of Agrobacterium were transf-ormed into the native plant tartary buckwheat by explant infection,and 3 transgenic tartary buckwheat hairy root lines were successfully obtained.Conclusion:Under the induction of blue light,FtHY5 transcription factor can increase the expression of flavonoid metabolism pathway related enzyme groups by combining with the tartary buckwheat flavonoid metabolism pathway related enzyme gene promoters CHS2,FLS1,CHI,F3H,F3’H and FtMYB7 transcription factor In order to improve the biosynthesis of flavon-oids,it positively regulates the flavonoid inferior pathway.