Cloning And Functional Study Of MYB63 Gene Related To Flavonoid Biosynthesis In Safflower

Safflower(Carthamus tinctorius L.)is a traditional Chinese herbal medicine,with a bright red color,so it is called safflower.Safflower flavonoids(Safflower flavonoids)are the main active ingredient of safflower,which are widely used in coronary heart disease,hypertension,cerebral infarction,anti-tumor,diabetes complications and other diseases,and they are in great demand.In recent years,although the area of safflower planting area in China has been expanding in China,the problem has not been solved.Therefore,it is of great significance to increase the content of own flavonoids and cultivate new strains of safflower with high flavonoid content.Transcription factors play an important regulatory role in the synthesis of plant secondary metabolites,and the relevant literature indicates that the transcription factors in the biosynthesis of flavonoids mainly include MYB,b HLH,WD40,and b ZIP,etc.This study focused on MYB transcription factors in safflower,and on the basis of safflower genome sequencing,screened and identified safflower MYB family members,analyzed the expression pattern of its families,and selected the CtMYB63 gene;cloning and subcellular localization of the CtMYB63 gene were analyzed,and we further investigated the function of the gene in flavonoid accumulation by overexpressing CtMYB63 in Arabidopsis and by overexpressing and silencing CtMYB63 in the safflower transient transformation system.The specific study results are presented as follows:1.We selected 440 MYB proteins from the genome database and constructed a phylogenetic tree,dividing the safflower MYB gene family into 12 subgroups.Gene structure analysis showed that R2R3-MYB protein has a conservative domain;Cis-acting elements such as light-responsive and methyl jasmonate-responsive are present in the CtMYB gene promoter.Protein interaction network prediction analysis was performed to verify that safflower MYB proteins can interact with other proteins.2.Analysis of CtMYB gene expression patterns showed that seven of these MYB genes were highly expressed in the petal fading stage,17 MYB genes were highly expressed in stems and 9 MYB genes were highly expressed in roots,among them,CtMYB63 and CtMYB77 were highly expressed in leaves,while CtMYB63 showed the highest expression.3.A coding region of 912b pCtMYB63 gene was successfully cloned.The pCAMBIA1302-CtMYB63 expression vector was constructed,and this gene localization in the nucleus was demonstrated by subcellular localization experiments.4.The plant expression vector pCAMBIA3301-CtMYB63 was constructed and transformed into Arabidopsis,and the OE-8 and OE-9 strains and COM4 and COM8 strains.Comparing the phenotypes between different Arabidopsis strains,the deepest and the largest leaf size,and the mutant strains recovered in both leaf color and size.The Me JA stress treatment indicates that it promotes flavonoids accumulation and the CtMYB63 gene has some sensitivity to exogenous methyl jasmonate(Me JA)stress.Under UV-B stress,the survival rate was the highest,followed by the wild type strain,and the mutant strain was the lowest;through the change of malondialdehyde(MDA)content and DAB and NBT staining,it showed that the overexpressed Arabidopsis was the least damage under the UV-B stress,indicating that the CtMYB63 gene has certain tolerance to UV light(UV-B).5.Construction of a transient transformation system for safflower leaves,using overexpression and gene silencing techniques(VIGS)to infect safflower leaves,followed by Me JA stress and UV-B stress,showed that the CtMYB63 gene was significantly upregulated during transient overexpression,positively correlated with the expression of downstream structural genes(CtCHI1,CtDFR1,CtFLS1,and Ct4CL1)in the flavonoid metabolism pathway,and the contents of total flavonoids,anthocyanins,and proanthocyanidins were significantly increased,while DAB NBT has the shallowest staining degree and the smallest damage.Silent strains are the opposite.The overall results are similar to those of Arabidopsis,confirming again that the CtMYB63 gene promotes flavonoids accumulation and has certain sensitivity and tolerance to these two stresses.