Cloning And Expression Analysis Of R2R3-MYB Genes Related To Flavonoid Biosynthesis In Glycyrrhiza Uralensis Fisch

Licorice flavonoids are very important kind of components in licorice,and its content directly affects the quality of licorice.A large number of studies have shown that flavonoids have various biological activities and pharmacological effects,such as antiviral,antioxidant,anticancer,antibacterial,scavenging free radicals and so on.The biosynthesis of flavonoid in plants comes from the phenylpropane metabolic pathway.MYB transcription factor is one of the largest families of transcription factors in plants,which plays an important role in the process of plant growth and development,and are the main family of transcription factors that regulate the synthesis of flavonoids.Among them,R2R3-MYB is the largest class of MYB transcription factors in plants.MYB gene expression is obviously induced by some growth regulator,such as the signal molecule jasmonate.Methyl jasmonate is a plant endogenous growth regulator that can induce the jasmonate signaling pathway and regulate synthesis of secondary metabolites(especially flavonoids).Previous studies showed that the synthesis of licorice flavonoids was increased significantly and 11 differentially expressed MYB transcription factors were screened with the addition of exogenous methyl jasmonate by RNA-seq.Using Glycyrrhiza uralensis Fisch as the materials,we cloned two R2R3-MYB transcription factors.Through bioinformatics analysis,qPCR,subcellular localization,over expression and detection of flavonoids,it was showed that GlMYBs are related gene regulating flavonoids synthesis in licorice,which responded to methyl jasmonate induction.The results were listed as below:(1)Cloning and bioinformatics analysis of GlMYB51 and 88: The results showed that both of them belong to the R2R3-MYB gene family.(2)Spatiotemporal expression pattern analysis of GlMYBs gene: GlMYB51,GlMYB88,GlMYB84 and GlMYB4.To determine the expression of the GlMYBs in different organizations and growth periods of licorice plants,the tube seedling at 2,3,4and 5 weeks of age was used for qPCR experiments.The expression levels of GlMYBs are all higher in licorice root than in other organs,and increases with plant growth.(3)Subcellular localization of the GlMYBs: The recombinant plasmid pJG054-GlMYBs was constructed and transformed into Agrobacterium tumefaciens GV3101 cells.Tobacco leaves were injected by transformed Agrobacterium tumefaciens and observed under laser confocal fluorescence microscope after 60 hours.GlMYB88,GlMYB84 and GlMYB4 are located on the nucleus.(4)Preliminary analysis of the function of GlMYBs gene: GlMYB88,GlMYB84 and GlMYB4 are overexpressed in licorice cells.As a result,the expression level of GlMYBs in the over expression group was clearly higher than in the control group,and the expression levels of C4 H and CHS genes,which are important enzyme genes for flavonoid synthesis,were clearly higher than in the control group.Using ultraviolet spectrophotometry and high-performance liquid chromatography,contents of flavonoids and isoliquiritigenin in transgenic cells with stable growth were measured.The over expression group was higher than the control group in both cases,and the effect of GlMYB88 transcription factor was obvious.