Molecular Mechanism Of R2R3-MYB Transcription Factor Regulating Flavonol Synthesis In Wild Jujube Seed

Jujube(Ziziphus jujuba Mill.)is a plant of the genus Ziziphus Mill.(Rhamnaceae),and is a traditional characteristic economic forest fruit in China.Wild jujube(Ziziphus jujuba var.spinosa Hu)is varietas and wild type of jujube,mostly a shrub,mainly distributed in northern China.Its seeds can be used as medicine,rich in flavonol compounds.It is a traditional Chinese medicine with calming and tranquilizing effects.Jujube is a traditional characteristic economic forest fruit in China.In order to clarify the molecular mechanism of flavonol biosynthesis and regulation in wild jujube seed,this study investigated the regulatory mechanism of transcription factors on flavonol biosynthesis in wild jujube seed by using q RT-PCR,transient overexpression and silencing,dual fluorescein enzyme assay,and GUS activity detection,using two types of wild jujube(’Qingjiansuanzao’,’Tiansuanzao’)and one cultivar of jujube(’Xiangfenyuanzao’),In order to provide theoretical basis for the development and utilization of the nutritional value of wild jujube seed.The main research results are as follows:(1)The flavonol content test showed that during the development and maturation process,five flavonols were detected in the seed and pulp of ’Qingjiansuanzao’,’Tiansuanzao’ and’Xiangfenyuanzao’,namely Quercetin,Quercetin-3-rutinoside,Quercetin-3-rhamnoside,Quercetin-3-galactoside and Quercetin-3-glucoside.The flavonol content in the seed and pulp of jujube and wild jujube showed a downward trend,and the flavonol content in the seed and pulp of the wild jujube was higher than that of the jujube.During the expansion stage,the content of flavonols in the pulp is higher than that in the seed.As the fruit grows and develops,the content of flavonols in the seed is generally higher overall than that in the pulp from the beginning of red stage.(2)Four candidate transcription factors(ZjMYB3 a,ZjMYB3 b,ZjMYB12 and ZjMYB308)were obtained by screening from the transcriptome of ’Tiansuanzao’ seed.Amino acid sequence alignment and phylogenetic tree analysis showed that ZjMYB3 a,ZjMYB3 b,ZjMYB12 and ZjMYB308 had typical R2 and R3 conservative domains,and were homologous with transcription factors regulating flavonol biosynthesis in other species.q RTPCR and correlation analysis showed that the expression patterns of the four transcription factors were significantly positively correlated with changes in flavonol content.(3)The results of transient overexpression in jujube fruits showed that ZjMYB3 a,ZjMYB12 and ZjMYB308 significantly increased flavonol content,while ZjMYB3 b did not promote flavonol accumulation.The virus induced silencing test showed that reducing the expression levels of ZjMYB3 a,ZjMYB12 and ZjMYB308 resulted in a significant reduction in flavonol content.Gene expression analysis showed that ZjMYB3 a,ZjMYB12 and ZjMYB308 had an activation effect on the Zj4 CL gene in the phenylpropanoid metabolic pathway,and had a positive regulatory effect on the ZjF3 H,ZjF3’H and ZjFLS in the flavonol synthesis pathway.ZjF3’5’H significantly decreased when overexpressed with ZjMYB3 a,ZjMYB12 and ZjMYB308.At the same time,ZjMYB3 a,ZjMYB12 and ZjMYB308 have a negative regulatory effect on the Zj ANS of the anthocyanin synthesis pathway,and positively regulate Zj LAR.ZjMYB12 and ZjMYB308 positively regulate Zj DFR,while ZjMYB3 a inhibits Zj DFR expression.In the tobacco transient overexpression test,a flavonol substance was detected as Quercetin-3-rutinoside.Instantaneous overexpression of ZjMYB3 a,ZjMYB12 and ZjMYB308 in tobacco also significantly increased flavonol content.(4)Dual luciferase results showed that ZjMYB3 a,ZjMYB12 and ZjMYB308 can significantly activate the promoter activity of the key flavonol synthesis genes ZjFLS1 and ZjFLS2.The results of the GUS reporter gene detection test were consistent with the results of the dual luciferase test.In summary,the flavonol content in the seed and pulp of jujube and wild jujube showed an overall downward trend,and the content in wild jujube was significantly higher than that in jujube.The overall content of flavonols in seed is higher than that in the pulp from the beginning of red period;the R2R3-MYB transcription factors ZjMYB3 a,ZjMYB12 and ZjMYB308 that positively regulate the synthesis of flavonols in wild jujube seed were screened and identified,revealing the molecular regulatory mechanism of flavonol biosynthesis in wild jujube seed,providing a basis for the directional selection of specific components such as flavonols from wild jujube seed and utilization.