Function Analysis And Application Of MYB12 And MYB308 Genes Of Mulberry In Regulating Flavonoid Synthesis

Flavonoids are a class of important secondary metabolites,which not only have important physiologyogical functions in plants,but also have extensive pharmacological activities and important application values.Due to the shortage of plant resources rich in flavonoids,its development and utilization are faced with difficulties in extraction and separation,also with high production costs.On the other hand,transgenic hairy roots have some advantages in terms of high transformation efficiency,stable genetic characteristics,fast growth and low culture cost,which led them increasingly become an important material in producing medicinal secondary compounds that derived from many plants.In addition,mulberry root is an ideal material for extracting flavonoids because of its rich content of flavonoids and its high volume of biomass.Surprisingly,it is found that there is no existing research report regarding biosynthesis of flavonoids with mulberry transgenic hairy roots.Since R2R3-MYB transcription factor plays an important role in the regulation of flavonoid biosynthesis in plants,MbMYB12 and MbMYB308 genes regulating flavonoid biosynthesis were screened and cloned from mulberry in this study.The roles and mechanisms of MbMYB12 and MbMYB308 genes regulating flavonoid biosynthesis were explored using mulberry transient expression system and targeted metabolomics technology.Meanwhile,the transgenic MbMYB12 or MbMYB308 mulberry hairy roots were obtained by Agrobacterium-mediated transformation of mulberry hairy roots,the effects of overexpression of MbMYB12 or MbMYB308 on the synthesis of flavonoids in mulberry hairy roots were explored.The study laid a foundation for further research of the regulation and mechanism of MYB genes on flavonoid synthesis.Moreover,it provides a reference for the research on the efficient synthesis of flavonoids using mulberry hairy roots,which is of great significance to promote the diversified development of mulberry industry.The main results of this study are as follows:（1）Screening and cloning of MYB genes involved in flavonoid biosynthesis in mulberry.Through the combined analysis of transcriptome and flavonoid content in mulberry fruits at different developmental stages,five MYB genes（MbMYB6,MbMYB8,MbMYB12,MbMYB14 and MbMYB308）that may regulate flavonoid synthesis were selected.Furthermore,phylogenetic tree analysis showed that the five genes belonged to the R2R3-MYB category,and MbMYB6,MbMYB8 and MbMYB308 belonged to the SG4 subgroup which was confirmed to negatively regulate flavonoid biosynthesis.It is indicated that the expression trends of the three genes were opposite to the change of flavonoid content.While MbMYB12 and MbMYB14 belonged to SG7 and SG6 subgroups that were confirmed to positively regulate flavonoid synthesis,and their expression trends were consistent with the variation trend of flavonoid content in the mulberry fruits.（2）Identification of the regulation of MYB gene on flavonoid synthesis using tobacco transient expression system.The flavonoid content in the tobacco leaves transiently expressing the genes was determined,respectively,and the results showed that the transient expression of MbMYB12 and MbMYB14 genes in tobacco leaves could improve the flavonoid content in the leaves,and the content of flavonoids in the tobacco leaves transiently expressing MbMYB12 gene was the highest.On the contrary,the transient expression of MbMYB6,MbMYB8 and MbMYB308 in tobacco leaves decreased the flavonoid content in the leaves,and the flavonoid content in tobacco leaves transiently expressing MbMYB308 was the lowest.（3）Expression characteristics of MbMYB12 and MbMYB308 genes.The promoter sequences of MbMYB12 and MbMYB308 genes（p MbMYB12 and p MbMYB308）contain multiple cis-acting elements associated with plant hormones.The results of RT-q PCR showed that the expression of MbMYB12 gene was induced by ABA,SA and Me JA,while the expression of MbMYB308 gene was induced by ABA and Me JA.（4）Analysis of the regulation and mechanism of MbMYB12 and MbMYB308 on flavonoid synthesis with mulberry leaf transient expression system.The transient expression system of mulberry leaf was established,and the leaves transiently expressing MbMYB12 or MbMYB308 genes were successfully obtained.The content of flavonoids in the mulberry leaves transiently expressing MbMYB12 was increased,while the content of flavonoids in the leaves transiently expressing MbMYB308 was decreased.The species and relative contents of flavonoids in the mulberry leaves transiently expressing MbMYB12 or MbMYB308 gene were identified and analyzed by targeted metabonomics technology.A total of 30 flavonoid metabolites,which abundances were significantly up-regulated,were detected in the mulberry leaves transiently expressing MbMYB12 gene.Most of these metabolites belonged to the upstream metabolites of flavonoid synthesis pathway and flavones.Fluorescence quantitative PCR analysis showed that the expression level of Mb F3 H gene,which was the upstream gene of flavonoid synthesis pathway,and the Mb FLS gene,which was in the flavonol branching pathway,was up-regulated by the overexpression of MbMYB12.A total of 106 flavonoid metabolites,which abundances were significantly decreased in the transgenic MbMYB308 mulberry leaves,were detected.These metabolites include various upstream metabolites and downstream metabolites of flavonoid synthesis pathway,such as chalcone and anthocyanins.Meanwhile,fluorescence quantitative PCR analysis showed that the expression of Mb CHS gene in the upstream of flavonoid synthesis pathway,and Mb FLS,Mb DFR,Mb ANS genes in the downstream,were down-regulated.（5）Obtainment and flavonoid content analysis of the transgenic hairy roots of mulberry.Mulberry transgenic MbMYB12 or MbMYB308 gene hairy roots were successfully obtained by Agrobacterium tumefaciens K599 mediated root transformation system.It was showed that the content of flavonoids in the transgenic MbMYB12 gene mulberry hairy roots was significantly higher than that in the transgenic empty vector hairy roots,while that in the transgenic MbMYB308 mulberry hairy roots was lower than that in the transgenic empty vector hairy roots.