Analysis Of Flavonoid Biosynthetic Enzyme Genes And The Regulatory Roles Of MiR828 In Salvia Miltiorrhiza

Flavonoids are a class of important secondary metabolites.They not only play important roles in the biological processes such as disease resistance,abiotic stress tolerance and organ coloring,but also have diverse pharmacological activities in anti-inflammatory,anti-bacteria,and anti-oxidation.miR828 is well known as a miRNA involved in regulating flavonoid biosynthesis by miR828-MYB cascade or miR828-ta-siRNA-MYB cascade.Salvia miltiorrhiza is a perennial herbaceous plant.It is widely used for treatment of cardiovascular and cerebrovascular diseases.As a model medicinal plant,dissection for the molecular mechanism of secondary metabolism in Salvia miltiorrhiza has attracted wide attention from many scientists.The capacity of anti-oxidation and anti-inflammatory activities of Salvia miltiorrhiza correlates with flavonoid content.In addition,flavonoids and phenolic acid compounds share the same upstream of phenylpropanoid pathway.Their crosstalk remains to be elucidated.Arabidopsis miR828 can regulate the expression of A tMYB 7 5/PAPA1 and AtMYB90/PAP2 via miR828-ta-siRNA-MYB cascade.Over-expression of AtMYB75/PAPA1 and AtMYB90/PAP2 can up-regulate the accumulation of phenolic acids.Therefore,identification of flavonoid biosynthetic enzyme genes and discussion of miR828-mediated regulation is of great importance to understand the crosstalk of complex metabolic networks.Besides,it can improve the abiotic and biotic stress resistance,active compounds and molecular breeding of Salvia miltiorrhiza.Systematic analysis of flavonoid biosynthetic enzyme genes and function identification of miR828 were carried out in Salvia miltiorrhiza.The main conclusions obtained from this study are as follows:1.Identification,cloning and analysis of flavonoid biosynthetic enzyme genes in Salvia miltiorrhizaTotal 26 flavonoid biosynthesis related genes were identified,cloned and analyzed.They can be divided into 9 families,including CHS,CHI,FN.S,F3H,F3 ’H,F3’5’H.FLS,DFR and ANS.Integrated analysis of sequence features,phylogenetic relationships,differential expression revealed that 20 genes are novel and 14 genes are most likely to be involved in flavonoid biosynthesis.The results provides valuable information for further understanding the biosynthetic pathway of flavonoids in salvia miltiorrhiza.2.Identification and analysis of UGT genes in Salvia miltiorrhizaTotal 149 UGT genes were identified.We investigated their physical and chemical characteristics,phylogenetic relationships,tissue-differential expression,and GO annotation.The results showed that SmUGTs were clustered into 16 phylogenetic groups(A-P).Based on gene co-expression with flavonoid biosynthetic enzyme genes and GO annotation,15 UGTs were predicted to be involved in the glycosylation of flavonoids.3.Identification,cloning and analysis of target genes of Sm-miR828SmTAS4、SmMYB36、SmMYB97、SmMYB111 and SmMYB113 were identified as the target genes of Sm-miR828 through psRNATarget prediction and degradome analysis.SmTAS4 and SmMYB36 were successfully verified by RLM-5 ’ RACE experiments.SmTAS4 were triggered to produce phased ta-siRNA.The most abundant ta-siRNA was the fourth phased siRNA(Sm-TAS4-siR81(-))in antisense strand.Sm-TAS4-siR81(-)targets SmMYB112 and SmMYB114 for cleavage.Comprehensive analysis of phylogenetic relationships,sequence features and conserved functional domains suggested that identified MYB genes were all R2R3-MYBs involved in flavonoid biosynthesis.4.Functional analysis of Sm-miR828To investigate the function of Sm-miR828,we over-expressed SmMIR828 in transgenic Salvia miltiorrhiza.Three transgenic lines were tested and analyzed by transcriptome and metabolome.The results indicate miR828 was involved in the biosynthesis of flavonoids,phenolic acids and tanshiones in Salvia miltiorrhiza.Intriguingly,the content of various tanshione were up-regulated in transgenic Salvia miltiorrhiza plants over-expressing MIR828.Furthermore,a simple genetic regulatory network was proposed.In conclusion,we systematically investigated flavonoid biosynthetic enyzme genes and the biological function of Sm-miR828.The results showed that various secondary metabolites,including tanshione,phenolic acids and flavonoids,were regulated by miR828-SmMYB36/SmMYB111/SmMYB113 cascade in roots,and miR828-TAS4-ta-siRNA-SmMYB112 cascade or miR828-SmMYB111 cascade in leaves.Over-expression of Sm-miR828 enhanced the biosynthesis of tanshione in roots and leaves of Salvia miltiorrhiza.This study provided valuable information for understanding the synthesis pathway of flavonoids and the function of miR828 in Salvia miltiorrhiza.