The Study Of The Transcription Factor SmMYB111 Of Salvia Miltiorrhiza On The Regulation Of The Synthesis Of Phenolic Acids

Salvia miltiorrhiza Bunge is a well-known traditional Chinese medicinal plant belonging to Labiatae,whose dry root and rhizome can be used as medicine.The main active ingredient is composed of lipid-soluble tanshinones and water-soluble phenolic acids.S.miltiorrhiza is an ideal material for studying secondary metabolites and is called a model medicinal plant.The water-soluble phenolic acid is considered to be the main active ingredient of Danshen.Its synthesis pathway is composed of two metabolic pathways,tyrosine and phenylpropane.At present,the application of genetic engineering methods to increase the content of salvianolic acid has become a hot topic in recent years.In addition to affecting plant growth and development,the MYB transcription factor family plays an important role in the regulation of plant primary and secondary metabolism.The R2R3-MYB transcription factors are the most abundant and most widely studied transcription factors in the MYB family.In our laboratory,we obtained a R2R3-MYB transcription factor SmMYB111,and preliminarily speculated that SmMYB111 affected the synthesis of phenolic acids in Smiltiorrhiza.In view of this,this research is based on the transcription factor SmMYB111.The changes of total phenolic acids,total flavonoids,anthocyanins,rosmarinic acid and salvianolic acid B in SmMYB111 transgenic lines were studied.The molecular mechanism of SmMYB111 regulation of phenolic acid synthesis was preliminarily determined.This study provides a theoretical basis for improving the content of salvianolic acid by genetic engineering.The main results were listed as follows:1.Bioinformatics analysis of R2R3-MYB transcription factor SmMYB111 revealed that the N-terminus contained a conserved motif[DE]Lx2[RK]x3Lx6Lx3R interacting with bHLH protein.Multiple sequence alignment showed that SmMYB111 had relatively high homology with VvMYBPA1,PtoMYB115 and DkMYB4,which regulated the synthesis of tlavonoids.Gene structure analysis showed that SmMYB111 contained only one intron and was highly conserved.It was preliminarily speculated that SmMYB111 might involve in the regulation of phenylpropane secondary metabolic pathway.2.The 698 bp sequence of the SmMYB111 promoter region was cloned,and the promoter elements of the promoter region were analyzed by the PlantCARE online site.The results indicated that the promoter region contained a MYB transcription factor binding site,a bHLH-like transcription factor binding site and an abiotic stress response element.The SmbHLH51 promoter region contained two MYB transcription factor binding sites of MBS and MBSII.It was speculated that SmMYB111 not only participated in the response of plant abiotic stress,but also had a transcriptional regulatory relationship with SmbHLH51.3.Prokaryotic expression analysis of SmMYB111.The results of Coomassie blue staining and Western Blot showed that the relative molecular mass of SmMYB111 protein was about 29.3 kDa,which could be expressed in highest amount at 37℃ for 6 h.Some proteins were soluble in the cells,but most of them were inclusion body.Prokaryotic expression of SmMYB111 provided experimental conditions for subsequent purification of SmMYB111 protein.4.The SmMYB111 overexpression vector and interference vector were constructed.Overexpressing and interfering transgenic lines of SmMYBlll in S.miltiorrhiza were obtained by Agrobacterium-mediated transformation.Eight positive transgenic lines were obtained by DNA and RNA molecular level verification.According to the expression level of SmMYB111 in the transgenic lines,overexpressing lines(OM-1,OM-2)and interfering lines(iM-1,iM-2)were used for the following experiments to study the function of SmMYB111 in S.miltiorrhiza.5.The contents of total phenolic acids,total flavonoids and anthocyanins in SmMYB111 transgenic lines were measured.The results showed that the total phenolic acids,total flavonoids and anthocyanins contents in the overexpressing lines were significantly different from the control lines,and the total phenolic acid content was 1.77 and 1.83 times higher than that of the control lines,respectively.The total flavonoids content was 1.92 and 1.57 times higher while anthocyanins were 1.80 and 1.63 times higher,respectively.There was a significant reduction in total phenolic acids and total flavonoids in the interferening lines compared with the control lines,but no significant difference was detected in anthocyanins content.It indicated that SmMYB111 played a positive role in the accumulation of total phenolic acids and total flavonoids in S.miltiorrhiza.6.Rosmarinic acid and salvianolic acid B in the SmMYB111 overexpressing and interfering transgenic lines were determined.The content of rosmarinic acid in overexpressing lines OM-1 and OM-2 was found to be 3.03 and 3.05 times higher than that of the control lines,respectively;while the content of salvianolic acid B was 3.26 and 2.36 times higher,respectively.The contents of rosmarinic acid and salvianolic acid B in the interfering lines iM-1 and iM-2 were significantly reduced compared with the control lines,indicating that SmMYB111 positively regulated the synthesis of salvianolic acids.7.The expression levels of 16 enzyme genes in the salvianolic acid synthesis pathway of SmMYB111 transgenic lines were determined.Quantitative results indicated that 10 enzyme genes SmTAT1,SmTAT2,SmPAL1,SmPAL3,SmC4H1,Sm4CL2,Sm4CL3,SmRAS1,SmRAS5 and SmCYP98A14 were significantly up-regulated in the SmHYB111 overexpressing lines,while the expression in the interfering lines was supressed.It indicated that the SmMYB111 transcription factor promoted the accumulation of salvianolic acids in S.miltiorrhiza by activating expression of multiple enzyme genes such as SmTAT1,SmRAS5 and SmCYP98A14 in the salvianolic acid synthesis pathway.8.SmMYB111 target genes and transcriptional regulation activity were preliminarily verified.After analyzing the MYB binding site in the promoter regions of genes involving in the salvianolic acid synthesis pathway and the expression of the enzyme gene in the SmMYB111 transgenic lines,yeast one-hybrid was used to explore the interaction of SmMYB111 with the promoter regions of SmTAT1 and SmCYP98A14,respectively.Lyciferase assay indicated that SmMYB111 activated the expression of SmTAT1 and SmCYP98A14.It was preliminarily determined that the regulation of SmMYB111 was that SmMYB111 bound and activated the target genes SmTAT1 and SmCYP98A14 directly,and thereby promoted the accumulation of salvianolic acids.