The Mechanisms Of The Transcription Factor SmMYB36 Regulates Tanshinone And Salvianolic Acid Biosynthesis

Salvia miltiorrhiza Bunge is classified in the Flora of China as an erect perennial herb of the family Lamiaceae.Its medicinal components are concentrated in the rhizomes,the mature and dried rhizomes are widely used as a traditional Chinese herb with medicinal effects in cardiovascular diseases,neurasthenia,insomnia,tumors and so on.The accumulation of the medicinal components tanshinone and salvianolic acid is regulated by the transcription factor SmMYB36.The aim of this study was to determine the downstream target genes of SmMYB36 in the tanshinone and salvianolic acid pathways and the biological functions of SmMYB36 self-activating sequence（amino acids 154-160）,and to establish the mechanism by which SmMYB36 regulates the tanshinone and tannic acid pathways.The results are as follows.1.SmMYB36 can bind and promote the promoter expression of Sm DXS2,Sm GGPPS1,Sm CPS1 and Sm ERF6,and bind and inhibit the promoter expression of Sm RAS,Sm GAPC and Sm ERF115,as verified by Dual luciferase reporter assays and EMSA.2.It was verified by Dual luciferase reporter assays that SmMYB36 had both promotive and inhibitory regulatory functions,while SmMYB36^1-153retained the inhibitory function but lost the promotive function,indicating that SmMYB36 self-activation sequence"LELDEDE"was responsible for activating the expression of target genes and had nothing to do with inhibitory function.3.Overexpression vectors p K7WG2R-SmMYB36 and p K7WG2R-SmMYB36^1-153were constructed and two overexpressed transgenic hairy roots were obtained.The contents of tanshinone and salvianolic acid in this two hairy roots and control hairy roots were detected by HPLC.The results showed that the contents of tanshinones in over-expressed SmMYB36hairy roots increased and salvianolic acids decreased,while there was no difference in the content of tanshinones in overexpressed SmMYB36^1-153hairy roots,but the content of salvianolic acids were aslo reduced.4.The expression levels of target genes in overexpressed hairy roots were detected by fluorescence quantitative PCR.The results showed that the expression levels of tanshinone pathway Sm DXS2,Sm GGPPS1,Sm CPS1 and transcription factor Sm ERF6 were increased in p K7WG2R-SmMYB36 hairy roots,and unchanged in p K7WG2R-SmMYB36^1-153hairy roots.The expression levels of salvianolic acid pathway Sm RAS,transcription factor Sm ERF115 and primary metabolic pathway Sm GAPC were significantly decreased in both the hairy roots of p K7WG2R-SmMYB36 and p K7WG2R-SmMYB36^1-153.