Molecular Mechanism Of SmWRKY44 Promotes Tanshinone Biosynthesis In Salvia Miltiorrhiza Bunge

Salvia miltiorrhiza is a traditional medicinal material in China.It taking medicine with roots and rhizomes,which has a significant effect in the treatment of cardiovascular diseases.Tanshinone and salvianolic acid are its main active ingredients.Among them,tanshinone belongs to abietane-type diterpenoids.Previous studies have confirmed that SmCPS1 is the key to influence the production of tanshinone.In this study,the promoter of Salvia miltiorrhiza SmCPS1 was used as a bait to screen the Salvia miltiorrhiza c DNA library through yeast single hybrid,and the WRKY transcription factor SmWRKY44 interacting with it was obtained.In this experiment,the function and mechanism of SmWRKY44 were mainly analyzed.The genetic transformation test showed that SmWRKY44 promoted the accumulation of tanshinone in the hairy root of Salvia miltiorrhiza;and the q RT-PCR,Y1 H,EMSA,Ch IP-q PCR and Dual-Luc report test revealed the molecular mechanism of SmWRKY44 in promoting the biosynthesis of tanshinone in the hairy root of Salvia miltiorrhiza.The main results are as follows: 1.The 1771 bp promoter of SmCPS1 was cloned from Salvia miltiorrhiza,and used to screen c DNA library of Salvia miltiorrhiza.The identified WRKY transcription factor SmWRKY44 was cloned with a CDS of 798 bp and encodes 265 amino acids.It expressed mainly in root and stem,as well as responding to JA,SA,chilling,salt and drought stresses.SmWRKY44 was localized in the nucleus.2.Overexpression and RNAi hairy roots of SmWRKY44 were obtained through A.rhizogenes(C58C1)mediated genetic transformation.Cryptotanshinone,tanshinone I,and tanshinone IIa were all improved significantly compared with wild-type hairy roots while decreased significantly in the RNAi hairy roots.Furthermore,expression analysis of key genes involving in tanshinone biosynthetic pathway showed that the expression patterns of SmCPS1,SmCPS5 and SmKSL1 are consistent with the SmWRKY44.In order to further analyze the molecular mechanism of SmWRKY44 promoting tanshinone biosynthesis,Y1 H,Ch IP-q PCR,EMSA and Dual-Luc tests were used to verify that SmWRKY44 can directly bind to the W-box in the promoters of SmCPS1,SmCPS5 and SmKSL1 in vitro and in Salvia miltiorrhiza,And can activate the transcription of these three genes.In summary,this study revealed the molecular mechanism of SmWRKY44 by directly binding the W-box in the promoters of SmCPS1,SmCPS5 and SmKSL1 and activating their transcription,thereby promoting the biosynthesis of tanshinone.