Screening And Identification Of Transcription Factors Interacting With SmJAZ8 Protein In Salvia Miltiorrhiza

Salvia miltiorrhiza is a perennial herb belonging to the Salvia genus of Lamiaceae.Its dry roots or rhizomes have been widely used in the prevention and treatment of coronary heart disease,chronic renal failure,arteriosclerosis and other diseases as traditional medicinal materials in China.In this study,SmJAZ8 was used as a bait protein to screen for transcription factors interacting with it by yeast two-hybrid(Y2H)system,which laid a foundation for further study on the interaction between JAZ proteins and transcription factors to regulate the synthesis of tanshinones and phenolic acids.The following research results have been obtained in this study:1.The full-length normalized AD library was prepared by obtaining total RNA from different tissues of S.miltiorrhiza.The titer of the yeast library constructed in this study was more than 1×10~7 cfu/mL,and the yeast library capacity was 3.1×10~7 cfu/mL.The average insert is larger than 1.0 kb.2.BD-SmJAZ8 was used as a bait vector to screen yeast homogenization library by Y2H and289 clones were obtained.Using BLASTX analysis,we found that one of the clones labeled 8-6 showed it to be a bHLH transcription factor,and it was named as SmbHLH128.3.We verified the interaction between SmJAZ8 and SmbHLH128 by BiFC.Subsequently,the domain of SmJAZ8 was truncated and Y2H assays showed that the interaction with SmbHLH128was depended on the Jas domain of SmJAZ8.4.The transcriptional activation domain of SmbHLH128 was verified.We constructed the overexpression vector and dominant SRDX expression vector of SmbHLH128,and obtained 10 overexpressing transgenic hairy root lines and 11 dominant inhibiting transgenic hairy root lines,respectively.The positive transgenic hairy roots were confirmed by fluorescence microscopy and qPCR analysis.