| Salvia miltiorrhiza is one of the traditional bulk medicinal materials in China,which has significant curative effect on cardiovascular diseases.The mainly active ingredients of salvia miltiorrhiza include tanshinone and salvianolic acid..Jasmonate(JA)can significantly promote the accumulation of active ingredients of Salvia miltiorrhiza.By regulating JA biosynthesis and further regulating the accumulation of active ingredients of Salvia miltiorrhiza,new ideas and approaches can be provided for improving the quality of medicinal materials.Although the biosynthetic pathway of JA has been clarified,the regulatory mechanism of JA biosynthesis in Salvia miltiorrhiza Bunge is little known.In this study,firstly,a key enzyme gene for JA biosynthesis of Salvia miltiorrhiza,namely malondialdehyde oxidase gene(SmAOS)and its promoter,were cloned.Using the promoter as bait,a c DNA library of Salvia miltiorrhiza was screened by yeast single hybridization,and a b HLH transcription factor SmbHLH74 interacting with SmbHLH 74 was obtained.Further,Y1 H,EMSA,ChIP-q PCR and Dual-LUC tests,the molecular mechanism of SmbHLH74 transcription factor participating in JA biosynthesis by regulating AOS transcription was proved.The main results are as follows:1.SmAOS gene was cloned from Salvia miltiorrhiza Bge.CDS was 1599 bp,encoding 532 amino acids.It is expressed at a high level in roots and at a low level in stems and leaves.Exogenous Me JA treatment can rapidly induce SmAOS expression.Subcellular localization showed that SmAOS was localized on chloroplasts.2.The SmAOS overexpression vector was constructed.Through genetic transformation mediated by Agrobacterium rhizogenes and Agrobacterium tumefaciens,only a small number of regenerated plants and hairy roots were obtained.The regenerated buds could not take root,while the hairy roots grew slowly.With the culture process,they were easy to brown and die.It is presumed that the increase of JA content severely inhibited the growth of roots.3.The promoter 2079 bp of SmAOS was cloned.Using the promoter as bait,yeast single hybridization was used to screen the c DNA library of Salvia miltiorrhiza,and two WRKY and two b HLH transcription factors were obtained.SmbHLH74 was the main research object in this study.4.In order to further analyze the transcriptional regulation of SmbHLH74 and SmAOS,through Y1 H,EMSA,ChIP-q PCR and Dual-Luc reports and other tests,it was found that SmbHLH74 directly binds to the promoter of SmAOS and inhibits its transcription.Moreover,in SmbHLH74 overexpressed plants,SmAOS expression level was down-regulated and JA content was significantly reduced.To sum up,this study proves that SmbHLH74 in Salvia miltiorrhiza directly binds to the promoter of SmAOS and inhibits its transcription.So as to negatively regulate the jasmonic acid biosynthesis. |
