Research On The Technology Of Improving Tanshinone Content By Transcription Factor SmbHLH61

Salvia miltiorrhiza Bunge,as a traditional Chinese medicinal herb,is often incorporated with dried roots and rhizomes.Among them,tanshinone,a lipid soluble phenanthrene quinone compound,is the major pharmacologically active component of Salvia miltiorrhiza and has shown significant efficacy in the treatment of heart and cerebrovascular diseases.In our study,the SmbHLH61,which is highly expressed in the root of Salvia miltiorrhiza and selected in our laboratory in the previous period,was investigated to explore its mechanism in regulating tanshinone synthesis and the technology to improve tanshinone content using transgenic technology.The main results are as follows:1.Gene cloning: using the c DNA from the root of Salvia miltiorrhiza as a template,primers were designed to amplify the fragment of interest,and the SmbHLH61 gene with a full length of 1008 bp was obtained by gene cloning.2.Analysis of tissue expression: RNA was extracted from four different tissues(root,stem,leaf and flower)of Salvia miltiorrhiza respectively,and performed q RT-PCR after reverse transcription to c DNA,analysis identified that SmbHLH61 expression levels were greatly different in the four tissues of Salvia miltiorrhiza,with the highest expression levels in roots and lower expression levels in stems,leaves,and flowers.3.Inducible expression analysis: the expression abundance of SmbHLH61 was down regulated to various degrees within 0-24 hours after treatment with exogenous hormones of Me JA and SA.It was illustrated that the induction of exogenous hormones Me JA and SA would inhibit the gene expression of SmbHLH61 in the root of Salvia miltiorrhiza.4.Subcellular localization: the subcellular localization vector of SmbHLH61 was successfully constructed by using PBI121 EGFP,and the localization of SmbHLH61 to the nucleus was observed under the confocal laser scanning microscopy.5.Obtaining transgenic hairy roots: the overexpression vector and RNAi vector of SmbHLH61 were constructed,and successfully induced positive hairy root plants for SmbHLH61 overexpression and RNAi through an agrobacterium rhizogenes of Ar.Qual mediated genetic transformation system.6.Gene analysis of key enzymes related to the tanshinone synthesis pathway: The expression levels of six related enzyme genes(Sm GGPPS,Sm DXS2,Sm KSL1,Sm CYP76AK1,Sm CYP71D375,Sm CYP71D411)in the tanshinone synthesis pathway were analyzed in SmbHLH61 using q RT-PCR.The results showed that the expression levels of all six related enzyme genes were significantly up-regulated in the overexpression strain compared with the Ar.Qual control,with the expression levels of Sm GGPPS,Sm DXS2,Sm KSL1,Sm CYP76AK1,Sm CYP71D375 and Sm CYP71D411 being 9.79,7.86,2.35,4.22,and 2.10 times,higher than the control,respectively.While the expression levels of the six related enzyme genes in the RNAi strain were significantly down-regulated,and even in Sm DXS2 the amount of expression approximates zero.The above results illustrate that SmbHLH61 is a key positive regulator in the tanshinone synthesis pathway.7.Analysis of the content of tanshinone compounds: The changes of four tanshinones(Tanshinone Ⅰ,Tanshinone ⅡA,Dihydrotanshinone,and Cryptotanshinone)contents in the transgenic strains were determined by Ultra Performance Liquid Chromatography,and the results showed that: compared with the control strain of wild type,the contents of the four tanshinones in the overexpressing SmbHLH61 strain increased respectively by 3.15,4.71,22.52 and 25.93 times on average,However,compared with the control strain,the contents of the four tanshinones in the RNA interference strains were 0.47,0.11,0.49 and 0.14 times on average,respectively.It follows that SmbHLH61 overexpression positively regulates tanshinones synthesis and accumulation,whereas RNAi inhibits tanshinones synthesis and accumulation.