Functional Study Of PnbHLH1 Transcription Factor Involved In Biosynthetic Regulation Of Panax Notoginseng Saponins

Panax notoginseng(Burk.)F.H.Chen,which roots and rhizomes can be uesd as medicines,belongs to Panax genus of the Araliaceae family and has nearly 600 years of application history used as a traditional Chinese medicine.Panax notoginseng has triple effects of hemostasis,activating blood and replenishing blood,which is the vital component of many Chinese patent medicines and preparations.P.notoginseng saponins(PNS)are the main medicinal active ingredients in P.notoginseng.Currently,raw material of P.notoginseng is obtained mainly through artificial cultivation,which is labor-intensive,long duration and low efficiency.PNS has complicated structures and the chemical synthesis of which is critically difficult.It has important research values and application potential by using techniques of bioengineering to carry out homologous or heterologous expression of PNS.Several function-related genes of key enzymes in secondary metabolic pathway are often positively or negatively regulated by the same transcription fator.To promote the synthesis of end-products in the secondary metabolic pathway,the genetic modifications of transcription fator genes are easier than the operations of several key enzymes genes.Based on the full-length cDNA of PnbHLHl transcription fator gene,the vivo and vitro functional analysis of PnbHLH1 transcription fator was peformed in this study,and which regulatory function in the biosynthesis of PNS was also studied.The main findings are listed as follows:1.The ORF of PnbHLH1 transcription fator gene was cloned by taking the cDNA of P.notoginseng cell as a template;then the prokaryotic expression vector pET-28a-PnbHLHl and the plant overexpression vector pCAMBIA1300S-PnbHLH1 were constructed.2.The prokaryotic expression vector pET-28a-PnbHLH1 was transferred into prokaryotic expression strain BL21(DE3),then the foreign protein was expressed with the induction of IPTG,and the biologically active PnbHLH1 transcription factor was obtained by inclusion body extraction,denaturation,purification,refolding,concentrating and other steps.The obtained PnbHLH1 transcription factor was incubated with probes and the EMSA experiment was carried out.Results showed that PnbHLH1 transcription factor could specifically bind to E-box cis-acting element.3.The plant overexpression vector pCAMBIA1300S-PnbHLH1 was transferred into Agrobacterium tumefaciens EHA105;with the mediation of EHA105,the recombinant vector was transferred into P.notoginseng cell.By means of antibiotics and molecular detection methods,the P.notoginseng cell was screened to get the positive PnbHLH1 transgenic cell lines.The expressions of key enzymes genes involved in the biosynthetic pathway of PNS were dectected by RT-PCR and qRT-PCR methods.The results indicated that,compared with the non-transgenic cells of P.notoginseng(as the control),the expressions of PnbHLH1,DS,SS and SE genes were increased significantly,the expression of FPS gene also had a certain amount of increase;and there was no obvious change of HMGR gene expression,which might be related to the position of HMGR gene that was in the upstream of PNS biosynthetic pathway and PnbHLH12 transcription fator had no effect on HMGR expression;in addition,the expression of CAS gene was increased slightly.The contents of total saponins,monomer saponins and phytosterols were detected on the PnbHLH1 transgenic cell lines of P.notoginseng,respectively.Results showed that,compared with the control,the PNS content has been improved obviously,approximately two times of the control.The contents of four major monomer saponins including Rg1,Re,Rbl and Rd had different amplitude of rise.It was worth mentioning that PnbHLH1 transcription fator might regulate the expressions of relevant genes of post-modification enzymes in the synthesis of Rd.The content of phytosterols has been increased slightly.Combining with the results of qPCR,it can be presumed that the metabolic flux mainly flowed into the synthesis branch of triterpenoids and the production of PNS was rised when the gene expressions of key enzymes in PNS biosynthsis pathway have been increased.In addition,CAS should be the key enzyme involved in the synthesis of phytosterols in P.notoginseng.Above studies have demonstrated that PnbHLH1 can achieve multipoint and positive regulations in PNS biosynthesis.Such results will provide theoretical and scientific basis for the establishment of efficient homologous or heterologous expression systems of PNS.