Synergistic Effect On The Biosynthesis Of Panax Notoginseng Saponins By Overexpressing A Transcription Factor PnbHLH And RNA Interference Of Cycloartenol Synthase Gene

Panax notoginseng is a medicinal plant of Panax genus,which has a long history in medicine.Panax notoginseng saponins(PNS)are the main active components in P.notoginseng and play a significant role in the prevention and treatment of cardiovascular and cerebrovascular diseases.Since P.notoginseng belongs to perennial plants,and which cultivation needs crop rotation with low land utilization,it is necessary to study the technology of biosynthetic regulation of PNS,and provide theoretical and technical basis for the production of medicinal saponins by synthetic biology.Panax notoginseng saponins are mainly biosynthesized by the mevalonate(MVA)pathway which is also the biosynthetic pathway of phytosterols and the saponin anabolic flow will be shared by the biosynthesis of by-products phytosterols.Thus,although the first key enzyme “cycloartenol synthase(CAS)” in phytosterols biosynthetic pathway has no direct effect on the biosynthesis of PNS,it shares the common precursor 2,3-oxidosqualene with dammarenediol-II synthase(DS),the regulation of CAS expression could indirectly affect PNS biosynthesis.In addition,our previous research indicated that transcription factors could affect the expressions of key enzyme genes involved in PNS biosynthesis,and then achieve the regulation of saponin anabolic flow.This study utilized the advantage of unique "multi-point regulation" of transcription factors,combined with the down-regulation of the by-product branch,to maximize the biosynthesis efficiency of PNS.The overexpression vector pCAMBIA1300S-PnbHLH was constructed according to the known transcription factor gene sequence of PnbHLH,and the overexpression of PnbHLH was realized in the P.notoginseng cell lines achieved CAS RNAi,thus to explore the synergistic effect of PnbHLH overexpression and CAS RNAi on the regulation of PNS biosynthesis.In this study,the transcription factor gene PnbHLH was cloned firstly,the open reading frame(ORF)of PnbHLH was 966 bp in length and encoded 321 amino acids.Homologous sequence alignment and phylogenetic analysis indicated that the PnbHLH belonged to the bHLH TFs family.The recombinant vector pCAMBIA1300S-PnbHLH,which was obtained by ligating the target gene PnbHLH with pCAMBIA1300 S vector,was introduced into Agrobacterium EHA105 by freeze-thaw method,and the T-DNA containing the PnbHLH fragment was integrated into the genome of P.notoginseng which covered CAS interference fragment to get PnbHLH/CAS-transgenic cell lines.Positive cell lines of PnbHLH/CAS were screened out by resistance genes(npt II and hpt II)PCR detection after several rounds of double antibiotic culture.The results of qPCR showed that the PnbHLH gene was successfully overexpressed and four PnbHLH/CAS positive cell lines were selected and numbered T1,T2,T3 and T4 respectively.The contents of PNS in the four transgenic positive cell lines were significantly increased compared with those in both the wild type(WT)cells and the cells which covered CAS RNAi fragment(Ti);while the contents of phytosterols were all decreased in the positive cells.Overexpression of PnbHLH in P.notoginseng cells could enhance the biosynthesis of PNS;CAS RNAi led to the decrease content of phytosterols and its synergistic effect with PnbHLH overexpression would increase the metabolic flux of the PNS biosynthesis more significantly.High-performance liquid chromatography(HPLC)was used to detect the contents of main monomer saponins in all P.notoginseng cell lines.It was found that the contents of five monomer saponins(Rd,Rb1,Re,Rg1 and R1)were increased to some extents in PnbHLH/CAS cells.Moreover,qPCR showed that the relative expression levels of some key enzyme genes involved in the biosynthetic pathway of triterpenoid saponins such as DS,SS and SE were increased compared with WT and Ti cell lines.In order to verify the function of PnbHLH in the biosynthesis of PNS,primers were designed based on the promoter sequences known before and the promoters of PnDS,PnSS,PnSE and PnCAS were successfully cloned using genome DNA of P.notoginseng as the template.The four promoter sequences were inserted into the multiple cloning sites of the bait plasmid pAbAi respectively to get recombinant plasmids pAbAi-PnDSP,pAbAi-PnSSP,pAbAi-PnSEP,and pAbAi-PnCASP.The recombinant prey vector pGADT7-PnbHLH was obtained by ligating PnbHLH with the yeast prey vector pGADT7.The recombinant vector pGADT7-PnbHLH was co-transformed into yeast cells Y1 HGold with four recombinant bait plasmids respectively,and cultured on the selective medium SD/-Leu/AbA.The results showed that the yeast cells containing the recombinant plasmids pAbAi-PnDSP,pAbAi-PnSSP and pAbAi-PnSEP were able to grow on the selective medium,whereas the yeast cells containing pAbAi-PnCASP could not grow.It was meant that the PnbHLH transcription factor could specifically bind to the promoter sequences of DS,SS and SE.Therefore,the PnbHLH transcription factor could enhance the biosynthesis of PNS indirectly by multi-point regulation of the key enzyme genes involved in the PNS biosynthetic pathway;and its synergistic effect with the RNA interference of CAS further increased the content of PNS.