Effect On The Synthesis Of Panax Japonicus Saponins By RNAi-mediated Silencing Of CAS Gene

Panax japonicus is a kind of valuable medicinal herb used commonly in China,which belongs to the Panax genus,Araliaceae family.It has many pharmacological activities such as removing blood stasis,anti-inflammatory,analgesic,anti-tumor,immune regulation and hemostasis.P.japonicus saponins are the main active ingredients in P.japonicus,including dammarane-type and oleanane-type saponins.Nowadays,most of P.japonicus herb is from wild resource,and their growth cycle is long.Generally,they can be used as medicine for 6-years growth period.With the continuous increase of market demand,the gap between supply and demand is getting bigger.For such high-value,low-yield natural medicines,in order to solve the problem of medicinal resources,it is necessary to use modern molecular biology and genetic engineering technique to explore the biosynthesis pathway of P.japonicus saponins,and then carry out research on the synthetic biology of P.japonicus saponins.Triterpenoid saponins are the main active ingredients in P.japonicus.Compared with the medicinal herb“Panax notoginseng”,which only contains dammarane-type saponins,P.japonicus contains not only dammarane-type saponins but also oleanane-type saponins.Therefore,the clinical usage of P.japonicus is significantly different from that of P.notoginseng.P.japonicus saponins is mainly synthesized by the mevalonate(MVA)pathway,in which dammarenedion-II synthase(DS)and?-amyrin synthase(?-AS)are key enzymes in the synthesis of dammarane-type saponins and oleanane-type saponins respectively,and 2,3-oxidosqualene is the common precursor of DS and?-AS.Moreover,2,3-oxidosqualene is also the precursor of phytosterol synthesis.Phytosterols are by-products of P.japonicus saponins biosynthesis,which is synthesized by a series of enzymatic reactions.Cycloartenol synthase(CAS)is first enzyme in the synthetic branch of phytosterols,which catalyzes 2,3-oxidosqualene to form cycloartenol.Therefore,there is a competitive consumption of 2,3-oxidosqualene between CAS,DS and?-AS in the biosynthetic pathway of phytosterols and P.japonicus saponins.In this study,RNAi technology was used to down-regulate the expression of CAS gene(PjCAS)in order to weaken the synthesis of phytosterol,so that the metabolic flux could flow more to the biosynthetic pathway of triterpenoid saponins,and finally achieved the purpose of improving the biosynthesis of P.japonicus saponins.In this study,P.japonicus RNA was reverse-transcribed into cDNA by reverse-transcription PCR,and the RNAi fragment of PjCAS gene was amplified based on this template.Then the primers with attB site were designed for PCR amplification of RNAi fragments with attB recombination sites at both ends.Subsequently,the RNAi fragment with the attB site was cloned into the binary expression vector pHellsgate 2 by BP reaction using Gateway~TMM technology to complete the construction of the RNAi expression vector pHellsgate-CAS.The successfully constructed recombinant vector pHellsgate-CAS was introduced into Agrobacterium tumefaciens EHA105 by freeze-thaw method,and the foreign gene was successfully introduced into the genomic DNA of P.japonicus by A.tumefaciens.Six positive cell lines of PjCAS RNAi P.japonicus were obtained by screening of kanamycin resistance and PCR of resistant gene.Quantitative real-time PCR showed that the expression level of PjCAS gene was significantly down-regulated in PjCAS RNAi cells compared with the normal cell line,which proved that the RNAi cell lines were successfully constructed.In order to clarify the role of PjCAS gene in the biosynthesis pathway of P.japonicus saponins,the total saponins and phytosterols in 6transgenic cell lines was detected.The result showed that the phytosterols content in PjCAS RNAi cell lines were significantly lower than that in normal cells,and the total saponins content was higher than that in normal cells,indicating that RNAi of PjCAS could influence the metabolic flux of P.japonicus saponins.Six important monomer saponins in P.japonicus,ginsenoside Rd,Rb1,Re,R0,chikusetsusaponin IV and??a were detected by high performance liquid chromatography,it was found that all those monomer saponins have different levels of raise.In order to determine the mechanism of the increase of saponins content,the expression levels of key enzyme genes PjDS and PjAS in the biosynthetic pathway of P.japonicus saponins was analyzed,which revealed that the expression levels of PjDS and PjAS were increased in the PjCAS RNAi cell lines.This indicate that the decrease of PjCAS expression might reduce the metabolic flux of phytosterols synthesis branch,which indirectly increased the flux of saponins synthesis branch.In order to dispose the increased metabolic flux,the two branches of P.japonicus saponins synthesis passively increase the expression of two genes of key enzymes PjDS and PjAS;therefore,the content of the P.japonicus saponins was increased.To sum up,this study indicated that PjCAS played an important regulatory role in the synthesis of triterpenoid saponins in P.japonicus.The down-regulation of PjCAS expression may indirectly increase the metabolic flux of saponins biosynthesis and then promote the biosynthesis of saponins.