Molecular Cloning And Functional Study Of PnHMGR Gene In The Biosynthetic Pathway Of Panax Notoginseng Saponins

Panax notoginseng belongs to the genus Panax of the family Araliaceae,and the root of which is the main medicinal part.Panax notoginseng saponins(PNS),which belong to dammarane-type tetracyclic triterpenoid saponins,are the major active components in P.notoginseng and exhibit multiple biological activities.Studies have shown that PNS have good physiological activities in cardio-cerebrovascular system,central nervous system,immune system,blood system,anti-tumor,anti-fibrosis,anti-aging,etc.Because PNS is P.notoginseng secondary metabolites and the plants themselves capacity of production secondary metabolites is limited.The PNS application and popularization has been hampered seriously.It requires a long maturation period before they can be used as medicines.What is worse,because of the harsh growth environment,low land-capability,and plant diseases and pests,the gap between supply and demand has been broadened significantly.In recent years,plant secondary metabolism engineering has become the hot and difficult research of molecular biology.PNS are known as of the main active components in plant secondary metabolism,which yield and composition were determined by the key enzymes of biosynthesis.With the deep study of saponin biosynthetic pathway,the control of PNS biosynthesis through metabolic engineering has been gradually become possible.In PNS biosynthetic pathway,3-hydroxy-3-methylglutaryl-Coenzyme A reductase(HMGR)has been proposed to play pivotal roles in regulating the carbon flux into triterpene saponins.In this project,the technology of molecular biology was used to reveal the regulatory function of PnHMGR in PNS metabolic pathway.PnHMGR full-length cDNA was amplified from the P.notoginseng callus by the method of homologous cloning and analyzed.Sequence analysis showed that the cDNA sequence of obtained fragment(PnHMGR)is 1893 bp,containing an ORF spaning 1725 bp,and exhibited 98%,93%and 80%sequence identity with HMGR in Panax ginseng,Eleutherococcus senticosus and Eucommia ulmoides.The cDNA is a new one as searching in the Genbank.The cDNA sequence was submitted to Genbank and abtained accession number:KJ578757.The bioinformatic analysis showed that PnHMGR-encoding protein contained two transmembrane regions and one HMGR catalytic domain,without signal peptide.The constitutive plant expression vectors pCAMBIA1300s-PnHMGR was constructed,and transferred into Agrobacterium EHA105 via a freeze-thawing method.P.notoginseng cells,which had overexpressed SS gene,were infected by Agrobacterium EHA105 in order to imported PnHMGR gene and intergrated it into the genome of SS transformed cell lines.The genetic transformation system was established using P.notoginseng callus.The transgenic cell lines were screened by 50 mg/L kanamycin and 25 mg/L hygromycin.The cell lines which could grow continuously were used to process genomic DNA PCR.The results suggested that fifteen PnHMGR-SS transgenic cell lines were obtained.Then,the qRT-PCR analysis was carried out to analyze PnHMGR,SS genes expression levels in the six PnHMGR-SS transgenic cell lines(HS-1,HS-2,HS-3,HS-4,HS-5 and HS-6)which were growing faster and in good condition.In addition,PnHMGR activities were detected in the six cell lines.The results showed that PnHMGR was overexpressed in the PnHMGR-SS transformant and the expressions were about 7-11 times higher than that in the SS-transgenic cells,and 6-10 fold higher than that in the wild-type(WT)cell lines.In PnHMGR-SS transformant,the expressions of SS were about 6-11 times higher than that in the WT cell lines.Such results indicated that PnHMGR has been successfully intergrated into the genome of SS transformed cell lines;PnHMGR and SS were co-overexpressed in transgenic P.notoginseng cells.The PnHMGR activity was significantly increased in PnHMGR-SS transgenic cells,which were up to 2-3 times higher than that in SS-transgenic cells.In order to define the role of PnHMGR gene and the synergistic effect of PnHMGR and SS in P.notoginseng saponins biosynthetic pathway,the contents of PNS and phytosterols were detected in the six PnHMGR-SS transgenic cell lines.The results indicated that the contents of phytosterols and PNS in the PnHMGR-SS transformant were higher than those in SS-transgenic cells,and significantly elevated compared to those in WT cell lines.In the study,six main monomer saponins(Re,Rbl,Rg1,Rh1,Rh2 and F1)were chosen to detect their contents in the transgenic cell lines.Compared to the SS-transgenic cell lines,monomer saponins were increased remarkably when co-overexpression of PnHMGR and SS in four PnHMGR-SS transgenic cell lines(HS-1,HS-3,HS-4 and HS-5).This demonstrated that there was a synergistic effect between PnHMGR and SS in PNS biosynthetic pathway.Co-overexpression of PnHMGR and SS in P.notoginseng also led to a significant increase in the accumulation of phytosterols.