The Effect Of Cycloartenol Synthase Gene Silencing Mediated By RNA Interference On The Biosynthesis Of Saponins In Panax Notoginseng

Panax notoginseng total saponins (PNS), the major bioactive gradient of Panax notoginseng, are all belong to tetracyclic triterpenoid saponins. The previous studies have shown that the Panax notoginseng total saponins are responsible for biological activities of anticancer, antivirus, lower cholesterol, improving immunity, and can effectively prevent and treat the cardiovascular and cerebrovascular diseases. However, the habitat of P. notoginseng is narrow, mainly in Wenshan of Yunnan, China. And it requires a long maturation period (>3years) before they can be used as medicines. What is worse, because of the harsh growth environment, plant diseases and pests, crop rotation needing, and low land-capability, the gap between supply and demand has been broadened significantly. In recent years, gene regulation of plant secondary metabolic pathway has become the hot and difficult research of molecular biology. With the deep research of triterpene saponins biosynthetic pathway, the regulation of PNS biosynthesis through metabolic engineering has gradually become possible.It is known that the enzyme dammarenediol-II synthase (DS) and cycloartenol synthase (CAS) catalyze a common precursor,2,3-oxidosqualene to synthesize triterpenoid saponins and phytosterols, respectively. In this study, suppression of CAS gene expression in P. notoginseng, which was achieved by RNAi interference, led to more precursors available for triterpenoid saponins biosynthesis, therefore resulted in the enhanced P. notoginseng saponins levels. A568bp RNAi fragment was amplified through RT-PCR with primers designed based on the P. notoginseng CAS gene in GenBank, and then the RNAi fragment was cloned into a pMD18-T vector. RNAi fragments which had attB connectors at both ends were obtained by the attB-PCR with the pMD18-T-CAS plasmid DNA as the template. Based on the homologous recombination, RNAi fragments with attB connectors were subcolned into the plant binary expression vector pHellsgate2by the way of GatewayTM technology, and constructed the RNAi expression vector pHellsgate-CAS.The RNAi expression vector was transferred into Agrobacterium LBA4404and EHA105competent cells by CaCl2freeze-thawed method. The exogenous genes were transferred and integrated into the P. notoginseng genome mediated by the Agrobacterium tumefaciens, and established a genetic transformation system based on P. notoginseng callus. The callus obtained by transformation were screened by the kanamycin, then eight cell lines which were growing faster and in good condition were used to conduct the genomic DNA PCR. Five transgenic cell lines (E5, E8, E16, E24and E30) were identified to be positive initially based on the PCR results. The real-time PCR was selected to detect the expression level of CAS and DS gene in the five transgenic cell lines. The real-time PCR result showed that except the cell line E24, the expression of CAS in the remaining four transgenic cell lines was lower than that in the non-transgenic cell line, while the expression of DS in the remaining four transgenic cell lines was higher than that in the non-transgenic cell line.In order to define the role of CAS gene in P. notoginseng saponins biosynthetic pathway, the contents of P. notoginseng total saponins and phytosterols in the four transgenic positive cell lines were detected. The results showed that the silencing of CAS expression in the four transgenic cell lines led to a reduction of phytosterols but an increase in the content of P. notoginseng saponins in the four transgenic cell lines. Almost each monomer saponin in the P. notoginseng was believed to have different pharmacological effects. In the study, nine triterpenoid saponins (Re, Rb1, Rg1, Rg3, Rh2, F2, Rh1, F1and Rd) which were reported as major constituents were chosen to detect their contents in the transgenic cell lines. Compared to the non-transgenic cell line, except the saponin Rd, the contents of the other eight monomer saponins were all higher in the transgenic cell lines. These results indicate that the CAS gene played a vital role in the biosynthesis of saponins in P. notoginseng, and the reduction of CAS expression was benefit to the accumulation of P. notoginseng saponins.