Simultaneous Overexpressions And Regulation Study Of SS,DS Genes 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 total saponins(PNS)are the major bioactive gradients in Panax notoginseng.PNS have good pharmacological activities in central nervous system,cardiovascular system,blood system,immune system,anti-fibrotic,anti-aging and anti-tumor.Panax notoginseng is a kind of perennial herb.The long growth cycle and the status of low land utilization restrained the development of Panax notoginseng industry.With the research of PNS biosynthetic pathway,it is possible that metabolic engineering can be used to regulate the biosynthetic pathway of PNS.In order to obtain a large number of low-productivity,high valuable natural medicine quickly,combinatorial biosynthesis will be used to establish homology(plant cell culture)or heterologous(genetically engineered bacteria)expression system for the production of pharmaceutical active ingredients.The technology of molecular biology was used in this project to research the synergy of SS(Squalene synthase gene)and DS(dammarenediol-? synthase gene)genes in PNS biosynthetic pathway.Squalene synthase(SS)and dammarenediol-II synthase(DS)are key enzymes in PNS biosynthetic pathway.In this study,SS gene was amplified from total RNA of Panax notoginseng using reverse transcription-PCR,and pGEM-T-SS cloning vector was constructed.The cloning vector and pCAMBIA1300S empty vector were digested by two specific restriction endonuclease enzymes respectively,and the SS fragment was recombinated in pCAMBIA1300S to construct pCAMBIA1300S-SS plant overexpression vector.The plant overexpression vector was transferred into Agrobacterium LBA4404 and EHA105 competent cells by CaCl2 freeze-thawed method.Panax notoginseng cells,which had overexpressed DS gene,were infected by Agrobacterium tumefaciens in order to imported SS gene and integrated it into the genome of Panax notoginseng.The genetic transformation system was established using Panax notoginseng callus.The transgenic cells were screened by the kanamycin and hygromycin B.The cell lines which could grow continuously were used to process genomic DNA PCR.Five transgenic cell lines were identified to be positive based on the PCR results.Realtime-PCR was used to detect genes expression levels of SS and DS in three transgenic cell lines.The results showed that DS expressed slightly higher(1.41 times and 1.39 times)than the control in two cell lines,and DS expressed higher(2 times)than the control in other one cell line.It is considered that the expression of the DS gene was not affected obviously during the overexpression of the other gene.SS gene in three cell lines expressed significantly higher(5.66 times,7.37 times and 7.37 times)than the control.It follows that two genes could overexpress simultaneously in Panax notoginseng successfully.To further clarify the effects of the two genes(SS and DS)overexpression in the biosynthetic pathway of PNS,two cell lines which grew fast,were used to measured the contents of PNS and monomer saponins.The results showed that the PNS average content in transgenic cells was 2.4 times and 1.3 times of the wild type cells and the control,respectively.This suggested that two genes overexpression can improve the content of PNS further more.SS and DS gene overexpression is in favor of the accumulation of PNS.