Regulated Expression For Key Synthase In2,3-oxidosqualene Metabolism Pathways Of Panax Ginseng

Ginsenosides are the key pharmacologically active ingredients in ginseng. Previousstudies have suggested that ginsenosides and phytosterols share the same precursor,2,3-oxidosqualene in Panax quinquefolium and Panax ginseng biosynthetic pathway.The main metabolic pathway of ginsenosides is composed of two branches. Onepathway leads to synthesis of ginsenosides contained dammarane-type ginsennosidesand oleanane-type ginsenosides, while the other produces lanosterols and cycloartenols.The enzymes cycloartenol synthase (CAS) and dammarenediol synthase (DS) are thekey enzymes responsible for ginsenoside and phytosterol biosynthesis from2,3-oxidosqualene, respectively. In this research, we found another key enzyme inphytosterol synthesis pathway——lanosterol synthase(LAS), which catalyzed2,3-oxidosqualene together with cycloartenol synthase. Traditional cultivation ofginseng has some inconvenient factors, such as long growth periods, continuousharvesting obstacles, and susceptibility to pests and pathogens. In this research, weestablished rapid growth ginseng hairy roots asepsis system with MS medium, andanalyzed ginseng hairy roots extracts with HPLC quantitatively.Here, we first regulated and identified LAS、CAS and DS gene in ginseng hairyroots by RNAi technique, then regulated the metabolic pathways of ginsenosides atmolecular level in order to improve its production.The key contents and conclusions are as follows:1. There are two phytosterol synthesis pathways in ginseng, leading to synthesisof lanosterol and cycloartenols catalyzed by LAS and CAS respectively.2. Sub-culture ginseng hairy roots system suited to this study was established. Theroot tips of ginseng hairy roots were sterilized by2.0%NaClO for90seconds, thendistributed into culture flasks (1.0g/flask). After30days cultivation, the experimentalmaterials for this research were obtained.3. Selecting core regions of LAS、DS and CAS, composed their RNAi elementswith PT-PCR respectively. The recombinant plasmids——pBI121-LAS-RNAi、pBI121-DS-RNAi and pBI121-CAS-RNAi——were constructed by connecting theseelements with plant transformation carrier pBI121respectively. RNAi engineeredbacteria were constructed by transferred RNAi expression vectors to Agrobacteriumrhizogenes A4. Then new ginseng hairy roots were induced by these engineered bacteria respectively.4. We compared contents of total ginsenoside、ginsenoside (Rg1、Re and Rb1)、total phytosterols、 lanosterols、 cycloartenols and dammarenediols between LASRNAi group and control group. Results of these comparisons indicated that RNAi coulddown-regulated expression of LAS. The results quantity of phytosterol decreased alongwith shrink of lanosterols indicated the existence of lanosterols metabolic pathway inginseng. While increasing production of total ginsenoside and ginsenoside indicated thatsuppression of LAS gene could promote yields of ginsenosides.5. Contents of total ginsenoside and ginsenoside(Rg1、Re and Rb1)、 totalphytosterols、 lanosterols、 cycloartenols and dammarenediols between DS RNAigroup and control group were also compared. A sharp declined of dammarenediol、totalginsenoside and ginsenoside in DS RNAi group demonstrated that DS was the keyenzyme in biosynthesis of ginsenosides. On the contrary, production of totalphytosterol、 lanosterols and cycloartenols were increased in DS RNAi group. Thisphenomenon indicated that the biosynthesis of ginsenosides and phytosterols competeda common precursor——2,3-oxidosqualene.6. We also compared contents of total ginsenoside and ginsenoside (Rg1、Re andRb1)、 total phytosterols、 lanosterols、 cycloartenols and dammarenediols betweenCAS RNAi group and control group. Reduction of cycloartenols in CAS RANi groupindicated the successful interference of CAS gene. While increase of total ginsenosideand ginsenoside illustrated that suppression of CAS gene could promote synthesis ofginsenosides.7. RNA interference could not influence morphology of ginseng hairy roots.8. By RNAi technique, we established two kinds of new ginseng hairy rootssystems. One possesses a54.93%increase of ginsenosides, while the other possesses a67.15%increase of phytosterols.