Impact On Content Of Ginseng Triterpenoid By Interference Expression Of Ginseng CS Gene

Ginseng triterpenoid ingredients includes ginsenoside and plant sterols, whichshare a common precursor in ginseng secondary metabolites. Ginsenoside is the keypharmacologically active ingredient in ginseng, which has effects on anti-aging,anti-fatigue, enhance immunity, etc. In this study, we improved production of qualityof ginsenoside via metabolic engineering means to regulate the biosynthetic pathwayof triterpenoid ingredients. With molecular biology techniques, the cole consensussequence of cycloartenol synthase gene(CS) was cloned. The highly efficient RNAiexpression system of CS gene was constructed. Then with Agrobacterium rhizogenesA4as media, we introduced the RNAi expression vector into ginseng through explantinoculation, in order to regulate the production of ginsenoside in ginseng hairy roots.The main results of study are as follows:1. Ginseng genome cDNA were gained through reverse transcription with totalRNA as template extracted from ginseng hairy roots conserved by our labrotary. Thecole consensus fragments of CS gene were cloned by PCR. Sequencing analysisshowed that the size of these cole gene fragments were390bp, which was consistentwith the enunciated sequnece in GenBank.2. RNAi structual elements of CS gene were construced with recombinant PCRtechnology. The elements were named as CS-RNAi. Sequencing analysis showed thatthe size of the RNAi elements were8484bp. Then constucted RNAi plant expressionvector by connected the elements to plant expression vector pBI121.3. RNAi engineered bacteria was constructed by transferred RNAi expressionvectors to Agrobacterium rhizogenes A4. The engineered bacteria were named asA4-CS-RNAi.4. New ginseng hairy roots were induced by A4-CS-RNAi engineered bacteriaand Agrobacterium rhizogenes A4with empty pBI121plasmid.5. Content of cycloartenol and dammarenediol in transgenic ginseng hairy rootswere evaluated by HPLC. The detection results show that compared to control, thecontent of cycloartenol in CS-RNAi hairy roots were decreased by80.64%, and thecontent of dammarenediol in CS-RNAi hairy roots were increased slightly.6. Content of total ginsenoside and plant sterols in transgenic ginseng hairy rootswere evaluated by untraviolet spectrophotometry method. The detection results indicate that compared to control, the content of plant sterols in CS-RNAi hairy rootswere decreased by53.61%.7. Content of monomer saponin Rg1, Re and Rb1in transgenic ginseng hairyroots were evaluated by HPLC. The detection results indicate that compared tocontrol, the content of monomer saponin Rg1,Re, and Rb1in CS-RNAi hairy rootswere decreased by58.33%,39.30%,51.21%, respectively. This result were consistentwith trends of total ginsenoside.