| Panax ginseng C. A. Meyer is a famous and widely used oriental herb. Ginseng saponins,secondary metabolites of the plant, are its pharmacological active components. In the recentyears, ginseng has been increasingly used as a health tonic formulated into a variety ofcommercial health products,including ginseng cap sules, soups, drinks, and cosmetics, whichare marketed in the Asian as well as many other countries around the world; However, naturalsources of wild ginseng have been overexploited and very limited. The current supply ofginseng mainly depends on field cultivation, which is an extremely long-lasting andlabor-intensive process. So far, total chemical synthesis of ginsenoside has not yet been realized;Therefore, biotechnology and gene regulation method has been exploited for more efficientproduction of ginseng saponins; Since the1980s, researchers at home and abroad have studiedthe production of ginsenosides by tissue and cell cultures, such as callus tissues, cellsuspensions, normal roots, but due to the lower growth rate, with little success; Ginseng hairroot transformed by Agrobacterium rhizogenes of ginseng tissue cells, have fast growth rateand stable genetic traits, is considered to be the most potential ginsenoside production methodsbut the productivityof ginseng saponins production is quite low; therefore, based on thebiosynthetic pathway of ginsenoside, by means of metabolic engineering to enhance the abilityof the ginseng root saponin biosynthesis is an effective way to solve this problem.The ginseng saponin derived from the same precursor2,3-oxidosqualene, beta-amyrinsynthase (βAS) and dammarenediol synthase (DS) are key enzymes of ginseng saponinbiosynthesis, DS is responsible for the synthesis of dammarenediol providing tetracyclicskeletons for dammarane-type ginsenosides, and βAS is involved in oleanane-type ginsenosidebiosynthesis; the intermediates, dammarenediol and b-amyrin, are transformed into ginsenosides through a series of hydroxylation and glycosylation reactions; For Ro is only oneoleanane-type ginsenoside, and its pharmacological activity is very small, and the content isvery low, belonging to the minor saponins; Therefore, we generally considered that the mainactive ingredients of ginseng is dammarane-type ginsenosides,which was synthesised under theaction of DS.This study inhibited ginseng βAS gene expression through RNA interference technology,then oleanane-type ginsenoside synthesis pathway wil be inhibited, so more precursor flow tothe dammarane-type ginsenoside synthesis pathway, thereby the ginseng hair root saponincontent was increased; Through the determination of the total saponin and monomer saponinfrom ginseng hairy root, to explore the effect of ginsenoside biosynthesis by the RNAinterference of βAS gene, and lay the foundation for ginsenoside metabolic engineering andfurther clarify of the ginsenoside biosynthesis mechanisms.The main contents and conclusions are as follows:1The design and construction of RNA interference components: we determined the RNAicomponents region of the βAS gene through sequence alignment between βAShomologous genes and βAS and the other three key enzyme gene DS, LS, CAS, and thenin view of the design of the restriction sites and the help of bioinformatics software tofinalize the primer sequences, and we build ginseng βAS gene RNAi components byrecombinant PCR technology;2The constrction of RNA interference plant expression vectors and engineered bacteria: webuilt the βAS gene RNAi plant expression vector through enzyme digestion and ligationtechnology, then transferred into Agrobacterium rhizogenes strain A4by freeze-thawmethod, and we got the RNAi plant expression vector engineered bacteria; 3Genetic transformation of ginseng hairy root: infected three years ginseng root explants bythe prepared engineered bacteria, then got the ginseng hairy root with βAS gene RNAiplant expression vector; Both interference and control groups showed typicalcharacteristics of the hair root and no significant difference in the form of growthmorphology;4Total saponin: compared with the control group, total saponin content of the interferencegroup in different periods increaed, which rose to more than10%after20d, the highestnumber is at30d, reached16.31%; For this reason, we selected the30d interference group,as the DS and monomer saponin content sample solution which were tested by HPLC;5DS, monomer saponin: DS content of the control group was0.158mg/g, the content ofinterference group was0.227mg/g, it can be seen that DS content of interference group hada marked increase, reached43.67%; The Rg1, Re, Rb1content in interference groupsample solution is about1.0-2.5mg/g, compared with the control group, the content of theinterference group increased by more than10%, between them Rb1reached themaximum(11.88%); The Ro content of the sample solution was much smaller, less than0.4mg/g, the content of the control group was0.392mg/g, the content of interference groupwas0.375mg/g; Compared with the control group, the content of Ro in interference groupdecreased4.53%;... |
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