Construction Of The Yeast Two-hybrid Library Of Ginseng And Screening Of Ginseng Proteins Interacted With E₀Protein

Panax ginseng C.A. Meyer (Araliaceae) has been used in traditional medicine sinceancient times in China. Ginsenosides the primary bioactive components of ginseng have manyphysiological functions. Ginsenoside mainly isolated from ginseng and the yield was very low.This has seriously hindered the use and research of ginsenosides.In vitro tissue culture, hairy root technology can reduce the stress of the lack of resources ina certain extent. But the content of ginsenosides in ginseng explants or ginseng hairy roots arevery low. Li Ran et al. induced E0gene into ginseng. By HPLC analysis, the content ofginsenosides (Rc, Rd, Rg1, Re, Rf and Rg2) had significant improment. But the mechanism isstill unknown. In this experiment, the yeast two hybrid systems were used to screen theinteraction proteins of E0in ginseng cDNA library.1. The construction of the bait plasmid pGBKT7-E0. The E0gene was inserted intopGBKT7vector. Then, the recombinant plasmid was introduced into the yeast strain Y2H Goldby lithium acetate method. At last, transformants were selected on the synthetic medium lackingTrp. The result shown that pGBKT7-E0has no self-activity and no yeast toxicity. This laid asolid foundation for screening the interaction proteins of E0in ginseng.2. The construction of ginseng cDNA library. The ginseng cDNA library was quickly madeby SMART method. The resulting cDNAs were subcloned into pGADT7vector and introducedinto yeast strain Y187. The capacity of ginseng cDNA library is7×106cfu while recombinationrate is100%. The length of insert section is0.3kb-2kb. All these suggested that the ginsengcDNA library was successfully constructed and the library could be used in the followingexperiments.3. Screening of Ginseng Proteins Interacted with BVDV E0protein. Through the yeast twohybrid systems, we got the eukaryotic translation initiation factor5A (eIF5A) which wasinteracted with BVDV E0protein.4. In this study, the eIF5A was successfully expressed in BL21strain induced by IPTG.5. The eukaryotic translation initiation factor5A (eIF5A) which was interacted with BVDVE0protein could inhibit the synthesis and accumulation of ginseng saponins.6. The E0gene promoted the synthesis and accumulation of ginseng saponins by relievingthe inhibitory effect of eIF5A.