Study On Human Basic Fibroblast Growth Factor2(FGF2) Expressed By Ri-plasmid Mediating Ginseng Hairy Roots

Ginseng (Panax ginseng CA Meyer) is a perennial herb of Araliaceae, containingsaponins, carbohydrates, proteins, peptides amino acids, vitamins and other activesubstances. Ginseng has moisturizing emollient, anti-oxidation, anti-UV damage tothe skin and other cosmetic effect, its extracts is often added as an importantingredient in many cosmetics. Fibroblast growth factor(FGF2), a very small amountof active substance in the human body, is a multifunctional growth factor, havinggreat potential clinical application in promoting tissue growth and development, newblood vessel formation, repairing process and other participating organizations. Thisexperiment carried out the plant-preferenced codon transformation of the FGF-2gene,then constructed binary plant expression vector by recombinant-PCR. Thenmediated by rhizogenes A4, transfered the plant expression vectors which contain theFGF-2gene into ginseng cells, finally getting the transgenic ginseng root hair thatcontain FGF2gene. Through the identification and analysis of heterologousexpression of FGF2protein of hairy roots of transgenic ginseng, we can use theginseng root to produce large amounts of pharmaceutical proteins, Ginseng root as abioreactor for the production of drugs laid the foundation for industrial development,this laid the foundation for industrial development that make the ginseng root as abioreactor to produce drugs. Meanwhile, provides a theoretical basis for the massproduction of FGF2gene.In this subject, I mainly did these following work and got my conclusions:1. The construction of plant binary expression vectors and engineering bacteria:With pET16+FGF2vector as the template, we made the plant-preferred codonstransformation of the FGF-2gene, Then build the binary plant expression vector thatnamed pBI121-FGF2by recombinant PCR methods and digested connectiontechnology. After that we transferred the plant expression vector pBI121-FGF2intoAgrobacterium rhizogenes strain A4through the method of freeze-thaw.,constructed the engineering bacteria and named that A4-pBI121-FGF2. 2. Genetic transformation of ginseng root: Infect the slices of triennial ginsengroot by prepared good engineering bacteria A4-pBI121-FGF2and empty pBI121plasmids, genetically transformed out the ginseng hair roots with plant expressionvector FGF2;Although transgenic and control groups exhibited typical characteristicsof the hair roots in the morphology, there was no significant difference in morphologybetween them.3. The identification of FGF-2, the heterologous expression of Ginseng root:With the transgenic hairy root of ginseng that transformed from the inducing ofA4-pBI121-FGF2engineered bacteria as materials, Using molecular biologicalmethods for detecting FGF2gene and integrated it into the genome of ginseng roots;Extract out the protein in ginseng root, Use SDS-PAGE and Western blot to prove thatthe exogenous gene FGF-2has been translated and got expressed in ginseng root.4.Detect of total saponins and part single saponin in the hairy roots of transgenicginseng: With the accumulation of time, the total saponin content was graduallyincreasing，and it tend to be stable30days later. The total saponin content in thecontrol group and the transgenic group are in the range of15-22mg/g, the totalsaponin content of hairy roots of transgenic ginseng (22.51) was higher than that ofthe blank control hairy root of ginseng (20.47), the difference was not significant (P>0.05), As measured by high performance liquid we found that the content of threemonomers saponin in the hairy roots of transgenic ginseng was higher than that inthe control roots.5.The polysaccharides detection of hairy roots of transgenic ginseng:Thepolysaccharide content of hairy roots of transgenic ginseng (11.5) is higher than thatof blank control ginseng root (9.5),the difference was significant(P <0.05).