Impact On Content Of Ginsenoside By Over-expression Of Ginseng D12H Gene

Belonging to Araliaceae ginseng species,Ginseng is a traditional precious herb.Itwas found that ginsenoside is the main secondary metabolites of ginseng andginsenosides are major active ingredients of ginseng.The pharmacologic effects ofginsenosides are various.Because the content of ginsenosides is low in ginseng,so theusing of ginseng saponin is limited.In recent years,with the developting of plantbioengineering, the study on improving the content of ginsenosides in ginseng byplant bioengineering has been a new hotspot.2,3-oxidosqualene is the precursor ofginsenoside biosynthesis.2,3-oxidosqualene generates dammarenediol in the catalyticconversion of DS,and dammarenediol generates protopanaxadiol in the catalyticconversion of D12H,finally protopanaxadiol further is glycosylated to form a varietyof ginsenosides by the GT.Objective to study the effects of content ginsenoside inginseng hairy toots which the ginsenoside biosynthesis process are regulated withgenetic technology.In this study,we cloned the key genes,D12H in ginseng hairy rootsby PCR,and D12H was connected with over-expression vector pBI121.Then withAgrobacterium rhizogenes A4as media,we introduced the over-expression vectorpBI121-D12H into ginseng through explant inoculation, in order to regulate theproduction of ginsenoside in ginseng hairy roots.The main results of study are as follows:1.Ginseng D12H gene PCR amplification:Ginseng genome cDNA were gainedthrough reverse transcription with total RNA as template extracted from ginseng hairyroots conserved by our labrotary.Primers were designed according to the known keyenzyme genes D12H(JN604536) in ginseng saponin biosynthesis.Using cDNA as atemplate,D12H gene were successfully cloned. 2.The constrction of plant over-expression vectors:We built the D12H plantover-expression vector through enzyme digestion and ligation,The over-expressionvectors were constructed and named pBI121-D12H.3.The constrction of plant engineered bacteria:Transferred D12H over-expressionvector into Agrobacterium rhizogenes strain A4by freeze-thaw method.Theengineered bacteria were named asA4-pBI121-D12H.4.Genetic transformation of ginseng hairy root:Infected ginseng root explants bythe prepared engineered bacteria,then got the ginseng hairy roots with D12H geneplant over-expression vector.RT-PCR analysis of the total RNA extracted fromginseng hairy roots,and showed that ginseng was induced successfully.5.Analysis of ginsenoside:Compared with the control group, total saponincontent of the interference group in different periods increaed.Content of monomersaponin Rg1, Re and Rb1in transgenic ginseng hairy roots were evaluated by HPLC.The detection results indicate that compared to control, the content of monomersaponin Rg1,Re, and Rb1in D12H over-expresssion hairy roots were increased.