Double-stranded RNA-mediated Gene Silencing And Over-expression Of Fm-STS In Polygonum Multilforum Thunb Hairy Roots

In the2010Chinese Pharmacopoeia,2,3,5,4’-tetrahydroxy-stilbene-2-beta-D-glucoside(TSG) was determined as the quality control indicators composition of Fallopia multifloraThunb. TSG contain more than three phenolic hydroxyl group, and modern pharmacologicaland clinical studies have demonstrated that TSG possess very effective activities for treatmentof anti-oxidation, anti-aging, lowering blood pressure and cardiovascular protection. Sothe demands are very bigger.(resveratrol with the same structure of the nucleus has beenJapan and other countries as a daily dietary supplement, the recommended adult dosage of4mg/d). The content of secondary metabolites in natural medicine is very small and thewildlife resources dwindling as well as the wild germplasm degradation, that make thecontradiction between supply and demand, However, the rapid development of modernbiotechnology to solve this contradiction. In Fallopia multiflora Thunb, firstly we must knowthe biosynthesis path and the key enzyme gene of TSG, but there has no reports on the thisuntil now. So there need for us to do this.The purpose of this paper is to research the function of the gene Fm-STS that wasamplified by RACE in Fallopia multiflora Thunb from the conserved sequence of the stilbenesynthase. and to establish a methodology to study of plant gene function. We used the baseplants express plasmid pBIN-35S-the GFP that contained the CaMV35S promoter-driven andGFP gene, to build over-expression plasmid vector pBIN-35S-STS-GFP (positive) anddouble-stranded RNA-mediated RNA interference plasmid vector pBIN the-35S-forward-reverse-GFP (negative). Both those plasmids and also the blank expression plasmidpBIN-35S-GFP (blank) were introduced into wild-type Agrobacterium rhizogenesATCC15834, to obtain the positive, negative and blank strains, and then used them infectedthe Polygonum multiflorum explants (no vaccine leaves) to induce the hairy root and culturethem. In the end we measurement the compounds TSG by HPLC of each group of hairy rootand used Real-time PCR to quantitative the stilbene synthase gene (Fm-STS) expressionlevels.The main findings are as follows:1) The Over-expression recombinant plasmid pBIN-35S-STS-GFP (positive) and the double-stranded RNA-mediated RNA interference recombinant plasmid vectorpBIN-35S-forward-reverse-GFP (negative) was successfully constructed. They werecheckout by PCR amplification, restriction enzyme digestion and sequencing verification.2) The target gene fragments were effective import hairy root’s nuclear genome, andexpressed in the hairy roots. Because the T-DNA of Agrobacterium rhizogenes Riplasmid can put fragments into genome, and ZEISS microscope detected under290nmGFP gene expression of the hairy roots, as the target gene and GFP gene connected with aCaMV35S promoter-driven.3) Used HPLC to detect the content of TSG in the hairy root of over-expression, the RNAinterference group and the blank group, The result are as followed:4.67±0.11mg/g and0.65±0.07mg/g and2.18±0.12mg/g. So positive correlation between the stilbenesynthase Fm-STS and TSG, we can explain the stilbene synthase Fm-STS to participatein the biosynthesis of stilbene glucoside.4) Real-Time PCR technology in the above three groups of hairy root in mRNA levels. Wefound that in the over-expression group the stilbene synthase Fm-STS was expressed thehighest, in which the over-expression group was2.41times that in the blank group, in theRNAi group is1/433times that in the blank group. In the three groups there weresignificant differences in the level of RNA expression, and connect the TSG measured byHPLC, they show that the accumulation of gene expression and product are positivecorrelation, and the stilbene synthase Fm-STS expression significantly impact theaccumulation of TSG. Description of the stilbene synthase Fm-STS is a key enzyme inthe biosynthesis of TSG.5) Double-stranded RNA-mediated RNA interference can effectively silence the purpose ofstilbene synthase Fm-STS. Because of in the interference group Fm-STS gene expressionlevels of only1/433of the blank group, and in the hairy root growth process, RNAinterference group of hairy root grow the slowest, followed by the blank group,over-expression group is the fastest. They both can explain mediated RNA interferencecan effectively silence the purpose of stilbene synthase Fm–STS.6) Hairy root of the RNA interference group, there was still has TSG exist, almost1/4ofTSG in the blank group, but the genes Fm-STS expressed just1/433times in the blank group, the difference is bigger, we also inferred there may be has other stilbene synthasegene or Fm-STS’ family gene.7) Use of HPLC and Real-Time PCR to detect the content of TSG and the stilbene synthaseFm-STS’s experssion in one Polygonum multiflorum Thunb root, stem and leaves. Foundthat TSG in the root is highest, stem is followed and the leaves is the lowest (root:14.62±0.09mg/g,; stem:1.78±0.12mg/g,; leaf:0.47±0.07mg/g,); and we analysisstilbene synthase Fm-STS expression level that the gene in Polygonum multiflorumThunb leaves the highest, the root of the expression level was about1/10in leaf; the stemexpression level was about1/64in leaves. We can drew the conclude that in Polygonummultiflorum Thunb TSG biosynthesis in the part of the leaf, and then enrich in the root.