双链RNA干扰技术在毛状根体系中对何首乌芪合酶基因Fm STS功能研究中的应用

[目的]将双链RNA介导的基因沉默与发根农杆菌瞬间表达渗入相结合,为植物功能基因的研究提供一套有效的方法。[方法]构建含gfp基因、CaMV 35S启动子及基于何首乌中芪合酶Fm STS基因序列设计的双链RNA的干扰重组质粒pBIN-35S-正向-反向-GFP(干扰),并将其与空白表达质粒pBIN-35S-GFP(空白)分别导入野生型发根农杆菌MSU440中,转化何首乌无菌苗的叶片,诱导生成毛状根。[结果]双链RNA介导的芪合酶Fm STS基因表达沉默,空白对照组二苯乙烯苷含量为2.18 mg/g,是双链RNA干扰组(0.65 mg/g)的3倍多。[结论]应用dsRNA介导的RNAi能有效沉默何首乌Fm STS基因,芪合酶Fm STS基因是何首乌中二苯乙烯苷合成代谢的关键酶基因。

Double-stranded RNA-mediated Gene Silencing of FmSTS in Hairy Roots of Polygonum multiflorum Thunb

[Objective] To construct an effective method for studying the plant gene function by the way of binding the double-stranded RNA-mediated gene silencing with the transient expression of Agrobacterium rhizogenes.[Method] A novel expression vector pBIN-35S-forward-reverse-GFP was constructed from the plasmids which included gfp gene driven by the CaMV 35S promoter and the forward/reverse direction sequence based on double stranded RNA.The hairy roots of Polygonum multiflorum Thunb.were induced by wild-type Agrobacterium rhizogenes MSU440.[Result] The double-stranded RNA induced the gene silencing of FmSTS.The content of 2,3,5,4’-tetrahydroxy-stilbene-2-O-β-D-glycoside in blank group(2.18 mg/g dry weight) was more than three times that in RNAi group(0.65 mg/g dry weight).[Conclusion] The gene silencing of FmSTS was effectively induced by dsRNA-mediated RNA interference,and the gene FmSTS is the key enzyme gene in the synthesis of 2,3,5,4’-tetrahydroxy-stilbene-2-O-β-D-glycoside.