芪合酶基因Fm-STS在何首乌毛状根中的过量表达及dsRNA干扰

目的:建立一套探究植物基因功能的方法体系,验证由芪合酶基因保守序列通过RACE扩增技术在何首乌中得到的基因Fm -STS的功能.方法:由含CaMV 35S启动子驱动的gfp基因的植物转基因表达基础质粒pBIN-35S-GFP构建过表达质粒pBIN-35S-STS-GFP(阳性)和双链RNA干扰重组质粒pBIN-35S-正向-反向-GFP(阴性),并同空白表达质粒pBIN-35S-GFP(空白)均导入野生型发根农杆菌ATCC15834中,转化何首乌外植体,诱导生成毛状根并培养,对毛状根进行高效液相色谱分析以及实时荧光定量检测.结果:在过表达组、空白组和干扰组中毛状根中发根农杆菌Ri质粒中的rolB基因和外源基因gfp均有表达,高效液相色谱法分析芪合物二苯乙烯苷含量依次为4.67mg/g、2.18mg/g和0.65 mg/g,在mRNA水平上测试荧光定量检测基因Fm-STS表达量:RNAi组是空白组的1/433.53,过表达组是空白组的2.41倍.结论:结果表明过量表达与双链RNA干扰相结合在植物基因功能研究中有良好的应用,何首乌中芪合酶Fm-STS是二苯乙烯苷主要的合成酶.

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

Objective: To construct a method to research the gene FM-STS’ function that the gene come by RACE amplified from the stilbene synthase gene’s consensus sequence in Polygonum multiflorum Thunb.Method: The over-expression vector pBIN-35S-STS-GFP was constructed from the blank plasmids pBIN-35S-GFP and the gene FM-STS sequence and also the interference expression vector pBIN-35S-forward-reverse-GFP was constructed from the blank plasmids and the forward/ reverse direction sequence on account of double stranded RNA.Sent the over-expression plasmid/the interference plasmid and the blank plasmid into wild-type Agrobacterium rhizogenes ATCC15834,then induced hairy roots and cultured them.In the end the hairy roots were analyzed by HPLC and Real-time PCR.Result: The PCR results showed that the gene rolB and gfp are both expressed in hairy roots,and the content of stilbene glucoside in blank group is 2.18mg/g,in the over-expression group is 4.67mg/g and in the interference group is 0.65mg/g,At the mRNA level to detect gene Fm-STS expression level,in the over-expression group is the highest,it’s 3.17 times in the blank group and 101.44 times in the interference group.Conclusion: The method binding both technique that double stranded RNA-mediated gene silencing and over-expression is made good use of researching the gene’ s function,and the gene FM-STS is the key enzyme gene in synthesis stilbene glucoside.