芪合酶基因Fm-STS在何首乌毛状根中的过量表达及dsRNA干扰  

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

作　　者：朱宽鹏[1,2] 赵树进[1] 

机构地区：[1]广州军区广州总医院,广州510010 [2]华南理工大学生物科学与工程学院,广州510640

出　　处：《中国生物工程杂志》2012年第8期41-48,共8页China Biotechnology

基　　金：广东省自然科学基金(10151001002000012);广东省科技计划(00697862100303016)资助项目

摘　　要：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0.65mg/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。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 mult^orum 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 glueoside in blank group is 2.18mg/g,in the over-expression group is 4.67mg/g and in the interference group is O. 65rag/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.

关 键 词：过量表达 DSRNA RNA干扰 何首乌 毛状根 荧光定量PCR 高效液相色谱法 

分 类 号：Q94-3[生物学—植物学]