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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 establish a method system for exploring plant gene function and to verify the function of Fm-STS gene in Polygonum multiflorum obtained by RACE amplification using the conserved sequence of stilbene synthase gene. METHODS: The recombinant plasmid pBIN-35S-GFP (positive) and the double stranded RNA were constructed by overexpression plasmid pBIN-35S-GFP containing the gfp gene driven by CaMV 35S promoter. (Reverse) -GFP (negative), and the same plasmid pBIN-35S-GFP (blank) was introduced into wild type Agrobacterium rhizogenes ATCC15834, transformed Polygonum multiflorum explants, induced hairy roots and cultured, hairy Roots for high performance liquid chromatography and real-time fluorescence quantitative detection. Results: The rolB gene and the exogenous gene gfp of Agrobacterium rhizogenes were all expressed in hairy roots of overexpression group, blank group and interference group, and the content of stilbene stilbene glycoside was determined by high performance liquid chromatography Followed by 4.67mg / g, 2.18mg / g and 0.65mg / g, respectively. The mRNA expression levels of Fm-STS were detected in mRNA level: 1 / 433.53 in the RNAi group and 2.41 in the overexpression group Times Conclusion: The results showed that overexpression and double-stranded RNA interference in the study of plant gene function has a good application, Polygonum multiflorum STH Fm-STS is a stilbene glycoside the main synthase.