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目的:通过过量表达探究在何首乌中得到的芪合酶基因Fm-STS的功能。方法:由含CaMV 35S启动子驱动以及荧光标记蛋白(Green fluorescent protein,GFP)基因的植物转基因基础表达质粒pBIN-35S-GFP构建过量表达质粒pBIN-35S-STS-GFP(阳性),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(无菌苗叶片),诱导生成毛状根并培养,对毛状根进行高效液相色谱分析芪合物二苯乙烯苷含量变化以及实时荧光定量检测基因Fm-STS表达差异。结果:在过表达组和空白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,芪合物二苯乙烯苷含量依次为4.67 mg/g和2.18 mg/g(干重),在mRNA水平上检测基因Fm-STS表达量:过表达组是空白组的2.41倍。结论:基因FM-STS是何首乌中芪合物二苯乙烯苷生物合成过程中的酶基因,过量表达在基因功能研究中有良好的应用。
OBJECTIVE: To explore the function of stilbene synthase gene Fm-STS obtained from Polygonum multiflorum by overexpression. Methods: The overexpression plasmid pBIN-35S-STS-GFP (positive) was constructed from plant transgenic basic expression plasmid pBIN-35S-GFP driven by CaMV 35S promoter and green fluorescent protein (GFP) The expression plasmid pBIN-35S-GFP (blank) was introduced into wild Agrobacterium rhizogenes ATCC15834, which was transformed into Polygonum multiflorum explants (sterile leaf) to induce hairy root formation and culture. Hairy roots were subjected to high performance liquid chromatography Analysis of stilbene stilbene glycoside content changes and real-time fluorescent quantitative detection of Fm-STS expression differences. Results: Both rolB gene and exogenous gene GFP were expressed in the Ri plasmid of A. rhizogenes in the overexpression group and the blank group. The contents of stilbene glucoside in the rhizobium were 4.67 mg / g and 2.18 mg / g (dry weight). The mRNA expression level of Fm-STS was detected in mRNA level: 2.41-fold in the overexpression group. Conclusion: FM-STS gene is an enzyme gene in the biosynthesis of stilbene glycoside in Polygonum multiflorum Thunb. The overexpression of FM-STS has a good application in the study of gene function.