增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)是一种优化的突变型GFP,DFL是从甘菊中分离出的LFY基因的同源序列。为了研究DFL基因的功能和表达模式,研究利用小片段克隆法将linker序列插入到EGFP基因5′端启始密码子前面,在pBI121载体的CaMV35S启动子的3′端后面插入一段多克隆位点,成功地构建了pBI-DFL-EGFP表达载体。通过设计特异引物,利用PCR技术扩增得了到拟南芥LFY基因的启动子序列,用粘性末端PCR技术将pBI-DFL-EGFP表达载体中CaMV35S启动子替换成LFY基因启动子,构建成了pLFY-DFL-EGFP表达载体。用含有pBI-DFL-EGFP和pLFY-DFL-EGFP质粒的农杆菌侵染洋葱表皮细胞,在荧光显微镜下分别用蓝光激发,均观测到了荧光。这一结果表明,融合蛋白DFL∷EGFP表达载体构建成功,同时还证明了通过PCR技术克隆到的LFY启动子序列具有启动子功能。
Real-Lime fluorescent quantitative PCR is a method for quantitative analysis of gene expression developed in recent years, which has been widely used in various fields such as basic scientific research, clinical diagnosis, disease study, drug research and development since its appearance. It starts relatively late in study on plants, but has already been used for analysis of gene expression in plants and gene identification of exogenous genes. The principles or advantages and dis- advantages of real-time fluorescent quantitative PCR, or its potential problems and condition optimizations in tests were introduced in this study, and then the appli- cation and prospect of real-time fluorescent quantitative PCR in study on plants were also been discussed.
为探索甘菊[Chrysanthemum lavandulifolium(Fisch. ex Trautv.)Makino]在非生物胁迫下以及营养物质代谢中精准的生理、基因表达变化机理,研究通过筛选外植体的种类和营养液的浓度,建立了甘菊水培体系。结果表明:水培中,播种苗的成活率和生根率极显著高于1年生大苗和茎段插条;在长时间的水培中,使用标准Hoagland和1.5倍Hoagland培养液,其畸形叶率极显著低于1/2和1/4Hoagland培养液,并且株高极显著高于另外2个浓度。结合科研需求,认为组培播种苗、标准Hoagland营养液是甘菊水培的最佳外植体和培养条件。
