【作者】 曾志锋；

【导师】 王莹；

【作者基本信息】 暨南大学 ， 遗传学， 2005， 硕士

【摘要】 RNA干涉是近年来发现的小片段双链RNA分子能够特异地沉默基因表达的现象,它被广泛应用于功能基因组的研究、疾病治疗的研究、干细胞的研究及植物抗病毒等领域,各类型的实验室及研究机构广泛开展RNA干涉实验。RNA干涉实验首要的步骤是对候选的siRNA序列进行筛选,但这一过程耗时、繁琐。 为了使候选siRNA分子的筛选过程更加快捷及有效,利用统计学x~2检验方法,分析已发表的高效沉默目的基因的siRNA分子,得到关于siRNA分子的一些分子特性的信息:①siRNA21位点中第1、2、20和21位点A/T碱基频率显着高于G/C碱基频率;②第3、4和6位点G/C碱基频率显着高于A/T碱基频率:③5’端第2—7位G/C碱基含量大于3’端第15—19位点G/C碱基含量;④siRmA片段平均Tm值为31.2°C其可信区间为[25.1°C,37.3°C]。结合前人的研究成果,我们推测这些分子特性与RNA干涉发生过程中涉及的各种酶如Dicer酶、RISC复合体、RNA依赖的RNA聚合酶(RNA depending RNA polymerase,RdRp)等的作用效率有关。 同时,以上述siRNA分子特性为指导,筛选候选siRNA分子,通过载体介导的RNA干涉技术成功地沉默人巨细胞病毒UL49基因表达,由此验证了以这些分子特性为参考,可以快捷有效地筛选出沉默基因表达的siRNA分子,对于候选siRNA分子的筛选具有参考价值。更多还原

【Abstract】 RNA interference is a new way of specific gene silence,that induced by small duble strand RNA.RNAi is used widely in kinds of research fields , including detection of gene function , diseases therapy ,stem cell, plant resistance to virus and so on.The primary step involved in RNA interference experiment is to screen out the efficient siRNA induceing gene silence from many potential siRNA sequences.That is a work taking time and burdensome.To make the screening work at top speed and feasible, we analysed the efficient siRNA published by Chitest and found that the siRNA has the following features: i A/U at position 1 2 20 and 21; ii G/C at position 3 4 and 6;iiiG/C content of 3’is lower than that of 5’; iv Tm is 31.2℃,with E value[25.1℃,37.3℃].Concerned with others research we supposed these features are tied with the enzymes efficiency involving RNA interference such as Dicer, RISC and RNA depending RNA polymerase.To identify wether these features are available for screening out siRNA,we carried out an experiment that screened out siRNA by following these features.We found that the siRNA could silence the U149 efficeincy.更多还原