III型内部核糖体进入位点（IRES）介导核糖体对不同翻译起始区识别敏感性的研究

项目名称： III型内部核糖体进入位点（IRES）介导核糖体对不同翻译起始区识别敏感性的研究

项目编号： No.31302100

项目类型： 青年科学基金项目

立项/批准年度： 2014

项目学科： 农业科学

项目作者： 周建华

作者单位： 中国农业科学院兰州兽医研究所

项目金额： 24万元

中文摘要： 内部核糖体进入位点(IRES)介导的翻译起始机制存在于多种正链RNA病毒中。翻译起始序列内部核苷酸类型和密码子使用模式对于病毒基因的翻译起始效率十分重要，不同类型IRES对翻译起始区识别的敏感性是不同的。然而，目前III型IRES所优先识别的翻译起始区及其对翻译起始效率的影响还未见系统研究报道。本实验室在前期研究RNA病毒进化的过程中初步确定了在翻译起始区内部影响翻译起始效率的关键位点。本项目在此基础上通过PCR融合技术和定点突变技术构建系列具有不同翻译起始序列和内参报告基因的真核表达质粒，依靠真核细胞瞬时表达和蛋白质检测技术来检测翻译起始序列区关键核苷酸和密码子使用模式的改变对靶基因翻译起始效率的影响，确定III型IRES介导核糖体所识别的最优翻译起始序列及不同翻译起始序列对基因表达效率的影响程度。为探索翻译起始序列对RNA病毒翻译效率的影响等科学问题提供理论依据。

中文关键词： 核糖体内部进入位点；定点突变；基因；翻译起始序列；表达

英文摘要： For single-stranded positive-sense RNA viruses, 5'-UTR recognition by the 40S ribosomal subunit occurs and is driven by an RNA structure located within the message itself. This structure is termed as the internal ribosomal entry site (IRES). The specific initiation translation region has an important effect on the translational initiation efficiency, and each type of IRES has a tendency to choose the initiation translation region which has the specific nucleotides and codon usage pattern. However, there are few reports about effects of combination of type III IRES with an optimal initiation translation region on translational efficiency. During the analysis of the feature of genome of single-stranded positive-sense RNA virus, we have found some key positions in the initiation translation region, which may affect translational efficiency. Therfore, depending on the overlap PCR and site-directed mutagenesis,we plan to construct a series of eukaryotic expression systems which is composed of two reporter genes, IRES element and various initiation translation regions. By detection of translational efficiencies of the report gene with various initiation translation regions through transient transfection and western-blotting, the role of each initiation translation region leaded by the type III IRES in the translation

英文关键词： IRES；site-specific mutagenesis；gene；translation initiation region；expression