银杏等木本植物端粒结合蛋白对端粒保护的分子机制研究

Telomere-associated proteins have recently been recognized as necessary components in telomere structure as well as in telomere function. The regulation of telomere length is carried out not only by the telomerases but also by the telomere binding proteins. Based on our previous research work, the woody plants of Ginkgo biloba L. would be selected as experimental materials to study on the telomere maintaining mechanism in this project. First of all, the gene sequences of the telomeric binding proteins would be identified. In the second place, the gene expression patterns would be investigated in different tissue groups. Furthermore, the interaction between the telomeric binding proteins and the telomeric DNA would be analyzed by several important methods, for instants, the gel retardation assay, the yeast one-hybridization assay and the chromatin immunoprecipitation assay. Additionally, the telomere length and the telomerase activity would be measured in transgenic ginkgo suspension cells and transgenic regenerated seedlings, respectively. We will propose the crucial function of the telomere binding proteins in the telomere maintaining mechanism. This research work will be advantageous to us to reveal the biological function of the telomere in woody plants.

端粒结构的稳定、端粒长度的维持、端粒功能的正常运行与端粒蛋白的协调作用密切相关。深入研究端粒结合蛋白对端粒保护的分子机制是揭示植物端粒生物学功能的重要结点。本项目拟在已有工作的基础上，以银杏等木本植物为实验材料，筛选鉴定银杏等木本植物端粒结合蛋白基因，在分析其序列结构的基础上，进一步分析其在银杏等木本植物各组织器官的表达模式；同时通过凝胶阻滞分析、酵母单杂、染色质免疫沉淀等技术检测该蛋白与端粒DNA相互作用；并运用转基因技术，通过银杏悬浮细胞体系和转基因再生苗体系，从细胞和个体发育两个不同层面，检测端粒结合蛋白过量表达或缺失时的端粒变化。项目从木本植物端粒结合蛋白的基因表达、与端粒DNA相互作用、对端粒DNA的识别、结合、保护和稳定等方面阐述木本植物端粒结合蛋白在保护端粒DNA中的分子作用机制，为进一步揭示木本植物端粒的生物学功能奠定重要的理论基础。

端粒的稳定、端粒功能的正常运行与端粒蛋白的协调作用密切相关。端粒长度的维持，一方面依赖于端粒酶， 另一方面则依赖于端粒结合蛋白。本项目在已有工作的基础上，以银杏、油松等木本植物为实验材料，通过酵母单杂交文库筛选、同源克隆等分子生物学方法，获得了一些可以和端粒重复序列相结合的端粒结合蛋白，进一步通过凝胶阻滞分析、酵母单杂等技术检测该蛋白与特异DNA序列的识别能力和结合能力。此外，课题组还以银杏、油松的成熟胚为外植体，进行愈伤组织培养，获得银杏、油松的悬浮细胞，进一步通过农杆菌侵染法获得转基因细胞以期研究端粒结合蛋白功能。本项目分别从木本植物端粒结合蛋白的基因表达、与端粒 DNA 相互作用、对端粒DNA的识别、保护和稳定等方面阐述木本植物端粒结合蛋白在保护端粒DNA中的分子作用机制，为进一步揭示木本植物端粒的生物学功能奠定重要的理论基础。