精子介导转基因过程中外源基因整合机制的研究

项目名称： 精子介导转基因过程中外源基因整合机制的研究

项目编号： No.31460282

项目类型： 地区科学基金项目

立项/批准年度： 2015

项目学科： 生物科学

项目作者： 张顺

作者单位： 桂林医学院

项目金额： 50万元

中文摘要： 精子介导转基因是目前主要的生产转基因动物方法之一，但其分子机制目前尚未明确。文献报道显示外源基因由蛋白质介导转运进入精子内部，我们前期通过非变性单向聚丙烯酰胺电泳、质谱技术以及生物信息学分析，初步认为精子介导外源基因转运的蛋白质（DNA Binding Protein，DBP）应为IAM38。为了完善精子介导转基因的分子机制，本课题拟进一步通过非变性双向聚丙烯酰胺凝胶电泳和抗体封闭等对精子结合外源基因的DBP进行鉴定并验证；并对结合了外源基因的蛋白质复合物的转运方式进行深入探讨；在报告基因EGFP片段内部插入内含子，追踪并探讨进入精子核内部的外源基因的整合方式，揭示出外源基因进入宿主基因组的路径；最后，采用Genome Walking方法对转基因进行整合位点的鉴定，分析整合位点的特异性和规律。本课题拟通过上述探讨从而阐明外源基因进入宿主基因组的分子机制，为转基因技术的发展奠定理论基础。

中文关键词： 精子介导转基因；精子结合外源基因蛋白质；外源基因；整合；分子机制

英文摘要： Sperm-mediated gene transfer (SMGT) is one of the important techniques for generating transgenic animals. However, the underlying molecular mechanisms remain undefined. It has been demonstrated that exogenous genes was transferred via a protein named DNA binding protein（DBP）into sperm genome. Our previous data by using Blue Native Polyacrylamide Gel Electrophoresis, mass-spectrometric and bioinformatics preliminarily showed that the protein which response for gene transfer should be IAM38. To further investigate the molecular mechanisms of SMGT, firstly, we will identify and verify the DNA binding protein by Two-Dimensional Blue Native Polyacrylamide Gel Electrophoresis and blocking antibody. Moreover, the transport route for the compound exogenous DNA and protein will also be explored. Secondly, to uncover the mechanisms of exogenous DNA integration, the plasmid vector harboring a enhanced green fluorescent protein (EGFP) will be interrupted by an intron. Finally, the integration sites will be analyzed by genome walking and the specificity will be explored via blasting searches against the published genomic database.The object of the study was to clarify the molecular mechanisms of exogenous genes into the host genome, which will lay the theoretical foundation for the development of transgenic research.

英文关键词： sperm-mediated gene transfer(SMGT);DNA Binding Protein (DBP);exgenous DNA;integration;molecular mechanism