ALTERING GENE EXPRESSION WITH DESIGNED ZINC FINGERS

描述 这个项目的目标是开发一种新的静音方法 基因在植物中的表达。多指锌指蛋白将被 从锌指的文库中构建，并被选中以绑定唯一的 要沉默的基因中包含的DNA序列。锌指结构域 将被融合到转录抑制因子结构域并在 转基因植物沉默目标基因的表达。An In 体外实验将被用来鉴定最有效的转录 植物中的阻遏结构域，并确定其最适位置 要捆绑的锌手指。基因沉默将通过以下方法在体内得到证实 用阻遏子构建物瞬时导入烟草原生质体。 转基因植物将产生一种表达锌指抑制物 设计用来沉默内源基因的结构。最后，锌指 抑制蛋白将被设计成沉默病毒的表达 复制所必需的蛋白质。这些构造将用于 培育出对病毒抵抗力增强的转基因植物 感染。这项技术将为基因沉默提供一种有用的方法 在植物中表达以增加它们的抗病能力，增强它们的 营养价值，或改变其他重要的农业特性。这个 在这个项目中开发的概念也可以扩展到改变基因 在其他真核生物中的表达。

DESCRIPTION The objective of this project is to develop a novel method of silencing the gene expression in plants. Polydactyl zinc finger proteins will be constructed from a library of zinc fingers and selected to bind a unique DNA sequence contained in the gene to be silenced. The zinc finger domain will be fused to a transcriptional repressor domain and expressed in transgenic plants to silence the expression of the target gene. An in vitro assay will be used to identify the most effective transcriptional repression domains in plants and to determine the optimum position for the zinc finger to bind. Gene silencing will be demonstrated in vivo by transiently transfecting tobacco protoplasts with the repressor construct. Transgenic plants will be generated the express a zinc finger repressor construct designed to silence an endogenous gene. Finally, zinc finger repressor proteins will be designed to silence the expression of viral proteins necessary for replication. These constructs will be used to generate transgenic plants that have increased resistance in viral infection. This technique will provide a useful method for silencing gene expression in plants to increase their disease resistance, enhance their nutritional value, or alter other agriculturally important properties. The concepts developed in this project can also be extended to alter gene expression in other eukaryotes.