Transgene Silencing: New Insight to Genetic Mechanisms

转基因沉默是指通过重组工程引入生物体的基因意外缺乏表达。许多证据表明，基因沉默反映了一系列策略，这些策略保护生物体中存在的遗传信息不受外来遗传信息的干扰。其中一些策略与众所周知的防御机制有关，这些防御机制提供了对感染生物的保护，但另一些策略之前没有得到承认，其机制只是刚刚被研究。这些考虑反映在这个项目的目标中，该项目的目标是识别与转基因沉默机制有关的基因，并描述它们在正常发育中的运作。除了为转基因表达的遗传控制系统提供新的见解外，这种理解还将导致对未来转基因构建的战略设计，这些构建不会被受体基因组感觉到是外来的，从而更可靠地表达。进行实验的一个前提是，负责沉默机制的基因失活将重新激活沉默的基因。已经开发出三种不同的水稻品系，其中的转基因被转录沉默，并表现出非常适合于复活研究的特征。然而，由于其快速的生长周期和完全测序的基因组，拟南芥将被用作加快水稻基因靶标选择的模式植物。宿主沉默系统(S)的失活主要是通过RNA干扰的方法完成的，即表达与基因靶标对应的双链RNA。最初的基因靶点包括水稻甲基转移酶、含染色域的基因和组蛋白脱乙酰酶基因。这是因为已知染色质结构的甲基化和改变与许多基因沉默事件有关。一旦目标基因在拟南芥中产生了有趣的敲除(沉默失活)表型，将在现有的数据库中搜索水稻中的类似基因。将构建基于RNAi的这些基因的敲除结构，并研究它们在沉默水稻品系中恢复转基因表达的能力，以评估敲除基因作为沉默基因的作用。

Transgene silencing is the unexpected lack of expression of a gene introduced into an organism through recombinant engineering. Many lines of evidence have led to the realization that gene silencing reflects a series of strategies that protect the genetic information present in an organism from interference by extraneous genetic information. Some of these strategies are related to well-known defense mechanisms that provide protection against infectious organisms, but others have not previously been recognized and their mechanisms are only just now being investigated. These considerations are reflected in the goal of this project, which is to identify genes involved in transgene silencing mechanisms and to characterize their operation in normal development. In addition to providing new insight to genetic control systems of transgene expression, this understanding will lead to the strategic design of future transgene constructs that will not be sensed by the recipient genome as being alien and thus be expressed more reliably.A premise for the experiments to be undertaken is that inactivation of genes that are responsible for the silencing machinery will re-activate the silenced gene. Three different sets of rice lines, in which a transgene is transcriptionally silenced, have been developed and characterized that are eminently suitable for reactivation studies. However, because of its rapid growth cycle and its fully-sequenced genome, Arabidopsis will be used as a model plant to expedite selection of gene targets for rice. Inactivation of the host silencing system(s) will be accomplished primarily by an RNA interference (RNAi) approach in which double-stranded RNA corresponding to the gene target is expressed. Initial gene targets include rice methyltransferase, chromo domain-containing genes and histone deacetylase genes. This is because methylation and alteration of chromatin structure is known to be associated with many gene silencing events. Once a target gene yields an interesting knock-out (silencing inactivation) phenotype in Arabidopsis, existing databases will be searched for similar genes in rice. RNAi-based knock-out constructs of these genes will be made and their ability to restore transgene expression in the silenced rice lines investigated to evaluate the role of the knocked-out gene as being one responsible for silencing.