Transgene Silencing: New Insight to Genetic Mechanisms

转基因沉默是通过重组工程引入生物体的基因的意想不到的表达缺失。许多证据使人们认识到，基因沉默反映了一系列保护生物体中存在的遗传信息不受外来遗传信息干扰的策略。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性生物的保护，但其他策略以前没有被认识到，它们的机制只是现在才被研究。这些考虑反映在这个项目的目标，这是确定基因参与转基因沉默机制，并在正常发育的运作特点。除了为转基因表达的遗传控制系统提供新的见解外，这一认识还将导致未来转基因构建体的战略设计，这些构建体不会被受体基因组感知为外源基因，从而更可靠地表达。进行实验的前提是，负责沉默机制的基因失活将重新激活沉默基因。三套不同的水稻品系，其中的转基因是转录沉默，已经开发和特点是非常适合再激活研究。然而，由于其快速的生长周期和完整的基因组序列，拟南芥将被用作模式植物，以加快水稻基因靶的选择。宿主沉默系统的失活将主要通过RNA干扰（RNAi）方法完成，其中表达对应于基因靶标的双链RNA。最初的基因靶标包括水稻甲基转移酶、含染色体结构域的基因和组蛋白脱乙酰酶基因。这是因为已知甲基化和染色质结构的改变与许多基因沉默事件相关。一旦一个目标基因在拟南芥中产生一个有趣的敲除（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。将制备这些基因的基于RNAi的敲除构建体，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.