转基因植物中外源基因的沉默及应对策略

随着转基因技术在作物育种领域的应用,转基因植物中外源基因表达量低的现象较为普遍。导致外源基因表达量低的主要原因是基因沉默。外源基因沉默可分为转录水平的基因沉默和转录后水平的基因沉默。如何应对基因沉默,提高外源基因的表达量,是转基因技术发展亟待解决的问题。     

转基因植物转录后基因沉默的克服对策

人们对植物进行遗传转化的目的是让转基因在植物基因组中能够稳定整合并在当代及其子代中能够有效、稳定的表达 ,但是由于多种因素的影响 ,转基因在植物中的表达并不很理想。自从 1 986年Ｐｅｅｒｂｏｔｔｅ报道转基因烟草中转基因发生沉默以来 ,很多学者也都发现大量的转基因植株不能正常表达[1]。经过多年的研究 ,发现导致转基因沉默的机理有多种 ,根据其作用机理和水平的不同 ,人们通常把转基因沉默分为[2 ,3]:位置依赖性基因沉默 (Ｐｏｓｔｉｏｎ -ｄｅｐｅｎｄｅｎｔｇｅｎｅｓｉｌｅｎｃｉｎｇ ,ＰＤＧＳ)、转录水平的基因沉默 (Ｔｒ…     

转基因植物中外源基因沉默机制及防止对策

系统论述了诱发转基因沉默的因素、植物转基因沉默发生水平并提出了基因沉默的防止对策,基因沉默现象是导致外源基因不能在转化植物中正常表达的重要原因。甲基化、重复序列、反式失活和共抑制是基因沉默的主要诱因。其作用水平主要有三种：位置效应、转录水平的基因沉默和转录后水平的基因沉默,避免基因间的同源性、避免重复序列的出现、消除甲基化的影响、使用MAR以及使用诱导型启动子,可以提高外源基因的表达水平,有效防止基因沉默。     

核基质附着区与转基因表达

核基质附着区 (matrixattachmentregions,MARs)是与核基质 (或核骨架 )特异结合的DNA序列 ,属于非编码序列 ,富含AT。通过与核基质的结合 ,它能使染色质形成独立的环状结构 ,调控基因的转录和表达 ,减少由于位置效应引起的转基因沉默。MARs在提高转基因表达水平、消除转基因个体间表达水平的差异、抑制转基因沉默等方面起着重要的作用。就MAR的一些结构功能特征及其在基因工程中的应用等方面进行了阐述。     

核基质结合区与转基因沉默

核基质结合区(matrix attachment region,MAR)又叫支架附着区(scaffold attachment region,SAIL)是染色质被限制酶消化后仍与核基质或核骨架结合的DNA序列。转基因沉默主要发生在两个水平上,一是转录水平,二是在转录后水平。位置效应是引起转录水平基因沉默的重要因素。研究发现,用MAR构建的载体能够提高转基因的表达水平,能在一定程度上克服基因沉默．降低转基因在不同转基因系间的表达差异,增强外源基因表达的稳定性。     

植物转基因沉默及控制

植物基因工程的目的就是获取外源基因能够按照设计要求正常表达和稳定遗传的转基因植物。近几年来,广泛报道了转基因植物中存在转基因沉默现象。主要阐述了两种转基因沉默的机理即转录水平的基因沉默和转录后水平的基因沉默,各种机理都涉及DNA DNA,DNA RNA,RNA RNA的相互作用。同时,还讨论一些控制转基因沉默现象的策略,特别是MAR在转基因植物中具有增强基因表达和减少株间差异的作用。     

植物转基因沉默研究进展、对策及应用

转基因沉默 (ｔｒａｎｓｇｅｎｅｓｉｌｅｎｃｉｎｇ)是导入并整合进受体基因组中的外源基因在当代转化体或其后代中表达受到抑制的现象。自Ｐｅｅｒｂｏｌｔｅ在 1986年首次报道发现转基因沉默现象以来 ,相关报道不断发表。如今 ,转基因沉默现象已成为遗传转化技术实用化 ,商品化过程中的巨大障碍。从九十年代开始 ,人们就致力于转基因沉默方面的研究。随着研究的不断深入 ,转基因沉默的各种机理不断被揭示 ,并研究出相应的对策。1.植物转基因沉默研究进展转基因沉默的各种机理都涉及到各种核酸之间的相互作用 ,包括转录水平的基因沉默 (ｔ…     

CMV抑制PVYN CP基因沉默及其介导的抗病性

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T,代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVY^N CP基因的沉默,而且CMV对PVY^N CP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVY^N CP基因沉默则未受到影响。采用ELISA方法对CMV PVY^N复合接种的转基因植株进行PVY^N检测,结果表明,接种叶及下部叶没有检测到PVY^N,植株叶片对PVY^N表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVY^N,植株叶片对PVY^N表现为感病。该文报道了在表达PVY^N CP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。     

SAR与植物转基因沉默的消除

随着转基因技术在各个应用领域的深入和发展,转基因沉默现象已引起越来越多的人关注。转基因沉默主要发生在转录或转录后水平,涉及核酸的三种相互作用,即：ＤＮＡ－ＤＮＡ、ＤＮＡ－ＲＮＡ、ＲＮＡ－ＲＮＡ。这是植物基因表达调控的具体表现之一,也是外源基因插入后生物体的一种自我保护。ＳＡＲ协同转基因进行转化,可以消弱宿主ＤＮＡ对外源基因的不利影响。实验证明,在稳定整合的转基因植物中,ＳＡＲ提高了整体表达水平,并降低了不同转化体之间的差异。这是消除植物转基因沉默的一条有效途径。     

Stable expression of silencing‐suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA‐guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co‐evolved viral silencing‐suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA‐regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three‐step transgenic pathway for the synthesis of arachidonic acid (AA), an ω‐6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP‐AA‐transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.     

Position effect variegation and imprinting of transgenes in lymphocytes

Sequences proximal to transgene integration sites are able to deregulate transgene expression resulting in complex position effect phenotypes. In addition, transgenes integrated as repeated arrays are susceptible to repeat-induced gene silencing. Using a Cre recombinase-based system we have addressed the influence of transgene copy number (CN) on expression of hCD2 transgenes. CN reduction resulted in a decrease, increase or no effect on variegation depending upon the site of integration. This finding argues that repeat-induced gene silencing is not the principle cause of hCD2 transgene variegation. These results also suggest that having more transgene copies can be beneficial at some integration sites. The transgenic lines examined in this report also exhibited a form of imprinting, which was manifested by decreased levels of expression and increased levels of variegation, upon maternal transmission; and this correlated with DNA hypermethylation and a reduction in epigenetic chromatin modifications normally associated with active genes.     

植物转基因沉默研究进展

随着转基因技术在植物中的广泛应用,转基因沉默受到越来越多的重视。转基因沉默可发生在转录和转录后两种水平,其基本特征就是依赖于同源的重复序列。转基因的重复拷贝间,转基因与同源的内源基因间及RNA病毒与同源转基因间都会发生基因沉默。可能有不同的机制导致转基因沉默,本文综述了转基因沉默的机理研究及转基因沉默在植物抗病基因工程和植物功能基因组学方面的应用 。     

Silencing of transgene expression in mammalian cells by DNA methylation and histone modifications in gene therapy perspective

DNA methylation and histone modifications are vital in maintaining genomic stability and modulating cellular functions in mammalian cells. These two epigenetic modifications are the most common gene regulatory systems known to spatially control gene expression. Transgene silencing by these two mechanisms is a major challenge to achieving effective gene therapy for many genetic conditions. The implications of transgene silencing caused by epigenetic modifications have been extensively studied and reported in numerous gene delivery studies. This review highlights instances of transgene silencing by DNA methylation and histone modification with specific focus on the role of these two epigenetic effects on the repression of transgene expression in mammalian cells from integrative and non-integrative based gene delivery systems in the context of gene therapy. It also discusses the prospects of achieving an effective and sustained transgene expression for future gene therapy applications.