基因沉默及其克服策略

基因沉默现象是导致转基因不能正常表达的主要原因,基因沉默发生在两种水平上,一种是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平上的基因沉默,另一种是转录后基因沉默,有活跃的启动子,能转录外源基因,核内有正常水平的mRNA,细胞质不积累mRNA。消除甲基化的影响﹑避免使用同源和重复序列和使用核基质结合序列可有效克服基因沉默。     

Advances of Researches on the Mechanism of Transgene Silencing in Transgenic Plants

转基因沉默是导致外源基因不能在转化植株中正常表达的重要原因。在此，从转录水平和转录后水平两个方面，综述了转基因植物中外源基因沉默的机制。DNA甲基化是引起外源基因沉默的主要原因。人们目前用以下几种模型来解释转录后水平基因沉默的机制，如RNA 阈值模型、异常RNA和异位配对模型及双链RNA 模型。对外源基因沉默机制的探讨是顺利开展植物基因工程的迫切要求，也将促进植物功能基因组学研究的进一步深入。     

农作物转基因技术研究进展及存在问题

就转基因应用所面临的主要技术问题--外源基因的逃逸、失活和沉默进行了分析,解读了该现象发生的机理,并指出可以在操作过程中通过时空隔离、避免基因间的同源性、避免重复序列的出现、消除DNA甲基化的影响、使用MAR以及使用诱导型启动子等方法,对上述现象加以控制.最后,对转基因技术的应用前景作了积极的展望.     

植物转基因沉默机制及消除对策

从位置效应、转录水平的基因沉默和转录后水平的基因沉默等三个方面阐述了植物转基因沉默的作用机制,并提出了相应的消除对策,以期为寻找控制植物转基因失活技术提供帮助,促进植物基因工程的发展。     

转基因拟南芥中外源基因的Southern blot检测

外源基因在转基因植物的稳定表达是获得理想转基因植物的首要条件。在我们进行的转基因植物研究中，根据转基因植物所带有的转基因成分和标记基因的DNA序列，采用Southemblot技术对转基因植物中的猪α-乳清蛋白基因和标记基因的拷贝数进行了分析。结果表明：外源基因的拷贝数和外源基因的稳定表达相关。     

拟南芥中AtACO3基因启动子DNA甲基化的调控机制

RNA介导的DNA甲基化途径(RdDM)在基因表达调控过程中起重要作用。沉默抑制因子(repressor of silencing 1, ROS1)能负调控RdDM途径。定量RT-PCR(quantitative RT-PCR, RT-qPCR)被利用检测拟南芥(Arabidopsis thaliana)中ABA途径相关基因AtACO3表达水平,结果表明非生物胁迫诱导后AtACO3表达水平显著上调。重亚硫酸盐测序结果证明非生物胁迫诱导该基因启动子重复序列的DNA去甲基化,并导致该基因的转录激活。进一步研究证实ROS1在诱导该基因启动子DNA去甲基化的过程中起作用。本研究揭示了非生物胁迫诱导逆境响应基因AtACO3表达的分子机制,为探索植物适应非生物胁迫的表观遗传调控机制提供了科学依据。     

应用RNA沉默技术获取抗黄瓜花叶病毒(CMV)和烟草花叶病毒(TMV)转基因烟草

RNA沉默是迄今最为有效的抗病毒策略,利用该策略不但能获得免疫转基因植株,且所得植株不易与其他病毒基因重组或异源包壳、生物安全性较高。将本实验室已构建的携带有烟草花叶病毒(TMV)部分移动蛋白基因(ΔMP)和黄瓜花叶病毒(CMV)部分复制酶基因(ΔRep)反向重复结构的植物表达载体pBIN438-MP-Rep(i/r),用农杆菌浸润法转化普通烟草品种K326,共得196株转化植株,经卡那霉素筛选和PCR检测发现128株为阳性转基因株;PCR-Southern和RT-PCR分析表明外源基因已整合到烟草基因组并在转录水平上得到表达;ELISA结果显示20.3%的转基因植株对CMV和TMV复合侵染表现免疫性。本结果为利用RNA沉默技术进行植物抗多种病毒育种提供重要数据,为防治其他多种病毒复合侵染提供借鉴。     

基因沉默技术及其在棉花中的应用

基因沉默是指生物体内基因表达调控的重要手段,主要表现为特定基因的表达量下调或表达受到抑制。基因沉默主要有两种,转录水平上的基因沉默和转录后基因沉默。前者主要包括基因甲基化、位置效应、同源基因的反式失活、后成修饰作用以及重复序列引起的基因沉默;后者主要包括共抑制和RNA干扰引起的基因沉默。基因沉默广泛存在于植物体中,是一种基因表达调控和抵御病毒侵害的重要机制。文中介绍了基因沉默的类型和作用,重点评述了基因沉默技术在植物基因功能研究,尤其在棉花种质创新与品种改良方面的应用进展。     

农杆菌渗入法介导的基因瞬时表达技术及应用

农杆菌渗入法是在植物中瞬时表达外源基因的一种方法,该方法通过在植物叶片上注射农杆菌,使目的基因转入植物细胞中进行快速表达。本文综述了农杆菌渗入法的原理、技术流程、影响因素及其应用,其应用主要涉及外源基因的表达、检测转基因的表达、启动子分析、转录后基因沉默、抑制子功能鉴定、细胞定位以及基因相互作用等方面。随着技术的进一步完善,我们相信该技术将成为转基因育种领域的重要的手段。     

蚜虫基因RNAi载体的构建及其在转基因烟草中dsRNA的表达分析

RNAi机制可以通过dsRNA诱导同源mRNA的降解,从而导致该基因转录后沉默。RNA干扰作用从发现以来就具有了利用转录后基因沉默（PTGS）来进行抗虫的一种潜在可能性。针对dsRNA的这种作用,研究中提取蚜虫RNA,克隆出蚜虫Cathepsin B基因的干扰片段并构建出RNAi载体,转入本氏烟草中,在转基因烟草体内表达dsRNA。成功构建RNAi载体后,通过农杆菌转化和叶盘法将RNAi载体导入烟草中并采用组织培养方法培养出转基因烟草,通过PCR鉴定显示15株生根的转基因烟草中14株显示阳性。提取转基因烟草的RNA进行RT-PCR和Northern表达分析显示转基因烟草植株中dsRNA表达的存在。实验结果说明了在转基因植物体内表达RNAi载体可以产生dsRNA,从而在蚜虫进食植物时达到利用dsRNA抗蚜虫的作用。     

35S启动子甲基化引起棉花转基因沉默

 用带PBI121质粒的农杆菌LBA4404菌株转化泗棉3号胚性愈伤组织,获得的再生植株进一步对gus和nptⅡ基因进行PCR跟踪检测,共得到97株阳性转基因植株。GUS组织化学检测发现,97株转基因幼苗中有10株GUS检测阴性,嫁接后,只成活一株。利用来源于同一愈伤系的GUS检测阳性植株作为对照,对这一GUS检测阴性的植株进行gus基因沉默机理研究。Southern分析表明,该GUS检测阴性植株与GUS检测阳性植株有相同拷贝数。GUS组织化学检测和RT-PCR分析显示,gus基因在GUS检测阴性植株中没有表达,而nptⅡ基因在这两株转基因棉花中都表达。用限制性内切酶-PCR法分析35S启动子区甲基化发现：GUS检测阴性植株35S启动子区TATA box的HapII/MspI酶切位点发生甲基化,而GUS检测阳性植株该位点没有甲基化。以上研究表明,这株gus沉默的转基因棉花可能是由于其35S启动子区甲基化引起的。     

Analysis of DNA Cytosine Methylation on Cotton under Salt Stress

DNA methylation,especially methylation of cytosine in eukaryotic organisms,has been implicated in gene regulation,genomic imprinting,the timing of DNA replication,and determination of chromatin structure.It was reported that 6.5% of the whole cytosine residues in the nuclear DNA in higher plants were methylated.The methylation of cytosine in plant nuclear DNA occurs usually in both CpG and CpNG sequences,and the methylation state can be maintained through the cycles of DNA replication and is likely to play an integral role in regulating gene expression.     

转Bt基因棉花选择标记基因nptⅡ转录后沉默分析

本研究利用病毒诱导基因沉默技术对转Bt基因棉花选择标记基因nptⅡ进行沉默,分析标记基因转录后沉默的可行性。以烟草脆裂病毒载体为骨架构建nptⅡRNAi载体,利用农杆菌浸染棉花子叶,获得nptⅡ干涉的转Bt基因棉花植株,然后涂抹卡那霉素进行检测。结果表明nptⅡ沉默植株叶片出现黄斑症状,RT-PCR和q RT-PCR检测表明叶片、根和茎中nptⅡ基因转录分别被抑制了99.25%、99.05%和98.65%,沉默后期的转录抑制也达到98%。本研究结果为解决已经在环境中释放转基因生物发生意外扩散和不可预料的生物安全事件提供了快速有效的应对方法和新思路。     

抗甘蔗花叶病毒的无标记反向重复转基因玉米

构建了无标记基因的源自玉米矮花叶病的主要病原——甘蔗花叶病毒(sugarcane mosaic virus, SCMV)复制酶基因的反向重复序列表达载体,通过农杆菌介导法以该表达载体和除草剂标记基因表达载体共转化玉米自交系综3的幼胚,用除草剂梯度筛选,获得了可育的再生株。对T₁和T₂代群体作PCR和DNA点杂交,结合温室和田间人工接种病毒进行抗病性鉴定,获得了比较稳定的对SCMV高抗的无标记转基因玉米株系。     

The cell biology of plant transformation: Current state,problems, prospects and the implications for the plant breeding

Summary The DNA delivery systems which are routinely used to introduce genes into crop plants are Agrobacterium tumefaciens, electroporation and particle bombardment. The differences and similarities between these different transformation techniques are outlined. The influence of the cell biological approach, and more specifically the impact of the state of the plant cell at the moment of transformation, on the genotype and phenotype of the regenerated transgenic plant is analysed. In this respect phenomena such as position effects, gene silencing, co-suppression, epistasis, co-transformation and somaclonal variation are discussed. The relevance of these factors for plant breeders is discussed.     

CspB基因植物表达载体的构建及转化玉米自交系的研究

利用来自Bacillus subtilis细菌的CspB基因(Gene ID:936224)构建了Ubiquitin启动子驱动的CspB基因植物表达载体PBPC-CspB-bar,以bar基因为抗性筛选标记,通过花粉管通道法将构建的表达载体转化到玉米自交系京501、京517和吉444,通过喷洒除草剂筛选得到60株草丁膦抗性植株,用PCR检测得到48株bar基因阳性植株,将获得的转基因植株进行CspB基因PCR鉴定,获得13株同时整合CspB和bar的转基因株系。     

Genetic issues and pitfalls in transgenic plant breeding

Transgene technology provides a powerful tool for developing traits that are otherwise difficult to achieve through conventional breeding. In order to effectively apply the technology to breeding, we need to understand how transgenes behave in plants. Transgenes may or may not follow Mendelian segregation; their expression can be significantly affected by integration positions and structures of the transgenic DNA in host genomes; transgenes may become unstable over generations, genetic background sand environmental conditions; and they may have significantly negative impact on expression of endogenous genes. If not well understood, the sehurdles could become significant barriers in transgenic breeding. This paper reviews some genetic issues and pitfalls that are often encountered in transgenic breeding. Because of the necessity of being brief, transgene expression, silencing, and breeding are the three areas of focuses in this discussion. While molecular mechanisms underlying many of the transgenic phenomena have not been completely understood, some practical ‘rules’ are now available for creating, evaluating and selecting desirable transgenic transformants. It can be certain that with more transgenic plants generated and characterized our knowledge of transgene genetics at both molecular and plant levels will continue to accumulate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Field analyses of horizontal gene flow among Vigna angularis complex plants

Recent studies have indicated that spontaneous interspecies crossing commonly occurs among vascular plants, and therefore that horizontal gene flow from transgenic plants into wild relatives is unavoidable. Few surveys, however, have been conducted to determine an actual flow frequency for individual plant species. For the present estimation of gene flow among Vigna angularis complex (small red bean) plants, randomly amplified polymorphic DNA was analysed, the patterns of which differ between a cultivar, var. angularis and its wild counterpart, var. nipponensis. Cultivars and wild‐type plants were planted alternately and approximately 1% of the screened F₁ beans of wild type were hybrid. These F₁ hybrids were selected, allowed to self to produce F₂ beans, and subsequent crossing between the F₂ plants and wild type resulted in that 3.7% of the F₃ generation possessed DNA fragments specific to the cultivar. These results indicate that gene flow actually occurs among V. angularis complex plants, and that transferred genes might be stably maintained by the offspring. The present observation cannot be directly applied to transgenic plants due to the lack of information as to whether or not artificially introduced genes behave as do native genes after horizontal movement. However, as gene flow is found to be inevitable in this species, specific assessment of whether or not the gene to be introduced confers a significant selective advantage to the host is critical for utilization of genetically modified plants in future.