转基因植物中RNA介导病毒抗性

１９８６年以来,利用病毒外壳蛋白及其它基因转化植物,获得具抗病毒能力的植株,已有大量成功的报道。以前,一直认为是病原体来源的基因引发抗性,但在实验过程中,发现转基因植物中转化基因的表达水平与病毒抗性程度之间没有直接联系,因此有人提出转基因植物抗性获得与病毒ＲＮＡ特异性降解有关的机制。本文对ＲＮＡ介导抗性机制进行讨论     

多种外源因素对转基因植物抗性影响的研究进展

病毒病对植物的危害在近些年日益严重,培育抗病毒转基因植物是众多防治方法中较为有效的一种。转基因植物在不同机制介导下会产生抗性,尤其是病毒基因介导的抗性试验最为普遍。众多研究证明,抗性受多种因素作用且表现为不同水平。对插入序列来源、转录后hpRNA长度、茎环比和拷贝数等多个因素在近些年的研究进展进行了综述,重点对各自在转基因植物抗性产生中的特点及影响程度进行了分析,并对其他因素进行了展望。     

基因沉默

基因沉默 (ｇｅｎｅｓｉｌｅｎｃｉｎｇ)是指生物体中特定基因由于种种原因不表达。一方面 ,基因沉默是遗传修饰生物 (ｇｅｎｅｔｉｃａｌｌｙｍｏｄｉｆｉｅｄｏｒｇａｎｉｓｍｓ)实用化和商品化的巨大障碍 ,另一方面 ,基因沉默是植物抗病毒的一个本能反应 ,为用抗病毒基因植物工程育种提供了具有较大潜在实用价值的策略———ＲＮＡ介导的病毒抗性 (ＲＮＡ ｍｅｄｉａｔｅｄｖｉｒｕｓｒｅｓｉｓｔａｎｃｅ ,ＲＭＶＲ) [1～ 3] 。基因沉默现象首先在转基因植物中发现 ,接着在线虫、真菌、昆虫、原生动物以及老鼠中陆续发现。大量的研究表…     

双链RNA和植物对病毒的抗性

双链RNA能诱导转录后的基因沉默,是生物抵御病毒入侵、维持自身基因稳定的一种自我保护机制.把源自病毒的基因构建成反向重复结构转入植物体内,其转录出的RNA会通过分子内序列互补形成双链,将入侵病毒的同源序列降解,使转基因植株获得对病毒的高抗性.RNA干扰型抗病毒转基因植株中,转病毒基因的mRNA不存在或存在量很少,也不会翻译成有功能的病毒蛋白,因此不存在病毒RNA重组、异源包装及协生作用的潜在风险,具有较高的生物安全性.双链RNA抗病毒转基因正在成为一种高效、安全的植物抗病毒策略.     

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。     

外源RNA干涉基因在烟草中的转化及表达

依据RNA干涉机制,以TMV复制酶基因为靶标基因,针对TMV 5个株系复制酶基因间高度同源序列设计引物,经RT-PCR反应获得靶序列,构建靶序列反向重复结构的RNA干涉双元载体.用根癌农杆菌介导将外源基因转化至烟草品种K326基因组中,培育RNA干涉转基因烟草.人工接种病毒验证转基因烟草中外源基因在植物抗病毒能力方面的表达效果,实时荧光定量PCR分析转基因烟草抗病毒能力.结果表明,实验培育的RNA干涉转基因烟草67%对TMV呈现高度抗性;荧光定量PCR分析显示,对TMV具高度抗性的转基因烟草中病毒复制酶基因转录产物mRNA存在很大程度的降解,证实了RNA干涉技术在培育抗病毒烟草品种中的效果.     

植物抗病毒分子机制

在与植物病毒的长期斗争中,植物进化出多种抗病毒机制,其中RNA沉默和R基因介导的病毒抗性是最受人们关注的两种机制.一方面,RNA沉默是植物抵抗病毒侵染的重要手段.植物在病毒侵染过程中可形成病毒来源的双链RNA,经过DCL蛋白的切割、加工形成sRNA,与AGO蛋白结合形成RISC指导病毒RNA的沉默,用于清除病毒.相应地,病毒在与植物的竞争中进化出RNA沉默抑制子,抑制宿主RNA沉默系统以逃避宿主RNA沉默抗病毒反应,增强致病能力.另一方面,植物也进化出R基因介导植物对包括病毒在内的多类病原的抗性.R蛋白直接或间接识别病毒因子,通过一系列的信号转导途径激活植物防御反应,限制病毒的进一步侵染.对植物抗病毒的研究有助于人们对植物抗病分子基础的理解,有重要的科学意义和潜在应用价值.本文综述了植物抗病毒分子机制的重要进展.     

转基因植物中RNA介导病毒抗性

1986年以来,利用病毒外壳蛋白及其它基因转化植物,获得具抗病毒能力的植株,已有大量成功的报道。以前,一直认为是病原体来源的基因引发抗性,但在实验过程中,发现转基因植物中转化基因的表达水平与病毒抗性程度之间没有直接联系,因此有人提出转基因植物抗性获得与病毒RNA特异性降解有关的机制。本文对RNA介导抗性机制进行讨论。     

SAR与植物转基因沉默的消除

随着转基因技术在各上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     

植物遗传工程

920268抗病毒植物的遗传工程〔英〕/Godoni,F一ZAreh.Virol一2990,115(i一2)一i~22〔译 自DBA,1991,10(9),91一05050] 讨论的主要内容包括:用于交叉保护的外壳蛋 白序列对植物的转化,表达病毒外壳蛋白的转基因植物的田间试验,抗病毒侵染的反义RNA技术,病毒卫星RNA在植物中的表达,ribozyme和锤头RNA机制,作为受体专性抗病毒荆的抗个体基因型的抗体;人干扰素基因在转基因植物中的克隆和表达。构建了抗下列病毒的转基因植物,烟草花叶病毒、首藉花叶病毒、烟草线条病毒、马铃薯X病毒、马铃薯Y病毒等。外壳蛋白介导的保护是最广泛采用的…     

Citrus tristeza virus (CTV) resistance in transgenic citrus based on virus challenge of protoplasts

Summary A strategy for the sereening of candidate virus-derived sequences to provide RNA-mediated citrus tristeza virus (CTV) resistance and early selection of virus-resistant citrus is presented. The system is based on the polyethylene glycol-(PEG) mediated cotransformation of protoplasts using virus-derived sequences and green fluorescent protein as a single selectable marker, followed by an in vitro assay of virus inoculation into transgenic protoplasts to determine the level of citrus tristeza virus replication. A cotransformation rate higher than 20% allowed selection of several clones carrying the desired transgenes. Efficient in vitro inoculation of virus in transgenic protoplasts was performed. Tobacco mosaic virus virions were used as a control in order to check eitrus protoplast viability. Different CTV replication levels were detected in transgenic clones. Only one clone showed no replication of CTV. Considerations regarding selection of candidate virusderived sequences and virus challenge of transgenic cells are presented.     

Hairpin RNA-mediated strategies for silencing of tomato leaf curl virus AC1 and AC4 genes for effective resistance in plants

RNA interference (RNAi) using short interfering RNAs (siRNAs) has been widely explored for the suppression of intracellular viral target mRNAs. On the basis of our previous work with stable silencing of Tomato leaf curl virus, in vivo by the antisense replicase gene (AC1) of the virus and characterizing AC4, as a small RNA regulator, besides its role in pathogenicity, we used four different plasmid vector-based siRNA generation strategies to silence viral genes (AC1 and AC4) of tomato leaf curl viruses. The RNAi target sequence were chosen from DNA A of the Tomato leaf curl virus (ToLCV) on the basis of conserved regions in AC1 with an overlapping sequences of the AC4 gene. Different hairpin RNA-mediated strategies like antisense, self-complementary inverted repeats, intron-spliced hairpin RNAs, and small hairpin RNAs were deployed for efficient and predictable resistance to the viruses. Here we present that appropriately designed siRNAs not only prevents RNAi suppression but also help in developing trait-stable transgenics. These strategies imply that ToLCV rep-driven RNAi, targeting AC4 and conserved viral sequences, provides a promising approach to suppress a wide spectrum ToLCV infection in the tomato.     

Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance

Plant microRNAs (miRNAs) regulate the abundance of target mRNAs by guiding their cleavage at the sequence complementary region. We have modified an Arabidopsis thaliana miR159 precursor to express artificial miRNAs (amiRNAs) targeting viral mRNA sequences encoding two gene silencing suppressors, P69 of turnip yellow mosaic virus (TYMV) and HC-Pro of turnip mosaic virus (TuMV). Production of these amiRNAs requires A. thaliana DICER-like protein 1. Transgenic A. thaliana plants expressing amiR-P69(159) and amiR-HC-Pro(159) are specifically resistant to TYMV and TuMV, respectively. Expression of amiR-TuCP(159) targeting TuMV coat protein sequences also confers specific TuMV resistance. However, transgenic plants that express both amiR-P69(159) and amiR-HC-Pro(159) from a dimeric pre-amiR-P69(159)/amiR-HC-Pro(159) transgene are resistant to both viruses. The virus resistance trait is displayed at the cell level and is hereditable. More important, the resistance trait is maintained at 15 degrees C, a temperature that compromises small interfering RNA-mediated gene silencing. The amiRNA-mediated approach should have broad applicability for engineering multiple virus resistance in crop plants.     

Engineering resistance to geminiviruses--review and perspectives

Following the conceptual development of virus resistance strategies ranging from coat protein-mediated interference of virus propagation to RNA-mediated virus gene silencing, much progress has been achieved to protect plants against RNA and DNA virus infections. Geminiviruses are a major threat to world agriculture, and breeding resistant crops against these DNA viruses is one of the major challenges faced by plant virologists and biotechnologists. In this article, we review the most recent transgene-based approaches that have been developed to achieve durable geminivirus resistance. Although most of the strategies have been tested in model plant systems, they are ready to be adopted for the protection of crop plants. Furthermore, a better understanding of geminivirus gene and protein functions, as well as the native immune system which protects plants against viruses, will allow us to develop novel tools to expand our current capacity to stabilize crop production in geminivirus epidemic zones.     

Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants

Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil. ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes. We determined that in N. benthamiana, the primary targets corresponded to the 5' end of AC1 and the embedded AC4, the intergenic region and 5' end of AV1 and overlapping central part of AC5. Subsequently, transgenic N. benthamiana plants were generated that were preprogrammed to express double-stranded RNA corresponding to this most targeted portion of the virus genome by using an intron-hairpin construct. These plants were shown to indeed produce ToCMoV-specific siRNAs. When challenge inoculated, most transgenic lines showed significant delays in symptom development, and two lines had immune plants. Interestingly, the levels of transgene-produced siRNAs were similar in resistant and susceptible siblings of the same line. This indicates that, in contrast to RNA viruses, the mere presence of transgene siRNAs corresponding to DNA virus sequences does not guarantee virus resistance and that other factors may play a role in determining RNA-mediated resistance to DNA viruses.     

Broad virus resistance in transgenic plants

Viruses are significant threats to agricultural crops worldwide and the limited sources of natural resistance warrant the development of novel resistance sources. Several methods of transgenic protection have been successfully applied, including protein- and RNA-mediated approaches. Increased understanding of the molecular biology of virus infection is starting to bear fruit, enabling specific strategies to be designed for virus resistance in crops.