基于RNA干扰原理抗病毒转基因作物的应用及安全性

基因沉默是广泛存在于各种生物中的一种古老现象,是生物抵抗外来入侵者的保护机制。RNA干扰（RNA interference,RNAi）则是近年来发现的一种重要基因沉默现象。此策略已在植物抗病毒育种等研究中应用,如对水稻、大麦、大豆、玉米、马铃薯、番茄、辣椒、木瓜、南瓜、李、烟草等的抗病毒研究。在转基因抗病毒展现出诱人的前景时,对转基因抗病毒植物释放的安全性问题的关注也越来越多。本文介绍了RNAi的作用机制,在转基因抗病毒育种中的应用,并探讨了以RNAi为基础的转基因抗病毒作物的食用安全性和环境安全性等问题。     

RNA干扰及其在水稻抗病毒基因工程中的应用

RNA干扰(RNA interference,RNAi)是一种基因沉默机制。RNAi作为新兴的基因阻断技术具有明显的优势,已被广泛应用到动植物功能基因组和植物抗病研究中。在抗病毒研究中,人为地将与病毒或宿主基因同源的双链RNA分子导入转基因植株,引起与其同源的基因发生沉默,达到抗病毒的作用。本文主要综述了RNA干扰的相关知识以及在水稻抗病毒基因工程研究中的应用进展。     

植物抗病毒的遗传工程

植物抗病毒道传工程中利用病毒衣壳蛋白基因、弱毒株系的完整基因组、病毒反义RNA序列和RNA随体序列等方法获得作物抗病毒转基因植物,本文列举了这些方法的最新应用实例。     

RNAi基因沉默技术及其在植物抗病性改良中应用的策略

RNAi (RNA interference) 是一种由dsRNA参与、对靶基因表达进行干扰或沉默的现象。由此发展起来的RNAi基因沉默技术已成为当今植物基因功能研究和遗传改良的一个重要手段。该技术已经在靶向病原物(真菌、细菌、病毒和线虫)基因沉默方面得到了广泛的应用,并且产生了一批抗病性增强的转基因植物。人工设计和合成的amiRNAs和ata siRNAs的成功研发加快了RNAi技术的应用。本文对RNAi基因沉默机制、RNAi技术研发进展及其在植物抗病性遗传改良中的应用进行综述,并对其应用策略进行探讨。     

正向和反向重复RNA介导的抗马铃薯Y病毒基因工程比较研究

 RNA介导的病毒抗性与RNA沉默现象密切相关。反向重复cDNA序列(IR)的转录产物往往形成双链RNA结构,而双链RNA是诱发RNA沉默的有效因子。据此,本研究通过体外合成马铃薯Y病毒坏死株系衣壳蛋白基因(PVY^N-CP)5'端反向重复cDNA序列和正向重复cDNA序列(DR),分别构建植物表达载体pROK-IR和pROK-DR,利用农杆菌介导方法转化烟草NC89,比较这2种转基因烟草在RNA介导抗病性方面的差异。抗病性检测表明,转化IR和DR的转基因烟草均可获得抗病程度达到免疫的植株,但转化IR序列可大大提高抗病植株在转基因植株中的比例。分析结果表明所获得的抗病性为RNA介导的抗病性,是RNA沉默的结果。这一研究结果为利用IR策略进行抗病毒遗传育种提供了理论依据,并为讲一步开展RNA介导抗病性的机制研究奠宗了基础。     

植物蛋白酶抑制剂抗虫基因工程研究进展

蛋白酶抑制剂抗虫基因工程为提高植物的抗虫能力提供了一种新的策略。与其它来源的抗虫蛋白相比，植物蛋白酶抑制剂有很多独特的优点。蛋白酶抑制剂的抗虫机理尚不完全清楚，影响其抗虫效果的因素也很多。文中列举分析了PI抗虫作用的机理以及目前研究的新进展，提出植物蛋白酶抑制剂抗虫基因工程中存在的问题和进一步解决的办法。     

抗性基因工程在植物医学上的应用

采用基因工程技术 ,将外源抗性基因导入作物 ,育成抗性品种 ,抵抗病虫草害 ,减少农药用量 ,防止环境污染 ,促进农业可持续发展成效显著 ,前景广阔。目前 ,全世界已获得转基因植物１２０类３０００多种 ,面积４０００多万ｈｍ２ ,美国转基因棉占５０ % ,转基因大豆占３０ % ,转基因玉米占２５ %。我国已获得转基因作物４７类１０３种 ,其中抗病虫草害基因６２种（抗病毒３３种、抗细菌８种、抗真菌７种、抗虫１１种、抗除草剂３种）。一、抗虫转基因作物目前 ,抗虫基因工程有两类 :一类是Ｂｔ 杀虫蛋白基因 ,来自苏云金杆菌 ,杀虫毒性为伴孢…     

植物病毒基因沉默抑制因子研究进展

RNAi是近年来发现的一种重要的基因沉默现象,可以介入植物的整体防御体系,在植物细胞中产生一种不确定的流动信号,使远距离组织的特异RNA序列得到降解。为利于病毒的侵染,植物、动物和昆虫的病毒同时也编码一种蛋白来对抗RNAi,这类蛋白可以抑制RNA沉默的各个步骤,称为RNAi抑制因子,本文对几个研究较清楚的植物病毒抑制因子,从其发现到主要特点、作用机制等方面进行了阐述,并且依据其特点及前景进行归类与展望。     

抗同翅目昆虫的相关基因及其转基因抗虫植物研究进展

植物抗虫基因工程为控制害虫的危害提供了新的途径。目前,对同翅目害虫具抗虫活性的基因有三种来源,(1)植物:如植物凝集素基因、番茄抗线虫基因Mi等;(2)微生物:如异戊烯转移酶抗性基因;(3)动物:如来自一些昆虫的蛋白酶抑制素基因。其中一些基因已被成功地转入植物体内,并且获得的转基因抗虫植物对同翅目害虫的生长、发育、繁殖能力等方面都具有一定的抑制作用,表现出这些抗虫基因在防治这类害虫中的应用潜力。雪花莲凝集素可通过人工饲料或转基因作物进入昆虫体内,并通过营养级传递于天敌,进而对天敌造成直接或间接的影响。     

有关转基因植物农药的安全问题

转基因植物农药是指利用分子生物学和基因工程技术将外派目标基因插入受体植物的基因组，使其遗传组成改变；以达到防浪病、虫、草、鼠害目的而产生的植物农药产品。在美国这种农药被称为“pjantpesti－”，这与一般意义的生物农药是有区别的。为完善基因工程工作的安全管理，国家科委于1993年颁发了（基因工程安全管理办法》，农业部又针对我国农业生物基因工程领域的研究和开发，于1996年制订了（农业生物基因工程安全管理实施办法》。转基因植物农药的安全性问题还涉及到昆虫抗性的控制等一系列问题。历史上，常规育种技术在提高作物抗虫…     

植物抗病毒的可能机制--基因沉默

根据目前RNA介导的植物病毒抗性(RMVR)、转录后基因沉默(PTGS)的研究成果,综述了基因沉默可能是植物抵抗病毒的一种机制,深入研究基因沉默不仅在抗病机制上有重要的理论意义,而且对彻底解决植物病毒问题有较大的潜在实用价值.     

RNAi‐mediated plant protection against aphids

Aphids (Aphididae) are major agricultural pests that cause significant yield losses of crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring harmful plant viruses. Expression of double‐stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through plant‐mediated RNA interference (RNAi). Thus, as a potential alternative and effective strategy for insect pest management in agricultural practice, plant‐mediated RNAi for aphid control has received close attention in recent years. In this review, the mechanism of RNAi in insects and the so far explored effective RNAi target genes in aphids, their potential applications in the development of transgenic plants for aphid control and the major challenges in this regard are reviewed, and the future prospects of using plant‐mediated RNAi for aphid control are discussed. This review is intended to be a helpful insight into the generation of aphid‐resistant plants through plant‐mediated RNAi strategy. © 2016 Society of Chemical Industry     

转基因烟草中PVYN CP基因沉默及其介导的抗病性受温度的调控

 本研究以转不可翻译的马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)烟草的T₃代植株为材料,在获得高度抗病植株并证明转基因植株的抗病性是由RNA沉默介导的基础上,采用Northern杂交及ELISA检测病毒的方法,分析了温度对转基因烟草中RNA沉默以及转基因植株抗病水平的影响。结果表明,低温可以改变转基因植株中已发生的RNA沉默和转基因植株的抗病状态。在15℃低温下生长的转基因植株,转基因产生的RNA沉默被抑制,转基因植株失去了对PVY^N高度抗病的特性,表现感病症状;而在25℃或以上高温(30℃、35℃)下生长的转基因植株,转基因产生的RNA沉默没有发生被抑制的现象,转基因植株对PVY^N病毒的侵染仍保持高度抗病性。     

高通量测序技术在植物病毒检疫中的应用与发展

高通量测序技术是当前主流的测序技术,具有快速、灵敏、高通量以及非序列依赖性等特点。本文详细阐述了高通量测序技术的发展以及检测植物病毒的流程,并探讨了该技术应用于植物病毒检测的优势和不足。同时,介绍了利用该技术发现的新的病毒或类病毒,尤其是一些已被欧洲和地中海国家植物保护组织列入检疫性有害生物名录的病毒。目前,有些国家已应用该技术筛查进口植物材料携带的病毒,这对我国的口岸检疫工作提供了新思路。     

RNA silencing against viruses: molecular arms race between Cucumber mosaic virus and its host

Plants have developed RNA silencing as an antiviral defense mechanism. To escape from the plant host’s defenses, viruses have countered their host’s antiviral silencing by producing RNA silencing suppressor proteins (RSSs). Although the mode of action of the majority of viral RSSs has been found to be through double-stranded RNA-binding, viruses have different strategies to counteract the host’s antiviral silencing pathways. The 2b protein of Cucumber mosaic virus, which is one of the most extensively studied viral RSSs, is reviewed here to provide insights on the molecular arms race between viruses and their host plants.     

Turnip crinkle virus with nonviral gene cancels the effect of silencing suppressors of P19 and 2b in Arabidopsis thaliana

In plants, green fluorescent protein (GFP) has become a preferred molecular marker for gene expression and cellular localization, and plant viral vectors are valuable tools for heterologous gene expression. Some plant viruses have been used for expression of GFP, and the activities of these viruses are barely affected by the extra GFP gene. In contrast, the packaging and the length of Turnip crinkle virus (TCV) genome is strictly limited when foreign genes are inserted into the coding sequences of TCV genome. In this report, we removed the silencing suppressor p38 from TCV, and constructed GFP derivatives of TCV. Then the resulting TCV mutants were used to infect Arabidopsis plants containing mutations in key silencing pathway genes, including triple dcl2/dcl3/dcl4, dcl2, dcl4 and ago mutant plants. Our results demonstrate that the activity of TCV is affected by nonviral GFP insert in Arabidopsis plants, and RNA silencing appears not play an important role. AGOs appear to be more efficient at slicing RNAs of viral origin, especially AGO2 and AGO7. Although the viral suppressors of RNA silencing (VSRs) P19 and 2b can enhance the accumulation of viral RNAs, neither P19 nor 2b can significantly increase the expression of TCV mutants with nonviral genes. TCV is an example of an RNA virus that is recalcitrant to add nonviral gene sequences.     

Robust RNAi-based resistance to mixed infection of three viruses in soybean plants expressing separate short hairpins from a single transgene

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.