RNA干扰技术治疗疾病

RNA干扰(RNA interference,RNAi)现象最早发现于秀丽隐杆线虫(Caenorhabditis elegans),随后发现该现象普遍存在于真菌、植物和哺乳动物等真核生物,并行使基因调控和抵御外源基因片段侵袭的作用。目前,RNAi分子机制和RNAi在基因功能方面的研究已经取得了突破性的进展。鉴于RNAi在基因沉默中的特异性、高效性和易操作,其在药物筛选和疾病治疗等方面有着广泛的应用前景。然而,RNAi技术用于治疗疾病的安全性尚待确定,分子传递途径也有待进一步的研究。     

植物RNAi的特点及其应用研究进展

RNAi是真核生物中普遍存在的抵抗病毒复制表达、抑制转座子转座及调控基因表达的监控机制.清楚植物体内的RNAi途径,将对认识植物基因组功能和转基因研究工作具有积极意义.就国内外与植物RNAi有关的研究作了综述,重点介绍了植物RNAi特征和该技术在植物基因工程中的应用.     

灰葡萄孢丝裂原活化蛋白激酶编码基因bmp1和bmp3的功能

【背景】植物病原真菌丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)信号途径参与病菌有性生殖、细胞壁完整、菌丝侵染、致病力、胁迫响应等过程,灰葡萄孢MAPK信号途径参与病菌生长发育、致病力以及胁迫响应,但MAPK信号途径基因在灰葡萄孢中的功能尚未完全阐明,该信号途径对灰葡萄孢的生长发育和致病力的调控机制尚不明确。【目的】明确灰葡萄孢MAPK编码基因bmp1、bmp3在病菌生长发育、致病力以及氧化胁迫响应过程的功能,为进一步阐明MAPK信号途径调控灰葡萄孢生长发育和致病力的分子机制奠定基础。【方法】利用RNAi技术构建灰葡萄孢MAPK编码基因bmp1和bmp3的RNAi突变体,并以野生型BC22菌株为对照,对bmp1和bmp3基因的RNAi突变体的表型、致病力以及对氧化胁迫的敏感性进行分析。【结果】灰葡萄孢bmp1和bmp3基因的RNAi突变体其菌落形态、菌丝形态均与野生型BC22菌株没有明显差别;bmp1基因的RNAi突变体生长速率明显减慢,分生孢子产量明显降低;bmp3基因的RNAi突变体的生长速率与野生型BC22菌株没有明显差别,不能产生分生孢子。bmp1和bmp3基因的RNAi突变体在番茄果实的表面均不能产生明显的致病症状,而且不能穿透玻璃纸。bmp1基因的RNAi突变体在含有H_2O_2的培养基上受抑制的程度显著低于野生型,而在含甲萘醌的培养基上受抑制的程度显著高于野生型;bmp3基因的RNAi突变体在含有H_2O_2和甲萘醌的培养基受抑制的程度均显著高于野生型。【结论】灰葡萄孢bmp1基因正调控病菌生长、分生孢子形成、致病力和穿透能力,参与调控病菌对氧化胁迫的响应;灰葡萄孢bmp3基因正调控病菌分生孢子形成、致病力、穿透能力以及对氧化胁迫的响应。     

甲壳动物RNAi技术研究与应用进展

RNA干扰(RNA interference,RNAi)是指由双链RNA(double-stranded RNA,ds RNA)诱发的一种使特定基因沉默的现象。作为一种研究基因功能、发现新基因和抗病毒的新型手段,近年来RNAi技术在昆虫、真菌、植物和哺乳动物等的研究中已被广泛应用,但是在甲壳动物研究中尚处于起步阶段。对甲壳动物RNAi技术实施方法做了简要介绍;着重综述了RNAi技术在甲壳动物CHH家族神经肽类基因功能、蜕皮和生长调控机制、配子及性腺发育调控和甲壳动物抗病毒机制研究上的应用进展。     

青狗尾草RNAi途径相关基因的全基因组鉴定和表达分析

RNAi途径是RdDM途径的衍生途径,其中的AGO、DCL和RDR蛋白在植物的生长发育和响应非生物/生物胁迫过程中发挥重要作用。为了研究RNAi途径的3种主要蛋白在青狗尾草中的序列及结构特征,利用比较基因组学方法,在青狗尾草中鉴定到13个AGO基因、7个DCL基因和4个RDR基因,并对其蛋白质亚细胞定位、系统发育关系、保守结构域进行预测。同时,利用转录组数据分析RNAi途径的3类相关基因在青狗尾草的16种不同生长时期、不同生长条件下的表达模式。蛋白质结构域分析发现,SvDCL3b和SvRDR3缺少重要的结构域。转录组分析发现,SvAGO1b、SvDCL1a和SvRDR1在各家族中表达量较高,可能在RNAi途径中发挥主要作用,且大多数青狗尾草和谷子的同源基因间的表达模式基本一致。综上,本研究为理解RNAi途径的3种主要基因在调控青狗尾草的表观遗传修饰中的功能和作用提供初步的理论依据,为青狗尾草和谷子之间驯化的分子机制提供 参考。     

植物萜类生物合成与抗虫反应

植物次生代谢在植物的环境适应,尤其是与微生物和动物的互作和防御反应中起重要作用。萜类是植物次生代谢物中最丰富的一类化合物,许多成分具有重要生理功能和应用价值。腺毛和腺体是植物次生代谢物合成、储藏和分泌的重要器官。我们实验室在植物倍半萜和单萜的生物合成途径及其调控机制、表皮毛发育与萜类代谢调控等方面取得了一系列研究进展。此外,还分析了棉铃虫对棉酚的耐受性(适应性)反应,利用棉铃虫防御基因,发展了一种植物介导的RNAi抗虫技术,可以有效、特异地抑制昆虫基因的表达,在植物抗虫技术研究领域有应用前景。     

植物系统性RNAi的研究进展

RNAi作为反向遗传学手段,以其序列特异性、高效性等特点,在植物基因功能验证、抗病和品种改良等方面发挥了积极作用,RNAi的系统性也同时受到研究者的广泛关注.系统性RNAi(systemic RNA in-terference)传统定义是指沉默信号从一个细胞或一种组织,传递到另外的细胞或同一个体的另外组织中去的现象.本文主要综述植物基因工程研究中应用系统性RNAi技术的研究现状,总结了植物系统性RNAi的特征及沉默效应的抑制.对植物系统性RNAi的研究方法与应用,特别是在植物抗性研究和遗传改良等方面作了重点介绍,并分析了技术存在的限制因素.     

RNAi技术在作物品质改良中的应用

RNA干涉(RNAi)是由同源性内源或外源dsRNA引起的序列特异性基因沉默现象,在动、植物和真菌中广泛存在并被证实。目前已应用RNAi技术在改善油脂的品质、改良淀粉品质、提高营养物质或降低有害物质含量、提高抗褐化能力、提高果实耐贮性、进行代谢调控以获得目的次生代谢物等方面进行了作物品质改良研究。作为一种下调表达技术,该技术在研究植物基因功能和改良作物品质等领域有良好的应用前景。本文重点从上述六个方面对近年来应用RNAi技术在作物品质改良研究方面进行了回顾并对其存在的问题作了初步探讨。     

RNA干扰技术及其在植物研究中的应用

RNA干扰(RNA interference,RNAi)是最近几年发现和发展起来的一门新兴的在转录水平上的基因阻断技术,它是生物体内由双链RNA(double-stranded RNA,dsRNA)介导同源mRNA降解的现象。RNAi广泛存在于从真菌到高等植物、从无脊椎动物到哺乳动物各种生物中。研究表明通过转入目的基因序列的双链RNA可以诱导产生基因沉默现象。同时,RNAi能监控异常的或外源的遗传物质在机体内的水平,并调控基因的表达,是生物体抵御外在感染的一种重要的保护机制,这使得RNA干扰技术具有十分诱人的应用前景。介绍了RNAi的研究历史、作用机制、特点及其在植物研究中的应用。     

RNA干扰及其在增强作物抵抗有害真核生物研究中的应用

RNAi是一种序列特异的同源依赖型基因沉默现象,在真核生物中普遍存在,是生物体抵抗核酸入侵、调控自身基因表达的重要途径。RNAi发现以后,很快就作为一种反向遗传学手段广泛应用于基因功能鉴定,并且在作物改良中得到了广泛应用,在作物抗病毒及品种改良方面得到了成功应用。近年来,随着对RNAi机制认识的不断加深,RNAi技术作为一种增强植物抵抗线虫、草食昆虫、真菌等有害真核生物的新策略的研究逐渐展开,并取得了一定成果,展现出良好的发展前景。对RNAi及其在增强植物抵抗有害真核生物方面的研究进展进行了综述,并对RNAi作为一种持久抗病虫育种策略的前景进行了展望。     

RNA干扰及其在植物代谢工程中的应用

RNA干扰(RNA interference,RNAi)是一种高效的、序列特异的基因沉默现象。本文介绍了RNA干扰的发现及其干扰机制,阐明RNA干扰可在发现代谢途径中的新基因并验证其功能、改善植物营养价值、提高植物抗性、创造新种质等方面发挥重要作用。     

A transient RNA interference assay system using Arabidopsis protoplasts

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.     

Development of plants resistant to tomato geminiviruses using artificial trans‐acting small interfering RNA

RNA interference (RNAi), a conserved RNA‐mediated gene regulatory mechanism in eukaryotes, plays an important role in plant growth and development, and as an antiviral defence system in plants. As a counter‐strategy, plant viruses encode RNAi suppressors to suppress the RNAi pathways and consequently down‐regulate plant defence. In geminiviruses, the proteins AC2, AC4 and AV2 are known to act as RNAi suppressors. In this study, we have designed a gene silencing vector using the features of trans‐acting small interfering RNA (tasiRNA), which is simple and can be used to target multiple genes at a time employing a single‐step cloning procedure. This vector was used to target two RNAi suppressor proteins (AC2 and AC4) of the geminivirus, Tomato leaf curl New Delhi virus (ToLCNDV). The vector containing fragments of ToLCNDV AC2 and AC4 genes, on agro‐infiltration, produced copious quantities of AC2 and AC4 specific siRNA in both tobacco and tomato plants. On challenge inoculation of the agro‐infiltrated plants with ToLCNDV, most plants showed an absence of symptoms and low accumulation of viral DNA. Transgenic tobacco plants were raised using the AC2 and AC4 tasiRNA‐generating constructs, and T₁ plants, obtained from the primary transgenic plants, were tested for resistance separately against ToLCNDV and Tomato leaf curl Gujarat virus. Most plants showed an absence of symptoms and low accumulation of the corresponding viruses, the resistance being generally proportional to the amounts of siRNA produced against AC2 and AC4 genes. This is the first report of the use of artificial tasiRNA to generate resistance against an important plant virus.     

RNA interference and its role in the regulation of eucaryotic gene expression

Several years ago it was discovered that plant transformation with a transcribed sense transgene could shut down the expression of a homologous endogenous gene. Moreover, it was shown that the introduction into the cell of dsRNA (double-stranded RNA) containing nucleotide sequence complementary to an mRNA sequence causes selective degradation of the latter and thus silencing of a specific gene. This phenomenon, called RNA interference (RNAi) was demonstrated to be present in almost all eukaryotic organisms. RNAi is also capable of silencing transposons in germ line cells and fighting RNA virus infection. Enzymes involved in this process exhibit high homology across species. Some of these enzymes are involved in other cellular processes, for instance developmental timing, suggesting strong interconnections between RNAi and other metabolic pathways. RNAi is probably an ancient mechanism that evolved to protect eukaryotic cells against invasive forms of nucleic acids.     

A Transient RNA Interference Assay System Using Arabidopsis Protoplasts

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.     

RNA干扰与植物抗病毒

RNA干扰是多种生物体内由双链RNA介导的同源mRNA降解现象,是植物体内天然的抗病毒机制。然而病毒在长期进化过程中也获得了通过编码沉默抑制蛋白来对抗植物体RNAi系统的能力。本文对RNA干扰过程、病毒编码的沉默抑制蛋白及利用干扰技术进行抗病毒基因工程研究进行简要综述。     

pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants

RNA interference (RNAi) is a powerful tool for functional gene analysis, which has been successfully used to down-regulate the levels of specific target genes, enabling loss-of-function studies in living cells. Hairpin (hp) RNA expression cassettes are typically constructed on binary plasmids and delivered into plant cells by Agrobacterium-mediated genetic transformation. Realizing the importance of RNAi for basic plant research, various vectors have been developed for RNAi-mediated gene silencing, allowing the silencing of single target genes in plant cells. To further expand the collection of available tools for functional genomics in plant species, we constructed a set of modular vectors suitable for hpRNA expression under various constitutive promoters. Our system allows simple cloning of the target gene sequences into two distinct multicloning sites and its modular design provides a straightforward route for replacement of the expression cassette's regulatory elements. More importantly, our system was designed to facilitate the assembly of several hpRNA expression cassettes on a single plasmid, thereby enabling the simultaneous suppression of several target genes from a single vector. We tested the functionality of our new vector system by silencing overexpressed marker genes (green fluorescent protein, DsRed2, and nptII) in transgenic plants. Various combinations of hpRNA expression cassettes were assembled in binary plasmids; all showed strong down-regulation of the reporter genes in transgenic plants. Furthermore, assembly of all three hpRNA expression cassettes, combined with a fourth cassette for the expression of a selectable marker, resulted in down-regulation of all three different marker genes in transgenic plants. This vector system provides an important addition to the plant molecular biologist's toolbox, which will significantly facilitate the use of RNAi technology for analyses of multiple gene function in plant cells.     

Argonaute蛋白与小RNA的作用机制

RNA干扰(RNA interference, RNAi)是在植物、动物、线虫、真菌以及昆虫等生物体中普遍存在的通过双链RNA(double strand RNA, dsRNA)诱导的抑制同源基因表达的一种保守的调控机制.小分子RNA通过特异性地识别结合RNA诱导的沉默复合体（RNA-induced silencing complex, RISC）对目标mRNA的表达在转录和翻译水平进行抑制.作为RISC的重要组成成分,Argonaute蛋白（Ago）发挥了至关重要的作用.为了进一步阐明Ago蛋白在RNA干扰中对小分子RNA的作用机制,本文介绍了Ago蛋白的结构、分类及其在RNA干扰机制中的作用,并着重阐述了目前已知的植物Ago蛋白对小分子RNA的几种作用机制,以及目前研究发现的Ago蛋白的功能作用,从而更进一步证实Ago蛋白对小分子RNA的作用是一个复杂的过程.     

A chemical-regulated inducible RNAi system in plants

Constitutive expression of an intron-containing self-complementary 'hairpin' RNA (ihpRNA) has recently been shown to efficiently silence target genes in transgenic plants. However, this technique cannot be applied to genes whose silencing may block plant regeneration or result in embryo lethality. To obviate these potential problems, we have used a chemical-inducible Cre/loxP (CLX) recombination system to trigger the expression of an intron-containing inverted-repeat RNA (RNAi) in plants. A detailed characterization of the inducible RNAi system in transgenic Arabidopsis thaliana and Nicotiana benthamiana plants demonstrated that this system is stringently controlled. Moreover, it can be used to induce silencing of both transgenes and endogenous genes at different developmental stages and at high efficiency and without any detectable secondary affects. In addition to inducing complete silencing, the RNAi can be produced at various times after germination to initiate and obtain different degrees of gene silencing. Upon induction, transgenic plants with genetic chimera were obtained as demonstrated by PCR analysis. Such chimeric plants may provide a useful system to study signaling mechanisms of gene silencing in Arabidopsis as well as other cases of long-distance signaling without grafting. The merits of using the inducible CLX system for RNAi expression are discussed.