RNA干扰机制研究进展

RNAi是多种生物体内由dsRNA介导的同源mRNA降解现象。这是一个高度特异化的过程,涉及多种蛋白质的共同参与。在这一过程中,siRNA的结构影响其两条链装配到RISC中去的能力。除了与RISC结合外,siRNA还引导了RITS复合物结合到同源染色质,介导异染色质化过程。干扰效应的扩散,即系统性沉默可能依赖于跨膜蛋白的转运,并且很可能是在多因素调控下完成的。Abstract: RNA interference (RNAi) is a phenomenon that the double-stranded RNA (dsRNA) intermediates the degradation of complementary mRNA found in many organisms. This is a specifically mechanism involved in kinds of proteins to complete the interference function. Structure of siRNA affects which strand will be assembled into RISC. Another role of siRNA is directing RITS complex to bind with homologue chromosome, and then induces heterochromatinization. Although systemic silence induced by dsRNA is observed in Caenorhabditis elegans and plants, this progress is probably transmembrane protein-dependent, and mostly, the systemic silencing is controlled by multi-factors.     

Nmnoparticle-mediated double-stranded RNA delivery system: A promising approach for sustainable pest management

RNA interference(RNAi)targeting lethal genes in insects has great potential for sustainable crop protection.Compared with traditional double-stranded(ds)RNA delivery systems,nanoparticles such as chitosan,liposomes,and cationic dendrimers offer advantages in delivering dsRNA/small interfering(si)RNA to improve RNAi efficiency,thus promoting the development and practice of RNAi-based pest management strategies.Here,we illustrate the limitations of traditional dsRNA delivery systems,reveal the mechanism of nanoparticle-mediated RNAi,summarize the recent progress and successful applications of nanoparticle-mediated RNAi in pest management,and finally address the prospects of nanoparticle-based RNA pesticides.     

Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence, dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3‘ overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNAduplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0-450 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5‘ end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.     

RNA干涉分子机制研究进展

RNA干涉（RNA interference,RNAi）是生物体内的一种通过双链RNA(dsRNA)来抵抗病毒入侵和抑制转座子活动的自然机制.双链RNA与同源mRNA互补结合而使特定基因失活,这一过程已经在包括拟南芥、线虫和真菌等多种模式生物中得到揭示.近来研究表明,21～25 nt的小干涉RNA（small interference RNA, siRNA）可介导哺乳动物细胞特异性基因沉默.RNAi具有高效性和高度特异性,可能成为关闭基因的新技术而在基因功能研究和疾病基因治疗中发挥重要作用.     

Oral delivery of dsHvlwr is a feasible method for managing the pest Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae)

RNA interference(RNAi)techniques have emerged as powerful tools that facilitate development of novel management strategies for insect pests,such as Henosepilachna vigintioctopunctata(Coleoptera:Coccinellidae),which is a major pest of solanaceous plants in Asia.In this study,the potential of oral delivery of in vvYro-synthesized and bacterially expressed double-stranded H.vigintioctopunctata lesswright(Iwr)gene(dsHvlwr)to manage of H.vigintioctopunctata was investigated.Our results showed that the gene Hvlwr had a 480-bp open reading frame and encoded a 160-amino acid protein.Hvlwr expression levels were greater in the fat body than other tissue types.Hvlwr silencing led to greater H.vigintioctopunctata mortality rates and appeared to be time-and partially dose-dependent,likely as a result of the number of hemocytes increasing with dsRNA concentration,but decreasing with time.Bacterially expressed dsHvlwr that was applied to leaf discs caused 88%,66%,and 36%mortality in 1st instars,3rd instars,and adults after 10,10,and 14 d,respectively;when applied to living plants,there was greater mortality in 1 st and 3rd instars,but there was no effect on adults.Furthermore,dsHvlwr led to improved plant protection against H.vigintioctopunctata.Our study shows an effective dietary RNAi response in H.vigintioctopunctata and that Hvlwr is a promising RNAi target gene for control of this pest species.     

Gene silencing in Xenopus laevis by DNA vector-based RNA interference and transgenesis

A vector-based RNAi expression system was developed using the Xenopus tropicalis U6 promoter, which transcribes small RNA genes by RNA polymerase Ⅲ. The system was first validated in a Xenopus laevis cell line, designing a short hairpin DNA specific for the GFP gene. Co-transfection of the vector-based RNAi and the GFP gene into Xenopus XR1 cells significantly decreased the number of GFP-expressing cells and overall GFP fluorescence. Vector-based RNAi was subsequently validated in GFP transgenic Xenopus embryos. Sperm nuclei from GFP transgenic males and RNAi construct-incubated-sperm nuclei were used for fertilization, respectively. GFP mRNA and protein were reduced by -60% by RNAi in these transgenic embryos compared with the control. This transgene-driven RNAi is specific and stable in inhibiting GFP expression in the Xenopus laevis transgenic line. Gene silencing by vector-based RNAi and Xenopus transgenesis may provide an alternative for ＇repression of gene function＇ studies in vertebrate model systems.     

RNA介导的转基因沉默:原理及应用

RNA介导的转基因沉默是有dsRNA参与指导的,以外源和内源mRNA为降解目标的转基因沉默现象。本文对植物中RNA介导的转基因沉默的机理做了详细的阐述,并且将各种生物中在此过程中起关键作用的蛋白及其功能进行了总结,最后介绍dsRNA作为一种基因组学研究手段的优势和它实际的应用前景。 Abstract:RNA-based transgene silencing is a phenomenon that endogenous and exogenous mRNAs are degraded specifically directed by double-stranded RNA.Here we review the recent advances on its mechanism and summarize related proteins and expatiate their functions.Furthermore,we introduce the enormous potential of dsRNA as a tool in functional genomics research and practical biotechnology.     

RNA/DNA嵌合分子介导的高效基因修复

本文介绍了RNA/DNA嵌合分子介导的高效基因修复技术。这一技术是1996年开始发展起来的全新技术,它通过人工合成的双链开环RNA/DNA嵌合分子转染细胞而使特定基因靶位点产生单碱基改变,从而修复突变基因。这一技术高效（目前最高可达50％以上）、特异性强、安全、无随机插入致变的危险、无免疫反应、无明显毒性,能够用于定点突变、基因敲除、动植物功能基因组学、药物遗传学等很多方面的研究,在不久的将来能够应用于人类基因治疗,具有很高的应用价值和医学前景。 Abstract：We introduce a new technique?targeted gene correction directed by chimeric RNA/DNA oligonucleotides which began at 1996.It uses synthetic double?stranded non?circular RNA/DNA chimeric oligonucleotides to transfect cells and make a single?based change at the targeted site of the target gene.It is highly efficient (the highest efficiency is more than 50％),highly special,safe,without danger of mutation caused by random insertion,without immune response,and without obvious toxicity.It can be used to make point mutation,or gene knock?out plants and animals,and is very likely to be used in human gene therapy in the near future.It is also valuable in the study of functional genomics,pharmacogenetics,and medicine.     

真菌RNA沉默机制研究进展

RNA沉默(RNA silencing)或者RNA干扰(RNA interference)是由dsRNA(double-stranded RNA)介导的序列特异性降解或者抑制与起始dsRNA一样或者高度相似RNA的一种机制,在进化上存在保守性,具有基因调控、防御外源核苷酸入侵的功能。真菌RNA沉默依据sRNA来源不同,分为多种途径,主要有quelling、qiRNA、MSUD。参与不同途径的主要有Argonaute、Dicer、RdRP3种核心蛋白质,并且在不同真菌中数目变化很大,反映出真菌RNA沉默的多样性。有些真菌的RNA沉默在进化的过程中丢失,在抗病毒机制上进化出"互换"的killer系统。