RNA干扰技术及其应用

RNA干扰(RNA in terference,RNA i)技术是抑制和逆转基因表达的新型生物学研究工具.论述了RNA i的作用机制及其在基因组学、基因治疗、药物探查等方面的应用情况.d sRNA在酶切作用下产生siRNA,后者与RNA诱导沉默复合物R ISC结合,共同降解靶mRNA,从而达到阻断特异基因表达的目的.该技术已对基因功能、基因治疗和药物筛选等方面的体外实验产生了深刻的影响,但由于存在一些尚未解决的问题,这项技术尚未进行大规模临床应用.     

高等真核生物中重叠基因研究

基因重叠是高等真核生物中一种普遍存在的基因组排现象．综述了高等真核生物中的重叠基因的分类、数量、互补或自然反义转录本（NATs）可能的生物特性和功能及重叠基因与人类疾病的关系．     

Subcellular localization and inductive expression of the dsRNA-dependent protein kinase PKR from Japanese flounder, Paralichthys olivaceus

The double-stranded RNA (dsRNA)-dependent protein kinase (PKR) belongs to the eIF2α kinase family and plays a critical role in interferon (IFN)-mediated antiviral response. Recently, in Japanese flounder (Paralichthys olivaceus), a PKR gene has been identified. In this study, we showed that PoPKR localized to the cytoplasm, and the dsRNA-binding motifs (dsRBMs) played a determinative role in protein localization. In cultured FEC cells, PoPKR was detected at a low level of constitutive expression but was highly induced after treatment with UV-inactivated grass carp hemorrhagic virus, active SMRV and Poly I:C although with different expression kinetics. In flounder, PoPKR was ubiquitously distributed in all tested tissues, and SMRV infection resulted in significant upregulation at mRNA and protein levels. In order to reveal the role of PoPKR in host antiviral response, its expression upon exposure to various inducers was characterized and further compared with that of PoHRI, which is another eIF2α kinase of flounder. Interestingly, expression comparison revealed that all inducers stimulated upregulation of PoHRI in cultured flounder embryonic cells and fish, with a similar kinetics to PoPKR but to a less extent. These results suggest that, during antiviral immune response, both flounder eIF2α kinases might play similar roles and that PoPKR is the predominant kinase.     

葡萄卷叶病病毒双链RNA(dsRNA)提纯分析研究*

以传统的检测方法为对照经过一系列的筛选后,在国内首次将病毒的ｄｓＲＮＡ检测分析引入果树无病毒苗的检测方法之中,确立了葡萄病ｄｓＲＮＡ的提取步骤,包括试剂选择、浓度的设定、操作方法的确立,与国外文献相比有多方面的改进,使其更加简单、实用,并在实际的应用当中取得成功。在确立方法的基础上对生产中广泛发生的葡萄卷叶病病毒（ＧＬＲｖ）进行检测并确立检测标准,为今后葡萄病毒病的诊断检测奠定了良好的基础。     

RNA干扰及其应用

RNA干扰（RNA interference，RNAi）是真核生物中普遍存在的一种自然现象，是由双链RNA（double—stranded RNA，dsRNA）启动的序列特异的转录后基因沉默过程．在这一过程中，双链RNA被核酸内切酶切割成小干扰RNA（small interfering RNA，siRNA），小干扰RNA与相关的蛋白质结合成RNA诱导沉默复合体（siRNA—induced silencing complex，RISC），RISC识别并降解靶mRNA．现在认为RNA干扰是真核生物共有的抗病毒、抗转座子、抗转基因和抗异常RNA的基因组免疫系统．随着RNA干扰的研究，产生了一种新的技术-RNA干扰技术，并得到了广泛的应用．     

RNA干涉的研究进展

RNA干涉(RNA interfering)是目前分子生物学领域的研究热点之一,RNAi是一种极为保守的生物机制,而且已在很多种生物中发现了该现象,或是获得了成功的干涉实验。由于RNAi具有良好的技术优越性,使其在很多领域,如基因功能验证、疾病治疗方面有着广阔的应用前景。     

Hairpin RNA: a secondary structure of primary importance

An RNA hairpin is an essential secondary structure of RNA. It can guide RNA folding, determine interactions in a ribozyme, protect messenger RNA (mRNA) from degradation, serve as a recognition motif for RNA binding proteins or act as a substrate for enzymatic reactions. In this review, we have focused on cis-acting RNA hairpins in metazoa, which regulate histone gene expression, mRNA localization and translation. We also review evolution, mechanism of action and experimental use of trans-acting microRNAs, which are coded by short RNA hairpins. Finally, we discuss the existence and effects of long RNA hairpin in animals. We show that several proteins previously recognized to play a role in a specific RNA stem-loop function in cis were also linked to RNA silencing pathways where a different type of hairpin acts in trans. Such overlaps indicate that the relationship between certain mechanisms that recognize different types of RNA hairpins is closer than previously thought. Received 21 November 2005; received after revision 3 January 2006; accepted 11 January 2006     

抗口蹄疫病毒siRNA在HEK-293细胞诱导β-干扰素的研究

利用shRNA（shon-hairpinRNA）表达质粒在HEK-293细胞上评估了多个dsRNA诱发产生干扰素（IFN）的能力．结果显示,p3D3（19nt）,pPol（56nt）,p21NT（21nt）和p63NT（63nt）能够诱发IFN-β的产生,其中p3D3和p63NT能强烈诱导IFN-β;而p3D1（19nt）,p3D2（19nt）和pLacZ（83nt）没有明显的IFN-β诱发能力．结果显示,诱发干扰素产生的能力与dsRNA的片段长度没有关系,可能与其序列有一定相关性．     

RNAi作用机制及其应用研究进展

在Dicer酶作用下,dsRNA形成siRNA,组装成具有活性的蛋白质复合物RISC,触发染色质固缩,DNA甲基化、基因沉默和干扰蛋白质合成等。RNAi基因沉默高效、特异和简捷等优点,为细胞内基因表达研究提供了新思路、新方法,为临床治疗遗传性基因扩增型疾病提供极具潜力的全新策略。文章就RNAi机制及其应用研究做一综述。