The Occurrence of dsRNA Species in Apparently Healthy and Virus-infected Ribes Cultivars, and Evidence That One Such Species Originates from a Member of the Virus Family Totiviridae

Analysis was made of dsRNA in 37 cultivars and species of Ribes, that were healthy, naturally affected with the virus-like diseases, blackcurrant yellows, blackcurrant infectious variegation, gooseberry veinbanding or blackcurrant reversion, or graft-inoculated with scions from such diseased plants. Various dsRNA species, differing in size (from ca. 2 to 11kbp), number and staining intensity in gels, were detected in some or all assays of all plants, including those held as virus-tested stock. In different plant tissues from individual plants, the dsRNA species were usually similar in size and number but, in some sources, the dsRNA profile from flowers and/or bark differed greatly from the profiles of dsRNA obtained from leaves. No dsRNA species were associated consistently with any of these diseases. A 499kbp cDNA probe was obtained that in Northern blot analysis was specific to a ca. 5kbp dsRNA species present in the blackcurrant cv. Baldwin. It also detected a similarly sized dsRNA species in plants of many other blackcurrant cultivars, but it did not react with a similarly sized dsRNA species in redcurrant and gooseberry tissues. The 156 amino acid sequence encoded by the cDNA was very similar to sequences in the RNA-directed RNA polymerases of virus species in the family Totiviridae, especially Saccharomyces cerevisiae viruses L-1 and L-A. The significance of these findings and the possible origin of these dsRNA species are discussed.     

美洲型猪繁殖与呼吸综合征病毒荧光RT-PCR检测方法的建立

参照美洲型猪繁殖与呼吸综合征病毒（PRRSV）M基因序列，设计引物和探针，并建立其荧光RT-PCR检测方法，以检测严重危害我国养猪业的此型PRRSV。该法能检测到1 TCID50的PRRSV。用此法检测猪瘟病毒、乙脑病毒、伪狂犬病毒、马动脉炎病毒、圆环病毒Ⅱ型和MARC145细胞的RNA，结果均为阴性。对126份临床样品进行检测应用，检测结果（7份阳性，119份阴性）与病毒分离完全符合。此法中的阳性对照为体外转录的dsRNA，具有高度的稳定性，解决了此类检测方法所用的RNA阳性对照所普遍存在的易于降解的难题。     

RNA干扰的研究进展

RNA干扰(RNA interference,RNAi)现象是1998年在对秀丽线虫的研究中发现的.RNAi利用双链RNA(dsRNA)特异性地降解相应序列的mRNA.从而特异性地阻断相应基因的表达.本文介绍了RNA干扰现象的发现、分子机制、生物学意义及其技术的应用发展.     

源于马铃薯Y病毒CP基因的dsRNA原核表达及提取

以马铃薯Y病毒(Potato virus Y,PVY)为基础构建了含PVY CP基因3’端253bp反向重复片段的双链RNA(dsRNA)原核表达载体,并转化大肠杆菌HT115(DE3),经IPTG诱导产生dsRNA,用LiCl沉淀法提取,获得了高质量的dsRNA。     

RNA干扰技术及其应用

RNA干扰(RNAinterference,RNAi)即通过双链RNA的介导特异性降解相应序列的mRNA,从而导致转录后水平的基因沉默的现象。作为一种简单有效的影响基因表达的工具,RNA干扰已经被广泛应用于许多领域,同时也为基因功能的研究开辟了一条新途径。文章综述了RNA干扰的机制及其应用方面的最新进展。     

食用菌病毒研究进展

食用菌病毒作为真菌病毒的一部分,有多种形态,多数为分段的dsRNA基因组形式,也有正义单链RNA,其中主要编码衣壳蛋白和依赖于RNA的RNA聚合酶。目前,已有食用菌病毒基因组序列被测定,并分析了其基因组的结构与功能。dsRNA技术和酶学方法使dsRNA食用菌病毒的研究很容易的深入到分子水平,特别是dsRNA技术,已在平菇得到应用。食用菌病毒有其特有的复制模式,分两个阶段进行,其原始的起源可能是自我复制的细胞内mRNA,因为细胞内mRNA明显的原始性。综述了上述内容,并对食用菌病毒与其寄主在共进化这方面做了进一步展望,对食用菌病毒的进一步研究,以及选育脱毒菌株,都有重要的意义。     

Ingested RNA interference for managing the populations of the Colorado potato beetle, Leptinotarsa decemlineata

BACKGROUND: RNA interference (RNAi) is a breakthrough technology for conducting functional genomics studies and also as a potential tool for crop protection against insect pests. The major challenge for efficient pest control using RNAi in the field is the development of efficient and reliable methods for production and delivery of double‐stranded RNA (dsRNA). In this paper, the potential of feeding dsRNA expressed in bacteria or synthesized in vitro to manage populations of Colorado potato beetle, Leptinotarsa decemlineata (Say) (CPB), was investigated. RESULTS: Feeding RNAi successfully triggered the silencing of all five target genes tested and caused significant mortality and reduced body weight gain in the treated beetles. This study provides the first example of an effective RNAi response in insects after feeding dsRNA produced in bacteria. CONCLUSION: These results suggest that the efficient induction of RNAi using bacteria to deliver dsRNA is a possible method for management of CPB. This could be also a promising bioassay approach for genome‐wide screens to identify effective target genes for use as novel RNAi‐based insecticides. Copyright © 2010 Society of Chemical Industry     

褐飞虱呼肠孤病毒干扰水稻锯齿叶矮缩病毒的初步研究

褐飞虱(Nilaparvata lugens Stal)可携带多种病毒,其中褐飞虱呼肠孤病毒(Nilaparvata lugens reovirus,NLRV)和水稻锯齿叶矮缩病毒(Rice ragged stunt virus,RRSV)均为ds RNA基因组的呼肠孤病毒。通过接种、ds RNA小量提取及RT-PCR等方法分析NLRV对RRSV传播和增殖的影响,初步探讨这两种呼肠孤病毒之间的关系。结果表明,褐飞虱携带NLRV比率高低影响褐飞虱接种RRSV效率;携带NLRV的褐飞虱种群接种RRSV后两种病毒的含量差异较大,NLRV的含量远远大于RRSV的含量,推测褐飞虱体内的NLRV某种程度上干扰了RRSV的传播和增殖。     

应用dsRNA测序技术检测草鱼呼肠孤病毒的混合感染

从草鱼(Ctenopharyngodon idellus)出血病疑似病料提取物感染的草鱼肾细胞(CIK)中提取病毒基因组dsRNA,电泳显示出水生呼肠孤病毒基因组的典型特征。应用简化的FLAC(full-length amplification of cDNA)技术扩增得到病毒的4个全长基因组片段进行测序分析。核酸序列BLAST分析表明:其中3个基因组片段与GCRV-873第5、9、10片段高度同源,另外1个基因组片段与GCRV-HZ08第11片段高度同源;因此推测病料中同时存在两种不同的病毒核酸,但也可能存在一种杂合病毒。根据已发表的GCRV-HZ08第5、9、10片段设计了3对引物对分离的病毒总RNA进行RT-PCR检测并测序,结果显示这3个片段与GCRV-HZ08第5、9、10片段均具有99%同源性,表明是由于混合感染而存在两株不同病毒的核酸dsRNA,两株病毒分别命名为GCRV-JX01和GCRV-JX02。首次运用dsRNA测序法检测出了GCRV病毒的混合感染,为草鱼呼肠孤病毒的流行病学和防控提供了一种新的技术选择。     

RNAi技术防治害虫的研究进展

利用RNAi技术来防治害虫成了人们研究的热点之一。RNAi可分为细胞自主性的和非细胞自主性的。对应用RNAi技术防治害虫来讲,研究的焦点集中在非细胞自主性RNAi,即昆虫能通过取食来内化目标基因的dsRNA。RNAi技术防治害虫的特异性极高,不会出现残留污染问题。可以通过注射、饲喂和转基因dsRNA来进行害虫的防治。本文还介绍了昆虫吸收dsRNA的机制。