黄瓜花叶病毒致弱卫星RNA对辅助病毒含量的影响

为研究黄瓜花叶病毒(CMV)弱病毒株的基因组RNA和卫星RNA的含量变化,将一个卫星RNA介导的CMV弱病毒株NDM-1与CMV强毒株在4种寄主系统上进行对照实验.采用RNA点杂交方法,检测从早期接种病毒到大田释放阶段（病毒接种后5到40天）,寄主植物叶组织中的基因组RNA和卫星RNA的相对含量变化.结果显示：弱病毒株NDM-1基因组RNA和卫星RNA负荷量具有寄主效应和时间效应,趋势一致但程度不同.它们在番茄上的变化规律不同于其他寄主而且病毒的致弱效果最明显：在接种后的5～10天 (生产上接种育苗的保护期) ,基因组RNA和卫星RNA相对含量上升,到40天(大田释放期) 时回落至最低.比较NDM-1于不同时间在各寄主上的卫星RNA和基因组RNA的相对比值：在40天时,寄主番茄上的相对比值达15.8,与在其他寄主上和其他时间段比较均有显著差异,即此时卫星RNA达到含量优势.说明弱病毒NDM-1在番茄上的致弱机制为:卫星RNA的大量复制对CMV基因组RNA产生抑制作用.此弱病毒株在烟草和心叶烟中的变化规律基本一致.     

黄瓜花叶病毒卫星RNA XJs1侵染性cDNA克隆构建及其生物学功能初步研究

利用RT_PCR的方法,获得了黄瓜花叶病毒卫星RNA XJs1的全长侵染性cDNA克隆pMSC20。序列分析显示,XJs1全长384nt(GenBank登录号:DQ070748),比较XJs1与具有代表性的CMV卫星RNA的序列结构表明,在XJs1核苷酸序列的325nt~350nt间,具有典型的坏死型卫星RNA保守序列。通过体外转录,将XJs1与不含卫星RNA的辅助病毒分离物CMV_AH组合接种普通烟和心叶烟并进行检测。初步研究结果表明,XJs1为一致弱卫星RNA。     

黄瓜花叶病毒卫星RNA人工突变体的构建及其侵染性测定

从1个369nt的黄瓜花叶病毒(Cucumbermosaicvirus,CMV)卫星RNA的cDNA出发,采用DNA改组技术构建人工突变体,经过体外转录,将其与不携带卫星RNA的黄瓜花叶病毒株进行假重组,鉴定突变体的生物活性,结果显示所获得的4个卫星RNA的点突变子MS1、MS5、MS6和MS11中,只有MS11仍然具有复制能力;而其他3个点突变子,尽管均只有1个位点的替换,却不能在辅助病毒作用下复制。序列比较分析发现MS11的突变位点位于卫星RNA变异区内,发生的突变与自发突变一致,其他3个突变子的突变位点发生在卫星RNA的高度保守区。而且通过侵染性试验证实突变子MS11与野生型Yi没有明显的差异。由此可推测卫星RNA序列中的高度保守区与卫星RNA的生物活性密切相关,个别碱基的突变会导致RNA二级结构的改变,进而引起其复制能力或稳定性的完全丧失。     

在系统侵染的烟草中假重组体病毒FMF的时序变化

构建了由黄瓜花叶病毒Fny株系RNA1、RNA3与M株系RNA2组合得到的假重组体病毒FMF,并用其侵染烟草幼苗,接种5 d后烟草幼叶上出现坏死环斑,接种5-30 d新生叶上没有呈现任何明显症状;30 d后新生叶上突然出现轻微绿斑驳。采用实时荧光定量PCR(FQ-PCR)方法,监测不同时期展开的幼叶中病毒粒子含量和病毒基因组RNA含量的动态变化,结果显示,接种后5-25 d新生叶中病毒粒子含量保持较低水平,30 d时含量突然大幅度增长;接种后5-30 d新生叶中病毒基因组RNA1、RNA3和RNA4含量均经历骤减期和增长期;RNA2含量变化情况平缓,没有明显先减后增的趋势。病毒粒子、病毒基因组RNA含量及病症严重程度的动态变化规律及其相关性将为研究FMF侵染机制、病毒与寄主互作及病毒防控提供依据。     

黄瓜花叶病毒CB7株系引起心叶烟坏死反应与RNA2相关

采用RT-PCR获得黄瓜花叶病毒CMV-CB7株系全长基因组cDNA,经克隆测序发现该CMV的RNA1、2和3分别为3356nt、3045nt和2218nt(序列登录号为:EF216866、DQ785470和EF216867).CMV-CB7基因组cDNA克隆体外转录RNA接种心叶烟引起坏死症状,而CMV-Fny则产生典型花叶.由CMV-CB7和CMV-Fny基因组RNA相互交换而构建6个假重组型病毒(C1C2F3、C1F2C3、F1C2C3、F1F2C3、F1C2F3和C1F2C3)活性分析表明:CMV-CB7基因组RNA2决定其在寄主上的症状反应.嵌合型RNA2(RNA2F5C3和RNA2C3F5)的寄主侵染活性测定表明:2b基因或RNA23′端非编码序列决定CMV-CB7在心叶烟坏死症状.RNA印迹分析结果显示:CMV-CB7和CMV-FnyF5C3引起寄主坏死与基因组RNA积累没有直接关系.     

黄瓜花叶病毒互补型载体可与CP转基因发生重组

为了探讨利用黄瓜花叶病毒(CMV)作为表达载体的可行性,克隆了山东株(SD)CMV RNA3的全长cDNA,并测定了全序列.采用定点突变的方法在衣壳蛋白(CP)基因起始密码子处改造出一个NsiⅠ位点.以绿色荧光蛋白(GFP)基因置换SD-CMV RNA3 cDNA的CP基因.将Fny株CMV RNA1,RNA2和嵌合SD-CMV RNA3的cDNA分别克隆在35 S启动子和终止子之间构建成表达载体.在烟草原生质体中验证了该表达载体可以表达GFP.然后将其接种到表达SD CP的转基因烟草上,试图构建成一个互补型载体.接种10d后,18棵植株中,有5棵的接种叶和其中1棵的系统叶上可检测到表达的GFP.然而1个月后,所有接种植株中都检测不到GFP.通过RT-PCR及序列分析,证实在互补系统中CMV载体与CP转基因发生了重组.上述结果对这种CMV互补型载体的可行性提出了质疑,同时也揭示了转CMV CP基因抗病毒植物的生物安全性中存在的新的问题.     

卫星RNA研究新进展：结构与功能表达

植物病毒卫星通常是指那些如果没有特定辅助病毒的帮助就不能复制,而它本身对于辅助病毒的复制是不必需的,且与辅助病毒的基因组无序列同源性的一类RNA分子。到目前为止,已报道有六组共26种植物病毒带有卫星。这些植物病毒卫星有的具有编码自身外壳蛋白的遗传信息,称卫星病毒;有的无此编码功能称为病毒卫星RNA。病毒卫星RNA在其发现的早期,其生物学功能尚不为人重视。然而,七十年代早期,在法国阿尔萨斯爆发的由黄瓜花叶病毒(Cucumber Mosaic Virus,CMV)侵染引起的番茄作物坏死病被确定与CMV卫星RNA有关,至此,关于卫星RNA的生物     

CMV抑制PVYN CP基因沉默及其介导的抗病性

以前曾报道用RNA介导的抗病毒策略,获得了高度抗病的表达马铃薯Y病毒坏死株系外壳蛋白基因(PVY^N CP)的转基因烟草,并对T1、T2代转基因植株进行了遗传和抗病性分析。此次以T,代转基因植株为试验材料,在筛选高度抗病植株并证明其抗病性是基于转基因沉默的基础上,采用Northern杂交的方法,证明CMV侵染抑制了转基因植株中PVY^N CP基因的沉默,而且CMV对PVY^N CP基因沉默的抑制部位是发生在接种后的新生叶上,接种叶及其下部叶片中PVY^N CP基因沉默则未受到影响。采用ELISA方法对CMV PVY^N复合接种的转基因植株进行PVY^N检测,结果表明,接种叶及下部叶没有检测到PVY^N,植株叶片对PVY^N表现为抗病。而在CMV接种后植株新生叶中则检测出了高滴度的PVY^N,植株叶片对PVY^N表现为感病。该文报道了在表达PVY^N CP基因的RNA介导抗性转基因植株中,异源病毒侵染抑制了转基因的沉默,并导致转基因植株的抗病性丧失。     

中国番茄黄化曲叶病毒DNAβ缺失突变体与异源病毒的互作研究

与一些单组份双生病毒伴随的卫星DNAβ是辅助病毒在自然寄主上引起典型症状所必需的致病相关分子。迄今发现的所有DNAβ分子在互补链上都含有一个位置和大小保守的βC1基因。对中国番茄黄化曲叶病毒的缺失βC1基因的DNAβ突变体(DNAΔC1β)与异源病毒接种后的致病性及稳定性进行了研究。PCR和Southern杂交证实该突变体能利用赛葵黄脉病毒、烟草曲茎病毒、中国胜红蓟黄脉病毒、假马鞭曲叶病毒等多种异源双生病毒复制并系统侵染本氏烟。接种后65 d内的PCR检测和序列分析显示,该突变体与其它辅助双生病毒接种后在寄主细胞中的复制是稳定的。     

系统侵染寄主中黄瓜花叶病毒及其卫星RNA的动态变化

以³²P标记的黄瓜花叶病毒（CMV）RNA3 cDNA片段和卫星RNA全长cDNA作为探针,定量测定CMV基因组RNA和卫星RNA的含量变化,结果显示：二者均具有明显的寄主效应和时间效应.在16～20℃条件下,接种不携带卫星RNA的分离物CMV-R3,15天、30天和75天时,CMV基因组RNA负荷量呈显著下降的趋势.在第15天,RNA3的负荷量以烟草>心叶烟>克里夫兰烟>番茄的顺序表现为不同寄主的显著性差异.相同条件下接种携带高拷贝卫星RNA的分离物CMV-RS,在5天和15天之间基因组RNA和卫星RNA负荷量均呈现上升的趋势,同时测得其基因组RNA和卫星的负荷量具有相似的寄主效应和时间效应,但程度不同.第15天时,二者负荷量以烟草＞心叶烟＞番茄的顺序表现寄主效应的显著性差异.在18～21℃条件下,接种携带坏死卫星RNA的CMV强毒株HC4,第5天、第10天和第15天时,基因组RNA和卫星RNA的负荷量均以番茄＞心叶烟＞烟草的顺序表现出显著性差异,并表现出明显的时间效应.不同来源CMV分离物还存在寄主选择性差异.     

Recombination with coat protein transgene in a complementation system based on Cucumber mosaic virus (CMV)

In order to study the feasibility of Cucumber mosaic virus (CMV) as an expression vector, the full-length cDNA of RNA 3 from strain SD was cloned and the sequence around the start codon of the coat protein (CP) gene was modified to create an Nsi I site for insertion of foreign genes. The CP gene was replaced by the green fluorescent protein (GFP) gene. The cDNAs of Fny RNAs 1 and 2 and the chimeric SD RNA 3 were cloned between the modified 35S promoter and terminator. Tobacco protoplasts were transfected with a mixture of the viral cDNAs containing 35S promoter and terminator as a replacement vector and expressed GFP. A complementation system was established when the replacement vector was inoculated onto the transgenic tobacco plants expressing SD-CMV CP. GFP was detected in the inoculated leaves in 5 of 18 tested plants and in the first upper systemic leaf of one of the 5 plants ten days after inoculation. However, no GFP could be detected in all the plants one month after inoculation. Recombination be     

Plant virus satellite RNAs and their role in engineering resistance to virus diseases

The mechanism of satellite RNA (Sat-RNA) mediated attenuation of virus disease was suggested to be the result of competition between satellite RNAs and their helper viral RNAs for replication. Subcellular distribution of viral coat protein in cucumber mosaic virus (CMV) infected leaf tissues shows that the presence of Sat-RNAs interferes with the movement of CMV coat protein into chloroplants. As a strategy for controlling virus diseases, the expression of Sat-RNAs in transgenic plants confers some extent of resistance that may not be strong enough to protect the plants from natural virus infections. Transgenic plants expressing both the Sat-RNA and the coat protein of CMV exhibit enhanced resistance to the virus.     

Support of a cucumber mosaic virus satellite RNA maps to a single amino acid proximal to the helicase domain of the helper virus.

Cucumber mosaic virus (CMV) is a tripartite RNA virus that can support the replication of satellite RNAs, small molecular parasites of the virus. Satellite RNAs can have a dramatic effect on the helper virus and the host plant in a manner specific to the helper, satellite, and host. Previously, we showed that the Sny-CMV strain is not able to support the replication of the WL1 satellite RNA in zucchini squash and that this phenotype maps to RNA 1. In the present study, we use recombinant cDNA clones of Fny- and Sny-CMV RNA 1 and a site-directed mutant of Fny-CMV RNA 1 to demonstrate that the inability to support WL1 satellite RNA maps to a single amino acid at residue 978 in the 1a protein, proximal to the helicase domain VI. Support of satellite RNA in whole plants and in protoplasts of zucchini squash is analyzed.     

Resistance of Pepper Lines to the Movement of Cucumber Mosaic Virus

The multiplication and the migration of cucumber mosaic virus (CMV) were studied in greenhouse conditions in one susceptible ‘Yolo wonder’ and two resistant ‘Milord’ and ‘Vania’ pepper varieties. DAS-ELISA tests have revealed that the virus is replicated in inoculated leaves of the resistant varieties as high as in the susceptible variety. In the susceptible variety ‘Yolo wonder’, CMV migrated from the leaf lamina to the petiole two days after inoculation and it became systemic three days later regardless the season. In ‘Milord’ the virus migrated from the leaf lamina to the petiole five days after inoculation and it became systemic during the winter 16 days after inoculation. Whereas plants of the same genotype were not infected systemically during the summer. In ‘Vania’, during the two seasons, CMV spread from the blade to the petiole five days after inoculation, but the virus was not detected beyond the inoculated leaf. These results show that ‘Milord’ and ‘Vania’ are resistant to CMV migration. Therefore, the resistance to CMV migration is affected by plant genotype and temperature. The study of effect of pepper plant phenology on infection has revealed that resistance to CMV migration is also affected by the development stage of the plants.