三重RT-PCR同步检测马铃薯多种病毒影响因素*

根据病毒外壳蛋白区序列设计PVX、PVS特异性引物对 ,根据P1基因区序列设计PVA特异性引物对 ,应用三重RT PCR同步检测马铃薯X病毒 ,马铃薯A病毒及马铃薯S病毒 ,分别得到 5 62bp、 2 5 5bp、 1 82bp大小的扩增片段。试验从反转录反应、PCR反应及循环条件 3方面讨论了试剂和循环条件对三重RT PCR同步检测 3种病毒的影响。结果表明反转录反应中dNTPs浓度、 3种病毒下游引物浓度比例对整个反应影响较大 ;其次是PCR反应中MgCl₂ 浓度和退火温度 ;     

一种快速提取植物病毒DNA的方法

介绍在PCR检测体系中一种快速提取病毒DNA的方法。利用高盐缓冲液溶液释放病毒DNA,同时利用葡聚糖凝胶微柱纯化提取液,有效消除样品中PCR抑制物质并直接作为PCR反应模板扩增检测病毒。该法无需任何特殊设备,适合对大量植株进行大通量的检测分析。     

三重RT-PCR同步检测马铃薯多种病毒影响因素

根据病毒外壳蛋白区序列设计PVX、PVS特异性引物对,根据P1基因区序列设计PVA特异性引物对,应用三重RT-PCR同步检测马铃薯X病毒,马铃薯A病毒及马铃薯S病毒,分别得到562bp、255bp、182bp大小的扩增片段。试验从反转录反应、PCR反应及循环条件3方面讨论了试剂和循环条件对三重RT-PCR同步检测3种病毒的影响。结果表明反转录反应中dNTPs浓度、3种病毒下游引物浓度比例对整个反应影响较大;其次是PCR反应中MgC12浓度和退火温度;反转录时间,循环条件对RT-PCR影响较小。     

BST-2抑制HIV-1病毒样颗粒释放的研究

BST-2是最近发现的可以抑制成熟HIV-1(human immunodeficiency virus,HIV)病毒颗粒从哺乳动物细胞表面释放的宿主因子,随之发现其也可以抑制多种包膜病毒的释放。本研究采用密码子优化的表达HIV-1 gag和gag-pol蛋白的质粒所形成的病毒样颗粒作为研究对象,观测BST-2对这两种病毒样颗粒(Virus-like particle,VLP)的释放抑制情况及其作用机制。结果发现,瞬时表达和稳定表达的BST-2均可以显著抑制病毒样颗粒从哺乳动物细胞释放,同时发现这两种病毒样颗粒(gag/gag-pol)的释放都可以被BST-2抑制;而且,HIV-1中Vpu蛋白可以拮抗BST-2抑制HIV病毒样颗粒释放的作用,另外,通过化学试剂和酶学方法处理,确证BST-2可以被包装进病毒样颗粒中。     

烟草环斑病毒的定量检测技术研究

目的:建立特异、灵敏、快速的TaqMam实时荧光定量PCR方法,用于烟草环斑病毒(TRSV)的定量检测。方法:用纳米磁珠法提取病毒RNA,构建包含烟草环斑病毒全CP序列的质粒标准品。根据CP保守序列设计特异性的引物和TaqMam荧光探针,构建标准曲线,建立TRSV的实时荧光绝对定量PCR方法,并对该方法的特异性、灵敏度和重复性进行评估。结果:建立的方法特异性好,与南芥菜花叶病毒、马铃薯X病毒和马铃薯Y病毒均无交叉反应;至少能检测到767个病毒拷贝,灵敏度比普通PCR高100倍;同一样品试验内及试验间重复性实验的变异系数均小于3%,重复性好;检测结果准确可靠,构建的标准曲线有较好的线性关系(R2=0.997)。结论:建立的TRSV TaqMan实时荧光定量PCR检测方法可满足口岸高通量、快速、准确的检验检疫要求。     

马铃薯无病毒原种繁育会议召开

1976年9月3日至7日中国科学院在内蒙古自治区察右后旗召开了马铃薯无病毒原种繁育会议。参加会议的有内蒙、甘肃、宁夏、云南、江西、北京各有关单位的代表,共七十多人。会议以阶级斗争为纲,认真学习了毛主席一系列重要指示,批判了修正主义科研路线,总结交流了马铃薯无病毒原种繁育的经验。马铃薯退化是影响增产的一个重要因素。解放以     

马铃薯S病毒RT-PCR检测技术的研究

从感染PVS病毒的马铃薯病叶组织中提取出病毒的RNA,进行反转录cDNA的合成,用特异性引物进行PCR扩增,得到一条长度约为199 bp的特异性PCR扩增产物,与理论设计的外壳蛋白基因大小一致。在基因水平上为PVS的检测提供了一种快速、灵敏、简便的新方法,为PVS的防治提供了有效手段。     

马铃薯Y病毒外壳蛋白基因的克隆及序列分析

本文报道应用聚合酶链式反应(PCR)技术,在体外扩增马铃薯 Y 病毒外壳蛋白基因及其克隆和序列分析的结果。病毒 RNA 从马铃薯 Y 病毒感染的烟草叶片中提取,用合成的PCR 3引物及 AMV 逆转录酶合成了单链的 cDNA。利用 PCR 技术,经30个循玎的扩增。得到了一特异的0.8kb 片段。克隆后对此片段进行了限制性内切酶物理图谱分析,并测定了其全序列。实验结果证明,我们克隆到的是完整的马铃薯 Y 病毒的外壳蛋白基因。与国外报道的马铃薯 Y 病毒 N 株相比,其核苷酸序列及推测的氨基酸序列的同源率分别为97.8%和97%。将该基因导入马铃薯以期获得抗 Y 病毒马铃薯的工作正在进行。本文还对 PCR 技术用于扩增植物 RNA 病毒的方法以及用基因工程方法培育抗病毒作物新品种的可行性等进行了讨论。     

犬细小病毒的PCR诊断试剂盒的研制

目的　建立检测犬细小病毒的PCR诊断试剂盒。方法　通过在犬细小病毒 (CPV)的基因组中设计的特异性寡核苷酸引物 ,利用PCR技术研制犬细小病毒的PCR诊断试剂盒。结果　在特定的反应条件下 ,使用该试剂盒能特异地扩增含有犬细小病毒的样品 ,且能检出痕量 (10ng)的犬细小病毒DNA ,被测粪样只需进行简单煮沸就能进行PCR反应 ,该试剂盒能在常温下保存 1周以上、4℃保存 1年以上。同血凝试验 (HA〕及单克隆抗体ELISA方法比较 ,对 6 6份样品进行检测 ,显示该PCR试剂盒具有特异、灵敏等优点。结论　成功研制了犬细小病毒的PCR诊断试剂盒 ,利用该试剂盒能从DNA水平上对犬细小病毒进行监控     

一个侵染山东大葱的胡葱黄条病毒分离物的鉴定

在山东章丘大葱上发现了一种引起畸形黄条症状的线状病毒。该病毒容易摩擦接种侵染大葱和胡葱,但不侵染其它22种测试植物,能经由豌豆蚜传播。病毒粒子形态和细胞病理特征具有马铃薯Y病毒科成员的典型特征。Western blot分析表明,提纯病毒制备的抗血清能与病毒自身外壳蛋白起特异性的强反应,与薤花叶病毒、晚香玉轻斑驳病毒、小西葫芦黄花叶病毒和芋花叶病毒外壳蛋白有较弱的反应。采用马铃薯Y病毒科简并引物PCR技术扩增了该病毒的基因组3′-末端部分序列,序列比较表明它为胡葱黄条病毒(SYSV),系统进化树分析表明世界范围内的SYSV分离物可以区分为4个主要群体,在一定程度上与寄主植物种类及地理分布相关。     

Gene amplification and expression by RNA viruses and potential for further application to plant gene transfer

The great majority of plant viruses encapsidate messenger-sense ssRNA and have no natural DNA phase in their life cycle. Despite their RNA nature, essentially any desired change can be introduced into such genomes by using recombinant DNA techniques with suitably constructed, expressible viral cDNA clones. For some viruses such as brome mosaic virus, these methods have been used to define the sequences controlling RNA-directed genomic RNA replication and the expression of internal genes via subgenomic mRNAs. The results suggest a surprising degree of genetic flexibility, which appears to be reflected in the varied gene complements and genetic organizations of presumably related plant and animal RNA viruses sharing conserved replication genes. Foreign genes inserted in such RNA virus genomes can be amplified and expressed to a high level in transfected plant cells. In addition to the potential use of such viruses as episomal expression vectors, it should be possible to couple the viral pathways of RNA-dependent RNA synthesis to amplify and to further regulate the expression of genes transformed into plant chromosomes.     

Cryotherapy of shoot tips: a technique for pathogen eradication to produce healthy planting materials and prepare healthy plant genetic resources for cryopreservation

Cryotherapy of shoot tips is a new method for pathogen eradication based on cryopreservation techniques. Cryopreservation refers to the storage of biological samples at ultra-low temperature, usually that of liquid nitrogen (−196°C), and is considered as an ideal means for long-term storage of plant germplasm. In cryotherapy, plant pathogens such as viruses, phytoplasmas and bacteria are eradicated from shoot tips by exposing them briefly to liquid nitrogen. Uneven distribution of viruses and obligate vasculature-limited microbes in shoot tips allows elimination of the infected cells by injuring them with the cryo-treatment and regeneration of healthy shoots from the surviving pathogen-free meristematic cells. Thermotherapy followed by cryotherapy of shoot tips can be used to enhance virus eradication. Cryotherapy of shoot tips is easy to implement. It allows treatment of large numbers of samples and results in a high frequency of pathogen-free regenerants. Difficulties related to excision and regeneration of small meristems are largely circumvented. To date, severe pathogens in banana ( Musa spp.), Citrus spp., grapevine ( Vitis vinifera ), Prunus spp., raspberry ( Rubus idaeus ), potato ( Solanum tuberosum ) and sweet potato ( Ipomoea batatas ) have been eradicated using cryotherapy. These pathogens include nine viruses (banana streak virus, cucumber mosaic virus, grapevine virus A, plum pox virus, potato leaf roll virus, potato virus Y, raspberry bushy dwarf virus, sweet potato feathery mottle virus and sweet potato chlorotic stunt virus), sweet potato little leaf phytoplasma and Huanglongbing bacterium causing 'citrus greening'. Cryopreservation protocols have been developed for a wide variety of plant species, including agricultural and horticultural crops and ornamental plants, and can be used as such or adjusted for the purpose of cryotherapy.     

Comparing the plant virus spread patterns of non-persistently transmitted virus and persistently transmitted virus using an individual-based model

To compare the spread patterns between two types of plant viruses, non-persistent virus (NPV) and persistent virus (PV), we developed a spatially-explicit individual-based model. Our probability-based model is driven by the actions of insect vectors that are affected by interactions with host plants and plant viruses, considering both biological and behavioral components of their relationship. As a model system, we used potato virus y and potato leafroll virus, respectively for NPV and PV, potato for host plant, and Myzus persicae for the insect vector; empirical results from previous studies were acquired and adjusted to be used as our parameter values. Our simulation results showed that initial infection of PV in the field resulted in over 1.3 times greater number of insect vectors while causing approximately 7 times greater number of virus-infected plants compared to NPV by the end of simulation. Furthermore, spatial analysis showed that PV-infected plants showed greater aggregation in the field, forming larger patches compared to NPV-infected plants. Our results demonstrated the importance of host plant and insect vector manipulation by plant viruses as well as biological properties such as infectious period in the insect on the difference in overall spread pattern.     

Development of immunochromatographic test systems for express detection of plant viruses

Express immunochromatographic test-strip assays were developed for detection of five plant viruses varying in shape and size of virions: spherical carnation mottle virus, bean mild mosaic virus, rodshaped tobacco mosaic virus, and filamentous potato viruses X and Y. Multimembrane composites (test strips) with immobilized polyclonal antibodies against viruses and colloidal gold-conjugated antibodies were used for the analysis. The immunochromatographic test strips were shown to enable the detection of viruses both in purified preparations and in leaf extracts of infected plants with a sensitivity from 0.08 to 0.5 μg/ml for 10 min. The test strips may be used for express diagnostics of plant virus diseases in field conditions.     

The impact of local assembly rules on RNA packaging in a T = 1 satellite plant virus

The vast majority of viruses consist of a nucleic acid surrounded by a protective icosahedral protein shell called the capsid. During viral infection of a host cell, the timing and efficiency of the assembly process is important for ensuring the production of infectious new progeny virus particles. In the class of single-stranded RNA (ssRNA) viruses, the assembly of the capsid takes place in tandem with packaging of the ssRNA genome in a highly cooperative co-assembly process. In simple ssRNA viruses such as the bacteriophage MS2 and small RNA plant viruses such as STNV, this cooperative process results from multiple interactions between the protein shell and sites in the RNA genome which have been termed packaging signals. Using a stochastic assembly algorithm which includes cooperative interactions between the protein shell and packaging signals in the RNA genome, we demonstrate that highly efficient assembly of STNV capsids arises from a set of simple local rules. Altering the local assembly rules results in different nucleation scenarios with varying assembly efficiencies, which in some cases depend strongly on interactions with RNA packaging signals. Our results provide a potential simple explanation based on local assembly rules for the ability of some ssRNA viruses to spontaneously assemble around charged polymers and other non-viral RNAs in vitro.     

CHARACTERIZATION OF HaRNAV,A SINGLE‐STRANDED RNA VIRUS CAUSING LYSIS OF HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)1

HaRNAV, a novel virus that infects the toxic bloom‐forming alga Heterosigma akashiwo (Hada) Hada ex Hada et Chihara, was characterized based on morphology, pathology, nucleic acid type, structural proteins, and the range of host strains that it infects. HaRNAV is a 25‐nm single‐stranded RNA (ssRNA) virus with a genome size of approximately 9100 nucleotides. This is the first report of an ssRNA virus that causes lysis of a phytoplankton species. The virus particle is sensitive to chloroform and contains at least five structural proteins ranging in apparent size from 24 to 34 kDa. HaRNAV infection causes swelling of the endoplasmic reticulum and progeny virus particles assemble in the cytoplasm of the host, frequently in crystalline arrays. The infectivity of HaRNAV was tested against 15 strains of H. akashiwo isolated from Japanese waters, the Northeast Pacific, and the Northwest Atlantic. HaRNAV caused lysis of three strains from the Northeast Pacific and two strains from Japan but none from the Northwest Atlantic. The characterization of HaRNAV demonstrates that HaRNAV is a novel type of phytoplankton virus but has some similarities with plant viruses belonging to the Sequiviridae and to other known ssRNA viruses. Further genomic analysis, however, is necessary to determine any phylogenetic relationships. The discovery of HaRNAV emphasizes the diversity of H. akashiwo viral pathogens and, more importantly, algal–virus pathogens and the complexity of virus–host interactions in the environment.     

Isolation of Viruses by Electro-Extraction of Infected Plants

The isolation of viruses from infected plant material by a process termed electro-extraction appeared to be a convenient and simple method of obtaining viruses in a fair state of purity. The method has the advantage over the conventional methods of virus purification that the infected plant tissue is not disintegrated and that organic solvents such as chloroform and butanol are avoided. The procedure used was demonstrated on the extraction of tobacco mosaic virus (TMV) from infected tobacco and turnip yellow mosaic virus (TYMV) from Chinese cabbage plants. To obtain the virus it was found advisable to freeze and thaw the plants prior to extraction.     

Identification and study of tobacco mosaic virus movement function by complementation tests

The phenomenon of trans-complementation of cell-to-cell movement between plant positive-strand RNA viruses is discussed with an emphasis on tobamoviruses. Attention is focused on complementation between tobamoviruses (coding for a single movement protein, MP) and two groups of viruses that contain the triple block of MP genes and require four (potato virus X) or three (barley stripe mosaic virus) proteins for cell-to-cell movement. The highlights of complementation data obtained by different experimental approaches are given, including (i) double infections with movement-deficient (dependent) and helper viruses; (ii) infections with recombinant viral genomes bearing a heterologous MP gene; (iii) complementation of a movement-deficient virus in transgenic plants expressing the MP of a helper virus; and (iv) co-bombardment of plant tissues with the cDNAs of a movement-dependent virus genome and the MP gene of a helper virus.