山东烟台小麦土传病毒病由小麦黄花叶病毒和土传小麦花叶病毒相关病毒复合侵染所致

在山东省烟台地区的小麦上发生一种由土壤中禾谷多粘菌Polymyxa graminis传播的病毒病,感病小麦植株表现矮化褪绿和花叶症状.我们于1997年4月从病区采集感病小麦植株,进行了病毒种类鉴定.直接电镜观察发现有二种病毒粒子,一种粒子呈棒状,占大多数,其长度约为300nm和150nm; 另一种粒子呈线状,数量较少,长度为500nm～700nm.免疫电镜结果表明,棒状病毒粒子仅与土传小麦花叶病毒(soil-borne wheat mosaic virus, SBWMV)抗血清反应,而不与小麦黄花叶病毒(wheat yellow mosaic virus,WYMV)抗血清和小麦梭条斑花叶病毒(wheat spindle streat mosaic virus,WSSMV)抗血清反应;反之,线状病毒仅与WYMV、WSSMV抗血清反应,而不与SBWMV抗血清反应.用WYMV和SBWMV两种抗血清同时进行修饰时,线状病毒粒子和棒状病毒粒子均发生反应.     

A Point Mutation in the Coat Protein Gene Affects Long-Distance Transport of the Tobacco Mosaic Virus

A mutation resulting in substitution of positively charged Lys53 with negatively charged Glu in the coat protein was introduced in the infectious cDNA copy of the genome of wild-type tobacco mosaic virus strain U1. Kinetic analysis of long-distance virus transport in plants showed that systemic spread of the mutant virus was delayed by 5–6 days as compared with the wild-type one. On evidence of RNA sequencing in the mutant progeny, Glu50 of the coat protein was substituted with Lys after passage I to compensate for the loss of the positive charge at position 53. Electron microscopy revealed atypical inclusions (rodlike structures, multiple electron-dense globular particles) in the nuclear interchromatin space of leaf mesophyll cells infected with the mutant but not with the wild-type virus.     

番茄花叶病毒单克隆抗体的制备及检测应用

用番茄花叶病毒(ToMV)免疫的BAL B/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,经筛选克隆,获得4株能稳定传代并分泌抗ToMV单克隆抗体(Mab)的杂交瘤细胞,其中2株能同时检测ToMV和烟草花叶病毒(TMV),各单克隆抗体腹水ELLSA效价在1∶32 000～1∶1 024 000之间。经TASELISA测定,4株单克隆抗体检测病汁液的稀释度均能达到1∶2 000倍以上。4株单克隆抗体与其他病毒无交叉反应。Westernblot分析表明,其中两株与ToMV176kD的外壳蛋白亚基有特异反应,而另两株无反应,推测它们是针对构象决定簇的抗体。     

黄瓜绿斑驳花叶病毒辽宁分离物全基因组序列测定

以感病组织总RNA为模板,采用RT-PCR方法扩增并测定黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)辽宁分离物(CGMMV-LN)的基因组全序列。CGMMV-LN基因组全长6 422 nt,5'非编码区(noncoding region,NCR)和3'NCR分别为59 nt和175 nt。CGMMV-LN编码的4个蛋白依次是186 kD和129kD的复制酶,29 kD的移动蛋白和17.4 kD的外壳蛋白。CGMMV-LN与其他4个CGMMV分离物基因组核苷酸序列同源性为97.6%～99.3%,与同属其他3种病毒基因组核苷酸序列同源性仅为61.7%～62.8%。基于186kD复制酶和外壳蛋白氨基酸序列的同源树显示:侵染葫芦科作物的烟草花叶病毒属病毒可分为2个亚组,亚组I包括所有CGMMV分离物,亚组II包括Kyuri绿斑驳花叶病毒(Kyuri green mottle mosaic virus,KGMMV)、黄瓜果实斑驳花叶病毒(Cucumber fruit mottle mosaic virus,CFMMV)和小西葫芦绿斑驳花叶病毒(Zucchini ...     

西瓜花叶病毒中国分离株全基因组核苷酸序列测定

西瓜花叶病毒(Watermelon mosaic virus,WMV)是马铃薯Y病毒属(Potyvirus)成员,主要危害西瓜和甜瓜,引起花叶病。在田间,该病害主要由蚜虫以非持久性方式传播。西瓜和甜瓜花叶病在国内陕西、山东、云南、辽宁、山西、新疆、河南和黑龙江等地广泛发生[1-6]。从20世纪80年代中期开始发生,逐渐上升为普遍发生的主要病害。我国大部分地区因西瓜和甜瓜病毒病造成的损失为30%~50%,甚至会绝产,西瓜花叶病毒已经成为制约西瓜和甜瓜高产稳产最主要的因素之一[7]。到目前为止,多数工作集中在对西瓜和甜瓜病毒病的鉴定,在分子生物学上仅限于对CP基因…     

Structural comparisons of the aggregates of tobacco mosaic virus protein

The coat protein of tobacco mosaic virus forms numerous aggregates, including the small A-protein, the disk, and two helical forms. The structures of the disk, the helical protein forms, and the virus are compared. Most of the differences are in the conformation of the chain between residues 89 and 113, which lies in the region of protein at the center of the virus, inside the RNA. It is disordered in the disk, but has a fixed conformation in the virus and the protein helices. The differences between the virus and the two helical protein forms are largely in the conformations of arginines and carboxylic acids in this region.     

Translation of the RNAs of brome mosaic virus: The monocistronic nature of RNA1 and RNA2

Each of the two largest brome mosaic virus RNAs, RNA1 and RNA2, directs the synthesis of a large protein in cell-free extracts derived from wheat embryo. The size of each protein represents the translation of almost the entire length of the corresponding RNA. It was shown previously that brome mosaic virus RNA4 directs the synthesis of the coat protein and that brome mosaic virus RNA3, although it also contains the coat protein cistron, is translated mostly into a single product unrelated to the coat protein (Shih & Kaesberg, 1973). Thus, the brome mosaic virus genome encodes a total of four proteins.     

应用免疫胶体金技术定位感病大麦细胞中的大麦和性花叶病毒

本文报道免疫胶体金标记技术的建立,并用此技术定位大麦叶和根组织超薄切片中大麦和性花叶病毒(BaMMV)。在感染病毒的大麦叶和根细胞中,病毒束、游离病毒颗粒和病毒外壳蛋白多分布于细胞质丰富的细胞中,且以液泡和叶绿体(仅叶组织)周围较多。在细胞器已解体的病根表皮细胞中,有时也可检测到大量游离病毒粒子。少数风轮体或板状集结体上也存在病毒或病毒外壳蛋白。细胞核、叶绿体、线粒体、细胞膜以及其他细胞器上都未见有特异性金颗粒标记。     

Biological and Molecular Characterization of Taiwanese Isolates of Cucumber mosaic virus Associated with Banana Mosaic Disease

Banana mosaic disease (BMD) caused by Cucumber mosaic virus (CMV) has become an important threat to the banana industry. We collected and characterized 10 CMV isolates associated with BMD in Taiwan and compared their biological characteristics and coat protein sequences. The isolates fell into four pathotypes on the basis of the symptoms they induce on banana, Nicotiana glutinosa and Vigna unguiculata (cowpea). Double-stranded RNA analysis revealed that the different pathotypes are not related to the presence of CMV satellite RNA. Phylogenetic analysis of worldwide CMV coat protein sequences revealed that among the currently known CMV subgroups IA, IB and II, subgroup IB is phylogenetically unresolved. Our CMV isolates form a new subgroup, IT, within subgroup I. In addition, we resolved another new CMV subgroup, IS, within subgroup I. The analysis also revealed that isolates within different subgroups can infect the banana.     

Temperature dependence of the structure of aggregates of tobacco mosaic virus protein at pH 7.2. Static synchrotron small-angle X-ray scattering

The small-angle X-ray scattering (SAXS) method using a synchrotron radiation source was applied to the study of the self-aggregation process of tobacco mosaic virus protein (TMVP) at a concentration of 5.0 or 12.0 mg ml-1 in 50 mM or 100 mM-phosphate buffer (ionic strengths approx. 0.1 and 0.2, respectively) at pH 7.2 in the temperature region of 4.8 to 25.0 degrees C. This paper presents the results of static measurements of SAXS. Sedimentation velocity experiments were performed simultaneously under the same conditions. These results are qualitatively parallel to those of the SAXS measurements, although the size of stacked disks derived from the SAXS measurements is larger than that derived from the sedimentation experiments, suggesting a change in the equilibrium conditions in the centrifugal field. Qualitative analysis of the SAXS data with model simulation calculations implies that the aggregation of TMVP consists of two steps: (1) the aggregation of A-protein comprising a few subunits to form double-layered disks; and (2) the random polymerization of double-layered disks by disk-stacking. Increase in temperature, ionic strength or protein concentration induced TMVP to polymerize to form a double-layered disk or a quadruple-layered short rod with consumption of A-proteins, accompanied by a small number of multi-layered short rods. The SAXS results indicate that the A-protein and the multilayered short rods are polydisperse with respect to size and shape, i.e. the mixture of A-protein, double-layered disks and multi-layered short rods coexists in the equilibrium state without pressure-induced partial dissociation of TMPV as observed during normal ultracentrifugation, and even under solution conditions in which the formation of double-layered disks or higher-order aggregates is favored.     

外壳蛋白羧端序列缺失对烟草花叶病毒侵染性的影响

对野生型烟草花叶病毒(TMV-U1)的外壳蛋白羧端序列进行系列缺失突变,观察到TMV-U1株系的外壳蛋白羧端序列缺失6个氨基酸(保留152个氨基酸),仍能较强系统侵染烟草并高水平表达外壳蛋白,且能在新生叶里复制大量完整的病毒粒子。该研究结果表明：外壳蛋白羧端6个氨基酸序列并非烟草花叶病毒感染和复制所必需,并对利用外壳蛋白羧端缺失型病毒载体表达外源多肽具有一定的启示性。     

Coat Protein-mediated Resistance to Pea Enation Mosaic Virus in Transgenic Pisum Sativum L.

Pea (Pisum sativum L.) plants were transformed in planta by injection/electroporation of axillary meristems with a chimeric pea enation mosaic virus (PEMV) coat protein gene contruct. R1 progenies of these plants were shown to harbor the transgene by polymerase chain reaction (PCR) and genomic Southern analysis, while transgene expression was demonstrated by western blot analysis. Transgenic R2, R3 and R4 plants displayed delayed or transient PEMV multiplication and attenuated symptoms as compated to control inoculated individuals.     

Macroscopic Aggregation of Tobacco Mosaic Virus Coat Protein

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry melting curves, TMV CP in the form of a trimer–pentamer mixture (4S-protein) has very low thermal stability, with a transition temperature at about 40°C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial -helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.     

Desiccation Alters the Stability and Distribution of Pea Seed-borne Mosaic Virus in Pea (Pisum sativum) Cotyledon Cells

As a seed transmitted pathogen, pea seed-borne mosaic vires (PSbMV) not only replicates in embryonic cells but can also withstand seed desiccation. To understand the mechanism of PSbMV tolerance to seed desiccation, the authors compared the stability of viral coat protein (CP) and the distribution of viral particles in the cotyledon cells of pea ( Pisum sativum L. ) embryos collected before and after the dehydration process. Before dehydration, when the embryo was fresh and immature, degradation of CP was observed and a predominantly perinuclear distribution of viral particles in the cotyledon cells was evident. After dehydration, when the embryo was dry and mature, degradation of CP did not occur and the perinuclear viral distribution disappeared. Instead, aggregates containing PSbMV CP were found in the cytoplasm. Electron microscopy showed that these aggregates were composed of PSbMV particles. The formation of PSbMV particle aggregates is apparently triggered by seed dehydration and may be favorable to the virus survival in the desiccated embryonic cells.     

紫藤脉花叶病毒cp基因在大肠杆菌中的表达及抗血清的制备

采用RT-PCR方法自紫藤脉花叶病毒北京分离物(WVMV-BJ)的基因组中分离出其CP基因,连接到原核表达载体pET22b( )上。获得的重组子pET-WVMVCP转化大肠杆菌BL21(DE3)后,用IPTG进行诱导表达。SDS-PAGE和Western blot分析表明,cp基因在大肠杆菌中获得了高效表达,融合蛋白分子量约为34．4kDa。将融合蛋白纯化后免疫兔子,获得了特异性较高的抗血清。微量免疫沉淀法测定该抗血清的效价为1／1024,酶联法(enzyme-linked immunosorbant assay,ELISA)测定的效价为1／8192。     

Ultrastructural Alteration of Host Plants Infected with Tomato Mosaic Virus

The ultrastructural aheration of two host plants infected with tomato mosaic virus (ToMV) were studies with transmission electron microscopy. A large number of virus particles were found being accumulated in different cells such as epidermis, parenchyma cells and vascular bundle cells of Lycopersicon esculentum Mill. grown at 25℃ Crystalline inclusions and paracrystal inclusions composed of ToMV particles were observed in the cytoplasm or vacuoles. Some muhivesicular bodies and myeloid bodies protming into the vacuole and vires-specific vesicles associated with the tonoplast were also observed. The ultrastructuml alteration of Nicotiana tabacum L. tv. Xanthinn was similar to that in tomato infected by ToMV grown at 25 cE. In addition to the aggregate inclusions described above, some cytoplasmic angularly-layered aggregates and abnormal chloroplasts with small peripheral vesicles were observed in the parenchyma cells. The densely stained amorphous material was seen in the cytoplasm of N. tabacum L. cv. Xanthiun grown at 35℃. No X- body was observed in the cytoplasm of the ToMV infected tomato and tobacco grown at 25℃ or 35℃. The authors' results suggest a significant difference between the cytopathological effects of ToMV and tobacco mosaic virus (TMV). These characteristic difference may be useful in the virus diagnosis and identification virus infections in plants.     

芜菁花叶病毒(TuMV)特性的研究进展

芜菁花叶病毒(TuMV)是世界范围内广泛分布的重要病毒。从生物学和理化特性、基因组结构、侵染和繁殖、变异及利用转基因技术提高病毒抗性等方面对TuMV的国内外研究现状进行了阐述。     

杭州地区发生的玉米花叶病由甘蔗花叶病毒引起

从杭州地区呈现玉米矮花叶典型症状的玉米病组织中提纯得到大量线状病毒粒子,大多数长度为750?nm。病组织中含有大量风轮状内含体和板状集结体。病毒外壳蛋白为33.6 kD。病毒RNA13’端序列(1.8 kb)与甘蔗花叶病毒(SCMV)同源性最高,达71.5%～99.1%,与高梁花叶病毒(SrMV)同源性次之,为67.8%～68.5%,与玉米矮花叶病毒(MDMV)同源性最低,仅为38.4%～48.4%,从而初步认为此病害由SCMV引起。根据已发表的SCMV外壳蛋白氨基酸序列作亲缘性分析,表明SCMV可分为美国、南非、澳大利亚;德国和中国三大类。