豇豆退绿斑驳病毒(CCMV)突变体的混合侵染研究

 本研究采用PCR点突变的方法,对豇豆退绿斑驳病毒(CCMV) RNA3的cDNA克隆进行突变,突变体TP7是在外壳蛋白基因之前突变形成一个独特的Sal I酶切点;TP8是在3a基因之前突变形成一个独特的Bam H I酶切点;AG 1是在外壳蛋白基因之后突变形成一个独特的Not I酶切点。TP7、TP8、AG1体外转录产物分别与CCMV RNA1、RNA2的c DNA克隆的体外转录产物混和后接种豇豆,结果表明,接种后第6 d TP7植株首先表现出症状,9d后TP8、AG1和野生型CCMV接种的植株表现出症状,TP7的症状发展最快。
采用TP7、TP8、AG1接种20 d的毒源植株,按TP7+TP8、TP7+AG1、TP8+AG1等量混合汁液接种豇豆,接种20 d,通过RT-PCR分析接种植物,在TP7+TP8和TP7+AG1混合侵染中,TP7在混合侵染中占优势,在TP8+AG1的混合侵染中,TP8和AG1的种群十分接近。     

Defective Movement of Viruses in the Family Bromoviridae Is Differentially Complemented in Nicotiana benthamiana Expressing Tobamovirus or Dianthovirus Movement Proteins

ABSTRACT Taxonomically distinct tobacco mosaic tobamovirus (TMV), red clover necrotic mosaic dianthovirus (RCNMV), cucumber mosaic cucumovirus (CMV), brome mosaic bromovirus (BMV), and cowpea chlorotic mottle bromovirus (CCMV) exhibit differences in their host range. Each of these viruses encodes a functionally similar nonstructural movement protein (MP) that is essential for cell-to-cell movement of a progeny virus. Despite the lack of significant amino acid identity among the MPs of CMV, TMV, and RCNMV, movement-defective CMV (CMVFnyDeltaMP-DeltaKPN) was able to move locally and systemically in transgenic Nicotiana benthamiana expressing either TMV MP (NB-TMV-MP(+)) or RCNMV MP (NB-RCNMV-MP(+)). These observations contrast with those of previous studies in which transgenic N. tabacum cv. Xanthi plants expressing TMV MP supported only the cell-to-cell movement of CMVFnyDeltaMP-DeltaKPN. To verify whether similar complementation could be observed for movement-defective bromoviruses, NB-TMV-MP(+) and NB-RCNMV-MP(+) plants were inoculated independently with movement-defective variants of BMV (B3DeltaMP) and CCMV (CC3DeltaMP). Neither NB-TMV-MP(+) nor NB-RCNMV-MP(+) was able to rescue the defective cell-to-cell and long-distance movement of B3DeltaMP. In contrast, NB-RCNMV-MP(+) complemented the cell-to-cell, but not the long-distance, movement of CC3DeltaMP. Taken together, these studies suggest that virus movement is a complex process and that, in some cases, the host species plays a major role in determining the long-distance movement function of a virus.     

siRNA对烟草原生质体中TMV RNA积累的干扰作用研究(英文)

 RNA干扰被认为是转录后基因沉默的一种机制。RNA干扰通过小干扰RNA特异性降解目标mRNA来沉默基因表达。本文以烟草花叶病毒126kD蛋白为靶蛋白,在原生质体水平上研究了小干扰RNA对病毒侵染的干扰和抑制作用。ELISA和Northern杂交的实验结果表明,共转染小干扰RNA和TMV的原生质体内检测到较低的病毒含量。在枯斑寄主上,叶片接种小干扰RNA和TMV共转染原生质体后,与对照叶片相比,仅有很少量的病斑产生。这说明,小干扰RNA能够在原生质体水平对病毒起到干扰和抑制作用。因此认为,烟草原生质体系统有利于快速和定量分析小干扰RNA介导对植物病毒的抑制作用。     

A Variant of Cucumber mosaic virus Is Restricted to Local Lesions in Inoculated Tobacco Leaves with a Hypersensitive Response

A variant of Cucumber mosaic virus, CMV(Y/GM2), was isolated from a tobacco plant with mild green mosaic symptoms that was regenerated in vitro from a yellow strain of CMV [CMV(Y)]-infected tobacco leaves by tissue culture. CMV(Y/GM2) has two amino acid substitutions at 36 and 111 positions in the coat protein encoded on RNA3. CMV, assembled by mixing in vitro transcribed CMV(Y) RNA1 and RNA2 plus infectious RNA3 transcribed in vitro from cDNA to RNA3 of CMV(Y/GM2), was prepared and designated as CMV(Y/GM2)tr. When tobacco (Nicotiana tabacum cv. Xanthi nc) plants were inoculated with CMV(Y/GM2)tr, large necrotic local lesions in which the virus was localized, developed on the inoculated leaves. This host response unique to CMV(Y/GM2)tr was similar to the hypersensitive response (HR), which is a common resistance response to avirulent pathogens and was observed in five cultivars of Nicotiana tabacum and eight Nicotiana species. The revertant virus, however, accumulated to quite different levels in the various hosts. CMV(Y/GM2)tr induced pathogenesis-related 1 (PR-1) protein accumulation and systemic acquired resistance (SAR) which were generally observed in the HR. However, when tobaccos were inoculated with CMV(S36P)tr and CMV(V111I)tr, which have an amino acid substitution at either the 36 or 111 position in the coat protein of CMV(Y), respectively, CMV(S36P)tr was restricted to the primary infection site without necrotic local lesion formation and PR-1 protein and SAR induction. CMV(V111I)tr, however, systemically spread and induced mild green mosaic symptoms, while the host had the HR to CMV(Y/GM2)tr. The localization of CMV(Y/GM2)tr at the primary infection site may not only be caused by the HR, but also by the restriction of virus systemic movement resulting from the amino acid substitution at position 36 in the coat protein of CMV(Y). Received 15 December 1999/ Accepted in revised form 18 April 2000     

稻瘟菌双分病毒MoPV2特性研究

稻瘟病是稻瘟菌引起的一种世界性病害,每年造成水稻大幅减产。真菌病毒是指能够在真菌体内进行复制和繁殖的病毒,可以作为生物防治的资源。本实验从发病的水稻叶片上采用单孢分离的方法获得纯化培养的稻瘟菌菌株YC13。菌株YC13携带多条dsRNA片段,其中片段F1与Magnaporthe oryzae virus 2(MoV2)的Coat Protein(CP)和RNA-dependent RNA polymerase(RdRp)的氨基酸序列均具有99%的同源性,确定为MoV2的基因组;片段F4、F5序列与双分病毒Penicillium stoloniferum virus F(Ps V-F)的RdRp和CP的氨基酸序列分别具有69%和63%的同源性,命名为Magnaporthe oryzae partitivirus virus 2(MoPV2)。MoPV2的基因组含有2条dsRNA:dsRNA 1编码RdRp,dsRNA 2编码CP。根据MoPV2的RdRp编码的氨基酸序列将MoPV2归属于双分病毒科(Partitiviridae)G ammapartitivirus属,是一种新的真菌病毒。通过病毒粒子转染,将MoPV2转到稻瘟菌菌株RB11。与菌株RB11相比,带毒菌株RB11T25的分生孢子产生量显著减少,并且对大麦活体的致病性也有所降低,但其他生物学特征均无明显的影响。本研究在稻瘟菌中发现了一种新的真菌病毒,该病毒能够使稻瘟菌致病力下降,为稻瘟病的防治提供了潜在的病毒资源。     

Interference between potyviruses during aphid transmission

Single aphids ( Myzus persicae ) were allowed sequential access to two leaves which were either free from infection or infected with potato virus Y° (PVY°). PVY^N. beet mosaic virus (BMV), or tobacco mosaic virus (TMV). Aphids given prior or subsequent access to PVY^N-infected leaves transmitted PVY° and BMV less frequently than aphids given prior or subsequent access to virus-free leaves; those given prior or subsequent access to BMV also transmitted PVY° less frequently. However, prior or subsequent access to PVY° did not decrease transmission of either BMV or PVY^N, and access to BMV did not decrease transmission of PVY^N. Access to the non-aphid-transmissible TMV did not affect transmission of either PVY° or PVY^N. PVY^N had the greatest electrophoretic mobility. BMV was least mobile and PVY° was intermediate, so ability to decrease transmission was not directly related to the total anionic charge on the virus particles.     

植物病毒病选择性治疗药剂的筛选研究

 超氧自由基(O₂^·-)清除剂甘露醇和抗坏血酸在离体条件下对稻瘟菌丝生长有一定抑制作用,但它们施用到水稻上后却会加重稻瘟病的发生。O₂^·-清除剂对烯丙异表现拮抗作用。经烯丙异处理的水稻幼苗接种稻瘟菌后能刺激O₂^·-的产生。外源H₂O₂和超氧自由基对稻瘟病菌和水稻细胞有直接毒害。本研究表明:烯丙异刺激水稻体内产生的O₂^·-在药剂诱导水稻对稻瘟病抗性反应中发挥作用。     

High resistance to rice yellow mottle virus in two cultivated rice cultivars is correlated with failure of cell to cell movement

Rice yellow mottle virus (RYMV) accumulation in protoplasts and whole plants was investigated in two highly resistant cultivars, Tog5681 (Oryza glaberrima) and Gigante (Oryza sativa). Three susceptible cultivars, i.e. one O. glaberrima Tog5673 and two O. sativa (IR64, Ac. 2428), and a partially resistant cultivar (Azucena) were used as control. After inoculation, accumulation of coat protein (CP) and viral RNA were monitored on protoplasts, inoculated leaves, sheaths of inoculated leaves and newly infected leaves by serological and Northern blot analysis. Viral RNA accumulated to a similar extent in protoplasts from all cultivars studied. In contrast, three distinct in planta behaviors were noted. In susceptible plants (IR64, Tog5673 and Ac. 2428), there was high CP and RNA accumulation at 5 d.p.i. in whole plants, suggesting that cell to cell and vascular movements occurred before 5 d.p.i. in inoculated leaves. The second behavior concerned Azucena, which showed a delay (around 7 d.p.i.) of viral accumulation in inoculated leaves. The third behavior involved the highly resistant cultivars Tog5681 and Gigante. CP and viral RNA were not detected in these cultivars. The comparison of viral accumulation in protoplasts and plants suggested that resistance of the highly resistant cultivars Tog5681 (O. glaberrima) and Gigante (O. sativa) was not due to the inhibition of virus replication but rather to the failure of cell to cell movement.     

A novel member of the genus Nepovirus isolated from Cucumis melo in Japan

An unusual virus was isolated from a Japanese Cucumis melo cv. Prince melon plant showing mild mottling of the leaves. The virus had a broad experimental host range including at least 19 plant species in five families, with most infected plants showing no symptoms on inoculated and uninoculated systemically infected leaves. The virus particles were spherical, approximately 28 nm in diameter, and the coat protein (CP) had an apparent molecular mass of about 55 kDa. The virus possessed a bi-partite genome with two RNA species, of approximately 8,000 and 4,000 nucleotides. Both genome components for the new virus were sequenced. Amino acid sequence identities in CP between the new virus and previously characterized nepoviruses were found to be low (less than 27%); however, in phylogenetic reconstructions the closest relationship was revealed between the new virus and subgroup A nepoviruses. These results suggest that the new virus represents a novel member of the genus Nepovirus. A new name, Melon mild mottle virus, has been proposed for this new virus.     

Characterization of an isolate of beet mosaic virus from South Kazakhstan

A filamentous virus isolated from a sugar-beet plant showing systemic mosaic collected in South Kazakhstan was identified as an isolate of beet mosaic virus (BMV-K). BMV-K was transmitted by the green peach aphid Myzus persicae in a non-persistent manner, and by sap inoculation to 11 out of 19 species from seven families tested. The virus could not be transmitted to Nicotiana tabacum, N. debneyi, N. glutinosa and N. clevelandii, cither mechanically or with M. persicae. The thermal inactivation point of BMV-K in sugar-beet sap was 55-60 C, dilution end point 1:1000 and longevity in vitro 2 days at 20 C. A purification procedure produced 1-5-3 mg of purified virus from 100 g of infected Stellaria media plants. Purified virus contained a single protein species of molecular weight 34 700 Da. In ELISA tests, BMV-K reacted positively with BMV-specifc antisera obtained from Japan. Germany and Portugal. By competitive DAS- ELISA, the virus isolate was shown to be closely serologically related to all the three isolates of BMV, and very distantly related to bean yellow mosaic and soy bean mosaic viruses.     

黄瓜花叶病毒外壳蛋白质进入叶绿体与症状发生的关系

 由感染黄瓜花叶病毒(CMV)、健康和转外壳蛋白(CP)基因的烟叶以原生质体法制备纯化的叶绿体。经SDS-PAGE电泳,银染色,比较其蛋白质图谱。用CMV外壳蛋白游离亚基制备的抗血清进行Western blotting。结果发现:1.CP存在于CMV侵染的烟叶绿体中。2.叶绿体中CP的浓度和花叶症状严重程度呈正相关;3.表达CP的转基因烟草叶绿体中未测出CP存在。根据以上结果,认为CMV侵染的烟花叶症状产生与CP进入叶绿体有直接相关性。     

The coat protein of <Emphasis Type="Italic">Tomato mosaic virus</Emphasis> L<Subscript>11</Subscript>Y is associated with virus-induced chlorosis on infected tobacco plants

The L₁₁Y strain of Tomato mosaic virus (ToMV) causes severe chlorosis on infected tobacco leaves. Sequencing analysis for the genome showed that L₁₁Y contained multiple nucleotide changes and that some led to amino acid substitutions, when compared with that of the common L strain of ToMV. The chimeric virus, which has the CP of L₁₁Y in the context of the L strain RNA genome, caused severe chlorosis on infected tobacco plants, suggesting that the CP of L₁₁Y containing three amino acid changes (E33S, A86T and E97K) was the determinant of the chlorosis. Two of these amino acid changes (A86T and E97K) were associated with the induction of chlorosis when present together in the CP. Severe destruction and deformation of chloroplasts and the formation of discrete dark-staining materials adjacent to chloroplasts were observed with electron microscopy in L₁₁Y-infected plants. Fewer virus particles accumulated in the cytoplasm of L₁₁Y-infected plant cells. The level of accumulation of CP subgenomic RNA and CP in the infected protoplasts was similar between L and L₁₁Y. Fewer virus particles accumulated in L₁₁Y-infected protoplasts, and many of them were shorter-than-full-length. The nucleotide sequence data reported is available in DDBJ/EMBL/GenBank databases as accession AB355139.     

小麦黄花叶病毒Nib蛋白介导的体外转录体系的创建

 本研究是利用异源表达的小麦黄花叶病毒(Wheat yellow mosaic virus, WYMV)复制酶Nib蛋白创建了病毒核酸的体外复制系统。将WYMV的Nib编码序列扩增并插入到大肠杆菌表达载体pMAL-C2X中以构建原核表达质粒pMAL-WY-Nib。0.4 mmol·L^-1 IPTG诱导可特异性表达分子量约为100 kDa的MBP-Nib融合蛋白。根据温度梯度测定,20℃下诱导MBP-Nib的可溶性比例较高,约为30％,可满足MBP标签的亲和层析。通过与WYMV的其他蛋白和烟草丛顶病毒的RdRp进行比较,纯化的MBP-Nib融合蛋白可以特异性识别WYMV RNA1和RNA2的3′末端区域并体外合成其互补链,此体外转录体系可用于WYMV复制调控的研究。     

烟草花叶病毒丁香分离物的分离与鉴定

 从表现花叶症状的丁香病株上获得一病毒分离物,其在电镜下为约300 nm×18nm的杆状粒子;电泳分析表明感病组织中ds RNA大约为6.4kbp,而其外壳蛋白分子量约为17.6k Da。以上实验结果初步将该病毒分离物鉴定为烟草花叶病毒属(Tobamovirus)。根据该属病毒复制酶基因序列设计通用引物,进行RT-PCR检测,扩增出约1000 bp的预期特异片段(Gen Bank AY566703)。将PCR产物克隆后测序,序列分析表明,与从蚕豆中分离的TMV-B株系序列(Gen Bank AJ011933.1)同源性为99.90%。根据烟草花叶病毒(Tobacco mosaic virus,TMV)的RNA CP基因序列设计引物,进行RT-PCR,扩增出约800 bp的预期特异片段(Gen Bank AY56672),序列分析表明,与TMV-B株系序列(Gen Bank AJ011933.1)同源性达99%,上述实验结果表明,该病毒分离物为TMV。由于该分离物与TMV-B在指示植物上的症状存在明显差异,所以,作者把该分离物暂命名为TMV-S。     

黄瓜绿斑驳花叶病毒低风险参照物质的研制

采用RT-PCR方法扩增黄瓜绿斑驳花叶病毒的外壳蛋白基因与3,非编码区,并将其构建到马铃薯X病毒(PVX)载体中.重组质粒经线性化及体外转录后接种烟草,获得含有病毒外壳蛋白基因的感病植株.感病组织可用于分子检测的质控,也可作为毒源来繁殖阳性参照物质.基于PVX载体制备的参照物质可降低检疫性病毒的生物安全风险,尤其对稳定性强、可造成严重经济损失的高风险病毒的检疫更具实用价值.