雀麦花叶病毒缺陷型RNA_（3b）的研究

采用ＰＥＧ沉淀和差速离心的方法提纯雀麦花叶病毒的Ｇ和Ｔ分离物。利用蛋白酶Ｋ和两相酚法制备雀麦花叶病毒的总ＲＮＡｓ。将Ｇ和Ｔ分离物ＲＮＡｓ进行琼脂糖凝胶电泳,结果发现Ｂｒ－ＭＶ－Ｇ除含有正常的ＲＮＡ组分外,还出现了另一新的ＲＮＡ＿（３ｂ）组分,其分子量为０．５０×１０￣６。ＲＮＡ＿（３ｂ）只出现在大麦寄主中,而在昆诺基上缺失。ＲＮＡ＿（３ｂ）仅依靠于其来源的Ｇ分离物的ＲＮＡｓ进行复制。以ＲＮＡ＿（３ｂ）为模板合成￣（３２）Ｐ－ｃＤＮＡ探针,和ＢｒＭＶ－Ｇ分离物的ＲＮＡｓ进行分子杂交试验表明：ＲＮＡ＿（３ｂ）属ＲＮＡ＿３的缺陷型组分,它依赖于ＢｒＭＶ－Ｇ－ＲＮＡ＿３的帮助才能在大麦寄主中复制。ＲＮＡ＿（３ｂ）的出现和缺失对ＢｒＭＶ的症状表现没有影响。     

大麦黄花叶病毒(BaYMV)的提纯和生化性质的研究

大麦黄花叶病是由禾谷多粘菌(polymyxa graminis)传布的一类麦类病毒病。是上海市效区及长江中下游及沿海大麦产区的重要病害。大麦黄花叶病毒(BaYMV)在国内外虽已有不少研究,但对国内分离株的生化性质尚未有详细报道。本文报道以上海郊区的BaYMV为研究材料,成功地建立了一套分离提纯的方法,并在此基础上,研究了该病毒的结构蛋白和核酸性质,并和国外的一些研究结果进行了比较,从而为今后应用生物技术防治大麦黄花叶病毒打下了基础。     

用DNA杂交技术测定烟草花叶病毒与长叶车前花叶病毒的关系

从油菜上分离的属于烟草花叶病毒组的油菜花叶病毒15(YMV15)和从大白菜上分离到的白菜花叶病毒(ccMV)的外壳蛋白中,都含有组氨酸和甲硫氨酸,类似于长叶车前花叶病毒(RMV)。用YMV15一、RMV一和番茄花叶病毒(ToMV)突变体Nc广RNA的cDNA与Ymv_RNA、RMV—RNA和ccMV．RNA进行了同源和异源的分子杂交试验。 从分子杂交的R0t曲线和杂交产物的Tm值看,YMV-,和ccMV都不同于RMV,其饱和杂交率表明,ccMV与YMV,,有一定核苷酸序列的同源性,两者都与RMV没有核苷酸序列同源性,说明它们属于烟花叶病毒组的不同亚组。     

利用RT_PCR、DNA序列分析及原核基因表达对我国大豆花叶病毒进行分子鉴定(英文)

大豆花叶病毒（ＳＭＶ） 在大豆（ Ｇｌｙｃｉｎｅ ｍａｘ Ｌ．） 上引起严重病害。利用ＲＴ＿ＰＣＲ 扩增并克隆了ＳＭＶ＿ＺＫ（ 一个中国ＳＭＶ 分离株） 基因组中全部蛋白质编码区的ｃＤＮＡ。通过对ＨＣ＿ＰＲＯ、ＮＩｂ 和ＣＰ编码区进行序列测定与分析,发现ＳＭＶ＿ＺＫ 与ＳＭＶ＿Ｇ２ 高度同源,从而在分子水平上证明在我国大豆作物中存在ＳＭＶ＿Ｇ２ 类似株系。将ＳＭＶ＿ＺＫｃＤＮＡ克隆于细菌表达载体,获得并提纯了６ 种ｃＤＮＡ 的表达产物。这项工作将为进一步研究ＳＭＶ 基因组的功能奠定基础。     

小麦黄花叶病毒和小麦梭条斑花叶病毒的生物学和分子生物学研究

线状土传小麦花叶病毒病在欧洲、亚洲和北美洲等地均有发生.这类病毒由根肿菌纲的禾谷多粘菌(Polymyxa graminis)传播,并归类于马铃薯Y病毒科(Potyviridae)的大麦黄花叶病毒属(Bymovirus).     

芜菁花叶病毒外壳蛋白在寄主植物叶绿体中的积累及其对光系统ll活性的影响

分别以接种感染芜菁花叶病毒(TuMV)和健康对照的青菜苏州青品种(Brassica chinens L．cv．Suzhou)和芥菜温州芥菜品种(B,jucea L．cv．Wenzhou)叶片为材料提取完整叶绿体,用胰蛋白酶消除其表面蛋白后,抽提总蛋白,经SDS—PAGE电泳和Western blot检测,发现TuMV的外壳蛋白(CP)存在于感病寄主的叶绿体中。免疫金标记电镜实验显示TuMV—CP定位在感病青菜和芥菜的细胞质和叶绿体中。对两种寄主植物叶片的叶绿素荧光动力学参数测定结果显示,青菜、芥菜的Fv／Fo、Fv／Fm、φpsⅡ、qp值都有不同程度的降低,qn值增大。实验结果表明TuMV侵染后在寄主细胞叶绿体中积累的CP,抑制了光系统Ⅱ(PS Ⅱ)的光化学活性,这可能是影响寄主植物光合作用的一个重要原因。     

南方菜豆花叶病毒外壳蛋白基因序列的分析

用重组DNA技术及序列分析法测定了南方菜豆花叶病毒RNA基因组3′端1,000个碱基的序列,以及由此序列推导出的整个外壳蛋白的氨基酸顺序,它与巳报导的基本上一致。介绍了用DNA的寡核苷酸水解混合物作为起始引物,以3′端不含PolyA尾巴且不能加上PolyA的病毒RNA作为模板合成互补DNA,及进一步无性繁殖此cDNA的方法。     

印度木薯花叶双生病毒外壳蛋白的研究Ⅱ．计算机分析外壳蛋白的一些特性

用NWGCG软件系统分析了印度木薯花叶双生病毒外壳蛋白的一些性质：(1)外壳蛋白是碱性蛋白,其等电点为10．91;(2)在外壳蛋白氨基酸序列中分布有α—helcies,β—Sheets及turns等二级结构;(3)分析了外壳蛋白中可能的表面氨基酸区域,亲水性和抗原决定簇区域;(4)在外壳蛋白的氨基酸序列中存在两个糖基化位点HNT,同时分析了双生病毒外壳蛋白氨基酸遗传密码的利用频率。     

芜菁花叶病毒外壳蛋白在寄主植物叶绿体中的积累及其对光系统II活性的影响

分别以接种感染芜菁花叶病毒(TuMV)和健康对照的青菜苏州青品种(Brassica chinensis L. cv.Suzhou)和芥菜温州芥菜品种(B.juncea L. cv.Wenzhou)叶片为材料提取完整叶绿体,用胰蛋白酶消除其表面蛋白后,抽提总蛋白,经SDS-PAGE电泳和Western blot检测,发现TuMV的外壳蛋白(CP)存在于感病寄主的叶绿体中。免疫金标记电镜实验显示TuMV-CP定位在感病青菜和芥菜的细胞质和叶绿体中。对两种寄主植物叶片的叶绿素荧光动力学参数测定结果显示,青菜、芥菜的Fv/Fo、Fv/Fm、φPSII、qp值都有不同程度的降低,qN值增大。实验结果表明TuMV侵染后在寄主细胞叶绿体中积累的CP,抑制了光系统II(PS II)的光化学活性,这可能是影响寄主植物光合作用的一个重要原因。     

Molecular Variability and Distribution of Cassava Mosaic Begomoviruses in Nigeria

Several begomovirus species and strains causing Cassava mosaic disease (CMD) have been reported from cassava in Africa. In Nigeria, African cassava mosaic virus (ACMV) was the predominant virus in this important crop, and East African cassava mosaic virus (EACMV), first reported from eastern Nigeria in 1999, was also found occasionally. A survey was conducted in 2002 to resolve the diversity of the virus types present in cassava in Nigeria and to further understand the increasing complexity of the viruses contributing to CMD. A total of 234 leaf samples from cassava with conspicuous CMD symptoms were collected in farmers’ fields across different agroecological zones of Nigeria and subjected to polymerase chain reaction (PCR) with type‐specific primers. In addition and, to provide a full characterization of the viruses present, DNA‐A genome components of several viruses and informative genome fragments were sequenced. In Nigeria, ACMV proved to be the dominant virus with 80% of all samples being positive for ACMV. The East African cassava mosaic Cameroon virus (EACMCV) prevalent in Cameroon and Ivory Coast was detected in single infections (2%) and in mixed infections (18%) with ACMV. There was no indication for other virus strains of EACMV present in the country. The EACMCV samples collected showed a high nucleotide sequence identity >98% and resembled the described sequence of a Cameroon isolate (EACMCV‐CM) more than an Ivory Coast isolate, EACMCV‐CM[CI]. Evidence is provided that the EACMCV has reached epidemiological significance in Nigeria.     

Field Studies of Cross Protection with Cassava Mosaic Geminiviruses in Uganda

The effect of cassava mosaic virus disease (CMD) was compared on plants grown from cuttings that were initially virus‐free at planting and those infected with a mild strain of East African cassava mosaic virus‐Uganda (EACMV‐UG). All initially healthy plants developed CMD symptoms within 5 months of planting (MAP) at both trial sites in Uganda, although spread was more rapid at Kamuli than at Serere. Significantly (P < 0.001) higher symptom severity scores were recorded in initially healthy plants, which had average scores of 3.6 and 3.5 at Kamuli and Serere, respectively, compared with 2.8 for mildly diseased plants at each location. Severity scores of 4 and 5 were more frequent in initially healthy plants, accounting for 77 and 39% of the total infections recorded in comparison with 47 and 11% in mildly diseased plants at Kamuli and Serere, respectively. Mildly diseased plants were significantly taller than initially healthy plants 8 and 12 MAP at both locations. However, the converse was true 4 MAP although differences were significant at Serere but not at Kamuli. Mildly diseased plants yielded significantly more tuberous roots than initially healthy plants at Kamuli but not at Serere. Average total weights of tuberous roots per plant were 2.48 and 1.63 kg for mildly diseased and initially healthy plants at Kamuli and 4.46 and 4.61 kg at Serere, respectively. These results may help to explain the increased prevalence in recent years of mildly diseased plants of local CMD‐susceptible cultivars in eastern Uganda, from where these varieties virtually disappeared following the severe CMD epidemic in the 1990s. The results also provide the first field evidence of a cross protective effect of mild strains of a cassava mosaic geminivirus.     

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.     

Viruses Associated with Cassava Mosaic Disease in Senegal and Guinea Conakry

A survey in Senegal and Guinea Conakry established the presence and incidence of cassava mosaic virus disease (CMD) in both countries. CMD occurred in all the fields surveyed, although its incidence was higher in Senegal (83%) than in Guinea (64%). Populations of the whitefly vector, Bemisia tabaci, were low in both countries averaging 1.7 adults per shoot in Guinea and 3.2 in Senegal. Most infections were attributed to the use of infected cuttings, 86 and 83% in Senegal and Guinea, respectively, and there was no evidence of rapid current‐season, whitefly‐borne infection at any of the sampled locations. Disease severity was generally low in the two countries and averaged 2.5 in Guinea and 2.3 in Senegal. No plants with unusually severe CMD symptoms characteristic of the CMD pandemic in East and Central Africa were observed. Restriction fragment length polymorphism (RFLP)‐based diagnostics revealed that African cassava mosaic virus (ACMV) is exclusively associated with CMD in both the countries. Neither East African cassava mosaic virus (EACMV), nor the recombinant Uganda variant (EACMV‐UG2) was detected in any sample. These survey data indicate that CMD could be effectively controlled in both countries by phytosanitation, involving the use of CMD‐free planting material and the removal of diseased plants.     

不同CMV分离物侵染寄主的超微结构变化

应用电镜观察了黄瓜花叶病毒CMV不同分离物侵染寄主的细胞 超微结构变化。来自一串红(Salvia splendens)的不含卫星RNA分离物M-22侵染心叶烟,病毒粒子散布于细胞质,在液泡中形成大片病毒粒子结晶,液泡膜边缘产生小泡结构,完整的病毒粒子穿过胞间连丝在细胞间运转,胞间连丝中央部分有扩张现象。自然感染三生烟的含坏死卫星RNA分离物8-S1侵染普通烟,病毒粒子分散于细胞质,在液泡中未观察到结晶体,叶绿体产生囊泡结构,部分病毒粒子处在叶绿体空泡中。田间寄主上受8-S1侵染的三生烟细胞质中分布着大量球形病毒粒子,叶绿体也产生含有病毒粒子的囊泡结构。表明含有卫星RNA和不含卫星RNA的CMV分离物引起的细胞病变特征存在差别,可能是CMV卫星RNA参与病理变化的依据之一。     

不同CMV分离物侵染寄主的超微结构变化

应用电镜观察了黄瓜花叶病毒ＣＭＶ不同分离物侵染寄主的细胞超微结构变化。来自一患红（Ｓａｌｖｉａｓｐｌｅｎｄｅｎｓ）的不含卫星ＲＮＡ分离物Ｍ－２２侵染心叶烟,病毒粒子散布于细胞质,在液泡中形成大片病毒粒子结果,液泡膜边缘产生小泡结构,完整的病毒粒子穿过胞间连丝在细胞间运转,胞间连丝中央部分有扩张现象。     

Spatial and Temporal Spread of Cassava Mosaic Virus Disease in Cassava Grown Alone and when Intercropped with Maize and/or Cowpea

The spread of cassava mosaic disease (CMD) and populations of the whitefly vector (Bemisia tabaci) were recorded in cassava when grown alone and when intercropped with maize and/or cowpea. The trials were planted under conditions of high inoculum pressure in 1995 and 1996 at a site in the lowland rainforest zone of southern Cameroon. In the 1995 experiment, the maize and cowpea intercrops reduced the final incidence of CMD in the cassava cvs. Dschang White and Dschang Violet, but not in the more resistant cv. Improved. In the 1996 experiment with cv. Dschang Violet, the maize and cowpea intercrops grown alone or together decreased adult whitefly populations on cassava by 50% and CMD incidence by 20%. The monomolecular population growth model generally provided the best fit for disease progress. Areas under the disease progress curves (AUDPCs) and incidences expressed as multiple infection units were significantly (P<0.05) less for cassava intercropped with maize and/or cowpea than in cassava alone; times to 50% CMD incidence were significantly (P<0.05) longer in all intercrop systems. In 1995 the basic infection rates (r) were similarly low (0.010 per month) in the moderately resistant cv. Dschang Violet intercropped with maize and in all treatments in the more resistant cv. Improved. By contrast, rates were significantly higherfor cv. Dschang Violet alone or with cowpea and in all treatments for the less resistant cv. Dschang White (0.030–0.060). In 1996, r values in cassava grown alone (0.077) were significantly larger (P<0.05) than in the other cropping systems (0.042–0.052). There were no significant differences in the symptom severity in the different cropping systems. Disease foci were isodametric and more compact in plots containing cowpea than in other cropping systems.     

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。     

The Nucleotide Sequence and Genome Structure of Mung Bean Yellow Mosaic Geminivirus

Complete nucleotide sequences of the infectious cloned DNA components (DNA 1 and DNA 2) of mung bean yellow mosaic virus (MYMV) were determined. MYMV DNA 1 and DNA 2 consists of 2,723 and 2,675 nucleotides respectively. DNA 1 and DNA 2 have little sequence similarity except for a region of approximately 200 bases which is almost identical in the two molecules. Analysis of open reading frames revealed nine potential coding regions for proteins of mol. wt. > 10,000, six in DNA 1 and three in DNA 2. The nucleotide sequence of MYMV DNA was compared with that of bean golden mosaic virus (BGMV), tomato golden mosaic virus (TGMV) and African cassava mosaic virus (ACMV). The 200-base region common to the two DNAs of each virus had little sequence similarity, except for a highly conserved 33-36 base sequence potentially capable of forming a stable hairpin structure. The potential coding regions in the MYMV DNAs had counterparts in the BGMV, TGMV and ACMV, suggesting an overall similarity in genome organization, except for absence of 1L3 in MYMV DNA 1. The most highly conserved ORFs, MYMV 1R1, BGMV 1R1, TGMV 1R1 and ACMV 1R1, are the putative genes for the coat proteins of MYMV, BGMV, TGMV and ACMV, respectively. MYMV 1L1 has also a high degree of sequence similarity with BGMV 1L1, TGMV 1L1 and ACMV 1L1.     

Cloning and sequencing of partial DNA-A of Cassava mosaic virus

Abstract

Polymerase chain reaction of Cassava mosaic virus revealed that out of the 50 samples analysed only two samples, one from Musiri (Trichy district) and other from Mallur (Salem district), were detected with ICMV infection as 904 bp fragment of DNA-A amplified. All the other samples from various districts of Tamil Nadu were detected invariably with SLCMV as they amplified 599 bp of DNA-A. A 599 bp fragment of DAN-A was cloned and sequenced from the sample collected from Mallur. The nucleotide sequence has been submitted to GenBank under the accession number DQ303479. The nucleotide sequence was compared with other cassava infecting geminiviruses and other geminiviruses in GenBank. Cluster dendrogram revealed that the cloned sequence was most closely related to ICMV, Maharastra strain rather than SLCMV, forming one cluster. Comparative sequence analyses showed that the cloned fragment shared a maximum sequence identity with ICMV at nucleotide levels (93%) than with SLCMV (88%).