黄瓜花叶病毒的株系鉴定研究进展

黄瓜花叶病毒的株系鉴定研究进展李华平,胡晋生,范怀忠（华南农业大学植保系,广州５１０６４２）（夏威夷大学植病系,美国夏威夷９６８２２）关键词黄瓜花叶病毒,株系种类,株系鉴定ＡｄｖａｎｃｅｓｉｎＩｄｅｎｔｉｆｉｃａｔｉｏｎｏｆＣｕｃｕｍｂｅｒＭｏｓａｉ...     

黄瓜花叶病毒香蕉株系的衣壳蛋白基因克隆和序列分析

对侵染香蕉的黄瓜花叶病毒广东３个株系的衣壳蛋白（ＣＰ）基因,进行了克隆和序列分析,以提纯病毒ＲＮＡ为模板,应用ＲＮＡ反转录酶（ＡＭＶ）合成ＣＰ基因ｃＤＮＡ再用ＴａｑＤＮＡ聚合酶进行ＰＣＲ扩增,通过常规基因克隆法扩增的ＣＰ基因克隆入载体,选取每一株系的ＣＰ基因与载体表面方向相同和相反的各一个克隆,进行插入片段的全序列分析,结果表明,每一重组克隆测序长约７５０个核苷酸,任一株系其插入方向相反的两个重组     

黄瓜花叶病毒香蕉株系衣壳蛋白转基因烟草的研究

构了侵染香蕉的黄瓜花叶病毒两个株系的衣壳蛋白基因的植物表达载体,并通过农杆菌共培养法的基因枪法,分别将两个株系的ＣＰ基因转化入了两种烟草植株。其ＣＰ基因转化频率及植株再生率研究结果表明,农杆菌共培养法比基因枪法,土耳其烟比本生烟,农杆菌１：１０倍稀释液比培养原液和１：１００倍稀释液,具有更高的转化频率和植株再生能力。Ｓｏｕｔｈｅｍ ｂｌｏｔ,ＰＣＲ－Ｓｏｕｔｈｅｍ ｂｏｌｔ检测ＣＰ基因整合研究结果     

含卫星RNA的黄瓜花叶病毒弱株系的分离鉴定及在病毒病防治上的应用

从豌豆上分离获得黄瓜花叶病毒分离物ＣＭＶＰ１,摩擦接种９科２９种植物,ＣＭＶＰｌ在大多数植物上症状很轻或无任何症状,提纯的病毒颗粒为球形,直径约２８ｎｍ,病毒衣壳蛋白亚基分子量约２７．５ｋＤ,所含核酸有五个组份,即ＣＭＶＰ１含有卫星ＲＮＡ。ＣＭＶＰ１接种三生烟后８－１２天内呈轻花叶,此时组织中病毒含量最高,随后症状消失,去除卫星ＲＮＡ能加重ＣＭＶＰ１在番茄上的症状,因而是卫星ＲＮＡ减轻了ＣＭＶＰｌ的病状。当ＣＭＶＰ１保护接种番茄后攻强毒,番茄发病率低,病情轻,保护率达９０％以上,并有一定的刺激生长作用,还能提早开花４天,植株结果数增多,成熟果实的颜色、形态、品质和重量均正常。ＣＭＶＰ１对烟草亦具有很好的保护效果。保护接种的植株能明显减少强毒株侵染,可减少９０％以上。     

广东烟草花叶病毒株系研究

烟草花叶病毒（TMV）引起的烟草花叶病在广东不同的烟草主产区存在着症状的差异。从广东梅州和南雄烟区采集的138个TMV标样中,选取6个不同症状的分离物作为研究对象,发现它们在不同种属寄主和不同烟草品种上表现明显的TMV普通株特征症状,虽然在某些寄主上其症状也有一些差异,但未能反映株系间的差异。这6个分离物具有几乎相同的病毒粒体形态、电泳迁移率、钝化温度以及紫外吸收特征。经琼脂双扩散法和间接ELISA法的血清学反应,表明它们具较强的血清学关系。因而它们有很强的同源性。经病毒粒体外壳蛋白的氨基酸分析,在氨基酸总数上,分离物GD1和GD3各有156个氨基酸．GD2和GD4各有159个,GD5和GD6分别有158和157个氨基酸。上述分析试验结果初步表明,在广东不同烟草主产区引起的症状有所差异的6个分离物均属IMV普通株。     

黄瓜花叶病毒香蕉株系（CMV—Xb）RNA3cDNA的克隆和序列分析

通过PR－PCR方法,设计两对引物,克隆了黄瓜花叶病毒香蕉株系（CMV-Xb)RNA3,并进行了核苷酸和蛋白质水平上的分析,结果表明Xb株系RNA3全长2205nt,具有两个蛋白编码阅读框型架（ORF）,其中5'端（97－936nt)编码一个279aa的3a蛋白;3'端（1225－1871nt)编码一个2188aa的CP蛋白。5'非编码域为96nt;其因间隔区（IR）长288nt;3'NR含有324nt。通过与亚组Ⅱ其它株系RNA3核苷酸和所编码产物推导的氨基酸序列分析发现,亚组Ⅱ株系无论在编码区还是非编码区的核苷酸同源性都相对较高;亚组Ⅱ株系在进化过程中具有连续性。     

侵染番茄的黄瓜花叶病毒（CMV）株系特性的比较研究

从山东省主要番茄种植区病毒样本中分离到111个黄瓜花叶病毒(CMV)分离物,根据病毒的寄主范围和症状,初步鉴定为三个株系:番茄蕨叶株系(CMV-ToF)、番茄花叶株系(CMV-ToM)和番茄轻花叶株系(CMV-ToL)。三株系除在生物学特性存在明显差异外,其病毒粒体形态大小,电泳迁移率,病毒外壳蛋白亚基分子量、核酸组分以及病毒粒体血清学特性亦存在差别。     

黄瓜花叶病毒香蕉株系(CMV-Xb)RNA3 cDNA的克隆和序列分析

通过RT-PCR方法,设计两对引物,克隆了黄瓜花叶病毒香蕉株系(CMV-Xb)RNA3,并进行了核苷酸和蛋白质水平上的分析.结果表明Xb株系RNA3全长2205nt,具有两个蛋白编码阅读框架(ORF),其中5'端(97～936nt)编码一个279aa的3a蛋白;3'端(1225～1871nt)编码一个218aa的CP蛋白.5'非编码区域为96nt;基因间隔区(IR)长288nt;3'NR含有324nt.通过与亚组Ⅱ其它株系RNA3核苷酸和所编码产物推导的氨基酸序列分析发现,亚组Ⅱ株系无论在编码区还是非编码区的核苷酸同源性都相对较高;亚组Ⅱ株系在进化过程中具有连续性.     

侵染菊花的黄瓜花叶病毒的初步鉴定和血清学检测

侵染菊花的黄瓜花叶病毒的初步鉴定和血清学检测张海保,朱西儒,张云开（中国科学院华南植物研究所广州５１０６５０）关键词菊花,黄瓜花叶病毒,间接ＥＬＩＳＡ菊花（Ｃｈｒａｙｓａｎｔｈｅｍｕｍｍｏｒｉｆｏｌｉｕｍ）病毒病是危害菊花的一类主要病害。国外文献报道...     

辣椒上CMV株系鉴别寄主的筛选与应用

根据“基因对基因”理论和日本小室与都凡按寄主的科属关系及被害症状划分株系的方法,研究了辣椒CMV的“基因型株系”和“致病型株系”。从373个甜、辣椒品种(系)中,筛选出一套抗性不同的差别品种,编号为:LS-8501(HR)、LS-8502(R)、LS-8503(T)、LS-8504(S)、LS-8505(HS)。用这套差别品种做“基因型”株系鉴别寄主,将59个CMV分离物划分为5个株系,命名为:CMV-P0,CMV-P1,CMV-P2,CMV-P3,CMV-P4。又从7科39种不同科属寄主值物中,筛选出一套“致病型”株系的鉴别寄主谱7种,用这套鉴别寄主将59个CMV分离物划分为5个株系群,即十字花科株系群,藜科株系群,茄科、葫芦科株系群,豆科株系群,普通黄色花叶株系群。文中比较了两种方法划分的株系致病性与辣椒病症表现型之间的关系,以及各株系的分布。还讨论了“基因型”鉴别寄主谱及“基因型”株系划分方法盼学术价值和实用性,比较了5个株系与国内外已分化的CMV株系的异同点。     

Effects of Light Quality on the Interaction between Cucumber Mosaic Virus and Nicotiana tabacum

Light is an important environment factor controlling plant growth, development, and nutritional quality and is also one of the most important factors inducing plant defence. In this study, we assayed the potential effects of light quality on the interaction between Nicotiana tabacum and cucumber mosaic virus (CMV). Our results indicated that white light‐treated N. tabacum plants displayed obvious symptoms at early stage postinoculation, while the symptoms were significantly inhibited under red light and blue light. Western blotting and quantitative real‐time PCR (qRT‐PCR) analyses showed that blue light and red light can effectively delay the replication of CMV compared with white light. The activities of various reactive oxygen species (ROS)‐scavenging enzymes and reducing substances [reduced glutathione (GSH) and ascorbic acid (ASA)] were increased under blue light and red light. In addition, hormone measurements and qRT‐PCR analyses revealed that salicylic acid (SA)‐mediated signalling pathway plays positive role in the related regulation, and cytokinin (CTK) may also participate in them. Furthermore, we found that the formation of dark green islands (DGIs) was significantly suppressed in plants under red light and blue light at 30 days postinoculation (Dpi). However, the accumulation of virus in plants under different light conditions had no notable differences at later stage of postinoculation. Taken together, these results indicated that red light and blue light could effectively delay symptom expression and replication of CMV on N. tabacum at the relatively earlier stage postinoculation.     

黄瓜花叶病毒及抗病转基因烟草研究进展

黄瓜花叶病毒(Cucumber mosaic virus,CMV)是世界上分布最广的植物病毒之一,其株系繁多,给烟草生产造成很大的损失。近些年来,各国学者对CMV开展了大量研究。该文主要介绍了黄瓜花叶病毒的基因组结构、亚组以及抗CMV的烟草基因工程的研究进展。     

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.     

黄瓜花叶病毒2b蛋白对寄主光合速率和叶绿体结构的影响

黄瓜花叶病毒(Cucumber mosaic virus,CMV)2b蛋白作为重要致病因子的角色在茄科模式植物上已为广泛描述,但其致病机理尚未廓清.从寄主叶绿体结构和光合特征的角度探索了病毒侵染过程中2b蛋白对心叶烟(Nicotiana glutinosa)的影响.首先利用PCR方法导入突变位点,构建了2b蛋白不表达的人工突变体Fny-CMV!2bpro,然后通过平行接种野生型Fny-CMV和Fny-CMV"2bpro,分析二者侵染后心叶烟的症状表现、叶绿素含量、光合速率以及叶绿体超微结构变化.结果显示:接种30天,Fny-CMV侵染的心叶烟表现重花叶、畸形和矮化症状,植株的光合速率和叶绿素含量显著降低,叶绿体形态和结构发生明显病变.但是,Fny-CMV#2bpro侵染的心叶烟植株仅表现轻微花叶或不表现明显症状,与健康对照相比,其光合速率和叶绿素含量没有显著差异,叶绿体形态和结构亦未出现明显病变.由此可见,野生型病毒侵染导致的相对低的光合速率和叶绿素含量,与2b蛋白引起的叶绿体形态和结构的破坏有一定的相关性.RNA杂交分析结果显示:2b蛋白的致病性与CMV子代RNA在寄主体内的积累量有关,不表达2b蛋白使得CMV基因组RNA1和RNA2含量显著下降,其亚基因组RNA4(编码CP蛋白)含量降低尤为显著.     

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。     

黄瓜花叶病毒衣壳蛋白基因转化辣椒研究

The plant expression vector of the coat protein(CP) gene of cucumber mosaic virus (CMV) BS strain was used to transform three kinds of pepper (Capsicum annuum) tissues (cotyledon, stem and root) by agrobacterium-mediated co-cultivation. 53%-68.4% of the total tissues (639) can be induced to be calli, but only cotyledon calli can be further regenerated to form shoots (regenerated efficiency 39.7%). 70%(42/60) of the putative transformed plants were confirmed to have CP gene in their genomes by Southern blot. The mRNAs and the CP were respectively found in 80% of transgenic plants by Northern blot and DAS-ELISA. 24 of the transgenic plants expressing CP gene of BS strain showed three kinds of resistant level (severe symptom, delay of symptomatic development, no symptom) to infection of CMV-BS and of CMV-P. However, there was distinctly higher resistance to inoculation of CMV-BS than that with CMV-P in these transgenic plants.