【作者】 王卫兵；

【导师】 洪健；

【作者基本信息】 浙江大学 ， 植物病理学， 2004， 硕士

【副题名】Ⅰ：BBWV-2在寄主体内的复制和转运机制探讨 Ⅱ：SCMV和SrMV的细胞病理变化比较

【摘要】 蚕豆萎蔫病毒-2(BBWV-2)、甘蔗花叶病毒(SCMV)和高粱花叶病毒(SrMV)在我国广泛发生，对许多经济作物造成严重危害。本实验运用电子显微镜和免疫胶体金标记技术系统研究了这三个病毒分别侵染寄主后产生的一系列细胞病理变化，分析讨论了病毒的复制场所、胞间转运机制等问题，获得的主要结果如下： 1．BBWV-2的B935分离物(分离自中国)分别接种豌豆、蚕豆和昆诺藜，三种寄主上均呈现典型的花叶症状。采集不同侵染程度的感病叶片，经过化学固定、脱水和包埋后制作超薄切片，切片经染色后在透射电镜下观察。感病豌豆和蚕豆叶片细胞的主要病变特征包括：(1)细胞质中膜结构明显增生，形成大块含有膜结构、小泡结构以及电子致密物质的增生区域，该区域可能是病毒的复制区：(2)在细胞质和膜增生区域周围大量存在管状结构和结晶体，管状结构横切面显示由个17-19个粒子组成，直径约160nm左右，免疫金标记显示管状结构和结晶体均由病毒粒子组成；(3)线粒体数目明显增加，体积膨大变形，有的线粒体内含有结晶结构，在线粒体之间常夹有一些晶格状物质，免疫金标记证明为正在组装的病毒粒子，线粒体之间可能是病毒的装配部位；(4)在叶肉细胞的胞间连丝中观察到病毒样颗粒排列在小管中，该小管部分伸到细胞质内，免疫金标记显示这些颗粒可能为转运中的完整病毒粒子，BBWV-2的胞间运动模式与CPMV相似。 2．应用Li-6400便携式光合作用系统测定了接种BBWV-2的B935分离物的蚕豆和健康蚕豆叶片的光合速率、二氧化碳浓度等生理指标，结果显示，受病毒感染后，寄主的光合速率有大幅度的下降，二氧化碳浓度显著升高，蒸腾速率和气孔导度变化不大。蚕豆叶片叶绿体结构的发生一些异常变化，叶绿体形状改变，基质明显增加，片层紊乱等。光合速率的大幅下降可能与叶绿体结构被破坏相关。 3．BBWV-2的PV131分离物(分离自意大利)分别接种到蚕豆和昆诺藜健康幼叶上，10天左右嫩叶显现典型花叶症状。取显症叶片经树脂包埋后超薄切片，染色后透射电镜下观察。与B935分离物侵染蚕豆和豌豆相类似，PV131分离物在昆诺藜叶肉细胞中引起明显的膜结构增生，形成复制区，大块梭形结晶体和管状结构分布在细胞质中，但管状结构的横切面显示由个9粒子组成，直径约80nm左右，与B935分离物产生的管状结构不同。在蚕豆叶肉细胞中，仅仅观察到膜增生区域的存在，始终未观察到管子和结晶体等结构。 4．运用超薄切片电镜技术比较观察了甘蔗花叶病毒(SCMV)和高粱花叶病毒(SrMV)侵染玉米叶片细胞的超微结构变化，结果显示：线状SCMV和SrMV粒子分散或成束分布在感病细胞的细胞质内，同时在细胞质中产生大量柱状内含体。SCMV引起风轮体、卷筒体和片层聚集体，属于Edw别心son等划分的第m型，一些成束的病毒粒子和内含体存在于筛管中。SrMV引起风轮体和卷筒体，属于第I型，一些内含体分布于细胞壁附近，有的直接与细胞壁胞间连丝相接，完整的柱状内含体存在筛管中，推测内含体在病毒的胞间运动中起定位和传输作用。两种病毒在细胞病理学效应方面的差异也可以作为诊断鉴定的一个参考依据。更多还原

【Abstract】 Bean broad wilt virus (BBWV), Sugarcane mosaic virus (SCMV) and Sorghum mosaic virus (SrMV), reported in many regions of China, caused serious loses in economic crops. In this paper, the ultrastructural alteration of several host plants respectively infected with BBWV, SCMV and SrMV were systemically studied by transmission electron microscope (TEM) and immunological technique.1. The typical mosaic symptoms occurred on the leaves of Vicia faba, Pisum sativum and Chenopodium quinoa inoculated with the B935 isolate of BBWV2. Under the TEM, several similar ultrastructural changes were observed in the thin sections of the infected leaves of V. faba and P. sativum, such as membranous proliferation composed of the fibril-containing vesicles, virus-form tubular structures 160nm in diameter, crystals composed of virus particles, virion-containing plasmodesmata, as well as the lineable virus particles between the proliferous mitochondria. In addition, some chloroplasts were often disrupted in the B935-infected cells of P. sativum leaves. By contraries, the similar cytopathological changes never been found in thin sections of C.quinoa leaf tissue infected with B935 isolates.2. Several physiological parameters of health and B935-infected V.faba were measured, and the statistical data revealed that B935 infection caused the significant reduce of net photosynthetic rate, the noted increase of intercellular CO2 concentration, and little influences on transpiration rate and stomata conductance.3. The leaves of V.faba and C.quinoa were infected with PV131, one of the BBWV2 isolates from Italy, and the mosaic symptoms developed in 10d after infection. Checked the thin sections of infected leaf tissue by TEM, the membranous proliferation, virus-formed crystals and tubular structures 80nm in diameter, were found in parenchyma cells. In addition, the thin sections of PV131-infected leaves of V.faba were examined, howerve, only proliferation of membranes located in the cytoplasm.4. The ultrastructural alterations of maize infected with SrMV and SCMV, respectively, were examined under transmission electron microscope. A large number of filamentous SrMV and SCMV particles were observed to be present as scattered or in more or less dense bundles in the cytoplasm of infected cells. Three types of cylindrical inclusions (CIs), pinwheels, scrolls and laminated aggregates, located in the SCMV-infected cytoplasm, andthe similar CIs occur in the SrMV-infected cytoplasm except for the absence of laminated aggregates. According to Edwardson, SrMV and SCMV were placed in subdivision I and III. In this paper, not only cytopathological alterations of maize infected with SrMV and SCMV were compared with each other, but also some ultrastructures possibly associated with the virus movement were analyzed.更多还原