植物病毒协生作用及其分子机理

植物病毒协生作用分布广,是造成农作物减产的重要原因之一.抗病毒转基因植物中协生作用的出现,严重限制了基因工程植物的商品化生产.本文对植物病毒协生作用的类型和特点、协生作用中病毒与病毒、病毒与环境间的相互作用及其分子机制进行了阐述.     

马铃薯Y病毒HC-Pro中心区域在病毒协生作用中的主导地位

利用PCR方法获得了马铃薯病毒中国株系（PVY－C）HC－Pro基因的5个缺失突变体,构建了相应的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens)介导法转化了烟草品种K326(Nicotina tabacum cv.k326)。PCR和Southern blot分析证明了HC－Pro基因及其缺失突变体已整合到烟草基因组中,Western blot表明它们在转基因烟草中得到了表达。侵染性试验发现HC－Pro中心区域介导转基因烟草中PVC－C和黄瓜花叶病毒（CMV）、PVY－C和马铃薯X病毒（PVX）之间的协生作用,从而明确了PVY－C HC－Pro中心区域为病毒协生作用的功能区域。     

马铃薯Y病毒HC-Pro中心区域在病毒协生作用中的主导地位

利用PCR方法获得了马铃薯病毒中国株系(PVY-C)HC-Pro基因的5个缺失突变体,构建了相应的植物表达载体。通过土壤农杆菌(Agrobacterium tumefaciens)介导法转化了烟草品种K₃₂₆(Nicotina tabacum cv.K₃₂₆)。PCR和Southern blot分析证明了HCPro基因及其缺失突变体已整合到烟草基因组中,Western blot表明它们在转基因烟草中得到了表达。侵染性试验发现HCPro中心区域介导转基因烟草中PVY-C和黄瓜花叶病毒(CMV)、PVYC和马铃薯X病毒(PVX)之间的协生作用,从而明确了PVY-C HC-Pro中心区域为病毒协生作用的功能区域。     

双链RNA和植物对病毒的抗性

双链RNA能诱导转录后的基因沉默,是生物抵御病毒入侵、维持自身基因稳定的一种自我保护机制.把源自病毒的基因构建成反向重复结构转入植物体内,其转录出的RNA会通过分子内序列互补形成双链,将入侵病毒的同源序列降解,使转基因植株获得对病毒的高抗性.RNA干扰型抗病毒转基因植株中,转病毒基因的mRNA不存在或存在量很少,也不会翻译成有功能的病毒蛋白,因此不存在病毒RNA重组、异源包装及协生作用的潜在风险,具有较高的生物安全性.双链RNA抗病毒转基因正在成为一种高效、安全的植物抗病毒策略.     

国家自然科学基金委员会生命科学部1998年度资助青年科学基金项目一览表

项目名称负责人单位名称1微生物学学科（7项）植物病毒协生作用的分子机理研究鲁瑞芳中国科学院微生物研究所石蕊科地农的分子系统学研究郭守玉中国科学院微生物研究所利用GFP对根瘤菌识别和侵染Neptunia的分王文军中山大学子机制的研究参与烟草花叶病毒胞间转运的寄主因子基因的克隆凌建群浙江农业大学微生物降解褐煤产生黄腐酸的研究袁红莉中国农业大学棉花疫苗elicitin基因的致病功能域研究王源超浙江农业大学中国鹅膏菌科真菌分类与系统研究杨祝良中国科学院昆明植物研究所2植物学学科（6项）茨藻目植物花器官发生和系统发育研究孙坤…     

运动蛋白介导的病毒在植物体中的传播

综述了病毒在植物寄主内扩散中的运动蛋白的作用。由病毒基因组编码的运动蛋白与病毒核酸形成运动蛋白核酸复合物,介导病毒扩散。在病毒复制与扩散过程中,运动蛋白与宿主细胞内质网、高尔基体、细胞骨架、胞间连丝发生作用,并受细胞果胶甲基脂酶、包含体、β-1,3-葡聚糖酶、磷酸化等因素的影响,形成了植物体内遗传物质系统性运输的一个模式。     

番茄环纹斑点病毒与马铃薯Y病毒复合侵染烟草的细胞病理特征

在自然情况下, 番茄环纹斑点病毒(TZSV)和马铃薯Y病毒(PVY)通常复合侵染同一植株。该文首次报道了TZSV和PVY复合侵染烟草(Nicotiana sp.)植株的细胞病理特征, 并与单独侵染进行了比较分析。复合侵染的烟草植株细胞中, TZSV病毒颗粒明显增多, 并聚集于囊膜内形成包涵体, 与PVY的风轮状及片层状内含体和病毒颗粒聚集体交叉分布于细胞质(内含线粒体明显增多)中, 线粒体、叶绿体和细胞核结构较完整; 两种病毒的颗粒、包涵体和内含体数量均较单一侵染增多, 且对寄主亚细胞结构的破坏较单一侵染为轻, TZSV和PVY及其与寄主的互作可能存在协生作用。     

植物病毒的运动蛋白（Movement Protein）

植物病毒的运动蛋白是由病毒编码的一种蛋白,在病毒的细胞间运动中起重要作用。现在,发现的运动蛋白越来越多,对其一级结构、在植物体内的表达、定位和功能日益清楚。但运动蛋白在体内的修饰及其与运动蛋白功能的关系的研究还刚开始,对与运动蛋白作用的寄主因子了解很少。植物运动蛋白的研究对植物病毒细胞间运动和植物体内特有的胞间连丝的研究提供了很好的突破口。     

DNA甲基化在植物与双生病毒互作中的作用

DNA甲基化是一种重要的表观遗传修饰,在维持基因组稳定、调控基因表达、转座子沉默以及植物抗逆等过程中具有重要作用.基因组为单链环状DNA的双生病毒复制过程中形成的双链DNA与组蛋白结合形成微染色体,可诱导并成为DNA甲基化的靶标.而相应地,双生病毒在与植物长期斗争中,进化出多种不同类型的抑制子并且通过不同的策略来抑制DNA甲基化以逃脱植物的防御反应.本文将结合植物与病毒互作中的研究重点,简述DNA甲基化在植物抗双生病毒方面的研究进展,并围绕双生病毒编码的蛋白抑制DNA甲基化的机制进行综述,以更好地理解植物中DNA甲基化介导的表观遗传调控在植物与双生病毒互作中的作用.     

病毒与宿主互利共生的研究

在病毒与其宿主的相互作用中,病毒所扮演的角色不仅仅是病原体,它们也是宿主保持健康的重要共生体。病毒与微生物、植物、昆虫和哺乳动物之间存在互利共生作用,有的甚至涉及共生功能体的多个生物体。随着新病毒的不断发现,越来越多的互利共生关系被发掘,也还有许多有待发掘。本文旨在突出一些近年来关于有益病毒的典型例子,阐明为何需要重新认识病毒——病毒不仅是病原体,同时也是共生功能体中的一个完整个体。     

Development of transgenic sweet potato with multiple virus resistance in South Africa (SA)

Multiple infections of Sweet potato feathery mottle virus (SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato virus G (SPVG) and Sweet potato mild mottle virus (SPMMV) cause a devastating synergistic disease complex of sweet potato (Ipomoea batatas Lam.) in KwaZulu-Natal, South Africa. In order to address the problem of multiple virus infections and synergism, this study aimed to develop transgenic sweet potato (cv. Blesbok) plants with broad virus resistance. Coat protein gene segments of SPFMV, SPCSV, SPVG and SPMMV were used to induce gene silencing in transgenic sweet potato. Transformation of apical tips of sweet potato cv. Blesbok was achieved by using Agrobacterium tumefaciens strain LBA4404 harboring the expression cassette. Polymerase chain reaction and Southern blot analyses showed integration of the transgenes occurred in six of the 24 putative transgenic plants and that all plants seemed to correspond to the same transformation event. The six transgenic plants were challenged by graft inoculation with SPFMV, SPCSV, SPVG and SPMMV-infected Ipomoea setosa Ker. Although virus presence was detected using nitrocellulose enzyme-linked immunosorbent assay, all transgenic plants displayed delayed and milder symptoms of chlorosis and mottling of lower leaves when compared to the untransformed control plants. These results warrant further investigation on resistance to virus infection under field conditions.     

Environmental risk assessment of releases of transgenic plants containing virus-derived inserts

Sequences derived from the genomes of plant viruses are being used to provide virus resistance in transgenic crop plants. Although the environmental hazards associated with the release of such plants have been discussed widely, it has not been possible to reach generally acceptable conclusions about their safety. A case-by-case approach to the risk assessment of real examples is recommended as a means of building up confidence and of indicating areas of uncertainty. A logical framework for risk assessment is suggested, a key feature of which is identification of the viruses in the release environment that may infect the transgenic plants. Each of these is considered in relation to each of the three main classes of hazard (transcapsidation, recombination and synergism), and the risk associated with each event is analysed.     

马铃薯Y病毒蚜传辅助成分介导PVX/PVY协生作用

构建了马铃薯Y病毒中国株系（PVY－C）蚜传辅助成分（HC－Pro）基因的正义、反义和缺失三种植物表达载体,通过农杆菌介导法转化烟草品种NC89。Southern blot分析表明,HC－Pro基因及其突变体已经整合到烟草染色体中,Western blot分析证明,正义HC－Pro基因及其缺失突变体在转基因烟草中有表达产物,攻毒试验结果表明,转正义,HC－Pro基因及其缺失突变体不仅能够提高T1转基因烟草中PVY－C的病毒积累和致病,而且对异源病毒PVX具有同样的作用,而转反义HC－Pro基因烟草对PVY－C和PVX的致病性无影响,因此,PVY－C HC－Pro基因介导PVX／PVY的协作作用。     

Combination of Trichoderma harzianum endochitinase and a membrane-affecting fungicide on control of Alternaria leaf spot in transgenic broccoli plants

Progeny from transgenic broccoli (cv. Green Comet) expressing a Trichoderma harzianum endochitinase gene were used to assess the interaction between endochitinase and the fungicide Bayleton in the control of Alternaria brassicicola. In vitro assays have shown synergistic effects of endochitinase and fungicides on fungal pathogens. Our study examined the in planta effects of endochitinase and Bayleton, individually and in combination. Two month old transgenic and non-transgenic plants were sprayed with ED50 levels of Bayleton and/or inoculated with an A. brassicicola spore suspension. Disease levels in non-sprayed transgenic plants were not statistically different from sprayed transgenic plants nor from sprayed non-transgenic controls. Thus endochitinase-transgenic plants alone provided a significant reduction of disease severity, comparable to the protection by fungicide on non-transgenic plants. Comparison of the expected additive and observed effects revealed no synergism between endochitinase and Bayleton (at ED50 level), and usually less than an additive effect. Some transgenic lines sprayed with fungicide at doses higher than ED50 showed resistance similar to the non-sprayed transgenic lines, again suggesting no synergistic effect. Lack of synergism may be due to incomplete digestion of the cell wall by endochitinase, so that the effect of Bayleton at the cell membrane is not enhanced.     

Transcomplementation and synergism in plants: implications for viral transgenes?

In plants, viral synergisms occur when one virus enhances infection by a distinct or unrelated virus. Such synergisms may be unidirectional or mutualistic but, in either case, synergism implies that protein(s) from one virus can enhance infection by another. A mechanistically related phenomenon is transcomplementation, in which a viral protein, usually expressed from a transgene, enhances or supports the infection of a virus from a distinct species. To gain an insight into the characteristics and limitations of these helper functions of individual viral genes, and to assess their effects on the plant-pathogen relationship, reports of successful synergism and transcomplementation were compiled from the peer-reviewed literature and combined with data from successful viral gene exchange experiments. Results from these experiments were tabulated to highlight the phylogenetic relationship between the helper and dependent viruses and, where possible, to identify the protein responsible for the altered infection process. The analysis of more than 150 publications, each containing one or more reports of successful exchanges, transcomplementation or synergism, revealed the following: (i) diverse viral traits can be enhanced by synergism and transcomplementation; these include the expansion of host range, acquisition of mechanical transmission, enhanced specific infectivity, enhanced cell-to-cell and long-distance movement, elevated or novel vector transmission, elevated viral titre and enhanced seed transmission; (ii) transcomplementation and synergism are mediated by many viral proteins, including inhibitors of gene silencing, replicases, coat proteins and movement proteins; (iii) although more frequent between closely related viruses, transcomplementation and synergism can occur between viruses that are phylogenetically highly divergent. As indicators of the interoperability of viral genes, these results are of general interest, but they can also be applied to the risk assessment of transgenic crops expressing viral proteins. In particular, they can contribute to the identification of potential hazards, and can be used to identify data gaps and limitations in predicting the likelihood of transgene-mediated transcomplementation.     

抗病毒基因工程与转基因植物释放的环境风险评估

全面介绍了抗病毒转基因植物释放的风险性,包括重组（ｒｅｃｏｍｂｉｎａｔｉｏｎ）、衣壳转移（ｔｒａｎｓｃａｐ－ｓｉｄａｔｉｏｎ）和协生作用（ｓｙｎｅｒｇｉｓｍ）等,并指出,我国应对抗病毒转基因植物释放产生的风险进行系统评估,在缺乏对可能产生的风险的认识之前,应控制进行大规模的田间试验。     

转基因植物根系分泌物对土壤微生态的影响

随着转基因植物商品化进程的加快,对其进行生态风险性评价日益引起学者的重视。诸如转基因逃逸到其它亲缘物种中、产生超级杂草和病毒、昆虫产生耐受性及生物多样性遭受破坏等问题已在部分转基因作物中显现。本文综述了转基因植物中根系分泌物对土壤微生态的影响。     

苜蓿花叶病毒外壳蛋白基因对红三叶的遗传转化及转基因植株的抗病性分析

以红三叶(Trifolium pratense L．)子叶为转化体,用农杆菌介导法将外源的苜蓿花叶病毒外壳蛋白基因AMV4转入红三叶,经过筛选、分化和再生,得到了具有卡拉霉素抗性的转基因植株。对这些植株进行PCR、Southern印迹杂交和Northern杂交分析,结果表明,外源目的基因已经整合到红三叶基因组中并且得到了表达。对Northern分析呈阳性的植株进行了抗病性检测,结果表明,表达苜蓿花叶病毒外壳蛋白基因的植株病症减轻,发病率、病情指数及病毒积累量都明显低于对照,有的甚至不表现症状,达到了免疫的程度。