病毒/细胞同步侵染体系筛选抗植物病毒剂的研究

 本文采用病毒/细胞同步侵染体系筛选3种植物抽提物抗TMV增殖及对叶绿体的保护作用,其中射干抽提物处理转染烟草细胞96h后对病毒增殖抑制率为76.0%,吴茱萸抽提物抑制病毒对叶绿体的破坏作用达到89.58%,筛选出了作用方式不同的抗病毒物质。用植物整株法和大田试验比较病毒/细胞同步侵染体系测定的结果相近,因此病毒/细胞同步侵染体系用以筛选抗病毒物质具有可行性。     

植物小RNA在病毒致病过程中的作用研究进展

目前,我国正遭受多种外来有害生物的入侵,给我国的农业生产以及生态环境造成了严重影响。其中检疫性植物病毒危害大、隐蔽性强,是重要的外来生物。掌握病毒致病的相关机制是有效防控入侵病毒病害的基础。微小RNA(microRNA,miRNA)以及病毒小干扰RNA(small interfering RNA,siRNA)是植物体内重要的非编码小RNA,在病毒致病过程中扮演着重要角色。病毒在侵染过程中,可以利用植物抗病毒免疫反应产生的siRNA进行致病,同时可以直接或间接干扰miRNA的代谢途径,导致相关症状的产生。本文根据植物小RNA的近期研究进展,对miRNA和病毒siRNA的产生途径、生物学特性以及作用分子机制进行了综述,并对其在病毒侵染过程中的作用进行了探讨,初步从小RNA的角度阐述了植物病毒的致病机制,以期为检疫性病毒病害的防控提供理论基础。     

蚜虫传播非持久性病毒的取食行为调控机制

蚜虫能够传播上百种植物病毒,是最重要的农业介体昆虫之一。蚜虫在刺探和取食植物过程中,唾液组分会连同附着在口针中的病毒粒子一同被分泌进入植物内,在调节植物诱导抗性、病毒侵染扩散、介体昆虫行为等过程中均有重要作用。本文围绕蚜虫传播病毒和获取病毒2个关键过程,总结分析了蚜虫独特的刺吸取食行为与传毒效率和获毒效率之间的联系;针对取食活动中关键的唾液蛋白在调控植物免疫抗性、帮助病毒侵染过程中的功能,阐述了蚜虫高效传播非持久病毒的分子基础;针对蚜虫的获毒过程,综述了病毒侵染植物间接调控蚜虫趋向和行为的作用方式。这些研究的开展将为解释蚜虫和病毒协同侵染的分子机制以及有效开展基于蚜虫取食行为调控的病虫害防控新技术提供思路。     

马铃薯Y病毒侵染对叶绿体超微结构、光合和荧光参数的影响

 马铃薯Y病毒(PVY)侵染导致马铃薯叶片叶绿体个体变小,部分叶绿体的结构遭到破坏,淀粉粒的体积变小而粒数密度提高。随着病毒侵染,叶片中的叶绿素含量逐步减少,光合系统电子传递速率和净光合速率显著下降,而光系统Ⅱ的最大光化学效率则没有显著变化。PVY导致叶绿体结构破坏和碳同化酶活性下降,可能是造成光合作用下降的主要原因。     

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。     

病毒侵染后植物叶绿体光合作用变化的分子机制

 当植物遭受病毒侵染后通常会引起植物重要生理功能的改变,包括光合作用与呼吸作用的变化、核酸与蛋白质的变化、酚类代谢的变化、水分生理的变化以及体内激素的紊乱和寄主膜结构的破坏等,其中对光合作用的影响是最关键的因素。     

百合无症病毒对百合光合生理、酶活性及叶绿体超微结构的影响

 以东方百合“西伯利亚”为试验材料,研究百合无症病毒(LSV)侵染百合对其叶片生理生化以及叶绿体超微结构的影响。检测结果表明:叶片中叶绿素a、b以及总叶绿素含量与健康对照相比分别下降了28.6%、33.3%和23.5%,净光合速率、气孔导度及胞间CO₂浓度分别下降33.3%、25%和13.8%;超氧化物歧化酶(SOD)、过氧化物酶(POD)、多酚氧化酶(PPO)和苯丙氨酸解氨酶(PAL)与健康对照相比,分别增加了16.6%、29.4%、16.7%和22.2%。电镜观察发现:感病植株叶绿体膨胀变形,基质片层散乱,叶绿体内淀粉粒肿大且数目增多,从而证明LSV侵染破坏叶绿体结构,影响植株的光合作用。     

TMV侵染后烟叶叶绿体的荧光光谱与生理学特性

作者研究了ＴＭＶ侵染后烟草叶片中完整叶绿体的荧光光谱与生理学特性：６８５ｎｍ与７４０ｎｍ荧光强度之比升高，放氧放降低，完整叶绿体数目下降，结合ＴＭＶ侵染后烟叶荧光主特征变化，分析讨论了ＴＭＶ侵染过程引起的病理变化及其机制。     

抗植物病毒剂作用机制研究进展

根据植物病毒侵染寄主、体内复制和症状表达的过程,综述了目前所开发的抗病毒剂的作用机制,以及药剂、病毒(TMV)对寄主(烟草)基因表达差异的研究情况,分析了抗病毒剂研究中存在的问题,并进行了展望。     

miRNA在植物病毒基因组中的靶基因预测及分析

microRNA(miRNA)是一类长约22nt的内源性的非编码小RNA,在植物的生长发育和胁迫响应中起重要调控作用。本文通过生物信息学的方法,在miRBase中下载了12种植物的miRNA序列;在DPVweb和VIDE病毒数据库中查找侵染这些植物的病毒,并在NCBI数据库中搜索其基因组序列,共获得173种病毒的基因组序列。用psRNATarget在线软件预测植物miRNA在植物病毒中的靶基因,发现植物miRNA可能参与调控包括聚合蛋白、衣壳蛋白、逆转录酶、复制相关基因、通读蛋白、依赖RNA的RNA聚合酶等多种植物病毒基因的表达。     

Selbst oder Nicht-Selbst �C die Rezeptoren des pflanzlichen Immunsystems

Plants recognize conserved molecular structures from microorganisms, which triggers active immune responses. Successful pathogens have to overcome this level of immunity; however, plants in turn can adapt their immune system, thus plants and pathogens are in an evolutionary arms race. As being sessile organisms, plants need to integrate and adapt to changing environmental conditions such as light, temperature, drought, or microorganisms. Plants protect themselves against diseases through sensitive recognition of potential pathogens and effective defense systems. The first level of the plant immune system provides recognition of a broad spectrum of microorganisms leading to defense activation (Bittel and Robatzek 2007). The second level of the plant immunity allows certain plant cultivars to detect of specific pathogen strains??a phenomenon also referred to as ??gene-for-gene resistance?? (Jones and Dangl 2006). The first level of immunity occurs rapidly and triggers active defenses normally without harm to the plant cell. The second level of plant immunity develops over days and deploys a local cell death, which prevents pathogens from further spread into tissues. In addition to these cell-autonomous defense systems, plants have also evolved strategies of systemic immunity.     

Drought as a modulator of plant–virus–vector interactions: Effects on symptom expression,plant immunity and vector behaviour

Plant–virus interactions are affected by environmental conditions that determine plant vulnerability to pathogens and the population dynamics of insect vectors. We hypothesize that drought enhances horizontal transmission by dampening the basal immunity of plants, which triggers symptom expression and vector manipulation. The potato yellow vein virus (PYVV) causes potato yellow vein disease (PYVD), a re-emerging epidemic of potato crops in South America, and is transmitted horizontally by the greenhouse whitefly (GWF), Trialeurodes vaporariorum (Hemiptera: Aleyrodidae), or vertically through infected seed tubers. We investigated the role of drought and temperature as modulators of PYVD symptom expression, plant immune response, and vector survival, development and host preference. We found that drought induced symptom expression, suppressed the salicylic acid pathway and increased PYVV replication. GWF survival was reduced on PYVV-infected hosts and development was slowest when they fed on plants with PYVD symptoms, which also triggered adults’ attraction to PYVV-infected plants. However, adults previously fed on infected plants showed the opposite effect, being more attracted to PYVV-free plants. We propose a theoretical model that explains the role of drought in modulating potato–PYVV–GWF interactions and provides new insights into plant–virus–vector coevolution.     

Antiphytoviral activity of bruceine-D from Brucea javanica seeds

BACKGROUND: Brucea javanica (L.) Merr. is widely distributed throughout the southern parts of China and has been used in traditional medicine to treat a variety of diseases. The objective of the present study was to identify the active antiphytoviral compound in the seeds of B. javanica and evaluate the inhibitory activity of the compound against plant virus. RESULTS: Bioassay-guided fractionation of the most active extract from the seeds led to the isolation of an antiphytoviral compound which was identified as bruceine-D by conventional spectroscopy methods. The compound exhibited significant inhibitory activity against the infection and replication of tobacco mosaic virus (TMV), with IC(50) values of 13.98 and 7.13 mg L(-1) respectively. The compound also showed a strong inhibitory effect on the infectivity of potato virus Y (PVY) and cucumber mosaic virus (CMV). Furthermore, the compound could effectively inhibit systemic TMV infection in the host tobacco plant under glasshouse conditions.CONCLUSION: The results suggested that bruceine-D from Brucea javanica may have the potential to be used as a natural viricide, or a lead compound for new viricides.     

Selection of Beet Necrotic Yellow Vein Virus Specific Single-chain Fv Antibodies from a Semi-synthetic Combinatorial Antibody Library

Methods for the generation of monoclonal antibodies against plant viruses are limited because current hybridoma techniques do not allow efficient exploitation of the immune repertoire. Moreover, the immunization procedures often lead to a bias towards an immunodominant contaminant in the immunogen preparation and not to the plant virus itself. The selection of six different single-chain antibody variable fragments (scFv) against beet necrotic yellow vein virus from a semi-synthetic human combinatorial antibody library showed the feasibility of the phage display system. No bias towards minor contaminants in the purified virus preparation was observed in ELISA, as all the selected scFvs reacted only with beet necrotic yellow vein virus infected plant homogenates. In addition, two of the isolated beet necrotic yellow vein virus-specific scFvs could be produced in E. coli as a scFv fusion protein with alkaline phosphatase, and were applied in ELISA as specific ready to use antibody-enzyme conjugates. Because of their specificity, these antibodies have potential to be used as reagents in sensitive diagnostic assays for routine testing for beet necrotic yellow vein virus in sugar beets.     

利用RNA介导的抗病性获得抗2种病毒的转基因烟草

 RNA介导的病毒抗性(RMVR)是近年发展起来的一种新的植物抗病毒基因工程策略,具有抗病性强、抗性持久、生物安全性高等特点。利用该策略培育多抗病毒植株具有广阔的应用前景和重要的实践意义。本研究将非翻译的马铃薯X病毒的衣壳蛋白(PVX-CP)基因和非翻译的马铃薯Y病毒的衣壳蛋白(PVY-CP)基因组成嵌合基因,构建植物表达载体pROKXY,利用农杆菌介导法转化烟草NC89,获得6株对PVX和PVY的混合侵染表现为免疫的转基因植株。分子分析表明,这种抗性为RNA介导的病毒抗性。这一研究结果为利用RMVR进行植物多抗病毒育种提供了重要数据和资料。     

蝉拟青霉侵染小菜蛾幼虫SSH文库的构建及免疫相关基因分析

为探讨小菜蛾Plutella xylostella对病原真菌入侵的免疫防御机制,利用抑制消减杂交技术构建蝉拟青霉Paecilomyces cicadae侵染的小菜蛾幼虫的抑制性差减文库,并对文库进行鉴定。ESTs序列聚类分析共获得412个独立基因。通过同源比对,28.24%ESTs为未知功能基因,71.76%ESTs与已知功能基因序列相似,包括免疫相关、金属离子结合和加工、核酸和蛋白代谢及加工、细胞信号、细胞结构、形状和流动、能量代谢、胁迫和解毒以及其它基因。鉴定出39个可能参与小菜蛾免疫蝉拟青霉侵染的免疫基因,包括：识别分子、蛋白酶及蛋白酶抑制剂、效应因子及其它免疫基因。qRT-PCR结果表明肽聚糖识别蛋白、器官芽生长因子、葛佬素、溶菌酶、酚氧化酶原激活蛋白酶3以及转铁蛋白基因均可诱导表达。免疫相关基因在不同微生物诱导下的表达模式存在不同。结果表明当病原真菌蝉拟青霉侵染寄主小菜蛾后,小菜蛾体内发生了一系列的复杂反应,小菜蛾抵御病原真菌侵染是一个多途径共同作用的复杂过程。该研究为进一步研究小菜蛾抵御病原真菌入侵的分子机制提供基础信息。     

水稻病毒与植物寄主互作研究新进展

水稻是维持世界一半以上人口生存的主要粮食作物,其产量和品质关系到社会稳定。近年来,水稻病毒引起的病毒病严重影响作物的产量及品质。植物在与病毒长期的互作过程中进化出了多种有效的防御机制来抑制病毒侵染。本文围绕近年来国内外关于水稻抗病毒的研究进展,对植物激素、自噬与水稻病毒的互作关系进行总结,并提出了水稻病毒研究过程中所涉及到的一些问题,以期为开展水稻抗病育种相关研究提供参考。     

靶向G-四链体的抗番茄丛矮病毒卟啉类衍生物的筛选

植物病毒病给蔬菜生产带来很大影响,目前蔬菜病毒防控主要采用非药剂措施,广谱抗病毒剂效果有限,需要基于新的药物靶点开发新的病毒抑制剂。G-四链体(G4s)是一种特殊的核酸高级结构,其在病毒基因组中的形成或解链可调节基因的复制、转录和翻译等过程,进而影响病毒增殖。一些可调节G-四链体结构稳定性的小分子呈现出抗病毒活性,使得G-四链体有望成为新的抗病毒药物的靶标。番茄丛矮病毒(tomato bushy stunt virus, TBSV)是一种分布广泛的植物病毒,有关其基因组的复制、转录和翻译等分子机制已研究得较为成熟,是一种研究病毒与寄主互作的模式病毒。揭示TBSV中G-四链体的结构及功能,有望为植物病毒基因中G-四链体的研究奠定基础。本研究通过生物信息学分析,在TBSV基因中鉴定出两条保守的、潜在的G-四链体可形成序列(putative G-quadruplex sequences, PQS)——TBSV-PQS2和TBSV-PQS4;通过紫外、荧光光谱和圆二色光谱(CD光谱)方法筛选出与TBSV-PQS2和TBSV-PQS4互作的G4配体;通过烟草体内侵染性克隆试验发现,G-四链体配体...