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为明确银川番茄(Lycopersicon esculentum)是否遭受了番茄斑萎病毒(TSWV)的危害, 采用国家标准TSWV RT- PCR检测技术对银川番茄上采集的14份疑似感染TSWV病叶样本进行分子鉴定, 对克隆得到的核衣壳蛋白基因N (Nucleocapsid)序列进行多序列比对和系统进化树分析, 随后对PCR阳性样本进行蛋白检测。结果表明, 14份病叶样本中有8份扩增出长度为394 bp的TSWV N基因序列, 且8条序列完全一致; 获得的银川番茄TSWV分离物与云南番茄、中国莴苣(Lactuca sativa)、中国鸢尾(Iris tectorum)和重庆辣椒(Capsicum annuum) TSWV分离物相对近缘, 与山东、黑龙江和北京等地及国外TSWV分离物相对远缘; 利用TSWV的抗体通过Western blot对8个PCR阳性样本进一步检测, 结果也证实8个阳性样本中存在TSWV感染。该研究首次通过分子鉴定及蛋白检测证明银川番茄上存在TSWV感染, 需要加快抗TSWV番茄品种的选育工作。 相似文献
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接种番茄斑萎病毒番茄植株对西花蓟马生物学特性的影响 总被引:1,自引:0,他引:1
西花蓟马Frankliniella occidentalis(Pergande)是我国的一种重要入侵害虫。本文研究了西花蓟马在番茄3种处理(健康CK、机械接种番茄斑萎病毒MI、机械损伤MD)叶片上的生长发育、存活及种群增长。结果表明:健康、机械接种番茄斑萎病毒、机械损伤叶片上的发育历期依次为12.68、12.99和11.79d。雌雄成虫寿命和雌虫繁殖能力在各处理叶片上差异不显著(P>0.05)。健康、机械接种番茄斑萎病毒、机械损伤叶片上的内禀增长率依次为0.1362、0.1526和0.1292d-1。本研究表明,接种番茄斑萎病毒的番茄叶片未缩短西花蓟马发育历期,也不能延长寿命及提高产卵量,不能明显加速种群数量增长。这意味着番茄斑萎病毒对西花蓟马在番茄叶片上的生物学特性未能产生明显的有利作用。 相似文献
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[目的]西花蓟马Frankliniella occidentalis (Pergande)是番茄斑萎病毒(Tomato spotted wilt orthotospovirus,TSWV)的主要传播媒介,以循环增殖方式传播TSWV,但在传播过程中参与的互作蛋白尚不清楚。本研究以TSWV的外鞘糖蛋白GN (GlycoproteinN)为诱饵筛选与西花蓟马互作的蛋白,为阐明TSWV与西花蓟马的互作机制提供依据。[方法]构建以pPR3-N为载体的西花蓟马泛素分裂酵母双杂交膜系统cDNA文库,利用Gene Ontology (GO)通路注释互作蛋白,分析其生物学功能。[结果]西花蓟马cDNA文库库容为3.2×10~6,除去重复序列、载体序列和移码序列,筛选到74个与G_N互作的蛋白,参与了细胞过程、代谢过程、生物调节过程等12种生物过程。[结论]成功构建了西花蓟马酵母双杂交膜体系cDNA文库,筛选到与TSWV GN互作的蛋白,为进一步研究TSWV与西花蓟马的互作机制奠定了基础。 相似文献
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目的:基于荧光偏振技术建立靶向番茄斑萎病毒(Tomato spotted wilt virus,TSWV)核蛋白(NP)与核酸相互作用的药物筛选体系,并应用该体系展开药物筛选。方法:将目的基因克隆到pGEX-6p-1表达载体上,并采用大肠杆菌表达系统进行目的蛋白的异源表达。建立靶向TSWV NP与核酸相互作用的荧光偏振药物筛选体系,对体系的结合时间、DMSO耐受、变异性和稳定性进行研究,并展开药物筛选。结果:成功构建重组质粒pGEX-6p-1-NP,在大肠杆菌中表达并分离纯化出高质量的核蛋白。基于荧光偏振技术建立了信噪比为8∶1,Z因子为0. 82的稳定的靶向TSWV NP与核酸相互作用的药物筛选体系,并对化合物库中1 000种化合物展开药物筛选,经过初步筛选获得了1种IC_(50)为4. 15μmol/L的化合物。结论:建立了稳定的荧光偏振筛选体系,适用于靶向NP与核酸相互作用的药物的筛选。筛选到的化合物为番茄斑萎病毒的预防和控制提供参考。 相似文献
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为了明确对番茄斑萎病毒(tomato spotted wilt virus,TSWV)免疫的番茄YNAU335自交系表现出TSWV感病症状(抗性被打破)的原因,在排除YNAU335自交系不纯和或混杂其它感病番茄材料的因素外,选取96172I(感病)和YNAU335(抗性被打破)自交系感病植株,进行TSWV核衣壳蛋白(nucleocapsid protein,NP)和运动蛋白(movement protein,MP)基因的RT-PCR检测,并对阳性克隆进行基因测序分析。结果表明:(1)2个自交系中均克隆出TSWV的NP和MP基因,共获得5条NP(登录号:MK628735~MK628739)和3条MP(登录号:MK883723、MK883724和MK887284)多态性基因序列。(2)96172I感病材料中克隆出以上所有基因序列,YNAU335感病材料中克隆出其中的3条NP和1条MP基因序列。(3)对YNAU335中TSWV特有的NP和MP氨基酸突变位点进行分析,结果发现4个特异突变位点可能与打破YNAU335抗性有关,4个突变位点分别为:NP 18位氨基酸G突变为V,36位T突变为I,39位L突变为R,MP 274位E突变为K。(4)系统发育分析显示,5条NP和3条MP与云南省已登记的NP和MP聚类在不同的分支,表明云南省昆明地区TSWV存在丰富的遗传多样性。 相似文献
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[目的]研究番茄斑萎病毒(Tomato spotted wilt virus,TSWV)运动蛋白NSm的细胞定位.[方法]将NSm基因融合GFP后构建到植物双元表达载体pCHF3中,农杆菌介导浸润本氏烟叶片,同时将融合GFP的NSm基因利用Bac-to-Bac杆状病毒表达体系转染昆虫Tn细胞.在激光共聚焦显微镜下观察NSm-GFP在烟草表皮细胞和昆虫Tn细胞中的定位情况.[结果]观察发现与单独表达GFP在细胞壁周围和细胞核处均匀分布不同,NSm-GFP融合蛋白会在植物细胞内扩散,能够在细胞壁边缘定位,并且在胞间连丝处呈不连续的绿色荧光小点,偶尔成对出现在相邻的两个细胞之间;NSm蛋白在昆虫Tn细胞表面产生数量众多的管状结构并向外延伸.[结论]研究结果表明NSm能够特异性定位在植物细胞的胞间连丝处,并能在昆虫Tn细胞内表达,在细胞表面产生运动蛋白小管. 相似文献
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寄主植物接种番茄斑萎病毒对西花蓟马种群的影响 总被引:1,自引:0,他引:1
【目的】西花蓟马Frankliniella occidentalis (Pergande)是一种入侵我国的重要害虫, 而番茄斑萎病毒是以西花蓟马传播为主的一种极具危害性的世界性病毒, 通过研究西花蓟马与番茄斑萎病毒之间的互作将有助于进一步深入理解西花蓟马以及番茄斑萎病毒的发生与猖獗机制, 同时也将为制定合理、可持续的控制西花蓟马及其传播的植物病毒防控策略提供理论依据。【方法】利用应用特定年龄-龄期及两性生命表方法, 研究了西花蓟马在辣椒3种处理(健康CK、机械损伤MD、机械接种番茄斑萎病毒MI)叶片上的生长发育、存活及种群增长。【结果】健康、机械损伤和机械接毒叶片上的发育历期依次为12.45, 11.97和11.18 d。雌雄成虫寿命和雌虫产卵量在不同处理植株叶片上差异显著(P<0.05), 在机械接毒叶片上寿命最长(雌13.51 d, 雄12.69 d); 繁殖能力最强, 产子代数高达33.01头1龄若虫/雌。健康、机械损伤和机械接毒叶片上西花蓟马内禀增长率分别为-0.009, 0.153和0.190 d-1, 净生殖率依次为0.84, 14.54和21.79。【结论】番茄斑萎病毒诱导寄主植物辣椒反应使西花蓟马发育历期缩短, 成虫寿命延长, 繁殖能力提高, 种群增长加速。 相似文献
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Dr. Константпн Тэръщэ Dr. Эманупл Менчер Dipl.-Gartenbau-Ing. Инге Гропе Doz. Dr. sc. Хаiiнц Aцaм 《Archives Of Phytopathology And Plant Protection》2013,46(6):483-494
TSWV belongs to the genus Tospovirus which was established in the family Bunyaviridae, a family of animal viruses. Besides TSWV, Impatiens necrotic spot virus (INSV) and ground nut bud necrosis virus (GBNV) were established as different Tospovirus species. Tospoviruses have quasispherical particles of 85 nm diametre which are surrounded by a membrane and contain 3 RNA species and 4 structural proteins. In Tospovirus infected plant cells virions were detected in cavaties of the endoplasmatic reticulum and additionally amorphous electron dense material accumulates in infected cells. Defective forms of TSWV lack the ability to form complete virus particles. TSWV is the only plant pathogenic virus that is transmitted by thrips which transmit the virus with different efficiency. The virus has an extensive plant host range of more than 360 different species. The developing symptoms depend on the Tospovirus species, the virulence of the virus strains and the environmental conditions. Based on the reaction of TSWV isolates with N‐specific polyclonal antisera, 3 serogroups were established. The most frequently used technique for serologically based diagnosis of Tospoviruses is DAS ELISA with N‐specific or preadsorbed antisera against complete virus. For TSWV epidemiology distinct weeds and cultural host plants play an important role for the survival of virus and vector. Breeding for resistance is the most important preventive measure of control. 相似文献
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Keisuke Komoda Masanori Narita Isao Tanaka Min Yao 《Acta Crystallographica. Section F, Structural Biology Communications》2013,69(6):700-703
Tomato spotted wilt virus (TSWV), which causes severe damage to various agricultural crops such as tomato, pepper, lettuce and peanut, is a negative‐stranded RNA virus belonging to the Tospovirus genus of the Bunyaviridae family. Viral genomic RNA molecules are packaged in the form of ribonucleoprotein complexes, each of which contains one viral RNA molecule that is coated with many nucleocapsid (N) proteins. Here, the expression and crystallization of TSWV N protein are reported. Native and selenomethionine‐substituted crystals of N protein belonged to the same space group P21. Their unit‐cell parameters were a = 66.8, b = 97.2, c = 72.0 Å, β = 112.8° and a = 66.5, b = 96.3, c = 72.1 Å, β = 113.4°, respectively. 相似文献
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Phoebe Rapando Ann Wangai Rose Ramkat 《Archives Of Phytopathology And Plant Protection》2013,46(6):579-586
Abstract Tomato spotted wilt virus (TSWV) vectored by thrips is one of the major diseases affecting cucumber yield. Control of thrips is an underlying factor in its management. A study was conducted to determine the effect of time of inoculation, variety and mulch on disease incidence. Four varieties were inoculated with TSWV at cotyledon, 3 – 4 leaf and flower bud stages in a RCBD experiment replicated four times in a greenhouse. In the field, a 2×8 factorial design where two cucumber varieties were raised on seven types of mulches (red, yellow, silver, clear, black, white, and straw) with unmulched plots as controls was used. Variety Marketer was more tolerant to the disease compared to other varieties. Most varieties were generally tolerant to TSWV at cotyledonous but susceptible at 3 – 4 leaf and flower bud stages. Silver and clear mulches significantly suppressed thrip populations, yield and quality under field conditions. 相似文献
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Tomato spotted wilt virus (TSWV) is the type member of the tospovirus genus and causes significant losses in a wide range of economically important ornamental and vegetable crops worldwide. The nucleocapsid gene, located on the ambisense S RNA segment of TSWV was expressed in Escherichia coli using pET-32a as vector and correct expression of recombinant protein was confirmed by Western blot using an anti-TSWV monoclonal antibody (MAb). The recombinant protein was purified using Ni-NTA agarose and the purified protein was used for the production of MAbs. Three murine MAbs against the recombinant nucleocapsid protein were produced. Triple antibody sandwich enzyme-linked immunosorbent assay and immunocapture RT-PCR methods were then established for reliable and efficient detection of TSWV using the produced MAbs. 相似文献
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S. Matsuura S. Ishikura N. Shigemoto S. Kajihara K. Hagiwara 《Journal of Phytopathology》2004,152(4):219-223
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At present, the so-called \"substantial equivalence\" is the only widely accepted criterion for deciding whether or not a transgenic food is, from an alimentary point of view, to be considered totally correspondent to the \"traditional\" one from which it derives. Although never exactly defined, it deals with a comparison between the chemical composition of the two foods. A more in-depth analysis can be performed by one of the most suitable methods that allows for the simultaneous screening of many components without prior identification, the analysis of the proteome. As a model for testing this kind of approach, we compared protein expression of two types of tomato plants, having the same genetic background, except for a virus resistance trait introduced by genetic engineering. When proteins extracted from seedlings of the two types were analyzed by two-dimensional electrophoresis, no significant differences, either qualitative or quantitative, were detected, indicating that in this case the expression of major proteins was unmodified by the genetic manipulation. Fifteen proteins were identified by peptide mass fingerprinting. 相似文献
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I. Macharia D. Backhouse E.M. Ateka S.‐B. Wu J. Harvey M. Njahira R.A. Skilton 《The Annals of applied biology》2015,166(3):520-529
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Ying Huang Hao Hong Min Xu Jiaoling Yan Jing Dai Jianyan Wu Zhike Feng Min Zhu Zhongkai Zhang Xuefeng Yuan Xinshun Ding Xiaorong Tao 《Molecular Plant Pathology》2020,21(7):985-998
Tomato spotted wilt virus (TSWV) is one of the most devastating plant viruses and often causes severe crop losses worldwide. Generally, mature plants become more resistant to pathogens, known as adult plant resistance. In this study, we demonstrated a new phenomenon involving developmentally regulated susceptibility of Arabidopsis thaliana to TSWV. We found that Arabidopsis plants become more susceptible to TSWV as plants mature. Most young 3-week-old Arabidopsis were not infected by TSWV. Infection of TSWV in 4-, 5-, and 6-week-old Arabidopsis increased from 9%, 21%, and 25%, respectively, to 100% in 7- to 8-week-old Arabidopsis plants. Different isolates of TSWV and different tospoviruses show a low rate of infection in young Arabidopsis but a high rate in mature plants. When Arabidopsis dcl2/3/4 or rdr1/2/6 mutant plants were inoculated with TSWV, similar results as observed for the wild-type Arabidopsis plants were obtained. A cell-to-cell movement assay showed that the intercellular movement efficiency of TSWV NSm:GFP fusion was significantly higher in 8-week-old Arabidopsis leaves compared with 4-week-old Arabidopsis leaves. Moreover, the expression levels of pectin methylesterase and β-1,3-glucanase, which play critical roles in macromolecule cell-to-cell trafficking, were significantly up-regulated in 8-week-old Arabidopsis leaves compared with 4-week-old Arabidopsis leaves during TSWV infection. To date, this mature plant susceptibility to pathogen infections has rarely been investigated. Thus, the findings presented here should advance our knowledge on the developmentally regulated mature host susceptibility to plant virus infection. 相似文献