湖北省小麦黄花叶病病原的部分序列鉴定

 从湖北省真菌传小麦黄花叶病病毒分离物中抽提病毒总RNA,应用逆转录-聚合酶链式反应(RT-PCR)方法,合成病毒外壳蛋白(CP)基因cDNA。对所获得的cDNA克隆进行序列测定及分析,结果表明,湖北省真菌传小麦黄花叶病病毒分离物与小麦梭条斑花叶病毒(WSSMV)、大麦黄花叶病毒(BaYMV)、大麦和性花叶病毒(BaMMV)等病毒CP基因相应序列的同源性均低于70%,而与报道的小麦黄花叶病毒(WYMV)分离物CP基因核苷酸及编码氨基酸序列同源性均超过97%。表明湖北省真菌传小麦黄花叶病病原应为小麦黄花叶病毒(WYMV)。     

小麦黄花叶病毒Nib蛋白介导的体外转录体系的创建

 本研究是利用异源表达的小麦黄花叶病毒(Wheat yellow mosaic virus, WYMV)复制酶Nib蛋白创建了病毒核酸的体外复制系统。将WYMV的Nib编码序列扩增并插入到大肠杆菌表达载体pMAL-C2X中以构建原核表达质粒pMAL-WY-Nib。0.4 mmol·L^-1 IPTG诱导可特异性表达分子量约为100 kDa的MBP-Nib融合蛋白。根据温度梯度测定,20℃下诱导MBP-Nib的可溶性比例较高,约为30％,可满足MBP标签的亲和层析。通过与WYMV的其他蛋白和烟草丛顶病毒的RdRp进行比较,纯化的MBP-Nib融合蛋白可以特异性识别WYMV RNA1和RNA2的3′末端区域并体外合成其互补链,此体外转录体系可用于WYMV复制调控的研究。     

小麦FtsH2蛋白与WYMV CP互作研究

 小麦黄花叶病毒(Wheat yellow mosaic virus, WYMV)是马铃薯Y病毒科大麦黄花叶病毒属成员,主要危害冬小麦。实验室前期以WYMV外壳蛋白(coat protein,CP)为诱饵,通过酵母双杂交筛选小麦cDNA文库,发现FtsH2蛋白部分片段与WYMV CP互作。FtsH2蛋白是AAA蛋白酶家族成员,参与植物叶绿体光损伤修复和类囊体发育进程。本研究利用酵母双杂交和双分子荧光互补技术进一步对FtsH2蛋白全长与WYMV CP进行互作验证,并鉴定互作结构域;利用荧光蛋白标记技术研究FtsH2蛋白与CP的亚细胞定位。实验结果表明FtsH2蛋白全长与CP互作;两者互作的关键结构域包含FtsH2蛋白低复杂区、跨膜区及AAA结构域和WYMV CP的中段区域(61-293 aa)。FtsH2蛋白单独表达时定位在细胞质、细胞核和叶绿体;CP单独表达时定位在细胞质;两者共同表达时亚细胞定位均没有发生明显变化,且主要共定位于细胞质中。WYMV CP与小麦FtsH2蛋白的互作可能会干扰植物绿叶体的发育和功能。本研究对了解WYMV的症状形成机制具有一定意义。     

小麦FtsH2蛋白与WYMV CP互作研究

 小麦黄花叶病毒(Wheat yellow mosaic virus, WYMV)是马铃薯Y病毒科大麦黄花叶病毒属成员,主要危害冬小麦。实验室前期以WYMV外壳蛋白(coat protein,CP)为诱饵,通过酵母双杂交筛选小麦cDNA文库,发现FtsH2蛋白部分片段与WYMV CP互作。FtsH2蛋白是AAA蛋白酶家族成员,参与植物叶绿体光损伤修复和类囊体发育进程。本研究利用酵母双杂交和双分子荧光互补技术进一步对FtsH2蛋白全长与WYMV CP进行互作验证,并鉴定互作结构域;利用荧光蛋白标记技术研究FtsH2蛋白与CP的亚细胞定位。实验结果表明FtsH2蛋白全长与CP互作;两者互作的关键结构域包含FtsH2蛋白低复杂区、跨膜区及AAA结构域和WYMV CP的中段区域(61-293 aa)。FtsH2蛋白单独表达时定位在细胞质、细胞核和叶绿体;CP单独表达时定位在细胞质;两者共同表达时亚细胞定位均没有发生明显变化,且主要共定位于细胞质中。WYMV CP与小麦FtsH2蛋白的互作可能会干扰植物绿叶体的发育和功能。本研究对了解WYMV的症状形成机制具有一定意义。     

siRNA对烟草原生质体中TMV RNA积累的干扰作用研究(英文)

 RNA干扰被认为是转录后基因沉默的一种机制。RNA干扰通过小干扰RNA特异性降解目标mRNA来沉默基因表达。本文以烟草花叶病毒126kD蛋白为靶蛋白,在原生质体水平上研究了小干扰RNA对病毒侵染的干扰和抑制作用。ELISA和Northern杂交的实验结果表明,共转染小干扰RNA和TMV的原生质体内检测到较低的病毒含量。在枯斑寄主上,叶片接种小干扰RNA和TMV共转染原生质体后,与对照叶片相比,仅有很少量的病斑产生。这说明,小干扰RNA能够在原生质体水平对病毒起到干扰和抑制作用。因此认为,烟草原生质体系统有利于快速和定量分析小干扰RNA介导对植物病毒的抑制作用。     

RNA干扰及其在水稻抗病毒基因工程中的应用

RNA干扰(RNA interference,RNAi)是一种基因沉默机制。RNAi作为新兴的基因阻断技术具有明显的优势,已被广泛应用到动植物功能基因组和植物抗病研究中。在抗病毒研究中,人为地将与病毒或宿主基因同源的双链RNA分子导入转基因植株,引起与其同源的基因发生沉默,达到抗病毒的作用。本文主要综述了RNA干扰的相关知识以及在水稻抗病毒基因工程研究中的应用进展。     

玉米弯孢霉叶斑病菌生物学特性的研究

 利用小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV)和小麦梭条花叶病毒(wheat spindle streak mosaic virus,WSSMV)的特异性序列为引物,逆转录-PCR方法并配合免疫电镜观察,对采自我国四川陕西、河南、安徽、江苏5省9个县、市的27份感病田小麦样品进行检测。除2份样品未检测到病毒外,其余25份样品均为小麦黄花叶病毒所侵染,没有发现小麦梭条花叶病毒,由此表明在中国长江中、下游、淮河及渭河流域严重危害小麦的真菌传线状病毒应主要为小麦黄花叶病毒。     

小麦黄花叶病毒(WYMV)2个山东分离物的全基因组序列及进化分析

利用5'RACE结合一步法RT-PCR分别从山东泰安与临沂冬小麦上克隆了小麦黄花叶病毒(Wheat yellow mosaic virus,WYMV)的全基因组序列。这2个分离物(TADWK和LYJN)基因组间核苷酸一致性分别为97.21%(RNA1)和95.12%(RNA2)。通过对目前已报道的共14个分离物的基因组不同部分的分析,表明5'UTR是WYMV基因组变化幅度最大的区域,而编码区(ORF)及3'UTR的序列一致性较高且变动幅度小。另外,发现LYJN RNA2的5'UTR与已报道的所有分离物RNA2的核苷酸一致率仅为90%左右。综合分析RNA1和RNA2的系统发生树,表明WYMV各基因组片段呈单独进化特征,不同分离物间存在RNA重排。RNA重组分析显示在LYJN RNA1和TADWK RNA2发现了RNA重组。此研究说明WYMV在山东地区存在分离物分化现象,WYMV的5'UTR是基因组中的突变热点。     

利用RNA干扰介导抗病性获得兼抗四种病毒的转基因马铃薯

为获得兼抗马铃薯X病毒(Potato virus X,PVX)、马铃薯Y病毒(Potato virus Y,PVY)、马铃薯卷叶病毒(Potato leaf roll virus,PLRV)和马铃薯潜隐花叶病毒(Potato virus S,PVS)4种病毒的转基因马铃薯新材料,分别以这4种病毒全长CP基因为模板,通过设计PCR引物和亚克隆获得4种病毒CP基因相对保守区段的基因片段,并将其拼接成融合基因,以载体pHANNIBAL和pBI121为基础,构建RNA干扰(RNA interference,RNAi)载体,利用农杆菌介导的转基因体系进行马铃薯遗传转化,并对获得的转基因马铃薯进行病毒抗性检测。结果表明,所获得的融合基因片段RH1和RH2,酶切鉴定分别得到长度为1 200 bp的条带,与预期片段相符;构建了含pdk内含子和RH1、RH2融合基因的RNAi植物表达载体,经Bam H I/Sac I双酶切,获得长度约3 200 bp的片段,表明RNAi植物表达载体pBI121-pRH构建成功;转化易感病毒马铃薯品种陇薯11号,PCR检测和PCRSouthern杂交分析表明融合基因已整合到陇薯11号马铃薯基因组中;抗病性检测显示4株转基因马铃薯植株对4种病毒均免疫。表明利用RNAi可筛选出抗多种病毒的转基因马铃薯新种质。     

江苏小麦条纹病毒病和中国小麦花叶病的初步鉴定

江苏位于长江中下游,地处中国的南北交界处,小麦上病毒病种类较多。先后发现的有小麦丛矮病(病原为北方禾谷花叶病毒,Northern cereal mosaic virus,NCMV)、小麦梭条花叶病(病原为小麦黄花叶病毒,Wheat yekllow mosaic virus, WYMV)、小麦黄矮病(病原为大麦黄矮病毒, Barley yellow dwarf virus,BYDV)、小麦“绿矮”病 (病原为水稻黑条矮缩病毒,Rice black streaked dwarf virus,RBSDV),其中小麦梭条花叶病曾大面积流行。2005年春天以来,江苏小麦上又发生了多种病毒病,除了以上4种,还出现了2种新的     

Viruses of tomato in plastic houses in Crete

Tomatoes grown in plastic houses in Crete have been inspected since 1980 for virus diseases. Plants with virus-like symptoms were checked by sap inoculation to test plants and the isolated viruses were identified by host reaction and serology. The most common viruses were, in order of frequency, tomato mosaic virus (ToMV), potato virus X, tomato bushy stunt virus (TBSV), potato virus Y and cucumber mosaic virus. The large use of ToMV-resistant cultivars reduces gradually the importance of ToMV while TBSV tends to become a serious problem of tomato in Crete.Samenvatting Van 1980–1984 werden op Kreta de in plasticfolie-kassen geteelde tomaten geïnventariseerd op de aanwezigheid van virussen. Verdachte plante werden door sapinoculatie op toetsplanten onderzocht. De daarbij geïsoleerde virussen werden daarna via de symptomen op de waardplanten en serologisch geïdentificeerd. In volgorde van belangrijkheid werden de volgende virussen het meest aangetroffen: tomatemozaïekvirus, aardappelvirus X, tomatedwerggroeivirus, aardappel Y virus en komkommermozaïekvirus, De laatste jaren is door de toenemende teelt van tomaterassen met resistentie tegen tomatemozaïekvirus het belang van dit virus sterk verminderd, terwijl het tomatedwerggroeivirus een steeds ernstiger probleem lijkt te worden.     

云南怒江番茄种植新区番茄病毒检测及病毒种传特性

病毒病对番茄生产造成严重危害, 近年来在番茄种植新区发生严重, 疑似为种子带毒传播。本研究通过对云南省怒江州番茄种植新区的番茄病毒病样品采用RNA-seq高通量测序, RT-PCR验证的方法检测病毒种类;对番茄病果种子进行超薄切片制样透射电子显微镜观察, 将病果种子播种后对种苗进行RT-PCR带毒检测。结果表明, RNA-seq高通量测序及RT-PCR检测到的病毒有番茄环纹斑点病毒(tomato zonate spot orthotospovirus, TZSV)、番茄黄斑驳相关病毒(tomato yellow mottle-associated virus, TYMaV)、辣椒脉斑驳病毒(chili veinal mottle virus, ChiVMV)、南方番茄病毒(southern tomato virus, STV)。透射电镜观察到种胚细胞及胚乳细胞中分布典型的正番茄斑萎病毒属Orthotospovirus病毒粒体。病果种子播种28 d后的种苗具有病毒病症状, 通过RT-PCR检出TZSV、ChiVMV、STV, 检出率分别为60%、100%、80%。上述研究结果为TZSV通过种子传播提供了有利的证据, 并为源头防控番茄病毒病提供了依据。     

Viruses of pepper in plastic houses in Crete

During a survey of virus diseases affecting pepper grown in plastic houses in Crete, during 1984–1986, tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV) were detected. The most common virus was TMV, being present in samples of alle pepper cultivars carrying the L₁ resistance gene, while ToMV was isolated only from susceptible pepper cultivars. According to responses ofCapsicum spp. the isolates from 640 samples checked were classified into three pathotypes: P₀, P_1.2 and P_1.2.3. Results of this study show that P_1.2 represents at present the major threat to the Cretan pepper industry.Samenvatting Bij een in de jaren 1984–1986 gehouden inventarisatie van virusziekten in paprika in plastic-foliekassen op Kreta werd zowel het tabaksmozaïekvirus (TMV) als het tomatemozaïekvirus (ToMV) aangetoond. TMV kwam zeer algemeen voor: het werd aangetroffen in alle monsters van de paprikarassen met het resistentiegen L₁. ToMV werd alleen geïsoleerd uit planten van vatbare paprikarassen. Volgens de waargenomen symptomen die de isolaten van 640 monsters opCapsicum spp. vertoonden, konden de isolaten in drie pathotypen, nl. P₁, P_1,2 en P_1,2,3, worden geklassificeerd. Uit het onderzoek is gebleken dat P_1,2 de belangrijkste bedreiging vormt voor de teelt van paprika op Kreta.     

Pelargonium flower-break and pelargonium line pattern viruses in the Netherlands; purification,antiserum preparation,serological identification,and detection in pelargonium by ELISA

Two viruses, detected frequently in the Netherlands in pelargonium, were identified by serology and test plant reactions. Antisera were prepared and an ELISA procedure was developed to detect the viruses in pelargonium.One of the viruses, PFBV-N, proved to be pelargonium flower-break virus. With the antiserum to PFBV-N, it could be detected reliably throughout the year inPelargonium zonale Springtime Irene.The other virus, PLPV-N, was serologically closely related to pelargonium line pattern virus (PLPV) and to pelargonium ring pattern virus (PRPV), as were an old virus isolate from Saturnus, collected in the Netherlands in 1971 (L128), and PLPV isolates from Yugoslavia (PLPV-Y) and Denmark (PLPV-D). There were only minor differences in host-plant reactions between the virus isolates. Based on these tests, PLPV and PRPV are considered as isolates of the same virus, for which, for practical reasons, the name pelargonium line pattern virus is proposed.PLPV could be reliably detected by ELISA inP. zonale Springtime Irene and Amanda throughout the year with only a few exceptions. InPelargonium peltatum Tavira, however, reslts were erratic due to uneven distribution of virus in the plant. Best results were obtained when petioles of fully expanded leaves were tested.     

Yellow tailflower mild mottle virus and Pelargonium zonate spot virus co‐infect a wild plant of red‐striped tailflower in Australia

Isolates of an Australian indigenous virus, Yellow tailflower mild mottle virus (YTMMV‐Kalbarri), and an exotic virus, Pelargonium zonate spot virus (PZSV‐SW13), are described from Anthocercis ilicifolia subsp. ilicifolia (red‐striped tailflower, family Solanaceae), a species endemic to Western Australia. This is the first report of either virus from this plant species. The complete genome sequences of YTMMV‐Kalbarri and of PZSV‐SW13 were obtained. YTMMV‐Kalbarri shared 97% nucleotide pairwise identity with the sequence of the type isolate YTMMV‐Cervantes. The sequence PZSV‐SW13 shared greatest sequence identity with the partial sequence of an Australian isolate of PZSV, also from a wild plant, and with a sunflower‐derived isolate of PZSV from Argentina. An experimental host range study was done of YTMMV‐Kalbarri using cultivated and wild solanaceous and non‐solanaceous plants. Most solanaceous plants became systemically infected, with symptoms of systemic infection ranging from symptomless to whole plant necrosis. Based on these studies, it is suggested that YTMMV has the potential to become a pathogen of commercial species of Solanaceae. This study provides further evidence that PZSV is present in wild plants in Australia, in this case an indigenous host species, and possible routes by which it invaded Australia are discussed.     

Inhibition of different cowpea viruses by non-virulent cowpea mosaic virus is dependent on the type of immunity of the plant to the inhibitory virus

Samenvatting In cowpea-planten die onvatbaar zijn voor het cowpea-mozaïekvirus (CPMV) kan dit virus, mits infectieus, infectie door vier andere, onderling geheel verschillende virussen remmen. Dit vermogen tot aspecifieke remming komt tot uiting in cowpea-planten met onvatbaarheid die bepaald wordt door één dominant gen, maar niet in planten met onvatbaarheid op basis van één recessief gen. De beide typen van onvatbaarheid laten dus in verschillende mate expressie van de genetische informatie van CPMV toe.     

烟草品种对烟草花叶病毒和黄瓜花叶病毒的抗性鉴定

 2010-2011年采用大田人工接种鉴定的方法,对生产中大面积推广使用的24份烟草品种进行了烟草花叶病毒（TMV）和黄瓜花叶病毒（CMV）的抗性鉴定。结果表明,供试品种对TMV和CMV的抗病性存在较大差异,对TMV表现抗病的有Coker86、吉烟5号、Coker176、CV87、辽烟8号、CV91、中烟90等7份材料;表现中抗的有秦烟98、双抗70、C151等3份材料;表现中感的有秦烟96、G80、金星6007、龙江981、K326、秦烟201、NC89等7份材料;表现感病的有G28、云烟97、净叶黄、红花大金元、RG11、云烟85、云烟87等7份材料。对CMV表现抗病的有Coker86、龙江981、C151、秦烟201、云烟87等5份材料;表现中抗的材料是金星6007;表现中感的有CV91、RG11、Coker176、中烟90、K326、红花大金元、净叶黄、G80、G28等9份材料;表现感病的有秦烟98、云烟85、秦烟96、NC89、双抗70、云烟97、CV87、辽烟8号、吉烟5号等9份材料。其中兼抗TMV和CMV两种病毒病的材料有2份,分别是Coker86和C151。同时研究还发现,抗病性不同的烟草品种在受到病毒危害以后,对烟叶的产量和品质的影响也不同。明确了中国24个烟草品种的抗病性水平,为抗病品种的利用与品种合理布局提供科学依据,同时为烟草抗病毒病育种的亲本选择提供抗源信息。     

云南美人蕉病毒病病原鉴定

In this study,the causal agents were identified from Canna indica viral diseased plants in Yunnan Province.The diseased C.indica plants mainly exhibited the symptoms like veinal chlorosis and yellowing,streak mosaic or interveinal chlorosis,while older leaves always showed veinal necrosis as well as chlorosis.Viral pathogens were detected by RT-PCR/PCR in 24 diseased C.indica samples collected from Kunming and Yuxi City in Yunnan Province.The results indicated that the main C.indica-infecting viruses were canna yellow mottle virus (CaYMV),bean yellow mosaic virus (BYMV),sugarcane mosaic virus (SCMV).CaYMV showed the highest detection rate of 87.5 %,whereas,the BYMV had the lowest rate of 16.7% in the 24 samples.Co-infections of CaYMV+SCMV,CaYMV+BYMV and CaYMV+SCMV+BYMV were also detected in the diseased samples.However,cucumber mosaic virus (CMV),tobamovirus,luteovirus,orthotospovirus,begomovirus and umbravirus were not detected in these samples.This is the first report of CaYMV and SCMV infecting C.indica in Yunnan province.     

云南美人蕉病毒病病原鉴定

In this study,the causal agents were identified from Canna indica viral diseased plants in Yunnan Province.The diseased C.indica plants mainly exhibited the symptoms like veinal chlorosis and yellowing,streak mosaic or interveinal chlorosis,while older leaves always showed veinal necrosis as well as chlorosis.Viral pathogens were detected by RT-PCR/PCR in 24 diseased C.indica samples collected from Kunming and Yuxi City in Yunnan Province.The results indicated that the main C.indica-infecting viruses were canna yellow mottle virus (CaYMV),bean yellow mosaic virus (BYMV),sugarcane mosaic virus (SCMV).CaYMV showed the highest detection rate of 87.5 %,whereas,the BYMV had the lowest rate of 16.7% in the 24 samples.Co-infections of CaYMV+SCMV,CaYMV+BYMV and CaYMV+SCMV+BYMV were also detected in the diseased samples.However,cucumber mosaic virus (CMV),tobamovirus,luteovirus,orthotospovirus,begomovirus and umbravirus were not detected in these samples.This is the first report of CaYMV and SCMV infecting C.indica in Yunnan province.     

北京地区辣椒上黄瓜花叶病毒和甜菜西方黄化病毒复合侵染的分子鉴定

辣椒是我国重要的蔬菜和经济作物,受多种病毒危害。2014年在北京市顺义区调查时发现部分种植的辣椒植株上叶片大面积黄化,边缘症状明显,个别植株叶片轻微上卷。提取典型症状样品的总RNA,反转录得到cDNA,分别用黄瓜花叶病毒(Cucumber mosaic virus,CMV)特异引物和马铃薯卷叶病毒属(Polerovirus)通用引物进行PCR检测,CMV特异引物和马铃薯卷叶病毒属通用引物分别扩增得到约650bp和1 400bp的特异条带。测序和核苷酸序列比对表明,其分别与CMV和甜菜西方黄化病毒(Beet western yellows virus,BWYV)序列同源性最高为99%和96%。这是对我国种植的辣椒上发生的CMV和BWYV复合侵染的首次报道。