黄瓜花叶病毒株系间交叉保护作用与卫星RNA干扰作用的比较研究

用除了卫星RNA和含有卫星RNA的CMV分离物在烟草和甜椒上进行了两组不同设计的温室实验。一组在不同保护接种后,在不同时间攻强毒CMV;另一组在不同保护接种后,在同一时间攻强毒CMV。攻毒后进行寄主症状调查、病情指数统计以及强毒CMV的A蛋白夹层ELISA测定,发现交叉保护和卫星RNA干扰作用在体内都存在,但其作用时间和程度不同。交叉保护在保护接种后10—15d以前明显,15d以后很弱;而卫星RNA干扰作用则很强,尤其在保护接种10d以后保护很完全,并且维持到30d以后。     

卫星RNA对黄瓜花叶病毒基因组RNA体外合成的影响

卫星ＲＮＡ对黄瓜花叶病毒基因组ＲＮＡ体外合成的影响杨海花,康良仪,赵大健,田波（中国科学院微生物研究所,北京１０００８０）关键词卫星ＲＮＡ,黄瓜花叶病毒,依赖ＲＮＡ的ＲＮＡ聚合酶,体外合成利用卫星ＲＮＡ生防制剂控制田间的番茄、青椒、烟草等由黄瓜花叶病...     

卫星RNA作为黄瓜花叶病毒病生防因子的研究III．卫星RNA对黄瓜花叶病毒感染的烟叶组织中病毒双链RNA含量的影响

用3H尿嘧啶核苷标记、聚丙烯酰胺凝胶电泳分析、放射自显影等技术以及根据抗RNA酶和DNA酶的性质比较了感染黄瓜花叶病毒(CMV)及其带有卫星RNA的黄瓜花叶病毒(CMV-S52)的三生烟叶组织中病毒ds RNA含量的变化。结果表明,随接种后时间的延长,CMV-S32感染的组织中卫星RNA的ds RNA含量呈直线增长,第10天达最高峰,而病毒基因组RNA的ds RNA的含量则随时间延长而降低,这一趋势一直延续到最后一次测定。卫星RNA的双链RNA含量也比病毒基因组ds RNA高得多,从接种后的第二天的2．5倍到第10天的20倍及第l{天的近40倍,但不含卫星RNA的cMV接种后在所测定的14天内,各基因组RNA的ds RNA含量比较衡定。     

带卫星RNA黄瓜花叶病毒保护接种的植物中病毒积累与基因组RNA合成及病状表达

本文报道了三生烟在接种带卫星RNA的黄瓜花叶病毒(CMV-S52)后,或接种后用强毒CMV攻击,其病状表现、组织中病毒含量,病毒基因组RNA合成与卫星RNA合成的关系.植物在接种CMV-S52后7～10天,所有接种植物都表现出不同程度的轻度花叶症状,此期正是组织内病毒含量最高,而卫星dsRNA占总dsRNA百分比较低时期.接种后不同时间的测定证明,组织内病毒基因组dsRNAl和dsRNA2的合成水平均较低,而卫星dsRNA则始终保持较高的合成水平;到接种后15天病状消失时,卫星dsRNA占总dsRNA合成百分比的84.79％之多. 用CMV-S52保护接种后再用强病毒攻击,植物中的病毒含量、病毒ssRNA合成不增加或稍有增加;而基因组dsRNA和卫星dsRNA合成都有增加,但卫星dsRNA合成仍占绝对优势.保护作用的效果,取决于攻强病毒时已存在于组织内的卫星RNA与基因组RNA合成量的相对比例。讨论了卫星RNA的保护机理。     

含卫星RNA的黄瓜花叶病毒弱株系的分离鉴定及在病毒病防治上的应用

从豌豆上分离获得黄瓜花叶病毒分离物ＣＭＶＰ１,摩擦接种９科２９种植物,ＣＭＶＰｌ在大多数植物上症状很轻或无任何症状,提纯的病毒颗粒为球形,直径约２８ｎｍ,病毒衣壳蛋白亚基分子量约２７．５ｋＤ,所含核酸有五个组份,即ＣＭＶＰ１含有卫星ＲＮＡ。ＣＭＶＰ１接种三生烟后８－１２天内呈轻花叶,此时组织中病毒含量最高,随后症状消失,去除卫星ＲＮＡ能加重ＣＭＶＰ１在番茄上的症状,因而是卫星ＲＮＡ减轻了ＣＭＶＰｌ的病状。当ＣＭＶＰ１保护接种番茄后攻强毒,番茄发病率低,病情轻,保护率达９０％以上,并有一定的刺激生长作用,还能提早开花４天,植株结果数增多,成熟果实的颜色、形态、品质和重量均正常。ＣＭＶＰ１对烟草亦具有很好的保护效果。保护接种的植株能明显减少强毒株侵染,可减少９０％以上。     

黄瓜花叶病毒卫星RNA致弱辅助病毒的机理

分析了卫星RNA致弱的黄瓜花叶病毒 (CMV)侵染的烟草叶绿体内的CP浓度与花叶症状的严重程度成正相关 .用番茄不孕病毒 (TAV)接种表达CMV卫星RNA的转基因烟草叶绿体中 ,TAV- CP含量明显比不含卫星RNA的TAV株系侵染的低 ,而在细胞质中TAV -CP含量无差别 .用完整游离叶绿体进行体外跨膜运输试验 ,CMV- CP能快速进入离体叶绿体 ,CP降低叶绿体动力学荧光光谱 .将CMV的CP基因与 1 ,5 二磷酸核酮糖羧化酶小亚基引导肽基因进行体外拼接 .借助农杆菌转化烟草 .转基因烟草表现出黄化、根系发育受抑制 ,产生类似病毒侵染的症状 .     

系统侵染寄主中黄瓜花叶病毒及其卫星RNA的动态变化

以³²P标记的黄瓜花叶病毒（CMV）RNA3 cDNA片段和卫星RNA全长cDNA作为探针,定量测定CMV基因组RNA和卫星RNA的含量变化,结果显示：二者均具有明显的寄主效应和时间效应.在16～20℃条件下,接种不携带卫星RNA的分离物CMV-R3,15天、30天和75天时,CMV基因组RNA负荷量呈显著下降的趋势.在第15天,RNA3的负荷量以烟草>心叶烟>克里夫兰烟>番茄的顺序表现为不同寄主的显著性差异.相同条件下接种携带高拷贝卫星RNA的分离物CMV-RS,在5天和15天之间基因组RNA和卫星RNA负荷量均呈现上升的趋势,同时测得其基因组RNA和卫星的负荷量具有相似的寄主效应和时间效应,但程度不同.第15天时,二者负荷量以烟草＞心叶烟＞番茄的顺序表现寄主效应的显著性差异.在18～21℃条件下,接种携带坏死卫星RNA的CMV强毒株HC4,第5天、第10天和第15天时,基因组RNA和卫星RNA的负荷量均以番茄＞心叶烟＞烟草的顺序表现出显著性差异,并表现出明显的时间效应.不同来源CMV分离物还存在寄主选择性差异.     

黄瓜花叶病毒卫星RNA人工突变体的构建及其侵染性测定

从1个369nt的黄瓜花叶病毒(Cucumbermosaicvirus,CMV)卫星RNA的cDNA出发,采用DNA改组技术构建人工突变体,经过体外转录,将其与不携带卫星RNA的黄瓜花叶病毒株进行假重组,鉴定突变体的生物活性,结果显示所获得的4个卫星RNA的点突变子MS1、MS5、MS6和MS11中,只有MS11仍然具有复制能力;而其他3个点突变子,尽管均只有1个位点的替换,却不能在辅助病毒作用下复制。序列比较分析发现MS11的突变位点位于卫星RNA变异区内,发生的突变与自发突变一致,其他3个突变子的突变位点发生在卫星RNA的高度保守区。而且通过侵染性试验证实突变子MS11与野生型Yi没有明显的差异。由此可推测卫星RNA序列中的高度保守区与卫星RNA的生物活性密切相关,个别碱基的突变会导致RNA二级结构的改变,进而引起其复制能力或稳定性的完全丧失。     

表达黄瓜花叶病毒卫星RNA的转基因烟草耐烟草花叶病毒

表达黄瓜花叶病毒(CMV)的卫星RNA的转基因烟草可以抗CMV的侵染.为了决定这些植物对其它病毒,如烟草花叶病毒(CMV)的安全性,我们对这些植物接种CMV.结果发现 卫星RNA可减弱TMV引起的症状,井降低病情指数,然而却对TMV在植物中的积累没有明显影响.这一发现有助于对卫星RNA抗病机制的理解,有助于含卫星RNA的生防制剂及表达病毒卫星RNA的转基因植物的应用.本文在国际上首次报道卫星RNA可减弱非相关病毒引起的症状.1 材料和方法1.1 病毒及植物烟草花叶病毒(TMV),烟草G140种子及枯班三生烟种子均为中国科学院微生物研究所病毒室保存;含黄瓜花叶病毒卫星RNA-R1基因的烟草G140(烟Sat-G140)为中国科学院微生物研究所病毒室培育,所用材料为第三代种子.     

Plant resistance to fungal diseases induced by the infection of cucumber mosaic virus attenuated by satellite RNA

Biological control agents (BCAs) composed of attenuated cucumber mosaic (CMV) and its satellite RNA for controlling CMV diseases were found to induce plant resistance to a number of fungal diseases. Tests conducted in both the field and greenhouse showed evident protective effects against fungal infections by the BCAs. Artificial inoculation with a fungal spore suspension using BCA-treated plants, satellite transgenic plants and plants infected with CMV alone indicated that the resistance to fungi was induced by the virus infection, not by the presence of satellite RNA.     

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02-0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

Anti-sense RNAs of cucumber mosaic virus in transgenic plants assessed for control of the virus

Three synthetic genes for the production of anti-sense RNA to different regions of the cucumber mosaic virus (CMV) genome were constructed using virus-derived double-stranded cDNA coupled to a promoter sequence from cauliflower mosaic virus. The genes were used to transform tobacco plants by a Ti plasmid vector. Transgenic plants obtained with the three constructs produced anti-sense RNA at different levels. Plants expressing each of the three anti-sense RNAs were inoculated with CMV and their sensitivity to the virus infection was compared with the non-transformed plants. Only one plant line which expressed relatively low levels of one of the anti-sense RNAs showed resistance to CMV but other plants expressing the same or the other two antisense RNAs had similar sensitivity to CMV infection as the non-transformed plants.     

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02–0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

Evolutionary relationship of alfalfa mosaic virus with cucumber mosaic virus and brome mosaic virus

The amino acid sequences of the non-structural protein (molecular weight 35,000; 3a protein) from three plant viruses — cucumber mosaic, brome mosaic and alfalfa mosaic have been systematically compared using the partial genomic sequences for these three viruses already available. The 3a protein of cucumber mosaic virus has an amino acid sequence homology of 33.7% with the corresponding protein of brome mosaic virus. A similar protein from alfalfa mosaic virus has a homology of 18.2% and 14.2% with the protein from brome mosaic virus and cucumber mosaic virus, respectively. These results suggest that the three plant viruses are evolutionarily related, although, the evolutionary distance between alfalfa mosaic virus and cucumber mosaic virus or brome mosaic virus is much larger than the corresponding distance between the latter two viruses.     

Plant growth-promoting rhizobacterial mediated protection of tomato in the field against cucumber mosaic virus and its vector Aphis gossypii

Studies evaluated the effect of rhizobacterial interactions on a virus under natural conditions of high levels of vector–virus pressure on tomato plants. The rhizobacterium Bacillus subtilis was isolated from the rhizosphere soil of healthy tomato plant and the strain is referred to as BS3A25. The BS3A25 treatment of seeds significantly enhanced tomato seed germination rates to 99% compared to the control seeds at 78%. Combination of both seed and foliar spray treatments significantly protected the plants from cucumber mosaic virus (CMV) and also significantly enhanced plant growth. Strain BS3A25 possesses many positive traits such as phosphate solubilization ability, ACC utilization as the sole source of nitrogen and production of significantly higher concentrations of indoles, IAA and the cytokinin, IPA. The growth parameters of the aphid Aphis gossypii Glover such as development time, time from birth to adult (t_D) and prereproduction time (t_d) were longer, whereas the relative growth rate (RGR) and intrinsic rate of natural increase (r_m), were found to be lower in the BS3A25 supernatant treatment compared to commercial insecticide imidacloprid treatment. Noticeably, the present study recorded higher activity of phenylalanine ammonia lyase, Peroxidase, total phenols, and polyphenol oxidase in plants treated with BS3A25 generating the speculation of induced defence responses in plants. The results obtained in the field study showed that treatment by BS3A25 formulation as seed and foliar treatment proved highly effective in reducing the aphid population and CMV incidence compared to a commercially available insecticide. The highest cost:benefit ratio (1:2.5) was obtained in BS3A25 formulation treatment followed by imidacloprid (1:2.0) and control plants (1:1.4). These constitute an important step towards the production of BS3A25 as a commercially-available induced resistance agent.     

Satellite RNA-mediated reduction of cucumber mosaic virus genomic RNAs accumulation in Nicotiana tabacum

Satellite RNAs(satRNAs)are molecular parasites that interfere with the pathogenesis of thehelper viruses.In this study,the relative accumulation of cucumber mosaic virus(CMV)-Fny genomicRNAs with or without satRNAs were quantitatively analyzed by real-time RT-PCR.The results showed thatsatRs apparently attenuated the symptoms of CMV-Fny on Nicotiana tabacum by depressing the accumu-lation of CMV-Fny genomic RNAs,tested as open reading frames.The accumulation of CMV-Fny 1a,2a,2b,3a,and CP genes was much higher than that of CMV-Fny with satRs added(CMV-Fsat),at differentinoculation times.CMV-FnyΔ2b,in which the complete 2b gene and 41 amino acids at the C-terminal of the2a gene were deleted,caused only a slight mosaic effect on N.tabacum seedlings,similar to that of CMV-Fsat,but the addition of satRs to CMV-FnyΔ2b showed further decrease in the accumulation of CMV-FnyΔ2b genomic RNAs.Our results indicated that the attenuation of CMV,by adding satRs or deleting the2b gene,was due to the low accumulation of CMV genomic RNAs,and that satRNA-mediated reduction ofCMV genomic RNAs accumulation in N.tabacum was possibly related to the 2b gene.     

抗烟草黄瓜花叶病毒多糖筛选及其对烟草防御酶活性的影响

[目的]筛选出对烟草黄瓜花叶病毒病有良好抑制作用的多糖并探索其对烟叶防御酶活性的影响.[方法]采用半叶法,测定了安络小皮伞多糖等21种真菌多糖在枯斑三生烟上对CMV的钝化、预防及治疗效果,并测定了抗病毒多糖处理后普通烟NC-89体内防御酶的变化.[结果]安络小皮伞多糖对CMV具有较好的钝化及预防效果,其200倍液与等量供试病毒液混合30 min后接种,钝化效果为83.41%;喷施安络小皮伞多糖200倍液24 h后接毒处理,预防效果可高达93.15%.安络小皮伞多糖对CMV防治机理的研究表明,多糖处理后烟草相关防御酶POD、PAL和PPO活性增强,其中喷施安络小皮伞多糖24 h后接毒处理的酶活增加最为显著,该处理烟苗的POD、PAL和PPO的酶活峰值分别可增加至对照的2.74、3.45和2.82倍.[结论]安络小皮伞多糖通过增强烟草体内防御酶活性而提高烟草对烟草黄瓜花叶病毒病的抗性.