一种食用菌提取物y3对烟草花叶病毒的钝化作用及其机制

测定了从食用菌毛头鬼伞(Coprinus comatus)中提纯的y3蛋白对烟草花叶病毒(Tobacco mosaic virus,TMV)的钝化作用,结果表明y3对TMV有较强的体外钝化作用,在心叶烟枯斑寄主上对TMV的抑制中浓度约为2．0μg／mL;y3在pH9．0时较稳定;TMV与y3混合后刚RNase处理,测得侵染率为61．74％,比未用RNase处理的对照降低了38．26％,说明y3具有一定的体外脱衣壳作用;另外电镜观察发现y3可使部分TMV粒体发生裂解,变短。     

壳寡糖对烟草花叶病毒的体外钝化作用

壳寡糖是一种由脱乙酰基几丁质经酶降解后制备的氨基葡萄糖,它具有多方面的生物活性,在调节植物的生理机能、调控植物形态发生、诱导植物抗病性、抗逆性以及抑制真菌、细菌的生长发育等方面的作用多有报道,但关于壳寡糖对植物病毒的抑制作用未见报道.     

检测烟草中烟草花叶病毒的RNA斑点杂交法

用普通烟草花叶病毒OM株3′-端约2kb的cDNA为探针,探索了用RNA斑点杂交法对烟草组织中烟草花叶病毒RNA进行检测的条件。这些条件包括用分子杂交法观察云南烟区和上海烟草上分到的烟草花叶病毒与OM株的同源性,从烟草组织中提取烟草花叶病毒的几种方法的比较,使RNA有效地固定在硝酸纤维素滤膜上的方法,烟草组织中是否有干扰RNA固定和杂交的物质,斑点杂交方法检测烟草花叶病毒的特异性、灵敏度等。     

侵染蚕豆的烟草花叶病毒研究

１９９３年４月从杭州蚕豆田病株上分离获得病毒分离物Ｂ－９３５Ａ,汁液摩擦接种５科１９种植物,Ｂ－９３５Ａ能侵染３科１２种植物。被侵染的蚕豆产生黄斑、花叶,后期叶片黄化并有坏列;在菜豆上产生局部枯斑。Ｂ－９３５Ａ的钝化温度为８５－９０摄氏度,稀释限点为１０＾－８－１０＾－７。病毒粒体杆状,平均长度约３０ｎｍ,病毒的衣壳蛋白亚基只有一条多肽链,分子量约为１７．５ＫＤ,包裹的核酸为单链ＲＮＡ,长约６．４     

烟草花叶病毒单克隆抗体杂交瘤细胞株的建立

用TMV免疫的BALB/c鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,获得10株能稳定传代並分泌抗烟草花叶病毒(TMV)单克隆抗体(McAb)的杂交瘤细胞株,其中T73McAb滴度最高,ELISA滴度高达1/655,360,能被TMV兔免疫血清所阻断,能中和TMV的感染性。但与其它植物病毒无交叉反应,与TMV不同毒株均有反应。     

广东烟草花叶病毒株系研究

烟草花叶病毒（TMV）引起的烟草花叶病在广东不同的烟草主产区存在着症状的差异。从广东梅州和南雄烟区采集的138个TMV标样中,选取6个不同症状的分离物作为研究对象,发现它们在不同种属寄主和不同烟草品种上表现明显的TMV普通株特征症状,虽然在某些寄主上其症状也有一些差异,但未能反映株系间的差异。这6个分离物具有几乎相同的病毒粒体形态、电泳迁移率、钝化温度以及紫外吸收特征。经琼脂双扩散法和间接ELISA法的血清学反应,表明它们具较强的血清学关系。因而它们有很强的同源性。经病毒粒体外壳蛋白的氨基酸分析,在氨基酸总数上,分离物GD1和GD3各有156个氨基酸．GD2和GD4各有159个,GD5和GD6分别有158和157个氨基酸。上述分析试验结果初步表明,在广东不同烟草主产区引起的症状有所差异的6个分离物均属IMV普通株。     

转望江南核糖体失活蛋白基因cassin烟草及其对烟草花叶病毒的抗性

通过构建植物表达载体,由农杆菌介导,将望江南核糖体失活蛋白基因cassin转入烟草。PCR和Southern blot杂交结果证明：外源基因已经以单拷贝整合到烟草基因组内,并且在后代发生遗传分离。RT—PCR和Northern blot杂交结果显示：外源基因可以正常转录。用不同浓度的TMV机械摩擦接种转基因T1、T2代各3个自交株系,以非转基因烟草为阴性对照,实验结果表明转基因烟草对TMV表现出不同程度的抗性。     

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.     

Evaluation of the Antiviral Activity of Extracts from Plants Grown in the Qinling Region of China Against Infection by Tobacco mosaic virus (TMV)

To investigate their antiviral potential, extracts from 126 plants grown in the Qinling region of China belonging to 103 plant species of 36 families were evaluated for antiviral activity against Tobacco mosaic virus. The activity of each plant extract on infection and replication was determined by local lesion and leaf‐disc methods. Extracts from nine species [Lactuca tatarica (Linn.) C. A. Mey, Rubus flosculosus Focke, Thermopsis lanceolata R. Br, Cotinus coggygria Scop Pulsatilla chinensis (Bunge) Regel, Thlaspi rvense Linn, Rodgersia podophylla A. Gray, Achnatherum splendens (Trin.) Nevski and Rhodiola eurycarpa (Frod.) S. H. Fu] strongly inhibited both infection and viral replication.     

毛头鬼伞(Coprinus comatus)中一种碱性蛋白的纯化及其活性

用离子交换层析(CM-sepharose FF)和凝胶层析(Superdex^TM 75)方法,从新鲜食用菌毛头鬼伞(Coprinus omatus)子实体中分离纯化出一碱性蛋白y3,经SDS-PAGE初步确定其分子量约为14．4kD。活性检测结果显示：当其浓度为12．5μg／mL时,对烟草花叶病毒(TMV)在心叶烟枯斑寄主上的侵染抑制率达83．0%;y3对兔血凝集活性滴度为2^5,对人血凝集活性滴度为26,其浓度分别为1．562μg／mL和0．78lμg／mL;利用胃癌细胞株MGC-803检测y3体外抗肿瘤活性,其IC50为12μg／mL。y3 N-端序列为NRDVAACARFIDDFCDTLTP,为一新的蛋白序列。在SWISS-PORT上登录号为P83477。     

Proteasomes (prosomes) inhibit the translation of Tobacco mosaic virus RNA by preventing the formation of initiation complexes

Proteasomes (prosomes) are large multiprotein complexes. They are involved in protein degradation of ubiquitin-conjugated proteins and in the generation of MHC class I peptides. We gave further evidence that they interfere within vitro protein synthesis. Proteasomes inhibit the translation of Tobacco mosaic virus RNA. Analysis of cell-free systems by sucrose gradient centrifugation revealted that they prevent the formation of 80S initiation complexes but not the early phase of initiation.     

Plasma Membrane NADPH Oxidase in Tobacco tobacco mosaic virus Interaction

The plasma membrane NADPH oxidase and its regulatory role in the production of reactive oxygen species (ROS) in tobacco (Nicotiana tabacum L. )-tobacco mosaic virus (TMV) interaction was examined by using tobacco cv. "Samsun NN" (incompatible with TMV, containing the N gene for resistance to TMV) and tobacco cv. "3002" (compatible with TMV) as experimental materials. Plasma membrane (PM) vesicles were isolated from leaves of tobacco by a biphasic aqueous system. The membrane preparations were sealed, highly purified and largely in right-side-out orientation as detected by marker enzyme assays and latency studies of the PM marker, vanadate-sensitive ATPase with non-ionic detergent Triton X-100. The oxidase activity was assayed by the rate of SOD-sensitive Cyt c reduction in PM system. The oxidase activity could be increased about 80% when adding 0.01% Triton X-100 in the reactive system. This result showed that the binding-site of NADPH was on the cytosolic side of the plasma membrane and the production of O2- is on the apoplastic side. DPI (diphenylene iedonium), a specific inhibitor of the NADPH oxidase in neutrophils, also inhibited the NADPH oxidase activity in tobacco. Furthermore, the oxidase activity increased in incompatible interaction, but not in compatible interaction. The role of NADPH oxidase in the production of reactive oxygen species and stimulation of hypersensitive reaction were discussed.     

Characterization of Synergy between Cucumber mosaic virus and Tobacco necrosis virus in Nicotiana benthamiana

Mixed infections of Nicotiana benthamiana plants by Cucumber mosaic virus (CMV) and Tobacco necrosis virus (TNV) exhibit a synergistic interaction and result in symptom enhancement. Accumulation of CMV(+) RNA as well as capsid protein (CP) in mixed infection was considerably higher than that of singly‐infected plants. There was also a slight increase in TNV(+) RNA and CP levels in doubly infected plants. Synergistic infection by CMV‐ and TNV‐induced higher increase in the levels of malonyldialdehyde, hydrogen peroxide (H₂O₂) and more decline in the activities of catalase than singly infected ones. Both peroxidase and superoxide dismutase activities increased rapidly for the first 10 days post inoculation (dpi) in doubly‐infected plants and then declined, whereas the enzyme activities continued to increase after 10 dpi in singly infected plants and had higher enzyme activities in the late stages than that of co‐infected plants. These results suggest that synergistic infection by CMV and TNV produced severes oxidative stress in N. benthamiana plants and the synergy between the two viruses was mutual.     

榆黄蘑中一种抗病毒蛋白的纯化及其抗TMV和HBV的活性

采用阴离子交换层析和凝胶层析方法从新鲜食用菌榆黄蘑(Plearotus citrinopileatus)中进行了抗病毒蛋白的纯化,结果获得了一个纯化蛋白YP46-46,经SDS-PAGE可确定其分子量为27.4kD.以半叶法在枯斑寄主心叶烟上检测该蛋白对烟草花叶病毒(TMV)的抑制率,发现有较好的抗TMV活性,其抑制TMV的中浓度为0.24μg/mL.同时以HepG2.2.2.15细胞株为模型,对所获得的蛋白进行体外抗乙型肝炎病毒效果的评价.结果表明该蛋白对HBsAg的50%抑制时的浓度为0.08μg/mL,但对HBeAg效果不大.     

Induction of systemic resistance in tobacco against Tobacco mosaic virus by Bacillus spp.

Bacillus pumilus strain EN16 and Bacillus subtilis strain SW1 were tested for their systemic resistance and protection abilities against tobacco mosaic virus disease under greenhouse conditions. The results showed that strain EN16 and SW1 treatment significantly reduced mosaic symptoms and disease severity, resulting in 52 and 71% protection at 14 days of inoculation, respectively. A decreased amount of virus was detected in EN16- or SW1-treated tobacco plants by ELISA. Moreover, 5- and 7-day intervals between inducer treatment and pathogen inoculations were respectively required for strain EN16 and SW1 to induce optimal resistance. Further analysis on phenylalanine ammonia-lyase, peroxidase, polyphenol oxidase and pathogenesis-related (PR) proteins in tobacco showed that the amounts of defense enzymes and PR proteins significantly increased in Bacillus-treated plants challenged with pathogen when compared to control.     

云南五个不同地区烟草花叶病毒外壳蛋白基因的序列比较

Five Tobacco mosaic virus isolates, obtained from tobacco leaves showing typical symptom in Qujing, Honghe, Dali, Chuxiong and Yuxi in Yunnan province, were selected and studied from 637 TMV samples. Using a pair of primers specific for TMV-U1 strain, a specific fragment of 530bp including the TMV coat protein gene was amplified using IC-PCR. The products of PCR were cloned and sequenced. The nucleotide and amino acid sequences of the coat protein of the five isolates were found to be very similar each other and have more than 90% sequence identity with TMV-U1, TMV-B,TMV-P, TMV-FUJIAN, although minor differences existed among them. The results showed that the five isolates from Yunnan province belong toTMV-U1 strain.