【作者】 朱春玉；

【导师】 吴元华；

【作者基本信息】 沈阳农业大学 ， 植物病理学， 2005， 博士

【摘要】 嘧肽霉素是由链霉菌产生的新型抗病毒农用抗生素,对烟草花叶病毒(Tobacco Mosaic Virus,TMV)引起的植物病毒病害有很好的防治效果。本文采用生理生化及分子生物学等手段对嘧肽霉素抗TMV的作用机制进行了深入研究,结果如下: 1、根据嘧肽霉素理化性质和已摸索的分离纯化条件,筛选了一套适合于该抗生素的、经济实用的分离纯化工艺。用吸附法、离子交换法及薄层层析法获得了嘧肽霉素的纯品。本文选用嘧肽霉素的纯品及其制剂进行抗病毒作用机制的研究。 2、嘧肽霉素对不同寄主上由TMV引起的病毒病害具有很好的防治效果。嘧肽霉素对烟草花叶病毒病害防治效果最好,该药剂对TMV具有较强的体外抑制作用,枯斑抑制率达96.2%;对寄主有一定的保护和治疗作用,枯斑抑制率分别为80.4%和39.7%。对TMV引起的辣椒、番茄花叶病毒病害也具有很好的防治效果,可延缓症状出现、减轻发病程度。另外,嘧肽霉素能降低植株体内TMV的浓度,接种前24h喷施嘧肽霉素处理的效果好于接种后24h喷药处理的,药剂与病毒混合1h后再接种的效果最好,抑制率高达96%。 3、嘧肽霉素对TMV粒体具有较强的体外抑制作用和一定的体内抑制作用,抑制率分别为92.1%和32.8%。体外抑制作用在测定的时间段内随着作用时间的增加而增强,并且不能通过稀释药剂来恢复病毒粒子的侵染能力。在电镜下观察,与药剂混合不同时间的病毒粒体结构受到了不同程度的破坏,病毒粒体出现断裂现象。另外药剂与病毒粒体混合液的紫外吸收光谱也发生了很大的变化,出现了没有规律的峰值,这些试验结果都充分说明嘧肽霉素对TMV具有很强的抑制作用。 4、采用生物学,同位素示踪和定量竞争—PCR三种方法系统地研究了嘧肽霉素对TMV—RNA的影响。试验结果表明,嘧肽霉素对离体TMV—RNA具有很强的降解作用,嘧肽霉素处理TMV—RNA后接种心叶烟,平均枯斑抑制率为92.6%;利用~3H—尿苷作为标记物结合放射性免疫分析法研究了嘧肽霉素对烟草花叶病毒RNA合成的影响,结果表明,嘧肽霉素可以抑更多还原

【Abstract】 Cytosinpeptidemycin is a newly reported antiphytoviral agricultural antibiotic. It was produced by fermentation broth of Streptomyces achygroscopicus var.liaoningensis, which was isolated from soil of Tianzhu hill in Shenyang. Cytosinpeptidemycin has no harmful effects to animals and environment. It can be used to inhibit plant disease caused by tobacco mosaic virus (TMV), and it has gained the pesticide register. Physiological, biochemical and molecular biological methods were adopted to study the action mechanism of cytosinpeptidemycin on TMV thoroughly by TMV/tobacco system. The results are as follows:1. According to its physical and chemical properties and known techniques of extraction and purification, a set of suitable craft was chosen to separate and purify the antibiotic,which is both economic and practical. Cytosinpeptidemycin was separated and purified using methods of adsorption with active charcoal and ion-exchange chromatography combining with the method of TLC, and the effects were obvious. Cytosinepeptide as an effective compoment was used for this research.2. Cytosinpeptidemycin is a kind of extensive antiphytoviral agricultural antibiotics, and it can prevent and cure virus diseases of various kinds of hosts caused by TMV effectively. The influence of cytosinpeptidemycin on the concentration of TMV in Plant with different treatments were tested by several methods, such as biological methods, UV absorption, and ELISA. Results indicated that cytosinpeptidemycin could decrease the concentration of TMV in plants. The effect of spraying 4% cytosinpeptidemycin 24h before TMV inoculation is better than that of spraying after inoculation. The treatment which virus was mixed with the 4% cytosinpeptidemycin for lh at the equal volume before inoculation got the best effect, and the rate of inhibition reached 96%;Theeffect of spraying 4% cytosinpeptidemycin 24h after inoculation was not distinct.3. The inhibition of cytosinpeptidemycin on TMV was studied by biological and Electronic Microscope method combining with UV absorption analytic method. The results showed that cytosinpeptidemycin could inactive strongly virions of TMV in vitro and in viro, and the inhibition rate reached 92.1% and 32.8%, respectively. The infectivity of TMV was not resumable by diluting the mixture of TMV and cytosinpeptidemycin. The virions treated with cytosinpeptidemycin appeared ruptures and abnormalities through Electron Microscopy, which showed that cytosinpeptidemycin destructed the structure of the TMV.4. Cytosinpeptidemycin degraded strongly TMV-RNA in vitro. TMV-RNA was treated with cytosinpeptidemycin before inoculation and the inhibition rate reached 92.6%.Tracing of ~3H-uridine was used to test the antiviral mechanism of cytosinpeptidemycin against tobacco mosaic virus also. The result showed that cytosinpeptidemycin inhibited the use of the precursor of viral RNA in the test of leaf discs and seedlings. A new method, quantitative competitive PCR with software of Image Master ED was used in quantitative detecting TMV-RNA in infected plant tissues and those treated with 4% cytosinpeptidemycin. Three different treatments were adopted. The results indicated that in all treatments TMV-RNA accumulation was reduced in TMV-infected tobacco leaves treated with 4% cytosinpeptidemycin. TMV-RNA accumulation was reduced greatly when TMV was mixed with cytosinpeptidemycin at the equal volume. The results were also confirmed by Indi-ELISA technique. Cytosinpeptidemycin inhibited the replication of TMV-RNA to reduce the harm to host caused by TMV. In conclusion, cytosinpeptidemycin inhibited the infection of TMV by reducing RNA accumulation in infected plants. The effect of cytosinpeptidemycin on the RNase activity was studied in order to find the action mode of cytosinpeptidemycin on replication of TMV-RNA. The results indicted that cytosinpeptidemycin inhibited replication of TMV-RNA by increasing RNase更多还原