【作者】 翟熙伦；

【导师】 王凤龙；

【作者基本信息】 中国农业科学院 ， 作物， 2012， 硕士

【摘要】 为了筛选出对烟草花叶病毒（Tobacco mosaic virus，TMV）和马铃薯Y病毒（Potato virus Y，PVY)有双重高效、高活性的生物源抗病毒物质，从山东诸城等植烟区采集烟草病毒病高发区健康烟株中烟100（Nicotiana tabacum var. Zhongyan100）的根际土壤，分离得到抗TMV的活性菌株，LB培养基培养纯化。温室条件下用半叶法接种枯斑三生烟（Nicotiana tabacum var.samsun NN），对TMV的抑制效果可达98%以上，该菌株命名为4A1。为进一步验证该菌株的抗病毒活性，本研究采用Real-time PCR技术定量检测了该菌株对TMV和PVY的抑制作用。检测结果显示其发酵液对烟草TMV、PVY具有显著的抑制活性，抑制率分别达到91.4%-100%和93.1%-100%。田间试验表明，处理I菌液与病毒混合2h后接种可有效抑制大部分病毒侵染，TMV、PVY发病率分别为5.3%、7.7%，病情指数分别为0.8和2.9，都远远低于其他处理。分子鉴定结果表明该菌株16S rDNA序列与Pseudomonas monteilii的16s rDNA序列同源度最高，同源率达到99%。生理生化测定菌株4A1为革兰氏阴性，氧化酶、淀粉水解VP、吲哚等反应为阴性，明胶液化、葡萄糖反应为阳性，能运动，确定该菌株为蒙氏假单胞菌（Pseudomonas monteilii）。将鉴定出的拮抗菌株从培养基和发酵条件的优化等方面进行摇瓶发酵研究。4A1菌株适宜的摇瓶发酵条件是：适宜温度28℃，时间为48h，接种量3%，初始pH=10，装液量200mL/500mL，葡萄糖和玉米粉作为混合碳源，酵母膏和蛋白胨为混合氮源，能够达到理想效果。同时，本研究对菌株发酵液的活性物质进行了分离纯化。通过发酵液预处理、液-液萃取后，浓缩得到活性粗提物，利用D-101大孔树脂吸附法、硅胶色谱法进行分离纯化活性物质，最后将所得的浓缩物采用气相色谱-质谱联用(GC/MS)的方法进行化学分析和结构鉴定。萃取得到的活性物质经生物学测定，结果表明乙酸乙酯萃取后液相有抗TMV活性，应用GC/MS技术进行结构鉴定和化学分析后，共得到4种化合物，分别是2，4，6(1H，3H，5H)-Pyrimidinetrione，5-butyl-5-ethyl-1，3-bis(trimethylsilyl)；Di-n-octyl phenyl phosphate；1，2-Benzenedicarboxylic acid,mono(2-ethylhexyl)ester；Hexanedioic acid，bis(2-ethylhexyl) ester。此外，本研究对活性小分子物质对TMV和PVY的拮抗机制做了探索。电镜观察表明活性小分子物质使病毒粒体断裂并且排列凌乱无序，破坏了病毒粒体的完整性。经过聚丙烯酰胺凝胶电泳证明活性物质能够降解病毒外壳蛋白，使病毒粒体失去侵染性。更多还原

【Abstract】 In order to develop antivirus products, strain4A1with high efficiency and high activity againstTMV and PVY was selected from healthy tobacco(Nicotiana tabacum var. Zhongyan100) field fromthe area infected TMV in Zhucheng. The antiviral rate of4A1is up to98%by half-leaf method in lab.Real-time quantitative PCR was used to test the inhibition of TMV and PVY for further verifying theanti-viral activity. The strain was identified by the physiological and biochemical and molecularidentification. Sequence analysis results showed that the nucleotide sequence of the16S rDNA of thestrain had99%identity with that of Pseudomonas monteiliis. The strain4A1is gram-negative. Enzyme,starch hydrolysis reaction such as VP and indole were negative, Gelatin, liquefaction, glucose responsewere positive and can move.4A1was identified as P. monteilii based on the above physiological,biochemical and molecular test results. Field trials were handled. Treatment I mixed the virus juice withfermentation for2h can effectively inhibit most viral infection. TMV incidence rate was5.3%. PVYincidence rate was7.7%. Disease index respectively was0.8and2.9. Those are much lower than othertreatments.The results of strains’ culture medium and fermentation conditions showed conditions were asfollowing: the culture temperature:28℃, the culture time48h, inoculation amount3%, initial pH=10,ventilation volume100mL/250mL, glucose and corn powder as the carbon source, yeast extract andpeptone as mixed nitrogen sources. It may reach the ideal effect.Active constituents from bacterial strain4A1was isolated and purified through pretreatment,solvent extraction, D-101macroporous adsorption resin and silica gel chromatography adsorption.Liquid-liquid extraction was proved that the liquid phase has anti-TMV activity through biologicaldetermination. We analyze the chemical composition and structure identification by GC/MS. Weseparated4kinds of compounds which identified by GC/MS. There were2,4,6(1H,3H,5H)–Pyrimidinetrione,5-butyl-5-ethyl-1,3-bis(trimethylsilyl); Di-n-octyl phenyl phosphate;1,2-Benzenedicarboxylic acid, mono(2-ethylhexyl) ester; Hexanedioic acid, bis(2-ethylhexyl) ester.We made study about the mechanism of small molecular against TMV and PVY. The resultsshowed that TMV particles can be inactivated by the activity substances from4A1broth.Polyacrylamide gel electrophoresis demonstrated active material can crack virus coat protein. the virusparticle lose infectivity.更多还原