【作者】 潘桂宇；

【导师】 李明；

【作者基本信息】 贵州大学 ， 农药学， 2006， 硕士

【摘要】 烟草花叶病毒病是农业生产上的重要病害之一，由于植物病毒的绝对寄生性，使其难于防治，目前国内外缺少高效的抗病毒药剂。随着人们生活水平的提高，环境问题备受关注。植物源抗病毒剂低毒、低残留、高效、与环境相和谐，是今后抗病毒剂发展方向。本文从中草药——天葵(Semiaquilegia adoxoides)块根中提取分离抗烟草花叶病毒(Tobacco mosaic virus，TMV)的活性物质，为抗病毒剂活性成分的筛选提供重要的理论依据与实践指导。 在师姐毕亚玲对天葵提取物对TMV活性研究基础上，改进了提取方法；采用半叶枯斑法以及成功摸索的酶联免疫吸附法进行了生物活性测定，筛选出天葵萃取物中抗TMV的活性成分；通过测定寄主植物体内几种酶活性的变化来研究天葵萃取物对TMV的作用机理。取得如下结果： 1．本研究明确了对天葵化学成分的初提取(萃取)方法，先用95％无水乙醇冷凝回流提取天葵根粉，提取液依次萃取，得到天葵石油醚萃取物、氯仿萃取物、乙酸乙酯萃取物、正丁醇萃取物和水部分，其萃取率分别为38.0％、7.5％、6.5％、2.4％、27.5％。 2．用烟叶半叶枯斑法进行对TMV活性测定，发现浓度为10.0mg／ml天葵萃取物对侵染心叶烟的13.0μg／ml TMV具有一定的抑制效果。试验结果表明：天葵萃取物有较强的治疗作用，其中乙酸乙酯萃取物治疗效果最好，离体效果、盆栽效果分别达71.41％、72.36％，均与对照药液病毒A差异极显著；石油醚萃取物与氯仿萃取物的盆栽抑制效果分别为50.26％、47.01％，均与对照药液病毒A差异显著；但氯仿萃取物离体治疗效果为52.69％，与对照药剂差异不显著。钝化效果表明：乙酸乙酯萃取物的钝化效果最好，达到69.67％，与病毒A差异极显著；氯仿萃取物、石油醚萃取物钝化效果分别为53.39％、49.86％，与对照药液效果差异不显著。保护作用中，离体效果较好的氯仿萃取物、乙酸乙酯萃取物，抑制效果分别是41.01％、35.4％，与对照药剂差异极显著；盆栽实验中，氯仿萃取物、乙酸乙酯萃取物、石油醚萃取物抑制效果分别为42.04％、41.83％、37.14％，均与对照药剂病毒A差异极显著。 3．用酶联免疫吸附法进行天葵萃取物对TMV生物活性测定结果进一步表明：处理后寄主植物病毒侵染量达到最高值的时间推迟；处理后对寄主植物症状有较好的减轻效果。本试验在保护作用中，石油醚萃取物、氯仿萃取物、乙酸乙酯萃取物、正丁醇萃取物、病毒A处理寄主植物后病毒OD(于492nm光密度)值在第5天达到最大值，而阳性对照在3天出现最大值，说明处理能延迟TMV侵染时间。氯仿萃取物保护效果较好，且优于其他更多还原

【Abstract】 Tobacco mosaic virus（TMV） is one of the important diseases in agriculture productions, almost all of agronomic crops are endangered by Tobacco mosaic virus（TMV）. It is difficult to control, because of the plant virus’ absolute parasitism. At present, there is a lack of profitable antiviral medicament products both here and abroad. With the development of the living standard of the people, they pay more and more attention on the environmental problem. Planting pesticides are the way of the future, for its characteristics such as low toxicity, low residual, highly active and in agreement with the environment. Passing the method of biological activity track, getting the bioactivity substance TMV from the earthnut of Semiaquilegia adoxoides （a kind of herbal medicine） by extraction and separation, it afford the important academic theory and practical direction for the filtration on antiviral agent’s active component.The study is based on Bi yalin’ s （Studies on Bioactivity of the Extracts from semiaquilegia adoxoides to TMV）, and is ameliorated on the method of extraction. Based on bioactivity determination by using the half a leaf withered spot method, I used Enzyme linked Immunosorbent assay （short for ELISA）, and determined several enzyme activity in infected plants, which were sprayed TMV, to filtrate the active component in extracts of Semiaquilegia adoxoides .The results as follow:1. In the study, I explored the way how to distill the chemical composition from Semiaquilegia adoxoides, I used the method of condensation circumfluence to distill the powder of earthnut; then used petroleum ether, chloroform, ethyl acetate and n-butanol extracted the extraction in order; after that, I got the extraction of them and water. At last, I counted those data, which we got, then got the rate of extraction differently was 38.0%、1.5%、 6.5%、 2.4%、 27.5 %.2. I discovered that the extraction of Semiaquilegia adoxoides （TMV）, which concentration was 10.0 mg·ml^-1, had definite inhibitory effect on the infected Nicotiana glutinosa for the first time, when I used the half a leaf withered spot method to do the experiment about the resisting TMV determination of activity. The experimental results show that: the extractions of Semiaquilegia adoxoides have good therapeutic actions. There into, the extraction of ethyl acetate is best on treatment, and its effect of in vitro is 71.41%, its effect of potted plant is 72.36%, both of them are evidently better than antithetic liquid medicine virus A; the potted plant inhibitory effects of extraction of petroleum ether and chloroform are 50.26% and 47.01%, the difference between extraction of petroleum ether and chloroform and control solution virus A is quiet prominence; but the difference between extraction of chloroform and virus A isn’t prominence, the effect of chloroform’s extraction is 52.69%. In protective actions, the extraction of chloroform and ethyl acetate both have a good effect on in vitro, the C of them are 41.01% and 35.4%, and the difference between the extraction of chloroform and ethyl acetate and control solution is quiet prominence. In the experiments of potted plant, the inhibitory effects of chloroform extraction, ethyl acetate extraction and petroleum ether extraction are 42.04% 41.83% and 37.14%, all of them have quietly distinct difference with virus A. On inactivation, the effect of ethyl acetate extraction is best, it come up to 69.67%, which is distinct better than virus A; but chloroform extraction and petroleum ether extraction don’t have distinct difference with virus A in inactivation, the results of them are 53.39% and 49.86%. in the experiment, we find that, both of in vitro inoculation method and living inoculation method have definite effect on the results of biological assay, but the trend of them are accordant.更多还原