几种植物源活性物质的抑菌、抗病毒效果及其机理研究

发布时间：2018-03-21 02:57

本文选题：植物源活性物质　切入点：烟草花叶病毒　出处：《西南大学》2017年硕士论文　论文类型：学位论文

【摘要】：寻找植物源活性物质是天然产物农药研发的重要途径。本文一方面对常用的抗病毒物质筛选以及评价方法进行优化和探索,另一方面结合相关研究成果和报道,广泛搜集可能具有抗病毒或抑菌活性的植物源物质,研究其对烟草赤星病菌[Alternaria alternata(Freis)Keissler]和烟草花叶病毒(Tobacco mosaic virus,TMV)的抑制效果,并初步探讨其作用方式和机制。得到的主要研究结果如下:1.以本氏烟和几种常用的植物病毒抑制剂为实验材料,分别将药剂注射于烟叶的五个点和整片烟叶,利用农杆菌介导法接种TMV-GFP,2 d后在紫外灯下观察并统计不同处理后的荧光斑面积与斑点数量。结果显示,各处理组荧光强度均低于对照组,荧光斑的统计情况与ELISA检测结果相近,表明荧光强弱情况能够大致反映叶片内病毒含量。并且本体系对几种病毒抑制剂的室内评价结果与田间药效结果基本一致,说明本文建立的2种筛选方式可以用于抗病毒物质的活性评价和筛选。与筛选抗病毒物质常用的枯斑法和叶碟法相比,该体系不仅能够快速评价抗病毒物质的活性还能直观的反映药剂在病毒扩散和复制过程中的作用特点,具有操作简单,现象直观,周期短的优点。2.以烟草花叶病毒为靶标,对收集到的15种植物源物质进行抗病毒活性测定,经过初筛、复筛并结合防御酶活性情况,筛选出熊果酸与4-甲氧基香豆素2种能够降低病毒初侵染含量的活性物质。研究其作用方式与机制,结果显示,熊果酸与4-甲氧基香豆素能够不同程度提高烟草中SOD、PPO、POD、CAT这几种防御酶活性,降低烟叶中过氧化物含量;在病毒侵染前期可以降低植物细胞的膜脂过氧化程度,减轻细胞膜受损;并且能够上调PR1、NPR1和PR2等防卫反应相关基因表达量,促进病程相关蛋白的合成,从而降低病毒初侵染含量,具有保护和延缓病程的作用。体外作用研究发现,2种活性物质与TMV混合1 h后,病毒粒体发生了明显的聚集现象,而溶剂处理后的病毒分散均匀无聚集,其中熊果酸处理后的聚集程度较4-甲氧基香豆素更为明显。将处理后的病毒摩擦接种至本氏烟,荧光的斑点数量和病毒含量都有所降低,说明2种物质能在体外直接作用病毒粒体,降低病毒的侵染活性或体内复制水平,对病毒具有一定钝化作用。3.利用平板抑菌法,以烟草赤星病菌为靶标,测定15种植物源物质的抑菌活性,发现丁香油与丁香油酚具有较好的抑菌作用。丁香油含量为4.13%时能够强效抑制赤星病菌孢子的萌发,丁香油含量为0.52%时显著降低菌丝的生长速度,减少菌丝的生长量。进一步研究作用机理发现,丁香油的作用位点之一是细胞膜,其能破坏赤星病菌的细胞膜结构,增加质膜通透性,使细胞内含物外渗,并提高膜脂过氧化程度,增加丙二醛含量,导致菌丝细胞死亡,抑制病原菌的生长和繁殖。
[Abstract]:It is an important way to find plant active substances in the research and development of natural products pesticides. On the one hand, the selection and evaluation methods of common antiviral substances are optimized and explored, on the other hand, combined with the related research results and reports, To study the inhibitory effect of phytoplasm on Alternaria alternata(Freis)Keissler and Tobacco mosaic virus (TMV), which may have antiviral or antimicrobial activity, The main results are as follows: 1. Using Burns tobacco and several common plant virus inhibitors as experimental materials, the drug was injected into the tobacco leaf at five points and the whole leaf, respectively. After inoculating TMV-GFPN with Agrobacterium tumefaciens for 2 days, the fluorescent spot area and the number of spots were observed and counted under ultraviolet lamp. The results showed that the fluorescence intensity of each treatment group was lower than that of the control group, and the statistical results of fluorescent spot were similar to that of ELISA. The results showed that the fluorescence intensity could roughly reflect the virus content in leaves, and the results of laboratory evaluation of several virus inhibitors were basically consistent with the results of field efficacy. The results show that the two screening methods can be used to evaluate and screen the activity of antiviral substances, compared with the methods of spotting and leaf disc, which are commonly used to screen antiviral substances. The system can not only evaluate the activity of antiviral substances quickly, but also reflect the function characteristics of insecticides in the process of virus diffusion and replication. It has the advantages of simple operation, intuitive phenomenon and short period. 2. The tobacco mosaic virus is used as the target. The antiviral activity of 15 plant materials collected was determined, and the activity of defense enzymes was combined with the primary screening and re-screening. Two active substances, ursolic acid and 4-methoxy-coumarin, were selected to reduce the initial infection content of the virus. Ursolic acid and 4-methoxy-coumarin could increase the activities of several defense enzymes, such as PODPODcat in tobacco, decrease the content of peroxides in tobacco leaves, reduce the degree of lipid peroxidation and reduce the damage of cell membrane in the early stage of virus infection. Moreover, it can up-regulate the expression of defense response-related genes such as PR1, NPR1 and PR2, and promote the synthesis of pathogenesis-related proteins, thus reducing the initial infection content of the virus. In vitro, it was found that virion agglomeration occurred after two active substances were mixed with TMV for 1 h, but the virus dispersed uniformly without aggregation after solvent treatment. The aggregation of ursolic acid was more obvious than that of 4-methoxy-coumarin. Inoculation of the treated virus with Bentworth smoke reduced the number of fluorescent spots and the virus content. The results showed that the two substances could directly act on the virion in vitro, reduce the infection activity or the replication level of the virus in vivo, and have a certain passivation effect on the virus. The antimicrobial activity of 15 plant materials was determined, and it was found that clove oil and eugenol had better bacteriostatic effect. The clove oil content of 4.13 could inhibit the germination of the spores. When the content of clove oil was 0.52, the growth rate of hyphae was significantly decreased and the growth of hyphae was reduced. Further study showed that one of the action sites of clove oil was cell membrane, which could destroy the cell membrane structure of A. It can increase the permeability of plasma membrane, the exosmosis of cell inclusions, increase the degree of membrane lipid peroxidation, increase the content of malondialdehyde, lead to the death of hyphal cells, and inhibit the growth and reproduction of pathogenic bacteria.
【学位授予单位】：西南大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S482.292

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