本文選題：植物源活性物質(zhì)　切入點(diǎn)：煙草花葉病毒　出處：《西南大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：尋找植物源活性物質(zhì)是天然產(chǎn)物農(nóng)藥研發(fā)的重要途徑。本文一方面對(duì)常用的抗病毒物質(zhì)篩選以及評(píng)價(jià)方法進(jìn)行優(yōu)化和探索,另一方面結(jié)合相關(guān)研究成果和報(bào)道,廣泛搜集可能具有抗病毒或抑菌活性的植物源物質(zhì),研究其對(duì)煙草赤星病菌[Alternaria alternata(Freis)Keissler]和煙草花葉病毒(Tobacco mosaic virus,TMV)的抑制效果,并初步探討其作用方式和機(jī)制。得到的主要研究結(jié)果如下:1.以本氏煙和幾種常用的植物病毒抑制劑為實(shí)驗(yàn)材料,分別將藥劑注射于煙葉的五個(gè)點(diǎn)和整片煙葉,利用農(nóng)桿菌介導(dǎo)法接種TMV-GFP,2 d后在紫外燈下觀察并統(tǒng)計(jì)不同處理后的熒光斑面積與斑點(diǎn)數(shù)量。結(jié)果顯示,各處理組熒光強(qiáng)度均低于對(duì)照組,熒光斑的統(tǒng)計(jì)情況與ELISA檢測(cè)結(jié)果相近,表明熒光強(qiáng)弱情況能夠大致反映葉片內(nèi)病毒含量。并且本體系對(duì)幾種病毒抑制劑的室內(nèi)評(píng)價(jià)結(jié)果與田間藥效結(jié)果基本一致,說明本文建立的2種篩選方式可以用于抗病毒物質(zhì)的活性評(píng)價(jià)和篩選。與篩選抗病毒物質(zhì)常用的枯斑法和葉碟法相比,該體系不僅能夠快速評(píng)價(jià)抗病毒物質(zhì)的活性還能直觀的反映藥劑在病毒擴(kuò)散和復(fù)制過程中的作用特點(diǎn),具有操作簡(jiǎn)單,現(xiàn)象直觀,周期短的優(yōu)點(diǎn)。2.以煙草花葉病毒為靶標(biāo),對(duì)收集到的15種植物源物質(zhì)進(jìn)行抗病毒活性測(cè)定,經(jīng)過初篩、復(fù)篩并結(jié)合防御酶活性情況,篩選出熊果酸與4-甲氧基香豆素2種能夠降低病毒初侵染含量的活性物質(zhì)。研究其作用方式與機(jī)制,結(jié)果顯示,熊果酸與4-甲氧基香豆素能夠不同程度提高煙草中SOD、PPO、POD、CAT這幾種防御酶活性,降低煙葉中過氧化物含量;在病毒侵染前期可以降低植物細(xì)胞的膜脂過氧化程度,減輕細(xì)胞膜受損;并且能夠上調(diào)PR1、NPR1和PR2等防衛(wèi)反應(yīng)相關(guān)基因表達(dá)量,促進(jìn)病程相關(guān)蛋白的合成,從而降低病毒初侵染含量,具有保護(hù)和延緩病程的作用。體外作用研究發(fā)現(xiàn),2種活性物質(zhì)與TMV混合1 h后,病毒粒體發(fā)生了明顯的聚集現(xiàn)象,而溶劑處理后的病毒分散均勻無聚集,其中熊果酸處理后的聚集程度較4-甲氧基香豆素更為明顯。將處理后的病毒摩擦接種至本氏煙,熒光的斑點(diǎn)數(shù)量和病毒含量都有所降低,說明2種物質(zhì)能在體外直接作用病毒粒體,降低病毒的侵染活性或體內(nèi)復(fù)制水平,對(duì)病毒具有一定鈍化作用。3.利用平板抑菌法,以煙草赤星病菌為靶標(biāo),測(cè)定15種植物源物質(zhì)的抑菌活性,發(fā)現(xiàn)丁香油與丁香油酚具有較好的抑菌作用。丁香油含量為4.13%時(shí)能夠強(qiáng)效抑制赤星病菌孢子的萌發(fā),丁香油含量為0.52%時(shí)顯著降低菌絲的生長(zhǎng)速度,減少菌絲的生長(zhǎng)量。進(jìn)一步研究作用機(jī)理發(fā)現(xiàn),丁香油的作用位點(diǎn)之一是細(xì)胞膜,其能破壞赤星病菌的細(xì)胞膜結(jié)構(gòu),增加質(zhì)膜通透性,使細(xì)胞內(nèi)含物外滲,并提高膜脂過氧化程度,增加丙二醛含量,導(dǎo)致菌絲細(xì)胞死亡,抑制病原菌的生長(zhǎng)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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S482.292

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本文編號(hào)：1641956