【作者】 吕印谱；

【导师】 宋宝安；

【作者基本信息】 贵州大学 ， 农药学， 2007， 博士

【摘要】 植物病毒病害的化学防治是目前植物病理学科研究的热点和难点。本文以烟草为材料，对贵州大学精细化工研究开发中心研制的GU188[3-甲硫基-3-（2-硝基苯氨基）-2-氰基丙烯酸乙酯]新化合物及病毒星抗烟草花叶病毒（TMV）的生物活性、生化机制以及GU188抗TMV的分子生物学机理进行了较为全面深入的研究，其结果如下：1、对TMV的生物活性新化合物GU188对烟草花叶病毒（TMV）具有较好的预防效果，防效优于宁南霉素。在普通烟上的预防效果达58.33％，高于在心叶烟上的防效；在心叶烟上的治疗效果为38.94％，在普通烟上的治疗效果达44.44％，显著高于宁南霉素。与离体钝化试验相比，新化合物GU188在心叶烟活体叶片上的枯斑抑制率明显高于离体的枯斑抑制率，预防及治疗作用的枯斑抑制率分别达31.04％和53.31％。30％病毒星WP250 mg.L^-1处理对烟草花叶病毒（TMV）具有较好的预防和治疗效果，与GU188 250 mg.L^-1处理及对照药剂宁南霉素处理的效果相当。2、药物活性的生化机制分析对防御酶的影响：接种后药剂处理，GU188 250 mg·L^-1、CK1（宁南霉素）处理烟草叶片内的过氧化物酶（POD）活性高峰与对照活性高峰出现时间相同，活性峰值以GU188 250mg·L^-1最高；接种后药剂处理，GU188药物250 mg·L^-1和CK1（宁南霉素）处理烟草叶片的POD活性值提前达到峰值，这可能是由于接种后用GU188 250 mg·L^-1、CK1（宁南霉素）处理，引起酚类物质氧化、木质素及木栓质合成速度加快，提前达到高峰，进而也能引起多酚氧化酶（PPO）活性提前达到高峰。药剂诱导的烟草叶片内的苯丙氨酸解氨酶（PAL）活性和对照烟草叶片的PAL活性都在接种后6d达到高峰，且GU188 250 mg·L^-1处理的活性峰值最高。这可能是因为药剂诱导刺激了细胞，控制PAL防御体系的基因表达量增加，促进PAL催化的代谢产物大量合成，抵抗烟草花叶病毒（TMV）的侵入、扩展。药剂诱导后接种，烟草叶片内的PPO活性高峰出现时间推迟，从接种后4d开始，各药剂处理的PPO活性均明显高于对照；接种后药剂处理，各处理的PPO活性明显高于对照的PPO活性，活性高峰比对照提前出现。经药剂诱导后，除GU188药物250 mg·L^-1处理过氧化氢酶（CAT）活性在接种后0d时提前达到高峰外，其它处理及对照的CAT活性均在接种后8d达到活性高峰。对烟草抗病性的激发：理想的植物病毒抑制剂不仅能够钝化病毒的活性，更为重要的是能够通过激发植物的抗病毒机制，来限制病毒在体内的复制、扩展和症状的产生。本研究中发现具有良好治疗作用的GU188 250 mg·L^-1处理能够导致接种TMV的叶片蛋白质表达谱发生变化。大多数新出现的蛋白质可能涉及多种病程相关蛋白（PR蛋白），特别是PR-2a、PR-2b、PR-3组中的成员，此外还可能涉及PR-1、PR-4、PR-5b组中的成员。在没有N基因背景的普通烟上，GU188似乎也可以导致接种TMV的叶片蛋白质表达谱发生变化，但变化的总体趋势是蛋白质浓度和种类减少，至处理后24h达到最低，处理后48h的蛋白质浓度与处理前相当。在普通烟中新出现的3条带，很可能就是PR-1、PR-4组中的酸性蛋白成员，这一现象具有重要意义。3、药物活性的分子机理分析为了揭示GU188抑制烟草花叶病毒（TMV）在分子水平上的活性机理，本研究共设三种处理：处理A（treatment A），单独接种TMV；处理B（treatment B），烟草叶片接种TMV10h后再涂抹GU188药剂(500 mg·L^-1)；处理C（treatment C），单用GU188药剂(500 mg·L^-1)涂抹烟草叶片。对照叶片涂抹磷酸缓冲液（PBS，pH7.2），采用基于拟南芥基因组的寡聚核苷酸芯片对GU188所诱导的烟草叶片基因的表达进行了探测。结果表明：A处理（单独接种TMV）有260个基因有效表达，上调基因为180个，下调基因为80个；B处理（TMV接种后再施用GU188药剂）有175个基因有效表达，上调基因为55个，下调基因为120个；C处理（单独施用GU188药剂）有83个基因有效表达，上调基因10个，下调基因73个。三种处理间的基因表达具有明显的规律性：即被TMV诱导的大部分上调表达基因，施用药剂后表达为下调，相反被TMV诱导的大部分下调表达基因，施用药剂后则表达为上调；单独旌药处理也表现出相同的趋势，但有效表达基因数量比接种TMV后再施用药剂的少。初步从基因水平上证明GU188药剂具有抵消或部分抵消TMV引起的寄主不正常表达以及治疗效果优于预防效果的作用机理。GU188药剂的主要活性机理是纠正由TMV所诱导的烟草与RNA合成相关的基因的上调表达，从而实现对TMV利用寄主RNA合成机器复制自己的抑制，这是GU188提高寄主对烟草TMV侵染抗性的关键之所在。基因功能分析还表明TMV与寄主间存在着及其复杂的调控关系：TMV侵染后可以上调寄主细胞核蛋白、RNA合成、蛋白质合成、泛素介导的蛋白质分解途径相关的基因表达；TMV干扰了寄主体内激素相关基因的正常表达，较为突出的是与乙烯合成和发挥效应的基因被上调而赤霉素的相关基因被下调；与光合和呼吸作用相关的基因被TMV下调；细胞壁和细胞骨架合成相关的基因被下调；植物正常的抗逆途径的基因如热激蛋白、活性氧清除蛋白等的表达被下调；TMV在植物体内的信号传导途径可能是以钙信号传导途径为主，因为钙信号途径中的主要成员在TMV的侵染后其基因表达差异最大；此外芯片还检测到DNA脱甲基化基因、miRNA基因及组蛋白脱乙酰化酶基因的上调表达，显示了TMV可能在多种水平上实现对寄主的基因表达的调控。总之，通过本研究初步得知GU188抗TMV的生物活性机理主要通过控制寄主RNA合成系统的基因表达来抑制TMV的复制，这一结论对新药的筛选具有重要的参考意义。从基因表达水平上初步得出了GU188对TMV的治疗效果优于其预防效果，其整体防效应在50％左右。同时表明，基因芯片技术在药物机理研究中所具有的简单、快速和明确的作用，这一尝试为今后药物活性筛选和药物作用机理的研究提供了借鉴。此外，还检测到67个目前还未知功能的有效差异表达的基因，随着对这些基因功能的逐步诠释，对TMV诱导的寄主基因的表达以及抗TMV药物靶标的研究将会提供更好的依据。更多还原

【Abstract】 The control of viral disease in plants has become an importance-area of research in the current-phytopathology. Biological activity, physiological achieve and inhibitory mechanistic actions of Gu188 [2-cyano-3-（methylthio）-3-（2-nitrophenylamino）acrylate], synthesized by Center for Research and Development of Fine Chemicals of Guizhou University are useful for further development of this kind of antivirus chemicals.1、Biological activities against TMVPrevention effects of GU188 on inhibition of TMV infecting tobacco were either higher than or equal to that of the control chemical, Ningnanmycin. Controlling effects of GU188 on common tobacco had reached 58.33% and higher than that on Nicotiana glutinosa. Compared with the result of the isolated inactivation experiment, blight inhibition rate of tested chemicals in living tobacco leaves was remarkably higher. The blight inhibition rates for preventive are curative effects reached up to 30.04% and 53.31% respectively. The results also showed that 250 mgL^-1 of Bingduxing WP30% had preventive and curative effects, similar to those of 250 mgL^-1 of GU188 and control （Ningnanmycin）.2、Analysis of biochemical activitiesActivities of defense enzymes: Activities of several defense enzymes in the tobacco plant treated with the tested compounds were studied. When inoculated after being induced by the compound, the activity peak value of the POD in tobacco leaf appeared at the same time as the CK after treatment with GU188 activity peak value was found to be the highest. When treated with the compounds after inoculation, the activity of POD in the tobacco leaves treated with D2 and CK1 reached the peak value in the beginning. The PAL activity in the compound-induced tobacco leaf reached the highest level 6d after inoculation similar to the control treated tobacco leaf with the D2 treated are assuring the highest value. The PAL activity in the tobacco leaf treated with the tested compound after inoculation equally reached the highest level 6 days after being treated and was much higher than that of CK, which reached the peak value 2 days after inoculation. When inoculated after being induced by the tested compounds, the appearance of PPO activity peak in the tobacco leaf was delayed. From 4th day onward after being inoculated, the PPO activities of all the tested compounds were found to be remarkably higher than that of CK. When treated with the tested compounds after being inoculated, the PPO activities of all the treatments were significantly higher than that of CK and the peak value appeared earlier than that of CK. When induced by the tested compounds, except for D2’s, CAT activity initially reached the highest level 0 day after inoculation, other CAT activities of CK achieved the peak value 8 days after inoculation. At the same time, the D treatment could cause some changes in the protein expression spectrum in the tobacco leaf inoculated with TMV.Stimulatory of resistance of tobacco: A better antivirus compound should not only inactivate action of virus but should also be able to stimulate ability of host’s antivirus mechanism to inhibit virus’ replication, spread and syndrome. In this study the compound D2 (250 mg·L^-1 of GU188), showed better preventive and curative effects, could change the profile of protein expression after TMV infection. Most of the new protein bands the appeared should related to various PR proteins, especially the members of the group of PR-2a, PR-2b, PR-3 and members of the group of PR-1, PR-4, PR-5b. GU188 could also change the profile of protein expression in common tobacco in which there was no background of N gene after TMV infection. A decreasing trend was noticed with 24 h after treatment of GU188 reached the lowest point and 48 h resuming to the level before treatment. 3 bands appeared at common tobacco might be the acidic members of PR-1 and PR-4 group. The phenomenon is of enormous significance.3、Analysis of mechanism action of tested compound at the molecular levelIn order to study the mechanistic aspects pertaining to GU188 in gene expression for host plant leaves were detected by the analysis of microarrays based on Arabidopsis thaliana genome sequence. The results showed that the 260 genes in tobacco leaves inoculated by TMV alone （treatment A） were induced to exhibit significant expression changes, including 180 up-regulated genes and 80 down-regulated genes. There were 175 genes in tobacco leaves treated by inoculation of TMV and spray of GU188 at same time （treatment B） to display effective expression, which consisted of 55 up-regulated genes and 120 down-regulated genes. 83 genes in leaves treated by the compound but without inoculation of TMV （treatment C） were induced to exhibit effective expression, including 10 up-regulated genes and 73 down-regulated genes. Remarkable phenomena of the gene expression differences in 3 treatments were observed: almost entire up-regulated genes by TMV were down-regulated by the usage of the tested chemical, while most all of the down-regulated genes by TMV were found to be up-regulated by GU188. Similar results were obtained in treatment C except for the face that fewer genes were expressed as compared with that in treatment B. Analysis of gene functions showed that TMV-infection up-regulated expression of the genes in relation to the host nucleoproteins, synthesis of RNAs and proteins, and the protein degradation pathways mediated by ubiquitin and SUMO; TMV disturbed the normal gene expression associated with phytohormones metabolism in its host, down-regulated expression of the genes related to photosynthesis, respiration, construction of cell wall and cytoskeleton, normal anti-stress pathways such as heat shock protein and active oxygen scavenger protein; gene expression of main components in calcium signal pathway was altered sharply due to TMV infection; the cDNA microarray also identified the up-regulated genes of DNA demethylation, miRNA, HDACs, which demonstrated that TMV might regulate the host gene expression at various levels.To sum up, this paper explained that role of GU188 in improving tobacco resistance against TMV infection was probably achieved by counteraction or correction of abnormal gene expression in the host leaves induced by TMV. The key mechanism underlined that inhibition of GU188 against TMV reproduction was down-regulated the expression of genes relation to RNA synthesis in leaf cells because the RNA synthesis of TMV completely depends on the host’s machine of RNA synthesis. And the judgment of the results also indicated that curing effects of tested chemical was probably better than its’ preventing effect by judgment at gene transcription level. In addition, out of the 260 significant expression genes, there were 57 whose function could not be ascertained. We believe that with continuous of these gene functions, a clearer profile of gene expression induced by TMV infection in tobacco leaf would be achieved.更多还原