西瓜食酸菌（Acidovorax citrulli）Ⅲ型分泌系统抑制剂的高通量筛选

发布时间：2018-06-01 01:34

本文选题：甜瓜果斑病 + Ⅲ型分泌系统　；参考：《中国农业大学》2016年博士论文

【摘要】：西瓜食酸菌(Acidovorax citrulli)能够侵染甜瓜等作物引起果斑病(Bacterial Fruit Blotch,BFB),Ⅲ型分泌系统(Type Three Secretion System, TTSS)在其侵染寄主的过程中发挥关键作用。如果TTSS功能受到抑制,A. citrulli的致病性会明显减弱甚至完全丧失。本研究将TTSS作为药剂作用靶点,构建了筛选TTSS抑制剂的报告载体、明确了A. citrulli MH21中TTSS的诱导条件,建立了TTSS抑制剂高通量筛选体系。完成了对12000个化合物筛选试验,发现了对TTSS有抑制作用的丙二腈肟醚类化合物,并且对该类化合物开展了甜瓜果斑病温室防效、hrp/hrc基因表达qRT-PCR检测、构效关系分析等研究。将食酸菌Ⅲ型分泌系统效应子Aave_3502 (Pseudomonas syringae中TTSS Effector蛋白HopAO1同源蛋白)的N端信号肽和β-内酰胺酶基因翻译融合构建报告载体,转化A. citrulli MH21野生型及TTSS突变体。利用胞外p-内酰胺酶水解头孢硝噻吩导致的颜色变化来判断TTSS是否正常发挥功能。采用上述报告体系,系统检测了TTSS诱导培养基、培养温度、培养基酸碱度、离子浓度等条件,最终确定pH5.8以及含有10 mM镁离子的LB培养基能够诱导西瓜食酸菌TTSS表达。在此基础上,建立了以96孔细胞培养板为载体、TTSS效应物信号肽融合β-内酰胺酶为报告的TTSS抑制剂高通量筛选体系。通过对12000个人工合成化合物的初筛,发现了463个可能有抑制TTSS活性的化合物,其中的丙二腈肟醚类化合物对TTSS的抑制作用明显优于其他化合物。构效分析结果表明,2个丙二腈肟醚取代基团位于苯环对位的化合物活性较高,用卤素或者硝基替换其中一个丙二腈肟醚结构,能够进一步提高化合物的抑制活性。活体试验中,部分丙二腈肟醚类化合物对A. citrulli MH21在植物体上的侵染发病有明显的抑制作用,化合物33962对甜瓜果斑病的防效达到80%以上。采用qRT-PCR技术对经过丙二腈肟醚类化合物处理的菌株进行检测,发现TTSS编码基因簇hrp/hrc中的保守基因hrcU、hrcC、hrcJ,外泌装置基因hrpE以及泌出物基因hopDl的表达量有明显影响,部分基因的表达量减少80%以上。综合各阶段研究结果进行分析,丙二腈肟醚类化合物有很大可能性是一种新的TTSS抑制剂。本研究发现的丙二腈肟醚类TTSS抑制剂,可以通过对病原细菌关键致病系统的抑制作用发挥其防病效果。由于该类化合物与传统细菌病害防治药剂作用机制完全不同,因此对于病原细菌的抗药性治理和病害防治具有积极意义。
[Abstract]:Watermelon acid bacteria (Acidovorax citrulli) can infect melon and other crops to cause fruit spot disease (Bacterial Fruit Blotch, BFB). Type III secretory system (Type Three Secretion System, TTSS) plays a key role in the process of infecting host. If the TTSS function is inhibited, the pathogenicity will be significantly weakened or even completely lost. In this study, TTSS was used as a target for drug action. A report carrier for screening TTSS inhibitors was constructed. The induction conditions for TTSS in A. citrulli MH21 were clarified. A high throughput screening system for TTSS inhibitors was established. The screening tests for 12000 compounds were completed. The two nitrile oxime ether compounds with inhibitory effects on TTSS were found and the combination of these compounds was found. The greenhouse effect of melon fruit spot disease, the hrp/hrc gene expression qRT-PCR detection and the structure-activity relationship analysis were carried out. The N terminal signal peptide and beta lactamase gene of the acid bacteria type III secretory system effector Aave_3502 (Pseudomonas syringae protein HopAO1 homologous protein) were translated and fused by the beta lactamase gene, and the A. citrull was converted to A. citrull. I MH21 wild type and TTSS mutant. Using extracellular p- lactamase hydrolysis of cefitine thiophene to determine the normal function of TTSS. Using the above-mentioned reporting system, the TTSS induced medium, culture temperature, medium pH, ion concentration and so on were systematically detected, and pH5.8 and L containing 10 mM magnesium ions were finally determined. B medium can induce the expression of watermelon acid eating bacteria TTSS. On this basis, a high throughput screening system for TTSS inhibitors was established with 96 pore cell culture plate as carrier and TTSS effector signal peptide fused beta lactamase. By screening of 12000 synthetic compounds, 463 compounds that might have inhibition of TTSS activity were developed. The inhibitory effect of C two nitrile oxime ether on TTSS is obviously better than that of other compounds. The results of structure-activity analysis show that the 2 C two nitrile oxime ether substituents are more active in the benzene ring counterpart compound, and the substitution of one of the one two nitrile oxime ether with halogen or nitro can further improve the inhibitory activity of the compound. In the experiment, some two nitrile oxime ether compounds have obvious inhibitory effect on the infection of A. citrulli MH21 on plant body, and the control effect of compound 33962 on melon fruit spot is more than 80%. QRT-PCR technique is used to detect the strains treated by propane oxime ether compounds, and the protection of TTSS coding gene cluster hrp/hrc is found. The expression of gene hrcU, hrcC, hrcJ, exocrine gene hrpE and secretory gene hopDl was significantly affected, and the expression of some genes was reduced by more than 80%. The comprehensive results of various stages of study were analyzed. The propane two nitrile oxime ether compounds were highly likely to be a new TTSS inhibitor. This study found the TTSS inhibition of the class of malonitrile oxime ether. The preparation can play its preventive effect by inhibiting the key pathogenic bacteria of pathogenic bacteria. Because the mechanism of this kind of compound is completely different from that of the traditional bacterial disease control agent, it is of positive significance to the resistance control and disease control of the pathogenic bacteria.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S432.4

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