植物源杀松材线虫活性成分的分离鉴定及杀线机理研究

发布时间：2018-05-25 01:15

本文选题：松材线虫 + 杀线活性　；参考：《青岛大学》2016年博士论文

【摘要】：松材线虫病,又称松萎蔫病(Pine wilt disease,PWD),是松树的一种极具毁灭性的病害,松树一经感染,蔓延迅速,涉及数十种松属植物,包括我国在内的世界多个国家存在疫情,该病已发展为世界性的严重森林病害。松材线虫(Bursaphelenchus xylophilus)是松材线虫病主要的病原,松材线虫的防控对于控制松材线虫病至关重要。目前防控松材线虫最直接、有效的杀线虫剂主要是广谱性合成药剂,线虫易产生耐药性,且存在高毒、高残留、污染环境、对非靶标有益生物构成威胁等缺陷,这使得生态友好的天然杀线剂的开发显得尤为迫切。自然界植物资源丰富,植物源杀线剂近些年一直是松材线虫病研究的热点。本实验选择小茴香根、小茴香籽、无花果叶、无花果根、花椒、茼蒿根、独活、苍耳全草、槟榔、鹤虱、艾叶、使君子、石榴皮、百部、蒺藜籽等成本低廉或在开发利用时常被作为副产物的植物原料作为受试材料,采用药物浸渍法对15种原料的乙醇浸提物进行了杀松材线虫活性测试,结果表明无花果叶、无花果根、独活、石榴皮的乙醇提取物具有较强杀松材线虫活性。通过活性追踪,利用柱层析、制备高效液相色谱、重结晶等分离纯化手段从4种植物原料的粗提物中分离得到具有杀松材线虫活性的9种化合物,经质谱、核磁共振波谱等技术鉴定为:补骨脂素1、佛手柑内酯2、蛇床子素3、二氢欧山芹醇当归酸酯4、花椒毒素5、鞣花酸6、安石榴林7、安石榴苷8、柯里拉京9,化合物2-9的杀松材线虫活性均为首次报道。其中补骨脂素1、佛手柑内酯2、蛇床子素3、二氢欧山芹醇当归酸酯4、花椒毒素5和安石榴苷8具有强杀线活性,72 h的LC50值依次为463.32μM,430.08μM,489.17μM,406.74μM,435.66μM,307.08μM。结合强杀松材线虫化合物补骨脂素1、佛手柑内酯2、蛇床子素3、二氢欧山芹醇当归酸酯4、花椒毒素5和安石榴苷8的化学结构和杀线活性进行分析,推测香豆素母核结构苯骈α-吡喃酮为杀线活性的关键;安石榴苷8的强杀松材线虫活性可能是gallagyl基和六羟基联苯二酰基的协同效应所致;6种杀线化合物化学结构中均具有α,β-不饱和羰基,该基团可能对化合物的杀线活性有重要贡献。本实验对分离得到的强杀松材线虫活性化合物补骨脂素1、佛手柑内酯2、蛇床子素3、二氢欧山芹醇当归酸酯4、花椒毒素5和安石榴苷8进行了杀线机理研究。借助体视显微镜、扫描电镜和透射电镜技术,分析了以上6种强杀线活性化合物对松材线虫形态及生理结构的影响:(1)显微镜下观察,补骨脂素1、佛手柑内酯2、蛇床子素3、二氢欧山芹醇当归酸酯4、花椒毒素5对线虫的影响相似,最初活动趋于迟缓但身体严重蜷曲或扭曲,后随时间延长身体逐渐展开,最终趋于僵直,死亡线虫体内多出现异常泡状物或空腔;安石榴苷8处理的线虫,最初较对照身体扭摆加剧,而后活动逐渐迟缓并渐渐趋于僵直,体内多呈现一连串的水泡状结构;(2)扫描电镜结果显示,经6种杀线化合物处理的松材线虫较对照体壁均出现明显的皱缩,线虫虫体经过固定化处理后较对照容易断裂;(3)透射电镜结果表明,杀线化合物处理的线虫较对照出现体内组织与体壁分离现象或异常空腔,细胞不同程度的解体。通过体内、体外试验研究了补骨脂素1、佛手柑内酯2、蛇床子素3、二氢欧山芹醇当归酸酯4、花椒毒素5和安石榴苷8对松材线虫淀粉酶、纤维素酶、乙酰胆碱酯酶、谷胱甘肽S-转移酶等重要催化酶的影响:(1)体外实验表明,6种杀线化合物对线虫淀粉酶、纤维素酶、乙酰胆碱酯酶均有不同程度的抑制作用,安石榴苷8的抑制作用最强,IC50值分别为0.96 m M,1.24 m M,0.60 m M;对于谷胱甘肽S-转移酶,安石榴苷8具有明显的抑制作用,二氢欧山芹醇当归酸酯4具有弱抑制作用,其他4种杀线化合物无明显作用;(2)体内实验表明,受试化合物对于松材线虫体内乙酰胆碱酯酶及谷胱甘肽S-转移酶活性的影响较大。为了更深入了解本研究中最具杀线潜力的安石榴苷8的杀线机理,采用Illumina测序技术对由其处理的松材线虫及对照线虫的转录组进行高通量测序,经过分析,2575条unigenes表达出现显著差异,其中1428条表达上调,1147条表达下调。综合Nr,GO,KOG,KEGG注释结果,推测:松材线虫被安石榴苷8处理后主要通过细胞内噬路径、吞噬体路径、过氧化物酶体路径、MAPK信号通路等途径启动了应急反应;NADH脱氢酶、细胞色素c氧化酶、细胞色素b和电子转移黄素蛋白等呼吸电子传递链相关蛋白,ATP合成酶和β-葡萄糖苷酶等能量代谢相关酶,热休克蛋白,颤搐蛋白以及松材线虫的表皮角质层胶原蛋白等的编码基因是安石榴苷8的重要调控位点,这可能是其杀线的重要原因。采用实时荧光定量PCR(Quantitative real-time PCR,q RT-PCR)技术对通过转录组测序分析筛选出的部分与松材线虫生命活动相关的差异表达基因进行了表达验证,结果与测序数据一致,证实了转录组分析的可靠性。上述研究结果发现为植物源杀松材线虫剂的开发提供了物质基础和理论依据。
[Abstract]:Pine wood nematode disease, also known as Pine wilt disease (PWD), is a devastating disease of pine trees. The pine tree is infected and spread rapidly, involving dozens of species of pine plants, including a number of countries in the world, including our country. The disease has developed into a serious forest disease of the world. Pine wood nematode (Bursaphelenchus xylophil). Us) is the main pathogen of pine wood nematode disease. The prevention and control of pine wood nematode is very important for controlling pine wood nematode disease. At present, the most direct control of pine wood nematode, effective nematode is broad-spectrum synthetic agent, nematode is susceptible to drug resistance, and there are high toxicity, high residue, pollution environment, and threat to non target organisms, such as threat, and so on. The development of ecological friendly natural wire killing agents is particularly urgent. The natural plant resources are rich in nature. Plant source nematode has been a hot spot in the study of pine wood nematode disease in recent years. This experiment selected fennel root, fennel seed, fig leaf, fig root, pepper, chrysanthemum root, single live, whole grass of Xanthium, areca, crane plant, AI leaf, Artemisia, stone. Pomegranate, 100, Tribulus seeds, and other plant raw materials which are often used as by-products in development and utilization, were used as tested materials. The ethanol extracts of 15 raw materials were tested by drug impregnation. The results showed that the ethanol extracts from the leaves of the fig, fig root, single live and pomegranate skin had strong pine wood. By tracing the nematode, using column chromatography, preparation of high performance liquid chromatography and recrystallization, 9 compounds with the activity of Nematicidal nematode were isolated from the crude extracts of 4 plant materials, and were identified by mass spectrometry and nuclear magnetic resonance spectroscopy as: bonalin 1, bergamot 2, osthole 3, two Ooe. Apigenic acid Angelica 4, zanthoxylin 5, tannin 6, pomegranate 7, pomegranate 8, korralin 9, and compound 2-9 of pine wood nematode were all reported for the first time. Among them, psoralen 1, citrus actone 2, osthole 3, two parsley Angelica 4, zanthoxylin 5 and pomegranate 8 have strong line activity, and the LC50 value of H is in turn. 463.32 mu M, 430.08 mu, 489.17 mu M, 406.74 mu M, 435.66 mu M, 307.08 mu M. combined with pinewood nematode compound of psoralen 1, bergamot lactone 2, osthole 3, two hydrogen ozonoside 4, zanthoxinin 5 and anoroside 8, the chemical structure and nematide activity were analyzed. The key of sex is that the activity of pinewood nematode 8 in pomegranate glycoside 8 may be caused by the synergistic effect of the two acyl group and six hydroxyl biphenyl group. The chemical structures of the 6 kinds of nemicidal compounds have alpha, beta unsaturated carbonyl groups, and this group may have important contribution to the activity of the compounds. Combination of psoralen 1, citrus analactone 2, osthole 3, two hydrogen ozonoside 4, zanthoxylin 5 and pomegranate 8 were used to study the line killing mechanism. The effects of the above 6 strong nematode compounds on the morphological and physiological structure of pine wood nematode were analyzed with the stereomicograph, scanning electron microscopy and transmission electron microscopy. (1) microscopes Under observation, psoralen 1, bergamot lactone 2, osthole 3, two diosorcicin 4, the effect of zanthoxylin 5 on the nematode is similar, the initial activity tends to slow but the body is seriously curled up or twisted, and then the body gradually expands, eventually tends to be stiff, and the death nematode has abnormal bubbles or cavity in the body of the nematode; pomegranate The nematode treated by glucoside 8 was initially stronger than the control body torsion pendulum, and then the activity gradually slowed down and gradually became rigid, and the body showed a series of vesicular structures. (2) the scanning electron microscope showed that the pine wood nematode treated by 6 kinds of nematidal compounds showed obvious shrinkage compared with the control body wall, and the nematode body was compared to the control after immobilization. (3) the transmission electron microscope showed that the nematode treated nematodes were separated from the body and the body wall or the abnormal cavity, and the cells were disintegrated in different degrees. Through the body, the psoralen 1, the citrus lactones 2, the osthole 3, the two parsley angelicol 4, the zanthoxylin 5 and the pomegranate were studied in vitro. The effect of glucoside 8 on amylase, cellulase, acetylcholinesterase, glutathione S- transferase, and other important catalytic enzymes in pine wood nematode: (1) in vitro experiments showed that 6 kinds of nematidal compounds have different inhibitory effects on nematode amylase, cellulase and acetylcholinesterase. The inhibitory effect of pomegranate 8 is the strongest, and the value of IC50 is 0.96 m M, 1. 24 m M and 0.60 m M; for glutathione S- transferase, pomegranate 8 had obvious inhibitory effect, two HMP 4 had weak inhibitory effect, and the other 4 kinds of line killing compounds had no obvious effect. (2) in vivo experiments showed the effects of the tested compounds on the activity of acetylcholinesterase and glutathione S- transferase in pine wood nematode. In order to understand the mechanism of the killing line of pomegranin 8, which has the most potential to kill line in this study, Illumina sequencing technology was used to sequence the high flux sequence of the transcriptional group of the pine wood nematode and the control nematode. After analysis, there were significant differences in the expression of 2575 unigenes, of which 1428 were up and 1147 were downregulated. Integrated Nr, GO, KOG, KEGG annotation results, we speculated that pine wood nematode was treated mainly by intracellular phagocytosis, phagocytic pathway, peroxisome pathway, MAPK signaling pathway, and other respiratory electron transport chains, such as NADH dehydrogenase, cytochrome c oxidase, cytochrome b and electron transfer flavin protein, after treatment with pomegranate 8. Related protein, ATP synthetase and beta glucosidase, energy metabolism related enzymes, heat shock protein, twitching protein, and collagen of epidermal cuticle of pine wood nematode are important regulatory sites of pomegranate 8. This may be an important reason for its line killing. Real-time fluorescent quantitative PCR (Quantitative real-time PCR, Q RT) is used. -PCR) verifying the differentially expressed genes related to the life activity of pine wood nematode through the sequence analysis of the transcriptional sequence analysis. The results were consistent with the sequencing data and confirmed the reliability of the transcriptional analysis. The results of the study found the material basis and theoretical basis for the development of plant source pine wood nematode.
【学位授予单位】：青岛大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S763.18

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