發(fā)布時(shí)間：2018-05-25 01:15

  本文選題：松材線蟲(chóng) + 殺線活性��； 參考：《青島大學(xué)》2016年博士論文

【摘要】：松材線蟲(chóng)病,又稱松萎蔫病(Pine wilt disease,PWD),是松樹(shù)的一種極具毀滅性的病害,松樹(shù)一經(jīng)感染,蔓延迅速,涉及數(shù)十種松屬植物,包括我國(guó)在內(nèi)的世界多個(gè)國(guó)家存在疫情,該病已發(fā)展為世界性的嚴(yán)重森林病害。松材線蟲(chóng)(Bursaphelenchus xylophilus)是松材線蟲(chóng)病主要的病原,松材線蟲(chóng)的防控對(duì)于控制松材線蟲(chóng)病至關(guān)重要。目前防控松材線蟲(chóng)最直接、有效的殺線蟲(chóng)劑主要是廣譜性合成藥劑,線蟲(chóng)易產(chǎn)生耐藥性,且存在高毒、高殘留、污染環(huán)境、對(duì)非靶標(biāo)有益生物構(gòu)成威脅等缺陷,這使得生態(tài)友好的天然殺線劑的開(kāi)發(fā)顯得尤為迫切。自然界植物資源豐富,植物源殺線劑近些年一直是松材線蟲(chóng)病研究的熱點(diǎn)。本實(shí)驗(yàn)選擇小茴香根、小茴香籽、無(wú)花果葉、無(wú)花果根、花椒、茼蒿根、獨(dú)活、蒼耳全草、檳榔、鶴虱、艾葉、使君子、石榴皮、百部、蒺藜籽等成本低廉或在開(kāi)發(fā)利用時(shí)常被作為副產(chǎn)物的植物原料作為受試材料,采用藥物浸漬法對(duì)15種原料的乙醇浸提物進(jìn)行了殺松材線蟲(chóng)活性測(cè)試,結(jié)果表明無(wú)花果葉、無(wú)花果根、獨(dú)活、石榴皮的乙醇提取物具有較強(qiáng)殺松材線蟲(chóng)活性。通過(guò)活性追蹤,利用柱層析、制備高效液相色譜、重結(jié)晶等分離純化手段從4種植物原料的粗提物中分離得到具有殺松材線蟲(chóng)活性的9種化合物,經(jīng)質(zhì)譜、核磁共振波譜等技術(shù)鑒定為:補(bǔ)骨脂素1、佛手柑內(nèi)酯2、蛇床子素3、二氫歐山芹醇當(dāng)歸酸酯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的殺松材線蟲(chóng)活性均為首次報(bào)道。其中補(bǔ)骨脂素1、佛手柑內(nèi)酯2、蛇床子素3、二氫歐山芹醇當(dāng)歸酸酯4、花椒毒素5和安石榴苷8具有強(qiáng)殺線活性,72 h的LC50值依次為463.32μM,430.08μM,489.17μM,406.74μM,435.66μM,307.08μM。結(jié)合強(qiáng)殺松材線蟲(chóng)化合物補(bǔ)骨脂素1、佛手柑內(nèi)酯2、蛇床子素3、二氫歐山芹醇當(dāng)歸酸酯4、花椒毒素5和安石榴苷8的化學(xué)結(jié)構(gòu)和殺線活性進(jìn)行分析,推測(cè)香豆素母核結(jié)構(gòu)苯駢α-吡喃酮為殺線活性的關(guān)鍵;安石榴苷8的強(qiáng)殺松材線蟲(chóng)活性可能是gallagyl基和六羥基聯(lián)苯二�；�的協(xié)同效應(yīng)所致;6種殺線化合物化學(xué)結(jié)構(gòu)中均具有α,β-不飽和羰基,該基團(tuán)可能對(duì)化合物的殺線活性有重要貢獻(xiàn)。本實(shí)驗(yàn)對(duì)分離得到的強(qiáng)殺松材線蟲(chóng)活性化合物補(bǔ)骨脂素1、佛手柑內(nèi)酯2、蛇床子素3、二氫歐山芹醇當(dāng)歸酸酯4、花椒毒素5和安石榴苷8進(jìn)行了殺線機(jī)理研究。借助體視顯微鏡、掃描電鏡和透射電鏡技術(shù),分析了以上6種強(qiáng)殺線活性化合物對(duì)松材線蟲(chóng)形態(tài)及生理結(jié)構(gòu)的影響:(1)顯微鏡下觀察,補(bǔ)骨脂素1、佛手柑內(nèi)酯2、蛇床子素3、二氫歐山芹醇當(dāng)歸酸酯4、花椒毒素5對(duì)線蟲(chóng)的影響相似,最初活動(dòng)趨于遲緩但身體嚴(yán)重蜷曲或扭曲,后隨時(shí)間延長(zhǎng)身體逐漸展開(kāi),最終趨于僵直,死亡線蟲(chóng)體內(nèi)多出現(xiàn)異常泡狀物或空腔;安石榴苷8處理的線蟲(chóng),最初較對(duì)照身體扭擺加劇,而后活動(dòng)逐漸遲緩并漸漸趨于僵直,體內(nèi)多呈現(xiàn)一連串的水泡狀結(jié)構(gòu);(2)掃描電鏡結(jié)果顯示,經(jīng)6種殺線化合物處理的松材線蟲(chóng)較對(duì)照體壁均出現(xiàn)明顯的皺縮,線蟲(chóng)蟲(chóng)體經(jīng)過(guò)固定化處理后較對(duì)照容易斷裂;(3)透射電鏡結(jié)果表明,殺線化合物處理的線蟲(chóng)較對(duì)照出現(xiàn)體內(nèi)組織與體壁分離現(xiàn)象或異�？涨�,細(xì)胞不同程度的解體。通過(guò)體內(nèi)、體外試驗(yàn)研究了補(bǔ)骨脂素1、佛手柑內(nèi)酯2、蛇床子素3、二氫歐山芹醇當(dāng)歸酸酯4、花椒毒素5和安石榴苷8對(duì)松材線蟲(chóng)淀粉酶、纖維素酶、乙酰膽堿酯酶、谷胱甘肽S-轉(zhuǎn)移酶等重要催化酶的影響:(1)體外實(shí)驗(yàn)表明,6種殺線化合物對(duì)線蟲(chóng)淀粉酶、纖維素酶、乙酰膽堿酯酶均有不同程度的抑制作用,安石榴苷8的抑制作用最強(qiáng),IC50值分別為0.96 m M,1.24 m M,0.60 m M;對(duì)于谷胱甘肽S-轉(zhuǎn)移酶,安石榴苷8具有明顯的抑制作用,二氫歐山芹醇當(dāng)歸酸酯4具有弱抑制作用,其他4種殺線化合物無(wú)明顯作用;(2)體內(nèi)實(shí)驗(yàn)表明,受試化合物對(duì)于松材線蟲(chóng)體內(nèi)乙酰膽堿酯酶及谷胱甘肽S-轉(zhuǎn)移酶活性的影響較大。為了更深入了解本研究中最具殺線潛力的安石榴苷8的殺線機(jī)理,采用Illumina測(cè)序技術(shù)對(duì)由其處理的松材線蟲(chóng)及對(duì)照線蟲(chóng)的轉(zhuǎn)錄組進(jìn)行高通量測(cè)序,經(jīng)過(guò)分析,2575條unigenes表達(dá)出現(xiàn)顯著差異,其中1428條表達(dá)上調(diào),1147條表達(dá)下調(diào)。綜合Nr,GO,KOG,KEGG注釋結(jié)果,推測(cè):松材線蟲(chóng)被安石榴苷8處理后主要通過(guò)細(xì)胞內(nèi)噬路徑、吞噬體路徑、過(guò)氧化物酶體路徑、MAPK信號(hào)通路等途徑啟動(dòng)了應(yīng)急反應(yīng);NADH脫氫酶、細(xì)胞色素c氧化酶、細(xì)胞色素b和電子轉(zhuǎn)移黃素蛋白等呼吸電子傳遞鏈相關(guān)蛋白,ATP合成酶和β-葡萄糖苷酶等能量代謝相關(guān)酶,熱休克蛋白,顫搐蛋白以及松材線蟲(chóng)的表皮角質(zhì)層膠原蛋白等的編碼基因是安石榴苷8的重要調(diào)控位點(diǎn),這可能是其殺線的重要原因。采用實(shí)時(shí)熒光定量PCR(Quantitative real-time PCR,q RT-PCR)技術(shù)對(duì)通過(guò)轉(zhuǎn)錄組測(cè)序分析篩選出的部分與松材線蟲(chóng)生命活動(dòng)相關(guān)的差異表達(dá)基因進(jìn)行了表達(dá)驗(yàn)證,結(jié)果與測(cè)序數(shù)據(jù)一致,證實(shí)了轉(zhuǎn)錄組分析的可靠性。上述研究結(jié)果發(fā)現(xiàn)為植物源殺松材線蟲(chóng)劑的開(kāi)發(fā)提供了物質(zhì)基礎(chǔ)和理論依據(jù)。
[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.
【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S763.18

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9 賁愛(ài)玲;戚建光;張宏娟;陳玲;韓正敏;;松材線蟲(chóng)與其攜帶細(xì)菌關(guān)系[A];第三屆全國(guó)生物入侵大會(huì)論文摘要集——“全球變化與生物入侵”[C];2010年

10 齊文靜;閆凡峰;張宗俠;曹陽(yáng);劉振宇;;松材線蟲(chóng)乙酰膽堿酯酶的活力測(cè)定及基因克隆[A];第三屆全國(guó)生物入侵大會(huì)論文摘要集——“全球變化與生物入侵”[C];2010年

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1 祝日耀 劉達(dá)富 劉允義;江山連續(xù)30年無(wú)松材線蟲(chóng)疫情發(fā)生[N];中國(guó)綠色時(shí)報(bào);2013年

2 張愷邋廣一;南京入境板材中首次發(fā)現(xiàn)松材線蟲(chóng)[N];中國(guó)國(guó)門時(shí)報(bào);2007年

3 記者 甘勇 通訊員 周衛(wèi)平;松材線蟲(chóng)快速取樣技術(shù)試驗(yàn)成功[N];湖北日?qǐng)?bào);2007年

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6 記者 齊聯(lián);檢疫不嚴(yán)放跑松材線蟲(chóng)[N];中國(guó)綠色時(shí)報(bào);2007年

7 于飛;惠州口岸發(fā)現(xiàn)活體擬松材線蟲(chóng)[N];中國(guó)國(guó)門時(shí)報(bào);2008年

8 本報(bào)記者 馬文生;都是松材線蟲(chóng)惹的禍[N];中國(guó)國(guó)門時(shí)報(bào)(中國(guó)出入境檢驗(yàn)疫報(bào));2002年

9 記者 楊懷周 通訊員 劉長(zhǎng)實(shí);綜合防治松材線蟲(chóng)[N];連云港日?qǐng)?bào);2009年

10 通訊員陳向 姚麗 記者湯曉峰;國(guó)檢嚴(yán)防松材線蟲(chóng)“偷渡”[N];南通日?qǐng)?bào);2010年

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3 王璇;松材線蟲(chóng)CYP450基因致病機(jī)理研究[D];中國(guó)林業(yè)科學(xué)研究院;2016年

4 丁曉磊;基于高通量測(cè)序的松材線蟲(chóng)致病機(jī)理與毒力差異研究[D];南京林業(yè)大學(xué);2016年

5 郭群群;植物源殺松材線蟲(chóng)活性成分的分離鑒定及殺線機(jī)理研究[D];青島大學(xué);2016年

6 潘紅偉;松材線蟲(chóng)（Bursaphelenchus xylophilus）在我國(guó)的潛在分布區(qū)研究[D];中國(guó)林業(yè)科學(xué)研究院;2009年

7 賁愛(ài)玲;松材線蟲(chóng)體表優(yōu)勢(shì)細(xì)菌的分離及定殖能力研究[D];南京林業(yè)大學(xué);2010年

8 張鍇;受松材線蟲(chóng)侵染的馬尾松抑制消減文庫(kù)構(gòu)建與表達(dá)譜分析[D];中國(guó)林業(yè)科學(xué)研究院;2010年

9 田雪亮;松材線蟲(chóng)和擬松材線蟲(chóng)伴生細(xì)菌多樣性及生態(tài)功能研究[D];西北農(nóng)林科技大學(xué);2010年

10 劉寶軍;松材線蟲(chóng)擇偶對(duì)策及擇偶偏向的轉(zhuǎn)錄組分析[D];中國(guó)林業(yè)科學(xué)研究院;2014年

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2 劉穎;松材線蟲(chóng)和擬松材線蟲(chóng)的差異蛋白質(zhì)組學(xué)研究[D];廈門大學(xué);2007年

3 齊文靜;松材線蟲(chóng)運(yùn)動(dòng)行為及其神經(jīng)遞質(zhì)乙酰膽堿的相關(guān)研究[D];山東農(nóng)業(yè)大學(xué);2012年

4 黃瑞芬;中國(guó)不同地理種群松材線蟲(chóng)的耐寒性研究[D];北京林業(yè)大學(xué);2015年

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10 王林松;松材線蟲(chóng)山梨醇脫氫酶基因的克隆與功能性研究[D];青島大學(xué);2016年

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