自噬在煙粉虱與雙生病毒互作中的機制
發(fā)布時間：2018-08-28 14:40
【摘要】：煙粉虱Bemisia tacbaci (Gennadius)作為雙生病毒科(Geminiviridae)菜豆金黃花葉病毒屬(Begomovirus)傳播的唯一媒介昆蟲,在病毒傳播過程中起著至關重要的作用。近年來煙粉虱-雙生病毒互作方面的研究相繼增多,但在分子水平上闡述煙粉虱與病毒的互作關系還不深入。本實驗室已有研究表明,番茄黃曲葉病毒侵染后,能在煙粉虱體內(nèi)檢測自噬的現(xiàn)象,而自噬的激活又反過來抑制病毒在煙粉虱體內(nèi)的積累。但是對煙粉虱傳播病毒的特異性機理的探討,還有一些問題需要明確,如：(1)煙粉虱體內(nèi)自噬如何發(fā)生?(2)病毒又是如何利用自噬?(3)是否只有非選擇性的自噬發(fā)生在病毒侵染的煙粉虱體內(nèi)?(4)是否所有的雙生病毒侵染的煙粉虱都能檢測到自噬?明確這四個問題能夠為進一步探究煙粉虱先天免疫機制提供參考,從而為闡明煙粉虱傳播病毒的特異性機理提供依據(jù)。本研究探討TYLCV、TYLCCNV、 PaLCuCNV三種雙生病毒侵染對MEAM1煙粉虱自噬途徑的影響,以明確自噬在煙粉虱抵御病毒侵染過程中的作用；采用透射電鏡從形態(tài)學上分析TYLCV侵染的MEAM1煙粉虱中自噬“fluz”的出現(xiàn)；采用抑制劑和RNA干擾阻斷自噬通路,來驗證病毒和自噬的關系；采用特異性誘導劑和抑制劑處理ME AMI煙粉虱,分析病毒在煙粉虱體內(nèi)的積累以及煙粉虱傳播病毒的效率,最后驗證自噬在TYLCV與寄主昆蟲互作過程中所起的作用。本研究還通過探索不同的雙生病毒在MEAM1煙粉虱體內(nèi)的積累,來探究病毒增殖是否與煙粉虱自噬相關,為自噬與病毒增殖的研究提供了方向。最后,本研究初步探索了TYLCV侵染MEAM1煙粉虱后疑似選擇性自噬的出現(xiàn),為選擇性自噬可能出現(xiàn)在昆蟲中這一現(xiàn)象提供證據(jù)。(1) TYLCV侵染對ME AMI煙粉虱自噬"flux"的影響TYLCV侵染煙粉虱后,TEM檢測結(jié)果表明自噬"flux"出現(xiàn);Western Blot檢測結(jié)果進一步證明自噬不僅僅是某一點的現(xiàn)象,而是一個通量的變化；免疫熒光結(jié)果顯示TYLCV-CP與自噬體的關聯(lián)；說明MEAM1煙粉虱受到TYLCV侵染后,自噬通路被激活。(2)自噬對TYLCV在ME AMI煙粉虱體內(nèi)積累的影響自噬抑制劑處理ME AMI煙粉虱后,qPCR、Western Blot檢測結(jié)果都顯示TYLCV-DNA和TYLCV-CP在煙粉虱體內(nèi)的積累量顯著增多。傳毒實驗也表明,自噬被抑制后傳毒效率顯著提高。RNAi實驗也證明,在阻斷自噬通路后,TYLCV在MEAMl煙粉虱體內(nèi)的積累量也顯著增多。以上實驗結(jié)果都說明,抑制自噬通路能夠促進TYLCV在MEAMl煙粉虱體內(nèi)的積累及傳毒效率的提高。(3) TYLCCNV、PaLCuCNV對MEAMl煙粉虱自噬途徑的影響Western Blot結(jié)果顯示MEAMl煙粉虱在被TYLCCNV感染后,MEAMl煙粉虱中自噬標記蛋白LC3-Ⅱ周轉(zhuǎn)明顯提升,與TYLCV侵染后的結(jié)果相似。結(jié)果表明TYLCCNV侵染MEAMl煙粉虱也同樣能夠激活自噬途徑。但是PaLCuCNV侵染MEAMl煙粉虱并不能激活自噬。同時TYLCV不能穿透溫室白粉虱的中腸屏障,也不能激活自噬途徑。(4) TYLCV侵染MEAMl煙粉虱體內(nèi)疑似選擇性自噬的出現(xiàn)TYLCV持續(xù)侵染MEAMl煙粉虱后,TEM檢測結(jié)果發(fā)現(xiàn),受損的線粒體被自噬體吞噬,線粒體自噬體的出現(xiàn)表明TYLCV侵染MEAMl煙粉虱中可能出現(xiàn)線粒體自噬。此外,免疫熒光共定位分子伴侶介導的自噬標記蛋白LAMP-2A與TYLCV-CP,為TYLCV侵染MEAMl煙粉虱中可能出現(xiàn)分子伴侶介導的自噬提供初步證據(jù)。
[Abstract]:Bemisia tacbaci (Gennadius), as the only vector insect of the Geminiviridae (Begomovirus), plays an important role in the transmission of Begomovirus. Studies in our laboratory have shown that autophagy can be detected in B. tabaci after infection, and the activation of autophagy in turn inhibits the accumulation of the virus in B. tabaci. (1) How does autophagy occur in B. tabaci? (2) How does the virus utilize autophagy? (3) Does non-selective autophagy occur only in B. tabaci infected with the virus? (4) Can autophagy be detected in all B. tabaci infected with geminiviruses? Clear understanding of these four questions can provide further information on the innate immune mechanism of B. tabaci. The aim of this study was to investigate the effects of TYLCV, TYLCCNV and PaLCuCNV infection on the autophagy pathway of meaM1 Whitefly in order to clarify the role of autophagy in the resistance of B. tabaci to virus infection. Autophagy "fluz" appeared in Bemisia tabaci; inhibition of autophagy pathway by inhibitors and RNA interference was used to verify the relationship between virus and autophagy; specific inducers and inhibitors were used to treat Bemisia tabaci with ME AMI to analyze the accumulation of the virus in the body of Bemisia tabaci and the transmission efficiency of the virus by Bemisia tabaci, and finally to verify the autophagy the relationship between TYLCV and host insects. This study also explored the accumulation of different geminiviruses in BEAM 1 tobacco whitefly to explore whether the proliferation of the virus is related to the autophagy of B. tabaci, providing a direction for the study of autophagy and virus proliferation. Finally, this study preliminarily explored the emergence of suspicious selective autophagy after TYLCV infection of B. tabaci. (1) Effect of TYLCV infection on autophagy flux of Bemisia tabaci in ME AMI. TEM results showed that autophagy flux appeared after TYLCV infection. Western Blot results further proved that autophagy was not only a point phenomenon, but a flux change. Fluorescence results showed that TYLCV-CP was associated with autophagy, indicating that the autophagy pathway was activated after TYLCV infection. (2) Effects of autophagy on the accumulation of TYLCV in Bemisia tabaci treated with autophagy inhibitors in ME AMI. The results of qPCR and Western Blot showed that TYLCV-DNA and TYLCV-CP accumulated in B. tabaci. Virus transmission experiments also showed that the transmission efficiency of TYLCV was significantly improved after autophagy was inhibited. RNAi experiments also showed that the accumulation of TYLCV in BET was significantly increased after autophagy pathway was blocked. (3) Effect of TYLCCNV and PaLCuCNV on the autophagy pathway of MEAMl whitefly Western Blot results showed that the turnover of autophagy marker protein LC3-II in MEAMl whitefly increased significantly after infection with TYLCCNV, similar to the results after infection with TYLCV. Meanwhile, TYLCV could not penetrate the midgut barrier of the greenhouse whitefly, and could not activate the autophagy pathway. (4) Suspected selective autophagy appeared in the body of the whitefly infected by TYLCV. TEM results showed that the damaged mitochondria were engulfed by autophagy and mitochondrial autophagy. In addition, immunofluorescent co-localized chaperone-mediated autophagy marker proteins LAMP-2A and TYLCV-CP provide preliminary evidence for the presence of chaperone-mediated autophagy in TYLCV-infected whitefly.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S433;S432.41
，
本文編號：2209681
[Abstract]:Bemisia tacbaci (Gennadius), as the only vector insect of the Geminiviridae (Begomovirus), plays an important role in the transmission of Begomovirus. Studies in our laboratory have shown that autophagy can be detected in B. tabaci after infection, and the activation of autophagy in turn inhibits the accumulation of the virus in B. tabaci. (1) How does autophagy occur in B. tabaci? (2) How does the virus utilize autophagy? (3) Does non-selective autophagy occur only in B. tabaci infected with the virus? (4) Can autophagy be detected in all B. tabaci infected with geminiviruses? Clear understanding of these four questions can provide further information on the innate immune mechanism of B. tabaci. The aim of this study was to investigate the effects of TYLCV, TYLCCNV and PaLCuCNV infection on the autophagy pathway of meaM1 Whitefly in order to clarify the role of autophagy in the resistance of B. tabaci to virus infection. Autophagy "fluz" appeared in Bemisia tabaci; inhibition of autophagy pathway by inhibitors and RNA interference was used to verify the relationship between virus and autophagy; specific inducers and inhibitors were used to treat Bemisia tabaci with ME AMI to analyze the accumulation of the virus in the body of Bemisia tabaci and the transmission efficiency of the virus by Bemisia tabaci, and finally to verify the autophagy the relationship between TYLCV and host insects. This study also explored the accumulation of different geminiviruses in BEAM 1 tobacco whitefly to explore whether the proliferation of the virus is related to the autophagy of B. tabaci, providing a direction for the study of autophagy and virus proliferation. Finally, this study preliminarily explored the emergence of suspicious selective autophagy after TYLCV infection of B. tabaci. (1) Effect of TYLCV infection on autophagy flux of Bemisia tabaci in ME AMI. TEM results showed that autophagy flux appeared after TYLCV infection. Western Blot results further proved that autophagy was not only a point phenomenon, but a flux change. Fluorescence results showed that TYLCV-CP was associated with autophagy, indicating that the autophagy pathway was activated after TYLCV infection. (2) Effects of autophagy on the accumulation of TYLCV in Bemisia tabaci treated with autophagy inhibitors in ME AMI. The results of qPCR and Western Blot showed that TYLCV-DNA and TYLCV-CP accumulated in B. tabaci. Virus transmission experiments also showed that the transmission efficiency of TYLCV was significantly improved after autophagy was inhibited. RNAi experiments also showed that the accumulation of TYLCV in BET was significantly increased after autophagy pathway was blocked. (3) Effect of TYLCCNV and PaLCuCNV on the autophagy pathway of MEAMl whitefly Western Blot results showed that the turnover of autophagy marker protein LC3-II in MEAMl whitefly increased significantly after infection with TYLCCNV, similar to the results after infection with TYLCV. Meanwhile, TYLCV could not penetrate the midgut barrier of the greenhouse whitefly, and could not activate the autophagy pathway. (4) Suspected selective autophagy appeared in the body of the whitefly infected by TYLCV. TEM results showed that the damaged mitochondria were engulfed by autophagy and mitochondrial autophagy. In addition, immunofluorescent co-localized chaperone-mediated autophagy marker proteins LAMP-2A and TYLCV-CP provide preliminary evidence for the presence of chaperone-mediated autophagy in TYLCV-infected whitefly.
【學位授予單位】：浙江大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S433;S432.41
，
本文編號：2209681
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