自噬在烟粉虱与双生病毒互作中的机制
发布时间：2018-08-28 14:40
【摘要】：烟粉虱Bemisia tacbaci (Gennadius)作为双生病毒科(Geminiviridae)菜豆金黄花叶病毒属(Begomovirus)传播的唯一媒介昆虫,在病毒传播过程中起着至关重要的作用。近年来烟粉虱-双生病毒互作方面的研究相继增多,但在分子水平上阐述烟粉虱与病毒的互作关系还不深入。本实验室已有研究表明,番茄黄曲叶病毒侵染后,能在烟粉虱体内检测自噬的现象,而自噬的激活又反过来抑制病毒在烟粉虱体内的积累。但是对烟粉虱传播病毒的特异性机理的探讨,还有一些问题需要明确,如：(1)烟粉虱体内自噬如何发生?(2)病毒又是如何利用自噬?(3)是否只有非选择性的自噬发生在病毒侵染的烟粉虱体内?(4)是否所有的双生病毒侵染的烟粉虱都能检测到自噬?明确这四个问题能够为进一步探究烟粉虱先天免疫机制提供参考,从而为阐明烟粉虱传播病毒的特异性机理提供依据。本研究探讨TYLCV、TYLCCNV、 PaLCuCNV三种双生病毒侵染对MEAM1烟粉虱自噬途径的影响,以明确自噬在烟粉虱抵御病毒侵染过程中的作用；采用透射电镜从形态学上分析TYLCV侵染的MEAM1烟粉虱中自噬“fluz”的出现；采用抑制剂和RNA干扰阻断自噬通路,来验证病毒和自噬的关系；采用特异性诱导剂和抑制剂处理ME AMI烟粉虱,分析病毒在烟粉虱体内的积累以及烟粉虱传播病毒的效率,最后验证自噬在TYLCV与寄主昆虫互作过程中所起的作用。本研究还通过探索不同的双生病毒在MEAM1烟粉虱体内的积累,来探究病毒增殖是否与烟粉虱自噬相关,为自噬与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烟粉虱后疑似选择性自噬的出现,为选择性自噬可能出现在昆虫中这一现象提供证据。(1) TYLCV侵染对ME AMI烟粉虱自噬"flux"的影响TYLCV侵染烟粉虱后,TEM检测结果表明自噬"flux"出现;Western Blot检测结果进一步证明自噬不仅仅是某一点的现象,而是一个通量的变化；免疫荧光结果显示TYLCV-CP与自噬体的关联；说明MEAM1烟粉虱受到TYLCV侵染后,自噬通路被激活。(2)自噬对TYLCV在ME AMI烟粉虱体内积累的影响自噬抑制剂处理ME AMI烟粉虱后,qPCR、Western Blot检测结果都显示TYLCV-DNA和TYLCV-CP在烟粉虱体内的积累量显著增多。传毒实验也表明,自噬被抑制后传毒效率显著提高。RNAi实验也证明,在阻断自噬通路后,TYLCV在MEAMl烟粉虱体内的积累量也显著增多。以上实验结果都说明,抑制自噬通路能够促进TYLCV在MEAMl烟粉虱体内的积累及传毒效率的提高。(3) TYLCCNV、PaLCuCNV对MEAMl烟粉虱自噬途径的影响Western Blot结果显示MEAMl烟粉虱在被TYLCCNV感染后,MEAMl烟粉虱中自噬标记蛋白LC3-Ⅱ周转明显提升,与TYLCV侵染后的结果相似。结果表明TYLCCNV侵染MEAMl烟粉虱也同样能够激活自噬途径。但是PaLCuCNV侵染MEAMl烟粉虱并不能激活自噬。同时TYLCV不能穿透温室白粉虱的中肠屏障,也不能激活自噬途径。(4) TYLCV侵染MEAMl烟粉虱体内疑似选择性自噬的出现TYLCV持续侵染MEAMl烟粉虱后,TEM检测结果发现,受损的线粒体被自噬体吞噬,线粒体自噬体的出现表明TYLCV侵染MEAMl烟粉虱中可能出现线粒体自噬。此外,免疫荧光共定位分子伴侣介导的自噬标记蛋白LAMP-2A与TYLCV-CP,为TYLCV侵染MEAMl烟粉虱中可能出现分子伴侣介导的自噬提供初步证据。
[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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S433;S432.41
，
本文编号：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.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S433;S432.41
，
本文编号：2209681
本文链接：https://www.wllwen.com/shoufeilunwen/jckxbs/2209681.html
