MAPK途径在烟粉虱—双生病毒互作中的作用

发布时间：2018-05-23 06:58

本文选题：烟粉虱 + 番茄黄曲叶病毒　；参考：《浙江大学》2016年硕士论文

【摘要】：烟粉虱Bemisia tacbaci (Gennadius)是传播双生病毒科(Geminiviridae)菜豆金黄花叶病毒属(Begomovirus)的唯一媒介昆虫,在病毒传播过程中起着至关重要的作用。随着烟粉虱和双生病毒在世界范围内的危害日益加重,对两者之间的互作机制研究也越来越重要。本实验室已有研究表明,双生病毒在侵染烟粉虱后诱导了虫体内的自噬免疫反应,而自噬反应途径的激活又反过来抑制了病毒在烟粉虱体内的积累。昆虫主要依靠非特异的先天性免疫来抵抗病原菌的侵染,而烟粉虱中相关免疫机制还不清楚,明确烟粉虱的免疫机制能够为进一步探究烟粉虱传播双生病毒的特异性机理提供参考。本论文通过研究TYLCV、TYLCCNV、PaLCuCNV三种双生病毒侵染对MEAM1烟粉虱中丝裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)途径的影响,以明确MAPK途径在烟粉虱抵御病毒侵染过程中的作用；通过c-Jun氨基末端激酶(c-JunN-terminalkinase, JNK)特异性诱导剂和抑制剂处理MEAM1烟粉虱,分析病毒在烟粉虱体内的积累情况以及烟粉虱传播病毒的效率,验证MEAM1烟粉虱中MAPK家族成员JNK信号通路在TYLCV与寄主昆虫互作过程中所起的作用；采用RNAi手段阻断JNK信号通路,来进一步验证JNK信号通路的作用。本研究还通过JNK抑制剂和诱导剂处理MEAM1烟粉虱,探究了烟粉虱中JNK信号通路与细胞凋亡的关系,为烟粉虱中细胞凋亡免疫途径的研究提供了方向。研究结果如下：(1) TYLCV侵染对MEAM1烟粉虱JNK信号通路的影响TYLCV侵染烟粉虱后,Western Blot检测结果表明JNK及JNK下游的转录因子c-Jun磷酸化水平都显著提升；免疫荧光结果显示c-Jun还呈现明显的进入细胞核的现象。说明MEAM1烟粉虱受到TYLCV感染后,JNK信号通路被激活,以抵御病毒的侵染。(2) JNK信号通路对TYLCV在MEAM1烟粉虱体内积累的影响JNK抑制剂处理MEAM1烟粉虱后,RT-PCR、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减少。传毒实验也表明,JNK被诱导后传毒效率显著提高,而抑制JNK后传毒效率下降很显著。RNAi实验也证明,在阻断JNK信号通路后,TYLCV在MEAM1烟粉虱体内的积累量也显著减少。以上实验结果都说明,JNK信号通路的激活能够促进TYLCV在MEAM1烟粉虱体内的积累及传毒效率的提高。(3) TYLCCNV、PaLCuCNV对MEAM1烟粉虱MAPK途径的影响Western Blot结果显示MEAM1烟粉虱在被TYLCCNV和PaLCuCNV感染后,MEAM1烟粉虱中JNK的磷酸化水平也明显提升,与TYLCV侵染后的结果相似。结果表明TYLCVNV和PaLCuCNV侵染MEAM1烟粉虱后也同样能够激活其MAPK途径。(4) MEAM1烟粉虱体内JNK信号通路与细胞凋亡的关系JNK抑制剂处理MEAM1烟粉虱,TUNEL方法检测结果发现,JNK受到抑制后细胞凋亡现象发生明显增强,表明MEAM1烟粉虱中JNK与细胞凋亡是相互抑制的关系。
[Abstract]:Bemisia Tabaci Bemisia tacbaci Gennadius is the only vector insect for the transmission of Geminiviridae, the genus Begomovirus, which plays an important role in the transmission of Begomovirus. With the increasing harm of Bemisia Tabaci and Geminiviruses in the world, it is more and more important to study the interaction mechanism between them. Previous studies in our laboratory have shown that Geminiviruses induce autophagy immune response after infection with Bemisia Tabaci and the activation of autophagy pathway in turn inhibits the accumulation of the virus in Bemisia Tabaci. Insects rely mainly on non-specific innate immunity to resist the infection of pathogenic bacteria, but the immune mechanism of whitefly is not clear. Understanding the immune mechanism of Bemisia Tabaci can provide a reference for further exploring the specific mechanism of Bemisia Tabaci transmission. In this paper, we studied the effect of TYLCV (TYLCV) and TYLCCNV (TYLCCNV) on the mitogen activated protein kinase, MAPK) pathway of mitogen-activated protein kinase (mitogen) in MEAM1 whitefly (Bemisia Tabaci), in order to clarify the role of MAPK pathway in the resistance of whitefly to the infection of Bemisia Tabaci (Bemisia Tabaci). MEAM1 Bemisia Tabaci was treated with c-Jun amino-terminal kinase c-JunN-terminalkinase (c-Jun) specific inducers and inhibitors. The accumulation of the virus in Bemisia Tabaci and the efficiency of Bemisia Tabaci transmission virus were analyzed. To verify the role of JNK signaling pathway of MAPK family members in MEAM1 whitefly interaction with host insects and to block JNK signaling pathway by RNAi to further verify the role of JNK signaling pathway. In this study, MEAM1 whitefly was treated with JNK inhibitor and inducer, and the relationship between JNK signaling pathway and apoptosis in Bemisia Tabaci was explored, which provided a direction for the study of the immune pathway of apoptosis in Bemisia Tabaci. The results were as follows: (1) the effect of TYLCV infection on the JNK signaling pathway of MEAM1 whitefly; the results of Western Blot detection after TYLCV infection showed that the phosphorylation level of transcription factor c-Jun in the downstream of JNK and JNK was significantly increased. Immunofluorescence results showed that c-Jun also appeared to enter the nucleus. These results suggest that the MEAM1 Bemisia Tabaci signal pathway is activated after TYLCV infection. Effects of JNK signaling Pathway on the accumulation of TYLCV in MEAM1 Bemisia Tabaci; the results of JNK inhibitor treatment with MEAM1 Bemisia Tabaci showed that the accumulation of TYLCV-DNA and TYLCV-CP in Bemisia Tabaci was significantly decreased. The results also showed that the transmission efficiency of JNK was significantly increased after induction, and the decrease of transmission efficiency after inhibition of JNK was significant. RNAi experiments also showed that the accumulation of TYLCV in MEAM1 whitefly after blocking the JNK signaling pathway was also significantly reduced. These results indicate that the activation of JNK signaling pathway can promote the accumulation of TYLCV in MEAM1 Bemisia Tabaci and improve the efficiency of transmission. 3) the effect of TYLCCNV Pal CuCNV on MAPK pathway of MEAM1 whitefly Western Blot results show that MEAM1 Bemisia Tabaci is infected by TYLCCNV and PaLCuCNV. The phosphorylation level of JNK in Bemisia Tabaci MEAM1 was also significantly increased. The results were similar to those after TYLCV infection. The results showed that TYLCVNV and PaLCuCNV could also activate the MAPK pathway of MEAM1 whitefly. 4) the relationship between JNK signaling pathway and apoptosis in MEAM1 whitefly was detected by Tunel method after JNK inhibitor treatment of MEAM1 whitefly. The phenomenon of apoptosis was obviously enhanced. The results showed that JNK and apoptosis in MEAM1 whitefly inhibited each other.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S432.41;S433

【相似文献】