本文選題：煙粉虱 + 番茄黃曲葉病毒��； 參考：《浙江大學》2016年碩士論文

【摘要】：煙粉虱Bemisia tacbaci (Gennadius)是傳播雙生病毒科(Geminiviridae)菜豆金黃花葉病毒屬(Begomovirus)的唯一媒介昆蟲,在病毒傳播過程中起著至關重要的作用。隨著煙粉虱和雙生病毒在世界范圍內的危害日益加重,對兩者之間的互作機制研究也越來越重要。本實驗室已有研究表明,雙生病毒在侵染煙粉虱后誘導了蟲體內的自噬免疫反應,而自噬反應途徑的激活又反過來抑制了病毒在煙粉虱體內的積累。昆蟲主要依靠非特異的先天性免疫來抵抗病原菌的侵染,而煙粉虱中相關免疫機制還不清楚,明確煙粉虱的免疫機制能夠為進一步探究煙粉虱傳播雙生病毒的特異性機理提供參考。本論文通過研究TYLCV、TYLCCNV、PaLCuCNV三種雙生病毒侵染對MEAM1煙粉虱中絲裂原活化蛋白激酶(mitogen activated protein kinase, MAPK)途徑的影響,以明確MAPK途徑在煙粉虱抵御病毒侵染過程中的作用；通過c-Jun氨基末端激酶(c-JunN-terminalkinase, JNK)特異性誘導劑和抑制劑處理MEAM1煙粉虱,分析病毒在煙粉虱體內的積累情況以及煙粉虱傳播病毒的效率,驗證MEAM1煙粉虱中MAPK家族成員JNK信號通路在TYLCV與寄主昆蟲互作過程中所起的作用；采用RNAi手段阻斷JNK信號通路,來進一步驗證JNK信號通路的作用。本研究還通過JNK抑制劑和誘導劑處理MEAM1煙粉虱,探究了煙粉虱中JNK信號通路與細胞凋亡的關系,為煙粉虱中細胞凋亡免疫途徑的研究提供了方向。研究結果如下：(1) TYLCV侵染對MEAM1煙粉虱JNK信號通路的影響TYLCV侵染煙粉虱后,Western Blot檢測結果表明JNK及JNK下游的轉錄因子c-Jun磷酸化水平都顯著提升；免疫熒光結果顯示c-Jun還呈現(xiàn)明顯的進入細胞核的現(xiàn)象。說明MEAM1煙粉虱受到TYLCV感染后,JNK信號通路被激活,以抵御病毒的侵染。(2) JNK信號通路對TYLCV在MEAM1煙粉虱體內積累的影響JNK抑制劑處理MEAM1煙粉虱后,RT-PCR、Western Blot檢測結果都顯示TYLCV-DNA和TYLCV-CP在煙粉虱體內的積累量顯著減少。傳毒實驗也表明,JNK被誘導后傳毒效率顯著提高,而抑制JNK后傳毒效率下降很顯著。RNAi實驗也證明,在阻斷JNK信號通路后,TYLCV在MEAM1煙粉虱體內的積累量也顯著減少。以上實驗結果都說明,JNK信號通路的激活能夠促進TYLCV在MEAM1煙粉虱體內的積累及傳毒效率的提高。(3) TYLCCNV、PaLCuCNV對MEAM1煙粉虱MAPK途徑的影響Western Blot結果顯示MEAM1煙粉虱在被TYLCCNV和PaLCuCNV感染后,MEAM1煙粉虱中JNK的磷酸化水平也明顯提升,與TYLCV侵染后的結果相似。結果表明TYLCVNV和PaLCuCNV侵染MEAM1煙粉虱后也同樣能夠激活其MAPK途徑。(4) MEAM1煙粉虱體內JNK信號通路與細胞凋亡的關系JNK抑制劑處理MEAM1煙粉虱,TUNEL方法檢測結果發(fā)現(xiàn),JNK受到抑制后細胞凋亡現(xiàn)象發(fā)生明顯增強,表明MEAM1煙粉虱中JNK與細胞凋亡是相互抑制的關系。
[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.
【學位授予單位】：浙江大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S432.41;S433

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