發(fā)布時間：2018-06-27 03:03

  本文選題：馬鈴薯Y病毒屬 + 病毒復(fù)制　； 參考：《山東農(nóng)業(yè)大學(xué)》2017年博士論文

【摘要】：馬鈴薯Y病毒屬(Potyvirus)是最大植物病毒屬,包含200多種病毒。該屬病毒寄主范圍廣泛,發(fā)生普遍,給作物生產(chǎn)帶來嚴重損失。種植抗病品種是防治作物病毒病最為經(jīng)濟有效的方式。但目前生產(chǎn)中缺乏有效的抗病毒品種。植物病毒需要借助寄主蛋白來完成侵染循環(huán)。沉默或敲除病毒依賴的寄主蛋白可以使植物產(chǎn)生抗性。同時,理解病毒蛋白的分子功能以及病毒與寄主互作的分子機制是研發(fā)新的抗病毒策略的前提。本研究揭示了馬鈴薯Y病毒屬的煙草脈帶花葉病毒(Tobacco vein banding mosaic virus,TVBMV)在復(fù)制和移動兩個病毒侵染關(guān)鍵環(huán)節(jié)中與寄主的相互作用機制。主要研究結(jié)果如下:1、病毒寄主蛋白復(fù)合體6K1-6K2-PsbO1調(diào)控病毒復(fù)制的分子機制:第一6kDa蛋白(6K1)包含一個保守的RSD基序。丙氨酸掃描實驗證明該基序中任何一個氨基酸的改變都使TVBMV復(fù)制的復(fù)制水平下降。亞細胞定位實驗證明TVBMV 6K1單獨瞬時表達時分布在整個細胞之中,然而在病毒侵染過程中被招募到葉綠體并與TVBMV復(fù)制相關(guān)第二6 kDa蛋白(6K2)和復(fù)制酶核內(nèi)涵體b共定位。6K1能與6K2在體內(nèi)相互作用并被6K2招募到位于葉綠體的病毒復(fù)制復(fù)合體。免疫共沉淀和質(zhì)譜鑒定表明本氏煙光系統(tǒng)II放氧復(fù)合體蛋白(Photosystem II oxygen evolution complex protein of Nicotiana benthamiana,NbPsbO1)存在于6K1-6K2復(fù)合體上。NbPsbO1能與TVBMV 6K2蛋白互作但不與6K1互作。利用病毒誘導(dǎo)基因沉默降低NbPsbO1基因的表達抑制了馬鈴薯Y病毒屬病毒TVBMV和馬鈴薯Y病毒基因組RNA和外殼蛋白在植株中的積累量,但并不影響馬鈴薯X病毒屬的馬鈴薯X病毒基因組RNA和外殼蛋白的積累量。光系統(tǒng)II放氧復(fù)合體的另外兩個元件NbPsbP1和NbPsbQ1不能與6K2互作,而且沉默NbPsbP1和NbPsbQ1基因不影響TVBMV復(fù)制。2、病毒寄主蛋白復(fù)合體P3N-PIPO-CI-NbDREPP調(diào)控TVBMV細胞間移動的作用機制:TVBMV的PIPO結(jié)構(gòu)域由60個氨基酸位點組成。含有由7個、20個或43個氨基酸組成的PIPO結(jié)構(gòu)域的TVBMV突變體喪失了細胞間移動和系統(tǒng)侵染能力。但這些突變體的復(fù)制水平與野生型TVBMV一致。本氏煙發(fā)育調(diào)控質(zhì)膜蛋白(Developmentally regulated plasma membrane protein of N.benthamiana,NbDREPP)能與TVBMV P3N-PIPO和柱狀內(nèi)涵體蛋白CI互作。沉默NbDREPP基因抑制了TVBMV的細胞間移動和系統(tǒng)侵染。NbDREPP自身定位在細胞壁上,能與胞間連絲定位蛋白PDLP1共定位,而且能與P3N-PIPO和CI共定位于胞間連絲。早期分泌途徑抑制劑Brefeldin A處理或瞬時過表達顯性負抑制突變體ADP核糖基化因子1(ADP-ribosylation factor1)-T31N或分泌相關(guān)ras超家族相關(guān)基因1b(Secretion-associated and ras superfamily-related gene1b)-H74L都破壞了NbDREPP的胞間連絲定位。微絲骨架抑制劑Latrunculin B處理或過表達顯性負抑制突變體肌球蛋白XI-2尾部結(jié)構(gòu)域也破壞了NbDREPP的胞間連絲定位。3、通過新一代測序技術(shù)比較野生型TVBMV和弱毒突變體侵染植物后的轉(zhuǎn)錄組變化揭示了TVBMV的癥狀形成機制。輔助成分蛋白酶(helper component proteinase,HCpro)是TVBMV的RNA沉默抑制子。通過定點突變技術(shù)在TVBMV侵染性克隆pCamTVBMV-GFP(pCamT-WT)HCpro編碼區(qū)引入突變,獲得突變體pCamTVBMV-GFP-HCproD198K+DE250-251(pCamT-HCm)。這些氨基酸的改變破環(huán)了HCpro的RNA沉默抑制活性而且使T-HCm在本氏煙上的癥狀明顯減輕。我們通過轉(zhuǎn)錄組測序比較了T-WT和T-HCm侵染本氏煙1天、2天和10天后的基因表達變化。在T-WT或T-HCm侵染后1天和2天,與蛋白翻譯相關(guān)的基因上調(diào)表達;而與脂質(zhì)代謝以及胞內(nèi)刺激反應(yīng)相關(guān)基因下調(diào)表達。在病毒接種后10天,T-WT的侵染抑制了光合合成相關(guān)基因的表達,而T-HCm的侵染對光合合成相關(guān)基因無明顯影響。T-WT的侵染使RNA沉默相關(guān)途徑的關(guān)鍵基因DCL2、DCL4、RDR1和AGO1上調(diào)表達;而T-HCm的侵染對這些基因的表達無影響。T-WT的侵染也使水楊酸和乙烯信號途徑中的相關(guān)基因上調(diào)表達,但使茉莉酸信號途徑的相關(guān)基因下調(diào)表達。T-WT和T-HCm的侵染差異調(diào)控了生長素信號轉(zhuǎn)導(dǎo)途徑中相關(guān)基因的表達,這與TVBMV引起的矮化癥狀有關(guān)。綜上所述,TVBMV劫持寄主因子NbDREPP和NbPsbO1用于自身細胞間移動和復(fù)制,DREPP和PsbO1可以作為抗TVBMV的新靶標。TVBMV的侵染干擾了植物的卡爾文循環(huán)、葉綠素代謝以及生長素信號轉(zhuǎn)導(dǎo)途徑導(dǎo)致植物產(chǎn)生褪綠和矮化癥狀。
[Abstract]:Potato Y virus (Potyvirus) is the largest plant virus and contains more than 200 viruses. It has a wide range of host viruses and is widespread, causing serious loss to crop production. Planting resistant varieties is the most economical and effective way to prevent and control crop virus diseases. However, there are no effective antiviral varieties in production. Plant viruses need to be used. The host protein is the host protein to complete the infection cycle. The host protein that is silent or knocked out of the virus can cause resistance to plants. At the same time, understanding the molecular function of the virus protein and the molecular mechanism of the interaction between the virus and the host are the prerequisite for the development of the new antiviral strategy. This study revealed the tobacco vein zone mosaic virus (Tobacco) of the potato Y virus. Vein banding mosaic virus, TVBMV) interaction mechanism with the host in the replication and movement of two viral infection key links. The main results are as follows: 1, the molecular mechanism of viral replication by the host protein complex 6K1-6K2-PsbO1: the first 6kDa protein (6K1) package contains a conservative RSD motif. The alanine scanning experiment proved that The changes in any amino acid in the sequence reduced the replication level of TVBMV replication. The subcellular localization experiment showed that TVBMV 6K1 was distributed in the whole cell in a single transient expression. However, the chloroplasts were recruited during the virus infection process and were associated with the TVBMV replication related second 6 kDa egg white (6K2) and the replicating enzyme nucleus endosomal B to co localize.6K1. It can interact with 6K2 in the body and be recruited by 6K2 to the replication complex of the virus in the chloroplast. Immunoprecipitation and mass spectrometry identification show that the II oxygen complex protein (Photosystem II oxygen evolution complex protein of Nicotiana benthamiana) exists on the complex. Protein interaction but not interacted with 6K1. Using virus induced gene silencing to reduce the expression of NbPsbO1 gene inhibits the accumulation of genomic RNA and shell protein of potato Y virus TVBMV and potato Y virus, but does not affect the accumulation of genomic RNA and shell protein of potato X virus in potato X. The other two components of the II oxygen complex, NbPsbP1 and NbPsbQ1, cannot interact with 6K2, and the silence of NbPsbP1 and NbPsbQ1 genes does not affect TVBMV replicating.2, and the host protein complex P3N-PIPO-CI-NbDREPP regulates the mechanism of intercellular movement of TVBMV: TVBMV PIPO domain consists of 60 amino acid sites. 7, 20, or 43 contain The TVBMV mutant of the PIPO domain of the amino acid lost the intercellular movement and the ability to infect the system. However, the replication level of these mutants is the same as that of the wild type TVBMV. The plasma membrane protein (Developmentally regulated plasma membrane protein of N.benthamiana, NbDREPP) can be associated with TVBMV and columnar connotations. Somatic protein CI interacts. Silencing the NbDREPP gene inhibits the intercellular movement of TVBMV and the system infect.NbDREPP itself on the cell wall, can co localize with the cytoplasmin PDLP1, and can co locate with P3N-PIPO and CI in the intercellular plasmods. The early secretory pathway inhibitor Brefeldin A treatment or transient overexpression of dominant negative inhibition mutation ADP ribonucleic factor 1 (ADP-ribosylation factor1) -T31N or secretory related Ras superfamily associated gene 1B (Secretion-associated and RAS superfamily-related gene1b) -H74L both destroyed the localization of intercellular hyfiles. The domain also disrupted the NbDREPP intercellular localization.3. The transcriptional changes of the wild type TVBMV and the weakly toxic mutants revealed by a new generation sequencing technology revealed the mechanism of the TVBMV's symptom formation. The auxiliary component protease (helper component proteinase, HCpro) is a RNA silencing suppressor of TVBMV in RNA. The mutant pCamTVBMV-GFP-HCproD198K+DE250-251 (pCamT-HCm) was obtained by the introduction of mutation in the TVBMV infected clone pCamTVBMV-GFP (pCamT-WT) HCpro coding region. The changes of these amino acids changed the RNA silencing inhibitory activity of HCpro and reduced the symptoms of T-HCm on the tobacco. We compared T-WT and T-HCm infection through the sequence of transcriptional sequences. Gene expression changes in 1 days, 2 days and 10 days after 1 days. The genes related to protein translation were up-regulated at 1 and 2 days after T-WT or T-HCm infection. The genes related to lipid metabolism and intracellular stimulation were down regulated. The infection of T-WT inhibited the expression of photosynthetic related genes on the 10 day after the virus inoculation, and the infection of T-HCm The genes related to photosynthetic synthesis had no obvious effect on the infection of.T-WT, the key genes of RNA silencing related pathways, DCL2, DCL4, RDR1 and AGO1, were up-regulated, while T-HCm infection on these genes had no effect on the infection of.T-WT and related genes in salicylic acid and ethylene signal pathways, but related genes for jasmonic acid signaling pathways. The differential expression of.T-WT and T-HCm regulates the expression of related genes in the auxin signal transduction pathway, which is related to the dwarf symptoms caused by TVBMV. To sum up, TVBMV hijacking host factor NbDREPP and NbPsbO1 are used for intercellular migration and replication, and DREPP and PsbO1 can be interfered with the infection of.TVBMV of the new target for anti TVBMV. The Calvin cycle, chlorophyll metabolism and auxin signal transduction pathways cause the plants to produce chlorosis and dwarfing symptoms.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：S432.41

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