本文關(guān)鍵詞：水稻矮縮病毒非結(jié)構(gòu)蛋白Pns10的介體互作因子的篩選與功能驗(yàn)證　出處：《福建農(nóng)林大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

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【摘要】：水稻矮縮病毒(Rice dwarf virus,RDV)屬于持久增殖型病毒,主要由介體黑尾葉蟬傳播。在葉蟬內(nèi),病毒利用自身編碼的非結(jié)構(gòu)蛋白Pns10形成包裹病毒粒體的小管通道,采取"小管運(yùn)輸病毒"的策略進(jìn)行安全擴(kuò)散,以抵御葉蟬的各種免疫攻擊。這種安全運(yùn)輸病毒策略需要介體蛋白共同參與才能順利完成,先前研究表明Pns10通過(guò)酵母雙雜交篩選到介體互作蛋白肌動(dòng)蛋白(actin)、肌球蛋白(myosin)等,黑尾葉蟬胞質(zhì)型actin與Pns10的特異性互作決定黑尾葉蟬專化性傳播RDV,這些為Pns10互作介體蛋白的篩選提供了基礎(chǔ)。鑒于此,本研究繼續(xù)利用核蛋白互作的Clontech酵母雙雜交系統(tǒng)篩選數(shù)量相等的成蟲(chóng)和若蟲(chóng)的黑尾葉蟬cDNA庫(kù),獲取陽(yáng)性克隆。在NCBI Blastx進(jìn)行序列比對(duì)分析后,預(yù)測(cè)篩選的基因功能多為與昆蟲(chóng)生長(zhǎng)發(fā)育、細(xì)胞成分等相關(guān),挑選6個(gè)可能互作的蛋白:原肌球調(diào)節(jié)蛋白(tropomodulin,Tmod),線粒體孔蛋白(mitochondrial porin,Mito P),運(yùn)脂蛋白前體(lipophorin precusor,LP),高密度脂蛋白結(jié)合蛋白(Vigilin),凋亡誘導(dǎo)因子(apoptosis inducing factor,AIF),卵黃原蛋白(vitellogenin,Vg),將這6種蛋白的酵母質(zhì)粒與pGBKT7-Pns10再次回轉(zhuǎn)酵母感受態(tài)細(xì)胞AH109,確定Pns10與候選蛋白互作。雙分子免疫熒光互補(bǔ)技術(shù)(BiFC),證明Tmod、Vg和LP與Pns10互作,而Mito P、AIF和vigilin與Pns10不互作。在本氏煙共定位表達(dá)系統(tǒng)中,Tmod 和 Vg,vigilin,LP 均能與 RDV Pns10 共定位,而 Mito P、AIF不與Pns10共定位。為進(jìn)一步明確6個(gè)候選蛋白在RDV擴(kuò)散中發(fā)揮的作用,利用RT-qPCR實(shí)驗(yàn)檢測(cè)6個(gè)候選蛋白基因在帶毒培養(yǎng)細(xì)胞和黑尾葉蟬中的相對(duì)表達(dá)量。結(jié)果表明除Mito P與對(duì)照組無(wú)明顯差別外,其余5個(gè)基因的相對(duì)表達(dá)量均高于對(duì)照組。由于Tmod作為actin慢生長(zhǎng)端的唯一蓋帽蛋白,是actin的相關(guān)蛋白,而包裹病毒Pns10小管運(yùn)動(dòng)是沿著actin纖維絲進(jìn)行病毒的運(yùn)輸。利用GST-Pull down實(shí)驗(yàn)證明Tmod能與RDVPns10在體外發(fā)生特異性互作;Tmod熒光抗體免疫標(biāo)記帶毒培養(yǎng)細(xì)胞和黑尾葉蟬消化道,Tmod定位在微絨毛上,與Pns10共定位,Pns10小管能夠穿出actin形成的微絨毛。RT-qPCR分析Tmod、Pns10、RDVP8在帶毒葉蟬中的表達(dá)趨勢(shì)相似。利用dsTmod抑制Tmod在介體中的表達(dá),發(fā)現(xiàn)Tmod、Pns10和P8表達(dá)量明顯下降。并且干擾Tmod表達(dá)的第14天,蟲(chóng)體的帶毒率也下降了一半。因此,Tmod對(duì)Pns10行使功能有正調(diào)控作用,Pns10小管依賴于actin的小管動(dòng)力(ABTM)充分利用Tmod在介體中的調(diào)控作用,以至于actin能夠無(wú)限延伸,反向便捷了Pns10小管插入鄰近細(xì)胞,甚至為突破介體組織膜屏障提供動(dòng)力,最終實(shí)現(xiàn)病毒擴(kuò)散。Vg為卵黃蛋白前體,是卵發(fā)育的營(yíng)養(yǎng)物質(zhì),在脂肪體上合成釋放到血淋巴中,最終被卵巢吸收。利用GST-Pulldown實(shí)驗(yàn)進(jìn)一步確定Pns10與Vg互作。免疫熒光抗體標(biāo)記培養(yǎng)細(xì)胞,發(fā)現(xiàn)Vg可與Pns10共定位。此外,Pns10與Vg還可在卵巢濾泡細(xì)胞、菌胞和卵巢管柄處共定位,但當(dāng)Pns10與Vg大量共定位于卵巢管柄時(shí),卵內(nèi)并未標(biāo)記到Pns10蛋白,因此推測(cè)卵巢管柄是RDV侵入卵的初侵染點(diǎn),Vg與Pns10互作幫助了病毒在卵內(nèi)的擴(kuò)散。
[Abstract]:Rice dwarf virus (Rice dwarf, virus, RDV) belong to the persistent virus proliferation, mainly by the mediator cincticeps spread. In the use of their own within the leafhopper, virus encoding the non structural protein Pns10 formation of tubular channel virus particles package, take the "tubular transport virus" strategy for safe diffusion in various immune attack against the virus. The security strategy of leafhopper transport needs mediator protein to participate in the completion of Pns10, the yeast two hybrid screening to mediated protein interaction with actin (actin), previous research has shown that myosin (myosin), specific cincticeps cytoplasmic actin and Pns10 interaction decided cincticeps designed of the RDV transmission, which provides the basis for screening Pns10 interaction mediated protein. In view of this, this study continues the Clontech interaction of nuclear proteins by yeast two hybrid screening system is equal to the number of adults and nymphs The leafhopper cDNA library, to obtain positive clones. The sequences of NCBI in Blastx after the screening of gene function prediction for the growth and development of insects, and cellular components, select 6 protein may interaction: tropomodulin (tropomodulin, Tmod), mitochondrial porin (mitochondrial porin, Mito, P) transport of lipoprotein precursor (lipophorin precusor, LP), high density lipoprotein binding protein (Vigilin), apoptosis inducing factor (apoptosis inducing, factor, AIF), vitellogenin (vitellogenin, Vg), the yeast plasmid and pGBKT7-Pns10 of these 6 proteins again turning yeast competent AH109 cells, Pns10 and determination of the candidate protein interaction. Double immunofluorescence molecular complementary technology (BiFC), Tmod, Vg and LP and Pns10 interaction, and Mito P, AIF and vigilin and Pns10 interaction. In n.benthamiana co expression system, Tmod and Vg, vigilin, LP can and RDV Pns10 and Mito P, CO localization, AIF co localized with Pns10. In order to further clarify the 6 candidate proteins play the role in the diffusion of RDV, using RT-qPCR test to detect 6 candidate protein genes in infected cells and relative expression of leafhoppers in culture. The results showed that except Mito P and control group obvious difference, the relative expression of other 5 genes were higher than the control group. The Tmod as the only protein blocks actin slow growing end, is related to protein actin, and wrapped the virus Pns10 movement is carried out along the tubular virus actin filament transport. Using GST-Pull down experiments show that Tmod can specifically with RDVPns10 in vitro interaction; Tmod fluorescent antibody virus immune markers in cultured cells and leafhoppers in digestive tract, Tmod was localized in the microvilli, CO localization with Pns10, Pns10 can actin the formation of microvilli were tubular.RT-qPCR analysis Tmod, Pns 10, similar to RDVP8 in the expression. The expression trend of poison leafhopper, inhibition of Tmod mediated by dsTmod in Tmod, Pns10 and P8 expression decreased significantly. The expression of Tmod and interference for fourteenth days, the virus carrying rate of worms also fell by half. Therefore, the Tmod of Pns10 has positive regulation function effect of tubular actin dependent Pns10 tubule (ABTM) Regulation to make full use of Tmod in the medium, so that actin can be extended indefinitely, reverse Pns10 tubular insertion of adjacent cells is convenient, even to provide power for the breakthrough of mediator membrane barrier, and ultimately realize the spread of the virus.Vg yolk protein precursor, nutrition the material egg development, synthesis and release of blood in Bazhong with fat body, ovary was eventually absorbed. Experiments using GST-Pulldown Pns10 to further determine the interaction with Vg. Immunofluorescence antibody labeled cells, Vg was co localized with Pns10. In addition, Pns10 And Vg can also in ovarian follicular cells, bacteria and ovarian tube handle of the co localization of Pns10 and Vg, but when a large number of colocalized in ovarian tube handle, the egg does not mark to the Pns10 protein, suggesting that ovarian tube handle is RDV into eggs of primary infection, Vg interacts with Pns10 to help spread the virus in the egg.

【學(xué)位授予單位】：福建農(nóng)林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 齊敏;董淮富;;輪狀病毒感染及腸外擴(kuò)散機(jī)制的研究進(jìn)展[J];中國(guó)臨床藥理學(xué)與治療學(xué);2016年08期

2 陳倩;張玲華;黃海寧;魏太云;謝聯(lián)輝;;干擾水稻矮縮病毒(RDV)非結(jié)構(gòu)蛋白Pns11的表達(dá)可抑制病毒在介體黑尾葉蟬內(nèi)的復(fù)制[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2015年11期

3 徐雷;馮若飛;馬忠仁;;病毒受體研究進(jìn)展[J];動(dòng)物醫(yī)學(xué)進(jìn)展;2015年02期

4 何桂明;劉偉良;;水稻矮縮病的發(fā)生與防控技術(shù)[J];北京農(nóng)業(yè);2012年30期

5 向毓;廖志華;;水稻矮縮病的發(fā)生與防控技術(shù)[J];現(xiàn)代農(nóng)村科技;2011年12期

6 尹哲;吉栩;吳云鋒;李毅;;水稻矮縮病毒外殼蛋白P9具有體內(nèi)轉(zhuǎn)錄激活活性[J];中國(guó)農(nóng)業(yè)科技導(dǎo)報(bào);2007年03期

7 陳茂;葉恭銀;李毅;姚洪渭;胡萃;;水稻矮縮病毒基因組及毒粒三維結(jié)構(gòu)的研究進(jìn)展[J];植物保護(hù)學(xué)報(bào);2005年02期

8 邵承華,Z. H.Zhou,盧光瑩;水稻矮縮病毒內(nèi)殼層顆粒的三維結(jié)構(gòu)[J];中國(guó)科學(xué)(C輯:生命科學(xué));2001年02期

9 肖錦,李毅,張凈,劉勁鋒,陳章良;水稻矮縮病毒第一號(hào)組份基因和編碼蛋白的序列分析[J];微生物學(xué)報(bào);1998年05期

10 謝聯(lián)輝,林奇英,郭景榮;傳帶水稻矮縮病毒的二點(diǎn)黑尾葉蟬[J];福建農(nóng)業(yè)科技;1982年03期

相關(guān)博士學(xué)位論文 前1條

1 賈東升;SRBSDV和RRSV在介體飛虱體內(nèi)的侵染機(jī)理[D];福建農(nóng)林大學(xué);2013年

相關(guān)碩士學(xué)位論文 前2條

1 任堂雨;水稻矮縮病毒非結(jié)構(gòu)蛋白Pns10與介體葉蟬肌動(dòng)蛋白的互作關(guān)系[D];福建農(nóng)林大學(xué);2013年

2 章松柏;水稻矮縮病毒的檢測(cè)及部分基因的比較分析[D];福建農(nóng)林大學(xué);2004年

，

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