曲瀟玲，焦裕冰，羅健達(dá)，宋麗云，李瑩，申莉莉，楊金廣，王鳳龍

本氏煙的克隆及對(duì)馬鈴薯Y病毒侵染的抑制作用

曲瀟玲，焦裕冰，羅健達(dá)，宋麗云，李瑩，申莉莉，楊金廣，王鳳龍

中國(guó)農(nóng)業(yè)科學(xué)院煙草研究所，山東青島 266101

【】馬鈴薯Y病毒（potato virus Y，PVY）是危害我國(guó)煙草生產(chǎn)的最重要病毒之一，NAC轉(zhuǎn)錄因子與植物的抗病、抗逆密切相關(guān)，本論文克隆進(jìn)行生物信息學(xué)分析，并研究其在PVY侵染過(guò)程中的作用，為煙草抗病毒藥劑的開(kāi)發(fā)提供靶標(biāo)。以本氏煙（）為材料克隆，利用MEGA、UniProt、SMART、TMHMM Server 2.0、Sol Genomics Network、PlantCARE等技術(shù)進(jìn)行生物信息學(xué)分析；利用激光共聚焦與實(shí)時(shí)熒光定量PCR（quantitative real-time PCR，qRT-PCR）明確PVY侵染前后NbNAC062蛋白定位及mRNA表達(dá)量變化；基于病毒介導(dǎo)的基因沉默（virus-induced gene silencing，VIGS）和過(guò)表達(dá)技術(shù)，構(gòu)建pTRV::NbNAC062沉默載體與pEarleyGate100::RFP::NbNAC062過(guò)表達(dá)載體，采用qRT-PCR和Western blot檢測(cè)在本氏煙中沉默與過(guò)表達(dá)后，PVY的積累量變化及未折疊蛋白應(yīng)答（unfolded protein response，UPR）相關(guān)基因的表達(dá)差異。編碼646個(gè)氨基酸，N端28—179 aa為NAC結(jié)構(gòu)域，129—185 aa為DNA結(jié)合區(qū)域，C末端621—643 aa為疏水跨膜結(jié)構(gòu)，系統(tǒng)進(jìn)化樹(shù)與蛋白序列分析表明本氏煙與漸狹葉煙草親緣關(guān)系最近。啟動(dòng)子中包含脫落酸、茉莉酸甲酯、水楊酸以及逆境響應(yīng)相關(guān)的多種順式作用元件。PVY侵染激活NbNAC062從細(xì)胞膜轉(zhuǎn)移至細(xì)胞核，且誘導(dǎo)上調(diào)表達(dá)。PVY侵染本氏煙5、7 d，處理組mRNA水平分別為對(duì)照組的2.52、1.95倍；PVY侵染3 d，mRNA表達(dá)量為對(duì)照組的2.39倍，PVY侵染7 d，表達(dá)量極顯著低于對(duì)照組，下調(diào)表達(dá)56.77%。本氏煙沉默并接種PVY，接種后3、5、7 d，與對(duì)照組相比，沉默組PVYmRNA上調(diào)表達(dá)，分別為對(duì)照組的2.12、2.41、1.38倍，mRNA表達(dá)量則下調(diào)，分別下調(diào)28.19%、58.11%、10.77%，接種后5、7 d沉默組PVY CP蛋白含量亦顯著高于對(duì)照組。過(guò)表達(dá)并接種PVY，接種后24、48、72 h，與對(duì)照組相比，過(guò)表達(dá)組PVYmRNA分別下調(diào)22.60%、34.51%、36.21%，接種48、72 h，mRNA上調(diào)表達(dá)，分別為對(duì)照組的1.56、1.35倍，過(guò)表達(dá)組PVY CP蛋白含量亦低于對(duì)照組。NbNAC062屬于NAC類(lèi)膜結(jié)合轉(zhuǎn)錄因子，可被PVY侵染激活轉(zhuǎn)移至細(xì)胞核，可能通過(guò)調(diào)控UPR相關(guān)基因的表達(dá)，促進(jìn)細(xì)胞生存，抑制PVY早期侵染。

；馬鈴薯Y病毒；基因沉默；瞬時(shí)過(guò)表達(dá)

0 引言

【研究意義】馬鈴薯Y病毒（potato virusY，PVY）是一種單鏈正義RNA病毒[1]，寄主范圍廣泛，能侵染34個(gè)屬170余種植物[2]。煙草馬鈴薯Y病毒病會(huì)造成煙株花葉、脈壞死、莖壞死、點(diǎn)刻條斑等癥狀[3-4]，目前已成為危害我國(guó)煙草生產(chǎn)最主要的病毒病之一[5-7]。NAC類(lèi)轉(zhuǎn)錄因子與植物抗病、抗逆相關(guān)[8]，研究其在PVY侵染中的調(diào)控作用，可為煙草抗病毒藥劑的開(kāi)發(fā)提供靶標(biāo)，對(duì)防控馬鈴薯Y病毒病具有重要意義?！厩叭搜芯窟M(jìn)展】NAC是一類(lèi)植物特有的轉(zhuǎn)錄因子[9]，在整個(gè)生命周期中起到重要作用。在植物生長(zhǎng)發(fā)育中，NAC類(lèi)轉(zhuǎn)錄因子參與調(diào)控種子萌發(fā)[10]、次生細(xì)胞壁生長(zhǎng)[11]、葉片與果實(shí)衰老[12-13]等過(guò)程。在植物抗逆與激素信號(hào)傳導(dǎo)中，NAC類(lèi)轉(zhuǎn)錄因子參與調(diào)控植物抗病、冷脅迫、干旱脅迫以及水楊酸（salicylic acid，SA）[14]、脫落酸（abscisic acid，ABA）[15]、茉莉酸（jasmonic acid，JA）[16]等應(yīng)答反應(yīng)。內(nèi)質(zhì)網(wǎng)是真核生物蛋白形成加工的重要場(chǎng)所[17]，蛋白的折疊合成過(guò)程復(fù)雜，易受外界環(huán)境影響。當(dāng)植物處于逆境，細(xì)胞內(nèi)未折疊蛋白與錯(cuò)誤蛋白累積過(guò)量時(shí)，內(nèi)質(zhì)網(wǎng)會(huì)啟動(dòng)未折疊蛋白應(yīng)答（unfolded protein response，UPR），調(diào)節(jié)一系列下游基因如分子伴侶的表達(dá)，幫助蛋白正確折疊，維持內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)[18]。植物膜結(jié)合轉(zhuǎn)錄因子在UPR信號(hào)傳導(dǎo)過(guò)程中起到重要作用，NAC062則是一種參與UPR的NAC類(lèi)膜結(jié)合轉(zhuǎn)錄因子。以往研究發(fā)現(xiàn)，NAC062主要在干旱、寒冷、高鹽等逆境過(guò)程中起調(diào)控作用。擬南芥中NAC062蛋白在A(yíng)BA介導(dǎo)的抗旱反應(yīng)中起調(diào)節(jié)作用，過(guò)表達(dá)的植株表現(xiàn)出強(qiáng)的抗旱性[19]。另外NAC062還能將冷信號(hào)整合到植物防御反應(yīng)中。當(dāng)受到冷脅迫時(shí)，NAC062蛋白發(fā)生水解[20]，直接與病程相關(guān)蛋白（pathogenesis-related protein，PR）、、結(jié)合，激發(fā)病原體抗性反應(yīng)[21]，促使植株對(duì)丁香假單胞菌（）的侵染表現(xiàn)出較強(qiáng)的抗性。【本研究切入點(diǎn)】目前，關(guān)于NAC062的研究多數(shù)集中在模式植物擬南芥的冷害和干旱等非生物脅迫中，而在生物脅迫尤其是在病毒侵染中的功能研究較少。生物信息學(xué)分析表明啟動(dòng)子序列中包含抗逆、抗病等多種脅迫響應(yīng)相關(guān)的順式作用元件，但目前尚無(wú)基因克隆及其在PVY侵染中作用的研究，鑒于NAC062在內(nèi)質(zhì)網(wǎng)脅迫應(yīng)答以及抗逆過(guò)程中的重要作用，本文探究煙草NAC062轉(zhuǎn)錄因子在PVY侵染中的功能及相關(guān)的調(diào)控信號(hào)?！緮M解決的關(guān)鍵問(wèn)題】以本氏煙（）為材料，克隆，進(jìn)行生物信息學(xué)分析與亞細(xì)胞定位，構(gòu)建沉默與過(guò)表達(dá)載體，研究其在PVY侵染過(guò)程中的生物學(xué)功能。

1 材料與方法

試驗(yàn)于2019—2020年在中國(guó)農(nóng)業(yè)科學(xué)院煙草研究所完成。

1.1 試驗(yàn)材料

植物材料：本氏煙，中國(guó)農(nóng)業(yè)科學(xué)院煙草研究所保存，試驗(yàn)用煙均為5—6葉期溫室土培煙苗，溫室培養(yǎng)條件為溫度（25±1）℃，光周期16 h光照/8 h黑暗，光照強(qiáng)度2 000 lx，相對(duì)濕度65%；病毒：馬鈴薯Y病毒，中國(guó)農(nóng)業(yè)科學(xué)院煙草研究所病毒課題組保存于枯斑三生煙（var. Samsun NN）活體上；侵染性克?。篜VY-GFP[22]由中國(guó)農(nóng)業(yè)科學(xué)院煙草研究所病毒課題組提供，于TE緩沖液中，-80℃保存。

1.2 NbNAC062克隆與分析

根據(jù)National Center for Biotechnology Information（https://www.ncbi.nlm.nih.gov）網(wǎng)站預(yù)測(cè)的（序列號(hào)：XM_019370781.1）序列設(shè)計(jì)引物NbNAC062 F/R（表1），以本氏煙cDNA為模板，利用Phanta Max Master Mix高保真酶（Vazyme）進(jìn)行PCR擴(kuò)增，擴(kuò)增產(chǎn)物連接至pCE2-TA/Blunt-Zero（Vazyme）載體并轉(zhuǎn)化1-T1（TransGen Biotech）大腸桿菌感受態(tài)細(xì)胞，陽(yáng)性克隆送派森諾生物科技有限公司測(cè)序。利用MEGA7（https://www.megasoftware.net）、DNAMAN（https://www.lynnon.com/qa.html）、UniProt（https:// www.uniprot.org）、SMART（http://smart.embl.de）、TMHMM Server 2.0（http://www.cbs.dtu.dk/services/ TMHMM）、Sol Genomics Network（https://solgenomics. net/）、PlantCARE（http://bioinformatics.psb.ugent.be）等生物信息學(xué)工具對(duì)序列進(jìn)行分析。

1.3 PVY侵染對(duì)NbNAC062的影響

取PVY侵染的枯斑三生煙病葉1 g，加入PBS緩沖液（0.01 mol·L-1，pH 7.2—7.4）40 mL，于滅菌研缽中充分研磨后濾殘?jiān)?，得病毒接種液。對(duì)本氏煙第3—4片真葉浸潤(rùn)病毒接種液，每片葉200 μL[23]，以浸潤(rùn)PBS緩沖液為對(duì)照，接種后1、3、5、7 d取接種葉進(jìn)行qRT-PCR。

1.4 NbNAC062沉默載體的構(gòu)建

根據(jù)序列設(shè)計(jì)含有RⅠⅠ酶切位點(diǎn)的引物TRV-NbNAC F/R（表1），以本氏煙cDNA為模板，PCR擴(kuò)增沉默片段。利用ClonExpress II One Step Cloning Kit（Vazyme）將連接至pTRV載體，構(gòu)建pTRV:: NbNAC062重組載體；將含有pTRV::NbNAC062載體的農(nóng)桿菌（OD600=0.5）浸潤(rùn)本氏煙下表皮，每片葉500 μL，以浸潤(rùn)含有pTRV::PDS載體的農(nóng)桿菌為陽(yáng)性對(duì)照，浸潤(rùn)含有pTRV00空載的農(nóng)桿菌為陰性對(duì)照，浸潤(rùn)15 d檢測(cè)沉默效率并浸潤(rùn)PVY接種液。接種后1、3、5、7 d取接種葉，進(jìn)行qRT-PCR與Western blot檢測(cè)PVY CP積累量及UPR相關(guān)基因表達(dá)量。另取部分沉默植株，浸潤(rùn)PVY-GFP侵染性克隆，5 d后在手持紫外燈下觀(guān)察葉片熒光情況。

1.5 NbNAC062瞬時(shí)過(guò)表達(dá)載體的構(gòu)建

根據(jù)序列設(shè)計(jì)含有Ⅰ、RⅠ酶切位點(diǎn)的引物Fu-NbNAC F/R（表1），以本氏煙cDNA為模板，PCR擴(kuò)增，擴(kuò)增產(chǎn)物連接Fu46- RFP載體，構(gòu)建入門(mén)載體Fu46::RFP::NbNAC062。利用LR ClonaseTMII enzyme mix（Invitrogen）將入門(mén)載體同源重組至pEarleyGate100載體，最終構(gòu)建pEarleyGate100::RFP::NbNAC062表達(dá)載體[24]。將含有pEarleyGate100::RFP::NbNAC062載體的農(nóng)桿菌（OD600=0.5）浸潤(rùn)本氏煙下表皮，每片葉500 μL，以浸潤(rùn)含有pEarleyGate100::RFP的農(nóng)桿菌為對(duì)照。浸潤(rùn)農(nóng)桿菌12 h后，統(tǒng)一浸潤(rùn)PVY接種液，接種后24、48、72 h取接種葉進(jìn)行qRT-PCR與Western blot檢測(cè)PVY CP積累量及UPR相關(guān)基因表達(dá)量。

1.6 NbNAC062蛋白的亞細(xì)胞定位

將含有pEarleyGate100::RFP::NbNAC062載體的農(nóng)桿菌（OD600=0.5）浸潤(rùn)本氏煙下表皮，每片葉500 μL，12 h后接種PVY，溫室培養(yǎng)48 h。利用DAPI細(xì)胞核藍(lán)色染液（358 nm/461 nm，Solarbio）、BBcellProbe M01細(xì)胞膜綠色染液（488 nm/500 nm，Bestbio），進(jìn)行細(xì)胞染色后，激光共聚焦觀(guān)察NbNAC062蛋白亞細(xì)胞定位。

1.7 qRT-PCR與Western blot

取各時(shí)間段處理組與對(duì)照組樣品，利用RNA isolater Total RNA Extraction Reagent（Vazyme）與Plant Protein Extraction Kit（CWBIO）提取總RNA與總蛋白。根據(jù)不同樣品的序列設(shè)計(jì)特異性熒光定量檢測(cè)引物（表1），總RNA反轉(zhuǎn)錄成cDNA（Vazyme），利用ChamQTMUniversal SYBR? qPCR Master Mix（Vazyme）試劑盒進(jìn)行qRT-PCR。以為內(nèi)參基因，2-ΔΔCt法計(jì)算相對(duì)表達(dá)量，反應(yīng)程序：95℃ 30 s；95℃10 s，60℃30 s，40個(gè)循環(huán)；95℃15 s，60℃60 s，95℃15 s?？偟鞍捉?jīng)SDS-PAGE電泳（180 V、32 min），濕法轉(zhuǎn)膜（100 V、90 min）后，分別4℃過(guò)夜孵育PVY抗體（Agdia，兔源），Actin抗體（CWBIO，鼠源），對(duì)應(yīng)二抗Goat Anti-Rabbit IgG（CWBIO），Goat Anti-Mouse IgG（CWBIO）25℃孵育2 h后觀(guān)察Western blot結(jié)果。

2 結(jié)果

2.1 NbNAC062克隆與分析

以本氏煙cDNA為模板，NbNAC062 F/R為引物（表1），PCR擴(kuò)增出1 944 bp CDS序列，編碼646個(gè)氨基酸。CDS序列于NCBI全網(wǎng)比對(duì)，發(fā)現(xiàn)與漸狹葉煙草（）、栽培煙草（）、林煙草（）、絨毛狀煙草（）的序列最為相似，相似度分別為96.04%、95.88%、95.88%、94.91%，對(duì)應(yīng)蛋白序列相似度為95.83%、94.28%、94.28%、93.04%。其他物種核苷酸序列相似度為83.71%—85.27%，對(duì)應(yīng)蛋白序列相似度為74.73%—79.35%。利用MEGA7，鄰接法、自舉抽樣各分支置信度為1 000構(gòu)建系統(tǒng)發(fā)育樹(shù)（圖1）[25]。利用DNAMAN進(jìn)行氨基酸序列比對(duì)（圖2-A），結(jié)果均顯示，擴(kuò)增序列與漸狹葉煙草序列相似度最高，表明擴(kuò)增序列為本氏煙的同源序列，命名為。

表1 本試驗(yàn)所用引物

圖1 NbNAC062系統(tǒng)發(fā)育分析

利用UniProt[26]、SMART[27-29]、TMHMM Server 2.0[30]對(duì)NbNAC062蛋白進(jìn)行序列分析，發(fā)現(xiàn)第28—179 aa為NAC結(jié)構(gòu)域，第129—185 aa為DNA結(jié)合區(qū)域，第621—643 aa為C端疏水跨膜域（圖2-B）。

序列于Sol Genomics Network網(wǎng)站比對(duì)獲取其啟動(dòng)子。PlantCARE分析發(fā)現(xiàn)啟動(dòng)子序列中包含多種脅迫反應(yīng)順式作用元件，包括脫落酸反應(yīng)順式作用元件ABRE、茉莉酸甲酯反應(yīng)順式作用元件TGACG-motif和CGTCA-motif、參與轉(zhuǎn)錄表達(dá)的水楊酸響應(yīng)元件as-1，以及參與干旱、高鹽、低溫等脅迫應(yīng)答相關(guān)的順式作用元件DRE、MYB、MYC。

A：NbNAC062蛋白氨基酸序列對(duì)比分析。深藍(lán)色為氨基酸完全相同，粉色為有一個(gè)氨基酸不同，淺藍(lán)色為兩個(gè)氨基酸不同。紅、黃、綠色箭頭線(xiàn)分別代表NAC結(jié)構(gòu)域、DNA結(jié)合區(qū)域、TMD跨膜結(jié)構(gòu)域Comparative analysis of NbNAC062 protein amino acid sequence. Dark blue indicates that the amino acids are identical, pink indicates that there is only one amino acid difference, light blue indicates that there are two amino acids differences. The red, yellow, and green arrow lines represent the NAC domain, the DNA binding region, and the TMD transmembrane domain in turn；B：NbNAC062蛋白結(jié)構(gòu)域分析Protein domain analysis of NbNAC062

2.2 PVY侵染對(duì)NbNAC062的影響

本氏煙接種PVY后1、3、5、7 d取接種葉進(jìn)行qRT-PCR與統(tǒng)計(jì)分析。結(jié)果表明PVY侵染導(dǎo)致表達(dá)量上調(diào)，其中接種PVY 5 d，mRNA水平顯著高于接種PBS對(duì)照組，為對(duì)照組的2.52倍；接種PVY 7 d，mRNA水平極顯著高于對(duì)照組，為對(duì)照組的1.95倍（圖3-A）。qRT-PCR檢測(cè)UPR相關(guān)基因發(fā)現(xiàn)，接種PVY后1、3、5 d，mRNA表達(dá)量逐漸上調(diào)，其中接種PVY 3 d，mRNA水平極顯著高于對(duì)照組，為對(duì)照組的2.39倍，接種后7 d，表達(dá)量極顯著低于對(duì)照組，下調(diào)表達(dá)56.77%（圖3-B），說(shuō)明及可能在PVY侵染中發(fā)揮作用。

含pEarleyGate100::RFP::NbNAC062載體的農(nóng)桿菌介導(dǎo)浸潤(rùn)本氏煙下表皮，12 h后接種PVY，溫室培養(yǎng)48 h進(jìn)行激光共聚焦觀(guān)察。結(jié)果表明，PVY侵染前，NbNAC062蛋白定位于細(xì)胞膜，PVY侵染后部分NbNAC062蛋白轉(zhuǎn)移至細(xì)胞核（圖4）。

*表示差異顯著P＜0.05，**表示差異極顯著P＜0.01。下同

A：NbNAC062蛋白亞細(xì)胞定位，BBcellProbe M01為細(xì)胞膜綠色染料，激發(fā)波長(zhǎng)/發(fā)射波長(zhǎng)為488 nm/500 nm Subcellular localization of NbNAC062 protein, BBcellProbe M01 is cell membrane green dye, excitation wavelength/emission wavelength is 488 nm/500 nm；B：PVY侵染后NbNAC062蛋白亞細(xì)胞定位，DAPI為細(xì)胞核藍(lán)色染料，激發(fā)波長(zhǎng)/發(fā)射波長(zhǎng)為358 nm/461 nm Subcellular localization of NbNAC062 protein after PVY infection, DAPI is nuclear blue dye, excitation wavelength/emission wavelength is 358 nm/461 nm

2.3 沉默NbNAC062對(duì)PVY侵染的影響

含pTRV::NbNAC062沉默載體的農(nóng)桿菌介導(dǎo)浸潤(rùn)本氏煙第7天，沉默組與陰性對(duì)照組pTRV00表型無(wú)明顯變化，陽(yáng)性對(duì)照組pTRV::PDS葉片開(kāi)始出現(xiàn)白化現(xiàn)象。第15天，qRT-PCR檢測(cè)沉默效率為84.25%（圖5）。接種PVY后，1、3、5、7 d取接種葉進(jìn)行qRT-PCR與Western blot檢測(cè)PVY CP積累量。qRT-PCR結(jié)果顯示，3、5、7 d沉默組本氏煙PVYmRNA顯著高于對(duì)照組，分別為對(duì)照組的2.12、2.41、1.38倍。Western blot顯示5、7 d沉默組PVY CP蛋白含量顯著高于對(duì)照組。通過(guò)PVYmRNA和蛋白在沉默植株中積累的分析表明，表達(dá)的下調(diào)顯著促進(jìn)了PVY的積累。qRT-PCR檢測(cè)表達(dá)，3、5、7 d沉默組mRNA表達(dá)量逐漸下調(diào)，分別下調(diào)表達(dá)28.19%、58.11%、10.77%，說(shuō)明沉默抑制了的表達(dá)（圖6）。手持紫外燈下觀(guān)察，沉默組PVY-GFP熒光強(qiáng)度高于對(duì)照組（圖5），也說(shuō)明本氏煙沉默促進(jìn)了PVY的侵染。

A：沉默第7天處理組pTRV::NbNAC062、陰性對(duì)照組pTRV00、陽(yáng)性對(duì)照組pTRV::PDS表型On the 7th day of silence, the phenotype of pTRV::NbNAC062 treatment group, pTRV00 negative control group and pTRV::PDS positive control group；B：沉默NbNAC062第15天沉默效率Silencing efficiency of NbNAC062 on the 15th day；C：NbNAC062沉默植株接種PVY-GFP第5天葉片熒光情況Fluorescence of leaves on the 5th day after silencing NbNAC062 inoculated with PVY-GFP

2.4 過(guò)表達(dá)NbNAC062對(duì)PVY侵染的影響

含過(guò)表達(dá)載體的農(nóng)桿菌介導(dǎo)浸潤(rùn)本氏煙12 h后，浸潤(rùn)PVY接種液，接種后24、48、72 h取接種葉進(jìn)行qRT-PCR與Western blot檢測(cè)PVY CP積累量，結(jié)果表明，過(guò)表達(dá)組PVYmRNA含量基本持平，對(duì)照組呈上升趨勢(shì)，接種72 h，過(guò)表達(dá)組PVYmRNA含量顯著低于對(duì)照組，下調(diào)表達(dá)36.21%。Western blot結(jié)果顯示，接種48、72 h，過(guò)表達(dá)組PVY蛋白含量低于對(duì)照組，說(shuō)明過(guò)表達(dá)抑制PVY的侵染。qRT-PCR檢測(cè)表達(dá)，接種48、72 h，過(guò)表達(dá)組mRNA表達(dá)量極顯著高于對(duì)照組，分別為對(duì)照組的1.56、1.35倍，說(shuō)明過(guò)表達(dá)促進(jìn)了的表達(dá)（圖7）。

3 討論

NAC062是一種膜結(jié)合轉(zhuǎn)錄因子，屬于NAC類(lèi)轉(zhuǎn)錄因子，N端有NAC保守結(jié)構(gòu)域，C末端有疏水跨膜結(jié)構(gòu)[31]，主要參與植物抗逆反應(yīng)、激素信號(hào)傳導(dǎo)以及生長(zhǎng)發(fā)育等過(guò)程。擬南芥中，被發(fā)現(xiàn)可被高鹽等非生物脅迫以及ABA等植物激素觸發(fā)，參與植物抗旱反應(yīng)；冷脅迫可使AtNAC062蛋白從細(xì)胞膜釋放并進(jìn)入細(xì)胞核，調(diào)節(jié)的表達(dá)，增強(qiáng)植株對(duì)病原菌的抗性反應(yīng)。本研究以本氏煙cDNA為模板，擴(kuò)增得到序列，編碼646個(gè)氨基酸，N端28—179 aa為NAC結(jié)構(gòu)域，129—185 aa為DNA結(jié)合區(qū)域，C末端621—643 aa為疏水跨膜結(jié)構(gòu)，與AtNAC062結(jié)構(gòu)相同。啟動(dòng)子序列中包含多種脅迫反應(yīng)順式作用元件，例如轉(zhuǎn)錄表達(dá)的水楊酸響應(yīng)元件as-1，脫落酸反應(yīng)順式作用元件ABRE以及DRE、MYB、MYC等參與干旱、高鹽、低溫等脅迫誘導(dǎo)相關(guān)的順式作用元件，說(shuō)明同類(lèi)似，可參與抗逆、抗病反應(yīng)。

A：沉默NbNAC062后PVY侵染1、3、5、7 d，qRT-PCR檢測(cè)PVY CP mRNA變化Changes of PVY CP mRNA are detected by qRT-PCR when PVY infection was 1, 3, 5, 7 days after silencing NbNAC062；B：沉默NbNAC062后PVY侵染1、3、5、7 d，qRT-PCR檢測(cè)BiP變化Changes of BiP are detected by qRT-PCR when PVY infection was 1, 3, 5, 7 days after silencing NbNAC062；C：沉默NbNAC062后PVY侵染1、3、5、7 d，western blot檢測(cè)PVY蛋白量變化。每天取樣組中左側(cè)為對(duì)照右側(cè)為處理Changes of PVY protein are detected by western blot when PVY infection was 1, 3, 5, 7 days after silencing NbNAC062. In the daily sampling group, the left side is the control and the right side is the treatment；D：C對(duì)應(yīng)的Actin western blot蛋白雜交圖The western blot protein hybridization map of Actin corresponding to C

除冷脅迫外，內(nèi)質(zhì)網(wǎng)脅迫（endoplasmic reticulum stress，ER stress）也可激活NAC062發(fā)揮作用。許多RNA病毒可以利用內(nèi)質(zhì)網(wǎng)的蛋白合成功能附著在內(nèi)質(zhì)網(wǎng)上，完成自身的復(fù)制組裝[32-34]。作為RNA病毒的PVY，其進(jìn)入宿主細(xì)胞后，誘導(dǎo)內(nèi)質(zhì)網(wǎng)形成囊泡狀結(jié)構(gòu)，以構(gòu)建病毒復(fù)制復(fù)合體（viral replication complex，VRC），完成自身增殖，因而PVY的復(fù)制增殖與內(nèi)質(zhì)網(wǎng)密切相關(guān)[35]。與此同時(shí)，大量病毒蛋白的聚集會(huì)造成內(nèi)質(zhì)網(wǎng)脅迫[36]，細(xì)胞啟動(dòng)UPR，以維持內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)。NAC類(lèi)轉(zhuǎn)錄因子在UPR信號(hào)傳導(dǎo)中起到重要作用。擬南芥中，AtNAC062通常以休眠形式定位于細(xì)胞膜，內(nèi)質(zhì)網(wǎng)脅迫誘導(dǎo)劑tunicamycin，）處理后，引發(fā)ER stress，AtNAC062被激活從細(xì)胞膜轉(zhuǎn)移至細(xì)胞核，調(diào)節(jié)下游UPR相關(guān)基因等的表達(dá)[37]。分子伴侶是蛋白質(zhì)折疊過(guò)程中十分重要的輔助因子，能與非自然狀態(tài)的蛋白短暫互作，幫助它們穩(wěn)定原生狀態(tài)，防止錯(cuò)誤折疊。BiP（binding protein）蛋白是一種常見(jiàn)的分子伴侶，在內(nèi)質(zhì)網(wǎng)腔中含量較高，是UPR途徑中維持內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)的重要基因。本研究發(fā)現(xiàn)PVY侵染本氏煙前后，激光共聚焦觀(guān)察NbNAC062蛋白從細(xì)胞膜轉(zhuǎn)移至細(xì)胞核，PVY侵染前期，UPR相關(guān)基因表達(dá)量逐漸上調(diào)，接種PVY 3 d，表達(dá)量為對(duì)照組的2.39倍，接種7 d后，表達(dá)量開(kāi)始下調(diào)，下調(diào)表達(dá)56.77%。說(shuō)明PVY侵染會(huì)激活NbNAC062蛋白至細(xì)胞核發(fā)揮轉(zhuǎn)錄作用，及UPR相關(guān)基因在PVY侵染中發(fā)揮作用。伴隨的沉默與過(guò)量表達(dá)，的表達(dá)量也相應(yīng)發(fā)生下調(diào)和上調(diào)，分別下調(diào)10.77%—58.11%，上調(diào)1.35—1.56倍，說(shuō)明NbNAC062轉(zhuǎn)錄因子可能調(diào)控的表達(dá)，內(nèi)質(zhì)網(wǎng)脅迫時(shí)，通過(guò)加強(qiáng)轉(zhuǎn)錄達(dá)到維持內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)的作用，本研究還發(fā)現(xiàn)PVY侵染引起表達(dá)上調(diào)，綜上推測(cè)，PVY利用內(nèi)質(zhì)網(wǎng)完成自身復(fù)制增殖時(shí)造成ER stress，寄主通過(guò)NbNAC062轉(zhuǎn)錄因子，上調(diào)表達(dá)維持內(nèi)質(zhì)網(wǎng)穩(wěn)態(tài)，調(diào)節(jié)細(xì)胞穩(wěn)定性來(lái)增強(qiáng)寄主基礎(chǔ)抗性。

A：過(guò)表達(dá)NbNAC062后PVY侵染1、3、5、7 d，qRT-PCR檢測(cè)PVY CP mRNA變化Changes of PVY CP mRNA are detected by qRT-PCR when PVY infection was 1, 3, 5, 7 days after overexpression NbNAC062；B：過(guò)表達(dá)NbNAC062后PVY侵染1、3、5、7 d，qRT-PCR檢測(cè)BiP變化Changes of BiP are detected by qRT-PCR when PVY infection was 1, 3, 5, 7 days after overexpression NbNAC062；C：過(guò)表達(dá)NbNAC062后PVY侵染1、3、5、7 d，western blot檢測(cè)PVY蛋白量變化。每天取樣組中左側(cè)為對(duì)照右側(cè)為處理Changes of PVY protein are detected by Western blot when PVY infection was 1, 3, 5, 7 days after overexpression NbNAC062. In the daily sampling group, the left side is the control and the right side is the treatment；D：C對(duì)應(yīng)的Actin western blot蛋白雜交圖The western blot protein hybridization map of Actin corresponding to C

目前，關(guān)于的研究多數(shù)集中在非生物脅迫中，關(guān)于其增強(qiáng)病原菌抗性的研究也是基于冷脅迫誘導(dǎo)，其在生物脅迫中的功能研究較少，尤其是在病毒侵染方面。本研究利用病毒介導(dǎo)的基因沉默（virus- induced gene silencing，VIGS）與瞬時(shí)過(guò)表達(dá)技術(shù)，研究在PVY侵染過(guò)程中的作用，發(fā)現(xiàn)的表達(dá)量與PVY的積累量呈負(fù)相關(guān)，證明對(duì)PVY侵染具有抑制作用，并調(diào)節(jié)下游相關(guān)基因的表達(dá)，后續(xù)研究會(huì)通過(guò)敲除和過(guò)表達(dá)突變體，進(jìn)一步確認(rèn)這一結(jié)果。本試驗(yàn)為研究PVY侵染過(guò)程中寄主自我防衛(wèi)機(jī)制提供了新思路。目前，我國(guó)傳統(tǒng)的農(nóng)業(yè)、物理和化學(xué)防治方法對(duì)于病毒病的防治效果并不理想[38]，鑒于對(duì)PVY的抑制作用，未來(lái)可考慮利用納米材料包裹，開(kāi)發(fā)抗病毒納米藥劑。

4 結(jié)論

NbNAC062屬于NAC類(lèi)膜結(jié)合轉(zhuǎn)錄因子，PVY侵染本氏煙導(dǎo)致表達(dá)上調(diào)，激活其從細(xì)胞膜轉(zhuǎn)移至細(xì)胞核，調(diào)控UPR相關(guān)基因表達(dá)，維持細(xì)胞穩(wěn)態(tài)。本氏煙沉默導(dǎo)致PVY積累量上升；過(guò)表達(dá)則使PVY積累量下降，說(shuō)明對(duì)PVY侵染有一定抑制作用，未來(lái)或可作為抗病毒藥劑開(kāi)發(fā)的靶標(biāo)。

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Cloning ofand Its Inhibitory Effect on Potato Virus Y Infection

Qu XiaoLing, Jiao YuBing, Luo JianDa, SONG LiYun, LI Ying, Shen LilLi, Yang JinGuang, WANG FengLong

Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong

【】Potato Y virus (PVY) is one of the most important viruses that endanger the tobacco production in China. NAC transcription factors are closely related to plant disease resistance and stress resistance. The objective of this study is to clone, analyze its bioinformatics and research its role in the process of PVY infection, and to provide a target for the development of tobacco antiviral agents. 【】was used as the material to clone, and MEGA, UniProt, SMART, TMHMM Server 2.0, Sol Genomics Network, PlantCARE and other technologies were used for bioinformatics analysis. Laser confocal microscope and quantitative real-time PCR (qRT-PCR) were used to clarify the localization of NbNAC062 protein and the change ofmRNA expression before and after PVY infection. Based on virus-induced gene silencing (VIGS) technology and over-expression technology, the pTRV::NbNAC062 silencing vector and the pEarleyGate100::RFP::NbNAC062 over-expression vector were constructed. qRT-PCR and Western blot were used to detect the changes of PVY accumulation and the expression of unfolded protein response (UPR) related geneafter silencing and over-expression in.【】encodes 646 amino acids, the N-terminal 28-179 aa is the NAC domain, 129-185 aa is the DNA binding region, and the C-terminal 621-643 aa is a hydrophobic transmembrane structure. Phylogenetic tree and protein sequence analysis show thatis closely related to. Thepromoter contains a variety of cis-acting elements related to abscisic acid, methyl jasmonate, salicylic acid and stress response. PVY infection activates NbNAC062 to transfer from cell membrane to nucleus and inducesup-regulation of expression. For 5 and 7 days after PVY infection, themRNA level in the treatment group was 2.52 and 1.95 times of that of the control group, respectively. For 3 days after PVY infection, themRNA expression was 2.39 times of that of the control group, and for 7 days after PVY infection, the expression ofwas significantly lower than that of the control group, which was down-regulated by 56.77%.was silenced and PVY was inoculated, compared with the control group, the expression of PVYmRNA was up-regulated in the silence group at 3, 5, and 7 days after inoculation, which was 2.12, 2.41, and 1.38 times of that of the control group, respectively. However, the expression ofmRNA was down-regulated by 28.19%, 58.11%, and 10.77%, respectively. The PVY CP protein content of the silence group was also significantly higher than that of the control group at 5 and 7 days after vaccination.was over-expressed and PVY was inoculated, compared with the control group, the expression of PVYmRNA in the over-expression group at 24, 48, 72 hours after inoculation was down-regulated by 22.60%, 34.51%, and 36.21%, respectively, andmRNA was up-regulated at 48 and 72 hours after inoculation, which was 1.56 and 1.35 times of that of the control group, respectively. The content of PVY CP in the over-expression group was also lower than that of the control group.【】NbNAC062 belongs to the NAC class of membrane-bound transcription factors, which can be activated by PVY infection and transferred to the nucleus. It may regulate the expression of the UPR-related geneto promote cell survival and inhibit early PVY infection.

; potato virus Y (PVY); gene silencing; transient over-expression

10.3864/j.issn.0578-1752.2021.19.007

2021-02-22；

2021-04-14

中國(guó)農(nóng)業(yè)科學(xué)院農(nóng)業(yè)科技創(chuàng)新工程（ASTIP-TRIC04）、煙草綠色防控重大專(zhuān)項(xiàng)（110202001033（LS-02））、中國(guó)煙草總公司四川省公司科技項(xiàng)目（SCYC202008）、中國(guó)煙草總公司貴州省公司科技項(xiàng)目（201921）

曲瀟玲，E-mail：812718023@qq.com。通信作者申莉莉，E-mail：shenlili@caas.cn。通信作者楊金廣，E-mail：yangjinguang@caas.cn

（責(zé)任編輯 岳梅）