轉(zhuǎn)錄因子NbMYB1R1通過促進(jìn)活性氧積累抑制病毒侵染

姜興林，于連偉，付涵，艾妞，崔熒鈞，李好海，夏子豪，袁虹霞，李洪連,3,4，楊雪,2，施艷

轉(zhuǎn)錄因子NbMYB1R1通過促進(jìn)活性氧積累抑制病毒侵染

姜興林1，于連偉1，付涵1，艾妞1，崔熒鈞5，李好海6，夏子豪7，袁虹霞1，李洪連1,3,4，楊雪1,2，施艷

1河南農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院，鄭州 450002；2河南農(nóng)業(yè)大學(xué)作物學(xué)博士后流動站，鄭州 450002；3河南省糧食作物協(xié)同創(chuàng)新中心，鄭州 450002；4小麥玉米作物學(xué)國家重點實驗室，鄭州 450002；5河南省植物保護(hù)新技術(shù)推廣協(xié)會，鄭州 450002；6河南省植物保護(hù)檢疫站，鄭州 450002；7沈陽農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院，沈陽 110866

【背景】黃瓜綠斑駁花葉病毒（cucumber green mottle mosaic virus，CGMMV）是我國重要的檢疫性植物病毒，嚴(yán)重降低了世界范圍內(nèi)蔬菜以及瓜類的產(chǎn)量。MYB蛋白家族龐大，功能多樣，存在于所有真核生物中。大多數(shù)MYB作為轉(zhuǎn)錄因子控制植物的發(fā)育和代謝，并對植物應(yīng)對生物和非生物脅迫反應(yīng)起重要的調(diào)控作用。前期研究顯示CGMMV侵染后可以顯著上調(diào)寄主轉(zhuǎn)錄因子基因的表達(dá)?！灸康摹棵鞔_NbMYB1R1參與CGMMV侵染的機制，為CGMMV病害防控提供理論依據(jù)?！痉椒ā窟\用MEGA 7.0構(gòu)建系統(tǒng)進(jìn)化樹，對NbMYB1R1的氨基酸序列進(jìn)行系統(tǒng)進(jìn)化分析；通過構(gòu)建的熒光表達(dá)載體，轉(zhuǎn)化GV3101農(nóng)桿菌后浸潤本氏煙葉片，激光共聚焦顯微鏡觀察其亞細(xì)胞定位；利用qRT-PCR技術(shù)分析在CGMMV侵染不同時期的轉(zhuǎn)錄水平及沉默煙草植株中活性氧（reactive oxygen species，ROS）相關(guān)基因的轉(zhuǎn)錄變化；通過沉默及下游調(diào)控基因和，分析及下游調(diào)控基因和在CGMMV侵染過程中的作用；瞬時過表達(dá)NbMYB1R1和NbMYB1R1關(guān)鍵氨基酸突變體，分析NbMYB1R1對CGMMV侵染的影響；使用臺盼藍(lán)染色以及DAB染色觀察瞬時過表達(dá)NbMYB1R1造成的細(xì)胞死亡是否與程序性細(xì)胞死亡（programmed cell death，PCD）以及ROS的積累有關(guān)；運用酵母雙雜交技術(shù)驗證NbMYB1R1是否與CGMMV相關(guān)蛋白互作。【結(jié)果】系統(tǒng)進(jìn)化樹分析表明，NbMYB1R1屬于1R MYB大類并與多種煙草的MYB轉(zhuǎn)錄因子同源性極高；亞細(xì)胞定位結(jié)果顯示NbMYB1R1定位于細(xì)胞核；在CGMMV侵染的煙草植株中，的轉(zhuǎn)錄水平對比健康植株有明顯變化，在CGMMV侵染8、12 d時的轉(zhuǎn)錄水平顯著上調(diào)；在沉默內(nèi)源基因的植株上接種CGMMV，3 d后沉默植株系統(tǒng)葉出現(xiàn)斑駁、卷曲癥狀，而對照植株在3.5 d時才出現(xiàn)癥狀；同時CGMMV CP mRNA水平和蛋白水平檢測結(jié)果也表明沉默可以有效促進(jìn)CGMMV的積累；在本氏煙葉片瞬時過表達(dá)NbMYB1R1及其突變體3 d時檢測CGMMV蛋白水平表達(dá)量，結(jié)果顯示過表達(dá)NbMYB1R1可以有效抑制CGMMV的侵染，DNA結(jié)合結(jié)構(gòu)域缺失后會減輕NbMYB1R1對CGMMV的抑制；臺盼藍(lán)和DAB染色結(jié)果表明，在瞬時過表達(dá)NbMYB1R1蛋白后導(dǎo)致ROS積累并引起細(xì)胞死亡；在沉默內(nèi)源基因的植株葉片上檢測ROS相關(guān)基因的轉(zhuǎn)錄水平，發(fā)現(xiàn)交替氧化酶基因、轉(zhuǎn)錄水平顯著上調(diào)；在沉默內(nèi)源基因和的植株上接種CGMMV，4 d后和沉默植株系統(tǒng)葉出現(xiàn)斑駁、卷曲癥狀，而對照植株在3.5 d時就出現(xiàn)癥狀；同時CGMMV CP mRNA水平和蛋白水平檢測結(jié)果也表明沉默和可以有效抑制CGMMV的積累；酵母雙雜交結(jié)果顯示NbMYB1R1不與CGMMV編碼蛋白直接相互作用?！窘Y(jié)論】隨著CGMMV侵染，防御相關(guān)基因表達(dá)上調(diào)，從而抑制下游基因、的表達(dá)并激活細(xì)胞內(nèi)產(chǎn)生ROS抑制病毒侵染，但NbMYB1R1不是通過與CGMMV病毒蛋白直接互作而產(chǎn)生此作用。由此可見，NbMYB1R1在CGMMV侵染過程中發(fā)揮了重要作用。

黃瓜綠斑駁花葉病毒；；轉(zhuǎn)錄因子；致病機制；活性氧

0 引言

【研究意義】我國是世界上最大的蔬菜生產(chǎn)國之一，黃瓜等葫蘆科作物是我國重要的經(jīng)濟(jì)作物[1]。黃瓜綠斑駁花葉病毒（cucumber green mottle mosaic virus，CGMMV）引起的病害是我國葫蘆科作物上重要的檢疫性病毒病。葫蘆科作物感染CGMMV后會導(dǎo)致減產(chǎn)，從而影響經(jīng)濟(jì)價值[2]。挖掘植物中參與病毒侵染的寄主因子對抗病機制解析及抗病品種選育具有重要意義。【前人研究進(jìn)展】MYB（v-myb avian myeloblastosis viral oncogene homolog）家族是真核生物中一個龐大的、普遍存在的轉(zhuǎn)錄因子（TF）家族。MYB轉(zhuǎn)錄因子最初是在禽髓母細(xì)胞中發(fā)現(xiàn)的[3]。1987年，在玉米中首次發(fā)現(xiàn)植物MYB轉(zhuǎn)錄因子（）[4]。之后，許多植物中的MYB轉(zhuǎn)錄因子被鑒定出來，例如擬南芥、棉花、小麥、水稻和番茄等[5-8]。MYB轉(zhuǎn)錄因子具有一個高度保守的DNA結(jié)合域（DNA binding domain，DBD）[9]。DBD是區(qū)分MYB家族的重要區(qū)域，MYB轉(zhuǎn)錄因子就是通過該區(qū)域的空間結(jié)構(gòu)與DNA結(jié)合[10]。MYB家族蛋白也是通過該區(qū)域的3個不完全重復(fù)序列，即R1、R2和R3進(jìn)行分類。根據(jù)MYB結(jié)構(gòu)域的數(shù)量和位置，MYB轉(zhuǎn)錄因子被分為4組：1R-MYB（R1/R2, R3-MYB）、2R-MYB（R2R3-MYB）、3R-MYB（R1R2R3-MYB）和4R-MYB（R1/R2-MYB）[11]。1R-MYB是一種重要的端粒結(jié)合因子，負(fù)責(zé)維持染色體結(jié)構(gòu)的完整性和調(diào)節(jié)基因轉(zhuǎn)錄[12]。2R-MYB又稱R2R3-MYB，在植物中2R-MYB含量最多，功能也最豐富。R2R3-MYB參與細(xì)胞分化、器官形成、葉片形態(tài)改變、調(diào)節(jié)植物激素、次生代謝以及對各種應(yīng)激源的響應(yīng)[11]。3R-MYB主要調(diào)控細(xì)胞周期、細(xì)胞分化和植物對脅迫的耐受性[13]。4R-MYB僅在擬南芥、葡萄和毛楊中發(fā)現(xiàn)，其功能還未被發(fā)掘[14-16]。在植物中許多MYB轉(zhuǎn)錄因子的轉(zhuǎn)錄被誘導(dǎo)以應(yīng)對病原體的攻擊。例如番茄MYB基因在轉(zhuǎn)基因煙草中的表達(dá)提高了對尖鐮孢（）和灰霉病菌（）的抗性[17]，GhMYB36在擬南芥和棉花中均可增強其對黃萎病菌（）的抗性[18]；GhMYB4也可增強棉花對黃萎病菌的抗性[19]；PnMYB2能夠增強三七對根腐病病原體茄病鐮孢（）的抗性[20]；番茄MYB49增強植物對致病疫霉（）的抗性[21]；SmMYB44能夠提高茄類植物對青枯病菌（）的耐受性[22]。同時一些MYB轉(zhuǎn)錄因子在植物對抗病原物的過程中起著負(fù)調(diào)控的作用。例如AtMYB44負(fù)調(diào)控植物對軟腐病的耐受性[23]。MYB轉(zhuǎn)錄因子在植物防御病毒入侵反應(yīng)中也起到了重要作用。例如在煙草花葉病毒（tobacco mosaic virus，TMV）感染期間，觀察到和轉(zhuǎn)錄上調(diào)，和的表達(dá)可以增強煙草對于TMV的抗性[24-25]；SlMYB28作為負(fù)調(diào)節(jié)因子參與了番茄黃化曲葉病毒（tomato yellow leaf curl virus，TYLCV）感染的調(diào)控[26]；在感染番茄曲葉病毒（tomato leaf curl virus，ToLCV）的番茄植株中，觀察到MYB33轉(zhuǎn)錄水平較低[27]；在擬南芥中表達(dá)花椰菜花葉病毒（cauliflower mosaic virus，CaMV）基因VI（P6）后，誘導(dǎo)了基因的上調(diào)[28]；在黃瓜中，CGMMV感染后已經(jīng)發(fā)現(xiàn)6種差異表達(dá)的MYB轉(zhuǎn)錄因子，在侵染早期這些MYB轉(zhuǎn)錄因子在表達(dá)上出現(xiàn)上調(diào)的趨勢，暗示它們對在植物響應(yīng)CGMMV過程中起重要的調(diào)節(jié)作用[29]。植物為了避免受到病原體的侵害，進(jìn)化出了多重防御系統(tǒng)來保護(hù)自身。這些防御機制中最重要的一步是快速識別入侵者和調(diào)配資源[30]。過敏性細(xì)胞壞死（hypersensitive response，HR）是植物免疫反應(yīng)中最有效、最快速的防御反應(yīng)之一[31]。HR反應(yīng)是直接與病原體接觸或靠近病原體的植物細(xì)胞迅速死亡從而將病原體限制在壞死細(xì)胞內(nèi)阻止病原菌的擴散[32]。有研究表明MYB轉(zhuǎn)錄因子可以通過誘導(dǎo)HR反應(yīng)來增強植物的抗病性。如在擬南芥中，AtMYB30作為HR反應(yīng)的正調(diào)節(jié)因子來增強植物抗病性[32-33]；辣椒中的CaPHL8正向調(diào)節(jié)辣椒防御反應(yīng)，并伴隨HR反應(yīng)[34]；以及油菜中BnaMYB111L誘導(dǎo)HR反應(yīng)，并誘導(dǎo)一系列防御相關(guān)基因上調(diào)[35]。HR反應(yīng)會誘導(dǎo)活性氧（reactive oxygen species，ROS）爆發(fā)從而引起細(xì)胞死亡[36]。BnaMYB111L表達(dá)誘導(dǎo)油菜籽原生質(zhì)體中ROS積累以及細(xì)胞死亡[35]。因此，筆者推測MYB轉(zhuǎn)錄因子可以通過誘導(dǎo)ROS積累以及HR來增強自身抗病?！颈狙芯壳腥朦c】通過RNA-Seq數(shù)據(jù)分析發(fā)現(xiàn)，CGMMV侵染后會特異性上調(diào)本氏煙（）內(nèi)源基因表達(dá)，該基因?qū)儆贛YB轉(zhuǎn)錄因子家族。目前暫無研究報道NbMYB1R1蛋白參與病毒侵染。【擬解決的關(guān)鍵問題】探索轉(zhuǎn)錄因子NbMYB1R1參與CGMMV侵染的機制，為CGMMV防治提供理論依據(jù)。

1 材料與方法

試驗于2022年5月至2023年10月在河南農(nóng)業(yè)大學(xué)植物保護(hù)學(xué)院完成。

1.1 材料

本氏煙及核定位H2B轉(zhuǎn)基因煙草種植于光周期為16 h光照/8 h黑暗、相對濕度為60%、溫度為25 ℃的溫室。含CGMMV侵染性克隆菌株、GV3101菌株保存于本實驗室-80 ℃超低溫冰箱。大腸桿菌DH5感受態(tài)購自擎科生物，DAB和臺盼藍(lán)染料購自索萊寶生物科技有限公司。載體TRV-RNA1、TRV-RNA2、pEG103、pGADT7和pGBKT7均由本實驗室保存。供試引物見表1。

1.2 NbMYB1R1同源進(jìn)化樹的構(gòu)建

在NCBI網(wǎng)站查找不同植物中MYB的氨基酸序列，利用DNAMAN軟件對NbMYB1R1的氨基酸序列進(jìn)行Blast比對，對同源性高的氨基酸序列采用極大似然法（maximum likelihood）進(jìn)行系統(tǒng)進(jìn)化分析。利用MEGA 7.0軟件建立系統(tǒng)發(fā)育樹。用于建立系統(tǒng)發(fā)育樹的基因登錄號見表2。

1.3 亞細(xì)胞定位

使用引物-GFP-F/-GFP-R，以本氏煙cDNA為模板進(jìn)行RT-PCR擴增，將擴增出的片段利用Gateway方法克隆至pEG103載體，構(gòu)建好的質(zhì)粒電擊轉(zhuǎn)化GV3101農(nóng)桿菌感受態(tài)，將含有NbMYB1R1-GFP質(zhì)粒的農(nóng)桿菌懸浮于瞬時轉(zhuǎn)染緩沖液（10 mmol·L-1MgCl2，10 mmol·L-1MES，200 μmol·L-1乙酰丁香酮，pH 5.6），調(diào)節(jié)菌液濃度OD600為0.5，挑選28 d H2B轉(zhuǎn)基因本氏煙植株浸潤，進(jìn)行瞬時表達(dá)。農(nóng)桿菌浸潤48 h后，在共聚焦顯微鏡下進(jìn)行觀察，綠色熒光蛋白GFP的激發(fā)光為488 nm，紅色熒光蛋白RFP的激發(fā)光為560 nm。

1.4 VIGS系統(tǒng)沉默

通過TRV侵染性克隆介導(dǎo)的VIGS技術(shù)對、和進(jìn)行系統(tǒng)沉默。以和與的基因同源區(qū)域為模板，利用網(wǎng)站SGN VIGS Tool（http://vigs.solgenomics. net）預(yù)測該基因上一個300 bp左右的片段，根據(jù)該片段設(shè)計引物VIGS-F/VIGS-R和VIGS-F/VIGS-R，利用Gateway方法將該片段克隆至TRV-RNA2載體，構(gòu)建好的質(zhì)粒電擊轉(zhuǎn)化農(nóng)桿菌GV3101，得到含有TRV-RNA2和TRV-RNA2載體的農(nóng)桿菌，搖菌TRV-RNA1、TRV-RNA2、TRV-RNA2-和TRV-RNA2-農(nóng)桿菌，用瞬時轉(zhuǎn)染緩沖液將其懸浮OD600為1.0。1﹕1混合浸潤，TRV-RNA1和TRV-RNA2-農(nóng)桿菌以及TRV-RNA1和TRV-RNA2-/農(nóng)桿菌共浸為處理組，TRV-RNA1和TRV-RNA2農(nóng)桿菌共浸為對照組。每組處理至少8個生物學(xué)重復(fù)。

1.5 病毒接種

將含有CGMMV侵染性克隆的農(nóng)桿菌菌液在含Kan+Rif（50 μg·mL-1）的LB平板上劃線活化，28 ℃培養(yǎng)箱倒置培養(yǎng)1.5—2 d。挑取多克隆于5 mL含Kan+Rif（50 μg·mL-1）抗性的LB液體培養(yǎng)基中，28 ℃，220 r/min搖床振蕩培養(yǎng)過夜。用瞬時轉(zhuǎn)染緩沖液將其懸浮OD600調(diào)為1.0，浸潤健康本氏煙植株，等待6—8 d后發(fā)病。將發(fā)病的系統(tǒng)葉取下用石英砂磨碎加水混勻，并摩擦接種到本氏煙植株上。

1.6 突變體的構(gòu)建

以為模板，利用網(wǎng)站預(yù)測該基因上的關(guān)鍵功能的氨基酸位點為第121—127位氨基酸。在該位點的前端以及后端設(shè)計引物121-127VIGS-F/121-127VIGS-R，以為模板擴增片段，利用同源重組的方法將片段克隆到GFP載體上，得到缺失121—127位氨基酸的載體121-127-GFP。

1.7 qRT-PCR分析

植物總RNA的提取使用Trizol法，利用HiScript&III RT SuperMix for qPCR（+gDNA wiper）試劑盒將提取的總RNA反轉(zhuǎn)錄為cDNA。在無酶的八連排中配制如下混合液：2×ChamQ Universal SYBR qPCR Master Mix 5 μL，正向與反向引物各0.2 μL，cDNA 1 μL，ddH2O 3.6 μL。在實時熒光定量PCR儀中進(jìn)行反應(yīng)，反應(yīng)程序如下：預(yù)變性95 ℃ 30 s；95 ℃ 10 s、56 ℃ 30 s、72 ℃ 30 s，進(jìn)行40個循環(huán)；添加熔解曲線。得到數(shù)據(jù)后，采用2-ΔΔCT方法進(jìn)行分析。

1.8 Western blot分析

將樣品裝入2 mL離心管中，液氮降溫并置于研磨儀上破碎。植物組織變成粉末后加入適量的蛋白裂解液，冰上充分裂解30 min，低溫離心后取上清液并加入5×Loading buffer，放入沸水中變性。將變性后的蛋白樣品進(jìn)行聚丙烯酰胺凝膠電泳。采用半干轉(zhuǎn)的方法進(jìn)行轉(zhuǎn)膜，25 V電壓轉(zhuǎn)膜20—30 min。5 %脫脂牛奶，NC膜正面朝上室溫封閉1 h。根據(jù)目的蛋白所帶的標(biāo)簽不同，選擇不同的抗體，將封閉后的NC膜加入一抗，4 ℃過夜。二抗室溫孵育1 h。使用ECL發(fā)光液顯色。

表1 本研究所用引物

表2 用于建立系統(tǒng)發(fā)育樹的基因登錄號

1.9 臺盼藍(lán)染色

臺盼藍(lán)染色時，將葉片置于臺盼藍(lán)溶液（1 mg·mL-1）中25 ℃黑暗浸泡12 h。反應(yīng)結(jié)束后，將葉片置于沸水3 min，靜置2 min，重復(fù)兩次以去除多余的臺盼藍(lán)染料。冷卻后，將葉片置于水合氯醛（1 mg·mL-1）2 d去除葉綠素。

1.10 DAB染色

DAB染色時，將葉片置于DAB（2 mg·mL-1）溶于Tris-HCl（pH 3.8）中，25 ℃黑暗浸泡12 h。反應(yīng)結(jié)束后，將葉片置于沸騰的95%乙醇中去除葉綠素。冷卻后，將葉片置于75%乙醇中2—3 h。觀察其產(chǎn)生的棕黃色物質(zhì)即為產(chǎn)生的H2O2量。

1.11 酵母雙雜交試驗

利用同源重組的方法將載體pGBKT7與CP、MP、129K、MET、HEL和P2 PCR產(chǎn)物，載體pGADT7與PCR產(chǎn)物連接。將pGADT7-NbMYB1R1+ pGBKT7-CP、pGADT7-NbMYB1R1+pGBKT7-MP、pGADT7-NbMYB1R1+pGBKT7-129K、pGADT7- NbMYB1R1+pGBKT7-MET、pGADT7-NbMYB1R1+ pGBKT7-HEL和pGADT7-NbMYB1R1+pGBKT7-P2載體共轉(zhuǎn)化酵母感受態(tài)Y2H，以pGADT7-T+ pGBKT7- 53為陽性對照，pGADT7-T+pGBKT7-Lam為陰性對照。將上述組合在二缺固體培養(yǎng)基SD-L-T和SD-L-T- H-Ade平板上梯度稀釋培養(yǎng)。觀察SD-L-T-H-Ade平板上菌落是否變藍(lán)證明是否互作。

1.12 數(shù)據(jù)處理與分析

每項試驗設(shè)置至少3個生物學(xué)重復(fù)，采用T測驗進(jìn)行數(shù)據(jù)分析。數(shù)據(jù)均用平均值±標(biāo)準(zhǔn)差表示。采用GraphPad Prism 8.0.2軟件制作柱形圖和折線圖。

2 結(jié)果

2.1 本氏煙NbMYB1R1同源進(jìn)化樹分析

同源進(jìn)化樹分析是鑒定轉(zhuǎn)錄因子蛋白功能的一種方法，聚在同一支上的同源蛋白結(jié)構(gòu)相似，可能具有相似的功能。對NbMYB1R1進(jìn)行同源進(jìn)化樹分析，由圖1可知，NbMYB1R1屬于MYB分類中的1R MYB大類并與多種煙草的MYB轉(zhuǎn)錄因子相聚成簇，與擬南芥和辣椒中的MYB1R1轉(zhuǎn)錄因子親緣關(guān)系較遠(yuǎn)。

2.2 NbMYB1R1轉(zhuǎn)錄因子亞細(xì)胞定位

構(gòu)建了NbMYB1R1-GFP，通過共聚焦顯微鏡觀察NbMYB1R1亞細(xì)胞定位。結(jié)果顯示，對照GFP分布在整個細(xì)胞中，而MYB1R1-GFP與H2B-RFP共定位于細(xì)胞核中（圖2）。推測NbMYB1R1在細(xì)胞核中作為轉(zhuǎn)錄因子調(diào)控下游基因的轉(zhuǎn)錄，進(jìn)而影響病毒的侵染。

2.3 CGMMV侵染對NbMYB1R1轉(zhuǎn)錄水平的影響

CGMMV侵染植株在8 d時出現(xiàn)癥狀，因此取8、10和12 d的發(fā)病植株的系統(tǒng)葉為處理組，健康植株的系統(tǒng)葉為對照組，提取RNA并反轉(zhuǎn)錄。qRT-PCR結(jié)果顯示，在CGMMV侵染8、12 d時，的轉(zhuǎn)錄水平顯著上調(diào)；10 d轉(zhuǎn)錄水平與健康植株無顯著差異。表明CGMMV侵染初期及中后期會誘導(dǎo)的轉(zhuǎn)錄表達(dá)（圖3）。

2.4 NbMYB1R1沉默促進(jìn)CGMMV的侵染

為了明確在CGMMV侵染中的作用，通過VIGS技術(shù)研究沉默后對CGMMV侵染的影響。將TRV-RNA2-與TRV-RNA1農(nóng)桿菌菌液等體積混合后，浸潤本氏煙進(jìn)行基因沉默，標(biāo)記為TRV:；TRV-RNA1菌液與TRV-RNA2菌液混合后浸潤本氏煙作為對照組，標(biāo)記為TRV:00?；虺聊?0 d后，觀察發(fā)現(xiàn)與對照植株相比，沉默后植株無表型變化（圖4-A）。檢測沉默效率為81%（圖4-B）。

對沉默10 d的TRV:和TRV:00植株進(jìn)行摩擦接種CGMMV。第3.5天TRV:植株開始發(fā)病，第4天TRV:00植株開始發(fā)病，因此每0.5 d觀察并統(tǒng)計兩組處理的發(fā)病植株，發(fā)現(xiàn)從第3—6天，TRV:組的發(fā)病植株數(shù)量始終多于TRV:00（圖4-C）。第6天，沉默植株系統(tǒng)葉上病毒癥狀明顯較重（圖4-A）。利用qRT-PCR和Western blot檢測病毒積累量，沉默植株的CGMMV CP無論在轉(zhuǎn)錄水平（圖4-D）還是蛋白水平（圖4-E），其積累量均顯著高于對照植株。證明沉默促進(jìn)了CGMMV CP積累，即促進(jìn)CGMMV的侵染。

2.5 NbMYB1R1瞬時過表達(dá)抑制CGMMV侵染

為進(jìn)一步明確NbMYB1R1對CGMMV侵染的影響，在CGMMV侵染的葉片上浸潤NbMYB1R1-GFP，并以GFP為對照（OD600=1.0），浸潤在同一葉片的左右兩側(cè)，72 h后，在激光共聚焦顯微鏡下觀察綠色熒光均正確表達(dá)后進(jìn)行取樣。western blot結(jié)果顯示，NbMYB1R1-GFP和GFP均正常表達(dá)，與對照相比，NbMYB1R1-GFP過表達(dá)后的CGMMV CP積累量較少（圖5-A），表明NbMYB1R1瞬時過表達(dá)抑制CGMMV的侵染。對NbMYB1R1關(guān)鍵結(jié)構(gòu)域進(jìn)行預(yù)測，其中121—127位氨基酸為MYB DNA結(jié)合結(jié)構(gòu)域，在CGMMV侵染的葉片上浸潤NbMYB1R1-GFP和NbMYB1R1121-127-GFP，并以GFP為對照（OD600= 1.0），72 h后western blot結(jié)果表明，NbMYB1R1-GFP關(guān)鍵氨基酸缺失后會緩解NbMYB1R1對CGMMV的抑制作用（圖5-B）。

圖1 基于氨基酸序列的NbMYB1R1進(jìn)化樹分析

2.6 NbMYB1R1促進(jìn)ROS積累及細(xì)胞壞死

NbMYB1R1瞬時過表達(dá)葉片出現(xiàn)細(xì)胞壞死癥狀，推測由于NbMYB1R1表達(dá)調(diào)控ROS的積累導(dǎo)致葉片壞死。對NbMYB1R1瞬時過表達(dá)葉片進(jìn)行了臺盼藍(lán)和DAB染色。結(jié)果顯示，NbMYB1R1-GFP的浸潤區(qū)域出現(xiàn)壞死，臺盼藍(lán)染色顯示藍(lán)色；DAB染色后會有棕黃色物質(zhì)積累（圖6），表明NbMYB1R1過表達(dá)會誘導(dǎo)ROS積累并出現(xiàn)細(xì)胞壞死的癥狀。

圖2 NbMYBIR1的亞細(xì)胞定位

*：差異顯著significant difference (P＜0.05)。下同The same as below

2.7 沉默NbMYB1R1激活了交替氧化酶基因NbAOX1a和NbAOX1b的轉(zhuǎn)錄

為探究如何調(diào)控ROS的積累，檢測了沉默植株中ROS負(fù)調(diào)控相關(guān)基因的轉(zhuǎn)錄水平，結(jié)果顯示交替氧化酶基因和轉(zhuǎn)錄水平顯著上調(diào)，而其余ROS清除相關(guān)基因、、、和[37]的轉(zhuǎn)錄水平無顯著變化（圖7）。上述結(jié)果表明可特異性調(diào)控交替氧化酶基因和，推測可能通過抑制和的轉(zhuǎn)錄從而促進(jìn)ROS積累。

2.8 NbAOX1a和NbAOX1b沉默抑制CGMMV的侵染

為了明確和在CGMMV侵染中的作用，通過VIGS技術(shù)研究沉默和對CGMMV侵染的影響。將TRV-RNA2-/與TRV-RNA1農(nóng)桿菌菌液等體積混合后，浸潤本氏煙進(jìn)行基因沉默，標(biāo)記為TRV:/；TRV-RNA1菌液與TRV-RNA2菌液混合后浸潤本氏煙作為對照組，標(biāo)記為TRV:00?；虺聊?0 d后，發(fā)現(xiàn)與對照植株相比，/沉默后植株有很明顯的矮化（圖8-A）。和的沉默效率分別為71%和64%（圖8-B）。

A：TRV:NbMYB1R1和TRV:00在CGMMV侵染前后的生物學(xué)表型Biological phenotypes of TRV:NbMYBIR1 and TRV:00 before and after CGMMV infection；B：VIGS NbMYBIR1的沉默效率Silencing efficiency of VIGS NbMYBIR1；C：TRV:NbMYBIR1和TRV:00的植株被CGMMV侵染后系統(tǒng)侵染植株占比Proportion of systemic infected plants after infection with CGMMV in plants of TRV:NbMYBIR1 and TRV:00；D：TRV:NbMYBIR1對CGMMV CP轉(zhuǎn)錄水平的影響Effect of TRV:NbMYBIR1 on the transcription level of CGMMV CP；E：TRV:NbMYBIR1對CGMMV CP蛋白水平的影響 Effect of TRV:NbMYBIR1 on the protein level of CGMMV CP

A：NbMYB1R1瞬時過表達(dá)對CGMMV CP蛋白水平的影響Effect of transient overexpression of NbMYB1R1 on the protein level of CGMMV CP；B：NbMYB1R1 DNA結(jié)合結(jié)構(gòu)域缺失突變對CGMMV CP蛋白水平的影響Effect of NbMYB1R1 DNA binding domain deletion mutation on the protein level of CGMMV CP

對沉默10 d的TRV:/和TRV:00植株進(jìn)行摩擦接種CGMMV。第4天TRV:00植株開始發(fā)病，第4.5天TRV:/植株開始發(fā)病，因此每0.5 d觀察并統(tǒng)計兩組處理的發(fā)病植株，發(fā)現(xiàn)從第3.5—7天，TRV:00組的發(fā)病植株數(shù)量始終多于TRV:/（圖8-C）。第7天，/沉默植株系統(tǒng)葉上病毒癥狀明顯較輕（圖8-A）。利用qRT-PCR和Western blot檢測病毒積累量，/沉默植株的CGMMVCP無論在轉(zhuǎn)錄水平（圖8-D）還是蛋白水平（圖8-E），其積累量都比對照植株顯著降低。證明/沉默減少了CGMMV CP積累，即抑制CGMMV的侵染。

圖6 NbMYB1R1過表達(dá)引起活性氧積累與細(xì)胞死亡

2.9 NbMYB1R1不與CGMMV編碼蛋白互作

為了解NbMYB1R1是否與CGMMV編碼蛋白之間存在互作關(guān)系，進(jìn)而直接影響CGMMV的侵染，利用酵母雙雜交系統(tǒng)驗證NbMYB1R1與CGMMV相關(guān)蛋白之間的互作。構(gòu)建重組質(zhì)粒pGADT7-NbMYB1R1、pGBKT7-CP、pGBKT7-MP、pGBKT7-129K、pGBKT7- MET、pGBKT7-HEL和pGBKT7-P2。將pGADT7- NbMYB1R1+pGBKT7-CP、pGADT7-NbMYB1R1+ pGBKT7-MP、pGADT7-NbMYB1R1+pGBKT7-129K、pGADT7-NbMYB1R1+pGBKT7-MET、pGADT7- NbMYB1R1+pGBKT7-HEL和pGADT7-NbMYB1R1+ pGBKT7-P2載體共轉(zhuǎn)化酵母感受態(tài)Y2H，以pGADT7- T+pGBKT7-53為陽性對照，pGADT7-T+pGBKT7-Lam為陰性對照。將上述組合涂布于二缺培養(yǎng)基。3 d后，所有組合均能在二缺培養(yǎng)基上正常生長，說明質(zhì)粒成功轉(zhuǎn)入酵母菌株。將上述組合在二缺固體培養(yǎng)基SD-L-T和SD-L-T-H-Ade平板上梯度稀釋培養(yǎng)。結(jié)果顯示，僅pGADT7-T+pGBKT7-53陽性對照生長且變藍(lán)色，其余包含NbMYB1R1組合在SD-L-T-H-Ade平板上生長的菌落均未顯現(xiàn)藍(lán)色（圖9），表明酵母雙雜交試驗中NbMYB1R1不與CGMMV編碼蛋白直接互作。

圖7 NbMYBIR1沉默對ROS相關(guān)基因轉(zhuǎn)錄水平的影響

3 討論

3.1 NbMYB1R1作為轉(zhuǎn)錄因子調(diào)控病毒侵染

MYB轉(zhuǎn)錄因子在調(diào)控植物生長發(fā)育過程和其對生物及非生物脅迫的反應(yīng)等方面發(fā)揮著重要作用[38]。自1987年在玉米中首次發(fā)現(xiàn)植物MYB轉(zhuǎn)錄因子（ZmMYBC1）后[4]，越來越多的研究表明MYB作為轉(zhuǎn)錄因子在植物生物脅迫和非生物脅迫中發(fā)揮重要作用[39]。CiMYB4作為轉(zhuǎn)錄因子可以顯著降低鎘脅迫對煙草和野菊生長發(fā)育的影響[40]；過表達(dá)轉(zhuǎn)錄因子VvMYB30可增強轉(zhuǎn)基因擬南芥的耐鹽性[41]；轉(zhuǎn)錄因子CitMYB20可以增強柑橘株系對柑橘潰瘍病的抗性[42]；NtMYB1可能參與病程相關(guān)（PR）基因的轉(zhuǎn)錄激活進(jìn)而增強煙草對于TMV的抗性[24]；番茄黃化曲葉病毒侵染植株后，SlMYB28作為轉(zhuǎn)錄因子激活多個防御相關(guān)基因的表達(dá)進(jìn)而增強植株自身抗病能力[26]。前期研究表明，CGMMV侵染上調(diào)本氏煙表達(dá)[43]，進(jìn)一步探索發(fā)現(xiàn)在CGMMV侵染初期及中后期，其轉(zhuǎn)錄水平明顯上調(diào)（圖3）。過表達(dá)和沉默均能對CGMMV的侵染產(chǎn)生影響（圖4、圖5），且NbMYB1R1中DNA結(jié)合結(jié)構(gòu)域是影響CGMMV侵染的關(guān)鍵結(jié)構(gòu)域（圖5-B）。利用其氨基酸序列進(jìn)行系統(tǒng)發(fā)育分析，表明NbMYB1R1與多種煙草中的MYB1R1具有高度相似性（圖1），且NbMYB1R1主要定位于煙草細(xì)胞核（圖2）。因此，筆者推測NbMYB1R1可能作為轉(zhuǎn)錄因子參與病毒的侵染過程。

A：TRV:NbAOX1a/b和TRV:00在CGMMV侵染前后的生物學(xué)表型Biological phenotypes of TRV:NbAOX1a/b and TRV:00 before and after CGMMV infection；B：VIGS NbAOX1a和NbAOX1b的沉默效率Silencing efficiency of VIGS NbAOX1a and NbAOX1b；C：TRV:NbAOX1a/b和TRV:00的植株被CGMMV侵染后系統(tǒng)侵染植株占比Proportion of systemic infected plants after infection with CGMMV in plants of TRV:NbAOX1a/b and TRV:00；D：TRV:NbAOX1a/b對CGMMV CP的轉(zhuǎn)錄水平影響Effect of TRV: NbAOX1a/b on the transcription level of CGMMV CP；E：TRV:NbAOX1a/b對CGMMV CP蛋白水平的影響Effect of TRV:NbAOX1a/b on the protein level of CGMMV CP

圖9 酵母雙雜交分析NbMYB1R1與CGMMV編碼蛋白的互作

3.2 NbMYB1R1通過抑制NbAOX1a和NbAOX1b相關(guān)基因的轉(zhuǎn)錄激活細(xì)胞內(nèi)ROS積累從而抑制病毒侵染

細(xì)胞內(nèi)ROS信號轉(zhuǎn)導(dǎo)或ROS激活的防御機制可抑制病原物侵染。例如，在植株中沉默內(nèi)源基因后可以增強寄主植物活性氧積累從而提高植株抗病能力[44]；可能通過調(diào)控植物體內(nèi)活性氧產(chǎn)生誘導(dǎo)病程相關(guān)蛋白基因的表達(dá)，從而提高植株對赤霉菌的抗性[45]；H2O2參與StRab5b介導(dǎo)的對馬鈴薯疫霉的抗病過程[46]；柑橘衰退病毒（citrustristeza virus，CTV）侵染會引發(fā)本氏煙和天然宿主中ROS爆發(fā)，進(jìn)而增強植物對CTV的抗性。本研究結(jié)果也顯示NbMYB1R1增強細(xì)胞內(nèi)ROS積累導(dǎo)致細(xì)胞壞死從而抑制病毒侵染。同時檢測了ROS清除酶相關(guān)的基因交替氧化酶（和）、超氧化物歧化酶（）、抗壞血酸過氧化物酶（）、過氧化氫酶（）、谷胱甘肽過氧化物酶（）和谷胱甘肽轉(zhuǎn)移酶（）[37]。其中和活性可以降低和NO的產(chǎn)生，從而減少細(xì)胞內(nèi)ROS的產(chǎn)生[47]。本研究結(jié)果顯示沉默可以特異性地上調(diào)和的轉(zhuǎn)錄（圖7）。已經(jīng)有研究證明MYB轉(zhuǎn)錄因子可以與AOX基因的啟動子相結(jié)合進(jìn)而影響植株的抗性。轉(zhuǎn)錄因子MYB29是的負(fù)調(diào)節(jié)因子，MYB29通過抑制的表達(dá)增強植株的脅迫防御反應(yīng)[48]；MYB30結(jié)合的啟動子以調(diào)節(jié)植物耐鹽性[49]。本研究結(jié)果顯示沉默和能抑制CGMMV的侵染（圖8）。推測NbMYB1R1作為轉(zhuǎn)錄因子與和的啟動子相結(jié)合，通過抑制和相關(guān)基因的轉(zhuǎn)錄促進(jìn)細(xì)胞內(nèi)ROS積累從而抑制病毒侵染。

4 結(jié)論

NbMYB1R1作為1R MYB家族轉(zhuǎn)錄因子在細(xì)胞核中行使功能，其在CGMMV侵染初期及中后期表達(dá)量顯著上調(diào)。NbMYB1R1在CGMMV侵染過程中發(fā)揮重要作用，其可能原因是NbMYB1R1作為轉(zhuǎn)錄因子抑制和的表達(dá)，從而促進(jìn)細(xì)胞內(nèi)ROS積累，抑制了病毒的積累。

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The Transcription Factor NbMYB1R1 Inhibits Viral Infection by Promoting ROS Accumulation

JIANG XingLin1, YU LianWei1, Fu Han1, Ai Niu1, Cui YingJun5, Li HaoHai6, XIA ZiHao7, Yuan HongXia1, LI HongLian1,3,4, YANG Xue1,2, SHI Yan1

1College of Plant Protection, Henan Agricultural University, Zhengzhou 450002;2Crop science postdoctoral programme of Henan Agricultural University, Zhengzhou 450002;3Collaborative Innovation Centre of Henan Grain crops, Zhengzhou 450002;4State Key Laboratory of Wheat and Maize Crop Science, Zhengzhou 450002;5Henan Association for the promotion of new plant protection techniques, Zhengzhou 450002;6Henan plant protection and quarantine station, Zhengzhou 450002;7College of Plant Protection, Shenyang Agricultural University, Shenyang 110866

【Background】Cucumber green mottle mosaic virus (CGMMV) is an important quarantine plant virus in China, which has seriously reduced the production of vegetables and melons worldwide. The MYB protein family is large, multifunctional and present in all eukaryotes. Most MYB proteins act as transcription factors that control development, and metabolism in plants, and regulate biotic and abiotic stress responses. Previous studies have shown that during CGMMV infection, the expression of transcription factor genewas significantly up-regulated.【Objective】The objective of this study is to clarify the mechanism of NbMYB1R1 involved in CGMMV infection, and to provide a theoretical basis for controlling CGMMV infection.【Method】MEGA 7.0 was used to construct a phylogenetic tree to analyze the amino acid sequence of NbMYB1R1 protein. The expression vector NbMYB1R1-GFP was constructed, transformed intoGV3101 and infiltrated the tobacco leaves to observe the subcellular localization of NbMYB1R1 by confocal microscopy. The transcriptional levels ofat different stages of CGMMV infection and ROS related genes in silencedplants were analyzed using qRT-PCR technology. TRV-mediated gene silencing (VIGS) was utilized to analyze the function of,, andduring CGMMV infection. transient overexpression of NbMYB1R1 and NbMYB1R1 mutant was conducted to analyze the effect of NbMYB1R1 on CGMMV infection. trypan blue staining and DAB stainingwereused toobserve whether the cell death caused by the transient overexpression of NbMYB1R1 was related to the programmed cell death (PCD) and the accumulation of ROS. Yeast two-hybrid system was used to verify whether NbMYB1R1 interacted with CGMMV related proteins.【Result】Phylogenetic tree analysis showed that NbMYB1R1 belonged to the 1R MYB category and had high homology with MYB transcription factors in a variety of tobaccos. The results of subcellular localization showed that NbMYB1R1 was localized in the nucleus. In CGMMV-infected tobacco plants, the transcript level ofwas significantly changed compared with healthy plants, which was significantly up-regulated at 8 and 12 d of CGMMV infection. CGMMV was inoculated into TRV:and TRV:00 plants. The-silenced plants showed the mottled and curled symptoms at 3 dpi, while the control plant appeared the symptoms at 3.5 dpi. Silencingcould effectively promote the accumulation of CGMMV at mRNA and protein levels. the CGMMV accumulation in NbMYB1R1 and NbMYB1R1 mutant transiently overexpression leaves was detected, and the results showed that NbMYB1R1 overexpression could effectively inhibit CGMMV infection and DNA binding domain deletion mutant reduced the inhibition of CGMMV by NbMYB1R1. The results of trypan blue and DAB staining showed that NbMYB1R1 overexpression could induce the ROS accumulation and cell death. the transcription level of ROS-related genes in TRV:and TRV:00 plant was also detected, and the results showed that silencingcould specifically up-regulate the transcription ofand. After CGMMV inoculation on silencing endogenous genesand, the systemic leaves of silencedandplants showed mottled and curled symptoms at 4 dpi, while the control plants appeared symptoms at 3.5 d. Meanwhile, the results of CGMMV CP mRNA and protein levels also indicated that silencingandcould effectively inhibit the accumulation of CGMMV. The yeast two-hybridresults showed that NbMYB1R1 did not directly interact with the CGMMV proteins.【Conclusion】During the CGMMV infection, the defense-related geneis up-regulated. It speculates that the upregulation ofinhibits the transcription of the downstream genesandand activates the production of ROS in cells, thereby inhibiting viral infection. However, NbMYB1R1 does not produce this effect through a direct interaction with the CGMMV viral protein. NbMYB1R1 plays an important role in CGMMV infection.

cucumber green mottle mosaic virus (CGMMV);; transcription factor; pathogenic mechanism; reactive oxygen species (ROS)

10.3864/j.issn.0578-1752.2024.08.006

2023-12-06；

2024-02-04

國家自然科學(xué)基金（3210170372）、河南農(nóng)業(yè)大學(xué)科技創(chuàng)新項目（KJCX2021A13）

姜興林，E-mail：1442295312@qq.com。于連偉，E-mail：ylwnet972@163.com。姜興林和于連偉為同等貢獻(xiàn)作者。通信作者楊雪，E-mail：yangxuepphappy@126.com。通信作者施艷，E-mail：shiyan00925@126.com

（責(zé)任編輯 岳梅）