翟玉山 趙 賀 張 海 鄧宇晴 程光遠(yuǎn) 楊宗桃 王 彤 彭 磊 徐 倩 董 萌 徐景升

甘蔗NAD(P)H脫氫酶復(fù)合體O亞基基因克隆及其與甘蔗花葉病毒VPg互作

翟玉山 趙 賀 張 海 鄧宇晴 程光遠(yuǎn) 楊宗桃 王 彤 彭 磊 徐 倩 董 萌 徐景升*

福建農(nóng)林大學(xué)國家甘蔗工程技術(shù)研究中心 / 農(nóng)業(yè)部福建甘蔗生物學(xué)與遺傳育種重點(diǎn)實(shí)驗(yàn)室 / 教育部作物遺傳育種與綜合利用重點(diǎn)實(shí)驗(yàn)室, 福建福州 350002

NAD(P)H脫氫酶(NDH)復(fù)合體介導(dǎo)循環(huán)電子傳遞, 對于維持葉綠體高效的光合作用具有重要作用。甘蔗(spp. hybrid)中NDH復(fù)合體應(yīng)答及參與甘蔗花葉病毒(, SCMV)的侵染尚未見報(bào)道。本研究克隆了甘蔗NDH復(fù)合體的O亞基, 命名為, 其開放讀碼框(open reading frame, ORF)長度為471 bp, 編碼長度為156 aa的蛋白。生物信息學(xué)分析表明, ScNdhO為穩(wěn)定的親水性蛋白, 不存在信號肽, 無跨膜結(jié)構(gòu)域; 蛋白二級結(jié)構(gòu)元件多為無規(guī)則卷曲, 具有典型的NDH復(fù)合體O亞基結(jié)構(gòu)域; 系統(tǒng)進(jìn)化樹分析表明, 該蛋白屬于NDH復(fù)合體O亞基蛋白家族。實(shí)時(shí)熒光定量 PCR分析發(fā)現(xiàn),基因的表達(dá)具有明顯的組織特異性, 在成熟甘蔗葉片中的表達(dá)量最高, 在莖中的表達(dá)量最低, 在根中幾乎不表達(dá);基因在SCMV侵染早期上調(diào)表達(dá), 后期下調(diào)表達(dá)。亞細(xì)胞定位分析表明, ScNdhO定位于葉綠體。酵母雙雜交(yeast two hybrid, Y2H)和雙分子熒光互補(bǔ)(bimolecular fluorescence complementation, BiFC)實(shí)驗(yàn)表明, ScNdhO與SCMV-VPg互作。推測ScNdhO被SCMV選擇性利用, 可能參與SCMV基因組復(fù)制及花葉病癥狀的產(chǎn)生。

甘蔗; SCMV; 葉綠體; NAD(P)H脫氫酶復(fù)合體; O亞基

甘蔗(spphybrid)是我國乃至全世界最重要的糖料作物和能源作物之一, 甘蔗糖占我國食糖總量的92％, 占世界食糖總量的74%, 甘蔗乙醇占世界生物質(zhì)燃料乙醇的60%[1-2]。甘蔗花葉病是嚴(yán)重危害甘蔗生產(chǎn)的病毒性病害之一, 在全世界蔗區(qū)包括我國甘蔗主產(chǎn)區(qū)廣西、云南、廣東、海南等地普遍發(fā)生[3-9]。甘蔗感染花葉病后, 葉片出現(xiàn)黃綠相間、大小不一的條紋或斑駁, 植株矮小, 分蘗減少,蔗糖結(jié)晶率下降, 甘蔗產(chǎn)量損失3%~50%, 蔗糖損失6%~14%[6,9-11]。甘蔗花葉病的病原主要為甘蔗花葉病毒(, SCMV)、高粱花葉病毒(, SrMV)和甘蔗條紋花葉病毒(, SCSMV), 均屬于馬鈴薯Y病毒科()[6,8,12-19], 其中SCMV和SrMV屬于馬鈴薯Y病毒屬()[20-21], SCSMV是新發(fā)現(xiàn)的病原, 2012年國際病毒分類委員會(International Committee on Taxonomy of Viruses, ICTV)將其立為該科新屬, 即禾病毒屬()[9,22-23]。SCMV、SrMV和SCSMV都是單鏈正義RNA病毒, 長度約為10 kb, 編碼2個(gè)多聚蛋白, 經(jīng)自身編碼的P1、HC-Pro和NIa酶解后形成11個(gè)功能成熟的蛋白, 分別為P1、HC-Pro、P3、P3N-PIPO、6K1、CI、6K2、VPg、NIa、NIb和CP[12-17,22-24]。此類病毒結(jié)構(gòu)簡單, 其基因組5′端沒有帽子結(jié)構(gòu)(m7GpppN, N為任意核酸), 病毒要完成復(fù)制, 必須通過其編碼的VPg與真核翻譯起始因子eIF4E共價(jià)連接, 才能啟動病毒RNA翻譯[25-26]。VPg是馬鈴薯Y病毒科病毒的多功能蛋白, 除了參與病毒的翻譯, 還參與病毒在寄主體內(nèi)的復(fù)制、胞間移動、長距離運(yùn)輸及自噬等過程[25,27-28]。

葉綠體中的光合系統(tǒng)由光合系統(tǒng)I (PSI)和光合系統(tǒng)II (PSII)組成。PSII反應(yīng)中心被光能激發(fā)后, 裂解水分子釋放氧氣, 將來自水分子的電子經(jīng)過PSII、質(zhì)醌(PQ)、細(xì)胞色素b6/f復(fù)合體、質(zhì)藍(lán)素、PSI、鐵氧還蛋白線性傳遞, 最終使NADP+還原, 并偶聯(lián)產(chǎn)生跨類囊體膜質(zhì)子梯度(ΔpH), 驅(qū)動ATP合成酶合成ATP[29]。除了這條主要的線性光合電子傳遞鏈, 葉綠體中還有一條與線粒體復(fù)合體I介導(dǎo)的呼吸電子傳遞鏈類似的由NAD(P)H脫氫酶(NDH)復(fù)合體和質(zhì)子末端氧化酶共同參與的圍繞PSI循環(huán)電子途徑, 循環(huán)電子途徑只產(chǎn)生ATP, 不積累NADPH[30-37]。葉綠體NDH復(fù)合體位于類囊體膜上[38-40], 在調(diào)節(jié)ATP合成促進(jìn)碳同化產(chǎn)物形成、防止光系統(tǒng)過還原、減少活性氧、光保護(hù)抗逆等方面具有重要的生物學(xué)功能[41-47]。葉綠體NDH復(fù)合體至少有29個(gè)亞基, 構(gòu)成NDH的5個(gè)亞復(fù)合體[25,48-56]。NDH復(fù)合體O亞基屬于亞復(fù)合體A成員, 對于維持NDH復(fù)合體的穩(wěn)定和正常功能具有重要作用[53,57]。

葉綠體是綠色植物光合作用的場所, 同時(shí)也是馬鈴薯Y病毒的復(fù)制場所, 馬鈴薯Y病毒的6K2誘導(dǎo)內(nèi)質(zhì)網(wǎng)形成囊泡, 移動到葉綠體形成葉綠體-6K2復(fù)合物, 為病毒基因組復(fù)制提供場所[58]。在前期研究工作中, 我們以SCMV編碼蛋白VPg為誘餌, 利用酵母雙雜交(yeast two hybrid, Y2H)技術(shù)從甘蔗葉片的cDNA酵母文庫[59]中篩選到了一個(gè)具有完整開放讀碼框(open reading frame, ORF)的基因; 該基因的ORF為471 bp, 編碼長度為156 aa的蛋白。根據(jù)Blastp同源比對分析推測該基因編碼葉綠體NDH復(fù)合體的O亞基, 命名為。本研究克隆了該基因, 并進(jìn)行了生物信息學(xué)、組織特異性與應(yīng)答SCMV侵染的表達(dá)模式分析, 利用Y2H和BiFC研究了ScNdhO與SCMV-VPg的互作關(guān)系, 并對其在SCMV侵染中的作用做了探討, 以期為甘蔗抗花葉病育種提供基礎(chǔ)數(shù)據(jù)和實(shí)驗(yàn)依據(jù)。

1 材料與方法

1.1 材料及處理方法

SCMV-FZ1病毒株系、甘蔗品種和本氏煙由福建農(nóng)林大學(xué)農(nóng)業(yè)部福建甘蔗生物學(xué)與遺傳育種重點(diǎn)實(shí)驗(yàn)室提供。采用腋芽快繁技術(shù)培養(yǎng)組培苗。組培苗長至15~25 cm、完全展開葉出現(xiàn)4~5片時(shí), 摩擦接種SCMV, 設(shè)置3個(gè)重復(fù), 每個(gè)重復(fù)3株, 對照植株使用磷酸緩沖液(pH 7.0)摩擦接種, 使用基因特異引物(表1)檢測接種是否成功。分別在接種后0 h、4 h、8 h、12 h、1 d、2 d、5 d、7 d取樣, 研究目的基因應(yīng)答SCMV侵染的表達(dá)模式。組培苗分蘗期時(shí)隨機(jī)選取9株長勢一致的植株, 分成3組, 每組3株。取其心葉、+1葉(甘蔗植株由上到下第一個(gè)有可見肥厚帶的葉片)、+1葉的葉鞘、側(cè)芽、蔗莖和白色嫩根, 用于研究目的基因的空間表達(dá)特性。取樣后立即用液氮速凍, 置–80℃冰箱保存?zhèn)溆谩?/p>

表1 本研究使用的引物

1.2 總RNA提取和cDNA合成

采用TRIzol (Invotrigen, USA)試劑按照說明書提取樣品總RNA, 使用1.0%瓊脂糖凝膠電泳檢測RNA質(zhì)量。使用Nanodrop (Thermo Scientific, USA)測定RNA的濃度, 按照Prime Script RT Reagent Kit使用說明書, 將RNA反轉(zhuǎn)錄成cDNA。

1.3 ScNdhO基因的克隆及生物信息學(xué)分析

挑取陽性酵母克隆測序, 獲得目的基因的序列。利用NCBI的ORF finder (https://www.ncbi.nlm. nih.gov/orffinder/)軟件, 對所獲的基因序列進(jìn)行開放閱讀框分析。根據(jù)序列信息設(shè)計(jì)特異PCR引物, 以葉片的cDNA模板, 擴(kuò)增該基因的ORF并測序驗(yàn)證。然后將該基因克隆至pMD19-T載體, 轉(zhuǎn)化DH5α 感受態(tài)細(xì)胞。

利用ProtParam (http://expasy.org/tools/protparam. html)預(yù)測蛋白的一級結(jié)構(gòu)、理化性質(zhì); 利用Prabi (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_hnn.html)、SignalP 4.1 (http://www. cbs.dtu.dk/services/SignalP/)和TMHMM 2.0 (http:// www.cbs.dtu.dk/services/TMHMM/)分別預(yù)測分析其二級結(jié)構(gòu)、信號肽和跨膜特性; 通過NCBI 中的CDD (conserved domain database)數(shù)據(jù)庫(https:// www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)預(yù)測蛋白保守結(jié)構(gòu)域; 用 Blastp在線工具查找ScNdhO的同源氨基酸序列, 并使用DNAMAN7.0軟件多重比對同源氨基酸序列, 使用ClustalX和MEGA 6.0的ML (maximum likelihood, LG+G)法構(gòu)建系統(tǒng)進(jìn)化樹。

1.4 ScNdhO的RT-qPCR表達(dá)分析

根據(jù)基因的ORF序列, 設(shè)計(jì)特異性實(shí)時(shí)熒光定量PCR引物, 以供試材料的cDNA為模板, 以基因和基因(表1)為內(nèi)參基因[60], 按照SYBR Green PCR Master Mix Kit (Roche)說明書配制反應(yīng)體系。實(shí)時(shí)熒光定量PCR擴(kuò)增程序?yàn)?0℃ 2 min; 95℃ 10 min; 95℃ 15 s、60℃ 1 min, 循環(huán)40次。設(shè)置每個(gè)樣品3次重復(fù), 以無菌水作對照, 采用2–ΔΔCt算法進(jìn)行基因表達(dá)水平分析, 使用統(tǒng)計(jì)軟件SPSS 12.0對基因表達(dá)差異進(jìn)行顯著性分析。

1.5 載體構(gòu)建

用目的片段, 以綠色熒光蛋白(green fluorescence protein, GFP)為標(biāo)記, 構(gòu)建ScNdhO蛋白的亞細(xì)胞定位載體ScNdhO-GFP。用SCMV的VPg編碼序列, 以紅色熒光蛋白(mCherry fluorescence protein, mCherry)為標(biāo)記, 構(gòu)建VPg的亞細(xì)胞定位載體VPg-mCherry。將基因構(gòu)建到酵母表達(dá)載體 pGADT7中, 獲得ScNdhO的捕獲載體(GAL4 activation domain, AD) pGADT7-ScNdhO。利用Gateway技術(shù), 將基因構(gòu)建到BiFC的YC載體中, 得到ScNdhO-YC載體。所有構(gòu)建的載體都經(jīng)過測序驗(yàn)證。SCMV-VPg的Y2H誘餌載體(GAL4 DNA binding domain, BD) pGBKT7-SCMV-VPg、BiFC的YN載體SCMV-VPg-YN來自本課題組前期研究工作[17]。

1.6 ScNdhO的亞細(xì)胞定位

將含有ScNdhO-GFP植物表達(dá)載體的農(nóng)桿菌注射入健康的本氏煙葉片, 48 h后在激光共聚焦顯微鏡(Leica TCS SP5II)下觀察本氏煙葉片表皮細(xì)胞中ScNdhO蛋白的定位, GFP的激發(fā)光波長為488 nm, 捕獲波長為505~555 nm。mCherry的激發(fā)光波長為552 nm, 捕獲波長為590~630 nm。葉綠素自熒光的激發(fā)光波長為552 nm, 捕獲波長為650~680 nm。

1.7 Y2H驗(yàn)證ScNdhO與SCMV-VPg的互作

將pGADT7-ScNdhO、pGBKT7-SCMV-VPg組合, pGADT7-T、pGBKT7-53組合(陽性對照), pGADT7-T、pGBKT7-Lam組合(陰性對照), 分別轉(zhuǎn)化酵母菌株, 30℃ 250 r min-1培養(yǎng)至OD546為0.6~0.8。將轉(zhuǎn)化后的酵母細(xì)胞按照1、10-1和10-2梯度稀釋, 分別在缺少亮氨酸(Leu)和色氨酸(Trp)的固體酵母合成限定基本培養(yǎng)基(SD/?Leu/?Trp)固體培養(yǎng)基上培養(yǎng), 30℃倒置培養(yǎng)4~5 d。待菌落長到直徑為3~4 mm左右后, 挑取單菌落轉(zhuǎn)移到1 mL缺少亮氨酸(Leu)、色氨酸(Trp)、組氨酸(His)和腺嘌呤(Ade)的液體酵母合成限定基本培養(yǎng)基(SD/?Leu/? Trp/?His/?Ade)中培養(yǎng)。如果酵母生長, 將其轉(zhuǎn)移到SD/?Leu/?Trp/?His/?Ade固體培養(yǎng)基上培養(yǎng), 菌落生長表明蛋白之間存在互作。

1.8 BiFC驗(yàn)證ScNdhO與SCMV-VPg的互作

參照朱海龍等[18]的方法將ScNdhO-YC與SCMV-VPg-YN等比例共轉(zhuǎn)農(nóng)桿菌菌株, 將其注射入健康的本氏煙葉片中。正常條件下培養(yǎng)2~3 d后取樣, 在激光共聚焦顯微鏡(Leica TCS SP5II)下觀察拍照。YFP的激發(fā)光波長為514 nm, 捕獲波長為530~590 nm?

2 結(jié)果與分析

2.1 ScNdhO基因的生物信息學(xué)分析

在前期研究中, 以SCMV-VPg為誘餌從甘蔗葉片cDNA酵母文庫中篩選到1個(gè)具有全長ORF的基因, ORF長度為471 bp, 編碼156個(gè)氨基酸。根據(jù)該基因序列設(shè)計(jì)特異PCR引物(表1), 從甘蔗中克隆了該基因, 測序結(jié)果與陽性酵母質(zhì)粒測序結(jié)果相同。使用BlastP進(jìn)行氨基酸序列比對, 發(fā)現(xiàn)該基因編碼蛋白與高粱葉綠體NAD(P)H脫氫酶復(fù)合體O亞基(XP_002456968.1)同源性高達(dá)89%, 與玉米的O亞基(NP_001349312.1)同源性為89%, 因此將該基因命名為(GenBank登錄號為MK280691)。

ProtParam分析表明, ScNdhO蛋白的分子量為16.93 kD, 等電點(diǎn)為9.30; 不穩(wěn)定系數(shù)為39.54, 為穩(wěn)定蛋白; 脂溶指數(shù)為88.46, 總平均疏水性是–0.164, 可能是親水蛋白。二級結(jié)構(gòu)預(yù)測表明, ScNdhO中無規(guī)則卷曲結(jié)構(gòu)所占的比例最高, 為57.69%; 延伸鏈所占比例最低, 為18.59%; α-螺旋占23.72%。SignalP 4.1分析表明, ScNdhO蛋白不含信號肽, 是非分泌蛋白。TMHMM 2.0分析表明, ScNdhO蛋白無明顯的跨膜區(qū)域。保守結(jié)構(gòu)域分析表明,屬于NDH復(fù)合體O亞基超基因家族, 佐證了ScNdhO為甘蔗NDH復(fù)合體的O亞基。

2.2 ScNdhO的氨基酸同源性分析和系統(tǒng)進(jìn)化樹分析

使用NCBI網(wǎng)站的Blastp搜索ScNdhO的同源序列, 對甘蔗及其他物種NDH復(fù)合體O亞基的氨基酸序列的同源性分析表明, ScNdhO蛋白的物種分化比較明顯, 地錢()及單細(xì)胞的集胞藍(lán)藻()與高等植物明顯分離, 形成群I; 雙子葉植物番茄()、煙草()、擬南芥()、葡萄()、三葉楊()、大豆()、苜蓿()形成群II; 單子葉植物高粱()、玉米()、谷子()、水稻()、二穗短柄草()、大麥()形成群III; 在單子葉植物群III中, C4植物形成明顯的亞群III-2, C3植物形成亞群III-1 (圖1)。這表明, ScNdhO在遺傳進(jìn)化上具有明顯的種屬差異性, 與物種進(jìn)化程度相關(guān)。

2.3 ScNdhO的亞細(xì)胞定位

由圖2可知, 對照GFP在煙草表皮細(xì)胞中大量表達(dá), 綠色熒光信號在細(xì)胞質(zhì)、質(zhì)膜和細(xì)胞核中均有分布; ScNdhO-GFP融合蛋白的綠色熒光信號與葉綠體的自熒光信號重合, 表明其主要定位于葉綠體; ScNdhO-GFP與VPg-mCherry共定位于葉綠體, 表明SCMV-VPg有可能與ScNdhO-GFP互作于葉綠體。

2.4 ScNdhO基因的組織特異性表達(dá)及應(yīng)答SCMV侵染的表達(dá)模式

圖3表明,基因的表達(dá)具有明顯的組織特異性, 在不同組織中的表達(dá)量差異顯著: 在成熟葉片中相對表達(dá)量最高, 心葉和葉鞘中次之, 側(cè)芽較少, 莖中微量表達(dá), 根中幾乎不表達(dá)。使用基因特異引物(表1)檢測SCMV接種甘蔗, 擴(kuò)增出目的片段, 表明接種成功。SCMV侵染對基因表達(dá)影響顯著, 與對照相比,基因在侵染早期顯著上調(diào), 然后明顯下調(diào)表達(dá), 第7天的表達(dá)量低于對照(圖4)。

圖1 ScNdhO蛋白的氨基酸序列同源性分析和系統(tǒng)進(jìn)化樹分析

圖2 ScNdhO-GFP在本氏煙表皮細(xì)胞中的定位

ScNdhO-GFP的定位如箭頭所示; Up row: GFP對照; Middle row: ScNdhO-GFP定位; Down row: ScNdhO-GFP與VPg-mCherry共定位; bar = 50 μm。

The ScNdhO-GFP was labled by arrow; Up row: GFP control; Middle row: localization of ScNdhO-GFP; Down row: colocalization of ScNdhO-GFP with VPg-mCherry; bar = 50 μm.

圖3 ScNdhO基因在甘蔗不同組織中的表達(dá)模式

誤差線為每組處理的標(biāo)準(zhǔn)誤差(= 3)。Leaf roll: 心葉; Leaf: 正一葉; Sheath: 葉鞘; Bud: 腋芽; Stem: 莖; Root: 根。

The error bars represent the standard error of each treating group (= 3).

圖4 ScNdhO基因應(yīng)答SCMV侵染的表達(dá)模式

誤差線為每組處理的標(biāo)準(zhǔn)誤差(= 3)。

The error bars represent the standard error of each treating group (= 3).

2.5 ScNdhO與SCMV-VPg的互作驗(yàn)證

Y2H實(shí)驗(yàn)結(jié)果顯示, 在四缺培養(yǎng)基上, pGADT7-ScNdhO和pGBKT7-SCMV-VPg共轉(zhuǎn)的酵母菌株可以生長并呈現(xiàn)藍(lán)色, 陽性對照可以正常生長并呈現(xiàn)藍(lán)色, 陰性對照無菌落(圖5), 說明ScNdhO與SCMV-VPg互作。BiFC實(shí)驗(yàn)結(jié)果與Y2H實(shí)驗(yàn)結(jié)果一致, ScNdhO-YC與SCMV-VPg-YN互作產(chǎn)生黃色熒光信號, 并與葉綠體的紅色熒光信號重疊, 表明互作于葉綠體(圖6)。

3 討論

抗花葉病是甘蔗育種的重要指標(biāo)之一[61-63], 但是有關(guān)甘蔗花葉病病原即SCMV、SrMV和SCSMV侵染甘蔗及誘發(fā)花葉病癥狀的分子機(jī)制研究卻鮮見報(bào)道[12,17-18]。本研究首次克隆了甘蔗葉綠體NAD(P)H脫氫酶(NDH)復(fù)合體的O亞基基因, 首次報(bào)道了ScNdhO與SCMV-VPg互作并應(yīng)答SCMV侵染。本研究對于闡明SCMV侵染甘蔗導(dǎo)致花葉病癥狀的分子機(jī)制研究具有重要意義。

葉綠體NDH復(fù)合體在碳水化合物同化、逆境應(yīng)答等過程中具有重要作用[31,36,41-42,44,57]。線性電子傳遞中ATP/NADPH比值是固定的, 低于或接近卡爾文循環(huán)所需的比值1.5[64], 但是在逆境或者同化物大量合成的情況下, 循環(huán)電子途徑必須介入供應(yīng)ATP。對于C4植物而言, 光合作用所需的ATP/ NADPH比值至少要達(dá)到2.5。與C3光合作用相比, C4光合作用需要額外的2分子ATP用于1,5 -二磷酸核酮糖羧化酶/加氧酶(Rubisco)濃縮CO2, 這2分子額外的ATP就是由圍繞PSI的循環(huán)電子傳遞提供的, 現(xiàn)已在玉米、黃頂菊等C4植物中鑒定了NDH復(fù)合體介導(dǎo)的循環(huán)電子傳遞和生產(chǎn)ATP[33-34,65-67]。進(jìn)化分析也表明, C3植物與C4植物的NdhO分成不同的亞群。甘蔗葉片是主要的光合器官, 完全展開的正一葉是光合作用最旺盛的葉片, 本研究中在正一葉中的表達(dá)量最高, 推測葉綠體NDH復(fù)合體通過循環(huán)電子傳遞為CO2同化提供能量。NDH復(fù)合體O亞基對NDH復(fù)合體的正確組裝和行使功能很重要, 敲除煙草或擬南芥的突變體, 亞復(fù)合體A無法組裝, 進(jìn)而完全瓦解NDH介導(dǎo)的循環(huán)電子傳遞[57,68]。在SCMV侵染早期上調(diào)表達(dá), 一方面有可能通過這種互作使SCMV復(fù)制復(fù)合體定位于葉綠體, 完成病毒基因組復(fù)制; 另一方面則是寄主對SCMV侵染的抗性反應(yīng)。在SCMV侵染后期下調(diào)表達(dá), 本研究推測SCMV侵染影響了NDH復(fù)合體的穩(wěn)定, 干擾了循環(huán)電子傳遞, 使光合作用處于逆境狀態(tài), 導(dǎo)致葉綠素減少[10], 產(chǎn)生花葉癥狀, 進(jìn)而影響碳水化合物合成。

圖5 Y2H檢測ScNdhO與SCMV-VPg的互作

pGADT7-T和pGBKT7-53組合作為陽性對照, pGADT7-T和pGBKT7-Lam組合作為陰性對照。SD/?Leu/?Trp: 缺少亮氨酸(Leu)和色氨酸(Trp)的酵母合成限定基本培養(yǎng)基; SD/?Leu/?Trp/?His/?Ade: 缺少亮氨酸(Leu)、色氨酸(Trp)、組氨酸(His)和腺嘌呤(Ade)的酵母合成限定基本培養(yǎng)基。

The positive and negative controls are yeast cotransformants with pGADT7-T plus pGBKT7-53 and pGADT7-T plus pGBKT7-Lam, respectively. SD/?Leu/?Trp: synthetic defined yeast minimal medium lacking Leu and Trp; SD/?Leu/?Trp/?His/?Ade: synthetic defined yeast minimal medium lacking Leu, Trp, His, and Ade.

圖6 BiFC檢測ScNdhO與SCMV-VPg的互作

ScNdhO融合于YFP的C末端, SCMV-VPg融合于YFP的N末端, 然后在本氏煙葉片中瞬時(shí)表達(dá), 48 h后激光共聚焦觀察。bar = 50 μm。

ScNdhO was fused to the C-terminal half of YFP, while SCMV-VPg was fused to the N-terminal half of YFP. ScNdhO-YC and SCMV-VPg-YN were transiently coexpressed inleave. The fluorescent signal was monitored by confocal microscopy at 48 hpi. bar = 50 μm.

植物病毒結(jié)構(gòu)簡單, 必須與寄主因子互作才能完成系統(tǒng)性侵染[25-26]。本研究發(fā)現(xiàn)ScNdhO與SCMV-VPg互作, 因此推測ScNdhO被SCMV選擇性利用。NDH復(fù)合體約有30個(gè)亞基, SrMV和SCSMV與SCMV的基因組結(jié)構(gòu)非常相似, 他們的VPg有可能與ScNdhO或者其他亞基互作執(zhí)行相應(yīng)的生物學(xué)功能。病毒與寄主是協(xié)同進(jìn)化的, 病毒對寄主侵染的最終目的是繁殖和傳播[69]。由昆蟲作為媒介傳播的植物病毒中至少50%是由蚜蟲以非持續(xù)性方式傳播的[70-71]。蚜蟲普遍嗜好發(fā)病植株, 比如雀麥花葉病毒屬()的菜豆黃花葉病毒(, BYMV)和蠶豆斑駁病毒(, BBMV), 豌豆耳突花葉病毒屬()的豌豆耳突花葉病毒(, PEMV), 侵染后造成葉片黃化, 吸引豌豆蚜[72], 與SCMV同屬的小西葫蘆黃花葉病毒(, ZYMV)侵染造成的葉片黃化植株吸引棉蚜[73]。SCMV是由多種蚜蟲非持續(xù)性方式傳播的[6], SCMV-VPg與ScNdhO互作并在侵染后期降低其表達(dá), 可能對葉綠體產(chǎn)生脅迫, 造成葉片黃化, 吸引蚜蟲便于病毒傳播。病毒與寄主的互作是十分復(fù)雜的, 花葉病癥狀的產(chǎn)生可能是多方面因素的結(jié)果, 比如SCSMV-P3與甘蔗 Rubisco大亞基互作可能干擾Rubisco的功能而影響葉綠體的穩(wěn)定性[18]。SCMV-VPg與ScNdhO互作的具體生物學(xué)功能還有待進(jìn)一步研究。

4 結(jié)論

從甘蔗中克隆到葉綠體NAD(P)H脫氫酶(NDH)復(fù)合體的O亞基基因(GenBank登錄號為MK280691), 其ORF長度為471 bp, 編碼長度為156 aa的蛋白。ScNdhO蛋白定位于葉綠體, 在完全展開的功能葉片中表達(dá)量最高。ScNdhO蛋白與SCMV-VPg互作?；蝽憫?yīng)SCMV侵染, 在侵染早期上調(diào)表達(dá), 在侵染后期則下調(diào)表達(dá)。

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Cloning of NAD(P)H complex O subunit gene and its interaction with VPg of

ZHAI Yu-Shan, ZHAO He, ZHANG Hai, DENG Yu-Qing, CHENG Guang-Yuan, YANG Zong-Tao, WANG Tong, PENG Lei, XU Qian, DONG Meng, and XU Jing-Sheng*

SugarcaneResearch & Development Centre, China Agricultural Technology System, Fujian Agriculture and Forestry University/Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops, Fuzhou 350002, Fujian,China

NAD(P)H dehydrogenase (NDH) complex mediates cyclic electron transports, playing key role in efficient photosynthesis in chloroplast. The involvement of NDH complex in(SCMV) infection of sugarcane (spp. hybrid) has not been reported. In this study, we isolated the coding sequence of the subunit of the NAD(P)H dehydrogenase complex from sugarcane and designated it as. The open reading frame (ORF) ofis 471 bp and encodes a 156 aa length protein. Bioinformatics analysis showed that ScNdhO is a stable hydrophilic protein with no signal peptide and transmembrane domain. The secondary structure of ScNdhO is composed of mostly random coilα-helices, with a typical domain of NDH complex O subunit. Phylogenetic tree analysis showed that ScNdhO belongs to the NDHO supperfamily. Real-time quantitative PCR analysis showed thatgene was tissue specific in sugarcane, with the lowest expression level in roots or stem, and the highest in leaf. The expression ofwas upregulated in the early stage of SCMV infection, but downregulated with time going. Subcellular location assays showed that ScNdhO was located in chloroplast. ScNdhO interacted with the VPg from SCMV as demonstrated by yeast two hybrid and bimolecular fluorescence complementation assays. We proposed that ScNdhO should be selectively employed by SCMV and involved in the mosaic symptom.

sugarcane; SCMV; chloroplast; NAD(P)H dehydrogenase complex; O subunit

本研究由國家自然科學(xué)基金項(xiàng)目(31371688)資助。

This study was supported by the National Natural Science Foundation of China (31371688).

徐景升, E-mail: xujingsheng@126.com

E-mail:yushanzhai@126.com

2018-12-31;

2019-01-19;

2019-03-08.

10.3724/SP.J.1006.2019.94002

URL: http://kns.cnki.net/kcms/detail/11.1809.S.20190307.1645.004.html