張 海 程光遠(yuǎn) 楊宗桃 劉淑嫻 商賀陽 黃國強 徐景升

甘蔗PsbR亞基應(yīng)答SCMV侵染及其與SCMV-6K2的互作

張 海 程光遠(yuǎn) 楊宗桃 劉淑嫻 商賀陽 黃國強 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)農(nóng)村部福建甘蔗生物學(xué)與遺傳育種重點實驗室 / 教育部作物遺傳育種與綜合利用重點實驗室, 福建福州 350002

光系統(tǒng)II (Photosystem II, PSII)的PsbR亞基對于放氧復(fù)合體(oxygen-evolving complex)的組裝和穩(wěn)定具有至關(guān)重要的作用。本課題組前期克隆了甘蔗(spp. hybrid)的PsbR亞基編碼基因, 命名為, 并利用酵母雙雜交技術(shù)(yeast two hybrid, Y2H)驗證了ScPsbR與甘蔗花葉病毒(, SCMV)編碼蛋白6K2的互作。本研究通過生物信息學(xué)分析表明, ScPsbR具有典型的PsbR亞基結(jié)構(gòu)域, 無信號肽, 具有1個跨膜結(jié)構(gòu)域, 為穩(wěn)定的疏水性蛋白。系統(tǒng)進(jìn)化樹分析表明, 該蛋白在C3和C4植物中存在明顯的分化。亞細(xì)胞定位試驗表明, ScPsbR定位于葉綠體且與SCMV-6K2共定位。雙分子熒光互補(bimolecular fluorescence complementation, BiFC)試驗進(jìn)一步驗證了ScPsbR與SCMV-6K2的互作。實時熒光定量PCR檢測表明,基因表達(dá)具有顯著的組織特異性, 在根和莖中表達(dá)極少, 未成熟葉片和初衰葉中次之, 成熟葉片中相對表達(dá)量最高; SCMV侵染顯著影響基因表達(dá),基因在侵染0~12 h顯著上調(diào), 侵染1~5 d下調(diào)至略低于對照的水平, 但差異不顯著, 侵染7~15 d顯著下調(diào)。

PsbR亞基; 甘蔗花葉病毒; 6K2; 蛋白互作

甘蔗花葉病毒(, SCMV)屬于馬鈴薯Y病毒屬()[1], 是甘蔗花葉病的主要病原物之一[2-7]。甘蔗花葉病嚴(yán)重影響甘蔗(spp. hybrid)的產(chǎn)量和品質(zhì)[2,7-9]。SCMV的基因組為單鏈正義RNA, 長度約為10 Kb, 編碼11個功能蛋白: P1、HC-Pro、P3N-PIPO、P3、6K1、CI、6K2、VPg、NIa-Pro、NIb和CP[4,10-14]。其中6K2在馬鈴薯Y病毒屬病毒侵染寄主的過程中起關(guān)鍵作用。6K2蛋白能夠誘導(dǎo)內(nèi)質(zhì)網(wǎng)形成復(fù)制囊泡, 既可以與葉綠體外膜融合參與病毒高效復(fù)制[15-21], 也可以介導(dǎo)病毒胞間移動, 侵染相鄰細(xì)胞[22-24]; 另外, 該蛋白還可以拮抗或利用寄主的自噬系統(tǒng), 促進(jìn)病毒侵染[25-26]。

光合放氧來自光合作用的光反應(yīng)[27], 由位于類囊體腔內(nèi)側(cè)放氧復(fù)合體(oxygen-evolving complex, OEC)執(zhí)行[28-29]。在植物和藻類中, OEC由光系統(tǒng)II (photosystem II, PSII)的Mn4O5Ca團(tuán)簇及其配體、4個外在蛋白PsbO (33 kD)、PsbP (23 kD)、PsbQ (17 kD)和PsbR (10 kD)組成[28,30-35]。OEC的4個外在蛋白由核基因編碼, 在氧氣釋放中起關(guān)鍵作用[31,35]。研究表明, PsbR對于維持PSII復(fù)合體的構(gòu)象及穩(wěn)定PsbP和PsbQ的結(jié)合是必需的[28,30,32]。因此, 敲除降低了氧氣釋放和醌再氧化速率, 進(jìn)而影響光合效率[28,30,35]。OEC亞基廣泛應(yīng)答逆境[36], 但是在甘蔗中還鮮見報道[7,36]。

在前期工作中, 本課題組克隆了甘蔗PSII的PsbR亞基編碼基因, 命名為(GenBank登錄號為QHD26873.1), 并利用酵母雙雜交技術(shù)(yeasttwo hybrid, Y2H)驗證了ScPsbR與SCMV-6K2的互作[36]。本研究利用雙分子熒光互補技術(shù)(bimolecular fluorescent complimentary, BiFC)進(jìn)一步佐證了ScPsbR與SCMV-6K2的互作, 利用RT-qPCR技術(shù)研究了在不同組織部位的表達(dá)情況以及應(yīng)答SCMV侵染的表達(dá)模式, 并深入探討了在SCMV侵染中的作用, 旨在為研究甘蔗花葉病癥狀形成分子機理及甘蔗抗花葉病育種提供基礎(chǔ)數(shù)據(jù)和試驗依據(jù)。

1 材料與方法

1.1 材料及處理方法

甘蔗熱帶種、SCMV-FZ1病毒株系[4]和本氏煙()由福建農(nóng)林大學(xué)國家甘蔗工程技術(shù)研究中心提供。在光周期為16 h光照/8 h暗, 光照強度為200 μmol m-2s-1, 溫度28℃, 空氣濕度60%的條件下, 采用腋芽快繁技術(shù)培養(yǎng)組培苗。待其出現(xiàn)四至五片完全展開的葉片時, 挑選健壯且長勢一致的組培苗光培1 h后摩擦接種SCMV, 設(shè)置3個重復(fù), 每個重復(fù)3株, 使用磷酸緩沖液(pH 7.0)摩擦接種的植株作為對照。在接種0 h、4 h、8 h、12 h、1 d、3 d、5 d、7 d、15 d后取接種葉片, 使用SCMV-CP基因特異性引物(表1)檢測接種成功與否。從福建農(nóng)林大學(xué)隔離網(wǎng)室中選取伸長期健壯且長勢一致熱帶種植株, 取未成熟葉心葉、成熟葉片正一葉(甘蔗植株由上到下第1個有可見肥厚帶的葉片)、初衰葉片正七葉、第三節(jié)間、第八節(jié)間和根, 用于基因的組織特異性表達(dá)實驗, 選取3株混合作為1個生物學(xué)重復(fù), 設(shè)3個生物學(xué)重復(fù)。取樣后用錫箔紙包好液氮速凍, 然后置-80℃冰箱保存?zhèn)溆谩?/p>

表1 本研究使用的引物

1.2 ScPsbR蛋白的生物信息學(xué)分析

利用ProtParam (http://expasy.org/tools/prot-param.html)預(yù)測ScPsbR蛋白的一級結(jié)構(gòu)和理化性質(zhì); 使用NCBI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; 利用SignalP 5.0 (http://www.cbs.dtu.dk/services/SignalP/)、ChloroP (http://www.cbs.dtu.dk/services/ChloroP/)、GOR4 (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl? page=npsa_gor4.html)和TMHMM 2.0 (http://www. cbs.dtu.dk/services/TMHMM/)分別預(yù)測ScPsbR蛋白的信號肽、轉(zhuǎn)運肽、二級結(jié)構(gòu)和跨膜特性; 利用NCBI和Phytozome網(wǎng)站的Proteome Blastp在線工具查找ScPsbR的同源氨基酸序列, 使用DNAMAN 8.0進(jìn)行氨基酸序列比對分析; 參照張海等[37]方法, 使用ClustalX和MEGA 7.0的ML (maximum likelihood, LG+G)法構(gòu)建系統(tǒng)進(jìn)化樹。

1.3 RNA提取和cDNA合成

在液氮中將采集的樣品充分研磨至粉末狀, 使用TRIpure (北京艾德萊生物科技有限公司, 中國)試劑提取總RNA (參照TRIpure說明書進(jìn)行), 使用1.0%瓊脂糖凝膠電泳檢測提取RNA質(zhì)量。利用Evo-MLV反轉(zhuǎn)錄試劑盒(湖南艾科瑞生物工程有限公司)并按其說明書將不同組織樣品RNA反轉(zhuǎn)錄成cDNA。

1.4 載體構(gòu)建

參照Cheng等[11]的方法構(gòu)建ScPsbR的亞細(xì)胞定位載體ScPsbR-YFP及用于BiFC試驗的載體ScPsbR-YC、YN-ScPsbR, 委托福州白鯨生物科技有限公司測序驗證。SCMV-6K2-CFP[36]、YN-SCMV- 6K2和SCMV-6K2-YC載體來自本課題組前期研究工作。

1.5 共聚焦熒光觀察

將上述載體分別轉(zhuǎn)入農(nóng)桿菌GV3101中, 參照Cheng等[11]農(nóng)桿菌侵染方法, 將含有ScPsbR-YFP的農(nóng)桿菌GV3101, 含有組合SCMV-6K2-CFP和ScPsbR-YFP、組合YN-SCMV-6K2和ScPsbR-YC、組合YN-ScPsbR和SCMV-6K2-YC的農(nóng)桿菌GV3101等比例混合, 分別注射健康的本氏煙葉片。正常條件下(16 h光/8 h暗, 22℃)培養(yǎng)48 h后在激光共聚焦顯微鏡(Leica TCS SP5II)下觀察照相。CFP的激發(fā)波長為442 nm, 采集波長為450~500 nm; YFP的激發(fā)波長為514 nm, 采集波長為530~590 nm; 葉綠素自熒光的激發(fā)光波長為552 nm, 采集波長為650~680 nm。采用數(shù)字采集圖像, 使用LSM 2.6.3軟件進(jìn)行處理。

1.6 ScPsbR基因的RT-qPCR表達(dá)分析

根據(jù)基因序列設(shè)計特異性實時熒光定量PCR引物(表1), 以供試材料的cDNA為模板, 以基因和基因(表1)為內(nèi)參基因[38], 使用SYBR Green PCR Master Mix Kit (Roche, 日本)試劑盒, 按其說明書配制反應(yīng)體系, 包含SYBR Green Master Mix 10.0 μL、上下游引物各1.0 μL、模板1.0 μL、DEPC處理的ddH2O 7.0 μL。RT-qPCR在7500型實時熒光定量PCR儀(ABI, USA)上進(jìn)行, 擴增程序為50℃ 2 min; 95℃ 10 min, 95℃ 15 s, 60℃ 1 min, 40個循環(huán)。每個樣品設(shè)置3次技術(shù)重復(fù)和生物學(xué)重復(fù), 以DEPC處理的ddH2O作為對照, 采用2–ΔΔCt算法計算基因的相對表達(dá)量, 使用統(tǒng)計軟件SPSS 12.0對基因表達(dá)差異進(jìn)行顯著性分析。

2 結(jié)果與分析

2.1 ScPsbR蛋白的生物信息學(xué)分析

ProtParam分析表明, ScPsbR蛋白的分子量為13.42 kD, 等電點為9.55; 脂溶指數(shù)為88.63, 總平均親水性是0.051, 可能是疏水性蛋白; 不穩(wěn)定系數(shù)為18.06, 為穩(wěn)定蛋白; ScPsbR的二級結(jié)構(gòu)預(yù)測顯示, 無規(guī)則卷曲、α-螺旋、延伸鏈所占比例分別為55.73%、18.32%、25.95%, 無β-折疊結(jié)構(gòu); 信號肽預(yù)測表明, ScPsbR蛋白無信號肽, 屬非分泌蛋白。保守結(jié)構(gòu)域分析表明, ScPsbR蛋白包含1個保守結(jié)構(gòu)域, 位于第17~131位。PsbR亞基由核基因編碼, 借助其N端轉(zhuǎn)運肽進(jìn)入葉綠體, 然后經(jīng)基質(zhì)蛋白酶剪切折疊形成成熟蛋白, 并通過C端跨膜區(qū)域靶向類囊體腔[39-41]。轉(zhuǎn)運肽預(yù)測分析表明, ScPsbR亞基N端具有一個長度為33 aa的轉(zhuǎn)運肽(圖1); 跨膜結(jié)構(gòu)域預(yù)測分析表明, ScPsbR亞基具有一個跨膜結(jié)構(gòu)域, 分布在104~126, 膜向性為i→o (圖1)。

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

通過NCBI網(wǎng)站的Blastp和Phytozome網(wǎng)站的Proteome BLASTp搜索PsbR不同物種的同源序列發(fā)現(xiàn), ScPsbR蛋白與高粱(, XP_002444002.1)、玉米(, XP_020408082.1)、谷子(S, XP_004972630.1)、黍(, XP_025821160.1)、二穗短柄草(, XP_010234446.1)和水稻(, AAB46718.1)的PsbR相似性分別為93.18%、87.88%、90.84%、90.77%、76.34%和79.03% (圖2)。

*: 終止密碼子。黑色下畫線部分為轉(zhuǎn)運肽。紅色下畫線部分為跨膜結(jié)構(gòu)域。轉(zhuǎn)運肽和成熟蛋白之間的邊界用箭頭標(biāo)出。

*: stop codon. The sequence underlined in black indicates the transit peptide and the sequence underlined in red indicates the transmembrane domains. ↓ indicates the digestion site. The border between transit sequence and mature protein is marked with an arrowhead.

SbPsbR (XP_002444002.1): 高粱; ZmPsbR (XP_020408082.1): 玉米; PhPsbR (XP_025821160.1): 黍; SiPsbR (XP_004972630.1): 谷子; BdPsbR (XP_010234446.1): 二穗短柄草; OsPsbR (AAB46718.1): 水稻。

SbPsbR (XP_002444002.1):; ZmPsbR (XP_020408082.1):; PhPsbR (XP_025821160.1):; SiPsbR (XP_004972630.1):; BdPsbR (XP_010234446.1):; OsPsbR (AAB46718.1):.

以地錢(, OAE29880.1)的PsbR蛋白序列作為外源序列構(gòu)建系統(tǒng)進(jìn)化樹, 分析ScPsbR蛋白與其他物種PsbR蛋白的進(jìn)化關(guān)系。進(jìn)化樹分析結(jié)果表明, 單子葉植物甘蔗、高粱、玉米、谷子、黍、水稻、二穗短柄草、小麥(, AAP72269.1)和毛竹(, ABC02754.1)形成群I; 其中C4植物甘蔗、高粱、玉米、谷子和黍形成亞群I-1, C3植物水稻、二穗短柄草、小麥和毛竹分別形成亞群和I-2; 雙子葉植物陸地棉(, AHK23268.1)、煙草(, XP_016498221.1)、擬南芥(, NP_178025.1)、楊梅(, KAB1216962.1)、馬鈴薯(, P_001275173.1)、大豆(, NP_001235442.1)和碧桃(, XP_007212214.1)形成群II (圖3)。表明, 在單子葉C3植物和C4植物之間, 以及單子葉植物和雙子葉植物之間, PsbR蛋白在遺傳進(jìn)化上存在明顯分化。

2.3 ScPsbR的亞細(xì)胞定位

我們前期研究表明, SCMV-6K2蛋白定位于內(nèi)質(zhì)網(wǎng)和葉綠體[36], PsbR也被前人報道定位于葉綠體[30-31]。亞細(xì)胞定位試驗表明, ScPsbR-YFP融合蛋白的黃色熒光信號(綠色)與葉綠體的自熒光信號重合, 表明其定位于葉綠體(圖4-A); 將SCMV- 6K2-CFP和ScPsbR-YFP蛋白在健康的煙草葉片中表達(dá), ScPsbR-YFP與SCMV-6K2-CFP融合蛋白的青色熒光信號重合, 表明SCMV-6K2和ScPsbR共同定位, 且2種融合蛋白的熒光信號與葉綠體的自發(fā)熒光信號部分重合(圖4-B), 但主要定位于內(nèi)質(zhì)網(wǎng)。表明SCMV-6K2和ScPsbR共同定位, SCMV-6K2的表達(dá)影響ScPsbR在細(xì)胞中的分布。

A: ScPsbR-YFP亞細(xì)胞定位; B: ScPsbR-YFP和6K2-CFP亞細(xì)胞共定位。白色箭頭表示葉綠體, 標(biāo)尺為25 μm。

A: subcellular localization of ScPsbR-YFP; B: subcellular colocalization of ScPsbR-YFP with 6K2-CFP. White arrows indicate the chloroplasts, Bar: 25 μm.

2.4 ScPsbR與SCMV-6K2的互作驗證

BiFC試驗結(jié)果表明, 共注射的組合ScPsbR-YC與YN-SCMV-6K2、組合YN-ScPsbR與SCMV- 6K2-YC都產(chǎn)生黃色熒光信號(綠色) (圖5), 進(jìn)一步證明了SCMV-6K2與ScPsbR互作, 與我們前期Y2H試驗結(jié)果一致[36]。

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

熒光定量PCR結(jié)果表明,基因在不同組織中的表達(dá)差異顯著(圖6), 在成熟葉片正一葉中相對表達(dá)量最高, 未成熟葉心葉和初衰葉正七葉中次之, 根和莖的第三節(jié)間、第八節(jié)間中表達(dá)極少。

使用SCMV-CP基因特異性引物(表1)檢測SCMV接種甘蔗組培苗葉片, 擴增出目的片段, 表明接種成功。熒光定量PCR結(jié)果表明, SCMV侵染對基因表達(dá)影響顯著, 與對照相比,基因在侵染0~12 h顯著上調(diào), 1~5 d下調(diào)至略低于對照的水平, 但是差異不顯著, 7 d和15 d顯著下調(diào)(圖7)。

A: YC融合于ScPsbR的C末端, YN融合于SCMV-6K2的N末端; B:YN融合于ScPsbR的N末端, YC融合于SCMV-6K2的C末端。將YN-6K2和ScPsbR-YC(A), YN-ScPsbR和6K2-YC(B)分別共注射到本氏煙葉片中進(jìn)行瞬時表達(dá), 48 h后激光共聚焦觀察。白色箭頭表示具有互作熒光信號的葉綠體。標(biāo)尺為25 μm。

A: the C-terminal half of YFP was fused to the C-terminal of ScPsbR to generate ScPsbR-YC, while the N-terminal half of YFP was fused to the N-terminal of SCMV-6K2 to generate YN-6K2; B: the N-terminal half of YFP was fused to the N-terminal of ScPsbR to generate YN- ScPsbR, while the C-terminal half of YFP was fused to the C-terminal of SCMV-6K2 to generate 6K2-YC. Plasmids combination of YN-6K2 plus ScPsbR-YC (A), YN-ScPsbR plus SCMV-6K2-YC (B) were individually co-injected intoleaves for transient expression. White arrows indicate the chloroplasts with interacting fluorescent signals. The fluorescent signal was monitored by confocal microscopy at 48 hours post infiltration. Bar: 25 μm.

誤差線為每組處理的標(biāo)準(zhǔn)誤差(= 3)。LR: 心葉; +1 L: 正一葉; +7 L: 正七葉; +3 I: 第三節(jié)間; +8 I: 第八節(jié)間; R: 根。柱上不同小寫字母表示在5%水平差異顯著。

The error bars represent the standard error of each treating group (= 3). LR: leaf roll; +1 L: 1st leaf; +7 L: 7th leaf; +3 I: 3rd internode; +8 I: 8th internode; R: root. Different lowercase letters above the bars are significantly different at the 5% probability level.

誤差線為每組處理的標(biāo)準(zhǔn)誤差(= 3)。柱上不同小寫字母表示在5%水平差異顯著。

The error bars represent the standard error of each treating group (= 3). Different lowercase letters above the bars are significantly different at the 5% probability level.

3 討論

絕大多數(shù)病毒以葉綠體為靶標(biāo), 與葉綠體組分廣泛互作, 或利用葉綠體進(jìn)行高效復(fù)制或移動, 或改變?nèi)~綠體的結(jié)構(gòu)和功能產(chǎn)生病癥, 而葉綠體也會介導(dǎo)植物對病毒產(chǎn)生抗性[35,42-43]。本課題組在前期工作中也鑒定了一些與SCMV互作的葉綠體因子, 如與VPg互作的ScNdhO[44], 與6K2互作的ScSULTR3-3、ScPPT2、ScVTE3、ScPsbS和ScPsbR[36-37]。OEC亞基PsbR與馬鈴薯Y病毒屬病毒SCMV編碼蛋白6K2互作, 為本課題組首次報道[36], 本研究進(jìn)一步利用BiFC技術(shù)驗證了ScPsbR與6K2的互作。

OEC亞基廣泛應(yīng)答病毒侵染, 可能介導(dǎo)了植物對病毒的抗性, 比如過表達(dá)抑制苜蓿花葉病毒(, AMV,)的復(fù)制[45], 沉默促進(jìn)水稻條紋病毒(, RSV,)積累并加劇病癥[46], 沉默促進(jìn)煙草花葉病毒(, TMV,)復(fù)制[47]。本研究中SCMV侵染試驗表明,基因在侵染早期顯著上調(diào)表達(dá), 推測寄主應(yīng)答SCMV逆境以維持正常的生理功能。但是由于是SCMV的天然寄主, 隨著侵染時間的加長,基因逐漸下調(diào), 以便SCMV建立系統(tǒng)性侵染。本氏煙易受番茄黃葉卷曲病毒(侵染, 其在5 d與對照0 h水平相當(dāng), 在15 d則顯著低于對照, 與本研究中應(yīng)答SCMV侵染的表達(dá)模式極為相似[49]以熱帶種為受體的抗花葉病轉(zhuǎn)基因材料B-48[49]在接種SCMV后, PSII的10 kD蛋白編碼基因顯著上調(diào)表達(dá)[7], 檢索該研究的參考基因組發(fā)現(xiàn), 該基因為[50]。因此, 我們推測基因參與調(diào)節(jié)了甘蔗對花葉病的抗性。

盡管葉綠體是包括SCMV在內(nèi)的病毒高效復(fù)制的場所[19,51], 但是SCMV-6K2與ScPsbR的互作可能并沒有決定6K2復(fù)制囊泡的葉綠體定位。研究表明,病毒6K2復(fù)制囊泡的葉綠體定位由SNARE蛋白Syp71介導(dǎo)的, 抑制表達(dá)嚴(yán)重阻礙了6K2復(fù)制囊泡的葉綠體定位, 且Syp71并不與6K2直接互作[52]。6K2與ScPsbR互作的主要生物學(xué)意義可能是隔離PsbR, 抑制其介導(dǎo)的抗性, 并促進(jìn)花葉病癥狀的產(chǎn)生。比如, AMV-CP、RSV-SP分別與PsbP互作于細(xì)胞質(zhì), 改變了PsbP的葉綠體定位, 借此隔離PsbP的抗性[45-46], 而單組分雙生病毒()的致病因子βC1與PsbP互作, 抑制后者非特異性結(jié)合病毒DNA, 促進(jìn)病毒侵染[53]。本研究的BiFC試驗表明, SCMV-6K2與ScPsbR互作于葉綠體, 但是改變了葉綠體的分布, 所有帶有互作熒光信號的葉綠體均分布在細(xì)胞周質(zhì); 共定位試驗也表明, 熒光信號主要位于內(nèi)質(zhì)網(wǎng)。鑒于PsbR對OEC的重要作用, SCMV-6K2與ScPsbR互作可能會影響OEC乃至PSII的結(jié)構(gòu)和功能, 促進(jìn)花葉病癥狀的產(chǎn)生, 借此吸引蚜蟲等媒介促進(jìn)病毒傳播[3,54-56]。

4 結(jié)論

ScPsbR蛋白定位于葉綠體, 與SCMV-6K2互作?；蛟诟?、莖和葉中都有表達(dá), 在成熟葉中表達(dá)量最高。在SCMV侵染條件下,基因表達(dá)早期上調(diào)而后期下調(diào)。

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Sugarcane PsbR subunit response to SCMV infection and its interaction with SCMV-6K2

ZHANG Hai, CHENG Guang-Yuan, YANG Zong-Tao, LIU Shu-Xian, SHANG He-Yang, HUANG Guo-Qiang, and XU Jing-Sheng*

National Engineering Research Center for Sugarcane / Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs / Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops / Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China

The PsbR subunit of photosystem II (PSII) plays a vital role in the assembly and stability of the oxygen-evolving complex. In the previous study, we cloned the coding sequence of the PsbR subunit from sugarcane (spp. hybrid) and designated it as. The interaction between ScPsbR and 6K2 protein encoded by(SCMV) was verified by yeast two-hybrid technology. In this study, bioinformatics analysis indicated ScPsbR protein was found to possess a canonical subunit domain of PsbR and a transmembrane domain without signal peptide, and be a stable hydrophobic protein. Phylogenetic tree analysis indicated obvious divergence between C3and C4plants for the PsbRs. Subcellular localization experiments suggested that ScPsbR was localized and co-localized with SCMV-6K2 to the chloroplast. The interaction of ScPsbR with the SCMV-6K2 was further verified by bimolecular fluorescence complementation assays. Real-time quantitative PCR results indicated thatgene was tissue-specific in sugarcane plants. There was almost no expression ofgene in the roots or stems, with increased expression level in the senescing leaves and immature leaves, and the highest expression level in mature leaves. However, the expression level ofwas significantly changed under the challenged of SCMV. During the infection of SCMV,was significantly up-regulated at 0–12 hour(s) and reduced to a level slightly lower than that of the control at 1–5 days with no significant differences, then significantly down-regulated in 7–15 days.

PsbR subunit;; 6K2; protein interaction

10.3724/SP.J.1006.2021.04194

本研究由國家自然科學(xué)基金項目(31971991), 福建農(nóng)林大學(xué)科技創(chuàng)新基金項目(CXZX2018026)和福建省科技廳引導(dǎo)性項目(2017N0003)資助。

This study was supported by the National Natural Science Foundation of China (31971991), the Science and Technology Innovation Project of Fujian Agriculture and Forestry University (CXZX2018026), and the Guiding Project of Science and Technology Department of Fujian Province (2017N0003).

徐景升, E-mail: xujingsheng@126.com

E-mail: zhanghai940410@163.com

2020-08-25;

2020-11-13;

2020-12-22.

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20201222.1536.004.html