干旱、高鹽及低溫脅迫下植物生理及轉(zhuǎn)錄因子的應(yīng)答調(diào)控

王 冰，程憲國

（中國農(nóng)業(yè)科學(xué)院農(nóng)業(yè)資源與農(nóng)業(yè)區(qū)劃研究所/農(nóng)業(yè)部植物營養(yǎng)與肥料重點實驗室，北京 100081）

干旱、高鹽及低溫脅迫下植物生理及轉(zhuǎn)錄因子的應(yīng)答調(diào)控

王 冰，程憲國*

（中國農(nóng)業(yè)科學(xué)院農(nóng)業(yè)資源與農(nóng)業(yè)區(qū)劃研究所/農(nóng)業(yè)部植物營養(yǎng)與肥料重點實驗室，北京 100081）

干旱、高鹽及低溫等非生物脅迫是限制植物生長發(fā)育的主要環(huán)境因子。這些環(huán)境脅迫因子通常導(dǎo)致植物體內(nèi)生理代謝改變，并參與非生物脅迫調(diào)控轉(zhuǎn)錄因子的差異表達(dá)。植物抵御上述非生物逆境的能力與轉(zhuǎn)錄因子調(diào)控逆境相關(guān)功能基因的表達(dá)密不可分。近年來，發(fā)掘植物非生物脅迫相關(guān)轉(zhuǎn)錄因子的功能及揭示轉(zhuǎn)錄因子介導(dǎo)植物非生物脅迫響應(yīng)的調(diào)控機制，已成為植物營養(yǎng)分子生物學(xué)關(guān)注的熱點之一。因此，了解植物非生物脅迫下的生理應(yīng)答及轉(zhuǎn)錄因子參與的調(diào)控機制，對建立植物適應(yīng)性改良途徑具有重要科學(xué)意義。本文從干旱、高鹽和低溫三方面闡述了非生物脅迫下植物生理生化的適應(yīng)性變化，概述了MYB、bZIP、AP2/EREBP、WRKY和NAC五類與植物抗逆相關(guān)的轉(zhuǎn)錄因子的結(jié)構(gòu)與功能特征，著重論述了轉(zhuǎn)錄因子介導(dǎo)植物抵御非生物脅迫的分子調(diào)控機制。植物遭遇非生物脅迫時，通常表現(xiàn)為生長速率、葉面積和葉片數(shù)量下降，蒸騰及光合速率降低。同時，植物體內(nèi)活性氧逐漸累積，使細(xì)胞膜脂過氧化程度加劇，造成細(xì)胞損傷。為適應(yīng)不利環(huán)境，在生理上植物表現(xiàn)為體內(nèi)抗氧化酶活性增強，滲透調(diào)節(jié)物數(shù)量增多；在分子水平上，植物對非生物脅迫適應(yīng)性的增強，通常與轉(zhuǎn)錄因子識別抗逆基因啟動子特異性元件及調(diào)控逆境防御基因的轉(zhuǎn)錄有關(guān)。本文對于深入闡明干旱、高鹽及低溫脅迫下植物生理生化應(yīng)答與轉(zhuǎn)錄因子的分子調(diào)控機制提供了全新的科學(xué)啟示。

干旱脅迫；鹽脅迫；低溫脅迫；生理應(yīng)答；轉(zhuǎn)錄因子

干旱、鹽、低溫等非生物脅迫嚴(yán)重限制了植物的生長發(fā)育。世界主要農(nóng)作物每年有近50%的產(chǎn)量損失都與非生物脅迫有關(guān)[1]。了解植物非生物脅迫下的生理生化應(yīng)答特征是評價作物對非生物脅迫抗性效果的重要指標(biāo)。為了適應(yīng)環(huán)境變化，植物形成了一系列防御機制以抵抗各種逆境傷害。其中，植物抗逆基因的轉(zhuǎn)錄調(diào)控 (transcription regulation) 對植物抵御逆境脅迫發(fā)揮著重要的調(diào)節(jié)作用。轉(zhuǎn)錄調(diào)控主要通過特定的轉(zhuǎn)錄因子與相應(yīng)的順式作用元件 (cisacting element) 相互作用來實現(xiàn)。轉(zhuǎn)錄因子是指能夠與基因啟動子區(qū)域中順式作用元件發(fā)生特異性相互作用的DNA結(jié)合蛋白，通過它們之間或與其它蛋白之間的互作，激活或抑制轉(zhuǎn)錄發(fā)揮調(diào)控作用。與植物抗逆相關(guān)的轉(zhuǎn)錄因子主要包括禽成髓細(xì)胞瘤病毒致癌基因同源物類 (v-myb avian myeloblastosis viral oncogene homolog, MYB)、堿性域亮氨酸拉鏈 (basicdomain leucine-zipper, bZIP)、乙烯應(yīng)答元件組合蛋白/因子 (APELATA2/ethylene-responsive element binding proteins/factors, AP2/EREBP)、WRKY和NAC等五類。研究植物非生物脅迫相關(guān)轉(zhuǎn)錄因子的功能及其在植物非生物脅迫過程中的響應(yīng)與調(diào)控機制，已成為研究者關(guān)注的熱點。

1 非生物脅迫下植物的生理應(yīng)答

1.1 干旱脅迫下植物生理生化的適應(yīng)性變化

干旱脅迫嚴(yán)重限制植物生長并降低作物的產(chǎn)量和品質(zhì)[2]。由于干旱導(dǎo)致細(xì)胞周期蛋白依賴性激酶活性降低、細(xì)胞分裂緩慢，因此缺水情況下植株的生長速率顯著降低，特別是苗期階段[3]。此外，干旱通常引起植株葉面積、葉伸展速率及數(shù)量的下降。干旱脅迫下，植物葉片通常表現(xiàn)出不同程度的萎蔫甚至變黃現(xiàn)象[4]，成熟植物能通過關(guān)閉葉片氣孔及老葉的加速衰老和脫落，減少葉片蒸騰作用，以降低其造成的水分損失[5]。葉片氣孔關(guān)閉的同時還降低了植物光合作用，使葉片中類胡蘿卜素含量、總?cè)~綠素含量明顯下降[6]。另外，干旱脅迫下，植物細(xì)胞因失水萎縮可導(dǎo)致細(xì)胞膜的機械損傷。脫水的細(xì)胞因體積減小，使得細(xì)胞內(nèi)溶物變粘稠，增加了蛋白質(zhì)間互作的可能性，致使其聚集和變性[7]。植物體內(nèi)活性氧 (ROS) 在干旱脅迫時的積累還可引起膜脂質(zhì)的過氧化，丙二醛 (MDA) 作為質(zhì)膜過氧化的最終產(chǎn)物，亦能造成細(xì)胞膜上蛋白質(zhì)等的失活，破壞生物膜的結(jié)構(gòu)及功能[4]。研究表明，干旱脅迫時植物抗氧化酶，如過氧化氫酶 (CAT)、過氧化物酶 (POD) 和超氧化物歧化酶 (SOD) 的活性以及滲透調(diào)節(jié)物質(zhì)，如脯氨酸、谷氨酸及可溶性糖等的含量均有所增加[4]。溶質(zhì)的積累在一定程度上降低了細(xì)胞的滲透勢，可以提高植物抵御干旱的能力。植物中次生代謝物的含量也隨著干旱脅迫程度發(fā)生變化，如小麥葉片中總黃酮的含量[8]及楊樹中酚類化合物的濃度等[9]都表現(xiàn)出增加趨勢。

1.2 鹽脅迫下植物生理生化的適應(yīng)性變化

鹽脅迫是造成幾乎所有陸生植物生長和產(chǎn)量下降的主要脅迫因素之一[10]。其中，Na鹽和Ca鹽對植物傷害較嚴(yán)重，尤其Na鹽的傷害更為普遍。鹽脅迫不僅影響植物生長發(fā)育、光合作用及呼吸作用等重要的代謝過程，且對植物體內(nèi)離子含量、酶活性、激素水平等均有影響。鹽脅迫的初始階段植物葉面積擴展速率降低，隨著鹽濃度的升高，葉面積減少，葉片相對含水量、水勢、蒸騰速率降低，且植物莖、根的鮮重及干重亦有不同程度的下降[11]。高鹽環(huán)境造成葉片氣孔因缺水而關(guān)閉，使葉綠體類囊體膜膨脹、基粒消失且出現(xiàn)巨型淀粉粒，葉綠體結(jié)構(gòu)遭破環(huán)。高鹽脅迫還可導(dǎo)致植物中與光合作用相關(guān)的酶變性失活，降低葉片光合反應(yīng)速率、影響同化產(chǎn)物合成[12]。通常，鹽脅迫初期植物的呼吸作用增強，以產(chǎn)生抵御脅迫所需的能量，但隨著脅迫強度的升高和時間的延長，植物呼吸強度呈現(xiàn)下降的趨勢[13]。鹽脅迫下的植物因經(jīng)常處于“生理干旱”狀態(tài)，使得體內(nèi)ROS過度積累，干擾植物正常代謝、破壞體細(xì)胞膜完整性并增加質(zhì)膜透性，導(dǎo)致植物體中抗氧化酶的活性隨外界鹽濃度的增加呈先上升后下降的趨勢[12]，無法持續(xù)維持較高水平，從而影響植物細(xì)胞的穩(wěn)態(tài)[14]。研究發(fā)現(xiàn)，鹽脅迫還能引起葉片硝酸還原酶活性 (NRA) 降低，造成含氮化合物代謝紊亂，影響植物總體新陳代謝水平[15]。另外，植物吸收礦質(zhì)元素時，環(huán)境中過多的鹽離子能與其相互競爭造成植物礦質(zhì)營養(yǎng)脅迫。多種植物的鹽脅迫實驗表明，隨處理中Na+和Cl-含量的增加，Ca2+、K+和Mg2+的含量呈降低趨勢，且高濃的Na+能嚴(yán)重阻礙植物對K+的吸收和運輸[16]。同時，高鹽能影響植物激素水平的分布，如脫落酸 (ABA) 能在滲透脅迫下積累，并參與修飾因鹽脅迫上調(diào)的基因的表達(dá)[17]。

1.3 低溫脅迫下植物生理生化的適應(yīng)性變化

低溫對植物的傷害分為冷害 (0～15℃) 和凍害 (＜0℃)。低溫脅迫下，植物葉片通常表現(xiàn)為萎縮、褪綠甚至壞死，且植物生長發(fā)育受到嚴(yán)重影響[5]。與干旱及鹽脅迫類似，低溫脅迫能減少植物對水分的吸收，破壞細(xì)胞膜完整性，使細(xì)胞喪失區(qū)室化[18]，并干擾植物的呼吸作用、光合作用等過程。光合作用對低溫較敏感[19]，其對光合作用的抑制主要體現(xiàn)在兩方面：一是，直接影響葉綠素合成及葉綠體結(jié)構(gòu)，低溫脅迫下，葉綠素a、b及類胡蘿卜素含量降低，葉綠體基質(zhì)、基粒片層松散，雙層膜完整性被破壞[20]；二是，通過減少葉片對CO2的吸收、阻礙光合產(chǎn)物運輸、引起水分脅迫等[19]生理過程，降低光合速率。另外，低溫脅迫下植物對氧的利用率降低，多余的氧在代謝過程中被轉(zhuǎn)化為ROS。但是，植物可通過調(diào)節(jié)酶促防御體系來限制并清除體內(nèi)過多的ROS，進(jìn)而避免或減輕低溫脅迫造成的傷害。此外，植物體中的可溶性蛋白、可溶性糖和游離脯氨酸等作為防凍劑和膜穩(wěn)定劑，能參與細(xì)胞滲透調(diào)解，從而提高植物低溫耐受性。

2 轉(zhuǎn)錄因子結(jié)構(gòu)功能及在非生物脅迫下的應(yīng)答調(diào)控

2.1 轉(zhuǎn)錄因子的結(jié)構(gòu)與功能特征

2.1.1 MYB類轉(zhuǎn)錄因子的結(jié)構(gòu)與功能特征 MYB家族轉(zhuǎn)錄因子存在于所有真核生物中，由于具有保守的MYB結(jié)構(gòu)域而得名。植物中MYB轉(zhuǎn)錄因子的共同特征是MYB結(jié)構(gòu)域通常由1～4個不完全重復(fù)的R結(jié)構(gòu)組成。根據(jù)R結(jié)構(gòu)的數(shù)目和位置，可將MYB家族分為4個亞家族[21]：1)4R-MYB(包含4個重復(fù)的R1/R2)，是最小的MYB亞家族。目前對該亞族成員的功能了解甚少，所發(fā)現(xiàn)的4R-MYB蛋白僅能在極少數(shù)植物 (擬南芥、葡萄和楊樹) 中進(jìn)行編碼。2)3R-MYB(R1R2R3-MYB)，在植物中其家族成員較少。擬南芥和水稻中僅5個左右基因編碼3RMYB蛋白。3)2R-MYB(R2R3-MYB)，是植物中最大的一類MYB亞家族。水稻中有近90個2R-MYB亞家族成員，擬南芥中也有120個以上。2R-MYB蛋白能參與細(xì)胞分化、代謝調(diào)節(jié)、激素應(yīng)答以及植物對逆境脅迫的響應(yīng)等過程。4)R-MYB(MYB-related)，是植物中第二大類MYB亞家族，它們既能調(diào)節(jié)植物生長發(fā)育也參與植物對逆境脅迫的響應(yīng)[21–22]。

2.1.2 bZIP類轉(zhuǎn)錄因子的結(jié)構(gòu)與功能特征 bZIP是一類廣泛分布于真核生物中以自身結(jié)構(gòu)域命名的轉(zhuǎn)錄因子。bZIP結(jié)構(gòu)域由堿性氨基酸域和亮氨酸拉鏈域構(gòu)成。植物bZIP蛋白含約60～80個氨基酸，其N端包含由20個左右氨基酸殘基構(gòu)成的堿性結(jié)構(gòu)域，該區(qū)域通過固定的N-x7-R/K-x9結(jié)構(gòu)直接結(jié)合DNA，除決定DNA結(jié)合的特異性外，還起到核定位信號的作用[23]。另外，其N末端還具有酸性激活區(qū)，能以二聚體的形式結(jié)合DNA。bZIP轉(zhuǎn)錄因子的堿性結(jié)構(gòu)域與亮氨酸拉鏈域緊密結(jié)合，亮氨酸拉鏈區(qū)域位于bZIP結(jié)構(gòu)域C端，由7個或9個氨基酸組成一個重復(fù)單位，每個重復(fù)單位的第7(9) 位含有一個亮氨酸，此位置的亮氨酸也可被疏水性氨基酸代替。這種重復(fù)單位能形成兩親性α螺旋結(jié)構(gòu)，該螺旋疏水側(cè)的亮氨酸殘基通過范德華力相互作用疊加成卷曲線圈，即亮氨酸拉鏈[24]。α螺旋結(jié)構(gòu)參與bZIP蛋白與DNA結(jié)合前的二聚化，也能影響蛋白質(zhì)二聚體的形成。植物bZIP類轉(zhuǎn)錄因子優(yōu)先結(jié)合到具有ACGT核心序列的順式作用元件，如G-box(CACGTG)、C-box(GACGTC)、A-box(ACGTA) 等[24]。

2.1.3 AP2/EREBP類轉(zhuǎn)錄因子的結(jié)構(gòu)與功能特征AP2/EREBP類轉(zhuǎn)錄因子是植物特有的轉(zhuǎn)錄因子超家族。該家族成員具有約60個氨基酸殘基組成的保守AP2/ERF結(jié)構(gòu)域，該結(jié)構(gòu)域能特異性結(jié)合DRE(dehydration-responsive element)/C-repeat順式元件或GCC-box順式元件。這兩個順式元件的共同核心序列為CCGNC，分別存在于受乙烯和脫水脅迫誘導(dǎo)表達(dá)的目標(biāo)基因啟動子序列中[25–26]。AP2/EREBP超家族可分為AP2、ERF(ethylene-responsive factor) 和DREB(dehydration-responsive element-binding protein)等亞家族，ERF和DREB又可統(tǒng)稱為EREBP家族。AP2亞族具有2個AP2結(jié)構(gòu)域，可負(fù)責(zé)調(diào)控花、胚珠和種子的發(fā)育；EREBP亞族具有1個AP2結(jié)構(gòu)域，主要參與植物生物和非生物脅迫應(yīng)答反應(yīng)[27]。

2.1.4 WRKY類轉(zhuǎn)錄因子的結(jié)構(gòu)與功能特征 WRKY轉(zhuǎn)錄因子是植物轉(zhuǎn)錄因子家族中較大的一類，也是最具特點的一類。WRKY家族成員因均含有與DNA結(jié)合的WRKY結(jié)構(gòu)域而得名，該結(jié)構(gòu)域由60個左右高度保守的氨基酸序列組成，在其N端具有WRKYGQK核心序列，C端包含一個鋅指結(jié)構(gòu)域C2H2(Cx4-5Cx22-23HxH) 和 C2HC(Cx7Cx23HxC)[28–29]。根據(jù)WRKY結(jié)構(gòu)域的數(shù)量和鋅指結(jié)構(gòu)域的類型，WRKY轉(zhuǎn)錄因子可分成3個亞家族：亞家族Ⅰ具有兩個WRKY結(jié)構(gòu)域和一個C2H2型鋅指結(jié)構(gòu)；亞家族Ⅱ具有一個WRKY結(jié)構(gòu)域和一個C2H2型鋅指結(jié)構(gòu)；亞家族Ⅲ具有一個WRKY結(jié)構(gòu)域和一個C2HC型鋅指結(jié)構(gòu)。受WRKY蛋白調(diào)控的基因啟動子區(qū)中的TTGACT/C保守片段為W-box。W-box是目標(biāo)基因與WRKY轉(zhuǎn)錄因子特異性結(jié)合的區(qū)域，目標(biāo)基因與WRKY轉(zhuǎn)錄因子中的WRKY結(jié)構(gòu)域和鋅指結(jié)構(gòu)域?qū)崿F(xiàn)特異性結(jié)合，致使WRKY轉(zhuǎn)錄因子在植物響應(yīng)非生物脅迫的過程中發(fā)揮重要作用[30]。

2.1.5 NAC類轉(zhuǎn)錄因子的結(jié)構(gòu)與功能特征 NAC轉(zhuǎn)錄因子是植物中特有的一類轉(zhuǎn)錄因子。因在矮牽牛NAM、擬南芥ATAF1/ATAF2和CUC2基因編碼蛋白的N端，均發(fā)現(xiàn)一段高度保守的氨基酸序列，故以這三類基因的首字母將其命名為NAC結(jié)構(gòu)域，包含NAC結(jié)構(gòu)域的蛋白即NAC轉(zhuǎn)錄因子。NAC結(jié)構(gòu)域一般由150～160個氨基酸組成，并分為5個亞結(jié)構(gòu)域[31]。NAC蛋白單體可通過谷氨酸和精氨酸間的氫鍵或鹽橋形成二聚體，二聚體所帶的正電荷可與DNA結(jié)合[32]。NAC轉(zhuǎn)錄因子的C端為氨基酸序列具有高度多樣性的轉(zhuǎn)錄調(diào)控區(qū)，能激活轉(zhuǎn)錄也能抑制轉(zhuǎn)錄。由于轉(zhuǎn)錄調(diào)控區(qū)的結(jié)構(gòu)具有不穩(wěn)定性，使得NAC蛋白能夠與其他目標(biāo)蛋白互作[33]，從而使NAC轉(zhuǎn)錄因子在植物應(yīng)答非生物脅迫過程中起到重要的調(diào)控作用。

2.2 轉(zhuǎn)錄因子對非生物脅迫的應(yīng)答

2.2.1 轉(zhuǎn)錄因子對干旱脅迫的應(yīng)答 許多MYB家族成員均能參與植物對干旱脅迫的應(yīng)答反應(yīng)。Liao等[34]從前人研究的大豆基因中獲得了156個MYB家族基因，利用酵母單雜交的方法篩選出了40個左右與逆境相關(guān)的基因，將這些基因轉(zhuǎn)入擬南芥進(jìn)行功能驗證，發(fā)現(xiàn)轉(zhuǎn)GmMYB177提高了擬南芥耐旱性。丁震乾等[35]在陸地棉中克隆了MYB轉(zhuǎn)錄因子基因GhRAX3，發(fā)現(xiàn)其表達(dá)在干旱脅迫0.5 h后即表現(xiàn)為顯著上調(diào)，且48 h內(nèi)持續(xù)高表達(dá)水平，而抑制GhRAX3的表達(dá)，加快了棉花植株的失水率、細(xì)胞質(zhì)膜過氧化和細(xì)胞受損程度，降低了棉花對干旱脅迫的耐受性。目前發(fā)現(xiàn)的大多MYB類轉(zhuǎn)錄因子基因均表現(xiàn)為提高植物抗旱能力，但亦有例外，如Zhou等的研究指出，將JcMYB001轉(zhuǎn)入擬南芥后，其過表達(dá)反而增加了擬南芥對干旱的敏感性[36]。水稻和白松樹中過表達(dá)的AtbZIP60基因可以通過調(diào)節(jié)相關(guān)Ca2+-依賴性蛋白激酶基因來提高植物對干旱脅迫的耐受性[37]。玉米ZmbZIP72在擬南芥中的過表達(dá)提高了轉(zhuǎn)基因株系抵御干旱的能力[38]。另外，Tu等發(fā)現(xiàn)葡萄VlbZIP36基因在擬南芥中的過表達(dá)能減輕干旱脅迫下的細(xì)胞損傷和水分流失，使其在種子萌發(fā)、幼苗和成熟階段都顯示出較強的耐脫水性[39]。干旱脅迫下DREB基因的過表達(dá)使得轉(zhuǎn)基因小麥葉片仍保持綠色而野生型葉片失綠，恢復(fù)澆水10天后，轉(zhuǎn)基因株系存活率顯著提高，說明DREB轉(zhuǎn)錄因子能有效提高小麥耐旱性[40]。Chen等報道GmDREB2受干旱脅迫誘導(dǎo)，可激活轉(zhuǎn)基因擬南芥中下游基因的表達(dá)，提高轉(zhuǎn)基因株系對干旱的耐受性[41]。Jiang等發(fā)現(xiàn)番茄SlDREB1基因可受干旱的強烈誘導(dǎo)，將其轉(zhuǎn)入擬南芥后，轉(zhuǎn)基因擬南芥耐旱能力明顯提高[26]。Yang等指出干旱脅迫可持續(xù)誘導(dǎo)柑橘CitERF的表達(dá)進(jìn)而增加其抗旱能力[42]。Wang等報道了10個WRKY基因，發(fā)現(xiàn)將TaWRKY44轉(zhuǎn)入煙草后其脯氨酸含量、可溶性糖含量、抗氧化酶活性均高于野生型，且MDA和H2O2含量顯著降低，說明TaWRKY44的表達(dá)增強了轉(zhuǎn)基因煙草對干旱的耐受性[43]。Niu等指出小麥TaWRKY2蛋白和TaWRKY19蛋白可通過與基因啟動子的直接結(jié)合或間接機制來調(diào)節(jié)下游基因，進(jìn)而提高小麥抗旱能力，且轉(zhuǎn)TaWRKY2和TaWRKY19基因的擬南芥表現(xiàn)出較強的耐旱性[44]。玉米的ZmNAC55基因受干旱脅迫誘導(dǎo)，且ZmNAC111基因在玉米中的過表達(dá)能提高轉(zhuǎn)基因植株的水分利用率，使干旱應(yīng)答基因的表達(dá)上調(diào)，從而提高植物耐旱性[45–46]。Zhao等報道了MlNAC9過表達(dá)的轉(zhuǎn)基因擬南芥中抗氧化酶活性提高、MDA含量顯著降低，且干旱脅迫應(yīng)答基因的表達(dá)在MlNAC9過表達(dá)株系中顯著增加，說明MlNAC9過表達(dá)提高了擬南芥對干旱的耐受性[47]。

2.2.2 轉(zhuǎn)錄因子對鹽脅迫的應(yīng)答 研究發(fā)現(xiàn)，AtMYB-41、AtMYB20、GmMYB76、GmMYB92、GmMYB177等MYB類轉(zhuǎn)錄因子基因均受鹽脅迫誘導(dǎo)[21,34]，且轉(zhuǎn)入GmMYB76和GmMYB177的轉(zhuǎn)基因擬南芥在高鹽處理下的存活率顯著高于野生型[34]。Chen等報道，鹽脅迫下花生中4個R2R3-MYB基因，AhMYB1、AhMYB2、AhMYB6和AhMYB7的表達(dá)顯著上升；4個MYB-related基因，AhMYB12、AhMYB18、AhMYB28和AhMYB30的mRNA豐度增加[48]。這說明花生中至少有8個MYB基因受到鹽脅迫的誘導(dǎo)。Liu等指出，擬南芥AtbZIP17在鹽脅迫下轉(zhuǎn)錄水平增加，并通過蛋白水解作用使其N端進(jìn)入核內(nèi)發(fā)揮轉(zhuǎn)錄激活作用，誘導(dǎo)下游逆境響應(yīng)基因AtRD29A和AtRD20的表達(dá)，從而提高植株抗鹽性[49]。Xiang等的研究表明，水稻OsbZIP23過表達(dá)的轉(zhuǎn)基因水稻耐鹽性顯著提高，敲除該基因的突變體植株對鹽耐受性明顯降低，而將OsbZIP23轉(zhuǎn)化到突變體后，其抗鹽性得到恢復(fù)，說明OsbZIP23在植物響應(yīng)鹽脅迫中發(fā)揮重要作用[50]。大豆GmDREB2基因在轉(zhuǎn)基因擬南芥中的過表達(dá)提高了轉(zhuǎn)基因株系對高鹽脅迫的抵御能力，且不影響擬南芥的生長周期[41]。水稻OsDREB1F基因、OsDREB2A基因均受高鹽脅迫誘導(dǎo)，二者的轉(zhuǎn)基因株系都表現(xiàn)出較強抗鹽能力[51–52]，且水稻OsDREB1A在擬南芥中的過表達(dá)誘導(dǎo)了擬南芥DREB1A基因的表達(dá)，從而提高了植株耐鹽性[53]。Yao等發(fā)現(xiàn)楊樹ERF76基因在鹽脅迫下表達(dá)上調(diào)，轉(zhuǎn)ERF76基因煙草的種子發(fā)芽率、株高、根長、鮮重以及脯氨酸含量、SOD活性等生理生化指標(biāo)均高于野生型，說明楊樹ERF76在轉(zhuǎn)基因煙草的抗鹽性中起關(guān)鍵的調(diào)控作用[54]。鹽脅迫下，小麥TaWRKY44的表達(dá)水平顯著上升，且轉(zhuǎn)TaWRKY44基因的煙草提高了對鹽脅迫的耐受性[43]。Qin等認(rèn)為小麥TaWRKY93為鹽誘導(dǎo)轉(zhuǎn)錄因子基因，TaWRKY93過表達(dá)的轉(zhuǎn)基因擬南芥主根及側(cè)根長度、脯氨酸含量、相對含水量、存活率等在鹽脅迫環(huán)境下明顯提高[55]。Zhou等指出，與野生型相比，在鹽脅迫下陸地棉GhWRKY34在擬南芥中的過表達(dá)使其有較高的發(fā)芽率、根長和葉綠素含量，證明轉(zhuǎn)GhWRKY34擬南芥的耐鹽性更強[56]；而Jia等則報道陸地棉GhWRKY68基因降低了轉(zhuǎn)基因煙草的抗鹽能力[57]，這說明WRKY轉(zhuǎn)錄因子在植物應(yīng)對鹽脅迫中既能發(fā)揮正調(diào)控功能也能發(fā)揮負(fù)調(diào)控功能。Mohammed等[58]研究發(fā)現(xiàn)，鹽脅迫下硬粒小麥的TtNAC-B60、TtNAC-A7、TtNAC-B35、TtNAC-B27和TtNAC-A51均受鹽脅迫誘導(dǎo)并表現(xiàn)出上調(diào)趨勢，小麥中的TaNAC29基因也參與了植物對鹽脅迫的響應(yīng)與信號通路，并能增強植株抗氧化物酶活性、減少H2O2積累和膜損傷[59]。Zhao等[47]發(fā)現(xiàn)MlNAC9過表達(dá)的轉(zhuǎn)基因擬南芥能通過ABA依賴途徑提高抵御鹽脅迫的能力。而Wei等[60]對甜瓜NAC轉(zhuǎn)錄因子家族全基因組的研究則表明，雖然鹽脅迫下的12h內(nèi)CmNAC14的表達(dá)持續(xù)增加，但CmNAC14過表達(dá)的轉(zhuǎn)基因擬南芥卻增加了對鹽脅迫的敏感性。

2.2.3 轉(zhuǎn)錄因子對低溫脅迫的應(yīng)答 Gopal等鑒定了大白菜中475個MYB基因，發(fā)現(xiàn)與正常條件相比，低溫脅迫下R2R3-MYB亞家族成員：BrMYB2、BrMYB13、BrMYB77、BrMYB81、BrMYB88、BrMYB121、BrMYB166、BrMYB169和 BrMYB217的表達(dá)成倍上調(diào)；M Y B-r e l a t e d亞家族成員：BrMYB1R25、BrMYB1R44、BrMYB1R70、BrMYB1R77、BrMYB1R171和BrMYB1R178也表現(xiàn)出上調(diào)[61]。研究發(fā)現(xiàn)，水稻的OsMYB30也受低溫脅迫誘導(dǎo)，但OsMYB30在水稻中的過表達(dá)降低了水稻的低溫耐受性，而OsMYB30敲除突變體水稻反而具有較好的抵御低溫脅迫的能力[62]。Hwang等通過對蕪菁bZIP轉(zhuǎn)錄因子的研究指出，Bra000256、Bra003320、Bra004689、Bra011648、Bra020735 和Bra023540在低溫脅迫下轉(zhuǎn)錄水平顯著上調(diào)，它們編碼的蛋白質(zhì)可能參與植株對冷脅迫的響應(yīng)[63]。甘藍(lán)bZIP轉(zhuǎn)錄因子基因Bol008071、Bol033132和Bol042729受低溫脅迫誘導(dǎo)而升高，表明其可能在植物響應(yīng)低溫脅迫時發(fā)揮重要作用[64]。Lee等分析了馬鈴薯中乙烯應(yīng)答元件組合蛋白，發(fā)現(xiàn)冷誘導(dǎo)的StEREBP1基因過表達(dá)可誘導(dǎo)含有GCC-box順式元件的基因表達(dá)，參與逆境響應(yīng)[25]，說明StEREBP1通過對GCC-box順式元件的轉(zhuǎn)錄調(diào)節(jié)，在逆境脅迫中發(fā)揮作用。

Niu等發(fā)現(xiàn)黃花苜蓿MfDREB1和MfDREB1s基因在低溫處理1 h后的表達(dá)水平均上調(diào)且6 h時達(dá)到峰值[65]，推測二者是黃花苜蓿抵御低溫的重要影響因子。小麥TaWRKY19能激活Cor6.6 (cold-regulated gene) 的表達(dá)并能結(jié)合其啟動子，使轉(zhuǎn)TaWRKY19的擬南芥表現(xiàn)出較好的低溫耐受力[44]。水稻OsWRKY71可以通過調(diào)節(jié)下游靶基因增強轉(zhuǎn)基因植株的耐寒性[66]。Wang等研究了葡萄基因組中59個VvWRKYs，發(fā)現(xiàn)有22個受冷脅迫誘導(dǎo)上調(diào)且VvWRKY55的漲幅最大[67]。黃瓜中CsWRKY46基因在寒冷脅迫下的表達(dá)上調(diào)，且其過表達(dá)的轉(zhuǎn)基因擬南芥在低溫脅迫下的幼苗存活率明顯提高[68]。番茄SlNAC1過表達(dá)可以減少ROS積累并使CBF1的表達(dá)上調(diào)，從而提高植株對低溫的適應(yīng)能力[69]，且Li等發(fā)現(xiàn)番茄中新型轉(zhuǎn)錄因子基因SlNAM1過表達(dá)的轉(zhuǎn)基因煙草能減輕細(xì)胞膜在寒冷環(huán)境下的氧化損傷[70]。Zhao等將MlNAC9轉(zhuǎn)入擬南芥后，發(fā)現(xiàn)轉(zhuǎn)基因株系中低溫脅迫應(yīng)答基因的表達(dá)和ROS的清除能力增強[47]。

部分轉(zhuǎn)錄因子在干旱、高鹽和低溫脅迫下的應(yīng)答調(diào)控見表1。

3 參與植物逆境調(diào)控的轉(zhuǎn)錄因子分子應(yīng)答機制

植物抗逆能力的提高與轉(zhuǎn)錄因子調(diào)控逆境相關(guān)的功能基因表達(dá)密不可分?；蛲ㄟ^不同信號途徑進(jìn)行特異性表達(dá)是不同基因啟動子中順式作用元件存在差異的結(jié)果，這些元件與相應(yīng)的轉(zhuǎn)錄因子結(jié)合從而激活或改變轉(zhuǎn)錄效率。已發(fā)現(xiàn)的一系列順式作用元件包括：GCC-box、G-box、C-box、W-box、DRE、MRE、ABRE、ERE等。不同轉(zhuǎn)錄因子中保守的DNA結(jié)合域能與下游基因的順式作用元件結(jié)合并相互作用，以實現(xiàn)對基因表達(dá)的調(diào)控，其轉(zhuǎn)錄調(diào)控域可同其他轉(zhuǎn)錄因子或DNA作用以提高或抑制基因表達(dá)調(diào)控的效率和靈活性。

ABA作為逆境脅迫響應(yīng)的內(nèi)源激素，在植物應(yīng)對干旱、鹽等脅迫中發(fā)揮重要作用[71]。植物響應(yīng)干旱脅迫的信號轉(zhuǎn)導(dǎo)途徑包括ABA依賴途徑和非ABA依賴途徑。ABA依賴途徑中，許多ABA誘導(dǎo)基因的啟動子區(qū)都含有ABRE(ABA-responsive cis-acting elements) 順式作用元件，如小麥Em、擬南芥RD29B的啟動子區(qū)等[72]。bZIP轉(zhuǎn)錄因子中的ABF(ABA-responsive element binding factors)/AREB(ABA responsive element binding protein) 家族，能與ABA依賴基因的ABRE元件結(jié)合并激活這些基因的表達(dá)。另外，MYB等轉(zhuǎn)錄因子中的成員，也能受ABA誘導(dǎo)表達(dá)。非ABA依賴途徑中，在基因的啟動子區(qū)含有DRE等其他核心元件，其作為蛋白識別位點受到相應(yīng)因子的誘導(dǎo)。如AP2/EREBP類轉(zhuǎn)錄因子中的一個亞家族就能與DRE序列相結(jié)合，進(jìn)而誘導(dǎo)相關(guān)基因的表達(dá)。植物對鹽脅迫的響應(yīng)機制主要通過Ca2+信號轉(zhuǎn)導(dǎo)和SOS(salt overly sensitive) 信號途徑。當(dāng)植物遭受鹽脅迫時，經(jīng)Ca2+信號轉(zhuǎn)導(dǎo)使SOS信號途徑發(fā)揮作用，使其結(jié)合細(xì)胞質(zhì)內(nèi)過多的Ca2+，并通過離子轉(zhuǎn)運功能在限制Na+進(jìn)入細(xì)胞的同時，促進(jìn)胞內(nèi)過多的Na+外排[73]。植物對低溫脅迫的抵御主要依靠信號轉(zhuǎn)導(dǎo)途徑來實現(xiàn)對低溫誘導(dǎo)基因的表達(dá)調(diào)控。當(dāng)植物受低溫脅迫時，ICE-CBFCOR轉(zhuǎn)錄級聯(lián)反應(yīng)在感知上游低溫信號和調(diào)控下游特異基因的表達(dá)中發(fā)揮重要作用[74]。根據(jù)CBF是否參與轉(zhuǎn)錄，此調(diào)控機制可分為CBF依賴途徑和非CBF依賴途徑。CBF依賴途徑中，低溫信號誘導(dǎo)激酶使ICE(inducer of CBF expression) 磷酸化，且與CBF(C-repeat Binding Factor) 啟動子順式作用元件結(jié)合，激活CBF基因的表達(dá)。有活性的CBF作為反式作用因子進(jìn)而與COR(cold responsive) 的順式作用元件結(jié)合誘導(dǎo)COR基因的表達(dá)，增強植物對低溫的耐受性[75]。一些低溫響應(yīng)基因，如擬南芥同源結(jié)構(gòu)域轉(zhuǎn)錄因子基因HOS9[76]、水稻OsMYB3R-2[77]基因不受CBF轉(zhuǎn)錄因子調(diào)控，而是通過非CBF依賴途徑來提高植株的抗低溫能力。

總的來說，植物受到外界環(huán)境 (干旱、高鹽、低溫等) 刺激后，會通過一系列抗逆脅迫信號的傳導(dǎo)激活轉(zhuǎn)錄因子，使其與下游靶基因啟動子區(qū)的相應(yīng)順式作用元件結(jié)合，調(diào)控靶基因表達(dá)，并通過基因產(chǎn)物的作用對內(nèi)、外界信號作出調(diào)節(jié)，進(jìn)而誘導(dǎo)相關(guān)功能基因的高表達(dá)，形成相應(yīng)的基因產(chǎn)物以應(yīng)答脅迫信號。

4 展望

干旱、土壤鹽漬化及低溫脅迫嚴(yán)重影響農(nóng)作物生產(chǎn)，而植物對干旱、高鹽和低溫的耐受性強弱通常受許多因子影響。轉(zhuǎn)錄因子作為調(diào)控基因表達(dá)的關(guān)鍵因素在植物抵抗非生物逆境脅迫方面發(fā)揮重要作用。一個轉(zhuǎn)錄因子能夠調(diào)控多個與同類性狀有關(guān)的基因表達(dá)，其通過增強某些關(guān)鍵調(diào)節(jié)因子的作用，促進(jìn)抗逆基因資源發(fā)揮其功能，從根本上使植物的抗逆性得到改良。雖然現(xiàn)有研究已報道了許多與抗逆境相關(guān)的不同轉(zhuǎn)錄因子家族成員，但仍有大量轉(zhuǎn)錄因子家族成員的相關(guān)功能未得到驗證，且目前對轉(zhuǎn)錄因子的研究側(cè)重于單一逆境條件的調(diào)控，

對其在不同信號途徑相互作用的調(diào)控機制尚不十分明確。

表 1 干旱、高鹽及低溫脅迫下部分轉(zhuǎn)錄因子的應(yīng)答調(diào)控Table 1 Response of some transcription factors in drought, high-salt and low temperature stresses

另外，目前尚未見干旱、高鹽及低溫脅迫下轉(zhuǎn)錄因子直接參與調(diào)控植物養(yǎng)分方面的報道。對于植物吸收氮、磷、鉀等營養(yǎng)元素的分子機制研究，主要集中于轉(zhuǎn)運蛋白及其相關(guān)基因的調(diào)節(jié)。如Duan等研究了氮充足和不施氮條件下，冬小麥根系中硝酸鹽轉(zhuǎn)運蛋白 (NRT) 基因TaNRT2.1、TaNRT2.2、TaNRT2.3、TaNRT1.1和TaNRT1.2和氨轉(zhuǎn)運蛋白(AMT)基因TaAMT1.1、TaAMT1.2、TaAMT2.1對土壤干旱脅迫的響應(yīng)，發(fā)現(xiàn)氮充足情況下冬小麥對干旱脅迫更敏感，因為氮低效小麥品種中低親和力的TaNRT1.1和TaNRT1.2基因表達(dá)受干旱脅迫誘導(dǎo)，而它們在氮高效小麥品種中的表達(dá)則受抑制，且干旱脅迫下，無論氮充足與否，高親和力的TaNRT2.1基因表達(dá)均受抑制。另外TaAMT1.1和TaAMT2.1的表達(dá)也受干旱脅迫抑制[78]。Shen等[79]指出，高鹽脅迫能顯著誘導(dǎo)水稻高親和力鉀轉(zhuǎn)運蛋白基因OsHAK21的表達(dá)。與野生型相比，OsHAK21功能遭破壞的水稻突變體對鹽脅迫敏感，其芽和根中的K+積累較少且K+凈吸收率顯著降低，而Na+凈吸收率增加。OsHAK21在K+攝取缺陷型酵母和擬南芥中的功能表征進(jìn)一步證明了OsHAK21具有K+轉(zhuǎn)運蛋白活性。說明OsHAK21在鹽脅迫下對維持水稻體內(nèi)Na+/K+動態(tài)平衡起重要作用[79]。對于磷轉(zhuǎn)運蛋白而言，Song等提出水稻磷轉(zhuǎn)運蛋白基因OsPT8在煙草中的過表達(dá)能提高轉(zhuǎn)基因煙草對磷和硒的吸收，且增強了轉(zhuǎn)基因煙草對低磷脅迫的耐受性[80]。董旭等對植物磷轉(zhuǎn)運子PHT1家族的表達(dá)模式和功能等進(jìn)行了總結(jié)，發(fā)現(xiàn)在擬南芥、水稻、大豆及茄科等植物中，多數(shù)PHT1家族成員受低磷信號調(diào)控且主要在根部表達(dá)，并行使相應(yīng)的磷轉(zhuǎn)運功能[81]。

在未來的研究中，應(yīng)基于全基因組層面的科學(xué)分析，進(jìn)一步發(fā)掘轉(zhuǎn)錄因子家族成員的相關(guān)功能，研究轉(zhuǎn)錄因子在植物多種信號途徑相互作用中的調(diào)控機制，這對深入理解植物對干旱等非生物逆境應(yīng)答及轉(zhuǎn)錄因子的調(diào)控過程具有科學(xué)價值，同時對逆境脅迫作物分子改良提供理論與材料支撐具有現(xiàn)實意義。

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Physiological responses and regulatory pathways of transcription factors in plants under drought, high-salt, and low temperature stresses

WANG Bing, CHENG Xian-guo*
( Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences/Key Laboratory of Plant Nutrition and Fertilizer, Ministry of Agriculture, Beijing 100081, China )

Drought, high-salinity and low-temperature, three major environmental stresses, are important adverse factors limiting plant growth and development. These environment stressful factors usually trigger the physiological changes and the differential expressions of the transcription factors, which are involved in the biological regulations in the plants under abiotic stresses. While the functional gene expression are closely associated with these transcription factors, and are essential for an enhanced tolerance ability of plants to the environmental stresses. In recent years, most studies were mainly focused on discovering functional genes and the regulatory mechanisms of the transcription factors in plants in response to the environmental stresses. Therefore,uncovering on the physiological responses and the regulatory mechanisms of the transcription factors in plants under these environmental stresses has an important scientific significance in establishing molecular acclimation pathways of plants. This paper systemically characterized the physiological-biochemical changes in plants under environmental stresses including drought, high salinity, and low temperature, and profiled the structural andfunctional characteristics of MYB, bZIP, AP2/EREBP, WRKY and NAC, which commonly were related to plant stress resistance. Meanwhile, we separately characterized the responsive models and molecular regulatory mechanism of these transcription factors in response to these three environmental stresses. Under these environmental stresses, the growth rate, leaf area, leaf number, transpiration rate, and photosynthesis of plants were usually reduced, and the accumulation of reactive oxygen in plants led to the enhancement of membrane lipid peroxidation, thus resulting in severe damages of the plant cells. When the plants were exposed to these adverse environmental stresses, the antioxidant enzymatic activity and the contents of osmolytes in plants were naturally increased to establish adaptive acclimation mechanisms, which were usually exhibited at the physiological and molecular levels. The enhancement magnitude of the plant adaptability to the abiotic stresses are mainly regulated through the pathways of the interactions between the transcription factors and the specific recognition elements in the promoters of the stress-resistant genes or in the target proteins. This review provides a complete insight into the physiological-biochemical responses and molecular regulatory mechanisms of the transcription factors in plants under drought, high salinity, and low temperature stresses.

drought stress; salt stress; low temperature stress; physiological response; transcription factors

2017–08–08 接受日期：2017–10–15

國家重點研發(fā)項目（2016YFC0501203）；973項目（2015CB150800）資助。

王冰（1991—），女，北京人，博士研究生，主要從事植物逆境生理與分子應(yīng)答研究。E-mail：wangbingyajd@126.com

* 通信作者 程憲國（1962—），遼寧大連人，博士，研究員，主要從事植物逆境生理與分子應(yīng)答研究。E-mail：chengxianguo@caas.cn