陳 鑫, 馬 超, 楊永娟, 王紅玲, 黃 盈, 張曉霞, 黃開封, 趙 卓, 張素芝**

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轉(zhuǎn)堿蓬基因煙草的耐鹽抗旱性研究*

陳 鑫1, 馬 超1, 楊永娟2, 王紅玲1, 黃 盈1, 張曉霞1, 黃開封1, 趙 卓1, 張素芝1**

(1. 四川農(nóng)業(yè)大學(xué)玉米研究所/農(nóng)業(yè)部玉米生物與遺傳改良重點實驗室 成都 611130; 2. 沂南縣銅井鎮(zhèn)農(nóng)業(yè)綜合服務(wù)中心 沂南 276300)

煙草是重要的模式植物和經(jīng)濟(jì)作物, 鹽害和干旱兩種環(huán)境因子對其生長發(fā)育、產(chǎn)量和品質(zhì)都危害很大。為了提高煙草的耐鹽抗旱性, 本研究利用農(nóng)桿菌介導(dǎo)的遺傳轉(zhuǎn)化法在煙草中過量表達(dá)了堿蓬液泡膜Na+/H+逆向轉(zhuǎn)運基因, 對轉(zhuǎn)基因煙草的耐鹽及抗旱性進(jìn)行表型鑒定和各項生化指標(biāo)的檢測, 以期得到耐鹽抗旱表性良好的轉(zhuǎn)基因煙草。表型分析發(fā)現(xiàn),基因過表達(dá)株系L1和L5的抗鹽能力比野生型顯著提高, 表現(xiàn)為鹽脅迫條件下仍能保持旺盛的生長且根系的伸長未受抑制。過表達(dá)株系在葉片和根系中積累了更多的Na+和K+, 同時Na+含量增長速率較快, 而K+含量降低速率較緩, 并可維持較高的葉片相對含水量和葉綠素含量, 及較低的丙二醛含量和相對電導(dǎo)率。干旱脅迫發(fā)現(xiàn), 過表達(dá)株系受干旱脅迫程度更小, 并在復(fù)水后迅速恢復(fù)正常生長。同時, 過表達(dá)株系的丙二醛含量和相對電導(dǎo)率顯著低于野生型, 且維持了較高的葉片相對含水量及葉綠素含量。這些結(jié)果說明基因在煙草中過量表達(dá)后, 降低了鹽脅迫和干旱脅迫對煙草根系及細(xì)胞膜的損傷, 并通過調(diào)節(jié)離子含量、降低細(xì)胞的滲透勢, 維持了葉片較高的相對含水量和葉綠素含量, 最終提高了煙草的抗鹽和抗旱性。

基因; 轉(zhuǎn)基因; 煙草; 耐鹽性; 抗旱性; 表型; 生化指標(biāo)

煙草(L.)是重要的模式植物和經(jīng)濟(jì)植物, 鹽害和干旱兩種環(huán)境因子對其生長發(fā)育、產(chǎn)量和品質(zhì)都危害很大。通過基因工程手段可以改善植物對這兩種脅迫的抗逆性。

植物耐鹽抗旱機制有很多種, 包括滲透調(diào)節(jié)和有害離子區(qū)室化等。Na+區(qū)室化是一個緩解植物體內(nèi)因滲透勢引起的鹽脅迫危害的關(guān)鍵方法[1]。Na+/H+逆向轉(zhuǎn)運蛋白(Na+/H+exchange, NHX)是Na+外排和區(qū)室化的主要載體蛋白[2], 通過Na+螯合作用作為鹽耐受性的重要決定因素[3-5], 也有其他方式可以調(diào)節(jié)如反轉(zhuǎn)錄酶[6-7]。液泡NHX的主要作用是維持膜系統(tǒng)的pH值, 控制K+和Na+體內(nèi)平衡, 并調(diào)節(jié)葉片的發(fā)育。擬南芥(L.)的基因是典型的液泡NHX;的過表達(dá)使酵母突變體具有耐鹽性[8], 并導(dǎo)致液泡NHX的活性增加, 兩者結(jié)合使NHX蛋白水平表達(dá)提高[9]。通過液泡膜[9-10]將Na+區(qū)室化到液泡中, 并在蛋白重構(gòu)脂質(zhì)體中催化Na+/H+的逆向轉(zhuǎn)運[11]。在鹽脅迫下, 液泡中Na+濃度隨著表達(dá)量的增加而持續(xù)增加, 說明植物液泡NHX有區(qū)室化Na+的作用, 這為研究NHX類蛋白的功能和應(yīng)用其改善植物的耐鹽性提供了思路。在擬南芥[12]、玉米(L.)[13]、歐洲油菜(L.)[14]、棉花(L.)[15]、番茄(Mill.)[16]、水稻(L.)[17]、煙草[18]楊樹(spp.)[19]等植物中的過表達(dá)都增加了這些植物的耐鹽性。鹽生植物堿蓬(L.)的基因編碼對Na+/H+逆向轉(zhuǎn)運蛋白具有很高的活性, 在鹽脅迫下的表達(dá)會提高, 且在根系中比在葉片的表達(dá)量更高[19]。與野生型相比,基因過表達(dá)的轉(zhuǎn)基因水稻耐鹽性更強, 表明在堿蓬的鹽耐受性中起了重要作用[20]。轉(zhuǎn)基因擬南芥在高鹽處理條件下能保持正常的發(fā)芽率且能正常生長并且完成其整個生命周期, 而野生型的擬南芥則生長和發(fā)育遲緩。

煙草是我國的一大經(jīng)濟(jì)作物, 但是長期以來煙草生產(chǎn)嚴(yán)重受干旱和鹽害的影響。由于基因轉(zhuǎn)化擬南芥和水稻后具有明顯的耐鹽作用, 因此本試驗將轉(zhuǎn)化煙草, 并對其轉(zhuǎn)基因煙草的耐鹽及干旱性進(jìn)行表型鑒定和各項生化指標(biāo)的檢測, 以期得到耐鹽抗旱表性良好的轉(zhuǎn)基因煙草。

1 材料與方法

1.1 植物生長條件

1.1.1 煙草幼苗在營養(yǎng)液中的培養(yǎng)

野生煙草(‘Wisconsin 38’)和轉(zhuǎn)基因煙草T3代株系L1、L2、L3、L4、L5的種子在28 ℃暗發(fā)芽3 d后, 將幼苗轉(zhuǎn)移到Hoagland的營養(yǎng)液中, 在人工氣候室(14 h光照/10 h黑暗, 18~25 ℃, RH為60%~80%)進(jìn)行培養(yǎng), 每天更換一次營養(yǎng)液。

1.1.2 煙草幼苗在土壤中的培養(yǎng)

將野生型煙草和轉(zhuǎn)基因煙草T3代株系(L1、L2、L3、L4、L5)的種子植入裝有普通土∶營養(yǎng)土為3∶1的塑料營養(yǎng)缽中, 在人工氣候室(14 h光照/10 h黑暗, 18 ℃/25 ℃, RH為60%~80%)進(jìn)行培養(yǎng), 待幼苗長至3~4葉時移栽。

1.2過表達(dá)轉(zhuǎn)基因煙草的分子鑒定

水培煙草幼苗長到5~6葉期, 用含5 g?L-1NaCl的Hoagland營養(yǎng)液處理24 h, 提取葉片DNA以及Trizol法提取葉片RNA?；蛲ㄟ^I和I酶切位點連接在植物表達(dá)載體pCPB(改自pCambia3300)的35S啟動子之后(圖1a), 用于檢測轉(zhuǎn)基因植物中的片段的引物是tSsNHX1-F(5￠-AGGGAGCAAA GACAAGAG-3￠)和tSsNHX1-R(5￠-TCTTCTATCTGAGC GGAATT-3￠)。并使用引物qSsNHX1-F(5￠-GTCATTTGGT GGGCTGGTCTC-3￠)和qSsNHX1-R(5￠-TGAAAAGGAC AACGGTTATGGTG-3￠)進(jìn)行RT-PCR檢測, 篩選出基因高表達(dá)株系。

1.3 鹽脅迫下轉(zhuǎn)基因煙草耐鹽性分析

將溶液培養(yǎng)的轉(zhuǎn)基因株系L1和L5及野生型煙草幼苗, 用0 g?L-1、2.5 g?L-1、5 g?L-1、7.5 g?L-1NaCl進(jìn)行處理, 每個株系3株煙草, 每個鹽濃度處理重復(fù)3次, 處理7 d, 每2 d更換一次營養(yǎng)液。處理結(jié)束取樣, 用根系掃描儀掃描根系, 觀察煙草幼苗的生長狀況并測量根系的長度; 隨后用火焰分光光度計分別測定野生型和轉(zhuǎn)基因煙草株系的根系和葉片中Na+和K+離子含量。

1.4 干旱脅迫下轉(zhuǎn)基因煙草耐旱性分析

土壤培養(yǎng)的轉(zhuǎn)基因株系L1和L5及野生型幼苗長至9~10葉期, 每個株系3株煙草, 每個處理天數(shù)重復(fù)3次, 進(jìn)行斷水12 d干旱處理后再復(fù)水3 d, 從每株葉片中間部分取樣, 觀測其在斷水0 d、3 d、6 d、9 d、12 d及復(fù)水1 d、3 d的生長情況。

1.5 鹽脅迫和干旱脅迫下轉(zhuǎn)基因煙草生理生化指標(biāo)測定

分別將鹽脅迫和干旱脅迫處理后的野生型和轉(zhuǎn)基因株系L1和L5煙草進(jìn)行生理生化指標(biāo)的鑒定, 包括: 相對含水量(relative water content, RWC)[21]、葉綠素含量[22]、丙二醛(malondialdehyde, MDA)含量[23]、相對電導(dǎo)率[24]。所得數(shù)據(jù)用SPSS 13.0軟件分析, 并用Microsoft Excel軟件作圖。

2 結(jié)果與分析

2.1過表達(dá)轉(zhuǎn)基因煙草的分子鑒定

基因轉(zhuǎn)化煙草后, 得到35S啟動子驅(qū)動下過量表達(dá)的煙草轉(zhuǎn)基因株系(圖1a), 通過將基因過表達(dá)煙草株系L1、L2、L3、L4、L5以及野生型進(jìn)行PCR檢測, 發(fā)現(xiàn)除野生型煙草外, 5個轉(zhuǎn)基因株系均有明顯的目的條帶, 說明基因已整合至這些轉(zhuǎn)基因株系中(圖1b)。為了后續(xù)的表型分析及鹽和干旱脅迫下的生理生化指標(biāo)測量, 對上述5個轉(zhuǎn)基因陽性株系以及野生型煙草又進(jìn)行了RT-PCR檢測, 發(fā)現(xiàn)L1、L4、L5的表達(dá)量最高, 因此選用其中的L1和L5轉(zhuǎn)基因株系用于后續(xù)試驗(圖1c)。

a: 將開放閱讀框(ORF)插入CaMV 35S啟動子和胭脂堿合酶終止子(Nos)區(qū)域之間, Bar基因用作選擇性標(biāo)記。b: 轉(zhuǎn)基因煙草的PCR檢測; M表示DNA marker DL2000; (-)表示H2O; (+)表示質(zhì)粒DNA; WT表示用空載體轉(zhuǎn)化的野生型煙草植物; L1-L5表示獨立的轉(zhuǎn)基因煙草品系。c: 轉(zhuǎn)基因株系和WT植物中基因的表達(dá)分析。a: theopen reading frame (ORF) was inserted between the CaMV 35S promoter and nopaline synthase terminator (Nos) regions. The bargene was used as the selective marker. b: PCR detection of the transgenic maize plants; M is DNA marker DL2000; (-) means H2O; (+) means plasmid DNA; WT means wild-type tobacco plant transformed with empty vector; L1-L5 are independent transgenic tobacco lines. c: expression analysis oftransgenic tobacco lines and WT plants.

2.2轉(zhuǎn)基因煙草在鹽脅迫下的表型分析

由圖2a可以看出, 在鹽脅迫處理前, 野生型和轉(zhuǎn)基因煙草之間表型沒有顯著差異。用2.5 g?L-1NaCl處理7 d后野生型和轉(zhuǎn)基因煙草的生長發(fā)育都仍未受抑制。但在5 g?L-1NaCl脅迫條件下, 野生型煙草的生長相對于轉(zhuǎn)基因煙草受到明顯抑制: 根系數(shù)量減少、根長變短、葉片變少、變小。當(dāng)NaCl濃度高達(dá)7.5 g?L-1時, 野生型煙草在脅迫的第5 d時葉片開始發(fā)黃、融爛, 最終死亡。而轉(zhuǎn)基因株系L1和L5的植株生長雖然隨著鹽脅迫濃度的升高逐漸變緩, 但依然能保持生長狀態(tài), 比野生型表現(xiàn)出更好的抗鹽性。

由于鹽脅迫對植物根長具有顯著的抑制作用, 本試驗對轉(zhuǎn)基因煙草與野生型在鹽脅迫下的根長進(jìn)行了研究。在無鹽脅迫下, 兩者總根長無顯著性差異, 但在鹽脅迫下, 轉(zhuǎn)基因煙草L1和L5與野生型的總根長差異顯著(圖2b)。且隨著鹽處理濃度的升高, 野生型的總根長明顯變短, 而轉(zhuǎn)基因株系L1和L5的總根長降低幅度較小, 如在7.5 g?L-1NaCl濃度處理下轉(zhuǎn)基因株系L1和L5根長分別是野生型煙草的2.64和2.72倍, 差異顯著。說明鹽脅迫雖然都抑制著煙草根系的生長, 但轉(zhuǎn)基因株系受到的抑制作用相對較輕, 基本不影響其根系的生長發(fā)育。

a: 幼苗在0 g?L-1、0.25 g?L-1、0.50 g?L-1和0.75 g?L-1的NaCl梯度濃度霍格蘭溶液中培養(yǎng), 直到三葉期; b: 經(jīng)過NaCl濃度梯度處理后的根長。WT為野生型煙草; L1和L5為煙草轉(zhuǎn)基因株系; *表示差異顯著, 誤差線表示標(biāo)準(zhǔn)方差。a: seedlings were hydrated with Hoagland nutrient solution with an increased NaCl gradient of 0 g?L-1, 2.5 g?L-1, 5 g?L-1and 7.5 g?L-1(/) until the three-leaf stage; b: root length after immersion in 0 g?L-1, 2.5 g?L-1, 5 g?L-1, and 7.5 g?L-1NaCl. WT is wild type tobacco; L1 and L5 aretransgenic tobacco lines. * indicates significant difference. Values of error bar are means standard deviation.

2.3轉(zhuǎn)基因煙草在鹽脅迫條件下根系和葉片的Na+和K+含量

植物體吸收過高的Na+, 會導(dǎo)致植物體營養(yǎng)離子的失衡, 特別是K+的吸收抑制和流失。在鹽脅迫條件下液泡Na+/H+逆向轉(zhuǎn)運蛋白則會將Na+轉(zhuǎn)運至液泡中, 減少K+散失[25]。在本試驗中, 經(jīng)NaCl處理后, 野生型和轉(zhuǎn)基因煙草植株葉片和根系中的Na+含量都迅速增加。例如, 在7.5 g?L-1NaCl處理時, 野生型煙草葉片中的Na+由15.84 mg?g-1上升至52.53 mg?g-1, 而轉(zhuǎn)基因煙草L1和L5則分別由18.09 mg?g-1上升至60.37 mg?g-1和由20.04 mg?g-1顯著上升至63.35 mg?g-1。同樣, 根系中野生型及轉(zhuǎn)基因煙草L1和L5 Na+含量分別顯著上升至40.48 mg?g-1、42.77 mg?g-1、53.75 mg?g-1(圖3a, 3b)。隨著NaCl處理濃度的增加, 葉片和根系中的K+含量降低。例如, 在7.5 g?L-1NaCl處理后的葉片中, 野生型葉片中的K+含量由79.76 mg?g-1降至46.13 mg?g-1, 而轉(zhuǎn)基因煙草L1和L5則分別由79.04 mg?g-1降至58.76 mg?g-1和由80.17 mg?g-1降至53.03 mg?g-1, 差異顯著。同樣, 在根系中野生型及轉(zhuǎn)基因煙草L1和L5的K+含量分別降低到29.44 mg?g-1、32.04 mg?g-1、38.69 mg?g-1, 差異顯著(圖3c, 3d)。K+和Na+根系中的變化趨勢與葉片中趨勢相同(圖3b, 3d)。與野生型相比, 轉(zhuǎn)基因煙草中K+含量隨鹽濃度的提高下降緩慢, 而Na+含量增加較快。因此, 轉(zhuǎn)基因煙草植株比野生型擁有更高的K+/Na+比。與根系相比, 葉片中的Na+和K+的含量更高, 尤其是在轉(zhuǎn)基因植物中, 表明脅迫條件下煙草的Na+的區(qū)室化主要發(fā)生在葉片中。

2.4轉(zhuǎn)基因煙草在鹽脅迫條件下生理指標(biāo)的變化

鹽脅迫可以誘導(dǎo)植物體內(nèi)一系列的生理變化, 如相對含水量、丙二醛含量、相對電導(dǎo)率和葉綠素含量的變化, 而這些變化通常與鹽脅迫具有一定的相關(guān)性。從圖4a可知, 當(dāng)NaCl處理6 d時, 野生型和轉(zhuǎn)基因煙草植株相對含水量都開始降低。從9 d開始到15 d時野生型和轉(zhuǎn)基因煙草的相對含水量下降幅度變大且差異顯著。對于葉綠素而言, 其在野生型煙草中的含量隨著時間的增加下降幅度越來越大, 并在15 d降到0.65 mg×g-1, 與轉(zhuǎn)基因煙草株系L1(1.51 mg×g-1)和L5的(1.68 mg×g-1)達(dá)到最大差值(圖4b)。

丙二醛含量和相對電導(dǎo)率的含量, 則與上述的相對含水量和葉綠素含量趨勢相反。在本試驗中都是隨著NaCl處理時間的增加而呈上升的趨勢, 且野生型的含量大于轉(zhuǎn)基因株系(圖4c, 圖4d)。例如, NaCl處理15 d時, 野生型煙草丙二醛的含量為9.71 nmol×g-1, 而轉(zhuǎn)基因株系L1和L5分別為8.75 nmol×g-1和7.34 nmol×g-1, 比野生型顯著降低。同樣, NaCl處理15 d后, 野生型的相對電導(dǎo)率為90.68%而轉(zhuǎn)基因株系L1和L5分別為75.52%和77.20%, 差異顯著。這些結(jié)果表明,轉(zhuǎn)基因植物的生理變化與其增強的耐鹽表型密切相關(guān)。

WT為野生型煙草, L1和L5為轉(zhuǎn)基因煙草株系; *表示差異顯著, 誤差線表示標(biāo)準(zhǔn)方差。 WT is wild type tobacco plant; L1 and L5 aretransgenic tobacco lines. * indicates significant difference. Values of error bar are means standard deviation.

WT為野生型煙草, L1和L5為轉(zhuǎn)基因株系; *表示差異顯著, 誤差線表示標(biāo)準(zhǔn)方差。WT is wild type tobacco plant; L1 and L5 aretransgenic tobacco lines. * indicates significant difference. Values of error bar are means standard deviation.

2.5轉(zhuǎn)基因煙草在干旱脅迫下的表型分析

很多研究的結(jié)果已表明抗鹽和抗旱相關(guān)聯(lián)。如圖5所示, 斷水處理3 d時,轉(zhuǎn)基因煙草和野生型并沒有明顯的差異(圖5a, b), 但在斷水處理6 d后, 野生型煙草開始有萎蔫的趨勢, 此時轉(zhuǎn)基因煙草仍能正常生長(圖5c)。斷水處理9 d時, 野生型煙草葉片縮小卷曲, 萎蔫嚴(yán)重, 而轉(zhuǎn)基因株系L1和L5只表現(xiàn)出輕微程度的萎蔫和葉片發(fā)黃(圖5d); 斷水處理12 d時, 野生型煙草以及轉(zhuǎn)基因煙草都嚴(yán)重萎蔫, 但相較之下轉(zhuǎn)基因株系L1萎蔫程度較輕(圖5e); 但經(jīng)過1 d復(fù)水后, 雖然轉(zhuǎn)基因煙草的葉片的葉綠素肉眼仍能可見其明顯減退, 卻能迅速地恢復(fù)到斷水前較為挺拔的生長狀態(tài), 野生型卻依然萎蔫, 甚至持續(xù)至復(fù)水3 d時, 說明其生理失水嚴(yán)重, 已不能靠自身調(diào)節(jié)恢復(fù)正常生長(圖5f, g)。

a: 干旱處理前; b: 干旱3 d; c: 干旱6 d; d: 干旱9 d; e: 干旱12 d; f: 復(fù)水0 d; g: 復(fù)水3 d。WT為野生型煙草, L1和L5為轉(zhuǎn)基因煙草。a: before drought treatment; b: three days of drought; c: six days of drought; d: nine days of drought; e: twelve days of drought; f: rehydrate for 0 day; g: rehydrated for three days. WT iswild type tobacco plant; L1 and L5 aretransgenic tobacco lines.

2.6轉(zhuǎn)基因煙草在干旱脅迫下生理指標(biāo)的變化

同其他脅迫因子一樣, 干旱脅迫同樣會對植物生理變化造成影響。干旱脅迫下轉(zhuǎn)基因煙草的的相對含水量和葉綠素含量變化總體趨勢與其在上述鹽脅迫下相當(dāng), 即隨著處理時間延長含量下降(圖6a, 6b)。當(dāng)斷水處理12 d時, 野生型煙草相對含水量降至63.40%, 而轉(zhuǎn)基因株系L1和L5的分別為70.56%和69.02%, 差異顯著。此時葉綠素含量野生型(0.94 mg×g-1)和轉(zhuǎn)基因煙草L1(1.77 mg×g-1)和L5(1.78 mg×g-1)的差異也達(dá)到最大值, 差異顯著。在斷水處理的過程中, 相對電導(dǎo)率以及丙二醛在野生型和轉(zhuǎn)基因煙草中都呈上升趨勢, 但在野生型中上升的趨勢更加明顯。處理12 d時, 野生型煙草丙二醛含量為4.67 nmol×g-1, 轉(zhuǎn)基因株系L1和L5分別為3.73 nmol×g-1和3.90 nmol×g-1(圖6c)。而相對電導(dǎo)率則為野生型植株大于轉(zhuǎn)基因植株, 如處理12 d時野生型的相對電導(dǎo)率為84.92%, 而轉(zhuǎn)基因煙草L1和L5分別為70.72%和72.92%(圖6d)。

3 討論與結(jié)論

為了有效地抗擊鹽和干旱脅迫以維持植物體內(nèi)Na+和K+等的離子平衡, 植物能將Na+外排, 吸收或區(qū)室化至液泡中, 以此增強它們對鹽和干旱脅迫的耐受性[26]。在該調(diào)節(jié)過程中, 液泡膜Na+/H+逆向轉(zhuǎn)運蛋白發(fā)揮著關(guān)鍵作用, 而編碼這類蛋白的NHX型基因的過量表達(dá)能改善許多物種轉(zhuǎn)基因植株的耐鹽性[27]?；蚴躯}生植物堿蓬的NHX型基因, 鹽脅迫下能提高表達(dá)水平, 并顯著改善水稻和擬南芥的耐鹽性[28-29]。然而, 關(guān)于在其他植物中的抗鹽和抗旱研究尚鮮見報道。因此, 本研究通過基因工程手段過量表達(dá)基因, 以改善煙草的耐鹽抗旱性。

植物耐鹽性與耐旱性密切相關(guān)。在許多轉(zhuǎn)基因品系中, 耐鹽性的提高通常伴隨耐旱性的增強。在本研究中,轉(zhuǎn)基因煙草與野生型相比, 在高鹽濃度下根部依然可以正常發(fā)育, 用遞增鹽濃度處理時表現(xiàn)出強耐鹽表型; 在干旱脅迫下表現(xiàn)出強耐旱表型, 并且在復(fù)水后能基本恢復(fù)缺水前的表型。說明轉(zhuǎn)基因煙草的耐鹽性和抗旱性都比野生型有所提高。與之相似,轉(zhuǎn)化擬南芥及轉(zhuǎn)化小麥時分別提高了轉(zhuǎn)基因植物的耐鹽抗旱性[30-31]。這些結(jié)果表明, 液泡基因的過量表達(dá)能提高轉(zhuǎn)基因植物應(yīng)對鹽脅迫和干旱脅迫的能力。

在鹽脅迫條件下, 液泡NHX蛋白不僅會排出Na+, 同時也會吸收K+, 以此來保持植物體內(nèi)Na+/K+的平衡。在本試驗中, 經(jīng)NaCl脅迫處理后, Na+含量在轉(zhuǎn)基因煙草的葉片和根系中明顯增加, 且葉片中的含量比根系中高。這可能是由于活性增強后, 將煙草葉片中的Na+直接區(qū)室化到液泡中所致[32-33]。這與液泡膜的Na+/H+逆向轉(zhuǎn)運蛋白表達(dá)量增加會增強Na+區(qū)室化的研究一致[34]。但是由于目前的研究技術(shù)尚無法從從細(xì)胞質(zhì)中分離液泡[4], 因此等液泡對Na+和K+在細(xì)胞質(zhì)和液泡中更精細(xì)地分配尚待進(jìn)一步的研究。保持植物體內(nèi)較低的Na+/K+可以有效地降低植物滲透勢以達(dá)到吸水的目的, 這與轉(zhuǎn)基因番茄[35]和轉(zhuǎn)基因矮牽牛[(J. D. Hooker) Vilmorin]中的發(fā)現(xiàn)相似[36]。這些結(jié)果說明液泡基因的過量表達(dá)提高轉(zhuǎn)基因煙草的耐鹽能力, 在一定程度上可能通過將Na+區(qū)室化有效地調(diào)節(jié)細(xì)胞內(nèi)滲透壓的平衡而實現(xiàn)煙草的耐鹽能力。

WT, 野生型煙草; L1和L5,轉(zhuǎn)基因株系。*表示差異顯著, 誤差線表示標(biāo)準(zhǔn)方差。WT, wild type plant; L1 and L5,transgenic tobacco lines. * indicates significant differences. Values of error bar are means standard deviation.

與轉(zhuǎn)基因煙草的生理指標(biāo)相比, 鹽害和干旱脅迫下野生型的相對水含量都較低, 表明轉(zhuǎn)基因煙草表現(xiàn)出改善的調(diào)節(jié)葉滲透壓和保水性的能力, 在這些脅迫條件下能保持適當(dāng)?shù)臐B透壓。轉(zhuǎn)基因煙草的葉綠素含量雖然在鹽脅迫和干旱脅迫下有所降低, 但仍高于野生型, 這可能與高濃度Na+和低含水量會抑制光合系統(tǒng)Ⅰ和Ⅱ, 導(dǎo)致光合作用和葉綠素合成速率降低有關(guān)[37-38]。此結(jié)果與轉(zhuǎn)基因棉花[14],轉(zhuǎn)基因水稻[39],[40]和[41]轉(zhuǎn)基因擬南芥以及轉(zhuǎn)基因煙草[42]的研究一致。在本試驗中,轉(zhuǎn)基因和野生型煙草中MDA含量和相對電導(dǎo)率均增加, 表明在鹽和干旱脅迫條件下質(zhì)膜受到損傷, 并且野生型比轉(zhuǎn)基因煙草受到的損傷更嚴(yán)重, 表明轉(zhuǎn)基因煙草在鹽害和干旱脅迫下降低了對質(zhì)膜的損害。

總之, 本研究的結(jié)果表明,基因編碼的液泡膜Na+/H+逆向轉(zhuǎn)運蛋白, 能通過改變轉(zhuǎn)基因煙草Na+和K+的比率降低脅迫對植物的損害程度。在煙草中的過表達(dá)增強了轉(zhuǎn)基因煙草的耐鹽性和抗旱性。由相對含水量、丙二醛含量、相對電導(dǎo)率和葉綠素含量的變化所反映的轉(zhuǎn)基因煙草生理的變化也與其改善的耐鹽抗旱性的能力相一致。

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Overexpression ofgene enhanced salt and drought tolerance of transgenic tobacco*

CHEN Xin1, MA Chao1, YANG Yongjuan2, WANG Hongling1, HUANG Ying1, ZHANG Xiaoxia1, HUANG Kaifeng1, ZHAO Zhuo1, ZHANG Suzhi1**

(1. Maize Research Institute, Sichuan Agricultural University / Key Laboratory of Biology and Genetic Improvement of Maize in Southwest China, Ministry of Agriculture, Chengdu 611130, China; 2. Agricultural Service Center of Tongjing Town, Yinan County, Yinan 276300, China)

Tobacco (L.) is an important economic and model plant. Salt and drought are two important environmental factors that are harmful to plant growth, development, production and quality of tobacco. In addition to conventional salt and drought prevention measures, genetic engineering of plants has also been proven to be effective. In order to improve salt and drought tolerance of tobacco, Na+/H+anti-porter genewas cloned fromsalt-tolerant plant and overexpressed in tobacco by-mediated genetic transformation. The differences in salt tolerance and drought resistance were compared by determining phenotypic and physiological indexes of wild and transgenic tobacco L1 and L5. Phenotypic analysis showed that salt tolerance oftransgenic tobacco lines L1 and L5 were significantly higher than that of the wild type. This was evident from the vigorously growth and uninhibited root elongation under salt stress condition. The overexpressed transgenic lines of tobacco accumulated more Na+and K+in the both leaves and roots and with faster rate of increase of Na+and slower decreasing rate of K+. The lines also maintained significantly higher contents of relative leaf water and chlorophyll, but lower malondialdehyde contents and relative conductivities. The results indicated that overexpression ofgene apparently promoted compartmentalization of Na+from vacuolar cells into vacuoles and improved salt tolerance of transgenic plants of tobacco. On the other hand,transgenic tobacco plants showed significant enhancement of drought tolerance than the wild type and restored normal growth after rehydration. Under drought stress, the contents of malondialdehyde and relative conductivities of transgenic lines were lower than those of the wild type, while the relative water and chlorophyll contents of leaves were maintained. The results suggested that under drought stress, overexpressedin tobacco reduced damage to cell membrane by reducing osmotic potential of cells, maintaining relative water and chlorophyll content of leaves, and finally improved drought resistance of tobacco.

gene; Gene transformation;L; Salt tolerance; Drought tolerance; Phenotype; Biochemical index

Mar. 16, 2017; accepted May 30, 2017

10.13930/j.cnki.cjea.170230

S188

A

1671-3990(2017)10-1518-09

2017-03-16

2017-05-30

* 國家自然科學(xué)基金項目(30800687, 31071434)資助

* This study was supported by the National Natural Science Foundation of China (30800687, 31071434).

** Corresponding author, E-mail: suzhi1026@163.com

**通訊作者:張素芝, 主要從事植物抗逆生理研究。E-mail: suzhi1026@163.com 陳鑫, 主要研究植物抗逆生理。E-mail: 446035286@qq.com