田文剛 朱雪峰 宋 雯 程文翰 薛 飛 朱華國,*

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異源表達(dá)棉花S-腺苷甲硫氨酸脫羧酶()基因提高了擬南芥抗鹽能力

田文剛1朱雪峰1宋 雯1程文翰2薛 飛1朱華國1,*

1石河子大學(xué)農(nóng)學(xué)院 / 新疆兵團(tuán)綠洲生態(tài)農(nóng)業(yè)重點(diǎn)實(shí)驗(yàn)室, 新疆石河子 832003;2荊楚理工學(xué)院, 湖北荊門 448000

以轉(zhuǎn)基因擬南芥研究了過量表達(dá)基因?qū)M南芥幼苗抗鹽能力的影響, 以及內(nèi)源多胺、過氧化氫(H2O2)、丙二醛(MDA)、葉綠素含量(Chl)、離子滲透率、抗氧化酶(SOD、CAT、POD)活性和表達(dá)量在鹽脅迫下的變化。結(jié)果表明, 過量表達(dá)基因能夠減少擬南芥內(nèi)源腐胺(Put)含量, 增加亞精胺(Spd)和精胺(Spm)含量。鹽脅迫下, 轉(zhuǎn)基因株系亞精胺合酶(、)和精胺合酶()基因表達(dá)量明顯高于野生型, Spd和Spm含量進(jìn)一步增加, H2O2、MDA、Chl以及離子滲透率顯著降低; 與野生型相比, 過氧化物酶(POD)活力無明顯差異, 但超氧化物歧化酶(SOD)和過氧化氫酶(CAT)活力明顯增加, 其表達(dá)水平與活力變化趨勢基本一致。因此, 鹽脅迫下,基因通過提高Spd和Spm合成相關(guān)基因的表達(dá), 增加了轉(zhuǎn)基因株系Spd和Spm含量, Spd和Spm直接或間接提高抗氧化系統(tǒng)相關(guān)酶的活力, 通過清除H2O2等活性氧的方式提高擬南芥的抗鹽能力。

擬南芥; 棉花S-腺苷甲硫氨酸脫羧酶基因; 鹽脅迫; 抗氧化酶

多胺(polyamine, PAs), 是生物代謝過程中產(chǎn)生的一類具有生物活性的低分子量脂肪族含氮堿, 廣泛存在于原核及真核生物中。高等植物中常見的多胺主要包括腐胺(putrescine, Put)、亞精胺(spermidine, Spd)和精胺(spermine, Spm), 參與植物細(xì)胞分化、形態(tài)建成、程序凋亡、脅迫響應(yīng)等生物學(xué)過程[1-2]。

目前植物體內(nèi)多胺的代謝過程已經(jīng)研究的比較清楚。研究發(fā)現(xiàn), 多胺的生物合成起始于Put的合成, 而Put的合成涉及鳥氨酸(Ornithine)和精氨酸(Arginine) 2條路徑。在動(dòng)物和真菌中, Put主要由鳥氨酸經(jīng)過鳥氨酸脫羧酶(ornithine decarboxylase, ODC)催化反應(yīng)而來。而在植物和細(xì)菌中, Put主要由精氨酸經(jīng)過精氨酸脫羧酶(arginine decarboxylase, ADC)、亞氨基脫氫酶(agmatine iminohydrolase, AIH)和N-氨甲?；孵０匪饷?N-carbamoyl putrescine amidohydrolase, CPA)三步催化反應(yīng)而來[1]。Put合成以后作為前體物質(zhì)在亞精胺合酶(spermidine synthase, SPDS)的催化下, 結(jié)合由S-腺苷甲硫氨酸脫羧酶(S-adenosylmethionine decarboxylase, SAMDC)催化S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)脫羧得到的反應(yīng)產(chǎn)物氨丙基進(jìn)而生成Spd。而Spd則進(jìn)一步在精胺合酶(spermine synthases, SPMS)的催化下, 結(jié)合S-腺苷甲硫氨酸脫羧反應(yīng)產(chǎn)物氨丙基進(jìn)而生成Spm[3]。目前為止, 除了基因以外, 所有參與多胺合成的基因均已經(jīng)在擬南芥中發(fā)現(xiàn)。

S-腺苷甲硫氨酸脫羧酶是調(diào)節(jié)多胺合成的關(guān)鍵限速酶。近年來已從辣椒[4]、曼陀羅[5]、番茄[6]、百脈根[7]、羊草[8]、棉花[9]、杜梨[10]、甘蔗[11]、高羊茅[12]等多種植物中克隆得到基因, 并證明基因可以通過調(diào)控植物體內(nèi)多胺含量來影響植物應(yīng)答逆境脅迫。過量表達(dá)羊草和辣椒基因通過積累更多的多胺提高擬南芥的耐寒、耐鹽性以及抗旱能力[4,13]。番茄中導(dǎo)入酵母基因, Spd和Spm含量顯著上升, 對高溫脅迫表現(xiàn)出明顯抗性[14]。相反, 在水稻中下調(diào), 顯著降低了的Spd和Spm含量, 多胺氧化酶(polyamine oxidase, PAO)活性下降, 降低了轉(zhuǎn)基因水稻的生育力和對非生物脅迫的耐受性[15]。同時(shí), 多胺參與逆境脅迫同樣通過外施多胺的方法得以證明。外源Spm能夠調(diào)節(jié)小麥幼苗過氧化氫酶(catalase, CAT)、谷胱甘肽還原酶(glutathione reductase, GR)、脫氫抗壞血酸還原酶(dehydroascorbate reductase, DHAR)、抗壞血酸過氧化物酶(ascorbic acid peroxidase, APX)和超氧化物歧化酶(superoxide dismutase, SOD)的活性, 并有效地調(diào)節(jié)它們的轉(zhuǎn)錄水平, 提高抗鹽能力, 表明Spm在調(diào)節(jié)植物的抗氧化酶活性、減輕鹽脅迫的氧化損傷中起關(guān)鍵作用[16]。在黃瓜中, 外源Spd可顯著緩解幼苗的鹽害, 提高幼苗的抗鹽性[17], 通過降低脫鎂葉綠酸A加氧酶(pheophorbide A oxygenase, PaO)途徑相關(guān)酶活性和轉(zhuǎn)錄水平、減緩Chl分解代謝和增加Chl濃度來增強(qiáng)高溫耐受性[18]。Liu等[19]通過研究發(fā)現(xiàn), 在鹽脅迫下, 大麥耐鹽品種J4比敏感品種K97幼苗積累更多的Spd、Spm和少量的Put; 抗鹽性較強(qiáng)的長春密刺比抗鹽性較弱的津春2號根系積累更多的的Spd和Spm[20]。表明Put向Spd和Spm的轉(zhuǎn)化, 并保持較高水平的Spd和Spm對植物耐鹽性是非常重要的。

鹽脅迫下, 植物細(xì)胞內(nèi)離子平衡被破壞, 各代謝途徑產(chǎn)生大量的活性氧(reactive oxygen species, ROS), 能迅速使酶失活, 破壞細(xì)胞器, 細(xì)胞質(zhì)膜、蛋白質(zhì)、脂質(zhì)和核酸, 導(dǎo)致植物細(xì)胞死亡[21]。過量的H2O2對植物來說是致命的。在遭受鹽脅迫時(shí), 植物通過高效的抗氧化酶防御系統(tǒng)清除ROS來保護(hù)細(xì)胞免受氧化損傷[22-23]。SOD是植物細(xì)胞中清除活性氧自由基最重要的酶類之一, 將超氧化物自由基轉(zhuǎn)變?yōu)镠2O2, H2O2由CAT和POD清除[24], 其他細(xì)胞器中產(chǎn)生的H2O2進(jìn)入過氧化體中也能夠被CAT清除[25]。SOD、POD與CAT這3種酶相互協(xié)調(diào), 使植物體內(nèi)的ROS處于相對穩(wěn)定的水平[26]。研究表明, 多胺同樣參與酶抗氧化防御系統(tǒng)的調(diào)節(jié), 主要通過直接或間接地調(diào)節(jié)抗氧化系統(tǒng)或抑制ROS生成來調(diào)節(jié)ROS的穩(wěn)態(tài)。外施Spd能提高結(jié)縷草中SAMDC等相關(guān)合成酶活性,增加Spd和Spm含量, 且顯著降低H2O2含量, 可以通過清除活性氧、穩(wěn)定細(xì)胞結(jié)構(gòu)、調(diào)節(jié)光保護(hù)機(jī)制、增加抗氧化酶蛋白活性和轉(zhuǎn)錄水平, 減輕鹽脅迫引起的氧化損傷[27-28]; 外施Spm增加了多胺的積累、CAT和SOD活性的增強(qiáng), 增加了擬南芥和水稻對滲透和鹽脅迫耐受能力[29-30]。因此, 多胺在刺激植物生長、調(diào)節(jié)植物生長發(fā)育、控制形態(tài)建成、提高植物抗逆性和延緩衰老等諸多方面發(fā)揮著重要作用。

在鹽脅迫下,的轉(zhuǎn)錄水平增加。為了確定鹽誘導(dǎo)在鹽脅迫應(yīng)答中的作用, 利用Gateway技術(shù)克隆了PGWB17-, 利用轉(zhuǎn)PGWB17-基因擬南芥探究基因功能, 通過檢測抗氧化活性和多胺含量發(fā)現(xiàn), PGWB17-的過量表達(dá)可以通過調(diào)節(jié)抗氧化酶活性增強(qiáng)轉(zhuǎn)基因擬南芥的耐鹽性。這對研究基因與多胺合成以及逆境脅迫下多胺含量與植物抗逆性的相互關(guān)系具有重要意義。

1 材料與方法

1.1 材料與試劑

野生型(col-0)及轉(zhuǎn)基因擬南芥種子均由本實(shí)驗(yàn)室保存。多胺(Put、Spd、Spm)純度≥99%, 甲醇為色譜純, 購自Sigma公司; H2O2(貨號A064)、MDA (貨號A003-3)、CAT (貨號A007-1)、SOD (貨號A001-3)和POD (貨號: A084-3)測定試劑盒均購自南京建成生物工程研究所; DAB染色(貨號CW0125M)試劑盒均購自康為世紀(jì)生物科技有限公司; 高氯酸、氫氧化鈉、氯化鈉、苯甲酰氯、乙醚等均為國產(chǎn)分析純。高效液相色譜儀為安捷倫1200型, 色譜柱為AgilentXDB-C18 (4.6 mm × 150.0 mm); 低溫離心機(jī)、常溫離心機(jī)及真空抽濾儀均為Eppendorf公司產(chǎn)品。

1.2 材料處理

將野生型及純合的轉(zhuǎn)基因擬南芥種子用2%的次氯酸鈉消毒10 min, 然后用無菌水沖洗3~4次。用1 mL槍頭分別點(diǎn)播于滅菌的1/2MS和1/2MS+100 mmol L-1NaCl培養(yǎng)基, 封口后置于4℃培養(yǎng)箱春化48 h, 然后放于人工氣候室(22±1)℃, 16 h/8 h光照培養(yǎng), 15 d后觀察表型、取樣, 備用。

1.3 多胺、葉綠素、H2O2含量測定及DAB染色

多胺含量測定和DAB染色參照程文翰等[31-32]的方法, 用高效液相色譜法測定多胺含量, DAB染色采用DAB染色試劑盒; 測定葉綠素含量參照李超等[33]的方法, 采用乙醇-丙酮混合液提取葉綠素; 測定H2O2含量采用H2O2含量測定試劑盒[34]。

1.4 離子滲透率測定

離子滲透率測定參考朱珍等[35]的方法, 略有改動(dòng)。取100 mmol L-1NaCl處理15 d的擬南芥幼苗葉片各0.1 g, 蒸餾水洗滌后, 用濾紙吸干表面水分, 剪成1 cm小段放入去離子水清洗的燒杯(50 mL)中, 加去離子水至20 mL, 立即測定電導(dǎo)率0。室溫下置水平震蕩器上溫和震蕩2 h, 測定電導(dǎo)率1。煮沸10 min, 冷卻至室溫, 再次加去離子水至刻度, 測電導(dǎo)率2。

離子滲透率(%) = (1P0)/(2P0)×100

1.5 MDA含量測定

采用MDA含量測定試劑盒測定MDA含量。準(zhǔn)確稱取植物組織, 按質(zhì)量體積比1∶9加入9倍體積的試劑五應(yīng)用提取液(按試劑五∶蒸餾水=1∶9的比例配置), 將樣品剪碎后用內(nèi)切式勻漿機(jī)冰水浴勻漿, 8000~10,000 ×, 每次10~15 s, 共3~5次, 再將勻漿吸入到離心管中, 3500~4000 ×, 離心10 min, 取上清液, 根據(jù)操作表加樣(具體方法參照說明書)。加樣結(jié)束后, 蓋上蓋, 渦旋混勻器混勻, 95℃以上水浴20 min, 取出后流水冷卻, 將酶標(biāo)板空板在530 nm處掃描, 準(zhǔn)確吸取0.25 mL各管反應(yīng)液加入到新的96孔板中, 以酶標(biāo)儀測定各孔吸光度(計(jì)算時(shí)要減去空板讀數(shù))。

樣本濃度=植物組織重(g)/所加提取液的量(mL)

1.6 CAT、SOD和POD活力檢測

1.6.1 CAT活力檢測 準(zhǔn)確稱取植物組織, 按質(zhì)量體積比1∶9加9倍體積的生理鹽水, 冰浴條件下, 制備10%的組織勻漿, 2500×, 離心10 min, 取上清液再用生理鹽水稀釋成最佳濃度, 待測。按照操作表加樣(具體方法參照說明書)。加樣后混勻, 波長405 nm, 光徑0.5 cm, 雙蒸水調(diào)零, 測定各管吸光度值。

式中, 271*為斜率的倒數(shù)。

1.6.2 SOD活力檢測 準(zhǔn)確稱取植物組織, 按質(zhì)量體積比1∶9加入9倍體積的磷酸鹽緩沖液(磷酸鹽緩沖液: 0.1 mol L-1, pH 7.0~7.4), 將植物組織剪碎后用勻漿機(jī)制備植物組織勻漿, 3500 ×, 離心10 min即為10%勻漿上清液, 再用磷酸鹽緩沖液稀釋成不同濃度進(jìn)行預(yù)實(shí)驗(yàn)。按照按照操作表加樣(具體方法參照說明書)。加樣結(jié)束后, 混勻, 37℃孵育20 min, 波長450 nm, 用酶標(biāo)儀測吸光度值。

1.6.3 POD活力檢測 準(zhǔn)確稱取植物組織, 按質(zhì)量體積比1∶9加入9倍體積的勻漿介質(zhì)(生理鹽水或磷酸鹽緩沖液0.1 mol L-1, pH 7.0~7.4), 冰浴條件下制備成10%的組織勻漿, 3500 ×, 離心10 min, 取上清液按照操作表加樣(具體方法參照說明書)。加樣后混勻, 3500 ×, 離心10 min, 取上清于波長420 nm處, 1 cm光徑, 雙蒸水調(diào)零, 測定各管吸光度值。

1.7 RNA提取及實(shí)時(shí)熒光定量PCR

提取RNA采用TransZol UP RNA提取試劑盒; 采用TransScriptAll-in-One First-Strand cDNA SynthesisSuperMix for qPCR試劑盒反轉(zhuǎn)錄, 并將反轉(zhuǎn)錄得到的cDNA模板稀釋10倍待用; 定量PCR系統(tǒng)采用羅氏Light Cycler 480系統(tǒng)(Roche, Switzerland), 反應(yīng)體系使用SYBR Premix Exkit (Takara, Japan)。PCR程序?yàn)?5℃預(yù)熱2 min; 94℃ 15 s, 56℃ 20 s, 72℃ 20 s, 40個(gè)循環(huán)。相對表達(dá)量的計(jì)算采用2–ΔΔCT法[36],基因作為內(nèi)參(, AT3G18780)。用Primer Premier 5.0設(shè)計(jì)qRT-PCR過程所用到的引物(表1)。

表1 qRT-PCR用到的引物

1.8 數(shù)據(jù)處理

利用Microsoft Excel 2007和SPSS 19.0統(tǒng)計(jì)分析數(shù)據(jù), 采用Duncan’s法進(jìn)行差異顯著性檢驗(yàn)(<0.05,<0.01), 用平均值±標(biāo)準(zhǔn)差表示結(jié)果, 以上實(shí)驗(yàn)均使用3次生物學(xué)重復(fù), 每次實(shí)驗(yàn)至少3次技術(shù)重復(fù)。

2 結(jié)果與分析

2.1 過量表達(dá)GhSAMDC1基因?qū)M南芥內(nèi)源多胺含量的影響

在NCBI中選取氨基酸同源性較高的擬南芥、水稻和玉米的SAMDC氨基酸序列與氨基酸序列構(gòu)建系統(tǒng)發(fā)育樹, 4種植物SAMDC的氨基酸序列可以分為2類, 而與水稻親緣關(guān)系較近(圖1-A)。根據(jù)擬南芥的序列為探針, 在雷蒙德氏棉基因組數(shù)據(jù)庫中Blast檢索, 獲得推定的棉花基因序列, 用特異性引物(正向5￠-CACCATGGAGCCTTCTCCTCGGT-3￠和反向5￠- CAAGATCGCTTCCGGAATG-3￠)進(jìn)行聚合酶鏈反應(yīng)(RT-PCR)擴(kuò)增cDNA, 并用Gateway技術(shù)克隆到PGWB17載體中(圖1-B)。通過電穿孔法將載體導(dǎo)入農(nóng)桿菌GV3101。通過花浸漬轉(zhuǎn)化擬南芥植株(col-0)。種子采自成熟植株, 在含卡那霉素50 mg L-1的1/2MS培養(yǎng)基中篩選。對卡那霉素抗性植物的后代進(jìn)行了抗性分離分析。進(jìn)一步培養(yǎng)具有3∶1分離比的植物的種子, 并再次分析所得后代以分離卡那霉素抗性, 鑒定T-DNA插入物為純合體。

為了探究基因在多胺合成中發(fā)揮的作用, 選取3個(gè)表達(dá)量較高的T4代轉(zhuǎn)基因株系(1-4、1-12、1-14)進(jìn)行后續(xù)研究(圖1-C~D)。通過檢測野生型及轉(zhuǎn)基因擬南芥葉片中內(nèi)源多胺含量發(fā)現(xiàn), 與野生型相比, 轉(zhuǎn)基因株系中多胺總量明顯上升, 增加了70%~116%, Put含量下降了23%~37%, Spd含量略有上升, 上升21%~33%, Spm含量顯著增加, 增加了79%~130% (圖2)。說明過量表達(dá)基因能夠改變擬南芥內(nèi)源多胺含量,基因可能在Put向Spd和Spm轉(zhuǎn)化中發(fā)揮重要作用。

2.2 過量表達(dá)GhSAMDC1基因?qū)M南芥的抗鹽能力的影響

基因在鹽脅迫下表達(dá)量明顯升高(附圖1)。為進(jìn)一步探究對擬南芥抗鹽能力的影響, 選取1-4、1-12和1-14作為研究對象。將純合轉(zhuǎn)基因種子與野生型置于含100 mmol L-1NaCl和不含NaCl的1/2MS培養(yǎng)基無菌培養(yǎng)(圖3-A)。正常培養(yǎng)下, 轉(zhuǎn)基因株系和野生型葉片數(shù)均為9~10片(圖3-B~D), 鮮重較野生型有所增加, 增加了6%左右, 但無顯著性差異(圖3-E); 在100 mmol L-1NaCl處理下, 轉(zhuǎn)基因株系生長明顯更好, 成活率為80%左右, 而野生型成活率僅為55% (圖3-C~F); 野生型擬南芥鮮重(30株)為0.08 g, 轉(zhuǎn)基因株系鮮重為0.10~0.12 g (圖3-G); 同時(shí), 轉(zhuǎn)基因株系葉片數(shù)為5.5片左右, 而野生型僅為4.5片(圖3-H)。此外, 轉(zhuǎn)基因擬南芥葉綠素含量明顯高于野生型, 葉綠素總量、葉綠素和葉綠素均為野生型的1.03~1.10倍左右(圖4)。表明轉(zhuǎn)基因株系較野生型有更強(qiáng)的耐鹽性, 過量表達(dá)基因能夠有效提高擬南芥的抗鹽能力。

圖1 陸地棉GhSAMDC1和其他物種同源蛋白進(jìn)化樹分析及轉(zhuǎn)基因鑒定

A: 陸地棉和其他物種同源蛋白進(jìn)化樹分析; B:表達(dá)載體; C: 轉(zhuǎn)基因擬南芥DNA鑒定及表達(dá)分析, M、1、2、3、4、5分別為marker、轉(zhuǎn)基因株系1-4、轉(zhuǎn)基因株系1-12、轉(zhuǎn)基因株系1-14、陽性對照、陰性對照, 目的基因片段大小為1035 bp; D: 轉(zhuǎn)基因擬南芥表達(dá)分析。

A: phylogenetic analysis of homologous proteins of upland cottonand other species; B: expression vector of; C: DNA identification of transgenic(), M, 1, 2, 3, 4, and 5 were marker, transgenic line 1-4, transgenic line 1-12, transgenic line 1-14, positive control, and negative control, respectively, the size of the target gene fragment was 1035 bp; D: expression analysis ofin.

(圖2)

A~D: 野生型和轉(zhuǎn)基因株系內(nèi)源的多胺總量、Put、Spd和Spm含量。選取正常培養(yǎng)30 d的擬南芥葉片, 利用高效液相色譜法檢測內(nèi)源多胺含量, 數(shù)據(jù)采用Duncan’s法進(jìn)行差異顯著性檢驗(yàn)(*< 0.05, **< 0.01), 結(jié)果用平均值±標(biāo)準(zhǔn)差表示, 以上實(shí)驗(yàn)均使用3次生物學(xué)重復(fù), 每次實(shí)驗(yàn)3次技術(shù)重復(fù)。

A–D: total endogenous polyamines, Put, Spd, and Spm contents in wild type and transgenic lines. The contents of endogenous polyamines inleaves were determined by high performance liquid chromatography (HPLC) after normal cultured for 30 days, and Duncan method was used to test the difference significance (*< 0.05, **< 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment.

圖3 過量表達(dá)GhSAMDC1基因?qū)M南芥抗鹽能力的影響

A: 種子分布示意圖; B~C: 正常和鹽脅迫下培養(yǎng)15 d后野生型及轉(zhuǎn)基因擬南芥表型; D~E: 正常培養(yǎng)15 d后野生型和轉(zhuǎn)基因擬南芥葉片數(shù)及鮮重; F~H: 100 mmol L-1NaCl處理15 d后野生型和轉(zhuǎn)基因擬南芥成活率、鮮重及葉片數(shù)。野生型和轉(zhuǎn)基因擬南芥在正常培養(yǎng)和100 mmol L-1NaCl處理15 d后統(tǒng)計(jì)鮮重、葉片數(shù)和成活率。數(shù)據(jù)采用Duncan法進(jìn)行差異顯著性檢驗(yàn)(*< 0.05, **< 0.01), 結(jié)果用平均值±標(biāo)準(zhǔn)誤表示, 以上實(shí)驗(yàn)均使用3次生物學(xué)重復(fù), 每次實(shí)驗(yàn)3次技術(shù)重復(fù)。

A: schematic diagram of seed distribution; B–C: phenotypes of wild type and transgeniccultivated under normal and salt stress for 15 days; D–E: fresh weight and leaf number of wild type and transgenicafter normal culture for 15 days; F–H: survival rate, fresh weight and leaf number of wild type and transgenicafter 100 mmol L-1NaCl treatment for 15 days. Duncan method was used to test the difference significance (*< 0.05, **< 0.01), the results were represented by mean (±standard), the above experiments were repeated three times biologically and three times technically in each experiment.

圖4 鹽脅迫下過量表達(dá)GhSAMDC1基因?qū)M南芥葉綠素含量影響

A~C: 鹽脅迫下野生型和轉(zhuǎn)基因株系葉綠素含量。選取100 mmol L-1NaCl處理15 d的擬南芥葉片, 檢測葉綠素和葉綠素含量。數(shù)據(jù)采用Duncan法進(jìn)行差異顯著性檢驗(yàn)(*< 0.05, **< 0.01), 結(jié)果用平均值±標(biāo)準(zhǔn)差表示, 以上實(shí)驗(yàn)均使用3次生物學(xué)重復(fù), 每次實(shí)驗(yàn)3次技術(shù)重復(fù)。

A–C: chlorophyll content of wild type and transgenic lines under salt stress. The leaves ofwere selected to detect the content of chlorophyllandafter treated with 100 mmol L-1NaCl for 15 days. Duncan method was used to test the difference significance (*< 0.05, **< 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment.

2.3 鹽脅迫下過量表達(dá)GhSAMDC1基因?qū)M南芥內(nèi)源多胺含量的影響

在鹽脅迫下, 野生型和轉(zhuǎn)基因擬南芥多胺含量均有明顯上升, 但轉(zhuǎn)基因株系上升更明顯。與野生型相比, 轉(zhuǎn)基因株系多胺總量上升42%~101%, Put含量略有上升, 但并無顯著性差異(除1-12外), 1-12 Put含量是野生型的4倍。Spd和Spm含量明顯上升,分別增加了15%~31%和42%~98% (圖5)。在鹽脅迫下, 轉(zhuǎn)基因株系、和的表達(dá)量明顯高于野生型(圖6)。這說明, 鹽脅迫下, 擬南芥通過積累多胺以抵御鹽脅迫, 而轉(zhuǎn)基因株系可以通過直接或間接誘導(dǎo)Spd和Spm合成相關(guān)基因的表達(dá)積累多胺, 尤其是Spd和Spm, 從而增強(qiáng)抗鹽能力。

圖5 鹽脅迫下過量表達(dá)GhSAMDC1基因?qū)M南芥內(nèi)源多胺含量的影響

A~D: 正常和鹽脅迫下野生型及轉(zhuǎn)基因株系內(nèi)源的多胺總量、Put、Spd和Spm含量對比。選取正常培養(yǎng)和100 mmol L–1NaCl處理15 d的擬南芥葉片, 利用高效液相色譜法檢測內(nèi)源多胺含量。數(shù)據(jù)采用Duncan法進(jìn)行差異顯著性檢驗(yàn)(*< 0.05, **< 0.01), 結(jié)果用平均值±標(biāo)準(zhǔn)差表示, 以上實(shí)驗(yàn)均使用3次生物學(xué)重復(fù), 每次實(shí)驗(yàn)3次技術(shù)重復(fù)。

A–D: comparison of total endogenous polyamines, Put, Spd, and Spm contents between wild type and transgenic lines under normal and salt stress conditions. The contents of endogenous polyamines inleaves were determined by high performance liquid chromatography (HPLC) after normal culture and treatment with 100 mmol L-1NaCl for 15 days. Duncan’s method was used to test the difference significance (*< 0.05, **< 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment.

圖6 鹽脅迫下過量表達(dá)GhSAMDC1基因?qū)M南芥內(nèi)源基因表達(dá)的影響

A~C: 野生型和轉(zhuǎn)基因擬南芥、和相對表達(dá)量。選取100 mmol L-1NaCl處理15 d的擬南芥葉片, 利用高效液相色譜法檢測內(nèi)源多胺含量, 數(shù)據(jù)采用Duncan法進(jìn)行差異顯著性檢驗(yàn)(*< 0.05, **< 0.01), 結(jié)果用平均值±標(biāo)準(zhǔn)差表示, 以上實(shí)驗(yàn)均使用3次生物學(xué)重復(fù), 每次實(shí)驗(yàn)3次技術(shù)重復(fù)。

A–C: relative expression of,, andin wild type and transgenic.The contents of endogenous polyamines inleaves were determined by high performance liquid chromatography (HPLC) after treated with 100 mmol L-1NaCl for 15 days. Duncan’s method was used to test the difference significance (*< 0.05, **< 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment.

2.4 鹽脅迫下過量表達(dá)GhSAMDC1基因?qū)M南芥內(nèi)源H2O2、MDA含量、離子滲透率、抗氧化酶活力及相對表達(dá)量的影響

鹽脅迫下, 各轉(zhuǎn)基因株系葉片中H2O2水平顯著減少, 降低40%~66% (圖7-A~B); MDA含量和離子滲透率也明顯低于野生型, 分別減少12%~ 40%和11%~25% (圖7-C~D)。與野生型相比, 轉(zhuǎn)基因株系CAT和SOD活力明顯上升(圖8-A~B), 但POD活力并未發(fā)生明顯變化(圖8-C),、和表達(dá)量的變化與其酶活變化趨勢相似, 轉(zhuǎn)基因株系表達(dá)均高于野生型(圖8-A~B),表達(dá)量與野生型無明顯差異(圖8-C)。這表明, 鹽脅迫下,基因不僅參與調(diào)節(jié)轉(zhuǎn)基因擬南芥內(nèi)源多胺含量, 且影響了H2O2和MDA的合成,基因可能通過調(diào)節(jié)多胺含量影響CAT、SOD和POD等酶的活力及表達(dá), 進(jìn)而通過消除活性氧的方式提高轉(zhuǎn)基因擬南芥的抗鹽能力。

圖7 鹽脅迫下過量表達(dá)GhSAMDC1基因?qū)M南芥葉片H2O2、MDA及離子滲透率影響

A: 鹽脅迫下野生型和轉(zhuǎn)基因株系DAB染色; B~D: 鹽脅迫下野生型和轉(zhuǎn)基因株系H2O2、MDA含量及離子滲透率; 選取100 mmol L-1NaCl處理15 d的擬南芥葉片, 分別進(jìn)行DAB染色, H2O2、MDA含量以及離子滲透率的檢測, 數(shù)據(jù)采用Duncan法進(jìn)行差異顯著性檢驗(yàn)(*< 0.05, **< 0.01), 結(jié)果用平均值±標(biāo)準(zhǔn)差表示, 以上實(shí)驗(yàn)均使用3次生物學(xué)重復(fù), 每次實(shí)驗(yàn)3次技術(shù)重復(fù)。

A: DAB staining of wild type and transgenic lines under salt stress; B–D: contents of H2O2, MDA and ion permeability of wild type and transgenic lines under salt stress. The leaves ofwere stained with DAB, and the contents of H2O2, MDA and ion permeability were measured after treated with 100 mmol L-1NaCl for 15 days. Duncan’s method was used to test the difference significance (*< 0.05, **< 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologically and three times technically in each experiment.

圖8 鹽脅迫下過量表達(dá)GhSAMDC1基因?qū)M南芥抗氧化酶活力和表達(dá)的影響

A~C: 鹽脅迫下野生型和轉(zhuǎn)基因株系CAT、SOD、POD活力; D~F: 鹽脅迫下野生型和轉(zhuǎn)基因株系CAT、SOD、POD表達(dá)分析。選取100 mmol L-1NaCl處理15 d的擬南芥葉片, 分別檢測CAT、SOD和POD的活力和相對表達(dá)量。數(shù)據(jù)采用Duncan法進(jìn)行差異顯著性檢驗(yàn)(*< 0.05, **< 0.01), 結(jié)果用平均值±標(biāo)準(zhǔn)差表示, 以上實(shí)驗(yàn)均使用3次生物學(xué)重復(fù), 每次實(shí)驗(yàn)3次技術(shù)重復(fù)。

A–C: enzyme activity of CAT, SOD, and POD in wild type and transgenic lines under salt stress; D-F: analysis of CAT, SOD, and POD expression in wild type and transgenic lines under salt stress. The leaves ofwere selected to detect the activity and relative expression of CAT, SOD, and POD after treated with 100 mmol L-1NaCl for 15 days, respectively. Duncan’s method was used to test the difference significance (*< 0.05, **< 0.01), the results were represented by mean (±standard deviation), the above experiments were repeated three times biologic ally and three times technically in each experiment.

3 討論

鹽脅迫能引起離子毒害和氧化脅迫, 從而導(dǎo)致植物生長減弱、失綠、萎蔫甚至死亡。目前關(guān)于多胺和鹽脅迫引起的氧化脅迫的關(guān)系的研究已經(jīng)取得了一些成果。S-腺苷甲硫氨酸脫羧酶是參與PAs生物合成的關(guān)鍵酶[37]。近年來, 不同物種基因被相繼克隆, 關(guān)于逆境脅迫下植株體內(nèi)多胺含量變化與抗逆性的關(guān)系已做了大量研究, 眾多研究發(fā)現(xiàn)基因可以通過增加Spd和Spm含量提高植物的抗逆能力[38-39]。本研究將棉花基因轉(zhuǎn)入擬南芥, 通過檢測內(nèi)源自由態(tài)多胺含量發(fā)現(xiàn), 轉(zhuǎn)基因株系Put含量減少, Spd、Spm含量明顯增加。此外, PGWB17-在擬南芥中的過量表達(dá)導(dǎo)致轉(zhuǎn)基因株系成活率、鮮重以及葉片數(shù)均高于野生型, 抗鹽能力明顯增強(qiáng)。鹽脅迫下植物體內(nèi)能否維持高含量的Spd與Spm是衡量其耐鹽性強(qiáng)弱的一個(gè)指標(biāo)[40]。Zapata等[41]發(fā)現(xiàn)在菠菜、香瓜、萵苣、辣椒、甘藍(lán)、甜菜、番茄中多胺含量隨著鹽脅迫而發(fā)生變化, 認(rèn)為在大多數(shù)情況下, Put含量的下降、Spd和Spm含量的上升或Spd+Spm/Put比值的增加能夠提高耐鹽性, 且抗性基因型的植物較敏感型的積累更多Spd和Spm, 而敏感型積累更多的Put[42]。這說明逆境脅迫下, 植物維持較高水平的Spd和Spm對提高其抗逆能力是非常重要的。100 mmol L-1NaCl處理下, 野生型擬南芥Spd和Spm含量明顯上升, 說明野生型擬南芥通過增加內(nèi)源自由態(tài)Spd和Spm含量以增加其抗鹽能力; 轉(zhuǎn)基因株系、(除1-12外)和表達(dá)均高于野生型, 內(nèi)源Spd和Spm含量進(jìn)一步增加。鹽脅迫下轉(zhuǎn)基因株系1-4和1-14 Put含量和野生型無顯著差異, 1-12株系Put含量是野生型的4倍, 這可能與1-12株系表達(dá)量較低有關(guān), 減少了Put向Spd的轉(zhuǎn)化, 但轉(zhuǎn)基因株系Spd含量的變化與的變化趨勢一致, Spm含量的變化與的變化趨勢相似,這表明轉(zhuǎn)基因擬南芥中Put向Spd和Spm轉(zhuǎn)化過程中和均發(fā)揮著重要作用?；蚩梢酝ㄟ^調(diào)節(jié)Spd和Spm合成相關(guān)基因的表達(dá)響應(yīng)鹽脅迫, 進(jìn)而積累更多的Spd和Spm, 直接或間接參與脅迫反應(yīng)。光合色素水平被認(rèn)為是評價(jià)植物耐鹽性的生化指標(biāo)[22]。研究表明, 鹽脅迫下轉(zhuǎn)基因株系葉綠素含量高于野生型, 表明轉(zhuǎn)基因系對鹽脅迫具有更強(qiáng)的耐受性。上述結(jié)果與前人關(guān)于過量表達(dá)基因提高抗鹽、寒冷、干旱的研究結(jié)果基本一致[38-39,43]。

ROS和MDA含量被認(rèn)為是氧化應(yīng)激的指標(biāo)。在脅迫條件下, calvin循環(huán)酶活性受到抑制, 所吸收的光能無法正常循環(huán), 促使ROS的產(chǎn)生[40]。植物具有有效的抗氧化防御系統(tǒng), SOD使超氧自由基歧化成H2O2, 隨后被CAT和POD清除[24], 通過清除活性氧的方式減少H2O2和DAB的積累, 降低離子滲透率, 保護(hù)細(xì)胞免受氧化損傷。本研究中, 在脅迫條件下, 轉(zhuǎn)基因擬南芥CAT和SOD活性和表達(dá)量均高于野生型; 轉(zhuǎn)基因擬南芥H2O2和MDA的積累也較少, 離子滲透率降低。這些結(jié)果表明, 轉(zhuǎn)基因擬南芥具有較高的抗氧化酶活性, 有助于它們更好地應(yīng)對逆境條件。非生物脅迫下, 多胺不僅可以穩(wěn)定分子的組成成分, 而且可以和多數(shù)蛋白結(jié)合, 從而維持細(xì)胞膜的完整性[42,44], 也能夠清除植物中的ROS[45], 減少脂質(zhì)過氧化, 保持膜的穩(wěn)定性, 以減少氧化脅迫引起的損傷[46]。因此, 過量表達(dá)PGWB17-誘導(dǎo)的鹽脅迫耐受性可能與、和的高表達(dá)水平有關(guān), 促進(jìn)了Put向Spd和Spm的轉(zhuǎn)化, 通過清除ROS的方式保護(hù)質(zhì)膜的完整性, 在增強(qiáng)植物抗逆性中發(fā)揮重要作用。

4 結(jié)論

基因能夠響應(yīng)鹽脅迫, 且參與了轉(zhuǎn)基因株系中多胺的合成。鹽脅迫下,基因通過提高Spd和Spm合成相關(guān)基因的表達(dá), 增加了Spd和Spm含量, 直接或間接提高抗氧化系統(tǒng)相關(guān)酶的活力, 通過清除H2O2等活性氧的方式, 保護(hù)細(xì)胞器、細(xì)胞質(zhì)膜、蛋白質(zhì)、脂質(zhì)和核酸等免受損傷, 提高擬南芥的抗鹽能力。這可能是植物應(yīng)對逆境, 提高抗逆性的一個(gè)重要方式。

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附圖1 300 mmol L-1 NaCl處理下棉花GhSAMDC1基因表達(dá)分析

0、1、3、6、12、24、48、72分別代表棉花幼苗(YZ-1)用300 mmol L-1NaCl處理0、1、3、6、12、24、48和72 h。培養(yǎng)30 d的棉花幼苗用300 mmol L-1NaCl處理, 分別在0、1、3、6、12、24、48和72 h取樣, 檢測不同處理時(shí)間段基因相對表達(dá)量。以上實(shí)驗(yàn)均使用3次生物學(xué)重復(fù), 每次實(shí)驗(yàn)3次技術(shù)重復(fù)。

0, 1, 3, 6, 12, 24, 48, and72 represent cotton seedlings (YZ-1) after normal culture for 30 days treated with 300 mmol L-1NaCl for 0, 1, 3, 6, 12, 24, 48, and 72 hours, respectively. Samples were taken at 0, 1, 3, 6, 12, 24, 48, and 72 hours of treatments to detect the relative expression ofin different treatment periods. The above experiments were repeated three times biologically and three times technically in each experiment.

Ectopic expression of S-adenosylmethionine decarboxylase () in cotton enhances salt tolerance in

TIAN Wen-Gang1, ZHU Xue-Feng1, SONG Wen1, CHENG Wen-Han2, XUE Fei1, and ZHU Hua-Guo1,*

1College of Agronomy, Shihezi University / Key Oasis Eco-Agriculture Laboratory of Production and Group, Shihezi 832003, Xinjiang, China;2Jingchu University of Technology, Jingmen 448000, Hubei, China

Transgenic() was used to study the effect of overexpression ofon salt tolerance ofseedlings, Contents of endogenous polyamines, hydrogen peroxide (H2O2), malondialdehyde (MDA), and chlorophyll, ion permeability, antioxidant enzymes (SOD, CAT, POD) activities and expression levels were investigated under salt stress. The overexpression ofdecreased the content of endogenous putrescine (Put) and increased spermidine (Spd) and spermine (Spm) contents in. Under salt stress, the expression levels of spermidine synthase (,) and spermine synthase () in transgenic lines were significantly higher than those in wild type, the contents of Spd and Spm were further increased, and the contents of H2O2, MDA, chlorophyll, and ion permeability were obviously decreased. Compared with the wild type, Transgenic lines had no remarkable difference in peroxidase (POD) activity, but significantly higher superoxide dismutase (SOD) and catalase (CAT) activities, with the same change trend as their expression levels. Therefore,increased the contents of Spd and Spm of transgenic plants by increasing the expression of genes related to Spd and Spm synthesis under salt stress, Spd and Spm directly or indirectly increased the activity of enzymes related to antioxidant system, and enhanced the salt tolerance ofby scavenging H2O2and other reactive oxygen species.

; cotton S-adenosylmethionine decarboxylase gene; salt stress; antioxidant enzyme

2018-11-05;

2019-01-19;

2019-03-15.

10.3724/SP.J.1006.2019.84142

朱華國, E-mail: 57530422@qq.com

E-mail: 631432853@qq.com

本研究由國家自然科學(xué)基金項(xiàng)目(31301363, 31660427), 新疆生產(chǎn)建設(shè)兵團(tuán)現(xiàn)代農(nóng)業(yè)科技攻關(guān)與成果轉(zhuǎn)化計(jì)劃項(xiàng)目(2015AC007)和湖北省自然科學(xué)基金項(xiàng)目(2017CFB162)資助。

This study was supported by the National Natural Science Foundation of China (31301363, 31660427), Science and Technology Development Program of Xinjiang Production and Construction Groups Project (2015AC007) and the Natural Science Foundation of Hubei Province (2017CFB162).

URL: http://kns.cnki.net/kcms/detail/11.1809.S.20190314.0901.002.html