异源表达棉花S-腺苷甲硫氨酸脱羧酶(GhSAMDC1)基因提高了拟南芥抗盐能力

田文刚 朱雪峰 宋 雯 程文翰 薛 飞 朱华国,*



异源表达棉花S-腺苷甲硫氨酸脱羧酶()基因提高了拟南芥抗盐能力

田文刚1朱雪峰1宋 雯1程文翰2薛 飞1朱华国1,*

1石河子大学农学院 / 新疆兵团绿洲生态农业重点实验室, 新疆石河子 832003;2荆楚理工学院, 湖北荆门 448000

以转基因拟南芥研究了过量表达基因对拟南芥幼苗抗盐能力的影响, 以及内源多胺、过氧化氢(H2O2)、丙二醛(MDA)、叶绿素含量(Chl)、离子渗透率、抗氧化酶(SOD、CAT、POD)活性和表达量在盐胁迫下的变化。结果表明, 过量表达基因能够减少拟南芥内源腐胺(Put)含量, 增加亚精胺(Spd)和精胺(Spm)含量。盐胁迫下, 转基因株系亚精胺合酶(、)和精胺合酶()基因表达量明显高于野生型, Spd和Spm含量进一步增加, H2O2、MDA、Chl以及离子渗透率显著降低; 与野生型相比, 过氧化物酶(POD)活力无明显差异, 但超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活力明显增加, 其表达水平与活力变化趋势基本一致。因此, 盐胁迫下,基因通过提高Spd和Spm合成相关基因的表达, 增加了转基因株系Spd和Spm含量, Spd和Spm直接或间接提高抗氧化系统相关酶的活力, 通过清除H2O2等活性氧的方式提高拟南芥的抗盐能力。

拟南芥; 棉花S-腺苷甲硫氨酸脱羧酶基因; 盐胁迫; 抗氧化酶

多胺(polyamine, PAs), 是生物代谢过程中产生的一类具有生物活性的低分子量脂肪族含氮碱, 广泛存在于原核及真核生物中。高等植物中常见的多胺主要包括腐胺(putrescine, Put)、亚精胺(spermidine, Spd)和精胺(spermine, Spm), 参与植物细胞分化、形态建成、程序凋亡、胁迫响应等生物学过程[1-2]。

目前植物体内多胺的代谢过程已经研究的比较清楚。研究发现, 多胺的生物合成起始于Put的合成, 而Put的合成涉及鸟氨酸(Ornithine)和精氨酸(Arginine) 2条路径。在动物和真菌中, Put主要由鸟氨酸经过鸟氨酸脱羧酶(ornithine decarboxylase, ODC)催化反应而来。而在植物和细菌中, Put主要由精氨酸经过精氨酸脱羧酶(arginine decarboxylase, ADC)、亚氨基脱氢酶(agmatine iminohydrolase, AIH)和N-氨甲酰基腐胺酰胺水解酶(N-carbamoyl putrescine amidohydrolase, CPA)三步催化反应而来[1]。Put合成以后作为前体物质在亚精胺合酶(spermidine synthase, SPDS)的催化下, 结合由S-腺苷甲硫氨酸脱羧酶(S-adenosylmethionine decarboxylase, SAMDC)催化S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)脱羧得到的反应产物氨丙基进而生成Spd。而Spd则进一步在精胺合酶(spermine synthases, SPMS)的催化下, 结合S-腺苷甲硫氨酸脱羧反应产物氨丙基进而生成Spm[3]。目前为止, 除了基因以外, 所有参与多胺合成的基因均已经在拟南芥中发现。

S-腺苷甲硫氨酸脱羧酶是调节多胺合成的关键限速酶。近年来已从辣椒[4]、曼陀罗[5]、番茄[6]、百脉根[7]、羊草[8]、棉花[9]、杜梨[10]、甘蔗[11]、高羊茅[12]等多种植物中克隆得到基因, 并证明基因可以通过调控植物体内多胺含量来影响植物应答逆境胁迫。过量表达羊草和辣椒基因通过积累更多的多胺提高拟南芥的耐寒、耐盐性以及抗旱能力[4,13]。番茄中导入酵母基因, Spd和Spm含量显著上升, 对高温胁迫表现出明显抗性[14]。相反, 在水稻中下调, 显著降低了的Spd和Spm含量, 多胺氧化酶(polyamine oxidase, PAO)活性下降, 降低了转基因水稻的生育力和对非生物胁迫的耐受性[15]。同时, 多胺参与逆境胁迫同样通过外施多胺的方法得以证明。外源Spm能够调节小麦幼苗过氧化氢酶(catalase, CAT)、谷胱甘肽还原酶(glutathione reductase, GR)、脱氢抗坏血酸还原酶(dehydroascorbate reductase, DHAR)、抗坏血酸过氧化物酶(ascorbic acid peroxidase, APX)和超氧化物歧化酶(superoxide dismutase, SOD)的活性, 并有效地调节它们的转录水平, 提高抗盐能力, 表明Spm在调节植物的抗氧化酶活性、减轻盐胁迫的氧化损伤中起关键作用[16]。在黄瓜中, 外源Spd可显著缓解幼苗的盐害, 提高幼苗的抗盐性[17], 通过降低脱镁叶绿酸A加氧酶(pheophorbide A oxygenase, PaO)途径相关酶活性和转录水平、减缓Chl分解代谢和增加Chl浓度来增强高温耐受性[18]。Liu等[19]通过研究发现, 在盐胁迫下, 大麦耐盐品种J4比敏感品种K97幼苗积累更多的Spd、Spm和少量的Put; 抗盐性较强的长春密刺比抗盐性较弱的津春2号根系积累更多的的Spd和Spm[20]。表明Put向Spd和Spm的转化, 并保持较高水平的Spd和Spm对植物耐盐性是非常重要的。

盐胁迫下, 植物细胞内离子平衡被破坏, 各代谢途径产生大量的活性氧(reactive oxygen species, ROS), 能迅速使酶失活, 破坏细胞器, 细胞质膜、蛋白质、脂质和核酸, 导致植物细胞死亡[21]。过量的H2O2对植物来说是致命的。在遭受盐胁迫时, 植物通过高效的抗氧化酶防御系统清除ROS来保护细胞免受氧化损伤[22-23]。SOD是植物细胞中清除活性氧自由基最重要的酶类之一, 将超氧化物自由基转变为H2O2, H2O2由CAT和POD清除[24], 其他细胞器中产生的H2O2进入过氧化体中也能够被CAT清除[25]。SOD、POD与CAT这3种酶相互协调, 使植物体内的ROS处于相对稳定的水平[26]。研究表明, 多胺同样参与酶抗氧化防御系统的调节, 主要通过直接或间接地调节抗氧化系统或抑制ROS生成来调节ROS的稳态。外施Spd能提高结缕草中SAMDC等相关合成酶活性,增加Spd和Spm含量, 且显著降低H2O2含量, 可以通过清除活性氧、稳定细胞结构、调节光保护机制、增加抗氧化酶蛋白活性和转录水平, 减轻盐胁迫引起的氧化损伤[27-28]; 外施Spm增加了多胺的积累、CAT和SOD活性的增强, 增加了拟南芥和水稻对渗透和盐胁迫耐受能力[29-30]。因此, 多胺在刺激植物生长、调节植物生长发育、控制形态建成、提高植物抗逆性和延缓衰老等诸多方面发挥着重要作用。

在盐胁迫下,的转录水平增加。为了确定盐诱导在盐胁迫应答中的作用, 利用Gateway技术克隆了PGWB17-, 利用转PGWB17-基因拟南芥探究基因功能, 通过检测抗氧化活性和多胺含量发现, PGWB17-的过量表达可以通过调节抗氧化酶活性增强转基因拟南芥的耐盐性。这对研究基因与多胺合成以及逆境胁迫下多胺含量与植物抗逆性的相互关系具有重要意义。

1 材料与方法

1.1 材料与试剂

野生型(col-0)及转基因拟南芥种子均由本实验室保存。多胺(Put、Spd、Spm)纯度≥99%, 甲醇为色谱纯, 购自Sigma公司; H2O2(货号A064)、MDA (货号A003-3)、CAT (货号A007-1)、SOD (货号A001-3)和POD (货号: A084-3)测定试剂盒均购自南京建成生物工程研究所; DAB染色(货号CW0125M)试剂盒均购自康为世纪生物科技有限公司; 高氯酸、氢氧化钠、氯化钠、苯甲酰氯、乙醚等均为国产分析纯。高效液相色谱仪为安捷伦1200型, 色谱柱为AgilentXDB-C18 (4.6 mm × 150.0 mm); 低温离心机、常温离心机及真空抽滤仪均为Eppendorf公司产品。

1.2 材料处理

将野生型及纯合的转基因拟南芥种子用2%的次氯酸钠消毒10 min, 然后用无菌水冲洗3~4次。用1 mL枪头分别点播于灭菌的1/2MS和1/2MS+100 mmol L-1NaCl培养基, 封口后置于4℃培养箱春化48 h, 然后放于人工气候室(22±1)℃, 16 h/8 h光照培养, 15 d后观察表型、取样, 备用。

1.3 多胺、叶绿素、H2O2含量测定及DAB染色

多胺含量测定和DAB染色参照程文翰等[31-32]的方法, 用高效液相色谱法测定多胺含量, DAB染色采用DAB染色试剂盒; 测定叶绿素含量参照李超等[33]的方法, 采用乙醇-丙酮混合液提取叶绿素; 测定H2O2含量采用H2O2含量测定试剂盒[34]。

1.4 离子渗透率测定

离子渗透率测定参考朱珍等[35]的方法, 略有改动。取100 mmol L-1NaCl处理15 d的拟南芥幼苗叶片各0.1 g, 蒸馏水洗涤后, 用滤纸吸干表面水分, 剪成1 cm小段放入去离子水清洗的烧杯(50 mL)中, 加去离子水至20 mL, 立即测定电导率0。室温下置水平震荡器上温和震荡2 h, 测定电导率1。煮沸10 min, 冷却至室温, 再次加去离子水至刻度, 测电导率2。

离子渗透率(%) = (1P0)/(2P0)×100

1.5 MDA含量测定

采用MDA含量测定试剂盒测定MDA含量。准确称取植物组织, 按质量体积比1∶9加入9倍体积的试剂五应用提取液(按试剂五∶蒸馏水=1∶9的比例配置), 将样品剪碎后用内切式匀浆机冰水浴匀浆, 8000~10,000 ×, 每次10~15 s, 共3~5次, 再将匀浆吸入到离心管中, 3500~4000 ×, 离心10 min, 取上清液, 根据操作表加样(具体方法参照说明书)。加样结束后, 盖上盖, 涡旋混匀器混匀, 95℃以上水浴20 min, 取出后流水冷却, 将酶标板空板在530 nm处扫描, 准确吸取0.25 mL各管反应液加入到新的96孔板中, 以酶标仪测定各孔吸光度(计算时要减去空板读数)。

样本浓度=植物组织重(g)/所加提取液的量(mL)

1.6 CAT、SOD和POD活力检测

1.6.1 CAT活力检测 准确称取植物组织, 按质量体积比1∶9加9倍体积的生理盐水, 冰浴条件下, 制备10%的组织匀浆, 2500×, 离心10 min, 取上清液再用生理盐水稀释成最佳浓度, 待测。按照操作表加样(具体方法参照说明书)。加样后混匀, 波长405 nm, 光径0.5 cm, 双蒸水调零, 测定各管吸光度值。

式中, 271*为斜率的倒数。

1.6.2 SOD活力检测 准确称取植物组织, 按质量体积比1∶9加入9倍体积的磷酸盐缓冲液(磷酸盐缓冲液: 0.1 mol L-1, pH 7.0~7.4), 将植物组织剪碎后用匀浆机制备植物组织匀浆, 3500 ×, 离心10 min即为10%匀浆上清液, 再用磷酸盐缓冲液稀释成不同浓度进行预实验。按照按照操作表加样(具体方法参照说明书)。加样结束后, 混匀, 37℃孵育20 min, 波长450 nm, 用酶标仪测吸光度值。

1.6.3 POD活力检测 准确称取植物组织, 按质量体积比1∶9加入9倍体积的匀浆介质(生理盐水或磷酸盐缓冲液0.1 mol L-1, pH 7.0~7.4), 冰浴条件下制备成10%的组织匀浆, 3500 ×, 离心10 min, 取上清液按照操作表加样(具体方法参照说明书)。加样后混匀, 3500 ×, 离心10 min, 取上清于波长420 nm处, 1 cm光径, 双蒸水调零, 测定各管吸光度值。

1.7 RNA提取及实时荧光定量PCR

提取RNA采用TransZol UP RNA提取试剂盒; 采用TransScriptAll-in-One First-Strand cDNA SynthesisSuperMix for qPCR试剂盒反转录, 并将反转录得到的cDNA模板稀释10倍待用; 定量PCR系统采用罗氏Light Cycler 480系统(Roche, Switzerland), 反应体系使用SYBR Premix Exkit (Takara, Japan)。PCR程序为95℃预热2 min; 94℃ 15 s, 56℃ 20 s, 72℃ 20 s, 40个循环。相对表达量的计算采用2–ΔΔCT法[36],基因作为内参(, AT3G18780)。用Primer Premier 5.0设计qRT-PCR过程所用到的引物(表1)。

表1 qRT-PCR用到的引物

1.8 数据处理

利用Microsoft Excel 2007和SPSS 19.0统计分析数据, 采用Duncan’s法进行差异显著性检验(<0.05,<0.01), 用平均值±标准差表示结果, 以上实验均使用3次生物学重复, 每次实验至少3次技术重复。

2 结果与分析

2.1 过量表达GhSAMDC1基因对拟南芥内源多胺含量的影响

在NCBI中选取氨基酸同源性较高的拟南芥、水稻和玉米的SAMDC氨基酸序列与氨基酸序列构建系统发育树, 4种植物SAMDC的氨基酸序列可以分为2类, 而与水稻亲缘关系较近(图1-A)。根据拟南芥的序列为探针, 在雷蒙德氏棉基因组数据库中Blast检索, 获得推定的棉花基因序列, 用特异性引物(正向5¢-CACCATGGAGCCTTCTCCTCGGT-3¢和反向5¢- CAAGATCGCTTCCGGAATG-3¢)进行聚合酶链反应(RT-PCR)扩增cDNA, 并用Gateway技术克隆到PGWB17载体中(图1-B)。通过电穿孔法将载体导入农杆菌GV3101。通过花浸渍转化拟南芥植株(col-0)。种子采自成熟植株, 在含卡那霉素50 mg L-1的1/2MS培养基中筛选。对卡那霉素抗性植物的后代进行了抗性分离分析。进一步培养具有3∶1分离比的植物的种子, 并再次分析所得后代以分离卡那霉素抗性, 鉴定T-DNA插入物为纯合体。

为了探究基因在多胺合成中发挥的作用, 选取3个表达量较高的T4代转基因株系(1-4、1-12、1-14)进行后续研究(图1-C~D)。通过检测野生型及转基因拟南芥叶片中内源多胺含量发现, 与野生型相比, 转基因株系中多胺总量明显上升, 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含量显著增加, 增加了79%~130% (图2)。说明过量表达基因能够改变拟南芥内源多胺含量,基因可能在Put向Spd和Spm转化中发挥重要作用。

2.2 过量表达GhSAMDC1基因对拟南芥的抗盐能力的影响

基因在盐胁迫下表达量明显升高(附图1)。为进一步探究对拟南芥抗盐能力的影响, 选取1-4、1-12和1-14作为研究对象。将纯合转基因种子与野生型置于含100 mmol L-1NaCl和不含NaCl的1/2MS培养基无菌培养(图3-A)。正常培养下, 转基因株系和野生型叶片数均为9~10片(图3-B~D), 鲜重较野生型有所增加, 增加了6%左右, 但无显著性差异(图3-E); 在100 mmol L-1NaCl处理下, 转基因株系生长明显更好, 成活率为80%左右, 而野生型成活率仅为55% (图3-C~F); 野生型拟南芥鲜重(30株)为0.08 g, 转基因株系鲜重为0.10~0.12 g (图3-G); 同时, 转基因株系叶片数为5.5片左右, 而野生型仅为4.5片(图3-H)。此外, 转基因拟南芥叶绿素含量明显高于野生型, 叶绿素总量、叶绿素和叶绿素均为野生型的1.03~1.10倍左右(图4)。表明转基因株系较野生型有更强的耐盐性, 过量表达基因能够有效提高拟南芥的抗盐能力。

图1 陆地棉GhSAMDC1和其他物种同源蛋白进化树分析及转基因鉴定

A: 陆地棉和其他物种同源蛋白进化树分析; B:表达载体; C: 转基因拟南芥DNA鉴定及表达分析, M、1、2、3、4、5分别为marker、转基因株系1-4、转基因株系1-12、转基因株系1-14、阳性对照、阴性对照, 目的基因片段大小为1035 bp; D: 转基因拟南芥表达分析。

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.

(图2)

A~D: 野生型和转基因株系内源的多胺总量、Put、Spd和Spm含量。选取正常培养30 d的拟南芥叶片, 利用高效液相色谱法检测内源多胺含量, 数据采用Duncan’s法进行差异显著性检验(*< 0.05, **< 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。

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.

图3 过量表达GhSAMDC1基因对拟南芥抗盐能力的影响

A: 种子分布示意图; B~C: 正常和盐胁迫下培养15 d后野生型及转基因拟南芥表型; D~E: 正常培养15 d后野生型和转基因拟南芥叶片数及鲜重; F~H: 100 mmol L-1NaCl处理15 d后野生型和转基因拟南芥成活率、鲜重及叶片数。野生型和转基因拟南芥在正常培养和100 mmol L-1NaCl处理15 d后统计鲜重、叶片数和成活率。数据采用Duncan法进行差异显著性检验(*< 0.05, **< 0.01), 结果用平均值±标准误表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。

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.

图4 盐胁迫下过量表达GhSAMDC1基因对拟南芥叶绿素含量影响

A~C: 盐胁迫下野生型和转基因株系叶绿素含量。选取100 mmol L-1NaCl处理15 d的拟南芥叶片, 检测叶绿素和叶绿素含量。数据采用Duncan法进行差异显著性检验(*< 0.05, **< 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。

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 盐胁迫下过量表达GhSAMDC1基因对拟南芥内源多胺含量的影响

在盐胁迫下, 野生型和转基因拟南芥多胺含量均有明显上升, 但转基因株系上升更明显。与野生型相比, 转基因株系多胺总量上升42%~101%, Put含量略有上升, 但并无显著性差异(除1-12外), 1-12 Put含量是野生型的4倍。Spd和Spm含量明显上升,分别增加了15%~31%和42%~98% (图5)。在盐胁迫下, 转基因株系、和的表达量明显高于野生型(图6)。这说明, 盐胁迫下, 拟南芥通过积累多胺以抵御盐胁迫, 而转基因株系可以通过直接或间接诱导Spd和Spm合成相关基因的表达积累多胺, 尤其是Spd和Spm, 从而增强抗盐能力。

图5 盐胁迫下过量表达GhSAMDC1基因对拟南芥内源多胺含量的影响

A~D: 正常和盐胁迫下野生型及转基因株系内源的多胺总量、Put、Spd和Spm含量对比。选取正常培养和100 mmol L–1NaCl处理15 d的拟南芥叶片, 利用高效液相色谱法检测内源多胺含量。数据采用Duncan法进行差异显著性检验(*< 0.05, **< 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。

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.

图6 盐胁迫下过量表达GhSAMDC1基因对拟南芥内源基因表达的影响

A~C: 野生型和转基因拟南芥、和相对表达量。选取100 mmol L-1NaCl处理15 d的拟南芥叶片, 利用高效液相色谱法检测内源多胺含量, 数据采用Duncan法进行差异显著性检验(*< 0.05, **< 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。

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 盐胁迫下过量表达GhSAMDC1基因对拟南芥内源H2O2、MDA含量、离子渗透率、抗氧化酶活力及相对表达量的影响

盐胁迫下, 各转基因株系叶片中H2O2水平显著减少, 降低40%~66% (图7-A~B); MDA含量和离子渗透率也明显低于野生型, 分别减少12%~ 40%和11%~25% (图7-C~D)。与野生型相比, 转基因株系CAT和SOD活力明显上升(图8-A~B), 但POD活力并未发生明显变化(图8-C),、和表达量的变化与其酶活变化趋势相似, 转基因株系表达均高于野生型(图8-A~B),表达量与野生型无明显差异(图8-C)。这表明, 盐胁迫下,基因不仅参与调节转基因拟南芥内源多胺含量, 且影响了H2O2和MDA的合成,基因可能通过调节多胺含量影响CAT、SOD和POD等酶的活力及表达, 进而通过消除活性氧的方式提高转基因拟南芥的抗盐能力。

图7 盐胁迫下过量表达GhSAMDC1基因对拟南芥叶片H2O2、MDA及离子渗透率影响

A: 盐胁迫下野生型和转基因株系DAB染色; B~D: 盐胁迫下野生型和转基因株系H2O2、MDA含量及离子渗透率; 选取100 mmol L-1NaCl处理15 d的拟南芥叶片, 分别进行DAB染色, H2O2、MDA含量以及离子渗透率的检测, 数据采用Duncan法进行差异显著性检验(*< 0.05, **< 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。

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.

图8 盐胁迫下过量表达GhSAMDC1基因对拟南芥抗氧化酶活力和表达的影响

A~C: 盐胁迫下野生型和转基因株系CAT、SOD、POD活力; D~F: 盐胁迫下野生型和转基因株系CAT、SOD、POD表达分析。选取100 mmol L-1NaCl处理15 d的拟南芥叶片, 分别检测CAT、SOD和POD的活力和相对表达量。数据采用Duncan法进行差异显著性检验(*< 0.05, **< 0.01), 结果用平均值±标准差表示, 以上实验均使用3次生物学重复, 每次实验3次技术重复。

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 讨论

盐胁迫能引起离子毒害和氧化胁迫, 从而导致植物生长减弱、失绿、萎蔫甚至死亡。目前关于多胺和盐胁迫引起的氧化胁迫的关系的研究已经取得了一些成果。S-腺苷甲硫氨酸脱羧酶是参与PAs生物合成的关键酶[37]。近年来, 不同物种基因被相继克隆, 关于逆境胁迫下植株体内多胺含量变化与抗逆性的关系已做了大量研究, 众多研究发现基因可以通过增加Spd和Spm含量提高植物的抗逆能力[38-39]。本研究将棉花基因转入拟南芥, 通过检测内源自由态多胺含量发现, 转基因株系Put含量减少, Spd、Spm含量明显增加。此外, PGWB17-在拟南芥中的过量表达导致转基因株系成活率、鲜重以及叶片数均高于野生型, 抗盐能力明显增强。盐胁迫下植物体内能否维持高含量的Spd与Spm是衡量其耐盐性强弱的一个指标[40]。Zapata等[41]发现在菠菜、香瓜、莴苣、辣椒、甘蓝、甜菜、番茄中多胺含量随着盐胁迫而发生变化, 认为在大多数情况下, Put含量的下降、Spd和Spm含量的上升或Spd+Spm/Put比值的增加能够提高耐盐性, 且抗性基因型的植物较敏感型的积累更多Spd和Spm, 而敏感型积累更多的Put[42]。这说明逆境胁迫下, 植物维持较高水平的Spd和Spm对提高其抗逆能力是非常重要的。100 mmol L-1NaCl处理下, 野生型拟南芥Spd和Spm含量明显上升, 说明野生型拟南芥通过增加内源自由态Spd和Spm含量以增加其抗盐能力; 转基因株系、(除1-12外)和表达均高于野生型, 内源Spd和Spm含量进一步增加。盐胁迫下转基因株系1-4和1-14 Put含量和野生型无显著差异, 1-12株系Put含量是野生型的4倍, 这可能与1-12株系表达量较低有关, 减少了Put向Spd的转化, 但转基因株系Spd含量的变化与的变化趋势一致, Spm含量的变化与的变化趋势相似,这表明转基因拟南芥中Put向Spd和Spm转化过程中和均发挥着重要作用。基因可以通过调节Spd和Spm合成相关基因的表达响应盐胁迫, 进而积累更多的Spd和Spm, 直接或间接参与胁迫反应。光合色素水平被认为是评价植物耐盐性的生化指标[22]。研究表明, 盐胁迫下转基因株系叶绿素含量高于野生型, 表明转基因系对盐胁迫具有更强的耐受性。上述结果与前人关于过量表达基因提高抗盐、寒冷、干旱的研究结果基本一致[38-39,43]。

ROS和MDA含量被认为是氧化应激的指标。在胁迫条件下, calvin循环酶活性受到抑制, 所吸收的光能无法正常循环, 促使ROS的产生[40]。植物具有有效的抗氧化防御系统, SOD使超氧自由基歧化成H2O2, 随后被CAT和POD清除[24], 通过清除活性氧的方式减少H2O2和DAB的积累, 降低离子渗透率, 保护细胞免受氧化损伤。本研究中, 在胁迫条件下, 转基因拟南芥CAT和SOD活性和表达量均高于野生型; 转基因拟南芥H2O2和MDA的积累也较少, 离子渗透率降低。这些结果表明, 转基因拟南芥具有较高的抗氧化酶活性, 有助于它们更好地应对逆境条件。非生物胁迫下, 多胺不仅可以稳定分子的组成成分, 而且可以和多数蛋白结合, 从而维持细胞膜的完整性[42,44], 也能够清除植物中的ROS[45], 减少脂质过氧化, 保持膜的稳定性, 以减少氧化胁迫引起的损伤[46]。因此, 过量表达PGWB17-诱导的盐胁迫耐受性可能与、和的高表达水平有关, 促进了Put向Spd和Spm的转化, 通过清除ROS的方式保护质膜的完整性, 在增强植物抗逆性中发挥重要作用。

4 结论

基因能够响应盐胁迫, 且参与了转基因株系中多胺的合成。盐胁迫下,基因通过提高Spd和Spm合成相关基因的表达, 增加了Spd和Spm含量, 直接或间接提高抗氧化系统相关酶的活力, 通过清除H2O2等活性氧的方式, 保护细胞器、细胞质膜、蛋白质、脂质和核酸等免受损伤, 提高拟南芥的抗盐能力。这可能是植物应对逆境, 提高抗逆性的一个重要方式。

[1] Alcázar R, Altabella T, Marco F, Bortolotti C, Reymond M, Koncz C, Carrasco P, Tiburcio A F. Polyamines: molecules with regulatory functions in plant abiotic stress tolerance., 2010, 231: 1237–1249.

[2] Kusano T, Berberich T C, Takahashi Y. Polyamines: essential factors for growth and survival., 2008, 228: 367–381.

[3] Slocum R D, Kaur-Sawhney R, Galston A W. The physiology and biochemistry of polyamines in plants., 1984, 235: 283–303.

[4] Soo W, Soo K, Woo K, Ky P. Constitutive S-adenosylmethionine decarboxylase gene expression increases drought tolerance through inhibition of reactive oxygen species accumulation in., 2014, 239: 979–988.

[5] Torrigiani P, Scaramagli S, Ziosi V, Mayer M, Biondi S. Expression of an antisenseS-adenosylmethionine decarboxylase cDNA in tobacco: changes in enzyme activity, putrescine-spermidine ratio, rhizogenic potential, and response to methyl jasmonate., 2005, 162: 559–571.

[6] Sinha R, Rajam M V. RNAi silencing of three homologues of S-adenosylmethionine decarboxylase gene in tapetal tissue of tomato results in male sterility., 2013, 82: 169–180.

[7] 路玉兰, 孙艳香, 冯雪, 赵学良. 百脉根S-腺苷甲硫氨酸脱羧酶基因克隆与表达分析. 华北农学报, 2013, 28(2): 78–85. Lu Y L, Sun Y X, Feng X, Zhao X L. Cloning and expression analysis of S-adenosylmethionine decarboxylase gene fromL, 2013, 28(2): 78–85 (in Chinese with English abstract).

[8] Peng X J, Zhang L X, Zhang L X, Liu Z J, Cheng L Q, Yang Y, Shen S H, Chen S Y, Liu G S. The transcriptional factorcooperates withto contribute to salt tole­rance in., 2013, 113: 245–256.

[9] 王凡龙, 朱华国, 程文翰, 刘永昌, 成新琪, 孙杰. 棉花S-腺苷蛋氨酸脱羧酶基因()的克隆及其诱导表达分析. 棉花学报, 2015, 27: 176–183. Wang F L, Zhu H G, Cheng W H, Liu Y C, Cheng X Q, Sun J. Cloning and induced expression analysis ofin cotton (L)., 2015, 27: 176–183 (in Chinese with English abstract).

[10] 张梅, 王然, 马春晖, 段艳欣, 李鼎立. 杜梨S-腺苷甲硫氨酸脱羧酶基因的克隆与生物信息学分析. 华北农学报, 2013, 28(1): 82–87. Zhang M, Wang R, Ma C H, Duan Y X, Li D L. Cloning and bioinformatics analysis of S-adenosylmethionine decarboxylase gene inBge., 2013, 28(1): 82–87 (in Chinese with English abstract).

[11] 文乐, 黄诚梅, 邓智年, 曹辉庆, 魏源文, 李楠, 吴凯朝. 甘蔗S-腺苷蛋氨酸脱羧酶基因的克隆和表达分析. 南方农业学报, 2015, 46: 1931–1936. Wen Y, Huang C M, Deng Z N, Cao H Q, Li N, Wu K C. Molecular cloning of sugarcane S-adenosylmethionine decarboxylase gene () and its expression analysis., 2015, 46: 1931–1936 (in Chinese with English abstract).

[12] 王小利, 刘晓霞, 王舒颖, 杨义成, 吴佳海. 高羊茅腺苷甲硫氨酸脱羧酶基因的克隆与差异表达分析. 草业学报, 2011, 20(4): 169–179. Wang X L, Liu X X, Wang S Y, Yang Y C, Wu J H. Cloning and differential expression analysis of S-adenosylmethionine decarboxylase genein tall fescue., 2011, 20(4): 169–179 (in Chinese with English abstract).

[13] Liu Z J, Liu P P, Qi D M, Peng X J, Liu G S. Enhancement of cold and salt tolerance of Arabidopsis by transgenic expression of the S-adenosylmethionine decarboxylase gene from., 2017, 211: 90–99.

[14] Cheng L, Zou Y J, Ding S L, Zhang J J, Yu X L, Cao J S, Lu G. Polyamine accumulation in transgenictomato enhances the tolerance to high temperature stress., 2009, 51: 489–499.

[15] Chen M, Chen J J, Fang J Y, Guo Z F, Lu S Y. Down-regulation of S-adenosylmethionine decarboxylase genes results in reduced plant length, pollen viability, and abiotic stress tolerance., 2014, 116: 311–322.

[16] Elsayed A I, Rafudeen M S, El-hamahmy M A M, Odero D C, Sazzad H M. Enhancing antioxidant systems by exogenous spermine and spermidine in wheat () seedlings exposed to salt stress., 2018, 45: 745, doi: 10.1071/FP17127.

[17] Wu J Q, Shu S, Li C C, Sun J, Guo S R. Spermidine-mediated hydrogen peroxide signaling enhances the antioxidant capacity of salt-stressed cucumber roots., 2018, 128: 152–162.

[18] Zhou H, Guo S R, An Y H, Shan X, Wang Y, Shu S, Sun J. Exogenous spermidine delays chlorophyll metabolism in cucumber leaves (L.) under high temperature stress., 2016, 38: 224.

[19] Liu J, Yu B J, Liu Y L. Effects of spermidine and spermine levels on salt tolerance associated with tonoplast H+-ATPase and H+-PPase activities in barley roots., 2006, 49: 119–126.

[20] Duan J J, Guo S R, Fan H F, Wang S P, Kang Y Y. Effects of salt stress on proline and polyamine metabolisms in the roots of cucumber seedlings., 2006, 26: 2486–2492.

[21] Halliwell B. Reactive species and antioxidants. Redox biology is a fundamental theme of aerobic life., 2006, 141: 312–322.

[22] Ma C Q, Wang Y G, Gu D, Nan J D, Chen S X, Li H Y. Overexpression of S-adenosyl-l-methionine synthetase 2 from sugar beet M14 increasedtolerance to salt and oxidative stress., 2017, 18: e847.

[23] Li J M, Hu L P, Zhang L, Pan X B, Hu X H. Exogenous spermidine is enhancing tomato tolerance to salinity-alkalinity stress by regulating chloroplast antioxidant system and chlorophyll metabolism., 2015, 15: 303, doi: 10.1186/s12870- 015-0699-7.

[24] Asada K. THE WATER-WATER CYCLE IN CHLOROPLASTS: Scavenging of active oxygens and dissipation of excess photons., 1999, 50: 601–639.

[25] Williamson G B, Richardson D. Bioassays for allelopathy: Measuring treatment responses with independent controls., 1988, 14: 181–187.

[26] Zhao F Y, Guo S L, Zhang H, Zhao Y X. Expression of yeast SOD2, in transgenic rice results in increased salt tolerance., 2006, 170: 216–224.

[27] Puyang X H, An M Y, Han L B, Zhang X Z. Protective effect of spermidine on salt stress induced oxidative damage in two Kentucky bluegrass (L) cultivars., 2015, 117: 96–106.

[28] Li S, Han J, Qiang Z. The effect of exogenous spermidine concentration on polyamine metabolism and salt tolerance in Zoysiagrass (Steud) subjected to short-term salinity stress., 2016, 7: 1221, doi: 10.3389/fpls. 2016.01221.

[29] Radhakrishnan R. Ameliorative effects of spermine against osmotic stress through antioxidants and abscisic acid changes in soybean pods and seeds., 2013, 35: 263–269.

[30] Zrig A, Tounelti T, Vadel A M, Mohamed H B, Valero D, Serrano M, Chtara C, Khemira H. Possible involvement of polyphenols and polyamines in salt tolerance of almond rootstocks., 2011, 49: 1313–1322.

[31] 程文翰, 朱华国, 李鹏飞, 王凡龙, 朱守鸿, 赵兰杰, 郭丽雪, 孙杰. 棉花多胺HPLC的测定方法优化及其在体细胞胚胎发生过程中的变化规律. 棉花学报, 2014, 26: 138–144. Cheng W H, Zhu H G, Li P F, Wang F L, Zhu S H, Zhao L J, Guo L X, Sun J. Method optimization of polyamine content by high-performance liquid chromatography and its changes in the process of somatic embryogenesis in cotton., 2014, 26: 138–144 (in Chinese with English abstract).

[32] Cheng W H, Wang F H, Cheng X Q, Zhu Q H, Sun Y Q, Zhu H G, Sun J. Polyamine and its metabolite H2O2play a key role in the conversion of embryogenic callus into somatic embryos in upland cotton (L.)., 2015, 6: 1063, doi: 10.3389/fpls.2015.01063.

[33] Li C, Zhang Y N, Zhang K, Guo D L, Cui B M, Wang X Y, Huang X Z. Promoting flowering, lateral shoot outgrowth, leaf development, and flower abscission in tobacco plants overexpressing cotton FLOWERING LOCUS T (FT)-like gene., 2015, 6: 454, doi: 10.3389/fpls.2015.00454.

[34] Crumbliss A L, Perine S C, Stonehuerner J, Tubergen K R, Zhao J, Henkens R W, Q’Daly J P. Colloidal gold as a biocompatible immobilization matrix suitable for the fabrication of enzyme electrodes by electrodeposition., 1992, 40: 483–490.

[35] 朱珍. 赤霉素调控采后番茄果实抗冷机制研究. 中国农业科学院硕士学位论文, 北京, 2016. Zhu Z. The Mechanism of Gibberellins in Regulation of Chilling Tolerance of Postharvest Tomato Fruit. MS Thesis of Chinese Academy of Agricultural Sciences, Beijing, China, 2016 (in Chinese with English abstract).

[36] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCTmethod., 2001, 25: 402–408.

[37] Hao Y J, Zhang Z L, Kitashiba H, Honda C, Ubi B, Kita M, Moriguchi T. Molecular cloning and functional characterization of two apple S-adenosylmethionine decarboxylase genes and their different expression in fruit development, cell growth and stress responses., 2005, 350: 41–50.

[38] Roy M, Wu R. Overexpression of S-adenosylmethionine decarboxylase gene in rice increases polyamine level and enhances sodium chloride-stress tolerance., 2002, 163: 987–992.

[39] Waie B, Rajam M V. Effect of increased polyamine biosynthesis on stress responses in transgenic tobacco by introduction of human S-adenosylmethionine gene., 2003, 164: 722–734.

[40] Sanchez D H, Cuevas J C, Chiesa M A, Ruiz O A. Free spermidine and spermine content inunder long-term salt stress., 2005, 168: 541–546.

[41] Zapata P J, Serrano M, Pretel M T, Amoros A, Botella M A. Polyamines and ethylene changes during germination of different plant species under salinity., 2004, 167: 781–788.

[42] Liu H P, Dong B H, Zhang Y Y, Liu Z P, Liu Y L. Relationship between osmotic stress and the levels of free, conjugated and bound polyamines in leaves of wheat seedlings., 2004, 166: 1261–1267.

[43] Wi S J, Kim W T, Park K Y. Overexpression of camation-adenosylmethionine decarboxylase gene generate a broad- spectrum tolerance to abiotic stresses in transgenenic tobacco plants., 2006, 25: 1111–1121.

[44] Liu H P, Zhu Z X, Liu Y L. Response of bound polyamines in the thylakoid membrane of wheat seedling to osmotic stress., 2007, 26: 58–60.

[45] Ha H C, Sirisoma N S, Kuppusamy P, Zweier J L, Woster P M, Casero R A. The natural polyamine spermine functions directly as a free radical scavenger., 1998, 95: 11140–11145.

[46] Tiburcio A F, Besford R T, Capell T, Borrell A, Testillano P S, Risueno M C. Mechanisms of polyamine action during senescence responses induced by osmotic stress., 1994, 45: 1789–1800.

附图1 300 mmol L-1 NaCl处理下棉花GhSAMDC1基因表达分析

0、1、3、6、12、24、48、72分别代表棉花幼苗(YZ-1)用300 mmol L-1NaCl处理0、1、3、6、12、24、48和72 h。培养30 d的棉花幼苗用300 mmol L-1NaCl处理, 分别在0、1、3、6、12、24、48和72 h取样, 检测不同处理时间段基因相对表达量。以上实验均使用3次生物学重复, 每次实验3次技术重复。

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

朱华国, E-mail: 57530422@qq.com

E-mail: 631432853@qq.com

本研究由国家自然科学基金项目(31301363, 31660427), 新疆生产建设兵团现代农业科技攻关与成果转化计划项目(2015AC007)和湖北省自然科学基金项目(2017CFB162)资助。

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