棉属野生种旱地棉苏氨酸醛缩酶基因<italic>GarTHA</italic> 的克隆及功能验证

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范昕琦, 刘章伟, 冯娟, 徐鹏, 张香桂, 沈新莲. 棉属野生种旱地棉苏氨酸醛缩酶基因GarTHA 的克隆及功能验证 . 作物学报, 2013, 39(11): 1962-1969[FAN Xin-Qi, LIU Zhang-Wei, FENG Juan, XU Peng, ZHANG Xiang-Gui, SHEN Xin-Lian. Cloning and Functional Analysis ofGarTHA Gene fromGossypium aridum . 作物学报, 2013, 39(11): 1962-1969] 复制到剪切板

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棉属野生种旱地棉苏氨酸醛缩酶基因GarTHA 的克隆及功能验证

范昕琦, 刘章伟, 冯娟, 徐鹏, 张香桂, 沈新莲^*

江苏省农业科学院经济作物研究所 / 农业部长江下游棉花与油菜重点实验室, 江苏南京210014

^*通讯作者(Corresponding author): 沈新莲, E-mail:xlshen68@126.com

收稿日期:2013-06-09

基金:本研究由国家转基因生物新品种培育科技重大专项(2011ZX08005-004-002)和江苏省农业科技自主创新基金项目(CX(11)1201)资助。

摘要

盐胁迫是影响作物生长和发育的重要因素之一。一些棉属野生种具有较好的耐盐性, 是开展棉花耐盐性机制研究以及改良陆地棉耐盐性的重要资源。本研究基于cDNA-AFLP技术分离获得的旱地棉(Gossypium aridum )盐胁迫下差异表达片段序列信息, 经电子克隆技术和RT-PCR方法克隆了旱地棉苏氨酸醛缩酶基因cDNA全长, 命名为GarTHA (GenBank登录号为KC167360)。该cDNA全长为1018 bp, 包含一个822 bp的完整ORF, 编码273个氨基酸残基, 蛋白质分子量为82.57 kD, 等电点为4.89。GarTHA 基因与杨树PtTHA 基因同源性最高, 为84.6%。为进一步验证其功能, 利用拟南芥逆境胁迫启动子rd29A构建植物表达载体, 将GarTHA 基因的完整ORF转入拟南芥中, 获得转基因植株并进行了耐盐性鉴定。结果表明, 在盐胁迫下转基因拟南芥种子的发芽率明显高于野生型, 且转基因植株的根长显著高于野生型。说明GarTHA 基因可能参与植物的盐胁迫反应, 从而提高植物抗逆性。

关键词: 旱地棉; 苏氨酸醛缩酶; 耐盐性; 结构特征; 功能分析

Cloning and Functional Analysis ofGarTHA Gene fromGossypium aridum

FAN Xin-Qi, LIU Zhang-Wei, FENG Juan, XU Peng, ZHANG Xiang-Gui, SHEN Xin-Lian^*

Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences / Key Laboratory of Cotton and Rapeseed (Nanjing), Ministry of Agriculture, Nanjing 210014, China

Abstract

Salinity stress is one of the most important factors that impede the growth and development of various crops. SomeGossypium wild species with tolerance to high salinity are valuable germplasm resource for studying salt tolerance mechanism inGossypium and improving salinity resistance in upland cotton. In the previous study, we obtained a differential expression fragment by comparing differential expression transcript under stress treatment in diploidG. aridum species using cDNA-AFLP technique. In this study, using the partial cDNA sequences as queries, theGossypium EST database was screened and the corresponding cDNA sequence containing a complete ORF was assembled. As a result, a novel gene, encodingG. aridum threonine aldolase, was cloned. The gene was designated asGarTHA (G. aridum THA ; GenBank accession number: KC167360). The ORF ofGarTHA was 822 bp encoding 273 amino acid residues with a predicted molecular weight of 82.57 kD and a predicted isoelectric point of 4.89. TheGarTHA showed the highest similarity of 84.6% with poplarPtTHA . To characterize its putative function, we transformed the ORF ofGarTHA gene driven by rd29A promoter intoArabidopsis . The growth of transgenic plants was observed under salinity stress. The seed germination rate and root length of transgenicArabidopsis were significantly higher than those of the wild type plants under salt stress. The results showed thatGarTHA gene could improve salt resistance in plants.

Keyword: Gossypium aridum; Threonine aldolase; Salt tolerance; Structural characterization; Functional analysis

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引言

土壤盐渍化是影响农业生产的一个重要因素, 世界上约有3.33亿公顷土地具有不同程度的盐渍化。我国现有盐渍土面积约34.70×10⁶hm², 有17.33×10⁶hm²盐碱荒地尚待开发^[1]。棉花属中等耐盐作物, 是盐碱地先锋作物。近年来, 随着人口增加、耕地面积减少, 粮棉油安全供给的矛盾日益突出。进一步发掘棉花抗胁迫能力, 将棉花生产向沿海滩涂及内陆盐碱地转移, 对缓解粮棉争地矛盾, 稳定植棉面积, 实现农业生产的可持续发展具有十分重要的意义。

从棉花中克隆耐盐相关基因、开展耐盐机制研究是改良棉花耐盐性的重要基础。植物中与盐胁迫相关的基因主要有以下几类, 即与信号转导途径相关的基因^{[2,3,4,5,6,7]}, 与离子转运及离子平衡相关的基因^[8,9], 参与渗透保护物质的生物合成类基因, 主要涉及水分胁迫如水通道蛋白^[10], 胚胎发育晚期丰富蛋白(LEA类蛋白)^{[11,12,13,14]}以及抗氧化胁迫如超氧化物歧化酶(SOD)、过氧化氢酶、抗坏血酸、谷胱甘肽合成酶基因等^{[15,16,17,18,19]}。

甜菜碱是植物抗非生物胁迫中一类重要的渗透保护性物质。当遇盐碱或水分胁迫时, 细胞质中积累大量有机渗透调节剂甜菜碱, 而将细胞质中的无机渗透调节剂被挤向液泡, 维持胞质与细胞内液泡渗透平衡, 从而避免了细胞质高浓度无机离子对酶和代谢的毒害。盐胁迫下植物体内甜菜碱的积累是一种有利于植物在胁迫下生长的重要生理现象, 其含量与植物耐盐性呈正相关^{[20,21,22,23]}。研究表明苏氨酸醛缩酶是由苏氨酸至甜菜碱生物合成过程中的关键酶之一。在高等植物中, 苏氨酸醛缩酶普遍存在, 它参与高等植物的氮素代谢过程, 苏氨酸在其作用下, 裂解成甘氨酸和乙醛, 乙醛可氧化成乙酸再生成乙酰辅酶A, 而乙酰辅酶A则参与了脂类的合成。甘氨酸在甘氨酸N-甲基化转移酶的作用下, 经过三步甲基化作用合成甜菜碱, 从而提高植株的抗逆性^[24,25,26]。

目前植物中有关苏氨酸醛缩酶在耐盐机制中作用的研究较少^[27], 在棉花中关于该基因的研究尚未见相关报道。在前期的研究中我们利用cDNA-AFLP技术从棉属野生种旱地棉( Gossypium aridum )中克隆了一个苏氨酸醛缩酶基因片段^[28], 本研究拟利用电子克隆及RT-PCR技术克隆该基因的ORF全长序列, 分析该基因在盐胁迫下的功能, 为棉花耐盐性的改良提供新的基因资源。

1 材料与方法

1.1 试验材料及盐处理方法

试验材料棉属野生种旱地棉( G. aridum )种子由中国农业科学院棉花研究所海南岛野生棉种植园提供。将旱地棉种子播于盆钵中, 待长至4~5片真叶时将小苗从土中取出, 洗净根部泥土, 水培缓苗3~5 d。然后用含200 mmol L^-1 NaCl (其中[Na⁺]∶[Ca²⁺]=15∶1)的营养液处理, 分别在处理0、1、3、6、12、24和48 h后, 取叶片以液氮速冻于-70℃保存备用。

拟南芥种子( Arabidopsis thaliana , wild-type, Col-0)由本实验室保存。将消毒灭菌处理的拟南芥种子播于1/2MS (含1.5%的蔗糖)培养基上, 4℃, 黑暗培养2~3 d后转移到培养箱培养(16 h光照/8 h黑暗) 5~7 d。再将一周龄幼苗转移到灭菌基质(草炭土∶蛭石=1∶1)中在22℃/18℃, 16 h光照/8 h黑暗下培养, 待植株长出花蕾时采用浸花法转化^[29]。下文所提到拟南芥植物的正常培养都是在22℃光照16 h/18℃黑暗8 h的条件下。

1.2 总RNA的提取

用改良的CTAB法提取旱地棉叶片RNA^[30], 采用TRIzol试剂盒提取拟南芥RNA。

1.3 目的基因的克隆

以本实验室通过cDNA-AFLP技术获得的一个苏氨酸醛缩酶基因片段(153 bp)为探针^[28], 利用电子克隆技术, 在NCBI中用BLAST程序(http://www. ncbi.nlm.nih.gov/genomes/PLANTS/PlantESTBLAST.shtml)对棉属的EST数据库进行Blastn比对, 检索出所有与查询探针高度同源的棉属EST序列(CD486154、DT567433、DN799983、DR463228、DR463773、DW477047、DW477048、DW513781、DW513782、ES828657、CO098604.1、CO090746.1、CO090745.1、FF403743.1、FG357312.1和CO 075985.1), 拼接后, 获得的序列经在线软件预测cDNA全长(http://genes.mit.edu/GENSCAN.html), 然后用在线软件ORF Finder (http://www.ncbi.nlm. nih.gov/gorf/gorf.html)预测完整开放阅读框(ORF)。

在ORF两端设计上游引物GarTHA-F: 5′-GAAA CAATGGTGACCAAAACAGTAG-3′和下游引物GarTHA-R: 5′-GTCTGACTTAGTTCCCATTTTCTT C-3′。以盐处理的旱地棉cDNA为模板采用TransGen Biotech公司的Easy Pfu DNA酶扩增目的基因。反应体系含2.5 U μL^-1Easy Pfu DNA聚合酶 0.5 μL、10×Easy Pfu buffer 2.5 μL、2.5 mmol L^-1 dNTPs 2.0 μL、模板1.0 μL, 上下游引物各1.0 μL, 加ddH₂O至终体积25 μL。扩增程序为94℃ 3 min; 94℃ 30 s, 60℃ 30 s, 72℃ 2 min, 34个循环; 72℃ 10 min, 4℃保存。回收扩增产物并连接到PTG19-T载体上, 转入大肠杆菌, 以菌液PCR筛选阳性克隆, 送上海英骏生物技术有限公司测序。

1.4 生物信息学分析

利用NCBI网站的Blast程序进行数据比对, 利用GENSCAN (http://genes.mit.edu/GENSCAN.html)在线软件进行基因预测, 利用ORF Finder (http: //www.ncbi.nlm.nih.gov/gorf/gorf.html)寻找完整的开放阅读框, 利用DNAMAN软件包与其他植物的THA氨基酸序列进行蛋白序列多重比对, 利用MEGA4.0.2构建系统进化树。利用Plants数据库(http://plantsp.genomics.purdue.edu/html/feature_scan.html)网站在线工具预测蛋白结构。

1.5 半定量RT-PCR检测

以半定量RT-PCR技术检测经200 mmol L^-1 NaCl处理0、1、3、6、12、24和48 h后旱地棉叶片中 GarTHA 的表达差异, 按照TaKaRa公司的反转录试剂盒说明书进行反转录操作。PCR反应扩增程序为94℃ 3 min; 95℃ 45 s, 55℃ 45 s, 72℃ 1 min, 36个循环; 72℃ 10 min, 4℃保存。以1.2%的琼脂糖凝胶电泳检测。扩增目的基因引物为GarTHA-F和GarTHA-R, 内参选用 Histone3 (约300 bp): 上游引物序列为5′-TCAAGACTGATTTGCGTTTCCA-3′, 下游引物序列为5′-GCGCAAAGGTTGGTGTCT TC-3′。

1.6 植物表达载体的构建

以pCAMBIA2301 (http://www.cambia.org/)为载体基本骨架,拟南芥逆境胁迫诱导型启动子rd29A表达载体pCAMBIA2301-rd29A-rbc的构建方法见冯娟等^[31]。

根据所得到的 GarTHA 的ORF序列设计构建载体所需的目的基因引物, 引物序列为VC-THA-F: 5′-GCTCTAGAGCGAAACAATGGTGACCAAAACAGTAG-3′ (下画线为 Xba I酶切位点); VC-THA-R: 5′-TCCCCCGGGGGAGTCTGACTTAGTTCCCATTTTCTTC-3′ (下画线为 Sma I酶切位点)。以盐处理12 h的旱地棉叶片cDNA为模板扩增 GarTHA 成熟蛋白编码区。将扩增产物回收, 克隆。提取测序正确的菌液的质粒, 进行 Xba I和 Sma I双酶切并回收目的片段, 同时对pCAMBIA2301-rd29A-rbc质粒也进行 Xba I和 Sma I双酶切并回收大片段, 然后将2次回收的片段连接构建植物表达载体pCAMBIA2301- rd29A-GarTHA。所有的效应质粒均经PCR、酶切鉴定正确。用电击法将pCAMBIA2301-rd29A-GarTHA质粒转入农杆菌菌株EHA105感受态细胞中, 获得用于植物转化实验的工程农杆菌。

1.7 转化植株的获得和检测

通过浸花法将含重组质粒pCAMBIA2301- rd29A- GarTHA 的工程农杆菌EHA105转入Col-0野生型拟南芥, 于培养箱中正常培养, 3~4周后收获T₀代成熟种子。将T₀代种子播种于含100 mg L^-1卡那霉素的1/2MS培养基上初筛选, 播种2周后, 将野生型和具有卡那抗性的T₁代植株分别转移到基质(泥炭土∶蛭石=1∶1)中单株培养, 然后进行GUS染色和目的基因PCR检测, 得到T₁代阳性转化植株, 收获种子。GUS染色过程是: 将新鲜的植物叶片放入GUS染色缓冲液[10 mL体系含0.5 mol L^-1磷酸钠缓冲液(pH 7.2) 1 mL、10% Triton X-100 0.2 mL、100 mmol L^-1亚铁氰化钾0.2 mL、100 mmol L^-1铁氰化钾0.2 mL、100 mmol L^-1 X-Gluc 0.2 mL、ddH₂O 8.2 mL], 37℃ 2~5 h, 然后用移液器吸净染色液, 室温条件下, 将染色样品依次在20%、35%和50%酒精中分别浸泡30 min脱色处理, 脱色后在室温下用FAA固定液(100 mL体系含38%甲醛5 mL、冰醋酸5 mL、70%酒精90 mL)浸泡30 min, 然后将植物样品放在显微镜下观察。

将阳性T₁代转基因植株的种子播于土质基质(蛭石∶泥炭土=1∶1)正常培养, 然后采集叶片进行RT-PCR验证。基于rd29A为逆境胁迫的诱导型启动子, 目的基因的RT-PCR检测在盐胁迫下进行, 具体方法为, 将PCR阳性的T₁代种子播于土质基质(蛭石∶泥炭土=1∶1), 3周左右将植株转移到含150 mmol L^-1 NaCl的营养液中处理12 h, 采集叶片进行RT-PCR验证。RT-PCR检测所使用的引物为GarTHA-F和GarTHA-R。RT-PCR反应采用TaKaRa的 r-Taq 酶进行扩增, 反应体系按照说明书配制, 扩增程序为94℃ 3 min; 95℃ 45 s, 55℃ 45 s, 72℃ 1 min, 36个循环; 72℃ 10 min, 4℃保存。以1.2%的琼脂糖凝胶电泳检测。

1.8 转基因植株的功能验证

将野生型和转基因拟南芥T₂代种子分别播于含0或150 mmol L^-1NaCl的1/2MS培养基上, 置光照培养箱正常培养, 3次重复, 每重复50粒种子, 10 d后观察植株表型并记录转基因株系的发芽率, 每重复调查20株苗的根长。采用SPSS软件统计分析。

2 结果与分析

2.1 GarTHA 基因的获得

通过cDNA-AFLP和电子克隆方法获得一长为1897 bp的苏氨酸醛缩酶基因的cDNA Contig。利用GENSCAN和ORF Finder软件预测该cDNA全长为1018 bp, 并发现该序列包含一个822 bp的完整ORF。在ORF两端设计引物, 以盐处理旱地棉叶片的cDNA为模板, 进行全长基因的扩增(图1)。通过TA克隆, 测序获得该基因在旱地棉中的cDNA序列, 测序结果经Blast比对, 证明所扩序列正确, 与电子克隆序列相比, 序列相似度为93.96%。将该基因命名为 GarTHA 并提交GenBank, 获得登录号为KC167360。

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	图1 GarTHA 全长ORF的克隆Fig. 1 Cloning for full-length ORF of GarTHA

2.2 生物信息学分析

旱地棉苏氨酸醛缩酶基因 GarTHA cDNA全长为1018 bp, 包含822 bp的完整ORF, 编码273个氨基酸残基, 蛋白质分子量为82.57 kD, 等电点为4.89。将旱地棉苏氨酸醛缩酶基因编码的氨基酸序列进行BlastP比对(图2), 并构建系统发育树(图3)发现, GarTHA 与拟南芥、杨树、马铃薯和玉米同源基因的氨基酸相似度分别为77.7%、84.6%、83.2%和75.8%。氨基酸序列多重比对结果表明, 旱地棉苏氨酸醛缩酶和其他物种在C端的保守性均较低。

在Plants数据库(http://plantsp.genomics.purdue. edu/html/feature_scan.html)中分析表明, 该基因蛋白包含氨基酸转移酶位点和9个N-肉豆蔻酰化位点, 表明该蛋白可能参与膜上蛋白间的相互作用, 并介导氨基酸的跨膜转运, 其催化作用为催化苏氨酸裂解为甘氨酸和乙醛。NCBI CDS程序分析(图4)显示 GarTHA 基因蛋白包含5′磷酸结合位点及四聚物结合位点, 这表明该类基因的表达可能受磷酸化影响。

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	图2 旱地棉苏氨酸醛缩酶(GarTHA)氨基酸序列与其他物种的氨基酸序列多重比对结果Fig. 2 Alignment of the deduced amino acid sequence of GarTHA with other THAs by DNAMAN programGarTHA (KC167360); AtTHA (NP_563822.1); PtTHA (Xp_002325334.1): StTHA (ABB72802.1); ZmTHA (ACG33933.1).

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	图3 旱地棉苏氨酸醛缩酶氨基酸序列与其他物种同源序列的进化树分析Fig. 3 Phylogenetic tree of putative GarTHA and other THAs by MEGA4.0.2 program

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	图4 GarTHA蛋白结构预测Fig. 4 Prediction of structure model in GarTHA

2.3 RT-PCR分析

为研究 GarTHA 基因在盐胁迫下的表达量变化, 分别提取不同盐处理时期的叶片RNA, 并反转录成cDNA, 进行了半定量RT-PCR检测。结果表明, 随着盐处理时间的增加, GarTHA 的表达量也呈递增的趋势, 24 h达到最大, 48 h后 GarTHA 的表达丰度开始下降(图5)。

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	图5 盐胁迫下 GarTHA 半定量PCR分析Fig. 5 Expression analysis of GarTHA by semi-quantitative RT-PCR in G.. aridum under salt stress

2.4 转基因拟南芥的筛选与PCR验证

将收获的T₀代种子播于含100 mg L^-1卡那霉素的1/2MS培养基上, 2周后转基因植株长出真叶并呈绿色, 表型正常, 而非转基因植株生长停留在子叶期, 并且呈黄色, 叶绿素含量较低。分别采集野生型植株和卡那霉素抗性的T₁代植株叶片进行GUS染色(图6)和PCR检测。获得GUS染色和PCR检测均为阳性的植株, 说明 GarTHA 基因已成功转入拟南芥基因组中。将T₁代阳性转基因植株的种子播于土质基质, 采集叶片进行RT-PCR验证。结果显示 GarTHA 基因在2个株系(T_2-1和T_2-6)中具有较强的表达(图7)。因此选取这2个株系用于进一步的基因功能研究。

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	图6 转 GarTHA 基因拟南芥的GUS染色Fig. 6 GUS staining of GarTHA transgenic Arabidopsis

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	图7 转 GarTHA 基因拟南芥RT-PCR检测+: 质粒; -: 非转基因植株; T_2-1、T_2-2、T_2-3、T_2-4、T_2-5、T_2-6; 转基因T₂代株系。Fig. 7 RT-PCR detection of GarTHA transgenic Arabidopsis+: plasmid; -: non-transgenic plant; T_2-1, T_2-2, T_2-3, T_2-4, T_2-5, T_2-6: transgenic lines of T₂ generation.

2.5 转基因拟南芥耐盐性鉴定

将野生型和2个转基因株系(T_2-1和T_2-6)的T₃代种子分别播于含0, 150 mmol L^-1 NaCl 1/2×MS上, 3次重复。10 d后, 野生型拟南芥种子平均萌发率分别为83%和35%, 而转 GarTHA 基因株系T_2-1的平均萌发率分别为87%和77%, T_2-6平均萌发率分别为91%和88% (图8)。统计分析表明盐胁迫下转基因株系的发芽率显著高于野生型(图9-A)。在不含NaCl的条件下, 野生型、T_2-1和T_2-6的平均根长分别为24.6、25.8和25.0 mm, 差异不显著。盐胁迫下转基因株系T_2-1、T_2-6的平均根长分别为7.45 mm和9.90 mm, 而野生型为5.05 mm, 差异达显著水平(图9-B)。且野生型植株出现的盐胁迫伤害和黄化现象较转基因植株明显, 侧根较少, 表明 GarTHA 基因可以增强拟南芥的耐盐性。

3 讨论

苏氨酸醛缩酶在高等植物中普遍存在, 是一类催化苏氨酸转化为甘氨酸和乙醛的酶, 参与高等植物的氮素代谢过程。研究表明植物在逆境下氨基酸的代谢较正常条件下更为活跃, 细胞内可溶性糖和氨基酸等含量明显提高, 并证明苏氨酸醛缩酶在这一过程中发挥了重要作用^[32]。甘氨酸可经3次甲基化而合成甜菜碱, 而乙酰辅酶A则参与了多种脂类的合成, 从而提高植物耐逆性^{[25,33,34,35,36]}。Waditee等^[25]研究表明在拟南芥中, 除胆碱两步氧化反应可生成耐逆物质甜菜碱外, 由甘氨酸经两种N-端甲基化转移酶的作用, 也可以产生甜菜碱。而甜菜碱是植物抗非生物胁迫中一类重要的渗透保护性物质。研究表明盐胁迫下植物体内甜菜碱的含量迅速上升, 这种积累是一种有利于植物在胁迫下生长的重要生理现象, 其含量与植物耐盐性呈正相关^{[20,21,22,23]}。本研究通过cDNA-AFLP和电子克隆技术从野生种旱地棉中克隆了 GarTHA 基因, 其结构与催化特性表明该基因编码苏氨酸醛缩酶。半定量PCR表明 GarTHA 基因受盐胁迫诱导表达。转 GarTHA 基因拟南芥发芽率和根长测定表明 GarTHA 基因能够提高植株的耐盐能力。另外, 我们利用 GarTHA 基因在拟南芥基因组中的同源基因 AtTHA1 (At1g08630)在ATTED (http://atted.jp/data/locus/At1g08630.shtml)中分析结果也表明该基因与脂类代谢酶类的共表达值最高, 且与逆境转录因子 MYB 家族中的 MYB13 (At1g 06180)基因有关联(数据未显示), 乔孟等^[37]研究表明 MYB13 基因受高盐胁迫诱导反应。虽然目前有关 THA 基因在植物中的功能报道不多, 但本研究中对 GarTHA 基因的结构与功能分析均表明该基因编码的蛋白可能参与逆境胁迫下的生理生化反应, 从而提高植物抗逆性。

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	图8 盐胁迫下野生型和转基因拟南芥的生长情况Fig. 8 Phenotype analysis of salt tolerance of GarTHA transgenic Arabidopsis

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图9 NaCl处理的野生型和转基因株系发芽率和根长统计分析图柱上方不同大写和小写字母分别表示在 P<0.01和 P<0.05水平上的差异显著性。Fig. 9 Statistical analysis of germination rate and root length in wild type and transgenic plants under salt stressBars with different capitals or lowercases are significantly different at P<0.01 and P<0.05, respectively. Data are means± SE of three experiments.

在植物抗逆基因工程研究中, 大多使用组成型启动子来驱动目的基因的表达, 但Kasuga等^[38]报道组成型启动子CaMV35S驱动 DREB 基因在拟南芥中表达, 虽提高了抗逆性, 但植株矮化、生长畸形, 而由诱导型启动子rd29A启动的 DREB 基因在拟南芥中的表达不仅提高了植株的抗逆性, 而且避免了对生长的负面影响。朱丽萍等^[39]报道用组成型启动子CaMV35S驱动 BADH 基因在烟草中表达, 转基因烟草的耐盐性提高了, 但生长延迟。Pellegrineschi等^[40]用诱导型启动子rd29A驱动 DREBA/CBF3 的表达, 提高了转基因小麦的耐旱性, 而且没有引起明显的畸形。表明组成型启动子在驱动外源基因表达的同时, 对植物的一些生理代谢活动却有抑制作用, 而rd29A启动子由于只在逆境诱导下表达, 对植株生长的影响较小。为了降低外源基因的表达对植株正常生长的影响, 提高该基因在植物耐盐性改良中的应用价值, 我们构建了逆境胁迫特异启动子rd29A的表达载体并将 GarTHA 成熟的蛋白编码区转入拟南芥进行功能验证。研究结果表明, 由rd29A启动的 GarTHA 基因能在盐胁迫下正常表达, 并表现了一定的耐盐功能, 为下一步该基因在棉花耐盐性改良中的应用提供了理论与实践依据。

本试验通过对转基因拟南芥植株的表型观察研究了 GarTHA 在高盐胁迫下的抗逆性, 证明 GarTHA 基因在逆境胁迫下可能参与一系列生理生化反应, 从而提高植物的耐盐抗逆性。而在逆境胁迫下, GarTHA 基因如何参与转基因植株的生理、生化反应以及这些生理、生化特性与抗逆性的关系及该基因所编码的蛋白在旱地棉逆境胁迫信号转导途径中的作用还需进一步研究。

4 结论

获得了旱地棉苏氨酸醛缩酶基因 GarTHA , 该基因包含一个822 bp完整的开放阅读框, 编码273个氨基酸残基。 GarTHA 基因受盐胁迫诱导表达量迅速上调, 该基因能提高植物的耐盐性。

The authors have declared that no competing interests exist.

作者已声明无竞争性利益关系。The authors have declared that no competing interests exist.

参考文献

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[1]	Zhao K-F(赵可夫), Feng L-T(冯立田). Halophytes Resources of China. Beijing: Science Press, 2001. pp32-43(in Chinese) [本文引用:1]
[2]	Guo Y H, Yu Y P, Wang D, Wu C A, Yang G D, Huang J G, Zheng C C. GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5. New Phytol, 2009, 183: 62-75 [本文引用:1] [JCR: 6.645]
[3]	Huang B, Jin L, Liu J Y. Identification and characterization of the novel gene GhDBP2 encoding a DRE-binding protein from cotton (Gossypium hirsutum). J Plant Physiol, 2008, 165: 214-223 [本文引用:1] [JCR: 2.791]
[4]	Yang Y-W(杨郁文), Ni W-C(倪万潮), Zhang B-L(张保龙), Shen X-L(沈新莲), Zhang X-G(张香桂), Xu Y-J(徐英俊), Yao S(姚姝). Molecular cloning and expression analysis of a serine/ threonine protein kinase gene in upland cotton. Cotton Sci(棉花学报), 2006, 18(3): 140-144 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.4495]
[5]	Haake V, Cook D, Riechmann J, Pineda O, Thomashow M F, Zhang J Z. Transcription factor CBF₄ is a regulator of drought adaptation in Arabidopsis. Plant Physiol, 2002, 130: 639-648 [本文引用:1] [JCR: 6.535]
[6]	Zhu J K. Salt and drought stress signal transduction in plants. Annu Rev Plant Biol, 2002, 53: 247-273 [本文引用:1] [JCR: 25.962]
[7]	Hu H H, You J, Fang Y J, Zhu X Y, Qi Z Y, Xiong L Z. Characterization of transcription factor gene SNAC₂conferring cold and salt tolerance in rice. Plant Mol Biol, 2008, 67: 169-181 [本文引用:1] [JCR: 4.15]
[8]	Shi H Z, Lee B H, Wu S J, Zhu J K. Overexpression of a plasma membrane Na⁺/H⁺ antiporter gene improves salt tolerance in Arabidopsis thaliana. Nat Biotechnol, 2003, 21: 81-85 [本文引用:1] [JCR: 23.268]
[9]	Wu C A, Yang G D, Meng Q W, Zheng C C. The cotton GhNHX1 gene encoding a novel putative tonoplast Na⁺/H⁺ antiporter plays an important role in salt stress. Plant Cell Physiol, 2004, 45: 600-607 [本文引用:1] [JCR: 4.702]
[10]	Kopka J, Pical C, Gray J E, Rober M B. Molecular and enzymatic characterization of three phosphoinositide-specific phospholipase C isoforms from potato. Plant Physiol, 1998, 116: 239-250 [本文引用:1] [JCR: 6.535]
[11]	Babu R C, Zhang J X, Blum A, David-Ho T H, Wu R, Nguyen H T. HVA1, a LEA gene from barley confers dehydration tolerance in transgenic rice (Oryza sativa L. ) via cell membrane protection. Plant Sci, 2004, 166: 855-862 [本文引用:1] [JCR: 2.945]
[12]	Imai R, Chang L, Ohta A, Bray E, Takagi M. A lea-class gene of tomato confers salt and freezing tolerance when expressed in Saccharomyces cerevisiae. Gene, 1996, 170: 243-248 [本文引用:1] [JCR: 2.341]
[13]	Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol, 1999, 17: 287-291 [本文引用:1] [JCR: 23.268]
[14]	Xu D, Duan X, Wang B, Hong B, Ho T H D, Wu R. Expression of a late embryogenesis abundant protein gene, HVA1, from barley confers tolerance to water deficit and salt stress in transgenic rice. Plant Physiol, 1996, 110: 249-257 [本文引用:1] [JCR: 6.535]
[15]	Guan L M, Zhao J, Scadalios J G. Cis-elements and transfactors that regulate expression of the maize Cat1 antioxidant gene in response to ABA and osmotic stress: H₂O₂ is the likely intermediary signaling molecule for the response. Plant, 2000, 22: 87-95 [本文引用:1]
[16]	Prasad T K, Anderson M D, Martin B A, Steward C R. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. Plant Cell, 1994, 6: 65-74 [本文引用:1] [JCR: 8.987]
[17]	Zhang X, Zhang L, Dong F, Gao J, Galbraith D W, Song C P. Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiol, 2001, 126: 1438-1448 [本文引用:1] [JCR: 6.535]
[18]	Zhao Z, Chen G, Zhang C. Interaction between reactive oxygen species and nitric oxide in drought-induced abscisic acid synthesis in root tips of wheat seedlings. Plant Physiol, 2001, 28: 1055-1061 [本文引用:1] [JCR: 6.535]
[19]	Zheng X, Pontes O, Zhu J, Miki D, Zhang F, Li W X, Iida K, Kapoor A, Pikaard C S, Zhu J K. ROS₃ is an RNA-binding protein required for DNA demethylation in Arabidopsis. Nature, 2008, 455: 1259-1262 [本文引用:1] [JCR: 36.28]
[20]	Hanson A D, May A M, Grumet R, Bode J, Jamieson G C, Rhodes D. Betaine synthesis in chenopods: localization in chloroplasts. Proc Natl Acad Sci USA, 1985, 82: 3678-3682 [本文引用:2] [JCR: 9.737]
[21]	McCue K F, Hanson A D. Drought and salt tolerance: towards understand ing and application. Trends Biotechnol, 1990, 8: 358-362 [本文引用:2] [JCR: 9.148]
[22]	Alloing G, Travers I, Sagot B, Le Rudulier D, Dupont L. Proline betaine uptake in Sinorhizobium meliloti: characterization of Prb, an Opp-like ABC transporter regulated by both proline betaine and salinity stress. J Bacteriol, 2006, 188: 6308-6317 [本文引用:2] [JCR: 3.825]
[23]	Ashraf M, Foolad M. Roles of glycine betaine and proline in improving plant abiotic stress resistance. Environ Exp Bot, 2007, 59: 206-216 [本文引用:2] [JCR: 2.985]
[24]	Li Q L, Gao X R, Yu X H, Wang X Z, An L J. Molecular cloning and characterization of betaine aldehyde dehydrogenase gene from Suaeda liaotungensis and its use in improved tolerance to salinity in transgenic tobacco. Biotechnol Lett, 2003, 25: 1431-1436 [本文引用:1] [JCR: 1.683]
[25]	Waditee R, Bhuiyan M N H, Rai V, Aoki K, Tanaka Y, Hibino T, Suzuki S, Takano J, Jagendorf A T, Takabe T. Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis. Proc Natl Acad Sci USA, 2005, 102: 1318-1323 [本文引用:3] [JCR: 9.737]
[26]	Luo X-L(罗晓丽), Xiao J-L(肖娟丽), Wang Z-A(王志安), Zhang A-H(张安红), Tian Y-C(田颖川), Wu J-H(吴家和). Overexpression of Spinacia oleracea betaine aldehyde dehydrogenase (SoBADH) gene confers the salt and cold tolerant in Gossypium hirsutum L. Chin J Biotech(生物工程学报), 2008, 24(8): 1464-1469 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.7549]
[27]	Liu J Q, Dairi T, Itoh N, Kataoka M, Shimizu S, Yamada H. Gene cloning, biochemical characterization and physiological role of a thermostable low-specificity L-threonine aldolase from Escherichia coli. Eur J Biochem, 1998, 255: 220-226 [本文引用:1] [JCR: 3.579]
[28]	Liu Z W, Feng J, Fan X Q, Xu P, Zhang X G, Shen X L. cDNA-AFLP analysis of differentially-expressed gene in response to salt stress in Gossypium airdum. Cotton Sci, 2012, 24: 435-443 [本文引用:2] [CJCR: 1.4495]
[29]	Harrison S J, Mott E K, Parsley K, Aspinall S, Gray J C, Cottage A. A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation. Plant Methods, 2006, 2: 19 [本文引用:1] [JCR: 2.833]
[30]	Hu G-H(胡根海), Yu S-X(喻树迅). Extraction of high-quality total RNA in cotton leaf with improved CTAB method. Cotton Sci(棉花学报), 2007, 19(1): 69-70 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.4495]
[31]	Feng J(冯娟), Fan X-Q(范昕琦)Xu P(徐鹏), Zhang X-G(张香桂), Shen X-L(沈新莲). Isolation and functional analysis of GarCIPK8 gene from Gossypium aridum. Acta Agron Sin(作物学报), 2013, 39(1): 1-9 (in Chinese with English abstract) [本文引用:1] [CJCR: 1.8267]
[32]	Luo Z-Y(罗宗雅), Tang Y-Z(汤玉璋). Nitrogen metabolism in higher plants. Biochem Biophys(生物化学与生物物理学报), 1964, 4(5): 391-454 (in Chinese) [本文引用:1]
[33]	Herbert D, Walker K A, Price L J, Cole D J, Pallett K E, Ridley S M, Harwood J L. Acetyl-CoA carboxylase—a graminicide target site. Pesticide Sci, 1997, 50: 67-71 [本文引用:1]
[34]	Nikolau B J, Ohlrogge J B, Wurtele E S. Plant biotin-containing carboxylases. Arch Biochem Biophy, 2003, 414: 211-222 [本文引用:1] [JCR: 2.935]
[35]	Gronwald J W. Lipid biosynthesis inhibitors. Weed Sci, 1991, 39: 435-449 [本文引用:1] [JCR: 1.733]
[36]	Turner J A, Pernich D J. Origin of enantiomeric selectivity in the aryloxyphenoxypropionic acid class of herbicidal acetyl coenzyme A carboxylase (ACCase) inhibitors. J Agric Food Chem, 2002, 50: 4554-4566 [本文引用:1] [JCR: 2.823]
[37]	Qiao M(乔孟), Yu Y-C(于延冲), Xiang F-N(向凤宁). The roles of the Arabidopsis R2R3-MYB transcription factors in the stress responses. Chin Bull Life Sci(生命科学), 2009, 21(1): 145-150 (in Chinese with English abstract) [本文引用:1]
[38]	Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K. Improving plant drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor. Nat Biotechnol, 1999, 17: 287-291 [本文引用:1] [JCR: 23.268]
[39]	Zhu L-P(朱丽萍), Yu Z(于壮), Zou C-X(邹翠霞), Li Q-L(李秋莉). Plant stress-inducible promotors and their function. Hereditas(遗传), 2010, 32(3): 229-234 (in Chinese with English abstract) [本文引用:1] [CJCR: 0.9787]
[40]	Pellegrineschi A, Reynolds M, Pacheco M, Brito R M, Almeraya R, Yamaguchi-Shinozaki K, Hoisington D. Stress induced expression in wheat of the Arabidopsis thaliana DREB1A gene delays water stress symptoms under greenhouse conditions. Genome, 2004, 47: 493-500 [本文引用:1] [JCR: 1.653]

2001

0.0

... 33×10⁶hm²盐碱荒地尚待开发^[1] ...

2009

6.645

0.0

New Phytol.. 2009, 183(1):62-75 DOI:10.1111/j.1469-8137.2009.02838.x

GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5.

Guo YH, Yu YP, Wang D, Wu CA, Yang GD, Huang JG, Zheng CC

State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai'an, Shandong, China.

* Zinc finger proteins are a superfamily involved in many aspects of plant growth and development. However, CCCH-type zinc finger proteins involved in plant stress tolerance are poorly understood. * A cDNA clone designated Gossypium hirsutum zinc finger protein 1 (GhZFP1), which encodes a novel CCCH-type zinc finger protein, was isolated from a salt-induced cotton (G. hirsutum) cDNA library using differential hybridization screening and further studied in transgenic tobacco Nicotiana tabacum cv. NC89. Using yeast two-hybrid screening (Y2H), proteins GZIRD21A (GhZFP1 interacting and responsive to dehydration protein 21A) and GZIPR5 (GhZFP1 interacting and pathogenesis-related protein 5), which interacted with GhZFP1, were isolated. * GhZFP1 contains two typical zinc finger motifs (Cx8Cx5Cx3H and Cx5Cx4Cx3H), a putative nuclear export sequence (NES) and a potential nuclear localization signal (NLS). Transient expression analysis using a GhZFP1::GFP fusion gene in onion epidermal cells indicated a nuclear localization for GhZFP1. RNA blot analysis showed that the GhZFP1 transcript was induced by salt (NaCl), drought and salicylic acid (SA). The regions in GhZFP1 that interact with GZIRD21A and GZIPR5 were identified using truncation mutations. * Overexpression of GhZFP1 in transgenic tobacco enhanced tolerance to salt stress and resistance to Rhizoctonia solani. Therefore, it appears that GhZFP1 might be involved as an important regulator in plant responses to abiotic and biotic stresses.

PMID: 19402879

... 植物中与盐胁迫相关的基因主要有以下几类, 即与信号转导途径相关的基因^{[2,3,4,5,6,7]}, 与离子转运及离子平衡相关的基因^[8,9], 参与渗透保护物质的生物合成类基因, 主要涉及水分胁迫如水通道蛋白^[10], 胚胎发育晚期丰富蛋白(LEA类蛋白)^{[11,12,13,14]}以及抗氧化胁迫如超氧化物歧化酶(SOD)、过氧化氢酶、抗坏血酸、谷胱甘肽合成酶基因等^{[15,16,17,18,19]} ...

2008

2.791

0.0

J. Plant Physiol.. 2008, 165(2):214-23

Identification and characterization of the novel gene GhDBP2 encoding a DRE-binding protein from cotton (Gossypium hirsutum).

Huang B, Jin L, Liu JY

Laboratory of Molecular Biology and MOE Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China.

A cDNA encoding one novel DRE-binding protein, GhDBP2, was isolated from cotton seedlings. It is classified into the A-6 group of DREB subfamily based on multiple sequence alignment and phylogenetic characterization. Using semi-quantitative RT-PCR, we found that the GhDBP2 transcripts were greatly induced by drought, NaCl, low temperature and ABA treatments in cotton cotyledons. The DNA-binding properties of GhDBP2 were analyzed by electrophoretic mobility shift assay (EMSA), showing that GhDBP2 successfully binds to the previously characterized DRE cis-element as well as the promoter region of the LEA D113 gene. Consistent with its role as a DNA-binding protein, GhDBP2 is preferentially localized to the nucleus of onion epidermal cells. In addition, when GhDBP2 is transiently expressed in tobacco cells, it activates reporter gene expression driven by the LEA D113 promoter. Taken together, our results indicate that GhDBP2 is a DRE-binding transcriptional activator involved in activation of down-stream genes such as LEA D113 expression through interaction with the DRE element, in response to environmental stresses as well as ABA treatment.

PMID: 17224201

2006

0.0

1.4495

2002

6.535

0.0

Plant Physiol.. 2002, 130(2):639-48

Transcription factor CBF4 is a regulator of drought adaptation in Arabidopsis.

Haake V, Cook D, Riechmann JL, Pineda O, Thomashow MF, Zhang JZ

Mendel Biotechnology, 21375 Cabot Boulevard, Hayward, CA 94545, USA.

In plants, low temperature and dehydration activate a set of genes containing C-repeat/dehydration-responsive elements in their promoter. It has been shown previously that the Arabidopsis CBF/DREB1 transcription activators are critical regulators of gene expression in the signal transduction of cold acclimation. Here, we report the isolation of an apparent homolog of the CBF/DREB1 proteins (CBF4) that plays the equivalent role during drought adaptation. In contrast to the three already identified CBF/DREB1 homologs, which are induced under cold stress, CBF4 gene expression is up-regulated by drought stress, but not by low temperature. Overexpression of CBF4 in transgenic Arabidopsis plants results in the activation of C-repeat/dehydration-responsive element containing downstream genes that are involved in cold acclimation and drought adaptation. As a result, the transgenic plants are more tolerant to freezing and drought stress. Because of the physiological similarity between freezing and drought stress, and the sequence and structural similarity of the CBF/DREB1 and the CBF4 proteins, we propose that the plant's response to cold and drought evolved from a common CBF-like transcription factor, first through gene duplication and then through promoter evolution.

PMID: 12376631

2002

25.962

0.0

Annu Rev Plant Biol. 2002, 53:247-73

Salt and drought stress signal transduction in plants.

Zhu JK

Department of Plant Sciences, University of Arizona, Tucson, Arizona 85721, USA. jkzhu@ag.arizona.edu

Salt and drought stress signal transduction consists of ionic and osmotic homeostasis signaling pathways, detoxification (i.e., damage control and repair) response pathways, and pathways for growth regulation. The ionic aspect of salt stress is signaled via the SOS pathway where a calcium-responsive SOS3-SOS2 protein kinase complex controls the expression and activity of ion transporters such as SOS1. Osmotic stress activates several protein kinases including mitogen-activated kinases, which may mediate osmotic homeostasis and/or detoxification responses. A number of phospholipid systems are activated by osmotic stress, generating a diverse array of messenger molecules, some of which may function upstream of the osmotic stress-activated protein kinases. Abscisic acid biosynthesis is regulated by osmotic stress at multiple steps. Both ABA-dependent and -independent osmotic stress signaling first modify constitutively expressed transcription factors, leading to the expression of early response transcriptional activators, which then activate downstream stress tolerance effector genes.

PMID: 12221975

2008

4.15

0.0

Plant Mol Biol. 2008, 67(1-2):169 - 181 DOI:10.1007/s11103-008-9309-5

Characterization of transcription factor geneSNAC2 conferring cold and salt tolerance in rice

Honghong Hu(1) Jun You(1) Yujie Fang(1) Xiaoyi Zhu(1) Zhuyun Qi(1) Lizhong Xiong(1)

1.Huazhong Agricultural University National Center of Plant Gene Research (Wuhan), National Key Laboratory of Crop Genetic Improvement Wuhan 430070 China

Plants respond to adverse environment by initiating a series of signaling processes including activation of transcription factors that can regulate expression of arrays of genes for stress response and adaptation. NAC (NAM, ATAF, and CUC) is a plant specific transcription factor family with diverse roles in development and stress regulation. In this report, a stress-responsive NAC gene (SNAC2) isolated from upland rice IRA109 (Oryza sativa L. ssp japonica) was characterized for its role in stress tolerance. SNAC2 was proven to have transactivation and DNA-binding activities in yeast and the SNAC2-GFP fusion protein was localized in the rice nuclei. Northern blot andSNAC2 promoter activity analyses suggest thatSNAC2 gene was induced by drought, salinity, cold, wounding, and abscisic acid (ABA) treatment. TheSNAC2 gene was over-expressed injaponica rice Zhonghua 11 to test the effect on improving stress tolerance. More than 50% of the transgenic plants remained vigorous when all WT plants died after severe cold stress (4–8°C for 5 days). The transgenic plants had higher cell membrane stability than wild type during the cold stress. The transgenic rice had significantly higher germination and growth rate than WT under high salinity conditions. Over-expression ofSNAC2 can also improve the tolerance to PEG treatment. In addition, theSNAC2-overexpressing plants showed significantly increased sensitivity to ABA. DNA chip profiling analysis of transgenic plants revealed many up-regulated genes related to stress response and adaptation such as peroxidase, ornithine aminotransferase, heavy metal-associated protein, sodium/hydrogen exchanger, heat shock protein, GDSL-like lipase, and phenylalanine ammonia lyase. Interestingly, none of the up-regulated genes in theSNAC2-overexpressing plants matched the genes up-regulated in the transgenic plants over-expressing other stress responsive NAC genes reported previously. These data suggest SNAC2 is a novel stress responsive NAC transcription factor that possesses potential utility in improving stress tolerance of rice.

2003

23.268

0.0

2004

4.702

0.0

Plant Cell Physiol.. 2004, 45(5):600-7

The cotton GhNHX1 gene encoding a novel putative tonoplast Na(+)/H(+) antiporter plays an important role in salt stress.

Wu CA, Yang GD, Meng QW, Zheng CC

Department of Biochemistry and Molecular Biology, College of Life Sciences, Shandong Agricultural University, Tai'an, Shandong 271018, P.R. China.

A cDNA clone was isolated from cotton (Gossypium hirsutum) cDNA library and characterized with regard to its sequence, regulation in response to salt stress and functions in yeast mutants and transgenic tobacco plants. The clone, designated as GhNHX1, contains 2485 nucleotides with an open reading frame of 1629 nucleotides, and the deduced amino acid sequence showed high identities with other plant vacuolar-type Na(+)/H(+) antiporters. Northern blot analysis indicated that the mRNA accumulation of GhNHX1 was strongly induced by salt stress and abscisic acid in cotton seedlings. The expression of GhNHX1 in yeast Na(+)/H(+) antiporter mutant showed function complementation. The transgenic tobacco plants overexpressing GhNHX1 also had higher salt tolerance than the wild-type plants. The salt-induced mRNA level of GhNHX1 was 3 and 7 times higher in the salt-tolerant cotton cultivar ZM3 than those in the salt-sensitive cotton cultivars ZMS17 and ZMS12, respectively. Together, these results suggest that the products of the novel gene, GhNHX1, function as a tonoplast Na(+)/H(+) antiporter and play an important role in salt tolerance of cotton.

PMID: 15169942

1998

6.535

0.0

2004

2.945

0.0

1996

2.341

0.0

1999

23.268

0.0

Nat. Biotechnol.. 1999, 17(3):287-91

Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor.

Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K

Biological Resources Division, Japan International Research Center for Agricultural Sciences, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki.

Plant productivity is greatly affected by environmental stresses such as drought, salt loading, and freezing. We reported previously that a cis-acting promoter element, the dehydration response element (DRE), plays an important role in regulating gene expression in response to these stresses. The transcription factor DREB1A specifically interacts with the DRE and induces expression of stress tolerance genes. We show here that overexpression of the cDNA encoding DREB1A in transgenic plants activated the expression of many of these stress tolerance genes under normal growing conditions and resulted in improved tolerance to drought, salt loading, and freezing. However, use of the strong constitutive 35S cauliflower mosaic virus (CaMV) promoter to drive expression of DREB1A also resulted in severe growth retardation under normal growing conditions. In contrast, expression of DREB1A from the stress inducible rd29A promoter gave rise to minimal effects on plant growth while providing an even greater tolerance to stress conditions than did expression of the gene from the CaMV promoter.

PMID: 10096298

1996

6.535

0.0

Plant Physiol.. 1996, 110(1):249-257

Expression of a Late Embryogenesis Abundant Protein Gene, HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice.

Xu D, Duan X, Wang B, Hong B, Ho T, Wu R

Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853 (D.X., X.D., B.W., R.W.).

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.

PMID: 12226181

2000

0.0

1994

8.987

0.0

2001

6.535

0.0

2001

6.535

0.0

2008

36.28

0.0

Nature. 2008, 455(7217):1259-62 DOI:10.1038/nature07305

ROS3 is an RNA-binding protein required for DNA demethylation in Arabidopsis.

Zheng X, Pontes O, Zhu J, Miki D, Zhang F, Li WX, Iida K, Kapoor A, Pikaard CS, Zhu JK

Center for Plant Cell Biology and Department of Botany and Plant Sciences, University of California, Riverside, California 92521, USA.

DNA methylation is an important epigenetic mark for transcriptional gene silencing (TGS) in diverse organisms. Recent studies suggest that the methylation status of a number of genes is dynamically regulated by methylation and demethylation. In Arabidopsis, active DNA demethylation is mediated by the ROS1 (repressor of silencing 1) subfamily of 5-methylcytosine DNA glycosylases through a base excision repair pathway. These demethylases have critical roles in erasing DNA methylation and preventing TGS of target genes. However, it is not known how the demethylases are targeted to specific sequences. Here we report the identification of ROS3, an essential regulator of DNA demethylation that contains an RNA recognition motif. Analysis of ros3 mutants and ros1 ros3 double mutants suggests that ROS3 acts in the same genetic pathway as ROS1 to prevent DNA hypermethylation and TGS. Gel mobility shift assays and analysis of ROS3 immunoprecipitate from plant extracts shows that ROS3 binds to small RNAs in vitro and in vivo. Immunostaining shows that ROS3 and ROS1 proteins co-localize in discrete foci dispersed throughout the nucleus. These results demonstrate a critical role for ROS3 in preventing DNA hypermethylation and suggest that DNA demethylation by ROS1 may be guided by RNAs bound to ROS3.

PMID: 18815596

1985

9.737

0.0

Proc. Natl. Acad. Sci. U.S.A.. 1985, 82(11):3678-82

Betaine synthesis in chenopods: Localization in chloroplasts.

Hanson AD, May AM, Grumet R, Bode J, Jamieson GC, Rhodes D

MSU-DOE Plant Research Laboratory, Michigan State University, East Lansing, MI 48824.

PLANTS FROM SEVERAL FAMILIES (CHENOPODIACEAE, GRAMINEAE, COMPOSITAE) ACCUMULATE BETAINE (GLYCINE BETAINE) IN RESPONSE TO SALT OR WATER STRESS VIA THE PATHWAY: choline --> betainal (betaine aldehyde) --> betaine. Betaine accumulation is probably a metabolic adaptation to stress. Intact protoplasts from leaves of spinach (Spinacia oleracea) oxidized [(14)C]choline to betainal and betaine, as did protoplast lysates. Upon differential centrifugation, the [(14)C]choline-oxidizing activity of lysates sedimented with chloroplasts. Chloroplasts purified from protoplast lysates by a Percoll cushion procedure retained strong [(14)C]choline-oxidizing activity (1-3 nmol/mg of chlorophyll per hr), although the proportion of the intermediate, [(14)C]betainal, in the reaction products was usually higher than for protoplasts. Isolated chloroplasts also readily oxidized [(14)C]betainal to betaine (20-100 nmol/mg of chlorophyll per hr). Light increased the oxidation of both [(14)C]choline and [(14)C]betainal by isolated chloroplasts approximately 3-fold; this light-stimulation was abolished by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Similar results were obtained with another chenopod (Beta vulgaris) but not with pea (Pisum sativum), a species that accumulates no betaine. The chloroplast site for betaine synthesis in chenopods contrasts with the mitochondrial site in mammals.

PMID: 16593569

... 盐胁迫下植物体内甜菜碱的积累是一种有利于植物在胁迫下生长的重要生理现象, 其含量与植物耐盐性呈正相关^{[20,21,22,23]} ...

... 研究表明盐胁迫下植物体内甜菜碱的含量迅速上升, 这种积累是一种有利于植物在胁迫下生长的重要生理现象, 其含量与植物耐盐性呈正相关^{[20,21,22,23]} ...

1990

9.148

0.0

... 盐胁迫下植物体内甜菜碱的积累是一种有利于植物在胁迫下生长的重要生理现象, 其含量与植物耐盐性呈正相关^{[20,21,22,23]} ...

2006

3.825

0.0

J. Bacteriol.. 2006, 188(17):6308-17

Proline betaine uptake in Sinorhizobium meliloti: Characterization of Prb, an opp-like ABC transporter regulated by both proline betaine and salinity stress.

Alloing G, Travers I, Sagot B, Le Rudulier D, Dupont L

Unité Interactions Plantes-Microorganismes et Santé Végétale, UMR6192 CNRS-INRA-Université de Nice Sophia Antipolis, Centre INRA Agrobiotech, 400 Route des Chappes, BP167, 06903 Sophia Antipolis Cédex, France. alloing@unice.fr

Sinorhizobium meliloti uses proline betaine (PB) as an osmoprotectant when osmotically stressed and as an energy source in low-osmolarity environments. To fulfill this dual function, two separate PB transporters, BetS and Hut, that contribute to PB uptake at high and low osmolarity, respectively, have been previously identified. Here, we characterized a novel transport system that mediates the uptake of PB at both high and low osmolarities. Sequence analysis of Tn5-luxAB chromosomal insertions from several PB-inducible mutants has revealed the presence of a four-gene locus encoding the components of an ABC transporter, Prb, which belongs to the oligopeptide permease (Opp) family. Surprisingly, prb mutants were impaired in their ability to transport PB, and oligopeptides were not shown to be competitors for PB uptake. Further analysis of Prb specificity has shown its ability to take up other quaternary ammonium compounds such as choline and, to a lesser extent, glycine betaine. Interestingly, salt stress and PB were found to control prb expression in a positive and synergistic way and to increase Prb transport activity. At low osmolarity, Prb is largely implicated in PB uptake by stationary-phase cells, likely to provide PB as a source of carbon and nitrogen. Furthermore, at high osmolarity, the analysis of prb and betS single and double mutants demonstrated that Prb, together with BetS, is a key system for protection by PB.

PMID: 16923898

... 盐胁迫下植物体内甜菜碱的积累是一种有利于植物在胁迫下生长的重要生理现象, 其含量与植物耐盐性呈正相关^{[20,21,22,23]} ...

2007

2.985

0.0

... 盐胁迫下植物体内甜菜碱的积累是一种有利于植物在胁迫下生长的重要生理现象, 其含量与植物耐盐性呈正相关^{[20,21,22,23]} ...

2003

1.683

0.0

Biotechnol lett. 2003, 25(17):1431 - 1436 DOI:10.1023/A:1025003628446

Molecular cloning and characterization of betaine aldehyde dehydrogenase gene fromSuaeda liaotungensis and its use in improved tolerance to salinity in transgenic tobacco

1.Liaoning Normal University
2.Dalian University of Technology
3.University of California at Berkeley
4.North east Normal University

cDNA encoding betaine aldehyde dehydrogenase (BADH) from the halophyteSuaeda liaotungensis has been cloned, sequenced and expressed in tobacco (Nictiana tabacum 89). The full-length cDNA is 1506 base pairs (bp) long and encodes a 502 amino-acid polypeptide. The cDNA fragment coding for the mature enzyme was cloned into vector pCAMBIA-1301 for expression in tobacco. Southern blotting analysis showed that that theBadh gene was integrated into the genome of tobacco. Tobacco expressing BADH survived on MS medium containing 200 mM NaCl, whereas the untransformed plants turned yellow after about 20 d and died.

... 甘氨酸在甘氨酸N-甲基化转移酶的作用下, 经过三步甲基化作用合成甜菜碱, 从而提高植株的抗逆性^[24,25,26] ...

2005

9.737

0.0

Proc. Natl. Acad. Sci. U.S.A.. 2005, 102(5):1318-23

Genes for direct methylation of glycine provide high levels of glycinebetaine and abiotic-stress tolerance in Synechococcus and Arabidopsis.

Waditee R, Bhuiyan MN, Rai V, Aoki K, Tanaka Y, Hibino T, Suzuki S, Takano J, Jagendorf AT, Takabe T, Takabe T

Research Institute, Graduate School of Environmental and Human Sciences, and School of Agriculture, Meijo University, Nagoya 468-8502, Japan.

Betaine is an important osmoprotectant, synthesized by many plants in response to abiotic stresses. Almost all known biosynthetic pathways of betaine are two-step oxidations of choline. Recently, a biosynthetic pathway of betaine from glycine, catalyzed by two N-methyltransferase enzymes, was found. Here, the potential role of N-methyltransferase genes for betaine synthesis was examined in a freshwater cyanobacterium, Synechococcus sp. PCC 7942, and in Arabidopsis plants. It was found that the coexpression of N-methyltransferase genes in Synechococcus caused accumulation of a significant amount of betaine and conferred salt tolerance to a freshwater cyanobacterium sufficient for it to become capable of growth in seawater. Arabidopsis plants expressing N-methyltransferase genes also accumulated betaine to a high level in roots, stems, leaves, and flowers and improved seed yield under stress conditions. Betaine levels were higher than those produced by choline-oxidizing enzymes. These results demonstrate the usefulness of glycine N-methyltransferase genes for the improvement of abiotic stress tolerance in crop plants.

PMID: 15665084

... 甘氨酸在甘氨酸N-甲基化转移酶的作用下, 经过三步甲基化作用合成甜菜碱, 从而提高植株的抗逆性^[24,25,26] ...

... 甘氨酸可经3次甲基化而合成甜菜碱, 而乙酰辅酶A则参与了多种脂类的合成, 从而提高植物耐逆性^{[25,33,34,35,36]} ...

... Waditee等^[25]研究表明在拟南芥中, 除胆碱两步氧化反应可生成耐逆物质甜菜碱外, 由甘氨酸经两种N-端甲基化转移酶的作用, 也可以产生甜菜碱 ...

2008

0.0

0.7549

... 甘氨酸在甘氨酸N-甲基化转移酶的作用下, 经过三步甲基化作用合成甜菜碱, 从而提高植株的抗逆性^[24,25,26] ...

1998

3.579

0.0

... 目前植物中有关苏氨酸醛缩酶在耐盐机制中作用的研究较少^[27], 在棉花中关于该基因的研究尚未见相关报道 ...

2012

0.0

1.4495

Cotton Sci. 2012, 24(5):435 - 443 DOI:1002-7807（2012）05-0435-09

cDNA-AFLP Analysis of Differentially-Expressed Genes in Response to Salt Stress inGosspyium aridum

盐胁迫下棉属野生种旱地棉（Gossypium aridum）差异表达基因的cDNA-AFLP分析

LIU Zhang-wei, FENG Juan，FAN Xin-qi, XU Peng, ZHANG Xiang-gui, SHEN Xin-lian^*

刘章伟，冯　娟，范昕琦，徐　鹏，张香桂，沈新莲^*

Institute of Industrial Crops,Jiangsu Academy of Agricultural Sciences/ Key Laboratory of Cotton and Rapeseed（Nanjing），Ministry of Agriculture,Nanjing210014,China

In this study, we used a salt-tolerant diploidG. aridum species and the salt-sensitiveG. hirsutum cultivar Sumian 12 to investigate differential expression in the presence of salt stress. Using cDNA-AFLP, 25 transcript-derived fragments(TDFs) were isolated and confirmed to be present only in salt tolerant speciesG. aridum under salt-stressed conditions. BLAST analysis with sequences assembled using anin-silico approach demonstrated that 23 of the cDNA fragments had homology to known proteins. The up-regulated genes were mainly involved in ion transport, ROS scavenging, cell signaling, cell division, transcription regulation, membrane protection, and penetration regulation. Quantitative real-time PCR(qRT-PCR) was used to analyze the expression patterns of nine of the 23 TDFs at different stages of salt stress in the tolerant speciesG. aridum. Their expression showed a significant increase and reached a peak between 12–24 h of stress. These TDFs may serve as useful resources for future research on molecular mechanisms of salt stress response in cotton.

以一个耐盐的二倍体野生种旱地棉和对盐敏感的陆地棉栽培种苏棉12号为材料，运用cDNA-AFLP技术，比较两个材料分别在盐胁迫前后的表达情况，获得了25个仅在旱地棉盐胁迫下特异表达的转录片段（TDF）。将这些片段进行电子克隆，延伸后的序列进行BLAST分析，结果显示23个转录片段推断的氨基酸序列与已知的蛋白同源，这些盐诱导表达的基因主要涉及离子转运、活性氧清除、细胞信号传导、细胞分裂、转录调节、膜保护、渗透调节等功能蛋白。从23个差异表达的转录片段中选择9个进行实时定量PCR（qRT-PCR）分析，结果表明这些基因在盐胁迫后表达显著增强，而且多数在12~24 h达到高峰。这些cDNA克隆是开展棉花耐盐性分子基础研究的重要资源。

... 在前期的研究中我们利用cDNA-AFLP技术从棉属野生种旱地棉(Gossypium aridum )中克隆了一个苏氨酸醛缩酶基因片段^[28], 本研究拟利用电子克隆及RT-PCR技术克隆该基因的ORF全长序列, 分析该基因在盐胁迫下的功能, 为棉花耐盐性的改良提供新的基因资源 ...

... 3 目的基因的克隆以本实验室通过cDNA-AFLP技术获得的一个苏氨酸醛缩酶基因片段(153 bp)为探针^[28], 利用电子克隆技术, 在NCBI中用BLAST程序(http://www ...

2006

2.833

0.0

Plant Methods. 2006, 2:19

A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation.

Harrison SJ, Mott EK, Parsley K, Aspinall S, Gray JC, Cottage A

Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK. Sam_Harrison@cantab.net

The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7-10 d and high seedling density and fungal contamination may result in failure to recover transformants.

PMID: 17087829

... 再将一周龄幼苗转移到灭菌基质(草炭土∶蛭石=1∶1)中在22℃/18℃, 16 h光照/8 h黑暗下培养, 待植株长出花蕾时采用浸花法转化^[29] ...

2007

0.0

1.4495

... 2 总RNA的提取用改良的CTAB法提取旱地棉叶片RNA^[30], 采用TRIzol试剂盒提取拟南芥RNA ...

2013

0.0

1.8267

Acta Agron Sin. 2013, 39(1):34 - 42 DOI:10.3724/SP.J.1006.2013.00034

Isolation and Functional Analysis ofGarCIPK8 Gene fromGossypium aridum

棉属野生种旱地棉蛋白激酶基因GarCIPK8的克隆与功能分析

FENG Juan,FAN Xin-Qi,XU Peng,ZHANG Xiang-Gui,SHEN Xin-Lian^*

冯娟,范昕琦,徐鹏,张香桂,沈新莲^*

Institute of Industrial Crops, Jiangsu Academy of Agricultural Sciences / Key Laboratory of Cotton and Rapeseed, Ministry of Agriculture, Nanjing 210014, China

CIPK (calcineurin B-like calcium sensor interacting protein kinase) is a typical group of serine / threonine protein kinase targeting calcium sensor calcineurin B-like protein-specific. In a previous study, we obtained the transcriptome ofGossypium aridum with a pooled RNA sample from root and leaf ofG. aridum under salt stress. From the transcriptome data, we founded that aCIPK related gene had high level of expression abundance. Real-time fluorescence quantitative PCR analysis showed its relative expression level was up-regulated in root induced by salt stress, indicating thisCIPK related gene might be involved in response to salt stress during cotton seedling stage. Byin silico cloning and RT-PCR technology, a full-length cDNA encoding a CIPKs homologue (GarCIPK8) was isolated from G. aridum. The deduced GarCIPK8 protein had 449 amino acids with a predicted molecular weight of 51.12 kD and a predicted isoelectric point of 8.13. The kinase N-terminal catalytic domain ofGarCIPK8 exhibited a typica1 serine / threonine protein kinase domain. The conserved 24 amino acid motif existed in C-terminal non-kinase regions of GarCIPK8 protein. Alignment of amino acid sequence showed that GarCIPK8 was 73.95% identical toOryza sativa OsCIPK8 protein. In order to characterize its putative function, theGarCIPK8 gene driven by CaMV35S promoter and stress-specific promoter rd29A respectively were transformed into tobacco byAgrobacterium-mediated transgenic technology. PCR and RT-PCR detection confirmed thatGarCIPK8 gene was integrated into tobacco genome and expressed normally. The growth of transgenic plants of T₁ generation was observed under salinity and drought stresses, showing that transgenic plants enhance tolerance to both high salt and osmotic stresses.

CIPK (calcineurin B-like calcium sensor interacting protein kinase)是植物钙感受器钙调磷酸酶B类似蛋白特定靶向的一类丝氨酸/苏氨酸蛋白激酶。在前期的研究中,我们将棉属野生种D亚组旱地棉(Gossypium aridum)盐胁迫前后RNA混合样品进行转录组测序,发现一CIPK相关基因存在较高的转录丰度。盐胁迫下荧光定量PCR分析表明该基因在根部表现诱导上调表达,推测该基因可能参与植物在高盐胁迫下的生理调控。利用电子克隆及RT-PCR方法从旱地棉中克隆了一个ORF全长为1350 bp的蛋白激酶基因GarCIPK8。序列分析表明该基因编码的蛋白含有449个氨基酸,分子量为51.12 kD,理论等电点为8.13,其N端催化结构域包含一个丝氨酸/苏氨酸蛋白激酶域,C端具有植物CIPK蛋白家族特有的24个氨基酸组成的NAF保守结构域;与水稻OsCIPK8蛋白的相似度为73.95%。为进一步验证其功能,利用组成型高效强启动子CaMV35S和拟南芥逆境胁迫启动子rd29A构建植物表达载体并转化烟草,经PCR和RT-PCR分子检测,证明GarCIPK8基因已经整合到烟草基因组中,并正常表达。T₁代转基因植株耐盐性鉴定结果表明GarCIPK8基因对提高转基因植株的耐盐性有较明显的作用,PEG干旱模拟试验也证明GarCIPK8基因可增强植株的抗旱性。

... org/)为载体基本骨架,拟南芥逆境胁迫诱导型启动子rd29A表达载体pCAMBIA2301-rd29A-rbc的构建方法见冯娟等^[31] ...

1964

0.0

... 研究表明植物在逆境下氨基酸的代谢较正常条件下更为活跃, 细胞内可溶性糖和氨基酸等含量明显提高, 并证明苏氨酸醛缩酶在这一过程中发挥了重要作用^[32] ...

1997

0.0

... 甘氨酸可经3次甲基化而合成甜菜碱, 而乙酰辅酶A则参与了多种脂类的合成, 从而提高植物耐逆性^{[25,33,34,35,36]} ...

2003

2.935

0.0

Arch. Biochem. Biophys.. 2003, 414(2):211-22

Plant biotin-containing carboxylases.

Nikolau BJ, Ohlrogge JB, Wurtele ES

Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA 50011, USA. dimmas@iastate.edu

Biotin-containing proteins are found in all forms of life, and they catalyze carboxylation, decarboxylation, or transcarboxylation reactions that are central to metabolism. In plants, five biotin-containing proteins have been characterized. Of these, four are catalysts, namely the two structurally distinct acetyl-CoA carboxylases (heteromeric and homomeric), 3-methylcrotonyl-CoA carboxylase and geranoyl-CoA carboxylase. In addition, plants contain a noncatalytic biotin protein that accumulates in seeds and is thought to play a role in storing biotin. Acetyl-CoA carboxylases generate two pools of malonyl-CoA, one in plastids that is the precursor for de novo fatty acid biosynthesis and the other in the cytosol that is the precursor for fatty acid elongation and a large number of secondary metabolites. 3-Methylcrotonyl-CoA carboxylase catalyzes a reaction in the mitochondrial pathway for leucine catabolism. The exact metabolic function of geranoyl-CoA carboxylase is as yet unknown, but it may be involved in isoprenoid metabolism. This minireview summarizes the recent developments in our understanding of the structure, regulation, and metabolic functions of these proteins in plants.

PMID: 12781773

... 甘氨酸可经3次甲基化而合成甜菜碱, 而乙酰辅酶A则参与了多种脂类的合成, 从而提高植物耐逆性^{[25,33,34,35,36]} ...

1991

1.733

0.0

... 甘氨酸可经3次甲基化而合成甜菜碱, 而乙酰辅酶A则参与了多种脂类的合成, 从而提高植物耐逆性^{[25,33,34,35,36]} ...

2002

2.823

0.0

J. Agric. Food Chem.. 2002, 50(16):4554-66

Origin of enantiomeric selectivity in the aryloxyphenoxypropionic acid class of herbicidal acetyl coenzyme A carboxylase (ACCase) inhibitors.

Turner JA, Pernich DJ

Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268-1054, USA. jaturner@dow.com

Molecular modeling was used to propose an "active conformation" for the R-2-phenoxypropionic acid portion of the aryloxyphenoxypropionic acid series of herbicidal acetyl CoA carboxylase (ACCase) inhibitors. This candidate active conformation is a low-energy conformer with the R-methyl distal to the phenoxy fragment, stabilized by the generalized anomeric effect around the propionate ether bond; the inactive S-enantiomer has difficulty accessing this conformation due to steric interaction of the S-methyl with the o-hydrogen of the phenyl. This candidate conformation was challenged by preparation of a series of novel rigid analogues. ACCase inhibition data suggest that the systems which contain a fused five-membered, but not a six-membered, ring present the necessary pharmacophore to the active site of ACCase, confirming the active conformation hypothesis and demonstrating that the precise placement of the carboxylate relative to the phenyl group is more critical than the placement of the methyl.

PMID: 12137475

... 甘氨酸可经3次甲基化而合成甜菜碱, 而乙酰辅酶A则参与了多种脂类的合成, 从而提高植物耐逆性^{[25,33,34,35,36]} ...

2009

0.0

... shtml)中分析结果也表明该基因与脂类代谢酶类的共表达值最高, 且与逆境转录因子MYB 家族中的MYB13 (At1g 06180)基因有关联(数据未显示), 乔孟等^[37]研究表明MYB13 基因受高盐胁迫诱导反应 ...

1999

23.268

0.0

Nat. Biotechnol.. 1999, 17(3):287-91

Improving plant drought, salt, and freezing tolerance by gene transfer of a single stress-inducible transcription factor.

Kasuga M, Liu Q, Miura S, Yamaguchi-Shinozaki K, Shinozaki K

Biological Resources Division, Japan International Research Center for Agricultural Sciences, Ministry of Agriculture, Forestry and Fisheries, Tsukuba, Ibaraki.

PMID: 10096298

... 在植物抗逆基因工程研究中, 大多使用组成型启动子来驱动目的基因的表达, 但Kasuga等^[38]报道组成型启动子CaMV35S驱动DREB 基因在拟南芥中表达, 虽提高了抗逆性, 但植株矮化、生长畸形, 而由诱导型启动子rd29A启动的DREB 基因在拟南芥中的表达不仅提高了植株的抗逆性, 而且避免了对生长的负面影响 ...

2010

0.0

0.9787

Hereditas. 2010, 32(3):229 - 234 DOI:10.3724/SP.J.1005.2010.00229

Plant stress-inducible promoters and their function

植物逆境相关启动子及功能

ZHU Li-Ping, YU Zhuang, ZOU Cui-Xia, LI Qiu-Li

朱丽萍, 于壮, 邹翠霞, 李秋莉

College of Life Sciences, Liaoning Normal University, Dalian 116029, China

Promoter is an important cis-regulatory element for gene expression and plays an important role in the process of plant gene expression and regulation. Constitutive promoters are being used to drive alien gene expression in most transgenic engineering. Although constitutive promoters can improve resistance of transgenic plants to abiotic stresses, over- and constitutive-expression of the alien genes have been shown to cause stunted growth and reduction of yield in transgenic plants. Therefore, inducible promoters, which are expressed only when exposed to stresses, are of importance. This paper reviews the types and functions of plant gene promoters induced by bio- and abio-stresses. The prospect of stress-induced promoters was discussed.

启动子是调控基因表达的重要顺式元件, 在植物基因表达调控过程中起着重要作用。目前植物抗逆基因工程中, 人们大多使用组成型表达启动子驱动目的基因的表达。组成型表达启动子虽然能提高转基因植株的抗逆性, 但是其持续过量地表达转化的外源基因会阻碍植物的生长且减少其产量。因此, 只在胁迫条件下才会驱动外源基因表达的诱导型启动子的研究显得尤其重要, 已成为目前研究的热点。文章综述了受非生物逆境和生物逆境胁迫诱导的植物基因启动子的种类和功能, 并展望了植物逆境诱导启动子的研究方向和前景。

... 朱丽萍等^[39]报道用组成型启动子CaMV35S驱动BADH 基因在烟草中表达, 转基因烟草的耐盐性提高了, 但生长延迟 ...

2004

1.653

0.0

... Pellegrineschi等^[40]用诱导型启动子rd29A驱动DREBA/CBF3 的表达, 提高了转基因小麦的耐旱性, 而且没有引起明显的畸形 ...